Biology subjects

Ali, J.

Publications and source records attributed to Ali, J..

At least 19 recordsLinked to original sources

bioRxiv2026

How a highly acidic SH3 domain binds to its intrinsically disordered partner through the formation of an encounter complex intermediate

Electrostatic interactions often play a role in determining the thermodynamic and kinetic properties of protein-protein interactions. However, the role of long-range electrostatic interactions in intrinsically disordered protein (IDP) binding is less clear, as they often bind in multiple steps including initial formation of a disordered encounter complex, followed by rearrangement into the bound state. We varied the salt concentration to probe the role of long-range electrostatic interactions in the binding of the highly charged AbpSH3 domain and the oppositely charged IDP ArkA. Using isothermal titration calorimetry, we observe that salt enthalpically destabilizes the bound complex. Molecular dynamics and NMR experiments reveal that salt has little effect on the bound state structure. However, simulations show that salt destabilizes the encounter complex intermediate, which primarily affects the association rate as measured by NMR. Consistent with these results, salt has the largest stabilizing effect on the apo SH3 domain, as cations substitute for the transient and long-range electrostatic interactions that can form with ArkA in the complex. We reveal a detailed picture of how a highly charged domain uses long-range, fuzzy, electrostatic interactions to help reach the bound state, a mechanism that is likely common among other highly charged domains that bind IDPs. TOC Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/741257v1_ufig1.gif" ALT="Figure 1000"> View larger version (21K): org.highwire.dtl.DTLVardef@dfc7acorg.highwire.dtl.DTLVardef@1ae0438org.highwire.dtl.DTLVardef@19704adorg.highwire.dtl.DTLVardef@1b40b15_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

From culture to clarity in four hours: accelerating clinical management of bloodstream infections using metagenomics

BackgroundMetagenomic next-generation sequencing (mNGS) has the potential to transform clinical diagnostics for bloodstream infections (BSIs). However, its clinical utility is currently limited by several challenges, including the extraction of DNA from blood cultures. Our aim was to develop, evaluate, and optimize an in-house method for host depletion and bacterial DNA extraction from positive blood cultures to enable rapid mNGS-based pathogen detection and antimicrobial resistance profiling, informing clinical management of BSIs. Methods151 clinical blood cultures (115 positive and 36 negative) were processed for DNA extraction using an in-house-developed SEPSINN method for host depletion and bacterial DNA extraction. mNGS on the MinION was performed, and the results for pathogen identification and antimicrobial susceptibility predictions were compared with the routine clinical workflow. SEPSINN was also evaluated against a commercial DNA extraction method to assess its effectiveness in depleting host DNA and recovering bacterial DNA. ResultsThe SEPSINN method achieved up to 1000-fold depletion of host DNA and outperformed the commercial DNA extraction method. At the sample level, mNGS achieved 100% accuracy, specificity, and sensitivity, identifying at least one pathogen in all 115 positive blood cultures. At the pathogen level, mNGS showed 98% accuracy (120/123), specificity, and sensitivity. For antimicrobial susceptibility predictions, mNGS achieved an accuracy of 95% (1382/1451), a sensitivity of 88% (203/230), and a specificity of 97% (1179/1221). Moreover, the method also identified fungi, indicating a wider taxonomic range. mNGS resulted in an approximately 4-hour turnaround time for pathogen identification and resistance profiling. ConclusionsThe method can provide information on BSI clinical management within approximately 24 hours of receiving the sample, including the time required for culture positivity. This represents an important advancement in the clinical management of BSIs, with the potential to save lives and promote antibiotic stewardship.

microbiology↗

bioRxiv2026

Structural Tuning of HEWL Amyloid Polymorphs Enhances Antibacterial Activity Against Gram-Positive and Gram-Negative Pathogens

Amyloid fibrils are traditionally associated with protein misfolding disorders; however, increasing evidence indicates that they can also perform beneficial biological functions, including antimicrobial defense. Here, we investigated whether structurally distinct amyloid polymorphs of hen egg white lysozyme (HEWL) exhibit enhanced antibacterial activity compared with the native protein. HEWL was converted into two amyloid polymorphs, flexible fibrils (FFs) and rigid fibrils (RFs), and their antibacterial activities were evaluated against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacteria Escherichia coli (Top10) and Salmonella Typhimurium. Fibril formation was confirmed by circular dichroism (CD) spectroscopy, thioflavin T (ThT) fluorescence, and transmission electron microscopy (TEM), demonstrating morphologically distinct amyloid assemblies with different secondary-structure organizations. Fluorescence-based bacterial growth assays showed that native HEWL exhibited only moderate antibacterial activity, whereas both amyloid polymorphs produced potent, concentration-dependent bacterial growth inhibition. FFs and RFs consistently displayed greater antibacterial efficacy than native HEWL across all tested strains, with FFs exhibiting slightly stronger activity against S. Typhimurium. At concentrations of 600-800 M, FFs achieved >90% growth inhibition for all bacterial species examined. Cytotoxicity studies using SH-SY5Y human neuroblastoma cells demonstrated minimal toxicity for native HEWL, modest effects for FFs, and substantially greater toxicity for RFs, indicating that amyloid polymorphism influences both antimicrobial activity and mammalian cell compatibility. Collectively, these findings establish a direct relationship between amyloid structure, antibacterial efficacy, and cytotoxicity. The combination of potent antibacterial activity and relatively low cytotoxicity identifies FFs as a promising functional amyloid biomaterial for the development of next-generation antimicrobial materials.

bioengineering↗

bioRxiv2026

Remote underwater photographs reveal environmental correlations and patterns in reef manta ray habitat use in Laamu Atoll, Maldives

Reef manta rays (Mobula alfredi) are threatened by fishing and other anthropogenic threats. Which, when coupled with conservative life history traits, have made this species vulnerable to extinction. Spatiotemporal ecological knowledge, such as site fidelity and visitation patterns to key aggregation sites, are imperative for effective conservation management of M. alfredi. A novel method of environmental sensing, remote underwater photo systems (RUPs), was employed to understand drivers of M. alfredi habitat use and resighting patterns. RUPs were deployed at four cleaning sites around Laamu Atoll, Maldives. Between March 2021 and May 2023, 455,458 photos were analysed. Generalised linear models revealed increases in M. alfredi presence in response to high chlorophyll-a concentrations, low illumination moon states, the Southwest Monsoon, and in the morning, while human presence had no effect. Branchial spot patterns allowed for 81 M. alfredi individuals to be identified, from 629 sightings, representing 51.59% of Laamu Atolls previously identified population (n = 157). Cleaning stations are visited more intensively during periods of increased productivity of the Southwest Monsoon, likely in response to greater foraging opportunities near the study areas. Additionally, moon state, used as a proxy for tidal strength, was associated with increased visitation during new moon periods, suggesting that weaker tidal states may facilitate presence. These data support integrating RUPs with observational surveys to improve inferences about habitat use and our understanding of cleaning sites frequented by M. alfredi. This study aims to inform the implementation of Laamu Atolls first marine protected area management plan.

ecology↗

bioRxiv2026

First occurrence of Corynespora cassiicola infecting chia plant in Bangladesh and its sensitivity to selected fungicides

Chia (Salvia hispanica L.) is an emerging crop in Bangladesh valued for its medicinal properties and economic significance. In March 2024, target spot-like symptoms were observed in an experimental chia field (24.75{degrees} N, 90.50{degrees} E) at Bangladesh Agricultural University in Mymensingh, Bangladesh with disease incidence ranging from 23% to 47% across approximately 0.25 ha. Initially appearing as brick-red spots, these symptoms developed into target-shaped concentric rings, affecting leaves, stems, and inflorescences. A total of 24 fungal isolates were recovered from infected tissue; two representative isolates (BGECh-3 and BGECh-4) were randomly selected for details characterization. Pathogen identity was established through morphological traits, multilocus phylogenetic analysis of internal transcribed spacer (ITS) and elongation factor 1-alpha (EF-1) genes sequence, and pathogenicity confirmation through Kochs postulates, collectively identifying the causal agent as Corynespora cassiicola. The isolates demonstrated a broad host range, successfully infecting brinjal, chili, bottle gourd, country bean, tomato, and soybean. In vitro fungicide sensitivity assays with seven commercial fungicides showed that both isolates were highly sensitive to Goldzim (50% carbendazim), which completely inhibited mycelial growth at 10 {micro}g mL-{superscript 1}. Conza (10% Hexaconazole) and Amister top (18.2% azoxystrobin + 11.4% difenoconazole) reduced growth by up to 85% and 67%, respectively at equal concentration. Other fungicides showed comparatively lower efficacy even at higher concentrations. This study represents the first report of target spot disease of chia caused by C. cassiicola in Bangladesh and provides insights for effective disease management strategies.

molecular biology↗

bioRxiv2026

Prefrontal brain-to-brain synchrony during human group hunting: Evidence from fNIRS hyperscanning

Orca, wolves, chimpanzees and humans share a similarly impressive capacity for group hunting, where individuals coordinate behaviour together to capture prey. Studying hunting behaviours has important implications for understanding how behaviour in group contexts may be indicative of cognitive decline. Despite growing interest in brain circuits for prey capture, the brain regions involved in tracking prey during a hunt and the behaviours in group hunt linked to success remain unclear. Here we combined functional near infrared spectroscopy (fNIRS) and a virtual minecraft world to examine behaviour, brain dynamics and brain synchrony involved in group hunting behaviour. We focused on the prefrontal cortex (PFC) due to its known role in planning and social coordination and recorded from pairs of individuals as they either cooperated to hunt another person (prey) or simply followed another person. Hunters were more successful if they managed to keep a smaller distance to the prey and moved at speeds that were more synchronised with their co-predator. At high-range frequencies for fNIRS (0.1-0.2Hz), we found greater brain-to-brain synchrony in lateral and medial (frontopolar) PFC regions during hunting compared with chance levels. Together, these findings provide insights into what behaviours and brain dynamics associated with successful group hunting.

neuroscience↗

bioRxiv2025

Extracellular ssDNA from P. tobira Exerts Strong Insecticidal Activity on C. hesperidum

Beyond its function as a carrier of hereditary information, recent research has uncovered novel properties of extracellular DNA, including its role in the adaptation to the environment when released from plants. The secreted DNA has been shown to exert insecticidal effects against insect pests, which play an adaptive role in plant-insect interactions, particularly in regulating populations of economically important sap-feeding insects. The molecular mechanisms underlying this insecticidal effect are underinvestigated and remain largely unknown. Therefore, there is a need for more efforts to uncover these mechanisms to better understand the plant-pest interactions, which would provide new insights into natural pest control strategies and inspire biotechnological applications. In the current study, we show that Pittosporum tobira (P. tobira) secretes single-stranded DNA (ssDNA) that exerts an insecticidal effect on Coccus hesperidum (C. hesperidum). We collected extracellular DNA from P. tobira leaves and tested its potential insecticidal effect by applying it to C. hesperidum, which is a well-known pest that causes damage to P. tobira. Our results revealed that the outermost layer of the leaf cuticle of P. tobira predominantly contains ssDNA of approximately 100 nt in length, originating from both chloroplast and nuclear genomes. This DNA exhibited pronounced insecticidal activity against C. hesperidum, with chloroplast-derived sequences significantly enriched compared to the total DNA in intact plant cells. These findings suggest that the microevolution of the P. tobira nucleome and plastome contributed to the formation of extracellular DNA with insecticidal properties (eci-DNA), which is part of its defence strategy against insect pests. Moreover, in this article for the first time we show that antisense DNA (oli-gonucleotide insecticide Coccus-11) is capable of activating insect retrotransposons and upregulating their RT-RNase H, a crucial enzyme for the DNA containment mechanism and successful action of oligonucleotide insecticides. Notably, the laboratory-developed ssDNA-based genetic zipper technology, designed for sustainable pest management, possesses characteristics similar to eci-DNA found in nature, highlighting a potential natural parallel to this biotechnological approach for sustainable pest management. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/689116v4_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1552668org.highwire.dtl.DTLVardef@14c84e3org.highwire.dtl.DTLVardef@a87493org.highwire.dtl.DTLVardef@17d624f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Optimizing Sample Preparation for Direct Nanopore Sequencing to Enable Rapid Pathogen and Antimicrobial Resistance Profiling in Bovine Mastitis

Long-read metagenomic sequencing allows for the rapid, culture-independent, and accurate identification of causative pathogens and antimicrobial resistance (AMR) profiles, supporting precise antibiotic use and reducing the spread of resistance. However, its application to mastitis milk is challenging due to the complex milk matrix, low bacterial count, and high somatic cell content. This study primarily aimed to further optimize our previously developed direct sequencing protocol for milk samples from mastitis cases. Additional optimizations included combining centrifugation, gradient centrifugation, and fat fraction treatment with Tween 20 and citric acid. Subsequently, four DNA extraction kits (Blood and Tissue, Molysis Complete5, HostZero, and SPINeasy Host depletion) were evaluated for their ability to remove host DNA and enrich bacterial DNA for long-read sequencing with Oxford Nanopore technologies. qPCR was used to quantify bacterial and bovine DNA, allowing comparison of host depletion efficiency among the kits. Our results show that simple centrifugation effectively concentrates bacterial cells, removing the need for chemical treatments. The HostZero kit consistently produced higher DNA yields, better DNA integrity, and more effective host DNA depletion. Using nanopore sequencing, both Gram-positive and Gram-negative mastitis pathogens, along with their AMR genes, were successfully detected. Overall, this study underscores the importance of an effective DNA extraction method for the direct sequencing of mastitis milk samples. Additionally, our findings support the potential of direct metagenomic sequencing as a rapid, culture-free approach for identifying mastitis pathogens and their resistance profiles.

microbiology↗

bioRxiv2025

Development and optimization of the host DNA depletion in blood cultures using a saponin and SAN nucleases-based method

Bloodstream infections and sepsis are major health issues causing millions of deaths annually. Early and accurate diagnosis of sepsis is crucial for reducing mortality and combating antimicrobial resistance. However, current diagnostic methods are slow and time-consuming. Nanopore sequencing has the potential to serve as a rapid diagnostic method for sepsis. However, extracting bacterial DNA from blood samples is challenging due to the high content of host DNA. This study aimed to develop and optimize a method using saponin and SAN nucleases for effective host DNA depletion in blood cultures. Different concentrations of saponin, HL-SAN, and M-SAN nucleases, combined with various salt conditions (NaCl and MgCl2), were tested for their ability to deplete host DNA in blood cultures spiked with E. coli and S. aureus. The impact of different bead beating durations on DNA fragment sizes was also examined. The efficiency of host DNA depletion and bacterial DNA extraction was assessed using quantitative PCR (qPCR) and nanopore sequencing. Results indicated that 4% of saponin effectively lysed host cells, releasing DNA while preserving bacterial cells. The host DNA is then efficiently depleted using 250 units (10 L) of HL-SAN and M-SAN nucleases. Although SAN in combination with higher salt concentrations (2.5 M NaCl and 50 mM MgCl2) showed somewhat better host DNA removal, the difference was not statistically significant. Reducing bead beating time to 6 minutes improved the recovery of longer DNA fragments compared to 10 minutes. These results demonstrate an effective method for host DNA depletion using saponin and SAN nucleases, which is compatible with downstream processes, such as nanopore sequencing and qPCR.

microbiology↗

bioRxiv2025

Low-Cost In-House Re-formulated Brain Heart Infusion Medium for Effective Planktonic Growth and Early Detection of Bloodstream Bacterial Pathogens

Sepsis, a clinically defined life-threatening condition, is a global contributor to high morbidity and mortality rates in humans. It is caused by systemic bloodstream bacterial infections, primarily involving aerobic pathogens such as Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae. Rapid and accurate identification of these pathogens is a high-demand task, as prolonged diagnosis may increase the mortality rate among sepsis patients. Worldwide, commercial blood culture systems, such as BD BACTEC PLUS Aerobic/F /F culture bottles (used in this study), are routinely used to monitor bloodstream infections. However, due to high costs ($10.00-$15.00/bottle), limited availability of culture media (especially in low- and middle-income countries, and war zones), and a lack of customization for antibiotic susceptibility assay and epidemiology research, there is a need for secondary alternatives to facilitate the growth and identification of bloodborne pathogens. Therefore, we developed a low-cost ($4-$5/bottle) in-house culture medium with a newly improved formulation of Brain Heart Infusion media that enhances bacterial growth from spiked human blood tested on a panel of bacteria (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecalis). The growth dynamics of these microbes in in-house formulated BHI-Blood+ culture media coincide with those in BACTEC Plus Aerobic/F culture vials, which primarily suggests the compatibility of bloodborne pathogens with this media and can be flagged positive <8h based on cellular growth rate. Additionally, conventional qPCR-based early detection (< 24h) and validation with the Oxford Nanopore MinION NGS platform highlight the value of this in-house culture media as an alternative to commercial culture media in terms of low-cost availability.

microbiology↗

bioRxiv2025

In vitro and in vivo synergy of Vancomycin and β-lactams against drug-resistant Mycobacterium tuberculosis and Non-tuberculous mycobacteria

The unabated increase in antimicrobial resistance has underlined the importance of identifying novel drug combinations which eliminate infections more potently and likely reduce the emergence of resistance. In this context, we have identified Vancomycin and many {beta}-lactams as being potently active against drug-resistant Mycobacterium tuberculosis and non-tuberculous mycobacteria including M. abscessus, emerging as pathogens of concern owing to their inherent drug resistance profile. In this study, we have identified combinations of Vancomycin, a glycopeptide and {beta}-lactams, especially Ceftriaxone, Ceftazidime and Meropenem, in the presence or absence of Sulbactam, a {beta}- lactamase inhibitor, as possessing potent antimicrobial activity against several drug-resistant mycobacterial strains. The combination of Vancomycin and {beta}-lactams exhibited potent bactericidal activity and reduced the bacterial load better than either drug alone. The molecular basis of synergy was mediated by increase in permeability of mycobacterial cell as demonstrated by ethidium bromide assay. In the murine model of mycobacterial infection, synergistic combination of Vancomycin and {beta}-lactams outperformed clinically utilized drugs including Isoniazid, Rifampicin and Ethambutol against M. tuberculosis and Amikacin, Clarithromycin against M. abscessus. The combinations caused a significant reduction in bacterial load in various organs in M. tuberculosis and M. abscessus infected mice. Thus, the synergistic combination of Vancomycin and {beta}-lactams could potentially be utilized for treatment of recalcitrant mycobacterial infection especially those caused due to drug-resistant pathogens.

microbiology↗

bioRxiv2025

Future flooding tolerant rice germplasm: resilience afforded beyond Sub1A gene

Developing high-yielding, flood-tolerant rice varieties is essential for enhancing productivity and livelihoods in flood-prone ecologies. We explored genetic avenues beyond the well-known SUB1A gene to improve flood resilience in rice. We screened a collection of 6,274 elite genotypes from IRRIs germplasm repository for submergence and stagnant flooding tolerance over multiple seasons and years. This rigorous screening identified 89 outstanding elite genotypes, among which thirty-seven exhibited high submergence tolerance, surpassing the survival rate of SUB1A introgression genotypes by 40-50%. Thirty-five genotypes showed significant tolerance to stagnant flooding, and 17 demonstrated dual tolerance capabilities, highlighting their adaptability to varying flood conditions. The genotypes identified have a broader genetic diversity and harbor 86 key QTLs and genes related to traits such as grain quality, grain yield, herbicide resistance, and various biotic and abiotic traits, highlighting the richness of the identified elite collection. Besides germplasm, we introduce an innovative breeding approach called Transition from Trait to Environment (TTE). TTE leverages a parental pool of high-performing genotypes with complete submergence tolerance to drive population improvement and enable genomic selection in the flood breeding program. Our approach of TTE achieved a remarkable 65% increase in genetic gain for submergence tolerance, with the resulting fixed breeding genotypes demonstrating exceptional performance in flood-prone environments of India and Bangladesh. The elite genotypes identified herein represent invaluable genetic resources for the global rice research community. By adopting the TTE approach, which is trait agonistic, we establish a robust framework for developing more resilient genotypes using advanced breeding tools. Plain Language SummaryTo address climate challenges, an urgent focus is necessary to identify and develop flood-tolerant rice varieties, particularly for flood-prone ecosystems across Asia and Africa. We screened 6,274 elite genotypes from IRRIs germplasm and identified 89 promising lines with improved tolerance to submergence and stagnant flooding. Among these, 37 demonstrated 40-50% greater submergence tolerance than SUB1A introgression lines, 35 exhibited stagnant flooding tolerance, and 17 showed dual tolerance. These genotypes contain 86 key QTLs and genes associated with yield, grain quality, and biotic and abiotic tolerance traits. A new breeding strategy, the Transition from Trait to Environment (TTE) approach, was developed. We achieved a genetic gain of 65% for submergence tolerance in rice using this method. The newly identified germplasm provides invaluable genetic resources for the global rice research community to develop flood-tolerant rice genotypes. Core ideas The SUB1A gene, enabling rice to survive underwater for 14 days, marked a significant breakthrough. We have identified elite genotypes with submergence tolerance significantly surpassing the SUB1A gene-mediated tolerance. The diverse elite genotypes identified harbor 86 key genes and QTLs that affect various traits positively. Developed a unique breeding strategy for implementing population improvement in challenging environments. The new breeding strategy demonstrated a genetic gain of 65% for submergence tolerance.

plant biology↗

bioRxiv2025

Anionic lipid catalyzes the generation of cytotoxic insulin oligomers

Misfolding and aggregation of proteins into amyloidogenic assemblies are key features of several metabolic and neurodegenerative diseases. Human insulin has long been known to form amyloid fibrils under various conditions, which affects its bioavailability and function. Clinically, insulin aggregation at recurrent injection sites poses a challenge for diabetic patients who rely on insulin therapy. Furthermore, decreased responsiveness to insulin in type 2 diabetic (T2D) patients may lead to its overproduction and accumulation as aggregates. Earlier reports have reported that various factors such as pH, temperature, agitation, and the presence of lipids or other proteins influence insulin aggregation. Our present study aims to elucidate the effects of non-micellar anionic DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) lipids on insulin aggregation. Distinct pathways of insulin aggregation and intermediate formation were observed in the presence of DMPG using a ThT fluorescence assay. The formation of soluble intermediates, alongside large insulin fibrils, was observed in insulin incubated with DMPG via TEM, DLS and NMR, as opposed to insulin aggregates generated without lipids. 13C magic angle spinning solid-state NMR and FTIR experiments indicated that lipids do not alter the conformation of insulin fibrils but do alter the time scale of motion of aromatic and aliphatic sidechains. Furthermore, the soluble intermediates were found to be more cytotoxic as compared to fibrils generated with or without lipids. Overall, our study elucidates the importance of anionic lipids in dictating the pathways and intermediates associated with insulin aggregation. These findings could be useful in determining various approaches to avoid toxicity and enhance the effectiveness of insulin in therapeutic applications.

biophysics↗

bioRxiv2024

Cleavage site heterogeneity at the pre-mRNA 3'-untranslated region regulates gene expression

Endonucleolytic cleavage step of the eukaryotic mRNA 3'-end processing is considered imprecise that leads to heterogeneity of cleavage site (CS) with hitherto unknown function. Contrary to the popular belief, we show that this imprecision in cleavage is tightly regulated resulting in CS heterogeneity (CSH) that controls gene expression in antioxidant response. CSH centres at a primary CS followed by a number of subsidiary cleavages that is determined by the position of the CS. Globally and by using reporter antioxidant NQO1 mRNA, we discovered an inverse relationship between the number of CS and the gene expression with the primary CS exhibiting highest cleavage efficiency. Strikingly, reducing CSH and increasing primary CS usage induces gene expression. Under oxidative stress condition (tBHQ, H2O2 or NaAsO2), there is a decrease in the CSH and an increase in the primary CS usage to induce antioxidant gene expression. Concomitantly, ectopic NQO1 expression from the primary CS or reduction in CSH imparts cellular oxidative stress tolerance. Genome-wide CS analysis of stress response genes also shows a concomitant result. We show that oxidative stress induces affinity/strength of cleavage complex assembly increasing the fidelity of cleavage at the primary CS thereby reducing CSH inducing antioxidant response.

molecular biology↗

bioRxiv2024

Detection of pathogens and antimicrobial resistant genes from urine within 5 hours using Nanopore sequencing

PurposeUrinary Tract Infection (UTI) is a prevalent global health concern accounting for 1-3% of primary healthcare visits. The current methods for UTI diagnosis have a high turnaround time of 3-5 days for pathogen identification and susceptibility testing. This work is a proof-of-concept study aimed at determining the detection limit by establishing a culture and amplification-free DNA extraction methodology from spiked urine samples followed by real-time Nanopore sequencing and data analysis. MethodsThis study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Pathogens were spiked at two clinically significant concentrations of 103 and 105 CFU/ml. Three commercial DNA extraction kits were investigated based on the quantity of isolated DNA, average processing time, elution volume and the average cost incurred per extraction. The outperforming kit was used for direct DNA extraction and subsequent sequencing on MinION and Flongle flowcells. ResultsThe Blood and Tissue kit outperformed the other kits. All pathogens were identified at a concentration of 105 CFU/ml within ten minutes, and the corresponding AMR genes were detected within three hours of the sequencing start. The overall turnaround time including the DNA extraction and sequencing steps was five hours. Moreover, we also demonstrate that the identification of some pathogens and antibiotic-resistance genes was possible at a spike concentration of 103 CFU/mL. ConclusionThis study shows great promise toward reducing the time required for making an informed antibiotic administration from approximately 48 hours to five hours thereby reducing the number of empirical doses and saving lives.

microbiology↗

bioRxiv2023

Genetic Gains in IRRIs Rice Salinity Breeding and Elite Panel Development as a Future Breeding Resource

Genetic gain is a crucial parameter to check the breeding programs success and help optimize future breeding strategies for enhanced genetic gains. In this work, IRRIs historical data from the Philippines and Bangladesh of the salinity breeding program was used to estimate the genetic gains and identify the best lines based on higher breeding values for yield as a future genetic resource. Two-stage mixed-model approach accounting for experimental design factors and pedigrees was adopted to obtain the breeding values for yield and estimate genetic trends under the salinity conditions. A positive genetic trend of 0.1% per annum with a yield advantage of 1.52 kg/ha for the Philippines and 0.31% per annum with a yield advantage of 14.02 kg/ha for Bangladesh datasets was observed. For the released varieties, genetic gain was 0.12% per annum with a yield advantage of 2.2 kg/ha/year and 0.14% per annum with a yield advantage of 5.9 kg/ha/year, respectively. Further, based on higher breeding values for grain yield, a core set of the top 145 genotypes with higher breeding values of >2400 kg/ha in the Philippines and >3500 kg/ha in Bangladesh with a selection accuracy >0.4 were selected for formulating the elite breeding panel as a future breeding resource. Conclusively, higher genetic gains are pivotal in IRRIs rice salinity breeding program, which requires a holistic breeding approach with a major paradigm shift in breeding strategies to enhance genetic gains. Key MessageEstimating genetic gains and formulating a future salinity elite breeding panel for rice pave the way for developing better high-yielding salinity tolerant lines with enhanced genetic gains.

genetics↗

bioRxiv2022

A glycine zipper motif governs translocation of type VI secretion toxic effectors across the cytoplasmic membrane of target cells

Type VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here we use the Agrobacterium tumefaciens DNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 binds to its adaptor Tap1 through the N-terminus, which harbours continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. Amino acid substitutions on G39xxxG43 motif does not affect Tde1-Tap1 interaction and secretion but abolish its membrane permeability and translocation of its fluorescent fusion protein into prey cells. The data suggest that G39xxxG43 governs the delivery of Tde1 into target cells by permeabilizing the cytoplasmic membrane. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore-forming toxins, we propose that glycine zipper mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes.

microbiology↗

bioRxiv2022

Multi-excitation Raman Spectroscopy Complements Whole Genome Sequencing for Rapid Detection of Bacterial Infection and Resistance in WHO Priority Pathogens

Current methods for diagnosing acute and complex infections mostly rely on culture-based methods and, for biofilms, fluorescence in-situ hybridization. These techniques are labor-intensive and can take 2-4 days to return a test result, especially considering an extra culturing step required for the antibiotic susceptibility testing (AST). This places a significant burden on healthcare providers, delaying treatment and leading to adverse patient outcomes. Here, we report the complementary use of our newly developed multi-excitation Raman spectroscopy (ME-RS) method with whole-genome sequencing (WGS). Four WHO priority pathogens are AST phenotyped and their antimicrobial resistance (AMR) profile determined by WGS. On application of ME-RS method we find high correlation with the WGS characterization. Highly accurate classification based on the species (98.93%), wild-type/non-wild type (99.45%), and presence or absence of thick peptidoglycan layers in cell walls (100%), as well as at the individual strain level (99.29%). These results clearly demonstrate the potential of ME-RS as a rapid and first-stage tool for species, resistance and strain-level classification which can be followed up by WGS for confirmation. Such a workflow can facilitate efficient antimicrobial stewardship to handle and prevent the spread of AMR.

microbiology↗