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Results for “Molecular Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 181 records · Page 10Linked to original sources

bioRxiv2017

Interplay between mitochondria and diet mediates pathogen and stress resistance in C. elegans

Diet is a crucial determinant of organismal biology. Here we demonstrate the dramatic impact of a subtle shift in diet on the ability of Caenorhabditis elegans to survive pathogenic or abiotic stress. Interestingly, this shift occurs independently of canonical host defense pathways, arising instead from improvements in mitochondrial health. Using a variety of assays, we reveal that the most common C. elegans food source (E. coli OP50) results in a vitamin B12 deficiency that compromises mitochondrial homeostasis. Increasing B12 supply by feeding on E. coli HT115 or by supplementing bacterial media with methylcobalamin restored mitochondrial function, even if the bacteria were dead. B12 supplementation also efficiently increased host health without adversely affecting lifespan. Our study forges a molecular link between a dietary deficiency (nutrition/microbiota) and a physiological consequence (host sensitivity), using the host-microbiota-diet framework. The ubiquity of B12 deficiency (~10-40% of US adults) highlights the importance of our findings.

molecular biology↗

bioRxiv2018

Toxoplasma gondii ROP18 Inhibits Human Glioblastoma Cell Apoptosis through Mitochondrial Pathway by Targeting Host Cell P2X1

It is known that Toxoplasma gondii infection both initiates and inhibits host cell apoptosis through different proapoptotic signaling cascades, but the parasitic factors involved in these processes remain unclear. T. gondii virulence factor ROP18 has been reported to regulate host cell apoptosis, but the results of this regulation are few reported and contradictory. In this study, we found that immune or neuro cells infected by any one of the T. gondii strains (RH-type I, ME49-type II, and VEG-type III) showed a significantly lower apoptosis index than their uninfected controls when apoptosis was induced by staurosporine (STS). We further found that ROP18 of RH strain inhibited ATP induced apoptosis in human glioblastoma cells (SF268) with endogenous expression of human proapoptotic protein purinergic receptor 1 (P2X1), but had no effects on the immune cells of RAW264.7 and THP-1 without detectable P2X1 expression, which may indicate that ROP18s inhibition of host cell apoptosis is related to P2X1. Interestingly, we further identified that ROP18 (RH strain) interacted with P2X1, and over-expression of ROP18 in COS-7 cells inhibited the cell apoptosis mediated by P2X1. We also found that ROP18 of RH strain inhibited P2X1-mediated Ca2+ influx, translocation of cytochrome C from mitochondria to cytoplasm, and 1 ATP-triggered caspases activation. Collectively, these findings supported that ROP18 inhibited the host cell apoptosis through the intrinsic mitochondria pathway by targeting host cell P2X1, thereby suggesting a sensor role of the host proapoptotic protein P2X1 in this process\n\nAuthor summaryThe obligate intracellular protozoan Toxoplasma gondii has been shown to modulate cell apoptosis through different apoptotic pathways. However, the consequences are various and even contradictory, and the parasite effectors and the precise biological mechanisms remain unclear. Herein we showed that T. gondii of type I, II, and III strains could inhibit the apoptosis of neuro cells and immune cells. Toxoplasma gondii ROP18 (RH strain) inhibited apoptosis of human glioblastoma cell SF268 by targeting C terminal of host cell P2X1 protein, but not through proteasome-dependent degradation of P2X1.

molecular biology↗

bioRxiv2018

An automated workflow for the discovery and docking simulation of the protein-protein complexes using in vivo chemical cross-linking

Introduction Introduction Results Discussion Materials and Methods Competing interests Contributions Materials and Correspondence References Chemical cross(X)-link mapping assisted by mass spectrometry (XL-MS, also CXMS and CLMS) is a low-resolution hybrid method of structural biology, yielding a set of pairwise distance restraints between reactive solvent-accessible amino acids1-9. Most commonly used X-linkers at present belong to the class of amino-reactive homo-bifunctional NHS-esters, which act as protein proximity sensors, connecting predominantly Lys residues (as well as far less common and informative N-terminal amines)10-14. Main structural application of XL-MS to date i ...

molecular biology↗

bioRxiv2018

Protein Structural Biology Using Cell-Free Platform from Wheat Germ

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high yield and high purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multistage eukaryotic wheat-germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also developed a robust purification procedure, which can produce highly-pure (>98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid generation of samples for structural characterization that may not be amenable to other recombinant approaches.

molecular biology↗

bioRxiv2018

Evaluation of the antifibrotic potency by knocking down SPARC, CCR2 and SMAD3

The genes of SPARC, CCR2, and SMAD3 are implicated in orchestrating inflammation and fibrosis in scleroderma and other fibrotic disorders. Aim of the studies was to examine synergistic effect of inhibition of these genes in treating fibrosis. The peptide nanoparticles were used to deliver the siRNAs in bleomycin-induced fibrotic mice. Triple combination of siRNAs targeting on Sparc, Ccr2 and Smad3 achieved favorable anti-inflammatory and anti-fibrotic effects. Inhibition of inflammation was evidenced by reduced inflammatory cells and proinflammatory cytokines in the BALF and/or the tissues. Activation of fibroblasts was suppressed in mouse tissues in which -Sma and collagens were significantly reduced. Aberrant expression of the genes in fibroblasts, monocytes/macrophage, endothelial and epithelial cells were reinstalled after the treatment. In addition, transcriptome profiles indicated that some bleomycin-induced alterations of multiple biological pathways were recovered to varying degrees by the treatment. The results indicated that the triple combination of siRNAs systemically reinstated multiple biopathways, probably through controlling on different cell types including fibroblasts, monocytes/macrophages, endothelial cells and others. The multi-target-combined therapeutic approach examined herein may represent a novel and effective therapy for fibrosis.

molecular biology↗

bioRxiv2018

Evolutionary analysis of base-pairing interactions in DNA and RNA secondary structures

Pairs of nucleotides within biologically functional nucleic acid secondary structures often exhibit evidence of coevolution that is consistent with the maintenance of canonical base-pairing. MESSI is a sequence evolution model that infers substitution rates associated with base-paired sites in alignments of DNA or RNA sequences. MESSI can estimate these whilst simultaneously accounting for the uncertainty associated with an unknown RNA or DNA secondary structure shared across an alignment of sequences. Moreover, the unknown structure can be predicted, or a base-pairing probability matrix calculated. MESSI optionally leverages CUDA GPU parallelism to accelerate inference. MESSI was used to infer coevolution rates associated with GC, AU (AT in DNA), GU (GT in DNA) pairs in non-coding RNA alignments, and single-stranded RNA and DNA virus alignments. Inferred rates of GU pair coevolution were found to be higher at base-paired sites in single-stranded RNA viruses and non-coding RNAs than those of GT pairs in single-stranded DNA viruses, suggesting that GT pairs do not stabilise DNA secondary structures to the same extent as GU pairs in RNA. The relative coevolution rates associated with GC, AU, and GU pairs were largely consistent with their relative chemical base-pairing stabilities (GC base-pairs being more stable than AU base-pairs, and AU base-pairs being more stable than GU base-pairs). Additionally, MESSI estimates the degrees of coevolution at individual base-paired sites in an alignment. These estimates were computed for a SHAPE-MaP-determined HIV-1 NL4-3 RNA secondary structure and two corresponding alignments. MESSIs estimates of coevolution were significantly more strongly correlated with experimentally-determined SHAPE-MaP pairing scores as compared to three non-evolutionary measures of base-pairing covariation. Finally, to assist researchers in prioritising substructures with potential biological functionality, MESSI automatically identifies substructures and ranks them by degrees of coevolution at base-paired sites within them. Such a ranking was created for an HIV-1 subtype B alignment, revealing an excess of top-ranking substructures that have been previously identified in the literature as having structure-related functional importance, and a number of top-ranking structures that have not yet been characterised.

molecular biology↗

bioRxiv2017

Rapid Sequential In Situ Multiplexing With DNA-Exchange-Imaging

To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 {degrees}C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

bioengineering↗

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↗

bioRxiv2016

PASP - a whole-transcriptome poly(A) tail length determination assay for the Illumina platform

The poly(A) tail, co-transcriptionally added to most eukaryotic RNAs, plays an important role in post-transcriptional regulation through modulating mRNA stability and translational efficiency. The length of the poly(A) tail is dynamic, decreasing or increasing in response to various stimuli through the action of enzymatic complexes, and changes in tail length are exploited in regulatory pathways implicated in various biological processes.\n\nTo date, assessment of poly(A) tail length has mostly relied on protocols targeting only a few transcripts. We present PASP ( poly(A) tail sequencing protocol), a whole-transcriptome approach to measure tail lengths -- including a computational pipeline implementing all necessary analyses. PASP uses direct Illumina sequencing of cDNA fragments obtained through G-tailing of poly(A)-selected mRNA followed by fragmentation and reverse transcription.\n\nAnalysis of reads corresponding to spike-in poly(A) tracts of known length indicated that mean tail lengths can be confidently measured, given sufficient coverage. We further explored the utility of our approach by comparing tail lengths estimated from wild type and {Delta}ccr4-1/pan2 mutant yeasts. The yeast whole-transcriptome tail length distributions showed high consistency between biological replicates, and the expected upward shift in tail lengths in the mutant samples was detected. This suggests that PASP is suitable for the assessment of global polyadenylation status in yeast.\n\nThe correlation of per-transcript mean tail lengths between biological and technical replicates was low (higher between mutant samples). Both, however, reached high values after filtering for transcripts with greater coverage. We also compare our results with those of other methods. We identify a number of improvements that could be used in future PASP experiments and, based on our results, believe that direct sequencing of poly(A) tails can become the method of choice for studying polyadenylation using the Illumina platform

Molecular Biology↗

bioRxiv2026

Male Age and Sexual Maturity: Lipopolysaccharide-induced tumor necrosis factor influences sperm quality and reproduction in Anopheles culicifacies

Elucidating the biological and molecular mechanisms that govern male fertility and mating behavior in mosquitoes is critical for optimizing genetic and sterile insect technique-based vector control strategies. Here, we examined age-related changes in male reproductive capacity in Anopheles culicifacies, using female egg output as an indirect indicator of male fertility. Our results demonstrated that male reproductive age follows a non-linear pattern of fertility. Morphometric analysis from emergence to day 13 post-eclosion revealed a strong correlation between seminal vesicle capacity and female fecundity, suggesting that age-dependent gonadal development directly influences reproductive potential. At the molecular level, we identified AcLITAF6 as a key regulator of male reproductive homeostasis. RNAi-mediated knockdown of AcLITAF6 impaired apoptosis-associated and phagocytic clearance, reduced sperm viability, and decreased female productive outcomes. Conclusively, we reveal a previously unrecognized role of LITAF in sperm quality control and male reproductive fitness, highlighting AcLITAF6 as a potential target for mosquito population suppression strategies.

developmental biology↗

bioRxiv2026

Spatial profiling and neurovascular communication in the developing and adolescent cortex following prenatal alcohol exposure

Fetal alcohol spectrum disorders (FASD) constitute a wide range of developmental, cognitive, and behavioral impairments caused by prenatal alcohol exposure (PAE). Although neuronal and vascular consequences of PAE have been studied, how alcohol affects the cerebrovasculature within the framework of the neurovascular unit (NVU) across development remains poorly understood. At minimum, the NVU comprises neurons, astrocyte endfeet, and endothelial cells (ECs), which coordinate to maintain brain homeostasis. Here, we used the NanoString Digital Spatial Profiling platform to characterize spatial transcriptomic data from neurons, astrocytes, and ECs from PAE and saccharin (SAC) control cortices at embryonic day 18 (E18) and postnatal day 28 (P28). Differentially expressed genes were then used for Ingenuity Pathway Analysis (IPA) to identify altered biological pathways and perform comparison analyses across developmental time points, while CellChat was used to infer cell cell communication networks. We uncovered thousands of differentially expressed genes and numerous altered pathways and biological processes in PAE cortices across development. Both IPA and CellChat analyses implicated dysregulation of vascular and extracellular matrix (ECM) remodeling, cell adhesion, and neuroinflammatory signaling. CellChat further predicted the loss of several key bidirectional relationships and altered ligand-receptor interactions among neurovascular cell types at E18 and P28. Overall, these findings identify PAE associated alterations in neurovascular gene expression and intercellular signaling across development, providing potential mechanisms by which PAE may disrupt neurodevelopment.

molecular biology↗

bioRxiv2016

Visualizing adenosine to inosine RNA editing in single mammalian cells

Conversion of adenosine bases to inosine in RNA is a frequent type of RNA editing, but important details about its biology, including subcellular localization, remain unknown due to a lack of imaging tools. We developed an RNA FISH strategy we called inoFISH that enables us to directly visualize and quantify adenosine-to-inosine edited transcripts in situ. Applying this tool to three edited transcripts (GRIA2, EIF2AK2 and NUP43), we found that editing of these transcripts is not correlated with nuclear localization nor paraspeckle association, and that NUP43 exhibits constant editing rates between single cells while the rates for GRIA2 vary.

molecular biology↗

bioRxiv2018

miRNAs in platelet-poor blood plasma and purified RNA are highly stable: a confirmatory study

The relative stability of microRNAs (miRNAs) as compared with other RNA molecules has been confirmed in many contexts. When bound to Argonaute (AGO) proteins, miRNAs are protected from degradation, even when released into the extracellular space in ribonucleoprotein complexes, and with or without the protection of membranes in extracellular vesicles (EVs). Purified miRNAs also appear to present less of a target for degradation than other RNAs. Although miRNAs are by no means immune to degradation, biological samples subjected to prolonged incubation at room temperature, multiple freeze/thaws, or collection in the presence of inhibitors like heparin, can typically be remediated or used directly for miRNA measurements. Here, we provide additional confirmation of early, well validated findings on miRNA stability and detectability. Our data also suggest that inadequate depletion of platelets from plasma may explain the occasional report that freeze-thaw cycles can adversely affect plasma miRNA levels. Overall, the repeated observation of miRNA stability is again confirmed.

molecular biology↗

bioRxiv2017

An open-source cryo-storage solution

The field of cryo-electron microscopy is a rapidly growing method in structural biology. With this development, access to cryo-EM facilities becomes a bottleneck that results in long wait times between sample preparation and data acquisition. To improve sample storage, we developed a cryo-storage system with a more efficient and larger storage capacity that enables cryo-sample storage in a highly organized manner. This system is simple to use, cost-effective and easily adaptable for any type of grid box and storage dewar and any size cryo-EM laboratory.

molecular biology↗

bioRxiv2018

Organ-specific translation elongation rates measured by in vivo ribosome profiling

Protein synthesis and degradation are intricate biological processes involving more than a hundred proteins operating in a highly orches-trated fashion. Despite the progress, few options are available to access translation in live animals as the increase in animals complexity limits the repertoire of experimental tools that could be applied to observe and manipulate processes within animals body, organs, and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type specific translation elongation rates. It is based on a time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ among mouse organs and determined them to be 6.8, 5.2, and 4.4 amino acids per sec for liver, kidney, and skeletal muscle, respectively.\n\nSignificanceProtein synthesis is a vital biological process. Modern methods of genome editing enable generation of sophisticated animal models to study the regulation of protein synthesis in health end disease. However, the methods that could track various steps of translation at a gene level resolution in vivo are lacking, particularly in complex vertebrates, such as mice and rats. Here, we measured the translation elongation rate in several organs by delivering inhibitors specific to certain phases of translation directly through the mouse bloodstream. This study lays out a path for interrogating translation in animals in response to various genetic and dietary interventions.

molecular biology↗

bioRxiv2018

Kinetic Pathways of Topology Simplification by Type-II Topoisomerases in Knotted Supercoiled DNA

The topological state of covalently closed, double-stranded DNA is defined by the knot type K and the linking-number difference {Delta}Lk relative to unknotted relaxed DNA. DNA topoisomerases are essential enzymes that control the topology of DNA in all cells. In particular, type-II topoisomerases change both K and {Delta}Lk by a duplex-strand-passage mechanism and have been shown to simplify the topology of DNA to levels below thermal equilibrium at the expense of ATP hydrolysis. It remains a puzzle how small enzymes are able to preferentially select strand passages that result in topology simplification in much larger DNA molecules. Using numerical simulations, we consider the non-equilibrium dynamics of transitions between topological states (K, {Delta}Lk) in DNA induced by type-II topoisomerases. For a biological process that delivers DNA molecules in a given topological state (K,{Delta}Lk) at a constant rate we fully characterize the pathways of topology simplification by type-II topoisomerases in terms of stationary probability distributions and probability currents on the network of topological states (K,{Delta}Lk). In particular, we observe that type-II topoisomerase activity is significantly enhanced in DNA molecules that maintain a supercoiled state with constant torsional tension. This is relevant for bacterial cells in which torsional tension is maintained by enzyme-dependent homeostatic mechanisms such as DNA-gyrase activity.

molecular biology↗

bioRxiv2018

Combinatorial recognition of clustered RNA elementsby a multidomain RNA-binding protein, IMP3

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We have established an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, providing a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

molecular biology↗

bioRxiv2018

Transcription activity contributes to the activation of non-constitutive origins to maintain the robustness of S phase duration in African trypanosomes

The cosynthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences have not yet been investigated. Here, using equations and computational analysis we demonstrated that the number of constitutive origins mapped in the Trypanosoma brucei genome is close to the minimum required to complete replication within S phase duration. However, taking into account the location of these origins in the genome, the replication in due time becomes virtually impossible, making it necessary to activate non-constitutive origins. Moreover, computational and biological assays pointed to transcription being responsible for activating non-constitutive origins. Together, our results suggest that transcription action through conflicts with replication contributes to the firing of non-constitutive origins, maintaining the robustness of S phase duration. The usage of this entire pool of origins seems to be of paramount importance for the survival of this parasite that infects million people around the world since it contributes to the maintenance of the replication of its DNA.

molecular biology↗