Splicing of branchpoint-distant exons is promoted by Cactin, Tls1 and the ubiquitin-fold-activated Sde2.

Intron diversity facilitates regulated gene expression and alternative splicing. Spliceosomes excise introns after recognizing their splicing signals: the 5'-splice site (5'ss), branchpoint (BP) and 3'-splice site (3'ss). The latter two signals are recognized by U2 small nuclear ribonucleoprotein (snRNP) and its accessory factors (U2AFs), but longer spacings between them result in weaker splicing. Here, we show that excision of introns with a BP-distant 3'ss (e.g. rap1 intron 2) requires the ubiquitin-fold-activated splicing regulator Sde2 in Schizosaccharomyces pombe. By monitoring splicing-specific ura4 reporters in a collection of S. pombe mutants, Cay1 and Tls1 were identified as additional regulators of this process. The role of Sde2, Cay1 and Tls1 was further confirmed by increasing BP-3'ss spacings in a canonical tho5 intron. We also examined BP-distant exons spliced independently of these factors and observed that RNA secondary structures possibly bridged the gap between the two signals. These proteins may guide the 3'ss towards the spliceosome's catalytic centre by folding the RNA between the BP and 3'ss. Orthologues of Sde2, Cay1 and Tls1, although missing in the intron-poor Saccharomyces cerevisiae, are present in intron-rich eukaryotes, including humans. This type of intron-specific pre-mRNA splicing appears to have evolved for regulated gene expression and alternative splicing of key heterochromatin factors.

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

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.

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

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 Schizosaccharomyces pombe 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.

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

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

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.

Intron specificity in pre-mRNA splicing.

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.

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.

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, CD56bright, CD56dim, 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, CD56bright 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.

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

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.

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

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.

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

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

BACKGROUND: TLR8 assists in antiviral approach by producing Type 1 INF via MyD88 dependent IRF7 pathway. However, over expression of INFα/ß 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 INFß 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 INFß. 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.

Incidence and Prognostic Significance of Androgen Receptors (AR) in Indian Triple-Negative Breast Cancer (TNBC).

Molecular sub-characterization of triple-negative breast cancer (TNBC) has great therapeutic and possibly prognostic implications. The primary aim of this study was to investigate the incidence of luminal androgen receptor (LAR) subtype of TNBC and secondary aims were sub-categorization and clinico-pathologic correlation of LAR breast cancers. Retrospective study (January 2008 and 31st of December 2018) consisting of 157 TNBC patients. Androgen receptor (AR) expression was measured by immunohistochemical analysis. One percent cutoff was set as a positive expression. Sub-categorization was done on the basis of EGFR (> 15% of tumor cells) and Ki-67 expression (low- < 11%, intermediate- 11-20%, and high- > 21%). AR expression was correlated with various clinico-pathologic features and outcomes of the patients. The incidence of AR expression in TNBC was 24.8%. Considering different thresholds of > 5%, > 10%, and > 20% immunostaining, the incidence of AR positivity was 18.4, 15.2, and 11.5% respectively. The incidence of Ki-67 (p = 0.89) and EGFR (p = 0.643) expression did not differ significantly in AR-positive and -negative TNBC. Based on EGFR expression 19, 67 and 14% patients were categorized as low, intermediate, and high risk respectively. Low-risk (p ≤ 0.001) and low-grade (p = 0.014) tumors were more likely to have > 10% AR expression. Clinico-pathological profile, response to neoadjuvant chemotherapy, disease-free survival (p = 0.458), and overall survival (p = 0.806) did not significantly differ between AR expressing and negative TNBC. On multivariate analysis, only tumor staging was a significant predictor of survival (p = 0.012) and AR expression of > 10% revealed a trend towards improved survival (p = 0.07). When considering only AR-positive TNBC, AR expression of > 10% (p = 0.038), distant metastases (p = 0.003), and EGFR status (p = 0.024) were significantly associated with survival. AR expression does not seem to very strongly correlate with prognosis in TNBC and further studies could focus more on its predictive role in deciding anti-androgen therapy.

A Rapid Method for Determination of Serum Methotrexate Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Application in Therapeutic Drug Monitoring.

Objectives Methotrexate (MTX) has anticancer therapeutic potential with multiple doses-related adverse effects and toxicities. Immunoassays for therapeutic monitoring of serum MTX have their own limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as the reference method; however, commercially availability of them is limited. We aimed to adapt/develop an in-house LC-MS/MS method for therapeutic monitoring of serum MTX. Materials and Methods Serum protein precipitation was performed using acetonitrile-water containing 250 µM solution of aminoacetophenone as internal standard (IS). Chromatographic separation was achieved on a C18 column with mobile phase of 0.1% solution of formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.4 mL/min. MS was performed under positive ion mode with mass transition for MTX and IS as m/z 455.1→308.1 and 136.2→94.1, respectively. The method was validated by following Bioanalytical Method Validation Guidance for Industry, 2018 and applied on leukemia patients' samples on MTX therapy. Results The correlation coefficient of eight serially diluted calibration standards of 0.09 to 12.5 µM was >0.99 and had linearity with > 95% precision and accuracy at analytical quality control levels. The lower limit of MTX quantification achieved was 0.09 µM with good intensity and sharp peak as compared with blank sample. The total run time of the assay was 5 minutes. The serum MTX levels obtained by this method in leukemia patients exhibited clinical correlation and an excellent agreement with commercial immunoassay used in parallel. Conclusion We were able to develop a rapid, sensitive, and cost-effective LC-MS/MS method suitable for therapeutic drug monitoring of MTX in routine clinical diagnostic laboratories.

The ubiquitin-like protein Hub1/UBL-5 functions in pre-mRNA splicing in Caenorhabditis elegans.

The ubiquitin-like protein Hub1/UBL-5 associates with proteins non-covalently. Hub1 promotes alternative splicing and splicing of precursor mRNAs with weak introns in yeast and mammalian cells; however, its splicing function has remained elusive in multicellular organisms. Here, we demonstrate the splicing function of Hub1/UBL-5 in the free-living nematode Caenorhabditis elegans. Hub1/UBL-5 binds to the HIND-containing splicing factors Snu66/SART-1 and PRP-38 and associates with other spliceosomal proteins. C. elegans hub1/ubl-5 mutants die at the Larval 3 stage and show splicing defects for selected targets, similar to the mutants in yeast and mammalian cells. UBL-5 complemented growth and splicing defects in Schizosaccharomyces pombe hub1 mutants, confirming its functional conservation. Thus, UBL-5 is important for C. elegans development and plays a conserved pre-mRNA splicing function.

Broader roles of the ubiquitin-like protein Hub1 indicated by its yeast two-hybrid interactors.

The conserved ubiquitin-like protein Hub1/UBL5 functions in RNA splicing, DNA repair and mitochondrial unfolding responses. It binds proteins specific to these pathways and modifies their functional properties. However, the identities of other Hub1 substrates remain unknown. We have found unreported interactors of Saccharomyces cerevisiae Hub1 from a yeast two-hybrid (Y2H) screen. Proteins containing SIMs (small ubiquitin-like modifier SUMO-interaction motifs) and ferulic acid decarboxylase Fdc1 are identified as potential Hub1 interactors. Further experiments are required to establish these interactions and their physiological relevance, nevertheless, data presented here point towards larger and intriguing roles of Hub1.

Molecular detection and characterization of dengue isolates circulating in north India.

Background and Objectives: In recent decades, the incidence of dengue has increased dramatically. In dengue-endemic countries, changes in dengue virus serotypes, genotypes, and lineages have been reported. This study was designed to detect and characterize the dengue virus isolates circulating in North India by serological and molecular techniques. Materials and Methods: This study was conducted at the Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. NS1 antigen and IgM antibody against dengue were detected by ELISA methods, viral RNA was extracted and amplified by conventional PCR and one-step single-tube multiplex PCR. The purified PCR products were cycle sequenced and a database search was implemented for the confirmation of the sequence product. Phylogenetic analysis was carried out with previously reported sequences. Results: Among 1509 samples, 205 (13.6%) were found positive for IgM antibodies with the highest number (n=67) among the 21 to 30 years age group with peak positivity during post-monsoon months. Among acute samples, NS1 antigen was positive in 62.9%. Seven patients out of 13 had dengue viral RNA in PCR. It comprised six DENV-2 serotypes and one DENV-3 serotype. On phylogenetic analysis, DENV-2 strains grouped with genotype IV and DENV-3 with genotype III. Conclusion: Dengue infection was found frequently during post-monsoon season. The positivity rate of the dengue NS1 antigen test was greater than that of the antibody test. The dengue isolates were characterized as genotype IV and genotype III of DENV-2 and DENV-3 respectively.

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

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.

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

PURPOSE: The primary aim of this study was to determine the incidence of estrogen receptor α (ERα), estrogen receptor ß (ERß), 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). ERα, ERß, 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 ERα, ERß, and HER-2 were 8.1 vs 16.3 vs 13.9% (p=0.15), 26.6 vs 11.5 vs 36.1% (p=0.002), and 12.9 vs 2.9 vs 0% (p=0.003) in papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and poorly differentiated thyroid carcinoma (PDTC), respectively. Overall ERα had significant correlation with distant metastases (0.038) and in case of PDTC with multicentricity (p=0.037). ERß had significant correlation with lymph node metastases (p=0.023) in FTC. HER-2 correlated with tumor size (p=0.027) only on univariate analysis. OS did not correlate with expression of any receptor. CONCLUSION: ERα, ERß, and HER-2 have differential expression and prognostic implications in different TC subtypes.

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

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.

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

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.