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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 523 records · Page 29Linked to original sources

bioRxiv2017

Evolutionary Insights On The Structure And Function Of Archaeal C/D Box sRNAs As Revealed By A Comprehensive Set From Six Pyrobaculum Species

Archaeal homologs of eukaryotic C/D box small nucleolar RNAs (C/D box sRNAs) guide precise 2'-O-methyl modification of ribosomal and transfer RNAs. Although C/D box sRNA genes constitute one of the largest RNA gene families in archaeal thermophiles, most genomes have incomplete sRNA gene annotation because reliable, fully automated detection methods are not available. We expanded and curated a comprehensive gene set across six species of the crenarchaeal genus Pyrobaculum, particularly rich in C/D box sRNA genes. Using high-throughput small RNA sequencing, specialized computational searches, and comparative genomics, we analyzed 526 Pyrobaculum C/D box sRNAs, organizing them into 110 families based on synteny and conservation of guide sequences which determine methylation targets. We examined gene duplications and rearrangements, including one family that has expanded in a pattern similar to retrotransposed repetitive elements in eukaryotes. New training data and inclusion of kink-turn secondary structural features enabled creation of an improved search model. Our analyses provide the most comprehensive, dynamic view of C/D box sRNA evolutionary history within a genus, in terms of modification function, feature plasticity, and gene mobility.

molecular biology↗

bioRxiv2017

Urine Glucose Levels Are Disordered Before Blood Glucose Levels Increase In Zucker Diabetic Fatty Rats

Diabetes mellitus is a type of metabolic disease marked by hyperglycemia, and 90% of diabetes cases are type 2 diabetes mellitus. More than half of patients are not diagnosed at all or are diagnosed too late to be effectively treated, resulting in nonspecific symptoms and a long period of incubation of the disease. Pre-diabetes mellitus, also known as impaired glucose regulation, is an early warning signal of diabetes, which has long been determinated by impaired fasting glucose and impaired glucose tolerance. In this study, Zucker diabetic fatty (ZDF) rats were used to test if there were changes in urine glucose before blood glucose increases. Six 8-week-old male ZDF rats (fa/fa) and Zucker lean (ZL) rats (fa/+) were fed with Purina 5008 high-fat diet and tested for fasting blood glucose and urine glucose. After 12 weeks of feeding, the urine glucose values of the ZL rats were normal (0-10 mmol/L), but the values of the ZDF model rats increased 10 weeks before their blood glucose levels elevated. The urine glucose values of the ZDF model rats showed a state of disorder that was frequently elevated (>10 mmol/L) and occasionally normal (0-10 mmol/L). This finding may provide an easy early diagnosis. Screening for human diabetes can be considered by frequently monitoring urine glucose levels: pre-diabetes may be revealed by frequently disordered urine glucose levels over a period.

molecular biology↗

bioRxiv2017

Independence Between Pre-mRNA Splicing And De Novo DNA Methylation In An Isogenic Minigene Resource

Actively transcribed genes adopt a unique chromatin environment with characteristic patterns of enrichment. Within gene bodies, H3K36me3 and cytosine DNA methylation are elevated at exons of spliced genes and have been implicated in the regulation of pre-mRNA splicing. H3K36me3 is further responsive to splicing, wherein splicing inhibition led to a redistribution and general reduction over gene bodies. In contrast, little is known of the mechanisms supporting elevated DNA methylation at actively spliced genic locations. Recent evidence associating the de novo DNA methyltransferase Dnmt3b with H3K36me3-rich chromatin raises the possibility that genic DNA methylation is influenced by splicing-associated H3K36me3. Here, we report the generation of an isogenic resource to test the direct impact of splicing on chromatin. A panel of minigenes of varying splicing potential were integrated into a single FRT site for inducible expression. Profiling of H3K36me3 confirmed the established relationship to splicing, wherein levels were directly correlated with splicing efficiency. In contrast, DNA methylation was equivalently detected across the minigene panel, irrespective of splicing and H3K36me3 status. In addition to revealing a degree of independence between genic H3K36me3 and DNA methylation, these findings highlight the generated minigene panel as a flexible platform for the query of splicing-dependent chromatin modifications.

molecular biology↗

bioRxiv2017

Early urinary candidate biomarker discovery in a rat thioacetamide-induced liver fibrosis model

Biomarker is the change associated with the disease. Blood is relatively stable because of the homeostatic mechanisms of the body. However, urine accumulates changes of the body, which makes it a better early biomarker source. Liver fibrosis, which results from the deposition of extracellular matrix (ECM) components, is a reversible pathological condition, whereas cirrhosis, the end-stage of liver fibrosis, is irreversible. Consequently, noninvasive early biomarkers for fibrosis are desperately needed. In this study, differential urinary proteins were identified in the thioacetamide (TAA) liver fibrosis rat model using tandem mass tagging and two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS). A total of 766 urinary proteins were identified, 143 and 118 of which were significantly changed in the TAA 1-week and 3-week groups, respectively. Multiple reaction monitoring (MRM)-targeted proteomics was used to further validate the abundant differentially expressed proteins in the TAA 1-week, 3-week, 6-week and 8-week groups. A total of 40 urinary proteins were statistically significant (fold change >2 and p<0.05), 15 of which had been previously reported as biomarkers of liver fibrosis, cirrhosis or other related diseases and 10 of which had been reported to be associated with the pathology and mechanism of liver fibrosis. These differential proteins were detected in urine before the alanine aminotransferase (ALT) and aspartate transaminase (AST) changes in the serum and before fibrosis was observed upon hematoxylin and eosin (HE) and Massons staining.

molecular biology↗

bioRxiv2017

Integrated Computational Guide Design, Execution, And Analysis Of Arrayed And Pooled CRISPR Genome Editing Experiments

CRISPR genome editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep sequencing data resulting from genome editing experiments. However, these tools are typically developed in isolation and oftentimes not readily translatable into laboratory-based experiments. Here we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome editing experiment. This protocol provides instructions for sgRNA design with CRISPOR, experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso. This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts as well as computational data analysis in 1-2 days that can be performed by both computational and non-computational biologists alike.

molecular biology↗

bioRxiv2017

A Cassava Drought Inducible CC-Type Glutaredoxin, MeGRX232, Negatively Regulates Drought Tolerance In Arabidopsis By Inhibition Of ABA-Dependent Stoma

CC-type glutaredoxins (GRXs) are a land plant-specific GRX subgroup that evolved from CGFS GRXs, and participate in organ development and stress responses through the regulation of transcription factors. Here, genome-wide analysis identified 18 CC-type GRXs in the cassava genome, of which six (MeGRX058, 232, 360, 496, 785, and 892) were induced by drought and ABA stress in cassava leaves. Furthermore, we found that overexpression of MeGRX232 results in drought hypersensitivity in soil-grown plants, with a higher water loss rate, but with increased tolerance of mannitol and ABA in Arabidopsis on the sealed agar plates. The ABA induced stomatal closure is impaired in MeGRX232-OE Arabidopsis. Further analysis reveals that the overexpression of MeGRX232 leads to more ROS accumulation in guard cells. MeGRX232 can interact with TGA5 from Arabidopsis and MeTGA074 from cassava in vitro and in vivo. The results of microarray assays show that MeGRX232-OE affected the expression of a set of drought and oxidative stress related genes. Taken together, we demonstrated that CC-type GRXs involved in ABA signal transduction and play roles in response to drought through regulating stomatal closure.\n\nNovelty statementWe found that drought and ABA stress induced the transcription of CC-type glutaredoxins (GRXs) in cassava leaves. Ectopic expression of one of them, MeGRX232 in Arabidopsis affected the sensitivity to abscisic acid (ABA) and mannitol, and caused drought hypersensitivity by impairment of ABA-dependent stomatal closure.

molecular biology↗

bioRxiv2017

Index Switching Causes "Spreading-Of-Signal" Among Multiplexed Samples In Illumina HiSeq 4000 DNA Sequencing

Illumina-based next generation sequencing (NGS) has accelerated biomedical discovery through its ability to generate thousands of gigabases of sequencing output per run at a fraction of the time and cost of conventional technologies. The process typically involves four basic steps: library preparation, cluster generation, sequencing, and data analysis. In 2015, a new chemistry of cluster generation was introduced in the newer Illumina machines (HiSeq 3000/4000/X Ten) called exclusion amplification (ExAmp), which was a fundamental shift from the earlier method of random cluster generation by bridge amplification on a non-patterned flow cell. The ExAmp chemistry, in conjunction with patterned flow cells containing nanowells at fixed locations, increases cluster density on the flow cell, thereby reducing the cost per run. It also increases sequence read quality, especially for longer read lengths (up to 150 base pairs). This advance has been widely adopted for genome sequencing because greater sequencing depth can be achieved for lower cost without compromising the quality of longer reads. We show that this promising chemistry is problematic, however, when multiplexing samples. We discovered that up to 5-10% of sequencing reads (or signals) are incorrectly assigned from a given sample to other samples in a multiplexed pool. We provide evidence that this \"spreading-of-signals\" arises from low levels of free index primers present in the pool. These index primers can prime pooled library fragments at random via complementary 3 ends, and get extended by DNA polymerase, creating a new library molecule with a new index before binding to the patterned flow cell to generate a cluster for sequencing. This causes the resulting read from that cluster to be assigned to a different sample, causing the spread of signals within multiplexed samples. We show that low levels of free index primers persist after the most common library purification procedure recommended by Illumina, and that the amount of signal spreading among samples is proportional to the level of free index primer present in the library pool. This artifact causes homogenization and misclassification of cells in single cell RNA-seq experiments. Therefore, all data generated in this way must now be carefully re-examined to ensure that \"spreading-of-signals\" has not compromised data analysis and conclusions. Re-sequencing samples using an older technology that uses conventional bridge amplification for cluster generation, or improved library cleanup strategies to remove free index primers, can minimize or eliminate this signal spreading artifact.

molecular biology↗

bioRxiv2017

STRT-seq-2i: dual-index 5′ single cell and nucleus RNA-seq on an addressable microwell array

Single-cell RNA-seq has become routine for discovering cell types and revealing cellular diversity, but currently no high-throughput platform has been used successfully on archived human brain samples. We present STRT-seq-2i, an addressable 9600-microwell array platform, combining sampling by limiting dilution or FACS, with imaging and high throughput at competitive cost. We applied the platform to fresh single mouse cortical cells and to frozen post-mortem human cortical nuclei, matching the performance of a previous lower-throughput platform.

molecular biology↗

bioRxiv2017

Intermolecular Disulfide-Bond Formation In Human FICD Modulates The Activity Of The Hyperactive E234G Mutant

Endoplasmic reticulum (ER) stress that leads to the accumulation of misfolded proteins in the ER initiates the unfolded protein response (UPR). This homeostatic response activates signaling pathways that seek to reinstate a proper ER protein folding balance or induce apoptosis if ER stress persists. Recently, we and others identified human FICD (Filamentation induced by cyclic AMP domain-containing protein), an enzyme with adenylyltransferase (aka AMPylation) activity, as a new UPR target. Here, we demonstrate that FICD is functionally linked to the UPR, as evidenced by the finding that the adenylyltransferase activity of the protein induces ER stress, while FICD silencing increases sensitivity to ER stress. We identify BiP, an abundant ER chaperone and key regulator of the UPR, as the main substrate of FICD AMPylation in ER-derived microsomes, further emphasizing close functional connection of FICD to the UPR and in line with recent reports that AMPylation inactivates BiP. Notably, BiP overexpression increased the levels of BiP AMPylation as well as FICD auto-AMPylation, suggesting a homeostatic response that balances the pool of active BiP to modulate its functions in protein folding as well as UPR signaling. Finally, we show that overexpressed FICD forms a disulfide-bonded homo-dimer through Cys51 and Cys75 and demonstrate that mutation of these two cysteines in the context of a hyperactive FICD mutant leads to increased BiP AMPylation. This latter finding opens up the possibility that FICD activity is redox regulated and closely connected with ER redox homeostasis.

molecular biology↗

bioRxiv2017

Gut Microbiota Composition Related With Clostridium difficile-Positive Diarrhea And C. Difficile Type (A+B+, A-B+, And A-B-) In ICU Hospitalized Patients

BackgroundGut microbiota composition of intensive care unit (ICU) patients suffering from Clostridium difficile-positive diarrhea (CDpD) is still poorly understood. This study aims to use 16S rDNA (and metagenome) sequencing to compare the microbiota composition of 58 (and 5) ICU patients with CDpD (CDpD group), 33 (and 4) ICU patients with C. difficile negative diarrhea (CDnD group), and 21 (and 5) healthy control subjects (control group), as well as CDpD patients in A+B+ (N=34; A/B: C. difficile TcdA/B), A-B+ (N=7), and A-B- (N=17) subgroups. For 16S rDNA data, OTU clustering (tool: UPARSE), taxonomic assignment (tool: RDP classifier), -diversity and {beta}-diversity analyses (tool: QIIME) were conducted. For metagenome data, metagenome assembly (tool: SOAP), gene calling (tools: MetaGeneMark, CD-HIT, and SoapAligner), unigene alignment (tool: DIAMOND), taxon difference analysis (tool: Metastats), and gene annotation (tool: DIAMOND) were performed.\n\nResultsThe microbial diversity of CDpD group was lower than that of CDnD and control groups. The abundances of 10 taxa (e.g. Deferribacteres, Cryptomycota, Acetothermia) in CDpD group were significantly higher than that in CDnD group. The abundances of Saccharomycetes and Clostridia were significantly lower in CDpD in comparison with control. A+B+, A-B+ and A-B- subgroups couldnt be separated in principal component analysis, while some taxa are significantly different between A+B+ and A-B- subgroups.\n\nConclusionCDpD might relate to the decrease of beneficial taxa (i.e. Saccharomycetes and Clostridia) and the increase of harmful taxa (e.g. Deferribacteres, Cryptomycota, Acetothermia) in gut microbiota in ICU patients. C. difficile type might be slightly associated with gut microbiota composition.

molecular biology↗

bioRxiv2017

Modulation of Genome Editing Outcomes by Cell Cycle Control of Cas9 Expression

Targeting specific chromosomal sequences for genome modification or regulation during particular phases of the cell cycle may prove useful in creating more precise, predictable genetic changes. Here, we present a system using a fusion protein comprised of a programmable DNA modification protein, Cas9, linked to a cell cycle regulated protein, geminin, as well as green fluorescent protein (GFP) for visualization. Despite the large size of Cas9 relative to geminin, cells were observed to express Cas9-GFP-geminin at levels which oscillate with the cell cycle. These fusion proteins are also shown to retain double-strand break (DSB) activity at specific chromosomal sequences to produce both indels and targeted integration of donor ssDNA. Most importantly, the ratio of ssDNA donor integration to non-homologous end joining (NHEJ) was observed to increase, suggesting that cell cycle control Cas9 expression may be an effective strategy to bias DNA repair outcomes.

molecular biology↗

bioRxiv2017

Autonomous Combinatorial Color Barcoding For Multiplexing Single Molecule RNA Visualization

Single molecular fluorescence in situ hybridization (smFISH) detects RNA transcripts with spatial information and digital molecular counting. However, the broad usage of smFISH is still hindered by the complex chemical probe conjugation or microscopy set-up, especially for investigating multiple gene expression. Here we present a multiple fluorophore enzymatic labeling method (termed HuluFISH) for smFISH probes to achieve flexible combinatorial color barcoding in single hybridization step. The multiplex capacity of HuluFISH follows an exponential growth with the increase of the number of fluorophore types. We demonstrate that this method can be used to detect cellular heterogeneity in embryonic mouse brain on single cell level.

molecular biology↗

bioRxiv2017

Autogenous Cross-Regulation Of Quaking mRNA Processing And Translation Balances Quaking Functions In Splicing And Translation

Quaking RNA binding protein (RBP) isoforms arise from a single Quaking gene, and bind the same RNA motif to regulate splicing, stability, decay, and localization of a large set of RNAs. However, the mechanisms by which the expression of this single gene is controlled to distribute appropriate amounts of each Quaking isoform to regulate such disparate gene expression processes are unknown. Here we explore the separate mechanisms that regulate expression of two isoforms, Quaking-5 (Qk5) and Quaking-6 (Qk6), in mouse muscle cells. We first demonstrate that Qk5 and Qk6 proteins have distinct functions in splicing and translation respectively, enforced primarily through differential subcellular localization. Using isoform-specific depletion, we find both Qk5 and Qk6 act through cis and trans post-transcriptional regulatory mechanisms on their own and each others transcripts, creating a network of auto- and cross-regulatory controls. Qk5 has a major role in nuclear RNA stability and splicing, whereas Qk6 acts through translational regulation. In different cell types the cross-regulatory influences discovered here generate a spectrum of Qk5/Qk6 ratios subject to additional cell type and developmental controls. These unexpectedly complex feedback loops underscore the importance of the balance of Qk isoforms, especially where they are key regulators of development and cancer.

molecular biology↗

bioRxiv2017

The LDB1 complex co-opts CTCF for erythroid lineage specific long-range enhancer interactions

Lineage-specific transcription factors are critical for long-range enhancer interactions but direct or indirect contributions of architectural proteins such as CTCF to enhancer function remain less clear. The LDB1 complex mediates enhancer-gene interactions at the {beta}-globin locus through LDB1 self-interaction. We find that a novel LDB1-bound enhancer upstream of carbonic anhydrase 2 (Car2) activates its expression by interacting directly with CTCF at the gene promoter. Both LDB1 and CTCF are required for enhancer-Car2 looping and the domain of LDB1 contacted by CTCF is necessary to rescue Car2 transcription in LDB1 deficient cells. Genome wide studies and CRISPR/Cas9 genome editing indicate that LDB1-CTCF enhancer looping underlies activation of a substantial fraction of erythroid genes. Our results provide a mechanism by which long-range interactions of architectural protein CTCF can be tailored to achieve a tissue-restricted pattern of chromatin loops and gene expression.

molecular biology↗

bioRxiv2017

Regulatory RNPs: A novel class of ribonucleoproteins that contribute to ribosome heterogeneity

Ribonucleoproteins (RNPs), which are comprised of non-coding RNA and associated proteins, are involved in essential cellular processes such as translation and pre-mRNA splicing. One class of RNP is the small Cajal body-specific RNP (scaRNP), which contributes to the biogenesis of small nuclear RNPs (snRNPs) that are central components of the spliceosome. Interestingly, three scaRNAs are internally processed, generating stable nucleolus-enriched RNAs of unknown function. Here we provide evidence that these RNAs become part of novel RNPs we term regulatory RNPs (regRNPs). We postulate that regRNPs can impact rRNA modifications via interactions with the guide RNA component of small nucleolar RNPs (snoRNPs). Most modifications within rRNA (predominantly pseudouridylation and ribose 2-O-methylation) are conducted by snoRNPs, and we hypothesize that the activity of at least some of these snoRNPs is under the control of regRNPs. Ribosome heterogeneity leading to specialized ribosomes is an exciting emerging concept. Because modifications within rRNA can vary in different physiological or pathological situations, rRNA modifications are thought to be the major source of ribosome heterogeneity. Our identification of regRNPs thus provides important and timely insight into how ribosome heterogeneity may be accomplished. This work also provides additional functional connections between the Cajal body and the nucleolus.\n\nSummary StatementProcessed scaRNAs give rise to a novel regulatory RNP which regulates the modification of ribosomal RNA. These findings provide insight into the mechanisms governing ribosome heterogeneity.

molecular biology↗

bioRxiv2017

Effects of the glucocorticoid drug prednisone on urinary proteome and candidate biomarkers

Urine is a good source of biomarkers for clinical proteomics studies. However, one challenge in the use of urine biomarkers is that outside factors can affect the urine proteome. Prednisone is a commonly prescribed glucocorticoid used to treat various diseases in the clinic. To evaluate the possible impact of glucocorticoid drugs on the urine proteome, specifically disease biomarkers, this study investigated the effects of prednisone on the rat urine proteome. Urine samples were collected from control rats and prednisone-treated rats after drug administration. The urinary proteome was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and proteins were identified using label-free proteome quantification. Differentially expressed proteins and their human orthologs were analyzed with bioinformatics methods. A total of 523 urinary proteins were identified in rat urine. Using label-free quantification, 27 urinary proteins showed expression changes after prednisone treatment. A total of 16 proteins and/or their human orthologs have been previously annotated as disease biomarkers. After functional analysis, we found that the pharmacological effects of prednisone were reflected in the urine proteome. Thus, urinary proteomics has the potential to be a powerful drug efficacy monitoring tool in the clinic. Meanwhile, alteration of the urine proteome due to prednisone treatment should be considered in future disease biomarker studies.

molecular biology↗

bioRxiv2017

Improved DOP-PCR (iDOP-PCR): a robust and simple WGA method for efficient amplification of low copy number genomic DNA

Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.

molecular biology↗

bioRxiv2017

Supercoiling DNA locates mismatches

We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.5 pN force and show that, under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.

molecular biology↗