Target Pathogens Research Articles

ISOLATION AND MOLECULAR IDENTIFICATION OF INDIGENOUS BACTERIOCIN-PRODUCING WEISSELLA CIBARIA

Globally, over 6.22 million deaths are associated with antibiotic resistance. Bacteriocins, a set of antimicrobial peptides synthesized on the ribosomes, are widely viewed as a potential answer to this issue. This is due to their pore-forming ability and antimicrobial activity against antibiotic-resistant pathogens. The aim of this study is to isolate bacteriocin-producing Weissella cibaria and evaluate its antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, Streptococcus sp., Candida sp. and Rhizopus stolonifer. Weissella cibaria man1 was isolated by inoculating deMan Rogosa Sharpe (MRS) broth with small pieces of ripe Mangifera indica (mango), 24-hour incubation at 370C, 10-fold serial dilution and plating on MRS agar. Molecular identification was achieved by DNA extraction, amplification of the 16S rRNA gene through polymerase chain reaction (PCR), agarose gel electrophoresis, gene sequencing, and BLASTN homology searches in the National Center for Biotechnology Information (NCBI). Antimicrobial activity of the bacteriocin was determined by agar well diffusion assay. Mangifera indica (mango) was found to harbor bacteriocin-producing Weissella cibaria man1. The bacteriocin (weissellicin man1) exhibited a broad spectrum of antimicrobial activity. Weissellicin man1 suppressed the growth of several target pathogens (Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, Candida sp. and Rhizopus stolonifer) but had no inhibitory action against Escherichia coli, Streptococcus sp., Staphylococcus aureus. In conclusion, weissellicin man1 from Weissella cibaria man1 has a broad-spectrum of antimicrobial action. These findings will facilitate further evaluation of the antimicrobial potency of weissellicin man1.

Uncovering the Impact of COVID-19 Mediated Bidirectional Dysregulation of CYP3A4 on Systemic and Pulmonary Drug Concentrations Using Physiologically Based Pharmacokinetic Modeling.

Several clinical studies have shown that COVID-19 increases the systemic concentration of drugs in hospitalized COVID-19 patients. However, it is unclear how COVID-19-mediated bidirectional dysregulation of hepatic and pulmonary CYP3A4 impacts drug concentrations, especially in the lung tissue which is most affected by the disease. Herein, PBPK modeling was used to demonstrate the differences in systemic and pulmonary concentrations of four respiratory infectious disease drugs when CYP3A4 is concurrently downregulated in the liver and upregulated in the lung based on existing clinical data on COVID-19 - CYP3A4 interactions at varying severity levels including outpatients, non-ICU, and ICU patients. The study showed that hepatic metabolism is the primary determinant of both systemic and pulmonary drug concentrations despite the concurrent bidirectional dysregulation of liver and lung CYP3A4. ICU patients had the most systemic and pulmonary drug exposure with a percentage increase in AUCplasma of approximately 44%, 56%, 114%, and 196% for clarithromycin, nirmatrelvir, dexamethasone, and itraconazole, respectively, relative to the healthy group. Within the ICU cohort, clarithromycin exhibited its highest exposure in lung tissue mass with a fold change of 1189, while nirmatrelvir and dexamethasone showed their highest exposure in the plasma compartment, with fold changes of about 126 and 5, respectively, compared to the maximum therapeutic concentrations for their target pathogens. Itraconazole was significantly under-exposed in the lung fluid compartment potentially explaining its limited efficacy for the treatment of COVID-19. These findings underscore the importance of optimizing dosing regimens in at risk ICU patients to enhance both efficacy and safety profiles. Significance Statement This study investigated whether COVID-19-mediated concurrent hepatic downregulation and pulmonary upregulation of CYP3A4 leads to differences in the systemic and pulmonary concentrations of four respiratory medicines. The study demonstrated that intercompartmental differences in drug concentrations were driven by only hepatic CYP3A4 expression. This work suggests that ICU patients with significant COVID-19 - CYP3A4 interactions may be at risk of clinically relevant COVID-19-drug interactions, highlighting the need for optimizing dosing regimens in this patient group to improve safety and efficacy.

TamGen: drug design with target-aware molecule generation through a chemical language model

Generative drug design facilitates the creation of compounds effective against pathogenic target proteins. This opens up the potential to discover novel compounds within the vast chemical space and fosters the development of innovative therapeutic strategies. However, the practicality of generated molecules is often limited, as many designs focus on a narrow set of drug-related properties, failing to improve the success rate of subsequent drug discovery process. To overcome these challenges, we develop TamGen, a method that employs a GPT-like chemical language model and enables target-aware molecule generation and compound refinement. We demonstrate that the compounds generated by TamGen have improved molecular quality and viability. Additionally, we have integrated TamGen into a drug discovery pipeline and identified 14 compounds showing compelling inhibitory activity against the Tuberculosis ClpP protease, with the most effective compound exhibiting a half maximal inhibitory concentration (IC50) of 1.9 μM. Our findings underscore the practical potential and real-world applicability of generative drug design approaches, paving the way for future advancements in the field.

Identification and Ranking of Binding Sites from Structural Ensembles: Application to SARS-CoV-2

Target identification and evaluation is a critical step in the drug discovery process. Although time-intensive and complex, the challenge becomes even more acute in the realm of infectious disease, where the rapid emergence of new viruses, the swift mutation of existing targets, and partial effectiveness of approved antivirals can lead to outbreaks of significant public health concern. The COVID-19 pandemic, caused by the SARS-CoV-2 virus, serves as a prime example of this, where despite the allocation of substantial resources, Paxlovid is currently the only effective treatment. In that case, significant effort pre-pandemic had been expended to evaluate the biological target for the closely related SARS-CoV. In this work, we utilize the computational hot spot mapping method, FTMove, to rapidly identify and rank binding sites for a set of nine SARS-CoV-2 drug/potential drug targets. FTMove takes into account protein flexibility by mapping binding site hot spots across an ensemble of structures for a given target. To assess the applicability of the FTMove approach to a wide range of drug targets for viral pathogens, we also carry out a comprehensive review of the known SARS-CoV-2 ligandable sites. The approach is able to identify the vast majority of all known sites and a few additional sites, which may in fact be yet to be discovered as ligandable. Furthermore, a UMAP analysis of the FTMove features for each identified binding site is largely able to separate predicted sites with experimentally known binders from those without known binders. These results demonstrate the utility of FTMove to rapidly identify actionable sites across a range of targets for a given indication. As such, the approach is expected to be particularly useful for assessing target binding sites for any emerging pathogen, as well as for indications in other disease areas, and providing actionable starting points for structure-based drug design efforts.

Green Medicine: Advancing Antimicrobial Solutions with Diverse Terrestrial and Marine Plant-Derived Compounds

Infectious diseases continue to cause global morbidity and mortality. The rise of drug-resistant pathogens is a major challenge to modern medicine. Plant-based antimicrobials may solve this issue; hence, this review discussed in detail plant-sourced antimicrobial drugs as an alternative toward bacterial, fungal, and viral pathogens. Plant-derived chemicals from various sources such as marine, medicinal, and non-medicinal sources have diverse antimicrobial properties. Complex chemical profiles from these sources allow these molecules to interact with several targets in the microbial pathogens. Due to their multi-component composition, these compounds are more effective and less likely to acquire resistance than single-target antibiotics. Medicinal herbs have long been used for their antimicrobial properties; however, non-medicinal plants have also been identified for their antimicrobial properties. Other interesting new pathways for the identification of antimicrobials include marine plants, which contain a wide variety of metabolites that are both distinct and varied. We have conducted a thorough literature search for the medicinal, non-medicinal, and marine plant-derived molecules with antimicrobial roles from databases which include Scopus, PubMed, Google Scholar, and Web of Science. The review also discussed the synergistic potential of combining these plant-derived compounds with traditional antimicrobial drugs to attenuate the microbial pathogenesis. Based on the existing research and advancements, the review article emphasizes the importance of continuing research into plant-based antimicrobials from these many sources and integrating them with existing therapies to combat the rising threat of drug-resistant infections.

Composition of biopesticides for crop protection – current state and prospects (review)

Aim. To summarise and analyse available information on research into biopesticide compositions aimed at enhancing their effectiveness and to assess prospects for the development of this field so as to determine the influence of components on the effectiveness of biopesticides for agricultural crop protection.This review article presents the results of studies of components included in the compositions of microorganism‐based biopesticides based on a system analysis. It is noted that the selection of biopesticide components must be carried out in accordance with the requirements for products recommended for use in ecological and organic farming systems. It is indicated that the choice of optimal formulation is associated with the characteristics of the producer strains, their trophic needs and resistance to biotic and abiotic factors and with the characteristics of the biopesticide application technology (seed treatment, application to the soil or treatment of plants during vegetation), as well as the type of equipment used.Knowledge of the basic requirements for biopesticide formulations will help maintain the viability and biological activity of microbial agents during storage of biopesticides, taking into account the sensitivity of producer strains to environmental conditions. Selection of optimal carriers, adhesives, stabilisers, thickeners and synergists, taking into account the technology of application of the biopesticide and the target pathogen, will facilitate the development and appearance on the market of new biopesticides for protecting agricultural crops from diseases.

Mitochondria and Alcohol-Associated Liver Disease: Pathogenic Role and Target for Therapy.

Alcohol-associated liver disease (ALD) is one of the leading causes of chronic liver disease and a major cause of liver-related death. ALD is a multifactorial disease triggered by the oxidative metabolism of alcohol which leads to the activation of multiple factors that promote the progression from steatosis to more advanced stages like alcohol-associated steatohepatitis (AH) that culminate in alcohol-associated cirrhosis and hepatocellular carcinoma. Poor understanding of the complex heterogeneous pathology of ALD has limited drug development for this disease. Alterations in mitochondrial performance are considered a crucial event in paving the progression of ALD due to the crucial role of mitochondria in energy production, intermediate metabolism, calcium homeostasis, and cell fate decisions. Therefore, understanding the role of mitochondria in eliciting steatosis and progression toward AH may open the door to new opportunities for treatment. In this review, we will cover the physiological function of mitochondria, its contribution to ALD in experimental models and human disease, and explore whether targeting mitochondria may represent a game changer in the treatment of ALD.

Little disease but lots of bites: social, urbanistic, and entomological risk factors of human exposure to Aedes aegypti in South Texas, U.S.

Aedes aegypti presence, human-vector contact rates, and Aedes-borne virus transmission are highly variable through time and space. The Lower Rio Grande Valley (LRGV), Texas, is one of the few regions in the U.S. where local transmission of Aedes-borne viruses occurs, presenting an opportunity to evaluate social, urbanistic, entomological, and mobility-based factors that modulate human exposure to Ae. aegypti. Mosquitoes were collected using BG-Sentinel 2 traps during November 2021 as part of an intervention trial, with knowledge, attitudes, and practices (KAP) and housing quality surveys to gather environmental and demographic data. Human blood samples were taken from individuals and a Bitemark Assay (ELISA) was conducted to quantify human antibodies to the Ae. aegypti Nterm-34kDa salivary peptide as a measure of human exposure to bites. In total, 64 houses were surveyed with 142 blood samples collected. More than 80% of participants had knowledge of mosquito-borne diseases and believed mosquitoes to be a health risk in their community. Our best fit generalized linear mixed effects model found four fixed effects contributed significantly to explaining the variation in exposure to Ae. aegypti bites: higher annual household income, younger age, larger lot area, and higher female Ae. aegypti abundance per trap night averaged over 5 weeks prior to human blood sampling. Most surveyed residents recognized mosquitoes and the threat they pose to individual and public health. Urbanistic (i.e., lot size), social (i.e., income within a low-income community and age), and entomological (i.e., adult female Ae. aegypti abundance) factors modulate the risk of human exposure to Ae. aegypti bites. The use of serological biomarker assays, such as the Bitemark Assay, are valuable tools for surveillance and risk assessment of mosquito-borne disease, especially in areas like the LRGV where the transmission of target pathogens is low or intermittent.

The inactivation of Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes using isochoric freezing in raw milk and carrot juice

Isochoric freezing is a novel food preservation method that may maintain the quality and safety of products by simultaneously applying low temperatures and high pressures. This work aims to determine whether isochoric freezing can improve food safety by inactivating Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes in raw milk and carrot juices. Raw milk and carrot juice samples were inoculated with cocktails of either STEC or L. monocytogenes and then subjected to isochoric freezing conditions in pressurized chambers set at −5, −10, and −15 °C and treated for 1, 5, 7, or 10 days. Following treatment, the product was sampled for surviving pathogens. The results demonstrated that a 5-log reduction of both STEC and L. monocytogenes can be achieved for raw milk and carrot juice without adversely affecting quality. For STEC inoculated in raw milk, both the linear and Weibull models suggest that 10 days is required to achieve 5-log reduction at −10 °C. Decreasing the temperature to −15 °C led to accelerated log reduction but induced phase separation in the milk. Listeria in raw milk demonstrated a biphasic reduction, indicating 1.3 days is required for a 5-log reduction at −10 °C. In comparison, pathogens demonstrated faster log reduction in carrot juice due to its intrinsic properties. The survival curves for STEC-inoculated carrot juice demonstrated that the 5-log reduction times were 6.9 days at −10 °C and 3.8 days at −15 °C. L. monocytogenes in carrot juice required 1.5 days at −10 °C for a 5-log reduction and was eliminated (7 log cfu/ml) within 24 h at −15 °C. Isochoric freezing could be an option for milk and carrot juice processors wishing to improve food safety without applying heat. This method is not a one-size-fits-all solution, and the time and temperature will need further optimization depending on the target pathogen and intended commodity.

Multiplex PCR detection of enteric pathogens in a community-based birth cohort in Ecuador: comparison of xTAG-GPP and TaqMan array card assays.

We compared the performance of two multiplex platforms, Luminex xTAG Gastrointestinal Pathogen PanelⓇ and TaqMan Array Card, against a panel of 14 enteric pathogen targets in a community-based birth cohort in Ecuador. We found high levels of agreement and similar prevalence estimates across most pathogens.

Exploring Structural and Molecular Features of Sciatic Nerve Lesions in Diabetic Neuropathy: Unveiling Pathogenic Pathways and Targets.

Lesioned fascicles (LF) in the sciatic nerves of individuals with diabetic neuropathy (DN) correlate with clinical symptom severity. This study aimed to characterize the structural and molecular composition of these lesions to better understand DN pathogenesis. Sciatic nerves from amputees with and without type 2 diabetes (T2D) were examined using ex vivo magnetic resonance neurography, in vitro imaging, and proteomic analysis. Lesions were only found in T2D donors and exhibited significant structural abnormalities, including axonal degeneration, demyelination, and impaired blood nerve barrier (BNB). While non-lesioned fascicles from T2D donors showed activation of neuroprotective pathways, lesioned fascicles lacked this response and instead displayed increased complement activation via the classical pathway. The detection of liver-derived acute-phase proteins suggests that BNB disruption facilitates harmful inter-organ communication between the liver and nerves. These findings reveal key molecular mechanisms contributing to DN and highlight potential targets for therapeutic intervention.

Antibody sequence determinants of viral antigen specificity.

Throughout life, humans experience repeated exposure to viral antigens through infection and vaccination, resulting in the generation of diverse, antigen-specific antibody repertoires. A paramount feature of antibodies that enables their critical contributions in counteracting recurrent and novel pathogens, and consequently fostering their utility as valuable targets for therapeutic and vaccine development, is the exquisite specificity displayed against their target antigens. Yet, there is still limited understanding of the determinants of antibody-antigen specificity, particularly as a function of antibody sequence. In recent years, experimental characterization of antibody repertoires has led to novel insights into fundamental properties of antibody sequences but has been largely decoupled from at-scale antigen specificity analysis. Here, using the LIBRA-seq technology, we generated a large data set mapping antibody sequence to antigen specificity for thousands of B cells, by screening the repertoires of a set of healthy individuals against 20 viral antigens representing diverse pathogens of biomedical significance. Analysis uncovered virus-specific patterns in variable gene usage, gene pairing, somatic hypermutation, as well as the presence of convergent antiviral signatures across multiple individuals, including the presence of public antibody clonotypes. Notably, our results showed that, for B-cell receptors originating from different individuals but leveraging an identical combination of heavy and light chain variable genes, there is a specific CDRH3 identity threshold above which B cells appear to exclusively share the same antigen specificity. This finding provides a quantifiable measure of the relationship between antibody sequence and antigen specificity and further defines experimentally grounded criteria for defining public antibody clonality.IMPORTANCEThe B-cell compartment of the humoral immune system plays a critical role in the generation of antibodies upon new and repeated pathogen exposure. This study provides an unprecedented level of detail on the molecular characteristics of antibody repertoires that are specific to each of the different target pathogens studied here and provides empirical evidence in support of a 70% CDRH3 amino acid identity threshold in pairs of B cells encoded by identical IGHV:IGL(K)V genes, as a means of defining public clonality and therefore predicting B-cell antigen specificity in different individuals. This is of exceptional importance when leveraging public clonality as a method to annotate B-cell receptor data otherwise lacking antigen specificity information. Understanding the fundamental rules of antibody-antigen interactions can lead to transformative new approaches for the development of antibody therapeutics and vaccines against current and emerging viruses.

Analytical and clinical validation of direct detection of antimicrobial resistance markers by plasma microbial cell-free DNA sequencing.

This manuscript is ideally suited for the Innovative Diagnostic Methods sections as it reports the analytical and clinical validation of a novel application of plasma microbial cell-free DNA sequencing for direct detection of seven selected antimicrobial resistance markers in 18 target pathogens. Clearly, it has potential clinical utility in optimizing therapy and was incorporated into the Karius test workflow in September 2023. In addition, the workflow could readily be adapted to expand the number of target bacteria and antimicrobial resistance markers as needed.

Loss of Gst1 enhances resistance to MMS by reprogramming the transcription of DNA damage response genes in a Rad53-dependent manner in Candida albicans

The DNA damage response is a highly conserved protective mechanism that enables cells to cope with various lesions in the genome. Extensive studies across different eukaryotic cells have identified the crucial roles played by components required for response to DNA damage. When compared to the essential signal transducers and repair factors in the DNA damage response circuitry, the negative regulators and underlying mechanisms of this circuitry have been relatively under-examined. In this study, we investigated Gst1, a putative glutathione transferase in the fungal pathogen Candida albicans. We found that under stress caused by the DNA damage agent MMS, GST1 expression was significantly upregulated, and this upregulation was further enhanced by the loss of the checkpoint kinases and DNA repair factors. Somewhat counterintuitively, deletion of GST1 conferred increased resistance to MMS, potentially via enhancing the phosphorylation of Rad53. Furthermore, overexpression of RAD53 or deletion of GST1 resulted in upregulated transcription of DNA damage repair genes, including CAS1, RAD7, and RAD30, while repression of RAD7 transcription in the GST1 deletion reversed the strain’s heightened resistance to MMS. Finally, Gst1 physically interacted with Rad53, and their interaction weakened in response to MMS-induced stress. Overall, our findings suggest a negative regulatory role for GST1 in DNA damage response in C. albicans, and position Gst1 within the Rad53-mediated signaling pathway. These findings hold significant implications for understanding the mechanisms underlying the DNA damage response in this fungal pathogen and supply new potential targets for therapeutic intervention.Graphical

Use of T2Bacteria Panel in Pediatric Hematopoietic Stem Cell Transplant Patients: A Single Center Experience

Abstract Background Bloodstream infection is a common complication following hematopoietic cell transplant (HCT), occurring in 10-40% of patients (1,2). Rapid identification of causative bacteria is imperative to provide proper empiric therapy and ensure optimal patient outcomes. The T2Bacteria Panel detects five bacterial pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli) using T2 magnetic resonance (T2MR, T2Biosystems, Lexington MA, USA) directly from blood specimens. Previous studies have reported per-patient sensitivity, specificity, and negative predictive value (NPV) of 84-90%, 85.9-90%, and 99.7%, respectively, as well as 100% positive agreement and 98.4% negative agreement when compared to blood culture (3-5). To date, there have been no studies on the use of this test in pediatric HCT recipients. Methods We reviewed all pediatric HCT patients at St. Jude Children’s Research Hospital who had a T2Bacteria Panel performed for fever and suspected infection between January 2019 and September 2022. A total of 706 T2Bacteria Panel results and concurrent blood culture results from 251 unique patients were analyzed to determine assay performance for detection of the 5 targeted pathogens. Results T2Bacteria PCR and blood culture showed concordant results in 97% of cases. Overall, our study revealed 65% positive percent agreement with blood culture and 95% negative percent agreement for bacteremia caused by any of the target pathogens. PPV, NPV, sensitivity and specificity for each organism detected by the panel, and the panel overall, are presented in Table 1. Positive predictive value ranged from 28-73%. Negative predictive value was high for all organisms (98-100%). Sensitivity was highest for Pseudomonas aeruginosa (88%), and lowest for Escherichia coli (44%). Specificity was high for all organisms (97-99%). Conclusion In pediatric HSCT patients, the T2Bacteria Panel was most useful for rapidly ruling out the presence of target pathogens, with high negative percent agreement, NPV, and specificity. Positive percent agreement was lower than noted in previous studies. Positive predictive value and sensitivity varied per pathogen.

Supramolecular Porphyrin Photosensitizers Based on Host-Guest Recognition for In Situ Bacteria-Responsive Near-Infrared Photothermal Therapy.

Antibiotic resistance resulting from the overuse of antibiotics sets a high challenge for brutal antimicrobial treatment. Although photothermal therapy (PTT) overcomes the awkward situation of antibiotic resistance, it usually mistakenly kills the beneficial bacteria strains when eliminating pernicious bacteria. Specifically recognizing and damaging the target pathogens is urgently required for PTT-mediated sterilization strategy. Based on the host-guest recognition between cucurbit[10]uril (CB[10]) and porphyrins, two water-soluble supramolecular porphyrins are designed and implement selective bactericidal effect via in situ bacteria-responsive near-infrared (NIR) PTT. With the help of CB[10], the π-π stacking and hydrophobic interactions of porphyrins are efficiently inhibited, thus contributing to a good photostability and a high photothermal conversion efficiency. Attributing to the matching reduction potential between facultative anaerobic Escherichia coli (E. coli) and porphyrins, they are selectively in situ reduced into supramolecular phlorin and supramolecular chlorin by E. coli, successfully achieving a selective sterilization against E. coli. In vivo, the in situ bacteria-responsive NIR PTT systems also promote the quick recovery of E. coli-infected abscesses and trauma on mice without inducing obvious systemic toxicity, providing a new alternative to the current antibiotics and helping relieve the global public health crisis of abusive antibiotics.

Fluorescent polymers for environmental monitoring: Targeting pathogens and metal contaminants with naphthalimide derivatives

Monitoring Hg2+ levels in aqueous environments is crucial to assess the potential methylmercury contamination via bacterial conversion, however, existing methods often require extensive sample treatment and expensive equipment. To mitigate this issue, this study examines the synthesis and application of three naphthalimide-based compounds, with significant fluorescent and solvatochromic behavior (C1, C2, and C3). Compounds C1 and C2 demonstrated a strong affinity for Hg2+ metal ions, with C2 showing selectivity and a strong antibacterial profile, particularly against S. aureus (MIC50 (C2) = 0.01 µg/mL). Moreover, these compounds were incorporated into three polymeric matrices, namely polyvinyl chloride (PVC), poly (methyl methacrylate-co-methacrylic acid) (PMMMA), and Starch, allowing for the development of solid-support sensors/surfaces with a strong antibacterial profile, highlighting the inherent dual-functionality of the compounds. Interestingly, the C2-doped Starch biopolymer detected low concentrations of Hg2+ ions, such as 23 nM in tap water (value within the WHO standards for drinking water), through a rapid spectroscopic evaluation without sample treatment. This biopolymer was generated via a sustainable, green-chemistry-oriented, temperature-dependent water/Starch synthetic route, without the addition of plasticizers and any associated ecotoxicity. The study used sustainable methods for environmental monitoring and antibacterial applications, advancing material science to offer effective, accessible, and eco-friendly solutions for detecting and mitigating mercury pollution and bacterial contaminations, enhancing environmental and health safety.

Recognition of phylogenetically diverse pathogens through enzymatically amplified recruitment of RNF213.

Innate immunity senses microbial ligands known as pathogen-associated molecular patterns (PAMPs). Except for nucleic acids, PAMPs are exceedingly taxa-specific, thus enabling pattern recognition receptors to detect cognate pathogens while ignoring others. How the E3 ubiquitin ligase RNF213 can respond to phylogenetically distant pathogens, including Gram-negative Salmonella, Gram-positive Listeria, and eukaryotic Toxoplasma, remains unknown. Here we report that the evolutionary history of RNF213 is indicative of repeated adaptation to diverse pathogen target structures, especially in and around its newly identified CBM20 carbohydrate-binding domain, which we have resolved by cryo-EM. We find that RNF213 forms coats on phylogenetically distant pathogens. ATP hydrolysis by RNF213's dynein-like domain is essential for coat formation on all three pathogens studied as is RZ finger-mediated E3 ligase activity for bacteria. Coat formation is not diffusion-limited but instead relies on rate-limiting initiation events and subsequent cooperative incorporation of further RNF213 molecules. We conclude that RNF213 responds to evolutionarily distant pathogens through enzymatically amplified cooperative recruitment.

A genetic study to identify pathogenic mechanisms and drug targets for benign prostatic hyperplasia: a multi-omics Mendelian randomization study

Benign prostatic hyperplasia (BPH) as a common geriatric disease in urology, the incidence and prevalence are rapidly increasing with the aging society, prompting an urgent need for effective prevention and treatment of BPH. However, limited therapeutic efficacy and higher risk of complications result in the treatment of BPH remaining challenging. The unclear pathogenic mechanism also hampers further exploration of therapeutic approaches for BPH. In this study, we used multi-omics methods to integrate genomics, transcriptomics, immunomics, and metabolomics data and identify biomolecules associated with BPH. We performed transcriptomic imputation, summary data-based Mendelian randomization (SMR), joint/conditional analysis, colocalization analysis, and FOCUS to explore high-confidence genes associated with BPH in blood and prostate tissue. Subsequently, three-step SMR was used to identify the DNA methylation sites regulating high-confidence genes to improve the pathogenic pathways of BPH. We also used cis-instruments of druggable genes to conduct SMR analysis to find potential drug targets for BPH. Finally, we used MR analysis to explore the immune pathways and metabolomics related to BPH. Multiple analytical methods identified BTN3A2 (Blood: TWAS Z score = 5.02912, TWAS P = 4.93 × 10–7; Prostate: TWAS Z score = 4.89, TWAS P = 1.01 × 10–6) and C4A (Blood: TWAS Z score = 4.90754, TWAS P = 9.22 × 10–7; Prostate: TWAS Z score = 5.084, TWAS P = 3.70 × 10–7) as high-confidence genes for BPH and identified the cg14345882-BTN3A2-BPH pathogenic pathway. We also used druggable gene data to identify 30 promising therapeutic target genes, including BTN3A2 and C4A. For MR analysis of immune pathways, we identified immune cell surface molecules as well as the inflammatory factor IL-17 (OR = 1.25, 95% CI = 1.09–1.43, PFDR = 0.12, Maximum likelihood) as risk factors for BPH. In addition, we found that disulfide levels of cysteinylglycine (OR = 1.11, 95% CI = 1.05–1.18, P = 5.18 × 10–4, Weighted median), oxidation levels of cysteinylglycine (OR = 1.09, 95% CI = 1.04–1.14, P = 3.87 × 10–4, Weighted median), and sebacate levels (OR = 1.05, 95% CI = 1.02–1.08, P = 3.0 × 10–4, Maximum likelihood) increase the risk of BPH. This multi-omics study explored biomolecules associated with BPH, improved the pathogenic pathways of BPH, and identified promising therapeutic targets. Our results provide evidence for future studies aimed at developing appropriate therapeutic interventions.

B-216 Ethanol/isopropanol-free and enzyme-free universal nucleic acid extraction and purification system for fast molecular detection from swab samples

Abstract Background Efficient nucleic acid (NA) extraction has been made technology advancement in molecular science. However, there are several challenges yet to address. Firstly, ethanol or isopropanol are important components of the buffers for the most commercially available NA extraction kits. When put on cartridge for POC applications, they can react with plastic materials, undermining their mechanical/physiochemical properties. They can also be problematic due to their volatility, flammability and potential to leak. Secondly, turn-around time for most commercial kits is long and procedure is rather tedious. Thirdly, most of the extraction buffers can only be used to extract either DNA or RNA from either viruses or bacteria. A universal buffer system is lacking. Lastly, special components in some extraction buffers require -20°C or 4°C storage for desired performance, especially for those kits containing proteinase for cell lysis and nuclease denaturation and carrier NA for low quantity of template, causing inconvenience in shipping and storage. Delta developed buffers and associated protocol to address the challenges listed above. This study is to evaluate the performance of the Delta TNA buffer system in NA extraction and purification from various target pathogens in swab samples when compared to that of the leading commercial products. Methods The stock strains of staphylococcus aureus, pseudomonas aeruginosa and H-coronavirus OC43 were diluted in Delta Molecular Transport Media containing swab sample matrix to the testing concentrations before performing NA extraction. Vircell SARS-CoV-2 RNA was spiked directly into PCR reactions to generate a standard curve with 10x serial dilutions for the recovery rate test using Delta TNA kit. 300ul of samples were used for the NA extraction and purification with 80 ul eluate generated. The manufacturers’ instructions were followed. The extracted RNA or DNA was amplified on BioRAD CFX96. Results The extraction efficiency on RNA from H-coronavirus OC43, DNA from pseudomonas aeruginosa and staphylococcus aureus in swab samples, was compared with Katsura Respi and MN Pathogen kits, Qiagen DNA and viral RNA kits respectively. Delta TNA kit outperformed in either earlier Cq value or higher detection rate for different concentrations of analytes in <10 min comparing to up to 95 min for the commercial kits tested. The RNA recovery rate of Delta TNA kit was comparable to that of the Qiagen Viral Kit with 37.54% vs 29.84% at 10 copies/rxn, 146.84% vs 140.76% at 100 cpies/rxn and 118.29% vs 111.76% at 1000 copies/rxn. Meanwhile, Delta Swab TNA buffer was compatible with large spectrum of magnetic beads possessing various coating groups and particle sizes. Conclusions We provided a stable, user- and environment-friendly total nucleic acid extraction and purification system which is ethanol/isopropanol-free and enzyme-free. There is no pre-treatment or drying steps in the protocol and no centrifugation required. Delta’s protocol has the least total number of steps and least amount of total time taken amongst the kits compared. It serves as a universal recipe for both DNA and RNA from pathogens in swab samples including viruses and bacteria in less than 10 min, with competitive performance in both PCR and LAMP.