Epidemiology of COVID-19 and the Utility of Cycle Threshold (Ct) Values in Predicting the Severity of Disease.

OBJECTIVES: Advanced molecular diagnostic methods like real-time polymerase chain reaction (PCR) play a vital role in the early recognition of viral infections, including the coronavirus disease 2019 (COVID-19). Therefore, in the context of the recent COVID-19 pandemic, this study aimed to determine the correlation of cycle threshold (Ct) values with symptoms in COVID-19-positive patients. MATERIALS AND METHODS: A retrospective study was conducted in a virus research diagnostic laboratory (VRDL) at a COVID-19-dedicated tertiary care hospital in South India. A total of 5563 COVID-19-positive patients were analyzed for symptom spectrum and duration of illness with Ct values of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RESULTS: Around 80% (n= 4401) of the patients were symptomatic and the rest were asymptomatic. Among the symptomatic patients, fever (66%) was the most common symptom. About 44% of symptomatic patients had a low Ct value (Ct ≤ 24). There was a significant difference in symptoms among patients with low, medium, and high Ct values. In the subpopulation of symptomatic patients analyzed for the association of Ct value and duration of illness, the mean duration of illness was three days and almost 88% of the patients were tested within five days of onset of symptoms. It has been observed that a shorter duration of illness lowers the Ct values. A significant association was seen between the mean Ct value and days since symptom onset (p-value = 0.016). CONCLUSION: Most of the symptomatic patients had lower Ct values in comparison to the asymptomatic patients. A significant association between low Ct values and the duration of symptoms observed in our study explains the viral dynamics, i.e., higher viral shedding at the onset of symptoms and declines thereafter.

Treatment Response to Diphenylcyclopropenone in Patients with Alopecia Totalis/Universalis.

Introduction: Alopecia totalis (AT) and Alopecia universalis (AU) are forms of Alopecia areata (AA) which represent the strongest predictor of poor prognosis since spontaneous regrowth is <10%. Topical immunotherapy agent, diphenylcyclopropenone (DPCP) has shown clinical efficacy with limited side effects in severe forms of AA. However, its specific role in AT/AU characterized by complete hair loss over the scalp can help highlight the efficacy of the drug with fewer confounders. Methodology: Data were collected from 18 patients diagnosed with AT/AU and treated with topical immunotherapy with DPCP as per protocol by Happle et al. Baseline Severity of Alopecia Tool (SALT) score and subclass was recorded. In the case of AU, baseline body hair loss score was also recorded. Patients were reassessed after 6 months of treatment in terms of change in SALT score and hair regrowth was assessed using the Global Assessment Score. The side effects during treatment were also assessed and recorded. Results: Eighteen patients of whom eleven (61.1%) were diagnosed as AU and seven (38.9%) as AT were treated. The mean age was 21.6, with a male: female ratio of 3:2. The comorbidities noted were atopy in six (33.3%), atopy and hypothyroidism in one (5.5%), Down's syndrome in two (11.1%), and hypothyroidism alone in one (5.5%) patient. The mean duration of disease at the time of presentation was 3 years and all patients had remained refractory to various other modalities of treatment. All patients had a baseline SALT score of 100 corresponding to S5. After 6 months of treatment, 27.7% of patients did not show any response (SALT score S5), 16.6% had a score of S4, 11.1% had a score of S3, 11.1% had a score of S2, 22.2% had a score of S1, and 11.1% had a score of S0. On assessing improvement in body hair loss score, 36.3% of patients showed no improvement, 36.3% showed partial improvement, and 27.2% of patients showed complete body hair regrowth. About 55.5% of patients developed notable side effects that included severe local reactions, cervical lymphadenopathy, acne and pigmentation at the site of application as well as untreated sites. Conclusion: The AT/AU subtypes of AA, was amenable to treatment with contact immunotherapeutic agent DPCP with a >75% hair regrowth in 33.3% of patients. The castling phenomenon was seen in 63.6% of AU patients. The adverse effects noted were not severe enough to deter treatment.

A Clinical Study of Premature Canities and Its Association with Hemoglobin, Ferritin and Calcium Levels.

Background and Objectives: Greying of hair is a regular feature of chronological aging that occurs in all regions and races. Premature canities is defined as minimum of five grey hairs in a person less than 20 years in Whites, 25 years in Asians, and 30 years in Africans. Premature canities is a common yet incompletely understood dermatological entity. This study aims at finding any association between premature hair greying (PHG) and parameters like hemoglobin (Hb), ferritin and calcium levels as well as its clinical profile. Methodology: This was a hospital-based cross-sectional analytical study conducted in the Department of Dermatology and Venereology, Trivandrum over one and half years. The study population consisted of 40 cases and 40 controls. Severity assessment, calculation of body mass index, and estimation of hemoglobin, serum ferritin, calcium, Random blood sugar, Anti Thyroid Peroxidase antibody, T3, T4, and TSH were done. Results: The mean age of the 40 patients was 17.14 years and most patients had onset of greying between 16 and 20 years. The male to female ratio was 1.2:1. A positive family history with a paternal predominance was noted. Vertex was the most common site of onset (42.5%), diffuse pattern was the most common clinical pattern (47.5%) and 60% had involvement of mild grade. Fourteen patients (35%) had abnormal investigations reports, in terms of low ferritin levels in 7 (17.5%), low calcium in 4 (10%) and a low Hb levels in 3 (7.5%) patients. Six (15%) patients had raised Anti TPO values. The association of PHG with low ferritin and raised anti-TPO levels were statistically significant. Conclusion: Low serum ferritin and raised Anti TPO levels may have a role in premature hair greying.

Publisher Correction: Structural Implications of Mutations Conferring Rifampin Resistance in Mycobacterium leprae.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Structural Implications of Mutations Conferring Rifampin Resistance in Mycobacterium leprae.

The rpoB gene encodes the ß subunit of RNA polymerase holoenzyme in Mycobacterium leprae (M. leprae). Missense mutations in the rpoB gene were identified as etiological factors for rifampin resistance in leprosy. In the present study, we identified mutations corresponding to rifampin resistance in relapsed leprosy cases from three hospitals in southern India which treat leprosy patients. DNA was extracted from skin biopsies of 35 relapse/multidrug therapy non-respondent leprosy cases, and PCR was performed to amplify the 276 bp rifampin resistance-determining region of the rpoB gene. PCR products were sequenced, and mutations were identified in four out of the 35 cases at codon positions D441Y, D441V, S437L and H476R. The structural and functional effects of these mutations were assessed in the context of three-dimensional comparative models of wild-type and mutant M. leprae RNA polymerase holoenzyme (RNAP), based on the recently solved crystal structures of RNAP of Mycobacterium tuberculosis, containing a synthetic nucleic acid scaffold and rifampin. The resistance mutations were observed to alter the hydrogen-bonding and hydrophobic interactions of rifampin and the 5' ribonucleotide of the growing RNA transcript. This study demonstrates that rifampin-resistant strains of M. leprae among leprosy patients in southern India are likely to arise from mutations that affect the drug-binding site and stability of RNAP.

Kinetochore Components Required for Centromeric Chromatin Assembly Are Impacted by Msc1 in Schizosaccharomyces pombe.

Eukaryotic chromosome segregation requires a protein complex known as the kinetochore that mediates attachment between mitotic spindle microtubules and centromere-specific nucleosomes composed of the widely conserved histone variant CENP-A. Mutations in kinetochore proteins of the fission yeast Schizosaccharomyces pombe lead to chromosome missegregation such that daughter cells emerge from mitosis with unequal DNA content. We find that multiple copies of Msc1-a fission yeast homolog of the KDM5 family of proteins-suppresses the temperature-sensitive growth defect of several kinetochore mutants, including mis16 and mis18, as well as mis6, mis15, and mis17, components of the Constitutive Centromere Associated Network (CCAN). On the other hand, deletion of msc1 exacerbates both the growth defect and chromosome missegregation phenotype of each of these mutants. The C-terminal PHD domains of Msc1, previously shown to associate with a histone deacetylase activity, are necessary for Msc1 function when kinetochore mutants are compromised. We also demonstrate that, in the absence of Msc1, the frequency of localization to the kinetochore of Mis16 and Mis15 is altered from wild-type cells. As we show here for msc1, others have shown that elevating cnp1 levels acts similarly to promote survival of the CCAN mutants. The rescue of mis15 and mis17 by cnp1 is, however, independent of msc1 Thus, Msc1 appears to contribute to the chromatin environment at the centromere: the absence of Msc1 sensitizes cells to perturbations in kinetochore function, while elevating Msc1 overcomes loss of function of critical components of the kinetochore and centromere.

Novel cell-based assay for detection of thyroid receptor beta-interacting environmental contaminants.

Even though the presence of endocrine disrupting chemicals (EDCs) with thyroid hormone (TH)-like activities in the environment is a major health concern, the methods for their efficient detection and monitoring are still limited. Here we describe a novel cell assay, based on the translocation of a green fluorescent protein (GFP)-tagged chimeric molecule of glucocorticoid receptor (GR) and the thyroid receptor beta (TRß) from the cytoplasm to the nucleus in the presence of TR ligands. Unlike the constitutively nuclear TRß, this GFP-GR-TRß chimera is cytoplasmic in the absence of hormone while translocating to the nucleus in a time- and concentration-dependent manner upon stimulation with triiodothyronine (T3) and thyroid hormone analogue, TRIAC, while the reverse triiodothyronine (3,3',5'-triiodothyronine, or rT3) was inactive. Moreover, GFP-GR-TRß chimera does not show any cross-reactivity with the GR-activating hormones, thus providing a clean system for the screening of TR beta-interacting EDCs. Using this assay, we demonstrated that Bisphenol A (BPA) and 3,3',5,5'-Tetrabromobisphenol (TBBPA) induced GFP-GR-TRß translocation at micro molar concentrations. We screened over 100 concentrated water samples from different geographic locations in the United States and detected a low, but reproducible contamination in 53% of the samples. This system provides a novel high-throughput approach for screening for endocrine disrupting chemicals (EDCs) interacting with TR beta.

Microtubule dynamics decoded by the epigenetic state of centromeric chromatin.

Cell division with accurate chromosome segregation is fundamental to cell survival of all organisms. The precise molecular mechanisms that ensure accurate chromosome segregation are still being discovered using a variety of experimental systems and approaches. Microtubule attachment to the kinetochore is a prerequisite for mitotic progression, failure of which activates the spindle assembly checkpoint (SAC). The dynamic tension generated by interaction of the centromere, kinetochore and microtubules is a key regulator of the SAC. Here, in the context of current literature we discuss our recent observation in fission yeast that epigenetic alterations in centromeric and pericentromeric chromatin can compensate for altered dynamics of kinetochore-microtubule attachment to permit escape from mitotic arrest. A role for the spatial configuration of the centromere to influence the finely tuned regulators of mitotic progression opens up new avenues for research.

Escape from Mitotic Arrest: An Unexpected Connection Between Microtubule Dynamics and Epigenetic Regulation of Centromeric Chromatin in Schizosaccharomyces pombe.

Accurate chromosome segregation is necessary to ensure genomic integrity. Segregation depends on the proper functioning of the centromere, kinetochore, and mitotic spindle microtubules and is monitored by the spindle assembly checkpoint (SAC). In the fission yeast Schizosaccharomyces pombe, defects in Dis1, a microtubule-associated protein that influences microtubule dynamics, lead to mitotic arrest as a result of an active SAC and consequent failure to grow at low temperature. In a mutant dis1 background (dis1-288), loss of function of Msc1, a fission yeast homolog of the KDM5 family of proteins, suppresses the growth defect and promotes normal mitosis. Genetic analysis implicates a histone deacetylase (HDAC)-linked pathway in suppression because HDAC mutants clr6-1, clr3∆, and sir2∆, though not hos2∆, also promote normal mitosis in the dis1-288 mutant. Suppression of the dis phenotype through loss of msc1 function requires the spindle checkpoint protein Mad2 and is limited by the presence of the heterochromatin-associated HP1 protein homolog Swi6. We speculate that alterations in histone acetylation promote a centromeric chromatin environment that compensates for compromised dis1 function by allowing for successful kinetochore-microtubule interactions that can satisfy the SAC. In cells arrested in mitosis by mutation of dis1, loss of function of epigenetic determinants such as Msc1 or specific HDACs can promote cell survival. Because the KDM5 family of proteins has been implicated in human cancers, an appreciation of the potential role of this family of proteins in chromosome segregation is warranted.

Identification of compounds by high-content screening that induce cytoplasmic to nuclear localization of a fluorescent estrogen receptor α chimera and exhibit agonist or antagonist activity in vitro.

We have completed a robust high-content imaging screen for novel estrogen receptor α (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen was very robust, with Z' values >0.7 and coefficients of variation (CV) <5%. The screen utilized a stably transfected green fluorescent protein-tagged glucocorticoid/estrogen receptor (GFP-GRER) chimera, which consisted of the N-terminus of the glucocorticoid receptor fused to the human ERα ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands and translocated to the nucleus in response to stimulation with ERα agonists and antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. We screened 224,891 samples from our synthetic, pure natural product libraries, prefractionated natural product extracts library, and crude natural product extracts library, which produced a 0.003% hit rate. In addition to identifying several known ER ligands, five compounds were discovered that elicited significant activity in the screen. Transactivation potential studies demonstrated that two hit compounds behave as agonists, while three compounds elicited antagonist activity in MCF-7 cells.

Prevalent glucocorticoid and androgen activity in US water sources.

Contamination of the environment with endocrine disrupting chemicals (EDCs) is a major health concern. The presence of estrogenic compounds in water and their deleterious effect are well documented. However, detection and monitoring of other classes of EDCs is limited. Here we utilize a high-throughput live cell assay based on sub-cellular relocalization of GFP-tagged glucocorticoid and androgen receptors (GFP-GR and GFP-AR), in combination with gene transcription analysis, to screen for glucocorticoid and androgen activity in water samples. We report previously unrecognized glucocorticoid activity in 27%, and androgen activity in 35% of tested water sources from 14 states in the US. Steroids of both classes impact body development, metabolism, and interfere with reproductive, endocrine, and immune systems. This prevalent contamination could negatively affect wildlife and human populations.

Dynamic access of the glucocorticoid receptor to response elements in chromatin.

Transcriptional activation as a rate-limiting step of gene expression is often triggered by an environmental stimulus that is transmitted through a signaling cascade to specific transcription factors. Transcription factors must then find appropriate target genes in the context of chromatin. Subsequent modulation of local chromatin domains is now recognized as a major mechanism of gene regulation. The interactions of transcription factors with chromatin structures have recently been observed to be highly dynamic, with residence times measured in seconds. Thus, the concept of static, multi-protein complexes forming at regulatory elements in the genome has been replaced by a new paradigm that envisages rapid and continuous exchange events with the template. These highly dynamic interactions are a property of both DNA-protein and protein-protein interactions and are inherent to every stage of the transcriptional response. In this review we discuss the dynamics of a nuclear receptor, and its transcriptional response in the chromatin context.

Regulation of transcript elongation through cooperative and ordered recruitment of cofactors.

We studied the regulation of murine CD80, a gene whose basal transcriptional status was characterized by the presence of a stalled RNA polymerase II complex on the promoter-proximal region. Stimulus-induced activation of productive elongation involved a complex interplay of regulated events that included a synergy between ordered cofactor recruitment. This cascade of recruitments was initiated through the engagement of transcription factor NF-kappaB, leading to the temporal association of histone acetyltransferases and the consequent selective acetylation of a transcription start site downstream nucleosome. This in turn culminated into the nucleosomal association of Brd4-associated P-TEFb, a protein complex containing kinase specific for serine 2 of Rbp 1, the largest subunit of the carboxyl-terminal domain of RNA polymerase II. The consequent phosphorylation of serine 2 residues in CTD by CDK9 in the P-TEFb complex then facilitated escape of polymerase II into the productive elongation phase. Thus, the cooperative mechanisms that integrate between independent pathways characterize regulation of the elongation step of transcription, thereby providing another level at which specificity of gene regulation can be achieved.

Sinonasal carcinoma masquerading as fungal sinusitis.

A 26-year-old man presented with swelling of his face and nose of three months duration. He had multiple hyperpigmented, hyperkeratotic plaques over the swelling, of one-month duration and an erythematous indurated plaque below the left nostril for two weeks. Based on a biopsy taken from the antral mass with special stain for fungus, he was treated as fungal sinusitis with intravenous amphotericin-B, but the lesion did not regress. Later a skin biopsy from the indurated lesion showed moderately differentiated squamous cell carcinoma. A diagnosis of sinonasal carcinoma was made and chemotherapy was started.

Transcription regulation from a TATA and INR-less promoter: spatial segregation of promoter function.

The mode of regulation of class II genes that lack the known core promoter elements is presently unclear. Here, we studied one such example, the murine CD80 gene. An unusual mechanism was revealed wherein the pre-initiation complex (PIC) first assembled on an upstream, NF-kappaB enhancer element. Notably, this assembly occurred independent of contributions from the core promoter domain, and resulted in a PIC that was competent for transcription initiation. Positioning was subsequently achieved by exploiting the intrinsic architecture of the promoter, by virtue of which the tethered PIC was spatially juxtaposed with the transcription initiation site. Bridging interactions then ensued, through protein-protein contacts, which then enabled the elongation phase of CD80 transcription.