Biology subjects

Alam, M. S.

Publications and source records attributed to Alam, M. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Experimental evolution and functional genetics identify KIC1 as a determinant of reduced artemisinin susceptibility in Bangladeshi Plasmodium falciparum

Bangladesh has greatly reduced malaria transmission, but persistent Plasmodium falciparum transmission in the Chittagong Hill Tracts (CHT), bordering Myanmar, remains a concern for elimination. Emerging artemisinin resistance could threaten remaining control efforts, making it important to identify determinants of reduced artemisinin susceptibility in CHT parasites. We combined long-term in vitro dihydroartemisinin (DHA) selection, whole-genome sequencing, functional genetics, and analysis of contemporaneous clinical isolates to define artemisinin-response variation in CHT P. falciparum. Starting with a 2018 patient-isolated, artemisinin-sensitive CHT clone, three independent cultures were exposed over 18 months to 27 cycles of stepwise DHA pressure up to 1600 nM. This generated CHT-R lines with elevated RSA survival relative to parental/sibling controls (4.0-6.5% versus <1%; 7-11-fold increase) and faster post-DHA recovery than CHT-S-sib (5.2 {+/-} 0.3 versus 10.1 {+/-} 0.57 days). Whole-genome sequencing identified convergent disruption of PF3D7_0606000, encoding KIC1 (Kelch13 Interacting Candidate1), a protein linked to the Kelch13-associated endocytic compartment; no pfkelch13 mutations emerged. Independent DHA-selected lines acquired distinct stop-gain or frameshift mutations predicted to truncate KIC1, and targeted pfkic1 disruption in parental CHT-S increased RSA survival and accelerated recovery, functionally validating KIC1 as a contributor to reduced artemisinin susceptibility. To link the in vitro selection findings to naturally circulating parasites, we tested whether pfkic1 variation was associated with artemisinin response in an independent set of CHT isolates. We integrated newly generated RSA measurements and whole-genome variation from lab adapted field isolates all carrying wild-type pfkelch13, with patient clearance values from our CHT artemether-lumefantrine efficacy study. Among 22 isolates, RSA survival ranged from 0.00 to 6.44%, with 12 exceeding the 1% in vitro ART-R threshold and was associated with PC50, the time required to clear 50% of the initial parasite density, but not parasite clearance half-life (PCt1/2). In this field-isolate dataset, targeted pfkic1 gene-score analysis and elastic-net modeling provided supportive evidence that natural variation at the pfkic1 locus is associated with PC50 and RSA survival; an exploratory genome-wide gene-score scan nominated additional candidate loci for future study. Together, these findings identify KIC1 as a functionally validated determinant of reduced artemisinin susceptibility in a Bangladeshi parasite background and suggest that artemisinin-response variation in CHT parasites may involve perturbation of the Kelch13-associated endocytic pathway beyond canonical pfkelch13 mutations.

microbiology↗

bioRxiv2026

Genomic Surveillance of Respiratory Syncytial Virus among Patients with Acute Respiratory Infection through Hospital-Based Influenza Surveillance Platforms in Bangladesh, August 2024-December 2025

BackgroundRSV is one of the major contributing factors of acute lower respiratory tract infection among younger children who are below five years old; over 95% of the burden falls on low- and middle-income countries. Bangladesh faces a substantial pediatric RSV burden yet lacks post-pandemic whole-genome data. Approval of nirsevimab and maternal vaccines (Arexvy, Abrysvo), underscores the need for region-specific genomic surveillance to inform prevention and control strategies. ObjectivesTo conduct the whole-genome sequencing (WGS) study of RSV in Bangladeshi population, characterizing viral genotypic diversity, phylogenetics, F and G protein mutations, glycosylation dynamics, while generating and depositing complete genomes in GISAID. MethodsBetween August 2024 and December 2025, of 1,390 RSV-positive specimens (Ct [≤]35), 59 high-viral-load samples (Ct [≤]25) were selected for whole-genome sequencing using Oxford Nanopore Technology (ARTIC primers), assembled with MIRA v2.0.0, and analyzed via Augur/Nextstrain, Nextclade, and NetNGlyc/NetOGlyc 4.0. ResultsOf 11,874 patients, 1,390 (11.7%) were RSV-positive (RSV-A 94.6%). From selected 59 RSV-positive cases, 49 high-quality genomes were generated (83.1% pass; 94.7% completeness; median depth 1,500x, range 443-4,948): 43 RSV-A (ON1; A.D.3.7 63%, A.D.3 19%, A.D.3.12 9%, A.D.3.1 7%, A.D.1.11 2%) and 6 RSV-B (BA9/B.D.E.1). S276N at antigenic site II (34.9% RSV-A) and S389P in all RSV-B were detected; neither confers resistance to nirsevimab or palivizumab. An F protein N75 N-glycosylation site was fixed in all RSV-A; RSV-B acquired HVR2 N256 glycan in 67%. All 49 genomes were deposited in GISAID. ConclusionThis WGS study of RSV in the Bangladeshi population confirms LMIC nanopore surveillance feasibility, multi-lineage co-circulation, and intact conservation of all vaccine and antibody targets. Progressive glycan remodeling warrants monitoring. These findings establish a genomic baseline to guide nirsevimab and maternal vaccine deployment in Bangladesh, with direct relevance for RSV surveillance programs across South Asia.

evolutionary biology↗

bioRxiv2026

A synonymous mutation in MSMEG_4729 occurs at a high frequency in spontaneous D29-resistant mutants of Mycobacterium smegmatis

Compassionate use of mycobacteriophage therapy highlights the promising potential of phage therapy as an alternative treatment option for antibiotic-resistant infections when conventional treatments fail. However, realizing the full potential of phage therapy requires addressing key challenges, including host immune responses, the limited arsenal of therapeutically-useful mycobacteriophages, and the emergence of phage resistance. Dissecting the mechanisms of phage resistance is critical for ensuring the effectiveness and sustainability of phage therapy. In this study, we demonstrate that exposure to the lytic mycobacteriophage D29 triggers diverse genetic changes in Mycobacterium smegmatis. A synonymous mutation in MSMEG_4729 arises frequently but is insufficient to confer D29 resistance on its own. Instead, we identified possible Lsr2-independent activation of the lipooligosaccharide (LOS) biosynthesis cluster in a D29-resistant mutant harboring this mutation. We have also detected the possible activity of MSMEG_3213, a type II methyltransferase associated with m6A modifications in M. smegmatis. Finally, we isolated defense escape mutants (DEMs) of D29 capable of overcoming resistance in a strain with the MSMEG_4729 synonymous mutation. This profiling of M. smegmatiss likely defensive arsenal against the therapeutically-useful mycobacteriophage D29 provides a roadmap for further investigations and rational engineering of next-generation mycobacteriophages to combat drug-resistant mycobacterial infections. Impact statementInterest in phage therapy has been gaining traction recently, which is largely due to the serious threat of antimicrobial resistance. However, the efficacy and sustainability of phage therapy is threatened by certain challenges, which includes the ever existent threat of phage resistance. In this study, we identified several likely factors involved in D29 interaction with the model mycobacterium M. smegmatis. These findings set a roadmap for future investigations that would guide rational phage engineering to expand the currently limited arsenal of therapeutically useful mycobacteriophages as well as improve the efficiency of existing ones.

microbiology↗

bioRxiv2026

A novel technique for monitoring Alzheimer's disease associated changes in brain-derived extracellular vesicle cargos in mouse models

Extracellular vesicles (EVs) are critical mediators of intercellular communication, carrying molecular cargos such as small noncoding RNAs (ncRNAs) that reflect the physiological and pathological state of their cells of origin. However, studying brain-derived EVs has been challenging due to the blood-brain barrier. Here, we optimized and validated an open-flow microdialysis (OFM) protocol for sampling EVs directly from brain interstitial fluid (ISF) in wild-type and APP/PS1 transgenic mice. Ex-vivo validation using plasma EVs demonstrated that OFM effectively captures the full EV population. In-vivo cerebral OFM (cOFM) enabled successful collection of brain ISF EVs, which were characterized by nanoparticle tracking analysis (NTA), electron microscopy, and western blotting, confirming their similarity to EVs isolated directly from brain tissue and plasma. Identification of small ncRNA cargos revealed that EVs sampled from brain ISF by cOFM were enriched in brain-specific signatures, many of which are associated with neuronal cell populations and biological functions. Furthermore, we observed a unique small ncRNA signature from the brain ISF EVs in the Alzheimers disease preclinical model compared to wild-type mice. These small ncRNAs were associated with genes considered important in biological functions associated with neurodegeneration. Our findings demonstrate that cOFM is a powerful tool for in-vivo sampling of brain EVs and highlight the unique molecular landscape of ISF EV small ncRNA cargos. This study offers new opportunities for biomarker discovery and mechanistic insights into neurodegenerative diseases, such as Alzheimers disease.

neuroscience↗

bioRxiv2026

Likely role of promoter reconstitution in Mpr-mediated D29 resistance by Mycobacterium smegmatis

The multi-copy phage resistance gene (mpr) of M. smegmatis is a major factor in resistance to the lytic mycobacteriophage D29. Mpr is a membrane-bound exonuclease that cleaves phage DNA post injection, hence blocking downstream stages in the phage infection cycle. The mechanism of resistance allows for adsorption, is non-abortive and independent of any mutation in the gene. Rather, it depends on overexpression of a wild type copy of the gene. However, the underlying factor behind mpr overexpression in spontaneous D29-resistant mutants of M. smegmatis remained elusive. Here, we report that D29 infection triggers insertion sequence (IS) rearrangements, including the transposition and integration of IS6120 directly upstream of mpr. Mutants with IS6120 integration upstream of mpr show highly elevated Mpr expression. Whole genome sequence analysis reveals that IS6120 introduced a putative transcription factor-binding site and a canonical -35 promoter element at the integration site, hence reconstituting a fuller promoter (rcp) than the original promoter (wtp) at the site. Promoter reporter assays suggest that rcp is a far stronger promoter than wtp, implying that elevated mpr expression in D29-resistant mutants with this transposition event could be due to promoter reconstitution. While strains with this transposition event appear to grow normally, rcp-driven, vector-borne Mpr overexpression appears to be toxic as it barely allows for colony formation on agar plates. This study reports a previously unknown factor likely behind mpr regulation in M. smegmatis, adding to the existing knowledge of mycobacterial anti-phage defense mechanisms and guiding rational phage engineering efforts for therapeutic applications.

microbiology↗

bioRxiv2025

A division-associated envelope protein, MAB_2363, drives intrinsic resistance and virulence in Mycobacterium abscessus

Mycobacterium abscessus exhibits intrinsic resistance to conventional antibiotics, significantly limiting treatment options. Our previous studies established that MAB_2362 (SteA) is a key regulator of cell division that contributes to intrinsic resistance and virulence. Considering that SteA-like proteins often act alongside SteB counterparts, we hypothesized that the adjacent gene MAB_2363 encodes the corresponding SteB-like division regulator. In this study, we found that deletion of MAB_2363 significantly increased susceptibility to multiple antibiotics and disrupted cell wall permeability. Microscopy revealed striking cell division defects in the mutant, including elongated cell morphology and multiple septa. Subcellular localization of a GFP-MAB_2363 fusion protein demonstrated its enrichment at division septa, confirming its direct involvement in cell division. Deletion of MAB_2363 resulted in attenuated virulence, as evidenced by reduced bacterial survival in macrophages and murine infection models. To assess its functional relation with MAB_2362, we compared the single-deletion mutant of MAB_2363 with the single-deletion mutant of MAB_2362 and the double-deletion mutant of MAB_2362-MAB_2363. Notably, the phenotypes of the MAB_2363 mutant, including cell division defects, antibiotic susceptibility, and virulence, were markedly milder than those of the other two mutants. Collectively, these findings indicate that MAB_2363 functions as a secondary but essential division-associated factor that operates during cell division, thereby influencing intrinsic resistance and virulence in M. abscessus.

microbiology↗

bioRxiv2025

A genomic toolkit for surveillance and elimination of the principal malaria vectors in Southeast Asia.

While substantial progress has been made toward malaria elimination in Southeast Asia, major challenges remain. The principal mosquito vectors, Anopheles dirus and Anopheles minimus, inhabit diverse ecological niches and exhibit a wide range of behavioural and physiological insecticide resistance phenotypes, complicating vector control efforts. In Africa, genomic surveillance has transformed our understanding of vector evolution and resistance, supported by open-access tools and data. Comparable resources for Southeast Asian vectors remain limited. Here, we release Adir1.0, a curated, analysis-ready catalog of 540 An. dirus whole-genome sequences, accessible for interactive cloud-based analysis via the malariagen-data-python application programming interface (API). Alongside, we provide an updated Amin1.0 API to enhance functionality and usability, enabling integrated analyses across historical and new datasets. We demonstrate these resources by performing the first exploratory population genomic analysis of An. dirus from Bangladesh, Thailand, and Cambodia, revealing population structure, candidate regions of physiological resistance, and genomic structural variation. Together, these resources provide a foundation for genomic surveillance studies to inform vector control and malaria elimination strategies in Southeast Asia.

genetics↗

bioRxiv2025

Targeting homologous recombination repair to potentiate fluoroquinolone efficacy in Mycobacterium abscessus

Synthetic antibiotics, fluoroquinolones (FQs), are extensively important treatment options for fast growing mycobacterial infections. However, their widespread use has led to decreased efficacy due to intrinsic or acquired resistance. FQs exhibit antimicrobial activity by stabilizing type II topoisomerases-generated DNA breaks, thereby inducing lethal double-strand breaks (DSBs) and leading to bacterial death. Escalating resistance among mycobacterial pathogens highlights the need to elucidate resistance mechanisms and identify novel strategies to restore or enhance the drug activity. Here, we revealed homologous recombination (HR) as a key determinant of FQ tolerance in Mycobacterium abscessus, a notoriously drug-resistant pathogen. Through a transposon mutagenesis screen, we identified that disruption of adnB - an HR-associated gene, markedly sensitized M. abscessus to FQs. This finding prompted a systematic dissection of DSB repair pathways in M. abscessus. Targeted deletion of HR core components (adnB, recO, recA, recR, ruvB) significantly increased FQ susceptibility in vitro, while inactivation of alternative DSB repair pathways - single-strand annealing (SSA) and non-homologous end joining (NHEJ) - had no effect, revealing a unique reliance on HR for FQ tolerance. Furthermore, the complementation of gene knock-out M. abscessus strains {Delta}adnB, {Delta}recO, {Delta}recA, {Delta}recR or {Delta}ruvB with the corresponding genes from M. abscessus, as well as homologous genes from Mycobacterium tuberculosis restored the resistance phenotype, indicating a conserved HR-dependent tolerance mechanism in M. abscessuss. In a murine infection model, genetic abrogation of HR significantly improved the therapeutic efficacy of FQs against M. abscessus, demonstrating translational relevance. Collectively, our findings position HR as a conserved and actionable vulnerability in M. abscessus, and provide a compelling rationale for developing HR-targeted adjuvant therapies to resensitize drug-refractory strains to FQ treatment. Impact statementFluoroquinolone (FQ) resistance poses a major challenge in the treatment of Mycobacterium abscessus infections. This study identifies homologous recombination (HR) as a key defense mechanism that limits FQ efficacy across multiple mycobacterial species. Genetic inhibition of HR not only sensitizes M. abscessus to FQs in vitro but also enhances antibiotic activity in vivo. These results highlight HR repair as a promising therapeutic target to potentiate FQ activity and combat drug resistance in M. abscessus infections.

molecular biology↗

bioRxiv2025

Variants in glycine decarboxylase activate mechanisms of mitochondrial energy metabolism in the brain.

Brain energy metabolism is produced from glucose by mitochondrial oxidative phosphorylation. Variants in the mitochondrial enzyme glycine decarboxylase (GLDC) cause a rare neurological disease, non-ketotic hyperglycinemia (NKH), with expected hallmarks of brain glycine elevation and responsiveness to folate deficiency that are equivalent to the severity of Gldc mutations. We remarkably find that brains of young- attenuated mutant mice with a 1.5-fold increase in glycine are reduced > 5-fold in GLDC, show a decline in both the mitochondrial lipoyl-transfer protein GCSH and lipoylation of the pyruvate dehydrogenase (PDH) complex, as well as concomitant rise in signatures of astrocyte mitochondrial {beta}-oxidation of fatty acids and activation of neuronal PDH. Our findings suggest a novel GLDC mechanism of remodeling mitochondrial energy systems throughout the brain, established early in and sustained throughout post-natal NKH disease.

neuroscience↗

bioRxiv2025

Chronic HDACi emends microglial differentiation and neurological disease in a mouse model of intellectual disability

Defect in lysine-specific methyltransferase 2D (KMT2D) underlies a rare intellectual disability disorder, Kabuki Syndrome (KS). We show that in addition to reduction in post-natal neurogenesis, KS mice present small, arrested hippocampal microglia which single cell RNA sequence analyses revealed as globally downregulated, occupying neither activated nor surveillance states. Weekly administration of a triple combination formulation (TCF) containing the FDA-approved HDAC inhibitor Vorinostat, HPBCD and PEG-400 for three months, boosted microglia. Transcriptomic, pseudotime cell trajectory analyses, imaging and behavioral data suggest that TCF differentiated KS microglia from arrested to non-diseased and non-activated surveillance forms by selective acetylation of histone H3, that restored the open chromatin mark H3K4me3 independent of Kmt2D, to improve cognitive and nociceptive responses. Wild-type microglia showed no substantial transcriptional or histone changes, intimating normal heterochromatin resisted TCF. These findings reveal a novel chromatin-mediated mechanism of microglial differentiation uncoupled from activation, with therapeutic potential for KS and related disorders.

neuroscience↗

bioRxiv2025

Globally prevalent Kelch13 mutations increase partial artemisinin resistance and fitness in Bangladeshi Plasmodium falciparum parasites

Artemisinin partial resistance (ArtR), mediated by Kelch13 (K13) gene mutations in Plasmodium falciparum, has caused delayed parasite clearance and, together with partner drug resistance, treatment failures in the Greater Mekong Subregion (GMS). The Chittagong Hill Tracts (CHTs), located in southeastern Bangladesh and bordering Myanmar and India, regions with prevalent K13 mutations, account for approximately 90% of the countrys malaria infections but have not yet reported ArtR-causing K13 mutations. Importantly, however, some isolates from the CHTs have demonstrated moderate in vitro ArtR in the absence of K13 mutations. To assess the potential threat of K13-mediated ArtR in Bangladesh, we proactively evaluated the impact of three prevalent neighboring K13 mutations (F446I, R561H, and C580Y) on ArtR and parasite fitness in parasites isolated from the CHTs. We edited these mutations into two distinct genetic backgrounds: an artemisinin-sensitive strain (CHT-S) and a K13-independent, moderately resistant strain (CHT-R). In these edited lines, we then evaluated ArtR levels and fitness using ring-stage survival assays (RSA), post-drug treatment recovery assays, and competitive fitness assays against isogenic control strains. Prior to genome editing, both isolates were characterized for baseline drug susceptibility, resistance-associated mutations, and population structure. We found that C580Y and R561H mutations, but not F446I, confer increased in vitro ArtR in the CHT-R background already exhibiting K13-independent moderate resistance. All three mutations incurred minimal or no fitness costs in both genetic backgrounds. Notably, R561H, the dominant allele at the Thai-Myanmar border and currently expanding in Rwanda, mediates extreme resistance in the CHT-R background, with mean RSA survival rates of 30.9{+/-}1.9%, an unprecedented resistance level among K13-engineered lines. R561H also showed fitness advantages (0.5% per generation) and the highest growth recovery post-treatment. This represents the first experimental study modeling this K13 mediated ArtR risk in Bangladeshi parasites. In conclusion, we found that indigenous P. falciparum isolates in the CHTs possess inherent genetic potential to sustain high-level ArtR and fitness advantages if K13 mutations emerge, raising urgent concerns for containment and surveillance strategies during Bangladeshs malaria elimination phase.

microbiology↗

bioRxiv2025

Astrocyte and neurogenic mechanisms of protective gene therapy prevent brain disease and death due to non-ketotic hyperglycinemia

Genetic defects in glycine decarboxylase (GLDC) cause non-ketotic hyperglycinemia (NKH), a rare and frequently fatal neurometabolic disease, which lacks FDA-approved therapies. We characterized CRISPR Cas9-edited humanized mice expressing a prevalent clinical mutation after administration with a single intraperitoneal dose of a novel recombinant of adeno-associated viral vector 9 expressing GLDC (rAAV9-GLDC). Long term biological activity of rAAV9-GLDC was first validated by assessment of its systemic efficacy over five and ten months in mice. Access of rAAV9 to the brain was confirmed by tracking green fluorescent protein (GFP) after a single intraperitoneal dose of rAAV9-GFP. Over five months, control mock treated GFP-mice showed reduction in astrocytes but not microglia, oligodendrocytes or neurons in the brain. 37% of these animals suffered long term neurological disease and/or death. rAAV9-GLDC boosts astrogenesis without triggering an inflammatory response and confers 100% protection against disease progression and fatality due to NKH.

neuroscience↗

bioRxiv2025

Topoisomerase IIIb protects from tumorigenesis and immune dysregulation

Topoisomerase III-beta (Top3b) reduces nucleic acid torsional stress and intertwining generated during RNA and DNA metabolism while protecting the genome from pathological R-loops, which otherwise result in DNA breakage and genome instability. By studying Top3b knockout mice (Top3b-KO), we find that the loss of Top3b accelerates the development of spontaneous lymphoid tumors arising in spleens and lymph nodes, the organs with prominent Top3b expression. Aging Top3b-KO mice also display splenomegaly and systemic immune alterations including neutrophilia and lymphopenia suggestive of chronic inflammation. At the molecular level, Top3b deficiency causes genome-wide R-loop accumulation in splenocytes as measured by CUT&Tag sequencing. Increased R-loops is associated with genomic DNA breaks and activation of immune signaling pathways including the IL-6 signaling, interleukin-7 signaling and cGAS-STING. Moreover, knocking-out Top3b promotes the rapid development of syngeneic EL4 T-cell lymphomas. In conclusion, our work implies that, in addition to its role in preserving the nervous system, Top3b protects from tumorigenesis and immune dysregulations. SignificanceTopoisomerase III-beta (TOP3B) is the only dual RNA-DNA topoisomerase. Its inactivation has previously been shown to be critical for neurodevelopmental disorders in humans and R-loop suppression in cell line models. Here, we demonstrate that loss of Top3b accelerates spontaneous lymphomagenesis and promotes syngeneic lymphoma growth in association with innate immune defects and R-loop accumulation in Top3b-KO mice. These findings provide evidence for a previously unappreciated role of Top3b as a tumor suppressor gene and immune regulator.

cancer biology↗

bioRxiv2025

The stress response factor SigH mediates intrinsic resistance to multiple antibiotics in Mycobacterium abscessus

Mycobacterium abscessus (Mab) causes pulmonary diseases with limited treatment options due to its high level of intrinsic resistance to available drugs. Mab possesses complex and poorly understood drug resistance mechanisms. Identifying new drug targets and gaining a deeper understanding of drug resistance mechanisms are essential for discovering novel therapeutic alternatives. Here, we investigated the role of a putative sigma factor SigH in intrinsic multi-drug resistance in Mab. Mab SigH shares an 84% peptide sequence identity with Mycobacterium tuberculosis (Mtb) SigH, a well-known stress response protein and global transcriptional regulator. We constructed a sigH gene deletion strain of Mab ({Delta}sigH) and complemented strains by expressing either Mab sigH (CPMabsigH) or Mtb sigH (CPMtbsigH) in {Delta}sigH. The {Delta}sigH strain exhibited hypersensitivity to a broad range of antibiotics, including levofloxacin, moxifloxacin, tigecycline, tetracycline, amikacin, vancomycin, and rifabutin and all complemented strains restored the drug resistance phenotype. Additionally, {Delta}sigH showed increased sensitivity to oxidative and heat stress compared to the wild-type Mab and complemented strains. Transcriptomic analysis revealed that deletion of sigH disrupted the balance of gene expression, primarily elevating the expression of genes encoding YrbE and MCE family proteins and downregulating genes expressing ABC-type transporters, sigma and anti-sigma factors and other genes associated with antimicrobial resistance. Collectively, our findings indicate that SigH is a key regulator of global gene expression in response to environmental stresses, including antimicrobial treatment, and is crucial for the intrinsic drug resistance of Mab. SigH represents a promising target for the development of novel therapeutic strategies against Mab infections.

molecular biology↗

bioRxiv2024

Fitness compatibility and dengue virus inhibition in a Bangladeshi strain of Aedes aegypti infected with the Wolbachia strain wAlbB

Dengue cases in Bangladesh have surged in recent years. The existing insecticide-based control program, implemented in parts of the country, is challenged by issues of insufficient household coverage and high levels of insecticide resistance in the primary dengue virus (DENV) vector, Aedes aegypti. A more sustainable, effective alternative could be the implementation of a Wolbachia-mediated disease management strategy. Infecting mosquitoes with Wolbachia can change their reproductive compatibility and their ability to transmit DENV. These new phenotypes can be exploited to suppress or replace wild-type Ae. aegypti populations. Such strategies require the development of well-characterised Wolbachia-infected strains with biological characteristics that are comparable with local mosquitoes. We created and characterised a Wolbachia-infected Ae. aegypti strain with a Dhaka wild-type genetic background, and compared its reproductive compatibility, maternal inheritance, fitness, and virus-blocking ability to the parental strains (Dhaka wild-type and wAlbB2-F4). The new Ae. aegypti strain wAlbB2-Dhaka demonstrated complete cytoplasmic incompatibility with the wild-type strain and complete maternal transmission, retaining levels of pyrethroid resistance of the Dhaka wild-type (70% survival to 10 times the dose of permethrin expected to kill susceptible mosquitoes). No significant fitness costs were detected during laboratory comparisons of fecundity, fertility, survival, mating competitiveness, or desiccation tolerance. Compared to the wild-type strain, wAlbB2-Dhaka mosquitoes had a significantly reduced number of DENV genome copies in the bodies (44.4%, p = 0.0034); two-fold reduction in dissemination to legs and wings (47.6%, p < 0.0001); and >13-fold reduction of DENV in saliva expectorates (proxy of transmission potential) (92.7%, p < 0.0001) 14 days after ingesting dengue-infected blood. Our work indicates that the wAlbB2-Dhaka strain could be used for Ae. aegypti suppression or replacement strategies for dengue management in Bangladesh. Author summaryBangladesh is currently grappling with a series of severe dengue outbreaks, resulting in more than 300 thousand infections, 1700 fatalities, and a significant influx of patients requiring hospitalisation in 2023. These outbreaks coincide with the emergence of high levels of insecticide resistance in the Ae. aegypti population, which is contributing to the failure of conventional, insecticidal control strategies. In search of more sustainable, effective dengue control strategies, approaches using the endosymbiotic bacteria Wolbachia that render mosquitoes resistant to arbovirus infection are being trialled in a number of countries. We transinfected Dhaka wild-type Ae. aegypti with the Wolbachia strain wAlbB2 by backcrossing it with a Wolbachia wAlbB2-infected Ae. aegypti strain and assessed the new strains suitability for field release. We compared the fitness and virus-blocking ability of the backcrossed strain with its parental strains. We demonstrated that the new strain, wAlbB2-Dhaka, is as fit as the Dhaka wild-type strain and blocked [~]92% of dengue virus transmission. This strain has great potential for Wolbachia-mediated dengue management strategies in Bangladesh.

zoology↗

bioRxiv2024

Disrupting Homologous Recombination or Single-Strand Annealing Significantly Hinders CRISPR-Cas12a-Assisted Nonhomologous End-Joining Gene Editing Efficiency in Mycobacterium abscessus

Mycobacterium abscessus, a fast-growing, non-tuberculous mycobacterium resistant to most antimicrobial drugs, causes many types of serious infections in humans, posing a significant public health challenge. Currently, effective genetic manipulation tools for M. abscessus are still being developed, which hampers research and therapeutic development. However, the clustered regularly interspaced short palindromic repeats (CRISPR) - associated protein (Cas) systems have emerged as promising methods for generating highly specific double-strand breaks (DSBs) in its genome. These DSBs can be repaired by the error-prone nonhomologous end joining (NHEJ) mechanism, facilitating targeted gene editing. Here, our study marks a pioneering application of the CRISPR-NHEJ strategy in M. abscessus. Additionally, we discovered that NrgA from Mycobacterium marinum is crucial for the repair of DSBs caused by the CRISPR-Cas system in M. abscessus. Finally, contrary to previous findings, our study also indicates that inhibiting or overexpressing homologous recombination/single-strand annealing significantly decreases the efficiency of NHEJ repair in M. abscessus. This discovery challenges established perspectives and suggests that the NHEJ repair in M. abscessus may require the involvement of components from homologous recombination and single-strand annealing, demonstrating the complex interactions among the three DSB repair pathways in M. abscessus. Impact statementThere are still very few genetic manipulation tools available for Mycobacterium abscessus. Here we report the successful application of CRISPR-Cas12a-assisted nonhomologous end joining (NHEJ) in efficient gene editing in M. abscessus. Contrary to previous research suggesting that homologous recombination (HR) inhibition may enhance such editing efficiency in other Mycobacterium species, our results showed that disruption or overexpression of either HR or single-strand annealing not only failed to enhance but also significantly reduced the gene editing efficiency in M. abscessus. This suggests that NHEJ repair in M. abscessus may require components from both HR and single-strand annealing, highlighting a complex interaction among the DSB repair pathways in M. abscessus.

microbiology↗

bioRxiv2023

Structural characterization of a novel luciferase-like-monooxygenase from Pseudomonas meliae an in silico approach

BackgroundLuciferase is a well-known oxidative enzyme that produces bioluminescence. The Pseudomonas meliae is a plant pathogen that causes wood rot on nectarine and peach and possesses a luciferase-like monooxygenase. After activation, it produces bioluminescence, and the pathogens bioluminescence is a visual indicator of diseased plants. MethodsThe present study aims to model and characterize the luciferase-like monooxygenase protein in P. meliae for its similarity to well-established luciferase. In this study, the luciferase-like monooxygenase from P. meliae infects chinaberry plants has been modeled first and then studied by comparing it with existing known luciferase. Also, the similarities between uncharacterized luciferase from P. meliae and template from Geobacillus thermodenitrificans were analyzed to find the novelty of P. meliae. ResultsThe results suggest that the absence of bioluminescence in P. meliae could be due to the evolutionary mutation in positions 138 and 311. The active site remains identical except for two amino acids; P. meliae Tyr138 instead of His138 and Leu311 instead of His311. Therefore, the P. meliae will have a potential future application, and mutation of the residues 138 and 311 can be restored luciferase light-emitting ability. ConclusionsThis study will help further improve, activate, and repurpose the luciferase from P. meliae as a reporter for gene expression.

bioinformatics↗

bioRxiv2022

Prediction of Electric Fields Induced by Transcranial Magnetic Stimulation in the Brain using a Deep Encoder-Decoder Convolutional Neural Network

Transcranial magnetic stimulation (TMS) is a non-invasive, effective, and safe neuromodulation technique to diagnose and treat neurological and psychiatric disorders. However, the complexity and heterogeneity of the brain composition and structure pose a challenge in accurately determining whether critical brain regions have received the right level of induced electric field. Numerical computation methods, like finite element analysis (FEA), can be used to estimate electric field distribution. However, these methods need exceedingly high computational resources and are time-consuming. In this work, we developed a deep convolutional neural network (DCNN) encoder-decoder model to predict induced electric fields, in real-time, from T1-weighted and T2-weighted magnetic resonance imaging (MRI) based anatomical slices. We recruited 11 healthy subjects and applied TMS to the primary motor cortex to measure resting motor thresholds. Head models were developed from MRIs of the subjects using the SimNIBS pipeline. Head model overall size was scaled to 20 new size scales for each subject to form a total of 231 head models. Scaling was done to increase the number of input data representing different head model sizes. Sim4Life, a FEA software, was used to compute the induced electric fields, which served as the DCNN training data. For the trained network, the peak signal to noise ratios of the training and testing data were 32.83dB and 28.01dB, respectively. The key contribution of our model is the ability to predict the induced electric fields in real-time and thereby accurately and efficiently predict the TMS strength needed in targeted brain regions.

bioengineering↗