How to do a journal club, a seminar and a webinar?

The topics for postgraduate teaching-learning tools are Journal club in-house with one speaker and a moderator, Seminars- with multiple speakers and a co-ordinator, and Webinars- online seminars with one or multiple speakers choosing multiple mediums of communication. They largely affect the working mechanism of a clinician as they help us upgrade with the recent development in our fields. Making them interesting for us as well as our colleagues is necessary. This article presents a few facts as well as tips and tricks to compile the literature in a manner, which includes all the necessary points for better learning.

Some data quality issues at ClinicalTrials.gov.

BACKGROUND: Clinical trial registries have been established as a form of public accountability. Sponsors ought to register their trials promptly and accurately, but this is not always done. Some of the problems include non-registration of trials, registration of trials with incomplete information, and non-reporting of trial results on time. In this study we enumerate or quantify some quality issues with respect to Principal Investigator (PI) and Responsible Party data. METHODS: We analyzed interventional trials registered with ClinicalTrials.gov. Using certain selection criteria, we started with 112,013 records, and then applied further filters. The trial had to (a) start between 1 January 2005 and 31 December 2014, (b) include a "drug" or "biological" in the "intervention" field, (c) be registered with an American authority, and (d) list a real person's name as investigator and also his or her role in the study. RESULTS: We identified four categories of errors in the ClinicalTrials.gov records. First, some data were missing. The name of the investigator, or his or her role, was missing in 12% of 35,121 trials. In examining 71,359 pairs of names and roles, 17% of the "names" were found to be not those of real persons, but instead junk information. Second, there were variations in a large number of names. We identified 19 categories of variants. We determined that 13% of the names had variants that could not be resolved using a program. Third, some trials listed many PIs each, although only one such person holds overall responsibility for the trial and therefore not more than one person should be listed as PI. Fourth, in examining whether the PI's name was available as part of the Responsible Party tag, we found that in 1221 (3.5%) of 35,121 trials, the Responsible Party tag is absent. CONCLUSIONS: We have outlined four categories of problems with data hosted by ClinicalTrials.gov and have quantified three of them. We also suggest how these errors could be prevented in future. It is important to carry out various kinds of audits of trial registries, in order to identify lacunae in the records, that they be addressed.

Management of patients who refuse blood transfusion.

A small group of people belonging to a certain religion, called Jehovah's witness do not accept blood transfusion or blood products, based on biblical readings. When such group of people are in need of health care, their faith and belief is an obstacle for their proper treatment, and poses legal, ethical and medical challenges for attending health care provider. Due to the rapid growth in the membership of this group worldwide, physicians attending hospitals should be prepared to manage such patients. Appropriate management of such patients entails understanding of ethical and legal issues involved, providing meticulous medical management, use of prohaemostatic agents, essential interventions and techniques to reduce blood loss and hence, reduce the risk of subsequent need for blood transfusion. An extensive literature search was performed using search engines such as Google scholar, PubMed, MEDLINE, science journals and textbooks using keywords like 'Jehovah's witness', 'blood haemodilution', 'blood salvage' and 'blood substitutes'.

Value of ancillary studies in the evaluation of fine-needle aspiration specimens: Our experience.

BACKGROUND: The cytological diagnosis of poorly differentiated tumors is challenging because the tumor cells may have morphologically difficult presentations in materials obtained by fine-needle aspiration cytology (FNAC). With the application of FNAC in primary diagnosis of malignant lesions, there has been a significant increase in the use of ancillary studies in the aspirated material. AIMS: We evaluated the value of ancillary studies, namely cell blocks, immunocytochemistry (ICC) and electron microscopy (EM), in the final interpretation of FNAC smears. MATERIALS AND METHODS: Sixty-nine cases of neoplastic swellings were subjected to FNAC. Material acquired was divided for ICC, consisting of immunoperoxidase staining of direct smears, and/or cellblocks and EM, in addition to routine light microscopy (LM). Correlation with the available histological material with immunohistochemistry and/or pertinent clinical information was used as a "gold" standard. RESULTS: Five (7.2%) cases were excluded from the study, the material being necrotic or insufficient. Cell blocks were available in 46/64 (71.8%) cases, ICC evaluation was performed in 41/64 cases (64%) and EM studies were done in 57/64 cases (89%). Diagnostic accuracy of LM alone was 32/64 (50%). Cell blocks improved the diagnoses in 8/46 (17%) cases. The ICC data were diagnostic in 18/41 (43.9%) cases, helpful in 8/41 (19.6%) cases and non-helpful in 15/41 (36.5%) cases. EM studies were diagnostic in 22/57 (38.5%) cases, helpful in 18/57 (31.5%) cases and non-helpful in 17/57 (30%) cases. In 34/64 (53.1%) cases, all ancillary techniques (cell blocks, ICC and EM) were applied and their diagnostic accuracy was compared. CONCLUSIONS: With appropriate case selection, ancillary studies performed on aspirated material can provide useful information in FNAC.

Cytology of hyalinising trabecular adenoma-like variant of medullary thyroid carcinoma.

Medullary thyroid carcinoma is a rare thyroid neoplasm that can be either sporadic or familial. It occurs in adults, presenting as a solitary cold nodule on thyroid scan. Most are solid, firm, and non-encapsulated, and occur in the mid portion or upper half of the thyroid gland, corresponding to areas with greater numbers of C cells. We present a case of a 36-year-old female with a swelling in the front of her neck for six years. Fine needle aspiration done elsewhere revealed spindle cells, suggestive of a 'spindle cell neoplasm'. The histopathology of the thyroidectomy specimen showed features of a hyalinizing trabecular adenoma-like variant of medullary carcinoma. Subsequently, we performed aspiration on the received specimen and studied the cytological findings. The cytological diagnosis of this variant requires identification of the dual spindle and ovoid cell population and the granular neuroendocrine chromatin.

Carbamazepine induced DRESS syndrome.

We present here an 18-yr-old male who presented with intermittent fever of moderate grade and of 15 days duration, followed by maculopapular erythematous rashes over upper and lower extremities, face, and trunk developing over 10-12 days. He was suffering from recurrent seizures since last 3 months for which he was started on carbamazepine 200mg twice daily for the past 6 weeks. He was febrile on admission. Generalized lymphadenopathy with discreet, non-matted, firm and tender inguinal lymph nodes. Patch test with 1% and 5% solution of carbamazepine was strongly positive.

Role of Lys-226 in the catalytic mechanism of Bacillus stearothermophilus serine hydroxymethyltransferase--crystal structure and kinetic studies.

Serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme catalyzes the reversible conversion of l-Ser and tetrahydropteroylglutamate (H(4)PteGlu) to Gly and 5,10-methylene tetrahydropteroylglutamate (CH(2)-H(4)PteGlu). Biochemical and structural studies on this enzyme have implicated several residues in the catalytic mechanism, one of them being the active site lysine, which anchors PLP. It has been proposed that this residue is crucial for product expulsion. However, in other PLP-dependent enzymes, the corresponding residue has been implicated in the proton abstraction step of catalysis. In the present investigation, Lys-226 of Bacillus stearothermophilus SHMT (bsSHMT) was mutated to Met and Gln to evaluate the role of this residue in catalysis. The mutant enzymes contained 1 mol of PLP per mol of subunit suggesting that Schiff base formation with lysine is not essential for PLP binding. The 3D structure of the mutant enzymes revealed that PLP was bound at the active site in an orientation different from that of the wild-type enzyme. In the presence of substrate, the PLP ring was in an orientation superimposable with that of the external aldimine complex of wild-type enzyme. However, the mutant enzymes were inactive, and the kinetic analysis of the different steps of catalysis revealed that there was a drastic reduction in the rate of formation of the quinonoid intermediate. Analysis of these results along with the crystal structures suggested that K-226 is responsible for flipping of PLP from one orientation to another which is crucial for H(4)PteGlu-dependent Calpha-Cbeta bond cleavage of l-Ser.

Mycobacterium tuberculosis Rv2118c codes for a single-component homotetrameric m1A58 tRNA methyltransferase.

Modified nucleosides in tRNAs play important roles in tRNA structure, biosynthesis and function, and serve as crucial determinants of bacterial growth and virulence. In the yeast Saccharomyces cerevisiae, mutants defective in N1-methylation of a highly conserved adenosine (A58) in the TPsiC loop of initiator tRNA are non-viable. The yeast m1A58 methyltransferase is a heterotetramer consisting of two different polypeptide chains, Gcd14p and Gcd10p. Interestingly, while m1A58 is not found in most eubacteria, the mycobacterial tRNAs have m1A58. Here, we report on the cloning, overexpression, purification and biochemical characterization of the Rv2118c gene-encoded protein (Rv2118p) from Mycobacterium tuberculosis, which is homologous to yeast Gcd14p. We show that Rv2118c codes for a protein of approximately 31 kDa. Activity assays, modified base analysis and primer extension experiments using reverse transcriptase reveal that Rv2118p is an S-adenosyl-l-methionine-dependent methyltransferase which carries out m1A58 modification in tRNAs, both in vivo and in vitro. Remarkably, when expressed in Escherichia coli, the enzyme methylates the endogenous E.coli initiator tRNA essentially quantitatively. Furthermore, unlike its eukaryotic counterpart, which is a heterotetramer, the mycobacterial enzyme is a homotetramer. Also, the presence of rT modification at position 54, which was found to inhibit the Tetrahymena pyriformis enzyme, does not affect the activity of Rv2118p. Thus, the mycobacterial m1A58 tRNA methyltransferase possesses distinct biochemical properties. We discuss aspects of the biological relevance of Rv2118p in M.tuberculosis, and its potential use as a drug target to control the growth of mycobacteria.

Antithrombin III assay using thrombin in disseminated intravascular coagulation (DIC), other thromboembolic disorders and hepatic diseases.

Fifty cases comprising 11 cases of disseminated intravascular coagulation (DIC), 16 cases of venous thromboses and 23 cases of hepatic diseases were studied for AT III levels using clotting assay. Twelve samples were subjected to ATIII estimation by the commercially available synthetic chromogenic assay. Twenty age and sex matched controls were also analysed to find out the reference value for the techniques. Low AT III levels, if present, were correlated with other markers of DIC, viz FDP and D-dimer assays. There was a decrease in the AT III levels in all the three disease categories with a significant difference between the AT III levels of the three disease categories. In DIC, lowest levels were observed which correlated well with FDP and D-dimer levels. There was no significant difference between the average AT III levels measured by both the clotting and synthetic chromogenic assay with the former procedure being relatively inexpensive.

von Willebrand Disease : A Clinico-haematological Spectrum.

BACKGROUND: Bleeding disorders are commonly seen in clinical practice. von Willebrand Disease (vWD), is the commonest and yet a profoundly under diagnosed cause, having a wide spectrum of clinical presentation. Of its three types, type 1 vWD (70% of the total vWD cases) has the mildest and a highly variable clinical and laboratory presentation. METHODS: A series of ten cases of vWD were comprehensively evaluated using recommended diagnostic parameters and therapeutic interventions. RESULTS: All major types of vWD were represented. A female preponderance, with primary presentation in the form of muco-cutaneous bleeds was observed. A positive history of consanguineous parental marriage and family history of bleeding disorder was elicited in two and three patients respectively. Nine patients were found to be anemic and thrombocytopenia was present in only one. Bleeding time by modified template (SIMPLATE) method, along with activated partial thromboplastin time (APTT) was increased in all ten cases and of these, nine had low factor VIII: C levels. Ristocetin induced platelet aggregation studies were abnormal in all the five cases it was performed. vWF:RCo activity determined in one individual was shown to be low. vWF:Ag assay was done in four cases revealing a near complete absence of von Willebrand factor antigen in one and mildly decreased levels in the other three. vWF multimer assay was advised in three cases. DDAVP, plasma derived vWF, blood products and local antifibrinolytics were used as primary modalities of treatment. CONCLUSION: Thus, strong clinical suspicion, thorough clinical evaluation and judicious use of investigations including repeated investigations at different times are needed for making a diagnosis of vWD.

Structure-function relationship in serine hydroxymethyltransferase.

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5'-phosphate (PLP)-dependent enzyme catalyzes the tetrahydrofolate (H(4)-folate)-dependent retro-aldol cleavage of serine to form 5,10-methylene H(4)-folate and glycine. The structure-function relationship of SHMT was studied in our laboratory initially by mutation of residues that are conserved in all SHMTs and later by structure-based mutagenesis of residues located in the active site. The analysis of mutants showed that K71, Y72, R80, D89, W110, S202, C203, H304, H306 and H356 residues are involved in maintenance of the oligomeric structure. The mutation of D227, a residue involved in charge relay system, led to the formation of inactive dimers, indicating that this residue has a role in maintaining the tetrameric structure and catalysis. E74, a residue appropriately positioned in the structure of the enzyme to carry out proton abstraction, was shown by characterization of E74Q and E74K mutants to be involved in conversion of the enzyme from an 'open' to 'closed' conformation rather than proton abstraction from the hydroxyl group of serine. K256, the residue involved in the formation of Schiffs base with PLP, also plays a crucial role in the maintenance of the tetrameric structure. Mutation of R262 residue established the importance of distal interactions in facilitating catalysis and Y82 is not involved in the formaldehyde transfer via the postulated hemiacetal intermediate but plays a role in stabilizing the quinonoid intermediate. The mutational analysis of scSHMT along with the structure of recombinant Bacillus stearothermophilus SHMT and its substrate(s) complexes was used to provide evidence for a direct transfer mechanism rather than retro-aldol cleavage for the reaction catalyzed by SHMT.

Restorative Proctocolectomy with Ileo-anal Reservoir, a Histopathological, Histochemical, and Electron Microscopic Study.

This study was conducted to investigate the nature of colonic metaplasia in ileo-anal pouches and incidence/frequency of pouchitis in the same. Biopsy specimens from 8 patients with functioning ileal pouches were studied using routine histology, mucin histochemistry and electron microscopy, over a 2 - year period. All 8 patients had villous abnormalities in the form of blunting of villi and sub total or partial villous atrophy. 6 patients had an increase in the goblet cell population and Paneth cell hyperplasia. These changes were supported by electron microscopic findings of a decrease in number and flattening of ileal type microvilli and their transformed morphologic resemblance to colonic type microvilli. All the ileal pouches also had acquired colorectal type sulphomucin, when sections stained with Alcian-blue and High Iron Diamine - Alcian blue, were studied. However, no case of pouchitis as defined in literature, was found in this study.

Different unfolding pathways for mesophilic and thermophilic homologues of serine hydroxymethyltransferase.

To determine how much information can be transferred from folding and unfolding studies of one protein to another member of the same family or between the mesophilic and thermophilic homologues of a protein, we have characterized the equilibrium unfolding process of the dimeric enzyme serine hydroxymethyltransferase (SHMT) from two sources, Bacillus subtilis (bsSHMT) and Bacillus stearothermophilus (bstSHMT). Although the sequences of the two enzymes are highly identical ( approximately 77%) and homologous (89%), bstSHMT shows a significantly higher stability against both thermal and urea denaturation than bsSHMT. The GdmCl-induced unfolding of bsSHMT was found to be a two-step process with dissociation of the native dimer, resulting in stabilization of a monomeric species, followed by the unfolding of the monomeric species. A similar unfolding pathway has been reported for Escherichia coli aspartate aminotransferase, a member of the type I fold family of PLP binding enzymes such as SHMT, the sequence of which is only slightly identical ( approximately 14%) with that of SHMT. In contrast, for bstSHMT, a highly cooperative unfolding without stabilization of any monomeric intermediate was observed. These studies suggest that mesophilic proteins of the same structural family even sharing a low level of sequence identity may follow a common unfolding mechanism, whereas the mesophilic and thermophilic homologues of the same protein despite having a high degree of sequence identity may follow significantly different unfolding mechanisms.

Purification and crystallization of CII: an unstable transcription activator from phage lambda.

The CII protein of the temperate bacteriophage lambda is a transcriptional activator involved in the lysis-lysogeny switch of the phage. It is an unstable protein of 97 amino acids and is known to exist as a tetramer in the native state. The cII gene has been cloned and expressed in Escherichia coli using a T7 promoter based over-expression system. The recombinant CII protein has been purified to homogeneity by ammonium sulfate fractionation followed by two steps of ion-exchange chromatography. The purified protein crystallized at pH 8.2 in hanging-drop vapor diffusion method at 293 K. The crystals diffract to a resolution of 2.8 A and belong to the space group C222 with unit-cell parameters a = 64.10, b = 106.95 and c = 120.16 A.

Crystal structure of Rv2118c: an AdoMet-dependent methyltransferase from Mycobacterium tuberculosis H37Rv.

Rv2118c belongs to the class of conserved hypothetical proteins from Mycobacterium tuberculosis H37Rv. The crystal structure of Rv2118c in complex with S-adenosyl-l-methionine (AdoMet) has been determined at 1.98 A resolution. The crystallographic asymmetric unit consists of a monomer, but symmetry-related subunits interact extensively, leading to a tetrameric structure. The structure of the monomer can be divided functionally into two domains: the larger catalytic C-terminal domain that binds the cofactor AdoMet and is involved in the transfer of methyl group from AdoMet to the substrate and a smaller N-terminal domain. The structure of the catalytic domain is very similar to that of other AdoMet-dependent methyltransferases. The N-terminal domain is primarily a beta-structure with a fold not found in other methyltransferases of known structure. Database searches reveal a conserved family of Rv2118c-like proteins from various organisms. Multiple sequence alignments show several regions of high sequence similarity (motifs) in this family of proteins. Structure analysis and homology to yeast Gcd14p suggest that Rv2118c could be an RNA methyltransferase, but further studies are required to establish its functional role conclusively.