Expression Profiling of EMT Transcriptional Regulators ZEB1 and ZEB2 in Different Histopathological Grades of Oral Squamous Cell Carcinoma Patients.

Background: Pakistan has a high burden of oral cancers, with a prevalence rate of around 9%. Oral Squamous Cell Carcinoma (OSCC) accounts for about 90% of oral cancer cases. Epithelial to Mesenchymal Transition (EMT) gets highly stimulated in tumor cells by adopting subsequent malignant features of highly invasive cancer populations. Zinc Finger E-Box binding factors, ZEB1 and ZEB2, are regulatory proteins that promote EMT by suppressing the adherent ability of cells transforming into highly motile cancerous cells. The present study aimed to analyze the expression of EMT regulators, ZEB1 and ZEB2, and their association with the clinicopathological features in different grades of OSCC patients. Methods: Tissue samples were collected for both case and control groups from the recruited study participants. Cancer tissues (cases) were collected from the confirmed OSCC patients, and healthy tissues (controls) were collected from third-molar dental extraction patients. The study participants were recruited with informed consent and brief demographic and clinical characteristics. The case group was further segregated with respect to the histological cancer grading system into well-differentiated (WD), moderately differentiated (MD), and poorly differentiated (PD) squamous cell carcinoma (SCC) groups. RNA was extracted from the tissue samples for expression profiling of ZEB1 and ZEB2 genes through quantitative real-time PCR (qRT-PCR). Results: All of the recruited participants had a mean age of 46.55 ± 11.7 (years), with most of them belonging to Urdu speaking ethnic group and were married. The BMI (kg/m2) of the healthy participants was in the normal range (18-22 kg/m2). However, BMI was found to be reduced with the proliferation in the pathological state of cancer. The oral hygiene of patients was better than the healthy participants, possibly due to the strict oral hygiene practice concerns of consultants. Every recruited OSCC patient had one or multiple addiction habits for more than a year. Patients reported health frailty (46.6%), unhealed mouth sores (40%), swallowing difficulties and white/reddish marks (80%), and restricted mouth opening (64.4%). Furthermore, 82.2% of the recruited patients observed symptoms within 1-12 months, and buccal mucosa was the most exposed tumor site among 55.6% of the patients. Expression profiling of EMT regulators showed gradual over-expressions of ZEB1 (8, 20, and 42 folds) and ZEB2 (4, 10, and 18 folds) in respective histological cancer grades. Conclusion: High expressions of ZEBs have been significantly associated with cancer progression and poor health. However, no association was found between OSCC with other clinicopathological features when compared to healthy controls.

Oxidative Stress in Beta-thalassemia Patients: Role of Enzymatic and Non-enzymatic Modulators.

BACKGROUND: Oxidative stress is a pathophysiological state that arises due to an imbalance created between ROS generation and the antioxidant potential of the host cell. Transfusion- dependent beta-thalassemia major patients are at high risk of cellular and molecular damages induced by ROS mainly due to iron overload caused by repetitive blood transfusion. OBJECTIVES: To analyze oxidative stress status levels in ß-thalassemia patients. To analyze the expression profile of enzymatic (NOS2, OGG1, HuR, SOD2) and non-enzymatic (VDR) redox regulators in ß-thalassemia patients. To assess polymorphism in VDR (rs2228570) and NOS2 (rs944725) in ß-thalassemia patients. To analyze serum vitamin D levels of ß-TM patients compared to healthy individuals. METHODS: The present case-control study aimed to identify Vitamin D levels in the serum of ß-thalassemia patients and compared it with healthy subjects. The study further analyzed VDR FOKI (rs2228570) polymorphism through ARMS-PCR. Expression profiling of VDR, anti-oxidant enzyme (SOD2 and GPx), and their respective regulator (HuR and NrF2) transcripts was done by the 2-ΔΔCt method. RESULTS: The study reports that there is no a significant difference between the Vitamin D levels among healthy and patients. VDR polymorphism analysis (rs2228570) demonstrates that although the C allele is prevalent in the study cohort, the frequency of the T allele is comparatively higher in ß-thalassemia patients as compared to healthy subjects. Furthermore, patients express lower levels of anti-oxidant enzymes despite having increased oxidative stress. CONCLUSION: The study reports that ß-thalassemia patients are at higher risk of cellular and molecular damages induced by oxidative stress and their associated pathologies inefficient enzymatic and non-enzymatic anti-oxidant defense systems.

Polymorphic Variants of ASS1 Gene Related to Arginine Metabolism and the Risk of HCC.

BACKGROUND: Hepatocellular carcinoma is a primary liver cancer and 6th most common cancer globally. Inefficient diagnostic strategies and the limited availability of treatments are the foremost reasons. Variable factors directly impact the disease burden, among them, molecular alterations have been found to play a significant role. In liver, argininosuccinate synthase-1 is a center of arginine metabolism and rate limiting enzyme of urea cycle. It also triggers multiple mechanisms that lead to HCC pathogenesis. OBJECTIVES: The aim of this study is to analyze the ASS1 gene expression, its polymorphic genotype and microsatellite instability among HCC patients from our Pakistani population. METHOD: Blood samples were collected from disease and healthy control individuals. Allele-Specific PCR was performed for SNP analysis. MSI of tri and tetra nucleotide repeats were analyzed by PCR. The differential expression of ASS1 gene was also investigated. Furthermore, the reactome database and STRING software were utilized for finding correlations between ASS1 gene with other associated gene/proteins. RESULTS: The GG wild-type genotype was more prevailed in the disease group as compared to the control. Significant downregulation in ASS1 and NOS2 genes was observed. Bioinformatics analysis reveals the correlation between ASS1 polymorphism and HCC development appears to be linked with the EMT pathway and polyamine production. Furthermore, MSI significantly resided in the disease group. Results were analyzed statistically to calculate the significance of obtained results. CONCLUSION: Study concludes that the insight of HCC mechanism through population-specific genetic mutations and altered gene expression of ASS1 might be helpful in early diagnostic and therapeutic purposes.

Association between Interleukin-1ß Gene Polymorphism and Chronic Periodontitis.

OBJECTIVES: Periodontitis is a pathological condition of the oral cavity, originating from multiple factors, including microbial, environmental and genetic factors. The vulnerability to several pathologies has been studied with the relationship to genetic polymorphisms, and one of the most prominent is the single nucleotide polymorphisms throughout the genome. The study aimed to find out the association of single nucleotide polymorphism (SNP) of interleukin-1ß +3954 gene with chronic periodontitis (CP) in Pakistan MATERIALS AND METHODS: This case-control study was conducted at Dow University of Health Sciences. DNA was extracted from the blood and amplified by using conventional polymerase chain reaction of respective genes followed by sequencing. Mann-Whitney test accessed the difference of clinical parameters between cases and controls, and Fisher's exact test was applied to access the association of alleles between subjects. Data entered and analyzed using SPSS 21. RESULTS: Significant differences were observed in clinical parameters in cases and controls (p < 0.001). In the IL-1ß +3954 gene, T alleles were significantly higher in cases as compared with controls (p < 0.001). Genotype CC was significantly dominant in the controls and genotype CT and TT in patients (Chi-square = 19.83, p < 0.001). CONCLUSION: Within the study's limits, IL-1ß +3954 gene polymorphism is associated with periodontitis and is expected to be among the several causes of respective pathology in Pakistan's population.

Whole-Exome Sequencing Identifies Small Mutations in Pakistani Muscular Dystrophy Patients.

Background: Muscular dystrophies are a heterogeneous group of inherited disorders that cannot be diagnosed clinically due to overlapping clinical phenotypes. Whole-exome sequencing is considered as the diagnostic strategy of choice in these cases. In this study we aimed to determine the mutational spectrum of multiplex ligation-dependent probe amplification (MLPA)-negative muscular dystrophy patients in Pakistan using whole-exome sequencing. Subsequently the mutations identified via WES were used to screen additional dystrophinopathy patients by Sanger sequencing. Materials and Methods: DNA extracted from the peripheral blood of three MLPA-negative muscular dystrophy patients was sent for whole-exome sequencing. The identified variants in these 3 patients were then checked in 18 dystrophinopathy patients using Sanger sequencing. Results: Four missense variants and one nonsense variant in the Duchenne muscular dystrophy (DMD) gene were detected. WES diagnosed a DMD patient carrying a nonsense variant c.4375C>T (rs398123953) who can benefit from Ataluren therapy. The other two patients carried missense variant (c.572G>T) in the YARS2 gene (rs11539445) labeling them as patients of MLASA (myopathy, lactic acidosis, and sideroblastic anemia). The identified missense and nonsense variants in the DMD gene were detected in 18 clinically diagnosed dystrophinopathy patients using Sanger sequencing. Three missense variants were detected in our cohort of 18 dystrophinopathy patients. One missense variant c.3406A>T (rs3827462) and a nonsense variant c.4375C>T (rs398123953) were not detected in our cohort of 18 dystrophinopathy patients. Conclusions: Whole-exome sequencing identified a nonsense variant in Pakistani muscular dystrophy patients, which is amenable to treatment by Ataluren and a missense variant in YARS2 gene responsible for causing MLASA.

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.

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.

A comparative study of masticatory performance in complete denture patients before and after application of soft liner.

BACKGROUND: To compare the masticatory performance in complete denture patients before and after application of soft liner and to assess the effect of aging of soft liner on masticatory performance at different intervals of time. METHODS: A total of 30 complete denture wearers between age of 64 and 80 years, were selected from both the sex (20 men, 10 women). This study evaluated the effect of before and after application of chair-side soft liner on masticatory performance of complete denture wearers by using raw carrots and roasted peanut as the test food. This study further evaluated the cushioning effect of chair-side soft liner material at the interval period of 1week, 4 weeks, 8 weeks, 12 weeks and 16 weeks. RESULTS: There were significant differences in masticatory performance without soft liner as compared to with soft liner at different intervals of time. The performance was found to be significantly higher for raw carrot as compared to roasted peanut (p < 0.001). For both the materials peak performance was recorded at 4th week. CONCLUSION: The use of soft liner helps in increasing the masticatory performance among complete denture wearers.

Opuntioside, opuntiol and its metallic nanoparticles attenuate adjuvant-induced arthritis: Novel suppressors of Toll-like receptors -2 and -4.

Rheumatoid arthritis (RA) is a chronic autoimmune disease of synovial inflammation and joint destruction. This study reports anti-arthritic potential of opuntioside-I opuntiol, and its gold and silver nanoparticles (NPs) against Complete Freund's Adjuvant (CFA)-induced arthritic rats. The mechanistic studies were performed targeting TLRs (TLR-2 and TLR-4) and cytokines (IL-1ß and TNF-α) expressions to validate their anti-inflammatory and immuno-modulatory response. The nano-formulations were successfully characterized employing Atomic Force Microscopy (AFM) and Dynamic Light Scattering (DLS) analysis. Opuntiol and opuntioside (OP and OPG: 10, 50 and 100 mg/kg) and opuntiol-coated silver and gold NPs (OP-AgNPs and OP-AuNPs: 0.5, 1 and 3 mg/kg) treatments in arthritic rat have shown minimal arthritic score exhibiting mild to moderate articular changes and tissue swelling in ankle joints. Radiographic examination reveals significant reduction in synovitis with improvement in joints degenarative changes in the presence of aforementioned treatments. Likewise, histology of rat ankle joints depicted comparatively lesser influx of inflammatory cells and diminished granulamatous inflammation. Moreover, treatment groups suppressed protein and mRNA expressions of TLRs (TLR-2 and TLR-4) and cytokines (IL-1ß and TNF-α) levels were also significantly declined in the presence of OPG, OP and its NPs comparing to arthritic control. This investigation concludes, the tested compounds and nano-formulations successfully restored the disease progression in CFA-induced arthritic rat owing to their immunomodulatory and anti-inflammatory potentials and can be considered for RA targeted therapy to address the utmost challenges of the disease.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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 (p = 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 (p = 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. CONCLUSIONS: 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.

Increased risk of breast cancer in multiparous and lactating women attending a breast care clinic in Pakistan: a paradigm shift?

AIMS AND OBJECTIVES: A changing paradigm shift with multiparity (MP) and breast feeding (BF) has been reported in recent years in breast cancer (BC). Our aim was to observe associations of parity, BF and other risk factors with BC among a local population attending a breast care clinic. MATERIALS AND METHODS: A total of 1,039 women (mean age 39 ± 15 years) attended for screening or presented with palpable breast lumps at KIRAN, Pakistan. The majority were in middle and low socioeconomic strata. As per American Cancer Society (ACS) guidelines 2003, mammography and ultrasound were performed, along with fine needle aspiration cytology (FNAC) in 195 women with Breast Imaging Reporting and Data Set (BIRADS) IV/V, high risk patients with BIRADS III on mammography and with suspicious ultrasound findings. RESULTS: The study population was stratified into two groups; one with BC on FNAC in 181 women (17%) and other including 858 healthy women after screening for cancer. The BC group had relative predominance of MP (86% p<0.001), BF (85% p<0.001), family history FH (8% p=0.106) and post-menopause PM (49% p<0.001) as compared to the healthy population. Estimated relative risk (RR) of BC in women with MP, BF, F/H and PM was 3.12 (95% CI=2.05-4.73; p<0.001), 2.47 (95%CI=1.69-3.61; p<0.001), 1.45 (95%CI=0.93-2.41; p=1.06) and 2.33 (95%CI=1.70-3.02; p<0.001) respectively. Higher incidence of BC was observed between 30-40 years 23% (p<0.001) and between 40-50 years 38% (p<0.001). CONCLUSION: MP, BF and PM have significant associations with BC in the studied Pakistani women and this possible paradigm shift now needs to be evaluated for confounding factors.