Impact of an Educational Measure on the Quality of Medical Certificate of Cause of Death at a Tertiary Care Teaching Hospital: A Pre-post Interventional Study.

Background A death certificate is an important document that serves as a tool for gathering epidemiological data and as an essential legal document. Although it is a mandatory document to be given for all deaths, the quality of its filling is often an ignored aspect and errors are frequently encountered. This documentation process can be mastered with minimal educational efforts. This study aimed to determine the utility of an educational measure in improving the accuracy of death certificate documentation. Methods and materials This pre- and post-interventional study was conducted at Maharaja Agrasen Medical College, Agroha, a tertiary care teaching hospital in Hisar, Haryana, India, wherein an audit of death certificates was done before and after an educational intervention on doctors responsible for filling death certificates. Errors in the death certificates were classified into major and minor errors and compared in the pre- and post-intervention groups. Results A total of 184 pre-intervention and 136 post-intervention death certificates were audited. In the pre-intervention certificates, at least one major and one minor error were present in 88% and 92.93% of the certificates, respectively, which was reduced to 33% (p < 0.01; relative risk (RR) = 3.62; 95% confidence interval (CI) = 2.69-4.91) and 38% (p < 0.01; RR = 3.33; 95% CI = 2.53-4.37), respectively, post-intervention. Reduction in all types of major and minor errors was statistically significant (p < 0.05). Conclusions Errors in death certification are a common but frequently ignored problem that can have a negative impact on epidemiological data and can be drastically reduced with simple educational measures, which need to be carried out regularly.

1,10-phenanthroline inhibits sumoylation and reveals that yeast SUMO modifications are highly transient.

The steady-state levels of protein sumoylation depend on relative rates of conjugation and desumoylation. Whether SUMO modifications are generally long-lasting or short-lived is unknown. Here we show that treating budding yeast cultures with 1,10-phenanthroline abolishes most SUMO conjugations within one minute, without impacting ubiquitination, an analogous post-translational modification. 1,10-phenanthroline inhibits the formation of the E1~SUMO thioester intermediate, demonstrating that it targets the first step in the sumoylation pathway. SUMO conjugations are retained after treatment with 1,10-phenanthroline in yeast that express a defective form of the desumoylase Ulp1, indicating that Ulp1 is responsible for eliminating existing SUMO modifications almost instantly when de novo sumoylation is inhibited. This reveals that SUMO modifications are normally extremely transient because of continuous desumoylation by Ulp1. Supporting our findings, we demonstrate that sumoylation of two specific targets, Sko1 and Tfg1, virtually disappears within one minute of impairing de novo sumoylation. Altogether, we have identified an extremely rapid and potent inhibitor of sumoylation, and our work reveals that SUMO modifications are remarkably short-lived.

IMPETUS Stroke: Assessment of hospital infrastructure and workflow for implementation of uniform stroke care pathway in India.

BACKGROUND: India accounts for 13.3% of global disability-adjusted life years (DALYs) lost due to stroke with a relatively younger age of onset compared to the Western population. In India's public healthcare system, many stroke patients seek care at tertiary-level government-funded medical colleges where an optimal level of stroke care is expected. However, there are no studies from India that have assessed the quality of stroke care, including infrastructure, imaging facilities, or the availability of stroke care units in medical colleges. AIM: This study aimed to understand the existing protocols and management of acute stroke care across 22 medical colleges in India, as part of the baseline assessment of the ongoing IMPETUS stroke study. METHODS: A semi-structured quantitative pre-tested questionnaire, developed based on review of literature and expert discussion, was mailed to 22 participating sites of the IMPETUS stroke study. The questionnaire assessed comprehensively all components of stroke care, including human resources, emergency system, in-hospital care, and secondary prevention. A descriptive analysis of their status was undertaken. RESULTS: In the emergency services, limited stroke helpline numbers, 3/22 (14%); prenotification system, 5/22 (23%); and stroke-trained physicians were available, 6/22 (27%). One-third of hospitals did not have on-call neurologists. Although non-contrast computed tomography (NCCT) was always available, 39% of hospitals were not doing computed tomography (CT) angiography and 13/22 (59%) were not doing magnetic resonance imaging (MRI) after routine working hours. Intravenous thrombolysis was being done in 20/22 (91%) hospitals, but 36% of hospitals did not provide it free of cost. Endovascular therapy was available only in 6/22 (27%) hospitals. The study highlighted the scarcity of multidisciplinary stroke teams, 8/22 (36%), and stroke units, 7/22 (32%). Lifesaving surgeries like hematoma evacuation, 11/22 (50%), and decompressive craniectomy, 9/22 (41%), were performed in limited numbers. The availability of occupational therapists, speech therapists, and cognitive rehabilitation was minimal. CONCLUSION: This study highlighted the current status of acute stroke management in publicly funded tertiary care hospitals. Lack of prenotification, limited number of stroke-trained physicians and neurosurgeons, relatively lesser provision of free thrombolytic agents, limited stroke units, and lack of rehabilitation services are areas needing urgent attention by policymakers and creation of sustainable education models for uniform stroke care by medical professionals across the country.

Unveiling foot tuberculosis as an under-recognized source of foot pain.

PURPOSE: Foot tuberculosis is a rare form of osteoarticular tuberculosis, accounting for less than 1% of cases. It presents unique diagnostic challenges due to its nonspecific clinical features and overlapping symptoms with other conditions. This study aimed to investigate the clinical presentation, radiographic findings, and prognosis of foot tuberculosis, with the goal of improving early recognition and appropriate intervention. METHODS: A prospective study was conducted between November 2016 and July 2021, involving 39 patients diagnosed with foot tuberculosis. Clinical examinations, laboratory tests, X-rays, and MRI evaluations were performed to aid in the diagnosis. Biopsy was conducted on patients with radiological lesions. Patients were treated with an 18-month course of antitubercular therapy (ATT). Foot Function Index (FFI) scores were recorded before and after treatment. Statistical analysis was conducted to assess factors impacting prognosis. RESULTS: Unilateral foot involvement was observed in all patients, with a male predominance (61.5%) and a mean age of 31.3 years. The most common symptoms were pain and edema, with sinus tracts present in 17.9% of patients. Radiographic findings showed cystic and sclerotic lesions, with the "spina ventosa" appearance primarily affecting the metatarsal bones. MRI played a valuable role in early detection. Histopathological examination confirmed tuberculosis in all cases, and acid-fast bacilli were found in 23% of patients. Most patients (79.4%) responded well to ATT without requiring surgery. Factors such as high initial ESR, delayed ATT initiation, multiple lesions, and tarsal involvement were associated with unfavourable outcomes. CONCLUSION: Foot tuberculosis presents with nonspecific symptoms, leading to misdiagnosis and delays in appropriate treatment. Clinical examination, radiographic evaluation, and biopsy are essential for accurate diagnosis. Early initiation of ATT is crucial for favourable outcomes. Factors such as high initial ESR, delayed treatment initiation, multiple lesions, and tarsal involvement negatively impact prognosis. This study highlights the importance of recognizing foot tuberculosis and provides insights into its clinical presentation, radiographic features, and treatment outcomes, facilitating timely intervention and improved patient management.

Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation.

BACKGROUND: Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts. RESULTS: We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes. CONCLUSION: Our results provide an evolutionary perspective on H3.3's conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.

Altered tRNA processing is linked to a distinct and unusual La protein in Tetrahymena thermophila.

Nascent pre-tRNAs are transcribed by RNA polymerase III and immediately bound by La proteins on the UUU-3'OH sequence, using a tandem arrangement of the La motif and an adjacent RNA recognition motif-1 (RRM1), resulting in protection from 3'-exonucleases and promotion of pre-tRNA folding. The Tetrahymena thermophila protein Mlp1 has been previously classified as a genuine La protein, despite the predicted absence of the RRM1. We find that Mlp1 functions as a La protein through binding of pre-tRNAs, and affects pre-tRNA processing in Tetrahymena thermophila and when expressed in fission yeast. However, unlike in other examined eukaryotes, depletion of Mlp1 results in 3'-trailer stabilization. The 3'-trailers in Tetrahymena thermophila are uniquely short relative to other examined eukaryotes, and 5'-leaders have evolved to disfavour pre-tRNA leader/trailer pairing. Our data indicate that this variant Mlp1 architecture is linked to an altered, novel mechanism of tRNA processing in Tetrahymena thermophila.

Serum Magnesium Levels During the Ictal and Interictal Phase in Patients of Migraine: A Prospective Observational Study.

Background: Migraine is one of the primary headaches having a global prevalence of 15%. It is characterized by neurovascular dysfunction and recurrent episodes of headache. The hyperexcitability of the cerebral cortex has been recognized as an important factor in the pathogenesis of migraine, and magnesium (Mg) being a regulator of neuronal excitability is thought to participate in migraine pathogenesis. Objectives: To determine the serum levels of Mg in patients of migraine during the attack and in between attacks as compared to healthy controls. Methods: A total of 50 patients of migraine who fulfilled inclusion criteria were enrolled in the study along with the same number of healthy controls. International Classification of Headache Disorders 3rd Edition, 2013 (ICHD-III) criteria was used for the diagnosis of migraine. Results: The mean serum Mg in migraine cases during the interictal phase was lower than healthy controls (1.849 ± 0.135 vs 2.090 ± 0.205, P < 0.001), which was statistically significant. It was also found that mean serum Mg during attacks was significantly lower than in between attacks (1.822 ± 0.149 vs 1.849 ± 0.135, P = 0.003). Serum Mg levels in migraine cases showed an inverse linear relationship with the frequency of attacks. Conclusion: Relatively low serum Mg in migraine cases when compared with healthy controls and inverse relation of serum Mg levels with the frequency of migraine attacks suggests that Mg is significantly involved in mechanisms underlying migraine pathogenesis, which can be explored as a therapeutic option.

Study Protocol: IMPETUS: Implementing a Uniform Stroke Care Pathway in Medical Colleges of India: IMPETUS Stroke.

Introduction: In India, a national program for stroke (national programme for the control of cardiovascular diseases, diabetes, cancer, and stroke) and stroke management guidelines exist. Its successful implementation would need an organized system of stroke care in practice. However, many challenges exist including lack of awareness, prehospital notification systems, stroke ready hospitals, infrastructural weaknesses, and rehabilitation. We present here a protocol to investigate the feasibility and fidelity of implementing a uniform stroke care pathway in medical colleges of India. Methods and Analysis: This is a multicentric, prospective, multiphase, mixed-method, quasi-experimental implementation study intended to examine the changes in a select set of stroke care-related indicators over time within the sites exposed to the same implementation strategy. We shall conduct process evaluation of the implementation process as well as evaluate the effect of the implementation strategy using the interrupted time series design. During implementation phase, education and training about standard stroke care pathway will be provided to all stakeholders of implementing sites. Patient-level outcomes in the form of modified Rankin Scale score will be collected for all consecutive patients throughout the study. Process evaluation outcomes will be collected and reported in the form of various stroke care indicators. We will report level and trend changes in various indicators during the three study phases. Discussion: Acute stroke requires timely detection, management, and secondary prevention. Implementation of the uniform stroke care pathway is a unique opportunity to promote the requirements of homogenous stroke care in medical colleges of India.

Epstein-Barr virus infection presenting as encephalitis in HIV-Phenomenon not seen frequently.

Epstein-Barr virus (EBV) infection can rarely present as encephalitis in HIV patients. We report a case of a 22-year-old female patient, diagnosed to have HIV infection 8 years back. She presented with headache and altered behavior for a week and focal fits for 2 days. Neurological examination was unremarkable. Cerebrospinal fluid (CSF) examination revealed lymphocytic pleocytosis with raised protein. EBV was detected in CSF using polymerase chain reaction test. Magnetic resonance imaging of the brain revealed T2/fluid-attenuated inversion recovery hyperintensities involving the left frontal cortex, left thalamus, and right medial temporal cortex. The patient was started on antiviral therapy considering the diagnosis of EBV encephalitis. The patient completely recovered over the next few weeks.

Drivers of air pollution variability during second wave of COVID-19 in Delhi, India.

To curb the 2nd wave of COVID-19 disease in April-May 2021, a night curfew followed by full lockdown was imposed over the National Capital Territory, Delhi. We have analyzed the observed variation in pollutants and meteorology, and role of local and transboundary emission sources during night-curfew and lockdown, as compared to pre-lockdown period and identical periods of 2020 lockdown as well as of 2018 and 2019. In 2021, concentration of pollutants (except O3, SO2, and toluene) declined by 4-16% during night-curfew as compared to the pre-lockdown period but these changes are not statistically significant. During lockdown in 2021, various pollutants decreased by 1-28% as compared to the night-curfew (except O3 and PM2.5), but increased by 31-129% compared to the identical period of 2020 lockdown except O3. Advection of pollutants from the region of moderate lockdown restrictions and an abrupt increase in crop-residue burning activity (120-587%) over Haryana and Punjab increased the air pollution levels over NCT during the lockdown period of 2021 as compared to 2020 in addition to a significant contribution of long-range transport. The increase in PM2.5 during the lockdown period of 2021 compared to 2020 might led to 5-29 additional premature mortalities.

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila.

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.

Functional proteomics protocol for the identification of interaction partners in Tetrahymena thermophila.

We describe an optimized protocol for one-step affinity purification of FZZ-tagged proteins followed by mass spectrometry analysis for the identification of protein-protein interactions in the ciliate protozoan Tetrahymena thermophila. The FZZ epitope tag contains 2 protein A moieties (ZZ) and a 3xFLAG separated by a TEV cleavage site, which can also be employed in tandem affinity purification. This protocol is versatile and is suitable to use for other common epitope tags and can be adapted for other ciliates. For complete details on the use and execution of this protocol, please refer to Garg et al. (2019).

Investigations of Huntington's Disease and Huntington's Disease-Like Syndromes in Indian Choreatic Patients.

BACKGROUND: The diagnostic workup for choreiform movement disorders including Huntington's disease (HD) and those mimicking HD like phenotype is complex. OBJECTIVE: The aim of the present study was to genetically define HD and HD-like presentations in an Indian cohort. We also describe HTT-CAG expansion manifesting as neuroferritinopathy-like disorder in four families from Punjab in India. MATERIALS AND METHODS: 159 patients clinically diagnosed as HD and HD-like presentations from various tertiary neurology clinics were referred to our centre (CSIR-IGIB) for genetic investigations. As a first tier test, CAG-TNR for HTT was performed and subsequently HD-negative samples were screened for JPH3 (HDL2), TBP (SCA17), ATN1 (DRPLA), PPP2R2B (SCA12) and GGGGCC expansion in C9orf72 gene. Four families presenting as neuroferritinopathy-like disorder were also investigated for HTT-CAG expansion. RESULTS: 94 of 159 (59%) patients were found to have expanded HTT-CAG repeats. Pathogenic repeat expansion in JPH3, TBP, ATN1 and C9orf72 were not found in HD negative cases. Two patients were positive for SCA12-CAG expansion in pathogenic length, whereas 5 cases harboured TBP-CAG repeats falling in reduced penetrance range of 41- 48 repeats for SCA17. Four unrelated families, presented with atypical chorea and brain MRI findings suggestive of basal ganglia abnormalities mimicking neuroferritinopathy were found to harbour HTT-CAG expansion. CONCLUSION: We present SCA12 as a new reported phenocopy of HD which should be considered for diagnostic workout along with SCA17 for HD-like syndromes. This study also illustrates the necessity, to consider evolving HD like phenotype, as a clinical diagnosis for cases with initial manifestations depicting neuroferritinopathy.

Posterior interosseous nerve entrapment due to bilobed parosteal lipoma.

Entrapment of posterior interosseous nerve (PIN) can be due to fracture dislocation of elbow, fibrous arcade of Frohse, neoplasms (lipoma, schwannoma), ganglion cysts and rheumatoid synovitis. Parosteal lipomas are extremely rare. These tumors grow slowly and as they grow, they can compress a nearby nerve producing sensory and motor disturbances. Till date less than 50 cases of PIN entrapment due to parosteal lipoma have been reported in literature. However, to the best of our knowledge, none was bilobed. A 54-year-old female patient presented with progressive weakness of the right-hand extensors including thumb for the last 5 months with no sensory loss. Clinico-radiological findings and electophysiological studies revealed parosteal lipoma causing entrapment of PIN. Surgical excision of the lesion was done through posterior approach. The excised mass was sent for histopathological examination which confirmed the diagnosis of lipoma. Appreciable recovery was first noticed at 3 months and complete recovery was seen at 7 months. No recurrence was seen until 2 years of follow up. Urgent surgical excision is necessary to prevent entrapment of this nerve and facilitate early functional and neurological recovery.

Nucleus-specific linker histones Hho1 and Mlh1 form distinct protein interactions during growth, starvation and development in Tetrahymena thermophila.

Chromatin organization influences most aspects of gene expression regulation. The linker histone H1, along with the core histones, is a key component of eukaryotic chromatin. Despite its critical roles in chromatin structure and function and gene regulation, studies regarding the H1 protein-protein interaction networks, particularly outside of Opisthokonts, are limited. The nuclear dimorphic ciliate protozoan Tetrahymena thermophila encodes two distinct nucleus-specific linker histones, macronuclear Hho1 and micronuclear Mlh1. We used a comparative proteomics approach to identify the Hho1 and Mlh1 protein-protein interaction networks in Tetrahymena during growth, starvation, and sexual development. Affinity purification followed by mass spectrometry analysis of the Hho1 and Mlh1 proteins revealed a non-overlapping set of co-purifying proteins suggesting that Tetrahymena nucleus-specific linker histones are subject to distinct regulatory pathways. Furthermore, we found that linker histones interact with distinct proteins under the different stages of the Tetrahymena life cycle. Hho1 and Mlh1 co-purified with several Tetrahymena-specific as well as conserved interacting partners involved in chromatin structure and function and other important cellular pathways. Our results suggest that nucleus-specific linker histones might be subject to nucleus-specific regulatory pathways and are dynamically regulated under different stages of the Tetrahymena life cycle.

The Med31 Conserved Component of the Divergent Mediator Complex in Tetrahymena thermophila Participates in Developmental Regulation.

Mediator is a large protein complex required for basal and regulated expression of most RNA polymerase II (RNAP II)-transcribed genes, in part due to its interaction with and phosphorylation of the conserved C-terminal domain (CTD) of Rpb1 [1, 2]. Mediator has been implicated in many aspects of gene expression including chromatin looping [3], higher-order chromatin folding [4], mRNA processing [5] and export [6], and transcriptional memory [7]. Mediator is thought to have played a major role during eukaryotic diversification [8, 9], although its function remains unknown in evolutionarily deep branching eukaryotes lacking canonical CTD heptad repeats. We used the ciliate protozoan Tetrahymena thermophila as a model organism whose genome encodes a highly divergent Rpb1 lacking canonical CTD heptad repeats. We endogenously tagged the Med31 subunit of the Mediator complex and performed affinity purification coupled with mass spectrometry (AP-MS) to identify Mediator subunits. We found that Med31 physically interacts with a large number of proteins (>20), several of which share similarities to canonical Mediator subunits in yeast and humans as well as Tetrahymena-specific proteins. Furthermore, Med31 ChIP-seq analysis suggested a global role for Mediator in transcription regulation. We demonstrated that MED31 knockdown in growing Tetrahymena results in the ectopic expression of developmental genes important for programmed DNA rearrangements. In addition, indirect immunofluorescence revealed Med31 localization in meiotic micronuclei, implicating Mediator in RNAPII-dependent ncRNA transcription. Our results reveal structural and functional insights and implicate Mediator as an ancient cellular machinery for transcription regulation with a possible involvement in global transcription of ncRNAs.

Functional Proteomics of Nuclear Proteins in Tetrahymena thermophila: A Review.

Identification and characterization of protein complexes and interactomes has been essential to the understanding of fundamental nuclear processes including transcription, replication, recombination, and maintenance of genome stability. Despite significant progress in elucidation of nuclear proteomes and interactomes of organisms such as yeast and mammalian systems, progress in other models has lagged. Protists, including the alveolate ciliate protozoa with Tetrahymena thermophila as one of the most studied members of this group, have a unique nuclear biology, and nuclear dimorphism, with structurally and functionally distinct nuclei in a common cytoplasm. These features have been important in providing important insights about numerous fundamental nuclear processes. Here, we review the proteomic approaches that were historically used as well as those currently employed to take advantage of the unique biology of the ciliates, focusing on Tetrahymena, to address important questions and better understand nuclear processes including chromatin biology of eukaryotes.