Development of a Spectral Flow Cytometry Analysis Pipeline for High-Dimensional Immune Cell Characterization.

Flow cytometry is a widely used technique for immune cell analysis, offering insights into cell composition and function. Spectral flow cytometry allows for high-dimensional analysis of immune cells, overcoming limitations of conventional flow cytometry. However, analyzing data from large antibody panels can be challenging using traditional bi-axial gating strategies. Here, we present a novel analysis pipeline designed to improve analysis of spectral flow cytometry. We employ this method to identify rare T cell populations in aging. We isolated splenocytes from young (2-3 months) and aged (18-19 months) female mice then stained these with a panel of 20 fluorescently labeled antibodies. Spectral flow cytometry was performed, followed by data processing and analysis using Python within a Jupyter Notebook environment to perform batch correction, unsupervised clustering, dimensionality reduction, and differential expression analysis. Our analysis of 3,776,804 T cells from 11 spleens revealed 34 distinct T cell clusters identified by surface marker expression. We observed significant differences between young and aged mice, with certain clusters enriched in one age group over the other. Naïve, effector memory, and central memory CD8 + and CD4 + T cell subsets exhibited age-associated changes in abundance and marker expression. Additionally, γδ T cell clusters showed differential abundance between age groups. By leveraging high-dimensional analysis methods borrowed from single-cell RNA sequencing analysis, we identified age-related differences in T cell subsets, providing insights into the immune aging process. This approach offers a robust, free, and easily implemented analysis pipeline for spectral flow cytometry data that may facilitate the discovery of novel therapeutic targets for age-related immune dysfunction.

Diagnostic characteristics and management outcome in patients with acromegaly: A 15-year experience at a tertiary care hospital in Pakistan.

OBJECTIVE: To assess the diagnostic features of acromegaly, and analyse its management outcomes over a 15-year period in a tertiary care setting. METHODS: The descriptive, cohort, retrospective study was conducted at the Aga Khan University Hospital, Karachi, and comprised data of adult patients of either gender diagnosed with acromegaly based on biochemical and radiological evidence between January 2005 and December 2019. Data was retrieved from the medical records. Data was analysed using SPSS 19. RESULTS: Of the 84 subjects, 54(64.3%) were males and 30(35.7%) were female. The overall mean age was 38.69±13.52 years. The patients presented 5.43±4.3 years after the onset of symptoms, with somatic growth features, such as enlarged hands and feet which was the most common complaint 81(96.4%). Of all the patients, 73(86.9%) underwent trans-sphenoidal surgery for the removal of the pituitary adenoma, while 11(13.1%) opted out of the surgical option. Further, 9(12.3%) patients showed biochemical and radiological remission 6 months post-surgery. Out of the remaining 64(87.7%) patients, 38(59.4%) received radiosurgery or radiotherapy, 15(23.4%) underwent repeat trans-sphenoidal surgery, and 11(17.2%) chose medical treatment. CONCLUSIONS: Majority of patients failed to achieve remission after trans-sphenoidal surgery, which is the first line of treatment. Radiotherapy/repeat surgery was generally the options taken by those with persistent disease.

Social intelligence in health professions educationists: how it helps to deal with challenges at the workplace? a mixed-method study.

Objectives: To identify the social intelligence of medical educationists, and the coping strategies used to deal with workplace challenges. METHODS: The mixed-method study with an explanatory sequential design was conducted from March 15 to July 30, 2021, after approval from the ethics review committee of Riphah International University, Rawalpindi, Pakistan, and comprised medical educationists working in medical and dental colleges and institutions across the country. Data was collected using Tromso social intelligence scale in the quantitative phase. The socially intelligent educationists were identified, and were interviewed. Qualitative data was subjected to thematic analysis to identify predominant themes explaining the coping strategies used. RESULTS: In the quantitative phase, there were 80 participants; 51(63.7%) females and 29(36.3%) males, with 24(30%) having >10 years of professional experience. Of them, 11(13.8%) scored low, 54(67.5%) moderate and 15(18.8%) high on the social intelligence scale. In the qualitative phase, there were 13 subjects; 9(69.2%) females and 4(30.8%) males. There were 4 themes identified as coping strategies; inspire respect and trust, bringing readiness before a change, a collaborative and inclusive approach and use of soft skills. CONCLUSIONS: Variation was seen in the levels of social intelligence among medical educationists working in academic institutions. Those with high levels of social intelligence used their non-cognitive soft skills to manage workplace challenges.

Association of eating habits with mitochondrial DNA copy number in eveningness chronotypes: Origin research.

OBJECTIVE: To determine the relationship between eating habits and mitochondrial deoxyribonucleic acid copy number in adult cases of eveningness chronotypes. Methods: The cross-sectional, analytical study was conducted from September 2022 to June 2023 at the Physiology Department of the Islamic International Medical College, Rawalpindi, in collaboration with the Genetic Resource Centre, Rawalpindi, Pakistan, and comprised adult subjects who were assessed using the Morningness-Eveningness Questionnaire. The participants' eating habits were assessed using the Healthy Eating Assessment Questionnaire, and on they were divided into those with healthy eating habits in group A and those with unhealthy eating habits in group B. Deoxyribonucleic acid was extracted using the Chelex method, the mitochondrial deoxyribonucleic acid copy number of all participants was quantified using quantitative polymerase chain reaction. Data was analysed using SPSS 27. RESULTS: Of the 80 subjects, 30(37.5%) were males and 50(62.5%) were females. The overall mean age was 24.27±6.91 years (range: 18-45 years). There were 40(50%) subjects in each group. The mean mitochondrial deoxyribonucleic acid copy number in group A was 2.74±0.14 compared to 2.26±0.25 in group B (p<0.001). Conclusion: Subjects with healthy eating habits exhibited higher mitochondrial deoxyribonucleic acid copy numbers, indicating reduced damage to mitochondrial deoxyribonucleic acid.

Effect of haemodialysis practice guideline on dialysis indicators and haemodynamic complications among patients undergoing haemodialysis.

To assess the effect of haemodialysis practice guidelines on dialysis indicators and haemodynamic complications, the comparative study was conducted at the dialysis unit of Sheikh Zayed Hospital, Lahore, Pakistan, and comprised patients undergoing haemodialysis who were divided into intervention group A in which updated haemodialysis practice guidelines were used, and control group B in which routine base dialysis was given. Data was collected using a self-structured tool. Data was analysed using McNemar test and Mann-Whitney U-test with p<0.05. Compared to baseline, there was a significant improvement in post-intervention ratio of effective removal of clearance (K) resulting from the treatment characterised by time (t) in the patient with a specific volume of distribution (V), or Kt/V, median & IQR 0.83(0.355) vs 1.21(0.11) and percentage of urea reduction ratio with median & IQR 49(12) vs. 66.5(18.65) (p<0.05). Intradialytic hypotension was found in 17(56.6%) subjects in group B and in 4(13.4%) in group A (p=0.002). Intradialytic hypertension was found in 8(25.6%) patients in group B and 1(3.4%) in group A (p=0.039). It is recommended that dialysis be performed in accordance with the most recent clinical guidelines in order to improve practices and to increase haemodialysis effectiveness.

A large, locally aggressive giant cell tumour arising from the laryngeal cartilage: A Rare Case Report.

Giant cell tumour is a growth predominantly found in long bones of the body. Giant cell tumour has a rare occurrence in the head and neck. A case of a 31 year old male with no known comorbidities at the ENT Department, Shifa International Hospital, Islamabad presented with anterior neck swelling and hoarseness of voice. Patient was diagnosed as having Giant Cell Tumour of Larynx (GTCL) proven on FNA cytology and post-operative biopsy. GCTL is an uncommon entity with only 45 reported cases in the world.

A histopathological and epidemiological study of urothelial carcinoma at a tertiary care centre in Peshawar, Pakistan.

Bladder cancer is the ninth leading cause of death worldwide and 14th leading cause of death in Pakistan. The objective of this study was to determine the frequency of urothelial carcinoma in various age groups, its gender distribution, and grades. A total of 131 cases of urothelial carcinoma, received at Department of Pathology, Peshawar Medical College, Peshawar, between January 2017 to December 2022, were included in the study; of them 107 (81.6%) were males while 24 (18.3%) were females with a mean age of 62±13 years. The most common histological subtype was papillary urothelial carcinoma in 117(89.3%) cases, followed by Squamous and Glandular in 5(3.8%) cases. Majority of the urothelial carcinoma with high grade showed a statistically significant relation with muscle invasion 38 (50.66%). Males were four times more likely to have urothelial carcinoma while older age groups were more likely to have high grade urothelial carcinoma.

Challenges in the management of idiopathic pulmonary fibrosis from a low- and middle-income country.

Idiopathic pulmonary fibrosis (IPF) is the most common progressive form of interstitial lung disease (ILD) that leads to gradual deterioration of lung function and ultimately death. Data from low- and middle-income countries (LMIC) on IPF is scarce. In this communication, we report the challenges encountered in managing IPF from Pakistan's largest tertiary care centre. A total of 108 patients with IPF were evaluated at the Aga Khan University Hospital in Karachi, Pakistan from January 2017 to March 2020. A significant concern was that most patients with IPF presented late during their disease. A bigger challenge encountered in clinical practice was the cost and nonavailability of antifibrotic therapy in the country until mid-2020. Successfully addressing these limitations, it is anticipated that better care will be available for the patients suffering from IPF in this part of the world.

Diagnostic evaluation of patients with epileptic spasms in the era of next-generation sequencing.

OBJECTIVE: Epileptic spasms (ES) can be caused by a variety of etiologies. However, in almost half of cases, the etiology is unidentified. With the advent of next-generation sequencing (NGS), the recognition of genetic etiologies has increased. METHODS: We retrospectively reviewed the medical records of patients with ES who were evaluated in the comprehensive epilepsy program at King Fahad Specialist Hospital Dammam between 2009 and 2022. RESULTS: Our data show that in 57.7% of patients with ES, the etiology was unidentified after a standard clinical evaluation and neuroimaging. Of these patients, n = 25 (35.2%) received a genetic diagnosis after some form of genetic testing, and 3.1% of patients from specialized metabolic work indicated the need for genetic testing to confirm the diagnosis. Karyotyping led to a diagnosis in 3.6% of patients, and chromosomal microarray led to a diagnosis in 7.1%. An NGS epilepsy gene panel (EP) was done for 45 patients, leading to a diagnosis in 24.4% (n = 11). Exome sequencing was done for 27 patients, including n = 14 with non-diagnostic panel testing; it led to a diagnosis in 37.3% (n = 10). Exome sequencing led to a diagnosis in 61.5% of patients without a previous panel test and in only two patients who had previously had a negative panel testing. SIGNIFICANCE: In this article, we present the diagnostic evaluations of ES for a cohort of 123 patients and discuss the yield and priority of NGS for evaluating ES. Our findings suggest that exome sequencing has a higher diagnostic yield for determining the etiology of ES in patients for whom the etiology is still unclear after an appropriate clinical assessment and a brain MRI.

Acquired Cytomegalovirus Retinitis in Preterm Infant Hospitalized in the NICU: A Noteworthy Case Report.

BACKGROUND: Acquired human cytomegalovirus (CMV) is a noteworthy disease in infants. This case study will highlight the influence of early diagnosis of CMV retinitis (CMVR) on avoid visual impairment. CLINICAL FINDINGS: We describe a preterm female infant with a birth weight of 2060 gr that was admitted for tracheostomy placement due to hypoxic-ischemic encephalopathy. There were no signs of CMV infection or sepsis in laboratory results upon admission such as serology (IgG, IgM antibodies), Toxoplasma gondii, Rubella virus, Herpes simplex virus, CMVR and urine polymerase chain reaction (PCR). PRIMARY DIAGNOSIS: Incidentally, upon screening for retinopathy of prematurity, diffuse occlusive vasculitis was detected in the retinal image on the 112th day of life. INTERVENTION: Intravenous and intraocular ganciclovir were administered for 4 weeks. OUTCOMES: In the follow-up visit 6 weeks after discharge from the hospital, visual impairment was detected on both sides. PRACTICE RECOMMENDATIONS: This is a report of a case of acquired CMVR, a silent finding, as an uncommon complication in preterm neonates during the hospital stay. This diagnosis should be taken into consideration in preterm infants, since early diagnosis and treatment are crucial to avoid visual impairment.

Crotalaria burhia Buch. -Ham.: a comprehensive review of its botany, traditional uses, phytochemistry, and pharmacology.

Crotalaria burhia (Family: Fabaceae) is an important medicinal plant widely distributed in arid parts of the world, including Pakistan, India, and Afghanistan. This plant has enormous ethnobotanical values and is used to treat various common ailments such as swelling, infections, cancer, hydrophobia, pain and skin diseases. Moreover, it is also utilised as food for goats, to make sheds for animals and as a suitable soil binder. This review article is an attempt to analyse critically and to provide updated and categorised information about C. burhia including comprehensive knowledge of the botanical description, traditional/folklore uses, phytochemistry, pharmacological/biological potential, and to facilitate scientific basis for future work. The phytochemical studies (qualitative and quantitative) on C. burhia have indicated the presence of important phytochemical classes, namely alkaloids, flavonoids, glycosides, saponins, phenolics, tannins, steroids, and terpenoids. Pharmacological studies such as anti-inflammatory/analgesic, antioxidant, anti-microbial, anti-tumour, anti-nociceptive, enzyme inhibition, and termiticidal activities were reported from different parts of this plant. Most of the bioassays from this plant have been done on the crude extract. Minimal information about the phytochemicals (responsible for biological activities), except a few compounds has been reported. The potential chemical compounds may need to be purified and tested for the biological potential from isolated compounds in future.

Why Evidence Generation Should Matter to Payers and How They Can Help.

Importance: The US leads the world in bringing new medical products to market, but the ability to generate evidence to inform clinical practice in postmarket settings needs improvement. Although a diverse group of stakeholders is working to improve postmarket evidence generation, the role of private payers has been underappreciated. Observations: Payers are crucial allies in improving evidence generation because better data would better inform coverage decisions, their policies and practices influence the conduct of care and research, and their claims data are a source of real-world evidence used in medical product evaluation. In addition, payers have a stake in improving evidence generation because the kinds of evidence needed to inform health care and coverage decisions are often not available when a product enters the market and may not be generated without their involvement. Here, we describe several key steps payers could take to improve evidence generation, including participating in efforts to reduce administrative and financial barriers to the conduct of clinical trials, directly incentivizing evidence generation on high-priority questions by funding potential cost-saving trials, increasing engagement with the medical products industry on evidentiary needs for coverage decisions, and improving usability of claims data by reducing data lags and routinely recording unique device identifiers. Broad payer engagement with US Food and Drug Administration recommendations regarding evidence generation will ensure that the opportunities to participate in clinical research are extended to all communities and that evidence needed to inform care is generated in trials and surveillance systems that reflect the clinical reality across the US. Conclusions and Relevance: Increasing payer involvement in evidence generation can benefit all participants in the medical innovation ecosystem. The importance of payers in these efforts will continue to grow in response to imperatives to increase integration of care and research, engage a diverse set of communities in clinical research, and move toward alternative payment models.

In vitro anti-breast cancer study of hybrid cinnamic acid derivatives bearing 2-thiohydantoin moiety.

Aim: To synthesize new hybrid cinnamic acids (10a, 10b and 11) and ester derivatives (7, 8 and 9) and investigate their anti-breast cancer activities. Materials & methods: Compounds 7-11 were evaluated (in vitro) for their cytotoxic activities against the MCF-7 cell line. A flow cytometry examination was performed. Protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2), topoisomerase II and caspase-9 were measured by qRT-PCR. Molecular docking studies were conducted. Results: Several components were discovered to be active, mainly component 11, which induced arrest in the cell cycle at phase S, greatly decreased the expression of Nrf2 and topoisomerase II; and upregulated the expression of caspase-9. Conclusion: The newly thiohydantoin-cinnamic acid hybrids can contribute to creating promising candidates for cancer drugs.

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Bias in published randomized trials that compare collagenase injection with percutaneous needle fasciotomy in the treatment of Dupuytren disease: a systematic review.

Purpose: Controversy exists regarding the comparative efficacy of collagenase injection and percutaneous needle fasciotomy in the treatment of Dupuytren contracture. The randomized controlled trials (RCTs) that have compared the two treatment methods have reported results mostly implying similar treatment efficacy, durability, and complications. We aimed to review these RCTs regarding methodical quality and risk of bias. Methods: We searched PubMed and Cochrane Library databases up to May 2023. All RCTs comparing collagenase injection with needle fasciotomy were included. Eligible articles were reviewed by two researchers, of whom one was blinded to each article's title, authors, year of publication, journal, and source of the studies. To assess methodical quality, we used the modified Jadad scale yielding a score of 0 (lowest quality) to 5 (highest quality). We assessed risk of bias with the Cochrane risk-of-bias tool (RoB 2). Results: Five studies were eligible, comprising 204 patients treated with collagenase injection and 209 patients treated with needle fasciotomy. The modified Jadad score ranged from 1 to 2 points in the five studies, and the overall risk of bias was high in all studies. Pretrial protocols could be retrieved for only two studies, revealing important discrepancies with the published articles. Conclusion: The published RCTs that have compared collagenase injection with needle fasciotomy in the treatment of Dupuytren contracture demonstrate a high risk of bias.

Genotypes, epidemiological variables and fungicides application associated with wheat leaf rust development and grain yield.

The present study was carried out at the Plant Pathology Hafizabad Research Station, the University of Layyah, during the crop seasons 2021-2022 and 2022-2023 to evaluate the response of various wheat genotypes against leaf rust severity (%), environmental conditions favourable for disease development and grain yield. Except for minimum temperature and minimum relative humidity, which had a negative association with disease development, there was a significant correlation between leaf rust severity (%) and all environmental conditions such as maximum temperature, maximum relative humidity, rainfall, and wind speed. All epidemiological variables such as maximum temperature, minimum temperature, minimum relative humidity, rainfall and wind speed significantly affect the disease progression. The disease predictive model accounted for 48-69 % variability in leaf rust severity. The model performance was evaluated using the coefficient of determination (R2 = 0.69) and RMSE, both demonstrated acceptable predictive results for leaf rust severity (%) management. Leaf rust severity (%) increased with an increase in maximum temperature (17.8-30 °C), maximum relative humidity (76.3-85 %), rainfall (2.2-10.85 mm) and wind speed 1.1-2.7 km/h and decreased with the increase of minimum temperature (7.91-16.71 °C) minimum relative humidity (47.15-56.45 %) during both rating seasons 2021-2022 and 2022-2023. The single and two applications of fungicides at the Zadok's scale 3, ZS 4.3, and ZS 5.4 stages led to a significant reduction in grain yield losses caused by leaf rust severity (%) in both the 2021-2022 and 2022-2023 crop seasons. Single and two sprays of prothioconazole, were found to be the first choice among all treatments to reduce the disease severity and increase grain production and maximum gross revenue (513.1-777.8$/ha), as compared to followed by single and two sprays of propiconazole (Progress), tebuconazole + trifloxystrobin, tebuconazole, bixafen + tebuconazole, and propiconazole (Tilt), respectively. These findings recommend the involvement of genotype resistance and weather predictors in wheat leaf rust development, along with fungicide application studies, to improve the predictability of host resistance to disease, future models, and the sustainability of disease control methods.

A reliable jumping-based classification methodology for environment sector.

Decision-makers have consistently developed a range of classification models, each possessing unique features within the domain of intelligent models. These endeavors are all directed toward achieving the highest levels of accuracy. In recent developments, two notable methodologies-reliable modeling and jumping modeling approaches-offer specific advantages in formulating cost functions and have been recognized for their role in enhancing classifier accuracy. Specifically, the jumping methodology is based on aligning the learning process with the discrete nature of the classification goal, while the reliable methodology integrates the reliability factor into the learning paradigm. However, their innovative combination, leveraging both accuracy and reliability factors in guiding learning processes, leads to the creation of a high-performing classifier. This addresses a research gap in tackling classification challenges, which remains the core focus of the present study. To evaluate the performance of the proposed reliable jumping-based intelligent classifier in environmental decision-making, we considered ten benchmark datasets spanning various application domains. The numerical results demonstrate that the proposed Reliable Jumping-based intelligent classifier consistently outperforms traditional intelligent classifiers across all studied cases. As a result, the proposed approach proves to be a viable and effective alternative to other intelligent methods in environmental applications.

Decoding children dental health risks: a machine learning approach to identifying key influencing factors.

Introduction and objectives: This study investigates key factors influencing dental caries risk in children aged 7 and under using machine learning techniques. By addressing dental caries' prevalence, it aims to enhance early identification and preventative strategies for high-risk individuals. Methods: Data from clinical examinations of 356 children were analyzed using Logistic Regression, Decision Trees, and Random Forests models. These models assessed the influence of dietary habits, fluoride exposure, and socio-economic status on caries risk, emphasizing accuracy, precision, recall, F1 score, and AUC metrics. Results: Poor oral hygiene, high sugary diet, and low fluoride exposure were identified as significant caries risk factors. The Random Forest model demonstrated superior performance, illustrating the potential of machine learning in complex health data analysis. Our SHAP analysis identified poor oral hygiene, high sugary diet, and low fluoride exposure as significant caries risk factors. Conclusion: Machine learning effectively identifies and quantifies dental caries risk factors in children. This approach supports targeted interventions and preventive measures, improving pediatric dental health outcomes. Clinical significance: By leveraging machine learning to pinpoint crucial caries risk factors, this research lays the groundwork for data-driven preventive strategies, potentially reducing caries prevalence and promoting better dental health in children.

Exploring the impact of occupational factors on low back pain in ride-sharing motorbike drivers in Bangladesh: A comprehensive cross-sectional analysis.

Objective: Low back pain (LBP) is a major global public health issue, prevalent among various occupational groups worldwide. However, existing studies have predominantly focused on sedentary workers in developed nations, leaving a gap in understanding LBP prevalence and associated factors among occupational cohorts in low- and middle-income countries like Bangladesh. This study aimed to determine the prevalence and occupational factors contributing to LBP among ride-sharing motorbike drivers (RSMD) in Bangladesh. Methods: A cross-sectional study was conducted in Dhaka city from May 20 to August 08, 2023. Data were collected from Bangladeshi adult RSMD using a paper-based questionnaire developed by Eriksen et al. Chi-square or Fisher's Exact tests compared categorical variables with and without LBP, and multiple logistic regression analyses were performed with LBP as the dependent variable and various predictors to compute adjusted odds ratios with a 95% confidence interval. Results: The one-month prevalence of LBP was 58.8%. Regression analysis revealed elevated adjusted odds of experiencing LBP among participants with hypertension, those using multiple ride-sharing operators, commuter and older bike users, and non-users of riding kits. Additionally, increased adjusted odds of LBP were observed among participants of higher age, higher body mass index, and those covering longer distances per week. Conclusion: This study underscores a significantly higher prevalence of LBP among RSMD in Bangladesh, with occupational factors strongly predicting LBP. Implementing strategies such as regular physical exercise, weight reduction, using sports and newer motorbikes, and reducing working hours per week may help mitigate the prevalence of LBP within this cohort.