Mismatch repair in T. brucei: roles in protection against oxidative damage

Cells are continuously exposed to different intracellular and extracellular mutagens, which can damage several different molecules, including DNA. To ensure survival, cells have evolved various defence and repair mechanisms. Mismatch repair (MMR) is the mechanism that serves to repair mismatched bases in DNA that are missed by the proof reading activity of DNA polymerases. Besides this, MMR also corrects base mismatches formed by altered bases modified by certain chemical mutagens. Thus, MMR is important to avoid mutagenesis and maintain genome fidelity. MMR is a complex, highly conserved pathway that involves a range of proteins along with several accessory proteins. In T. brucei, as in other eukaryotes, MMR core functions are carried out by bacterial MutS and MutL homologues, working as heterodimers: MSH2α (MSH2-MSH3) and MSH2β (MSH2-MSH6), and MLH1-PMS1, respectively. To date, only MSH2 and MLH1 function have been examined and only in bloodstream form (BSF) T. brucei cells. It was observed that MMR mutants are tolerant towards methylation damage, exhibit microsatellite instability and display elevated rates of homologous recombination between imperfectly matched DNA molecules. This confirmed the role of MMR in genome maintenance in BSF T. brucei. More recently, it was observed that BSF MSH2 mutants were sensitive towards oxidative damage, though the same phenotype was not observed in MLH1 mutants. This suggests that some aspect of the MMR machinery acts to protect BSF T. brucei cells against oxidative stress, but the machinery and mode of action is unknown. In this study we have generated null mutants of MSH2 and MLH1 in procyclic form (PCF) T. brucei cells, and MSH3 and MSH6 mutants in BSF cells. Characterization of tolerance to DNA methylation damage and evaluating microsatellite stability shows that each gene acts in MMR in both the life cycle stages, with the exception of MSH3, where null mutants show no discernible phenotypes. Mutants were also analyzed for their action towards oxidative stress in both the life stages and, remarkably, we find life cycle stage differences, with MSH2 mutants displaying hydrogen peroxide sensitivity and resistance in the BSF and PCF, respectively. The same phenotypes are not seen in MLH1 mutants, and we show that resistance to hydrogen peroxide in PCF cells is due to adaptation during the loss of MSH2. We have also shown that PCF MSH2 mutants may show a decrease in microsatellite instability when subjected to oxidative stress. This leads to the hypothesis that there might be an unidentified system, apart from MMR, present in T. brucei PCF cells that works as a defence in response to oxidative stress and can assume greater prominence when MSH2 is lost. Although we have tried to explore various cellular processes that might contribute this activity, our results are inconclusive. MSH2 and MLH1 have also been epitope tagged to explore the subcellular localization of these proteins and to ask if any changes in expression levels or changes in localisation are seen when subjected to oxidative stress. These preliminary data suggest that both factors are nuclear and cytoplasmic. We have also tried to ask if MSH2 and/or MLH1 co-localize with either MSH5 or MSH4, which are MutS-like factors that act in meiosis in other eukaryotes, but whose functions have not been explored in T. brucei. However, our attempts at this analysis have been unsuccessful

Primary hyperparathyroidism: Experience from a tertiary care centre in Pakistan

Objective: To study the clinical, biochemical and radiological features and management outcomes of patients with primary hyperparathyroidism.Methods: This retrospective study was conducted at the Aga Khan University Hospital, Karachi, Pakistan and comprised data of patients with primary hyperparathyroidism from 2008 to 2017.Results: Out of 103 patients, 83(80.6%) were female. Overall mean age was 59.3±16.2 years. Musculoskeletal manifestations were seen in 60(58.3%) patients and renal manifestations in 28(27.2%). Ostieits fibrosa cystica was found in 04(3.88%) patients. Overall, Ultrasound neck and sestamibi scan localized the lesion in 66 (64.1%) and 77 (76.2%) patients respectively. Among 79 patients who underwent surgery, 67 (84.8%) patients had an adenoma, 05 (6.3%) had hyperplasia and 02(2.53%) patients had parathyroid carcinoma whereas histopathology was inconclusive in 5 (6.32%) out of the 79 surgically treated patients. Disease recurrence was seen in 13 out of 79(16.45%) patients who underwent surgery.Conclusion: Primary hyperparathyroidism is associated with significant morbidity in our population. Targeted measures like improving patient awareness, routine calcium screening, vitamin D supplementation and a high index of suspicion by the clinician may help in early diagnosis of the condition and thus reduce morbidity

Evaluating the status of vitamin 25(OH) D levels among females of all age groups in Karachi, Pakistan.

Introduction: Vitamin D plays an important role in growth, metabolism and reproduction. Deficiency of this vitamin is highly prevalent globally and leads to various disorders besides different skeletal deformities. Women of different reproductive ages are at higher risk of developing bone diseases because of low vitamin D levels. Objective: This study aimed to assess the prevalence of vitamin D deficiencies in females of different ages in Karachi, Pakistan Methodology: A retrospective investigation of vitamin D levels during a six month period at the Clinical Lab, Al-Khidmat Diagnostic Center, Karachi; Pakistan was performed.  Serum vitamin 25(OH) D levels of 1035 female were measured by Electrochemiluminescence (ECL) assay. The data was analyzed through SPSS version 16. Results: Among 1035 participants of different reproductive ages, 26.1% (n= 270) showed severe vitamin D deficiency, 44.3% (n=458) displayed mild to moderate Vitamin D deficiency while 29.7% (n=307) were having normal levels of serum vitamin D. Results: The data of 1035 subjects was examined and the mean serum vitamin 25(OH) D levels of the studied subjects were found as 21.04±14.13 with the minimum and maximum range of 4.97ng/ml to 70.71ng/ml Conclusion: 70.4 % of our studied population had lower levels of vitamin D representing that the majority of Pakistani womenfolk have vitamin D Deficiency. Keywords: Vitamin 25 (OH) D levels, Female population, Karachi. &nbsp

Potential Predictors of Nodal Metastasis in Early Tongue Cancer Found Through Aperio Image Analysis

Introduction: Excessive use of cigarettes cigars/ pipe as well as smokeless tobacco is one of the most common cause of oral cancer. In Shaukat Khanum Memorial Center Hospital and Research Center (SKMCH&RC) in Pakistan, Squamous Cell Carcinoma of Tongue (SCCOT) is the second most common head and neck malignancy. Approximately 20-30% of patients with early oral tongue carcinoma will have occult neck nodal modal metastasis. Presently, elective neck dissection remains the only reliable way to predict regional and or distant metastasis. There is a pressing need to find reliable biomarkers and non-invasive predictors of metastasis and patient response in early squamous cell carcinoma of oral tongue.Retrospective analysis was performed in a double-blind manner on tissues microarray (5 cores/patient) created from paraffin-embedded specimens from 50 patients with well documented clinical history of the disease. A subset of 20 different proteins were elected as potential biomarkers of metastasis based on published literature on SCCOT and analyzed through immunohistochemistry. Four proteins, E-cadherin, Podoplanin, Microglobulin and Interleukin-8 were found as possible predicators of metastasis. These findings were validated using Aperio image analysis software. Digital analysis using Aperio confirmed results for two of the four proteins; E-cadherin and Podoplanin while Microglobulin and Interleukin-8 were found to have non-significant results. Specifically, Podoplanin was found to be upregulated while E-cadherin was downregulated in metastatic patients. Computer-aided image analysis provides a powerful tool to support clinical decisions while reducing inter-and intra-observer variability. Based on the results from Aperio, Podoplanin and E-cadherin may be used as potential biomarkers to predict nodal metastasis in early tongue cancer. Further studies are required to confirm these findings and to quantify the levels of these proteins in tissue and plasma of patients with cancer compared to non-tumoral controls

Predictors of metabolic response in propensity-matched lymphoma patients on interim 18F-fluorodeoxyglucose positron-emission tomography/computed tomography using standardized imaging and reporting protocol: Do we really have one?

The purpose of this prospective study was to determine metabolic response predictor(s) in propensity-matched patients having lymphomas who had baseline and interim 18fluorodeoxyglucose (FDG) positron-emission tomography/computed tomography (PET/CT) using strict standardized imaging and reporting protocols. This prospective study was conducted at PET/CT section of a JCI-accredited healthcare facility from April 2017 to February 2018. Patients with baseline and interim 18FDG PET/CT scans using standardized protocol were selected. Interim scans were performed not earlier than 2nd or later than 4th chemotherapy. During the study period, 97 of 112 consecutive patients with lymphomas (Hodgkin-HL: 32/97 and Non-Hodgkin-NHL: 65/97) were included in the study. Mean age of cohort was 45 ± 19 years (71% male and 29% female) having a mean body mass index (BMI) of 25.57 ± 5.54 Kg/m2 having Stage I (21%), Stage II (18%), Stage III (16%), and Stage IV (45%) disease. Bulky disease was found in 14% and 18FDG-avid marrow deposits in 33%. Standardized PET/CT imaging protocol as per EANM guidelines was strictly adopted for baseline and interim studies. %Δ changes in fasting blood sugar, 18FDG dose, uptake time, and liver SUV mean were 3.96%, 2.83%, 2.49%, and 12.15%, respectively. Based on Deauville\u27s scoring, cohort was divided into responders having Score 1-3 (49/97) and nonresponders having Score 4-5 (48/97). The demographic analysis found no significant difference between responders and nonresponders for age, gender, BMI, staging, bulky disease or marrow involvement, and study protocol. No significant coefficient or odd ratios were found on multivariate analysis for age, gender, maximum standardized uptake value (SUVmax), size, BMI, NHL, and advance disease (Stage III and IV) in both groups (χ2: 5.12; receiver operating characteristic [95% confidence interval]: 0.616 [0.51-0.713]; P =0.528). Among responders, baseline SUVmax and tumor size had a direct correlation with a metabolic response on iPET, more pronounced in NHL than HL groups (SUVmax: 13.4 vs. 19.5 and size: 52 vs. 87 mm; P \u3c 0.0001). We conclude that no significant predictor was found for response in propensity-matched patients with lymphomas (both HL and NHL) who had baseline and interim PET/CTs acquired with a standardized protocol. However, NHL responders were found to have higher baseline median SUVmax and larger lesion size as compared to HL responders. Although, these data are not in concordance with published findings but need to be validated with larger studies using standardized imaging and reporting protocols in propensity-matched patients with lymphoma

The utility of ultrasound in the preoperative localization of primary hyperparathyroidism: Insights from Pakistan

Objective: We aimed to evaluate and compare the diagnostic performance of ultrasound (US) and 99m-Tc sestamibi scintigraphy for the preoperative localization of primary hyperparathyroidism (PHPT).Methods: This retrospective study was conducted at the Aga Khan University Hospital in Karachi, Pakistan, and comprised the data of patients with PHPT who underwent parathyroidectomy from 2008 to 2017. Preoperative US and 99m-Tc sestamibi scintigraphy findings were recorded and compared to surgical and histological findings, which were taken as a reference standard.Results: The sensitivity of US in the preoperative localization of PHPT was 88.3%, positive predictive value (PPV) was 94.6%, and accuracy was 84.1%. The sensitivity of 99m-Tc sestamibi scintigraphy was 90.4%, PPV was 94.3%, and accuracy was 85.7%.Conclusion: US neck is an efficient tool for the preoperative localization of PHPT, demonstrating a comparable diagnostic yield with 99m-Tc sestamibi, and can serve as a credible first-line imaging modality in a resource-constrained healthcare setup

Higher event rate in patients with high-risk duke treadmill score despite normal exercise-gated myocardial perfusion imaging

his prospective study was carried out to find the negative predictive value of various Duke Treadmill Scores (DTSs) in patients with normal myocardial perfusion imaging (MPI). This study was conducted from August 2012 to July 2015, and 603 patients having normal exercise MPIs were included. Patients were followed for 2 years for fatal myocardial infarction (FMI) and nonfatal myocardial infarction (NFMI). Follow-up was not available in 23 patients, leaving a cohort of 583 participants. DTS was low risk (≥5) in 286, intermediate risk (between 4 and - 10) in 211, and high risk (≤-11) in 86 patients. Patients with high- and intermediate-risk DTS were significantly elder than low-risk DTS cohort. Patients with high-risk DTS had significantly higher body mass index with male preponderance compared to other groups. No significant difference was found among three groups regarding modifiable or nonmodifiable risk factors and left ventricular ejection fraction. On follow-up, single FMI was observed in high-risk DTS group (log-rank test value = 5.779, P = 0.056). Five NFMI events were observed in high-risk DTS (94.2% survival; log-rank test value = 19.398, P = 0.0001; significant) as compared to two events each in low- and intermediate-risk DTS (nonsignificant). We conclude that patients with normal exercise MPI and low-to-intermediate risk DTS have significantly low NFMI. High-risk DTS despite normal exercise MPI had high NFMI. Further, validation studies to find the predictive value of symptomatic and asymptomatic ST deviation resulting in high-risk DTS in patients with normal exercise MPI are warranted

Factors influencing the opinion of individuals in determining tumour spread after biopsy

<p>Abstract</p> <p>Background</p> <p>People often have concerns regarding tumour spread after biopsy which leads to a delay in seeking expert medical advice. The data regarding this perception is scanty. Therefore, we conducted this cross sectional study to explore the beliefs and perceptions of individuals regarding tumour spread after biopsy and the basis of those beliefs.</p> <p>Methods</p> <p>The survey was conducted in outpatient areas of two different tertiary care hospitals of Karachi namely Aga Khan University Hospital Karachi (AKUH) and Karachi Institute of Radiotherapy and Nuclear Medicine (KIRAN). We interviewed 600 individuals and documented their responses on a questionnaire. There were 400 responders from Aga Khan's Consulting Clinic and 100 each from Aga Khan's Oncology Clinic and KIRAN.</p> <p>Results</p> <p>Only 50% of the respondents chose biopsy as the best test for diagnosis of cancer. The level of education was statistically significant in making this choice of answer (<it>p </it>= 0.02) only in univariate analysis. Those individuals who were involved in the work up of cancer patients irrespective of their educational status gave more intelligent answers (<it>p </it>= 0.003). The tumour disturbance after biopsy was regarded as a major factor among 127 respondents (53%) who believed that biopsy could lead to spread of tumour.</p> <p>Conclusions</p> <p>Our study revealed that awareness regarding cancer diagnosis and biopsy is lacking among general public and it does not co-relate well with the level of formal education. These misconception and taboos need to be addressed in public seminars and in the media in order to increase the awareness which could facilitate prompt diagnosis.</p

Effects of a high-dose 24-h infusion of tranexamic acid on death and thromboembolic events in patients with acute gastrointestinal bleeding (HALT-IT): an international randomised, double-blind, placebo-controlled trial

Background: Tranexamic acid reduces surgical bleeding and reduces death due to bleeding in patients with trauma. Meta-analyses of small trials show that tranexamic acid might decrease deaths from gastrointestinal bleeding. We aimed to assess the effects of tranexamic acid in patients with gastrointestinal bleeding. Methods: We did an international, multicentre, randomised, placebo-controlled trial in 164 hospitals in 15 countries. Patients were enrolled if the responsible clinician was uncertain whether to use tranexamic acid, were aged above the minimum age considered an adult in their country (either aged 16 years and older or aged 18 years and older), and had significant (defined as at risk of bleeding to death) upper or lower gastrointestinal bleeding. Patients were randomly assigned by selection of a numbered treatment pack from a box containing eight packs that were identical apart from the pack number. Patients received either a loading dose of 1 g tranexamic acid, which was added to 100 mL infusion bag of 0·9% sodium chloride and infused by slow intravenous injection over 10 min, followed by a maintenance dose of 3 g tranexamic acid added to 1 L of any isotonic intravenous solution and infused at 125 mg/h for 24 h, or placebo (sodium chloride 0·9%). Patients, caregivers, and those assessing outcomes were masked to allocation. The primary outcome was death due to bleeding within 5 days of randomisation; analysis excluded patients who received neither dose of the allocated treatment and those for whom outcome data on death were unavailable. This trial was registered with Current Controlled Trials, ISRCTN11225767, and ClinicalTrials.gov, NCT01658124. Findings: Between July 4, 2013, and June 21, 2019, we randomly allocated 12 009 patients to receive tranexamic acid (5994, 49·9%) or matching placebo (6015, 50·1%), of whom 11 952 (99·5%) received the first dose of the allocated treatment. Death due to bleeding within 5 days of randomisation occurred in 222 (4%) of 5956 patients in the tranexamic acid group and in 226 (4%) of 5981 patients in the placebo group (risk ratio [RR] 0·99, 95% CI 0·82–1·18). Arterial thromboembolic events (myocardial infarction or stroke) were similar in the tranexamic acid group and placebo group (42 [0·7%] of 5952 vs 46 [0·8%] of 5977; 0·92; 0·60 to 1·39). Venous thromboembolic events (deep vein thrombosis or pulmonary embolism) were higher in tranexamic acid group than in the placebo group (48 [0·8%] of 5952 vs 26 [0·4%] of 5977; RR 1·85; 95% CI 1·15 to 2·98). Interpretation: We found that tranexamic acid did not reduce death from gastrointestinal bleeding. On the basis of our results, tranexamic acid should not be used for the treatment of gastrointestinal bleeding outside the context of a randomised trial

Understanding the concept of Purgation (Ishal) in Unani Medicine: A Review

The theory of humours (akhlat) is one of the fundamental pillars of the Unani System of Medicine (USM). The concept of health and disease depends on the quality (kaifiyat) and quantity (kammiyat) of humour (khilt). Health (sehat) lasts when humours remain in equilibrium and the main determinant of health is the balance in six essential factors (asbab-e-sittah zarooriya). These factors are highly modifiable and deviation in any of them leads to disequilibrium in humours either qualitatively or quantitatively which ultimately manifests in the form of the disease. Elimination (istifragh) of these morbid humours from the body becomes mandatory to treat the diseases or to restore health. One of the effective methods of elimination is purgation (ishal). It is a method by which morbid humours from the body are eliminated through the anal route. Before the elimination of any pathological humour especially in chronic diseases, it is mandatory to make the humour easily eliminable. This process of making the pathological humour eliminable is known as concoction (nuzj). The process of concoction is a regular and continuous process of the tabiyat (mediatrix naturae) of the body. In case of a minor deviation in humour, tabiyat itself eliminates it from the body after concoction. When the causative pathological humours are in abundance or grossly deviated from normalcy, tabiyat needs help from outside the body. This help of tabiyat can be done with some humour specific drugs which are known as concoctive medicines (munzij advia). Once, the humours become eliminable, the process of evacuation can be started. Classical Unani literature and published papers were explored to find the rationale of purgation therapy. Purgation is found to be advisable in the treatment of many chronic diseases. Tabiyat is the ultimate healer in the body and purgation helps it to overcome the diseases. Keywords: Istifragh; Munzij; Nuzj; Akhlat; Humours; Concoctio