Toward high-resolution modeling of small molecule-ion channel interactions.

Ion channels are critical drug targets for a range of pathologies, such as epilepsy, pain, itch, autoimmunity, and cardiac arrhythmias. To develop effective and safe therapeutics, it is necessary to design small molecules with high potency and selectivity for specific ion channel subtypes. There has been increasing implementation of structure-guided drug design for the development of small molecules targeting ion channels. We evaluated the performance of two RosettaLigand docking methods, RosettaLigand and GALigandDock, on the structures of known ligand-cation channel complexes. Ligands were docked to voltage-gated sodium (NaV), voltage-gated calcium (CaV), and transient receptor potential vanilloid (TRPV) channel families. For each test case, RosettaLigand and GALigandDock methods frequently sampled a ligand-binding pose within a root mean square deviation (RMSD) of 1-2 Å relative to the experimental ligand coordinates. However, RosettaLigand and GALigandDock scoring functions cannot consistently identify experimental ligand coordinates as top-scoring models. Our study reveals that the proper scoring criteria for RosettaLigand and GALigandDock modeling of ligand-ion channel complexes should be assessed on a case-by-case basis using sufficient ligand and receptor interface sampling, knowledge about state-specific interactions of the ion channel, and inherent receptor site flexibility that could influence ligand binding.

Enhanced effectiveness in preventing Nocardia seriolae infection utilizing heterologous prime-boost approach in orange-spotted grouper Epinephelus coioides.

Persistent nocardiosis has prompted exploration of the effectiveness of heterologous approaches to prevent severe infections. We have previously reported the efficacy of a nucleic acid vaccine in protecting groupers from highly virulent Nocardia seriolae infections. Ongoing research has involved the supplementation of recombinant cholesterol oxidase (rCho) proteins through immunization with a DNA vaccine to enhance the protective capacity of orange-spotted groupers. Recombinant rCho protein exhibited a maturity and biological structure comparable to that expressed in N. seriolae, as confirmed by Western blot immunodetection assays. The immune responses observed in vaccinated groupers were significantly higher than those observed in single-type homologous vaccinations, DNA or recombinant proteins alone (pcD:Cho and rCho/rCho), especially cell-mediated immune and mucosal immune responses. Moreover, the reduction in N. seriolae occurrence in internal organs, such as the head, kidney, and spleen, was consistent with the vaccine's efficacy, which increased from approximately 71.4 % to an undetermined higher percentage through heterologous vaccination strategies of 85.7 %. This study underscores the potential of Cho as a novel vaccine candidate and a heterologous approach for combating chronic infections such as nocardiosis.

ACLY alternative splicing correlates with cancer phenotypes.

ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA for protein acetylation. ACLY has two major splice isoforms: the full-length canonical "long" isoform and an uncharacterized "short" isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within an intrinsically disordered region and includes at least one dynamically phosphorylated residue. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the percent spliced in (PSI) of exon 14 is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types. This prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.

Clinical and Advocacy Implications of the Dobbs vs. Jackson Women's Health Organization Ruling on Trafficked Persons.

The United States Supreme Court decision on the case of Dobbs vs. Jackson Women's Health Organization abolished federal protections of abortion, leaving abortion legislation at the discretion of individual states. Trafficked persons are a population especially vulnerable to the impacts of this ruling. Because there is no existing literature describing the effects of restrictive abortion legislation on this group, we described some of the potential consequences of restrictive abortion laws for sex and labor trafficked persons, based on real case examples. We describe steps that should be taken to sufficiently protect and support pregnant trafficked women in relation to the Dobbs law.

Strategies for healthcare professionals to identify and assist migrant children at risk of labour exploitation or trafficking.

Increasingly large numbers of children and youth are migrating across international borders with many seeking employment in both formal and informal work sectors. These young people are at high risk of exploitation. Healthcare professionals need to be able to recognise vulnerable patients and advocate for their protection and safety, yet there is a paucity of literature that provides guidance on how to accomplish this. The goal of this paper is to provide guidance to clinicians on identifying and assisting migrant paediatric patients at risk of being exploited in the work sector, including conducting a risk assessment and making decisions about mandatory reporting. First, the best interest of the youth within their cultural context should be examined respecting their desires and goals, as well as immediate and longer-term physical health, mental health and safety issues. Second, clinicians should consider the best interest of the family, with attention to varying socioeconomic and psychosocial conditions including acculturation, immigration challenges, as well as cultural norms and values. Third, the situation must be evaluated within the legal framework of the host country regarding child labour, exploitation and trafficking. Cultural humility, open-mindedness, the active engagement of patients and families and an understanding of child labour within cultural contexts and legal statutes will empower healthcare professionals to identify and support patients at risk of exploitation in work settings. These recommendations serve to prioritise the best interests of vulnerable working migrant children and youth. The healthcare and migration systems of the USA will be used as a case for exploration.

Avian influenza A(H5) virus circulation in live bird markets in Vietnam, 2017-2022.

BACKGROUND: Highly pathogenic avian influenza A(H5) human infections are a global concern, with many A(H5) human cases detected in Vietnam, including a case in October 2022. Using avian influenza virus surveillance from March 2017-September 2022, we described the percent of pooled samples that were positive for avian influenza A, A(H5), A(H5N1), A(H5N6), and A(H5N8) viruses in live bird markets (LBMs) in Vietnam. METHODS: Monthly at each LBM, 30 poultry oropharyngeal swab specimens and five environmental samples were collected. Samples were pooled in groups of five and tested for influenza A, A(H5), A(H5N1), A(H5N6), and A(H5N8) viruses by real-time reverse-transcription polymerase chain reaction. Trends in the percent of pooled samples that were positive for avian influenza were summarized by LBM characteristics and time and compared with the number of passively detected avian influenza outbreaks using Spearman's rank correlation. RESULTS: A total of 25,774 pooled samples were collected through active surveillance at 167 LBMs in 24 provinces; 36.9% of pooled samples were positive for influenza A, 3.6% A(H5), 1.9% A(H5N1), 1.1% A(H5N6), and 0.2% A(H5N8). Influenza A(H5) viruses were identified January-December and at least once in 91.7% of sampled provinces. In 246 A(H5) outbreaks in poultry; 20.3% were influenza A(H5N1), 60.2% A(H5N6), and 19.5% A(H5N8); outbreaks did not correlate with active surveillance. CONCLUSIONS: In Vietnam, influenza A(H5) viruses were detected by active surveillance in LBMs year-round and in most provinces sampled. In addition to outbreak reporting, active surveillance for A(H5) viruses in settings with high potential for animal-to-human spillover can provide situational awareness.

An Integrated in silico and in vivo study of nucleic acid vaccine against Nocardia seriolae infection in orange-spotted grouper Epinephelus coioides.

Nocardiosis in aquatic animals caused by Nocardia seriolae is a frequently occurring serious infection that has recently spread to many countries. In this study, DNA vaccines containing potential bacterial antigens predicted using the reverse vaccinology approach were developed and evaluated in orange-spotted groupers. In silico analysis indicated that proteins including cholesterol oxidase, ld-transpeptidase, and glycosyl hydroxylase have high immunogenicity and are potential vaccine candidates. In vitro assays revealed the mature and biological configurations of these proteins. Importantly, when compared to a control PBS injection, N. seriolae DNA-based vaccines showed significantly higher expression of IL1ß, IL17, and IFNγ at 1 or 2 days, in line with higher serum antibody production and expression of other cellular immune-related genes, such as MHCI, CD4, and CD8, at 7 days post-immunization. Remarkably, enhanced immune responses and strong protective efficacy against a highly virulent strain of N. seriolae were recorded in DNA vaccine-cholesterol oxidase (pcD::Cho) injected fish, with a relative survival rate of 73.3%. Our results demonstrate that the reverse vaccinology approach is a valid strategy for screening vaccine candidates and pcD::Cho is a promising candidate that can boost both innate and adaptive immune responses and confer considerable protection against N. seriolae infection.

TSS Removal Efficiency and Permeability Degradation of Sand Filters in Permeable Pavement.

Permeable pavement is a highly effective technology in Low-Impact Development (LID) for managing stormwater runoff, which helps mitigate environmental impacts. Filters are essential components of permeable pavement systems as they prevent permeability reduction, remove pollutants, and enhance the system's overall efficiency. This research paper focuses on exploring the influence of three factors, including total suspended solids (TSS) particle size, TSS concentration, and hydraulic gradient, on the permeability degradation and TSS removal efficiency of sand filters. A series of tests were conducted using different values of these factors. The results demonstrate that these factors have an influence on permeability degradation and TSS removal efficiency (TRE). A larger TSS particle size results in higher permeability degradation and TRE than a smaller particle size. Higher TSS concentrations lead to higher permeability degradation and lower TRE. Additionally, smaller hydraulic gradients are associated with higher permeability degradation and TRE. However, the influence of TSS concentration and hydraulic gradient seems less significant than that of TSS particle size for the values of the factors considered in the tests. In summary, this study provides valuable insights into the effectiveness of sand filters in permeable pavement and identifies the main factors that influence permeability degradation and TRE.

Acetylcarnitine shuttling links mitochondrial metabolism to histone acetylation and lipogenesis.

The metabolite acetyl-CoA is necessary for both lipid synthesis in the cytosol and histone acetylation in the nucleus. The two canonical precursors to acetyl-CoA in the nuclear-cytoplasmic compartment are citrate and acetate, which are processed to acetyl-CoA by ATP-citrate lyase (ACLY) and acyl-CoA synthetase short-chain 2 (ACSS2), respectively. It is unclear whether other substantial routes to nuclear-cytosolic acetyl-CoA exist. To investigate this, we generated cancer cell lines lacking both ACLY and ACSS2 [double knockout (DKO) cells]. Using stable isotope tracing, we show that both glucose and fatty acids contribute to acetyl-CoA pools and histone acetylation in DKO cells and that acetylcarnitine shuttling can transfer two-carbon units from mitochondria to cytosol. Further, in the absence of ACLY, glucose can feed fatty acid synthesis in a carnitine responsive and carnitine acetyltransferase (CrAT)-dependent manner. The data define acetylcarnitine as an ACLY- and ACSS2-independent precursor to nuclear-cytosolic acetyl-CoA that can support acetylation, fatty acid synthesis, and cell growth.

Opening of capsaicin receptor TRPV1 is stabilized equally by its four subunits.

Capsaicin receptor TRPV1 is a nociceptor for vanilloid molecules, such as capsaicin and resiniferatoxin (RTX). Even though cryo-EM structures of TRPV1 in complex with these molecules are available, how their binding energetically favors the open conformation is not known. Here, we report an approach to control the number of bound RTX molecules (0-4) in functional rat TRPV1. The approach allowed direct measurements of each of the intermediate open states under equilibrium conditions at both macroscopic and single-molecule levels. We found that RTX binding to each of the four subunits contributes virtually the same activation energy, which we estimated to be 1.70 to 1.86 kcal/mol and found to arise predominately from destabilizing the closed conformation. We further showed that sequential bindings of RTX increase open probability without altering single-channel conductance, confirming that there is likely a single open-pore conformation for TRPV1 activated by RTX.

Facile preparation of porphyrin@g-C3N4/Ag nanocomposite for improved photocatalytic degradation of organic dyes in aqueous solution.

In the quest of improving the photocatalytic efficiency of photocatalysts, the combination of two and more semiconductors recently has garnered significant attention among scientists in the field. The doping of conductive metals is also an effective pathway to improve photocatalytic performance by avoiding electron/hole pair recombination and enhancing photon energy absorption. This work presented a design and fabrication of porphyrin@g-C3N4/Ag nanocomposite using acid-base neutralization-induced self-assembly approach from monomeric porphyrin and g-C3N4/Ag material. g-C3N4/Ag material was synthesized by a green reductant of Cleistocalyx operculatus leaf extract. Electron scanning microscopy (SEM), X-ray diffraction (XRD), FT-IR spectroscopy, and UV-vis spectrometer were utilized to analyse the properties of the prepared materials. The prepared porphyrin@g-C3N4/Ag nanocomposite showed well integration of porphyrin nanostructures on the g-C3N4/Ag's surface, in which porphyrin nanofiber was of the diameter in nanoscales and the length of several micrometers, and Ag NPs had an average particle size of less than 20 nm. The photocatalytic behavior of the resultant nanocomposite was tested for the degradation of Rhodamine B dye, which exhibited a remarkable RhB photodegrading percentage. The possible mechanism for photocatalysis of the porphyrin@g-C3N4/Ag nanocomposite toward Rhodamine B dye was also proposed and discussed.

Molecular determinants of µ-conotoxin KIIIA interaction with the human voltage-gated sodium channel NaV1.7.

The voltage-gated sodium (NaV) channel subtype NaV1.7 plays a critical role in pain signaling, making it an important drug target. Here we studied the molecular interactions between µ-Conotoxin KIIIA (KIIIA) and the human NaV1.7 channel (hNaV1.7). We developed a structural model of hNaV1.7 using Rosetta computational modeling and performed in silico docking of KIIIA using RosettaDock to predict residues forming specific pairwise contacts between KIIIA and hNaV1.7. We experimentally validated these contacts using mutant cycle analysis. Comparison between our KIIIA-hNaV1.7 model and the cryo-EM structure of KIIIA-hNaV1.2 revealed key similarities and differences between NaV channel subtypes with potential implications for the molecular mechanism of toxin block. The accuracy of our integrative approach, combining structural data with computational modeling, experimental validation, and molecular dynamics simulations, suggests that Rosetta structural predictions will be useful for rational design of novel biologics targeting specific NaV channels.

The Endoplasmic Reticulum and Its Contacts: Emerging Roles in Axon Development, Neurotransmission, and Degeneration.

The neuronal endoplasmic reticulum (ER) consists of a dynamic, tubular network that extends all the way from the soma into dendrites, axons, and synapses. This morphology gives rise to an enormous membrane surface area that, through the presence of tethering proteins, lipid transfer proteins, and ion channels, plays critical roles in local calcium regulation, membrane dynamics, and the supply of ions and lipids to other organelles. Here, we summarize recent advances that highlight the various roles of the neuronal ER in axonal growth, repair, and presynaptic function. We review the variety of contact sites between the ER and other axonal organelles and describe their influence on neurodevelopment and neurotransmission.

The dilemma of improving rational antibiotic use in pediatric community-acquired pneumonia.

Pneumonia is the number one cause of disease and deaths in children under five years old, outside the neonatal period, with the greatest number of cases reported from resource-limited settings. The etiology is variable, with not much information on the local etiology drug resistance profile in many countries. Recent studies suggest an increasing contribution from respiratory viruses, also in children with severe pneumonia, with an increased relative contribution in settings that have good vaccine coverage against common bacterial pathogens. Respiratory virus circulation was greatly reduced during highly restrictive measures to contain the spread of COVID-19 but rebounded once COVID-19 restrictions were relaxed. We conducted a comprehensive literature review of the disease burden, pathogens, case management and current available prevention of community acquired childhood pneumonia, with a focus on rational antibiotic use, since the treatment of respiratory infections is the leading cause of antibiotic use in children. Consistent application of revised World Health Organisation (WHO) guidance that children presenting with coryzal symptoms or wheeze can be managed without antibiotics in the absence of fever, will help to reduce unnecessary antibiotic use, as will increased availability and use of bedside inflammatory marker tests, such as C-reactive protein (CRP) in children with respiratory symptoms and fever.

In vitro and in silico antioxidant and α-glucosidase inhibitory potential of compounds isolated from Garcinia gaudichaudii.

Nine compounds including a new one, garcichaudiic acid (1), were isolated from the bark of G. gaudichaudii and their structures were characterized mainly by 1 D and 2 D NMR experiments. The antioxidant capacity of the isolated compounds was determined using DPPH radical scavenging assay and the anti-hyperglycemic activity was assessed by measuring the inhibitory effect against α-glucosidase. Among them, compound 4 showed higher antioxidant activity than the positive control, ascorbic acid, while both compounds 1 and 7 exhibited more significant α-glucosidase inhibitory activity than the reference drug acarbose. Molecular docking analysis of the bioactive compounds was also performed to examine the binding modes and key interactions with the catalytic site.

Automatic detection of Covid-19 from chest X-ray and lung computed tomography images using deep neural networks and transfer learning.

The world has been undergoing the most ever unprecedented circumstances caused by the coronavirus pandemic, which is having a devastating global effect in different aspects of life. Since there are not effective antiviral treatments for Covid-19 yet, it is crucial to early detect and monitor the progression of the disease, thereby helping to reduce mortality. While different measures are being used to combat the virus, medical imaging techniques have been examined to support doctors in diagnosing the disease. In this paper, we present a practical solution for the detection of Covid-19 from chest X-ray (CXR) and lung computed tomography (LCT) images, exploiting cutting-edge Machine Learning techniques. As the main classification engine, we make use of EfficientNet and MixNet, two recently developed families of deep neural networks. Furthermore, to make the training more effective and efficient, we apply three transfer learning algorithms. The ultimate aim is to build a reliable expert system to detect Covid-19 from different sources of images, making it be a multi-purpose AI diagnosing system. We validated our proposed approach using four real-world datasets. The first two are CXR datasets consist of 15,000 and 17,905 images, respectively. The other two are LCT datasets with 2,482 and 411,528 images, respectively. The five-fold cross-validation methodology was used to evaluate the approach, where the dataset is split into five parts, and accordingly the evaluation is conducted in five rounds. By each evaluation, four parts are combined to form the training data, and the remaining one is used for testing. We obtained an encouraging prediction performance for all the considered datasets. In all the configurations, the obtained accuracy is always larger than 95.0%. Compared to various existing studies, our approach yields a substantial performance gain. Moreover, such an improvement is statistically significant.

Acly Deficiency Enhances Myelopoiesis through Acetyl Coenzyme A and Metabolic-Epigenetic Cross-Talk.

Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit-enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor-treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation.

Computational design of peptides to target NaV1.7 channel with high potency and selectivity for the treatment of pain.

The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.

ß-sitosterol from Clinacanthus nutans Lindau enhances osteoblastogenic activity via upregulation of differentiation related genes and proteins.

ß-sitosterol derived from Clinacanthus nutans Lindau was tested for its in vitro osteogenic activity using MC3T3-E1 pre-osteoblasts. Our results indicated that ß-sitosterol was non-toxic to the cells cultured at a concentration <20 µg/mL. Treatment of the cells with ß-sitosterol significantly enhanced the alkaline phosphatase activity up to 210 and 204.6% at 5 and 10 µg/mL, respectively (P < .05). Similarly, the mineralization activity of the ß-sitosterol treated cells was elevated up to 134, 168, 118% at a concentration of 2.5, 5, and 10 µg/mL, respectively (P < .05). In addition, this compound up-regulated several marker genes for osteoblast differentiation, including runx2, osx and col I to 2, 2.5 and 5.6 folds at 10 µg/mL, respectively (P < .05). The expression of p38 and ERK proteins involved in the MAPK signal pathway related to mineralization and differentiation was also enhanced. Thus, the osteoblastogenic activity of ß-sitosterol was fully illustrated for the first time.

Prevalence and genetic basis of first-line drug resistance of Mycobacterium tuberculosis in Ca Mau, Vietnam.

Background and objective: Data on the prevalence of anti-tuberculous drug resistance and its association with genetic mutations in Mycobacterium tuberculosis are limited. Our study explores the genomics of tuberculosis in Ca Mau, Vietnam. Methods: Patients ≥15 years in Ca Mau Province, Vietnam, were screened annually for tuberculosis between 2014 and 2017. Isolates underwent drug susceptibility testing (DST) using the breakpoint method. DNA was extracted and whole genome sequencing (WGS) was performed. Results: We identified 365 positive sputum cultures for M. tuberculosis and processed 237 for DST and 265 for WGS. Resistance to isoniazid was present in 19.8% (95% CI 14.7 to 24.9%), rifampicin in 3.5% (1.1 to 5.7%) and ethambutol in 2.5% (0.9 to 5.4%) of isolates. Relevant mutations in rpoB gene were detected in 3.8% (1.8 to 6.8%). katG, inhA or fabG1 mutations were found in 19.6% (15.0 to 24.9%) with KatG being most common at 12.8% (9.1-17.5%). We found 38.4% of isolates were of Beijing lineage, 49.4% East-African-Indian lineage and 8.4% European-American lineage. There were no associations between resistance profiles and clinical features. Conclusion: The high burden of isoniazid resistance and the katG mutation highlights the challenges facing Vietnam in its efforts to achieve its EndTB goals.