Regulation of E2F1 in Keratinocytes During UV-Damage and Differentiation

The E2F1 transcription factor regulates the expression of key genes involved in cell proliferation and differentiation to maintain skin homeostasis. The expression of E2F1 is tightly regulated during cell cycle progression and when cells are committed to differentiate, as well as in response to DNA damage. In keratinocytes, E2F1 protein and transcript levels increase following UV-induced DNA damage, whereas, in response to Ca2+-induced differentiation, both E2F1 protein and transcript levels decrease. In this thesis, I examined in detail the mechanism that modulates E2F1 stability following DNA damage and during keratinocyte differentiation. I show that E2F1 associates with hHR23 and together these proteins participate in the nucleotide excision repair (NER) pathway. hHR23A stabilizes E2F1 by inhibiting its proteasomal degradation. Moreover, E2F1 regulates hHR23A subcellular localization and recruits hHR23A to sites of DNA photodamage to facilitate DNA repair. These results unveil a novel mechanism involving E2F1 and hHR23A to enhance the repair of damaged DNA, contributing to the maintenance of genomic stability following UV-induced DNA damage in epidermal cells. In contrast to DNA damage, proper keratinocyte differentiation occurs when E2F1 is down regulated. Although the regulation of E2F1 that leads to its nuclear export and degradation has been reported, the exact mechanisms are poorly characterized. When E2F1 Ser403 and/or Thr433 are substituted to an alanine (E2F1 ST/A), a significant increase in the half-life of this mutant in differentiated keratinocytes is observed. Furthermore, the E2F1 ST/A mutant does not form appreciable levels of K11- or K48-linked polyubiquitylation, which is associated with degradation. Moreover, I show that iii Cdh1, an activator protein of the anaphase-promoting complex/cyclosome (APC/C) complex, interacts with E2F1 in keratinocytes, and its inactivation prevents the turnover of E2F1 following Ca2+-induced differentiation. Collectively, these results reveal a new mechanism involving E2F1 and Cdh1 in coordinating its degradation through post-translational modification and nuclear export in differentiated keratinocytes. By elucidating the appropriate events required for the turnover of E2F1, we can better understand and treat various epidermal diseases such as non-melanoma skin carcinoma

CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes

The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation

E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair

Nucleotide excision repair (NER) is a major mechanism for removal of DNA lesions induced by exposure to UV radiation in the epidermis. Recognition of damaged DNA sites is the initial step in their repair, and requires multiprotein complexes that contain XPC and hHR23 proteins, or their orthologues. A variety of transcription factors are also involved in NER, including E2F1. In epidermal keratinocytes, UV exposure induces E2F1 phosphorylation, which allows it to recruit various NER factors to sites of DNA damage. However, the relationship between E2F1 and hHR23 proteins vis-à-vis NER has remained unexplored. We now show that E2F1 and hHR23 proteins can interact, and this interaction stabilizes E2F1, inhibiting its proteasomal degradation. Reciprocally, E2F1 regulates hHR23A subcellular localization, recruiting it to sites of DNA photodamage. As a result, E2F1 and hHR23A enhance DNA repair following exposure to UV radiation, contributing to genomic stability in the epidermis

Rhizosphere competence of native Rhizobium rhizogenes strain and its use in management of crown gall

Native Rhizobium rhizogenes strain UHFBA-212 [141/1A (NCBI: KC488174)]was isolated from rhizosphere soil of peach nursery plant of wild peach collected from Himachal Pradesh. In addition to this,159 isolates were also collected and were screened in vitro for their biocontrol potential against Agrobacterium tumefaciens. Out of these strain, UHFBA-212 showed maximum zone of inhibition i.e. 4.16 and 3.57cm without and after exposure to chloroform against C58.Sequence analysis (16SrDNA) of the strain showed nucleotide homology similar to Rhizobium sp. Amplification of total genomic DNA of the strain with Vir D2 andipt primers didn’t showed amplification with these virulence genes suggesting the absence of tumorigenic factors. In the field conditions, maximum population (329.33x106 cfu/g of soil) was observed in antibiotic resistant mutant of R. rhizogenes strain K84 applied on cherry rootstock Colt followed by 285.33 (x 106 ) cfu/g of soil in UHFBA-212 after 9 months at the time of uprooting of plants when applied alone as root dip. Minimum incidence of crown gall (2.00%) was observed in strain UHFBA- 212 co inoculated with strain C58 as seed treatment on behmi seeds. The data on population indices in rhizosphere and incidence of crown gall further suggested that for better management of disease R. Rhizogenes isolates should be either equal or more in population than that of A. tumefaciens isolates. Strain UHFBA-212 controls crown gall as effectively as strain K84 and can be exploited against tumorigenic isolates under field conditions

Modulation of type II TGF-β receptor degradation by integrin-linked kinase

Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-β (TGF-β) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-β and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-β receptor stimulation. We now show that ILK associates with type II TGF-β receptors (TβRII) in ligand- and receptor kinase activity-independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TβRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-β receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TβRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TβRII away from degradative pathways during their differentiation into myofibroblasts

Nations within a nation: variations in epidemiological transition across the states of India, 1990–2016 in the Global Burden of Disease Study

18% of the world's population lives in India, and many states of India have populations similar to those of large countries. Action to effectively improve population health in India requires availability of reliable and comprehensive state-level estimates of disease burden and risk factors over time. Such comprehensive estimates have not been available so far for all major diseases and risk factors. Thus, we aimed to estimate the disease burden and risk factors in every state of India as part of the Global Burden of Disease (GBD) Study 2016

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase 1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age  6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score  652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc = 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N = 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

THE H-NS PROTEIN REGULATES THE CHEMICAL STEPS IN TN10 TRANSPOSITION

Transposons are mobile DNA segments. The transposition of transposable elements can have important effects on genome structure and function. In this thesis, we demonstrate that a histone-like nucleoid structuring protein (H-NS), acts as an anti-channeling factor to limit self-destructive intramolecular transposition events and allows for the dissemination of the bacterial transposon TnlO in vitro. Evidence that H-NS competes with IHF for binding to the TnlO transpososome to block channelling, and that this event is relatively insensitive to the level of DNA supercoiling present in the Tnl0-containing substrate plasmid, are presented. We also demonstrate that H-NS rescues the transposition defect observed in a R182A transposase mutant reaction pathway by unfolding the transpososome to promote transposon excision and strand transfer. For the first time, we have provided the strongest evidence to date that in vivo H-NS promotes TnlO transposition by acting directly on the transpososome

Critical care management of the patient with an acute ischaemic stroke.

Acute ischaemic stroke is a leading cause of morbidity and mortality worldwide. In the UK alone, there are more than 100 000 strokes per year, causing 38 000 deaths. While the incidence remains high, there has been significant medical progress in reducing mortality following a stroke. Admission of patients to specialised stroke units has led to an improvement in clinical outcomes, but the role of intensive care is less well defined. This article reviews the current critical care management and neuro-therapeutic options after an acute ischaemic stroke