Publications by authors named "Shravan Kumar Mishra"

21 Publications

  • Page 1 of 1

Identification and prevalence of potentially therapeutic targetable variants of major cancer driver genes in ampullary cancer patients in India through deep sequencing.

Cancer Genet 2021 Aug 15;258-259:41-48. Epub 2021 Aug 15.

Department of Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, UP, India.

Ampulla is a complex region located at the confluence of pancreatic and common bile duct and intestinal epithelium. Tumors arising in this region are anatomically and morphologically heterogenous, however they show unique as well as overlapping molecular features. Cancers of both these anatomic sites share morphological as well as genetic profile despite having few unique differences. Targeted therapies are currently emerging as one of the demanding approaches for treatment in most cancer types especially for malignant epithelial tumors and therefore genetic profiling of cancers is the key for identification of potentially therapeutic targetable mutations to know their prevalence and prognostic impact. We studied 97 resected cases of formalin fixed paraffin-embedded AC by deep targeted sequencing using Ampliseq cancer hotspot panel comprising of 50 oncogenes and tumor suppressor genes. Potentially therapeutic targetable mutations were observed in 58/83 (70%) cases. Fourteen patients did not show any pathogenic mutation. TP53 (48.1%), KRAS (37.3%), APC (25.3%), SMAD4 (22.8%), MET (16.8%), CTNNB1 (15.6%) and PIK3CA (10.8%) were the major mutated potential therapeutic targets. KRAS mutation (43.2 Vs. 32.6%) was more prevalent in pancreatobiliary subtype, while TP53 (58.6 Vs 35.1), APC (36.9 Vs 10.8), SMAD4 (28.2 Vs 16.2), MET (21.7 Vs 10.8) and CTNNB1 (19.5 Vs 10.8) were more prevalent in intestinal subtype. WNT signaling pathway was the major altered pathway in intestinal subtype. These mutated genes and pathways may be targeted with currently available drugs and may be explored for future development of targetable agents to improve the disease course in patients of AC.
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http://dx.doi.org/10.1016/j.cancergen.2021.08.001DOI Listing
August 2021

Evaluation of Five International HBV Treatment Guidelines: Recommendation for Resource-Limited Developing Countries Based on the National Study in Nepal.

Pathophysiology 2020 Dec 19;27(1):3-13. Epub 2020 Nov 19.

National Public Health Laboratory (NPHL), Department of Health Services, Ministry of Health and Population, Teku, Kathmandu 44-600, Nepal.

Hepatitis B virus (HBV) infects the liver, causing cirrhosis and cancer. In developed countries, five international guidelines have been used to make a decision for the management of patients with chronic HBV infection. In this review, since the guidelines were established by clinical and epidemiological data of developed countries, we aimed to evaluate whether (1) HBV patient profiles of developing countries are similar to developed countries, and (2) which guideline can be applicable to resource-limited developing countries. First, as an example of the most recent data of HBV infections among developing countries, we evaluated the national HBV viral load study in Nepal, which were compared with the data from other developing countries. In Nepal, the highest number of patients had viral loads of 20-2000 IU/mL (36.7%) and belonged to the age group of 21-30 years; HBV epidemiology in Nepal, based on the viral loads, gender, and age groups was similar to those of not only other developing countries but also developed countries. Next, we reviewed five international HBV treatment guidelines of the World Health Organization (WHO), American Association for the Study of Liver Diseases (AASLD), National Institute for Health and Care Excellence (NICE), European Association for the Study of the Liver (EASL), and Asian Pacific Association for the Study of the Liver (APASL). All guidelines require the viral load and alanine aminotransferase (ALT) levels for decision making. Although four guidelines recommend elastography to assess liver cirrhosis, the WHO guideline alternatively recommends using the aspartate aminotransferase (AST)-to-platelet ratio index (APRI), which is inexpensive and conducted routinely in most hospitals. Therefore, in resource-limited developing countries like Nepal, we recommend the WHO guideline for HBV treatment based on the viral load, ALT, and APRI information.
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http://dx.doi.org/10.3390/pathophysiology27010002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8315108PMC
December 2020

Establishment of reference range of CD4 T-lymphocyte in healthy Nepalese adults.

BMC Res Notes 2020 Jul 2;13(1):316. Epub 2020 Jul 2.

Central Department of Biotechnology, Institute of Science and Technology, Tribhuvan University, Kathmandu, Nepal.

Objective: CD4 T lymphocytes are the most widely used cellular markers to assess the course of HIV infection, clinical staging and, monitoring the effect of antiretroviral therapy. The regional reference range for Eastern, Central and Western development region of Nepal had already been established whereas the same was still lacking in Mid-western and Far-western development region. The objective of this study was to establish reference range of CD4 T lymphocyte in the remaining two development regions and finally the national reference range using data from previous study.

Results: The average values (mean ± SD) of CD4 and CD3 T cell in present study was (819 ± 294) cells/μl and (1546 ± 532) cells/μl, respectively. The absolute CD4 T cell (914 ± 303) and CD3 T cell (1671 ± 560) count in female were significantly higher than those from male, CD4 (757 ± 270) and CD3 (1465 ± 499) (p value-0.000). National reference value of CD4 was determined to be (798 ± 335) cells/μl for healthy Nepalese adults.
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http://dx.doi.org/10.1186/s13104-020-05156-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330941PMC
July 2020

Distribution and Prognostic Significance of Estrogen Receptor (ER), Estrogen Receptor (ER), and Human Epidermal Growth Factor Receptor 2 (HER-2) in Thyroid Carcinoma.

J Thyroid Res 2020 5;2020:6935724. Epub 2020 May 5.

Department of Endocrine Surgery, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow 226014, India.

Purpose: The primary aim of this study was to determine the incidence of estrogen receptor (ER), estrogen receptor (ER), and human epidermal growth factor receptor 2 (HER-2) expression in various subtypes of thyroid carcinoma (TC) of follicular origin and the secondary aim was to correlate the expression with various clinicopathologic prognostic factors.

Methods: Immunohistochemistry analysis was performed on archival paraffin-embedded tissue sections (1991-2016). ER, ER and HER-2 expressions were correlated with clinicopathologic prognostic factors, disease recurrence, and overall survival (OS).

Results: A total of 264 TC patients were included in the study. Incidences of ER, ER and HER-2 were 8.1 vs 16.3 vs 13.9% (=0.15), 26.6 vs 11.5 vs 36.1% (=0.002), and 12.9 vs 2.9 vs 0% (=0.003) in papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and poorly differentiated thyroid carcinoma (PDTC), respectively. Overall ER had significant correlation with distant metastases (0.038) and in case of PDTC with multicentricity (=0.037). ER had significant correlation with lymph node metastases (=0.023) in FTC. HER-2 correlated with tumor size (=0.027) only on univariate analysis. OS did not correlate with expression of any receptor.

Conclusion: ER, ER and HER-2 have differential expression and prognostic implications in different TC subtypes.
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http://dx.doi.org/10.1155/2020/6935724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222548PMC
May 2020

Does genome surveillance explain the global discrepancy between binding and effect of chromatin factors?

FEBS Lett 2020 04 29;594(8):1339-1353. Epub 2020 Jan 29.

Department of Biological Sciences, Indian Institute of Science Education and Research (IISER)-Mohali, India.

Knocking out a chromatin factor often does not alter the transcription of its binding targets. What explains the observed disconnect between binding and effect? We hypothesize that this discrepancy could be associated with the role of chromatin factors in maintaining genetic and epigenetic integrity at promoters, and not necessarily with transcription. Through re-analysis of published datasets, we present several lines of evidence that support our hypothesis and deflate the popular assumptions. We also tested the hypothesis through mutation accumulation assays on yeast knockouts of chromatin factors. Altogether, the proposed hypothesis presents a simple explanation for the global discord between chromatin factor binding and effect. Future work in this direction might fortify the hypothesis and elucidate the underlying mechanisms.
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http://dx.doi.org/10.1002/1873-3468.13729DOI Listing
April 2020

An inquiry-based approach in large undergraduate labs: Learning, by doing it the "wrong" way.

Biochem Mol Biol Educ 2020 05 3;48(3):227-235. Epub 2020 Jan 3.

Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Sahibzada Ajit Singh Nagar, Punjab, India.

Undergraduate laboratory courses, owing to their larger sizes and shorter time slots, are often conducted in highly structured modes. However, this approach is known to interfere with students' engagement in the experiments. To enhance students' engagement, we propose an alternative mode of running laboratory courses by creating some "disorder" in a previously adopted structure. After performing an experiment in the right way, the students were asked to repeat the experiment but with a variation at certain steps leading to the experiment being done the "wrong" way. Although this approach led to fewer experiments being conducted in a semester, it significantly enhanced the students' involvement. This was also reflected in the students' feedback. The majority of students preferred repeating an experiment with a variant protocol than performing a new experiment. Although we have tested this inquiry-based approach only for an undergraduate laboratory course in molecular biology, we believe such an approach could also be extended to undergraduate laboratory courses of other subjects.
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http://dx.doi.org/10.1002/bmb.21331DOI Listing
May 2020

Molecular pathogenesis of gallbladder cancer: An update.

Mutat Res 2019 11 6;816-818:111674. Epub 2019 Jul 6.

Department of Pathology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, 226014, India. Electronic address:

Gallbladder carcinoma (GBC) is the most aggressive gastrointestinal malignancy throughout the world, with wide geographical variance. It is the subtype of biliary tract malignancy that has the poorest prognosis and lower survival among all biliary tract malignancies. Various factors are associated with GBC pathogenesis such as environmental, microbial, metabolic and molecular. Chronic inflammation of gallbladder due to presence of gallstone or microbial infection (eg. Salmonella or H. pylori) results in sustained production of inflammatory mediators in the tissue microenvironment, which can cause genomic changes linked to carcinogenesis. Genetic alterations are one of the major factors, associated with aggressiveness and prognosis. Researches have been done to explore suitable biomarker for early diagnosis and identify altered molecular pathways to develop appropriate biomarkers for early diagnosis, therapy and predicting prognosis. Different agents for targeted therapy against actionable mutations of molecules like EGFR, VEGF, mTOR, HER2, PDL-1, PD-1, MET, PI3K, N-cadherin, VEGFR, MEK1 and MEK2 are being tried. Despite these advancements, there is dismal improvement in the survival of GBC patients. Genetic aberrations other than actionable mutations and epigenetic modification including aberrant expressions of micro-RNAs, are also being studied both as diagnostic biomarker and therapeutic targets. Complex pathogenesis of GBC still needs to be unfolded. In this review we focus on the molecular pathogenesis of GBC elucidated till date along with future directions that can be explored to achieve better management of GBC patients.
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http://dx.doi.org/10.1016/j.mrfmmm.2019.111674DOI Listing
November 2019

Epidermal Growth Factor Receptor Mutation Frequency in Squamous Cell Carcinoma and Its Diagnostic Performance in Cytological Samples: A Molecular and Immunohistochemical Study.

World J Oncol 2019 Jun 29;10(3):142-150. Epub 2019 Jun 29.

Department of Radiodiagnosis, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India.

Background: Epidermal growth factor receptor (EGFR) mutation is the most frequent mutation tested in lung cancer for targeted therapy in the era of personalized medicine. Knowledge about EGFR mutation is constantly expanding regarding its frequency, clinicopathological association, advancements in testing methodology and sample requirement. We investigated EGFR mutation frequency in non-small cell lung cancer (NSCLC) in North Indian patients and evaluated its diagnostic performance in cytological samples.

Methods: Molecular EGFR testing was done in 250 cases of NSCLC by both real-time polymerase chain reaction (PCR) (Therascreen) and mutation-specific EGFR immunohistochemistry (IHC). Thirty cases had both cytology samples and biopsy including 20 pleural effusions and 10 fine-needle aspirates. EGFR mutation concordance between pleural effusion and biopsy was studied.

Results: EGFR mutation was overall 31.6% in NSCLC with 36.5% in adenocarcinoma and 15% in squamous cell carcinoma. L858R mutation accounted for 50.7% and DEL19 for 39.3% of total EGFR mutations. Complex mutations were seen in 2% of cases. Sensitivity of mutation-specific EGFR IHC was 48.3% and specificity was 92.3%. L858R showed higher sensitivity (55% vs. 33.3%) but similar specificity (93.2% vs. 91.3%) compared to DEL19. EGFR mutation was successful in 95% of pleural effusion and showed 83.3% concordance with tissue biopsy.

Conclusions: EGFR mutation frequency in North Indian patients was comparable to that of Asia-Pacific region and showed a similar pattern of histological distribution. EGFR mutation in squamous cell carcinomas is increasingly recognized which was 15% in our study. Mutation-specific EGFR IHC shows variable but generally low sensitivity and considering its significant pre- and post-analytical variables, it should be highly discouraged in patient management. Cytological samples may not only serve as suitable alternative but may be complementary to tissue biopsies.
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http://dx.doi.org/10.14740/wjon1204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615915PMC
June 2019

Emerging Roles of Ubiquitin-like Proteins in Pre-mRNA Splicing.

Trends Biochem Sci 2018 11 27;43(11):896-907. Epub 2018 Sep 27.

Max Planck-DST Partner Group, Centre for Protein Science Design and Engineering, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Sector 81, 140306 Punjab, India. Electronic address:

Ubiquitin-like proteins (UBLs) belong to the protein family whose members share a globular beta-grasp fold structure. The archetypal member, ubiquitin, is known for its function in proteasome-mediated protein degradation. UBLs have been shown to play several crucial roles besides protein turnover, including DNA damage response, cell cycle control, cellular signaling, protein trafficking, and innate immunity activation. In the past few years, accumulating evidence illustrates that four UBLs, namely, ubiquitin, SUMO, Hub1, and Sde2, are involved in eukaryotic pre-mRNA splicing. They modify the spliceosomes and promote splicing by adding new surfaces for intermolecular interactions, thereby refining the outcome of gene expression. In this review article, we highlight recent discoveries with an emphasis on the emerging roles of UBLs in splicing regulation.
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http://dx.doi.org/10.1016/j.tibs.2018.09.001DOI Listing
November 2018

The repressor and co-activator HsfB1 regulates the major heat stress transcription factors in tomato.

Plant Cell Environ 2019 03 11;42(3):874-890. Epub 2018 Oct 11.

Department of Biosciences, Molecular Cell Biology of Plants, Goethe University, Frankfurt am Main, Germany.

Plants code for a multitude of heat stress transcription factors (Hsfs). Three of them act as central regulators of heat stress (HS) response in tomato (Solanum lycopersicum). HsfA1a regulates the initial response, and HsfA2 controls acquired thermotolerance. HsfB1 is a transcriptional repressor but can also act as co-activator of HsfA1a. Currently, the mode of action and the relevance of the dual function of HsfB1 remain elusive. We examined this in HsfB1 overexpression or suppression transgenic tomato lines. Proteome analysis revealed that HsfB1 overexpression stimulates the co-activator function of HsfB1 and consequently the accumulation of HS-related proteins under non-stress conditions. Plants with enhanced levels of HsfB1 show aberrant growth and development but enhanced thermotolerance. HsfB1 suppression has no significant effect prior to stress. Upon HS, HsfB1 suppression strongly enhances the induction of heat shock proteins due to the higher activity of other HS-induced Hsfs, resulting in increased thermotolerance compared with wild-type. Thereby, HsfB1 acts as co-activator of HsfA1a for several Hsps, but as a transcriptional repressor on other Hsfs, including HsfA1b and HsfA2. The dual function explains the activation of chaperones to enhance protection and regulate the balance between growth and stress response upon deviations from the homeostatic levels of HsfB1.
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http://dx.doi.org/10.1111/pce.13434DOI Listing
March 2019

Intron specificity in pre-mRNA splicing.

Curr Genet 2018 Aug 3;64(4):777-784. Epub 2018 Jan 3.

Max Planck, DST Partner Group, Centre for Protein Science Design and Engineering, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Sector 81, Punjab, 140306, India.

The occurrence of spliceosomal introns in eukaryotic genomes is highly diverse and ranges from few introns in an organism to multiple introns per gene. Introns vary with respect to their lengths, strengths of splicing signals, and position in resident genes. Higher intronic density and diversity in genetically complex organisms relies on increased efficiency and accuracy of spliceosomes for pre-mRNA splicing. Since intron diversity is critical for functions in RNA stability, regulation of gene expression and alternative splicing, RNA-binding proteins, spliceosomal regulatory factors and post-translational modifications of splicing factors ought to make the splicing process intron-specific. We recently reported function and regulation of a ubiquitin fold harboring splicing regulator, Sde2, which following activation by ubiquitin-specific proteases facilitates excision of selected introns from a subset of multi-intronic genes in Schizosaccharomyces pombe (Thakran et al. EMBO J, https://doi.org/10.15252/embj.201796751 , 2017). By reviewing our findings with understandings of intron functions and regulated splicing processes, we propose possible functions and mechanism of intron-specific pre-mRNA splicing and suggest that this process is crucial to highlight importance of introns in eukaryotic genomes.
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http://dx.doi.org/10.1007/s00294-017-0802-8DOI Listing
August 2018

Sde2 is an intron-specific pre-mRNA splicing regulator activated by ubiquitin-like processing.

EMBO J 2018 01 25;37(1):89-101. Epub 2017 Sep 25.

Max Planck - DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India

The expression of intron-containing genes in eukaryotes requires generation of protein-coding messenger RNAs (mRNAs) via RNA splicing, whereby the spliceosome removes non-coding introns from pre-mRNAs and joins exons. Spliceosomes must ensure accurate removal of highly diverse introns. We show that Sde2 is a ubiquitin-fold-containing splicing regulator that supports splicing of selected pre-mRNAs in an intron-specific manner in Both fission yeast and human Sde2 are translated as inactive precursor proteins harbouring the ubiquitin-fold domain linked through an invariant GGKGG motif to a C-terminal domain (referred to as Sde2-C). Precursor processing after the first di-glycine motif by the ubiquitin-specific proteases Ubp5 and Ubp15 generates a short-lived activated Sde2-C fragment with an N-terminal lysine residue, which subsequently gets incorporated into spliceosomes. Absence of Sde2 or defects in Sde2 activation both result in inefficient excision of selected introns from a subset of pre-mRNAs. Sde2 facilitates spliceosomal association of Cactin/Cay1, with a functional link between Sde2 and Cactin further supported by genetic interactions and pre-mRNA splicing assays. These findings suggest that ubiquitin-like processing of Sde2 into a short-lived activated form may function as a checkpoint to ensure proper splicing of certain pre-mRNAs in fission yeast.
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http://dx.doi.org/10.15252/embj.201796751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753039PMC
January 2018

IL-17 and IFN-γ producing NK and γδ-T cells are preferentially expanded in synovial fluid of patients with reactive arthritis and undifferentiated spondyloarthritis.

Clin Immunol 2017 10 6;183:207-212. Epub 2017 Apr 6.

Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow Pin: 226014, India. Electronic address:

The IL-17/1L-23 axis is important in the pathogenesis of spondyloarthropathy. Innate cells produce IL-17 in addition to Th17 cells. We studied the frequencies of natural killer (NK) (total, CD56, CD56, perforin+ and granzyme+), NK-T, γδ-T, and IFN-γ+, IL-17+ NK and γδ-T cells in peripheral blood (PB) and synovial fluid (SF) of ReA/uSpA patients. PB from 45 patients and paired SF from 39 patients were studied, together with PB from 18 healthy controls (HC). The frequency of γδ-T cells was decreased (p<0.05) while IL-17 producing NK and γδ-T cells were increased (p<0.05) in PB of patients as compared to HC. In SF, CD56 NK cells were increased (p<0.001) but had reduced expression of perforin and granzyme (p<0.0001) as compared to PB. Frequency of IL-17+, IFN-γ+ NK and γδ-T cells was higher in SF as compared to PB (p<0.05). We suggest that innate cells by producing pro-inflammatory cytokines may contribute to pathogenesis.
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http://dx.doi.org/10.1016/j.clim.2017.03.016DOI Listing
October 2017

Evaluation of SD Bioline HIV/syphilis Duo rapid test kits in Nepal.

BMC Infect Dis 2016 Aug 26;16(1):450. Epub 2016 Aug 26.

Family Health Division, Teku, Kathmandu, Nepal.

Background: Accurate and prompt diagnosis of HIV and syphilis simultaneously has reinforcing effect on their control program because of their prevalent co-infection. Availability of a simple user-friendly two-pronged and affordable detection tools brings down the cost of health care. They are important in the antenatal clinics, with added opportunity for intervention and prevention of mother to child transmission. In cooperation with rapid test kit manufacturers, SD Bioline, NPHL and NCASC, an evaluation of commercially available HIV/syphilis Duo rapid test kit (SD Bioline) to assess its performance and operational characteristics was done in the present study.

Method: A prospective laboratory-based cross sectional study was conducted at a large Women's Hospital. Ten thousand pregnant women, visiting the Hospital for antenatal care or for delivery, were enrolled in study. Tests were performed by the SD Bioline HIV/Syphilis Duo kit as well as national algorithm for HIV and syphilis diagnosis which were considered gold standard. Sensitivity, Specificity, positive predictive value and negative predictive value along with kappa coefficient were calculated for the kit under evaluation.

Result: The sensitivity, specificity, Negative predictive value and Positive predictive value of the kit for HIV diagnosis were 100 % (95 % CI 83.18-100 %, 99.96-100 %, 83.18-100 %, and 99.96-100 %, respectively). Kappa value was found to be 1.0. Out of total cases, results of 9985 (99.85 %) cases were concordant with National algorithm for syphilis diagnosis. Thirteen (0.13 %) cases were found false positive while two were false negative. The sensitivity of the kit for syphilis diagnosis was found to be 95.45 % (95 % CI 84.86-98.74 %) and specificity was 99.87 % (95 % CI; 99.78-99.92 %). Positive predictive value was 76.36 % (95 % CI; 63.65-85.63 %) and Negative predictive value was 99.89 % (95 % CI; 99.39-99.99 %). Kappa value was found to be 0.85.

Conclusion: The performance characteristics of SD Bioline HIV/Syphilis duo kit were found almost concordant with the kits being used for HIV and Syphilis diagnosis separately. Its implementation in antenatal clinics/VCTs could be an added opportunity for simultaneous diagnosis of HIV and syphilis.
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http://dx.doi.org/10.1186/s12879-016-1694-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5002177PMC
August 2016

HsfA2 Controls the Activity of Developmentally and Stress-Regulated Heat Stress Protection Mechanisms in Tomato Male Reproductive Tissues.

Plant Physiol 2016 04 25;170(4):2461-77. Epub 2016 Feb 25.

Department of Biosciences, Molecular Cell Biology of Plants, Goethe University, D-60438 Frankfurt am Main, Germany (S.F., A.M., S.S., Y.H., P.P., S.K.M., E.S., K.-D.S.);Cluster of Excellence Frankfurt, Goethe University, D-60438 Frankfurt am Main, Germany (S.S., E.S.);Plant Breeding, Wageningen University, Wageningen 6708PB, The Netherlands (M.J.P., A.B.);Leibniz Institute of Plant Biochemistry, D-06120 Halle (Saale), Germany (B.T., K.-D.S.);Max Planck Institute for Plant Breeding Research, D-50829 Cologne, Germany (K.T.); andBuchmann Institute for Molecular Life Sciences, Goethe University, D-60438 Frankfurt am Main, Germany (E.S.)

Male reproductive tissues are more sensitive to heat stress (HS) compared to vegetative tissues, but the basis of this phenomenon is poorly understood. Heat stress transcription factors (Hsfs) regulate the transcriptional changes required for protection from HS In tomato (Solanum lycopersicum), HsfA2 acts as coactivator of HsfA1a and is one of the major Hsfs accumulating in response to elevated temperatures. The contribution of HsfA2 in heat stress response (HSR) and thermotolerance was investigated in different tissues of transgenic tomato plants with suppressed HsfA2 levels (A2AS). Global transcriptome analysis and immunodetection of two major Hsps in vegetative and reproductive tissues showed that HsfA2 regulates subsets of HS-induced genes in a tissue-specific manner. Accumulation of HsfA2 by a moderate HS treatment enhances the capacity of seedlings to cope with a subsequent severe HS, suggesting an important role for HsfA2 in regulating acquired thermotolerance. In pollen, HsfA2 is an important coactivator of HsfA1a during HSR HsfA2 suppression reduces the viability and germination rate of pollen that received the stress during the stages of meiosis and microspore formation but had no effect on more advanced stages. In general, pollen meiocytes and microspores are characterized by increased susceptibility to HS due to their lower capacity to induce a strong HSR This sensitivity is partially mitigated by the developmentally regulated expression of HsfA2 and several HS-responsive genes mediated by HsfA1a under nonstress conditions. Thereby, HsfA2 is an important factor for the priming process that sustains pollen thermotolerance during microsporogenesis.
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http://dx.doi.org/10.1104/pp.15.01913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825147PMC
April 2016

Negative feedback circuit for toll like receptor-8 activation in human embryonic Kidney 293 using outer membrane vesicle delivered bi-specific siRNA.

BMC Immunol 2015 Jul 23;16:42. Epub 2015 Jul 23.

Asian Institute of Technology & Management, Purbanchal University, Knowledge village, Khumaltar, Satdobato, Lalitpur, Nepal.

Background: TLR8 assists in antiviral approach by producing Type 1 INF via MyD88 dependent IRF7 pathway. However, over expression of INFα/β molecule poses threat by developing tolerance in chronic infection cases and enhancing inflammatory response. Here we report a bi-specific siRNA based complex which differentially activates and silences the TLR8 and MYD88 respectively in a negatively regulated fashion.

Results: Outer membrane vesicle from Escherichia coli used for siRNA delivery was observed more efficient when attached with invasive protein Ail along with OmpA (P<0.001) in HEK293-TLR8 cell line. siRNA complexed with p19 protein was efficient in activating TLR8, confirmed by the increment of INFβ molecules (P<0.001) in HEK293-TLR8 compared to its counterpart. Fusion of lipid bilayer of endosomal compartment was significant at pH 4.5 when fusogenic peptides (diINF-7) were incubated in membrane vesicle, thus facilitating the escape of siRNA complex to the host cytoplasm in order to silence MyD88 transcript (P<0.001).

Conclusions: We investigated the activation of TLR8 by bi-specific si-RNA for the production of INFβ. In the same setting we showed that bi-specific si-RNA was able to silence MyD88 transcript in a delayed manner. For the cases of auto immune disease and inflammation where over activation of endosomal TLRs poses serious threat, bi specific siRNA could be used as negative feedback controlled system.
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http://dx.doi.org/10.1186/s12865-015-0109-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510891PMC
July 2015

The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells.

J Mol Cell Biol 2014 Aug 28;6(4):312-23. Epub 2014 May 28.

Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins non-covalently. In Saccharomyces cerevisiae, Hub1 associates with spliceosomes and mediates alternative splicing of SRC1, without affecting pre-mRNA splicing generally. Human Hub1 is highly similar to its yeast homolog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast; however, unlike its S. cerevisiae homolog, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-like protein Hub1 is not a canonical spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing.
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http://dx.doi.org/10.1093/jmcb/mju026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4141198PMC
August 2014

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing.

Nature 2011 May 25;474(7350):173-8. Epub 2011 May 25.

Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1-HIND interaction, cannot use certain non-canonical 5' splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.
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http://dx.doi.org/10.1038/nature10143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587138PMC
May 2011

Functional dissection of the cytosolic chaperone network in tomato mesophyll protoplasts.

Plant Cell Environ 2009 Feb;32(2):123-33

J. W. Goethe-University, Molecular Cell Biology of Plants, Biocenter N200, 3OG, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany.

The heat stress response is universal to all organisms. Upon elevated temperatures, heat stress transcription factors (Hsfs) are activated to up-regulate the expression of molecular chaperones to protect cells against heat damages. In higher plants, the phenomenon is unusually complex both at the level of Hsfs and heat stress proteins (Hsps). Over-expression of both Hsfs and Hsps and the use of RNA interference for gene knock-down in a transient system in tomato protoplasts allowed us to dissect the in vivo chaperone functions of essential components of thermotolerance, such as the cytoplasmic sHsp, Hsp70 and Hsp100 chaperone families, and the regulation of their expression. The results point to specific functions of the different components in protection from protein denaturation and in refolding of denatured proteins.
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http://dx.doi.org/10.1111/j.1365-3040.2008.01902.xDOI Listing
February 2009

Heat stress response in plants: a complex game with chaperones and more than twenty heat stress transcription factors.

J Biosci 2004 Dec;29(4):471-87

Department of Molecular Cell Biology, Goethe University Frankfurt, Marie Curie Str. 9, D-60439 Frankfurt/M., Germany.

Compared to the overall multiplicity of more than 20 plant Hsfs, detailed analyses are mainly restricted to tomato and Arabidopsis and to three important representatives of the family (Hsfs A1, A2 and B1). The three Hsfs represent examples of striking functional diversification specialized for the three phases of the heat stress (hs) response (triggering, maintenance and recovery). This is best illustrated for the tomato Hsf system: (i) HsfA1a is the master regulator responsible for hs-induced gene expression including synthesis of HsfA2 and HsfB1. It is indispensible for the development of thermotolerance. (ii) Although functionally equivalent to HsfA1a, HsfA2 is exclusively found after hs induction and represents the dominant Hsf, the "working horse" of the hs response in plants subjected to repeated cycles of hs and recovery in a hot summer period. Tomato HsfA2 is tightly integrated into a network of interacting proteins (HsfA1a, Hsp17-CII, Hsp17-CI) influencing its activity and intracellular distribution. (iii) Because of structural peculiarities, HsfB1 acts as coregulator enhancing the activity of HsfA1a and/or HsfA2. But in addition, it cooperates with yet to be identified other transcription factors in maintaining and/or restoring housekeeping gene expression.
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http://dx.doi.org/10.1007/BF02712120DOI Listing
December 2004

In the complex family of heat stress transcription factors, HsfA1 has a unique role as master regulator of thermotolerance in tomato.

Genes Dev 2002 Jun;16(12):1555-67

Biocenter of the Goethe University, D-60439 Frankfurt am Main, Germany.

We generated transgenic tomato plants with altered expression of heat stress transcription factor HsfA1. Plants with 10-fold overexpression of HsfA1 (OE plants) were characterized by a single HsfA1 transgene cassette, whereas plants harboring a tandem inverted repeat of the cassette showed cosuppression (CS plants) by posttranscriptional silencing of the HsfA1 gene connected with formation of small interfering RNAs. Under normal growth conditions, major developmental parameters were similar for wild-type (WT), OE, and CS plants. However, CS plants and fruits were extremely sensitive to elevated temperatures, because heat stress-induced synthesis of chaperones and Hsfs was strongly reduced or lacking. Despite the complexity of the plant Hsf family with at least 17 members in tomato, HsfA1 has a unique function as master regulator for induced thermotolerance. Using transient reporter assays with mesophyll protoplasts from WT tomato, we demonstrated that plasmid-encoded HsfA1 and HsfA2 were well expressed. However, in CS protoplasts the cosuppression phenomenon was faithfully reproduced. Only transformation with HsfA2 expression plasmid led to normal expression of the transcription factor and reporter gene activation, whereas even high amounts of HsfA1 expression plasmids were silenced. Thermotolerance in CS protoplasts was restored by plasmid-borne HsfA2, resulting in expression of chaperones, thermoprotection of firefly luciferase, and assembly of heat stress granules.
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http://dx.doi.org/10.1101/gad.228802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC186353PMC
June 2002
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