This study aimed to investigate the appearance and frequencies of the booroola polymorphism of the bone morphogenetic protein receptor 1b (BMPR1B) gene (FecB) and the embrapa polymorphism of the growth differentiation factor 9 (GDF9) gene (FecGE) in sheep in Thailand. A total of 454 cross-bred sheep blood samples were collected from four provinces in Thailand during August 2022 to July 2023. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to identify the FecB and FecGE genotype. The history of ewe birth types was collected from the owners to analyze the association between Fecundity (Fec) genotypes and the history of birth types. The genotypic frequencies of FecB for homozygous genotype (B/B), heterozygous genotype (+/B) and wild-type (+/+) were 0.22%, 1.54%, and 98.24%, respectively. Meanwhile, the genotypic frequencies of FecGE for homozygous genotype (E/E), heterozygous genotype (+/E), and wildtype (+/+) were 0.00%, 2.42%, and 97.58%, respectively. Furthermore, three ewes exhibited both FecB and FecGE genotypes. Fisher’s exact test revealed that the possessor of FecB genotype was associated with multiple births (p<0.01). Both FecB and FecGE mutations were identified in crossbred sheep in Thailand. Sheep containing FecB allele is a better candidate to be selected to improve the prolificacy of crossbred sheep in Thailand.

Objective: To evaluate the value of otolith‑associated protein otoconin‑90 (OC90) and otolin-1 in the pathogenesis research and clinical treatment of benign paroxysmal positional vertigo (BPPV). Material and Method: The study included 50 patients with BPPV admitted to neurology and otorhinolaryngology departments and 30 healthy subjects with no history of dizziness as control group. Results: BPPV and controls were similar in terms of gender and age. Otolin-1 level was significantly greater in the BPPV group than controls (710.44 vs 280.45 ; p<0.001). No statistical significance was found although OC90 were higher in the BPPV group than controls. There was a strong positive correlation between otolin-1 and OC90, a moderate negative correlation between otolin-1 and vitamin D, and a strong negative correlation between OC90 and vitamin D in BPPV patient group. Otolin-1 had high specificity and AUC values for BPPV (AUC: 0.933; 95% CI: 0.881-0.986, 79.2% sensitivity, 100% specificity with a cutoff greater than 525). Conclusion: High serum levels of otolin-1 were associated with an increased risk of BPPV, but not CO90. Serum levels of otolin-1 can potentially be used as a biomarker for acute onset of inner ear disorders due to its significant increase in patients with BPPV. Vitamin D has high specificity and sensitivity in the patients with BPPV patients. It also provides evidence that BPPV patients with vitamin D deficiency may improve their complaints with replacement therapy. More large-scale prospective studies is required to confirm these association and clarify the exact mechanism.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people and caused tremendous morbidity and mortality worldwide. Effective treatment for coronavirus disease 2019 (COVID-19) due to SARS-CoV-2 infection is lacking and different therapeutic strategies are under testing. Host humoral and cellular immunity to SARS-CoV-2 infection is a critical determinant for patients’ outcome. SARS-CoV-2 infection results in seroconversion and production of anti-SARS-CoV-2 antibodies. The antibodies may suppress viral replication through neutralization but also might also participate in COVID-19 pathogenesis through a process termed antibody-dependent enhancement. Rapid progress has been made in the research of antibody response and therapy in COVID-19 patients including characterization of the clinical features of antibody responses in different populations infected by SARS-CoV-2, treatment of COVID-19 patients with convalescent plasma and intravenous immunoglobin products, isolation and characterization of a large panel of monoclonal neutralizing antibodies, as well as preliminary clinical results from several COVID-19 vaccine candidates. In this review, we summarize the recent progress and discuss the implications of these findings in vaccine development.

Background & aims It is documented that low protein and amino-acid dietary intake is related to poorer cognitive health and increased risk of dementia. Degradation of the neuromodulatory pathways, (comprising the cholinergic, dopaminergic, serotoninergic and noradrenergic systems) is observed in neurodegenerative diseases and impairs the proper biosynthesis of key neuromodulators from micro-nutrients and amino acids. How these micro-nutrients are linked to neuromodulatory pathways in healthy adults is less studied. The Locus Coeruleus–Noradrenergic System (LC-NA) is the earliest subcortical structure affected in Alzheimer’s disease, showing marked neurodegeneration, but is also sensitive for age-related changes. The LC-NA system is critical for supporting attention and cognitive control, functions that are enhanced both by tyrosine administration and chronic tyrosine intake. The purpose of this study was to 1) investigate whether the dietary intake of tyrosine, the key precursor for noradrenaline (NA), is related to LC integrity 2) whether LC integrity mediates the reported association between tyrosine intake and higher cognitive performance (measured with Trail Making Test – TMT), and 3) whether LC integrity relates to an objective measure of brain maintenance (BrainPAD). Methods The analyses included 398 3T MRIs of healthy participants from the Berlin Aging Study II to investigate the relationship between LC integrity and habitual dietary tyrosine intake-daily average (HD-Tyr-IDA). As a control procedure, the same analyses were repeated on other main seeds of the neuromdulatory subcortical system (Dorsal and Medial Raphe, Ventral Tegmental Area and Nucleus Basalis of Meynert). In the same way, the relationships between the five nuclei and BrainPAD were tested. Results Results show that HD-Tyr-IDA is positively associated with LC integrity. Similarly, LC integrity disproportionally relates to better brain maintenance (BrainPAD). Mediation analyses reveal that only LC, relative to the other nuclei tested, mediates the relationship between HD-Tyr-IDA I and performance in the TMT and between HD-Tyr-IDA and BrainPAD. Conclusions These findings provide the first evidence linking tyrosine intake with LC-NA system integrity and its correlation with neuropsychological performance. This study strengthens the role of diet for maintaining brain and cognitive health and supports the noradrenergic theory of cognitive reserve. Within this framework, adequate tyrosine intake might increase the resilience of LC-NA system functioning, by preventing degeneration and supporting noradrenergic metabolism required for LC function and neuropsychological performance.

Background & Objective. Perinatal hypoxia acutely triggers the release of cytokines and chemokines from neurons, astrocytes and microglia. In response to hypoxia-ischemia resting/ramified microglia proliferate and undergo activation, with the production of proinflammatory molecules. The brain damage extension seems to be related with both the severity of hypoxia and the balance between pro and anti-inflammatory host response. Methods: In this preliminary study we compared in 10 neonates with hypoxic ischemic encephalopathy (HIE), during therapeutic hypothermia (TH), plasmatic cytokines and other inflammatory molecules levels: monocytic chemotactic-protein-1 (CCL2/MCP-1), CXCL8, glial fibrillary acidic protein (GFAP), Interferon gamma (IFN y), Interleukin 10 (IL-10), IL-18, IL-6, CCL3, ENOLASE2, Colony Stimulating Factor(GM-CSF), IL-1b, IL-12p70, IL-33, Tumor Necrosis Factor alpha(TNF), collected at four different time points during TH (24 hours, 25-48 hours, 49-72 hours) , and after the rewarming (7-10 days from birth). Five of enrolled babies had pathological brain MRI (“cases”) and 5 had a normal examination (“controls”). Cytokines were measured by Magnetic Luminex Assay (Luminex SV 013-17 (14), instrument Biorad Bio-Plex 100 s.n. LX 10005039304). MRI images were classified according to the Barkovich score. Results: Mean levels of all cytokines and molecules at time T1 were not significantly different in the two groups of patients. Comparing samples paired by day of collection, the greatest differences between cases and controls were found at times T2 and T3, during TH. After rewarming, some molecules showed an upward trend. At T4, levels tended to be close again (with the exception of IL-6, IL10 and IL18). Infants with worse MRI showed plasmatic GFAP levels higher than those with normal MRI, while their IL-18 was lower. Mean levels of CCL3MIP1alpha, GMCSF, IL1BETA overlapped throughout the observation period in both the groups of neonates. Conclusion. In children with worse brain MRI we found high levels of GFAP and of IL-10 at T4, considered respectively an early biomarker of brain damage and a predictor of adverse outcome, according to the literature, and a trend towards low IL-18 levels. We found the greatest, although not significant, difference between molecules levels in cases and controls at time points T2 and T3, during TH. With the rewarming some molecules showed an upward trend. We cannot exclude that the small sample size may prevent significance in the differences from being achieved.

The Leave-One-Out (LOO) Green Fluorescent Protein (GFP) approach to biosensor design com-bines computational protein design with split protein reconstitution. LOO-GFPs reversibly fold and gain fluorescence upon encountering its target peptide, which can be redefined by computa-tional design of the LOO site. Such an approach can be used to create reusable biosensors for the early detection of emerging biological threats. Enlightening biophysical inferences for nine LOO-GFP biosensor libraries are presented, with target sequences from dengue, influenza, or HIV, replacing beta strands 7, 8 or 11. An initially low hit rate was traced to components of the energy function, manifesting in the over-rewarding of over-tight side chain packing. Also, screening by colony picking required a low library complexity, but designing a biosensor against a peptide of at least 12 residues requires a high complexity library. This double-bind was solved using a "piecemeal" iterative design strategy. Also, designed LOO-GFPs fluoresced in the unbound state due to unwanted dimerization, but this was solved by fusing a fully functional prototype LOO-GFP to a fiber-forming protein, Drosophila ultrabithorax, creating a biosensor fiber. One in-fluenza hemagglutinin biosensor is characterized here in detail, showing a shifted excita-tion/emission spectrum, a micromolar affinity for the target peptide, and an unexpected pho-to-switching ability.

Papain-like protease PLpro, a domain within a large polyfunctional protein nsp3, plays key roles in the life cycle of SARS-CoV-2, being responsible for the first events of cleavage of a polyprotein into individual proteins (nsp1-4) as well as for the suppression of cellular immunity. Here, we developed a new genetically encoded fluorescent sensor, named PLpro-ERNuc, for detection of PLpro activity in living cells using translocation-based readout. The sensor was designed as follows. A fragment of nsp3 protein was used to direct the sensor on the cytoplasmic surface of endoplasmic reticulum (ER) membrane, thus closely mimicking the natural target of PLpro. The fluorescent part included two bright fluorescent proteins - red mScarlet I and green mNeonGreen, separated by a linker with the PLpro cleavage site. Nuclear localization signal (NLS) was attached to ensure accumulation of mNeonGreen into the nucleus upon cleavage. We tested PLpro-ERNuc in the model of recombinant PLpro expressed in HeLa cells. The sensor demonstrated expected cytoplasmic reticular network in the red and green channels in the absence of protease, and efficient translocation of the green signal into nuclei in the PLpro-expressing cells (14-fold increase of the nucleus/cytoplasm ratio). Then, we used PLpro-ERNuc in the model of Huh7.5 cells infected with SARS-CoV-2 virus, where it showed robust ER-to-nucleus translocation of the green signal in the infected cells 24 h post infection. We believe that PLpro-ERNuc represents a useful tool for screening PLpro inhibitors as well as for monitoring virus spread in a culture.

Sporadic inclusion body myositis (sIBM) is the most common muscle disease of older people and is clinically characterized by slowly progressive asymmetrical muscle weakness, predominantly affecting quadriceps, deep finger flexors and foot extensors. At present there are no enduring treatments for this relentless disease that eventually leads to severe disability and wheelchair dependency. Although sIBM is considered a rare muscle disorder, its prevalence is certainly higher as the disease is often undiagnosed or misdiagnosed. The histopathogical phenotype of sIBM muscle biopsy includes muscle fiber degeneration and endomysial lymphocytic infiltrates that mainly consist of cytotoxic CD8+ T cells surrounding nonnecrotic muscle fibers expressing MHCI. Muscle fiber degeneration is characterized by vacuolization and accumulation of congophilic misfolded multi-protein aggregates, mainly in their non-vacuolated cytoplasm. Many players have been identified in sIBM pathogenesis including environmental factors, autoimmunity, abnormalities of protein transcription and processing, accumulation of several toxic proteins, impairment of autophagy and ubiquitin proteasome system, oxidative and nitrative stress, endoplasmic reticulum stress, myonuclear degeneration, and mitochondrial dysfunction. Aging has been also proposed as a contributor to the disease. However, the interplay between these processes and the primary event that leads to the coexistence of autoimmune and degenerative changes is still under debate. Here, we outline our current understanding of disease pathogenesis, focusing on degenerative mechanisms, and discuss the possible involvement of aging.

Plant-based proteins, like those derived from hemp and rapeseed can significantly contribute to a balanced diet and human daily nutritional requirements by providing essential nutrients such as protein, fiber, vitamins, minerals, and antioxidants. According to numerous recent research papers the consumption of plant-based proteins has been associated with numerous health benefits, including reduced risk of chronic diseases such as heart disease, diabetes, and certain cancers. Plant-based diets are often lower in saturated fat and cholesterol and higher in fiber and phytonutrients, which can support overall health and well-being. Present research investigates the nutritional attributes, functional properties, and potential food applications of hemp and rapeseed protein for a potential use in new food product development, with a certain focus on identifying anti-nutritional factors and bioactive compounds. Through comprehensive analysis, anti-nutritional factors and bioactive compounds were elucidated, shedding light on their impact on protein quality and digestibility. The study also delves into the functional properties of hemp and rapeseed protein, unveiling their versatility in various food applications. Insights from this research contribute to a deeper understanding of the nutritional value and functional potential of hemp and rapeseed protein, paving the way for their further utilization in innovative food products with enhanced nutritional value and notable health benefits.

Immobilized metal affinity chromatography (IMAC) is a popular and valuable method for the affinity purification of polyhistidine-tagged recombinant proteins. However, it often shows practical limitations, which might require cumbersome optimizations, additional polishing, and enrichment steps. Here, we present functionalized corundum particles for the efficient, economical, and fast purification of recombinant proteins in a column-free format. The corundum surface is first derivatized with the amino silane APTES, then EDTA dianhydride, and subsequently loaded with nickel ions. The Kaiser test, well-known in solid-phase peptide synthesis, was used to monitor amino silanization and the reaction with EDTA dianhydride. In addition, ICP-MS was performed to quantify the metal-binding capacity. His-tagged protein A/G (PAG), mixed with bovine serum albumin (BSA), was used as a test system. The PAG binding capacity was around 3 mg protein per gram of corundum. Cytoplasm obtained from different E. coli strains was examined as an example of a complex matrix. The imidazole concentration was varied in the loading and washing buffers. As expected, higher imidazole concentrations during loading are usually beneficial when higher purities are desired. Even when higher sample volumes, such as one liter, were used, recombinant protein down to a concentration of 1 µg/mL could be isolated selectively. Comparing the corundum material with standard Ni-NTA agarose beads indicated higher purities of proteins isolated using corundum. His6-MBP-mSA2, a fusion protein consisting of monomeric streptavidin and maltose-binding protein in the cytoplasm of E. coli, was purified successfully. To show that this method is also suitable for mammalian cell culture supernatants, purification of the SARS-CoV-2-S-RBD His8 expressed in human Expi293F cells was performed. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents for 1 gram of functionalized support or 10 cents per milligram of isolated protein. Another advantage of the novel system is the corundum particles' extremely high physical and chemical stability. The new material should be applicable in small laboratories and large-scale industrial applications. In summary, we could show that this new material is an efficient, robust, and cost-effective purification platform for the purification of His-tagged proteins, even in challenging, complex matrices and large sample volumes of low product concentration.

Neurodegenerative diseases of protein misfolding affect humans and animals. In humans, these diseases include Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Western Pacific amyotrophic lateral sclerosis and Parkinsonism-dementia complex (ALS/PDC). Mineral exposure may be important in the pathogenesis of protein misfolding cascades. The possible association of bentonite, montmorillonite, and mineral risk factors with Alzheimer’s disease, Parkinson’s disease, ALS, and Western Pacific ALS/PDC is analyzed and discussed.

In addition to the canonical loss-of-function mutations, mutations in proteins may additionally result in gain-of-function through the binary activation of cryptic ‘structural capacitance elements’. Our previous bioinformatic analysis allowed us to propose a new mechanism of protein evolution - structural capacitance – that arises via the generation of new elements of microstructure upon mutations that cause a disorder-to-order (DO) transition in previously disordered regions of proteins. Here we propose that the DO transition is a necessary follow-on from expected early codon-anticodon and tRNA acceptor stem-amino acid usage, via the accumulation of structural capacitance elements - reservoirs of disorder in proteins. We develop this argument further to posit that structural capacitance is an inherent consequence of the evolution of the genetic code.

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolizing cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L) identified by genome sequencing has been linked to lower testosterone levels and reduced P450 activities. We expressed POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant reduction of small molecule metabolism was observed with POR P228L while activities of CYP3A4 were reduced by 25%, and activities of CYP3A5, and CYP2C9 were reduced by more than 40% compared to WT POR. The 17,20 lyase activity of CYP17A1 responsible for production of main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in presence of P228L variant of POR. Based on in silico and in vitro studies we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterization of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.

Background: The rise of new SARS-CoV-2 mutations brought challenges and progress in the global fight against COVID-19. Mutations in spike and accessory genes affect transmission, vaccine efficacy, treatments, testing, and public health strategies. Monitoring emerging variants is crucial to halt virus spread. Methods: 44 nasopharyngeal/oropharyngeal swabs from Kenyan patients were sequenced with the Illumina platform. Galaxy's bioinformatic tools were used for genomic analysis. SARS-CoV-2 genome classification was done using PANGOLIN and mutation annotation with the COVID-19 Annotator tool. Results: The study showed 5 clades to be circulating in the region. 38(86%) were BA.1.1; 2(5%) were BA.1.1.1; 1(2%) was BA.1; 1(2%) was BA.1.14 and 2(5%) were AY.46. These clades had a cumulative of 173 mutations among them with 50 novel mutations. Forty-eight of these novel mutations occurred in a low frequency of 2.3% of the sequences tested while the other two, S:R214R, and NSP2:A555A, were for 43.2% and 18.2% of the cases respectively. Conclusions: The high-frequency novel mutations were synonymous mutations, a phenomenon that was previously viewed as phenotypically silent but recent studies indicate they can affect viral fitness with potential functional associations. These findings add to the understanding of the SARS-CoV-2 virus future evolutionary and immunological dynamics in the region

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, yet there is still much to be learnt on how NAGK is regulated in vivo. The use of unicellular green Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provided primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation.

The emergence of chemoresistance in neoplastic cells is one of the major obstacles in cancer therapy. Autophagy was recently reported as one of the mechanisms that promote chemoresistance in cancer cells by protecting from apoptosis and driving senescence. Thus, understanding the role of autophagy and its underlying signaling pathways is crucial for the development of new therapeutic strategies to overcome chemoresistance. We have previously reported that PKCη is a stress-induced kinase that confers resistance in breast cancer cells against chemotherapy by inducing senescence. Here we show that PKCη promotes autophagy induced by ER and oxidative stress and facilitates the transition from autophagy to senescence. We demonstrate that PKCη knockdown reduces both the autophagic flux and markers of senescence. Additionally, using autophagy inhibitors, such as chloroquine and 3-methyladenine, we show that PKCη and autophagy are required for establishing senescence in MCF-7 in response to oxidative stress. Different drugs used in the clinic are known to induce autophagy and senescence in breast cancer cells. Our study proposes PKCη as a target for therapeutic intervention, acting in synergy with autophagy-inducing drugs, to overcome resistance and enhance cell death in breast cancer.

Multidrug resistance protein 4 (MRP4) is an energy-dependent membrane transporter that is responsible for cellular efflux of a broad range of xenobiotics and physiological substrates. In this trial, we aimed to investigate the co-effects of aging and MRP4 deficiency using gene expression microarray and morphological and electrophysiological analyses of the mouse retina. Mrp4-knockout (null) mice and wild-type (WT) mice were reared in the same condition to 8–12 wk (young) or 45–55 wk (aged). Microarray analysis identified 186 differently expressed genes from the retinas of aged Mrp4-null mice as compared to that from aged WT mice, and subsequence gene ontology and KEGG pathway analyses showed that differently expressed genes were related to lens, eye development, vision, and transcellular barrier function that are involved in metabolic pathways or viral infection pathways. No significant change in thickness was observed for each retinal layer among young/aged WT mice and young/aged Mrp4-null mice. Moreover, immunohistochemical analyses of retinal cell type did not exhibit an overt change in the cellular morphology or distribution among the 4 age/genotype groups, and the electroretinogram responses showed no significant differences in the amplitude or the latency between aged WT mice and aged Mrp4-null mice. Aging would be an insufficient stress to cause some damage to the retina in the presence of MRP4 deficiency.

Centromeres, the chromosomal loci where spindle fibers attach during cell division to segregate chromosomes, are typically found within satellite arrays in plants and animals. Satellite arrays have been difficult to analyze because they comprise megabases of tandem head-to-tail highly repeated DNA sequences. Much evidence suggests that centromeres are epigenetically defined by the location of nucleosomes containing the centromere-specific histone H3 variant cenH3, independently of the DNA sequences where they are located; however, the reason that cenH3 nucleosomes are generally found on rapidly evolving satellite arrays has remained unclear. Recently, long read sequencing technology has clarified the structures of satellite arrays and sparked rethinking of how they evolve, while new experiments and analyses have helped bring both understanding and further speculation about the role these highly repeated sequences play in centromere identification.

Heavy metal contamination of drinking water is a public health concern that requires the development of more efficient bioremediation techniques. Absorption technologies, including biosorption, provide opportunities for improvements to increase the diversity of metal ions removed and overall binding capacity. Microorganisms are a key component in wastewater treatment plants and they naturally bind metal ions through surface macromolecules but with limited capacity. The long-term goal of this work is to engineer capsule polymerases to synthesize molecules with novel functionalities. In previously published work, we showed that the Neisseria meningitidis serogroup W (NmW) galactose-sialic acid (Gal—NeuNAc) heteropolysaccharide binds lead effectively, thereby demonstrating the potential for using this capsular polysaccharide in environmental decontamination applications. In this study, computational analysis of the NmW capsule polymerase galactosyltransferase (GT) domain was used to gain insight into how the enzyme could be modified to enable the synthesis N-acetylgalactosamine-sialic acid (GalNAc—NeuNAc) heteropolysaccharide. Various computational approaches, including molecular modeling with I-TASSER and molecular dynamics simulations (MD) with NAMD, were utilized to identify key amino acid residues in the substrate binding pocket of the GT domain that may be key to conferring UDP-GalNAc specificity. Through these combined strategies and using BshA, a UDP-GlcNAc transferase, as a structural template, several NmW active site residues were identified as mutational targets to accommodate the proposed N-acetyl group in UDP-GalNAc. Thus, a rational approach for potentially conferring new properties to bacterial capsular polysaccharides is demonstrated.

People with specific TREM2 gene variants are more prone to develop Alzheimer's disease (AD). The TREM2 receptor regulates the number of myeloid cells, phagocytosis, and the inflammatory response via interacting with apolipoproteins and amyloid. Higher TREM2 expression has been found to protect against AD. When TREM2 activity increases, the activity of genes involved in the activation of microglia cells decreases. This can improve the efficiency of phagocytosis. When TREM2 is highly expressed and the inflammasome is activated, the results are not always congruent. Therefore, this study aimed to discover how sTREM2 levels in CSF and plasma samples relate to other indices of AD pathology. We examined 98 AD plasma samples, 35 plasma samples of subjects with mild cognitive impairment (MCI), 11 healthy controls (HC) plasma samples, as well as 155 AD CSF samples, 90 MCI CSF samples, and 50 HC CSF samples. CSF sTREM2 levels were higher in the AD group than in the MCI and HC groups, in contrast to plasma sTREM2. This shows that CSF sTREM2 levels could be used to distinguish between healthy and AD patients. CSF sTREM2 levels were significantly correlated with neurofibrillary changes, cognitive decline, and inflammasome activity in AD patients. While our findings are consistent with previous research, future studies will need to include more patients and employ standardized methodological approaches to add CSF sTREM2 to the list of biomarkers for AD.

Our previous studies demonstrated that quercetin (Q) could be ingested and metabolized by macrophages and exerted prophylactic immuno-stimulatory activity and therapeutic anti-inflammatory effects on lipopolysaccharide (LPS)-treated macrophages ex vivo. To further clarify its possible anti-inflammatory mechanism, Q was selected to treat mouse peritoneal macrophages that obtained from female BALB/c mice exposed to LPS i.p. for 12 h. Relative gene expression of pro-/anti-inflammatory (TNF-α/IL-10) cytokines and components of inflammation-related intracellular signaling pathways (TLR2, TLR4, NF-κB, JAK2 and STAT3) was analyzed using two-step reverse transcription (RT) and real-time quantitative polymerase chain reaction (qPCR). STAT3 protein phosphorylation was determined using an in-cell ELISA method. As a result, Q and its metabolite quercetin-3-O-β-D-glucuronide (Q3G) decreased TNF-α gene expression amounts and ratios of pro-/anti-inflammatory (TNF-α/IL-10) cytokine gene expressions, but increased IL-10 gene expression amounts in activated inflammatory macrophages, supporting a substantial anti-inflammatory potential of Q and Q3G treatments. However, Q3G had lower effects than those of Q. Importantly, Q inhibited TLR2 gene expression and phosphorylation of STAT3 protein in the inflamed cells. Our results are the first report to suggest that Q inhibits LPS-induced inflammation ex vivo through suppressing TLR2 gene expression and STAT3 protein phosphorylation in activated inflammatory macrophages. Q has potential to further apply for treating inflammation-associated diseases.

Global warming poses a threat to plant survival, impacting growth and agricultural yield. Protein turnover, a critical regulatory mechanism balancing protein synthesis and degradation, is crucial for cellular response to environmental changes. We investigated the effects of elevated temperature on proteome dynamics in Arabidopsis thaliana seedlings using 15N-stable isotope labeling and ul-tra-performance liquid chromatography-high resolution mass spectrometry, coupled with the ProteinTurnover algorithm. Analyzing different cellular fractions from plants grown under 22°C and 30°C growth conditions, we found significant changes in the turnover rates of 571 proteins, with a median 1.4-fold increase, indicating accelerated protein dynamics under thermal stress. Notably, soluble root fraction proteins exhibited smaller turnover changes, suggesting tissue-specific adap-tations. Significant turnover alterations occurred with redox signaling, stress response, protein folding, secondary metabolism, and photorespiration, indicating complex responses enhancing plant thermal resilience. Conversely, proteins involved in carbohydrate metabolism and mito-chondrial ATP synthesis showed minimal changes, highlighting their stability. This analysis high-lights the intricate balance between proteome stability and adaptability, advancing our under-standing of plant responses to heat stress and supporting the development of improved thermo-tolerant crops.

Breast cancer (BC) is among the leading causes of mortality from cancer in women. Many of the available anticancer drugs have various side effects. Therefore, researchers are seeking novel anticancer agents particularly from natural compounds and in this regard, snake venom is still one of the main sources of drug discovery. Previous studies showed potential anticancer effects of Cytotoxin II (CTII) from Naja naja oxiana against the different types of cancers. In this study, a pET-SUMO-CTII vector was transformed into SHuffle® T7 Express, an Escherichia coli strain, for recombinant protein expression (rCTII) and the cytotoxic effects of this protein was assessed in MCF-7 cells. The flow cytometry assay was applied to measure the apoptosis and cell cycle. Also, mRNA levels of the Bax, Bcl2, P53, caspase-3, caspase-8, caspase-9, caspase-10, matrix metalloproteinases (MMP)-3, and MMP-9 were analyzed by quantitative real-time PCR to determine the underlying cellular pathways affected by rCTII. The results of this study showed that treatment with 4 µg mL-1 of rCTII enhanced apoptosis through the intrinsic and extrinsic pathways. Also, the increase of the cells' proportion in the sub-G1 phase as well as a reduction in S phase was observed. In addition, the expression of MMP-3 and MMP-9 was decreased in the treated group in comparison to the control group that may contribute to the reduced migratory ability of tumor cells. These experimental results indicate that rCTII has anti-proliferative potential, and so this protein could be a potential drug for BC therapy in combination with other drugs.

Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-years old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis, and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation.

Renal clear cell carcinoma (ccRCC) comprises over 75% of all renal tumors and arises in the epithelial cells of the proximal convoluted tubule. Molecularly ccRCC is characterized by copy number alterations (CNAs) such as the loss of chromosome 3p and VHL inactivation. Additional driver mutations (SETD2, PBRM1, BAP1, and others) promote genomic instability and tumor cell metastasis through the dysregulation of various metabolic and immune-response pathways. Many researchers identified mutation, gene expression, and proteomic signatures for early diagnosis and prognostics for ccRCC. Despite a tremendous influx of data regarding DNA alterations, gene expression, and protein expression, the incorporation of these analyses for diagnosis and prognosis of RCC into the clinical application has not been implemented yet. In this review, we focused on the molecular changes associated with ccRCC development, along with gene expression and protein signatures, to emphasize the utilization of these molecular profiles in clinical practice. These findings, in the context of machine learning and precision medicine, may help to overcome some of the barriers encountered for implementing molecular profiles of tumors into the diagnosis and treatment of ccRCC.

: Emaravirus (Order Bunyavirales; Family Fimoviridae) is a genus comprising over 20 emerging plant viruses with a worldwide distribution and economic impact. Emaraviruses infect a variety of host plants and have especially become prevalent in important long-living woody plants. These viruses are enveloped, with a segmented, single-stranded, negative-sense RNA genome and are transmitted by eriophyid mites or mechanical transmission. Emaraviruses have four core genome segments encoding an RNA-dependent RNA polymerase, a glycoprotein precursor, a nucleocapsid protein, and a movement protein. They also have additional genome segments, whose number varies widely. We report here that the proteins encoded by these segments form three main homology groups: a homolog of the sadwavirus Glu2 Pro glutamic protease; a protein involved in pathogenicity, which we named “ABC”; and a protein of unknown function, which we named “P55”. The distribution of these proteins parallels the emaravirus phylogeny and suggests, with other analyses, that emaraviruses should be split into at least two genera. Reliable diagnosis systems are urgently needed to detect emaraviruses, assess their economic and ecological importance, and take appropriate measures to prevent their spread (such as routine testing, hygiene measures, and control of mite vectors). Additional research needs include understanding the function of emaravirus proteins, breeding resistant plants and clarifying transmission modes.

C-reactive protein to lymphocyte ratio (CLR) has been shown to be associated with diseases characterized by chronic, low-grade inflammation. Since type 2 diabetes (T2DM) is also associated with inflammation, we aimed to study the association between CLR and T2DM. Patients with T2DM who presented to internal medicine outpatient clinics of our institution were divided into 2 groups according to their glycosylated hemoglobin (HbA1c) levels as well-controlled (HbA1c&lt;7%) and poorly controlled (HbA1c≥7%) T2DM groups. Subjects assigned as healthy in routine check-up were included as control group. CLR values of the well and poorly controlled diabetics and control cases were compared. CLR of T2DM group (3.51 (0.03-21.78)) was significantly higher than that of the controls (0.65 (0.02-2.92)) (p&lt;0.001). CLR was found to have a sensitivity of 63.2% and a specificity of 97.3% in predicting T2DM. The CLR value of patients with poor diabetic control was 4.76 (0.06-21.78), while the CLR value of patients with well controlled disease was 2.53 (0.03-12.07) (p&lt;0.001). The sensitivity and specificity of the CLR in demonstrating poor diabetic control was 41.2% and the 86.1%, respectively. In conclusion, elevated CLR in T2DM patients and even more increased levels in poorly controlled diabetics suggest that CLR could be a useful additional diagnostic tool in treatment follow-up of the T2DM population.

Evidence shows that weightloss improves insulin sensitivity but few studies have examined the effect of macronutrient composition independently of weight loss on direct measures of insulin sensitivity. We randomised 89 overweight or obese women to either a standard diet (StdD) that was intended to be low in fat and relatively high in carbohydrate (n=42) or to a relatively high protein (up to 30% of energy), relatively high fibre (>30g/day) diet (HPHFib) (n=47) for 10 weeks. Advice regarding strict adherence to energy intake goals was not given. Insulin sensitivity and secretion was assessed by a novel method - the Dynamic Insulin Sensitivity and Secretion Test (DISST). Although there were significant improvements in body composition and most cardiometabolic risk factors on HPHFib, insulin sensitivity was reduced by 19.3% (95% CI: 31.8, 4.5%; p=0.013) in comparison with StdD. We conclude that the reduction in insulin sensitivity after a diet relatively high in both protein and fibre, despite cardiometabolic improvements, suggests insulin sensitivity may reflect metabolic adaptations to dietary composition for maintenance of glucose homeostasis, rather than impaired metabolism.

Trauma to the spinal cord causes extensive neuronal death contributing to the loss of sensory-motor and autonomic functions below the injury level. Apoptosis affects neurons after spinal cord injury (SCI) and is associated with increased caspase activity. Cleavage of X-linked inhibitor of apoptosis protein (XIAP) after SCI may contribute to this rise of caspase activity. Accordingly, we have shown that the elevation of XIAP resulted in increased neuronal survival after SCI and improved functional recovery. Therefore, we hypothesize that neuronal overexpression of XIAP can be neuroprotective after SCI with improved functional recovery. In line with this, studies of a transgenic mouse with overexpression of XIAP in neurons revealed that higher levels of XIAP after spinal cord trauma favours neuronal survival, tissue preservation, and motor recovery after the spinal cord trauma. Using the human SH-SY5Y cells overexpressing XIAP we show further that XIAP reduced caspase activity and apoptotic cell death after pro-apoptotic stimuli. In conclusion, this study shows that the levels of XIAP expression are an important factor for the outcome after spinal cord trauma and identifies XIAP as an important therapeutic target for alleviating the deleterious effects of SCI.

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. We present here a shot-gun differential proteomic analysis on roots of two pldα1 mutants compared to the Col-0 wild type. Our data suggest new roles of PLDα1 in endomembrane transport, mitochondrial protein import and protein quality control and glucosinolate biosynthesis. Thus, we identified proteins involved in endocytosis, endoplasmic reticulum-Golgi transport and attachment sites of endoplasmic reticulum and plasma membrane (V-type proton ATPases, protein transport protein SEC13 homolog A, vesicle-associated protein 1-2, vacuolar protein sorting-associated protein 29, syntaxin-32, all upregulated in the mutants), mitochondrial import and electron transport chain (mitochondrial import inner membrane translocase subunits TIM23-2 and TIM13, mitochondrial NADH dehydrogenases, ATP synthases, cytochrome c oxidase subunit 6b-1, ADP,ATP carrier protein 2, downregulated in the mutants) and glucosinolate biosynthesis (3-isopropylmalate dehydrogenases 1, 2 and 3, methylthioalkylmalate synthase 1, cytochrome P450 83B1, Glutathione S-transferase F9, indole glucosinolate O-methyltransferase 1, adenylyl-sulfate kinase 1, all upregulated in mutants). Our results suggest broader biological roles of PLDα1 as anticipated so far.

The present study adopted a diverse approach to obtain antioxidant peptides from red seaweed, starting with an aqueous extraction of the entire seaweed followed by enzymatic hydrolysis of the solid residues from the first step. Additionally, the impact of three different pH levels (i.e. 3, 6, and 9) during the aqueous extraction phase was examined for their influence on the outcomes. The findings revealed that the solid fraction resulting from the sequential extraction process contained significantly higher levels of proteins and amino acids compared to other fractions (p < 0.05). Furthermore, the solid fractions (IC50 ranging from 2.29 to 8.15) proved to be significantly more effective free radical scavengers than the liquid fractions (IC50 either ranging from 9.03 to 10.41 or not obtained at the highest concentration tested) at both stages of extraction (p < 0.05). Among the solid fractions, those produced under alkaline conditions did not exhibit as potent radical scavenging abilities as those produced under acidic or nearly neutral conditions. The most effective metal ion chelating activity was found in the solid fractions derived from the subsequent enzymatic stage, particularly at pH 3 (IC50 = 0.63 ± 0.04) and pH 6 (IC50 = 0.89 ± 0.07), which were significantly more potent than those from the initial extraction stage (p < 0.05). Given that no significant difference was noted in the total phenolic content of these solid fractions compared to their corresponding liquid fractions (3.79 ± 0.05 vs. 3.48 ± 0.02 at pH 3 and 2.43 ± 0.22 vs. 2.51 ± 0.00 at pH 6) (p > 0.05), the demonstrated properties might be attributed to the potential roles of bioactive amino acids such as histidine, glutamic acid, aspartic acid, tyrosine, and methionine, whether present as free amino acids or integrated within the structure of proteins and peptides.

Ubiquitin-specific protease 2 (USP2) maintains mitochondrial integrity in culture myoblasts. In this study, we investigated molecular mechanisms underlying the protective role of USP2 on mitochondria. Knockout (KO) of the Usp2 gene or chemical inhibition of USP2 induced robust accumulation of mitochondrial reactive oxygen species (ROS), accompanied by defects in mitochondrial membrane potential, in C2C12 myoblasts. ROS removal by N-acetyl-L-cysteine restored the mitochondrial dysfunction induced by USP2 deficiency. Comprehensive RT-qPCR screening and following protein analysis indicated that both genetic and chemical inhibition of USP2 elicited a decrease of uncoupling protein 2 (UCP2) at mRNA and protein levels. Accordingly, the introduction of a Ucp2-expressing construct effectively recovered mitochondrial membrane potentials, entailing an increment of the intracellular ATP level in Usp2KO C2C12 cells. In contrast, USP2 deficiency also decreased peroxisome proliferator-activated receptor  coactivator 1 (PGC1 protein in C2C12 cells, while it upregulated Ppargc1a mRNA. Overexpression studies indicate that USP2 potentially stabilizes PGC1 in an isopeptidase-dependent manner. Given that PGC1 is an inducer of UCP2 in C2C12 cells, USP2 might ameliorate mitochondrial ROS by maintaining the PGC1–UCP2 axis in myoblasts.

The SARS-CoV-2 nucleocapsid protein (N protein) is critical to viral replication by undergoing liquid-liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in also suppressing both stress granules and processing bodies which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological neuronal and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependant on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modelling suggests that the biomolecular condensate that formed in the presence of RNA is composed of a N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated.

Introduction: This population-based retrospective cohort study assesses rates of adverse events (AEs) involving cerebral thromboembolism (CTE) after COVID-19 vaccines. Methods: Data were collected from the U.S. Centers for Disease Control and Prevention (CDC) and Food and Drug Administration (FDA) Vaccine Adverse Event Reporting System (VAERS) database from January 1, 1990 to December 31, 2023. CTE AEs after COVID-19 vaccines were compared to those after influenza vaccines and after all other vaccines using proportional reporting ratio (PRR) analysis by time. Results: There are 5137 cerebral thromboembolism AEs reported in the 3 years (36 months) after COVID-19 vaccines compared to 52 AEs for the influenza vaccines over the past 34 years (408 months) and 282 AEs for all other vaccines (excluding COVID-19) over the past 34 years (408 months). The PRR’s are significant when comparing AEs by time from COVID-19 vaccines to that of the influenza vaccines (p < 0.0001) or to that of all other vaccines (p < 0.0001). The CTE AEs PRR by time (95% confidence intervals) for the COVID-19 vaccine AEs vs influenza AEs is 1120 (95% confidence interval (723-1730), p < 0.0001) and for COVID-19 vaccines vs all others is 207 (95% confidence interval (144-296), p < 0.0001). Cerebral venous thromboembolism AEs are female predominant with a female/male odds ratio of 1.63 (95% confidence interval (1.52-1.74), p < 0.0001). Conversely, cerebral arterial thromboembolism has a nonsignificant male preponderance. Cerebral venous thromboembolism is far more common than cerebral arterial thromboembolism over 36 months with an odds ratio (OR) of 14.8 (95% confidence interval 14.0-15.5, p < 0.0001). Atrial fibrillation, the most common identifiable cause of cerebral arterial thromboembolism, occurs far more commonly after the COVID-19 as compared to all other vaccines with a PRR of 123 (95% CI 88.3-172, p < 0.0001) Conclusions: There is an alarming breach in the safety signal threshold concerning cerebral thrombosis AEs after COVID-19 vaccines compared to that of the influenza vaccines and even when compared to that of all other vaccines. An immediate global moratorium on the use of COVID-19 vaccines is necessary with an absolute contraindication in women of reproductive age.

Amino acid sequences of native proteins are generally not palindromic. The protein molecule obtained as a result of reading the amino acid sequence backwards, i.e., a retro-protein, having the same amino acid composition and the same hydrophobicity profile as the native sequence may behave as a different molecule. Here we have used R4, a directed evolution product of Scfv-13, a protein that binds β-galactosidase, and report the properties of the retro-R4 (rR4). rR4 remains a binding protein and its new target is Glutamine-fructose-6-phosphate aminotransferase (GLMS), a native E. coli protein. Additionally, a tight junction immunoglobulin domain- containing membrane protein, junctional adhesion molecule A (JAMA) retro-protein retains the ability to drive cell-cell interactions. Thus, protein engineering suggests a new potential application to retro-proteins of the immunoglobulin superfamily.

Vaccines are one of the most effective medical interventions, playing a pivotal role in treating infectious diseases. Although traditional vaccines comprising of killed, inactivated, or live attenuated pathogens have resulted in protective immune responses, negative consequences of their administration have been well-appreciated. Modern vaccines have evolved to contain purified antigenic proteins, epitopes, or antigen-encoding mRNAs, rendering them relatively safe. However, reduced humoral and cellular responses pose major challenges to modern vaccine platforms. Protein nanoparticle (PNP)-based vaccines have garnered substantial interest in recent years for their ability to present repetitive array of antigens for improving immunogenicity and enhancing protective responses. Discovery and characterization of naturally occurring PNPs from various living organisms such as bacteria, archaea, viruses, insects, and eukaryotes, as well as computationally designed structures and approaches to link antigens to the PNPs have paved the way for unprecedented advances in the field of vaccine technology. In this review, we focus on some of the widely used naturally occurring and optimally designed PNPs for their suitability as promising vaccine platforms for displaying native-like antigens from viral pathogens for protective immune responses. Such platforms hold great promise in combating emerging and re-emerging infectious diseases and enhancing vaccine efficacy and safety.

The severe and complex COVID-19 symptoms, often needing hospital care, occur suddenly, late in the virus replication stage of SARS-CoV-2 infection. Hospitalised patients have tissue damage due to micro-clots at multiple sites. Could long-covid, also clinically variable, be a milder form caused by “nano-clots”, too small to cause detectable tissue damage? The timing and suddenness of the onset of serious disease needs an explanation. A paper reported on the first 20 children needing urgent hospital care. No child had any symptoms prior to suddenly becoming seriously ill, only one had a positive PCR test but all had positive SARS-CoV-2 antibody test. That finding indicated to me that this onset occurred at the moment of sero-conversion. An emerging viral moiety, the virion-IgG-antibody complex, has concentrations rapidly increasing, then decreasing. It is known that influenza-IgG-antibody-complex consists of many virions bound together by the antibody. It seems a reasonable step to propose that SARS-2, being a large virus and having many spike proteins, can form larger clumps of virus-IgG-antibody complexes than with influenza, the former being sufficiently large to initiate micro-clots. Therefore, my hypotheses is that the SARS-CoV-2 virion-antibody complex initiates micro-clot formation. Either before or after sero-conversion, the complex will be at too low a concentration to initiate micro-clots. To be useful, a hypothesis should be testable, provide explanations and give predictions. My presentation will give examples. I like the way that several apparently separate pieces of a jig-saw puzzle have come together to form a single picture. [I suggest that the reader scrolls down to the addenda [10,11 & 12] before reading the ICAR Poster]

Mutations of PKD1 coding for polycystin-1 (PC1) account for most of the autosomal dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these is the leucine-rich repeats (LRR) in the far N-terminus of PC1. Using zebrafish (Danio rerio) as an in vivo model system, we explored the role of LRR in the function of PC1. Zebrafish expresses two human PKD1 paralogs, pkd1a and pkd1b. Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal axis curvature and pronephric cyst formation. We found that overexpression of LRR suppressed both phenotypes in pkd1-morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5β1γ1) in vitro. Mutations of amino acid residues in LRR structurally predicted to bind laminin-511 disrupted LRR-laminin interaction in vitro and neutralized the ability of LRR to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1-deficiency.

Oncostatin M (OSM), an inflammatory cytokine belonging to interleukin-6 superfamily, is recognized as an essential factor secreted by osteal macrophages to orchestrate bone remodelling. Osteoprotegerin (OPG) produced by osteoblasts regulates osteoclastogenesis. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates OPG synthesis in osteoblast-like MC3T3-E1 cells, and that SMAD1/5/8(9), p38 mitogen-activated protein kinase (MAPK), and p70 S6 kinase positively regulates the OPG synthesis. The present study is aimed to investigate the effect of OSM on BMP-4-stimulated OPG synthesis in osteoblasts. OSM suppressed the release and the mRNA expression of OPG upregulated by BMP-4 in MC3T3-E1 cells.  Neither the BMP-4-induced phosphorylation of SMAD1/5/9 nor that of p38 MAPK affected by OSM. On the other hand, the BMP-4-stimulated phosphorylation of p70 S6 kinase was considerably suppressed by OSM. These results strongly suggest that OSM suppresses the BMP-4-stimulated OPG synthesis via inhibition of the p70 S6 kinase-mediated pathway in osteoblasts.

Serine proteases regulate cell functions through G protein-coupled protease-activated receptors (PARs). Cleavage of one peptide bond of the receptor amino terminus results in the formation of a new N-terminus ("tethered ligand") that can specifically interact with the second extracellular loop of the PAR receptor and activate it. Activation of PAR1 by thrombin (canonical agonist) and activated protein C (APC, noncanonical agonist) was described as biased agonism. Here we have supposed that synthetic peptide analogues to the PAR1 tethered ligand liberated by APC could have neuroprotective effects like APC. To verify this hypothesis a model of the ischemic brain impairment based on glutamate (Glu) excitotoxicity in primary neuronal cultures of neonatal rats has been used. It was shown that nanopeptide NPNDKYEPF-NH2 (AP9) effectively reduced the neuronal death induced by Glu. The influence of AP9 on cell survival was comparable to that of APC. Both APC and AP9 reduced the dysregulation of intracellular calcium homeostasis in cultured neurons induced by excitotoxic Glu (100 µM) or NMDA (200 µM) concentrations. PAR1 agonist synthetic peptides might be a noncanonial PAR1-agonist and a basis of novel neuroprotective drugs for disorders related to Glu excitotoxicity such as brain ischemia, trauma and some neurodegenerative diseases.

A defining feature of a productive infection is the co-opting of host cell resources for viral replication. Despite their repertoire of molecular functions, viruses subvert host defenses to take control of cellular factors such as RNA binding proteins (RBPs). RBPs are involved in virtually all steps of mRNA life forming ribonucleoprotein complexes (mRNPs) in a highly ordered and regulated process to control RNA fate and stability in the cell. Thus, the hallmark of this viral takeover is to reshape RNA fate in the cell to modulate host gene expression and evade immune responses. Here we provide an extensive review of work in this area, particularly how the host-viral interplay influences RBP functions to modulate the host cell. Overall, in this review, we highlight the myriad of ways RBPs can regulate RNA stability in either a pro-viral or antiviral manner by gathering novel insights gained from research studies in this field.

Autoantibodies (Abs) to thyroid antigens are associated with autoimmune thyroid disease (AITD), although sometimes they have no clear clinical significance. The aim of this study was to evaluate the avidity of thyroid Abs in the sera of AITD patients and non-AITD Abs carriers. A multiplex microarray-based assay was developed to detect the avidity of thyroid disease-associated Abs, including Abs to thyroperoxidase (TPO), thyroglobulin (Tg), pendrin, thyroid hormones T3 and T4, pyruvate kinase, and carbonic anhydrase 2. The method is based on indirect multiplex assay with an additional step involving the chaotrope destruction of the autoantibody-antigen complexes. The avidity index (AI) is then calculated. The method was tested on clinical cohort (n =117) and then confirmed by studies with another independent cohort (n =123). For the confirmation study, the median AI for AbTg was 29.9% among healthy Abs carriers, 52.6% for AITD patients, and 92.7% for non-AITD type 1 diabetes (T1D) patients. Median AI for AbTPO were 39.9% in healthy carriers, 73.4% in AITD patients, 83.2% in T1D patients, and 98.5% in AITD patients with thyroid neoplasm. The presence of high-avidity AbTg in T1D patients was also confirmed by a modified ELISA. Identification of high-avidity autoantibodies to thyroid proteins in non-thyroid T1D patients requires further studies.

Colorectal cancer is a high-incidence tumor that has a high mortality rate due to its frequent metastasis to the liver. The difference in genes, proteins, and immune microenvironment between the primary and metastatic sites causes them to show different responses to treatment. Colorectal cancer liver metastasis patients also tend to show poorer treatment response and prognosis. Therefore, in this paper, we summarize the heterogeneity exhibited after colorectal cancer liver metastasis from five aspects (gene, transcriptome, protein, metabolism, and immunity), and we found that except for the genetic heterogeneity, the other four aspects exhibite significant heterogeneity, which might serve as a new therapeutic direction and a prognostic marker for patients with liver metastasis. Finally, the therapeutic modalities regarding tumors are rapidly evolving, and we have also summarize the new clinical therapeutic modalities currently proposed based on these heterogeneities, aiming to provide new therapeutic ideas for the clinical treatment of patients with colorectal cancer liver metastases.

In this study, 737 individual sheep milk samples were collected to evaluate the relationships between Brix refractometer measurements and milk constituents—particularly, protein and fat percentages—with the aim of verifying the ability of the refractometer to predict milk constituents. The Pearson's simple (rSP) and partial (rPP) correlations between milk constituents were calculated, and several first- and second-order regressions were tested to predict the protein and fat percentages. The results of the forecasts can be considered satisfactory only for the simple regression that predicted the percentage of milk protein through the measurements read with the Brix refractometer (PRT = -2.996 + 0.639*Brix), while the regression that predicted the percentage of fat + milk protein presented a weak forecasting capacity, which was probably due to the absence of partial correlations between the Brix refractometer measurements and the fat percentage.

The present study investigated the insulin-like growth factors (IGFs) and their receptors and binding proteins among different pig breeds during weaning. Sixty Duroc (DR), Taoyuan black (TB), and Xiangcun black (XB) piglets (20 piglets per breed) were selected at 21 and 24 days of age (three days of post-weaning) to analyze organ indices, plasma concentrations of IGF and IGF-binding proteins (IGFBPs) using ELISA kits, and gene expression of IGF system-related components in different tissues. The plasma IGFBP-3 level in TB piglets was higher (P > 0.05) than in the XB and DR piglets at 21 days of age. At 21 days of age, compared with the DR piglets, the IGF-1 expression was lower (P < 0.05) in the kidney but was higher (P < 0.05) in the spleen of XB and TB piglets. At 24 days of age, the IGF-1 expression was higher (P < 0.05) in the kidney of TB piglets than in the XB and DR piglets, while IGFBP-3 in the stomach and IGFBP-4 in the liver of XB and TB piglets were lower (P < 0.05) compared with the DR piglets. Weaning down-regulated (P < 0.05) the IGF-1 expression in the jejunum, spleen, and liver of piglets, while up-regulated (P < 0.05) IGFBP-3 expression in the stomach, IGFBP-4 in the liver, IGFBP-5 in the ileum, and IGFBP-6 in the jejunum of DR piglets. Spearman’s correlation analysis showed a negative correlation (P < 0.05) between plasma IGFBP-2 and IGFBP-5 and the organ index of piglets. Collectively, there were significant differences in IGF system components among different pig breeds. The IGF system components were altered during weaning, which might be involved in weaning stress to decrease the growth of piglets.

Fasting, a practice with historical roots in various cultures, has recently garnered significant interest in the field of medicine. In this article, we delve into the mechanisms underlying fasting-induced autophagy and its therapeutic applications for spike protein associated pathology. We explore the therapeutic potential of fasting on spike protein-related pathology and the role of interventions to upregulate autophagy, including compounds like spermidine, resveratrol, rapamycin, and metformin. In conclusion, fasting, coupled with an understanding of its nuances, holds promise as a therapeutic intervention for spike protein related diseases; with broad implications for human health. This review presents the therapeutic possibility of using autophagy to treat spike protein related diseases, and details the interventions to deploy this therapeutic modality.

Post-translational modifications (PTMs) represent a cornerstone in the complexity of the proteome, significantly contributing to diversifying protein structure and function. PTMs can significantly influence protein function, stability, localization, and interactions with other molecules. Therefore, it is important when choosing a protein expression system to ensure the precise incorporation of PTMs during protein synthesis, which is paramount for producing biologically active proteins. Cell-free protein synthesis (CFPS) system has emerged as a powerful protein synthesis platform and research toolkit in synthetic biology. The open nature of the system allows the reaction environment to be tailored to any protein of interest to promote specific PTMs, thus allowing for the production of a protein with the desired modifications. This review presents various PTMs achieved in the CFPS systems, providing insights into the current challenges, successes, and future prospects.

Fasting, a practice with historical roots in various cultures, has recently garnered significant interest in the field of medicine. In this article, we delve into the mechanisms underlying fasting-induced autophagy and its therapeutic applications for spike protein associated pathology. We explore the therapeutic potential of fasting on spike protein-related pathology. Additionally, we discuss factors that affect fasting, such as duration, type (dry vs. water), and the role of specific compounds like spermidine, resveratrol, rapamycin, and metformin. Furthermore, we analyse the interactions between fasting and other practices such as exercise, and highlight important considerations regarding participant characteristics, including pregnancy, breastfeeding, medication interactions, and metabolic disorders. In conclusion, fasting, coupled with an understanding of its nuances, holds promise as a therapeutic intervention with broad implications for human health.

Rapeseed meal (RM) is an important agroindustrial by-product produced in large quantities by oil extraction from seeds. However, the efficient utilization of RM as animal feed is limited by its low metabolizable energy, poor palatability, and high levels of fiber and anti-nutritional components. Here, we investigate the potential of enriching RM with single-cell protein by fermentation with conventional and unconventional yeasts, to make a nutritionally improved feed component. The process of simultaneous saccharification and fermentation improved the parameters of the waste biomass, especially the protein content, the amount of crude fiber, and the degree of biotransformation of isoflavone compounds present in the waste material. Fermentation yielded the highest protein gain for the Saccharomyces cerevisiae Ethanol Red strain (ΔN=2.38%) at a biomass load of 12.5 g and for Scheffersomyces stipitis (ΔN=2.34%) at an enzyme dose of 0.125ml/10g DM. Crude fiber content (CF) was reduced by 2.55–7.18%. The SSF process resulted in the conversion of isoflavones to forms with fewer adverse effects and lower estrogenic activity.

Lameness on dairy goat farms is a welfare concern and could negatively affect milk production. This study's objective was to evaluate the effects of clinical lameness on the daily milk production of dairy goats. Between July 2019 and June 2020, 11,847 test-day records were collected from 3,145 goats on three farms in New Zealand. Locomotion scoring of goats used a 5-point scoring system (0 to 4). The dataset was split into two groups by lactation type, where goats were classified as being in seasonal lactation (≤305 days in milk) or extended lactation (&gt;305 days in milk). A linear mixed model was used to analyze datasets using milk characteristics as the dependent variables. Clinically lame goats (score 3 or 4) in seasonal and extended lactation produced 2.63% and 4.66% less milk than goats not lame, respectively. Severely lame goats (score 4) in seasonal and extended lactation produced 7.10% and 8.56% less milk than goats not lame, respectively. The estimated average daily milk income lost was between NZD 26 and 104 per goat. This study established the negative impact of lameness on milk production and annual income in dairy goats on three farms.

The ever-growing repertoire of genomic techniques continues to expand our understanding of true diversity and richness of prokaryotic genomes. Riboproteogenomics laid the foundation for dynamic studies of previously overlooked genomic elements. Most strikingly, bacterial genomes were revealed to harbour robust repertoires of small open reading frames (sORFs) encoding a diverse and broadly expressed range of small proteins, or sORF-encoded polypeptides (SEPs). In recent years, continuous efforts led to great improvements in annotation and characterization of such proteins, yet many challenges remain to fully understand the pervasive nature of small proteins and their impact on bacterial biology. In this work we review recent developments in the dynamic field of bacterial genome reannotation, catalogue important biological roles carried out by small proteins and identify challenges obstructing the way to full understanding of these elusive proteins.

Enzymatically active nanocomposites are a perspective class of bioactive materials that finds their application in numerous fields of science and technology ranging from biosensors and therapeutic agents to industrial catalysts. Key properties of such systems are their stability and activity under various conditions, the problems that are addressed in any research devoted to this class of materials. Сomprehension ща the principles that affect these properties play the most important role in the development of the field, especially when it takes to a new class of bioactive systems. Recently, a new class of enzymatically doped magnetite-based sol-gel systems emerged and paved the way for a variety of potent bioactive magnetic materials with improved thermal stability. Such systems already showed themself as perspective industrial and therapeutic agents, but are still under intense investigation and many aspects are still unclear. Here we made a first attempt to describe the interaction of biomolecules with magnetite-based sol-gel materials and to investigate facets of protein structure rearrangements occurring within the pores of magnetite sol-gel matrix using dedicate Fourier-transform infrared spectroscopy.

Branched-chain amino acid (BCAA) levels are associated with skeletal muscle cross-sectional area (CSA). Serum BCAA levels are enhanced by whey protein supplementation (WPS), and evidence in clinical populations suggests an association of single nucleotide polymorphisms (SNPs) with BCAA metabolite levels. It is not known whether the same SNPs are associated with the ability to catabolise BCAAs from exogenous sources, such as WPS. The present study investigated whether possessing a higher number of alleles associated with increased BCAA metabolites correlates with muscle fiber CSA of m. vastus lateralis in physically active participants, and whether any relationship is enhanced by WPS. Endurance-trained participants (n=75) were grouped by self-reported habitual WPS consumption and genotyped for 5 SNPs (PPM1K rss1440580, APOA5 rs2072560, CBLN1 rs1420601, DDX19B rs12325419, TRMT61A rs58101275). Body mass, BMI and fat percentage were significantly lower and muscle mass higher in the WPS group (n=22) compared to Non-WPS (n=53). The number of BCAA-increasing alleles was correlated with fiber CSA in the WPS group (r=0.75, p&lt;0.0001) and was stronger for fast-twitch fibers (p=0.001) than slow-twitch fibers (p=0.048). Similar results remained when corrected for multiple covariates (age, physical activity, meat and dairy intake). No correlation was found in the Non-WPS group. This study presents novel evidence of a positive relationship between BCAA-increasing alleles and muscle fiber CSA in athletes habitually consuming WPS. We suggest that a high number of BCAA-increasing alleles improves the efficiency of WPS by stimulation of muscle protein synthesis, and contributes to greater fiber CSA.

G protein-coupled receptors (GPCRs) serve prominent roles in melanocyte lineage physiology, with an impact at all stages of development, as well as on mature melanocyte functions. GPCR ligands are present in the skin and regulate melanocyte homeostasis, including pigmentation. The role of GPCRs in the regulation of pigmentation and, consequently, protection against external aggression, such as ultraviolet radiation, has long been established. However, evidence of new functions of GPCRs directly in melanomagenesis has been highlighted in recent years. GPCRs are coupled, through their intracellular domains, to heterotrimeric G proteins, which induce cellular signaling through various pathways. Such signaling modulates essential cellular processes of melanomagenesis, such as proliferation and migration. GPCR-associated signaling in melanoma can be activated by the binding of paracrine factors to their receptors or directly by activating mutations. In this review, we present melanoma-associated alterations of GPCRs and their downstream signaling and discuss the various preclinical models used to evaluate new therapeutic approaches against GPCR activity in melanoma. Recent striking advances in our understanding of the structure, function, and regulation of GPCRs will undoubtedly broaden treatment options in melanoma in the future.

Our health requires continual protein synthesis for maintaining and repairing tissues. For protein synthesis to function, all the essential (indispensable) amino acids (IAA) that must be available in the diet, along with those AAs that the cells can synthesize, the dispensable amino acids. Here we review studies that have shown the location of the detector for IAA deficiency in the brain, specifically for recognition of IAA deficient diets (IAAD diets) in the anterior piriform cortex (APC), with subsequent responses in downstream brain areas. The APC is highly excitable, uniquely suited to serve as an alarm for reductions in IAAs. With a balanced diet, these neurons are kept from over-excitation by GABAergic inhibitory neurons. Because several transporters and receptors on the GABAergic neurons have rapid turnover times, they rely on intact protein synthesis to function. When an IAA is missing, its unique tRNA cannot be charged. This activates the enzyme General Control Nonderepressible 2 (GCN2) that is important in the initiation phase of protein synthesis. Without the inhibitory control supplied by GABAergic neurons, excitation in the circuitry is free to signal an urgent alarm. Studies in rodents have shown rapid recognition of IAA deficiency by quick rejection of the IAAD diet.

The current study evaluates the selection signals in the evolution of mitochondrial DNA of voles, subfamily Arvicolinae, during the colonization of subterranean environments. The comparative sequence analysis of mitochondrial protein-coding genes of eight subterranean vole species (Prometheomys schaposchnikowi, three species of the genus Ellobius: E. talpinus, E. fuscocapillus and E. lutescens, two species of the genus Terricola: T. subterraneus and T. daghestanicus, Lasiopodomys mandarinus and Hyperacrius fertilis) and their closest aboveground relatives using codon-substitution models was applied. The highest number of selection signatures was detected in genes ATP8 and CYTB. The relaxation of selection was observed in most mtDNA protein-coding genes. In mole voles (genus Ellobius) the signatures of adaptive evolution of mitochondrial genes related to subterranean niche were most pronounced. The number of selection signatures was found to be independent of the evolutionary age of the lineage but fits the degree of specialization to the subterranean niche. The common trends of selective pressures were observed among the evolutionary ancient and highly specialized subterranean rodent families and phylogenetically young lineages of voles. It suggests that the signatures of adaptations in individual mitochondrial protein-coding genes associated with the colonization of the subterranean niche may appear within a rather short evolutionary timespan.

The extrusion of food for human and animal consumption is a unit operation that includes mixing, shearing and force to the materials related to the rheological properties of the materials in the extruder. The present work aims to study the rheological behavior of an extruded fish diet incorporating hydrolyzed protein flour processed by extrusion. The measurement was carried out online with a slit die rheometer, defining the rheological models and parameters that fit the process. During the extrusion process, the raw materials used were hydrolyzed protein meal, fish meal and cassava starch. For the results, the evaluated treatments were adjusted to the Power Law, where an increase in the shear rate decreases the viscosity of the material, corresponding to a pseudoplastic behavior. The incorporation of hydrolyzed protein flour presented a significant effect on the value of n and Klp, increasing the viscosity with the increase in the percentage of inclusion of HPH. The models obtained for the prediction of the viscosity are adjusted to the changes in shear rate, temperature and humidity of the system.

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales; causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxyadenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins homologs to C. elegans NMAD-1 and ALKBH1, respectively. The function of the three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase of 6mA levels during the dimorphic transition, suggesting that 6mA regulates dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction of polar growth, suggesting that 6mA also has a role during the spore-macrophage interaction that could be important in the infection process.

The concentration of cerebrospinal fluid total protein (CSF-TP) is important for the diagnosis of neurological emergencies. Recently, some Western studies have shown that the current upper reference limit of CSF-TP is quite low for older patients. However, little is reported about the concentration of CSF-TP in older Asian population. In this study, we retrospectively analyzed the CSF-TP concentrations in Japanese healthy older volunteers. CSF samples in 69 healthy Japanese volunteers (age range: 55–73 years) were collected by lumbar puncture, and the data of CSF were retrospectively analyzed. The mean (standard deviation) CSF-TP was 41.7 (12.3) mg/dL. The older group (≥ 65 years old) had higher CSF-TP concentration than the younger group (55–64 years old). The 2.5th percentile and 97.5th percentile of CSF-TP were estimated as 22.5 and 73.2 mg/dL, respectively, which were higher than the current reference range in Japan (10–40 mg/dL).

The GAI‐RGA ‐ and ‐SCR (GRAS) proteins belong to the plant-specific transcription factor gene family and involved in several developmental processes, phytohormone and phytochrome signaling, symbiosis, stress responses etc. GRAS proteins have a conserved GRAS domain at C-terminal and hypervariable N-terminal. The C-terminal conserved domain directly affects the function of the GRAS proteins. For instance, in Arabidopsis, mutations in this domain in Slender rice 1 (SLR1) and Repressor of GA (RGA) proteins cause significant phenotypic changes. GRAS proteins have been reported in more than 30 plant species and till now it has been divided into 17 subfamilies. This review highlighted GRAS protein's importance during several biological processes in plants, structural features of GRAS proteins, their expansion and diversification in the plants, GRAS-interacting proteins complexes and their role in biological processes. We also summarized available recent research that utilized CRISPR-Cas9 technology to manipulate GRAS genes in a plant for different traits. Further, the exploitation of GRAS genes in crop improvement programs has also been discussed

With the global climate anomalies and the destruction of ecological balance, water shortage has become a serious ecological problem facing all mankind, and drought has become a key factor restricting the development of agricultural production. Therefore, it is essential to study the drought tolerance of crops. On the basis of previous studies, we reviewed the effects of drought stress on plant morphology and physiology, including the changes of external morphology and internal structure of root, stem and leaf, the effects of drought stress on osmotic regulation sub-stances, drought-induced proteins and active oxygen metabolism of plants. In this paper, the main drought stress signals and signal transduction pathways in plants are described, and the functional genes and regulatory genes related to drought stress are listed respectively. We summarize the above aspects in order to provide valuable background knowledge and theoret-ical basis for future agriculture and forestry breeding and cultivation.

Zinc finger proteins (ZFP) play important roles in cellular processes. The DNA binding region of ZFP consists of 3 zinc finger DNA binding domains connected by amino acid linkers, the sequence TGQKP connects ZF1 and ZF2, and TGEKP connects ZF2 with ZF3. Linkers act to tune the zinc finger protein in the right position to bind its DNA target, the type of amino acid residues and length of linkers reflect on ZF1-ZF2-ZF3 interactions and contribute to the search and recognition process of ZF protein to its DNA target. Linker mutations and the affinity of the resulting mutants to specific and nonspecific DNA targets were studied by MD simulations and MM_GB(PB)SA. The affinity of mutants to DNA varied with type and position of amino acid residue. Mutation of K in TGQKP resulted in loss in affinity due to the loss of positive K interaction with phosphates, mutation of G showed loss in affinity to DNA, WT protein and all linker mutants showed loss in affinity to a nonspecific DNA target, this finding confirms previous reports which interpreted this loss in affinity as due to ZF1 having an anchoring role, and ZF3 playing an explorer role in the binding mechanism. The change in ZFP-DNA affinity with linker mutations is discussed in view of protein structure and role of linker residues in binding.

The ongoing mutations in the structural proteins of SARS-CoV-2 is the major impediment for prevention and control of the COVID-19 disease. The envelope (E) protein of SARS-CoV-2 is a structural protein existing in both monomeric and homopentameric forms, associated with a multitude of functions including virus assembly, replication, dissemination, release of virions, infection, pathogenesis, and immune response stimulation. In the present study, 81,818 high quality E protein sequences retrieving from the GISAID were subjected to mutational analyses. Our analysis revealed that only 0.012 % (982/81818) stains possessed amino acid (aa) substitutions in 63 sites of the genome while 58.77% mutations in the primary structure of nucleotides in 134 sites. We found the V25A mutation in the transmembrane domain which is a key factor for the homopentameric conformation of E protein. We also observed a triple cysteine motif harboring mutations (L39M, A41S, A41V, C43F, C43R, C43S, C44Y, N45R) which may hinder the binding of E protein with spike glycoprotein. These results therefore suggest the continuous monitoring of each structural protein of SARS-CoV-2 since the number of genome sequences from across the world are continuously increasing.

Mammals have two insulin-like growth factors (IGF) that are key mediators of somatic growth, of tissue differentiation and cellular responses to stress. Thus, the mechanisms that regulate the bioavailability of IGFs are important in both normal and aberrant development. IGF-I levels are primarily controlled via the growth hormone-IGF axis, in response to nutritional status, and also reflect metabolic diseases and cancer. One mechanism that controls IGF bioavailablity is the binding of circulating IGF to a number of binding proteins that keep IGF in a stable, but receptor non-binding state. But even before IGF is released from the cells that produce it, it undergoes an obligatory association with a ubiquitous chaperone protein, GRP94. This binding is required for secretion of a properly folded, mature IGF. This chapter reviews the known aspects of the interaction and highlights the specificity issues yet to be determined. The IGF-GRP94 interaction provides a potential novel mechanism of idiopathic short stature, involving the obligatory chaperone and not just IGF gene expression. It also provides a novel target for cancer treatment, as GRP94 activity can be either inhibited or enhanced.

The integrated stress response (ISR) is an adaptational signaling pathway that is induced in response to different stimuli, such as accumulation of unfolded and misfolded protein, hypoxia, amino acid deprivation, viral infection and ultraviolet light. It has been known that viral infection can activate ISR, but the role of ISR during viral infection is still unclear. In some cases, ISR is a protective mechanism of host cell against infection with virus whilst ISR may be hijacked by viruses for facilitating its replication. In this review, we highlighted recent advances on induction of ISR upon viral infection and the downstream responses involved such as autophagy, apoptosis, formation of stress granules and innate immunity response. We then discussed the molecular mechanism of ISR regulating viral replication and how viruses antagonize this cellular stress response resulting from ISR.

Transcriptionally controlled tumor protein (TCTP) is a highly conserved protein performing a large number of cellular functions by binding with various partner proteins. The importance of its roles in many diseases requires an assay method to find regulatory compounds. However, the molecular characteristics of TCTP made it difficult to search for chemicals interacting with it. In this study, a tryptophan-based assay method was designed and Y151W mutant TCTP was constructed to search binding chemicals. Since there is no tryptophan in the native sequence of TCTP, the incorporation of tryptophan in the Y151W mutant was very effective to establish the method. A flavonoid library was employed to the assay with the method. With the native and Y151W mutant TCTPs, three flavonoids such as morin, myricetin and isobavachalcone have been found to interact with TCTP. Combined with native gel electrophoresis, the binding region of isobavachalcone was suggested to be the flexible loop of TCTP. This approach can be easily applicable to find binding compounds of proteins with similar molecular characteristics of TCTP.

The objective of this review is to do an overview about the current knowledge of Anti Iglon5 Syndrome, a disease that was first described in 2014. The IgLON proteins are a family of cell adhesion molecules and the presence of antibodies against IgLON5 are crucial for diagnosis of Anti IgLON5 Syndrome. This syndrome has an expanded clinical spectrum that involves prominent sleep disorder, progressive bulbar dysfunction, gait instability with abnormal eye movements reminiscent and cognitive deterioration sometimes associated with chorea. The main neuropathological finding is the neuronal loss with hyperphosphorylated tau (p-Tau) protein accumulation at hypothalamus, brainstem tegmentum, hippocampus, periaqueductal gray matter, medulla oblongata and upper cervical cord. The exact pathogenesis is still unclear and involves a neurodegenerative process and autoimmune response. The early diagnosis is important to avoid unnecessary tests and prevent complications. Important resources for diagnosis are the antibody testing of serum and CSF for IgLON5-IgG. The mortality of anti IgLON5 syndrome is high and new studies published described a good response to immune therapy. However, the response to immune therapy depends of some clinical and analytical characteristic. In addition, future studies are needed to thoroughly study the aspects of pathogenesis and treatment of this important pathological syndrome.

The most prevalent Ca2+-buffer proteins (CaBPs: parvalbumin—PV; calbindin—CaB; calretinin—CaR) are widely expressed by various neurons throughout the brain, including the retinal ganglion cells (RGCs). Even though their retinal expression has been extensively studied, a coherent assessment of topographical variations is missing. To examine this, we performed immunohistochemistry (IHC) in the mouse retina. We found variability in the expression levels and cell numbers for CaR, with stronger and more numerous labels in the dorso-central area. CaBP+ cells contributed to RGCs with all soma sizes, indicating heterogeneity. We separated 4-9 RGC clusters in each area based on expression levels and soma sizes. Besides the overall high variety in cluster number and size, the peripheral half of the temporal retina showed the greatest cluster number, indicating a better separation of RGC subtypes there. Multiple labels showed that 39% of the RGCs showed positivity for a single CaBP, 30% expressed two CaBPs, 25% showed no CaBP expression and 6% expressed all three proteins. Finally, we observed an inverse relation between CaB and CaR expression levels in CaB/CaR dually- and CaB/CaR/PV triple labeled RGCs, suggesting a mutual complementary function.

Amyloids are fibrillar protein aggregates that are associated with diseases such as Alzheimer’s disease, Parkinson’s disease, type II diabetes and Creutzfeldt–Jakob disease. The process of amyloid aggregation involves three pathological protein transformations; from natively-folded conformation to the cross-β conformation, from biophysically soluble to insoluble, and from biologically functional to non-functional. While amyloids share a similar cross-β conformation, the biophysical transformation can either take place spontaneously via a homogeneous nucleation mechanism (HON) or catalytically on an exogenous surface via a heterogeneous nucleation mechanism (HEN). Here, we postulate that the different nucleation pathways can serve as a mechanistic basis for an etiological classification of amyloidopathies, where hereditary forms generally follow the HON pathway, while sporadic forms follow surface-induced (including microbially-induced) HEN pathways. Furthermore, the conformational and biophysical amyloid transformation results in loss-of-function (LOF) of the original natively-folded and soluble protein. This LOF can, at least initially, be the mechanism of amyloid toxicity even before amyloid accumulation reaches toxic levels. By highlighting the important role of non-protein species in amyloid formation and LOF mechanisms of toxicity, we propose a generalized mechanistic framework that could help better understand the diverse etiology of amyloid diseases and offer new opportunities for therapeutic interventions including replacement therapies.

Lignin has interesting functionalities to be exploited in adhesives for medicine, foods and textiles. Nanoparticles (NPs) <100 nm coated with poly(L-lysine), PL and poly(L-glutamic acid) PGA were prepared from the laccase treated lignin to coat nanocellulose fibrils (CNF) with heat. NPs ca. 300 nm were prepared, β-casein coated and cross-linked with transglutaminase (Tgase) to agglutinate chamois specimens. Size exclusion chromatography (SEC) and Fourier-transform infrared (FTIR) spectroscopy were used to characterize polymerized lignin, zetapotential and dynamic light scattering (DLS) to ensure coating of colloidal lignin particles (CLPs). Protein adsorption on lignin was studied by quartz crystal microbalance (QCM). Atomic force microscopy (AFM) was exploited to examine interactions between different polymers and to image NPs with transmission electron microscopy (TEM). Tensile testing showed, when using CLPs for the adhesion, the stress improved ca. 10 and strain ca. 6 times compared to polymeric lignin. For the β-casein NPs the values were 20 and 8, respectively, and for the β-casein coated CLPs between these two cases. When NPs were dispersed in adhesive formulation, the Young's moduli confirmed significant improvement in the elasticity of the joints over the adhesive alone. Exploitation lignin in nanoparticulate morphology is a potential method to prepare bionanomaterials for advanced applications.

Although there are extensive literature reports on the use of gold nanoparticle (AuNP) based homogeneous assays for detection of biomolecules, very few experimental description and procedures involving their preparation are described. In this study, AuNPs conjugated to Bovine Serum Albumin or Envelope protein from Dengue II were developed as a homogeneous immunoassay for antibody detection. We report here optimization of key parameters to prepare an immunoassay like conjugation protein concentration, centrifugation time, electrolyte addition and assay temperature. We determined that saturating protein concentrations improved AuNPs surface coverage and uniformity of the assay and addition of sodium chloride improved sensitivity of the antibody detection method and assay stability. Furthermore, we showed that dynamic light scattering can be used to monitor changes in gold nanoparticles in the preparation and detection steps. Additionally, numerical simulations of the plasmonic optical response of AuNPs were carried out to scan for size-dependent response of the AuNPs. The AuNPs homogeneous immunoassay developed was further used in the detection of antibodies in vitro to detect Dengue virus infection.

The feed-forward loop (FFL) is an important and basic network motif to understand specific biological functions. Cyclic-AMP (cAMP) receptor protein (CRP), a transcription factor (TF), mediates catabolite repression and regulates more than 400 genes in response to changes in intracellular concentrations of cAMP in Escherichia coli. CRP participates in some FFLs like araBAD and araFGH operons and adapt to fluctuating environmental nutrients thus enhancing the survivability of E. coli. Although computational simulations have been used to explore the potential functionality of FFLs, a comprehensive study of the functions of all structural types based on in vivo data is lacking. Also, the regulatory role of CRP-mediated feed-forward loops (CRP-FFLs) remain unclear to date. Using EcoCyc and RegulonDB, we identified 393 CRP-FFLs in the E. coli. Dose-response genomic microarray of E. coli revealed dynamic gene expression of each target gene of CRP-FFLs in response to a range of cAMP dosages. All eight types of FFLs were present in CRP regulon with various expression patterns of each CRP-FFL, that were further divided into five functional groups. Microarray and reported regulatory relationships identified 202 CRP-FFLs which were directly regulated by CRP in these eight types of FFLs. Interestingly, 30% (147/482) of genes were directly regulated by CRP and CRP-regulated TFs, indicating that these CRP-regulated genes were also regulated by other CRP-regulated TFs responding to environmental signals through CRP-FFLs. Furthermore, we applied gene ontology annotation to reveal the biological functions of CRP-FFLs.

Abnormal Tau phosphorylation and aggregation into neuronal paired helical filaments and neurofibrillary tangles cause tauopathies, a class of neurodegenerative diseases, that include Alzheimer’s disease, frontotemporal dementia and parkinsonism linked to chromosome 17, progressive supranuclear palsy, Pick's disease, and corticobasal degeneration. Glycogen synthase kinase-3β (GSK-3β) is the most critical kinase to phosphorylate Tau. We have developed the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), with cyclopropane rings instead of cis-double bonds, as an anti-dementia drug. DCP-LA serves as a selective activator of PKCε and a potent inhibitor of protein tyrosine phosphatase 1B (PTP1B). DCP-LA prevents Tau phosphorylation due to PKCε-mediated direct inactivation of GSK-3β, to PKCε/Akt-mediated inactivation of GSK-3β, and to receptor tyrosine kinase-mediated inactivation of GSK-3β in association with PTP1B inhibition. DCP-LA targeting GSK-3β, thus, could become a valid drug for treatment of tauopathies.

The present investigation has been conducted on one hundred tissue fragments of breast cancer, collected and immediately cryopreserved following the surgical resection. The fragments were selected from patients with invasive ductal carcinoma of the breast, the most common and potentially aggressive type of mammary cancer, with the objective to increase the knowledge of breast cancer molecular markers, useful for diagnostic and prognostic categorization of patients, in assessing post-surgical therapeutic regimes. The proteomic screening, by 2D-IPG and mass spectrometry, allowed us to identify two main classes of protein clusters: proteins expressed ubiquitously at high levels in all patients, and proteins expressed sporadically among the same patients. Within the group of ubiquitous proteins, glycolytic enzymes and proteins with anti-apoptotic activity were predominant. Among the sporadic ones, proteins involved in cell motility, molecular chaperones and proteins involved in the detoxification appeared prevalent. The data of the present study indicates that the primary tumor growth is generally supported by two concurrent pathways: the inhibition of apoptosis and the stimulation of cellular proliferation. The second phase of the evolution of the tumor can be prematurely scheduled by the occasional presence of proteins involved in cell motility and in the defenses of the oxidative stress. To our knowledge this report on large-scales proteomics of breast cancer is currently a unique approach in the literature that offers the opportunity to evaluate the presence and recurrence of proteins to be used as prognostic indicators and susceptibility to metastasis in patients operated on for invasive ductal carcinoma of the breast.

The effects of hypothermia on neonatal encephalopathy may vary regionally, spatially, and cytopathologically in the gyrencephalic neocortex with manifestations potentially influenced by seizures that alter the severity and distribution of neuropathology. We developed a neonatal piglet survival model of hypoxic-ischemic (HI) encephalopathy and hypothermia with continuous encephalographic (cEEG) monitoring for seizures to study injury in neocortex. Neonatal piglets were randomized to naïve, HI-normothermia (NT), overnight hypothermia (HT) initiated 2 hours after HI, sham-NT, or sham-HT treatments. Some piglets within sham and HI groups received cEEG monitoring during recovery. Survival was 2-7 days (piglets were unmedicated and those with poor recovery and unresolving seizures were euthanized early); there was no differences in survival among groups (p&gt;0.078). Neuropathology was assessed by hematoxylin and eosin staining and immunohistochemistry for RNA Binding FOX-1 Homolog 3 (Rbfox3/NeuN). Normal and ischemic-necrotic neurons were counted (layers II-VI collectively) in somatosensory, motor, and prefrontal cortices, identified by cytoarchitecture and connectomics, and in inferior parietal cortex by layer. Seizure burden was determined. HI-NT piglets had reduced normal/total neuron ratio and increased ischemic-necrotic/total neuron ratio relative to naïve, sham-NT, and sham-HT piglets in anterior and posterior motor and somatosensory cortices. Frontal cortex was vulnerable in the prefrontal lateral bank after HI-NT with ischemic-necrosis. HI-HT piglets had higher normal/total neuron ratios and lower ischemic-necrotic/total neuron ratios than HI-NT piglets in anterior/posterior motor and somatosensory cortices. Total normal neuron density in layer III of inferior parietal cortex was reduced in HI-NT piglets compared to sham piglets and was protected by HT. Laminar analysis of Rbfox3 in somatosensory cortex revealed three types of neurons: Rbfox3-positive/normal, Rbfox3-positive/ischemic-necrotic, and Rbfox3-depleted. HI piglets had increased Rbfox3-depleted/total neuron ratio in layers II and III compared to sham-NT. Cortical neuron Rbfox3-depletion was partly rescued by HT. Seizure burden was more severe in HI piglets compared to sham piglets, but HI-NT and HI-HT piglets were similar. We conclude that 1) the neonatal piglet neocortex has a suprasylvian spatial vulnerability to HI and seizures; 2) HT protects against neuropathology in functionally different regions of the neonatal gyrencephalic neocortex; 3) neurons in neonatal neocortex have a limited cytopathology repertoire seen by H&amp;E staining; 4) higher seizure burden correlates with more ischemic-necrotic neurons in somatosensory cortex; 5) seizure presence associates with damage spread to inferior parietal cortex; 6) seizure presence is insensitive to HT, and 7) Rbfox3 immunophenotyping identifies a novel neuronal RNA splicing protein nuclear-depletion pathology that appears reversible by HT. This work demonstrates that HT protection of neocortex in neonatal HI is topographic and laminar, seizure unmitigating, and restores depleted neuronal RNA splicing factor.

It has not been reasonably explained still now, how the “chicken and egg relationship” between gene and protein was formed, although RNA world hypothesis was proposed to aim at explaining formation process of the relationship about 40 years ago. On the other hand, GADV hypothesis advocates that life emerged after five members of the fundamental life system; cell structure, metabolism, tRNA, genetic code and gene, were piled up one by one onto immature [GADV]-proteins, which were produced by random joining of [GADV]-amino acids on the primitive Earth. Furthermore, formation process of the first (GNC)n RNA gene encoding a mature [GADV]-protein can be also reasonably explained according to the hypothesis as follows. Double-stranded (ds)-(GNC)n RNA could be produced by complementary strand synthesis of a single-stranded (ss)-(GNC)n RNA, which was formed by random joining of GNC anticodons carried by four types of AntiC-SL tRNAs. Successively, the first (GNC)n RNA gene was generated upon maturation of an immature [GADV]-protein, which was produced through expression of codon sequence on one strand of ds-(GNC)n RNA, as accumulating necessary base substitutions and raising a weak catalytic activity of the immature protein to a more active state. It is considered that, consequently, a gene encoding a mature protein, which has the “chicken and egg relationship”, was generated for the first time. Therefore, it can be concluded that the “chicken and egg relationship” was built not by formation of either one, gene or protein, but by maturation of an immature gene, which was lead by improvement of catalytic activity of an immature protein. That is, it can be concluded that the reason, why it was unable to give an answer to the question on the formation process of the “chicken-egg relationship” thus far, is because it was an unanswered question.

Protein tyrosine phosphatases (PTPs) of the polymerase and histidinol phosphatases (PHP) superfamily with characteristic phosphatase activity dependent on divalent metal ions are found in many gram-positive bacteria. Although members of this family are co-purified with metal ions, they still require the exogenous supply of metal ions for full activation. However, the specific roles these metal ions play during catalysis are yet to be well understood. Here, we report the metal ion requirement for phosphatase activities of S. aureus Cap8C and L. rhamnosus Wzb. AlphaFold-predicted structures of the two PTPs suggest that they are members of the PHP family. Like other PHP phosphatases, the two enzymes have catalytic preference for Mn2+, Co2+, and Ni2+ ions. Cap8C and Wzb show an unusual thermophilic property with optimum activities over 75oC. Pre-incubation experiments show that the two enzymes are not inherently thermostable suggesting that certain catalytic step(s) in the reaction mechanism has a high activation energy. Consistent with this model, the activity-temperature profiles of the two enzymes are dependent on the divalent metal ion activating the enzyme.

Microbial immune escape represents the primary cause of induced pathogenesis in humans, and it represents a pivotal method used by viral agents to increase their load and suppress key mechanisms of the innate and adaptive immune system. This phenomenon represents the primary factor that led to the onset of the 1918-1920 A(H1N1) Influenza and 2020-2022 COVID-19 pandemics, and it possibly played a major role in the onset of the AIDS pandemic as well. Moreover, repeated incidents of immune evasion could be associated with higher rates of cellular aging (Jackson et al., 2017), most likely due to the consequent increased demands of energy consumption. Highly developed viral immune evasion ultimately indicates the high inner intelligence of human immunity due to reflective and imitative characteristics of reactions that are produced against initial actions. Ribonucleic acid-based viral genomes contain open reading frames, which consist of genes producing sixteen non-structural proteins. Such proteins play a considerable role in desensitizing first-line immunity during cellular infection, and non-structural proteins 1, 10 and 16 have the strongest effects against a healthy expression rate of Type I and Type III Interferon-encoding genes. Type I Interferons consist of IFN-alpha, -beta, -delta, -epsilon, -omega, -tau and -zeta, whilst Type III Interferons consist of IFN-lambda1, -lambda2 and -lambda3, and they act as stimulators of intracellular signalling cascades that in turn lead to the activation and expression of interferon-stimulated genes (Brown et al., 2022). The earlier the interferon-stimulated genes are activated, the lower the extent of pro-inflammatory mediation and overall, the more effective the antiviral immune response will be, given the exponential nature of the viral load increase. Non-structural protein 16 methylates the 5’ cap of the virus, making the pathogen-associated molecular patterns less recognisable by pattern-recognition receptors, and it requires activation by bonding with non-structural protein 10. It is preserved in the S-Adenosyl-L-Methionine pocket of the SARS-CoV-2 genome. Non-structural protein 1 (NS1) directly cleaves the host cell mRNA producing Type I and possibly Type III Interferons, thereby preventing a translation process of the immune proteins. NS1 has recently been found to often be packaged into exosomes once secreted by the viral genome in the cytosol, meaning that exocytosis and paracrine signalling to neighbouring cells before their actual infection is possible. As a result, NS1 is highly capable of silencing the first-line immune responses of uninfected neighbouring cells as well, thereby highlighting the need to adjust the focus of therapeutics and vaccinology toward first-line immunity and further indicating its foundational importance in the support for the development of precise and balanced defenses against microbial agents of concern (EL SAFADI et al., 2022).

Background: Although lower extremity revascularization is commonly performed in hemodialysis patients, poor prognosis remains a major problem. Protein-energy wasting is reportedly associated with chronic inflammation and advanced atherosclerosis in hemodialysis patients. We investigated the association between the geriatric nutritional risk index (GNRI) as a surrogate marker of protein-energy wasting, C-reactive protein (CRP), and their joint roles in the prediction of amputation and/or mortality. Methods: We enrolled 800 patients successfully underwent lower extremity revascularization. Patients were divided into low, middle and high tertile (T1, T2 and T3) according to GNRI and CRP levels, respectively. Results: Amputation-free survival rates for 8 years were 47.0%, 56.9%, and 69.5% in T1, T2, and T3 of the GNRI, and 65.8%, 58.7%, and 33.2% for T1, T2, and T3 of CRP, respectively (p<0.0001 for both). Declined GNRI [adjusted hazard ratio (aHR) 1.78, 95% confidence interval (CI) 1.24-2.59, p=0.0016 for T1 vs. T3] and elevated CRP (aHR 1.86, 95%CI 1.30-2.70, p=0.0007 for T3 vs. T1) were independent predictors of amputation and/or mortality. In the combined setting of both variables, the risk was 3.77-fold higher (95% CI 1.97-7.69, p<0.0001) in the T1 of GNRI with T3 of CRP than in the T3 of GNRI with T1 of CRP. Conclusions: Patients with pre-procedural decreased GNRI and elevated CRP levels frequently experienced amputation and/or mortality, and a combination of both variables could more accurately stratify the risk.

Plant viral nanoparticles (VNPs) are attractive to researchers in nanomedicine due to their safety, easy production, resistance, and straightforward functionalization. In this paper, we developed and successfully purified a VNP derived from turnip mosaic virus (TuMV), a well-known plant pathogen, that exhibits high affinity for immunoglobulins G (IgG) thanks to its functionalization with the Z domain from staphylococcal protein A via gene fusion. We selected cetuximab as a model IgG to prove the versatility of this novel TuMV VNP by developing a fluorescent nanoplatform to mark tumoral cells of the line Cal33, from tongue squamous cell carcinoma. We observed that the fluorescent VNP-cetuximab bound selectively to Cal33 and were internalized by confocal microscopy, revealing the potential of this nanotool in cancer research.

Nanofiber (NF) products exhibit outstanding performances in the materials science, textile, and medical fields, which cannot be realized using conventional technologies. However, the safety of such products is debated because of the potential risk of nanomaterials and lack of standardized guidelines for safely evaluating NF products. The global safety evaluations of nanomaterials focused on evaluating the cytotoxicity of zero-dimensional materials including nanoparticles and nanotubes based on OECD criteria. The NFs are one-dimensional materials with nanometer diameters and considerable lengths. Many fibers are applied in a densely woven web-like form, and therefore, assessing cellular penetration and fiber toxicity using the same methods is inappropriate. This study verifies the safety of polyurethane (PU) and polyvinylidene fluoride (PVDF) polymers, which are currently applied in filters and masks. To this end, polymer NFs were collected from each product, and the NFs were compared with reference samples considering their physical properties using FT-IR and Raman spectroscopy. For the safety evaluation, DMSO stocks of varying concentrations of PVDF and PU NFs (at 0.5, 1, 5, and 10 μg/mL) were prepared, and the cytotoxicity and inhibitory effects on nitric oxide production and protein expression obtained via Western blot were identified.

Scientists have observed amyotrophic lateral sclerosis (ALS) disease clusters around certain lakes in the regions of northern New England and northern Ohio. A Parkinson’s disease cluster has also been observed in Vancouver. Cyanobacteria toxin exposure has been considered as a potential risk factor to explain this association. It is reported here that these regions have several commonalities in their environment, including a notable geologic history, the presence of abundant glacial sediments, and possible mineral and bentonite exposures. The possible association with and significance of these risk factors in ALS and Parkinson’s disease is discussed.

(1) Background: Angiotensin II, a major culprit in cardiovascular disease, activates mediators that are also involved in pathological cardiac remodelling. We aimed at investigating the effects of two of them: aldosterone (Ald) and transforming growth factor-beta-1 (TGF-β1 ), on cardiac remodelling in an in vivo model. (2) Methods: Six-week-old male wild-type (WT) and TGF-β1 -overexpressing transgenic (TGF-β1 -TG) mice were infused with subhypertensive doses of Ald for 2 weeks, and/or treated orally with eplerenone from postnatal day 21. Heart’s ventricles were examined: by morphometry, immunoblotting to assess intracellular signalling pathways, RT qPCR to determine hypertrophy and fibrosis marker genes. (3) Results: TGF-β1 -TG mice spontaneously developed cardiac hypertrophy and interstitial fibrosis, exhibited higher baseline phosphorylation of p44/42 and p38 kinases, fibronectin and ANP mRNA expression. Ald induced a comparable increase in ventricular heart weight-to-body-weight ratio and cardiomyocyte diameter in both strains, but less pronounced increase in interstitial fibrosis in transgenic compared to WT mice (23.6% vs 80.9%, p&lt;0.005). Ald increased phosphorylation of p44/42 and p38 in WT but not TGF-β1 -TG mice. While eplerenone-enriched chow partially prevented Ald-induced cardiac hypertrophy in both genotypes and interstitial fibrosis in WT controls, it completely protected from additional fibrosis in transgenic mice. (4) Conclusions: Ald appears to induce cardiac hypertrophy independently of TGF-β1 , while in case of fibrosis downstream signalling pathways of these two factors probably converge

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. Pathological deposits of neurotoxin proteins within the brain, such as amyloid-Beta and hyperphosphorylated tau tangles, are prominent features in AD. The prion protein (PrP) is involved in neurodegeneration via its conversion from the normal cellular form PrPc, to the infection form PrP Sc. Some studies indicated that posttranslationally modified PrPc isoforms plays a fundamental role in AD pathological progression. Several studies have shown that interaction of Aβ oligomers with N-terminal residues of the PrPc protein region appears critical for neuronal toxicity. The PrPc-Aβ binding always occur in AD brains and is never detected in nondemented controls and the binding of Aβ aggregates to PrPc is restricted to the N-terminus of PrPc.

Lipophilic oligonucleotide derivatives are a potent approach to the intracellular delivery of nucleic acids. The binding of these derivatives to serum albumin is a major determinant of their fate in the body, as its structure contains several sites of high affinity for hydrophobic compounds. This study focuses on the features of self-association and non-covalent interactions with human serum albumin of novel self-penetrating oligonucleotide derivatives. The study revealed that the introduction of a triazinyl phosphoramidate modification bearing two dodecyl groups at the 3' end region of the oligonucleotide sequence has a negligible effect on its affinity for the complementary sequence. Dynamic light scattering verified that the amphiphilic oligonucleotides under study can self-assemble into micelle-like particles ranging from 8 to 15 nm in size. The oligonucleotides with dodecyl groups form stable complexes with human serum albumin with a dissociation constant of approximately 10-6 M. The oligonucleotide micelles are simultaneously destroyed upon binding to albumin. Using an electrophoretic mobility shift assay and affinity modification, we examined the ability of DNA duplexes containing triazinyl phosphoramidate oligonucleotides to interact with Ku antigen and PARP1, as well as the mutual influence of PARP1 and albumin or Ku antigen and albumin upon interaction with DNA duplexes. These findings, together with the capability of dodecyl-containing derivatives to effectively penetrate different cells, such as HEK293 and T98G, indicate that the oligonucleotides under study can be considered as a platform for the development of therapeutic preparations with a target effect.

The Duffy protein, a transmembrane molecule, functions as a receptor for various chemokines and facilitates attachment between the reticulocyte and the Plasmodium Duffy antigen-binding protein. Duffy expression correlates with the Duffy receptor gene for the chemokine, located on chromosome 1, and exhibits geographical variability worldwide. Traditionally, researchers have described the Duffy negative genotype as a protective factor against phenotypic malaria expression. However, recent studies suggest this microorganism's evolution potentially diminishes this protective effect. Nevertheless, there is currently insufficient global data to demonstrate this phenomenon. This study aimed to evaluate the relationship between the Duffy genotype/phenotype and the prevalence of Plasmodium vivax infection. The protocol for the systematic review was registered in PROSPERO as CRD42022353427 and involved reviewing published studies from 2012 to 2022. Medline, Web of Science, Scopus, and Scielo databases were consulted. Assessments of study quality were conducted using the STROBE and GRADE tools. A total of 34 studies were included, with Africa accounting for most recorded studies. The results varied significantly regarding the relationship between the Duffy genotype/phenotype and Plasmodium vivax invasion. Some studies predominantly featured the negative Duffy genotype yet reported no malaria cases. Other studies identified minor percentages of infections. Conversely, certain studies observed a higher prevalence (99%) of Duffy-negative individuals infected with Plasmodium vivax. In conclusion, no evidence of a gender-specific distribution of malaria between Duffy-negative men and women was found. However, evidence supports that the homozygous Duffy genotype positive for the A allele (FY*A/*A) is associated with a higher incidence of Plasmodium infection. Furthermore, the negative Duffy genotype does not confer protection against this disease.

Chronic hepatitis B (CHB) infection and the Hepatitis B virus protein X (HBx) are a major risk factor for the development of hepatocellular carcinoma (HCC). In CHB, HBx induces mitochondrial dysfunction, exhaustion and impaired function in hepatocytes. Restoring hepatocyte health along with reduction in virus replication could be an ideal treatment of CHB. Thiourea derivatives are well known for their antiviral property though their effect on mitochondrial and/ or hepatocyte health is obscure. The current pilot study focus on the repurposing of DSA-00, DSA-02, and DSA-09 on mitochondrial health and hepatocyte replenishment. HepG2.2.15 cells were treated with these, alongside Entecavir (ETV). The proteomics analysis showed DSA-00 and ETV were enriched with proteins associated with antiviral responses. DSA-00 additionally showed increase in proteins linked to mitochondrial response. DSA-02 showed association with innate immune system and citric acid cycle. DSA-09 displayed pathways similar to DSA-00 and ETV. The treated groups exhibited enhanced bio-energetic and antiviral response compared to the untreated group. FACS analysis, validated thiourea derivatives restored exhausted hepatocytes by targeting mitochondrial potential and function. Our findings suggest that (DSA-00, DSA-02, and DSA-09) may hold potential as a novel treatment strategy restoring mitochondrial healthy along with anti-viral response in CHB.

Abstract: Due to their rapid evolution and their impact on health care, be-ta-lactamases, protein degrading beta-lactam antibiotics, are used as generic model of protein evolution. Therefore, we investigated the mutation effects in two distant beta-lactamases TEM-1 and CTX-M-15. Interestingly we found a site with a complex pattern of genetic interactions. Mutation G251W in TEM-1 is in-activating the protein’s function just as the reciprocal mutation W251G in CTXM-15. Phylogenetic analysis revealed that mutation G has been entrenched in TEM-1 background: while rarely observed throughout the phylogeny it is es-sential in TEM-1. Using a rescue experiment in TEM-1 G251W mutant, we could identify sites that alleviates the deviation from G to W. While few of these muta-tions could potentially involve local interactions, most of them were found on distant residues in the 3D structure. Many well-known mutations having an impact on protein stability, such as M182T, were recovered. Our results there-fore suggest that entrenchment of an amino acid may rely on diffuse interactions among multiple sites with a major impact on protein stability.

The recent outbreak of the new virus in Wuhan city, China from the sea food market has led to the identification of a new strain called the corona virus and named as novel corona virus (2019-nCoV) belonging to Coronaviridae family. This has created major havoc and concern due to the mortality of 250 persons and affecting more than 10,000 people. This virus causes sudden fever, pneumonia and also kidney failure. In this study a computational approach is proposed for drug and vaccine design. The spike protein sequences were collected from a protein database and analysed with various bioinformatics tools to identify suitable natural inhibitors for the N-terminal receptor binding domain of spike protein. Also, it is attempted to identify suitable vaccine candidates by identifying B-Cell and T-cell epitopes. In the drug design, the tanshinone Iia and methyl Tanshinonate were identified as natural inhibitors based on the docking score. In the vaccine design, B-cell epitope VLLPLVSSQCVNLTTRTQLPPAYTN was found to have the highest antigenicity. FVFLVLLPL of MHC class-I allele and FVFLVLLPL of MHC class-II allele were identified as best peptides based on a number of alleles and antigencity scores. The present study identifies natural inhibitors and putative antigenic epitopes which may be useful as effective drug and vaccine candidates for the eradication of novel corona virus.

We present a case of a 47-year-old male who died unexpectedly from acute pulmonary hemorrhage 555 days after completing the BNT162b2 (Pfizer) COVID-19 vaccination primary series. Before death, he exhibited symptoms of a mild respiratory infection. Despite a healthy medical history and no medication use, the patient’s condition rapidly deteriorated and he experienced severe respiratory distress, followed by cardiopulmonary arrest with evidence of profuse pulmonary bleeding. Autopsy findings revealed massive lung congestion without embolism, normal heart size, moderate coronary atherosclerosis without myocardial infarction, and no evidence of other hemorrhagic events. The patient tested negative for COVID-19 and other respiratory pathogens at autopsy. Despite these findings, the medical examiner determined the cause of death was attributed to atherosclerotic and hypertensive cardiovascular disease, without considering the recent pulmonary hemorrhage and unremarkable medical history. Investigation into the vaccine batch indicated a higher-than-average number of serious adverse events, including fatalities. The patient's BNT162b2 batch was among the top 2.8% for reported deaths. Moreover, the autopsy failed to investigate potential contributions from the vaccine, such as the presence of the Spike protein or related antibodies. The evidence suggests that the pulmonary hemorrhage, exacerbated by a viral infection, was the immediate cause of death, with the COVID-19 vaccine potentially playing a role in the development of cardiopulmonary pathology and hemorrhage. We propose autopsy protocols for COVID-19 vaccine recipients to better investigate vaccine-related pathologies among those with one or more prior injections.

Calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) regulates bone remodeling through its effects on osteoblasts and osteoclasts. However, its role in osteocytes, the most abundant bone cell type, and the master regulator of bone remodeling, remains unknown. Here we report that the conditional deletion of CaMKK2 from osteocytes using Dentine matrix protein 1 (Dmp1)-8kb-Cre mice led to enhanced bone mass only in female mice owing to a suppression of osteoclasts. Conditioned media isolated from female CaMKK2-deficient osteocytes inhibited osteoclast formation and function in in vitro assays, indicating a role for osteocyte-secreted factors. Proteomics analysis revealed significantly higher levels of extracellular calpastatin, a specific inhibitor of calcium-dependent cysteine proteases calpains, in female CaMKK2 null osteocyte conditioned media, compared to media from female control osteocytes. Further, exogenously added non-cell permeable recombinant calpastatin domain I elicited a marked, dose-dependent inhibition of female wild-type osteoclasts and depletion of calpastatin from female CaMKK2-deficient osteocyte conditioned media reversed the inhibition of matrix resorption by osteoclasts. Our findings reveal a novel role for extracellular calpastatin in regulating female osteoclast function and unravel a novel CaMKK2-mediated paracrine mechanism of osteoclast regulation by female osteocytes.

Multiple myeloma (MM) accounts for about 10 to 15% of all diagnosed hematologic malignancies and about 1-2% of all cancer cases. Approximately 80-90% of MM patients develop bone disease and the changes rarely regress. It is only possible to stop or slow their progression. A major role in bone destruction in MM is attributed to the Wnt signaling pathway, and its action can be modified by various types of interventions including training and diet. Therefore, the aim of this project was to evaluate the effects of a Nordic Walking (NW) training cycle and intermittent fasting (IF) on the levels of selected bone turnover markers associated with the Wnt pathway in patients with MM. Results from 35 patients divided into training (NW and IF NW) and non-training (IF and control) groups were included in the analysis. A 6-week training cycle involving 60-minute workouts 3 times a week was conducted. Body mass and composition and the levels of: vitamin D, calcium and phosphorus, beta2-microglobulin and albumin were examined before and after completion of training cycle. Markers of bone turnover were also determined: sclerostin (SOST), Dickkopf-related protein 1 (DKK-1), osteoprotegrin (OPG), osteopontin (OPN) and Tartrate-resistant acid phos-phatase 5b (TRACP 5b). There was no negative effect of IF or combined training and fasting on the nutritional status of the patients (the level of albumines was unchanged). Both training groups showed an increase in serum concentrations of the active metabolite of vitamin D (IF NW and NW: p=0.001 and p=0.022, re-spectively). The change in the concentration of this vitamin negatively correlated with the con-centration of TRACP 5b (r=-0.413, p=0.014). Evaluating the concentrations of markers related to bone turnover, a reduction in the concentrations of SOST (time: p=0.026, time vs. group: p=0.033) and TRACP 5b (time: p

Medicinal plants possess a surplus of novel and biologically active secondary metabolites that are responsible for counteracting diseases. Traditionally, Gomphandra tetrandra (Wall.) Sleumer is used to treat mental disorders. The present research was designed to explore phytochemicals from the ethanol leaf extract of Gomphandra tetrandra (Wall.) Sleumer to identify the potential pharmacophore(s) in the treatment of neurological disorders. The chemical compounds of the experimental plant were identified through GC-MS analysis. Besides, in-vitro antioxidant activity was assessed using different methods. Furthermore, in-vivo neurological activity was assessed in Swiss-albino mice. Computer aided analysis was appraised to determine the best-fit phytoconstituent of a total of fifteen identified compounds in the experimental plant extract against beta-amyloid precursor protein. The experimental extract revealed fifteen compounds in GC-MS analysis and the highest content was 9, 12, 15-octadecatrienoic acid (Z, Z, Z). Also, the extract showed potent anti-oxidant activity in in-vitro assays. Furthermore, in in-vivo neurological assays, the extract disclosed significant (p<0.05) neurological activity. The most favorable phytochemicals as neurological agents were selected via ADMET profiling and molecular docking was studied with beta-amyloid precursor protein. Moreover, in the computer aided study, 1, 5-Diphenyl-2H-1, 2, 4-triazoline-3-thione (Pub Chem CID: 2802516) was more active than other identified compounds with strong binding affinity to beta-amyloid precursor protein. The present in vivo and in silico studies revealed neuropharmacological features of G. tetrandra leaves extract as a natural agent against neurological disorders, especially Alzheimer’s disease.

Currently, entire world is crumbled due to COVID-19 caused by novel SARS-CoV-2. Globally, over 5 million people are infected by SARS-CoV-2 with 6% fatality rate. The surface spike (S) protein plays a key role in the pathogenesis of SARS-CoV-2 by mediating viral entry through human angiotensin converting enzyme 2 (hACE2) receptors on the host cell and there is a big global race to find viral neutralizing antibodies and vaccine against S protein of SARS-CoV-2. Since SARS-CoV-2 evolved into 10 different clades in a very short span, a study on sipke protein mutation is essential to have effective vaccine coverage globally. Based on the mutation analysis of S protein from 166 Indian SARS-CoV-2 genome, a total of 40 different SNPs comprising of 14 synonymous and 26 non-synonymous mutations were observed, and notably, Indian S protein diverged into two major clusters, D614 and G614, with 11 different types. Majority of Indian strains fall in A2a and O clusters. Alarmingly, we have observed six SNPs at RBD and notably two of them at RBM (S438F and S494P). S494P SNP, similar to Bat–SARS like-CoV, may indicate a low ACE2 binding affinity. Interestingly 38% of Indian strains harbor a characteristic D614G SNP which was found predominantly in A2a cluster, mostly comprising USA and European strains with high disease severity. The association of disease severity with D614G SNP is well-correlated in states with high death rate except Maharashtra. Notably, more than 50% of D614G mutation were observed in Northern part of India and 14% in Southern part but not in Kerala and Tamil Nadu strains. Highly conserved motif, D614 (608-VAVLYQDVNCT-618) in upstream and also few downstream, of S1/S2 furin cleavage site may indicate specific key role in efficient interaction with host proteases in pathogenesis. Further studies are warranted to clarify the impact of SD614G SNP association to disease severity . Interestingly, C2367T (Y789Y) synonymous SNP is observed in 37% of Indian strains and notably similar SNPs with degeneracy bases were observed which is a key indication for the possibility of misdiagnosis by Real-Time PCR and revised strategies are needed for the precise diagnosis. Circulation of high number of signature SNPs [D614G and C2367T (Y789Y)] in certain states may be an early indication of emergence of community transmission in India. Further large genome sequence data from India will aid in deep understanding on the diversity of circulating SASR-Cov-2 and its impact on disease severity, origin of imported cases to India, community spread, effect on diagnosis and vaccine coverage.

Lipoxygenases are non-heme iron-containing enzymes that catalyze the oxidation of polyunsaturated fatty acids, resulting in the production of lipid hydroperoxides, which are precursors of inflammatory lipid mediators. The enzymes are widely distributed in humans, other eukaryotes, and cyanobacteria. Lipoxygenases hold promise as therapeutic targets for several human diseases, including cancer and inflammation-related disorders. Inhibitors of lipoxygenase have potential applications in pharmaceuticals, cosmetics, and food. Bioactive peptides are short amino acid sequences embedded within parent proteins, which can be released by enzymatic hydrolysis, microbial fermentation, and gastrointestinal digestion. A wide variety of bioactivities have been documented for protein hydrolysates and peptides derived from different biological sources. Recent findings indicate that protein hydrolysates and peptides derived from both edible and non-edible bioresources can act as lipoxygenase inhibitors. In this review, we explore current knowledge on the production of anti-lipoxygenase protein hydrolysates and peptides from millet grains, chia seeds, insects, milk proteins, fish feed, velvet antler blood, fish scales, and feather keratins. We delve into the anti-lipoxygenase activities and modes of action of these protein hydrolysates and peptides, while also highlighting the strengths and shortcomings of previous research. Additionally, we propose potential research directions and areas of improvement that can propel the discovery of anti-lipoxygenase peptides in the near future.

Background: Acute pancreatitis is a common emergency. Morbidity control requires early detection of disease severity. METHODS: A total of 131 AP patients were analyzed retrospectively. Patients were divided into two groups mild AP (MAP: Ranson score <3) and severe AP (SAP: Ranson score ≥3), according to Ranson’s criteria. Demographic data, hospitalization duration, PNI, CRP, and RDW levels were compared. Any p-value below 0.05 (p<0.05) was accepted as statistically significant. RESULTS: Study included 67 (51.15%) males and 64 (48.85%) females. The age average was 59.74 (19-90) years. 95 (72.52%) patients had MAP, and 36 (27.48%) patients had SAP. Mean hospitalization time, PNI, and CRP differed significantly between the two groups (p=0.010, p<0.05, p<0.05, respectively). The RDW (p=0.380) level difference was insignificant. For SAP prediction; the sensitivity, specificity and cut-off value according to Ranson code cut-off point for PNI were determined as 80.0% (95% CI:54.8-85.8), %72.2 (95% CI:70.5-87.5) and ≤45.6 (gr/L) + (mm3), respectively, and 94.7% (95% CI:57.8-87.9), %75.0 (95% CI:88.1-98.3) and >105,1 mg/L, respectively for CRP. CONCLUSION: PNI and CRP values (but not RDW values) were compatible with the disease severity determined by the Ranson criteria.

Protein phosphorylation and ADP-ribosylation (ADPr), as two types of post-translational modifications (PTM), are the process of adding phosphate group and ADP-ribose moieties to proteins, respectively. Although both PTM types can occur on many amino acid types, serine is the most common. Serine phosphorylation (pS), serine ADPr (SADPr), and their in situ crosstalks (pSADPr) play essential roles in biological processes. Although in silico classifiers have been developed for predicting pS and SADPr sites, the classifier for predicting pSADPr sites is unavailable. In this study, we developed classifiers to predict pSADPr sites. Specifically, we collected 3250 human pSADPr, 7520 SADPr, 151,227 pS and 80,096 unmodified serine sites. Based on them, we investigated the characteristics of pSADPr sites and constructed three classifiers to predict pSADPr sites from the pS dataset, the SADPr dataset and the protein sequences separately. We built and evaluated five deep-learning classifiers in ten-fold cross-validation and independent test datasets. Three of them (e.g. Convolutional Neural Network with the One-Hot encoding, dubbed CNNOH) performed better than the rest two. For instance, CNNOH had the AUC values of 0.700, 0.914 and 0.954 for recognizing pSADPr sites from the SADPr, pS and unmodified serine sites.Therefore, it is challenging to distinguish pSADPr sites from SADPr sites compared to the other two. It is consistent with our observation that pSADPr's characteristics are more similar to those of SADPr than the rest. Furthermore, we used the classifiers as base classifiers to develop a few stacking-based ensemble classifiers to improve performance. However, none of the ensemble classifiers showed better performances, suggesting that the base classifiers have good enough performances. Finally, we developed an online tool for extensively predicting human pSADPr sites based on the CNNOH classifier, dubbed EdeepSADPr. It is freely available through http://edeepsadpr.bioinfogo.org/.

Both grape pomace and whey are waste products from the food industry that are rich in valuable ingredients. The utilisation of these two by-products is becoming increasingly possible as consumer awareness of upcycling increases. The biological activities of grape pomace extract (GPE) are diverse and depend on its bioavailability, which is influenced by processes in the digestive system. In this work, goat whey protein (GW) was used as the primary coating to protect the phenolic compounds of GPE during the spray drying process. In addition, trehalose (T), sucrose (S), xylose (X) and maltodextrin (MD) were added to the goat whey proteins as co-coatings and protein stabilisers. All spray drying experiments resulted in microcapsules (MC) with high encapsulation efficiency (77.6—95.5%) and yield (91.5—99.0%) and almost 100% recovery of phenolic compounds during the release test. For o-coumaric acid, the GW-coated microcapsules (MC) showed a bioavailability index of up to 731.23%. A semi-crystalline structure and hydrophilicity are characteristics of the MC coated with 10% T, S, X or 5% MD. GW alone or in combination with T, S, MD or X has proven to be a promising carrier for polyphenols from grape pomace extract and ensures good bioavailability of these natural antioxidants.

We previously found that chronic adenosine A1 receptor stimulation with N6-Cyclopentyladenosine increased α-synuclein misfolding and neurodegeneration in a novel α-synucleinopathy model, a hallmark of Parkinson’s disease. Here, we aimed to synthesize a dimer caffeine-indan linked by a 6-carbon chain to cross the blood-brain barrier and tested its ability to bind α-synuclein, reducing misfolding, behavioural abnormalities, and neurodegeneration in our rodent model. Behavioural tests and histological stains assessed neuroprotective effects of the dimer compound. A rapid synthesis of the 18F-labelled analogue enabled Positron Emission Tomography and Computed Tomography imaging for biodistribution measurement. Molecular docking analysis showed that the dimer binds to α-synuclein N- and C-termini and the non-amyloid-β-component (NAC) domain, similar to 1-aminoindan, and this binding promotes a neuroprotective α-synuclein “loop” conformation. The dimer also binds to the orthosteric binding site for adenosine within the adenosine A1 receptor. Immunohistochemistry and confocal imaging showed the dimer abolished α-synuclein upregulation and aggregation in the substantia nigra and hippocampus, and the dimer mitigated cognitive deficits, anxiety, despair, and motor abnormalities. The 18F-labelled dimer remained stable post-injection and distributed in various organs, notably in the brain, suggesting its potential as a Positron Emission Tomography tracer for α-synuclein and adenosine A1 receptor in Parkinson’s disease therapy.

Respiratory syncytial virus (RSV) infection is a major cause of severe respiratory disease in infants and young children worldwide. Two vaccines targeting the elderly and pregnant women have been recently approved employing the prefusion form of the RSV-fusion protein (F). However, there are currently no vaccines for infants. Studies have shown a fusion-inactive prefusogenic F form is significantly more immunogenic and produces higher antibody titers to both the prefusion and postfusion structures of the F protein. Here we have developed RSV virus-like particles (RSV-VLPs) consisting of prefusogenic F, RSV glycoprotein and matrix proteins, produced them using the baculovirus expression system, and studied their protective efficacy in Balb/c mice. Morphology and successful assembly of VLPs were confirmed by transmission electron microscopy and western blot. Mice immunized with VLPs developed high levels of serum IgG and neutralizing antibodies as compared with mice immunized with inactivated virus. The VLP vaccine also induced higher levels of IFNγ and IL4, elicited limited proliferation of CD4+ and CD8+ T cells but higher cytotoxic-T-lymphocyte responses. VLP immunization abolished lung pathology in the mice after RSV challenge. Overall, our results indicate that RSV-VLPs consisting of prefusogenic F, glycoprotein and matrix proteins are a potential vaccine candidate against RSV.