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The accumulation of post-translational modifications (PTMs) in proteins throughout the lifecycle has been studied for decades, particularly more so with the advent of soft-ionization mass spectrometry-based proteomic techniques. However, particular PTMs, such as the deamidations of asparagine and glutamine residues, continue to accumulate in proteins that remain into the forensic, archaeological, and palaeontological records. The accurate measurement of these ancient ‘molecular timers’ has been proposed as a method to not only differentiate between exogenous and endogenous proteins within complex mixtures (i.e., contamination), but also as a method of providing relative age estimations into geological time. In this study we explored the extent to which deamidation varies with chronological age across different proteins in bones, as well as investigated differences between proteins across dental calculus and archaeological ceramics. We also analysed the relationships between the observed extent of deamidation and the protein primary structure. We found that collagen obtained from archaeological bones showed a chronological dependence on the extent of deamidation observed, but only when they were from similar environments, supporting prior suggestions about ‘thermal age’ being a major influence on the deamidation observed. Our study on non-collagenous proteins (NCPs) in archaeological bones showed that while biglycan, and to a lesser extent chondroadherin, showed positive correlations between geological age and the extent of deamidation, others including fetuin-A and serum albumin did not. However, despite the well-known dependence of deamidation on the three-dimensional structure of the peptides, we were unable to find any clear correlation between the structural motifs of the peptides in archaeological bones and the extent of deamidation observed. Our analysis of a set of food proteins obtained from Neolithic archaeological ceramics in Catalhoyuk also showed similar deamidation levels irrespective of the protein structure. Overall, our results suggest that deamidation in archaeological samples could be useful for obtaining additional information beyond identification of species and tissue type, be that as a measure of protein endogeneity and potential contamination, or a measure of protein degradation, or as an indicator of thermal age and for relative dating; however, further research needs to be undertaken to understand why particular proteins are better for this than others, going beyond simple consideration of their secondary structure.

Borassus flabellifer leaf extract has been used for rapid biogenic-synthesis of Zinc-Oxide nanoparticles (ZnO-NPs) due to rich source of bioactive compounds. The synthesized ZnO-NPs were preliminarily confirmed by UV-Visible spectroscopy adsorption peak range at 365nm. The XRD (X-ray diffraction) confirm purity of ZnO-NPs that were crystalline in nature. The analysis of FT-IR (Fourier-transform infrared spectroscopy) confirm the presence of following functional group such as alcohol, phenols, carboxylic acids, primary amides, secondary amides, alkyl halide. FE-SEM analysis indicated that ZnO-NPs were in spherical shape, followed by EDX analysis which confirmed the presence of Zn-element. Antimicrobial effect of ZnO-NPs was investigated using different clinical pathogens like bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella Pneumonia, Pseudomonas aeruginosa and fungi Aspergillus flavus, Candida albicans and Penicillium expansum and which confirmed ZnO-NPs efficiency as an antimicrobial agent. Antioxidant activity were ascertained to used for several biomedical applications. The ZnO-NPs was efficiently degraded the environmental toxic dyes (methylene blue and crystal violet) under sunlight. In support of photolight-degradation, the study was progressed to understand the ZnO-dye interaction stability using molecular mechanism and it was showing efficient bonding features in the NPs environment. Overall, this investigation have great potential for being an effective and eco-friendly materials was used in environmental applications.

Histone lysine methyltransferases and acetyltransferases are two classes of epigenetic enzymes that play pivotal roles in human gene regulation. Although they both recognise and posttranslationally modify lysine residues in histone proteins, their difference in histone peptide-based substrates and inhibitors remains to be firmly established. Here, we have synthesised lysine mimics that posses an amide bond linker in the side chain, incorporated them into histone H3 tail peptides, and examined synthetic histone peptides as substrates and inhibitors for human lysine methyltransferases and acetyltransferases. This work demonstrates that histone lysine methyltransferases G9a and GLP do catalyse methylation of the most similar lysine mimic, whereas they typically do not tolerate more sterically demanding side chains. In contrast, histone lysine acetyltransferases GCN5 and PCAF do not catalyse acetylation of the same panel of lysine analogues. Our results also identify potent H3-based inhibitors of G9a/GLP methyltransferases, providing a basis for development of peptidomimetics for targeting KMT enzymes.

Combining a bulky bis(amide) and a reactive one-coordinate TMP (2,2,6,6-tetramethylpiperidide) ligand, a new mixed K/Zn heteroleptic base has been developed for regioselective zincation of fluoroarenes. This special ligand set allows for trapping and structural authentication of the first intermediates of direct Zn-H exchange of fluoroarenes obtained via deprotonative metallation, providing mechanistic insights of the processes involved.

Mandipropamid (MDP) is a widely used chiral fungicide to control oomycete pathogens with two enantiomers. In this study, the enantioselective bioactivity, toxicity, and degradation of MDP were investigated for the first time. The bioactivity of S-MDP was 118-592 times higher than that of R-MDP and 1.14-1.67 times higher than that of Rac-MDP against six phytopathogens. Molecular docking found that S-MDP formed a strong halogen bond with HIS 693 of cellulose synthase and possessed a lower binding energy, which validated the results of the bioactivity assay. S-MDP showed lower toxicity toward Spirodela polyrhiza, while it exhibited higher toxicity in Danio rerio embryo and larva. S-MDP preferentially degraded in cowpea and pepper, while R-MDP preferentially degraded in soil. There is no significant difference between the two enantiomers in the toxicity of adult D. rerio and in cucumber degradation. Therefore, the development of the S-enantiomer was considered as a better option to exhibit high efficiency, which could reduce the residual risk of the pesticide and ensure environmental safety.

Amides derived from ferulic acid have a wide spectrum of pharmacological activities, including antitumor and antifungal activity. In the present study, a series of ten amides were obtained by coupling reactions using the reagents (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and N,N ′ -dicyclohexylcarbodiimide (DCC). All the compounds were identified on the basis of their IR, 1H- and 13C-NMR, HRMS data, and with yields ranging from 43.17% to 91.37%. The compounds were subjected to cytotoxic tests by the alamar blue technique and antifungal screening by the broth microdilution method to determine the minimum inhibitory concentration (MIC). The amides 10 and 11 displayed the best result in both biological evaluations, and compound 10 was the most potent and selective in HL-60 cancer cells, with no cytotoxicity on healthy cells. This amide had antifungal activity in all strains and had the lowest MIC against Candida albicans and Candida tropicalis. The possible mechanism of antifungal action occurs via the fungal cell wall. Molecular modeling suggested that compounds 10 and 11 interact with the enzymes GWT1 and GSC1, which are essential for the development of C. albicans. The findings of the present study demonstrated that compounds 10 and 11 may be used as a platform in drug development in the future.

Phosphoinositide-3-kinase γ (PI3Kγ) is highly expressed in immune cells and promotes the production and migration of inflammatory mediators. The inhibition of PI3Kγ has been shown to repolarize the tumor immune microenvironment to a more inflammatory phenotype, thereby controlling immune suppression in cancer. Herein, we report the structure-based optimization of an early lead series of pyrazolopyrimidine isoindolinones, which culminated in the discovery of highly potent and isoform-selective PI3Kγ inhibitors with favorable drug-like properties. X-ray cocrystal structure analysis, molecular docking studies, and detailed structure-activity relationship investigations resulted in the identification of the optimal amide and isoindolinone substituents to achieve a desirable combination of potency, selectivity, and metabolic stability. Preliminary in vitro studies indicate that inhibition of PI3Kγ with compound 56 results in a significant immune response by increasing pro-inflammatory cytokine gene expression in M1 macrophages.

Tetragonisca angustula honey was fractioned in a SiO2 column to furnish three fractions (A-C) in which four hydroxycinnamic acid-Spermidine amides (HCAAs), known as N', N″, N‴-tris-p-coumaroyl spermidine, N', N″-dicaffeoyl, N‴-coumaroyl spermidine, N', N″, N‴-tris-caffeoyl spermidine and N', N″-dicaffeoyl and N‴-feruloyl spermidine were identified in the fractions B and C by electrospray ionization tandem mass spectrometry. A primary culture model previously infected with Neospora caninum (72 h) was used to evaluate the honey fractions (A-C) for two-time intervals: 24 and 72 h. Parasitic reduction ranged from 38% on fraction C (12.5 µg/ml), after 24 h, to 54% and 41% with fractions B and C (25 µg/ml) after 72 h of treatment, respectively. Additionally, HCAAs did not show any cell toxicity for 24 and 72 h. For infected cultures (72 h), the active fractions B (12.5 µg/ml) and C (25 µg/ml) decreased their NO content. In silico studies suggest that HCAAs may affect the parasite's redox pathway and improve the oxidative effect of NO released from infected cells. Here, we presented for the first time, that HCAAs from T. angustula honey have the potential to inhibit the growth of N. caninum protozoa.

Directed evolution of heme proteins has opened access to new-to-nature enzymatic activity that can be harnessed to tackle synthetic challenges. Among these, reactions resulting from active site iron-nitrenoid intermediates present a powerful strategy to forge C−N bonds with high site- and stereoselectivity. Here we report a biocatalytic, intermolecular benzylic C−H amidation reaction operating at mild and scalable conditions. With hydroxamate esters as nitrene precursors, feedstock aromatic compounds can be converted to chiral amides with excellent enantioselectivity (up to >99 % ee) and high yields (up to 87 %). Kinetic and computational analysis of the enzymatic reaction reveals rate-determining nitrenoid formation followed by stepwise hydrogen atom transfer-mediated C−H functionalization.

Cellular senescence is a well-documented response to oncogene activation in many tissues. Multiple pathways are invoked to achieve senescence indicating its importance to counteract the transforming activities of oncogenic stimulation. We now report that the Rho-associated protein kinase (ROCK) signaling pathway is a critical regulator of oncogene-induced senescence in skin carcinogenesis. Transformation of mouse keratinocytes with oncogenic RAS upregulates ROCK activity and initiates a senescence response characterized by cell enlargement, growth inhibition, upregulation of senescence associated β-galactosidase (SAβgal) expression, and release of multiple pro-inflammatory factors comprising the senescence-associated secretory phenotype (SASP). The addition of the ROCK inhibitor Y-27632 and others prevents these senescence responses and maintains proliferating confluent RAS transformed keratinocyte cultures indefinitely. Mechanistically, oncogenic RAS transformation is associated with upregulation of cell cycle inhibitors p15Ink4b , p16Ink4a , and p19Arf and downregulation of p-AKT, all of which are reversed by Y-27632. RNA-seq analysis of Y-27632 treated RAS-transformed keratinocytes indicated that the inhibitor reduced growth-inhibitory gene expression profiles and maintained expression of proliferative pathways. Y-27632 also reduced the expression of NF-κB effector genes and the expression of IκBζ downstream mediators. The senescence inhibition from Y-27632 was reversible, and upon its removal, senescence reoccurred in vitro with rapid upregulation of cell cycle inhibitors, SASP expression, and cell detachment. Y-27632 treated cultured RAS-keratinocytes formed tumors in the absence of the inhibitor when placed in skin orthografts suggesting that factors in the tumor microenvironment can overcome the drive to senescence imparted by overactive ROCK activity.

Background Rho/ROCK signaling has been demonstrated to be involved in the vascular reactivity of many arterial networks. However, RhoA expression and the contribution of Rho/ROCK pathway to the control of perfusion pressure have not been investigated in the rat hind limb vascular bed as a skeletal muscle vascular network. Aims To investigate the contribution of the Rho/ROCK pathway in the control of perfusion pressure in the isolated-perfused rat hind limb vascular bed. Study design Animal experimentation. Methods Two Rho inhibitors (atorvastatin and C3 exoenzyme) and ROCK inhibitors (Y-27632 and fasudil) were tested on the phenylephrine-elevated perfusion pressure in the isolated-perfused rat hind limb vascular bed. Furthermore, we sought the expression of RhoA protein in the femoral, popliteal and saphenous arteries as well as quadriceps and gastrocnemius muscles by Western blotting. Results The ROCK inhibitors Y-27632 and fasudil (both 10-8 to 10-5 M) induced substantial vasodilatations. The maximum vasodilatations induced by Y-27632 and fasudil (both at 10-5 M) were 84.0 ± 6.9% and 76.9 ± 6.9%, respectively (P = .091). Y-27632 was not more potent than fasudil, as the EC50 values for Y-27632 and fasudil were 0.7 ± 2.1 μM and 2.5 ± 2.4 μM, respectively (P = .177). Atorvastatin (10-7 to 10-4 M) and C3 exoenzyme (3 × 10-8 M) also produced vasodilatation (maximum vasodilatation; 20.3 ± 1.7% and 13.7 ± 3.6%, respectively). The EC50 value for atorvastatin was 94.9 ± 1.2 μM. The western blot analysis showed that the femoral, saphenous, and popliteal arteries, as well as the gastrocnemius and quadriceps muscles, express RhoA protein. Conclusion The Rho/ROCK pathway contributes significantly to the control of perfusion pressure in the rat hind limb vascular bed.

Favipiravir is a broad-spectrum inhibitor of viral RNA-dependent RNA polymerase (RdRp) currently being used to manage COVID-19. Accumulation of mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRp may facilitate antigenic drift, generating favipiravir resistance. Focussing on the chain-termination mechanism utilized by favipiravir, we used high-throughput interface-based protein design to generate > 100 000 designs of the favipiravir-binding site of RdRp and identify mutational hotspots. We identified several single-point mutants and designs having a sequence identity of 97%-98% with wild-type RdRp, suggesting that SARS-CoV-2 can develop favipiravir resistance with few mutations. Out of 134 mutations documented in the CoV-GLUE database, 63 specific mutations were already predicted as resistant in our calculations, thus attaining Eœ 47% correlation with the sequencing data. These findings improve our understanding of the potential signatures of adaptation in SARS-CoV-2 against favipiravir.

Objectives The second-generation integrase strand transfer inhibitor (INSTI) bictegravir is becoming accessible in low- and middle-income countries (LMICs), and another INSTI, cabotegravir, has recently been approved as a long-acting injectable. Data on bictegravir and cabotegravir susceptibility in raltegravir-experienced HIV-1 subtype A- and D-infected patients carrying drug resistance mutations (DRMs) remain very scarce in LMICs. Patients and methods HIV-1 integrase (IN)-recombinant viruses from eight patients failing raltegravir-based third-line therapy in Uganda were genotypically and phenotypically tested for susceptibility to bictegravir and cabotegravir. Ability of these viruses to integrate into human genomes was assessed in MT-4 cells. Results HIV-1 IN-recombinant viruses harbouring single primary mutations (N155H or Y143R/S) or in combination with secondary INSTI mutations (T97A, M50I, L74IM, E157Q, G163R or V151I) were susceptible to both bictegravir and cabotegravir. However, combinations of primary INSTI-resistance mutations such as E138A/G140A/G163R/Q148R or E138K/G140A/S147G/Q148K led to decreased susceptibility to both cabotegravir (fold change in EC50 values from 429 to 1000×) and bictegravir (60 to 100×), exhibiting a high degree of cross-resistance. However, these same IN-recombinant viruses showed impaired integration capacity (14% to 48%) relative to the WT HIV-1 NL4-3 strain in the absence of drug. Conclusions Though not currently widely accessible in most LMICs, bictegravir and cabotegravir offer a valid alternative to HIV-infected individuals harbouring subtype A and D HIV-1 variants with reduced susceptibility to first-generation INSTIs but previous exposure to raltegravir may reduce efficacy, more so with cabotegravir.

Thirty-eight novel ferulic amide 1-aminocyclohexane carboxylic acid (Ac6c) derivatives D1-D19 and E1-E19 were designed and synthesized, and their antibacterial, antifungal, and insecticidal activities were tested. Most of the synthesized compounds displayed excellent activity againstXanthomonas oryzae pv. oryzae (Xoo), with EC50 values ranging from 11.6 to 83.1 μg/mL better than that of commercial bismerthiazol (BMT, EC50 = 84.3 μg/mL), as well as much better performance compared to that of thiediazole copper (TDC, EC50 = 137.8 μg/mL). D6 (EC50 = 17.3 μg/mL), D19 (EC50 = 29.4 μg/mL), E3 (EC50 = 29.7 μg/mL), E9 (EC50 = 27.0 μg/mL), E10 (EC50 = 18.6 μg/mL), and E18 (EC50 = 20.8 μg/mL) showed much higher activity on Xanthomonas oryzae pv. oryzicola compared with BMT (EC50 = 80.1 μg/mL) and TDC (EC50 = 124.7 μg/mL). In relation to controlling the fungus, Rhizoctonia solani, E1, E10, and E13 had much lower EC50 values of 0.005, 0.140, and 0.159 μg/mL compared to hymexazol at 74.8 μg/mL. Further in vivo experiments demonstrated that E6 and E12 controlled rice bacterial leaf blight disease better than BMT and TDC did. Scanning electron microscopy (SEM) studies revealed that E12 induced the Xoo cell membrane collapse. Moreover, D13 (73.7%), E5 (80.6%), and E10 (73.4%) also showed moderate activity against Plutella xylostella. These results indicated that the synthesized ferulic amide Ac6c derivatives showed promise as candidates for treating crop diseases.

BACKGROUND Cannabis sativa L. (hemp) is a medicinal plant producing various cannabinoids. Its consumption is legalized for medical use due to the alleged positive health effects of these cannabinoids. To satisfy the demand, C. sativa plants are propagated in contained growth chambers. During indoor propagation, pesticides usually are used to ensure efficient production. However, pesticide registration and safe application in C. sativa has not been investigated in detail. RESULTS With this study the metabolic degradation of pesticides in recently established C. sativa callus cultures was examined. Tebuconazole, metalaxyl-M fenhexamid, flurtamone and spirodiclofen were applied at 10 μm for 21 days. Results were compared with metabolism data obtained from Brassica napus L., Glycine max (L.) Merr., Zea mays L. and Tritium aestivum L. callus cultures as well as in metabolism guideline studies. The successfully established C. sativa callus cultures were able to degrade pesticides by oxidation, demethylation, and cleavage of ester bonds in phase I, as well as glycosylation and conjugation with malonic acid in phase II and III. Initial metabolites were detected after Day (D)7 and were traced at D21. CONCLUSION The resulting pathways demonstrate the same main degradation strategies as crop plants. Because metabolites could be the main residue, the exposure of consumers to these residues will be of high importance. We present here an in vitro assay for a first estimation of pesticide metabolism in C. sativa. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A bis-heteroleptic ruthenium(II) complex, 1[PF6 ]2 of benzothiazole amide substituted 2,2'-bipyridine ligand (bmbbipy) has been synthesized for the selective detection of G-quadruplex (GQ) DNA and luminescence-assay-based RNase H activity monitoring. Compound 1[PF6 ]2 exhibited aggregation-caused quenching (ACQ) in water. Aggregate formation was supported by DLS, UV-vis, and 1 H NMR spectroscopy results, and the morphology of aggregated particles was witnessed by SEM and TEM. 1[PF6 ]2 acted as an efficient GQ DNA-selective luminescent light-up probe over single-stranded and double-stranded DNA. The competency of 1[PF6 ]2 for selective GQ structure detection was established by PL and CD spectroscopy. For 1[PF6 ]2 , the PL light-up is exclusively due to the rigidification of the benzothiazole amide side arm in the presence of GQ-DNA. The interaction between the probe and GQ-DNA was analyzed by molecular docking analysis. The GQ structure detection capability of 1[PF6 ]2 was further applied in the luminescent 'off-on' RNase H activity detection. The assay utilized an RNA:DNA hybrid, obtained from 22AG2-RNA and 22AG2-DNA sequences. RNase H solely hydrolyzed the RNA of the RNA:DNA duplex and released G-rich 22AG2-DNA, which was detected via the PL enhancement of 1[PF6 ]2 . The selectivity of RNase H activity detection over various other restriction enzymes was also demonstrated.

To discover novel antibacterial agents, a series of novel 1,3,4-thiadiazole derivatives containing an amide moiety were designed and synthesized, and their antibacterial activities were tested. Compound 30 was designed and synthesized according to the CoMFA model. Compound 30 exhibited higher antibacterial activities against Xanthomonas oryzae pv. oryzicola and Xanthomonas oryzae pv. oryzae, with EC50 values of 2.1 and 1.8 mg/L, respectively, which were superior to those of thiodiazole copper (99.6 and 92.5 mg/L). The protective and curative activities of compound 30 against rice bacterial leaf blight were 51.3 and 46.1%, respectively, which were better than those of thiodiazole copper (37.8 and 38.5%). The protective and curative activities of compound 30 against rice bacterial leaf streak were 45.9 and 40.5%, respectively, which were better than those of thiodiazole copper (36.2 and 31.1%). In addition, the protective activity of compound 30 against rice bacterial leaf streak was related to increased activities of related defense enzymes and upregulated the differentially expressed proteins of the glycolysis/gluconeogenesis pathway.

The pandemic of SARS-CoV-2 has necessitated expedited research efforts towards finding potential antiviral targets and drug development measures. While new drug discovery is time consuming, drug repurposing has been a promising area for elaborate virtual screening and identification of existing FDA approved drugs that could possibly be used for targeting against functions of various proteins of SARS-CoV-2 virus. RNA dependent RNA polymerase (RdRp) is an important enzyme for the virus that mediates replication of the viral RNA. Inhibition of RdRp could inhibit viral RNA replication and thus new virus particle production. Here, we screened non-nucleoside antivirals and found three out of them to be strongest in binding to RdRp out of which two retained binding even using molecular dynamic simulations. We propose these two drugs as potential RdRp inhibitors which need further in-depth testing.

Background and Objective: Histone Deacetylases (HDACs) are important therapeutic targets for many types of human cancers. A derivative of valproic acid, N-(2-hydroxyphenyl)-2-propylpentanamide (HOAAVPA), has antiproliferative properties on some cancer cell lines and inhibits the HDAC1 isoform. Materials and Methods: In this work, HO-AAVPA was tested as an antiproliferative agent in U87-MG (human glioblastoma) and U-2 OS cells (human osteosarcoma), which are types of cancer that are difficult to treat, and its antiangiogenic properties were explored. Results: HO-AAVPA had antiproliferative effects at 48h with an IC50=0.655mM in U87-MG cells and an IC50=0.453mM in U-2 OS cells. Additionally, in the colony formation assay, HO-AAVPA decreased the number of colonies by approximately 99% in both cell lines and induced apoptosis by 31.3% in the U-2 OS cell line and by 78.2% in the U87-MG cell line. Additionally, HO-AAVPA reduced the number of vessels in Chorioallantoic Membranes (CAMs) by approximately 67.74% and IL-6 levels in both cell lines suggesting that the biochemical mechanism on cancer cell of HO-AAVPA is different compared to VPA. Conclusion: HO-AAVPA has antiproliferative effects on glioblastoma and osteosarcoma and antiangiogenic properties.

Mechanotransduction, the interplay between physical and chemical signaling, plays vital roles in many biological processes ranging from cell differentiation to metastasis. The state-of-the-art techniques to quantify cell forces employ deformable polymer films or molecular probes tethered to glass substrates. These types of flat substrates limit applications in investigating mechanotransduction on non-planar geometries where physiological activities such as phagocytosis and immunological synapse formation mostly occur. A second challenge is the low throughput of microscopy readout which limits the application of current assays in fundamental and clinical research. We address these challenges by developing a DNA-based microparticle tension sensor (μTS), which features a spherical surface and thus allows for investigation of mechanical events at curved interfaces or within groups of cells in suspension. Importantly, the micron-scale of μTS enables flow cytometry readout, which is rapid and high throughput. To demonstrate the scope of μTS, we applied the method to map and measure T-cell receptor (TCR) forces and platelet integrin forces at 12 and 56 pN thresholds. Furthermore, we quantified the inhibition efficiency of two anti-platelet drugs providing a proof-of-concept demonstration of μTS to screen drugs that modulate cellular mechanics.

Objectives HIV-1 group O (HIV-1/O) is one of the four HIV-1 groups and is endemic in Cameroon, representing 1% of HIV-1 infections in the population. Around 50% of the strains of this group naturally show a mutation (Y181C) providing them with resistance to NNRTIs and making therapeutic management more difficult. Today, the WHO recommends the use of integrase strand transfer inhibitors (INSTIs) as first-line treatment. Bictegravir and cabotegravir are the two most recent INSTIs. Because of the genetic polymorphism of HIV-1/O, studies are required to evaluate their phenotypic susceptibility to these two drugs. Patients and methods We performed a phenotypic study on a large panel including 41 HIV-1/O clinical isolates and other rare non-group M HIV-1 (2 HIV-1/N and 1 HIV-1/P) to evaluate in vitro susceptibility to bictegravir and cabotegravir. Results The results showed an overall susceptibility of non-group M strains to the two drugs compared with HIV-1 group M. There was no difference between the mean (min–max) IC50 of HIV-1/M [1.86 (0.93–4.12) and 5.24 (1.76–12.41) nM for bictegravir and cabotegravir, respectively] and HIV-1/non-M [2.17 (0.03–9.47) and 4.88 (0.02–15.64) nM for bictegravir and cabotegravir, respectively]. However, we found a significant difference between IC50 values for bictegravir and cabotegravir in the whole panel (P value Conclusions This study has shown encouraging results regarding the clinical use of these drugs in HIV-1/non-M-infected patients, which will need to be confirmed with clinical data.

As the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic engulfs millions worldwide, the quest for vaccines or drugs against the virus continues. The helicase protein of SARS-CoV-2 represents an attractive target for drug discovery since inhibition of helicase activity can suppress viral replication. Using in silico approaches, we have identified drugs that interact with SARS-CoV-2 helicase based on the presence of amino acid arrangements matching binding sites of drugs in previously annotated protein structures. The drugs exhibiting an RMSD of ≤ 3.0 Å were further analyzed using molecular docking, molecular dynamics (MD) simulation, and post-MD analyses. Using these approaches, we found 12 drugs that showed strong interactions with SARS-CoV-2 helicase amino acids. The analyses were performed using the recently available SARS-CoV-2 helicase structure (PDB ID: 5RL6). Based on the MM-GBSA approach, out of the 12 drugs, two drugs, namely posaconazole and grazoprevir, showed the most favorable binding energy, − 54.8 and − 49.1 kcal/mol, respectively. Furthermore, of the amino acids found conserved among all human coronaviruses, 10/11 and 10/12 were targeted by, respectively, grazoprevir and posaconazole. These residues are part of the crucial DEAD-like helicase C and DEXXQc_Upf1-like/ DEAD-like helicase domains. Strong interactions of posaconazole and grazoprevir with conserved amino acids indicate that the drugs can be potent against SARS-CoV-2. Since the amino acids are conserved among the human coronaviruses, the virus is unlikely to develop resistance mutations against these drugs. Since these drugs are already in use, they may be immediately repurposed for SARS-CoV-2 therapy.

In this study, we report the design and synthesis of a series of novel thiophene-arylamide compounds derived from the noncovalent decaprenylphosphoryl-β-d-ribose 2′-epimerase (DprE1) inhibitor TCA1 through a structure-based scaffold hopping strategy. Systematic optimization of the two side chains flanking the thiophene core led to new lead compounds bearing a thiophene-arylamide scaffold with potent antimycobacterial activity and low cytotoxicity. Compounds 23j, 24f, 25a, and 25b exhibited potent in vitro activity against both drug-susceptible (minimum inhibitory concentration (MIC) = 0.02–0.12 μg/mL) and drug-resistant (MIC = 0.031–0.24 μg/mL) tuberculosis strains while retaining potent DprE1 inhibition (half maximal inhibitory concentration (IC50) = 0.2–0.9 μg/mL) and good intracellular antimycobacterial activity. In addition, these compounds showed good hepatocyte stability and low inhibition of the human ether-à-go-go related gene (hERG) channel. The representative compound 25a with acceptable pharmacokinetic property demonstrated significant bactericidal activity in an acute mouse model of tuberculosis. Moreover, the molecular docking study of template compound 23j provides new insight into the discovery of novel antitubercular agents targeting DprE1.

Favipiravir is an antiviral agent effective against several RNA viruses that is converted into an inactive oxidative metabolite (M1), mainly by aldehyde oxidase, in humans. In the present study, the biotransformation of favipiravir into M1 in male and female humans, monkeys, rats, and mice was examined in an in vitro system using liver cytosolic fractions. The kinetics for M1 formation followed the Michaelis-Menten model in all species. The Km , Vmax , and CLint values in humans were 602 µM, 466 pmol/min/mg protein, and 776 nl/min/mg protein in males, respectively, and 713 µM, 404 pmol/min/mg protein, and 567 nl/min/mg protein in females, respectively. Species differences in CLint values were monkeys > humans > mice > rats in both males and females, and the variations for males and females were 120- and 96-fold, respectively. Sex differences in CLint values were males > females in humans and mice, females > males in monkeys and rats, and marked variation (4.3-fold) was noted in mice. This suggests that the roles of aldehyde oxidase in the hepatic metabolism of favipiravir differ extensively depending on the species and sex, and this study will aid in the assessment of the antiviral activities of favipiravir against novel and/or variant viruses.

Bronchopulmonary dysplasia (BPD) is caused primarily by oxidative stress and inflammation. To induce BPD, neonatal rat pups were raised in hyperoxic (>90% O2) environments from day one (P1) until day ten (P10) and treated with N-acetyl-lysyltyrosylcysteine amide (KYC). In vivo studies showed that KYC improved lung complexity, reduced myeloperoxidase (MPO) positive (+) myeloid cell counts, MPO protein, chlorotyrosine formation, increased endothelial cell CD31 expression, decreased 8-OH-dG and Cox-1/Cox-2, HMGB1, RAGE, TLR4, increased weight gain and improved survival in hyperoxic pups. EPR studies confirmed that MPO reaction mixtures oxidized KYC to a KYC thiyl radical. Adding recombinant HMGB1 to the MPO reaction mixture containing KYC resulted in KYC thiylation of HMGB1. In rat lung microvascular endothelial cell (RLMVEC) cultures, KYC thiylation of RLMVEC proteins was increased the most in RLMVEC cultures treated with MPO + H2O2, followed by H2O2, and then KYC alone. KYC treatment of hyperoxic pups decreased total HMGB1 in lung lysates, increased KYC thiylation of HMGB1, terminal HMGB1 thiol oxidation, decreased HMGB1 association with TLR4 and RAGE, and shifted HMGB1 in lung lysates from a non-acetylated to a lysyl-acetylated isoform, suggesting that KYC reduced lung cell death and that recruited immune cells had become the primary source of HMGB1 released into the hyperoxic lungs. MPO-dependent and independent KYC-thiylation of Keap1 were both increased in RLMVEC cultures. Treating hyperoxic pups with KYC increased KYC thiylation and S-glutathionylation of Keap1, and Nrf2 activation. These data suggest that KYC is a novel system pharmacological agent that exploits MPO to inhibit toxic oxidant production and is oxidized into a thiyl radical that inactivates HMGB1, activates Nrf2, and increases antioxidant enzyme expression to improve lung complexity and reduce BPD in hyperoxic rat pups.

Cystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-l-isoasparagine or a β-methoxy-l-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound l-asparagine. Furthermore, CysQ O-methylates hydroxyl-l-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis., The biosynthetic pathway leading to the production of cystobactamids, a group of myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity, remains unclear. Here, the authors report in vivo and in vitro evidence for unique steps in cystobactamid biosynthesis.

Background This is the first-in-human study of icenticaftor, an oral potentiator of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) channel. Restoration of CFTR activity has shown significant clinical benefits, but more studies are needed to address all CFTR mutations. Methods Safety, pharmacodynamics/pharmacokinetics of icenticaftor were evaluated in a randomized, double-blind, placebo-controlled study in healthy volunteers. Efficacy was assessed in adult CF patients with ≥1 pre-specified CFTR Class III or IV mutation (150 and 450 mg bid), or homozygous for F508del mutation (450 mg bid). Primary efficacy endpoint was change from baseline in lung clearance index (LCI2.5). Secondary endpoints included %predicted FEV1 and sweat chloride level. Results Class IV mutations were present in 22 patients, Class III in 2 (both S549N), and 25 were homozygous for F508del. Icenticaftor was well-tolerated in healthy and CF subjects with no unexpected events or discontinuations in the CF groups. The most frequent study-drug related adverse events in CF patients were nausea (12.2%), headache (10.2%), and fatigue (6.1%). Icenticaftor 450 mg bid for 14 days showed significant improvements in all endpoints versus placebo in patients with Class III and IV mutations; mean %predicted FEV1 increased by 6.46%, LCI2.5 decreased by 1.13 points and sweat chloride decreased by 8.36 mmol/L. No significant efficacy was observed in patients homozygous for a single F508del. Conclusions Icenticaftor was safe and well-tolerated in healthy volunteers and CF patients, and demonstrated clinically meaningful changes in lung function and sweat chloride level in CF patients with Class III and IV CFTR mutations. ClinicalTrials.gov: NCT02190604

The syntheses of aromatic monoamines and aliphatic polyamines (PAs) are responsive to environmental stresses, with some modulating aspects of plant defense. Conjugation of amines to hydroxycinnamic acids (HCAs) generates HCA amides (HCAAs), with the conjugates possessing properties from both compounds. Conjugation may reduce the polarity of the resulting metabolite and assist in translocation, stability, and compartmentalization. Recent metabolomic insights identified HCAAs as biomarkers during plant–pathogen interactions, supporting a functional role in defense. The conjugates may contribute to regulation of the dynamic metabolic pool of hydroxycinnamates. This review highlights the occurrence of aromatic amines (AAs) and PAs in stress metabolism, conjugation to HCAs, and the roles of HCAAs during host defense, adding emphasis on their involvement in hydrogen peroxide (H2O2) production and cell-wall strengthening.

PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1’s F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.

The non-nucleoside reverse transcriptase inhibitor VM1500A is approved for the treatment of HIV/AIDS in its N-acyl sulphonamide prodrug form elsulfavirine (Elpida®). Biochemical profiling against twelve human carbonic anhydrase (CA, EC 4.2.1.1) isoforms showed that while elsulfavirine was a weak inhibitor of all isoforms, VM1500A potently and selectively inhibited human (h) hCA VII isoform, a proven target for the therapy of neuropathic pain. The latter is a common neurologic complication of HIV infection and we hypothesise that by using Elpida® in patients may help alleviate this debilitating symptom., Graphical Abstract

To develop a new class of neutral fluorescent dyes with mitochondrial staining capacity, a series of functional dyes were obtained from Nile Red (2a–e) and coumarin (3a–e) with different amide compounds via Suzuki coupling reactions. The Nile Red derivatives (2a–e) emitted red fluorescence (590–660 nm) and coumarin derivatives (3a–e) showed blue emission (455–490 nm) in organic solvents. In addition, they exhibited high fluorescence quantum yields (0.27–0.98) in organic solvents and excellent photostability (>92%). Moreover, all of them possessed low cytotoxicity. More importantly, Nile Red borate (2) and coumarin borate (3) only accumulated in lipid droplets, while after being modified by different amide compounds, dyes 2a–e and 3a–e could successfully target mitochondria in HeLa cancer cells via confocal fluorescence experiments. This work provides a new strategy for the design of neutral cellular probes for mitochondrial staining.

Enzymatic deamidation, the conversion of glutamine (Gln) into glutamic acid (Glu) residues, mediated by tissue transglutaminase enzymes, can provoke autoimmunity by generating altered self-epitopes, a process well-known in celiac disease and more recently also described in type 1 diabetes (T1D). To identify deamidated proteins, liquid chromatography-tandem mass spectrometry is the method of choice. However, as nonenzymatic deamidations on asparagine (Asn) and to a minor extent on Gln are frequently induced in vitro during proteomics sample preparation, the accurate detection of in vivo deamidation can be hampered. Here we report on the optimization of a method to reduce in vitro generated deamidation by 70% using improved trypsin digestion conditions (90 min/pH 8). We also point to the critical importance of manual inspection of MS2 spectra, considering that only 55% of the high quality peptides with Gln deamidation were assigned correctly using an automated search algorithm. As proof of principal, using these criteria, we showed a significant increase in levels of both Asn and Gln deamidation in cytokine-exposed murine MIN6 β-cells, paralleled by an increase in tissue transglutaminase activity. These findings add evidence to the hypothesis that deamidation is occurring in stressed β-cell proteins and can be involved in the autoimmune process in T1D.

A series of halo-substituted mixed ester/amide-based analogues 4a-l have been prepared as jack bean urease inhibitor, which showed good to excellent inhibition of enzyme activity. The role of halo-substituted benzoyl moieties and alkyl substituted anilines in urease inhibitory kinetics was also investigated. The alkyl-substituted anilines 1a–b reacted with chloroacetyl chloride to afford intermediates 2a-b, which were then reacted with different halo-substituted benzoic acids 3a–f to prepare the title compounds 4a-l. The chemical structures of final products 4a-l were ascertained by FTIR, 1H NMR, 13C NMR, and mass spectra. The compound 4b showed remarkable activity with IC50 1.6 ± 0.2 nM, better than the standard thiourea having IC50 472.1 ± 135.1 nM. The 2-chloro-substituted phenyl ring on one side of compound 4b and 4-isopropyl-substituted benzene on the other side play an essential role in inhibition of urease activity. Lineweaver–Burk plots (kinetics study) indicated about 4b derivative as a mixed type of inhibitor. The virtual screening performed against urease enzyme (PDBID 4H9M) showed that compounds 4b and 4e have binding energies of −7.8 and −7.9 Kcal/mol, respectively. Based upon our results, it was found that derivative 4b is a highly potent urease inhibitor, better than the standard thiourea.

Endothelial cells injury and activation contribute to arteriovenous fistula (AVF) stenosis. Hirudin (Hiru) can inhibit the activity of thrombin, which was reported to enhance endothelial cell permeability and promote vascular inflammatory responses. RhoA/ROCK signaling pathway is also important in regulating vascular endothelial permeability. This study aimed to investigate the role of Hiru on the viability and permeability of human umbilical vein endothelial cells (HUVECs) following stimulation of serum from rat with chronic renal failure (CRF) and illustrated the effects of Hiru on RhoA/ROCK signaling. Wistar rats were randomly divided into control group and CRF group. Serum from each group was collected to stimulate HUVECs. Proliferation capability was estimated with Cell Count Kit-8 (CCK-8) assay. Transwell assay was performed to determine permeability. Cell apoptosis was examined using Tunel staining. Telomere length and telomerase activity were determined by qPCR. Moreover, the expression of RhoA, ROCK1 and ROCK2 was estimated via western blot. Results showed that the serum from CRF rat significantly inhibited cell viability while enhanced cell permeability and apoptosis. Different concentrations of Hiru prevented the above effects caused by CRF serum. Additionally, Hiru recovered the CRF serum-induced decreased telomere length and telomerase activity. Hiru also inhibited the protein expression of RhoA, ROCK1 and ROCK2, which were activated by CRF serum. Moreover, the ROCK inhibitor, Y27632, exhibited similar effects with Hiru. In conclusion, Hiru-restored HUVECs cell viability, telomere length and telomerase activity, suppressed permeability and apoptosis in the presence of CRF serum might depend on inactivating the RhoA/ROCK signaling.

The chemokine receptor CXCR7, also known as ACKR3, is a seven-transmembrane G-protein-coupled receptor (GPCR) involved in various pathologies such as neurological diseases, autoimmune diseases, and cancers. By binding and scavenging the chemokines CXCL11 and CXCL12, CXCR7 regulates their extracellular levels. From an original high-throughput screening campaign emerged hit 3 among others. The hit-to-lead optimization led to the discovery of a novel chemotype series exemplified by the trans racemic compound 11i. This series provided CXCR7 antagonists that block CXCL11- and CXCL12-induced s-arrestin recruitment. Further structural modifications on the trisubstituted piperidine scaffold of 11i yielded compounds with high CXCR7 antagonistic activities and balanced ADMET properties. The effort described herein culminated in the discovery of ACT-1004-1239 (28f). Biological characterization of ACT-1004-1239 demonstrated that it is a potent, insurmountable antagonist. Oral administration of ACT-1004-1239 in mice up to 100 mg/kg led to a dose-dependent increase of plasma CXCL12 concentration.

Activation of invariant natural killer T (iNKT) cells by α-galactosylceramides (α-GalCers) stimulates strong immune responses and potent anti-tumor immunity. Numerous modifications of the glycolipid structure have been assessed to derive activating ligands for these T cells with altered and potentially advantageous properties in the induction of immune responses. Here, we synthesized variants of the prototypical α-GalCer, KRN7000, with amide-linked phenyl alkane substitutions on the C4″-position of the galactose ring. We show that these variants have weak iNKT cell stimulating activity in mouse models but substantially greater activity for human iNKT cells. The most active of the C4″-amides in our study showed strong anti-tumor effects in a partially humanized mouse model for iNKT cell responses. In silico analysis suggested that the tether length and degree of flexibility of the amide substituent affected the recognition by iNKT cell antigen receptors of the C4″-amide substituted glycolipids in complex with their antigen presenting molecule CD1d. Our findings establish the use of stable C4″-amide linked additions to the sugar moiety for further exploration of the immunological effects of structural modifications of iNKT cell activating glycolipids and highlight the critical need for more accurate animal models to assess these compounds for immunotherapeutic potential in humans.

The amide bond with its planarity and lack of chemical reactivity is at the heart of protein structure. Chemical methylation of amides is known but was considered too harsh to be accessible to biology. Until last year there was no protein structure in the data bank with an enzymatically methylated amide. The discovery that the natural macrocyclic product, omphalotin is ribosomally synthesized, was not as had been assumed by non-ribosomal peptide synthesis. This was the first definitive evidence that an enzyme could methylate the amide bond. The enzyme, OphMA, iteratively self-hypermethylates its own C-terminus using SAM as cofactor. A second enzyme OphP, a prolyl oligopeptidase cleaves the core peptide from OphMA and cyclizes it into omphalotin. The molecular mechanism for OphMA was elucidated by mutagenesis, structural, biochemical and theoretical studies. This review highlights current progress in peptide N-methylating enzymes.

Sperm cryopreservation is a tool for the conservation of the genetic material of animals of genetic importance or for species preservation. In the case of domestic cats, this can be used to generate information about seminal harvest, evaluation and preservation, which is especially important due to its applicability to wild felids. This study evaluated seminal samples harvested by urethral catheterisation from 13 adult domestic cats. Samples were cryopreserved with experimental groups of extenders were defined by the penetrating cryoprotectant: 6% glycerol (GLY6%), 3% dimethylacetamide (DMA3%) and 3% dimethylformamide (DMF3%). The samples were thawed and evaluated by conventional microscopy and by computer-assisted sperm analysis (CASA). The structural and functional membrane integrity was assessed by supravital tests (EOS), hypoosmotic swelling tests (HOST) and flow cytometry (FC). There was a correlation (P 0.05) between total motility and EOS (r = 0.54), HOST and FC (r = -0.62) and total motility and flow cytometry (r = 0.63), indicating that these are complementary parameters that increase the accuracy of the feline sperm quality evaluation post-thaw. The results regarding the structural and functional integrity of the sperm plasma membrane did not differ (P 0.05) among groups. However, the DMA3% group had a lower (P 0.05) percentage of morphological changes in the sperm tail compared to samples cryopreserved with GLY6% and DMF3%. Additionally, DMA3% provided lower values of immobile sperm post-thaw when compared to DMF3%. DMA is an interesting alternative to GLY and superior to DMF for the cryopreservation of feline semen at the studied concentrations.

Proteins incorporating artificial moieties such as fluorophores and drugs have enjoyed increasing use in chemical biology and drug development research. Preparation of such artificial protein derivatives has relied mainly on native chemical ligation in which peptide/protein thioesters chemoselectively react with N-terminal cysteine (Cys) peptides to afford protein molecules. The protein thioesters derived from expressed proteins represent thioesters that are very useful for the preparation of artificial proteins by native chemical ligation with synthetic peptides with N-terminal Cys. We recently have developed a traceless thioester-producing protocol using carboxypeptidase Y (CPaseY) which is compatible with an expressed protein. The traceless character is ensured by CPaseY-mediated hydrazinolysis of C-terminal Xaa (X)-Cys-proline (Pro)-leucine (Leu)-OH sequence followed by an auto-processing of the Cys-Pro (CP) dipeptide unit, affording the corresponding X-thioester (X-SR). However, hydrazinolysis of the amide bond in the prolyl leucine junction depends significantly on the nature of X. In the case of hydrophobic X residues, the hydrazinolysis overreacts to give several hydrazides while the reaction of hydrophilic X residues proceeds slowly. In this research, we attempted to develop an X-independent CPaseY-mediated protocol and found that the incorporation of a triple CP sequence into the C-terminal end (X-(CP)3-Leu-OH) allows for efficient X-SR formation in a manner that is independent of X.

COVID-19 respiratory disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global health issue since it emerged in December 2019. While great global efforts are underway to develop vaccines and to discover or repurpose therapeutic agents for this disease, as of this writing only the nucleoside drug remdesivir has been approved under Emergency Use Authorization to treat COVID-19. The RNA-dependent RNA polymerase (RdRP), a viral enzyme for viral RNA replication in host cells, is one of the most intriguing and promising drug targets for SARS-CoV-2 drug development. Because RdRP is a viral enzyme with no host cell homologs, selective SARS-CoV-2 RdRP inhibitors can be developed that have improved potency and fewer off-target effects against human host proteins and thus are safer and more effective therapeutics for treating COVID-19. This review focuses on biochemical enzyme and cell-based assays for RdRPs that could be used in high-throughput screening to discover new and repurposed drugs against SARS-CoV-2.

General anesthetics interfere with dendritic development and synaptogenesis, resulting in cognitive impairment in the developing animals. RhoA signal pathway plays important roles in dendritic development by regulating cytoskeleton protein such as tubulin and actin. However, it's not clear whether RhoA pathway is involved in inhaled general anesthetics sevoflurane-induced synaptic development abnormalities and long-term cognitive dysfunction. Rats at postnatal day 7 (PND7) were injected intraperitoneally with RhoA pathway inhibitor Y27632 or saline 20 min before exposed to 2.8% sevoflurane for 4 h. The apoptosis-related proteins and RhoA/CRMP2 pathway proteins in the hippocampus were measured 6 h after sevoflurane exposure. Cognitive functions were evaluated by the open field test on PND25 rats and contextual fear conditioning test on PND32-33 rats. The dendritic morphology and density of dendritic spines in the pyramidal neurons of hippocampus were determined by Golgi staining and the synaptic plasticity-related proteins were also measured on PND33 rats. Long term potentiation (LTP) from hippocampal slices was recorded on PND34-37 rats. Sevoflurane induced caspase-3 activation, decreased the ratio of Bcl-2/Bax and increased TUNEL-positive neurons in hippocampus of PND7 rats, which were attenuated by inhibition of RhoA. However, sevoflurane had no significant effects on activity of RhoA/CRMP2 pathway. Sevoflurane disturbed dendritic morphogenesis, reduced the number of dendritic spines, decreased proteins expression of PSD-95, drebrin and synaptophysin, inhibited LTP in hippocampal slices and impaired memory ability in the adolescent rats, while inhibition of RhoA activity did not rescue the changes above induced by sevoflurane. RhoA signal pathway did not participate in sevoflurane-induced dendritic and synaptic development abnormalities and cognitive dysfunction in developing rats.

HIV has become a chronic disease despite the effective use of anti-retroviral therapy (ART). However, the mechanisms of tissue colonization, viral evolution, generation of viral reservoirs, and compartmentalization are still a matter of debate, and most of the underlying mechanisms behind these are still not well described due to the challenges involved in examining early events of infection at the cellular and molecular level. Thus, there is still an urgent need to explore these areas to develop effective HIV-cure strategies. Here, we describe the early events of tissue colonization and compartmentalization as well as the role of tunneling nanotubes (TNTs)-like structures (TLS) during viral spread in the presence and absence of effective anti-retroviral treatment. To examine these mechanisms, NOD/SCID IL2 RG−/− (NSG) humanized mice were either directly infected with HIV(ADA) or with low numbers of HIV(ADA)-infected leukocytes to limit tissue colonization in the presence and absence of TAK779, an effective CCR5 blocker of HIV-entry. We identify that viral seeding in tissues occurs early in a tissue and cell type-specific manner (24 to 72 h). Reduction in systemic HIV-replication by TAK779 treatment did not affect tissue seeding or spreading, despite reduced systemic viral replication. Tissue associated HIV-infected cells had different properties than cells in the circulation because the virus continues to spread in tissues in a TLS dependent manner, despite ART. Thus, understanding these mechanisms can provide new approaches to enhance the efficacy of existing ART and HIV-infection cure strategy.

Immune potentiators, termed adjuvant, trigger early innate immune responses to ensure the generation of robust and long‐lasting adaptive immune responses of vaccines. Here we present study that takes advantage of a self‐assembling small molecule library for the development of a novel vaccine adjuvant. Cell‐based screening of the library and subsequent structural optimization led to the discovery of a simple, chemically tractable deoxycholate derivative (molecule 6 , also named cholicamide) whose well‐defined nano‐assembly potently elicits innate immune responses in macrophages and dendritic cells. Functional and mechanistic analyses indicate that the virus‐like assembly is engulfed inside cells and stimulates the innate immune response through toll‐like receptor 7 (TLR7), an endosomal TLR that detects single‐stranded viral RNA. As an influenza vaccine adjuvant in mice, molecule 6 was as potent as Alum, a clinically used adjuvant. The studies described here paves the way for a new approach to discovering and designing self‐assembling small‐molecule adjuvants against pathogens, including emerging viruses., 自己集合性ワクチンアジュバントの発見. 京都大学プレスリリース. 2020-10-07., Vaccine ingredients could be hiding in small molecule libraries. 京都大学プレスリリース. 2020-10-07.

BACKGROUND Study 4030 was a phase 3, randomized, double-blinded study of 565 HIV-1 RNA-suppressed participants switching to bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) or dolutegravir (DTG)+F/TAF. Nucleoside reverse transcriptase inhibitor (NRTI), non-NRTI, and protease inhibitor resistance (-R) was allowed, but integrase strand transfer inhibitor-R was excluded. Here, we describe the detailed resistance analysis. METHODS Historical plasma HIV-1 RNA genotypes and baseline proviral DNA genotypes were analyzed. Documented or investigator-suspected NRTI-R was grouped for stratification into 3 categories of level of resistance. Viral blips were assessed through week 48. Virologic failures had genotypic and phenotypic resistance analyses at week 48, confirmed failure, or last visit, if HIV-1 RNA did not resuppress to

Expanding the repertoire of electrophiles with unique reactivity features would facilitate the development of covalent inhibitors with desirable reactivity profiles. We herein introduce bicyclo[1.1.0]butane (BCB) carboxylic amide as a new class of thiol-reactive electrophiles for selective and irreversible inhibition of targeted proteins. We first streamlined the synthetic routes to generate a variety of BCB amides. The strain-driven nucleophilic addition to BCB amides proceeded chemoselectively with cysteine thiols under neutral aqueous conditions, the rate of which was significantly slower than that of acrylamide. This reactivity profile of BCB amide was successfully exploited to develop covalent ligands targeting Bruton's tyrosine kinase (BTK). By tuning BCB amide reactivity and optimizing its disposition on the ligand, we obtained a selective covalent inhibitor of BTK. The in-gel activity-based protein profiling and mass spectrometry-based chemical proteomics revealed that the selected BCB amide had a higher target selectivity for BTK in human cells than did a Michael acceptor probe. Further chemical proteomic study revealed that BTK probes bearing different classes of electrophiles exhibited distinct off-target profiles. This result suggests that incorporation of BCB amide as a cysteine-directed electrophile could expand the capability to develop covalent inhibitors with the desired proteome reactivity profile.

Interaction of cancer cells with cancer‐associated fibroblasts (CAFs) plays critical roles in tumor progression. Recently we proposed a new tumor invasion mechanism in which invasive cancer cells individually migrate on elongate protrusions of CAFs (CAF fibers) in 3‐D collagen matrix. In this mechanism, cancer cells interact with fibronectin fibrils assembled on CAFs mainly through integrin‐α5β1. Here we tested whether this mechanism is applicable to the collective invasion of cancer cells, using two E‐cadherin‐expressing adenocarcinoma cell lines, DLD‐1 (colon) and MCF‐7 (breast). When hybrid spheroids of DLD‐1 cells with CAFs were embedded into collagen gel, DLD‐1 cells collectively but very slowly migrated through the collagen matrix in contact with CAFs. Epidermal growth factor and tumor necrosis factor‐α promoted the collective invasion, possibly by reducing the E‐cadherin junction, as did the transforming growth factor‐β inhibitor SB431542 by stimulating the outgrowth of CAFs. Transforming growth factor‐β itself inhibited the cancer cell invasion. Efficient collective invasion of DLD‐1 cells required large CAF fibers or their assembly as stable adhesion substrates. Experiments with function‐blocking Abs and siRNAs confirmed that DLD‐1 cells adhered to fibronectin fibrils on CAFs mainly through integrin‐α5β1. Anti‐E‐cadherin Ab promoted the single cell invasion of DLD‐1 cells by dissociating the E‐cadherin junction. Although the binding affinity of MCF‐7 cells to CAFs was lower than DLD‐1, they also collectively invaded the collagen matrix in a similar fashion to DLD‐1 cells. Our results suggest that the direct interaction with CAFs, as well as environmental cytokines, contributes to the collective invasion of cancers., This study established a simple collective cancer invasion model in which E‐cadherin‐expressing cancer cells collectively invaded the collagen matrix in contact with bundles of cancer‐associated fibroblast fibers through integrin‐α5β1/fibronectin interaction. The results suggest that the fibrosis and environmental cytokines contribute to the collective invasion of cancers in vivo.