Seasonal differences in physical activity, sedentary behaviour, and sleep patterns in people with type 1 diabetes in Kuwait.

AIMS: The main aim of the current study was to measure physical activity, sedentary behaviors and sleep levels across the different seasons in people with type 1 diabetes in Kuwait. METHODS: A prospective cross-sectional study was conducted from August 2021 to September 2022. Physical activity and sleep metrics were measured over a 7-day period with a wrist-worn accelerometer (GENEActiv). Overall physical activity was measured as a Euclidean Norm Minus One in milli gravitational units (mg). Accelerometer metrics were compared across the seasons and between the sex. RESULTS: A total of 784 people with type 1 diabetes participated. Mean daily physical activity was 25.2 mg (SD = 7.3). Seasonal differences were seen in overall physical activity (p = 0.05), inactivity (p = 0.04), light activity (p = 0.001), the intensity gradient (p = 0.001) and sleep efficiency (p = 0.02). Poorer metrics were generally seen in Spring and Summer. Overall physical activity, moderate and vigorous physical activity, and inactivity were significantly higher in males compared to females (p ≤ 0.02). Females had a longer sleeping duration (p = 0.02), and higher sleep efficiency (p = 0.04) and light physical activity (p = 0.01). Overall physical activity and the intensity gradient were negatively associated with HbA1c (both p = 0.01). CONCLUSIONS: Physical activity levels were generally low and sleep poor in people with type 1 diabetes in Kuwait and these varied by sex and season. The current data are useful to target and develop interventions to improve physical activity and glycemic control.

Prevalence and predictors of diabetes distress and depression in people with type 1 diabetes.

Background: People living with diabetes often encounter psychosocial challenges, including diabetes distress and depression. Despite this, little research has focused on the co-occurrence of these conditions. This study aimed to explore the prevalence of depressive symptoms and diabetes distress in people with type 1 diabetes in Kuwait and to identify clinical and demographic factors associated with these conditions. Methods: A total of 832 people with type 1 diabetes (females: 54.1%, mean age: 29 ± 8.5 years), were invited to participate in Dose Adjustment for Normal Eating (DAFNE) course. Diabetes distress was measured using the Problem Areas in Diabetes (PAID) scale and depressive symptoms were measured using the Patient Health Questionnaire-9 (PHQ-9). Depressive symptoms were defined as PHQ-9 scores ≥10. Data on biomedical outcomes, lifestyle factors, and sociodemographic information were collected. Results: The prevalence rates of diabetes distress and depressive symptoms were 27.8% and 38.3%, respectively. Notably, 19.6% of people experienced both conditions. In the regression analysis, PAID scale and PHQ-9 scores were significantly associated, patients with higher score on depressive symptoms scale were more likely to suffer diabetes distress (B= 2.65, p < 0.001). Female sex (odds ratio [OR]= 2.2, 95% CI= 1.5, 3.2), higher hemoglobin A1c levels (OR= 1.6, 95% CI= 1.0, 2.5), obesity (OR= 1.7, 95% CI= 1.1, 2.8), inactivity (OR= 2.4, 95% CI= 1.6, 3.6), microvascular complications (OR= 2.8, 95% CI= 1.5, 5.4), and lipohypertrophy (OR= 1.7, 95% CI= 1.1, 2.5) were associated with greater odds for the co-occurrence of diabetes distress and depressive symptoms (p< 0.05 for all). Conclusion: The majority of people with type 1 diabetes in Kuwait experience both diabetes distress and depressive symptoms. The strong correlation between diabetes distress and depressive symptoms suggests mutual predictability. The co-occurrence of both symptoms is associated with many sociodemographic and clinical factors.

Identification of potential regulatory mechanisms and therapeutic targets for lung cancer.

Lung cancer poses a significant health threat globally, especially in regions like India, with 5-year survival rates remain alarmingly low. Our study aimed to uncover key markers for effective treatment and early detection. We identified specific genes related to lung cancer using the BioXpress database and delved into their roles through DAVID enrichment analysis. By employing network theory, we explored the intricate interactions within lung cancer networks, identifying ASPM and MKI67 as crucial regulator genes. Predictions of microRNA and transcription factor interactions provided additional insights. Examining gene expression patterns using GEPIA and KM Plotter revealed the clinical relevance of these key genes. In our pursuit of targeted therapies, Drug Bank pointed to methotrexate as a potential drug for the identified key regulator genes. Confirming this, molecular docking studies through Swiss Dock showed promising binding interactions. To ensure stability, we conducted molecular dynamics simulations using the AMBER 16 suite. In summary, our study pinpoints ASPM and MKI67 as vital regulators in lung cancer networks. The identification of hub genes and functional pathways enhances our understanding of molecular processes, offering potential therapeutic targets. Importantly, methotrexate emerged as a promising drug candidate, supported by robust docking and simulation studies. These findings lay a solid foundation for further experimental validations and hold promise for advancing personalized therapeutic strategies in lung cancer.Communicated by Ramaswamy H. Sarma.

Effect of COVID-19 vaccine on blood glucose metrics in Arabic people with type 1 diabetes.

Introduction: People with diabetes are at a higher risk for coronavirus disease-19 (COVID-19) and hence are prioritized for vaccination. The aim of the current study was to investigate the effects of COVID-19 vaccination on blood glucose control in Arabic people with type 1 diabetes (T1D). Secondary aim was to compare the responses between the two vaccines approved for use in Kuwait. Method: This retrospective study compared ambulatory glucose metrics, using a continuous glucose monitoring device, measured for 14 days before, and 7 days and 14 days after, the first and second dose of the COVID-19 vaccine in Arabic people with Type 1 diabetes (T1D). We also explored possible links with vaccine type and other clinical characteristics. Glucose metrics calculated were time in range (TIR, 3.9-10 mmol/L), time above range (TAR, 10.1- 13.9 mmol/L or >13.9 mmol/L), time below range (TBR, 3- 3.9 mmol/L or <3 mmol/L) and glucose variability (CV). Results: We enrolled 223 participants in the study. Over the 7 days period after the first vaccination dose there was a decrease in TIR (mean difference (SD) -1.9% ± 14.8%; p = 0.05) and increase in TAR >10 mmol/L (2.2% ± 15.9%; p = 0.04), with no effects on TBR. These effects were not seen after the second dose or 14 days after either dose. There was a decrease in CV over the 7 days period after the first (-1.2% ± 7.4%; p = 0.02) and second vaccine doses (-1.1% ± 6.9%; p = 0.03), with no effects noted 14 days after either dose. In subgroup analysis similar effects on TIR and TAR were also seen in those who had received the viral vector-based vaccine, but not the mRNA-based vaccine, although the decrease in CV was seen in those who had received the mRNA based vaccine but not the viral vector-based vaccine. Conclusion: We found a temporary impairment in glucose control in the first 7 days, particularly among individuals receiving viral vector vaccines. The group receiving mRNA vaccine was likely to experience an increase in glucose levels above the target range. However, the temporary change in metrics appears to return to pre-vaccination levels after one-week post-vaccination. The effects on glycemic parameters were more neutral after the second dose.

Association Between Depression, Diabetes Self-Care Activity and Glycemic Control in an Arab Population with Type 2 Diabetes.

Purpose: Poor self-care behavior and depression are associated with worse glycemic control, but the relationship between these variables is poorly reported. The present study aimed to describe self-care behaviors and explore the association between depression, self-care and glycemic control in people with type 2 diabetes in an Arab population. Patients and Methods: This study recruited 446 patients with type-2 diabetes from an outpatient clinic in Kuwait and completed assessments of diabetes self-care using the Summary of Diabetes Self-Care Activities (SDSCA) questionnaire and depression using the Patients Health Questionnaire-9 (PHQ-9) scales. Multiple linear regression models were used to determine the associations between depression, self-care behaviors and glycemic control. Results: Self-care behaviors were highest for medication taking (92.9%), an average for foot care (61.4%), blood glucose testing (60%) and healthy diet (55.7%), and lowest for exercise (27.1%). Depression, poor self-care activities and poor HbA1c levels were inter-correlated in univariate analysis (p< 0.03). In regression analysis, following healthful eating (B= -0.27, p< 0.001), general eating plan (B= -0.21, p= 0.01), more space carbohydrates evenly throughout the day (B= - 0.17, p= 0.03) and exercise at least 30 minutes (B= -0.17, p= 0.02) behaviors were associated with better HbA1c levels. However, depression was associated with poor scores of following a general eating plan (B= -0.20, p= 0.03), space carbohydrates evenly throughout the day (B= -0.21, p= 0.05), and exercising at least 30 minutes (B= -0.24, p= 0.03) behaviors. Depression scores were also associated with poor HbA1c levels (B= 0.32, p= 0.03). However, this association no longer persists after mutual adjustment with following a healthy diet (B= 0.14, p= 0.38) and exercise (B = 0.26, p = 0.08) behaviors. Conclusion: Self-care activity was less than fifty percent in people with type 2 diabetes and higher activity was associated with better glycemic control. However, depression adversely affects self-care behaviors. To maintain and achieve glycemic control, diabetes educators might motivate people with type 2 diabetes to enhance their self-care activities, particularly those with depression symptoms and poor compliance with self-care activities.

Glucagon-like peptide-1 agonists combined with sodium-glucose cotransporter-2 inhibitors reduce weight in type 1 diabetes.

OBJECTIVE: This study evaluated whether adding sodium-glucose cotransporter-2 inhibitors (SGLT2i) and/or glucagon-like peptide-1 receptor agonists (GLP1-RA) to insulin reduced weight and glycemia in people with type 1 diabetes. METHODS: This retrospective analysis of electronic health records evaluated 296 people with type 1 diabetes over 12 months after medications were first prescribed. Four groups were defined: control n = 80, SGLT2i n = 94, GLP1-RA n = 82, and combination of drugs (Combo) n = 40. We measured changes at 1 year in weight and glycated hemoglobin (HbA1c). RESULTS: The control group did not have changes in weight or glycemic control. The mean (SD) percentage weight loss after 12 months was 4.4% (6.0%), 8.2%  (8.5%), and 9.0% (8.4%) in the SGLT2i, GLP1-RA, and Combo groups, respectively (p < 0.001). The Combo group lost the most weight (p < 0.001). The HbA1c reduction was 0.4% (0.7%), 0.3% (0.7%), and 0.6% (0.8%) in the SGLT2i, GLP1-RA, and Combo groups, respectively (p < 0.001). The Combo group had the biggest improvements in glycemic control and total and low-density lipoprotein cholesterol compared with baseline (all p < 0.01). Severe adverse events were similar between all the groups, with no increased risk of diabetic ketoacidosis. CONCLUSIONS: The SGLT2i and GLP1-RA agents on their own improved body weight and glycemia, but combining the medications resulted in more weight loss. Treatment intensification appears to result in benefits with no difference in severe adverse events.

Anticandidal Activity of Capsaicin and Its Effect on Ergosterol Biosynthesis and Membrane Integrity of Candida albicans.

Oral candidiasis is an infection of the oral cavity commonly caused by Candida albicans. Endodontic treatment failure has also been found to be persistent from C. albicans in the root canal system. Despite the availability of antifungal drugs, the management of Candida oral infection is difficult as it exhibits resistance to a different class of antifungal drugs. Therefore, it is necessary to discover new antifungal compounds to cure fungal infections. This study aimed to examine the antifungal susceptibility of Capsaicin, an active compound of chili pepper. The susceptibility of Capsaicin and Fluconazole was tested against the Candida species by the CLSI (M27-A3) method. The effect of Capsaicin on the fungal cell wall was examined by the ergosterol inhibitory assay and observed by the scanning electron micrograph. The MIC range of Capsaicin against Candida isolates from oral (n = 30), endodontic (n = 8), and ATCC strains (n = 2) was 12.5−50 µg/mL. The MIC range of Fluconazole (128- 4 µg/mL) significantly decreased (2- to 4-fold) after the combination with Capsaicin (MIC/4) (p < 0.05). Capsaicin (at MIC) significantly reduced the mature biofilm of C. albicans by 70 to 89% (p < 0.01). The ergosterol content of the cell wall decreased significantly with the increase in the Capsaicin dose (p < 0.01). Capsaicin showed high sensitivity against the hyphae formation and demonstrated a more than 71% reduction in mature biofilm. A fluorescence microscopy revealed the membrane disruption of Capsaicin-treated C. albicans cells, whereas a micrograph of electron microscopy showed the distorted cells' shape, ruptured cell walls, and shrinkage of cells after the release of intracellular content. The results conclude that Capsaicin had a potential antifungal activity that inhibits the ergosterol biosynthesis in the cell wall, and therefore, the cells' structure and integrity were disrupted. More importantly, Capsaicin synergistically enhanced the Fluconazole antifungal activity, and the synergistic effect might be helpful in the prevention of Fluconazole resistance development and reduced drug-dosing.

IMC based robust PI/PID controllers for time-delayed inverse response processes.

In the last decade, several works have been reported for stable and integrating processes to achieve a specified maximum sensitivity. Also, internal model control (IMC) is a popular controller design strategy as it has only one tuning parameter. IMC based controllers are available in literature for time-delayed inverse response processes but none of the reported works provide guidelines for selecting the tuning parameter. In the present work, IMC-PI/PID controllers for time-delayed inverse response processes are reported. To achieve a specified maximum sensitivity in the range of 1.4 to 2.0, set of tuning rules is proposed for the tuning parameter. Normalized form of the transfer functions are used in the present method which simplifies the design procedure. Novelty of the proposed approach is that the user can not only tune the tuning parameter for desired maximum sensitivity, but can also switch from smooth (Ms = 1.4) to tight (Ms = 2.0) control. Simulation results illustrate the effectiveness of the proposed tuning rules.

Effectiveness of virtual mindfulness-based interventions on perceived anxiety and depression of physicians during the COVID-19 pandemic: A pre-post experimental study.

Background: The outbreak of coronavirus disease 2019 (COVID-19) created unprecedented stress on physicians. Mindfulness is a type of meditation that focuses on being fully present, aware of senses, and emotions in the present moment without analyzing or judging them, and it may help reduce psychological distress in physicians. This study aimed to examine the effectiveness of virtual mindfulness-based intervention (MBI) on physicians' perceived anxiety and depression and different facets of mindfulness. Methods: During the COVID-19 pandemic, an online survey was administered to physicians to assess depression, anxiety, and awareness using the 9-item Patient Health Questionnaire (PHQ-9), 7-item General Anxiety Disorder (GAD-7), and Five-Facets Mindfulness Questionnaire (FFMQ), respectively. Physicians that received the virtual MBI sessions also completed post-questionnaires at a 3-week follow-up time point. Results: A total of 125 physicians responded to the online survey, with 56 completing the MBI. The prevalence of moderate to severe anxiety and depression was 45.0 and 46.7%, respectively. Mindfulness scores were negatively associated with depression (r = -0.38, P < 0.001) and anxiety (r = -0.36, p < 0.001). Mindfulness scores for the 56 physicians who received virtual MBI sessions were significantly improved (mean difference ± SD, 17.7 ± 16.1, p = 0.001). Significant reductions were also evidenced in anxiety (4.4 ± 4.2) and depression (4.5 ± 5.1) scores (p's < 0.001). There was also an improvement in mindfulness facets of observing (5.1 ± 4.7), describing (2.3 ± 4.3), acting with awareness (2.7 ± 5.3), non-judging of inner experience (3.6 ± 6.1), and non-reactivity to inner experience (3.9 ± 4.0) (p's < 0.001). A facet of mindfulness, acting with awareness was most efficiently associated with improved anxiety (B = -0.3, p = 0.02) and depression (B = -0.4, p = 0.01). Conclusion: This study has demonstrated that virtual MBI improved physicians' psychological wellbeing and mindfulness during the crisis. Regular mindfulness practice may help physicians to tolerate and handle unpleasant circumstances, such as future epidemics or pandemics.

A critical review of obstetric and gynecological physical examination videos available on YouTube: Content analysis and user engagement evaluation.

BACKGROUND: Video-sharing website "YouTube" is a growing source of healthcare information. But, the videos uploaded on this open platform are not peer reviewed, therefore, the information available needs to be sufficiently evaluated. No studies have been conducted to evaluate the authenticity and utility of obstetrics and gynecology (Obs/Gyne) physical examination YouTube videos. This study was performed to analyze the sources, contents, and quality of videos about the Obs/Gyne clinical examination available on YouTube. METHODS: A systematic search was performed on YouTube website using the following key words: "OBSTETRIC," "GYNECOLOGICAL," "SPECULUM OBSTETRIC," "OBSTETRIC CLINICAL," "BIMANUAL PELVIC," and "EXAMINATION" to analyze the sources, contents, and the quality of YouTube videos about the Obs/Gyne clinical examination during the period between November 2015 and March 2017. The videos were classified into educationally useful and useless based on the content, accuracy of the knowledge, and the demonstration. RESULTS: Out of total 457 screened videos, 176 (38.51%) videos met the pre-set inclusion criteria. After review, out of 176 pertinent videos, 84 (47.7%) videos were found educationally useful, and out of these 84 useful videos, only 29 (34.5%) were highly educational in nature. CONCLUSION: YouTube videos showed variable educational value. Only, a small number of videos were identified as useful and can be used by the medical students for self-directed learning and by the clinical teachers for educational purposes or other academic activities.

Correction: Self-reported cheating among medical students: An alarming finding in a cross-sectional study from Saudi Arabia.

[This corrects the article DOI: 10.1371/journal.pone.0194963.].

Immunopathogenesis of Liver Injury During Hepatitis C Virus Infection.

The present report describes current concepts about the mechanism of liver cell injury caused by host immune response against hepatitis C virus (HCV) infection in human beings. This report is based on the observations from experimental studies and follow-up actions on human liver diseases. The results from different investigations suggest that liver injury depends on the presentation of viral antigen and the level of host immune response raised against HCV-related peptides. Both innate and adaptive immunity are triggered to counter the viral onset. During development of host immunity, the cell-mediated immune response involving CD4+ Th1 cells and CD8+ cytotoxic T-lymphocyte (CTL) cells were found to play a major role in causing liver damage. The hepatic Innate lymphoid cells (ILCs) subsets are involved in the immune regulation of different liver diseases: viral hepatitis, mechanical liver injury, and fibrosis. Humoral immunity and natural killer (NK) cell action also contributed in liver cell injury by antibody-dependent cellular cytotoxicity (ADCC). In fact, immunopathogenesis of HCV infection is a complex phenomenon where regulation of immune response at several steps decides the possibility of viral elimination or persistence. Regulation of immune response was noted starting from viral-host interaction to immune reaction cascade engaged in cell damage. The activation or suppression of interferon-stimulated genes, NK cell action, CTL inducement by regulatory T cells (Treg), B cell proliferation, and so on was demonstrated during HCV infection. Involvement of HLA in antigen presentation, as well as types of viral genotypes, also influenced host immune response against HCV peptides. The combined effect of all these effector mechanisms ultimately decides the progression of viral onset to acute or chronic infection. In conclusion, immunopathogenesis of liver injury after HCV infection may be ascribed mainly to host immune response. Second, it is cell-mediated immunity that plays a predominant role in liver cell damage.

Prevalence of diabetic comorbidities and knowledge and practices of foot care among diabetic patients: a cross-sectional study.

BACKGROUND: Diabetes mellitus with comorbid complications constitute a major public health problem worldwide. The aim of this study was to evaluate the risk of comorbid complications with glycosylated hemoglobin levels and diabetes duration. Also assessed were patients' diabetic foot-care knowledge and practices. PATIENTS AND METHODS: This was a quasiexperimental study. A total of 360 type 2 diabetes mellitus patients were interviewed at a government health care center in Riyadh, Saudi Arabia. Diabetic complications and HbA1c-level data were collected from hospital records. A standard questionnaire was used to assess their diabetic foot-care knowledge and practice. RESULTS: Of the type 2 diabetes mellitus patients, 32.5% had highly uncontrolled glycosylated hemoglobin (HbA1c) levels (≥8.6%) and 62.8% had had diabetes >10 years. The patients had comorbid complications, such as hypertension (61.4%), dyslipidemia (58.6%), retinopathy (23.3%), heart disease (14.4%), and severe foot complications (3.9%). The majority of highly uncontrolled HbA1c-level patients had retinopathy (OR 8.90, P=0.0001), foot complications (OR 8.09, P=0.0001), dyslipidemia (OR 2.81, P=0.010), and hypertension (OR 2.0, P=0.028) compared to the controlled HbA1c-level (<7%) group. Patients with diabetes >10 years also had higher prevalence of foot complications (OR 2.92, P=0.0001), retinopathy (OR 2.17, P=0.011), and hypertension (OR 1.67, P=0.033). From patient responses, physicians examined only 34.2% of patient feet and 36.7% of patients received physicians advice for foot care. About 70% of patients had knowledge of diabetic foot care; however, only 41.7% of patients always examined their feet, 41.4% washed feet with warm water, 31.4% carefully dried between the toes, and 33.1% were using foot-moisturizing substances. CONCLUSION: The prevalence of comorbid complications was higher in the Saudi population compared to other international studies. Also, foot-care practice was not satisfactory. Therefore, there is need of improvement and increased awareness among physicians and patients to check regularly for proper care of the diabetic foot to avoid diabetic foot-related complications.

Self-reported cheating among medical students: An alarming finding in a cross-sectional study from Saudi Arabia.

Academic misconduct/dishonesty has become widespread behavior among many university students across the globe, and medical education is not an exception. Until recently, few efforts have been made to study the dishonest behavior in Middle-Eastern universities. This study examined the prevalence and predisposing factors of cheating among medical students in Saudi Arabia and suggests suitable preventive measures. A cross-sectional survey-based study was conducted at a government medical college during the 2014-2015 academic year. The response rate was 58.5% (421/720). The overall cheating behavior practiced by the participants was 29%, predominantly by male students. High GPA scoring students were the least likely to cheat. The participants living with their families were more likely to cheat compared to those who were living apart from their families. The reasons participants gave to justify their cheating behavior included getting better grades, passing the course, and lacking preparation while still recognizing that cheating is a 'mistake.' Overall, significant academic misconduct concerning cheating was found among the Saudi medical students; this misconduct is alarming in a reputable government institution. The implementation of strict punishments, requiring ethical courses and creating ethical awareness by exploiting the potential of Islamic religious belief might help to control this problem.

Prevalence and predictors of peripheral neuropathy in nondiabetic children with chronic kidney disease.

INTRODUCTION: This study sought to determine the prevalence and predictors of peripheral neuropathy in nondiabetic children with chronic kidney disease (CKD). METHODS: Fifty-one consecutive normally nourished children, 3-18 years of age, with CKD stages IV and V of nondiabetic etiology were enrolled from May to December 2012. Nerve conduction studies were performed in 50 children. Blood samples were analyzed for the biochemical parameters, trace elements, and micronutrients. RESULTS: The prevalence of peripheral neuropathy in our cohort was 52% (95% confidence interval 37.65, 66.34). The majority (80.8%) of the children had axonal neuropathy, and 11.5% had demyelinating neuropathy. Isolated motor neuropathy was identified in 92.3% of the children, and sensorimotor neuropathy was identified in 7.6%. The significant risk factors associated with peripheral neuropathy were older age, low serum copper, and dialysis therapy. DISCUSSION: Electrodiagnostic studies should be performed in children with CKD to assess for peripheral neuropathy for the purpose of optimizing medical care. Muscle Nerve 57: 792-798, 2018.

Effectiveness of longitudinal faculty development programs on MCQs items writing skills: A follow-up study.

This study examines the long-term impact of the faculty development programs on the multiple choice question (MCQ) items' quality leading to study its effect on the students' overall competency level during their yearly academic assessment. A series of longitudinal highly constructed faculty development workshops were conducted to improve the quality of the MCQs items writing skills. A total of 2207 MCQs were constructed by 58 participants for the assessment of 882 students' cognitive competency level during the academic years 2012-2015. The MCQs were analyzed for the difficulty index (P-value), discriminating index (DI), presence/absence of item writing flaws (IWFs), and non-functioning distractors (NFDs), Bloom's taxonomy cognitive levels, test reliability, and the rate of students' scoring. Significant improvement in the difficulty index and DI were noticed during each successive academic year. Easy and poor discriminating questions, NFDs and IWFs were decreased significantly, whereas distractor efficiency (DE) mean score and high cognitive level (K2) questions were increased substantially during the each successive academic year. Improved MCQs' quality leaded to increased competency level of the borderline students. Overall, the longitudinal faculty development workshops help in improving the quality of the MCQs items writing skills of the faculty that leads to students' high competency levels.

The natural compound magnolol affects growth, biofilm formation, and ultrastructure of oral Candida isolates.

The incidence of oral candidosis has increased in recent years due to the escalation in HIV-infection, cancer treatments, organ transplantation, and diabetes. In addition, corticosteroid use, dentures, and broad-spectrum antibiotic use have also contributed to the problem. Treatment of oral candidosis has continued to be problematic because of the potential toxicity of antifungals in clinical use, and, above all, development of drug resistance among patients. In this study, the antifungal effect of magnolol was investigated against 64 strains of Candida spp. (four standard and 60 oral isolates) through minimum inhibitory concentration (MIC) and growth curve assays. Insight into the mechanisms of the antifungal action has been gained through ultrastructural studies using confocal scanning laser microscopy (CSLM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Molecular docking was done for predicting the interactions of magnolol with ergosterol at supramolecular level. The toxicity of magnolol on human erythrocytes was measured by in vitro hemolytic assay. MIC values of magnolol ranged from 16-64 µg/ml, respectively. All tested isolates showed a marked sensitivity towards magnolol in growth curve assays. Biofilm results suggested that magnolol showed strong anti-biofilm activity. The results obtained for four different Candida spp. demonstrated that MBIC values of magnolol showed the average biofilm inhibition by 69.5%, respectively. CLSM experiments showed that cells exposed to magnolol (MIC) exhibited cell membrane disruption. SEM analysis of magnolol treated cells resulted in deformed cells. TEM micrographs showed rupturing of the cell wall and plasma membrane, releasing the intracellular content, and swelling of the cell wall. Hemolytic activity of magnolol is 11.9% at its highest MIC compared to an activity level of 25.4% shown by amphotericin B (Amp B) at 1 µg/ml. Lipinski's parameters calculated for magnolol suggested its good oral bioavailability. Docking studies indicated that magnolol might be interacting with ergosterol in the fungal cell membranes. Together, the present study provides enough evidence for further work on magnolol so that better strategies could be employed to treat oral candidosis.

A Single-step Multiplex Quantitative Real Time Polymerase Chain Reaction Assay for Hepatitis C Virus Genotypes.

BACKGROUND AND OBJECTIVES: The variable response of hepatitis C virus (HCV) genotypes towards anti-viral treatment requires prior information on the genotype status before planning a therapeutic strategy. Although assays for typing or subtyping of HCV are available, however, a fast and reliable assay system is still needed. The present study was planned to develop a single-step multiplex quantitative real time polymerase chain reaction (qPCR) assay to determine HCV genotypes in patients' sera. METHODS: The conserved sequences from 5' UTR, core and NS5b regions of HCV genome were used to design primers and hydrolysis probes labeled with fluorophores. Starting with the standardization of singleplex (qPCR) for each individual HCV-genotype, the experimental conditions were finally optimized for the development of multiplex assay. The sensitivity and specificity were assessed both for singleplex and multiplex assays. Using the template concentration of 102 copies per microliter, the value of quantification cycle (Cq) and the limit of detection (LOD) were also compared for both singleplex and multiplex assays. Similarly, the merit of multiplex assay was also compared with sequence analysis and restriction fragment length polymorphism (RFLP) techniques used for HCV genotyping. In order to find the application of multiplex qPCR assay, it was used for genotyping in a panel of 98 sera positive for HCV RNA after screening a total number of 239 patients with various liver diseases. RESULTS: The results demonstrated the presence of genotype 1 in 26 of 98 (26.53%) sera, genotype 3 in 65 (66.32%) and genotype 4 in 2 (2.04%) sera samples, respectively. One sample showed mixed infection of genotype 1 and 3. Five samples could not show the presence of any genotype. Genotypes 2, 5 and 6 could not be detected in these sera samples. The analysis of sera by singleplex and RFLP indicated the results of multiplex to be comparable with singleplex and with clear merit of multiplex over RFLP. In addition, the results of multiplex assay were also found to be comparable with those from sequence analysis. The sensitivity, specificity, Cq values and LOD values were compared and found to be closely associated both for singleplex and multiplex assays. CONCLUSION: The multiplex qPCR assay was found to be a fast, specific and sensitive method that can be used as a technique of choice for HCV genotyping in all routine laboratories.

Development of an Immunoassay for Detection of Torque Teno Virus (TTV) Antibodies Using the N22 Expression Product from TTV Genotype 2.

AIMS: This study describes an immunoassay to detect anti-torque teno virus (TTV) antibodies using a peptide obtained from expression of the N22 region of TTV genotype 2. METHODS: The N22 region (∼500 bp) of TTV genotype 2 was cloned in a pET-28a(+) vector and expressed in ZYM-5052 autoinduction medium. Following metal affinity chromatography, a purified polypeptide was used as an antigen for the development of an immunoassay to detect anti-TTV antibodies in human sera. RESULTS: Recombinant protein (∼25-kDa) was obtained after 24 h of incubation at 25°C in ZYM-5052 autoinduction medium. A blot assay developed using this polypeptide as an antigen and TTV-positive sera as the primary antibody produced a distinct spot on the nitrocellulose membrane. Serum samples from 36 of 42 patients with renal disease and 29 of 48 patients with liver diseases produced a positive signal using this immunoassay. Simultaneously, 18 of 48 healthy controls were also detected to be positive for anti-TTV antibodies. These results were found to be comparable with TTV detection using PCR, and the assay showed a high sensitivity and specificity (i.e., 97.44 and 91.67%, respectively). Moreover, this assay could detect TTV infection irrespectively of the genotype, including cases of mixed infection. CONCLUSION: The present immunoassay using the N22 expression product may be used as an alternative to PCR to detect TTV infection in large populations.

Molecular basis of hepatocellular carcinoma induced by hepatitis C virus infection.

Present study outlines a comprehensive view of published information about the underlying mechanisms operational for progression of chronic hepatitis C virus (HCV) infection to development of hepatocellular carcinoma (HCC). These reports are based on the results of animal experiments and human based studies. Although, the exact delineated mechanism is not yet established, there are evidences available to emphasize the involvement of HCV induced chronic inflammation, oxidative stress, insulin resistance, endoplasmic reticulum stress, hepato steatosis and liver fibrosis in the progression of HCV chronic disease to HCC. Persistent infection with replicating HCV not only initiates several liver alterations but also creates an environment for development of liver cancer. Various studies have reported that HCV acts both directly as well as indirectly in promoting this process. Whereas HCV related proteins, like HCV core, E1, E2, NS3 and NS5A, modulate signal pathways dysregulating cell cycle and cell metabolism, the chronic infection produces similar changes in an indirect way. HCV is an RNA virus and does not integrate with host genome and therefore, HCV induced hepatocarcinogenesis pursues a totally different mechanism causing imbalance between suppressors and proto-oncogenes and genomic integrity. However, the exact mechanism of HCC inducement still needs a full understanding of various steps involved in this process.