Stability Indicating Method Development and Validation for the Estimation of Bempedoic Acid by RP-HPLC.

BACKGROUND: Bempedoic acid (BEM) belongs to a category of drugs known as Adenosine triphosphate-citrate Lyase (ACL) inhibitors. It is a prodrug with intracellular activation that is administered orally. Bempedoic acid is used to treat existing atherosclerotic cardiovascular diseases, mainly hypercholesterolemia. METHODS: For the stability-indicating assay, the HPLC method was employed using a Kromasil 100-5-C8 column (100 mm × 4.6 mm), a UV detector set at 230 nm, and a mobile phase comprising a 70:30 v/v mixture of acetonitrile and 0.1% Orthophosphoric Acid (OPA) buffer. The method was operated at an ambient temperature with a flow rate of 1 mL/min. The method developed has been statistically validated according to ICH guidelines. RESULTS: The stability-indicating method was executed using a Kromasil 100-5-C8 (100 mm × 4.6 mm) column at a 1.0 mL/min flow rate. A mixture of acetonitrile and 0.1% Orthophosphoric Acid (OPA) buffer in a 70:30 v/v ratio made up the mobile phase. BEM's retention times were discovered to be 1.88 minutes each. The temperature was kept at room temperature. 234 nm was the ideal wavelength for BEM. According to ICH criteria, the approach developed has undergone statistical validation. BEM's % RSD was discovered to be 0.6, respectively. For BEM, the % recovery was determined to be 100.0%. Regression models for bempedoic acid yielded LoD and LoQ values of 3.3 and 10.1 g/mL, respectively. The method showed good reproducibility and recovery with a % RSD less than 2. Studies on forced degradation confirmed the method's capacity to indicate stability in the presence of stress conditions, such as acid, basic, peroxide, UV, heat, and humidity. Both the retention times and the run time were shortened. CONCLUSION: In accordance with ICH Q2 (R1) guidelines, this method was successfully tested with HPLC to confirm the chemical structures of newly produced degradation products of bempedoic acid.

Appearance and Prevalence of JN.1 SARS-CoV-2 Variant in India and Its Clinical Profile in the State of Maharashtra.

BACKGROUND: In August 2023, the BA.2.86 SARS-CoV-2 variant, with over 30 spike protein mutations, emerged amidst the global dominance of XBB sub-lineages. It evolved into JN.1 by late 2023, spreading across 71 countries. JN.1, distinct for its L455S mutation, significantly dominated global sequences, raising concerns over its transmission and clinical impact. The study investigates JN.1's clinical severity and its effect on hospital admissions in Maharashtra, India. METHODOLOGY: The present study involved 3,150 curated Indian SARS-CoV-2 whole genome sequences with collection dates between 1st August 2023 and 15th January 2024. Lineage and phylogenetic analysis of sequences was performed using Nextclade. Telephonic interviews were conducted to confirm the demographic details and obtain clinical information on the JN.1* (* indicates JN.1 and all its sub-lineages) cases. The obtained data were recorded and analyzed using Microsoft® Excel (Microsoft Corporation, Redmond, WA). RESULTS: Out of 3,150 sequences analyzed, JN.1* was the most common lineage (2377/3150, 75.46%), followed by XBB.2.3* (281/3150, 8.92%) and XBB.1.16* (187/3150, 5.94%). In India, it was first identified on 6th October 2023, in Kerala. The highest proportion of JN.1* sequences originated from Maharashtra (628/2377, 26.42%), followed by West Bengal (320/2377, 13.46%), Andhra Pradesh (293/2377, 12.33%), Kerala (288/2377, 12.12%), and Karnataka (285/2377, 11.99%). In Maharashtra, the JN.1* variant was first identified on 23rd November 2023. A total of 279 JN.1* cases were included in the clinical study. Of these, 95.34% (266/279) had symptomatic disease with mild symptoms; cold (187/279, 67.03%) being the most common symptom, followed by fever (156/279, 55.91%), cough (114/279, 40.86%), and headache (28/279, 15.64%). Of all the cases, 13.26% (37/279) required institutional quarantine or hospitalization, and the rest were isolated at home. Among the hospitalized patients, 54.05% (20/37) cases were given conservative treatment while 45.95% (17/37) cases required supplemental oxygen therapy. Regarding the vaccination status, 94.26% (263/279) of cases received at least one dose of the COVID-19 vaccine, while 5.02% (14/279) were not vaccinated, of which most were children aged zero to nine years (5/14, 35.71%). The overall recovery rate among JN.1* cases was 98.57% (275/279), with 1.43% (4/279) cases succumbing to the disease. CONCLUSION: The JN.1* variant, the dominant variant in India, exhibits clinical features similar to previous circulating variants in Maharashtra without increased severity. Its notable transmissibility underscores the importance of studying the ongoing viral evolution. The pressing necessity for swift identification and the clinical features of new variants is essential for effective public health response.

The Burden and Characteristics of Post-COVID-19 Conditions Among Laboratory-Confirmed Delta and Omicron COVID-19 Cases: A Preliminary Study From Maharashtra, India.

BACKGROUND: Post-COVID-19 conditions (PCC) have emerged as a significant global health concern due to their potential impact on patients' quality of life and healthcare resources. The present study aims to understand the burden and characteristics of PCC in Maharashtra, India, and compares its prevalence among cases infected with Delta and Omicron variants. MATERIAL AND METHODS: A retrospective observational study included 617 laboratory-confirmed Delta and Omicron variant cases. These cases were telephonically followed up to document persistent COVID-19 symptoms using a questionnaire based on the Post-COVID-19 Clinical Form from the Global COVID-19 Clinical Platform of the World Health Organization (WHO), and the results were analyzed. RESULTS: Out of 617 laboratory-confirmed COVID-19 cases, 82.97% and 17.03% were Omicron and Delta cases, respectively. The mean follow-up period for Delta and Omicron cases was 78.05 and 21.56 weeks, respectively. A total of 40 (6.48%) cases reported persistent symptoms at follow-up, with a higher prevalence among those infected with the Delta variant (12.38%) compared to the Omicron variant (5.27%). The most common long COVID symptoms reported were malaise (25%), dyspnea (20%), post-exertional fatigue (17.5%), joint pain (15%), and frequent episodes of cough and cold (15%). Additionally, 1.94% of participants developed a new medical condition following COVID-19 infection, most commonly hypertension (25%), lung fibrosis (16.67%), and asthma (8.33%). Factors such as more than five acute symptoms, a moderate to severe disease, the need for hospitalization, and hospitalization for more than five days were significantly associated with PCC. CONCLUSION: Long COVID results in extended disability and illness. The varying impacts of different COVID-19 variants highlight the complex nature of post-COVID-19 complications. Our findings highlight the need for strategic planning of healthcare resources to ensure optimal response and preparedness to manage the burden of PCC.

Chasing SARS-CoV-2 XBB.1.16 Recombinant Lineage in India and the Clinical Profile of XBB.1.16 Cases in Maharashtra, India.

Background SARS-CoV-2 has evolved rapidly, resulting in the emergence of lineages with a competitive advantage over one another. Co-infections with different SARS-CoV-2 lineages can give rise to recombinant lineages. To date, the XBB lineage is the most widespread recombinant lineage worldwide, with the recently named XBB.1.16 lineage causing a surge in the number of COVID-19 cases in India. Methodology The present study involved retrieval of SARS-CoV-2 genome sequences from India (between December 1, 2022 and April 8, 2023) through GISAID; sequences were curated, followed by lineage and phylogenetic analysis. Demographic and clinical data from Maharashtra, India were collected telephonically, recorded in Microsoft® Excel, and analyzed using IBM® SPSS statistics, version 29.0.0.0 (241). Results A total of 2,944 sequences were downloaded from the GISAID database, of which 2,856 were included in the study following data curation. The sequences from India were dominated by the XBB.1.16* lineage (36.17%) followed by XBB.2.3* (12.11%) and XBB.1.5* (10.36%). Of the 2,856 cases, 693 were from Maharashtra; 386 of these were included in the clinical study. The clinical features of COVID-19 cases with XBB.1.16* infection (XBB.1.16* cases, 276 in number) showed that 92% of those had a symptomatic disease, with fever (67%), cough (42%), rhinorrhea (33.7%), body ache (14.5%) and fatigue (14.1%) being the most common symptoms. The presence of comorbidity was found in 17.7% of the XBB.1.16* cases. Among the XBB.1.16* cases, 91.7% were vaccinated with at least one dose of vaccine against COVID-19. While 74.3% of XBB.1.16* cases were home-isolated; 25.7% needed hospitalization/institutional quarantine, of these, 33.8% needed oxygen therapy. Out of 276 XBB.1.16* cases, seven (2.5%) cases succumbed to the disease. The majority of XBB.1.16* cases who died belonged to an elderly age group (60 years and above), had underlying comorbid condition/s, and needed supplemental oxygen therapy. The clinical features of COVID-19 cases infected with other co-circulating Omicron variants were similar to XBB.1.16* cases. Conclusion The study reveals that XBB.1.16* lineage has become the most predominant SARS-CoV-2 lineage in India. The study also shows that the clinical features and outcome of XBB.1.16* cases were similar to those of other co-circulating Omicron lineage infected cases in Maharashtra, India.

Clinical Characteristics and Outcomes of Laboratory-Confirmed SARS-CoV-2 Cases Infected With Omicron Subvariants and the XBB Recombinant Variant.

Background SARS-CoV-2 has evolved to produce new variants causing successive waves of infection. Currently, six variants are being monitored by the World Health Organization that are replacing BA.5. These include BF.7 (BA.5 + R346T in spike), BQ.1 (and BQ.1.1, with BA.5 + R346T, K444T, N460K mutations in spike), BA.2.75 (including BA.2.75.2 and CH.1.1), and XBB (including XBB.1.5). BQ.1 and XBB variants are more immune evasive and have spread quickly throughout the world. Concerning the potential severity of infections caused by these variants, the present study describes the clinical characteristics and outcomes of these major variants in Maharashtra. Methodology A total of 1,141 reverse transcriptase-polymerase chain reaction (RT-PCR)-positive SARS-CoV-2 samples, with a cycle threshold (Ct) value of less than 25, were processed for SARS-CoV-2 whole genome sequencing between July 10, 2022, and January 12, 2023. All corresponding demographic and clinical data were recorded and analyzed using Microsoft® Excel and Epi Info™. Results Out of the 1,141 samples sequenced, BA.2.75* (63.78%) was the predominant Omicron variant, followed by the XBB* (18.88%), BA.2.38* (4.94%), BA.5* (4.06%), BA.2.10* (3.51%), and BQ.1* (1.65%). A total of 540 cases were contacted telephonically, of whom 494 (91.48%) were symptomatic with mild symptoms. Fever (77.73%) was the most common symptom, followed by cold (47.98%), cough (42.31%), and myalgia and fatigue (18.83%). Of the 540 cases, 414 (76.67%) cases recovered at home, and 126 (23.33%) were institutionally quarantined/hospitalized. Among the home-isolated and hospitalized cases, 416 (99.76%) and 108 (87.80%), respectively, recovered with symptomatic treatment, while one (0.24%) and 15 (12.20%), respectively, succumbed to the disease. Out of the 540 cases, 491 (90.93%) were vaccinated with at least one dose of the COVID-19 vaccine, 41 (7.59%) were unvaccinated, and for eight (1.48%) cases, vaccination data was not available. Conclusions The current study indicates that the XBB* variant is causing mild disease in India. However, as XBB* possesses both immune-escape and infectivity-enhancing mutations, it has the potential to spread to other parts of the world rapidly. Further, anti-SARS-CoV-2 vaccination improves survival rates in COVID-19.

Unraveling the Link: A Comprehensive Literature Review of Type 2 Diabetes and Menopause Onset.

Women with diabetes mellitus (DM), a metabolic endocrine illness, may experience a variety of reproductive problems. The age at menopause onset has been extensively studied as a major predictor of women's health in the future; however, its relationship to diabetes in Indian women has received less attention. This literature review looked at the consequences of diabetes in women as well as the association between diabetes and the age at which menopause begins. The average age at menopause onset among women with type 2 diabetes mellitus (T2DM) has decreased globally. According to one Indian study, the average menopause age dropped to 45 years for 26% of women with T2DM. In the current review, 10 studies indicated that women with T2DM displayed an imbalanced hormonal profile resulting in an extended anovulatory period. Two investigations highlighted the significance of altered body composition of women with T2DM, thereby suggesting obesity as the primary risk factor of ovarian aging and early climacteric symptoms. T2DM may lower the average age at menopause onset; however, further research on Indian women is necessary. There is a need of studies on T2DM in premenopausal women are needed to demonstrate how the changes in body composition impact the age at which menopause begins. Delaying the onset of menopause in women with T2DM necessitates diet and lifestyle interventions to minimize ovarian aging and hormonal imbalance.

Predictors of iron deficiency anaemia among children aged 6-59 months in Tanzania: Evidence from the 2015-16 TDHS-MIS cross-sectional household survey.

Iron deficiency anaemia remains a public health problem, particularly in children aged 6-59 months. This study assessed factors associated with iron deficiency anaemia among children aged 6-23 months, 24-59 months and 6-59 months in Tanzania. Data for this cross-sectional study were extracted from the 2015-16 Tanzania Demographic and Health Survey and Malaria Indicator Survey (2015-16 TDHS-MIS). The study covered 8014 children aged 6-59 months and their mothers. Iron deficiency anaemia was defined (haemoglobin < 11g/dL). Univariable and multivariable logistic regression analyses that adjust for clustering and sampling weights were conducted to describe the associations between anaemia and potential confounding variables. The prevalence of iron deficiency anaemia among children aged 6-23 months, 24-59 months and 6-59 months were 76%, 49% and 59%, respectively. Factors associated with increased odds of iron deficiency anaemia among children aged 6-23 months included a mother being employed, being a male child, child perceived to small size at birth by mothers, a mother being anaemic and children belonging to the poorest socio-economic quintile. In addition, being a mother with no schooling, children not being dewormed, a mother being anaemic, delivering a baby at home, child fever and stunting, were factors associated with increased odds of iron deficiency anaemia among children aged 24-59 months. Factors associated with increased odds of iron deficiency anaemia among children aged 6-59 months were: a mother being employed, being a mother with no schooling, being a male child, belonging to the 6-59 months age bracket, a mother having a BMI of between 19 and 25 kg/m2, a mother being anaemic, having a baby at home, children belonging to bigger households, child fever and stunting. Interventions to minimise the burden of iron deficiency anaemia in children should target employed and/or anaemic mothers, poor and rich households, as well as male children.

Temporal Proteomic Profiling During Differentiation of Normal and Dystrophin-Deficient Human Muscle Cells.

BACKGROUND: Myogenesis is a dynamic process involving temporal changes in the expression of many genes. Lack of dystrophin protein such as in Duchenne muscular dystrophy might alter the natural course of gene expression dynamics during myogenesis. OBJECTIVE: To gain insight into the dynamic temporal changes in protein expression during differentiation of normal and dystrophin deficient myoblasts to myotubes. METHOD: A super SILAC spike-in strategy in combination and LC-MS/MS was used for temporal proteome profiling of normal and dystrophin deficient myoblasts during differentiation. The acquired data was analyzed using Proteome Discoverer 2.2. and data clustering using R to define significant temporal changes in protein expression. RESULTS: sFour major temporal protein clusters that showed sequential dynamic expression profiles during myogenesis of normal myoblasts were identified. Clusters 1 and 2, consisting mainly of proteins involved mRNA splicing and processing expression, were elevated at days 0 and 0.5 of differentiation then gradually decreased by day 7 of differentiation, then remained lower thereafter. Cluster 3 consisted of proteins involved contractile muscle and actomyosin organization. They increased in their expression reaching maximum at day 7 of differentiation then stabilized thereafter. Cluster 4 consisting of proteins involved in skeletal muscle development glucogenesis and extracellular remodeling had a lower expression during myoblast stage then gradually increased in their expression to reach a maximum at days 11-15 of differentiation. Lack of dystrophin expression in DMD muscle myoblast caused major alteration in temporal expression of proteins involved in cell adhesion, cytoskeleton, and organelle organization as well as the ubiquitination machinery. CONCLUSION: Time series proteome profiling using super SILAC strategy is a powerful method to assess temporal changes in protein expression during myogenesis and to define the downstream consequences of lack of dystrophin on these temporal protein expressions. Key alterations were identified in dystrophin deficient myoblast differentiation compared to normal myoblasts. These alterations could be an attractive therapeutic target.

Validation of Chemokine Biomarkers in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease involving complex skeletal muscle pathogenesis. The pathogenesis is triggered by sarcolemma instability due to the lack of dystrophin protein expression, leading to Ca2+ influx, muscle fiber apoptosis, inflammation, muscle necrosis, and fibrosis. Our lab recently used two high-throughput multiplexing techniques (e.g., SomaScan® aptamer assay and tandem mass tag-(TMT) approach) and identified a series of serum protein biomarkers tied to different pathobiochemical pathways. In this study, we focused on validating the circulating levels of three proinflammatory chemokines (CCL2, CXCL10, and CCL18) that are believed to be involved in an early stage of muscle pathogenesis. We used highly specific and reproducible MSD ELISA assays and examined the association of these chemokines with DMD pathogenesis, age, disease severity, and response to glucocorticoid treatment. As expected, we confirmed that these three chemokines were significantly elevated in serum and muscle samples of DMD patients relative to age-matched healthy controls (p-value < 0.05, CCL18 was not significantly altered in muscle samples). These three chemokines were not significantly elevated in Becker muscular dystrophy (BMD) patients, a milder form of dystrophinopathy, when compared in a one-way ANOVA to a control group but remained significantly elevated in the age-matched DMD group (p < 0.05). CCL2 and CCL18 but not CXCL10 declined with age in DMD patients, whereas all three chemokines remained unchanged with age in BMD and controls. Only CCL2 showed significant association with time to climb four steps in the DMD group (r = 0.48, p = 0.038) and neared significant association with patients' reported outcome in the BMD group (r = 0.39, p = 0.058). Furthermore, CCL2 was found to be elevated in a serum of the mdx mouse model of DMD, relative to wild-type mouse model. This study suggests that CCL2 might be a suitable candidate biomarker for follow-up studies to demonstrate its physiological significance and clinical utility in DMD.

Absolute quantification of dystrophin protein in human muscle biopsies using parallel reaction monitoring (PRM).

The need for a reliable and accurate method to quantify dystrophin proteins in human skeletal muscle biopsies has become crucial in order to assess the efficacy of dystrophin replacement therapies in Duchenne muscular dystrophy as well as to gain insight into the relationship between dystrophin levels and disease severity in Becker's muscular dystrophy. Current methods to measure dystrophin such as western blot and immunofluorescence, while straightforward and simple, lack precision and sometimes specificity. Here, we standardized a targeted mass spectrometry method to determine the absolute amount of dystrophin in ng/mg of muscle using full-length 13 C6-Arg- and 13 C6,15 N2-Lys-labeled dystrophin and parallel reaction monitoring (PRM). The method was found to be reproducible with a limit of quantification as low as 30 pg of dystrophin protein per mg of total muscle proteins. The method was then tested to measure levels of dystrophin in muscle biopsies from a healthy donor and from Duchenne and Becker's muscular dystrophy patients.

Raman and Mossbauer spectroscopy and X-ray diffractometry studies on quenched copper-ferri-aluminates.

Four spinel ferrite compositions of the CuAl(x)Fe(2-x)O4, x = 0.0, 0.2, 0.4, 0.6, system prepared by usual double-sintering ceramic route and quenched (rapid thermal cooling) from final sintering temperature (1373 K) to liquid nitrogen temperature (80 K) were investigated by employing X-ray powder diffractometry, (57)Fe Mossbauer spectroscopy, and micro-Raman spectroscopy at 300 K. The Raman spectra collected in the wavenumber range of 100-1000 cm(-1) were analyzed in a systematic manner and showed five predicted modes for the spinel structure and splitting of A1g Raman mode into two/three energy values, attributed to peaks belonging to each ion (Cu(2+), Fe(3+), and Al(3+)) in the tetrahedral positions. The suppression of lower-frequency peaks was explained on the basis of weakening in magnetic coupling and reduction in ferrimagnetic behavior as well as increase in stress induced by square bond formation on Al(3+) substitution. The enhancement in intensity, random variation of line width, and blue shift for highest frequency peak corresponding to A1g mode were observed. The ferric ion (Fe(3+)) concentration for different compositions determined from Raman spectral analysis agrees well with that deduced by means of X-ray diffraction line-intensity calculations and Mossbauer spectral analysis. An attempt was made to determine elastic and thermodynamic properties from Raman spectral analysis and elastic constants from cation distribution.

No effect of atypical antipsychotic drugs on weight gain and risk of developing type II diabetes or lipid abnormalities among nursing home elderly patients with Alzheimer's disease.

AIM: Weight gain and the risk of developing alterations in lipid and glucose metabolism are possible side effects of atypical antipsychotic therapy in young and adult patients. The objective of this study was to examine whether elderly patients with Alzheimer's disease (AD) gain weight or develop disturbances in lipid and glucose metabolism while being treated with atypical antipsychotic drugs. METHODS: This retrospective study identified 36 out of 99 patients (mean age: 75.4+/-7.1, 27 female, 9 males) who were taking risperidone (N=9, mean dosage: 1.42+/-0.49 mg/day), olanzapine (N=17: 4.42+/-1.10 mg/day), and quetiapine (N=10: 75+/-27 mg/day) over a 12 months period. Anthropometric parameters, mini nutritional assessment (MNA), total, HDL and LDL cholesterol, triglycerides, glycaemia were assessed at baseline (T0) and 12 (T1) months. RESULTS: Body weight (BMI=23+/-5 vs 23+/-5), MNA score (21+/-4 vs 21+/-4), blood glucose (5.7+/-2 vs 4.9+/-0.9 mmol/L) or total cholesterol (4.9+/-1.1 vs 4.3+/-0.7 mmol/L), HDL cholesterol (1.3+/-0.3 vs 1.1+/-0.3 mmol/L), LDL cholesterol (3.3+/-0.7 vs 3 +/- 0.4 mmol/L), triglycerides (1.1+/-0 vs 1+/-0.3 mmol/L) did not reveal treatment-induced changes in the patients evaluated (T0 vs T1). CONCLUSION: These results suggest that the treatment with low-dose of atypical antipsychotic drugs is not associated with weight gain or increase the risk of developing type II diabetes or abnormalities of lipid metabolism among elderly patients with AD, who were residing in long-term nursing home.