Characterization of structural changes occurring in insulin at different time intervals at room temperature by surface-enhanced Raman spectroscopy.

BACKGROUND: Insulin storage above the temperature recommended by food and drug administration (FDA) causes decrease in its functional efficacy due to degradation and aggregation of its protein based active pharmaceutical ingredient (API) that results poor glycemic control in diabetic patients. The aggregation of protein causes serious neurodegenerative diseases such as type-2 diabetes, Huntington disease, Parkinson's disease, and Alzheimer's disease. Surface-enhanced Raman spectroscopy (SERS) has been employed for the denaturation study of many proteins at the temperature above the recommendations of food and drug administration (FDA) (above 30 °C) which indicates potential of technique for such studies. OBJECTIVE: SERS along with multivariate discriminating analysis techniques-based analysis of degradation of liquid pharmaceutical insulin protein after regular intervals of time at room temperature to analyze the structural changes in this protein during the storage of insulin pharmaceutical at room temperature. METHODS: Silver nanoparticles (Ag-NPs) prepared by chemical reduction method are used as SERS active substrate for the surface enhancement of the insulin spectral signal. SERS spectral measurements of insulin were collected from eight different samples of insulin in the time range of 7 pm to 7 am first at fridge temperature (5 °C), second after half hour and next six with the time difference of 2 h each time at room temperature. The acquired SERS spectral data was preprocessed and analyzed. SERS structural transformations detection and discrimination potential in insulin was further confirmed by applying multivariate discriminating analysis techniques including principal component analysis (PCA) and Partial least square regression analysis (PLSR). RESULTS: SERS significantly detects the structural changes produced in insulin even after 2 h of insulin placement at room temperature. PCA successfully differentiates the insulin spectral data obtained after regular intervals of time according to PC-1 (77 %) explained variance. Application of PLSR model provides quantitative confirmation of SERS efficiency, by providing insulin data regression coefficients plot, efficient prediction of time with calibration data set having 0.77 mean square absolute error of calibration (RMSAEC), validation data set with 0.80 mean square absolute error of prediction (RMSAEP) and 0.98 coefficient of determination (R2) for both calibration and validation data set. CONCLUSION: SERS is proved as a highly sensitive and discriminating technique to detect and discriminate insulin structural changes after regular intervals of time at room temperature.

Surface-enhanced Raman spectroscopy for monitoring antibacterial activity of imidazole derivative (1-benzyl-3-(sec­butyl)-1H-imidazole-3-ium bromide) against Bacillus subtilis and Escherichia coli.

BACKGROUND: Bacterial resistance against antibiotics remains a challenge and Raman Spectroscopy (SERS) may provide critical information concerning this. OBJECTIVES: In the current study, surface enhances Raman spectroscopy (SERS) has been used to determine the biochemical changes induced during the antibacterial activity of the in house synthesized imidazole derivative (1-benzyl-3-(sec­butyl)-1H-imidazole-3-ium bromide) in comparison to commercially available drugs (fasygien) against both gram-positive and gram-negative bacteria. METHODS: For this purpose, the antibacterial activity of this compound was assessed on Bacillus subtilis and Escherichia coli. The SERS spectral changes are detected which can be associated with the biochemical changes in the bacterial cells as a result of the application of both drugs, including fasygien and the imidazole derivative drug demonstrating the technique's potential for analyzing the antibacterial activities of drug candidates. RESULTS: The chemometric techniques such as Principal Component Analysis (PCA) and Partial Least Squares-Discriminant Analysis (PLS-DA) were performed for the differentiation of SERS spectral data sets of unexposed, exposed with imidazole derivative and commercially available antibacterial drugs for two different bacteria including E. coli and Bacillus. CONCLUSIONS: PCA was found helpful for the qualitative differentiation of all drug-treated E. coli and Bacillus in the form of separate clusters of spectral data sets and PLS-DA discriminated the unexposed and the exposed bacteria with imidazole derivative and commercially available drug with 93% sensitivity and 96% specificity for Bacillus and with 90% sensitivity and 89% specificity for E. coli.

Surface-enhanced Raman spectroscopy of centrifuged blood serum samples of diabetic type II patients by using 50KDa filter devices.

Blood serum contains essential biochemical information which are used for early disease diagnosis. Blood serum consisted of higher molecular weight fractions (HMWF) and lower molecular weight fractions (LMWF). The disease biomarkers are lower molecular weight fraction proteins, and their contribution to disease diagnosis is suppressed due to higher molecular weight fraction proteins. To diagnose diabetes in early stages are difficult because of the presence of huge amount of these HMWF. In the current study, surface-enhanced Raman spectroscopy (SERS) are employed to diagnose diabetes after centrifugation of serum samples using Amicon ultra filter devices of 50 kDa which produced two fractions of whole blood serum of filtrate, low molecular weight fraction, and residue, high molecular weight fraction. Furthermore SERS is employed to study the LMW fractions of healthy and diseased samples. Some prominent SERS bands are observed at 725 cm-1, 842 cm-1, 1025 cm-1, 959 cm-1, and 1447 cm-1 due to small molecular weight proteins, and these biomarkers helped to diagnose the disease early stage. Moreover, chemometric techniques such as principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) are employed to check the potential of surface-enhanced Raman spectroscopy for the differentiation and classifications of the blood serum samples. SERS can be employed for the early diagnosis and screening of biochemical changes during type II diabetes.

Frequency of complete remission after standard 3+7 induction therapy in patients with acute myeloid leukemia.

Objectives: To find the frequency of complete remission rate after standard 3+7 induction therapy in patients with Acute Myeloid Leukemia (AML) among different clinicopathological groups. Methods: Non-probability purposive sampling technique was used to collect data from July 2016 to Jan 2017, conducted at Department of Oncology, Jinnah Hospital Lahore. Sample size of 50 cases was calculated with 95% confidence level, 14% margin of error and taking expected percentage of complete remission (CR) 57% in AML patients after 3+7 induction therapy. Results: Out of 46 patients, majority had AML-M2. AML- Not Otherwise Specified (NOS) disease. Most common pathological presentation was Total Leucocyte Count (TLC) of <50,000/mm3 (60%) as compared to patients with hyperleukocytosis i.e., TLC >50,000/mm3 (40%). Twenty-two percent had low Lactate Dehydrogenase (LDH) level involvement and 78% had high LDH level involvement. As compared to target of 57 %, complete response was observed in 54% patients, (p<0.02) with better results in younger age group, male patients with low LDH and TLC level. Conclusion: It was concluded that 3+7 induction chemotherapy has 54% CR rates in patients with AML. Whereas, in AML-M5, AML-M6, AML-NOS patients especially, with high LDH and TLC and patients with advanced age, CR rate is low and needs more aggressive treatment.

Usefulness of convalescent plasma transfusion for the treatment of severely ill COVID-19 patients in Pakistan.

BACKGROUND: Convalescent plasma(CP) was utilized as potential therapy during COVID-19 pandemic in Pakistan. The study aimed at appraisal of CP transfusion safety and usefulness in COVID pneumonia. METHODS: Single arm, MEURI study design of non-randomized open label trial was conducted in five centers. Patients werecategorized as moderately severe, severe, and critical. The primary endpoint was a) improvement in clinical status and change in category of disease severity; secondary endpoint was b) CP ability to halt disease progression to invasive ventilation. CP transfused to hospitalized patients. Statistical tests including median (interquartile ranges), Mann-Whitney U test, Fisher's exact test using SPSS ver. 23, ANOVA and Chi-square test were applied for the analysis of results parameters before and after CP treatment. SOFA score was applied for multiorgan failure in severe and critical cases. RESULTS: A total of 50 adult patients; median age 58.5 years (range: 29-92 years) received CP with infusion titers; median 1:320 U/mL (Interquartile range 1:80-1:320) between April 4 to May 5, 2020. The median time from onset of symptoms to enrollment in trial was 3 to 7 days with shortness of breath and lung infiltration as severity criterion. In 35 (70%) recipients, oxygen saturation improved from 80 to 95% within 72h, with resolution of lung infiltrates. Primary endpoint was achieved in 44 (88%) recipients whereas secondary endpoint was achieved in 42 (84%). No patient experienced severe adverse events. A high SOFA score (> 7) correlated with deaths in severe and critical patients. Eight (16%) patients expired due to comorbidities; cardiac arrest in 2 (4%), multiorgan failure secondary to cytokine storm in 5 (10%) and ventilator associated complications in 1 (2%). CONCLUSION: CP transfusion can be used as a safe and useful treatment in moderately severe and severe patients. TRIAL REGISTRATION: The trial registration number is NCT04352751  ( https://www.irct.ir/search/result?query=IRCT20200414047072N1 ). Trial Registration date is 28th April 2020.

Knowledge, attitude and practices of medical undergraduates of Rawalpindi Medical University regarding potential organ donation.

OBJECTIVE: To determine the knowledge, attitude and practices of medical undergraduates regarding potential organ donation. METHODS: The descriptive cross-sectional study was conducted from April to July 2017 at Rawalpindi Medical University, Rawalpindi, Pakistan and comprised medical students of either gender selected from each of the five academic years. Data was collected using a structured questionnaire on knowledge, attitude and practices. Data was analysed using SPSS 22. RESULTS: Of the 400 subjects, there were 40(10%) male and 40(10%) female students from each of the five academic years. The mean age of the sample was 20.98+/-1.63 years (range: 18-27 years). Overall, 363(90.8%) students had knowledge of organ donation and 96(24%) knew about potential organ donation. Of the 184(46%) subjects willing for live donation, 86(46.7%) were males and 98(53.2%) were females. Of the 245(61.5%) subjects willing for potential organ donation, 62(77.5%) were from the 5th year and 30(37.5%) were from the 1st year. CONCLUSIONS: Medical students were found to have good knowledge regarding live organ donation, but they had very little information about potential organ donation.

Association of Hypertension with Non-HDL Cholesterol in Patients with Type 2 Diabetes Mellitus.

OBJECTIVE: To determine the association of hypertension with Non-HDL Cholesterol (Non-HDL.C) among patients with type 2 diabetes mellitus. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY:  Shifa Foundation Community Health Centre, Shifa International Hospital, Islamabad, from January to December 2019. METHODOLOGY:  This study comprised of 214 patients with type 2 diabetes. Detailed history, examination, anthropometric measurements, fasting lipid profiles and blood glucose levels were recorded for all patients. Non-HDL.C was determined in all patients by subtracting HDL Cholesterol (HDL.C) from total Cholesterol (Total.C). Association of hypertension with Non-HDL.C was analysed using Chi-square Fisher Exact test for qualitative data and post-stratification t-test for quantitative data. Correlation of rising levels of Non-HDL.C with Total.C, LDL.C, Triglycerides, HDL.C and fasting blood sugar was analysed using ANOVA. SPSS version 23 was used for statistical analysis. RESULTS: Non-HDL.C was raised in 169 (78.97%) patients with type 2 diabetes. However, there was no significant difference in the mean Non-HDL.C among patients with hypertension and without hypertension (156.35 +38.52 mg/dl versus 156.81 +45.82 mg/dl; p = 0.936). Furthermore, there was no significant difference in the Total.C, LDL.C, Triglycerides and the number of patients with raised Non-HDL.C among both groups. Increasing values of Non-HDL.C correlated significantly to Total.C, LDL.C and Triglycerides (p <0.001). CONCLUSION: Hypertension had no significant association with Non-HDL.C in patients with type 2 diabetes. However, the raised level of Non-HDL.C in majority of type 2 diabetic patients in addition to significant correlation with Total.C, LDL.C and Triglycerides necessitates its screening irrespective of the blood pressure status. Key Words: Non-HDL cholesterol, Dyslipidemia, Diabetes, Hypertension.

First report of Chikungunya fever in Rawalpindi, Pakistan.

BACKGROUND: Chikungunya shares many clinical features with dengue fever, but to date, no case has been reported in Rawalpindi and surrounding areas. AIMS: To detect the presence in Rawalpindi of chikungunya masquerading as dengue fever. METHODS: An observational study was conducted at Rawalpindi Medical University from July to December 2017. Patients with clinical features suggestive of dengue fever, but negative for dengue virus NS1 antigen were included and tested at the National Institute of Health Islamabad, Pakistan, for chikungunya using reverse transcription polymerase chain reaction. RESULTS: We tested 129 patients and 28 were positive for chikungunya. There were 17 (60.7%) men and 11 (39.3%) women, with a mean age of 32.53 years (range 16-60 years). All had fever at presentation. Other clinical features at presentation were noted, such as fever, chills, fatigue, headache, myalgia, arthralgia, retro-orbital pain, abdominal pain, nausea, and diarrhoea. No long-term sequelae or bleeding diatheses were seen and there was no mortality reported. CONCLUSIONS: The clinical features observed and investigated confirmed our reporting of the first case of chikungunya in Rawalpindi, Pakistan.

Perceptions and attitudes towards research amongst medical students at Shifa College of Medicine.

OBJECTIVE: To understand medical students' perceptions and attitudes towards research to help facilitators design specific courses according to their needs. METHODS: The cross-sectional study was conducted at Shifa College of Medicine, Islamabad, Pakistan, from May to November 2013, and comprised undergraduate medical students. A pre-tested questionnaire was used for data collection. Students' response was recorded on a Likert scale from 'strongly disagree' 1 to 'strongly agree' 5. Analysis was done using statistical SPSS17. RESULTS: Of the 195 students enrolled, 172(88%) responded. Overall, 78(45.3%) students said they were aware of research. Research was considered useful for their professional careers and relevant to their daily life by 133(65.7%) students, while 72(41.9%) did not consider it worthwhile to pursue research as a career. Besides, 71(41.3%) students enjoyed research, while 120(70%) perceived research as stressful and 107(62.2) complex. CONCLUSIONS: Most students considered research valuable but at the same time they perceived it as stressful and complex.