Effect of Phenol and Alkylamide Interaction on α-Glucosidase Inhibition and Cellular Antioxidant Activity during In Vitro Digestion: Using Szechuan Pepper (Zanthoxylum genus) as a Model.

Although recent evidence indicated significant phenol and alkylamide interaction in aqueous solutions, the gastrointestinal digestion influence of the combination remains unclear. This study aims to investigate phenol and alkylamide interaction during in vitro digestion, focusing on bioaccessibility and bioactivity, including α-glucosidase inhibition and cellular antioxidant activity. Additionally, the structural mechanism of phenol and alkylamide interaction during in vitro digestion was explored. The results indicated that the presence of phenols and alkylamides significantly increased or decreased their respective bioaccessibility, depending on the Zanthoxylum varieties. Furthermore, although antagonistic phenol/alkylamide interaction was evident during α-glucosidase inhibition, cellular oxidative stress alleviation, and antioxidant gene transcription upregulation, this effect weakened gradually as digestion progressed. Glycoside bond cleavage and the methylation of phenols as well as alkylamide isomerization and addition were observed during digestion, modifying the hydrogen bonding sites and interaction behavior. This study provided insights into the phenol/alkylamide interaction in the gastrointestinal tract.

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides.

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.

Copper-catalysed dehydrogenation or lactonization of C(sp3)-H bonds.

Cytochrome P450 enzymes are known to catalyse bimodal oxidation of aliphatic acids via radical intermediates, which partition between pathways of hydroxylation and desaturation1,2. Developing analogous catalytic systems for remote C-H functionalization remains a significant challenge3-5. Here, we report the development of Cu(I)-catalysed bimodal dehydrogenation/lactonization reactions of synthetically common N-methoxyamides through radical abstractions of the γ-aliphatic C-H bonds. The feasibility of switching from dehydrogenation to lactonization is also demonstrated by altering reaction conditions. The use of a readily available amide as both radical precursor and internal oxidant allows for the development of redox-neutral C-H functionalization reactions with methanol as the sole side product. These C-H functionalization reactions using a Cu(I) catalyst with loading as low as 0.5 mol.% is applied to the diversification of a wide range of aliphatic acids including drug molecules and natural products. The exceptional compatibility of this catalytic system with a wide range of oxidatively sensitive functionality demonstrates the unique advantage of using a simple amide substrate as a mild internal oxidant.

Favipiravir, remdesivir, and lopinavir: metabolites, degradation products and their analytical methods.

Severe acute respiratory syndrome 2 (SARS-CoV-2) caused the emergence of the COVID-19 pandemic all over the world. Several studies have suggested that antiviral drugs such as favipiravir (FAV), remdesivir (RDV), and lopinavir (LPV) may potentially prevent the spread of the virus in the host cells and person-to-person transmission. Simultaneously with the widespread use of these drugs, their stability and action mechanism studies have also attracted the attention of many researchers. This review focuses on the action mechanism, metabolites and degradation products of these antiviral drugs (FAV, RDV and LPV) and demonstrates various methods for their quantification and discrimination in the different biological samples. Herein, the instrumental methods for analysis of the main form of drugs or their metabolite and degradation products are classified into two types: optical and chromatography methods which the last one in combination with various detectors provides a powerful method for routine and stability analyses. Some representative studies are reported in this review and the details of them are carefully explained. It is hoped that this review will be a good guideline study and provide a better understanding of these drugs from the aspects investigated in this study.

Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor.

Pyrrolysine, the 22nd amino acid encoded by the natural genetic code, is essential for methanogenic archaea to catabolize methylamines into methane. The structure of pyrrolysine consists of a methylated pyrroline carboxylate that is linked to the ε-amino group of the l-lysine via an amide bond. The biosynthesis of pyrrolysine requires three enzymes: PylB, PylC, and PylD. PylB is a radical S-adenosyl-l-methionine (SAM) enzyme and catalyzes the first biosynthetic step, the isomerization of l-lysine into methylornithine. PylC catalyzes an ATP-dependent ligation of methylornithine and a second l-lysine to form l-lysine-Nε-methylornithine. The last biosynthetic step is catalyzed by PylD via oxidation of the PylC product to form pyrrolysine. While enzymatic reactions of PylC and PylD have been well characterized by X-ray crystallography and in vitro studies, mechanistic understanding of PylB is still relatively limited. Here, we report the first in vitro activity of PylB to form methylornithine via the isomerization of l-lysine. We also identify a lysyl C4 radical intermediate that is trapped, with its electronic structure and geometric structure well characterized by EPR and ENDOR spectroscopy. In addition, we demonstrate that SAM functions as a catalytic cofactor in PylB catalysis rather than canonically as a cosubstrate. This work provides detailed mechanistic evidence for elucidating the carbon backbone rearrangement reaction catalyzed by PylB during the biosynthesis of pyrrolysine.

A new and promiscuous α/ß hydrolase from Acinetobacter tandoii DSM 14970 T inactivates the mycotoxin ochratoxin A.

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-ß-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/ß hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/ß hydrolase core composed of a parallel, six-stranded ß-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/ß hydrolases based on our results. KEY POINTS: • AtABH is a promiscuous αß hydrolase with both esterase and amidohydrolase activities • AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα • Promiscuous αß hydrolases are a possible source of new OTA-degrading enzymes.

Comparative analysis of hippocampal extracellular space uncovers widely altered peptidome upon epileptic seizure in urethane-anaesthetized rats.

BACKGROUND: The brain extracellular fluid (ECF), composed of secreted neurotransmitters, metabolites, peptides, and proteins, may reflect brain processes. Analysis of brain ECF may provide new potential markers for synaptic activity or brain damage and reveal additional information on pathological alterations. Epileptic seizure induction is an acute and harsh intervention in brain functions, and it can activate extra- and intracellular proteases, which implies an altered brain secretome. Thus, we applied a 4-aminopyridine (4-AP) epilepsy model to study the hippocampal ECF peptidome alterations upon treatment in rats. METHODS: We performed in vivo microdialysis in the hippocampus for 3-3 h of control and 4-AP treatment phase in parallel with electrophysiology measurement. Then, we analyzed the microdialysate peptidome of control and treated samples from the same subject by liquid chromatography-coupled tandem mass spectrometry. We analyzed electrophysiological and peptidomic alterations upon epileptic seizure induction by two-tailed, paired t-test. RESULTS: We detected 2540 peptides in microdialysate samples by mass spectrometry analysis; and 866 peptides-derived from 229 proteins-were found in more than half of the samples. In addition, the abundance of 322 peptides significantly altered upon epileptic seizure induction. Several proteins of significantly altered peptides are neuropeptides (Chgb) or have synapse- or brain-related functions such as the regulation of synaptic vesicle cycle (Atp6v1a, Napa), astrocyte morphology (Vim), and glutamate homeostasis (Slc3a2). CONCLUSIONS: We have detected several consequences of epileptic seizures at the peptidomic level, as altered peptide abundances of proteins that regulate epilepsy-related cellular processes. Thus, our results indicate that analyzing brain ECF by in vivo microdialysis and omics techniques is useful for monitoring brain processes, and it can be an alternative method in the discovery and analysis of CNS disease markers besides peripheral fluid analysis.

Rapid Degradation of Hazardous Amides by Immobilized Engineered Pseudomonas putida KT2440 Based on a Novel Gene Expression Vector.

The amides 4-trifluoromethylnicotinamide, acrylamide, and benzamide are widely used in agriculture and industry, posing hazards to the environment and animals. Immobilized bacteria are preferred in wastewater treatment, but degradation of these amides by immobilized engineered bacteria has not been explored. Here, engineered Pseudomonas putida KT2440 pLSJ15-amiA was constructed by introducing a new amidase gene expression vector into environmentally safe P. putida KT2440. P. putida KT2440 pLSJ15-amiA had high amidase activity, even at 80 °C. P. putida KT2440 pLSJ15-amiA immobilized with calcium alginate exhibited a greater environmental tolerance than free cells. The amides were rapidly degraded by the immobilized cells, but the activity was inhibited by high concentrations of substrates. The substrate inhibition model revealed that the optimum initial concentrations of 4-trifluoromethylnicotinamide, acrylamide, and benzamide for degradation by immobilized cells were 197.65, 350.76, and 249.40 µmol/L, respectively. This study develops a novel and excellent immobilized biocatalyst for remediation of wastewater containing hazardous amides.

Reverse metabolomics for the discovery of chemical structures from humans.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.

Specific and rapid guanidinium CEST imaging using double saturation power and QUASS analysis in a rodent model of global ischemia.

PURPOSE: Guanidinium CEST is sensitive to metabolic changes and pH variation in ischemia, and it can offer advantages over conventional pH-sensitive amide proton transfer (APT) imaging by providing hyperintense contrast in stroke lesions. However, quantifying guanidinium CEST is challenging due to multiple overlapping components and a close frequency offset from water. This study aims to evaluate the applicability of a new rapid and model-free CEST quantification method using double saturation power, termed DSP-CEST, for isolating the guanidinium CEST effect from confounding factors in ischemia. To further reduce acquisition time, the DSP-CEST was combined with a quasi-steady state (QUASS) CEST technique to process non-steady-state CEST signals. METHODS: The specificity and accuracy of the DSP-CEST method in quantifying the guanidinium CEST effect were assessed by comparing simulated CEST signals with/without the contribution from confounding factors. The feasibility of this method for quantifying guanidinium CEST was evaluated in a rat model of global ischemia induced by cardiac arrest and compared to a conventional multiple-pool Lorentzian fit method. RESULTS: The DSP-CEST method was successful in removing all confounding components and quantifying the guanidinium CEST signal increase in ischemia. This suggests that the DSP-CEST has the potential to provide hyperintense contrast in stroke lesions. Additionally, the DSP-CEST was shown to be a rapid method that does not require the acquisition of the entire or a portion of the CEST Z-spectrum that is required in conventional model-based fitting approaches. CONCLUSION: This study highlights the potential of DSP-CEST as a valuable tool for rapid and specific detection of viable tissues.

Role of amide proton transfer imaging in maximizing tumor resection in malignant glioma: a possibility to take the place of 11C-methionine positron emission tomography.

BACKGROUND: Amide proton transfer (APT) imaging has been proposed as a technique to assess tumor metabolism. However, the relationship between APT imaging and other quantitative modalities including positron emission tomography (PET) has not been investigated in detail. This study aimed to evaluate the clinical usefulness of APT imaging in determining the metabolic status of malignant glioma and to compare findings with those from 11C-methionine (Met)-PET. METHODS: This research analyzed APT imaging data from 20 consecutive patients with malignant glioma treated between January 2022 and July 2023. Patients underwent tumor resection and correlations between tumor activity and intensity of APT signal were investigated. We also compared 11C-Met-PET and APT imaging for the same regions of the perifocal tumor invasion area. RESULTS: Clear, diagnostic APT images were obtained from all 20 cases. Mean APT intensity (APTmean) was significantly higher in the glioblastoma (GBM), IDH wild type group (27.2 ± 12.8%) than in other gliomas (6.0 ± 4.7%; p < 0.001). The cut-off APTmean to optimally distinguish between GBM and other malignant gliomas was 12.8%, offering 100% sensitivity and 83.3% specificity. These values for APTmean broadly matched the tumor-to-contralateral normal brain tissue ratio from 11C-Met-PET analysis (r = 0.66). The APT signal was also observed in the gadolinium non-contrast region on T1-weighted imaging, appearing to reflect the surrounding tumor-infiltrated area. CONCLUSIONS: APT imaging can be used to evaluate the area of tumor invasion, similar to 11C-Met-PET. APT imaging revealed low invasiveness in patients and was useful in preoperative planning for tumor resection, facilitating maximum tumor resection including the tumor invasive area.

A potent and selective cis-amide inhibitor of ryanodine receptor 2 as a candidate for cardiac arrhythmia treatment.

Ryanodine receptor 2 (RyR2) is a Ca2+ release channel mainly located on the sarcoplasmic reticulum (SR) membrane of heart muscle cells and regulates the concentration of Ca2+ in the cytosol. RyR2 overactivation causes potentially lethal cardiac arrhythmias, but no specific inhibitor is yet available. Herein we developed the first highly potent and selective RyR2 inhibitor, TMDJ-035, containing 3,5-difluoro substituents on the A ring and a 4-fluoro substituent on the B ring, based on a comprehensive structure-activity relationship (SAR) study of tetrazole compound 1. The SAR study also showed that the amide conformation is critical for inhibitory potency. Single-crystal X-ray diffraction analysis and variable-temperature 1H NMR revealed that TMDJ-035 strongly favors cis-amide configuration, while the inactive analogue TMDJ-011 with a secondary amide takes trans-amide configuration. Examination of the selectivity among RyRs indicated that TMDJ-035 displayed high selectivity for RyR2. TMDJ-035 suppressed abnormal Ca2+ waves and transients in isolated cardiomyocytes from RyR2-mutated mice. It appears to be a promising candidate drug for treating cardiac arrhythmias due to RyR2 overactivation, as well as a tool for studying the mechanism and dynamics of RyR2 channel gating.

Effect of carnosine synthesis precursors in the diet on jejunal metabolomic profiling and biochemical compounds in slow-growing Korat chicken.

The slow-growing Korat chicken (KR) has been developed to provide an alternative breed for smallholder farmers in Thailand. Carnosine enrichment in the meat can distinguish KR from other chicken breeds. Therefore, our aim was to investigate the effect of enriched carnosine synthesis, obtained by the ß-alanine and L-histidine precursor supplementation in the diet, on changes to metabolomic profiles and biochemical compounds in slow-growing KR jejunum tissue. Four hundred 21-day-old female KR chickens were divided into 4 experimental groups: a group with a basal diet, a group with a basal diet supplemented with 1.0% ß-alanine, 0.5% L-histidine, and a mix of 1.0% ß-alanine and 0.5% L-histidine. The feeding trial lasted 70 d. Ten randomly selected chickens from each group were slaughtered. Metabolic profiles were analyzed using proton nuclear magnetic resonance spectroscopy. In total, 28 metabolites were identified. Significant changes in the concentrations of these metabolites were detected between the groups. Partial least squares discriminant analysis was used to distinguish the metabolites between the experimental groups. Based on the discovered metabolites, 34 potential metabolic pathways showed differentiation between groups, and 8 pathways (with impact values higher than 0.05, P < 0.05, and FDR < 0.05) were affected by metabolite content. In addition, biochemical changes were monitored using synchrotron radiation-based Fourier transform infrared microspectroscopy. Supplementation of ß-alanine alone in the diet increased the ß-sheets and decreased the α-helix content in the amide I region, and supplementation of L-histidine alone in the diet also increased the ß-sheets. Furthermore, the relationship between metabolite contents and biochemical compounds were confirmed using principal component analysis (PCA). Results from the PCA indicated that ß-alanine and L-histidine precursor group was highly positively correlated with amide I, amide II, creatine, tyrosine, valine, isoleucine, and aspartate. These findings can help to understand the relationships and patterns between the spectral and metabolic processes related to carnosine synthesis.

The metabolic profile of the synthetic cannabinoid receptor agonist ADB-HEXINACA using human hepatocytes, LC-QTOF-MS and synthesized reference standards.

Synthetic cannabinoid receptor agonists (SCRAs) remain a major public health concern, with their use implicated in intoxications and drug-related deaths worldwide. Increasing our systematic understanding of SCRA metabolism supports clinical and forensic toxicology casework, facilitating the timely identification of analytical targets for toxicological screening procedures and confirmatory analysis. This is particularly important as new SCRAs continue to emerge on the illicit drug market. In this work, the metabolism of ADB-HEXINACA (ADB-HINACA, N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-hexyl-1H-indazole-3-carboxamide), which has increased in prevalence in the United Kingdom and other jurisdictions, was investigated using in vitro techniques. The (S)-enantiomer of ADB-HEXINACA was incubated with pooled human hepatocytes over 3 hours to identify unique and abundant metabolites using liquid chromatography-quadrupole time-of-flight mass spectrometry. In total, 16 metabolites were identified, resulting from mono-hydroxylation, di-hydroxylation, ketone formation (mono-hydroxylation then dehydrogenation), carboxylic acid formation, terminal amide hydrolysis, dihydrodiol formation, glucuronidation and combinations thereof. The majority of metabolism took place on the hexyl tail, forming ketone and mono-hydroxylated products. The major metabolite was the 5-oxo-hexyl product (M9), while the most significant mono-hydroxylation product was the 4-hydroxy-hexyl product (M8), both of which were confirmed by comparison to in-house synthesized reference standards. The 5-hydroxy-hexyl (M6) and 6-hydroxy-hexyl (M7) metabolites were not chromatographically resolved, and the 5-hydroxy-hexyl product was the second largest mono-hydroxylated metabolite. The structures of the terminal amide hydrolysis products without (M16, third largest metabolite) and with the 5-positioned ketone (M13) were also confirmed by comparison to synthesized reference standards, along with the 4-oxo-hexyl metabolite (M11). The 5-oxo-hexyl and 4-hydroxy-hexyl metabolites are suggested as biomarkers for ADB-HEXINACA consumption.

Amidation of glutamate residues in mycobacterial peptidoglycan is essential for cell wall cross-linking.

Introduction: Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to osmotic pressure. We previously demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. Here, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors. Methods: Synthetic fluorescent probes were used to assess PG remodelling dynamics in live bacteria. Fluorescence microscopy was used to assess protein localization in live bacteria and CRISPR-interference was used to construct targeted gene knockdown strains. Time-lapse microscopy was used to assess bacterial growth. Western blotting was used to assess protein phosphorylation. Results and discussion: In Mycobacterium smegmatis, we confirmed the essentiality for D-glutamate amidation in PG biosynthesis by labelling cells with synthetic fluorescent PG probes carrying amidation modifications. We also used CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation, and demonstrated that these genes are essential for mycobacterial growth. We show that MurT-rseGFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria. Furthermore, time-lapse microscopic analysis of MurT-rseGFP localization, in fluorescent D-amino acid (FDAA)-labelled mycobacterial cells during growth, demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during PG remodelling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and ß-lactam antibiotics. Cell growth inhibition was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, these data demonstrate the essentiality of D-glutamate amidation in mycobacterial PG precursors and highlight the MurT-GatD complex as a novel drug target.

Formation and microstructural characterization of scallop (Patinopecten yessoensis) male gonad hydrolysates/sodium alginate coacervations as a function of pH.

Studying the noncovalent interactions between proteins and polysaccharides is quite important mainly due to the wide number of applications such as developing pH-responsive complexes. Scallop Patinopecten yessoensis male gonad hydrolysates­sodium alginate (SMGHs-SA) was investigated as noncovalent complexes at pH from 1 to 10. The critical pH values pHC (around 6) and pHφ (around 4) were independent of the SMGHs-SA ratio, indicating the formation of soluble and insoluble complexes. The pH response of SMGHs-SA complexes was evaluated by investigating the rheological behavior, moisture distribution, functional group change and microstructure. Compared to the co-soluble and soluble complexes phases, the SMGHs-SA complexes had a higher storage modulus and viscosity as well as a lower relaxation time (T23) in the insoluble complexes phase (pHφ>3). Additionally, the amide I band and COO- stretching vibration peaks were redshifted and the amide A band vibration peaks were blueshifted by acidification. Electrostatic interactions and intermolecular/intramolecular hydrogen bonding led to SMGHs-SA agglomeration at pH 3, forming a uniform and dense gel network structure with strong gel strength and water-retention capacity. This study provides a theoretical and methodological basis for the design of novel pH-responsive complexes by studying SMGHs-SA complex coacervation.

Efficient establishment of an optimized culture condition for cashmere goat primary hair follicle stem cells.

Hair follicle stem cells (HFSCs) are an important basis for hair follicle morphogenesis and hair cycle growth. This cell type also represents an excellent model for studying the gene function and molecular regulation of the hair growth cycle, including proliferation, differentiation, and apoptosis. Basically, the functional investigation of hair growth-regulating genes demands a sufficient amount of HFSCs. However, efficient propagation of HFSCs in goats is a challenging process under the current culture conditions. Here, we investigated the effect of four components, including the Rho-associated protein kinase (ROCK) inhibitor Y-27632, leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and vitamin C, on cell growth and pluripotency in the basal culture medium (DMEM/F12 supplemented with 2% fetal bovine serum). We found that adding Y-27632, LIF, and bFGF independently increased the proliferation and pluripotency of goat HFSCs (gHFSCs), with Y-27632 having the most significant effect (P < 0.001). Fluorescence-activated cell sorting of the cell cycle revealed that Y-27632 promoted gHFSC proliferation by inducing the cell cycle from S to G2/M phase (P < 0.05). We further demonstrated that gHFSCs displayed superior proliferative capacity, clone-forming ability, and differentiation potential in the combined presence of Y-27632 (10 µM) and bFGF (10 ng/mL). We termed this novel culture condition as gHFEM, which stands for goat Hair Follicle Enhanced Medium. Taken together, these results indicate that gHFEM is an optimal condition for in vitro culture of gHFSCs, which will subsequently facilitate the study of HF growth and biology.

Hair follicle stem cells (HFSCs) are indispensable for skin repair, hair growth, development, and regeneration. One major challenge in primary cell culture is achieving efficient growth while maintaining stemness to achieve a high yield. Various factors, including the Rho-associated protein kinase inhibitor Y-27632, leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and vitamin C are known to regulate the growth of cells. Here, we investigated the optimal concentrations of Y-27632, LIF, bFGF, and vitamin C for promoting goat HFSCs (gHFSCs) proliferation and pluripotency. We further found that the combination of Y-27632 and bFGF exhibited optimal growth conditions. These findings offer valuable insights into the factors affecting gHFSC culture and potential applications for studying the cellular and molecular mechanisms underlying periodic HF growth and gene function associated with HF development.

Discovery of Novel Pentacyclic Triterpene Acid Amide Derivatives as Excellent Antimicrobial Agents Dependent on Generation of Reactive Oxygen Species.

Developing new agricultural bactericides is a feasible strategy for stopping the increase in the resistance of plant pathogenic bacteria. Some pentacyclic triterpene acid derivatives were elaborately designed and synthesized. In particular, compound A22 exhibited the best antimicrobial activity against Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac) with EC50 values of 3.34 and 3.30 mg L-1, respectively. The antimicrobial mechanism showed that the compound A22 induced excessive production and accumulation of reactive oxygen species (ROS) in Xoo cells, leading to a decrease in superoxide dismutase and catalase enzyme activities and an increase in malondialdehyde content. A22 also produced increases in Xoo cell membrane permeability and eventual cell death. In addition, in vivo experiments showed that A22 at 200 mg L-1 exhibited protective activity against rice bacterial blight (50.44%) and citrus canker disease (84.37%). Therefore, this study provides a paradigm for the agricultural application of pentacyclic triterpene acid.

Discovery of blood-brain barrier permeable and orally bioavailable caffeine-based amide derivatives as acetylcholinesterase inhibitors.

Caffeine is one of the privileged natural products that shows numerous effects on the central nervous system. Herein, thirty-one caffeine-based amide derivatives were synthesized and evaluated in vitro for their anticholinesterase activity. The introduction of the amide group to the caffeine core augmented its anticholinesterase activity from an IC50 value of 128 to 1.32 µM (derivative, 6i). The SAR study revealed that N7 substitution on caffeine core is favorable over N1, and the presence of amide 'carbonyl' as a part of the linker contributes to the biological activity. The caffeine core of 6i exhibits interactions with the peripheral anionic site, whereas the N-benzyl ring fits nicely inside the catalytic anionic site. Analog 6i inhibits AChE in a mixed-type mode (Ki 4.58 µM) and crosses the BBB in an in-vitro PAMPA assay. Compound 6i has a descent metabolic stability in MLM (>70% remaining after 30 min) and favorable oral pharmacokinetics in Swiss albino mice.

Characterization of a transitionally occupied state and thermal unfolding of domain 1.1 of σ A factor of RNA polymerase from Bacillus subtilis.

σ factors are essential parts of bacterial RNA polymerase (RNAP) as they allow to recognize promotor sequences and initiate transcription. Domain 1.1 of vegetative σ factors occupies the primary channel of RNAP and also prevents binding of the σ factor to promoter DNA alone. Here, we show that domain 1.1 of Bacillus subtilis σ A exists in more structurally distinct variants in dynamic equilibrium. The major conformation at room temperature is represented by a previously reported well-folded structure solved by nuclear magnetic resonance (NMR), but 4% of the protein molecules are present in a less thermodynamically favorable state. We show that this population increases with temperature and we predict its significant elevation at higher but still biologically relevant temperatures. We characterized the minor state of the domain 1.1 using specialized methods of NMR. We found that, in contrast to the major state, the detected minor state is partially unfolded. Its propensity to form secondary structure elements is especially decreased for the first and third α helices, while the second α helix and ß strand close to the C-terminus are more stable. We also analyzed thermal unfolding of the domain 1.1 and performed functional experiments with full length σ A and its shortened version lacking domain 1.1 ( σ A _ Δ 1.1 ). The results revealed that while full length σ A increases transcription activity of RNAP with increasing temperature, transcription with σ A _ Δ 1.1 remains constant. In summary, this study reveals conformational dynamics of domain 1.1 and provides a basis for studies of its interaction with RNAP and effects on transcription regulation.