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Results for “molecular biology”

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bioRxiv2024

Knockout of all nematode-specific NSPC genes expressed exclusively in the excretory gland cell results in transcriptomic signatures indicating an affected insulin signaling

The nematode Caenorhabditis elegans is one of the best-studied model organisms in molecular biology; however, many aspects of its physiology and the functions of many genes remain poorly understood. In this study, we investigated the role of nematode-specific NSPC proteins, whose mRNAs were recently identified as primary targets of the poly(A) polymerase TENT-5. Surprisingly, we found that NSPCs are exclusively expressed in the excretory gland cell, a cell with still unclear functionality. Using an optogenetic approach, we precisely ablated the excretory gland cell and observed that nematodes exhibited no transcriptomic or physiological changes in its absence. Additionally, we generated and thoroughly studied a strain with a deletion of all 18 nspc genes, which revealed that, despite previous indications, NSPCs do not influence the worms defense response. Instead, the transcriptomic analysis showed that the absence of NSPCs strongly impacts DAF-2/DAF-16 insulin signaling, suggesting that NSPCs may function as neuropeptides influencing key C. elegans signaling pathways. Although further studies are required to elucidate the physiological effects of this regulation, our findings provide new insights into this unexplored part of nematode physiology.

molecular biology↗

bioRxiv2024

Silencing of Hepatic AATF Ameliorates Metabolic Dysfunction Associated Steatohepatitis by Promoting Fatty Acid β-Oxidation in Experimental models

Background & aimsMetabolic dysfunction-associated steatohepatitis (MASH) is a multifactorial disease driven by complex molecular mechanisms. Identifying key regulators is critical for developing targeted therapies. Here, we demonstrate the impact of the loss of apoptosis antagonizing transcription factor (AATF) on hepatic lipid metabolism and MASH progression. MethodsA preclinical mouse model recapitulating human MASH was established by feeding C57Bl/6 mice either a chow diet (CD) or a western diet with sugar water (WD). Hepatic AATF silencing was achieved by tail vein injection of siAATF delivered by adeno-associated virus 8 (AAV8) with the liver-specific thyroxine-binding globulin (TBG) promoter. In addition to histological, biochemical, and molecular biology evaluations, the mechanistic insights were derived from whole transcriptomics and untargeted metabolomics analyses. ResultsAAV8-mediated specific knockdown of AATF in hepatocytes significantly reduced body weight, liver weight, and insulin resistance in mice fed with western diet (WD). However, no such effects were observed in mice fed with a chow diet (CD). Further analyses showed reduced liver injury, steatosis, and steatohepatitis in WDsiAATF mice. Transcriptomic analysis demonstrated that loss of AATF alleviated cellular stress, inflammation, and fibrosis in WD-fed mice. Moreover, AATF silencing led to alterations in lipid metabolism, notably by decreasing hepatic lipogenesis in the WD mice. Interestingly, untargeted metabolomics revealed an increase in the biosynthesis of glycerophospholipids and beta-oxidation of fatty acids in WDsiAATF mice. ConclusionOur findings reveal a previously unrecognized role of AATF as a central regulator of hepatic lipid metabolism in MASH, acting through the AKT-mTORC1 signaling pathway, and establish its inhibition as a promising therapeutic strategy for metabolic liver disease.

molecular biology↗

bioRxiv2019

Machine-driven parameter optimisation of biochemical reactions

The development of complex, multi-step methods in molecular biology is a laborious, costly, iterative and often intuition-bound process where an optimum is sought in a multidimensional parameter space through step-by-step optimisations. The difficulty of miniaturising reactions under the microliter volumes usually handled in multiwell plates by robots, plus the cost of the experiments, limit the number of parameters and the dynamic ranges that can be explored. Nevertheless, because of non-linearities of the response of biochemical systems to their reagent concentrations, broad dynamic ranges are necessary. Here we use a high-performance nanoliter handling platform and computer generation of liquid transfer programs to explore in quadruplicates more than 600 combinations of 4 parameters of a biochemical reaction, the reverse-transcription, which lead us to uncover non-linear responses, parameter interactions and novel mechanistic insights. With the increased availability of computer-driven laboratory platforms for biotechnology, our results demonstrate the feasibility and advantage of methods development based on reproducible, computer-aided exhaustive characterisation of biochemical systems.

molecular biology↗

bioRxiv2026

An Open-Source 3D-Printed Vacuum Manifold for Automated DNA Isolation in the Opentrons Flex and OT-2

Laboratory automation is increasingly being adopted in academic research to improve throughput, reproducibility, and integration with computational workflows. A common bottleneck in molecular biology workflows is the isolation of DNA, typically performed with spin-column kits relying on centrifugation or vacuum. Existing automation solutions depend on magnetic separation systems requiring costly additional modules and specialist kits, making them impractical for laboratories that perform large numbers of extractions only occasionally. Here we present an open-source, 3D-printed vacuum manifold compatible with the Opentrons family of liquid handling robots that enables automated spin-column DNA isolation for up to 24 samples per run (96 with four manifolds in series). The manifold can be manufactured for approximately $2 using standard ABS filament and is compatible with widely available spin-columns, requiring no additional specialist reagents. Using this system, hands-on time for 24 plasmid minipreps was reduced by 80% compared to manual centrifugation and vacuum protocols. The automated workflow demonstrated comparable or greater plasmid yields and eliminated failed recoveries. All design files, labware definitions, and Opentrons protocols are released under the CERN Open Hardware Licence or MIT License. This device lowers the cost barrier to automated DNA purification for academic laboratories worldwide.

molecular biology↗

bioRxiv2022

Protein Complexes in Bacillus subtilis by AI-assisted structural proteomics

Accurately modeling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Experimental data enables a candidate-based approach to systematically model novel protein assemblies. Here, we use a combination of in-cell crosslinking mass spectrometry, cofractionation mass spectrometry (CoFrac-MS) to identify protein-protein interactions in the model Gram-positive bacterium Bacillus subtilis. We show that crosslinking interactions prior to cell lysis reveals protein interactions that are often lost upon cell lysis. We predict the structures of these protein interactions and others in the SubtiWiki database with AlphaFold-Multimer and, after controlling for the false-positive rate of the predictions, we propose novel structural models of 153 dimeric and 14 trimeric protein assemblies. Crosslinking MS data independently validates the AlphaFold predictions and scoring. We report and validate novel interactors of central cellular machineries that include the ribosome, RNA polymerase and pyruvate dehydrogenase, assigning function to several uncharacterized proteins. Our approach uncovers protein-protein interactions inside intact cells, provides structural insight into their interaction interface, and is applicable to genetically intractable organisms, including pathogenic bacteria.

molecular biology↗

bioRxiv2021

DIRECT DETECTION OF LISTERIA MONOCYTOGENES BY RECOMBINASE POLYMERASE AMPLIFICATION

Listeria monocytogenes is one of the most common types of food poisoning bacteria which can cause serious foodborne diseases or even lethality. Generally, L. monocytogenes can be detected using traditional microbiology or molecular biology techniques, notably PCR. However, the application of these methods at the field is restricted due to the strict requirement of equipment and skilled personnel. In this study, recombinase polymerase amplification (RPA), an isothermal PCR assay was developed to rapidly detect L. monocytogenes in the crude samples. The results showed that the RPA reaction, without requiring complex thermal cycles, was well-performed in the optimal conditions of 39{degrees}C within only 25 minutes. The limit of detection was identified as 310 fg of L. monocytogenes genomic DNA, which was 1000-fold more sensitive than the conventional PCR. In addition, RPA also succeeded to directly detect L. monocytogenes cells at a concentration as low as 2.5 x 101 Colony Forming Unit (CFU)/ml in pure cultures and 2.5 x 102 CFU/ml in crude samples without sample extraction or processing. Therefore, RPA established in this study could be an alternative standard method to confirm the presence of L. monocytogenes in food. Accordingly, this rapid and sensitive method could be further applied to clinical testing for the diagnosis of L. monocytogenes infection, especially in areas with limited settings.

molecular biology↗

bioRxiv2025

Spatial Clustering of Interface Residues Enhances Few-Shot Prediction of Viral Protein Binding

AO_SCPLOWBSTRACTC_SCPLOWPredicting protein binding affinities across large combinatorial mutation spaces remains a critical challenge in molecular biology, particularly for understanding viral evolution and antibody interactions. While combinatorial mutagenesis experiments provide valuable data for training predictive models, they are typically limited due to experimental constraints. This creates a significant gap in our ability to predict the effects of more extensive mutation combinations, such as those observed in emerging SARS-CoV-2 variants. We present PROXICLUST, which strategically combines smaller combinatorial mutagenesis experiments to enable accurate predictions across larger combinatorial spaces. Our approach leverages the spatial proximity of amino acid residues to identify potential epistatic interactions, using these relationships to optimize the design of manageable-sized combinatorial experiments. By combining just two small combinatorial datasets, we achieve accurate binding affinity predictions across substantially larger mutation spaces (R2 {approx} 0.8), with performance strongly correlated with capture of high-order epistatic effects. We validated our method in five different protein-protein interaction datasets, including binding of SARS-CoV-2 receptor binding domain (RBD) to various antibodies and cellular receptors, as well as influenza RBD- antibody interactions. This work provides a practical framework for extending the predictive power of combinatorial mutagenesis beyond current experimental constraints, offering applications in viral surveillance and antibody engineering.

molecular biology↗

bioRxiv2018

The administration of high-mobility group box 1 fragment prevents deterioration of cardiac performance by enhancement of bone marrow mesenchymal stem cell homing in the delta-sarcoglycan-deficient hamster

ObjectivesWe hypothesized that systemic administration of high-mobility group box 1 fragment attenuates the progression of myocardial fibrosis and cardiac dysfunction in a hamster model of dilated cardiomyopathy by recruiting bone marrow mesenchymal stem cells thus causing enhancement of a self-regeneration system.\n\nMethodsTwenty-week-old J2N-k hamsters, which are {delta}-sarcoglycan-deficient, were treated with systemic injection of high-mobility group box 1 fragment (HMGB1, n=15) or phosphate buffered saline (control, n=11). Echocardiography for left ventricular function, cardiac histology, and molecular biology were analyzed. The life-prolonging effect was assessed separately using the HMGB1 and control groups, in addition to a monthly HMGB1 group which received monthly systemic injections of high-mobility group box 1 fragment, 3 times (HMGB1, n=11, control, n=9, monthly HMGB1, n=9).\n\nResultsThe HMGB1 group showed improved left ventricular ejection fraction, reduced myocardial fibrosis, and increased capillary density. The number of platelet-derived growth factor receptor-alpha and CD106 positive mesenchymal stem cells detected in the myocardium was significantly increased, and intra-myocardial expression of tumor necrosis factor stimulating gene 6, hepatic growth factor, and vascular endothelial growth factor were significantly upregulated after high-mobility group box 1 fragment administration. Improved survival was observed in the monthly HMGB1 group compared with the control group.\n\nConclusionsSystemic high-mobility group box 1 fragment administration attenuates the progression of left ventricular remodeling in a hamster model of dilated cardiomyopathy by enhanced homing of bone marrow mesenchymal stem cells into damaged myocardium, suggesting that high-mobility group box 1 fragment could be a new treatment for dilated cardiomyopathy.

molecular biology↗

bioRxiv2023

Detecting Protein-DNA Binding in Single Molecules using Antibody Guided Methylation

Characterization of DNA binding sites for specific proteins is of fundamental importance in molecular biology. It is commonly addressed experimentally by chromatin immunoprecipitation and sequencing (ChIP-seq) of bulk samples (103-107 cells). We have developed an alternative method that uses a Chromatin Antibody-mediated Methylating Protein (ChAMP) composed of a GpC methyltransferase fused to protein G. By tethering ChAMP to a primary antibody directed against the DNA-binding protein of interest, and selectively switching on its enzymatic activity in situ, we generated distinct and identifiable methylation patterns adjacent to the protein binding sites. This method is compatible with methods of single-cell methylation-detection and single molecule methylation identification. Indeed, as every binding event generates multiple nearby methylations, we were able to confidently detect protein binding in long single molecules. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/567792v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@dac365org.highwire.dtl.DTLVardef@522ecaorg.highwire.dtl.DTLVardef@17ddb38org.highwire.dtl.DTLVardef@1c371b0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO (i) ChAMP is added to fix and permeabilized cells, where it binds (ii) any antibody, and upon the addition of SAM, methylates nearby GpC sites, to be detected by sequencing (iii-iv). C_FIG

molecular biology↗

bioRxiv2018

Development of a papillation assay using constitutive promoters to find hyperactive transposases

BackgroundTransposable elements (TEs) form a diverse group of DNA sequences encoding functions for their own mobility. This ability has been exploited as a powerful tool for molecular biology and genomics techniques. However, their use is sometimes limited because their activity is auto-regulated to allow them to cohabit within their hosts without causing excessive genomic damage. To overcome these limitations, it is important to develop efficient and simple screening assays for hyperactive transposases.\n\nResultsTo widen the range of transposase expression normally accessible with inducible promoters, we have constructed a set of vectors based on constitutive promoters of different strengths. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. We observed the highest rate of transposition with the weakest promoters. We went on to investigate the effects of mutations in the Hsmar1 transposase dimer interface and of covalently linking two transposase monomers in a single-chain dimer. We also tested the severity of mutations in the lineage leading to the human SETMAR gene, in which one copy of the Hsmar1 transposase has contributed a domain.\n\nConclusionsWe generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. We also found that mutations in the Hsmar1 dimer interface provides resistance to overproduction inhibition in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.

molecular biology↗

bioRxiv2018

Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge-and column-based protocols require specialised equipment, often use toxic reagents and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA and total RNA from different sources, as well as environmental TNA and PCR amplicons. We also provide a bead-based protocol for bisulfite conversion, and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility and extreme cost-effectiveness of using magnetic beads. These open source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

molecular biology↗

bioRxiv2023

A C-terminal HaloTag Impairs AGO2 Function

A full understanding of RNA silencing requires appropriate molecular biology tools to explore the roles of Argonaute 2 (AGO2) and the RNA-induced silencing complex (RISC). Approaches relying on affinity tagging and antibodies have important limitations that can lead to artificial results. Both the N- and C-terminal domains of AGO2 have been shown to be important for correct activity and yet the consequences of appending tags to either terminus have not been fully investigated. N-terminal tags are frequently used to study AGO2 biology. Recently, an N-terminal HaloTag-Ago2 fusion was reported and examined in mice. While the versatile HaloTag provided new opportunities to study RISC biology, the tagged construct showed certain activity changes compared to unmodified AGO2. CRISPaint, a new CRISPR-Cas9 technique, permits the creation of endogenous C-terminal tag fusions. We used CRISPaint to generate the first reported recombinant AGO2 construct with a C-terminal tag: an endogenous C-terminal HaloTag fusion to AGO2 (AGO2HALO) in human (A549) cells. We found that the AGO2HALO fusion protein has a reduced capacity to interac with the key protein binding partner TNRC6A and that the C-terminal HaloTag does not affect cell viability. However, the AGO2HALO fusion significantly impairs RNA cleavage and RNA silencing activity compared to control cells and reduces nuclear localisation of the fusion protein. Using plasmid constructs and transient transfection, we compared AGO2 tagged with EGFP at the N- or C-terminus in siRNA and miRNA reporter gene assays, and cellular localisation. N-terminally tagged AGO2 functioned and localised similarly to WT untagged AGO2, whereas C-terminally tagged AGO2 was impaired in siRNA and miRNA silencing and exhibited poor nuclear and P-body localisation. We conclude that the fusion of a C-terminal HaloTag to AGO2 is not appropriate for studying AGO2 and RISC. Our results assert the importance of comprehensively validating recombinant tagging strategies to ensure that any experimental results generated do not arise from, or are not obscured by, critical functional defects.

molecular biology↗

bioRxiv2022

On-Ramp: a tool for rapid, multiplexed validation of plasmids using nanopore sequencing

Recombinant plasmid vectors are versatile tools which have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation, however this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids due to read-length limits. While next-generation sequencing (NGS) does provide full-plasmid sequencing at scale, it is impractical and costly when utilized outside of library-scale validation. Here we present OnRamp (Oxford nanopore-based Rapid Analysis of Multiplexed Plasmids), an alternative method for routine plasmid validation which combines the advantages of NGSs full plasmid coverage and scalability with Sangers affordability and accessibility by leveraging nanopores novel long-read sequencing technology. We include customized wet-lab protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is built into the OnRamp webapp (http://OnRamp.BoyleLab.org), which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views in a user-friendly manner, precluding the need for programming experience in analyzing nanopore results. Here we describe the OnRamp protocols and pipeline, and demonstrate our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure, at less than half the cost of equivalent Sanger sequencing.

molecular biology↗

bioRxiv2025

BLADE-R: streamlined RNA extraction for molecular diagnostics and high-throughput applications

Efficient nucleic acid extraction and purification are crucial for cellular and molecular biology research, yet they pose challenges for large-scale clinical RNA sequencing and PCR assays. Here, we present BLADE-R, a magnetic bead-based protocol that simplifies the process by combining cellular lysis and nucleic acid binding into a single step, followed by a unique on-bead rinse for nuclease-free separation of genomic DNA and RNA. The Agilent TapeStation and RT-qPCR analyses show that RNA extracted from HEK293T cell line using BLADE-R outperforms the TRIzol protocol in terms of time and cost. RNA sequencing reveals no differences in sequence quality or gene count variance between samples processed with BLADE-R and those processed with TRIzol followed by RNA kit clean-up. Additionally, BLADE-R outperformed TRIzol in RNA extraction from frozen tissue and whole blood samples, as confirmed by RT-qPCR. Our protocol can be adapted to a 96-well plate format, enabling RNA purification of up to 96 human blood samples in less time than a single-sample traditional extraction. Using BLADE-R in this format, we confirmed minimal well-to-well contamination in RNA purification, cDNA synthesis, and PCR. Therefore, our novel BLADE-R protocol, suitable for both low and high-throughput formats, is effective even in limited-resource settings for preparing clinical samples for PCR and sequencing assays. Thus, our new BLADE-R technique works well even in low-resource environments to prepare clinical samples for PCR and sequencing experiments. It can be adapted for both low- and high-throughput formats.

molecular biology↗

bioRxiv2016

RNA-DEPENDENT SYNTHESIS OF MAMMALIAN mRNA: IDENTIFICATION OF CHIMERIC INTERMEDIATE AND PUTATIVE END PRODUCT

Our initial understanding of the flow of protein-encoding genetic information, DNA to RNA to protein, a process defined as the \"central dogma of molecular biology\", was eventually amended to account for the information back-flow from RNA to DNA (reverse transcription), and for its \"side-flow\" from RNA to RNA (RNA-dependent RNA synthesis, RdRs). These processes, both potentially leading to protein production, were described only in viral systems, and although putative RNA-dependent RNA polymerase (RdRp) was shown to be present, and RdRs to occur, in most, if not all, mammalian cells, its function was presumed to be restricted to regulatory. Here we report the occurrence of protein-encoding RNA to RNA information transfer in mammalian cells. We describe below the detection, by next generation sequencing (NGS), of a chimeric doublestranded/pinhead intermediate containing both sense and antisense globin RNA strands covalently joined in a predicted and uniquely defined manner, whose cleavage at the pinhead would result in the generation of an endproduct containing the intact coding region of the original mRNA. We also describe the identification of the putative end product of RNA-dependent globin mRNA amplification. It is heavily modified, uniformly truncated at both untranslated regions (UTRs), terminates with the OH group at the 5 end, consistent with a cleavagegenerated 5 terminus, and its massive cellular amount is unprecedented for a conventional mRNA transcription product. It also translates in a cell-free system into polypeptides indistinguishable from the translation product of conventional globin mRNA. The physiological significance of the mammalian mRNA amplification, which might operate during terminal differentiation and in the production of highly abundant rapidly generated proteins such as some collagens or other components of extracellular matrix, with every genome-originated mRNA molecule acting as a potential template, as well as possible implications, including physiologically occurring intracellular PCR process, iPCR, are discussed in the paper.

Molecular Biology↗

bioRxiv2018

FilmArray Respiratory Panel Integrated in a field Point Of Care dispositive for the diagnosis of respiratory tract infections in rural areas in Senegal

The development of molecular syndrome-based kits for the diagnosis of respiratory infections offers rapid and sensitive detection of common respiratory pathogens and will have a significant impact on the care of patients. In this study, we present the results obtained after the introduction of the FilmArray respiratory panel in a field Point of care (POC) for the diagnosis of virus and bacteria responsible of respiratory tract infections in Senegal rural area. From February to August 2017, we collected nasal swabs from febrile patients that presented symptoms of respiratory tract infections in three health posts located in Niakhar. Specimens were tested on site by multiplex Polymerase Chain Reaction (PCR), using the FilmArray respiratory panel(R) that targets 20 pathogens, including 17 virus and 3 bacteria (bioMerieux). Nasal swabs were collected from 113 patients. The median age was 4 years (ranging from 4 months to 60 years) and 51 (45%) were males. The prevalence of respiratory pathogens was 37.5% (12/32) during the dry season and 54.3% (44/81) in the rainy season (p=0.16). The prevalence of respiratory pathogen carriage was higher in children under 5 years of age (38/55, 69.1%). The most prevalent micro-organisms detected were influenza B virus (16/113, 14%), human rhinovirus/enterovirus (10/113, 9%), parainfluenzae virus (9/113, 8%), respiratory syncytial virus (8/113, 7%), adenovirus (5/113, 4%), human metapneumovirus (3/113, 3%), Chlamydia pneumoniae (2/113, 2%) and Coronavirus (2/113, 2%). The study has demonstrated that the integration of the FilmArray respiratory panel into a field POC could significantly improve the management of respiratory tract infections in rural areas.\n\nAuthor summaryRespiratory tract infections are one of the leading causes of death worldwide. Populations in underdeveloped countries are the most affected, especially children under 5 years of age. Among the pathogens responsible for these infections, bacteria and viruses are the most identified. In poor countries, laboratory diagnosis can only be done in urban areas. They are generally based on bacteriological, immunological or molecular biology techniques. The concept of POC is not developed in underdeveloped countries. Its existence would have made it possible to carry out RDTs to confirm the clinicians diagnosis and allow rapid management of the patient. In the current situation, the time required to achieve results is often very long for patients. In this study, we sought to demonstrate the significant contribution of the FilmArray respiratory panel in the management of respiratory tract infections in rural areas. We also wanted to reduce the time it takes to deliver results in order to improve patient care. This dispositif was integrated in a field POC which was implemented in Niakhar since 2015, in order to improve the management of emerging diseases. The FilmArray respiratory panel gave us the opportunity to investigate the causes of respiratory tract infections in this area. For each patient, we systematically target 20 pathogens, including 17 viruses and 3 bacteria, with a single multiplex PCR. One of the main results of this study is that children under 5 years of age are the most affected by respiratory tract infections. Then we noted a lack of consultation among adults that could be explained by the banalization of respiratory problems or a preference for traditional care. The fact that children under 5 years of age are the most affected could also serve as a basis for implementing vaccination programmes directly targeting the most vulnerable age groups. It should be noted that for 51% of patients, the result of the diagnosis was negative. It would appear that some pathogens responsible for respiratory tract infections are not targeted by the multiplex PCR. Thus, it would be necessary to screen these pathogens in order to integrate them into a panel that would cover the most pathogens in circulation in this area.

molecular biology↗

bioRxiv2026

HDAC11 Regulates RNA Splicing via De-Fatty Acylation of SF3B2

Histone deacetylase 11 (HDAC11) is a lysine de-fatty acylase whose cellular substrates and mechanisms remain incompletely defined. Here, using metabolic labeling, mass spectrometry, click chemistry, and standard molecular biology, we show that SF3B2 is modified by lysine myristoylation at K10 and that HDAC11 efficiently removes this modification in cells, establishing SF3B2 as a direct enzymatic substrate. A de-myristoylation mimetic mutant (SF3B2 K10R) exhibits altered pre-mRNA binding activity in a context-dependent manner. In HCC cells, loss of SF3B2 lysine myristoylation enhances SF3B2 association with androgen receptor (AR) splice variant loci and promotes alternative splicing towards the AR-v7 variant. Consistently, HDAC11 overexpression increases, and HDAC11 knockdown decreases, the AR-v7/AR-FL splice isoform ratio in HCC cells in a manner requiring HDAC11 catalytic activity and recapitulated by SF3B2 K10R. In contrast, modulation of HDAC11 does not alter AR splicing in prostate cancer cells, indicating cell type specific regulation. Together, these findings establish lysine myristoylation as a reversible regulatory modification on a spliceosomal component and reveal HDAC11-catalyzed de-myristoylation of SF3B2 as a mechanism that can tune alternative splicing in liver cancer cells. In BriefClements et al. utilize metabolic labelling, mass spectrometry, click chemistry, and protein and RNA biochemistry to establish that a histone deacetylase enzyme, HDAC11, can influence RNA splicing through de-fatty acylation of the RNA splicing factor SF3B2. De-fatty acylation of SF3B2 at K10 by HDAC11 modulates SF3B2s pre-mRNA binding to AR splice variant loci, thereby driving alternative splicing of the AR-v7 variant in a cell type dependent manner. This work provides direct mechanistic evidence linking an HDAC to RNA splicing, identifies a reversible lipid modification on SF3B2, and expands current understanding of post-translational regulation of spliceosomal proteins and HDAC11 de-fatty acylation substrates. HighlightsO_LIHDAC11 de-fatty-acylates SF3B2 at K10, revealing a previously unrecognized modification on SF3B2. C_LIO_LISF3B2 de-fatty acylation enhances alternative splice-site binding in liver cancer cells. C_LIO_LIHDAC11 regulates RNA splicing through enzymatic de-fatty acylation of a spliceosomal protein. C_LI

molecular biology↗

bioRxiv2020

Molecular profiling of lipid droplets inside HuH7 Cells with Raman micro-spectroscopy

Raman imaging has become an attractive technology in molecular biology because of its ability to detect multiple molecular components simultaneously without labeling. Two major limitations in accurately accounting for spectral features, viz. background removal and spectral unmixing, have been overcome by employing a modified and effective routine in multivariate curve resolution (MCR). With our improved strategy, we have spectrally isolated seven structurally specific biomolecules without any post-acquisition spectral treatments. Consequently, the isolated intensity profiles reflected concentrations of corresponding biomolecules with high statistical accuracy. Our study reveals the changes in the molecular composition of lipid droplets (LD) inside HuH7 cells and its relation to the physiological state of the cell. Further, we show that the accurate separation of spectral components permits analysis of in vivo structural modification of molecules after cellular uptake. A detailed discussion is presented to highlight the potential of Raman spectroscopy with MCR in semi-quantitative molecular profiling of living cells.

bioengineering↗