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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 325 records · Page 18Linked to original sources

bioRxiv2016

Laboratory strains of Aedes aegypti are Competent to Brazilian Zika virus

Since the Zika outbreaks are unprecedented human threat in relation to congenital malformations and neurological/autoimmune complications as well as its high potential to spread in regions presenting the vectors, improvements in mosquito control is a top priority. Thus, Aedes aegypti laboratory strains will be fundamental to support studies in different research fields implicated on Zika-mosquito interactions which are the basis for the development of innovative control methods. In this sense, we determined the main infection aspects of the Brazilian Zika strain in reference Aedes aegypti laboratory mosquitoes.\n\nWe orally exposed Rockefeller, Higgs and Rexville mosquitoes to a Brazilian ZIKV (ZIKVBR) and qRT-PCR was applied to determine the infection and dissemination rates, and viral levels in mosquito tissues as well as in the saliva. The ZIKVBR kinetics was monitored during the infection in Rockefeller mosquitoes. Rockefeller strain was the most susceptible at 7 days post-infection but all strains presented similar infection levels at 14 days post-infection. Although variations in the saliva detection rates were observed, we confirmed that ZIKVBR was present in saliva from Rockefeller, Higgs and Rexville females at detectable levels at 14 days post-infection. The ZIKVBR kinetics in Rockefeller mosquitoes showed that the virus could be detected in the heads at 4 days post-infection but was more consistently detected late in infection. The viral levels peaked at 11 days post-infection in the mosquito bodies, remaining stable until 14 days post-infection, in contrast to the heads, where the mean viral levels only peaked at 14 days post-infection.\n\nOur study presents the first evaluation on how Brazilian Zika virus behaves in reference Aedes aegypti strains and shed light on how the infection evolves over time. Vector competence and basic hallmarks of the ZIKVBR development were revealed in laboratory mosquitoes. This study provides additional information to accelerate studies focusing on ZIKV-mosquito interactions.

Molecular Biology↗

bioRxiv2016

The dichotomous effects of caffeine on homologous recombination in mammalian cells

This study was initiated to examine the effects of caffeine on the DNA damage response (DDR) and homologous recombination (HR). An initial 2 h exposure to 5 mM caffeine slowed a fraction of the cells in G1, but thereafter, continued caffeine exposure permitted this cell fraction to progress through the cycle until they eventually stalled at G2/M and underwent apoptosis. This prolonged caffeine exposure also induced a strong DDR along with subsequent activation of wild-type p53 protein. An unexpected observation was the caffeine-induced depletion of Rad51 (and Brca2) proteins. Consequently, caffeine-treated cells were expected to be inefficient in HR. However, a dichotomy in the HR response of cells to caffeine treatment was revealed. Caffeine treatment rendered cells significantly better at performing the nascent DNA synthesis that accompanies the early strand invasion steps of HR. Conversely, the increase in nascent DNA synthesis did not translate into a higher level of gene targeting events. Levels of Rad51 appear to be irrelevant. Thus, prolonged caffeine exposure stalls the cell cycle, induces a p53-mediated apoptotic response and a down-regulation of critical HR proteins, and stimulates early steps of HR, but not the formation of complete repair products.

Molecular Biology↗

bioRxiv2016

Degradation of non-methylated DNA by MsPJI impairs its usefulness as epigenetic tool

DNA treatment with sensitive and/or dependent restriction enzymes, followed by PCR amplification is a widely used approach for testing CpG methylation. Recently, MspJI has been characterized as a promisory tool for epigenetic analyses. In the present report, we describe that MspJI shows significant activity against non-methylated DNA, suggesting that additional caution and improvements would be required before applying this enzyme as a routine epigenetic tool.

Molecular Biology↗

bioRxiv2016

Effects of Pol II catalytic mutants on in vivo elongation rate, processivity, gene expression, mRNA decay and response to nucleotide depletion

Here we employ a set of RNA Polymerase II (Pol II) activity mutants to determine the consequences of increased or decreased Pol II catalysis on gene expression in Saccharomyces cerevisiae. We find that alteration of Pol II catalytic rate, either fast or slow, leads to decreased Pol II occupancy and apparent reduction in elongation rate in vivo. However, we also find that determination of elongation rate in vivo by chromatin IP can be confounded by the kinetics and conditions of transcriptional shutoff in the assay. We identify promoter and template-specific effects on severity of gene expression defects for both fast and slow Pol II mutants. We show that mRNA half-lives for a reporter gene are increased in both fast and slow Pol II mutant strains and the magnitude of half-life changes correlate both with mutants growth and reporter expression defects. Finally, we tested a model that altered Pol II activity sensitizes cells to nucleotide depletion. In contrast to model predictions, mutated Pol II retains normal sensitivity to altered nucleotide levels. Our experiments establish a framework for understanding the diversity of transcription defects derived from altered Pol II activity mutants, essential for their use as probes of transcription mechanisms.

Molecular Biology↗

bioRxiv2016

An efficient electroporation protocol for the genetic modification of mammalian cells

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square wave generating devices, like Lonza{acute}s Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work we show that our in house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with Sleeping Beauty based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology.

Molecular Biology↗

bioRxiv2016

Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings support a model whereby MEX-5 functions as an mRNA sink to locally suppress MEG-3 phase separation and drive P granule asymmetry.\n\nHIGHLIGHTS- The intrinsically-disordered protein MEG-3 is essential for localized assembly of P granules in C. elegans zygotes.\n- MEG-3 binds RNA and RNA stimulates MEG-3 phase separation.\n- The RNA-binding protein MEX-5 inhibits MEG-3 granule assembly in the anterior cytoplasm by sequestering RNA.

Molecular Biology↗

bioRxiv2016

Fast algorithm for determining orientations using angular correlation functions and Bayesian statistics

Cryogenic electron microscopy (cryo-EM) method achieved revolutionary improvement in the past a few years, yet the orientation recovery for each particle remains computational challenging. Orientation determination can be solved using Bayesian approach, through which a model can be constructed to best match a whole set of experimental projections at their correct orientations. Without considering the centering of each particle projection, there are three degrees of freedoms (three Euler angles) that need to be fixed, usually resulting a computational complexity O(n3) where n is the number of discretization for each rotation angle. Here, we propose a method based on Maximum Likelihood approach with angular auto-correlation function of each projection, which is utilized to decouple the determination of three Euler angles to stepwise determination of two Euler angles and the subsequent third angle, the in-plane rotation. This approach reduces computational complexity from O(n3) to O(n2). Using simulation data, the accuracy and speed of the method is compared with the original maximum likelihood approach. We also investigated the impact of noise to the performance of this proposed method.

Molecular Biology↗

bioRxiv2016

PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions

Interactions between RNA binding protein (RBP) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using protein-centered (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest - in a gene-centered manner, yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA bait can be tested versus multiple RBP preys in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3'UTRs. PRIMA faithfully recapitulates numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored.

Molecular Biology↗

bioRxiv2016

GeRelion: GPU-enhanced parallel implementation of single particle cryo-EM image processing

Single particle cryo-EM emerges as a powerful and versatile method to characterize the structure and function of macromolecules, revealing the structural details of critical molecular machinery inside the cells. RELION is a widely used EM image processing software, and most of the recently published single particle cryo-EM structures were generated by using RELION. Due to the massive computational loads and the growing demands for processing much larger cryo-EM data sets, there is a pressing need to speed up image processing. Here we present GeRelion (https://github.com/gpu-pdl{not}nudt/GeRelion), an efficient parallel implementation of RELION on GPU system. In the performance tests using two cryo-EM data sets, GeRelion on 4 or 8 GPU cards outperformed RELION on 256 CPU cores, demonstrating dramatically improved speed and superb scalability. By greatly accelerating single particle cryo-EM structural analysis, GeRelion will facilitate both high resolution structure determination and dissection of mixed conformations of dynamic molecular machines.

Molecular Biology↗

bioRxiv2016

The RNA-binding protein RBP10 controls a regulatory cascade that defines bloodstream-form trypanosome identity

Gene expression control in the pathogen Trypanosoma brucei relies almost exclusively on post-transcriptional mechanisms, so RNA binding proteins must assume the burden that is usually borne by transcription factors. T. brucei multiply in the blood of mammals as bloodstream forms, and in the midgut of Tsetse flies as procyclic forms. We show here that a single RNA-binding protein, RBP10, defines the bloodstream-form trypanosome differentiation state. Depletion of RBP10 from bloodstream-form trypanosomes gives cells that can grow only as procyclic forms; conversely, expression of RBP10 in procyclic forms converts them to bloodstream forms. RBP10 binds to procyclic-specific mRNAs containing an UAUUUUUU motif, targeting them for translation repression and destruction. Products of RBP10 target mRNAs include not only the major procyclic surface protein and enzymes of energy metabolism, but also protein kinases and stage-specific RNA-binding proteins: consequently, alterations in RBP10 trigger a regulatory cascade.

Molecular Biology↗

bioRxiv2016

The Mstncmpt-Dl1abc mutation impairs secretion of promyostatin

Hypermuscularity of Compact mouse is caused by a 12-bp deletion in the myostatin gene, but the molecular basis of decreased myostatin activity is unclear since the deletion does not affect the integrity of the growth factor domain. In the present work we show that the deletion causes misfolding and impaired secretion of myostatin precursor with concomitant decrease in myostatin activity. We suggest that some modifier genes that influence the expression of the Compact phenotype of myostatin mutant MstnCmpt-dl1Abc mice may exert their action through their role in the Unfolded Protein Response.

Molecular Biology↗

bioRxiv2016

In−silico studies of facilitated VEGF(s) − VEGFR(s) bindings for assessment of Lysine as an indirect Low−Mol−Wt angiogen: Experimental validation of a potential synthetic Low−Mol−Wt angiogen.

In-vivo angiogenesis process is highly conserved and is mediated through a family of peptides having VEGF-A as the lead member. A respective receptor family comprising of members VEGFR-1, 2, 3 gets expressed on the endothelial cell membrane of the vascular bed in ischemic zone along with parallel expressions of VEGF-A, B, C, D and PlGF. Degree of ischaemia is the main regulator of these coupled expressions of angiogenic peptides/factors (AFs) and respective receptor(s) for a paracrine angiogenic process to take place. Physiological angiogenesis in intrauterine growth phase is the lead process in foetal growth, organogenesis and cellular specialization. Post birth and with aging, this process gets gradually inefficient and slow. In the present in-silico study, all angiogenic factors and receptor species are examined as for their binding stability in basal unaided condition and in presence of a possible Low-Mol-Wt linkage molecule-Lysine. Also a Lysine analogue 1,6-diaminohexanoic acid has been examined for its angiogenic potential both in dry docking experiment and in cell culture assay.

Molecular Biology↗

bioRxiv2016

AN INTERNATIONAL INTER-LABORATORY DIGITAL PCR STUDY DEMONSTRATES HIGH REPRODUCIBILITY FOR THE MEASUREMENT OF A RARE SEQUENCE VARIANT

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate labs. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using qPCR or NGS due to the presence of competing wild type sequences and the need for calibration. Using dPCR, eighteen laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration and could enable the reproducible application of molecular stratification to guide therapy, and potentially for molecular diagnostics.\n\nSIGNIFICANCE STATEMENTThe poor reproducibility of molecular diagnostic methods limits their application in part due to the challenges associated with calibration of what are relative measurement approaches. In this study we investigate the performance of one of the only absolute measurement methods available today, digital PCR (dPCR), and demonstrated that when compared across twenty-one laboratories, dPCR has unprecedented reproducibility. These results were achieved when measuring a challenging single nucleotide variant and without calibration to any reference samples. This opens the possibility for dPCR to offer a method to transform reproducibility in the molecular diagnostic field, both by direct use as well as in support of other currently used clinical methods.

Molecular Biology↗

bioRxiv2016

A Pif1-dependent threshold separates DNA double-strand breaks and telomeres

The natural ends of chromosomes resemble DNA double-strand breaks (DSBs) and telomeres are therefore necessary to prevent recognition by the DNA damage response. The enzyme telomerase can also generate new telomeres at DSBs, resulting in the loss of genetic information distal to the break. How cells deal with different DNA ends is therefore an important decision. One critical point of regulation is to limit telomerase activity at DSBs and this is primarily accomplished in budding yeast by the telomerase inhibitor Pif1. Here we use Pif1 as a sensor to gain insight into the cellular decision at DSB ends with increasing telomeric character. We uncover a striking transition point in which 34 bp of telomeric (TG1-3)n repeat sequence is sufficient to render a DNA end insensitive to Pif1, thereby facilitating extension by telomerase. This phenomenon is unlikely to be due to Pif1 modification and we propose that Cdc13 confers a unique property to the TG34 end that prevents Pif1 action. We identify novel Cdc13 mutations that resensitize DNA ends to Pif1 and discover that many Cdc13 telomerase-null mutations are dependent on Pif1 status. Finally, the observed threshold of Pif1 activity recapitulates several properties of both DSBs and telomeres and we propose that this is the dividing line between these entities.

Molecular Biology↗

bioRxiv2016

Evolving Notch polyQ tracts reveal possible solenoid interference elements

Polyglutamine (polyQ) tracts in regulatory proteins are extremely polymorphic. As functional elements under selection for length, triplet repeats are prone to DNA replication slippage and indel mutations. Many polyQ tracts are also embedded within intrinsically disordered domains, which are less constrained, fast evolving, and difficult to characterize. To identify structural principles underlying polyQ tracts in disordered regulatory domains, here I analyze deep evolution of metazoan Notch polyQ tracts, which can generate alleles causing developmental and neurogenic defects. I show that Notch features polyQ tract turnover that is restricted to a discrete number of conserved \"polyQ insertion slots\". Notch polyQ insertion slots are: (i) identifiable by an amphipathic \"slot leader\" motif; (ii) conserved as an intact C-terminal array in a 1-to-1 relationship with the N-terminal solenoid-forming ankyrin repeats (ARs); and (iii) enriched in carboxamide residues (Q/N), whose sidechains feature dual hydrogen bond donor and acceptor atoms. Correspondingly, the terminal loop and {beta}-strand of each AR feature conserved carboxamide residues, which would be susceptible to folding interference by hydrogen bonding with residues outside the ARs. I thus suggest that Notch polyQ insertion slots constitute an array of AR interference elements (ARIEs). Notch ARIEs would dynamically compete with the delicate serial folding induced by adjacent ARs. Huntingtin, which harbors solenoid-forming HEAT repeats, also possesses a similar number of polyQ insertion slots. These results strongly suggest that intrinsically disordered interference arrays featuring carboxamide and polyQ enrichment are coupled proteodynamic modulators of solenoids.\n\nSIGNIFICANCENeurodegenerative disorders are often caused by expanded polyglutamine (polyQ) tracts embedded in the disordered regions of regulatory proteins, which are difficult to characterize structurally. To identify functional principles underlying polyQ tracts in disordered regulatory domains, I analyze evolution of the Notch protein, which can generate polyQ-related alleles causing neurodevelopmental defects. I show that Notch evolves polyQ tracts that come and go in a few conserved \"polyQ insertion slots\". Several features suggest these slots are ankyrin repeat (AR) interference elements, which dynamically compete with the delicate solenoid formed by Notch. Huntingtin, whose polyQ expansions causes Huntingtons Disease in humans, also has solenoid-forming modules and polyQ insertion slots, suggesting a common architectural principle underlies solenoid-forming polyQ-rich proteins.

Molecular Biology↗

bioRxiv2016

Lysine as a potential low molecular weight angiogen: its clinical, experimental and in-silico validation- A brief study

Globally, the area of angiogenesis is dominated by investigations on anti-angiogenic agents and processes, due to its role in metastatic cancer treatment. Although, the area of ischemic tissue reperfusion is having much bigger demand and foot-mark. Following clinical failure of VEGF (Vascular endothelial growth factor) as a potential agent for induction of a controlled angiogenic response in ischemic tissues and organs, the progress is reasonably quiet as for new low molecular weight (LMW) angiogen molecules and their clinical applications are concerned. Basic amino acid Lysine has been observed to have profound angiogenic property in ischemic tissues, which is controlled, reproducible, time bound and without any accompanying reperfusion damage. In this study, the basic amino acid Lysine has been suggested as a LMW-angiogen, where it has been proposed to have a molecular binding property between VEGF and VEGF receptor (VEGFR). Here, the molecular adhesive hypothesis is being probed and confirmed both in the clinical and lab conditions through induced angiogenic response in tissue repair and in chick chorio allantoic membrane (CAM), respectively; and in dry-docking experiments (in-silico studies).

Molecular Biology↗

bioRxiv2016

SIRVs: Spike-In RNA Variants as External Isoform Controls in RNA-Sequencing

Spike-In RNA variants (SIRVs) enable for the first time the validation of RNA sequencing workflows using external isoform transcript controls. 69 transcripts, derived from seven human model genes, cover the eukaryotic transcriptome complexity of start- and end-site variations, alternative splicing, overlapping genes, and antisense transcription in a condensed format. Reference RNA samples were spiked with SIRV mixes, sequenced, and exemplarily four data evaluation pipelines were challenged to account for biases introduced by the RNA-Seq workflow. The deviations of the respective isoform quantifications from the known inputs allow to determine the comparability of sequencing experiments and to extrapolate to which degree alterations in an RNA-Seq workflow affect gene expression measurements. The SIRVs as external isoform controls are an important gauge for inter-experimental comparability and a modular spike-in contribution to clear the way for diagnostic RNA-Seq applications.

molecular biology↗

bioRxiv2016

Serum extracellular vesicle depletion processes affect release and infectivity of HIV-1 in culture

Extracellular vesicles (EVs, including exosomes and microvesicles) are involved in intercellular communication in health and disease and affect processes including immune and antiviral responses. Ultracentrifuged serum is depleted of EVs and, when used in culture media, reduces growth and viability of some cell types. In this study, we examined the effects of serum EV depletion processes on HIV-1 replication in primary cells and cell lines, including two HIV-1 latency models. Increased HIV-1 production was observed in certain EV-depleted conditions, along with cell morphology changes and decreased cell viability. Add-back of ultracentrifuge pellets rescued baseline HIV-1 production. Primary cells appeared to be less sensitive to EV depletion. ACH-2 and U1 latency models produced more HIV-1 under EV-depleted conditions, while virus produced under processed serum conditions was more infectious. Finally, changes in cellular metabolism and gene expression were associated with EV-depleted culture. In conclusion, the EV environment of HIV-1 infected cells has a substantial effect on virus production and infectivity. EV-dependence of cell cultures should be examined carefully along with other experimental variables. However, EVs may not be the only particles depleted by ultracentrifugation or other processes. Effects of EVs may be accompanied by or confused with those of closely associated or physically similar particles.

molecular biology↗