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bioRxiv2025

The ZNF512B-NuRD complex co-ordinates a neural-specific gene expression program

The nucleosome remodelling and deacetylase complex (NuRD) plays a key role in chromatin regulation and a wide range of biological processes including development, haemopoiesis, immunity and neurogenesis. Its interaction with tissue-enriched and sequence-specific transcription factors (TFs) leads to distinct functional outputs in the given tissue by targeting a specific set of genes. However, how NuRD dynamically and specifically regulates gene expression in a tissue-specific manner is poorly understood. Here, we refine an N-terminal specific NuRD interaction motif which enables direct engagement with many transcriptional regulatory proteins. Using a series of structural modelling and biochemical techniques, we show that ZNF512B, a poorly characterised neuronal-expressed zinc finger protein, directly binds to the RBBP4 subunit of the NuRD complex. Subsequent knockdown of ZNF512B results in the downregulation of several neural-related molecular pathways suggesting that ZNF512B may play a regulatory role during neurogenesis. We also show that in NTERA-2 neural cells, the expression of ZNF512B is necessary for cell growth and survival, and is markedly enhanced during neural progenitor cell (NPC) differentiation. In summary, our data suggest that ZNF512B might regulate neural-specific transcriptional programs via engagement with the NuRD complex.

molecular biology↗

bioRxiv2019

The Clinical Significance and Biological Function of DPEP1 in B-cell Acute Lymphoblastic Leukemia

Dehydropeptidase-1 (DPEP1) is a zinc-dependent metalloproteinase abnormally expressed in many cancers. However, its potential role in adults with B-cell acute lymphoblastic leukaemia (ALL) is unknown.\n\nWe found that in adults with common-B-cell ALL high DPEP1 transcript levels at diagnosis was independently-associated with an increased CIR and worse RFS compared with subjects with low transcript levels. We show an increased proliferation and pro-survival role of DPEP1 in B-cell ALL cells via regulation of phosphCREB and p53 which may be the biological basis of the clinical correlation we report. Our data implicate DPEP1 expression in the biology of common B-cell ALL in adults. We report clinical correlates and provide a potential biological basis for these correlations. If confirmed, analyzing DPEP1 transcript levels at diagnosis could help predict therapy-outcomes. Moreover, regulation of DPEP1 expression could be a therapy target in B-cell ALL.

molecular biology↗

bioRxiv2019

Diversity and complexity of the large surface protein family in the compacted genomes of various Pneumocystis species

Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multi-copy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins, and provide detailed description of the classification, unique characteristics and phylogenetic relatedness of five Msg families. We further describe the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests a wide variety of functions for this superfamily, not only conferring antigenic variation to allow immune evasion but also mediating life-stage development, optimizing cell mobility and adhesion, and adapting to specific host niches or environmental conditions. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.

molecular biology↗

bioRxiv2024

Activation loop phosphorylation of Cdk11 is restrained by PNUTS-PP1 and regulates Cdk11 activity and function

Organisation of the transcription cycle is facilitated by the reversible phosphorylation of the C-terminal domain of RNA Polymerase II (RNAPII-CTD) and its accessory factors. The PNUTS-PP1 protein phosphatase is crucial for mRNA synthesis and processing, yet the complete spectrum of its physiological targets in these processes remain elusive. Here, using quantitative phosphoproteomics, we discover that Cdk11, in addition to various spliceosomal and RNA processing factors, associates with PNUTS, and that disruption of PP1-binding results in hyperphosphorylation of Cdk11 at an evolutionarily conserved Serine residue, seven amino acid residues C-terminal to DFG residues in the activation loop. In vitro experiments reveal a role for Ser DFG+7 in modulating Cdk11 kinase activity towards RNAPII-CTD Ser5. Making use of a novel technique to conditionally disrupt PP1 binding, we show that PNUTS-PP1 normally serves to restrain Cdk11 phosphorylation in vivo. Mutational analysis shows that cdk11 is not only essential for survival but also plays a widespread role in regulating normal mRNA expression and splicing. Notably, we find that a phosphomimetic mutation in cdk11 exhibits distinct biological effects compared to loss of cdk11 function, including defective processing of intronic small nucleolar RNAs, diminished intronic RNA Pol II velocity, and a decrease in intergenic transcription. These findings underscore physiologically significant roles of Cdk11 dephosphorylation by PNUTS-PP1 in the regulation of mRNA transcription and processing.

molecular biology↗

bioRxiv2019

Early-life adversity is associated with differential gene expression in response to acute psychological stress: preliminary findings

ObjectiveExposure to early-life adversity (ELA) can result in long-term changes to physiological systems, which predispose individuals to negative health outcomes. This biological embedding of stress-responsive systems may operate via dysregulation of physiological resources in response to common stressors. The present study used a novel experimental design to test how young adults exposure to ELA influence neuroendocrine and inflammatory responses to acute stress.\n\nMaterials and methodsParticipants were 12 males (mean age= 21.25), half of whom endorsed at least three significant adverse events up to age 18 years ( ELA group), and half who confirmed zero ( controls). Using a randomized within-subjects, between-groups experimental design, we induced acute psychosocial stress (Trier Social Stress Test, TSST), and included a no-stress control condition one week apart. During these sessions, we obtained repeated measurements of physiological reactivity, gene expression of NR3C1, FKBP5 and NFKB1, and plasma levels of pro-inflammatory cytokines (IL-1{beta}, IL-6, IL-8 and TNF) over a 4-hour window post-test.\n\nResultsThe ELA group evinced significantly higher cortisol response and lower NR3C1 gene expression in response to the TSST compared with controls, while no differences were observed in the no-stress condition. Cortisol and group status interacted such that increase in cortisol predicted increase in both NR3C1 and NFKB1 expression among controls, but decrease in the ELA group. For pro-inflammatory cytokines, only IL-6 increased significantly in response to the TSST, with no differences between the two groups.\n\nConclusionOverall, we provide preliminary findings for the biological embedding of stress via a dynamic and dysregulated pattern evidenced in response to acute psychosocial stress. ELA may program physiological systems in a maladaptive manner more likely to manifest during times of duress, predisposing individuals to the negative health consequences of everyday stressors. Future studies with larger sample size including both males and females are needed to replicate these findings.

molecular biology↗

bioRxiv2026

Ubi-SCAPE enables deep exploration of the poly-ubiquitylome

Post-translational modification with chains of the 76-amino-acid protein ubiquitin ( poly-ubiquitylation) confers diverse fates to the targeted protein and non-protein substrates, including degradation, intracellular trafficking, and signal transduction. Despite being one of the most frequent modifications, the complexity of poly-ubiquitin chains adds methodological challenges to their characterization. Trypsin-resistant tandem-ubiquitin-binding entities (trTUBEs), engineered from natural ubiquitin-binding domains, can capture intact poly-ubiquitylated proteins with high cumulative avidity. However, such approaches have suffered from considerable co-eluting contaminants in proteomics applications. Here, we introduce an optimized trTUBE-based method for poly-ubiquitylated proteome purification, drastically depleting non-ubiquitylated protein contaminants and mono-ubiquitylated proteins. The method, termed Ubiquitylomics by Stringent, Cleavable, Affinity-based Proteome Extraction (Ubi-SCAPE), offers a streamlined and reproducible (median R2 > 0.98; CV < 8%) means of characterizing the poly-ubiquitylome. Over 7,800 poly-ubiquitylated proteins and 8,500 ubiquitin-modified peptides (diGly) were quantified with Ubi-SCAPE at a throughput of 40 samples per day, as well as over 6,000 from an equivalent of 33 g unstressed cell lysate. Upon acute stress by heat-shock, we identified 2,700 proteins and 8,000 diGly peptides with increased poly-ubiquitylation--offering similar biological insight as with far more material-, labor-, and cost-intensive peptide-based ubiquitin enrichment methods. Ubi-SCAPE therefore provides a simple and effective means of comprehensively quantifying a selective-enriched poly-ubiquitylome. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/703496v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@a28120org.highwire.dtl.DTLVardef@cb801aorg.highwire.dtl.DTLVardef@47577aorg.highwire.dtl.DTLVardef@1c030af_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

RNA G-quadruplex structure contributes to cold adaptation in plants

Nucleotide composition is suggested to infer gene functionality and ecological adaptation of species to distinct environments. However, the underlying biological function of nucleotide composition dictating environmental adaptations is largely unknown. Here, we systematically analyzed the nucleotide compositions of transcriptomes across 1000 plants (1KP) and their corresponding habitats. Intriguingly, we found that plants growing in cold climates have guanine (G)-enriched transcriptomes, which are prone to forming RNA G-quadruplex structures. Both immunofluorescence detection and in vivo structure profiling revealed that RNA G-quadruplex formation in plants was globally enhanced in response to cold. Cold-responsive RNA G-quadruplexes strongly enhanced mRNA stability, rather than affecting translation. Disruption of individual RNA G-quadruplex promoted mRNA decay in the cold, leading to impaired plant cold response. Therefore, we propose that plants adopted RNA G-quadruplex structure as a molecular marker to facilitate their adaptation to the cold during evolution.

molecular biology↗

bioRxiv2023

ENHANCED EFFICIENCY OF RNA-GUIDED CAS12a VERSUS CAS9 TRANSGENE KNOCK-IN AND ACTIVITY AT A SCHISTOSOMA MANSONI GENOME SAFE HARBOR

Recently, we reported programmed Cas9 mediated insertion of a reporter gene into a gene safe harbor site, GSH1, of Schistosoma mansoni via homology-directed repair (HDR) using overlapping guide RNAs. Here, we report efficient and precise CRISPR/Cas12a-mediated homology directed insertion (knockin, KI) of a 5 C6-PEG10-modified double-stranded transgene bearing microhomology arms, 50 nt in length, at GSH1. At the outset, we undertook bioinformatic and computational analysis following by experimental verification of the regulatory activity of endogenous schistosome ubiquitin (SmUbi) promoter and terminator, to drive strong reporter gene expression. Green fluorescent protein activity driven by SmUbi followed electroporation-mediated transfection of schistosome eggs. HDR induced by RNA-guided CRISPR/Cas12a, which releases overhanging DNA strands of 18-24, delivered more efficient KI than CRISPR/Cas9. In this non-model pathogen, programmed KI facilitated precise chromosomal integration of the reporter-gene with at GSH1. The approach advances schistosome transgenesis field and may also advance functional genomics and transfection methods in related parasitic and non-parasitic helminths, which hitherto lack these tools. Author summaryGenome editing (CRISPR) technology is revolutionizing advances in biology, medicine, and agriculture. Transgenesis approaches are integral in diverse applications including gene therapy, biotherapeutics, deciphering host-pathogen interactions, and enhancements in agricultural production. Parasitic worms that are responsible for infectious diseases including neglected tropical diseases (NTDs), which cause substantial morbidity and mortality. NTDs mainly occur in the Global South, and they are responsible for a disease burden that exceeds that caused by malaria and tuberculosis. Infections with parasitic helminths also are responsible for immense economic burden in the agriculture. Tools for functional genomics in parasitic helminths are limited. Access to CRISPR-based approaches can be expected to hasten development of drug and/or vaccine targets for these diseases. Here, we focused on the helminth Schistosoma mansoni, a water borne parasite of humans, and which is endemic in Africa, and northeastern South America. To advance the state of the art in laboratory techniques currently used to study the biology and pathogenesis of this and related pathogens, we evaluated a spectrum of technological approaches aimed at improved current lab practice in this field. The findings demonstrated that specific technical and chemical modifications, including deploying a DNA cutting enzyme termed Cas12a along with a transgene with chemically modified short flanking sequences (homology arms) provided improved gene editing efficiency for this schistosome.

molecular biology↗

bioRxiv2025

High throughput protein serial crystallography using a grease matrix and a large-area support film

Serial femtosecond crystallography (SFX) using ultrashort pulses from X-ray free- electron lasers (XFELs) enables the determination of crystal structures at room temperature while minimizing radiation damage to the samples. This method involves irradiating numerous crystals one by one with XFEL pulses, allowing even the capture snapshots of dynamical structures in biological macromolecules. To achieve this, an efficient sample delivery system is essential for acquiring a large number of diffraction patterns. The most common approach uses a highly viscous grease matrix containing sample crystals, injected into the XFEL path from a narrow nozzle. However, the injection often suffers from clogging issues inside the injector nozzle, resulting in additional challenges such as the need for suitably sized crystals, increased sample consumption and unstable flow rates. Alternatively, a fixed-target approach, which scans a two-dimensional substrate with dispersed samples, can circumvent these issues. However, it must ensure the integrity of biological samples and provide sufficient surface area for efficient data collection. We here present an approach that utilizes a grease matrix and a large-area support film specially designed to address these requirements. This system offers a fast and reliable solution for protein SFX, enabling high-quality structure determination while significantly reducing sample consumption.

molecular biology↗

bioRxiv2018

A transmissible RNA pathway in honey bees

One of the characteristics of RNA interference (RNAi) is systemic spread of the silencing signal among cells and tissues throughout the organism. Systemic RNAi, initiated by double-stranded RNA (dsRNA) ingestion, has been reported in diverse invertebrates, including honey bees, demonstrating environmental RNA uptake that undermines homologous gene expression. However, the question why any organism would take up RNA from the environment has remained largely unanswered. Here, we report on horizontal RNA flow among honey bees mediated by secretion and ingestion of worker and royal jelly diets. We show that ingested dsRNA spreads through the bees hemolymph associated with a protein complex. The systemic dsRNA is secreted with the jelly and delivered to larvae via ingestion. Furthermore, we demonstrate that transmission of jelly-secreted dsRNA to larvae is biologically active and triggers gene knockdown that lasts into adulthood. Finally, RNA extracted from worker and royal jellies harbor differential naturally occurring RNA populations. Some of these RNAs corresponded to honey bee protein coding genes, transposable elements, non-coding RNA and exogenous viruses. These results reveal an inherent property of honey bees to share RNA among individuals and generations. Thus, our findings suggest a transmissible RNA pathway, playing a role in social immunity and epigenetic dynamics among honey bees and potentially other closely interacting organisms.\n\nSIGNIFICANCEHoney bees are eusocial insects, living in a colony that is often described as a superorganism. RNA mobility among cells of an organism has been documented in plants and animals. Here we show that RNA spreads further in honey bees, and is horizontally transferred between individuals and across generations. We found that honey bees share biologically active RNA through secretion and ingestion of worker and royal jellies. Such RNA initiated RNA interference, which is a known defense mechanism against viral infection. Furthermore, we characterized diverse RNA profiles of worker and royal jelly, including fragmented viral RNA. Our findings demonstrate a transmissible RNA pathway with potential roles in social immunity and epigenetic signaling among members of the hive.

molecular biology↗

bioRxiv2018

Science Family skills: An Alexa Assistant Tailored for Laboratory Routine

Voice User Interfaces such as Amazon Alexa and Google Home are already widely available and used for personal purposes. These services could be used to improve experimental biology laboratory routine, facilitate troubleshooting and increase efficiency. Till date, no applications that are tailored to enhance laboratory routine have been made available. Here, we present a set of free-to-use, open source tools adapted to Alexa for application in the laboratory environment, with prospects of enhancing productivity and reducing work-related stress. All skills, 3D printer model and source codes are freely available in the Alexa app store and in GitHub.

molecular biology↗

bioRxiv2019

The tropical coral Pocillopora acuta has a mosaic DNA methylome, an unusual chromatin structure and shows histone H3 clipping

Pocillopora acuta is a hermatypic coral with a worldwide distribution and a strong ecological importance. Anthropogenic disturbances and global warming threaten it. Thermal stress can induce coral bleaching, a phenomenon in which the mutualistic symbiosis between the coral polyps host and its endosymbiotic unicellular algae is disrupted, and can lead to the death of entire colonies. Previous works have shown that soma clonal colonies display different levels of survival depending on the environmental conditions they previously faced. Epigenetic mechanisms are good candidates to explain this phenomenon. The clonal nature of a colony and the possibility of generating genetically identical colonies through propagation make corals an attractive model to study the impact of the environment on the epigenome. However, until now, no work had been published on the P. acuta epigenome. One of the main problems is caused by the intracellular location of Symbiodinium, which makes it complicated to isolate coral chromatin free of contamination by endiosymbiotic biological material. Here, (i) we describe a simple method to purify P. acuta chromatin, (ii) we provide the first description of a coral methylome, with a mosaic pattern of cytosine methylation principally in a CpG context (4% of all CpG), and (iii) we show that P. acuta, but not all corals, has an unusual chromatin structure, and displays histone H3 clipping.

molecular biology↗

bioRxiv2021

Improved Genome Packaging Efficiency of AAV Vectors Using Rep Hybrids

Recombinant Adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last two decades research focused primarily on the characterization and isolation of new cap genes resulting in hundreds of natural and engineered AAV capsid variants while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the ssDNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major byproduct of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids ~2-4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. ImportanceA major byproduct of all Adeno-associated virus (AAV) vector production systems are "empty" capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

molecular biology↗

bioRxiv2023

A chemical probe to modulate human GID4 Pro/N-degron interactions

The CTLH complex is a multi-subunit ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via the substrate receptor GID4. Recently, focus has turned to this complex as a potential mediator of targeted protein degradation, but the role GID4-mediated substrate ubiquitylation and proteasomal degradation plays in humans has thus far remained unclear. Here, we report PFI-7, a potent, selective, and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation enabled the identification of dozens of endogenous GID4-interacting proteins that bind via the GID4 substrate binding pocket, only a subset of which possess canonical Pro/N-degron sequences. GID4 interactors are enriched for nuclear and nucleolar proteins including RNA helicases. GID4 antagonism by PFI-7 altered protein levels of several proteins including RNA helicases as measured by label-free quantitative proteomics, defining proteins that are regulated by GID4 and the CTLH complex in humans. Interactions with GID4 via Pro/N-degron pathway did not result in proteasomal degradation, demonstrating that CTLH interactors are regulated through a combination of degradative and non-degradative functions. The lack of degradation of GID4 interactors highlights potential challenges in utilizing GID4-recruiting bifunctional molecules for targeted protein degradation. Going forward, PFI-7 will be a valuable research tool for defining CTLH complex biology and honing targeted protein degradation strategies.

molecular biology↗

bioRxiv2023

Fully automated workflow for integrated sample digestion and Evotip loading enabling high-throughput clinical proteomics

Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample-preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 (OT-2) robot that combines sample digestion, cleanup and Evotip loading in a fully automated manner, allowing the processing of up to 192 samples in 6 hours. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability even at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic Ti-IMAC beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.

molecular biology↗

bioRxiv2024

Profiling IOP-responsive genes in anterior and posterior ocular tissues in the rat CEI glaucoma model

PurposeThe rat Controlled Elevation of Intraocular pressure (CEI) model allows study of in vivo responses to defined intraocular pressures (IOP). In this study, we use Nanostring technology to investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals. MethodsMale and female rats (N=35) were subject to CEI for 8-hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5x IOP (CEI-50). Naive animals, receiving no anesthesia or surgical interventions, served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA isolated, and samples were analyzed with a Nanostring panel containing 770 genes. Post-processing, raw count data were uploaded to Rosalind(R) for differential gene expression analyses. ResultsFor the TM, 45 IOP-related genes were significant in the "CEI-50 vs. CEI-20" and "CEI-50 vs. naive" comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and TGF{beta} pathways to be the most up- and down-regulated KEGG pathways, respectively. For ONH, 22 significantly regulated genes were identified in the "CEI-50 vs. naive" comparison. Pathway analysis identified defense response and immune response as two significantly upregulated biological process pathways. ConclusionsThis study demonstrates the ability to assay IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGF{beta} pathway genes suggest that TM responses may prevent TGF{beta}-induced extracellular matrix synthesis. For ONH, the initial response to elevated IOP may be protective, with astrocytes playing a key role in these gene responses.

molecular biology↗

bioRxiv2026

A novel long-amplicon rpoB primer pair for high resolution microbiome analysis at the species-level

The 16S rRNA gene is the most widely used genetic marker for microbial community profiling, but its limited sequence divergence often prevents species-level identification. The RNA polymerase {beta}-subunit gene (rpoB) offers higher sequence variability, single-copy occurrence, and stronger phylogenetic consistency, yet its adoption in metataxonomic studies has been constrained by the lack of universal primer sets. Here, we present a novel universal primer pair that amplifies an [~]1,800 bp rpoB region (rpoB_MV) compatible with long-read sequencing platforms. In silico evaluation across 17683 bacterial reference genomes demonstrated high universality, with over 86% of genomes predicted to amplify. Compared with full-length and partial 16S rRNA gene markers, the rpoB_MV amplicon exhibited significantly greater inter-species sequence divergence and improved phylogenetic concordance with core-genome trees. Sequencing of two complementary mock communities confirmed superior species-level identification accuracy, with misclassification rates below 0.01% and no reads assigned to unresolved species clusters. These results establish rpoB_MV as a robust alternative to 16S rRNA gene-based profiling for high-resolution metataxonomic applications. IMPORTANCEMicrobial community studies increasingly require species-level resolution because species within the same genus can differ substantially in pathogenicity, ecological function, and metabolic capacity. Current 16S rRNA gene-based methods frequently fail to distinguish closely related species, collapsing biologically distinct organisms into the same taxonomic assignment and obscuring community differences that matter for clinical diagnostics, food safety, and environmental monitoring. The rpoB_MV primer pair presented here overcomes this limitation by targeting a longer, more variable region of the rpoB gene, enabling accurate species-level identification across diverse bacterial phyla. Combined with advances in long-read sequencing, this approach provides researchers with a practical tool to resolve microbial communities at the species-level.

molecular biology↗

bioRxiv2024

PTTG1-Mediated Pericyte Dysfunction Drives Diabetes-Induced Microvascular Dysfunction

BackgroundPericytes are crucial for the development, stabilization, and functional regulation of microvasculature, especially in the retina. In diabetic retinopathy (DR), early loss of pericytes is a key event that drives microvascular dysfunction. Despite their critical role, the mechanisms underlying the functional heterogeneity of pericytes in DR remain poorly understood, impeding the development of effective therapeutic strategies. MethodsWe employed single-cell RNA sequencing to construct a comprehensive single- cell atlas of non-diabetic and diabetic retinas. Using bioinformatic clustering and subcluster analysis, we identified a specific pericyte subcluster associated with diabetic microvascular complications. Differential gene expression analysis and immunofluorescence validation highlighted PTTG1 as a potential key regulator of pericyte dysfunction. To investigate its functional role, we emplyed CRISPR/Cas9 and adenoviral vectors to modulate PTTG1 expression in vitro and in vivo. Combined transcriptomic and metabolomic approaches were used to explore the mechanistic pathways through which PTTG1 influences pericyte biology and vascular function. ResultsWe identified a novel pericyte subcluster characterized by elevated expression of PTTG1, which was strongly correlated with diabetic microvascular dysfunction. Silencing PTTG1 using CRISPR/Cas9 and siRNA in vitro mitigated pericyte dysfunction under high- glucose conditions. Targeted knockdown of PTTG1 using viral vectors improved retinal vascular integrity and reduced neovascularization in diabetic mice. Transcriptomic and untargeted metabolomic analyses revealed that PTTG1 knockdown reprogrammed pericyte energy metabolism by modulating glycolysis pathway genes, reducing oxidative stress, and restoring pericyte function, ultimately alleviating microvascular dysfunction in DR. ConclusionsPTTG1 plays a critical role in regulating pericyte dysfunction and maintaining vascular homeostasis in diabetic retinopathy. By modulating key metabolic pathways and pericyte phenotypes, PTTG1 represents a promising therapeutic target for treating diabetic microvascular complications. These insights offer a novel molecular framework for developing targeted therapies aimed at restoring retinal vascular health in diabetic patients.

molecular biology↗