Deciphering the Role of Gfi1b Gene Target Rgs18 in Erythro-megakaryocytic Lineage Divergence

The molecular programs that govern the specification of erythroid and megakaryocytic lineages remain incompletely defined. Gene targeting experiments have shown the transcriptional repressor Gfi (Growth factor independence) 1b to be essential for the generation of both erythroid and megakaryocyte cells. Transcriptional repression of Gfi1b target genes is mediated mainly by the cofactors LSD (lysine demethylase) 1 and CoREST/Rcor1 (REST corepressor 1) or other Rcor factors. To understand the mechanism of Gfi1b action, its target genes were identified by chromatin immunoprecipitation (ChIP on Chip) screens. In this study we present the role of Rgs18 (Regulator of G protein signaling 18), a GTPase activating protein (GAP) and a transcriptional target of Gfi1b, in mediating erythro-megakaryocytic lineage specification in murine and human contexts. Following identification of Rgs18 as a potential Gfi1b and LSD1 target, its regulation by these factors was ascertained in erythro-megakaryocytic cells. Subsequently, the role of Rgs18 in erythro-megakaryocyte differentiation was interrogated by cDNA and shRNA mediated manipulation of expression in primary hematopoietic progenitors and cell lines. To determine the role of Rgs18 in vivo rgs18-/- mice have been generated and their phenotypes will be analyzed shortly. In parallel, to trace the underlying mechanistic alterations responsible for the phenotypes obtained by the above manipulations, the status of two branches of the MAPK (mitogen activated protein kinase) pathway and gene expression patterns of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1/ Klf1 (Kruppel like factor 1) were determined in Rgs18 manipulated cells. Also Rgs18 interacting proteins were identified in megakaryocytic cells. Rgs18 expression was found to be low in immature megakaryoblasts in keeping with strong Gfi1b and LSD1 expression, but was reciprocally upregulated in mature megakaryocytes following declining Gfi1b and LSD1 levels in cells and on the rgs18 promoter. In contrast, expression of Gfi1b was strong in immature erythroid cells and increased further in mature cells. Manipulation of Rgs18 expression in murine hematopoietic progenitors and a multi-potential human hematopoietic cell line produced opposite outcomes in the two lineages, with expression augmenting megakaryocytic, and potently suppressing erythroid differentiation and vice versa. These phenotypes resulted from differential impact of Rgs18 expression on the p38 and ERK branches of MAPK signaling in the erythroid and megakaryocytic lineages. Repercussions of these signaling changes impacted relative expression of the mutually antagonistic transcription factors Fli1 and Klf1 and were compensated by ectopic Fli1 expression demonstrating activity of this transcription factor downstream of Rgs18. These results identify Rgs18 as a critical downstream effector of Gfi1b and an upstream regulator of MAPK signaling and Klf1/Fli1 gene expression. Sustained Gfi1b expression during erythroid differentiation represses Rgs18 and limits megakaryocytic gene expression in these cells. However during the progress of megakaryocytic differentiation, declining Gfi1b levels result in robust expression of Rgs18 and lineage progression. Preliminary analysis of Rgs18 interactors in megakaryocytes indicates that Rgs18 likely modulates the MAPK pathway by impacting Gαi, cAMP, Ras signaling and certain cytoskeletal proteins. These results will be further extended and confirmed by phenotypic analysis of rgs18-/- mice, and by characterization of novel Rgs18 interacting proteins

Drug schedules: knowledge among undergraduate medical students in a government medical college in Eastern India

Background: In India many of the prescription only drugs (Schedule H) are available without prescription, leading to injudicious use, incidences of dangerous drug interactions, and unnecessary economic burden. Thus awareness among healthcare professionals and among common public are equally important. Objective was to assess the knowledge among undergraduate medical students in a government medical college in Eastern India regarding drug schedules in India.Methods: Willing 3rd semester and 5th semester students participated in the study. We used a pre-tested validated two-part questionnaire to assess the knowledge of undergraduate medical students regarding different drug schedules.Results: 5th semester students gave significantly higher correct answers (P<0.0001) regarding awareness about Drugs and Cosmetics Act 1940, different drug schedules in India, expiry period, guidelines for maintaining the details of standards for patent and proprietary medicines, guidelines regarding import and manufacture of new drugs, guidelines regarding import and manufacture of new drugs, any special license is required for the manufacture and sale of psychotropic drugs, drugs marketed only under “generic name”, guideline regarding pack size of drugs, and guidelines regarding good manufacturing practice (GMP)?. Analysis of second set of questionnaires revealed that the 5th semester students identified the different drug categories more correctly compared to the 3rd semester students.Conclusions: Doctors are one of the principle and reliable sources of drug information for the general population. Thus, it becomes relevant that the undergraduate medical students should regularly brush up their knowledge regarding drug regulations even after passing Pharmacology examination in the later years of training

DISSEMINATED TUBERCULOSIS PRESENTING AS HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS

ABSTRACTHemophagocytic lymphohistiocytosis (HLH) is an immune dysregulation syndrome which is characterized by widespread but ineffective activationof immune system of our body. This activation leads to release of a large pool of cytokines from the activated lymphocytes and macrophages. Thishypercytokinemia leads to the development of characteristic features of HLH such as fever, cytopenias, hepatosplenomegaly, raised serum ferritinlevel, hemophagocytosis in marrow/spleen/lymph nodes, low fibrinogen and or hypertriglyceridemia, low natural killer cell activity, and high-solubleCD25 [1]. Five out of the above eight features are required for the diagnosis. There are 2 variants of HLH, primary HLH; where the defect in theimmune system is hereditary and secondary HLH; where it is caused by other secondary diseases such as infections, hematological malignancies,autoimmune and auto-inflammatory diseases. In this article, we have reported a case of HLH, which was secondary to disseminated tuberculosis.There are only few case reports of HLH secondary to disseminated tuberculosis. Mortality may be as high as 50%. Although tuberculosis has variousmanifestations, our patient presented with fever, skin rash, cytopenias, splenomegaly, and very high ferritin. Marrow examination showed epithelioidgranuloma, hemophagocytosis, and positive Ziehl–Neelsen staining. At present, no definite treatment guidelines have been formulated becauseof multiple drug interactions and toxicities. We treated our patient with non-hepatotoxic anti-tubercular drugs and steroids, followed by additionof isoniazid, rifampicin, and pyrazinamide on improvement of hepatic profile. Thus, high index of clinical suspicion, prompt diagnosis, and earlymanagement may reduce the mortality in this devastating disease. Moreover, this is more common in immunocompromised patients, but here, wehave diagnosed this case in an immunocompetent man.Keywords: Erythematous rash, Fever, Disseminated tuberculosis

Differential Transcriptional Regulation of meis1 by Gfi1b and Its Co-Factors LSD1 and CoREST

Gfi1b (growth factor independence 1b) is a zinc finger transcription factor essential for development of the erythroid and megakaryocytic lineages. To elucidate the mechanism underlying Gfi1b function, potential downstream transcriptional targets were identified by chromatin immunoprecipitation and expression profiling approaches. The combination of these approaches revealed the oncogene meis1, which encodes a homeobox protein, as a direct and prominent target of Gfi1b. Examination of the meis1 promoter sequence revealed multiple Gfi1/1b consensus binding motifs. Distinct regions of the promoter were occupied by Gfi1b and its cofactors LSD1 and CoREST/Rcor1, in erythroid cells but not in the closely related megakaryocyte lineage. Accordingly, Meis1 was significantly upregulated in LSD1 inhibited erythroid cells, but not in megakaryocytes. This lineage specific upregulation in Meis1 expression was accompanied by a parallel increase in di-methyl histone3 lysine4 levels in the Meis1 promoter in LSD1 inhibited, erythroid cells. Meis1 was also substantially upregulated in gfi1b2/2 fetal liver cells along with its transcriptional partners Pbx1 and several Hox messages. Elevated Meis1 message levels persisted in gfi1b mutant fetal liver cells differentiated along the erythroid lineage, relative to wild type. However, cells differentiated along the megakaryocytic lineage, exhibited no difference in Meis1 levels between controls and mutants. Transfection experiments further demonstrated specific repression of meis1 promoter driven reporters by wild type Gfi1b but neither by a SNAG domain mutant nor by a DNA binding deficient one, thus confirming direct functional regulation of this promoter by the Gfi1b transcriptional complex. Overall, our results demonstrate direct yet differential regulation of meis1 transcription by Gfi1b in distinct hematopoietic lineages thus revealing it to be a common, albeit lineage specific, target of both Gfi1b and its paralog Gfi1

Initial Analysis o f the 3\u27 Spliced Region of the Receptor Tyrosine Kinase RET

The RET proto-oncogene is expressed in the developing kidney and enteric nervous system during vertebrate embryogenesis. It has two major splicing variants; RET9 and RET51. The expression pattern of the two variants appears to vary among the developing organisms. RET9 plays a major role in development of kidney and excretory system whereas the role of RET 51 seems to be of maintenance of these systems after development. The protein RET is a transmembrane receptor which expresses itself in the cell surface and initiates the signal transduction pathways for cellular differentiation and proliferation of the cell. The different expression patterns shown by RET 9 and RET51 isoforms and the work of other researchers indicating that different RET splice variants result in different phenotype when expressed independently, are demonstrations of the importance of these splice variations in RET functioning. In order to expand our understanding on the mechanism of RET splicing, the 3\u27 end of the RET 9 isoform was isolated and a new construct was created that contained the exons and introns involved in the RET9 and RET51 splice variations. This region was placed into a mammalian expression vector in both orientations and the ability to measure changes in splicing with these constructs was measured. As a sequential step, in this study two different constructs of a specific region of RET were designed and analyzed using RTPCR and Q-PCR to demonstrate the mechanism of splicing. These constructs are planned to be further used as a model for the study of conditions that lead to splicing of Human RET proto-oncogene. While no differences were seen in initial studies, the development of the constructs and the evaluation of these constructs will provide an opportunity to study this important splicing event in more detail

Transparent Glass/SU8-Based Microfluidic Device with on-Channel Electrical Sensors

This paper presents a transparent microfluidic chip designed for continuous-flow photochemistry applications with integrated electrical sensing. The transparent chip design allows for microscale photochemistry, and permits direct, real-time visual/electrical observation. The microchip uses optically transparent indium tin oxide (ITO) electrodes for reagent and phase tracking. High-speed videography was performed to validate the electrical measurement data