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bioRxiv2023

Multi-omics characterization of partial chemical reprogramming reveals evidence of cell rejuvenation

Partial reprogramming by cyclic short-term expression of Yamanaka factors holds promise for shifting cells to younger states and consequently delaying the onset of many diseases of aging. However, the delivery of transgenes and potential risk of teratoma formation present challenges for in vivo applications. Recent advances include the use of cocktails of compounds to reprogram somatic cells, but the characteristics and mechanisms of partial cellular reprogramming by chemicals remain unclear. Here, we report a multi-omics characterization of partial chemical reprogramming in fibroblasts from young and aged mice. We measured the effects of partial chemical reprogramming on the epigenome, transcriptome, proteome, phosphoproteome, and metabolome. At the transcriptome, proteome, and phosphoproteome levels, we saw widescale changes induced by this treatment, with the most notable signature being an upregulation of mitochondrial oxidative phosphorylation. Furthermore, at the metabolome level, we observed a reduction in the accumulation of aging-related metabolites. Using both transcriptomic and epigenetic clock-based analyses, we show that partial chemical reprogramming reduces the biological age of mouse fibroblasts. We demonstrate that these changes have functional impacts, as evidenced by changes in cellular respiration and mitochondrial membrane potential. Taken together, these results illuminate the potential for chemical reprogramming reagents to rejuvenate aged biological systems and warrant further investigation into adapting these approaches for in vivo age reversal.

molecular biology↗

bioRxiv2025

Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

Improved methods are needed to gain insights in how proteins exert their myriad roles in cells and organs. Multiplex in situ proximity ligation assay (misPLA), described herein, can provide a window into the functional states of proteins in cells and tissues by applying pairs of antibody-oligonucleotide conjugates to generate amplifiable DNA circles upon proximal binding. The analysis reveals interactions and modifications among sets of proteins, read out by recording the identity and location of the resulting localized DNA amplification products. We applied misPLA to both primary and cultured cells and to formalin-fixated paraffin-embedded (FFPE) tissues, to map dynamic changes in protein localizations, phosphorylations and interactions across surface markers, MAPK, immune-checkpoints, T- and B-cell receptors, and adhesion panels. Comparisons of single-plex versus nine-plex assays confirmed that misPLA maintains sensitivity and specificity while increasing throughput and spatial context. Across breast cancer, lymphomas and chronic myeloid leukemia (CML) misPLA uncovered shared and disease-specific signaling patterns, underscoring convergence of oncogenic networks. By preserving tissue architecture and enabling high-content functional spatial proteomics at single-cell resolution, misPLA offers a versatile platform for dissecting signaling heterogeneity, pathway crosstalk, and therapeutic responses, with broad applications in cell biology, biomarker discovery and in precision oncology.

molecular biology↗

bioRxiv2026

A Metabolic Labeling Strategy for Tracking Protein Synthesis in Complex Biological Systems

Protein synthesis supports most biological processes. In the brain in particular, protein synthesis plays a critical role in physiological and pathological states. Here, we describe Tellurophene-Alkyne Cycloaddition-mediated Amino acid Tagging (TeACAT), a versatile strategy for fast, facile, and flexible tagging of newly synthesized proteins in mice. TeACAT is based on metabolic incorporation of the non-canonical amino acid TePhe into proteins by the endogenous protein synthesis machinery. Due to their high similarity, TePhe can efficiently replace canonical Phe without dietary or genetic manipulation. The subsequent bio-orthogonal reaction of TePhe with either fluorescent dyes or affinity handles enables both visualization and affinity enrichment of proteins synthesized during TePhe exposure. TeACAT is compatible with immunofluorescence for cell-type specific visualization of protein synthesis with subcellular resolution and can be used in conjunction with routine proteomics to identify and quantify newly synthesized proteins. Robust incorporation into the mouse proteome was observed on the scale of hours to days, allowing the interrogation of various biological processes. In summary, TeACAT enables the visualization and quantification of protein synthesis with minimal perturbation for biological discoveries.

molecular biology↗

bioRxiv2026

Stop codon readthrough in Trichomonas is a mechanism for gene expression regulation and expanding protein function

Trichomonas vaginalis is the causative agent of trichomoniasis, a common sexually transmitted infection among women of reproductive and peri-menopausal age. The parasite has an unusually large genome, rich in complex repeats, including a vast repertoire of transposable elements and multi-copy gene families. Since very few T. vaginalis genes have introns, gene expression is usually straightforward, with ribosomal translational machinery proceeding from a start codon to the next in-frame stop codon of an unspliced poly(A)denylated mRNA. However, our previous studies raised the possibility of T. vaginalis gene expression involving stop codon readthrough (SCR), where transcription through in-frame stop codons produces longer-than-predicted mRNAs that translate to fully functional proteins. Here, we leverage long-read RNA-seq and new chromosome-scale assemblies of two T. vaginalis strains and two avian sister species to investigate and characterize ~1,400 long, mature mRNAs that contain more than one predicted protein-coding gene transcribed from what we call '' RT genes '', composites of adjacent predicted genes. We first identify RT genes in a second T. vaginalis strain and in close relatives T. vaginalis-like and T. stableri, indicating that this phenomenon is conserved among Trichomonas species and strains. Second, we find transcripts of RT genes to be more abundant by many orders of magnitude than monocistronic genes. Third, we found the distance between predicted genes within RT genes to be significantly shorter than between adjacent independent predicted genes. Fourth, functional annotation revealed that RT genes encode at least 50 distinct protein functions, suggesting that this unusual transcriptional mechanism has a role in an array of biological processes in Trichomonas. Our results from two Trichomonas species suggest that SCR is an important mechanism controlling gene expression and the diversity of protein function in this parasite.

molecular biology↗

bioRxiv2025

Public Cohort Analysis Identifies Thyroglobulin Variants as Hypothyroidism Risk Factors

Hypothyroidism is a prevalent endocrine disorder characterized by insufficient thyroid hormone (T3T4) production. Thyroglobulin (Tg) serves as the prohormone for T3 and T4 production, with many variants of uncertain clinical significance due to genetic diversity in the Tg gene. We leveraged the large-scale All of Us biobank to investigate the disease association of prevalent yet undercharacterized Tg variants. We related variant presence to thyroid-stimulating hormone levels and levothyroxine (LT4) usage as proxies for thyroid function. This identified R152H, Q870H, A993T, P1012L, and P1494L variants linked to increased LT4 usage and decreased thyroid function, while the R320C variant was associated with decreased thyroid function. Molecular characterization in Fisher rat thyroid cells revealed decreased secretion efficiency of R152H, Q870H, and R320C variants. Affinity purification-mass spectrometry demonstrated that secretion-deficient variants showed higher engagement with the protein homeostasis network, indicating protein quality control defects as the pathophysiology mechanism. In contrast, secretion-competent A993T and P1494L variants showed elevated interactions with degradation and antigen-presentation pathways, suggesting an alternative pathophysiology possibly linked to Hashimotos disease, an autoimmune condition with overproduction of autoantibodies that target thyroid proteins. In support, participants carrying the A993T or P1494L variants had elevated anti-TPO antibody levels. We estimate [~]150,000 US individuals currently taking levothyroxine could benefit from precision medicine targeting these variants, with [~]100,000 carrying Q870H. Our findings highlight the power of combining large public biobank data with molecular characterization to understand Tg genotype-to-phenotype relationships. Q870H represents a candidate for molecular therapies to restore secretion, offering precision medicine beyond LT4 replacement therapy. Significance StatementHypothyroidism affects millions of Americans who typically receive levothyroxine hormone replacement therapy, yet some patients continue experiencing symptoms despite treatment. Correlating genomic and health data from the large-cohort All of Us biobank identified specific variants in the thyroglobulin gene, which produces thyroid hormones, that contribute to thyroid dysfunction through distinct biological pathways. Molecular characterization and interactomics revealed that some variants impair hormone secretion due to protein misfolding, while others associate with immune presentation that may link to autoimmune thyroid disease. The Q870H variant emerged as a promising target for precision medicine, potentially benefiting [~]100,000 Americans currently taking levothyroxine. This research demonstrates how combining large-scale genomic data with molecular characterization can identify new therapeutic targets beyond standard hormone replacement therapy.

biochemistry↗

bioRxiv2017

Methyl donor deficient diets cause distinct alterations in lipid metabolism but are poorly representative of human NAFLD

Non-alcoholic fatty liver disease (NAFLD) is a global health issue. Dietary methyl donor restriction is used to induce a NAFLD/non-alcoholic steatohepatitis (NASH) phenotype in rodents, however the extent to which this model reflects human NAFLD remains incompletely understood. To address this, we undertook hepatic transcriptional profiling of methyl donor restricted rodents and compared these to published human NAFLD datasets.\n\nAdult C57BL/6J mice were maintained on control, choline deficient (CDD) or methionine/choline deficient (MCDD) diets for four weeks; the effects on methyl donor and lipid biology were investigated by bioinformatic analysis of hepatic gene expression profiles followed by a cross-species comparison with human expression data of all stages of NAFLD.\n\nCompared to controls, expression of the very low density lipoprotein (VLDL) packaging carboxylesterases (Ces1d, Ces1f, Ces3b) and the NAFLD risk allele Pnpla3 were suppressed in MCDD; with Pnpla3 and the liver predominant Ces isoform, Ces3b, also suppressed in CDD. With respect to 1-carbon metabolism, down-regulation of Chka, Chkb, Pcty1a, Gnmt and Ahcy with concurrent upregulation of Mat2a suggests a drive to maintain S-adenosylmethionine levels. There was minimal similarity between global gene expression patterns in either dietary intervention and any stage of human NAFLD, however some common transcriptomic changes in inflammatory, fibrotic and proliferative mediators were identified in MCDD, NASH and HCC.\n\nIn conclusion, this study suggests suppression of VLDL assembly machinery may contribute to hepatic lipid accumulation in these models, but that CDD and MCDD rodent diets are minimally representative of human NAFLD at the transcriptional level.\n\nSummary statementWe used transcriptional profiling of methyl donor restricted rodents to examine effects on methyl donor and lipid biology. We report novel mechanisms for lipid accumulation in this model and describe significant disparity between both dietary interventions and human disease.

molecular biology↗

bioRxiv2020

Loss of a subunit of vacuolar ATPase identifies unexpected biological signatures of reduced organelle acidification in vivo

The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases has been associated with various human diseases including heritable connective tissue disorders, neurodegenerative diseases and cancer. Multiple studies have investigated the pleiotropic effects of reduced acidification in vitro, but the mechanisms elicited by impaired endo(lyso)somal acidification in vivo remain poorly understood. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, hypotonia and epidermal structural defects, reminiscent of the phenotypic manifestations in cutis laxa patients carrying a defect in the ATP6V1E1 gene. Mechanistically, we found that in vivo genetic depletion of atp6v1e1b leads to N-glycosylation defects and reduced maturation of endosomal and lysosomal vesicles, but retains the hypoxia-mediated response. In order to gain further insights into the processes affected by aberrant organelle acidification, we performed an untargeted analysis of the transcriptome and metabolome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid and fatty acid metabolism with profound defects in mitochondrial respiration. Taken together, our results identify new complex biological effects of reduced organelle acidification in vivo, which likely contribute to the multisystemic manifestations observed in disorders caused by v-ATPase deficiency.

molecular biology↗

bioRxiv2021

Genome-wide screening of circadian and non-circadian impact of Neat1 genetic deletion

The functions of the long non-coding RNA, Nuclear enriched abundant transcript 1 (Neat1), are poorly understood. Neat1 is required for the formation of paraspeckles, but its respective paraspeckle-dependent or independent functions are unknown. Several studies including ours reported that Neat1 is involved in the regulation of circadian rhythms. We characterized the impact of Neat1 genetic deletion in a rat pituitary cell line. The mRNAs whose circadian expression pattern or expression level is regulated by Neat1 were identified after high-throughput RNA sequencing of the circadian transcriptome of wild-type cells compared to cells in which Neat1 was deleted by CRISPR/Cas9. The numerous RNAs affected by Neat1 deletion were found to be circadian or non-circadian, targets or non-targets of paraspeckles, and to be associated with many key biological processes showing that Neat1, interacting or independently of the circadian system, could play crucial roles in key physiological functions through diverse mechanisms.

molecular biology↗

bioRxiv2021

Automated ChIPmentation procedure on limited biological material of the human blood fluke Schistosoma mansoni

Automated ChIPmentation procedure is a convenient alternative to native chromatin immunoprecipitation (N-ChIP). It is now routinely used for ChIP-Seq. Using the human parasite Schistosoma mansoni, whose production requires scarifying animals and should therefore kept to a minimum, we show here that the automated ChIPmentation is suitable for limited biological material. We define as operational limit [≥]20,000 cells. We also present a streamlined protocol for the preparation of ChIP input libraries.

molecular biology↗

bioRxiv2024

Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

Pseudouridine ({Psi}) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. To address this challenge, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS) for quantitative profiling of {Psi} at single-base resolution. Based on novel bromoacrylamide cyclization chemistry, BACS enables a {Psi}-to-C transition. Compared to previous methods, BACS allowed the precise identification of {Psi} positions, especially in densely modified {Psi} regions and consecutive uridine sequences. BACS successfully detected all known {Psi} sites in human rRNA and spliceosomal snRNAs and generated the first quantitative {Psi} map of human snoRNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine (A-to-I) editing sites and N1-methyladenosine (m1A). Depletion of three key pseudouridine synthases (PUS) enabled us to elucidate the targets and sequence motifs of TRUB1, PUS7, and PUS1 in HeLa cells. We further applied BACS to Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) and identified a highly abundant {Psi}114 site in EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of {Psi} in their viral transcripts or genomes, shedding light on differences in pseudouridylation between virus families. We anticipate BACS to serve as a powerful tool to uncover the biological importance of {Psi} in future studies.

molecular biology↗

bioRxiv2025

Proximity Labeling of NIMA Kinase Complex Components in C. elegans

Proximity labeling has emerged as a powerful approach for identifying protein-protein interactions within living systems, particularly those involving weak or transient associations. Here, we present a comprehensive proximity labeling study of five conserved Caenorhabditis elegans proteins--NEKL-2, NEKL-3, MLT-2, MLT-3, and MLT-4--that form two NEKL-MLT kinase-scaffold subcomplexes involved in membrane trafficking and actin regulation. Using endogenously expressed TurboID fusions and a data-independent acquisition (DIA) mass spectrometry (MS) pipeline, we profiled NEKL-MLT interactomes across 23 experiments, including several methodological variations, applying stringent controls and filtering strategies. By analyzing and comparing experimental outcomes, we develop a set of intuitive quantitative metrics to assess experimental outcomes and quality. We demonstrate that DIA-based workflows produce sensitive physiologically relevant findings, even in the presence of experimental noise and variability across biological replicates. Our approach is validated through the identification of known NEKL-MLT binding partners and conserved genetic suppressors of nekl-mlt mutant phenotypes. Gene ontology enrichment further supports the involvement of newly identified NEKL-MLT interactors in processes including membrane trafficking, cytoskeletal regulation, and cell adhesion. Additionally, several novel proximate interactors were functionally validated using genetic assays. Our findings underscore the utility of DIA-MS in proximity labeling applications and highlight the value of incorporating internal controls, quantitative metrics, and biological validation to enhance confidence in candidate interactors. Overall, this study provides a scalable, organismal-level strategy for probing endogenous protein networks and offers practical guidelines for proximity labeling in multicellular systems.

molecular biology↗

bioRxiv2018

Distinct isoforms of Nrf1 diversely regulate different subsets of its cognate target genes

The single Nrf1 gene has capability to be differentially transcripted alongside with alternative mRNA-splicing and subsequent translation through different initiation signals so as to yield distinct lengths of polypeptide isoforms. Amongst them, three of the most representatives are Nrf1, Nrf1{beta} and Nrf1{gamma}, but the putative specific contribution of each isoform to regulating ARE-driven target genes remains unknown. To address this, we have here established three cell lines on the base of the Flp-In T-REx system, which are allowed for tetracycline-inducibly stable expression of Nrf1, Nrf1{beta} and Nrf1{gamma}. The RNA-Sequencing results have demonstrated that a vast majority of differentially expressed genes (i.e. >90% DEGs detected) were dominantly up-regulated by Nrf1 and/or Nrf1{beta} following induction by tetracycline. By contrast, other DEGs regulated by Nrf1{gamma} were far less than those regulated by Nrf1/{beta} (i.e. ~11% of Nrf1 and 7% of Nrf1{beta}). Further transcriptomic analysis revealed that tetracycline-induced expression of Nrf1{gamma} significantly increased the percentage of down-regulated genes in total DEGs. These statistical data were further validated by quantitative real-time PCR. The experimental results indicate that distinct Nrf1 isoforms make diverse and even opposing contributions to regulating different subsets of target genes, such as those encoding 26S proteasomal subunits and others involved in various biological processes and functions. Collectively, Nrf1{gamma} acts as a major dominant-negative competitor against Nrf1/{beta} activity, such that a number of DEGs regulated by Nrf1/{beta} are counteracted by Nrf1{gamma}.

molecular biology↗

bioRxiv2018

Co-translation drives the assembly of mammalian nuclear multisubunit complexes

AO_SCPLOWBSTRACTC_SCPLOWCells dedicate significant energy to build proteins often organized in multiprotein assemblies with tightly regulated stoichiometries. As genes encoding proteins assembling in the same multisubunit complexes are dispersed in the genome of eukaryotes, it is unclear how multisubunit complexes assemble. We show that mammalian nuclear transcription complexes (TFIID, TREX-2 and SAGA) composed of a large number of subunits but lacking precise architectural details are built co-translationally. We demonstrate that the dimerization domains and their positions in the interacting subunits determine the co-translational assembly pathway (simultaneous or sequential). Our results indicate that protein translation and complex assembly are linked in building mammalian multisubunit complexes and suggest that co-translational assembly is a general principle in mammalian cells to avoid non-specific interactions and protein aggregation. These findings will significantly advance structural biology by defining endogenous co-translational building blocks in the architecture of multisubunit complexes.

molecular biology↗

bioRxiv2018

Double-digest RAD-sequencing: do wet and dry protocol parameters impact biological results?

O_LINext-generation sequencing technologies have opened a new era of research in genomics. Among these, restriction enzyme-based techniques such as restriction-site associated DNA sequencing (RADseq) or double-digest RAD-sequencing (ddRADseq) are now widely used in many population genomics fields. From DNA sampling to SNP calling, both wet and dry protocols have been discussed in the literature to identify key parameters for an optimal loci reconstruction.\nC_LIO_LIThe impact of these parameters on downstream analyses and biological results drawn from RADseq or ddRADseq data has however not been fully explored yet. In this study, we tackled this issue by investigating the effects of ddRADseq laboratory (i.e. wet protocol) and bioinformatics (i.e. dry protocol) settings on loci reconstruction and inferred biological signal at two evolutionary scale using two systems: a complex of butterfly species (Coenonympha sp.) and populations of Common beech (Fagus sylvatica).\nC_LIO_LIResults suggest an impact of wet protocol parameters (DNA quantity, number of PCR cycles during library preparation) on the number of recovered reads and SNPs, the number of unique alleles and individual heterozygosity. We also found that bioinformatic settings (i.e. clustering and minimum coverage thresholds) impact loci reconstruction (e.g. number of loci, mean coverage) and SNP calling (e.g. number of SNPs, heterozygosity). We however do not detect an impact of parameter settings on three types of analysis performed with ddRADseq data: measure of genetic differentiation, estimation of individual admixture, and demographic inferences. In addition, our work demonstrates the high reproducibility and low rate of genotyping inconsistencies of the ddRADseq protocol.\nC_LIO_LIThus, our study highlights the impact of wet parameters on ddRADseq protocol with strong consequences on experimental success and biological conclusions. Dry parameters affects loci reconstruction and descriptive statistics but not biological conclusion for the two studied systems. Overall, this study illustrates, with others, the relevance of ddRADseq for population and evolutionary genomics at the inter- or intraspecific scales.\nC_LI

molecular biology↗

bioRxiv2019

A minimal CRISPR-Cas3 system for genome engineering

CRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilized a Cas3-based system featuring a processive nuclease, expressed endogenously or heterologously, for genome engineering purposes. Using an optimized and minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions ranging from 7 - 424 kb were generated in Pseudomonas aeruginosa with high efficiency and speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, and successfully and efficiently specified when present. The minimal Cas3 system is also portable; large deletions were induced with high efficiency in Pseudomonas syringae and Escherichia coli using an "all-in-one" vector. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to iteratively reduce a P. aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility of endogenous Cas3 systems (Type I-C and I-F) and develop an "anti-anti-CRISPR" strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes, genome minimization, and the analysis of large regions of unknown function.

molecular biology↗

bioRxiv2026

Noncanonical Circular RNAs and Potential Functions

Circular RNAs (circRNAs) are ubiquitous in eukaryotes; dysregulated circRNA expression is linked to diseases, including lung cancer. In contrast to canonical circRNAs arising from exon-intron boundaries, noncanonical circRNAs originating within exonic, intronic, and intergenic regions have typically been dismissed as transcriptional noise or technical artifacts. To explore circRNA diversity and appreciate their functions, we developed an algorithm to identify both canonical and noncanonical circRNAs without relying on genome annotation, enabling the identification of circRNAs of all types and in newly sequenced or poorly annotated species. Results from lung cancer cells revealed that noncanonical circRNAs constituted over two-thirds of the circRNA population and were expressed more abundantly than canonical circRNAs, and genes with fewer and shorter exons were hotspots for noncanonical circRNA and circRNA isoform production. Further analyses showed that many noncanonical circRNAs were indeed endogenous circRNAs transcribed within cells rather than experimental artifacts, were potentially translated into proteins or peptides, and were conserved across species. Moreover, we validated 65 noncanonical circRNAs in NCI-H23 cells using multiple bioassays and demonstrated that both exonic and intergenic noncanonical circRNAs influenced cell viability. CircRNA profiles in tumor and tumor-adjacent tissues of lung cancer patients revealed tissue-specific expression and differentially expressed canonical and noncanonical circRNAs from cognate genes involved in cancer-related pathways, indicating their potential clinical relevance. This study confirmed the authenticity of noncanonical circRNAs and provided the first experimental evidence that noncanonical circRNAs influence cancer cell phenotypes. These findings broaden our understanding of circRNA biology, highlighting their widespread genomic distribution, diverse functions, and potential clinical relevance.

molecular biology↗

bioRxiv2025

Metagenomic exploration of the bacteriome reveals natural Wolbachia infections in yellow fever mosquito Aedes aegypti and Asian tiger mosquito Aedes albopictus

BackgroundDengue and associated complications are spreading to non-endemic regions of Pakistan. Vector control, the foremost and widely adopted strategy for managing dengue has been implemented through various measures in Pakistan. Biological control through the use of Wolbachia, a bacterium naturally present in various insect genera, including Aedes, has demonstrated promising results globally. In this study we collected Aedes species and investigated their microbiomes with a particular focus on identifying the endosymbiont Wolbachia. MethodsMosquitoes were collected via Gravitraps in the Peshawar region of northwest Pakistan. The identity of the mosquitoes was initially confirmed through morphological characters followed by molecular identification using species-specific Cytochrome oxidase I (COI) primers. The DNA from female Ae. aegypti and Ae. albopictus was further subjected to 16S rRNA sequencing. The hypervariable regions V3/V4 of 16S rRNA were used for sequencing using the paired-end Illumina MiSeq platform. ResultsThe phylogenetic analysis of the COI gene in our samples demonstrated similarity to Aedes species previously documented in Pakistan. In comparative analysis of their microbiomes, Ae. albopictus was found to harbor 921 bacterial species, while Ae. aegypti only had 239 species. The metagenomic analysis revealed Wolbachia pipientis infection in Ae. aegypti while co-infection of Wolbachia pipientis and Wolbachia bourtzisii was detected in Ae. albopictus microbiota. ConclusionBoth Ae. aegypti and Ae. albopictus are present in Peshawar region of Khyber Pakhtoonkhwa province of Pakistan. Comparative analysis of the bacteriome showed higher bacterial diversity for Ae. albopictus as compared to Ae. aegypti. The investigation revealed Wolbachia infection in both Aedes mosquitoes species.

molecular biology↗

bioRxiv2020

Engineering highly multivalent sperm-binding IgG antibodies for potent non-hormonal female contraception

Many women risk unintended pregnancy due to dissatisfaction with available hormonal contraceptive methods. This led us to pursue topical sperm-binding monoclonal antibodies as a strategy for safe, non-hormonal contraception. Motivated by the greater agglutination potencies of polymeric immunoglobulins such as IgM and the exceptional bioprocessing ease in manufacturing IgG, we engineered IgGs possessing 6-10 Fabs against a unique surface antigen universally present on human sperm. These highly multivalent IgGs (HM-IgGs) are at least 10- to 16-fold more potent and faster than the parent IgG at agglutinating sperm, while preserving Fc-mediated trapping of individual spermatozoa in mucus. The increased potencies translate to effective (>99.9%) reduction of progressively motile sperm in the sheep vagina using 33 micrograms of the 10 Fab HM-IgG. HM-IgGs produce at comparable yields and possess identical thermal stability to the parent IgG, with greater homogeneity. HM-IgGs represent not only promising biologics for non-hormonal contraception but also a promising platform for generating potent agglutinating mAb for diverse medical applications.

molecular biology↗