Ustilaginoidea virens, an emerging pathogen of rice: the dynamic interplay between the pathogen virulence strategies and host defense.

MAIN CONCLUSION: The Ustilaginoidea virens -rice pathosystem has been used as a model for flower-infecting fungal pathogens. The molecular biology of the interactions between U. virens and rice, with an emphasis on the attempt to get a deeper comprehension of the false smut fungus's genomes, proteome, host range, and pathogen biology, has been investigated. Meta-QTL analysis was performed to identify potential QTL hotspots for use in marker-assisted breeding. The Rice False Smut (RFS) caused by the fungus Ustilaginoidea virens currently threatens rice cultivators across the globe. RFS infects rice panicles, causing a significant reduction in grain yield. U. virens can also parasitize other hosts though they play only a minor role in its life cycle. Furthermore, because it produces mycotoxins in edible rice grains, it puts both humans and animals at risk of health problems. Although fungicides are used to control the disease, some fungicides have enabled the pathogen to develop resistance, making its management challenging. Several QTLs have been reported but stable gene(s) that confer RFS resistance have not been discovered yet. This review offers a comprehensive overview of the pathogen, its virulence mechanisms, the genome and proteome of U. virens, and its molecular interactions with rice. In addition, information has been compiled on reported resistance QTLs, facilitating the development of a consensus genetic map using meta-QTL analysis for identifying potential QTL hotspots. Finally, this review highlights current developments and trends in U. virens-rice pathosystem research while identifying opportunities for future investigations.

Allosteric modulation of α1ß3γ2 GABAA receptors by farnesol through the neurosteroid sites.

In mammalian cells, all-trans farnesol, a 15-carbon isoprenol, is a product of the mevalonate pathway. It is the natural substrate of alcohol dehydrogenase and a substrate for CYP2E1, two enzymes implicated in ethanol metabolism. Studies have shown that farnesol is present in the human brain and inhibits voltage-gated Ca2+ channels at much lower concentrations than ethanol. Here we show that farnesol modulates the activity of γ-aminobutyric acid type A receptors (GABAARs), some of which also mediate the sedative activity of ethanol. Electrophysiology experiments performed in HEK cells expressing human α1ß3γ2 or α6ß3γ2 GABAARs revealed that farnesol increased chloride currents through positive allosteric modulation of these receptors and showed dependence on both the alcoholic functional group of farnesol and the length of the alkyl chain for activity. In silico studies using long-timescale unbiased all-atom molecular dynamics (MD) simulations of the human α1ß3γ2 GABAA receptors revealed that farnesol modulates the channel by directly binding to the transmembrane neurosteroid-binding site, after partitioning into the surrounding membrane and reaching the receptor by lateral diffusion. Channel activation by farnesol was further characterized by several structural and dynamic variables, such as global twisting of the receptor's extracellular domain, tilting of the transmembrane M2 helices, radius, cross-sectional area, hydration status, and electrostatic potential of the channel pore. Our results expand the pharmacological activities of farnesol to yet another class of ion channels implicated in neurotransmission, thus providing a novel path for understanding and treatment of diseases involving GABAA receptor dysfunction.

Membrane-Facilitated Receptor Access and Binding Mechanisms of Long-Acting ß2-Adrenergic Receptor Agonists.

The drugs salmeterol, formoterol, and salbutamol constitute the frontline treatment of asthma and other chronic pulmonary diseases. These drugs activate the ß2-adrenergic receptors (ß2-AR), a class A G protein-coupled receptor (GPCR), and differ significantly in their clinical onset and duration of actions. According to the microkinetic model, the long duration of action of salmeterol and formoterol compared with salbutamol were attributed, at least in part, to their high lipophilicity and increased local concentrations in the membrane near the receptor. However, the structural and molecular bases of how the lipophilic drugs reach the binding site of the receptor from the surrounding membrane remain unknown. Using a variety of classic and enhanced molecular dynamics simulation techniques, we investigated the membrane partitioning characteristics, binding, and unbinding mechanisms of the ligands. The obtained results offer remarkable insight into the functional role of membrane lipids in the ligand association process. Strikingly, salmeterol entered the binding site from the bilayer through transmembrane helices 1 and 7. The entry was preceded by membrane-facilitated rearrangement and presentation of its phenyl-alkoxy-alkyl tail as a passkey to an access route gated by F193, a residue known to be critical for salmeterol's affinity. Formoterol's access is through the aqueous path shared by other ß2-AR agents. We observed a novel secondary path for salbutamol that is distinct from its primary route. Our study offers a mechanistic description for the membrane-facilitated access and binding of ligands to a membrane protein and establishes a groundwork for recognizing membrane lipids as an integral component in the molecular recognition process. SIGNIFICANCE STATEMENT: The cell membrane's functional role behind the duration of action of long-acting ß2-adrenergic receptor (ß2-AR) agonists such as salmeterol has been a subject of debate for a long time. This study investigated the binding and unbinding mechanisms of the three commonly used ß2-AR agonists, salmeterol, formoterol, and salbutamol, using advanced simulation techniques. The obtained results offer unprecedented insights into the active role of membrane lipids in facilitating access and binding of the ligands, affecting the molecular recognition process and thus their pharmacology.

Mechanisms of Herb-Drug Interactions Involving Cinnamon and CYP2A6: Focus on Time-Dependent Inhibition by Cinnamaldehyde and 2-Methoxycinnamaldehyde.

Information is scarce regarding pharmacokinetic-based herb-drug interactions (HDI) with trans-cinnamaldehyde (CA) and 2-methoxycinnamaldehyde (MCA), components of cinnamon. Given the presence of cinnamon in food and herbal treatments for various diseases, HDIs involving the CYP2A6 substrates nicotine and letrozole with MCA (KS = 1.58 µM; Hill slope = 1.16) and CA were investigated. The time-dependent inhibition (TDI) by MCA and CA of CYP2A6-mediated nicotine metabolism is a complex process involving multiple mechanisms. Molecular dynamic simulations showed that CYP2A6's active site accommodates two dynamic ligands. The preferred binding orientations for MCA and CA were consistent with the observed metabolism: epoxidation, O-demethylation, and aromatic hydroxylation of MCA and cinnamic acid formation from CA. The percent remaining activity plots for TDI by MCA and CA were curved, and they were analyzed with a numerical method using models of varying complexity. The best-fit models support multiple inactivator binding, inhibitor depletion, and partial inactivation. Deconvoluted mass spectra indicated that MCA and CA modified CYP2A6 apoprotein with mass additions of 156.79 (142.54-171.04) and 132.67 (123.37-141.98), respectively, and it was unaffected by glutathione. Heme degradation was observed in the presence of MCA (48.5% ± 13.4% loss; detected by liquid chromatography-tandem mass spectrometry). In the absence of clinical data, HDI predictions were made for nicotine and letrozole using inhibition parameters from the best-fit TDI models and parameters scaled from rats. Predicted area under the concentration-time curve fold changes were 4.29 (CA-nicotine), 4.92 (CA-letrozole), 4.35 (MCA-nicotine), and 5.00 (MCA-letrozole). These findings suggest that extensive exposure to cinnamon (corresponding to ≈ 275 mg CA) would lead to noteworthy interactions. SIGNIFICANCE STATEMENT: Human exposure to cinnamon is common because of its presence in food and cinnamon-based herbal treatments. Little is known about the risk for cinnamaldehyde and methoxycinnamaldehyde, two components of cinnamon, to interact with drugs that are eliminated by CYP2A6-mediated metabolism. The interactions with CYP2A6 are complex, involving multiple-ligand binding, time-dependent inhibition of nicotine metabolism, heme degradation, and apoprotein modification. An herb-drug interaction prediction suggests that extensive exposure to cinnamon would lead to noteworthy interactions with nicotine.

Molecular Dynamics Simulations Provide Insight into the Loading Efficiency of Proresolving Lipid Mediators Resolvin D1 and D2 in Cell Membrane-Derived Nanovesicles.

Resolvins D1 and D2 (RvDs) are structural isomers and metabolites of docosahexaenoic acid, an omega-3 fatty acid, enzymatically produced in our body in response to acute inflammation or microbial invasion. Resolvins have been shown to play an essential role in the resolution of inflammation, tissue repair, and return to homeostasis and thus are actively pursued as potential therapeutics in treating inflammatory disorders and infectious diseases. However, effective in vivo delivery of RvDs continues to be a challenging task. Recent studies demonstrated that RvD1 or RvD2 loaded in cell membrane-derived nanovesicles significantly increased therapeutic efficacy in treating murine peritonitis and ischemic stroke, respectively. The mechanistic details of how the subtle structural difference between RvD1 and RvD2 alters their molecular interactions with the membrane lipids of the nanovesicles and thus affects the loading efficiency remain unknown. Here, we report the encapsulation profiles of the neutral and ionized species of both RvD1 and RvD2 determined with the cell membrane-derived nanovesicles at pH values 5.4 and 7.4, respectively. Also, we performed microsecond time-scale all-atom molecular dynamics (MD) simulations in explicit water to elucidate the molecular interactions of both neutral and ionized species of RvD1 and RvD2 with the lipid bilayer using a model membrane system, containing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol. We found that the differences in the position and chirality of hydroxyl groups in RvD1 and RvD2 affected their location, orientation, and conformations within the bilayer. Surprisingly, the deprotonation of their carboxyl group caused their orientation and conformation to change from a fully extended one that is oriented in parallel to the membrane plane to a J-shaped bent conformation that is oriented perpendicular to the bilayer plane. Our studies offer valuable insight into the molecular interactions of RvD1/D2 with the lipid bilayer in atomistic details and provide a mechanistic explanation for the observed differences in the encapsulation profiles of RvD1 and RvD2, which may facilitate the rational design of nanovesicle-based therapeutics for treating inflammatory diseases.

Does the Lipid Bilayer Orchestrate Access and Binding of Ligands to Transmembrane Orthosteric/Allosteric Sites of G Protein-Coupled Receptors?

The ligand-binding sites of many G protein-coupled receptors (GPCRs) are situated around and deeply embedded within the central pocket formed by their seven transmembrane-spanning α-helical domains. Generally, these binding sites are assumed accessible to endogenous ligands from the aqueous phase. Recent advances in the structural biology of GPCRs, along with biophysical and computational studies, suggest that amphiphilic and lipophilic molecules may gain access to these receptors by first partitioning into the membrane and then reaching the binding site via lateral diffusion through the lipid bilayer. In addition, several crystal structures of class A and class B GPCRs bound to their ligands offer unprecedented details on the existence of lipid-facing allosteric binding sites outside the transmembrane helices that can only be reached via lipid pathways. The highly organized structure of the lipid bilayer may direct lipophilic or amphiphilic drugs to a specific depth within the bilayer, changing local concentration of the drug near the binding site and affecting its binding kinetics. Additionally, the constraints of the lipid bilayer, including its composition and biophysical properties, may play a critical role in "pre-organizing" ligand molecules in an optimal orientation and conformation to facilitate receptor binding. Despite its clear involvement in molecular recognition processes, the critical role of the membrane in binding ligands to lipid-exposed transmembrane binding sites remains poorly understood and warrants comprehensive investigation. Understanding the mechanistic basis of the structure-membrane interaction relationship of drugs will not only provide useful insights about receptor binding kinetics but will also enhance our ability to take advantage of the apparent membrane contributions when designing drugs that target transmembrane proteins with improved efficacy and safety. In this minireview, we summarize recent structural and computational studies on membrane contributions to binding processes, elucidating both lipid pathways of ligand access and binding mechanisms for several orthosteric and allosteric ligands of class A and class B GPCRs.

A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine.

Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causes hemorrhagic fever with a high fatality rate. Viral protein 40 (VP40) is the major EBOV matrix protein and regulates viral budding from the plasma membrane. VP40 is a transformer/morpheein that can structurally rearrange its native homodimer into either a hexameric filament that facilitates viral budding or an RNA-binding octameric ring that regulates viral transcription. VP40 associates with plasma-membrane lipids such as phosphatidylserine (PS), and this association is critical to budding from the host cell. However, it is poorly understood how different VP40 structures interact with PS, what essential residues are involved in this association, and whether VP40 has true selectivity for PS among different glycerophospholipid headgroups. In this study, we used lipid-binding assays, MD simulations, and cellular imaging to investigate the molecular basis of VP40-PS interactions and to determine whether different VP40 structures (i.e. monomer, dimer, and octamer) can interact with PS-containing membranes. Results from quantitative analysis indicated that VP40 associates with PS vesicles via a cationic patch in the C-terminal domain (Lys224, 225 and Lys274, 275). Substitutions of these residues with alanine reduced PS-vesicle binding by >40-fold and abrogated VP40 localization to the plasma membrane. Dimeric VP40 had 2-fold greater affinity for PS-containing membranes than the monomer, whereas binding of the VP40 octameric ring was reduced by nearly 10-fold. Taken together, these results suggest the different VP40 structures known to form in the viral life cycle harbor different affinities for PS-containing membranes.

Impact of sunflower seed oil versus mustard seed oil on skin barrier function in newborns: a community-based, cluster-randomized trial.

BACKGROUND: Natural vegetable oils are widely used for newborn massage in many low resource settings. Animal models indicated that sunflower seed oil (SSO) can accelerate skin barrier recovery following damage, while other oils, including mustard oil (MO), may cause further skin barrier damage. The objective was to compare the effects of two SSO and MO used for routine massage on skin integrity in premature and full-term neonates. METHODS: This community-based cluster randomized controlled trial included 995 neonates assigned to full body massage with sunflower seed oil (SSO, intervention) or mustard seed oil (MO, standard practice) from July 2012-May 2014 in Sarlahi, Nepal. Skin integrity measures were evaluated over 28 days, including skin condition (erythema, rash, dryness), skin surface pH, stratum corneum (SC) cohesion/protein concentration, and transepidermal water loss (TEWL). Overall means and rates of change in these skin measures were compared between oil groups using bivariate random-effects models. RESULTS: 500 and 495 live born neonates received repeated massage with MO and SSO, respectively. Skin pH decreased more quickly for SSO than MO in the first week of life, with a difference in mean daily reductions of 0.02 (95% CI: 0.002-0.040). Erythema, rash and dryness increased (worsened) over days 1-14 then decreased by day 28, with no significant oil group differences. TEWL increased over time, with no significant oil group differences. Gestational age did not modify the effect; the slightly faster decrease in skin pH among SSO infants was similar in magnitude between term and preterm infants. CONCLUSIONS: Oil type may contribute to differences in skin integrity when neonates are massaged regularly. The more rapid acid mantle development observed for SSO may be protective for neonates in lower resource settings. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01177111); registered August 6th, 2010.

Prevalence and associated risk factors of Giardia duodenalis infection among school-going children in Nepal.

This study aimed to determine the prevalence of intestinal parasites and its associated risk factors among school-going children in Kathmandu, Nepal. Between August and September 2016, a total of 333 stool samples were collected from children at five public schools. The collected samples were subjected to formol-ether concentration, followed by conventional microscopic examination for intestinal parasites. The overall prevalence of intestinal parasites was 24.3% (81/333), with Giardia spp. showing the highest prevalence of 18.9% (63/333). Samples positive for Giardia spp. by microscopy were further subjected to quantitative polymerase chain reaction (qPCR) for G. duodenalis, resulting in a positive ratio of 100%. The positive ratio of Giardia spp. was considerably high among children consuming tanker water (27.3%), jar water (21.0%), and tap water (17.5%). Our results demonstrated that G. duodenalis remains predominant in school-going children in Nepal.

Prevalence and genotypic diversity of Entamoeba species in inhabitants in Kathmandu, Nepal.

In Nepal, gastrointestinal infections due to parasites including Entamoeba species are common. The main aim of this study was to identify species of Entamoeba using genotypic analysis. The prevalence of Entamoeba infections was examined by PCR in fecal samples from 143 inhabitants living close to wild rhesus macaques in Kathmandu, Nepal. The numbers of positive cases were one (0.7%) for E. histolytica, eight (5.6%) for E. dispar, seven (4.9%) for E. coli, and two (1.4%) for E. chattoni (E. polecki ST2). No infections with E. nuttalli, E. moshkovskii, and E. polecki ST1 were found. In E. dispar, at least seven different genotypes were detected from the eight samples by sequence analysis of tRNA-linked short tandem repeats. Different genotypes were found even in a couple from the same family. This is the first report demonstrating that E. dispar with high genotypic diversity is prevalent, rather than E. histolytica, in Kathmandu, and that zoonotic transmission of E. chattoni from rhesus macaques might occur in the inhabitants.

Etiology of Severe Acute Watery Diarrhea in Children in the Global Rotavirus Surveillance Network Using Quantitative Polymerase Chain Reaction.

Background: The etiology of acute watery diarrhea remains poorly characterized, particularly after rotavirus vaccine introduction. Methods: We performed quantitative polymerase chain reaction for multiple enteropathogens on 878 acute watery diarrheal stools sampled from 14643 episodes captured by surveillance of children <5 years of age during 2013-2014 from 16 countries. We used previously developed models of the association between pathogen quantity and diarrhea to calculate pathogen-specific weighted attributable fractions (AFs). Results: Rotavirus remained the leading etiology (overall weighted AF, 40.3% [95% confidence interval {CI}, 37.6%-44.3%]), though the AF was substantially lower in the Americas (AF, 12.2 [95% CI, 8.9-15.6]), based on samples from a country with universal rotavirus vaccination. Norovirus GII (AF, 6.2 [95% CI, 2.8-9.2]), Cryptosporidium (AF, 5.8 [95% CI, 4.0-7.6]), Shigella (AF, 4.7 [95% CI, 2.8-6.9]), heat-stable enterotoxin-producing Escherichia coli (ST-ETEC) (AF, 4.2 [95% CI, 2.0-6.1]), and adenovirus 40/41 (AF, 4.2 [95% CI, 2.9-5.5]) were also important. In the Africa Region, the rotavirus AF declined from 54.8% (95% CI, 48.3%-61.5%) in rotavirus vaccine age-ineligible children to 20.0% (95% CI, 12.4%-30.4%) in age-eligible children. Conclusions: Rotavirus remained the leading etiology of acute watery diarrhea despite a clear impact of rotavirus vaccine introduction. Norovirus GII, Cryptosporidium, Shigella, ST-ETEC, and adenovirus 40/41 were also important. Prospective surveillance can help identify priorities for further reducing the burden of diarrhea.

Membrane association and localization dynamics of the Ebola virus matrix protein VP40.

The Ebola virus matrix protein VP40 is a major structural protein that provides the scaffolding for new Ebola virus particles. For this, VP40 is first trafficked to the lower leaflet of the plasma membrane (PM) in its dimeric form. Once associated with the PM, the VP40 dimers undergo structural rearrangements and oligomerize into hexamers and filaments that make up the virus matrix. Therefore, association of the VP40 dimers and their stabilization at the PM is a crucial step in the Ebola life-cycle. To understand the molecular details of the VP40 dimer-PM interactions, we investigated the dimer association with the inner leaflet of the PM using detailed all-atom molecular dynamics (MD) simulations. The formation of the dimer-PM complex is facilitated by the interactions of the VP40 lysine residues and the anionic lipids POPS, POPI, and PIP2 in the PM. In contrast, the dimer fails to associate with a membrane without POPS, POPI, or PIP2 lipids. We explored the mechanisms of the association and identified important residues and lipids involved in localization and stabilization of VP40 dimers at the PM. MD simulations elucidate the role of a C-terminal α-helix alignment parallel to the lipid bilayer surface as well as the creation of membrane defects that allow partial insertion of the hydrophobic residue V276 into the membrane to further stabilize the VP40 dimer-PM complex. Understanding the mechanisms of the VP40 dimer-PM association that facilitate oligomerization can be important for potentially targeting the VP40 for small molecules that can interfere with the virus life-cycle.

PER-8, a Novel Extended-Spectrum ß-Lactamase PER Variant, from an Acinetobacter baumannii Clinical Isolate in Nepal.

A novel PER-type extended-spectrum ß-lactamase, PER-8, was identified in an Acinetobacter baumannii clinical isolate obtained in Nepal. The amino acid sequence of PER-8 has a substitution at position 39 (Gly to Glu) compared with that of PER-7. The kcat/Km ratio of PER-8 for aztreonam was lower than that of PER-7, while the kcat/Km ratio of PER-8 for imipenem was higher than that of PER-7. The genomic environment surrounding blaPER-8 was intI1 blaPSE-1qacEDI sulI ISCR1-blaPER-8gts sulI orfX on a 100-kb plasmid.

Pseudomonas aeruginosa Clinical Isolates in Nepal Coproducing Metallo-ß-Lactamases and 16S rRNA Methyltransferases.

A total of 11 multidrug-resistant Pseudomonas aeruginosa clinical isolates were obtained in Nepal. Four of these isolates harbored genes encoding one or more carbapenemases (DIM-1, NDM-1, and/or VIM-2), and five harbored genes encoding a 16S rRNA methyltransferase (RmtB4 or RmtF2). A novel RmtF variant, RmtF2, had a substitution (K65E) compared with the same gene in RmtF. To our knowledge, this is the first report describing carbapenemase- and 16S rRNA methyltransferase-coproducing P. aeruginosa clinical isolates in Nepal.

Emergence of Various NDM-Type-Metallo-ß-Lactamase-Producing Escherichia coli Clinical Isolates in Nepal.

Of 250 clinical isolates of Escherichia coli obtained in Nepal, 38 were carbapenem resistant, with MICs of imipenem or meropenem of ≥4 µg/ml. All 38 isolates harbored the following blaNDMs: blaNDM-1, blaNDM-3, blaNDM-4, blaNDM-5, blaNDM-7, blaNDM-12, and blaNDM-13 Most of these isolates also harbored the 16S rRNA methylase gene(s) armA, rmtB, and/or rmtC.

Graphene-VP40 interactions and potential disruption of the Ebola virus matrix filaments.

Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic.

The Ebola virus protein VP40 hexamer enhances the clustering of PI(4,5)P2 lipids in the plasma membrane.

The Ebola virus is a lipid-enveloped virus that obtains its lipid coat from the plasma membrane of the host cell it infects during the budding process. The Ebola virus protein VP40 localizes to the inner leaflet of the plasma membrane and forms the viral matrix, which provides the major structure for the Ebola virus particles. VP40 is initially a dimer that rearranges to a hexameric structure that mediates budding. VP40 hexamers and larger filaments have been shown to be stabilized by PI(4,5)P2 in the plasma membrane inner leaflet. Reduction in the plasma membrane levels of PI(4,5)P2 significantly reduce formation of VP40 oligomers and virus-like particles. We investigated the lipid-protein interactions in VP40 hexamers at the plasma membrane. We quantified lipid-lipid self-clustering by calculating the fractional interaction matrix and found that the VP40 hexamer significantly enhances the PI(4,5)P2 clustering. The radial pair distribution functions suggest a strong interaction between PI(4,5)P2 and the VP40 hexamer. The cationic Lys side chains are found to mediate the PIP2 clustering around the protein, with cholesterol filling the space between the interacting PIP2 molecules. These computational studies support recent experimental data and provide new insights into the mechanisms by which VP40 assembles at the plasma membrane inner leaflet, alters membrane curvature, and forms new virus-like particles.

Clinical epidemiology and molecular analysis of extended-spectrum-ß-lactamase-producing Escherichia coli in Nepal: characteristics of sequence types 131 and 648.

Recently, CTX-M-type extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli strains have emerged worldwide. In particular, E. coli with O antigen type 25 (O25) and sequence type 131 (ST131), which is often associated with the CTX-M-15 ESBL, has been increasingly reported globally; however, epidemiology reports on ESBL-producing E. coli in Asia are limited. Patients with clinical isolates of ESBL-producing E. coli in the Tribhuvan University teaching hospital in Kathmandu, Nepal, were included in this study. Whole-genome sequencing of the isolates was conducted to analyze multilocus sequence types, phylotypes, virulence genotypes, O25b-ST131 clones, and distribution of acquired drug resistance genes. During the study period, 105 patients with ESBL-producing E. coli isolation were identified, and the majority (90%) of these isolates were CTX-M-15 positive. The most dominant ST was ST131 (n = 54; 51.4%), followed by ST648 (n = 15; 14.3%). All ST131 isolates were identified as O25b-ST131 clones, subclone H30-Rx. Three ST groups (ST131, ST648, and non-ST131/648) were compared in further analyses. ST648 isolates had a proportionally higher resistance to non-ß-lactam antibiotics and featured drug-resistant genes more frequently than ST131 or non-ST131/648 isolates. ST131 possessed the most virulence genes, followed by ST648. The clinical characteristics were similar among groups. More than 38% of ESBL-producing E. coli isolates were from the outpatient clinic, and pregnant patients comprised 24% of ESBL-producing E. coli cases. We revealed that the high resistance of ESBL-producing E. coli to multiple classes of antibiotics in Nepal is driven mainly by CTX-M-producing ST131 and ST648. Their immense prevalence in the communities is a matter of great concern.

Correlation between genotypes of tRNA-linked short tandem repeats in Entamoeba nuttalli isolates and the geographical distribution of host rhesus macaques.

Several polymorphic markers, including serine-rich protein genes, have been used for the genotyping of isolates from the morphologically indistinguishable protozoan parasites Entamoeba histolytica, Entamoeba dispar, and Entamoeba nuttalli. Genotypes of tRNA-linked short tandem repeats (STRs) are highly polymorphic, but the correlation with geographical distribution is unknown. We have recently isolated 15 E. nuttalli strains from wild rhesus macaques in four locations in Kathmandu Valley, Nepal. The sequences of the serine-rich protein genes of the E. nuttalli strains differed among the four locations. In this study, we analyzed tRNA-linked STRs in six loci of the 15 strains. Two genotypes were found in loci N-K2, R-R, and S(TGA)-D, three in locus S-Q, and five in locus D-A. In locus A-L, one major genotype and ten minor genotypes were found, resulting in mixtures of two to six genotypes in eight strains. By combination of the main genotypes in the six loci, the 15 strains were divided into nine genotypes. The genotypes observed in E. nuttalli strains were quite different from those in E. histolytica and E. dispar. A phylogenetic tree constructed from tRNA-linked STRs in the six loci reflected the different places of isolation. These results suggest that sequence diversity of tRNA-linked STRs in E. nuttalli occurs with relatively high frequency and might be a marker of geographical distribution of host rhesus macaques, even in limited areas.

Application of Generative Artificial Intelligence in Predicting Membrane Partitioning of Drugs: Combining Denoising Diffusion Probabilistic Models and MD Simulations Reduces the Computational Cost to One-Third.

The optimal interaction of drugs with plasma membranes and membranes of subcellular organelles is a prerequisite for desirable pharmacology. Importantly, for drugs targeting the transmembrane lipid-facing sites of integral membrane proteins, the relative affinity of a drug to the bilayer lipids compared to the surrounding aqueous phase affects the partitioning, access, and binding of the drug to the target site. Molecular dynamics (MD) simulations, including enhanced sampling techniques such as steered MD, umbrella sampling (US), and metadynamics, offer valuable insights into the interactions of drugs with the membrane lipids and water in atomistic detail. However, these methods are computationally prohibitive for the high-throughput screening of drug candidates. This study shows that applying denoising diffusion probabilistic models (DDPMs), a generative AI method, to US simulation data reduces the computational cost significantly. Specifically, the models used only partial (one-third) data from the US simulations and reproduced the complete potential of mean force (PMF) profiles for three FDA-approved drugs (ß2-adrenergic agonists) and ∼20 biologically relevant chemicals with known experimentally characterized bilayer locations. Intriguingly, the model can predict the solvation-free energies for partitioning and crossing the bilayer, preferred bilayer locations (low-energy well), and orientations of the ligands with high accuracy. The results indicate that DDPMs can be used to characterize the complete membrane partitioning profile of drug molecules using fewer umbrella sampling simulations at select positions along the bilayer normal (z-axis), irrespective of their amphiphilic-lipophilic-cephalophilic characteristics.