Nuclear Organization and Dynamics of 7SK RNA in Regulating Gene Expression

We have identified 7SK RNA to be enriched in nuclear speckles. Knock-down of 7SK results in the mislocalization of nuclear speckle constituents, and the transcriptional up-regulation of a reporter gene locus. 7SK RNA transiently associates with the locus upon transcriptional down-regulation correlating with the displacement of pTEF-b

Functional annotation of human long noncoding RNAs via molecular phenotyping

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-todate lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.Peer reviewe

Identification of Wnt4 as the ovary pathway gene and temporal disparity of its expression vis-a-vis testis genes in the garden lizard, Calotes versicolor

Sex determination in the Indian garden lizard, Calotes versicolor, which lacks sex chromosomes and temperature-dependent sex determination, appears to be genically controlled, and previous studies have identified orthologues of Sox9, Dmrt1 and Androgen receptor (AR) as genes involved in testis differentiation in genetic males. In the pursuit of female pathway genes in this species, the present paper deals with the identification of Wnt4 and Dax1 genes in C. versicolor and their expression in embryonic gonad. CvDax1 is expressed throughout the development in certain embryos from day 5 onwards but shows no clear association with either testis or ovary. However, its preferred association with CvSox9 in early development and with CvWnt4 during later development suggests a role in the structuring of the gonads. CvWnt4 shows little expression in early development. It expresses prominently from day 20 onwards, and almost exclusively in those embryos that do not express CvSox9, demonstrating that CvWnt4 is the ovary differentiation gene in this species. This evidence leads us to suggest that temporal distinction of expression of ovary-specific (day 20 onwards) and testis-specific (day 5 onwards) genes could be an important part of the process of sex determination in C. versicolor. Taken together, the mechanism of sex determination in C. versicolor appears closer to the CSD in mammals than that in the ESD reptiles and birds

Weaning of mechanically ventilated chronic obstructive pulmonary disease patients by using non-invasive positive pressure ventilation: A prospective study

Background: Chronic obstructive pulmonary disease (COPD) patients frequently pose difficulty in weaning from invasive mechanical ventilation (MV). Prolonged invasive ventilation brings along various complications. Non-invasive positive pressure ventilation (NIPPV) is proposed to be a useful weaning modality in such cases. Objective: To evaluate the usefulness of NIPPV in weaning COPD patients from invasive MV, and compare it with weaning by conventional pressure support ventilation (PSV). Materials and Methods: For this prospective randomized controlled study, we included 50 COPD patients with type II respiratory failure requiring initial invasive MV. Upon satisfying weaning criteria and failing a t-piece weaning trial, they were randomized into two groups: Group I (25 patients) weaned by NIPPV, and group II (25 patients) weaned by conventional PSV. The groups were similar in terms of disease severity, demographic, clinical and biochemical parameters. They were compared in terms of duration of MV, weaning duration, length of intensive care unit (ICU) stay, occurrence of nosocomial pneumonia and outcome. Results: Statistically significant difference was found between the two groups in terms of duration of MV, weaning duration, length of ICU stay, occurrence of nosocomial pneumonia and outcome. Conclusion: NIPPV appears to be a promising weaning modality for mechanically ventilated COPD patients and should be tried in resource-limited settings especially in developing countries

Emblica officinalis-loaded poly(epsilon-caprolactone) electrospun nanofiber scaffold as potential antibacterial and anticancer deployable patch

Biological functions of nanofiber scaffolds can be engineered via the incorporation of plant extracts. In the present study, Emblica officinalis (EO) (Indian Gooseberry known as amla)-loaded poly(epsilon-caprolactone) (PCL) nanofibers were prepared via the electrospinning technique. The aim was to prepare nanofiber scaffolds with antibacterial and anti-cancerous properties. The scaffolds were characterized via SEM, XRD, small-angle X-ray scattering, FTIR spectroscopy, streaming potential, DSC, water contact angle and tensile measurements. Upon the incorporation of EO, the nanofiber surface became rougher and exhibited a braid-like' morphology. This is in contrast to the pristine PCL nanofibers, which exhibited a smooth fiber surface. The average fiber diameter increased, and the water contact angle of the nanofiber scaffolds decreased with an increase in the EO content in PCL. The crystalline structure of PCL was not affected; however, its crystallization temperature increased significantly and its melting temperature increased marginally with the addition of EO. The PCL scaffold showed a negatively charged surface and its -potential increased with an increase in EO content due to the presence of various functional groups. The antibacterial studies revealed that the PCL-EO nanofiber scaffolds exhibited efficient antibacterial properties against both Gram-positive and Gram-negative bacterial strains and the zone of inhibition increased with EO content. Furthermore, cellular behavior of MCF-7 cells was studied using the MTT assay, fluorescent staining and SEM, which indicated that cell proliferation was significantly inhibited by the incorporation of EO in the scaffolds

Long noncoding RNA MALAT1 controls cell cycle progression by regulating the expression of oncogenic transcription factor B-MYB.

The long noncoding MALAT1 RNA is upregulated in cancer tissues and its elevated expression is associated with hyper-proliferation, but the underlying mechanism is poorly understood. We demonstrate that MALAT1 levels are regulated during normal cell cycle progression. Genome-wide transcriptome analyses in normal human diploid fibroblasts reveal that MALAT1 modulates the expression of cell cycle genes and is required for G1/S and mitotic progression. Depletion of MALAT1 leads to activation of p53 and its target genes. The cell cycle defects observed in MALAT1-depleted cells are sensitive to p53 levels, indicating that p53 is a major downstream mediator of MALAT1 activity. Furthermore, MALAT1-depleted cells display reduced expression of B-MYB (Mybl2), an oncogenic transcription factor involved in G2/M progression, due to altered binding of splicing factors on B-MYB pre-mRNA and aberrant alternative splicing. In human cells, MALAT1 promotes cellular proliferation by modulating the expression and/or pre-mRNA processing of cell cycle-regulated transcription factors. These findings provide mechanistic insights on the role of MALAT1 in regulating cellular proliferation

p53 activation is essential for the S-phase defects observed in MALAT1-depleted human cells.

<p>(A–B) HDFs (WI-38) are incubated with control (scr) or MALAT1 antisense oligonucleotides (AS1, AS2), and total RNA and protein is isolated at indicated time points (12, 24 & 48 hr). The relative RNA and protein levels of indicated genes are determined by qPCR and immunoblot analyses. Note that the p53 target gene mRNA levels (p21, GADD45a) are increased within 12 hrs of MALAT1 knock down whereas E2F target mRNA (E2F1, CCNA2) levels are reduced only after 24 hrs of MALAT1 depletion. Similarly, MALAT1-depleted cells (all the time points) show increased levels of p53 and γH2AX. However Rb phosphorylation is reduced only 24 hrs after MALAT1 depletion. (C) Relative RNA levels of indicated genes in control (scr) and MALAT1-depleted (AS1 & AS2) HCT116-WT and HCT116-p53 −/− cells. MALAT1-depleted HCT116 p53−/− cells do not show reduction in the levels of E2F target gene mRNAs. Tubulin is used as a loading control. Mean ± SEM, *p<0.05, **p<0.01 and ***p<0.001.</p

MALAT1-depleted HDFs show defects in G1 to S transition.

<p>(A) Flow chart depicting the experimental design. HDFs (WI-38 cells) are G0 arrested by serum starvation, followed by MALAT1depletion (AS treat.) and released from quiescence (G0) by the addition of serum and further examined for G1/S progression in presence or absence of MALAT1. (B–C) Flow cytometry analyses and BrdU-incorporation assays of control (scr-oligo) and MALAT1-depleted cells (AS1 & AS2). (D–E) Effect of MALAT1 knockdown on serum-induced growth control gene expression. Serum-starved WI-38 cells are depleted of MALAT1 followed by serum stimulation, and relative mRNA and protein levels of indicated genes are determined by qPCR and immunoblot analyses. Mean ± SEM,**p<0.01 and ***p<0.001.</p

MALAT1 regulates mitotic gene expression by controlling the levels of B-MYB.

<p>(A) Top significant biological processes for genes whose alternative splicing was affected in MALAT1-depleted HDFs. (B) RT-PCR analysis using primers specific to exons in MALAT1-regulated alternative exons in indicated mRNAs show changes in alternative splicing in MALAT1-depleted WI-38 cells. Alternative exon-included (upper band) and -excluded bands (lower band) and the percentage change of alternative exon inclusion between scr-oligo/MALAT1-AS oligos (AS1 and AS2) observed is shown. Note that the alternative splicing of MGEA6 pre-mRNA is not altered upon MALAT1 depletion. (C) qRT-PCR analyses of SRSF1 RNA-IP samples from control and MALAT1-depleted (AS1) WI-38 cells reveal increased association of SRSF1 to B-MYB and CENPE mRNA. B-MYB-i-iii and CENPE-ii-iii correspond to separate sets of primers from independent exons of B-MYB and CENPE mRNA. (D) The relative expression and (E) serum-induced activation of B-MYB is determined by qRT-PCR and immunoblot analyses using RNA and protein from control (scr) and MALAT1-depleted (AS1 & AS2) WI-38 cells. (F) G1/S synchronization (0 hr) and release (2–8 hr) in control and MALAT1-depleted HeLa cells reveal reduced levels of B-MYB upon MALAT1 depletion as observed by qRT-PCR analyses. (G) The relative expression of B-MYB is determined by qRT-PCR analyses using RNA from control (scr) and MALAT1-depleted (AS1 & AS2) HCT116-WT and HCT116 p53 −/− cells. (H) Gene network analyses (Ingenuity pathways) indicate that a large number of mitotic specific genes that are downregulated upon MALAT1 depletion are regulated by B-MYB and FOXM1. Note that all the genes mentioned in this figure are downregulated in MALAT1-depleted fibroblasts. (I) Transient expression of B-MYB rescues the expression of mitotic specific genes in MALAT1-depleted WI-38 cells. (J) Relative expression of indicated genes is determined by qRT-PCR analyses in RNA from vector and MALAT1 overexpressed WI-38 cells. Note the increased B-MYB mRNA levels in MALAT1 overexpressed cells. Mean ± SEM, *p<0.05, **p<0.01 and ***p<0.001.</p