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Background: Pakistan is endemic to a diverse set of parasitic, mycobacterial and viral diseases. The recognition of BCG Trained Immunity (TI) led us to postulate that the continued presence of BCG-TI may play a protective role, previously reported for both infectious and noninfectious conditions. Most of the previous studies have addressed the issue of BCG-TI in the paediatric populations. This study addressed the key issue of maintenance of BCG-TI in a wider age range (adolescent and adults) to identify the strength and quality of the immune responses., Objective: To assess the BCG-induced recall responses in healthy individuals by cytokines secreted from the TI network and its potential role in providing cross-protection against COVID-19 and other viral infections., Study Design: In this cross-sectional study, healthy young adults and adolescents (n = 20) were recruited from 16-40 years of age, with no prior history of TB treatment, autoimmune, or chronic inflammatory condition., Methods: BCG-induced cytokine responses were assessed using prototypic markers for cells of the TI network [macrophages [M1 (TNFα, IFNγ), M2 (IL10)], NK (IL2), Gamma delta (γδ) T (IL17, IL4)] and SARS CoV2 IgG antibodies against RBD using short-term (12 hrs.) cultures assay., Results: Significant differences were observed in the magnitude of recall responses to BCG with macrophage cytokines showing the highest mean levels of TNFα (9148 pg/ml) followed by IL10 (488 pg/ml) and IFNγ (355 pg/ml). The ratio of unstimulated vs.BCG-stimulated cytokines was 132 fold higher for TNFα, 40 fold fo r IL10, and 27 fold for IFNγ. Furthermore, SARS-CoV-2 antibodies were also detected in unstimulated plasma which showed cross reactivity with BCG., Conclusion: The presence of cross reactive antibodies to SARS-CoV-2 and the relative ratio of pro- and anti-inflammatory cytokines secreted by activated TI cellular network may play a pivotal role in protection in the early stages of infection as observed during the COVID-19 pandemic in the younger age groups resulting in lower morbidity and mortality., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Iqbal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The brain is the central organ of adaptation to stress because it perceives and determines threats that induce behavioral, physiological, and molecular responses. In humans, chronic stress manifests as an enduring consistent feeling of pressure and being overwhelmed for an extended duration. This can result in a persistent proinflammatory response in the peripheral and central nervous system (CNS), resulting in cellular, physiological, and behavioral effects. Compounding stressors may increase the risk of chronic-stress-induced inflammation, which can yield serious health consequences, including mental health disorders. This review summarizes the current knowledge surrounding the neuroinflammatory response in rodent models of chronic stress-a relationship that is continually being defined. Many studies investigating the effects of chronic stress on neuroinflammation in rodent models have identified significant changes in inflammatory modulators, including nuclear factor-κB (NF-κB) and toll-like receptors (TLRs), and cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6. This suggests that these are key inflammatory factors in the chronic stress response, which may contribute to the establishment of anxiety and depression-like symptoms. The behavioral and neurological effects of modulating inflammatory factors through gene knockdown (KD) and knockout (KO), and conventional and alternative medicine approaches, are discussed.

Objective: To determine if the high-level personal protective equipment used in the treatment of high-consequence infectious diseases is effective at stopping the spread of pathogens to healthcare personnel (HCP) while doffing., Background: Personal protective equipment (PPE) is fundamental to the safety of HCPs. HCPs treating patients with high-consequence infectious diseases use several layers of PPE, forming complex protective ensembles. With high-containment PPE, step-by-step procedures are often used for donning and doffing to minimize contamination risk to the HCP, but these procedures are rarely empirically validated and instead rely on following infection prevention best practices., Methods: A doffing protocol video for a high-containment PPE ensemble was evaluated to determine potential contamination pathways. These potential pathways were tested using fluorescence and genetically marked bacteriophages., Results: The experiments revealed existing protocols permit contamination pathways allowing for transmission of bacteriophages to HCPs. Updates to the doffing protocols were generated based on the discovered contamination pathways. This updated doffing protocol eliminated the movement of viable bacteriophages from the outside of the PPE to the skin of the HCP., Conclusions: Our results illustrate the need for quantitative, scientific investigations of infection prevention practices, such as doffing PPE.

Several studies have shown an association between prostate carcinoma (PCa) and Epstein-Barr virus (EBV); however, none of the studies so far have identified the histopathological and genetic markers of cancer aggressiveness associated with EBV in PCa tissues. In this study, we used previously characterized EBV-PCR-positive (n = 39) and EBV-negative (n = 60) PCa tissues to perform an IHC-based assessment of key histopathological and molecular markers of PCa aggressiveness (EMT markers, AR expression, perineural invasion, and lymphocytic infiltration characterization). Additionally, we investigated the differential expression of key oncogenes, EMT-associated genes, and PCa-specific oncomiRs, in EBV-positive and -negative tissues, using the qPCR array. Finally, survival benefit analysis was also performed in EBV-positive and EBV-negative PCa patients. The EBV-positive PCa exhibited a higher percentage (80%) of perineural invasion (PNI) compared to EBV-negative PCa (67.3%) samples. Similarly, a higher lymphocytic infiltration was observed in EBV-LMP1-positive PCa samples. The subset characterization of T and B cell lymphocytic infiltration showed a trend of higher intratumoral and tumor stromal lymphocytic infiltration in EBV-negative tissues compared with EBV-positive tissues. The logistic regression analysis showed that EBV-positive status was associated with decreased odds (OR = 0.07; p-value < 0.019) of CD3 intratumoral lymphocytic infiltration in PCa tissues. The analysis of IHC-based expression patterns of EMT markers showed comparable expression of all EMT markers, except vimentin, which showed higher expression in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Furthermore, gene expression analysis showed a statistically significant difference (p < 0.05) in the expression of CDH1, AR, CHEK-2, CDKN-1B, and CDC-20 and oncomiRs miR-126, miR-152-3p, miR-452, miR-145-3p, miR-196a, miR-183-3p, and miR-146b in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Overall, the survival proportion was comparable in both groups. The presence of EBV in the PCa tissues results in an increased expression of certain oncogenes, oncomiRs, and EMT marker (vimentin) and a decrease in CD3 ITL, which may be associated with the aggressive forms of PCa., (© 2024. The Author(s).)

The study aimed to determine the in-vitro effect of metformin on total antioxidant capacity (TAC) of seminal samples of infertile male subjects. It was conducted from January to June 2022 on forty-four seminal plasma samples collected from male infertile patients, age ranging from 18 to 55 years. All 44 semen samples were treated as three distinct groups: (i) a control group (ii) a study group subjected to oxidative stress (OS) induction and (iii) a test group exposed to OS induction and subsequent treatment with metformin. OS was introduced by using commercially available 100μM hydrogen peroxide (H 2 O 2 ) and incubated for twenty-four hours at 37 º C. After that 1 ml of 100 mmol/l concentration of commercially available Metformin (PHR 1331, CAS: 461-58-5) was administered to test group samples for additional 24h at 37 º C. Low levels of TAC were observed after OS induction in comparison to the control group (p=0.01). In test samples (after treatment with Metformin), a positive correlation of TAC with sperm count, normal sperm morphology and sperm motility were observed however, results were not significant. The antioxidant effect of Metformin was shown to improve the antioxidant capacity of OS induced samples and their sperm parameters in seminal plasma of infertile male subjects.

Objective: Most families with heritable neuromuscular disorders do not receive a molecular diagnosis. Here we evaluate diagnostic utility of exome, genome, RNA sequencing, and protein studies and provide evidence-based recommendations for their integration into practice., Methods: In total, 247 families with suspected monogenic neuromuscular disorders who remained without a genetic diagnosis after standard diagnostic investigations underwent research-led massively parallel sequencing: neuromuscular disorder gene panel, exome, genome, and/or RNA sequencing to identify causal variants. Protein and RNA studies were also deployed when required., Results: Integration of exome sequencing and auxiliary genome, RNA and/or protein studies identified causal or likely causal variants in 62% (152 out of 247) of families. Exome sequencing alone informed 55% (83 out of 152) of diagnoses, with remaining diagnoses (45%; 69 out of 152) requiring genome sequencing, RNA and/or protein studies to identify variants and/or support pathogenicity. Arrestingly, novel disease genes accounted for <4% (6 out of 152) of diagnoses while 36.2% of solved families (55 out of 152) harbored at least one splice-altering or structural variant in a known neuromuscular disorder gene. We posit that contemporary neuromuscular disorder gene-panel sequencing could likely provide 66% (100 out of 152) of our diagnoses today., Interpretation: Our results emphasize thorough clinical phenotyping to enable deep scrutiny of all rare genetic variation in phenotypically consistent genes. Post-exome auxiliary investigations extended our diagnostic yield by 81% overall (34-62%). We present a diagnostic algorithm that details deployment of genomic and auxiliary investigations to obtain these diagnoses today most effectively. We hope this provides a practical guide for clinicians as they gain greater access to clinical genome and transcriptome sequencing., (© 2024 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Human studies of early brain development have been limited by extant neuroimaging methods. MRI scanners present logistical challenges for imaging young children, while alternative modalities like functional near-infrared spectroscopy have traditionally been limited by image quality due to sparse sampling. In addition, conventional tasks for brain mapping elicit low task engagement, high head motion, and considerable participant attrition in pediatric populations. As a result, typical and atypical developmental trajectories of processes such as language acquisition remain understudied during sensitive periods over the first years of life. We evaluate high-density diffuse optical tomography (HD-DOT) imaging combined with movie stimuli for high resolution optical neuroimaging in awake children ranging from 1 to 7 years of age. We built an HD-DOT system with design features geared towards enhancing both image quality and child comfort. Furthermore, we characterized a library of animated movie clips as a stimulus set for brain mapping and we optimized associated data analysis pipelines. Together, these tools could map cortical responses to movies and contained features such as speech in both adults and awake young children. This study lays the groundwork for future research to investigate response variability in larger pediatric samples and atypical trajectories of early brain development in clinical populations., (© 2024 The Authors. Human Brain Mapping published by Wiley Periodicals LLC.)

There is a need for schools that train medical and health professionals to reflect on whether their education program is aligned to current demands and challenges. Such a reflection is not a luxury but a necessity, as achieving minimum standards is not enough. A school should aim for excellence and incorporate best practice in their education program. The ASPIRE-to-Excellence award panels have elaborated on examples of excellence in a number of themes in medical and health professional education. These are presented in a series of articles to be published in Medical Teacher in 2024 and 2025. The frameworks and critical elements described in these articles may be used by institutions as a first step in an evaluation of their program. The frameworks and elements described and examples can be used as a resource for schools and other healthcare learning organizations to consider as they endeavor to improve their education program.

Darwin attributed the absence of species transitions in the fossil record to his hypothesis that speciation occurs within isolated habitat patches too geographically restricted to be captured by fossil sequences. Mayr's peripatric speciation model added that such speciation would be rapid, further explaining missing evidence of diversification. Indeed, Eldredge and Gould's original punctuated equilibrium model combined Darwin's conjecture, Mayr's model and 124 years of unsuccessfully sampling the fossil record for transitions. Observing such divergence, however, could illustrate the tempo and mode of evolution during early speciation. Here, we investigate peripatric divergence in a Miocene stickleback fish, Gasterosteus doryssus. This lineage appeared and, over approximately 8000 generations, evolved significant reduction of 12 of 16 traits related to armour, swimming and diet, relative to its ancestral population. This was greater morphological divergence than we observed between reproductively isolated, benthic-limnetic ecotypes of extant Gasterosteus aculeatus . Therefore, we infer that reproductive isolation was evolving . However, local extinction of G. doryssus lineages shows how young, isolated, speciating populations often disappear, supporting Darwin's explanation for missing evidence and revealing a mechanism behind morphological stasis. Extinction may also account for limited sustained divergence within the stickleback species complex and help reconcile speciation rate variation observed across time scales.

Background and Objectives : The pancreas, ensconced within the abdominal cavity, requires a plethora of sophisticated imaging modalities for its comprehensive evaluation, with ultrasonography serving as a primary investigative technique. A myriad of pancreatic pathologies, encompassing pancreatic neoplasia and a spectrum of inflammatory diseases, are detectable through these imaging strategies. Nevertheless, the intricate anatomical confluence and the pancreas's deep-seated topography render the visualization and accurate diagnosis of its pathologies a formidable endeavor. The objective of our paper is to review the best diagnostic imagistic tools for the pancreas. Materials and Methods : we have gathered several articles using Prisma guidelines to determine the best imagistic methods. The imperative of pancreatic scanning transcends its diagnostic utility, proving to be a pivotal element in a multitude of clinical specialties, notably surgical oncology. Within this domain, multidetector computed tomography (MDCT) of the pancreas holds the distinction of being the paramount imaging modality, endorsed for its unrivaled capacity to delineate the staging and progression of pancreatic carcinoma. In synergy with MDCT, there has been a notable advent of avant-garde imaging techniques in recent years. These advanced methodologies, including ultrasonography, endoscopic ultrasonography, contrast-enhanced ultrasonography, and magnetic resonance imaging (MRI) conjoined with magnetic resonance cholangiopancreatography (MRCP), have broadened the horizon of tumor characterization, offering unparalleled depth and precision in oncological assessment. Other emerging diagnostic techniques, such as elastography, also hold a lot of potential and promise for the future of pancreatic imaging. Fine needle aspiration (FNA) is a quick, minimally invasive procedure to evaluate lumps using a thin needle to extract tissue for analysis. It is less invasive than surgical biopsies and usually performed as an outpatient with quick recovery. Its accuracy depends on sample quality, and the risks include minimal bleeding or discomfort. Results, guiding further treatment, are typically available within a week. Elastography is a non-invasive medical imaging technique that maps the elastic properties and stiffness of soft tissue. This method, often used in conjunction with ultrasound or MRI, helps differentiate between hard and soft areas in tissue, providing valuable diagnostic information. It is particularly useful for assessing liver fibrosis, thyroid nodules, breast lumps, and musculoskeletal conditions. The technique is painless and involves applying gentle pressure to the area being examined. The resulting images show tissue stiffness, indicating potential abnormalities. Elastography is advantageous for its ability to detect diseases in early stages and monitor treatment effectiveness. The procedure is quick, safe, and requires no special preparation, with results typically available immediately. Results : The assembled and gathered data shows the efficacy of various techniques in discerning the nature and extent of neoplastic lesions within the pancreas. Conclusions : The most common imaging modalities currently used in diagnosing pancreatic neoplasms are multidetector computed tomography (MDCT), endoscopic ultrasound (EUS), and magnetic resonance imaging (MRI), alongside new technologies, such as elastography.

The advent of aquaculture has been one of the most significant shifts in world food supply during the last century. Aquaculture has rapidly expanded and become a global food industry, spurred by population expansion, increased seafood consumption, and decreased captured fisheries. Nonetheless, the exponential growth of aquaculture has emerged as a significant contributor to anthropogenic changes. Unexpectedly, the result has focused in the emergence and spread of new diseases. The Asian sea bass ( Lates calcarifer ) is an economically important species in aquaculture, contributing significantly to the global seafood market. However, bacterial diseases have emerged as a major concern, affecting both wild and cultured populations of this species. The most prevalent bacterial pathogens are streptococcus, vibriosis, nocardiosis, tenacibaculosis, and pot-belly disease. Therefore, this review aims to comprehensively analyze both emerging and non-emerging bacterial diseases affecting L. calcarifer and explore potential management approaches for their control. Through an extensive literature survey and critical evaluation of research findings, this review highlights the current understanding of bacterial diseases in L. calcarifer and proposes strategies for better disease management. In addition, this review looks at the rise and characteristics of aquaculture, the major bacterial pathogens of L. calcarifer and their effects, and the specific attributes of disease emergence in an aquatic rather than terrestrial context. It also considers the potential for future disease emergence in L. calcarifer due to aquaculture expansion and climate changes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

Background: MRI magnetization-prepared rapid acquisition (MPRAGE) is an easily available imaging modality for dementia diagnosis. Previous studies suggested that volumetric analysis plays a crucial role in various stages of dementia classification. In this study, volumetry, radiomics and demographics were integrated as inputs to develop an artificial intelligence model for various stages, including Alzheimer's disease (AD), mild cognitive decline (MCI) and cognitive normal (CN) dementia classifications., Method: The Alzheimer's Disease Neuroimaging Initiative (ADNI) dataset was separated into training and testing groups, and the Open Access Series of Imaging Studies (OASIS) dataset was used as the second testing group. The MRI MPRAGE image was reoriented via statistical parametric mapping (SPM12). Freesurfer was employed for brain segmentation, and 45 regional brain volumes were retrieved. The 3D Slicer software was employed for 107 radiomics feature extractions from within the whole brain. Data on patient demographics were collected from the datasets. The feed-forward neural network (FFNN) and the other most common artificial intelligence algorithms, including support vector machine (SVM), ensemble classifier (EC) and decision tree (DT), were used to build the models using various features., Results: The integration of brain regional volumes, radiomics and patient demographics attained the highest overall accuracy at 76.57% and 73.14% in ADNI and OASIS testing, respectively. The subclass accuracies in MCI, AD and CN were 78.29%, 89.71% and 85.14%, respectively, in ADNI testing, as well as 74.86%, 88% and 83.43% in OASIS testing. Balanced sensitivity and specificity were obtained for all subclass classifications in MCI, AD and CN., Conclusion: The FFNN yielded good overall accuracy for MCI, AD and CN categorization, with balanced subclass accuracy, sensitivity and specificity. The proposed FFNN model is simple, and it may support the triage of patients for further confirmation of the diagnosis.

Escherichia coli phytase (AppA) is widely used as an exogenous enzyme in monogastric animal feed mainly because of its ability to degrade phytic acid or its salt (phytate), a natural source of phosphorus. Currently, successful recombinant production of soluble AppA has been achieved by gene overexpression using both bacterial and yeast systems. However, some methods for the biomembrane immobilization of phytases (including AppA), such as surface display on yeast cells and bacterial spores, have been investigated to avoid expensive enzyme purification processes. This study explored a homologous protein production approach for displaying AppA on the cell surface of E. coli by engineering its outer membrane (OM) for extracellular expression. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of total bacterial lysates and immunofluorescence microscopy of non-permeabilized cells revealed protein expression, whereas activity assays using whole cells or OM fractions indicated functional enzyme display, as evidenced by consistent hydrolytic rates on typical substrates (i.e., p-nitrophenyl phosphate and phytic acid). Furthermore, the in vitro results obtained using a simple method to simulate the gastrointestinal tract of poultry suggest that the whole-cell biocatalyst has potential as a feed additive. Overall, our findings support the notion that biomembrane-immobilized enzymes are reliable for the hydrolysis of poorly digestible substrates relevant to animal nutrition.

Antibiotics are considered one of the most important contributions to clinical medicine in the last century. Due to the use and overuse of these drugs, there have been increasing frequencies of infections with resistant pathogens. One form of resistance, heteroresistance, is particularly problematic; pathogens appear sensitive to a drug by common susceptibility tests. However, upon exposure to the antibiotic, resistance rapidly ascends, and treatment fails. To quantitatively explore the processes contributing to the emergence and ascent of resistance during treatment and the waning of resistance following cessation of treatment, we develop two distinct mathematical and computer-simulation models of heteroresistance. In our analysis of the properties of these models, we consider the factors that determine the response to antibiotic-mediated selection. In one model, heteroresistance is progressive, with each resistant state sequentially generating a higher resistance level. In the other model, heteroresistance is non-progressive, with a susceptible population directly generating populations with different resistance levels. The conditions where resistance will ascend in the progressive model are narrower than those of the non-progressive model. The rates of reversion from the resistant to the sensitive states are critically dependent on the transition rates and the fitness cost of resistance. Our results demonstrate that the standard test used to identify heteroresistance is insufficient. The predictions of our models are consistent with empirical results. Our results demand a reevaluation of the definition and criteria employed to identify heteroresistance. We recommend that the definition of heteroresistance should include a consideration of the rate of return to susceptibility., Competing Interests: Competing interests statement:The authors declare no competing interest.

Modulating brain oscillations has strong therapeutic potential. Interventions that both non-invasively modulate deep brain structures and are practical for chronic daily home use are desirable for a variety of therapeutic applications. Repetitive audio-visual stimulation, or sensory flicker, is an accessible approach that modulates hippocampus in mice, but its effects in humans are poorly defined. We therefore quantified the neurophysiological effects of flicker with high spatiotemporal resolution in patients with focal epilepsy who underwent intracranial seizure monitoring. In this interventional trial (NCT04188834) with a cross-over design, subjects underwent different frequencies of flicker stimulation in the same recording session with the effect of sensory flicker exposure on local field potential (LFP) power and interictal epileptiform discharges (IEDs) as primary and secondary outcomes, respectively. Flicker focally modulated local field potentials in expected canonical sensory cortices but also in the medial temporal lobe and prefrontal cortex, likely via resonance of stimulated long-range circuits. Moreover, flicker decreased interictal epileptiform discharges, a pathological biomarker of epilepsy and degenerative diseases, most strongly in regions where potentials were flicker-modulated, especially the visual cortex and medial temporal lobe. This trial met the scientific goal and is now closed. Our findings reveal how multi-sensory stimulation may modulate cortical structures to mitigate pathological activity in humans., (© 2024. The Author(s).)

Homeodomains (HDs) are the second largest class of DNA binding domains (DBDs) among eukaryotic sequence-specific transcription factors (TFs) and are the TF structural class with the largest number of disease-associated mutations in the Human Gene Mutation Database (HGMD). Despite numerous structural studies and large-scale analyses of HD DNA binding specificity, HD-DNA recognition is still not fully understood. Here, we analyze 92 human HD mutants, including disease-associated variants and variants of uncertain significance (VUS), for their effects on DNA binding activity. Many of the variants alter DNA binding affinity and/or specificity. Detailed biochemical analysis and structural modeling identifies 14 previously unknown specificity-determining positions, 5 of which do not contact DNA. The same missense substitution at analogous positions within different HDs often exhibits different effects on DNA binding activity. Variant effect prediction tools perform moderately well in distinguishing variants with altered DNA binding affinity, but poorly in identifying those with altered binding specificity. Our results highlight the need for biochemical assays of TF coding variants and prioritize dozens of variants for further investigations into their pathogenicity and the development of clinical diagnostics and precision therapies., (© 2024. The Author(s).)

Background: The undergraduate bioethics curriculum introduced in a private medical college in Pakistan in 1988 and revised in 2009 has evolved over time to incorporate globally relevant innovations, including integration of bioethics spirally within an existing problem-based learning curricular framework. The present evaluation study shares the results of this integrated bioethics curriculum delivered for 10 years across the five-year undergraduate medical curriculum. The study assessed the effectiveness of the curriculum in terms of student achievement, appropriateness of course contents and efficiency of instructional methods., Methods: The study utilized a mixed method sequential explanatory design. The quantitative method was used in the first phase to gather data by utilizing a structured online questionnaire. This was followed by the second phase of qualitative methods to explain the findings of the first phase and enrich the data gathered. This phase was based on focus group discussions and document review., Results: Student and faculty responses showed the curriculum contents to be relevant, informative, and appropriate as per learning objectives and student achievement. Multi-modal instructional methods used were stated to be effective and engaging; small group teaching and shorter sessions suggested to be preferable for fostering discussion and maintaining student engagement and attention. Large class formats were stated to be less effective. Students affirmed the contribution of bioethics education to their personal and professional development and ethical positioning. The majority of students agreed that the curriculum contributed to their knowledge acquisition (60.3-71.2%), skill development (59.41-60.30%) and demonstration of ethical/professional behavior (62.54-67.65%). The ranges indicate agreement with related sets of questions. Participants suggested that the curriculum could be further strengthened by better integration in clinical years, role modelling and providing opportunities for application in clinical health care settings. Moreover, topics like ethical issues related to the use of social media, public health ethics and ethics and law were suggested as additions to the existing curriculum. These findings have regional and global relevance for the development and assessment of effective bioethics curricula., Conclusion: An effective bioethics curriculum for undergraduate medical education should run longitudinally across the 5 year curriculum and be integrated in the modules and clerkships. Basic acquisition of knowledge and skills takes place in Years 1 & 2 with reinforcement and application in Years 3-5. Learning embedded in an integrated curriculum can help students recognize, critically analyze and address ethical dilemmas. Involvement and commitment of the clinical faculty is essential for reinforcing the ethical principles and concepts learnt in the earlier years., (© 2024. The Author(s).)

Periodontitis, a prevalent oral condition, is facing difficulties in therapeutic approaches, sometimes leading to failure. This literature review was conducted to investigate the diversity of other therapeutic approaches and their potential contributions to the successful management of the disease. This research scrutinized the alterations in microbial diversity and imbalances in crucial microbial species, which contribute significantly to the pathogenesis of periodontitis. Within the limitations of this study, we highlight the importance of understanding the treatment plan's role in periodontitis disease, opening the way for further research and innovative treatment plans to mitigate the impact of periodontitis on oral health. This will aid both healthcare professionals and patients in preventing and effectively treating periodontitis, ultimately improving oral health outcomes and overall systemic health and well-being.

The coordinated action of multiple leg joints and muscles is required even for the simplest movements. Understanding the neuronal circuits and mechanisms that generate precise movements is essential for comprehending the neuronal basis of the locomotion and to infer the neuronal mechanisms underlying several locomotor-related diseases. Drosophila melanogaster provides an excellent model system for investigating the neuronal circuits underlying motor behaviors due to its simple nervous system and genetic accessibility. This review discusses current genetic methods for studying locomotor circuits and their function in adult Drosophila. We highlight recently identified neuronal pathways that modulate distinct forward and backward locomotion and describe the underlying neuronal control of leg swing and stance phases in freely moving flies. We also report various automated leg tracking methods to measure leg motion parameters and define inter-leg coordination, gait and locomotor speed of freely moving adult flies. Finally, we emphasize the role of leg proprioceptive signals to central motor circuits in leg coordination. Together, this review highlights the utility of adult Drosophila as a model to uncover underlying motor circuitry and the functional organization of the leg motor system that governs correct movement., (© 2024 Wiley Periodicals LLC.)

Advances in sequencing and imaging technologies enable enhanced assessment in the prenatal space, with a goal to diagnose and predict the natural history of disease, to direct targeted therapies, and to implement clinical management, including transfer of care, election of supportive care, and selection of surgical interventions. The current lack of standardization and aggregation stymies variant interpretation and gene discovery, which hinders the provision of prenatal precision medicine, leaving clinicians and patients without an accurate diagnosis. With large amounts of data generated, it is imperative to establish standards for data collection, processing, and aggregation. Aggregated and homogeneously processed genetic and phenotypic data permits dissection of the genomic architecture of prenatal presentations of disease and provides a dataset on which data analysis algorithms can be tuned to the prenatal space. Here we discuss the importance of generating aggregate data sets and how the prenatal space is driving the development of interoperable standards and phenotype-driven tools., (© 2024 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

The peptidase neurolysin (Nln) has been validated as a potential target for developing therapeutics for ischemic stroke (IS). Overexpression of Nln in a mouse model of IS provides significant cerebroprotection, leading to reduced infarction size and edema volume. Pharmacological inhibition of Nln in the post-stroke brain worsens neurological outcomes. A virtual screen identified dipeptide small-molecule activators of Nln. Optimization studies resulted in a class of peptidomimetic compounds with promising activity. However, these compounds still possessed an amide bond that compromised their stability in plasma and the brain. Herein, we report the synthesis and characterization of a series of amide bioisosteres based on our peptidomimetic leads. Imidazole-based bioisosteres afford scaffolds with increased potency to activate Nln combined with enhanced mouse plasma stability and significantly better brain permeability over the original dipeptide hits., Competing Interests: The authors declare the following competing financial interest(s): P.C.T., V.T.K. and T.J.A. are inventors on a patent describing compounds herein. US. Patent Appl. 17/186,489., (© 2024 American Chemical Society.)

Polyethylene terephthalate (PET), the most abundantly produced polyester plastic, can be depolymerized by the Ideonella sakaiensis PETase enzyme. Based on multiple PETase crystal structures, the reaction has been proposed to proceed via a two-step serine hydrolase mechanism mediated by a serine-histidine-aspartate catalytic triad. To elucidate the multi-step PETase catalytic mechanism, we use transition path sampling and likelihood maximization to identify optimal reaction coordinates for the PETase enzyme. We predict that deacylation is likely rate-limiting, and the reaction coordinates for both steps include elements describing nucleophilic attack, ester bond cleavage, and the "moving-histidine" mechanism. We find that the flexibility of Trp185 promotes the reaction, providing an explanation for decreased activity observed in mutations that restrict Trp185 motion. Overall, this study uses unbiased computational approaches to reveal the detailed reaction mechanism necessary for further engineering of an important class of enzymes for plastics bioconversion., (© 2024. The Author(s).)

This study presents a systematic review of the literature on individuals' health-related quality of life (HRQoL) following radical cystectomy for muscle-invasive bladder cancer (MIBC), utilizing the Short Form-36 Health Survey (SF-36) as a primary assessment tool. The review was designed as an exhaustive literature search across three major databases including PubMed, Scopus, and Embase up to December 2023, using the PRISMA guidelines. The selection process refined 2281 identified articles down to 11 studies meeting our inclusion criteria. These studies encompassed a diverse demographic and clinical profile of 774 participants, with follow-up durations ranging from 3 to 130 months, thereby offering insights into both short-term and long-term HRQoL outcomes. The results highlighted significant alterations in individuals' HRQoL across various domains post-radical cystectomy. Notably, the Physical Functioning (PF) and Bodily Pain (BP) domains generally scored higher, indicating a moderate to high perceived physical health status. However, the Role Physical (RP) and Role Emotional (RE) domains showed variability, reflecting the challenges in daily role fulfillment and emotional adjustment post-surgery. A marked variability in physical recovery was observed, with studies reporting significant differences in PF and RP scores between patient groups. The General Health (GH) and Vitality (VT) domains sometimes reflected perceived deteriorations, whereas the Mental Health (MH) scores suggested that many patients maintained or achieved high levels of well-being post-operatively. The conclusions drawn from this systematic review underscore the profound and multi-faceted impact of radical cystectomy on HRQoL, varying widely between studies, being influenced by geographic factors, surgical methods, and the time of evaluation. The findings emphasize the necessity for holistic patient care approaches that address both physical and emotional rehabilitation, aiming to improve HRQoL outcomes.

The World Health Organization's global initiative toward eliminating high-risk Human Papillomavirus (hrHPV)-related cancers recommends DNA testing over visual inspection in all settings for primary cancer screening and HPV eradication by 2100. However, multiple hrHPV types cause different types of cancers, and there is a pressing need for an easy-to-use, multiplex point-of-care diagnostic platform for detecting different hrHPV types. Recently, CRISPR-Cas systems have been repurposed for point-of-care detection. Here, we established a CRISPR -Cas multiplexed d iagnostic assay (CRISPRD) to detect cervical cancer-causing hrHPVs in one reaction (one-pot assay). We harnessed the compatibility of thermostable AapCas12b, TccCas13a, and HheCas13a nucleases with isothermal amplification and successfully detected HPV16 and HPV18, along with an internal control in a single-pot assay with a limit of detection of 10 copies and 100% specificity. This platform offers a rapid and practical solution for the multiplex detection of hrHPVs, which may facilitate large-scale hrHPV point-of-care screening. Furthermore, the CRISPRD platform programmability enables it to be adapted for the multiplex detection of any two nucleic acid biomarkers as well as internal control.

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the world and is characterized by ischemic necrosis of an area of the myocardium permanently devoid of blood supply. During reperfusion, reactive oxygen species are released and this causes further insult to the myocardium, resulting in ischemia-reperfusion (IR) injury. Since Nrf2 is a key regulator of redox balance, it is essential to determine its contribution to these two disease processes. Conventionally Nrf2 levels have been shown to rise immediately after ischemia and reperfusion but its contribution to disease process a week after the injury remains uncertain. Mice were divided into MI, IR injury, and sham surgery groups and were sacrificed 1 week after surgery. Infarct was visualized using H&E and trichrome staining and expression of Nrf2 was assessed using immunohistochemistry, Western blot, and ELISA. MI displayed a higher infarct size than the IR group (MI: 31.02 ± 1.45%, IR: 13.03 ± 2.57%; p < 0.01). We observed a significantly higher expression of Nrf2 in the MI group compared to the IR model using immunohistochemistry, spot densitometry of Western blot (MI: 2.22 ± 0.16, IR: 1.81 ± 0.10, Sham: 1.52 ± 0.13; p = 0.001) and ELISA (MI: 80.78 ± 27.08, IR: 31.97 ± 4.35; p < 0.01). There is a significantly higher expression of Nrf2 in MI compared to the IR injury group. Modulation of Nrf2 could be a potential target for therapeutics in the future, and its role in cardioprotection can be further investigated., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zuberi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma -infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase., Competing Interests: Dr. Hao Wu is a co-founder of Ventus Therapeutics. The other authors declare that they have no conflict of interest.

KRAS G12C inhibitors (adagrasib and sotorasib) have shown clinical promise in targeting KRAS G12C -mutated lung cancers; however, most patients eventually develop resistance. In lung patients with adenocarcinoma with KRAS G12C and STK11/LKB1 co-mutations, we find an enrichment of the squamous cell carcinoma gene signature in pre-treatment biopsies correlates with a poor response to adagrasib. Studies of Lkb1-deficient KRAS G12C and Kras G12D lung cancer mouse models and organoids treated with KRAS inhibitors reveal tumors invoke a lineage plasticity program, adeno-to-squamous transition (AST), that enables resistance to KRAS inhibition. Transcriptomic and epigenomic analyses reveal ΔNp63 drives AST and modulates response to KRAS inhibition. We identify an intermediate high-plastic cell state marked by expression of an AST plasticity signature and Krt6a. Notably, expression of the AST plasticity signature and KRT6A at baseline correlates with poor adagrasib responses. These data indicate the role of AST in KRAS inhibitor resistance and provide predictive biomarkers for KRAS-targeted therapies in lung cancer., Competing Interests: Declaration of interests A.J.A. has consulted for Anji Pharmaceuticals, Arrakis Therapeutics, AstraZeneca, Boehringer Ingelheim, Oncorus, Inc., Merck & Co., Inc., Mirati Therapeutics, Nimbus Therapeutics, Plexium, Revolution Medicines, Reactive Biosciences, Riva Therapeutics, Servier Pharmaceuticals, Syros Pharmaceuticals, T-knife Therapeutics. A.J.A. holds equity in Riva Therapeutics. A.J.A. has received research funding from Bristol Myers Squibb, Deerfield, Inc., Eli Lilly, Mirati Therapeutics, Novartis, Novo Ventures, Revolution Medicines, and Syros Pharmaceuticals. K.-K.W. has research funding and/or consulted for Janssen Pharmaceuticals, Pfizer, Bristol Myers Squibb, Zentalis Pharmaceuticals, Blueprint Medicines, Takeda Pharmaceuticals, Mirati Therapeutics, Novartis, Genentech, Merus, Bridgebio Pharma, Xilio Therapeutics, Allerion Therapeutics, Boehringer Ingelheim, Cogent Therapeutics, Revolution Medicines and AstraZeneca., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Objective: Previous germline studies on renal cell carcinoma (RCC) have usually pooled clear and non-clear cell RCCs and have not adequately accounted for population stratification, which might have led to an inaccurate estimation of genetic risk. Here, we aim to analyze the major germline drivers of RCC risk and clinically relevant but underexplored germline variant types., Methods: We first characterized germline pathogenic variants (PVs), cryptic splice variants, and copy number variants (CNVs) in 1436 unselected RCC patients. To evaluate the enrichment of PVs in RCC, we conducted a case-control study of 1356 RCC patients ancestry matched with 16 512 cancer-free controls using approaches accounting for population stratification and histological subtypes, followed by characterization of secondary somatic events., Key Findings and Limitations: Clear cell RCC patients ( n = 976) exhibited a significant burden of PVs in VHL compared with controls (odds ratio [OR]: 39.1, p  = 4.95e-05). Non-clear cell RCC patients ( n  = 380) carried enrichment of PVs in FH (OR: 77.9, p  = 1.55e-08) and MET (OR: 1.98e11, p  = 2.07e-05). In a CHEK2 -focused analysis with European participants, clear cell RCC ( n  = 906) harbored nominal enrichment of low-penetrance CHEK2 variants-p.Ile157Thr (OR: 1.84, p  = 0.049) and p.Ser428Phe (OR: 5.20, p  = 0.045), while non-clear cell RCC ( n  = 295) exhibited nominal enrichment of CHEK2 loss of function PVs (OR: 3.51, p  = 0.033). Patients with germline PVs in FH, MET, and VHL exhibited significantly earlier age of cancer onset than patients without germline PVs (mean: 46.0 vs 60.2 yr, p  < 0.0001), and more than half had secondary somatic events affecting the same gene ( n  = 10/15, 66.7%). Conversely, CHEK2 PV carriers exhibited a similar age of onset to patients without germline PVs (mean: 60.1 vs 60.2 yr, p  = 0.99), and only 30.4% carried somatic events in CHEK2 ( n  = 7/23). Finally, pathogenic germline cryptic splice variants were identified in SDHA and TSC1, and pathogenic germline CNVs were found in 18 patients, including CNVs in FH , SDHA , and VHL ., Conclusions and Clinical Implications: This analysis supports the existing link between several RCC risk genes and RCC risk manifesting in earlier age of onset. It calls for caution when assessing the role of CHEK2 due to the burden of founder variants with varying population frequency. It also broadens the definition of the RCC germline landscape of pathogenicity to incorporate previously understudied types of germline variants., Patient Summary: In this study, we carefully compared the frequency of rare inherited mutations with a focus on patients' genetic ancestry. We discovered that subtle variations in genetic background may confound a case-control analysis, especially in evaluating the cancer risk associated with specific genes, such as CHEK2 . We also identified previously less explored forms of rare inherited mutations, which could potentially increase the risk of kidney cancer., (© 2024 The Author(s).)

Introduction: The prevalence of female infertility in Pakistan is currently estimated at 22%, and emerging research suggests that vitamin D (VD) deficiency (VDD) may play a significant role in influencing female fertility. The focus of this study was to investigate the single nucleotide polymorphism (SNP) patterns within the VD binding protein (VDBP). The study aimed to explore dysregulated pathways and gene enrichment through an interaction network analysis, specifically focusing on the interplay between the VD receptor (VDR) and VDBP in females experiencing unexplained infertility (UI) coupled with VDD., Methods: A cross-sectional study was conducted on VD-deficient, fertile, and UI female subjects. VDBP and VDR were assessed by enzyme-linked immunoassay and genotyping performed. FunRich (version 3.1.3; http://funrich.org/index.html) was employed for analysis of the identified proteins: VDR and VDBP and with their mapped gene datasets, gene enrichment, and protein-protein interaction (PPI) network., Results: The mean VD and VDR values of infertile females were significantly lower than those of fertile females. VDBP in infertile females (median (IQR)): 296.05 (232.58-420.23)) was lower than that of fertile females (469.9 (269.57-875.55), (p=0.01)). On sequence analysis, a mutation rs 4588 SNP (Thr 436 Lys) was found in exon 11 of the VDBP gene of UI females, but no mutation in exons 8 and 9 of the VDR gene, with some insignificant intronic variants, was observed. The proteins such as plasma membrane estrogen receptor signaling pathway (p < 0.001), VDR, SMAD3, NCOR1, CREBBP, NCOA1, STAT1, GRB2, PPP2CA, TP53, and NCOA2 were enriched after biological pathway grouping when VDR was made the focused gene and directly interacting with VDBP., Conclusion: The females with UI exhibited significantly low VD, VDBP, and VDR. The plasma membrane estrogen receptor signaling pathway was enriched in VDD infertile females., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Rubab et al.)

Approaches to brain tumour diagnosis and detecting recurrence after treatment are costly and significantly invasive. Developing peripheral-sample liquid biopsy tools is the key to enhancing our ability to prognosticate brain tumour subtypes and molecular heterogeneity. The present scoping review was designed to discuss current updates in liquid biopsy tools for diagnosis and guiding clinical management of brain tumours; we evaluated the literature within the context of low-and-middle-income country challenges. Circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), cell-free DNA (cfDNA), extracellular vesicle-associated biomarkers, protein biomarkers, microRNAs, and serum metabolites are discussed with the collation of current data supporting their utility in liquid biopsy. Further challenges to implanting liquid biopsy tools at a systematic level are highlighted.

Deep burns sequelae involving the upper limb are challenging even for experienced surgeons, mainly because local reconstructive options and donor sites are often compromised. The use of free flaps for this type of reconstruction remains difficult due to the small recipient vessel diameter and tendency to vasospasm. Moreover, pediatric cases bring the challenge to another level. We present the case of a 13-year-old girl presenting major retractile sequelae of the upper left limb, including complete wrist immobilization combining wrist hyper-extension, ulnar deviation deformity, and a ulno-carpal dislocation. She was referred to our department where a two-stage reconstruction was performed using a pre-expanded free deep inferior epigastric artery perforator (DIEP) flap. The first surgery consisted of placing two kidney-shaped expanders in a subfascial plane in the hypogastric region. Four months later, after a bi-weekly expansion, an excision of the scar tissue, and the DIEP flap transfer were completed. At the 12-month follow-up evaluation, both aesthetic and functional results were satisfactory, with a good contour and regained mobility of the wrist., (Copyright © 2023. Published by Elsevier Masson SAS.)

Background: Laboratory resurrection of ancient coagulation factor (F) IX variants generated through ancestral sequence reconstruction led to the discovery of a FIX variant, designated An96, which possesses enhanced specific activity independent of and additive to that provided by human p.Arg384Lys, referred to as FIX-Padua., Objectives: The goal of the current study was to identify the amino acid substitution(s) responsible for the enhanced activity of An96 and create a humanized An96 FIX transgene for gene therapy application., Methods: Reductionist screening approaches, including domain swapping and scanning residue substitution, were used and guided by one-stage FIX activity assays. In vitro characterization of top candidates included recombinant high-purity preparation, specific activity determination, and enzyme kinetic analysis. Final candidates were packaged into adeno-associated viral (AAV) vectors and delivered to hemophilia B mice., Results: Five of 42 total amino acid substitutions in An96 appear sufficient to retain the enhanced activity of An96 in an otherwise human FIX variant. Additional substitution of the Padua variant further increased the specific activity 5-fold. This candidate, designated ET9, demonstrated 51-fold greater specific activity than hFIX. AAV2/8-ET9 treated hemophilia B mice produced plasma FIX activities equivalent to those observed previously for AAV2/8-An96-Padua, which were 10-fold higher than AAV2/8-hFIX-Padua., Conclusion: Starting from computationally inferred ancient FIX sequences, novel amino acid substitutions conferring activity enhancement were identified and translated into an AAV-FIX gene therapy cassette demonstrating high potency. This ancestral sequence reconstruction discovery and sequence mapping refinement approach represents a promising platform for broader protein drug and gene therapy candidate optimization., Competing Interests: Declaration of competing interests H.C.B., G.D., S.N.G., C.B.D., H.T.S., K.A.K., and C.W.C. are inventors on patents and patent applications describing the ancestral FIX technology filed by Expression Therapeutics, Emory University, Children’s Healthcare of Atlanta, and Georgia Institute of Technology. C.B.D., H.T.S., and H.C.B. are inventors on liver-directed codon-optimization and promoter technology filed by Emory University and Children’s Healthcare of Atlanta. H.T.S. and C.B.D. are cofounders of Expression Therapeutics, Inc, and own equity in the company. H.C.B., G.D., and S.N.G. are employees of Expression Therapeutics, Inc and own equity in the company. Expression Therapeutics, Inc has obtained licenses for FIX-An96, liver codon-optimized FIX, and synthetic liver-directed promoter intellectual property. K.C.C., P.C.S., and G.M.B. declare no conflicts of interest. The terms of these arrangements have been reviewed and approved by Emory University in accordance with its conflict of interest policies., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

The bacteriophage behavior of SARS-CoV-2 during the acute and post-COVID-19 phases appears to be an important factor in the development of the disease. The early use of antibiotics seems to be crucial to inhibit disease progression-to prevent viral replication in the gut microbiome, and control toxicological production from the human microbiome. To study the impact of specific antibiotics on recovery from COVID-19 and long COVID (LC) taking into account: vaccination status, comorbidities, SARS-CoV-2 wave, time of initiation of antibiotic therapy and concomitant use of corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs). A total of 211 COVID-19 patients were included in the study: of which 59 were vaccinated with mRNA vaccines against SARS-CoV-2 while 152 were unvaccinated. Patients were enrolled in three waves: from September 2020 to October 2022, corresponding to the emergence of the pre-Delta, Delta, and Omicron variants of the SARS-CoV-2 virus. The three criteria for enrolling patients were: oropharyngeal swab positivity or fecal findings; moderate symptoms with antibiotic intake; and measurement of blood oxygen saturation during the period of illness. The use of antibiotic combinations, such as amoxicillin with clavulanic acid (875 + 125 mg tablets, every 12 h) plus rifaximin (400 mg tablets every 12 h), as first choice, as suggested from the previous data, or azithromycin (500 mg tablets every 24 h), plus rifaximin as above, allows healthcare professionals to focus on the gut microbiome and its implications in COVID-19 disease during patient care. The primary outcome measured in this study was the estimated average treatment effect, which quantified the difference in mean recovery between patients receiving antibiotics and those not receiving antibiotics at 3 and 9 days after the start of treatment. In the analysis, both vaccinated and unvaccinated groups had a median illness duration of 7 days (interquartile range [IQR] 6-9 days for each; recovery crude hazard ratio [HR] = 0.94, p = 0.700). The median illness duration for the pre-Delta and Delta waves was 8 days (IQR 7-10 days), while it was shorter, 6.5 days, for Omicron (IQR 6-8 days; recovery crude HR = 1.71, p < 0.001). These results were confirmed by multivariate analysis. Patients with comorbidities had a significantly longer disease duration: median 8 days (IQR 7-10 days) compared to 7 days (IQR 6-8 days) for those without comorbidities (crude HR = 0.75, p = 0.038), but this result was not confirmed in multivariate analysis as statistical significance was lost. Early initiation of antibiotic therapy resulted in a significantly shorter recovery time (crude HR = 4.74, p < 0.001). Concomitant use of NSAIDs did not reduce disease duration and in multivariate analysis prolonged the disease (p = 0.041). A subgroup of 42 patients receiving corticosteroids for a median of 3 days (IQR 3-6 days) had a longer recovery time (median 9 days, IQR 8-10 days) compared to others (median 7 days, IQR 6-8 days; crude HR = 0.542, p < 0.001), as confirmed also by the adjusted HR. In this study, a statistically significant reduction in recovery time was observed among patients who received early antibiotic treatment. Early initiation of antibiotics played a crucial role in maintaining higher levels of blood oxygen saturation. In addition, it is worth noting that a significant number of patients who received antibiotics in the first 3 days and for a duration of 7 days, during the acute phase did not develop LC., (© 2024 Wiley Periodicals LLC.)

Bacterial leaf blight is a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo) which causes severe crop loss in rice. The molecular mechanism that initiates defense against such pathogens remains unexplored. Reports have suggested crucial role of several miRNAs in regulating immune responses in plants. Argonaute (AGO) proteins have been implicated in imparting immunity against pathogens by using small RNAs as guide molecules. Here, we show that phosphorylation of rice AGO1a by MAP kinases is required for miRNA expression regulation during Xoo infection. AGO1a is induced in response to pathogen infection and is under the control of SA signaling pathway. The pathogen responsive MAP kinases MPK3, MPK4 and MPK6, interact with AGO1a in planta and can phosphorylate the protein in vitro. Overexpression of AGO1a extends disease resistance against Xoo in rice and leads to a higher accumulation of miRNAs. Conversely, overexpression of a non phosphorylatable mutant protein aggravates disease susceptibility and remarkably suppresses the miRNA expression levels. At a molecular level, phosphorylation of AGO1a by MAP kinase is required for increased accumulation of miRNAs during pathogen challenge. Taken together, the data suggests that OsAGO1a is a direct phosphorylation target of MAP kinases and this phosphorylation is crucial for its role in imparting disease resistance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

When cultured together under standard laboratory conditions Pseudomonas aeruginosa has been shown to be an effective inhibitor of Staphylococcus aureus . However, P. aeruginosa and S. aureus are commonly observed in coinfections of individuals with cystic fibrosis (CF) and in chronic wounds. Previous work from our group revealed that S. aureus isolates from CF infections are able to persist in the presence of P. aeruginosa strain PAO1 with a range of tolerances with some isolates being eliminated entirely and others maintaining large populations. In this study, we designed a serial transfer, evolution experiment to identify mutations that allow S. aureus to survive in the presence of P. aeruginosa . Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa PAO1. After eight coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT , that were unique to evolved P. aeruginosa -tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompeted wild-type S. aureus when glutamate was absent from chemically defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two are cultured together.

Pre-mRNA splicing is a precise regulated process and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs) and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remains largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout (KO) zebrafish line and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 KO zebrafish line, we discovered that prpf31 KO zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31-deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell therapies involving c-kit+ progenitor cells (CPCs) and mesenchymal stem cells (MSCs) have been actively studied for cardiac repair. The benefits of such therapies have more recently been attributed to the release of small extracellular vesicles (sEVs) from the parent cells. These sEVs are 30-180 nm vesicles containing protein/nucleic acid cargo encapsulated within an amphiphilic bilayer membrane. Despite their pro-reparative effects, sEV composition and cargo loading is highly variable, making it challenging to develop robust therapies with sEVs. Synthetic alternatives have been developed to allow cargo modulation, including prior work from the laboratory, to design sEV-like vehicles (ELVs). ELVs are synthesized from the sEV membrane but allow controlled cargo loading. It is previously shown that loading pro-angiogenic miR-126 into CPC-derived ELVs significantly increases endothelial cell angiogenesis compared to CPC-sEVs alone. Here, they expand on this work to design MSC-derived ELVs  and study the role of the parent cell type on ELV composition and function. It is found that ELV origin does affect the ELV potency and that ELV membrane composition can affect outcomes. This study showcases the versatility of ELVs to be synthesized from different parent cells and highlights the importance of selecting ELV source cells based on the desired functional outcomes., (© 2023 Wiley-VCH GmbH.)

Vγ9Vδ2 T cells represent a promising cancer therapy platform because the implementation of allogenic, off-the-shelf product candidates is possible. However, intravenous administration of human Vγ9Vδ2 T cells manufactured under good manufacturing practice (GMP)-compliant, serum-free conditions are not tested easily in most mouse models, mainly because they lack the ability to migrate from the blood to tissues or tumors. We demonstrate that these T cells do not migrate from the circulation to the mouse bone marrow (BM), the site of many malignancies. Thus, there is a need to better characterize human γδ T-cell migration in vivo and develop strategies to direct these cells to in vivo sites of therapeutic interest. To better understand the migration of these cells and possibly influence their migration, NSG mice were conditioned with agents to clear BM cellular compartments, i.e., busulfan or total body irradiation (TBI), or promote T-cell migration to inflamed BM, i.e., incomplete Freund's adjuvant (IFA), prior to administering γδ T cells. Conditioning with TBI, unlike busulfan or IFA, increases the percentage and number of γδ T cells accumulating in the mouse BM, and cells in the peripheral blood (PB) and BM display identical surface protein profiles. To better understand the mechanism by which cells migrate to the BM, mice were conditioned with TBI and administered γδ T cells or tracker-stained red blood cells. The mechanism by which γδ T cells enter the BM after radiation is passive migration from the circulation, not homing. We tested if these ex vivo- expanded cells can migrate based on chemokine expression patterns and showed that it is possible to initiate homing by utilizing highly expressed chemokine receptors on the expanded γδ T cells. γδ T cells highly express CCR2, which provides chemokine attraction to C-C motif chemokine ligand 2 (CCL2)-expressing cells. IFNγ-primed mesenchymal stromal cells (MSCs) (γMSCs) express CCL2, and we developed in vitro and in vivo models to test γδ T-cell homing to CCL2-expressing cells. Using an established neuroblastoma NSG mouse model, we show that intratumorally-injected γMSCs increase the homing of γδ T cells to this tumor. These studies provide insight into the migration of serum-free, ex vivo- expanded Vγ9Vδ2 T cells in NSG mice, which is critical to understanding the fundamental properties of these cells., Competing Interests: HTS and CD have equity in Expression Therapeutics, which is developing cancer treatments based on engineered γδ T cells. BP is an employee of Expression Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Parwani, Branella, Burnham, Burnham, Bustamante, Foppiani, Knight, Petrich, Horwitz, Doering and Spencer.)

Pharmaceutical development of solid-state formulations requires testing active pharmaceutical ingredients (API) and excipients for uniformity and stability. Solid-state properties such as component distribution and grain size are crucial factors that influence the dissolution profile, which greatly affect drug efficacy and toxicity, and can only be analyzed spatially by chemical imaging (CI) techniques. Current CI techniques such as near infrared microscopy and confocal Raman spectroscopy are capable of high chemical and spatial resolution but cannot achieve the measurement speeds necessary for integration into the pharmaceutical production and quality assurance processes. To fill this gap, we demonstrate fast chemical imaging by epi -detected sparse spectral sampling stimulated Raman scattering to quantify API and excipient degradation and distribution.

Microplastic pollution has emerged as a new environmental concern due to our reliance on plastic. Recent years have seen an upward trend in scholarly interest in the topic of microplastics carrying contaminants; however, the available review studies have largely focused on specific aspects of this issue, such as sorption, transport, and toxicological effects. Consequently, this review synthesizes the state-of-the-art knowledge on these topics by presenting key findings to guide better policy action toward microplastic management. Microplastics have been reported to absorb pollutants such as persistent organic pollutants, heavy metals, and antibiotics, leading to their bioaccumulation in marine and terrestrial ecosystems. Hydrophobic interactions are found to be the predominant sorption mechanism, especially for organic pollutants, although electrostatic forces, van der Waals forces, hydrogen bonding, and pi-pi interactions are also noteworthy. This review reveals that physicochemical properties of microplastics, such as size, structure, and functional groups, and environmental compartment properties, such as pH, temperature, and salinity, influence the sorption of pollutants by microplastic. It has been found that microplastics influence the growth and metabolism of organisms. Inadequate methods for collection and analysis of environmental samples, lack of replication of real-world settings in laboratories, and a lack of understanding of the sorption mechanism and toxicity of microplastics impede current microplastic research. Therefore, future research should focus on filling in these knowledge gaps., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ferrets represent an invaluable model for the study of influenza virus pathogenicity and transmissibility. Ferrets are also employed for the study of bacterial pathogens that naturally infect humans at different anatomical sites. While viral and bacterial infection studies in isolation using animal models are important for furthering our understanding of pathogen biology and developing improved therapeutics, it is also critical to extend our knowledge to pathogen coinfections in vivo, to more closely examine interkingdom dynamics that may contribute to overall disease outcomes. We discuss how ferrets have been employed to study a diverse range of both influenza viruses and bacterial species and summarize key studies that have utilized the ferret model for primary influenza virus challenge followed by secondary bacterial infection. These copathogenesis studies have provided critical insight into the dynamic interplay between these pathogens, underscoring the utility of ferrets as a model system for investigating influenza virus-bacteria interactions., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2023.)

The outer membrane (OM) is an essential organelle of Gram-negative bacteria. Lipoproteins are key to building the OM, performing essential functions in several OM assembly machines. Lipoproteins mature in the inner membrane (IM) and are then trafficked to the OM. In Escherichia coli , the LolCDE transporter is needed to extract lipoproteins from the IM to begin trafficking. Lipoproteins are then transferred from LolCDE to the periplasmic chaperone LolA which ferries them to the OM for insertion by LolB. LolA recruitment by LolC is an essential trafficking step. Structural and biochemical studies suggested that two regions (termed Hook and Pad) within a periplasmic loop of LolC worked in tandem to recruit LolA, leading to a bipartite model for recruitment. Here, we genetically examine the LolC periplasmic loop in vivo using E. coli . Our findings challenge the bipartite interaction model. We show that while the Hook is essential for lipoprotein trafficking in vivo , lipoproteins are still efficiently trafficked when the Pad residues are inactivated. We show with AlphaFold2 multimer modeling that Hook:LolA interactions are likely universal among diverse Gram-negative bacteria. Conversely, Pad:LolA interactions vary across phyla. Our in vivo data redefine LolC:LolA recruitment into a hierarchical interaction model. We propose that the Hook is the major player in LolA recruitment, while the Pad plays an ancillary role that is important for efficiency but is ultimately dispensable. Our findings expand the understanding of a fundamental step in essential lipoprotein trafficking and have implications for efforts to develop new antibacterials that target LolCDE.IMPORTANCEResistance to current antibiotics is increasingly common. New antibiotics that target essential processes are needed to expand clinical options. For Gram-negative bacteria, their cell surface-the outer membrane (OM)-is an essential organelle and antibiotic barrier that is an attractive target for new antibacterials. Lipoproteins are key to building the OM. The LolCDE transporter is needed to supply the OM with lipoproteins and has been a focus of recent antibiotic discovery. In vitro evidence recently proposed a two-part interaction of LolC with LolA lipoprotein chaperone (which traffics lipoproteins to the OM) via "Hook" and "Pad" regions. We show that this model does not reflect lipoprotein trafficking in vivo . Only the Hook is essential for lipoprotein trafficking and is remarkably robust to mutational changes. The Pad is non-essential for lipoprotein trafficking but plays an ancillary role, contributing to trafficking efficiency. These insights inform ongoing efforts to drug LolCDE., Competing Interests: The authors declare no conflict of interest.

The vertebrate retina is made up of six specialized neuronal cell types and one glia that are generated from a common retinal progenitor. The development of these distinct cell types is programmed by transcription factors that regulate the expression of specific genes essential for cell fate specification and differentiation. Because of the complex nature of transcriptional regulation, understanding transcription factor functions in development and disease is challenging. Research on the Cone-rod homeobox transcription factor CRX provides an excellent model to address these challenges. In this review, we reflect on 25 years of mammalian CRX research and discuss recent progress in elucidating the distinct pathogenic mechanisms of four CRX coding variant classes. We highlight how in vitro biochemical studies of CRX protein functions facilitate understanding CRX regulatory principles in animal models. We conclude with a brief discussion of the emerging systems biology approaches that could accelerate precision medicine for CRX -linked diseases and beyond., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zheng and Chen.)

The genus Thraulus is widespread throughout much of the Eastern Hemisphere. Since Eaton established Thraulus in 1881, 62 species have, at one time or another, been placed in this genus. Thirty-eight of those species were eventually moved to other genera. Any comprehensive study of the remaining species, based on the published literature, is difficult as they were described by many authors, using different criteria, over a period of 142 years. The purpose of this study was to redescribe this genus, based on previously described species and nine new species, and to provide a format for future taxonomic and morphological studies of Thraulus. Redescriptions of most species were based on direct examination of external morphological characters. Descriptions or diagnoses of species, whose types were unavailable for study, were made using the original published description and additional information provided by authors of several of those species. The following species were studied: Thraulus amravati Vasanth, Subramanian & Selvakumar, 2022; T. bellus Eaton, 1881; T. bishopi Peters & Tsui, 1972; T. cuspidatus Vasanth, Subramanian & Selvakumar, 2022; T. demoulini Peters & Tsui, 1973; T. fasciatus (Kimmins, 1956); T. fatuus Kang & Yang, 1994; T. femoratus Li, Liu & Zhou, 2006; T. gopalani Grant & Sivaramakrishnan, 1985; T. jacobusi Isack, Srinivasan, Sivaruban & Barathy, 2022; T. macilentus Kang & Yang, 1994; T. malabarensis Vasanth, Subramanian & Selvakumar, 2022; T. mudumalaiensis Soman, 1991; T. plumeus Selvakumar, Vasanth & Subramanian, 2022; T. semicastaneus (Gillies, 1951); T. thiagarajani Balasubramanian & Muthukatturaja, 2019; T. thraker Jacob, 1988; T. torrentis (Gillies, 1964); T. turbinatus (Ulmer, 1909); T. umbrosus Kang & Yang, 1994; and T. vellimalaiensis Vasanth, Subramanian & Selvakumar, 2022. Nine new species of Thraulus are described: T. connubialis sp. nov., Malaysia; T. cursus sp. nov., Japan; T. eatoni sp. nov., Indonesia; T. ishiwatai sp. nov., Japan; T. madagasikarensis sp. nov., Madagascar; T. nihonensis sp. nov., Japan; T. opifer sp. nov., Australia; T. parentalis sp. nov., Malaysia; and T. petersorum sp. nov., Malaysia. Thraulus can be distinguished from all other genera of Leptophlebiidae by the following combination of characters: In the imagos, 1) upper portion of eyes oval-suboval, major axes diverge anteriorly; 2) vein MA fork of fore wings symmetrical; 3) vein MP fork of fore wings asymmetricala cross vein connects base of MP2 to MP1, MP fork closer to base of wing than Rs fork; 4) strongly oblique cross vein extends between veins R4+5 and MA1 just apical to fork of vein MA; 5) 2 cubital intercalary veins in fore wings; 6) costal projection on hind wings well-developed, bluntly rounded to acutely pointed; 7) claws dissimilarone blunt and pad-like, the other apically hooked; 8) penes long, relatively straight, narrow, parallel, usually contiguous mesally but not fused, apex may have lateral projections; 9) sternum 7 of female with posterior margin straight or shallowly concave or convex mesally; and 10) sternum 9 of females rounded apically. In addition, penile spines occur on most species. In the nymphs, 1) lateral margins of clypeus parallel; 2) width of labrum subequal to width of clypeus; 3) 2 dorsal rows of setae on labrum; 4) venter of labrum with 1 row of short stout setae on either side of midline near anterior margin, rows curve mesally; 5) hypopharynx with small, rounded, posterolateral projections on arms of superlingua; 6) large spine on posterolateral corners of terga 69, 79 or 89; 7) gills 17 dissimilar: gill 1 composed of 1 or 2 subulate lamellae or a dorsal subulate lamella and a ventral fimbriate oval lamella, and gills 27 composed of dorsal and ventral oval lamellae with fimbriate margins. Two species continue to be nomen dubiumT. siewertii (Weyenbergh, 1883) and T. vogleri (Weyenbergh, 1883). Thraulus grandis Gose, 1980 is considered nomen nudum. A review of published phylogenetic studies involving Thraulus is provided. With the species discussed in this paper, along with reports of additional new species to be described, Thraulus has the potential to be included among the more specious genera of Ephemeroptera.