Bacteria a Microscopic organisms are the most inexhaustible and flexible of microorganisms and constitute a huge division of the whole living earthly biomass, certain microorganisms were found to amass metallic components at a high limit Was Known as Bacterial Bio-sorption Due to their little size, capacity to become under controlled conditions, and their Accommodation to an extensive variety of ecological situations; Potent metal bio-sorbents among microorganisms, at low pH esteems, cell divider ligands are protonated and contend essentially with metals for official. With expanding pH, more ligands, such as amino and carboxyl groups, could be exposed, leading to attraction between these negative charges and the metals, and consequently increment bio-sorption onto the cell surface. Starting with Isolation and identification of heavy metal-resistant bacteria from rock Ore. Studying Factors Affecting Uranium Bio-sorption, Optimization of bacterial growth conditions and optimum for metal uptake by free and immobilized bacterial cells and Desorption ratio of uranium ions adsorbed by Coli. /alginate, All this evidence suggest that functions groups Represented in our study are responsible for metal uptake in our bacterial biomass beside change in peaks position which assigned for it's groups confirm bio-sorption of metal ions from waste due to ions charge interaction comparing with immobilized we found increase in no of binding sites indicate that immobilized bacterial have high efficiency for metal up take which also change in peaks position which assigned for its groups confirm bio-sorption of metal ions from waste due to ions charge interaction, Where the high bio-sorption yield obtained by bacteria, the Uranium & heavy metal bioremediation process expects microorganisms to be joined to a strong surface.

Magnetogenetics emerges as a transformative approach for modulating cellular signaling pathways through the strategic application of magnetic fields and nanoparticles. This technique leverages the unique properties of magnetic nanoparticles (MNPs) to induce mechanical or thermal stimuli within cells, facilitating the activation of mechano- and thermosensitive proteins without the need for traditional ligand-receptor interactions. Unlike traditional modalities that often require invasive interventions and lack precision in targeting specific cellular functions, magnetogenetics offers a non-invasive alternative with the capacity for deep tissue penetration and the potential for targeting a broad spectrum of cellular processes. This review underscores magnetogenetics' broad applicability, from steering stem cell differentiation to manipulating neuronal activity and immune responses, highlighting its potential in regenerative medicine, neuroscience, and cancer therapy. Furthermore, the review explores the challenges and future directions of magnetogenetics, including the development of genetically programmed magnetic nanoparticles and the integration of magnetic field-sensitive cells for in vivo applications. Magnetogenetics stands at the forefront of cellular manipulation technologies, offering novel insights into cellular signaling and opening new avenues for therapeutic interventions.

Currently, the textile industry is among the most rapidly expanding areas of the economy and is an important source of water pollution. There are many efficient chemical and physical methods for treating textile effluent, but they produce secondary pollutants. Therefore, there is a need to manage the textile waste wastewater. The potential EPS-producing and plant growth-promoting rhizobacteria Lysinibacillus capsici PB300 (T) bacteria strain has been isolated from the bark borer-affected Peltophorum pterocarpum plant and has been determined to be effective in > 95 decolorizing Reactive Red 120, and other azo dyes such as AB11385%, orange II 94.62%, Congo red 94.62%, phenol red 94.54%, and mixtures of azo dyes 81.66%. Physico-chemical factors were optimized manually, including Taguchi design. EPS production, PGPR strain, and fabric discoloration of L. capsici were qualitatively studied. The FTIR, GC-MS, and UV absorbance studies also confirmed that the dye had been broken into its amines. Research findings using enzyme assays have shown that the bacteria Lysinibacillus capsici PB300 (T) can utilize laccase and manganese peroxidase and are capable of degrading dyes significantly. According to this work, immobilised L. capsici cells and the studied four bacterial consortiums, Lysinibacillus capsici PB300 (T), Alcaligenes faecalis subsp. Phenolicus DSM 16503 (T), Acinetobacterbaumanni ATCC 19606 (T), and Pseudomonasaeruginosa JCM 5962 may be employed to degrade RR120 effectively, and it is concluded that L. capsici PB300 (T) bacteria is significantly efficient in textile effluent treatment.

Metal-supported fuel cells (MSCs) offer potential material cost and robustness advantage over anode supported cells (ASCs). Because of the very good thermal shock stability of such MSCs, these can be heated up very quickly. However the challenge co-firing of metal substrate and electrolyte stays still unsolved. The production of the core component, the tape casted metal substrate, with defined shrinkage resulting from modification of metal powders is presented in the paper. This approach leads to an adjustment of the shrinkage mismatch during sintering, between metal and ceramic components, and adjustment of target specifications like porosity, green density and layer thickness.

The paper presents the author’ original modification of the residual method in order to construct an efficient tool for assessing the condition of photovoltaic cells, in particular – panels made of amorphous cells which are characterized by significant random operation parameter changes. The solution constructed has been verified through experiments, which show the usefulness of the proposed methodology as it allows to minimize the risk of obtaining false diagnostic information in the process of assessing the condition of photovoltaic panels.

The seed habit is the most complex and successful method of sexual reproduction in vascular plants. It represents a remarkable moment in the evolution of plants that afterwards spread on land. In particular, seed size had a pivotal role as for evolutionary success and agronomic trait, especially in the field of crop domestication. Given that crop seeds constitute one of the primary products for consumption, it follows that seed size represents a fundamental determinant of crop yield. This adaptative feature is strictly controlled by genetic traits from both maternal and zygotic tissues, although seed development and growth are also affected by environmental cues. Despite being a highly exploited topic for both basic and applied research, there are still many issues to be elucidated as for developmental biology as well as for agronomic science. This review addresses a number of open questions related to cues that influence seed growth and size, and how developmental processes are influenced by them. Besides, new insights on the genetic-molecular control of this adaptive trait are presented.

Cell free systems are a widely used research tool in systems and synthetic biology and a promising platform for manufacturing of proteins and chemicals. In the past, cell free biology was primarily used to better understand fundamental biochemical processes. Notably, E. coli cell free extracts were used in the 1960s to decipher the sequencing of the genetic code. Since then, the transcription and translation capabilities of cell free systems have been repeatedly optimized to improve energy efficiency and protein yield. Today, cell free systems, in combination with the rise of synthetic biology, have taken on a new role as a promising technology for just in time manufacturing of therapeutically important biologics and high-value small molecules. They have also been implemented in an industrial scale for the production of antibodies and cytokines. In this review, we discuss the evolution of cell free systems, advancements in cell free protein synthesis, and cell free metabolic engineering, and conclude with discussing the importance and feasibility of mathematical modeling in cell free systems.

Female infertility is a global medical condition that can be caused by various disorders of the reproductive system, including premature ovarian failure (POF), polycystic ovary syndrome (PCOS), endometriosis, Asherman syndrome, and preeclampsia. It affects the quality of life of both patients and couples. Mesenchymal stem cells (MSCs) have received increasing attention as a potential cell-based therapy with several advantages over other cell sources, including greater abundance, fewer ethical considerations, and high capacity for self-renewal and differentiation. Clinical researchers have examined the therapeutic use of MSCs in female infertility. In this review, we discuss recent studies on the use of MSCs in various reproductive disorders that lead to infertility. We also describe the role of microRNAs (miRNAs) and exosomal miRNAs in controlling MSC gene expression and driving MSC therapeutic outcomes. The clinical application of MSCs holds great promise for the treatment of infertility or ovarian insufficiency and to improve reproductive health for a significant number of women worldwide.

The therapeutic, attenuative, and morphological effects of proprietary neural stem cell-derived extracellular vesicles (NSC EVs, AB126) produced by Aruna Bio, Inc. were assessed in a range of in vitro and in vivo models of stroke. We emphasize that modulation of neuroinflammation is a key target and suggest further studies to advance our understanding of the mechanisms of action and ultimately how best to dose and monitor the benefits of AB126 in stroke patients.

Natural carotenoids are secondary metabolites that exhibit antioxidant, anti-inflammatory and anti-cancer properties. These types of compounds are in high demand by pharmaceutical, cosmetic, textile and food industries, leading to the search for new natural sources of carotenoids. In recent years, the production of carotenoids from bacteria has become of great interest for industrial applications. In addition to carotenoids with C40-skeletons, some bacteria have the ability to synthesize characteristic carotenoids with C30-skeletons. In this regard, a great variety of methodologies for the extraction and identification of bacterial carotenoids has been reported and this is the first review that condenses much of this information. To understand the diversity of these carotenoids, we present their biosynthetic origin in order to focus on the methodologies employed in their extraction and characterization. Special emphasis has been made on high-performance liquid chromatography-mass spectrometry (HPLC-MS) for the analysis and identification of bacterial carotenoids. We end up this review showing their potential commercial use of bacterial carotenoids. This review is proposed as a guide for the identification of these metabolites, which are frequently reported in new bacteria strains.

The fuel cell particularly the Proton Exchange Membrane Fuel Cell (PEMFC) is a new energy technology that plays a critical role in achieving market viability. The PEMFC has high power density, zero CO₂ emissions, and high conversion efficiency. However, with uniform distribution of fuel in PEMFC, we can improve the performance and reduce the cost. The PEMFC will be more efficient and effective in terms of renewable energy as a byproduct only water. Metal foam is used as an alternative for the channels in PEMFC. The model of PEMFC was established by using the ANSYS Fluent 3D Model. Then we proposed four configurations of PEMFC. The outcome demonstrates that Case IV provided outstanding performance if it is compared with the other three cases. In terms of power density, Case IV provided a high power density that is double that of Case I. The porosity of metal foam was investigated. The 0.6 porosity value is found as the optimal value. The uniform distribution of the fuel can be achieved at this value which leads to the outstanding current density.

Background: Liquid biopsies are revolutionary tools to detect tumor-specific genetic alterations in body fluids, and cell-free DNA (cfDNA) can be used for molecular diagnosis in cancer patients. In brain tumors cerebrospinal fluid (CSF) cfDNA might be more informative than plasma cfDNA. Here, we assess the use of CSF cfDNA in pediatric embryonal brain tumors (EBT) for molecular diagnosis. Methods: CSF cfDNA of pediatric patients with medulloblastoma (n=18), ATRT (n=3), ETMR (n=1), CNS NB FOXR2 (n=2) and pediatric EBT NOS (n=1) (mean cfDNA concentration 48 ng/mL; range 4-442 ng/mL) and matched tumor genomic DNA were sequenced by WES, and/or a targeted sequencing approach, to determine single nucleotide variations (SNVs)/copy number alterations (CNA). A specific capture covering transcription start sites (TSS) of genes of interest was used for nucleosome footprinting in CSF cfDNA. Results: 15/25 CSF cfDNA samples yielded informative results, with informative CNA and SNVs in 11 and 15 cases, respectively. For cases with paired tumor and CSF cfDNA WES (n=15), a mean of 83 (range 1-160) shared SNVs was observed, including SNVs in classical medulloblastoma genes such as SMO and KMT2D. Interestingly, tumor-specific SNVs (mean 18; range 1-62) or CSF-specific SNVs (mean 5; range 0-25) were observed suggesting clonal heterogeneity. The TSS panel resulted in differential coverage profiles across all 112 studied genes in 7 cases, indicating distinct promoter accessibility. Conclusion: CSF cfDNA sequencing yielded informative results in 60% of cases (15/25), with WES feasible in 83% (15/18). These results pave the way for implementation of these novel approaches for molecular diagnosis and minimal residual disease monitoring.

Plant cells' ability to withstand abiotic stress is strongly linked to modifications in their mechanical characteristics. Nevertheless, the lack of a workable method for consistently tracking plant cell mechanical properties severely restricts our comprehension of the mechanical alterations in plant cells under stress. With Polyethylene Glycol (PEG6000), we created a drought-like environment, and we used the Double Resonator Piezoelectric Cytometry (DRPC) method to dynamically and non-invasively track changes in the stress (ΔS) created and viscoelasticity (storage modulus G' and loss modulus G") of protoplasts and suspension cells of rice during drought stress. The findings demonstrate that, rice suspension cells and protoplasts react mechanically differently to 5%–15% PEG6000 stress, implying distinct resistance mechanisms. However, neither of them can withstand 25% PEG6000 stress, they respond mechanically similarly to 25% PEG6000 stress. The results of DRPC are further corroborated by the morphological alterations of rice cells and protoplasts observed under an optical microscope. To sum up, the DRPC technique functions as a precise cellular mechanical sensor and offers novel research tools for the evaluation of plant cell adversity and differentiating between the mechanical reactions of cells and protoplasts under abiotic stress.

Circulating cell-free DNA (cfDNA) refers to small fragments of DNA molecules released after programmed cell death and necrosis in several body fluids such as blood, saliva, urine, and cerebrospinal fluid. The discovery of cfDNA has revolutionized the field of non-invasive diagnostics in the oncologic field, in prenatal testing, and in organ transplantation. Despite the potential of cfDNA and the solid results published in recent literature, several challenges remain, represented by low abundance, need for highly sensitive assays and analytical issues. In this review, the main technical advances in cfDNA analysis are presented and discussed, with a comprehensive examination of the current available methodologies applied in each field. Considering the potential advantages of cfDNA, this biomarker is increasing its consensus among clinicians, as it consents to monitor patients’ conditions in an easy and non-invasive way, offering a more personalized care. Nevertheless, cfDNA analysis is still considered a research marker to be further validated, and very few centers are implementing its analysis in the real-life assistance. As technical improvements are enhancing the performances of cfDNA analysis, its application will transversally improve patients’ quality of life.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and high-risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. Particularly, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, where many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed association with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.

Cell-free massive multiple input multiple output (MIMO) has the potential of providing joint services including joint initial access, efficient clustering of access points (APs) and pilot allocation to user equipments (UEs) over large coverage area with reduced interference. In cell-free massive MIMO, large coverage area corresponds to provision and maintenance of scalable quality of service requirements for infinitely large number of UEs. The research in cell free massive MIMO is mostly focused on time division duplex mode due to availability of channel reciprocity which aids in avoiding feedback overhead. However, frequency division duplex (FDD) protocol still dominates the current wireless standards and the provision of angle reciprocity aids in reducing this overhead. The challenge of providing a scalable cell-free massive MIMO system in FDD setting is also prevalent, since computational complexity regarding signal processing tasks such as channel estimation, precoding/combining and power allocation, becomes prohibitively high with increase in number of UEs. In this work, we consider an FDD based scalable cell-free network with angular reciprocity and dynamic cooperation clustering approach. We have proposed scalability for our FDD cell-free and perform comparative analysis with reference to channel estimation, power allocation and precoding/combining techniques. We present expressions for scalable spectral efficiency, angle based precoding/combining schemes and provide comparison of overhead between conventional and scalable angle based estimation as well as combining schemes. Simulations confirm that the proposed scalable cell-free network based on FDD scheme outperforms the conventional matched filtering scheme based on non-scalable precoding/combiming schemes. The angle based LP-MMSE in FDD cell-free network provides 14.3% improvement in spectral efficiency and 11.11% improvement in energy efficiency compared to non-scalable MF scheme.

This study propounds a sustainable alternative to petroleum-based polyurethane (PU) foams aiming to curtail this nonrenewable resource's continued and uncontrolled use. Coconut fatty acid distillate (CFAD) and crude glycerol (CG), both wastes from vegetable oil processes, were utilized for bio-based polyol production for rigid PU foam application. The raw materials were subjected to catalyzed glycerolysis with alkaline-alcohol neutralization and bleaching. The resulting polyol possessed properties suitable for rigid foam application with an average OH number of 215 mg KOH/g, acid number of 7.2983 mg KOH/g, and Gardner color value of 18. The polyol was used to prepare rigid PU foam, and its properties were determined using FTIR, TGA/DTA, and UTM. Additionally, the cell foam morphology was investigated by scanning electron microscope (SEM) in which most of its structure revealed an open-celled network and quantified at 92.71% open cell content using pycnometric testing. The PU foam thermal and mechanical analysis results showed an average compressive strength of 70.59 kPa, thermal conductivity of 46.50 mWm-1K-1, and density of 32.79 kgm-3. These properties showed its applicability as thermal insulating foam, thus, demonstrating the potential use of CFAD and CG in commercial polyol and PU foam production.

The reckless use of non-replenishable fuels by the growing population for energy and the resultant incessant emission of hazardous gases and waste products into the atmosphere insisted scientists to fabricate materials capable of managing these global threats at once. In recent studies, photo-catalysis employed focus on utilizing renewable solar energy to initiate chemical processes with the aid of semiconductors and highly selective catalysts. A wide range of nanoparticles have showcased promising photocatalytic properties. Ultrasmall metal nanoclusters (MNCs) with sizes below 2 nm, stabilized by ligands shows discrete energy levels, exhibits unique optoelectronic properties which are vital to photocatalysis. In this review, we intend to compile information on varying photocatalytic efficiency of metal NCs with respect to changes in their properties. Review discusses about the photocatalytic activity of atomically precise ligand-protected MNCs and its hybrids in the domain of in energy conversion processes like photodegradation of dyes, Oxygen Evolution Reaction (ORR), Hydrogen Evolution Reaction (HER) and CO2 Reduction Reaction (CO2RR), and photosensitizer in solar cell.

The mechanisms underlying the establishment of asymmetric structures during development remain elusive. The wing of Drosophila is asymmetric along the Anterior-Posterior (AP) axis, despite that its growth signals that drive its growth are presumably symmetric. Here, we investigated the contribution of cell recruitment, a process that drives wing fate differentiation in Drosophila, to the asymmetric shape and pattern of the adult wing. Genetic impairment of cell recruitment results in a significant reduction in adult wing asymmetry, primarily affecting the region between longitudinal vein 5 and the wing margin (L5-M). Morphometric analysis reveals that the lack of cell recruitment results in a more symmetric wing with respect to the AP, suggesting a contribution of cell recruitment to the establishment of asymmetry in the adult wing. A close examination of the expression of the wing selector gene vestigial (vg) in wing disc does not support this hypothesis. However, the circularity of the Vg pattern significantly increase in recruitment-impaired wing disc, suggesting that while the decrease of the L5-M region in the adult wing may not be attributed to cell recruitment, it could contribute to the overall asymmetry of the adult wing. We conclude that cell recruitment, a widespread mechanism that participates in growth and patterning of several developing systems, could drive, at least partially, the asymmetric shape of the Drosophila wing.

Methanotrophic bacteria that possess a unique ability of using methane as a sole source of carbon and energy have attracted considerable attention as potential producers of a single-cell protein. So far, this biotechnology implied using freshwater methanotrophs, although many regions of the world have limited freshwater resources. This study aimed at searching for novel methanotrophs capable of fast growth in saltwater comparable in composition with seawater. A methane-oxidizing microbial consortium containing Methylomarinum- and Methylotuvimicrobium-like methanotrophs has been enriched from a sediment of the river Chernavka (water pH 7.5, total salt content 30 g L-1), a tributary river of the hypersaline Lake Elton, southern Russia. This microbial consortium, designated Ch1, demonstrated stable growth on natural gas in a bioreactor in media with a total salt content of 23 to 35.9 g L-1 at a dilution rate of 0.19-0.21 h-1. The highest biomass yield of 5.8 g CDW/L with a protein content of 63% was obtained during continuous cultivation of the consortium Ch1 in a medium with a total salt content of 29 g L-1. Isolation attempts resulted in obtaining a pure culture of methanotrophic bacteria, strain Ch1-1. The 16S rRNA gene sequence of strain Ch1-1 displayed 97.09-97.24% similarity to the corresponding gene fragments of characterized representatives of Methylomarinum vadi, methanotrophs isolated from marine habitats. The genome of strain Ch1-1 was 4.8 Mb in size and encoded 3 rRNA operons, and about 4400 proteins. The genome contained the gene cluster coding for ectoine biosynthesis, which explains the ability of strain Ch1-1 to tolerate high salt concentration.

Microfluidics technology has not impacted the delivery and accessibility of point of care health services like diagnosis of infectious disease diagnosis, monitoring health or delivering interventions. Most microfluidics prototypes from academic research are not easy to manufacture with industrial scale fabrication techniques and cannot be operated without complex manipulations of supporting equipment and additives such as labels or reagents. We propose a label- and reagent-free inertial spiral microfluidic device to separate red blood, white blood and dendritic cells from blood fluid for applications in health monitoring and immunotherapy. We demonstrate that using larger channel widths in the range of 200 to 600 µm allows separation of cells into multiple streams according to different size ranges and we utilize a novel technique to collect the closely separated focused cell streams without constricting the channel. When tested on actual human blood cells, 77% of dendritic cells were separated and 80% of cells remained viable after our assay. Our contribution is a method to adapt spiral inertial microfluidic designs to separate more than two cell types in the same device which is robust against clogging, simple to operate and suitable for fabrication and deployment in resource-limited populations.

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.

Cantharidin, a toxic monoterpene secreted by blister beetles, has long been used in traditional Chinese and modern medicine for its unique properties. However, despite its widespread use, its effects on fish have not been studied in depth. The aim of this study was to evaluate the potential therapeutic applications of cantharidin in fish by examining its antioxidant, hemoagglutinating, hemolytic and cytotoxic activities at different concentrations (0, 0.625, 1.25, 2.5, 5 and 10 μg mL-1) in three different cell lines. In addition, the study explored the bactericidal and bacteriostatic properties of cantharidin against various fish pathogenic bacteria. The results revealed that there were no significant differences in antioxidant, hemagglutinating or hemolytic activities between the different concentrations of cantharidin tested. However, the study found that cantharidin exhibited dose- and time-dependent cytotoxicity in sea bream (Sparus aurata) erythrocytes and in SAF-1, PLHC-1 and Hela cell lines, resulting in morphological changes indicative of apoptosis. Interestingly, the highest dose of cantharidin tested demonstrated potent bactericidal activity against four marine fish opportunistic bacteria, including Vibrio harveyi, V. anguillarum, Photobacterium damselae and Tenacibaculum maritimum, but no statistically significant changes in bacteriostatic activity were observed against any of the bacteria tested. Overall, these results provide valuable information on the potential therapeutic applications of cantharidin in fish aquaculture. Further research is needed to fully understand the mechanisms of action and to explore possible preventive uses of cantharidin in fish.

Abstract: Patient blood samples are invaluable in clinical omics databases, yet current methodologies often fail to fully uncover the molecular mechanisms driving patient pathology. While Genome-scale Metabolic Models (GEMs) show promise in systems medicine by integrating various omics data, having only exometabolomic data remains a limiting factor. To address this gap, we introduce a comprehensive pipeline integrating GEMs with patient plasma metabolome. This pipeline constructs case-specific GEMs using literature-based and patient-specific metabolomic data. Novel computational methods, including adaptive sampling and an in-house developed algorithm for the rational exploration of the sampled space of solutions, enhance integration accuracy while improving computational performance. Model characterization involves task analysis in combination with clustering methods to identify critical cellular functions. The new pipeline was applied to a cohort of trauma patients to investigates shock-induced endotheliopathy using patient plasma metabolome data. By analyzing endothelial cell metabolism comprehensively, the pipeline identified critical substrates and contributed to the development of targeted therapeutic strategies. Our study demonstrates the efficacy of integrating patient plasma metabolome data into computational models to analyze endothelial cell metabolism in disease contexts. This approach offers a deeper understanding of metabolic dysregulations and provides insights into diseases with metabolic components and potential treatments.

Patient-derived tumor organoids (PDTOs) are a novel preclinical model of non-small cell lung cancer (NSCLC) for studying tumor biology and precision medicine, which recapitulates tumor morphology and gene expression profile. However, some issues such as low establishment rates, long-term production, and the loss of the immune microenvironment have hindered their widespread practical use. In this study, we investigated the efficacy of a recently introduced free-floating organoid production method to address these issues. The free-floating approach provided PDTO establishment success rate of over 90 % resulting in organoid formation within a short period of one week. These organoids retained the elements of the parental tumor morphology and contained stroma and immune cell populations, as confirmed by flow cytometry analysis. Importantly, we found that the cytokine and growth factor expression profiles of the free-floating organoids correlated with those of the original tumors in 58 % of cases, indicating the preservation of tumor-associated signaling pathways. Obtained organoids exhibited different responses to anticancer chemotherapeutics that might be a result of variable expression of drug resistance-associated genes. Considering the high establishment rate and short time of free-floating organoid production, this technique provides a valuable tool for a more accurate evaluation of therapeutic strategies and cancer biology studies.

Abstract: High step-up voltage gain nonisolated DC-DC converter have attracted much attention in photovoltaic, fuel cells and other renewable energy system applications. In this paper, by combining input current ripple-free boost cell with coupled-inductor voltage-doubler cell, an input current ripple-free high voltage gain nonisolated converter is proposed. In addition, passive lossless clamp circuit is adopted to recycle the leakage inductor energy and to reduce the voltage spike across the power switch. By utilizing voltage-doubler cell consisting of diode and capacitor, the voltage stress of switch is further reduced and the resonance between the leakage inductor and the stray capacitor of the output diode is eliminated. A low switch-on-resistance low-voltage-rated MOSFET can therefore be employed to reduce the conduction loss and cost. The reverse recovery loss of output diode is reduced, and the efficiency of converter can be improved. Furthermore, the proposed converter can achieve nearly zero input current-ripple and make the design of electromagnetic interference (EMI) filter easy. Steady state analysis and operation mode of the converter is performed. Finally, experimental results are presented to verify the analysis results of the proposed converter.

The optimal timing for actively discontinuing immune checkpoint inhibitor therapy in long-term responders with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) remains unresolved. We conducted a retrospective study of 246 patients with R/M HNSCC treated with nivolumab to determine the optimal timing to actively discontinue nivolumab therapy. We examined the point at which progression-free survival (PFS) plateaued in all cases. We compared the prognosis of 19 (7.7%) ongoing cases and 227 (92.3%) discontinued cases and analyzed treatment duration and treatment-free interval (TFI). The 6-year overall survival was 11.8% (median, 12.1), and the 6-year PFS was 15.3% (median, 3.0). The PFS curve remained stable for 3 years. The median duration of nivolumab treatment was 2.9 months (range, 0.03–81.9): Ongoing group, 41.8 (5.6–81.9); Decision group, 36.8 (4.0–70.1); Toxicity group, 30.6 (2.8–64.8); and progressive disease group, 2.0 (0.03–42.9). TFI in the Decision group was 15.1 months (0.6–61.6) and 30.6 months (2.8–64.8) in the Toxicity group. Limited number of long-term responders in R/M HNSCC cases treated with nivolumab exists. Given that PFS plateaued at 3 years, we propose that the optimal duration of treatment for this subset of patients is 3 years.

Blood-based circulating cell free DNA(cfDNA) detection offers a non-invasive and easily accessible way for early cancer detection. Despite the extensive utility of cfDNA, there are still many challenges to develop the clinical biomarkers. For example, cfDNA with genetic alterations often compose a small portion of the DNA circulating in plasma, which can be confounded by cfDNA contributed by normal cells. Therefore, filtering out the potential false-positive cfDNA mutations from health population will be important for the cancer-based biomarkers. Additionally, many low-frequency genetic alterations are easily overlooked in small amount of cfDNA-based cancer test. We hypothesize that, the combination of diverse types of cancer studies on cfDNA can provide us a new insight to identify low-frequency genetic variant across cancer types for early clinical detection of cancers. By building a standardized computational pipeline for 1358 cfDNA samples across seven cancer types, we prioritize 129 shard genetic variants in the major cancer types. Further functional analysis of the 129 variants found that they are mainly enriched in ribosome pathways such as cotranslational protein targeting to membrane, some of which are tumor suppressor, oncogene and related to cancer initiation. In summary, our integrative analysis revealed the important roles of ribosome proteins as the common biomarkers in early cancer diagnosis.

Abstract: Giant Cell Arteritis (GCA) is an autoimmune/autoinflammatory disease affecting large vessels in patients over 50 years old. The disease presents as an acute inflammatory response with two phenotypes, cranial-GCA and Large Vessel Vasculitis (LV)-GCA, involving the thoracic aor-ta and its branches. 18F-fluorodeoxyglucose positron emission tomography-computed tomogra-phy (18F-FDG PET-CT) is among the imaging techniques contributing to diagnosis of patients with systemic disease. However, its association with soluble inflammatory markers is still elu-sive. This proof-of-concept study aims to identify novel soluble serum biomarkers in PET/CT pos-itive patients with LV-GCA and associate them with active (0 months) and inactive disease (6 months following treatment), in sequential samples. The Most-Disease-Segment Target-to-Background Ratio (TBRMDS) was calculated for 9 LV-GCA patients, while 12 cranial-GCA and 7 Polymyalgia Rheumatica patients with negative initial PET/CT served as disease controls. Serum macrophage-related cytokines were evaluated by Cytometric Bead Array (CBA). Finally, previously published NMR-metabolomics data acquired at the same blood sampling were associated with PET/CT findings. TBRMDS was significantly increased in active versus inactive disease (3.45 vs. 2.55, p=0.008). The analysis identified 6 serum metabolites, as more sensitive to change from the active to inactive state. Among them, choline levels were exclusively altered in the LV-GCA group, but not the disease controls. Cytokine levels were not associated with PET/CT activity. Combining with CRP, ESR, and TBRMDS a composite index was generated to depict well the differences between active and inactive systemic LV-GCA (25.45 vs 11.45, p=0.0039). These preliminary results could pave the way for more extensive studies integrating serum metabolomic parameters with PET/CT imaging data to extract sensitive composite disease indexes useful for the everyday clinical practice.

Receptor–ligand interactions at cell interfaces initiate signaling cascades essential for cellular communication and effector functions. Specifically, T cell receptor (TCR) interactions with pathogen-derived peptides presented by the major histocompatibility complex (pMHC) molecules on antigen-presenting cells are crucial for T cell activation. The binding duration, or dwell time, of TCR–pMHC interactions correlates with downstream signaling efficacy, with strong agonists exhibiting longer lifetimes compared to weak agonists. Traditional surface plasmon resonance (SPR) methods quantify 3D affinity but lack cellular context and fail to account for factors like membrane fluctuations. In the recent years, single-molecule Förster resonance energy transfer (smFRET) has been applied to measure 2D binding kinetics of TCR–pMHC interactions in a cellular context. Here, we introduce a rigorous mathematical model based on survival analysis to determine exponentially distributed receptor–ligand interaction lifetimes, verified through simulated data. Additionally, we developed a comprehensive analysis pipeline to extract interaction lifetimes from raw microscopy images, demonstrating the model’s accuracy and robustness across multiple TCR–pMHC pairs. Our new software suite automates data processing to enhance throughput and reduce bias. This methodology provides a refined tool for investigating T cell activation mechanisms, offering insights into immune response modulation.

Although fine dust can cause various disorders, including cardiovascular, neurological, renal, reproductive, motor, systemic, respiratory, and cancerous diseases, there are not enough research for an oral disease. In this study we used various methods, including quantitative PCR, flow cytometry, immunocytochemistry, ELISA, and Alizarin O staining, to reveal the preventive effects of hemp-derived biomaterials in periodontal cells against fine dust. In this study, the two biomaterials (MSE; mature hemp stem extract and IE; induced exosomes derived from gingival cells exposed to MSE) revealed three main biological functions against fine dust. First, two materials suppressed the levels of inflammatory genes and activated anti-inflammatory genes in gingival cells. Second, these materials activated differentiation to osteoblast, periodontal ligament cells (PDLCs) and pulp progenitor cells (PPCs) from periodontal ligament stem cells (PDLSCs). Third, IE and MSE activated upregulation of LL-37 and down-regulation of MCP-1 in gingival cells and PDLSCs, respectively. Consequently, MSE and IE showed potential as a functional biomaterial in various fields, including pharmaceuticals, cosmetics, and foods.

Severe combined immunodeficiency disorders (SCID) are a genetically heterogeneous group of inherited defects characterized by severe abnormalities of immune system development and function that lead to a wide spectrum in clinical manifestations. A subgroup of patients presents a disabling and life-threatening clinical course. In these cases, allogeneic hematopoietic stem cell transplant provides a curative approach. The occurrence of SCID-associated lymphoproliferative disorders is rare and occur mostly in adenosine deaminase-deficient severe combined immunodeficiency disorders, after allogeneic hematopoietic stem cell transplant. Epstein Barr virus infection is present in the majority of individuals with primary immunodeficiency developing lymphoma, revealing compromised anti-tumour surveillance of virally transformed cells. Herein, we report a rare case of diffuse large B cell lymphoma occurring in an Epstein Barr virus negative, non-transplanted X-linked severe combined immunodeficiency 4-months-old infant, successfully treated with immunotherapy and allogeneic stem cell transplantation. This case suggests that Epstein Barr virus -independent mechanism of neoplastic transformation must take action in severe combined immunodeficiency associated lymphoma and unveils the curative potential of donor T cells after allogeneic transplantation.

Lactic acid bacteria (LAB) have an extracellular proteolytic system that includes a multi-domain, cell envelope protease (CEP) with a subtilisin homologous protease domain. These CEPs have different proteolytic activities despite having similar protein sequences. Structural characterization has previously been limited to CEP homologs of dairy and human derived LAB strains, excluding CEPs of plant derived LAB strains. CEP structures are a challenge to determine experimentally due to their large size and attachment to the cell envelope. This study aims to clarify the prevalence and structural diversity of CEPs by using the structure prediction software AlphaFold 2. Domain boundaries are clarified based on a comparative analysis of 21 three-dimensional structures, revealing novel domain architectures of CEP homologs that are not necessarily restricted to specific LAB species or ecological niches. The C-terminal flanking region of the protease domain is divided into fibronectin type-III-like domains with various structural traits. The analysis also emphasizes the existence of two distinct domains for cell envelope attachment that are preceded by an intrinsically disordered cell wall spanning domain. The domain variants and their combinations provide CEPs with different stability, proteolytic activity and potentially adhesive properties, making CEPs targets for steering proteolytic activity with relevance for both food development and human health.

The focus of this work lies in the construction of monitoring system designed in the form of a patch using a long-lasting potentiometric electrolyte concentration sensor from metal electrodes, in a galvanic cell configuration Mg(s)|NaCl(aq)|Cu(s). Initially, a mathematical analysis was conducted on the expected sensor output through the Nernst equation. The experimental phase involved conducting controlled tests in the laboratory using various molar concentrations of sodium chloride (NaCl) in aqueous solution. Results showed that the electrolyte sensor allows for monitoring electrolyte levels in NaCl in the range of 0.02 to 1 M/L, with electrode lifespan exceeding 10 days. On the other hand, when patch is using human sweat during controlled exercise routines, it showed the sensor's capability for monitoring changes in the subjects' hydration levels in real time. The sensor output potential is greater than 1.7 volts when the sensor electrodes are in contact with human sweat, which is equivalent to measuring molar concentrations at normal hydration and moderately high hydration levels. Sensor results indicate that the system can function in a self-powered manner without external batteries. Additionally, the extended lifespan of its electrodes allows for continuous monitoring of imbalances in the human body, showcasing the system capability for long-term health assessment.

Dengue virus is a global health problem most prevalent in tropical and subtropical regions. However, after the approval of two vaccines, Dengvaxia, TV003/TV005 of Takeda, for its prevention, problems were reported, like enhanced risks of infection or less efficiency of protection. Since then, more struggles have been in demand for the development of better vaccines. Here, we conducted a new design through an in-silico strategy. Initially, epitopes were chosen based on their antigenicity, immunogenicity, and binding affinity with MHC molecule, while excluded with allergenicity, toxicity and potential risk of antibody dependent enhancement. Subsequently, a core antigen was constructed with selected epitopes and linked with distinct adjuvant proteins to achieve three candidate vaccines, PSDV-1~3. PSDV-2 was chosen for further validation based on their advantage in physicochemical and structural properties. With a panel of simulations, this artificial protein showed tight binding with pattern recognition receptors, good stability, robust immune induction, and ultimately was confirmed as a high quality vaccine candidate. The plasmid for its recombinant expression was then designed accordingly. With our new design, one more choice was obtained for the effective protection from Dengue virus. Further experimental validations are still require to confirm it protection capacity and safety.

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders globally, and leads to an excessive loss of dopaminergic neurons in the substantia nigra of the brain. Circulating cell-free DNA (ccf-DNA) are double-stranded DNA fragments of different sizes and origins that are released into the serum and cerebrospinal fluid (CSF) due to cell death (i.e., necrosis and apoptosis) or are actively released by viable cells via exocytosis and NETosis. Methods: Using droplet digital polymerase chain reaction (ddPCR) we comprehensively analyzed and distinguished ccfDNA and ccf mtDNA in the serum and CSF of PD and control patients. Results: The quantitative analysis of serum ccf-DNA in PD patients demonstrated significant increase of ccf mtDNA and ccfDNA compared to healthy control patients and significantly higher copy of ccf mtDNA when compared to ccfDNA. Next, the serum ccf mtDNA levels significantly increased in male PD patients compared to healthy male controls. Furthermore, CSF ccf mtDNA in PD patients increased significantly compared to ccfDNA, and ccf mtDNA decreased in PD patients compared to healthy controls. These decreases were not statistically significant, but were in agreement with previous data. In-terestingly, ccf mtDNA increased in healthy control patients in both serum and CSF as compared to ccfDNA. Limitations: the small sample size of serum and CSF were the main limitations of this study. Conclusion: To the best of our knowledge, this is the first comprehensive study on serum and CSF of PD patients using ddPCR and indicating the distribution of the copy number of ccf mtDNA, as well as ccfDNA. If validated, we suggest that ccf mtDNA has greater than ccfDNA potential to lead the development of novel treatments for PD patients.

Solid oxide cells are capable of efficiently converting various chemical energy carriers to electricity and vice versa. The urgent challenge nowadays is the faster degradation rate compared with other fuel cell/electrolyzer technologies. To understand the degradation mechanisms, simulation of a solid oxide cell is helpful. Since most previous research developed models using commercial software, such as COMSOL and ANSYS Fluent, a gap for knowledge transfer is being gradually formed between academia and industry due to licensing issues. This paper introduces a multiphysics model, developed by a computational code, openFuelCell2. The code is implemented with an open-source library, OpenFOAM. It accounts for momentum transfer, mass transfer, electrochemical reactions and metal interconnect oxidation. The model can precisely predict I-V curves under different temperatures, fuel humidity and operation modes. Comparison between OpenFOAM and COMSOL simulations shows good agreement. The metal interconnect oxidation is modelled, which can predict the thickness of the oxide scale under different protective coatings. Simulations are conducted by assuming an ultra-thin film resistance on the rib surface. It is revealed that coatings fabricated by atmospheric plasma spraying can efficiently prevent metal interconnect oxidation, with a contribution of only 0.53 \% to the total degradation rate.

Lamin A, B and C, the nuclear intermediate-filament proteins, play a role in epigenetic regulation. While Lamin B is expressed in all nucleated cells studied, Lamin A/C are transcribed in most somatic cell types except mature B lymphocytes. Since Epstein-Barr virus (EBV), a human gammaherpesvirus, is associated with tumorigenic processes and is known to alter the epigenotype of its host cells, we studied the expression of the LMNA gene and its epigenetic marks in EBV-carrying human lymphoid cell lines. We observed a high lamin A/C mRNA and protein expression in EBV-immortalized lymphoblastoid cell lines (LCLs) and in group III Burkitt lymphoma (BL) lines where hypomethylated first exons were observed with activating histone marks. In most cell lines with low promoter activity a highly methylated first exon could be detected. Our data showed that methylation of the first exon of LMNA was associated with the downregulation of LMNA expression whereas euchromatic histone marks were enriched at active LMNA promoters in EBV-immortalized LCLs. These data suggest a role for viral latency products to activate LMNAp in EBV-infected latency type III B cells in vitro. Expression of lamin A/C may contribute to the establishment of activated B cell phenotype that needs further explorations.

At the end of 2023 the Whole Mouse Brain Atlas was announced, revealing that there are about 5,300 molecularly defined neuronal types in the mouse brain. We ask whether brain models exist that contemplate how this is possible. The conventional columnar model, implicitly used by the authors of the Atlas, is incapable of doing so with only 20 brain columns (5 brain vesicles with 4 columns each). We argue that definition of some 1,250 distinct progenitor microzones, each producing over time minimally 4-5 neuronal types, may be sufficient. This is nearly achieved presently by the prosomeric model amplified by secondary dorsoventral and anteroposterior microzonation of progenitor areas, plus clonal variation of cell types produced on average by each of them.

Recent advancements in molecular diagnostics have highlighted the potential of circulating cell-free DNA (ccfDNA) as a biomarker in various diseases. This study aims to explore the role of ccfDNA in the early diagnosis, prognosis, and therapeutic monitoring of diseases, with a focus on oncology. We conducted a comprehensive review and analysis of current literature, combined with experimental approaches involving advanced genomic sequencing and bioinformatics techniques. Our research systematically characterizes the molecular composition of ccfDNA, including its origins, size distribution, and methylation patterns, across different disease states. Furthermore, we investigate the dynamics of ccfDNA release and clearance in the bloodstream, elucidating mechanisms that could influence its diagnostic accuracy. In the context of cancer, we analyze the correlation between tumor-derived ccfDNA and disease progression, response to therapy, and overall survival. Our findings indicate that ccfDNA holds promise as a non-invasive biomarker for early disease detection, real-time monitoring of treatment efficacy, and potentially, predicting relapse. This study contributes to the growing field of liquid biopsies and opens new avenues for personalized medicine, emphasizing the need for standardized protocols in ccfDNA analysis to maximize its clinical utility. The implications of our findings extend beyond oncology, suggesting potential applications of ccfDNA in other pathological conditions.

This study describes the seasonal course of denitrifying and nitrate reducing bacteria in a dimictic mesotrophic lake (Lake Scharmützelsee, Brandenburg, Germany) within a three-year period from 2011 to 2013. The bacterial cell numbers were quantified by fluorescence microscopy, most probable number (MPN) and PCR dependent quantification of the chromosomal 16S rDNA and of the nirS and nirK gene copy number. The highest seasonal differences (up to three orders of magnitudes) has been measured by MPN in the epilimnion. This variation was not reflected by PCR dependent approaches or direct microscopical enumeration. At adverse conditions (low temperature and/or low nitrate concentrations), the differences between MPN and gene copy numbers increased up to five orders of magnitudes and decreased to one magnitude at favorable environmental conditions. These results can be explained best by an increasing ratio of viable but not cultivable (VBNC) cells or dead cells at impairing conditions. In the hypolimnion, the courses of MPN and nir gene copy numbers were similar. This can be explained by higher feeding pressure and therefore smaller amounts of dormant cells. In the pelagial in general, the total cell numbers enumerated by either microscopical or molecular approaches were similar. In the sediment, more than 99 % of the DNA was obviously not related to viable bacteria but was rather “dead DNA” in dead cells or adsorbed to particle surfaces.

Biohydrogen is a versatile energy carrier for the generation of electric energy from renewable sources. Hydrogenases can be used in enzymatic fuel cells to oxidize dihydrogen. The rate of electron transfer (ET) at the anodic side between the [NiFe]-hydrogenase enzyme distal iron-sulfur cluster and the electrode surface can be described by the Marcus equation. All parameters for the Marcus equation are accessible from DFT calculations. The distal cubane FeS-cluster has a three cysteine and one histidine coordination [Fe₄S₄](His)(Cys)₃ first ligation sphere. The reorganization energy (inner- and outer-sphere) is almost unchanged upon a histidine-to-cysteine substitution. Differences in rates of electron transfer between the wild-type enzyme and the all-cysteine mutant can be rationalized by a diminished electronic coupling between the donor and acceptor molecules in the [Fe₄S₄](Cys)₄ case. The fast and efficient electron transfer from the distal iron-sulfur cluster is realized by a fine-tuned protein environment which facilitates the flow of electrons. This study enables the design and control of electron transfer rates and pathways by protein engineering.

The DEAD-box RNA helicase DDX3X is a multifunctional protein involved in RNA metabolism and stress responses. In this study, we investigated the role of RG/RGG motifs in the dynamic process of liquid-liquid phase separation (LLPS) of DDX3X using cell-free assays and explored their potential link to cancer development through bioinformatic analysis. Our results demonstrate that the number, location, and composition of RG/RGG motifs significantly influence the ability of DDX3X to undergo phase separation and form self-aggregates. Mutational analysis revealed that the spacing between RG/RGG motifs and the number of glycine residues within each motif are critical factors in determining the extent of phase separation. Bioinformatic analysis of cancer genomic datasets uncovered a significant enrichment of DDX3X mutations in RG/RGG motifs across multiple cancer types, particularly in the N-terminal region of the protein. Furthermore, we found that DDX3X is co-expressed with the stress granule protein G3BP1 in several cancer types and can undergo co-phase separation with G3BP1 in a cell-free system, suggesting a potential functional interaction between these proteins in phase-separated structures. DDX3X and G3BP1 may interact through their RG/RGG domains and subsequently exert important cellular functions under stress situation. Collectively, our findings provide novel insights into the role of RG/RGG motifs in modulating DDX3X phase separation and their potential contribution to cancer pathogenesis.

Cadmium telluride (CdTe), a metallic dichalcogenide material, has been utilized as an absorber layer for thin film-based solar cells with appropriate configurations, and the SCAPS-1D structures program has been used to evaluate the results. In both known and developing thin film photovoltaic systems, a CdS thin film buffer layer has been frequently employed as a traditional n-type heterojunction partner. In this study, numerical simulation was used to find a suitable non-toxic material for the buffer layer instead of CdS, among various types of buffer layers (ZnSe, ZnO, ZnS, and In2S3), and carrier concentrations for the absorber layer (NA) and buffer layer (ND) were varied to determine the optimal simulation parameters. carrier concentrations (NA from 2 x 1012 cm-3 to 2 x 1017 cm-3 and ND from 1 x 1016 cm-3 to 1 x 1022 ??−3) have been differed. The results showed that the CdS as buffer layer based CdTe absorber layer solar cell has the highest efficiency (?%) of 17.43%. Furthermore, high conversion efficiencies of 17.42% and 16.27% have been found for ZnSe and ZnO based buffer layers, respectively. As a result, ZnO and ZnSe are potential candidates for replacing the CdS buffer layer in thin-film solar cells. Here, the absorber (CdTe) and buffer (ZnSe) layers were chosen to improve the efficiency by finding the optimal density of the carrier concentration (acceptor and donor). The simulation findings above provide helpful recommendations for fabricating high-efficiency metal oxide-based solar cells in the lab.

Addressing the pressing challenge of global warming, reducing greenhouse gas emissions in the transportation sector is a critical imperative. Battery and fuel-cell electric vehicles have emerged as promising solutions for curbing emissions in this sector. In this study, we conducted a comprehensive life cycle assessment (LCA) for typical passenger vehicles, heavy duty trucks and city buses using either proton-exchange membrane fuel cells or Li-ion batteries with different cell chemistries. To ensure accuracy, we supplemented existing studies with data from the literature, particularly for the recycling phase, as database limitations were encountered. Our results highlight that fuel cell and battery systems exhibit large emissions in the production phase. Recycling can significantly offset some of these emissions, but a comparison of the technologies examined revealed considerable differences. Overall, battery electric vehicles consistently outperform fuel cell electric vehicles regarding absolute greenhouse gas emissions. Hence, we recommend prioritizing battery electric over fuel cell vehicles. However, deploying fuel cell electric vehicles could become attractive in a hydrogen economy scenario where other factors, e. g. the conversion and storage of surplus renewable electricity via electrolysis, become important.

Sepsis is a life-threatening syndrome triggered by infection and accompanied by high mortality, with antimicrobial resistances (AMRs) further escalating clinical challenges. Rapid and reliable detection of causative pathogens and AMRs are key factors for fast and appropriate treatment, in order to improve outcome in septic patients. However, current sepsis diagnostics based on blood culture is limited by low sensitivity and specificity while current molecular approaches fail to enter clinical routine. Therefore, we developed a Suppression PCR-based selective enrichment sequencing approach (SUPSETS), providing a molecular method combining multiplex suppression PCR with Nanopore sequencing to identify most common sepsis-causative pathogens and AMRs using plasma cell-free DNA. Applying only 1 mL of plasma, we target eight pathogens across three kingdoms and ten AMRs in a proof-of-concept study. SUPSETS was successfully tested on first ten clinical samples and revealed comparable results to clinical metagenomics while clearly outperforming blood culture. Several clinically relevant AMRs could be detected additionally. Furthermore, SUPSETS provided first pathogen and AMR-specific sequencing reads within minutes of starting sequencing, thereby potentially decreasing time to results to 11 - 13 hours and suggesting diagnostic potential in sepsis.

Novel pH responsive semi-interpenetrating polymer hydrogels based on tamarind gum-co-poly(acrylamidoglycolic acid) (TMGA) polymers have been synthesized using simple free radical polymerization in the presence of bis[2-(methacryloyloxy)ethyl] phosphate as a crosslinker and potassium persulfate as a initiator. In addition, these hydrogels have been used as templates for green synthesis of silver nanoparticles (13.4±3.6 nm in diameter, TMGA-Ag) by using leaf extract of Teminalia bellirica as reducing agent. Swelling kinetics and equilibrium swelling behavior of the TMGA hydrogels have been investigated in various pH environment the maxium % equilibrium swelling behavior observed i.e., 2882±1.2. The synthesized hydrogels and silver nanocomposites have been characterized by the UV, FTIR, XRD, SEM and TEM. TMGA and TMGA-Ag hydrogels have been investigated to study the characteristics of drug delivery and antimicrobial study. Doxorubicin hydrochloride, a chemotherapeutic agent successfully encapsulated with maximum encapulstaion efficiency i.e., 69.20±1.2 and performed in vitro release studies in pH physiological and gastric environment at 37 ℃. The drug release behavior is examined with kinetic models such as zero order, first order, Higuchi, Hixson Crowell, Korsmeyer-Peppas. These release data was the best fitted with the Korsemeyer-Peppas transport mechanism with n=0.91. Treatment effect on HCT116 Cell, human colon cancer cells were assessed with cell viability and cell cycle analysis. Antimicrobial activity of TMGA-Ag hydrogels is studied against to Staphylococcus aureus and Klebsiella pneumonia. Finally, the results demonstrate that TMGA and TMGA-Ag are promising candidates for anti-cancer drug delivery and inactivation of pathogenic bacteria, respectively.

In the face of rising global demand and unsustainable production methods, cultivated crustacean meat (CCM) is proposed as an alternative means to produce delicious lobster, shrimp and crab products. Cultivated meat requires starting stem cells that may vary in terms of potency, and propensity to proliferate or differentiate into myogenic (muscle-related) tissues. We suggest that regenerating crustacean limbs harbor a range of stem cells suitable for cultivated meat production and present a humane tissue source because they can be accessed non-lethally. To investigate stem cell activity in this model, we have conducted RNA-Seq analysis across six stages of claw regeneration in the crayfish Cherax quadricarinatus (four pre-molt and two post-molt stages), along with histology and real time quantitative PCR (qPCR). Differential expression analysis revealed expression patterns of significantly up- or down-regulated genes in 14 functional categories. Relative expression of select stem cell target genes (including endocrine, myogenic, cell cycle and pluripotency factors) highlighted two growth peaks during pre-molt regeneration and limited stem cell activity after the molt. Histology and qPCR results support the overall finding that pre-molt limb regeneration stages harbor more stem cell activity, likely making them a more prolific cell source for CCM development.

Most research on colostrum quality and colostrum management focuses on maternal IgG transfer to the newborn calf. However, in addition to being a source of antibodies, colostrum has other components that influence passive immune transfer, nutrition, gut maturation, and thermoregulation. In this review, the constituents in the bovine colostrum, as well as their variation, are explored and it is discussed the biological role of colostrum on the newborn calf. Protein is the nutrient in higher concentration and fat is the nutrient with the most variation. There is a pronounced amplitude in the total solids content of colostrum, but the average concentration does not vary much between studies (24.7%). Bovine colostrum major components can vary with several factors, such as the region, season, breed, dry period length, and nutritional aspects of the dam. Bioactive components, cells, and microbiome variability are less understood, besides IgG. Regardless, they play crucial distinct roles in the physiological development of the newborn. However, a multi-interactive effect between these components, cells, and microorganisms is more likely to occur. Recently researched components in colostrum like miRNA, nucleo(t)sides, the milk fat globule membrane, oligosaccharides, minor proteins, and the microbiome are examples of promising interest areas of study.

Keywords: Cell-free DNA, sepsis, oxidative stress, immunoredox modulation, cecal ligation and puncture, toll-like receptor

Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including Ne-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS•tRNAPyl pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion. In the present study, we determined the crystal structure of the methanogenic archaeon ISO4-G1 PylRS (ISO4-G1 PylRS) and compared it with those of structure-known PylRSs. Based on the ISO4-G1 PylRS structure, we attempted the site-specific incorporation of Ne-(p-ethynylbenzyloxycarbonyl)-L-lysine (pEtZLys) into proteins, but it was much less efficient than that of TCO*Lys with M. alvus PylRS mutants. Thus, the first-layer mutations (Y125A and M128L) of ISO4-G1 PylRS, with no additional second-layer mutations, increased the protein productivity with pEtZLys up to 578% of that with TCO*Lys, at high enzyme concentrations in the cell-free protein synthesis.

Rapid isolation of white blood cells (WBCs) from whole blood is an essential part of any WBC examination platform. However, most conventional cell separation techniques are labor-intensive and low throughput, require large volumes of samples, need extensive cell manipulation, and have low purity. To address these challenges, we report the design and fabrication of a passive, label-free microfluidic device with a unique U-shaped cross-section to separate WBCs from whole blood using hydrodynamic forces that exist in a microchannel with curvilinear geometry. It is shown that the spiral microchannel with a U-shaped cross-section concentrates larger blood cells (e.g., WBCs) in the inner cross-section of the microchannel by moving smaller blood cells (e.g., red blood cells (RBCs) and platelets) to the outer microchannel section and preventing them from returning to the inner microchannel section. Therefore, it overcomes the major limitation of a rectangular cross-section where secondary Dean vortices constantly enforce particles throughout the entire cross-section and decrease its isolation efficiency. Under optimal settings, more than 95% of WBCs can be isolated from whole blood under high-throughput (6 ml/min), high-purity (88%), and high-capacity (180 ml of sample in 1 hour) conditions. High efficiency, fast processing time, and non-invasive WBC isolation from large blood samples without centrifugation, RBC lysis, cell biomarkers, and chemical pre-treatments make this method an ideal choice for downstream cell study platforms.

Background: Penile squamous cell carcinoma (PSCC) is classified into two prognostically distinct types: human papillomavirus (HPV)-associated and HPV-independent. Conversely, the impact of p53 status on prognosis remains controversial. We correlated HPV and p53 status with prognosis in a large series of PSCC patients. p53 was analysed according to a recently described immunohistochemical (IHC) pattern-based framework that includes two normal (scattered, mid-epithelial) and four abnormal patterns (diffuse, basal overexpression, cytoplasmic and null) and closely correlates with TP53 mutational status. Methods: A total of 122 patients with surgically treated PSCC in three hospitals in Barcelona, Spain, were included. Based on HPV in situ hybridization and p16 and p53 IHC expression, the tumors were classified into three molecular types: HPV-associated, HPV-independent/p53 normal, and HPV-independent/p53 abnormal. All patients were followed for at least 22 months (median 56.9 months), and disease-specific survival (DSS) was analysed. Results: Thirty-six tumors (29%) were HPV-associated, 35 (29%) were HPV-independent/p53 normal, and 51 (42%) were HPV-independent/p53 abnormal. Disease-related deaths were observed in 3/36 (8%), 0/35 (0%) and 14/51 (27%) of the patients, respectively (p<0.001). A total of 7/14 deaths in the latter group were patients with tumors showing p53 patterns not recognized in the classical p53 IHC interpretation (basal, null, cytoplasmic). According to our multivariate analysis, HPV-independent/p53 abnormal tumors and advanced stage were associated with impaired DSS (hazard ratio=23.4, 95% confidence interval [CI]= 2.7-3095.3; p=0.001 and 16.3, 95% CI=1.8-2151.5; p=0.008, respectively). CONCLUSION: Compared with patients with HPV-associated and HPV-independent/p53-normal PSCC, patients with HPV-independent/p53 abnormal PSCC have worse clinical outcomes. p53 IHC results define two prognostic categories in HPV-independent PSCC: HPV-independent/p53-normal tumors as low-risk tumors, whereas HPV-independent/p53-abnormal tumors as aggressive neoplasms.

Background: Metabolic flux analysis (MFA) is an indispensable tool in metabolic engineering. The simplest variant of MFA relies on an overdetermined stoichiometric model of the cell’s metabolism under the pseudo-steady state assumption, to evaluate the intracellular flux distribution. Despite its long history, the issue of model error in the overdetermined MFA, particularly misspecifications of the stoichiometric matrix, has not received much attention. Method: We evaluated the performance of statistical tests from linear least square regressions, namely Ramsey RESET test, F-test and Lagrange multiplier test, in detecting model misspecifications in the overdetermined MFA, particularly missing reactions. We further proposed an iterative procedure using the F-test to correct such an issue. Result: Using Chinese hamster ovary and random metabolic networks, we demonstrated that: (1) a statistically significant regression does not guarantee high accuracy of the flux estimates, (2) the removal of a reaction with a low flux magnitude can cause disproportionately large biases in the flux estimates, (3) the F-test could efficiently detect missing reactions, and (4) the proposed iterative procedure could robustly resolve the omission of reactions. Conclusion: Our work demonstrated that statistical analysis and tests could be used to systematically assess, detect and resolve model misspecifications in the overdetermined MFA.

This study focuses on enhancing controllable fibrin-based hydrogels for tissue engineering, addressing existing weaknesses. By integrating a novel copolymer, we improve the foundation for cell-based angiogenesis with adaptable structural features. Tissue engineering often faces challenges like waste disposal and nutrient supply beyond the 200 µm diffusion limit. Angiogenesis breaks through this limitation, allowing the construction of larger constructs. Our innovative scaffold combination significantly boosts angiogenesis, resulting in longer branches and more capillary network junctions. The copolymer attached to fibrin fibers enables precise adjustment of hydrogel mechanical-dynamic properties for specific applications. Our material proves effective for angiogenesis, even under suppression factors like suramin. In our study, we prepared fibrin-based hydrogels with and without the copolymer PVP12400-co-GMA10mol%. Using a co-culture system of human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), we analyzed angiogenetic behavior on and within the modified hydrogels. Capillary-like structures were reproducibly formed on different surfaces, demonstrating the general feasibility of three-dimensional endothelial cell networks in fibrin-based hydrogels. This highlights the biomaterial's suitability for in vitro pre-vascularization of biohybrid implants.

(1) Background: Double-strand breaks (DSBs) in a single nucleus are usually measured using the sperm chromatin structure assay (SCSA), sperm chromatin dispersion (SCD) test, and comet assay (CA). We developed mono-dimensional single-cell pulsed-field gel electrophoresis (1D-SCPFGE) and angle-modulated two-dimensional single-cell pulsed-field gel electrophoresis (2D-SCPFGE) to observe DNA fragmentation in separated motile sperm. (2) Methods: We set up comparative standards, calibration curves, the required sensitivity, and eligibility criteria for test sperm to validate their measurement principles. (3) Results: We revealed that the conventional methods overlooked the interference of nucleoproteins in their measurements. In-gel proteolysis improves the measurement accuracies of 1D- and 2D-SCPFGE. We recommend using naked DNA for comparative standards and test specimens. Moreover, we have observed several dysfunctions that may induce DNA damage in the separated motile sperm. Overall, our discussion highlights the need to revisit the conventional univariable analyses based on the SCSA, SCD test, and CA. (4) Conclusions: Human infertility is a complex syndrome, and the aim of quality control in intracytoplasmic sperm injection is to identify the underlying dysfunctions remaining in the separated motile sperm that render them ineligible for injection. Well-designed, multivariable analyses with special consideration to confounding factors are necessary in future cohort studies.

Coffee processing generates a huge amount of waste which contains many natural products. Here, we report the discovery of a panel novel cell-penetrating and metal-ion binding microproteins designated coffeetide cC1a-c and cL1-6 from the husk of two popular coffee plants, Coffea canephora and Coffea liberica, respectively. Combining sequence determination and database search, we showed that the prototypic coffeetide cC1a is a 37-residue, eight-cysteine microprotein with a hevein-like cysteine motif but without a chitin-binding domain. NMR determination of cC1a revealed a compact structure that confers it resistance to heat and proteolytic degradation. Disulfide mapping together with chemical synthesis revealed that cC1a has a ginsentide-like, and not a hevein-like, disulfide connectivity. In addition, transcriptomic analysis showed that the 98-residue micrcoproten-like coffeetide precursor contains a three-domain arrangement like ginsentide precursors. Molecular modeling together with experimental validation revealed a Mg2+ and Fe3+ binding pocket at the N-terminus formed by three glutamic acids. Importantly, cC1a is amphipathic with a continuous stretch of 19 apolar amino acids, and which enables its cell penetration to target intracellular proteins, despite being highly negatively charged. Our findings suggest that coffee by-products could provide a source of ginsentide-like bioactive peptides that have potential to target intracellular proteins.

The present work consisted of an exploratory study aiming to evaluate in vitro the potential of AuNPs during Radiation Therapy (RT) in human pancreatic adenocarcinoma cells. AuNPs coated with hyaluronic and oleic acids (HAOA-AuNPs) or with bombesin peptides (BBN-AuNPs) were used. AuNPs were characterized by Atomic Force Microscopy (AFM) and Dynamic Light Scattering. BxPC-3 tumour cells were irradiated with a 6 MV X-rays beam, in the absence or presence of AuNPs. AFM showed that HAOA-AuNPs and BBN-AuNPs are spherical with a mean size of 83+-20 nm and 49+-12 nm, respectively. For RT alone, a reduction in cell viability of up to 33+-12% was obtained compared to the control (p ≤ 0.0001). HAOA-AuNPs alone at 200 and 400 μM showed a reduction in cell viability of 20+-4% and 35+-4%, respectively, while for BBN-AuNPs, at 50 and 200 μM, a reduction in cell viability of 25+-3% and 37+-3% was obtained, respectively, compared to the control (p < 0.0001). At 72 h post-irradiation, a decrease in cell viability of 26+-3% and 22+-2% between RT+HAOA-AuNPs at 400 μM and RT+BBN-AuNPs at 50 μM, compared to RT alone, was obtained (p < 0.004). The combination of RT with AuNPs led to a significant decrease in cell viability compared to the control, or RT alone, thus representing an improved effect.

Cancer stem cells play a crucial role in tumor initiation, metastasis and therapy resistance. Cellular heterogeneity and plasticity challenge the isolation of cancer stem cells. The impact of intratumoral cellular heterogeneity in the context of treatment resistance using a label-free approach remains understudied. Here, we use the sedimentation field-flow fractionation technique to separate, without labeling, cell subpopulations of colorectal cancer cell lines and primary cultures according to their biophysical properties. One of the three cell subpopulations sorted by SdFFF exhibits cancer stem cell traits, including high tumorigenicity in vivo, and a higher frequency of tumor-initiating cells compared to the other subpopulations. In vitro two- and three-dimensional chemosensitivity assays emphasize the therapeutic relevance of this cancer stem cell-like subpopulation due to its chemoresistance. Therefore, our findings highlight a label-free cell sorting approach to reveal intratumoral cellular heterogeneity and its implication in therapy resistance. This approach enables the study of the individualized response of each sorted cell subpopulation by breaking down the tumor, thus offering new perspectives for personalized therapy.

Androgenetic alopecia is a genetic disorder caused by an excessive response to androgens. This alopecia is the most common cause of progressive hair loss in men and causes low self-esteem in patients. In this study we used various methods, including quantitative PCR, flow cytometry, ELISA, and immunocytochemistry, to evaluate the biological effects of germinated hemp seeds extract and exosomes derived from calli of germinated hemp seeds. This research reveals pre-ventive functions of two biomaterials (GE2000: 2000 ug/mL germinated hemp seeds extract; E40: 40 ng/mL exosomes derived from calli of germinated hemp seeds) for androgenetic alopecia through three biochemical categories: genetic modulation in hair follicle dermal papilla stem cells (HFDPSCs), cellular differentiation, and modulation of immune systems. Despite exposure to dihydrotestosterone (DT), two materials upregulated alopecia preventing genes including Wnt, β-catenin, and TCF in HFDPSCs. Conversely, alopecia activating genes (STAT1, 5α-reductase type 1, and IL-15R) were suppressed. Moreover, two materials suppressed alopecia genes (NKG2DL, IL2-Rβ, JAK1, and STAT1) in CD8+ T cells. Notably, E40 was effected more in-tensely in these biochemical categories than those of GE2000. Consequently, E40 and GE2000 ef-fectively prevented DT stress. Additionally, two materials activated mechanisms for hair for-mation through the biological activities of three categories.

Candidate vaccines against African swine fever virus (ASFV) based on naturally attenuated or genetically modified viruses have the potential to generate protective immune responses, although there is no consensus on what defines a protective immune response against ASFV. Studies, espe-cially in sensible host species, focused on unravelling protective mechanisms will contribute to the development of safer and more effective vaccines. The present study provides a detailed analysis of phenotypic and functional data on cellular responses induced by intradermal immunization and subsequent boosting of domestic pigs with the naturally attenuated field strain Lv17/WB/Rie1, as well as the mechanisms underlying protection against intramuscular challenge with the virulent genotype II Armenia/07 strain. The transient mild to moderate increase of IL-8 and IL-10 in serum observed after immunization might be directly correlated with survival. Protection was also associated with a robust ASFV-specific polyfunctional memory T cell response, where CD4CD8 and CD8 T cells were identified as the main cellular sources of virus-specific IFNγ and TNFα. In parallel to cytokine response, these T-cell subsets also showed specific cytotoxic activity as evidenced by the increased expression of the CD107a degranulation marker. Along with virus-specific multifunctional CD4CD8 and CD8 T responses, the increased levels of antigen experienced cytotoxic CD4 T cells observed after challenge in immunized pigs might also contribute to control virulent infection by killing mechanisms targeting infected antigen-presenting cells. Future studies should elucidate whether the memory T-cell responses evidenced in the present study persist and provide long-term protection against further ASFV infections.

One-Dimensional Solar Cell Capacitance Simulator (1D-SCAPS) and n-i-p planar configuration have been employed for the simulation of the caesium tin iodide (CsSnI₃)-based perovskite solar cell (PSC) with titanium (IV) oxide (TiO₂) and copper thiocyanate (CuSCN) as the electron and hole transport materials respectively. The results obtained for the initially modeled PSC compared well with similar devices in the literature but marred with high recombination rate. As such, the defect density in the CsSnI₃ absorber was varied from 10¹³ cm^-3 to 10¹⁷ cm^-3 and enriched with SnCl₂ (additive) and Br⁻(dopant) respectively. This strategy ensured a reduced concentration of Sn⁴⁺ vacancy (V_Sn) in the CsSnI₃ absorber with improved carrier lifetime and diffusion length beyond 0.05 ns and 1.1µm by a magnitude of the order of 10³ and 10² respectively for the enriched CsSnI₃ –based PSC. For the optimized PSC, we recorded V_OC=1.289 V, J_SC=32.60 mA.cm^-2, FF=83.56% and PCE=35.12%.

Autoantibodies against specific lung cancer-associated antigens have been suggested for lung cancer diagnosis. This study aimed to evaluate the diagnostic performance of anti-gen-autoantibody immune complex (AIC) against its free antigens for CYFRA21-1, ProGRP, NGAL, and NSE in non-small cell lung cancer (NSCLC). In total, 85 patients with NSCLC and 120 healthy controls (HCs) were examined using a 9G DNA chip method. The ratios of AICs to their antigens and combinations of ratios consisting of two to four markers were calculated. The levels of AICs for CYFRA21-1, ProGRP, NGAL, and NSE were higher than their free antigens in all participants. The levels of each free antigen distinguished patients with NSCLC from HCs. The ratios of AIC to its antigen and seven combinations consisting of two to four ratios were significantly higher in patients with NSCLC than those in HCs. Excellent diagnostic performance was observed for all combination ratios (C4-1), with 85.9% sensitivity and 86.7% specificity at cutoff 3.51. Higher sensitivity was observed in the very early stages (0–I) and adenocarcinoma than in stages II–IV and other pathological types. The combination of all ratios of AICs and their antigens for all four markers is useful in the diagnosis of NSCLC.

Research on the lead halide-based perovskite solar cells have obtained considerable interest in photovoltaic industry owing to their higher efficiency, easy manufacturing, light-weight and low cost. However, these lead halide-based solar cells are not suitable to manufacture commercially because of the toxicity of lead-based materials. In this context, a lead-free perovskite, cesium-bismuth iodide (Cs₃Bi₂I₉) is considered as a potential alternative to the lead halide-based cell due to their non-toxicity and stability, but this perovskite cannot be matched with random hole transport layer (HTL) and electron transport layer (ETL) materials compared to lead halide-based perovskite because of their crystal structure and band gap. Therefore, in this study, performance comparison of different ideal HTL and ETL materials for Cs₃Bi₂I₉ perovskite layer were studied using SCAPS-1D device simulation on the basis of open circuit voltage, short circuit current, power conversion efficiency (PCE) and fill factor (FF) as well as several novel PSC configuration model were designed that can direct for further experimental research for PSC device commercialization. Results from this investigation reveals that the maximum efficiency of 20.96% is obtained for the configuration ITO/WS₂/Cs₃Bi₂I_9­/NiO/Au with optimized parameters such as thickness 400 nm, band gap 2.1eV, absorber layer defect density 10¹² cm^-3, donor density of ETL 10¹⁸ cm^-3 and the acceptor density of HTL 10²⁰ cm^-3.

Background: The proven efficacy of mTOR inhibitors (mTORI), tyrosine kinase inhibitors (TKI) or immune checkpoint inhibitors (ICI) therapies in metastatic renal cell carcinoma (RCC), suggested that these agents should be investigated as adjuvant therapy with the aim of eliminating any residual undetectable microscopic disease after curative resection. Our study aimed to compare the efficacy of these treatments through the application of an innovative method that reconstructs individual patient data. Methods: Nine phase III studies describing different treatment options for adjuvant RCC were selected. Individual patient data were reconstructed from Kaplan–Meier (KM) curves through the “IPDfromKM method”. Disease-free survival (DFS) were compared among combination treatments and control arm (placebo). Results were summarized as multi-treatment KM curves. Standard statistical testing was used, including hazard ratio and likelihood ratio tests for heterogeneity. Results: In the overall population, this study showed that two ICIs (pembrolizumab and nivolumab plus ipilimumab) and one TKI (sunitinib) showed superiority over placebo, whereas both TKI and mTORI did not. As we assessed DFS as the primary endpoint for the adjuvant comparison, the overall survival benefit remains unknown.. Conclusion: This novel approach to investigating survival has allowed us to conduct all of the indirect head-to-head comparisons between these agents in a context where no "real" comparative trials have been conducted.

Alzheimer’s Disease (AD) is a debilitating neurodegenerative disease characterized by the accumulation of extracellular amyloid-β peptides (Aβ) within the cerebral parenchyma and vasculature, the latter termed cerebral amyloid angiopathy (CAA). This study utilized confocal imaging to investigate heparan sulfate (HS) within the cerebrovasculature and its potential associations with CAA, gender, and ApoE4 genotype. Our investigation revealed elevated levels of HS in the cerebrovasculature of AD patients with severe CAA. Additionally, these patients exhibited higher colocalization of HS with Aβ in the cerebrovasculature, including both endothelial and vascular smooth muscle cell compartments. Intriguingly, a reversal in the polarized expression of HS within the cerebral vasculature was detected in AD patients with severe CAA, suggesting a potential role in CAA development. Furthermore, male patients exhibited lower levels of both parenchymal and cerebrovascular HS. Additionally, ApoE4 carriers displayed heightened cerebrovascular Aβ expression and a tendency of elevated cerebrovascular HS levels in AD patients with severe CAA. Overall, these findings shed light on the intricate interplay between HS, Aβ, ApoE, and vascular pathology in AD, underscoring the potential roles of cerebrovascular HS in CAA development and AD pathology. Further exploring the underlying mechanisms may present novel therapeutic avenues for AD treatment.

To develop peptide drugs targeting integrin receptors, synthetic peptide ligands endowed with well-defined selective binding motifs are necessary. The snake venom KTS-containing disintegrins, that selectively block collagen α1β1 integrin, were used as lead compounds for the synthesis and structure-activity relationship of a series of linear peptides containing the KTS-pharmacophore and alternating natural amino acids and 3-aminobenzoic acid (MABA). To ensure a better stiffness and metabolic stability, one, two and three MABA residues, were introduced around the KTS pharmacophore motif. Molecular dynamics simulations have determined that the solution conformation of MABA peptide 4 is more compact, underwent larger conformational changes until convergence, and spent most of the time in a single cluster. The peptides’ binding affinity has been characterized by enzyme linked immunosorbent assay in which the most potent peptide 4 inhibited with IC50 of 324±8 µM and 550±45 µM the binding of GST-α1-A domain to collagen IV fragment CB3, and the cell adhesion to collagen IV using α1-overexpressor cells, respectively. Docking studies and MM-GBSA calculations confirmed that peptide 4 binds a smaller region of the integrin near the collagen- binding site and penetrated deeper into the binding site near Trp1. Peptide 4, inhibited tube formation by endothelial cell migration in the Matrigel angiogenesis in vitro assay. Peptide 4 was acutely tolerated by mice, showed stability in human serum, decreased tumor volume and angiogenesis, and significantly increased the survival of mice injected with B16 melanoma cells. These findings propose that MABA-peptide 4 can further serve as an α1β1-integrin antagonist lead compound for further drug-optimization in angiogenesis and cancer therapy.

The observation that certain therapeutic strategies for targeting inflammation benefit patients with distinct immune-mediated inflammatory diseases (IMIDs) is exemplified by the success of TNF blockade in conditions including rheumatoid arthritis, ulcerative colitis and skin psoriasis, albeit only for subsets of individuals with each condition. This suggests intersecting "nodes" in inflammatory networks at a molecular and cellular level may drive and/or maintain IMIDs, being “shared” between traditionally distinct diagnoses without mapping neatly to a single clinical phenotype. In line with this proposition, integrative tumour tissue analyses in oncology have highlighted novel cell states acting across diverse cancers, with important implications for precision medicine. Drawing upon advances in the oncology field, this narrative review will first summarise learnings from the Human Cell Atlas in health as a platform for interrogating IMID tissues. It will then review cross-disease studies to date that inform this endeavour, before considering future directions in the field.

A sustainable, scalable source of energy‐dense chemical fuel is urgently needed to ensure the security of our energy supply for future generations. Solar energy is the only renewable energy source of sufficient scale to replace fossil fuels and meet rising environmental demand. Hydrogen is expected to play a key role as an energy carrier in future energy systems of the world. As fossil fuel supplies become scarcer and environmental concerns increase, hydrogen is likely to become an increasingly important chemical energy carrier and eventually may become the principal chemical energy carrier. When most of the world’s energy sources become non-fossil based, hydrogen and electricity are expected to be the two dominant energy carriers for the provision of end-use services. Photocatalytic hydrogen evolution from water by solar energy over semiconductors offers a promising way for clean and renewable production of hydrogen. The TiO2-based materials have received considerable attention in recent years due to their extensive application in photocatalysis for hydrogen evolution. Though TiO2 is the most widely investigated photocatalyst for hydrogen evolution, it still provides low photocatalytic activity due to the wide bandgap that results in photocatalytic activity only under ultraviolet (UV) irradiation. Modification of TiO2 photocatalysts with several transition metals has been extensively studied to extend the absorbance capacity of TiO2 into the visible range. The effect of different photocatalytic deposition and reaction parameters also play major roles in enhancing the photostability of photoanode and increasing the hydrogen gas output, respectively.

Renal cell carcinoma (RCC) accounts for approximately 90-95% of all kidney cancers in adults, with the clear cell RCC (ccRCC) the most frequently identified subtype. RCC is known for its responsiveness to immunotherapy, making it an area of significant research interest. Immune checkpoint (IC) molecules, which regulate immune surveillance, are established therapeutic targets in RCC. The aim of this study was to analyze the influence of polymorphisms in genes HVEM and CD160 on susceptibility to ccRCC and patient overall survival (OS) with over a ten-year period of observation. We genotyped three HVEM single nucleotide polymorphisms (SNPs): rs1886730, rs2234167, and rs8725, and two CD160 SNPs: rs744877 and rs2231375, in 238 ccRCC patients and 521 controls. Our findings indicated that heterozygosity within rs2231375 and/or rs2234167 increases ccRCC risk. Furthermore, in women, heterozygosity within additional HVEM SNPs, rs8725 and rs1886730, is associated with an increased ccRCC risk. The presence of a minor allele for rs1886730, rs2234167, rs8725, and rs2231375 was also correlated with certain clinical features of the disease. Moreover, the rs1886730 SNP was found to be associated with patients' OS. In conclusion, our study highlights an association between HVEM and CD160 polymorphisms and the risk of developing ccRCC as well as patient’s OS.

The investigation of membrane proteins, key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage dependent anion channel (VDAC), a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function is proposed that quantifies the feasibility of formation of the translation initiation complex through the ribosome-mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome. The scoring function enables to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies. This scoring function is publicly available as webservice XenoExpressO at rna.tbi.univie.ac.at.

To elucidate mesenchymal stromal cells (MSC)-tumor intricate and ambiguous interactions, the reliable cell labeling and tracking techniques are imperative. Traditional methods of cell labelling have limitations mostly relevant to need for genetic modification what demands the exploration for alternative strategies. Here, we propose using fluorescent photoconvertible microcapsules containing Rhodamine B (RhB) to study mMSC migration in brain tumors. These 3 μm sized microcapsules are readily internalised by cells and undergo photoconversion under 561 nm laser exposure with fluorescence blue shift on demand. Optimal photoconversion was achieved with a laser irradiation duration of 0.4 ms, which provide a maximal brightness of photoconverted label without an excessive laser exposure on cells. Capsules modified with polyallylamine hydrochloride demonstrated the best potential for intracellular uptake without significantly affecting cell viability, motility, or proliferation. The optimal ratio of 20 capsules per mMSC was determined for labeling. Moreover, migration of individual mMSCs within 2D and 3D glioblastoma cell (EPNT-5) colonies over 2 days and in vivo tumor settings over 7 days was tracked. Our study unveils a robust platform for investigating MSC-tumor dynamics, offering insights into therapeutic strategies. Photoconvertible microcapsules could also become an indispensable tool for studying complex fundamental processes of cell-cell interactions for a wide range of problems in biology and medicine.

Viruses can infect a cell via one or both routes viz. cell-to-cell (c-c) or cell-free (c-f) . Pathogenesis studies of various viruses, including HIV, have shown that c-c transmission yields a significantly higher infection magnitude than the c-f route. Expectedly, potent antibodies inhibited c-f infection more efficiently than with c-cell transmission. To achieve a one-step, synchronous infection cycle that provides amplified infection, we have studied a consistent and efficient c-c HIV infection model since 1992. H9 cells persistently infected with HTLV-IIIB (H3B cells) and uninfected target CD4+ lymphocyte line (HuT78) were mixed in a ratio of 1:4 respectively. We have recently used this model to produce HIV designer antigens that have been shown to elicit monoclonal as well as polyclonal specific antibodies against novel epitopes that are formed post virus-cell engagement, but prior to fusion. The model can be extended for HIV neutralizing antibody assays or drug inhibitors against high multiplicity of infection.

Cell junction that is usually associated with dynamic cytoskeletons is essential for a wide range of cellular activities such as cell migration, cell communication, barrier function and signal transduction. Real-time observation of cell junction facilitates understanding of the mechanisms by which cell junction regulate relevant cellular activities. In this study, we examined the binding capacity of a modified tridecapeptide from Cx43 to cell junction protein ZO-1 and sought to create a fluorescent peptide that could be used to label cell junction. To introduce the fluorescent peptide into the cells, a cell-penetrating peptide was linked to the modified tridecapeptide and synthesized. The binding of the modified tridecapeptide was tested using pull down and immunoprecipitation assay. The ability of the peptide to label cell junction was assessed by adding it to fixed or cultured Caco-2 cells. Testing assay revealed that the Cx43-derived peptide can bind to ZO-1. Besides, the peptide was able to label cell junction of fixed cell, although no obvious cell junction labeling was observed clearly in living cells, probably due to the inadequate affinity. These results indicate that a peptide-based strategy for labeling cell junction may be feasible, and efforts to improve its affinity are warranted in the future.

(1) Background: donor-derived circulating free DNA (dd-cfDNA) is an innovative biomarker with great potential for the early identification and prevention of graft damage; (2) Methods: Prospective study to analyze dd-cfDNA plasma levels in 30 kidney transplant patients during their post-transplant follow-up (15 days, 3, 6 and 9 months), to determine if the result could be of interest in the identification of possible adverse events, specially allograft rejection. In addition, the aim is also to verify the obtained data on sensitivity, specificity, NPV and PPV; (3) Results: Plasma levels of dd-cfDNA could be considered a novel biomarker of graft rejection after first term post-transplant up to several months before its clinical presentation, mainly in TCMR or ABMR. In this study, it appears to have no positive predictive value for borderline rejection or TCMR IA. Our results show a low NPV of the technique because other pathologies or biological post-transplant complications can also affect dd-cfDNA levels and they must be considered in the interpretation of the result.; (4) Conclusions: dd cfDNA could allow, together with other additional biomarkers in the context of a future score, avoid invasive biopsies in most cases, mainly in the case of suspected TCMR or ABMR.

GCTs are developmental tumors and likely to reflect ontogenetic and teratogenetic determinants. The objective was to identify syndromes with or without congenital anomalies and non-syndromic defects as potential risk factors. Patients with extracranial GCTs (eGCTs) registered in MAKEI 96/MAHO 98 between 1996 and 2017 were used. According to Teilum's holistic concept malignant and benign teratomas were registered. We used a case control study design with Orphanet as a reference group for syndromic defects and the Mainz birth registry (EUROCAT) for congenital anomalies at birth. Co-occurring genetic syndromes and/or congenital anomalies were assessed accordingly. Odds ratios and 95% confidence intervals were calculated and p-values for Fisher’s exact test with Bonferroni correction if needed. A strong association was confirmed for Swyer (OR 338.6, 95% CI 43.7-2623.6) and Currarino syndrome (OR 34.2, 95% CI 13.2-88.6). We additionally found 17 isolated cases of eGCT with a wide range of syndromes, however not significantly associated following Bonferroni correction. All these cases pertained to girls. Regarding non-syndromic defects, no association with eGCTs could be identified. In our study we confirmed a strong association for Swyer and Currarino syndromes with additional congenital anomalies.

Blood cancers, including leukemia, lymphoma, and myeloma, pose significant diagnostic challenges due to their diverse and complex nature. Detecting these cancers early and accurately is crucial for timely intervention and improved patient outcomes. This review explores current and emerging detection methods for human blood cancers, focusing on their principles, advantages, limitations, and potential clinical applications.

Corneal ulcer (CU) is an ophthalmopathy characterized by depression of the corneal surface with at least one stromal loss. CU is common in canine and feline species and is usually caused, among others, by trauma, infections, toxic contamination and endocrine disorders. They usually result from an increased inflammatory response and are associated with some clinical signs such as blepharospasm, photophobia, epiphora, pain and loss of corneal transparency. Despite advances in conventional and pharmacological therapy, in many cases indolent and recurrent ulcer treatments still lead to loss of visual acuity of the animal. This paper aims to report the effect of topical application of canine adipose tissue-derived mesenchymal stem cell (cATMSCs) as treatment of recurrent CU in a Poodle dog breed that showed clear difficulty in the healing process associated with diabetes. The animal was submitted to two applications of cATMSCs and showed improvement in the blepharospasm, conjunctival hyperemia, mucopurulent ocular secretion, photophobia, corneal opacity, chemosis, pigmentation, neovascularization, and pain parameters. Besides, Fluorescein test, Schirmer test and ocular fundus exam also showed improvement in their values concomitantly with lesion resolution. Due this, we showed that cATMSC therapy contribute to the regeneration of corneal tissue in CU and may contribute to the treatment to others ophthalmopathies.

Background Glioblastoma (GBM) is a poor prognosis malignant grade 4 glioma. After surgical resection, standard therapy consists of concurrent radiotherapy (RT) and temozolomide (TMZ) followed by TMZ alone. The DC vaccination increased the amount of intratumoral-activated cytotoxic T lymphocytes and decreased the number of FoxP3-positive regulatory T cells. Based on these da-ta, we have developed a phase II protocol with the DC vaccine concomitant to standard RT-CT in patients undergoing surgery for GBM. Methods This is a single-arm, monocentric, phase II trial evaluating progression-free survival (PFS) and the safety of a DC vaccination integrated with standard therapy in resected GBM patients. The vaccine administration begins at the end of the RT-CT (Induction Phase) and is subsequently al-ternated with TMZ cycles (Maintenance Phase). The primary endpoints are PFS and safety. Among the secondary endpoints, are the in vitro and in vivo Immune response evaluations. Results By December 2022 , nine patients had been enrolled in the first step. Two patients progressed within three months after leukapheresis and none had experienced DC vaccine-related G3-4 tox-icity. Five patients had a positive DTH test against KLH. By June 2023. The median OS from leu-kapheresis was 23.1 months and 24.7 from surgery. Conclusions This combination therapy is well-tolerated, and we have achieved the two endpoints required for the first step. Therefore, the study will proceed to enroll the remaining 19 patients.

Delivering nucleic acid therapeutics across cell membranes is a significant challenge. Cell-penetrating peptides (CPPs) containing arginine (R), tryptophan (W), and histidine (H) show promise for siRNA delivery. To improve siRNA delivery and silencing a model STAT3 gene, we hypothesized that oleyl acylation to CPPs, specifically (WRH)n, would enhance STAT3 silencing efficiency in breast and ovarian cancer cells. Using Fmoc/tBu solid-phase peptide chemistry, we synthesized, purified, and characterized the oleyl-conjugated (WRH)n (n = 1-4) peptides. The peptide:siRNA complexes were non-cytotoxic at N/P 40 (~20 μM) against MDA-MB-231, MCF-7, SK-OV-3, and HEK-293 cells after 72 h incubation. All peptide:siRNA complexes showed serum stability at N/P ≥40. The synthesized conjugates, with a diameter of &lt;100 nm, formed nano complexes with siRNA and exhibited stable range of zeta potential values (13-18 mV at N/P 40). Confocal microscopy and flow cytometry analysis provided qualitative and quantitative evidence of successful cellular internalization of siRNA. Peptides oleyl-(WRH)3 and oleyl-(WRH)4 showed ~60% and ~75% cellular uptake of siRNA, respectively, in both MDA-MB-231 and SK-OV-3 cells. Western blot analysis of oleyl-(WRH)4 demonstrated effective silencing of the STAT-3 gene, with ~75% silencing in MDA-MB-231 cells and ~45% in SK-OV-3 cells.

Though early-stage colorectal cancer has a high 5-year survival rate of 65-92% depending on the specific stage, this probability drops to 13% after the cancer metastasizes. Frontline treatments for colorectal cancer such as chemotherapy and radiation often produce dose-limiting toxicities in patients and acquired resistance in cancer cells. Additional targeted treatments are needed to improve patient outcomes and quality of life. Immunotherapy involves treatment with peptides, cells, antibodies, viruses, or small molecules to engage or train the immune system to kill cancer cells. Preclinical and clinical investigations of immunotherapy for treatment of colorectal cancer including immune checkpoint blockade, adoptive cell therapy, monoclonal antibodies, oncolytic viruses, anti-cancer vaccines, and immune system modulators have been promising, but demonstrate limitations for patients with proficient mismatch repair enzymes. In this review, we discuss preclinical and clinical studies investigating immunotherapy for treatment of colorectal cancer and predictive biomarkers for response to these treatments. We also consider open questions including optimal combination treatments to maximize efficacy, minimize toxicity, and prevent acquired resistance and approaches to sensitize mismatch repair proficient patients to immunotherapy.

Angiogenesis, the sprout and growth of new blood vessels from existing vasculature, is an important process of tumor development for the supply of oxygen and nutrition to cancer cells. Endothelial cell is a critical player in angiogenic process by modulating cell proliferation, cell motility, and cell morphology in the response to pro-angiogenic factors and environments provided by tumor and cancer cells. Recent in vivo and in vitro studies have revealed that gap junction of endothelial cells also participates in the promotion of angiogenesis. Pro-angiogenic factors modulate gap junction function and connexins expression in endothelial cells, whereas endothelial connexins involve in angiogenic tube formation and cell migration of endothelial cells via both gap junction channel function dependent or independent mechanisms. In particular, connexin might have the potential to regulate cell mechanics such as cell morphology, cell migration, and cellular stiffness that are dynamically changed during angiogenic processes. Here, we review the implication for endothelial gap junction and cellular mechanics in vascular angiogenesis.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare severe hereditary skin disease characterized by skin and mucosa fragility, resulting in blister formation. The most severe complication in RDEB patients is the development of cutaneous squamous cell carcinoma (SCC) leading to premature death. Multiple evidences suggest a permissive tumor micro-environment (TME) as driver of SCC development in RDEB patients. In a cohort of RDEB patients, we characterized the immune profiles of RDEB-SCCs and compared them with clinical, histopathological, and prognostic features. RDEB-SCCs were subdivided into 4 groups based on their occurrence (first onset or recurrences) and grading according to their clinical-histopathological parameters of aggressiveness. Thirty-eight SCCs from 20 RDEB patients were analysed. Five RDEB patients experienced an unfavorable course af-ter the diagnosis of the first SCC, with early recurrence or metastasis, whereas 15 pa-tients developed multiple SCCs without metastasis. High-risk primary RDEB-SCCs showed a higher neutrophil-to-lymphocyte ratio in the tumor microenvironment, and an increased proportion of neutrophil extracellular traps (NET). Additionally, citrullinated histone H3, a marker of NETs, was increased in the serum of RDEB patients with high-risk primary SCC, suggesting that this modified form of histone H3 may serve as a potential blood marker of unfavorable prognosis in RDEB-SCCs.

Cell mechanics is a biophysical indicator of cell state, such as cancer metastasis, leukocyte activation, and cell cycle progression. Atomic force microscopy (AFM) is a widely used technique to measure cell mechanics, where the Young modulus of a cell is usually derived from the Hertz contact model. However, the Hertz model assumes that the cell is an elastic, isotropic, and homogeneous material and that the indentation is small compared to the cell size. These assumptions neglect the effects of the cytoskeleton, cell size and shape, and cell environment on cell deformation. In this study, we investigated the influence of cell size on the estimated Young’s modulus using liposomes as cell models. Liposomes were prepared with different sizes and filled with phosphate buffered saline (PBS) or hyaluronic acid (HA) to mimic the cytoplasm. AFM was used to obtain the force indentation curves and fit them to the Hertz model. We found that the larger the liposome, the lower the estimated Young’s modulus for both PBS-filled and HA-filled liposomes. This suggests that the Young modulus obtained from the Hertz model is not only a property of the cell material, but also depends on the cell dimensions. Therefore, when comparing or interpreting cell mechanics using the Hertz model, it is essential to account for cell size.

Buerger's disease or Thromboangiitis Obliterans (TAO) is a nonatherosclerotic segmental vascular disease which affects small and medium arteries and veins in the upper and lower extremities. Based on pathological findings, TAO can be considered as a distinct form of vasculitis that is most prevalent in young male smokers. There is no definitive cure for this disease as therapeutic modalities are limited in number and efficacy. Surgical bypass has limited utility and 24% of patients will ultimately require amputation. Recently, studies have shown that therapeutic angiogenesis and immunomodulatory approaches through the delivery of cells to target tissues are potential options for ischemic lesion treatment. In this review, we summarize the current knowledge of TAO treatment and provide an overview of stem cell-based treatment modalities.

Cell death resistance is a key feature of tumor cells. One of the main anti-cancer therapies is increasing the susceptibility of cells to death. Cancer cells have developed a capability of tumor immune escape. Hence, restoring the immunogenicity of cancer cells can be suggested as an effective approach against cancer. Accumulating evidence proposes that several anticancer agents provoke the release of danger-associated molecular patterns (DAMPs) that are determinants of immunogenicity and stimulate immunogenic cell death (ICD). It has been suggested that ICD inducers are two different types according to their various activities. Here, we review the well-characterized DAMPs and focus on the different types of ICD inducers and recent combination therapies that can augment the immunogenicity of cancer cells.

Background: Biological processes are based on complex networks of cells and molecules. Single cell multi-OMICs is a new tool aiming to provide new incites in the complex network of events controlling the functionality of the cell.; Methods: Since single cell technologies provide many sample measurements, they are the ideal environment for the application of deep learning and machine learning approaches. An autoencoder (AE) is composed of an encoder and a decoder sub-model. AE are very powerful in data compression and noise removal. However, the decoder model remains a black box from which is impossible to depict the contribution of the single input elements. We have recently developed a new class of autoencoders, called Sparsely Connected Autoencoders (SCA), which have the advantage of providing a controlled association among the input layer and the decoder module. This new architecture has the benefit that the decoder model is no anymore a black box and it can be used to depict new biologically interesting features from single cell data; Results: In this paper, we show that SCA hidden layer can grab new information usually hidden in single cell data, like as providing clustering on meta-features difficult, i.e. transcription factors expression, or impossible, miRNA expression, to depict in single cell RNAseq data. Furthermore, a SCA representation of cell clusters has the advantage of simulating a conventional bulk RNAseq, which is a data transformation allowing the identification of similarity among independent experiments; Conclusions: In our opinion, SCA represent the bioinformatics version of a “Swiss Army knife” for the extraction of hidden knowledgeable features from single cell OMICs data.

Abstract: The SARS-CoV-2 pandemic has reemphasized the urgent need for broad-spectrum antiviral therapies. We developed a computational workflow using scRNA-Seq data to assess cellular metabolism during viral infection. With this workflow we predicted the capacity of cells to sustain SARS-CoV-2 virion production in patients and found a tissue-wide induction of metabolic pathways that support viral replication. Expanding our analysis to influenza A and dengue viruses, we identified metabolic targets and inhibitors for potential broad-45 spectrum antiviral treatment. These targets were highly enriched for known interaction partners of all analyzed viruses. Indeed, phenformin, an NADH:ubiquinone oxidoreductase inhibitor, suppressed SARS-CoV-2 and dengue virus replication. Atpenin A5, blocking succinate dehydrogenase, inhibited SARS-CoV-2, dengue virus, respiratory syncytial virus, and influenza A with high selectivity indices. In vivo, phenformin showed antiviral activity against SARS-CoV-2 in a Syrian hamster model. Our work establishes host metabolism as druggable for broad-spectrum antiviral strategies, providing invaluable tools for pandemic preparedness.

Wild-type p53 cancer therapy-induced senescent cells frequently engulf and degrade neighboring ones inside a massive vacuole in their cytoplasm. After clearance of the internalized cell, the vacuole persists, seemingly empty, for several hours. Despite large vacuoles being associated with cell death, this process is known to confer a survival advantage to cancer engulfing cells, leading to therapy resistance and tumor relapse. Previous attempts to resolve the vacuolar structure and visualize their content using dyes were unsatisfying for lack of known targets and ineffective dye penetration and/or retain. Here, we overcame this problem by applying optical diffraction tomography and Raman spectroscopy to MCF7 doxorubicin-induced engulfing cells. We demonstrated a real ability of cell tomography and Raman to phenotype complex microstructures, such as cell-in-cells and vacuoles, and detect chemical species in extremely low concentrations within live cells in a completely label-free fashion. We show that vacuoles had a density indistinguishable to the medium, but were not empty, instead contained diluted cell-derived macromolecules, and we could discern vacuoles from medium and cells using their Raman fingerprint. Our approach is useful for the non-invasive investigation of senescent engulfing (and other peculiar) cells in unperturbed conditions, crucial for a better understanding of complex biological processes.

The main focus of the in vitro toxicity assessment methods is on the viability of the cells, which is usually based on metabolism changes. Yet, when exposed to toxic substances, the cell triggers multiple signals in response. With this in mind, we have developed a promising cell-based toxicity method that observes various cell responses when exposed to toxic substances (either death, division or remaining viable). Based on the collective cell response, we observed and predicted the dynamics of the cell population to determine the toxicity of the toxicant. The method was tested with two different conformations: In the first conformation, we exposed a monoculture model of blood macrophages to UV light, hydrogen peroxide, nutrient deprivation, tetrabromobisphenol A, fatty acids and 5-fluorouracil. In the second, we exposed a coculture liver model consisting of hepatocytes, hepatic stellate cells, Kupffer cells and liver sinusoidal endothelial cells to rifampicin, ibuprofen and 5-fluorouracil. The method showed good accuracy compared to established toxicity assessment methods. In addition, this approach provided more representative information on the toxic effects of the compounds as it considers the different cellular responses induced by toxic agents.

Our study explores the genetic mechanisms underlying the spotted leaf phenotype in rice, focusing on the rpt5a mutant characterized by persistent reddish-brown leaf spots from seedling stage to maturity, leading to extensive leaf necrosis. Through map-based cloning, we localized the responsible locus to a 330Kb region on chromosome 2 and identified LOC_Os02g56000 (named OsRPT5A) as the causative gene. A point mutation in OsRPT5A, substituting valine for glutamic acid, was identified as the critical factor for the phenotype. Functional complementation and generation of knockout lines in the IR64 background confirmed the central role of OsRPT5A in controlling this trait. Our study also revealed that OsRPT5A is constitutively expressed across various tissues, with its subcellular localization unaffected by mutations. Notably, we observed an abnormal accumulation of reactive oxygen species (ROS) in rpt5a mutants, suggesting a disruption in the ROS system. Complementation studies indicated OsRPT5A's involvement in ROS homeostasis and catalase activity regulation. Moreover, the rpt5a mutant exhibited enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo), highlighting OsRPT5A's role in rice's pathogen resistance mechanisms. These findings elucidate the genetic basis of the spotted leaf phenotype and emphasize the intricate interplay between plant defense responses and developmental pathways.

With ongoing colony losses driven in part by the Varroa mite and the associated exacerbation of virus load, there is an urgent need to protect honey bees (Apis mellifera) from fatal levels of virus infection and from nontarget effects of insecticides used in agricultural settings. A continuously replicating cell line derived from the honey bee would provide a valuable tool for study of molecular mechanisms of virus – host interaction, for screening of antiviral agents for potential use within the hive, and for assessment of the risk of current and candidate insecticides to the honey bee. However, the establishment of a continuously replicating, honey bee cell line has proved challenging. Here we provide an overview of attempts to establish primary and continuously replicating hymenopteran cell lines, methods (including recent results) for establishing honey bee cell lines, challenges associated with the presence of latent viruses (especially Deformed wing virus), in established cell lines and methods to establish virus-free cell lines. We also describe the potential use of honey bee cell lines in conjunction with infectious clones of honey bee viruses for examination of fundamental virology.

Although the first cancer immunotherapy was given in the clinic more than a century ago, this line of treatment has remained more of a distant goal than a practical therapy due to limited understanding of the tumor microenvironment and the mechanisms at play within it, which lead to failures of numerous clinical trials. However, in the last two decades, the immune checkpoint inhibitors and chimeric antigen receptor-T cell therapies have revolutionized the treatment of cancer and provided proof-of-concept that immunotherapies are a viable option. So far, immunotherapies have majoritarily focused on utilizing T cells, however T cells are not autonomous but rather function as part of, and therefore are influenced by, a vast cast of other immune cells, including innate lymphoid cells (ILCs). Here, we summarize the role of ILCs, especially helper ILCs, in tumor development, progression and metastasis, as well as their potential to be used as immunotherapy for cancer. By reviewing the studies that used helper ILCs as adoptive cell therapy, we highlight the rationale behind considering these cells as novel adoptive cell therapy for cancer as well as identify open questions and areas for future research.

Cell-culture meat is a novel concept to replace traditional meat produced from animals, and to satisfy rising food demands due to the world population growth. It is still a young field and requires to solve technical issues including cell sources and types, efficient culture for large scale production, taste and texture for customer satisfaction. The current review summarized traditional beef meat production and evaluation methods for customer satisfaction. In particular, serum biomarkers for beef meat evaluation are discussed, which can be applicated for cell-culture meat production.

The aim of this review was to provide an update on the potential of cell therapies to restore or replace damaged and/or lost cells in retinal degenerative and optic nerve diseases, describing the available cell sources and the challenges involved in such treatments when these techniques are applied in real clinical practice. Sources include human fetal retinal stem cells, allogenic cadaveric human cells, adult hippocampal neural stem cells, human CNS stem cells, ciliary pigmented epithelial cells, limbal stem cells, retinal progenitor cells (RPCs), human pluripotent stem cells (PSCs) (including both human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs)) and mesenchymal stem cells (MSCs). Of these, RPCs, PSCs and MSCs have already entered early-stage clinical trials since they can all differentiate into RPE, photoreceptors or ganglion cells, and have demonstrated safety, while showing some indicators of efficacy. Stem/progenitor cell therapies for retinal diseases still have some drawbacks, such as the inhibition of proliferation and/or differentiation in vitro (with the exception of RPE) and the limited long-term survival and functioning of grafts in vivo. Some other issues remain to be solved concerning the clinical translation of cell-based therapy, including (1) the ability to enrich for specific retinal subtypes; (2) cell survival; (3) cell delivery, which may need to incorporate a scaffold to induce correct cell polarization, which increases the size of the retinotomy in surgery and, therefore, the chance of severe complications; (4) the need to induce retinal detachment to perform the subretinal placement of the transplanted cell; and (5) the evaluation of the risk of tumor formation caused by the undifferentiated stem cells and prolific progenitor cells. Despite these challenges, stem/progenitor cells represent the most promising strategy for retinal and optic nerve disease treatment in the near future, and therapeutics assisted by gene techniques, neuroprotective compounds and artificial devices can be applied to fulfil clinical needs.

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19⁺ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

Cellular response to mechanical stimuli is a crucial factor for maintaining cell homeostasis. The interaction between extracellular matrix and mechanical stress plays a significant role in organ-izing the cytoskeleton and aligning cells. Tools that apply mechanical forces to cells and tissues, as well as those capable of measuring the mechanical properties of biological cells, have greatly contributed to our understanding of fundamental mechanobiology. These tools have been exten-sively employed to unveil the substantial influence of mechanical cues on the development and progression of various diseases. In this report, we present an economical and high-performance uniaxial cell stretching device. This paper reports the detailed operation concept of the device, ex-perimental design, and characterization. The device was tested with MDA-MB-231 breast cancer cells. Experimental results agree well with previously documented morphological changes re-sulting from stretching forces on cancer cells. Remarkably, our new device demonstrates compa-rable cellular changes within a 30-minute compared to the previous 2-hour stretching duration. Moreover, the device design incorporates an open-source software interface, facilitating conven-ient parameter adjustments such as strain, stretching speed, frequency, and duration. Its versatil-ity enables seamless integration with various optical microscopes, thereby yielding novel insights into the realm of mechanobiology.

Three-dimensional (3D) cell culture systems can be regarded as suitable platforms to bridge the huge gap between animal studies and two-dimensional (2D) monolayer cell culture to study chronic diseases such as cancer. In particular, the preclinical platforms for multicellular spheroid formation and culture can be regarded as ideal in vitro tumor models. The complex tumor microenvironment such as hypoxic region and necrotic core can be recapitulated in 3D spheroid configuration. Cells aggregated in spheroid structures can better illustrate the performance of anti-cancer drugs as well. Various methods have been proposed so far to create such 3D spheroid aggregations. Both conventional techniques and microfluidic methods can be used for generation of multicellular spheroids. In this review paper, we first discuss various spheroid formation phases. Then, the conventional spheroid formation techniques such as bioreactor flasks, liquid overlay and hanging droplet technique are explained. Next, a particular topic of the hydrogel in spheroid formation and culture is explored. This topic has received less attention in the literature. Hydrogels entail some advantages to the spheroid formation and culture such as size uniformity, the formation of porous spheroids or hetero-spheroids as well as chemosensitivity and invasion assays and protecting from shear stress. Finally, microfluidic methods for spheroid formation and culture are briefly reviewed.

Adult neurogenesis involves generation of functional neurons from neural progenitor cells, which have the potential to complement and restore damaged neurons and neural circuits. Therefore, the development of drugs that stimulate neurogenesis represents a promising strategy in stem cell therapy and neural regeneration, greatly facilitating the reconstruction of neural circuits in cases of neurodegeneration and brain injury. Our study reveals that compound A5, previously designed and synthesized by our team, exhibits remarkable neuritogenic activities, effectively inducing neurogenesis in neural stem/progenitor cells (NSPCs). Subsequently, transcriptome analysis using high-throughput Illumina RNA-seq technology was performed to further elucidate the underlying molecular mechanisms by which compound A5 promotes neurogenesis. Notably, comparative transcriptome analysis showed that the up-regulated genes were mainly associated with neurogenesis and the down-regulated genes were mainly concerned with cell cycle progression. Furthermore, we confirmed that compound A5 significantly affected the expression of transcription factors related to neurogenesis and cell cycle regulatory proteins. Collectively, these findings identify a new compound with neurogenic activity and may provide insights into drug discovery for neural repair and regeneration.

Regeneration is a fundamental process much attributed to functions of adult stem cells. In last decades delivery of suspended adult stem cells is widely adopted in regenerative medicine as a leading mean of cell therapy. However, adult stem cells can not complete the task of human body regeneration effectively by themselves as far as they need a receptive microenvironment (the niche) to engraft and perform properly. Understanding of mechanisms underlying mammalian regeneration lead us to an assumption that improved outcomes of cell therapy requires a specific microenvironment generated in damaged area prior to stem cell delivery. To certain extent it may be achieved by delivery of mesenchymal stromal cells (MSC), not in dispersed form, but rather self-organized in cell sheets (CS) – tissue-like structures comprising of viable cells and microenvironment components: extracellular matrix and soluble factors deposited in the matrix. In this communication we highlight a potential role of mesenchymal stromal cells (MSC) as regeneration organizers and speculate that this function emerges in CS. This concept shifts our understanding of therapeutic mechanism underlying a widely known CS-based delivery method for regenerative medicine.