The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed ...hypo-phosphorylation, to release E2F transcription factors. However, this model remains unproven biochemically and the biologically active form(s) of Rb remains unknown. In this study, we find that Rb is exclusively mono-phosphorylated in early G1 phase by cyclin D:Cdk4/6. Mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but show preferential E2F binding patterns. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by quantum hyper-phosphorylation. Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription, whereas cells undergoing differentiation utilize un-phosphorylated Rb. These observations fundamentally change our understanding of G1 cell cycle progression and show that mono-phosphorylated Rb, generated by cyclin D:Cdk4/6, is the only Rb isoform in early G1 phase.
Physical interactions between distal regulatory elements have a key role in regulating gene expression, but the extent to which these interactions vary between cell types and contribute to ...cell-type-specific gene expression remains unclear. Here, to address these questions as part of phase III of the Encyclopedia of DNA Elements (ENCODE), we mapped cohesin-mediated chromatin loops, using chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), and analysed gene expression in 24 diverse human cell types, including core ENCODE cell lines. Twenty-eight per cent of all chromatin loops vary across cell types; these variations modestly correlate with changes in gene expression and are effective at grouping cell types according to their tissue of origin. The connectivity of genes corresponds to different functional classes, with housekeeping genes having few contacts, and dosage-sensitive genes being more connected to enhancer elements. This atlas of chromatin loops complements the diverse maps of regulatory architecture that comprise the ENCODE Encyclopedia, and will help to support emerging analyses of genome structure and function.
Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in ...GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.
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•Systems-level approach reveals GATA4 roles in human cardiac development and function•Heterozygous GATA4 missense mutation impairs cardiac gene program•GATA4 G296S mutation disrupts TBX5 genome occupancy at cardiac super-enhancers•PI3K signaling is a key “hub” in the GATA4 gene regulatory network
A human missense mutation that causes congenital heart defects disrupts the cooperation between transcription factors at cardiac super-enhancers and gives rise to aberrant gene expression.
INTRODUCTION: Laparoscopic cholecystectomies are performed as daycare procedures. Postoperative pain associated with it may need multimodal opioid-free approaches to fast track the recovery. ...Intraoperative intravenous lignocaine infusion was employed in this study to observe it effect on analgesia, hemodynamic stress response, and recovery profile in patients undergoing laparoscopic cholecystectomies. METHODOLOGY AND RESULTS: This was a randomized placebo-controlled study with enrollment of 64 participants. The “L” group received preservative-free intravenous lignocaine 2% at 1.5 mg/kg bolus followed by continuous infusion at a rate of 1.5 mg/kg/h till skin closure. The “C” group received equal amounts of normal saline. No significant statistical difference was observed between the two groups with time to first analgesic, total analgesic consumption, and visual analog scale scores. No significant difference was seen with sedation, time to achieve modified Aldrete score, and time to return of bowel activity. Only significant difference in heart rate was observed in lignocaine group after intubation with no effects on mean arterial pressure. CONCLUSION: Lignocaine infusion did not prove any added benefits in terms of postoperative analgesia, opioid requirements, and functional recovery after laparoscopic cholecystectomy. Hemodynamic stress response were better maintained in lignocaine group. Bolus dose of lignocaine may prove beneficial and equipotent as fentanyl.
Progressive pseudorheumatoid dysplasia (PPD) is a skeletal dysplasia characterized by predominant involvement of articular cartilage with progressive joint stiffness. Here we report genetic ...characterization of a consanguineous family segregating an uncharacterized from of skeletal dysplasia. Whole-exome sequencing of four affected siblings and their parents identified a loss-of-function homozygous mutation in the
gene, leading to diagnosis of PPD in the affected individuals. The identified variant (Chr6: 112382301; WISP3:c.156C>A p.Cys52*) is rare and predicted to cause premature termination of the
protein.
The prevailing model of G1 cell cycle progression proposes Cyclin D:Cyclin-dependent kinase 4/6 (Cdk4/6) complexes inactivate the retinoblastoma tumor suppressor protein (Rb) during Early G1 phase by ...progressive multi-phosphorylation or "hypo-phosphorylation" to release E2F transcription factors, resulting in the gradual activation of Cyclin E:Cdk2 complexes. However, due to the use of supra-physiologic overexpression studies, absence of quantification of how many phosphates are present on "hypo-phosphorylated" Rb, and lack of clarity on what is the active isoform of Rb, this model remains largely unproven. Rb contains 16 Cdk phosphorylation sites and is thought to exist in three general isoforms: 1) un-phosphorylated Rb, 2) "hypo-phosphorylated" Rb, and 3) inactive hyper-phosphorylated Rb; however, the un-phosphorylated Rb and "hypo-phosphorylated" Rb isoforms cannot be resolved by 1D SDS-PAGE. Using highly synchronized primary and tumorigenic cells, performing biochemistry on physiologic levels of activities of proteins, and developing 2D isoelectric focusing (2D IEF) of Rb, I show that Rb is exclusively mono-phosphorylated on 14 different sites during Early G1 phase and that this phosphorylation is mediated by Cyclin D:Cdk4/6 complexes. Mono-phosphorylated Rb functioned to induce a G1 arrest, bind E2F transcription factors and regulate the global transcriptional profile, whereas un-phosphorylated Rb was non-functional, showing that mono-phosphorylated Rb is the biologically active isoform of Rb. These observations fundamentally change our understanding of G1 cell cycle regulation and show that Cyclin D:Cdk4/6 complexes activate Rb by mono-phosphorylation during Early G1 phase. Importantly, these observations point to the activation of Cyclin E:Cdk2 complexes as a likely key oncogenic step in the progression of cancer.
IntroductionTyrosine kinase inhibitors (TKIs) are known to induce cardiotoxicity and mitochondrial dysfunction. TKIs can cross the placenta, posing high risks for congenital heart defects. ...Mitochondria play important roles in the regulation of cardiac differentiation. Previous studies showed that mitochondrial dysfunction is correlated with a decrease of GATA4 expression and impairment of cardiac development. However, the relationship between GATA4 and mitochondrial function during cardiac development upon TKI-exposure remains poorly documented.HypothesisLow-dose exposure to TKIs alters GATA4-mediated regulatory network and metabolic remodeling during early cardiac development, leading to dysfunctions in differentiating cardiomyocytes (CMs).MethodsWe treated human stem cells with several TKI drugs (imatinib, sunitinib, and vandetanib at sublethal levels) during CM differentiation, and profiled the transcriptome and chromatin accessibility landscape in cardiac progenitors and CMs. The GATA4-mediated regulatory network was profiled using ChIP-seq. The functional influence of the early exposure to TKIs in differentiating CMs was evaluated, including Ca-handling and contractility, integrity of sarcomere structure, and mitochondrial respiration.ResultsDevelopmental exposure to TKIs induced impairment of mitochondrial respiration, disarrangement of sarcomere structure, increased calcineurin levels, and significant alterations in contractility and Ca-handling properties of differentiating CMs. Integrated genomic analyses revealed that TKI-exposure altered GATA4-mediated regulatory network, which was highly correlated with mitochondrial functions. Using a gain-of-function approach with CRISPR-activation, we observed that increases in GATA4 expression restored cardiac functions and mitochondrial respiration despite of TKI exposure.ConclusionsWe identified a novel crosstalk between GATA4 activity and mitochondrial respiration during CM differentiation. This study provides new insights into the relationship between gene-regulation and mitochondrial functions, and significantly enhances our understanding of sublethal TKI-induced cardiotoxicity during cardiac development.
Post-transcriptional gene silencing (PTGS)-mediated gene silencing exploits the cellular mechanism wherein transcripts having sequence similarity to the double-stranded RNA (dsRNA) molecules present ...in the cell will be subjected to degradation. PTGS is closely related to natural processes such as RNA-mediated virus resistance and cross-protection in plants. Gene silencing and the cellular machinery for affecting this phenomenon might have evolved as a natural protective measure against viral infection in plants. In PTGS, small interfering RNA (siRNA) molecules of 21–23 nucleotides length act as homology guides for triggering the systemic degradation of transcripts homologous to the siRNA molecules. PTGS phenomenon, first discovered in transgenic petunia plants harbouring chalcone synthase gene and termed co-suppression, has been subsequently exploited to target specific gene transcripts for degradation leading to manifestation of desirable traits in crop plants. Targeted gene silencing has been achieved either through the introduction of DNA constructs encoding dsRNA or antisense RNA or by deploying co-suppression constructs producing siRNAs against the transcript of interest. Understanding the mechanism of gene silencing has led to the development of several alternative strategies for inducing gene silencing in a precise and controlled way. This has paved the way for using PTGS as one of the chief functional genomics tools in plants and has helped in unraveling the mechanism of many cellular processes and identifying the focal points in pathways, besides, opening new vistas in genetic engineering of plants for human benefits. PTGS has shown great potential in silencing the deleterious genes efficiently so that value-added plant products could be obtained. Thus, PTGS has ushered in a new era in the genetic manipulation of plants for both applied and basic studies. In this review, we have outlined the basics of RNAi-mediated gene silencing and summarized the work carried out at our institute using this approach, as case studies. In particular, adopting RNAi-mediated gene silencing (a) as a method to restore fertility in transgenic male sterile lines developed based on
orfH522
gene from sunflower PET1-CMS source, (b) as a tool to suppress the production of toxic proteins, ricin and RCA, in castor, and (c) as an approach to induce bud necrosis virus resistance in sunflower has been discussed. Examples from other plant systems also have been mentioned to exemplify the concept and utility of gene silencing in crop plants.
Several secreted and surface-associated conserved microbial molecules are recognized by the host to mount the defence response. One such evolutionarily well-conserved bacterial process is the ...production of cell–cell signalling molecules which regulate production of multiple virulence functions by a process known as quorum sensing. Here it is shown that a bacterial fatty acid cell–cell signalling molecule, DSF (diffusible signal factor), elicits innate immunity in plants. The DSF family of signalling molecules are highly conserved among many phytopathogenic bacteria belonging to the genus Xanthomonas as well as in opportunistic animal pathogens. Using Arabidopsis, Nicotiana benthamiana, and rice as model systems, it is shown that DSF induces a hypersensitivity reaction (HR)-like response, programmed cell death, the accumulation of autofluorescent compounds, hydrogen peroxide production, and the expression of the PATHOGENESIS-RELATED1 (PR-1) gene. Furthermore, production of the DSF signalling molecule in Pseudomonas syringae, a non-DSF-producing plant pathogen, induces the innate immune response in the N. benthamiana host plant and also affects pathogen growth. By pre- and co-inoculation of DSF, it was demonstrated that the DSF-induced plant defence reduces disease severity and pathogen growth in the host plant. In this study, it was further demonstrated that wild-type Xanthomonas campestris suppresses the DSF-induced innate immunity by secreting xanthan, the main component of extracellular polysaccharide. The results indicate that plants have evolved to recognize a widely conserved bacterial communication system and may have played a role in the co-evolution of host recognition of the pathogen and the communication machinery.
Purpose
Total body irradiation (TBI) can be safely delivered on TomoTherapy (Accuray, Sunnyvale, CA, USA) in both pediatric and adult patients with proper imaging and planning despite the length ...constraint of 135 cm. To overcome this limitation, two CT (Computed Tomography) scans (CT1& CT2) are taken in patients above 135 cm in height. Adequate junction dose coverage is important in TBI. Presently, there is no clinical report with a focus on the quality of dose distribution at the CT junction in view of the guidelines on quality of coverage from the RTOG. Hence, our main objectives were to evaluate the dose distribution and quality of coverage at the junction in 16 patients who received TBI using TomoTherapy.
Methods
PTV(upper) and PTV(lower) along with a junction were created on CT1 and CT2, respectively. Subsequently, the 10 cm junction in the thigh region was divided into five target volumes of 2 cm thickness with dose prescription ranging from 10 to 90% to deliver a total dose equal to 100% when fused.
Results
The D
50
was equal to the prescribed dose in most of the cases ranging from 99.5 to 104% for PTV(upper), 100–103% for PTV(lower), and 99.5–108% for junctional PTVs (1PTV, 2PTV, 3PTV, 4PTV, and 5PTV). The average D
95
doses from PTV(upper) and PTV(lower) were 97 ± 1.4% and 96.7 ± 1.08%, respectively. The average D
95
doses for 1PTV, 2PTV, 3PTV, 4PTV, and 5PTV were 96.1 ± 1.88%, 91.6 ± 1.82%, 87.3 ± 1.5%, 91.6 ± 1.4%, and 96.2 ± 1.5% respectively. Q
RTOG
values ranged between 0.85 and 1.05 and were in concordance with RTOG guidelines.
Conclusion
Since junction-based planning was required for most TBI patients, it is essential to evaluate the quality of dose coverage in the junction for better TBI plans.
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