Autophagy-associated biomarkers ULK2, UVRAG, and miRNAs miR-21, miR-126, and miR-374: Prognostic significance in glioma patients.

As the pioneering study from Pakistan, our research distinctly focuses on validating the roles of autophagy-associated genes and MicroRNAs (miRs) in the unique context of our population for glioma prognosis. The study delves into the nuanced interplay of autophagy within a miR-modulated environment, prompting an exploration of its potential impact on glioma development and survival. Employing real-time PCR (qPCR), we meticulously assessed the expression profiles of autophagy genes and miRs in glioma tissues, complemented by immunohistochemistry on Formalin-fixed paraffin-embedded tissues from the same patients. Our comprehensive statistical analyses, including the data normality hypothesis Shapiro-Wilk test, the Mann-Whitney U-test, Spearman correlation test, and Kaplan-Meier survival analysis, were tailored to unravel the intricate associations specific to low- and high-grade glioma within our population. Clinicopathological analysis revealed a predominance of male patients (66%) with a median age of 35 years. Glioblastoma (32%) and Astrocytoma (36%) were the most prevalent histopathological subtypes. Molecular analysis showed significant correlations between prognostic markers (Ki-67, IDH-1, p53) and clinicopathological factors, including age, histological type, radiotherapy, and chemotherapy. In high-grade glioma, increased expression of AKT and miR-21, coupled with reduced ULK2 and LC3 expression was distinctly observed. While correlation analysis identified a strong positive correlation between ULK2 and UVRAG, PTEN, miR-7, and miR-100 in low-grade glioma, unveiling distinctive molecular signatures unique to our study. Furthermore, a moderate positive correlation emerged between ULK2 and mTOR, miR-7, miR-30, miR-100, miR-204, and miR-374, also between miR-21 and miR-126. Similarly, a positive correlation appeared between ULK2 and AKT, LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7 and miR-374. AKT positively correlated with LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7, miR-30, miR-204, miR-374, miR-126 and miR-21 weakly correlated with AKT and miR-30 in high-grade glioma, providing further insights into the autophagy pathway within our population. The enrichment analysis for miR-21, miR-126, and miR-374 showed MAPK pathway as a common pathway along with Ras, PI3K, and mTOR pathway. The low ULK2, UVRAG, and miR-374 expression group exhibited significantly poor overall survival in glioma, while miR-21 over-expression indicated a poor prognosis in glioma patients, validating it in our population. This study provides comprehensive insights into the molecular landscape of gliomas, highlighting the dysregulation of autophagy genes ULK2, and UVRAG and the associated miR-21, miR-126 and miR-374 as potential prognostic biomarkers and emphasizing their unique significance in shaping survival outcomes in gliomas within the specific context of the Pakistani population.

Differential analysis of histopathological and genetic markers of cancer aggressiveness, and survival difference in EBV-positive and EBV-negative prostate carcinoma.

Several studies have shown an association between prostate carcinoma (PCa) and Epstein-Barr virus (EBV); however, none of the studies so far have identified the histopathological and genetic markers of cancer aggressiveness associated with EBV in PCa tissues. In this study, we used previously characterized EBV-PCR-positive (n = 39) and EBV-negative (n = 60) PCa tissues to perform an IHC-based assessment of key histopathological and molecular markers of PCa aggressiveness (EMT markers, AR expression, perineural invasion, and lymphocytic infiltration characterization). Additionally, we investigated the differential expression of key oncogenes, EMT-associated genes, and PCa-specific oncomiRs, in EBV-positive and -negative tissues, using the qPCR array. Finally, survival benefit analysis was also performed in EBV-positive and EBV-negative PCa patients. The EBV-positive PCa exhibited a higher percentage (80%) of perineural invasion (PNI) compared to EBV-negative PCa (67.3%) samples. Similarly, a higher lymphocytic infiltration was observed in EBV-LMP1-positive PCa samples. The subset characterization of T and B cell lymphocytic infiltration showed a trend of higher intratumoral and tumor stromal lymphocytic infiltration in EBV-negative tissues compared with EBV-positive tissues. The logistic regression analysis showed that EBV-positive status was associated with decreased odds (OR = 0.07; p-value < 0.019) of CD3 intratumoral lymphocytic infiltration in PCa tissues. The analysis of IHC-based expression patterns of EMT markers showed comparable expression of all EMT markers, except vimentin, which showed higher expression in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Furthermore, gene expression analysis showed a statistically significant difference (p < 0.05) in the expression of CDH1, AR, CHEK-2, CDKN-1B, and CDC-20 and oncomiRs miR-126, miR-152-3p, miR-452, miR-145-3p, miR-196a, miR-183-3p, and miR-146b in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Overall, the survival proportion was comparable in both groups. The presence of EBV in the PCa tissues results in an increased expression of certain oncogenes, oncomiRs, and EMT marker (vimentin) and a decrease in CD3 ITL, which may be associated with the aggressive forms of PCa.

Colon Cancer Survival Among South Asian Americans: A Cross-Sectional Analysis of a National Dataset.

INTRODUCTION: Colon cancer (CC) is one of the most common cancers among South Asian Americans (SAAs). The objective of this study was to measure differences in risk-adjusted survival among SAAs with CC compared to non-Hispanic Whites (NHWs) using a representative national dataset from the United States. METHODS: A retrospective analysis of patients with CC in the National Cancer Database (2004-2020) was performed. Differences in presentation, management, median overall survival (OS), three-year survival, and five-year survival between SAAs and NHWs were compared. Kaplan-Meier analysis and multivariable Cox regression were used to assess differences in survival outcomes, adjusting for demographics, presentation, and treatments received. RESULTS: Data from 2873 SAA and 639,488 NHW patients with CC were analyzed. SAAs were younger at diagnosis (62.2 versus 69.5 y, P < 0.001), higher stage (stage III [29.0% versus 26.2%, P = 0.001] or Stage IV [21.4% versus 20.0%, P = 0.001]), and experienced delays to first treatment (SAA 5.9% versus 4.9%, P = 0.003). SAAs with CC had higher OS (median not achieved versus 68.1 mo for NHWs), three-year survival (76.3% versus 63.4%), and five-year survival (69.1% versus 52.9%). On multivariable Cox regression, SAAs with CC had a lower risk of death across all stages (hazard ratio: 0.64, P < 0.001). CONCLUSIONS: In this national study, SAA patients with CC presented earlier in life with more advanced disease, and a higher proportion experienced treatment delay compared to NHW patients. Despite these differences, SAAs had better adjusted OS than NHW, warranting further exploration of tumor biology and socioeconomic determinants of cancer outcomes in SAAs.

Bioethics curriculum for undergraduate medical students: an evaluation study utilizing mixed methods approach.

BACKGROUND: The undergraduate bioethics curriculum introduced in a private medical college in Pakistan in 1988 and revised in 2009 has evolved over time to incorporate globally relevant innovations, including integration of bioethics spirally within an existing problem-based learning curricular framework. The present evaluation study shares the results of this integrated bioethics curriculum delivered for 10 years across the five-year undergraduate medical curriculum. The study assessed the effectiveness of the curriculum in terms of student achievement, appropriateness of course contents and efficiency of instructional methods. METHODS: The study utilized a mixed method sequential explanatory design. The quantitative method was used in the first phase to gather data by utilizing a structured online questionnaire. This was followed by the second phase of qualitative methods to explain the findings of the first phase and enrich the data gathered. This phase was based on focus group discussions and document review. RESULTS: Student and faculty responses showed the curriculum contents to be relevant, informative, and appropriate as per learning objectives and student achievement. Multi-modal instructional methods used were stated to be effective and engaging; small group teaching and shorter sessions suggested to be preferable for fostering discussion and maintaining student engagement and attention. Large class formats were stated to be less effective. Students affirmed the contribution of bioethics education to their personal and professional development and ethical positioning. The majority of students agreed that the curriculum contributed to their knowledge acquisition (60.3-71.2%), skill development (59.41-60.30%) and demonstration of ethical/professional behavior (62.54-67.65%). The ranges indicate agreement with related sets of questions. Participants suggested that the curriculum could be further strengthened by better integration in clinical years, role modelling and providing opportunities for application in clinical health care settings. Moreover, topics like ethical issues related to the use of social media, public health ethics and ethics and law were suggested as additions to the existing curriculum. These findings have regional and global relevance for the development and assessment of effective bioethics curricula. CONCLUSION: An effective bioethics curriculum for undergraduate medical education should run longitudinally across the 5 year curriculum and be integrated in the modules and clerkships. Basic acquisition of knowledge and skills takes place in Years 1 & 2 with reinforcement and application in Years 3-5. Learning embedded in an integrated curriculum can help students recognize, critically analyze and address ethical dilemmas. Involvement and commitment of the clinical faculty is essential for reinforcing the ethical principles and concepts learnt in the earlier years.

Building excellence into medical and health professional education programs.

There is a need for schools that train medical and health professionals to reflect on whether their education program is aligned to current demands and challenges. Such a reflection is not a luxury but a necessity, as achieving minimum standards is not enough. A school should aim for excellence and incorporate best practice in their education program. The ASPIRE-to-Excellence award panels have elaborated on examples of excellence in a number of themes in medical and health professional education. These are presented in a series of articles to be published in Medical Teacher in 2024 and 2025. The frameworks and critical elements described in these articles may be used by institutions as a first step in an evaluation of their program. The frameworks and elements described and examples can be used as a resource for schools and other healthcare learning organizations to consider as they endeavor to improve their education program.

Humoral and T cell responses to SARS-CoV-2 reveal insights into immunity during the early pandemic period in Pakistan.

BACKGROUND: Protection against SARS-CoV-2 is mediated by humoral and T cell responses. Pakistan faced relatively low morbidity and mortality from COVID-19 through the pandemic. To examine the role of prior immunity in the population, we studied IgG antibody response levels, virus neutralizing activity and T cell reactivity to Spike protein in a healthy control group (HG) as compared with COVID-19 cases and individuals from the pre-pandemic period (PP). METHODS: HG and COVID-19 participants were recruited between October 2020 and May 2021. Pre-pandemic sera was collected before 2018. IgG antibodies against Spike and its Receptor Binding Domain (RBD) were determined by ELISA. Virus neutralization activity was determined using a PCR-based micro-neutralization assay. T cell - IFN-γ activation was assessed by ELISpot. RESULTS: Overall, the magnitude of anti-Spike IgG antibody levels as well as seropositivity was greatest in COVID-19 cases (90%) as compared with HG (39.8%) and PP (12.2%). During the study period, Pakistan experienced three COVID-19 waves. We observed that IgG seropositivity to Spike in HG increased from 10.3 to 83.5% during the study, whilst seropositivity to RBD increased from 7.5 to 33.3%. IgG antibodies to Spike and RBD were correlated positively in all three study groups. Virus neutralizing activity was identified in sera of COVID-19, HG and PP. Spike reactive T cells were present in COVID-19, HG and PP groups. Individuals with reactive T cells included those with and without IgG antibodies to Spike. CONCLUSIONS: Antibody and T cell responses to Spike protein in individuals from the pre-pandemic period suggest prior immunity against SARS-CoV-2, most likely from cross-reactive responses. The rising seroprevalence observed in healthy individuals through the pandemic without known COVID-19 may be due to the activation of adaptive immunity from cross-reactive memory B and T cells. This may explain the more favourable COVID-19 outcomes observed in this population.

Investigating the impact of prior COVID-19 on IgG antibody and interferon γ responses after BBIBP-CorV vaccination in a disease endemic population: A prospective observational study.

Background and Aims: COVID-19 vaccinations have reduced morbidity and mortality from the disease. Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) have been associated with immune protection. Seroprevalence studies revealed high immunoglobulin G (IgG) antibody levels to SARS-CoV-2 in the Pakistani population before vaccinations. We investigated the effect of BBIBP-CorV vaccination on circulating IgG antibodies and interferon (IFN)-γ from T cells measured in a cohort of healthy individuals, with respect to age, gender, and history of COVID-19. Methods: The study was conducted between April and October 2021. BBIBP-CorV vaccinated participants were followed up to 24 weeks. Antibodies to SARS-CoV-2 Spike protein and its receptor-binding domain (RBD) were measured. IFNγ secreted by whole blood stimulation of Spike protein and extended genome antigens was determined. Results: Study participants with a history of prior COVID-19 displayed a higher magnitude of IgG antibodies to Spike and RBD. IgG seropositivity was greater in those with prior COVID-19, aged 50 years or younger and in females. At 24 weeks after vaccination, 37.4% of participants showed IFN-γ responses to SARS-CoV-2 antigens. T cell IFN-γ release was higher in those with prior COVID-19 and those aged 50 years or less. Highest IFN-γ release was observed to extended genome antigens in individuals both with and without prior COVID-19. Conclusion: We found that IgG seropositivity to both Spike and RBD was affected by prior COVID-19, age and gender. Importantly, seropositive responses persisted up to 24 weeks after vaccination. Persistence of vaccine induced IgG antibodies may be linked to the high seroprevalence observed earlier in unvaccinated individuals. Increased T cell reactivity to Spike and extended genome antigens reflects cellular activation induced by BBIBP-CorV. COVID-19 vaccination may have longer lasting immune responses in populations with a higher seroprevalence. These data inform on vaccination booster policies for high-risk groups.

Experiences of Undergraduate Medical, Nursing Students and Faculty regarding Flipped Classroom: A Mixed Method Study at Private Medical University in Pakistan.

The 'flipped classroom (FCR)' is a teaching pedagogy where students are actively involved in the learning process. It reduces passivity, enables students to become active learners through reasoning and concept application and facilitates student interaction with their peers and instructors. This instructional approach enhances retention and decreases distraction by engaging students. OBJECTIVES: The purpose of this study was to train the faculty of the medical college and school of nursing in developing FCRs as an innovative teaching and learning strategy, to facilitate them in conducting flipped sessions for their students and to explore the experiences of medical, nursing students along with faculty members regarding the FCR they had attended and conducted. SETTING: Private medical college. PARTICIPANTS: A total of 442 students from medical college and school of nursing and midwifery participated in the evaluation survey with a female to male ratio of 339:103. Students who attended the flipped class sessions were included in the study sample. Students who did not complete the forms were excluded from the study. Nine faculty members who attended the workshop, agreed to facilitate the FCR session were invited to participate in the focus group discussion. RESULTS: Both medical and nursing students found FCR format stimulating. A significantly higher proportion of medical students (73%) found the FCR more engaging and interesting than a traditional lecture as compared with nursing students (59%) (p=0.009). Similarly, 73% of medical students believed the learning objectives of both the non-face-to-face and face-to-face sessions were shared with them as compared with the 62% of nursing students who believed the same (p=0.002). A significantly higher proportion of medical (76%) versus nursing (61%) students found the FCR format more useful for application of their theoretical knowledge into clinical practice (p=0.030). CONCLUSION: Students found the FCR more engaging and interesting in terms of applying theoretical knowledge into practice. Similarly, faculty found this strategy as effective but challenging in terms of involving and engaging students in the learning process. It is recommended to conduct more FCR sessions for an interactive and student-centred learning, but proper planning of the session and using variety of technological tools to engage learners is a key to success.

The Landscape of Stem Cell Research in Pakistan.

Objectives: The present study is a scoping review of the progress of the field of stem cell research (SCR) in Pakistan in the last two decades. METHODS: Data was extracted from electronic search engines, international clinical trial registry platforms, and PubMed and presented in tabular and graphical form. RESULTS: China, India and Iran are investing heavily in SCR. In Pakistan, reasonable growth in terms of the number of publications is observed in this area, however, clinical translation of the field does not demonstrate any considerable progress. The Government of Pakistan has developed the regulatory framework and initiated preliminary policymaking, adopting rules from international regulatory agencies like World Health Organization (WHO) and Federal Drug Authority (FDA), however, further clarity and policymaking are needed to address the growing trend of stem cell tourism in the country. CONCLUSIONS: The field of SCR is still in its infancy in Pakistan, and needs improvement; scientists, academia, policymakers, and funding agencies must come together to foster high-impact stem cell research in the country. This will aid in elevating the economic burden of many incurable diseases in the country. The outcomes of this study will be helpful for policymakers in their decision-making process.

Risk stratification and outcomes in 210 gynecologic perivascular epithelioid cell tumors (PEComas) cases.

OBJECTIVE: To better understand the risk stratification and outcomes of gynecologic PEComas. METHODS: Clinicopathological features and outcomes of gynecologic PEComas cases reported in both English and Chinese literature before September, 2020 were evaluated. The efficacy of three proposed criteria were compared to verify their practicability in gynecologic PEComas. The Chi-square test and Cox proportional hazard model were performed for statistical analysis. RESULTS: A total of 210 cases were retrieved: 95 from English literature and 115 from Chinese literature. The Flope criterion achieved an accuracy of 47% for detecting malignancy of gynecologic PEComas, 64.2% for the Schoolmeester criterion, and 63.8% for the WHO criterion. Both Chi-square test and uni-variate analysis showed that tumor size ≥ 5 cm, infiltrative growth pattern, mitotic rate ≥ 1/50 high per filed (HPF), high nuclear grade and cellularity, necrosis, and vascular invasion were significantly related to recurrence and/or metastasis (R/M) of gynecologic PEComas. Still only high mitotic rate (≥ 1/50 HPF), high nuclear grade and cellularity, and necrosis significantly influenced the long-term survival. Multi-variate analysis showed high nuclear grade and cellularity was an independent risk factor for R/M of gynecologic PEComas. No model was fitted for the death rate due to a small number of events. When defined malignant PEComas cases as meeting three or more out of six clinicopathologic features, the accuracy of such attempt was 62%, but the false-negative rate dropped by 37-55%. CONCLUSIONS: Gynecologic PEComas with three or more high-risk factors may be considered as malignant. Further efforts should be invested to look for new potential prognostic factors.

The outcomes of a mobile just-in-time-learning intervention for teaching bioethics in Pakistan.

INTRODUCTION: The study aimed to test the effectiveness and the feasibility of a mobile just-in-time-learning (m-JiTL) approach for teaching bioethics at a university in Pakistan. Over four months, a mobile app (EthAKUL) was used to enhance ethical reasoning among practising nurses, trainee physicians, and medical and nursing students utilising the m-JiTL approach. Participants used EthAKUL to access bioethics modules and participate in asynchronous discussions. METHODS: A mixed methods design was adopted. Pre- and post-knowledge tests were used to assess changes in participants' knowledge of bioethics concepts, while pre- and post-surveys were used to assess changes in participants' attitudes towards m-learning. After the intervention, focus group discussions with the participants were held. Analysis of the discussion posts and meeting notes was conducted. RESULTS: The learners had a favourable attitude toward using mobile devices for learning purposes at the start of the intervention, and the score remained positive afterwards. Bioethics knowledge test scores improved at the end of the intervention, with medical students experiencing the greatest improvement. However, because of the high drop-out rate and lack of participation after the initial phase, it is unclear whether the increase in score or positive attitude is the result of the intervention, making it difficult to draw firm conclusions about the intervention's success. CONCLUSIONS: EthAKUL is the first of its kind app for teaching bioethics, and the study has offered important insights into adopting new pedagogies and technologies for bioethics teaching. It has also identified issues with the design of the app and m-JiTL pedagogy that must be addressed before curriculum-wide adoption.

IgG antibodies to SARS-CoV-2 in asymptomatic blood donors at two time points in Karachi.

INTRODUCTION: An estimated 1.5 million cases were reported in Pakistan until 23 March, 2022. However, SARS-CoV-2 PCR testing capacity has been limited and the incidence of COVID-19 infections is unknown. Volunteer healthy blood donors can be a control population for assessment of SARS-CoV-2 exposure in the population. We determined COVID-19 seroprevalence during the second pandemic wave in Karachi in donors without known infections or symptoms in 4 weeks prior to enrollment. MATERIALS AND METHODS: We enrolled 558 healthy blood donors at the Aga Khan University Hospital between December 2020 and February 2021. ABO blood groups were determined. Serum IgG reactivity were measured to spike and receptor binding domain (RBD) proteins. RESULTS: Study subjects were predominantly males (99.1%) with a mean age of 29.0±7.4 years. Blood groups were represented by; B (35.8%), O (33.3%), A (23.8%) and AB (7%). Positive IgG responses to spike were detected in 53.4% (95% CI, 49.3-37.5) of blood donors. Positive IgG antibodies to RBD were present in 16.7% (95% CI; 13.6-19.8) of individuals. No significant difference was found between the frequency of IgG antibodies to spike or RBD across age groups. Frequencies of IgG to Spike and RBD antibodies between December 2020 and February 2021 were found to be similar. Seropositivity to either antigen between individuals of different blood groups did not differ. Notably, 31.2% of individuals with IgG antibodies to spike also had IgG antibodies to RBD. Amongst donors who had previously confirmed COVID-19 and were seropositive to spike, 40% had IgG to RBD. CONCLUSIONS: Our study provides insights into the seroprevalence of antibodies to COVID-19 in a healthy cohort in Karachi. The differential dynamics of IgG to spike and RBD likely represent both exposure to SARS-CoV-2 and associate with protective immunity in the population.

Detection and characterization of latency stage of EBV and histopathological analysis of prostatic adenocarcinoma tissues.

The pathophysiology of prostate cancer involves both genetic and acquired factors, including pathogens, such as viruses. A limited number of studies have shown the presence of Epstein-Barr virus (EBV) in prostate cancer tissues. However, there is a dearth of data exploring EBV latency profile in prostate cancer, and the relationship of EBV with histopathological features of prostate cancer. In this study, prostate cancer and benign prostatic hyperplasia (BPH) samples were screened for the presence of EBV, followed by the characterization of the EBV latency profile and analysis of histopathological parameters in EBV-positive and EBV-negative groups. A conventional PCR strategy was employed using virus-specific primers to screen EBV in 99 formalin-fixed paraffin-embedded (FFPE) prostate cancer and 33 BPH samples received for histopathological analysis during the years 2019-2020. Subsequently, cDNA samples were used in a qPCR array to analyze the expression of EBV latency-associated genes to map the latency profile EBV maintains in the samples. Finally, statistical analyses were performed to determine the correlation between EBV and several histopathological features of the samples. EBV was detected in 39% of prostate cancer and 24% of BPH samples. The histopathological analysis of prostate cancer samples identified all samples as prostatic adenocarcinoma of acinar type, while statistical analyses revealed EBV-positive samples to exhibit significantly higher (p < 0.05) Gleason major and total Gleason scores as compared to EBV-negative samples. In the EBV-positive samples, variable expression patterns of latency-associated genes were observed, where most of the samples exhibited EBV latency II/III-like profiles in prostate cancer, while latency-II-like profiles in BPH samples. This study suggests a high prevalence of EBV in prostate samples, where EBV exhibited latency II/III-like profiles. Furthermore, EBV-positive samples exhibited a higher Gleason score suggesting a possible link between EBV and the onset/progression of prostate cancers. However, future functional studies are required to understand the role of the EBV gene expression profile in the onset/progression of prostate cancer.

On pandemics and pivots: a COVID-19 reflection on envisioning the future of medical education.

The required adjustments precipitated by the coronavirus disease 2019 crisis have been challenging, but also represent a critical opportunity for the evolution and potential disruptive and constructive change of medical education. Given that the format of medical education is not fixed, but malleable and in fact must be adaptable to societal needs through ongoing reflexivity, we find ourselves in a potentially transformative learning phase for the field. An Association for Medical Education in Europe ASPIRE Academy group of 18 medical educators from seven countries was formed to consider this opportunity, and identified critical questions for collective reflection on current medical education practices and assumptions, with the attendant challenge to envision the future of medical education. This was achieved through online discussion as well as asynchronous collective reflections by group members. Four major themes and related conclusions arose from this conversation: Why we teach: the humanitarian mission of medicine should be reinforced; what we teach: disaster management, social accountability and embracing an environment of complexity and uncertainty should be the core; how we teach: open pathways to lean medical education and learning by developing learners embedded in a community context; and whom we teach: those willing to take professional responsibility. These collective reflections provide neither fully matured digests of the challenges of our field, nor comprehensive solutions; rather they are offered as a starting point for medical schools to consider as we seek to harness the learning opportunities stimulated by the pandemic.

A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum.

BACKGROUND: Individuals recovering from COVID-19 are known to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for the determination of virus-neutralizing activity in sera of individuals. Here we describe a PCR-based micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. METHODS: The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro-neutralization assay, 19 serum samples, with positive IgG titers against Spike Receptor-Binding Domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. RESULTS: Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated. CONCLUSION: We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses.

MicroRNAs as Potential Biomarkers for Exercise-Based Cancer Rehabilitation in Cancer Survivors.

Expression and functions of microRNAs (miRNAs) have been widely investigated in cancer treatment-induced complications and as a response to physical activity, respectively, but few studies focus on the application of miRNAs as biomarkers in exercise-based cancer rehabilitation. Research has shown that certain miRNA expression is altered substantially due to tissue damage caused by cancer treatment and chronic inflammation. MiRNAs are released from the damaged tissue and can be easily detected in blood plasma. Levels of the miRNA present in peripheral circulation can therefore be used to measure the extent of tissue damage. Moreover, damage to tissues such as cardiac and skeletal muscle significantly affects the individual's health-related fitness, which can be determined using physiologic functional assessments. These physiologic parameters are a measure of tissue health and function and can therefore be correlated with the levels of circulating miRNAs. In this paper, we reviewed miRNAs whose expression is altered during cancer treatment and may correlate to physiological, physical, and psychological changes that significantly impact the quality of life of cancer survivors and their role in response to physical activity. We aim to identify potential miRNAs that can not only be used for monitoring changes that occur in health-related fitness during cancer treatment but can also be used to evaluate response to exercise-based rehabilitation and monitor individual progress through the rehabilitation programme.

Level I Nodal Positivity as a Factor for Involvement of the Submandibular Gland in Oral Cavity Carcinoma: A Case Series Report.

Introduction The routine practice of neck dissection in the surgical management of oral carcinoma has evolved into a more functionally conservative approach. Over time, the rationale for removal of the submandibular gland has been questioned. Routine extirpation of the submandibular gland can aggravate the xerostomia experienced by many patients, significantly affecting their quality of life. Objective The objective of the present study was to determine the incidence of submandibular gland metastases in oral cavity carcinoma and to identify possible factors that may affect their involvement. Methods A total of 149 cases of oral carcinoma presenting at a private tertiary care hospital in Karachi, Pakistan, over the course of 1 year were reviewed retrospectively. Results Histopathological data showed that the submandibular gland was involved in 7 (4.7%) cases. Involvement of level I lymph nodes was found in all of the cases. Direct extension of primary tumor was noted in two cases when the primary tumor was in the floor of the mouth. Conclusion The results suggest that preservation of the submandibular gland during neck dissection for oral carcinoma can be practiced safely when there is no evidence of direct extension of the primary tumor toward the submandibular gland or when there is no clinical or radiological evidence of neck disease in level I. Presence of pathological lymph nodes in level I requires caution when contemplating preservation of the submandibular gland.

Level I Nodal Positivity as a Factor for Involvement of the Submandibular Gland in Oral Cavity Carcinoma: A Case Series Report

Abstract Introduction The routine practice of neck dissection in the surgical management of oral carcinoma has evolved into a more functionally conservative approach. Over time, the rationale for removal of the submandibular gland has been questioned. Routine extirpation of the submandibular gland can aggravate the xerostomia experienced by many patients, significantly affecting their quality of life. Objective The objective of the present study was to determine the incidence of submandibular gland metastases in oral cavity carcinoma and to identify possible factors that may affect their involvement. Methods A total of 149 cases of oral carcinoma presenting at a private tertiary care hospital in Karachi, Pakistan, over the course of 1 year were reviewed retrospectively. Results Histopathological data showed that the submandibular gland was involved in 7 (4.7%) cases. Involvement of level I lymph nodes was found in all of the cases. Direct extension of primary tumor was noted in two cases when the primary tumor was in the floor of the mouth. Conclusion The results suggest that preservation of the submandibular gland during neck dissection for oral carcinoma can be practiced safely when there is no evidence of direct extension of the primary tumor toward the submandibular gland or when there is no clinical or radiological evidence of neck disease in level I. Presence of pathological lymph nodes in level I requires caution when contemplating preservation of the submandibular gland.

The Role of K-Ras and P53 in Biliary Tract Carcinoma.

OBJECTIVE: To focus mainly on the role of proto-oncogene Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) and tumour-suppressor gene p53 which are among the most commonly mutated genes in biliary tract carcinomas. METHODS: The systematic review comprised research articles published between 2002 and 2019 on PubMed and Google Scholar databases which were searched using the terms 'TP53', 'K-Ras', 'mutation', 'biliary tract carcinoma', 'cholangiocarcinoma', and 'murine model'. Repetitions, duplicates and irrelevant articles were excluded. No data was retrieved from posters, presentations and symposiums, and experiments involving bile aspirations were also excluded. RESULTS: Of the 72 articles reviewed, 11(15.3%) were included. Of them, 3(27.3%) studies, conducted in China, Japan and Taiwan, reported a positive correlation between K-Ras mutation and biliary tract carcinoma. Only 1(9%) study, conducted in China, showed the sole correlation between p53 inactivation and biliary tract carcinoma. Also, 4(36.4%) studies, conducted in China, Japan and Europe, showed a positive association of both K-Ras mutation and p53 inactivation with biliary tract carcinoma. CONCLUSIONS: K-Ras and p53 mutation both contribute to biliary tract carcinoma. K-Ras mutation, however, has a much higher frequency compared to p53 inactivation in such cancers.