Background: The immunomodulatory role of 1,25-Dihydroxy vitamin D3 (1,25(OH)2D3) is exerted through its interaction with the Vitamin D receptor (VDR) present on pancreatic and immune cells. While a deficiency in Vitamin D has been linked to Type 1 Diabetes Mellitus (T1DM), the exact molecular mechanism driving this down-regulation in T1DM is yet to be fully understood. This study aimed to decipher differences in the expression of genes associated with Vitamin D metabolism in T1DM patients and to ascertain if there is a correlation between serum 1,25(OH)2D3 levels and the expression of these genes. We also sought to understand the influence of specific microRNAs (miRNAs) on the expression of Vitamin D metabolism genes in peripheral blood mononuclear cells (PBMCs) of T1DM patients. Furthermore, the study delved into the potential implications of altered Vitamin D metabolism genes and miRNAs on autoimmune processes. Methods: Utilizing real-time PCR, we assessed the expression profiles of genes encoding for 1-hydroxylases (CYP27B1) and 24-hydroxylases (CYP24A1), as well as related miRNAs, in PBMCs from 30 T1DM patients and 23 healthy controls. ELISA tests facilitated the measurement of 1,25(OH)2D3, GAD65, and IA-2 levels. Results: Our findings showcased down-regulated CYP27B1 mRNA levels, while CYP24A1 expression remained stable compared to healthy subjects (CYP27B1, p = 0.0005; CYP24A1, p = 0.205, respectively). In T1DM patients, the levels of has-miR-216b-5p were found to be increased, while the levels of has-miR-21-5p were decreased in comparison to the control group. Notably, no correlation was identified between the expression of CYP27B1 in T1DM patients and the levels of has-miR-216b-5p, has-miR-21-5p, and 1,25(OH)2D3. A significant negative correlation was identified between CYP27B1 mRNA levels in PBMCs of T1DM and IA2, but not with GAD65. Conclusions: The study highlights there were reduced levels of both CYP27B1 mRNA and has-miR-21-5p, along with elevated levels of has-miR-216b-5p in the PBMCs of T1DM. However, the absence of a correlation between the expression of CYP27B1, levels of has-miR-216b-5p, and the status of 1,25(OH)2D3 suggests the possible existence of other regulatory mechanisms. Additionally, the inverse relationship between IA2 autoantibodies and CYP27B1 expression in T1DM patients indicates a potential connection between this gene and the autoimmune processes inherent in T1DM.

Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase enzyme that catalyzes a key step in phenylpropanoid metabolism in plants. However, there is very limited information on the function of C4H in medicinal plants. In the present study, the C4H gene from the Ferula pseudalliacea genome (named FpC4H) was cloned and characterized. In addition, the expression levels of FpC4H in the roots, stems, leaves, flowers and seeds were investigated via real-time PCR. FpC4H showed the most similarity with C4H proteins from Daucus carota. The results predicted that FpC4H, as well as its orthologs, is an unstable and hydrophilic protein. A high genetic distance was observed between FpC4H and C4H from monocots based on evolutionary analysis. Moreover, a variable region related to the variable function of C4H was detected in conserved domain region of C4H proteins. Protein structure analysis revealed that isoleucine and leucine residues are frequently present in the binding region of FpC4H, and two ion binding regions, cysteine258 and asparagine217, were identified in the 3D structure of FpC4H. Expression analysis revealed that the FpC4H gene is expressed in all organs of F. pseudallicea. A low expression level of FpC4H was recorded in leaves, and a high expression level was observed in flower tissues. Based on the expression profile, FpC4H seems to be linked to a high content of phenolic compounds in flowers.

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis PCC 6803 (S.6803) and Synechococcus PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted at enhancing farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis or terpene precursors) from S.6803 or S.7002 did not increased farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified (GMO) strains of S.6803 we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene but decreased the production of farnesene in S.6803.

Cysteamine HCl has been established as a potent ulcerogenic agent of the gastrointestinal (GI) system. GI dysfunction and olfactory deficits are the most common clinical symptoms of many movement disorders including Parkinson's disease (PD). Cysteamine HCl has been shown to interfere with dopamine, a neurotransmitter crucial for motor, olfactory, and cognitive functions. However, the reports on the effect of cysteamine HCl treatment on the behavior and dopamine system appear to be inconsistent. Therefore, we revisited the impact of cysteamine HCl on motor function in experimental mice using a battery of behavioral tests such as pole test (PT), beam walking test (BWT), and rotarod test (RDT), while the olfactory ability and cognitive functions were examined through food buried test (FBT) and Y maze. Furthermore, we investigated the effect of cysteamine HCl on the density of dopaminergic tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) and olfactory bulb (OB) of experimental mice using immunohistochemistry. Results revealed that cysteamine HCl administration in mice induced significant impairments in motor balance and coordination, as their movement-related performance was markedly reduced in behavioral tasks. Mice exposed to cysteamine HCl showed a pronounced reduction in odor discrimination ability and cognitive impairments. Strikingly, the number of TH-positive neurons was found to be reduced in the SN and OB of the cysteamine HCl-treated group which is a bonafide neuropathogenic hallmark of PD. This study highlights the potential neurotoxic effects of cysteamine HCl in experimental brains and suggests further investigation into its role in pathogenesis of Parkinsonism.

Deficiency of Cytochrome P450 Steroid 21-hydroxylase (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction along with functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in the Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of 21OH deficiency.

Several studies suggests that ghrelin (GHRL) has neurobiological effects that extend beyond control of food intake. Our previous results confirmed that GHRL modulates the secretory activity of the pineal gland (PG) through nocturnal melatonin (MEL) secretion in sheep, the seasonally reproductive animals. Here we investigated the effects of GHRL (10 ng/ml) on the expression of enzymes limiting synthesis of MEL, including tryptophan 5-hydroxylase 1 (TPH1), serotonin N-acetyltransferase (AA-NAT) and its phosphorylated form p31T-AA-NAT in sheep PG explants (n = 72) during the 4-hour incubation in a gas-liquid interface, at a short (SD) and long (LD) photoperiods. After each hour of incubation selected explants were frozen in liquid nitrogen and stored at -80°C for subsequent analysis (real-time PCR, western-blotting, ELISA). Results show that GHRL regulates nightly MEL secretion in a TPH1-independent manner. The factor modulating GHRL activity was photoperiod. During SD photoperiod GHRL significantly reduced the expression of p31T-AA-NAT, AA-NAT and inhibited MEL secretion from PG explants. Whereas, during LD photoperiod no effect of GHRL on MEL secretion and expression of examined enzymes was noted. Studies indicate that GHRL directly affects PG under in vitro conditions and causes MEL secretion in animals which exhibit seasonality in reproductive and metabolic processes.

Parkinson’s disease (PD) is an aging-related and the second most common neurodegenerative disease after Alzheimer’s disease. The main symptoms of PD are movement disorders accompanied with deficiency of neurotransmitter dopamine (DA) in the striatum due to cell death of the nigro-striatal DA neurons. Two main histopathological hallmarks exist in PD: cytosolic inclusion bodies termed Lewy bodies that mainly consist of α-synuclein protein, the oligomers of which produced by misfolding are regarded to be neurotoxic, causing DA cell death; and black pigments termed neuromelanin (NM) that are contained in DA neurons and markedly decrease in PD. Synthesis of human NM is regarded to be similar to that of melanin in melanocytes; Melanin synthesis in skin is via DOPAquinone (DQ) by tyrosinase, whereas NM synthesis in DA neurons is via DAquinone (DAQ) by tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC). DA in cytoplasm is highly reactive and is assumed to be oxidized spontaneously or by an unidentified tyrosinase to DAQ and then synthesized to NM. Intracellular NM accumulation above a specific threshold was reported to be associated to DA neuron death and PD phenotypes. This review reports recent progress in biosynthesis and pathophysiology of NM in PD.

The mechanistic influences of dopamine (DA) signaling and impact on motor function is nearly always interpreted from changes in nigrostriatal neuron terminals in striatum. This is a standard practice in studies of human Parkinson’s disease (PD) and aging, and related animal models of PD and aging-related parkinsonism. However, despite dozens of studies indicating an ambiguous relationship between changes in striatal DA signaling and motor phenotype, this perseverating focus on striatum continues. Although DA release in substantia nigra (SN) was first reported almost 50 years ago. assessment of nigral DA signaling changes in relation to motor function is rarely considered. Whereas DA signaling has been well-characterized in striatum at all 5 steps of neurotransmission (biosynthesis and turnover, storage, release, reuptake, and post-synaptic binding) in the nigrostriatal pathway, the depth of such interrogations in the SN, outside of cell counts, is sparse. However, there is sufficient evidence that these steps in DA neurotransmission in the SN are operational and regulated autonomously from striatum, and are present in human PD and aging, and related animal models. To complete our understanding of how nigrostriatal DA signaling affects motor function, it is past time to include interrogation of nigral DA signaling. This brief review highlights evidence that changes in nigral DA signaling at each step in DA neurotransmission are autonomous from those in striatum and changes in the SN alone can influence locomotor function. Accordingly, for full characterization of how nigrostriatal DA signaling affects locomotor activity, interrogation of DA signaling in SN is essential.

Testosterone holds significant medical and economic importance, with the global market for testosterone replacement therapies valued at approximately $1.9 billion in 2023. This hormone is essential for the development and maintenance of male sexual characteristics as well as bone and muscle health. It plays a key role in conditions such as hypogonadism, muscle disorders, and andropause. However, the industrial production of testosterone often involves complex chemical processes that result in low yields, high costs, and environmental damage. Microbial biotransformation of steroids presents an eco-friendly alternative to traditional chemical synthesis. A knockout strain of Aspergillus nidulans deficient in steroid 11-α-hydroxylase activity was developed, rendering it incapable of hydroxylating androstenedione, progesterone, and testosterone. In this strain, two newly identified CYP450 enzymes—CYP68L1 from A. nidulans and CYP68L8 from Aspergillus ochraceus—were expressed to confirm their roles as steroid 11-α-hydroxylases. These enzymes successfully hydroxylated steroids such as androstenedione, progesterone, and testosterone. The availability of these 11-α-hydroxylases represents significant progress toward achieving efficient single-step steroid fermentation. Furthermore, the A. nidulans knockout strain is proposed as an effective model for studying the conversion of androstenedione to testosterone owing to its inability to hydroxylate the testosterone produced during bioconversion.