Gene expression of iron transporters, markers of vascularization and structural integrity in placenta of mothers with and without anemia.

OBJECTIVE: To compare the mRNA expression of placental iron transporters (TfR-1 and FPN), markers of placental vascularization (VEGF and sFLT1) and marker of structural integrity (LMN-A) in term women with and without iron deficiency anemia. MATERIALS AND METHODS: A total of 30 pregnant women were enrolled; 15 cases of iron deficiency anemia (Hb 7-10.9 gm/dL) and 15 gestational age matched healthy controls (Hb ≥ 11 gm/dL). Peripheral venous blood was collected for assessment of hemoglobin levels and serum iron profile. Placental tissue was used for assessing the mRNA expression of TfR-1, FPN, VEGF, sFLT-1 and LMN-A via real time PCR. RESULTS: Placental expression of TfR-1, VEGF and LMN-A was increased in pregnant women with anemia compared to healthy pregnant controls. Placental expression of sFLT-1 was decreased in pregnant women with anemia compared to healthy pregnant controls. There was no change in the placental expression of FPN. CONCLUSION: The increased expression of TfR-1, VEGF and LMN-A in cases of iron deficiency anemia are most likely to be compensatory in nature to help maintain adequate fetal iron delivery. WHAT DOES THIS STUDY ADDS TO THE CLINICAL WORK: Compensatory changes in the placenta aimed at buffering transport of iron to the fetus are seen in pregnant women with anemia compared to healthy pregnant controls.

Suppression of perioperative stress response in elective abdominal surgery: A randomized comparison between dexmedetomidine and epidural block.

Background and Aims: Stress response after surgery induces local and systemic inflammation which may be detrimental if it goes unchecked. Blockade of afferent neurons or inhibition of hypothalamic function may mitigate the stress response. Material and Methods: A total of 50 consenting adult ASA I/II patients undergoing elective abdominal surgery were randomized to receive either dexmedetomidine (Group D) or epidural bupivacaine (Group E) in addition to balanced general anesthesia. Laparoscopic surgery, contraindications to epidural administration, history of psychiatric disorders, obesity (BMI >30 kg/m2), on beta blockers or continuous steroid therapy for >5 days over last 1 year, and known case of endocrine abnormalities or malignancy were excluded. Serum cortisol, blood glucose, and blood urea were estimated. Hemodynamic parameters, total dose of dexmedetomidine, bupivacaine, emergence characteristics, and analgesic consumption over 24 h postoperatively were recorded. Statistical comparisons were done using Student's t-test, repeated measure analysis of variance followed by Dunnett's test, generalized linear model and Chi-square/Fisher's exact test. A P value <0.05 was considered significant. Results: Serum cortisol levels were significantly lower in group E than group D 24 h after surgery (P = 0.029). Intraoperative and postoperative glucose level was lower in group E compared with group D. Time to request of first rescue analgesic was longer in group E than group D (P = 0.040). There was no significant difference between the number of doses of paracetamol required in the postoperative period (P = 0.198). Conclusion: Epidural bupivacaine was more effective than intravenous dexmedetomidine for suppression of neuroendocrine and metabolic response to surgery. Dexmedetomidine provided better hemodynamic stability at the time of noxious stimuli and postoperatively.

Evaluation of maternal plasma platelet activating factor acetylhydrolase activity and mRNA expression in pre-eclampsia: a case control study.

Preeclampsia (PE) remains a leading cause of maternal morbidity and mortality all over the world. However, its aetiology and pathophysiology remain elusive. Platelet activating factor (PAF) is produced in response to oxidative stress and is a potent hypotensive agent. PAF acetylhydrolase (PAF-AH) inactivates PAF and is seen to decrease in normotensive women. The role of PAF-AH in preeclampsia has been in investigational literature, so far. The few studies done have shown a positive association of elevated levels of PAF-AH with preeclampsia. However, this marker has not been studied in the Indian population to-date and such studies are needed to elucidate the pathogenesis of this condition. Our study aimed to determine the PAF-AH activity by spectrophotometric assay in maternal plasma of 73 PE patients versus 73 normotensive controls and plasma PAF-AH mRNA expression to know the aberration of PAF-AH activity at the genetic level. Relative mRNA expression was calculated by Δ DCT method and a fold change was calculated by 2-ΔDCT. We found that the mean plasma PAF-AH activity levels among cases was significantly higher than the normotensive controls. However, the mRNA expression of the PAF-AH gene was similar between the cases and controls, as well as between severe and non-severe preeclampsia (true fold change =1). To conclude, PAF-AH appears to be increased in women with preeclampsia and hence may contribute to pathophysiology and severity. However, a larger sample size will be required to reiterate this association. Recently, PAF-AH inhibitors such as Darapladib has been tested as a therapeutic option in atherosclerosis. After studying the role of PAF-AH in the pathogenesis of PE, PAF-AH inhibitors may be used as a therapeutic tool in the future in PE.IMPACT STATEMENTWhat is already known on this subject? Platelet activating factor (PAF) is produced in response to oxidative stress and is a potent hypotensive agent. PAF acetylhydrolase (PAF-AH) hydrolyses and inactivates PAF and is seen to decrease in normotensive women. The role of platelet activating factor-acetylhydrolase (PAF-AH) in preeclampsia has been investigational so far. Few studies done have shown a positive association of elevated levels of PAF-AH in preeclamptic women.What do the results of this study add? Our study aimed to determine the activity of PAF-AH in maternal plasma of PE patients versus normal pregnancy and plasma PAF-AH mRNA expression to know the aberration of PAF-AH activity at the level of the gene. We found that plasma PAF-AH activity among preeclamptics was significantly higher than in the controls with a possible role in early-onset preeclampsia (<32 weeks), in the Indian population. This marker has never been studied in this population earlier. The results of our study re-emphasised its role in the pathogenesis of preeclampsia.What are the implications of these findings for clinical practice and/or further research? Such studies are important to not only give us a greater understanding of the various pathways involved in this multifactorial dreaded condition, but can also offer us a marker for early identification of women at risk. Recently, PAF-AH inhibitors like Darapladib has been tested as a therapeutic option in atherosclerosis. After studying the role of PAF-AH in the pathogenesis of PE, PAF-AH inhibitors may be used as a therapeutic tool in the future in PE.

Rapid transition to online practical classes in preclinical subjects during COVID-19: Experience from a medical college in North India.

BACKGROUND: COVID-19 pandemic compelled medical schools to opt for online mode in medical education. The competency-based curriculum started in India last year onwards allotted more hours to practical teaching than lectures. As the lockdown extended, there was a need to shift laboratory teaching to online mode. We describe our experience of developing and implementing a framework to rapidly shift practical lab teaching of preclinical subjects to online mode. METHODS: A mixed method study was conducted during the COVID-19 lockdown period in a public funded medical institute of India. A framework utilizing the principles of small group teaching using the available resources was developed and implemented. Online feedback was obtained from students, while in-depth telephonic interview was conducted for teachers. RESULTS: A Demonstrate-Engage-Assess framework for online Practical teaching of Preclinical subjects (DEAPP) was developed and implemented. Feedback was obtained from 103 first year students and six teachers from preclinical subjects. Around 62%-80% students were satisfied with online practical teaching or agreed with benefits of various online tools used in the teaching sessions. Teachers found the framework more planned, and resource efficient, while students found it to be more engaging, enjoyable, and motivated for learning. No face-to-face interaction, non-experiential learning, and adaptation to newer technology were the main barriers perceived in online practical laboratory teaching. CONCLUSION: DEAPP framework was found to be feasible for rapid online transition of practical lab teaching and reported by the students and teachers as engaging, enjoyable and motivated learning.

Role of Survivin and p53 Expression in Response of Primary Culture of Ovarian Cancer Cells to Treatment With Chemotherapeutic Agents.

BACKGROUND: Ovarian cancer is associated with a high relapse rate and is the fifth leading cause of cancer deaths in women. The genetic profile of a tumor is responsible for deciding response to chemotherapeutic agents. In this study, we investigate the relation between survivin and p53 expression and response to chemotherapeutic agents of primary cultures of ovarian cancer cells established from ascitic fluid. MATERIALS AND METHOD: Ascitic fluid and Dulbecco's modified Eagle medium was mixed in equal proportion in culture flasks and incubated to establish primary culture. The cells were treated with different combinations of carboplatin and paclitaxel with and without survivin small interfering RNA transfection. Cell survival was estimated by MTT assay. Survivin and p53 expression was quantified by real-time polymerase chain reaction. RESULTS: Out of 19 ascitic fluid samples, 13 primary cultures of ovarian cancer cells were established. The half maximal inhibitory concentration doses of carboplatin (≥70 µg/mL) and paclitaxel (≥18 µg/mL) were high for 10/13 and 5/13 patients, respectively. Survivin messenger RNA expression was significantly downregulated on treatment with carboplatin (100 µg/mL), paclitaxel (12.5 µg/mL), and a combination of carboplatin (50 µg/mL) and paclitaxel (6.25 µg/mL). Only paclitaxel-treated ovarian cancer cells showed decrease in expression of p53. Survivin small interfering RNA increased sensitivity of the primary cultures to chemotherapeutic agents. CONCLUSIONS: The present study highlights the fact that establishing primary cultures from ascitic fluid may help to develop personalized treatment regime for individual patients based on their molecular profile. Our study also shows that supplementing taxols drugs with survivin inhibitors may prove to be beneficial in the treatment of ovarian cancer patients.

Establishment of Primary Cell Culture From Ascitic Fluid and Solid Tumor Obtained From Epithelial Ovarian Carcinoma Patients.

OBJECTIVE: Ovarian cancer is the seventh leading cause of cancer death worldwide. This is mainly due to late diagnosis and high rate of relapse and resistance following chemotherapy. In the present study, we describe simple and cost-effective method to establish primary culture from ascitic fluid and solid tumor obtained from epithelial ovarian carcinoma patient, which may provide a better tool for in vitro testing of drug sensitivity and designing individualized treatment protocol. METHODS: Complete Dulbecco modified Eagle medium (DMEM) was prepared by supplementing DMEM with 10% fetal bovine serum and antibiotics (ciprofloxacin and amphotericin B). Establishment of primary culture of ovarian cancer cells from ascites fluid and solid tumor was done by using complete DMEM media. RESULTS: Primary cultures of ovarian cancer cells were established from ascitic fluid and solid tumor tissue. Of the 7 ascitic fluid samples, we were able to establish 5 primary cultures of ovarian cancer cells. All the 7 samples were diagnosed as serous papillary adenocarcinoma. Some fibroblasts were also attached to culture flask on day 4; they were removed by exposing them to trypsin for a brief period. On day 7, grape-like clusters were visualized under inverted microscope. The cells became confluent on the 10th and 11th day and showed cobblestone appearance, which is a hallmark of ovarian cancer cells. Senescent irregularly shaped cells that have ceased dividing were seen after 8 to 10 passages. CONCLUSION: This study highlights the fact that establishing primary cultures from ascitic fluid or solid tumor tissue may help us to understand the molecular profile of the cancer cells, which allow us to select the best chemotherapeutic agent for ovarian cancer patients and thus take a step toward patient-tailored therapy so that patients are not exposed to drugs to which they are not likely to respond.

Genetic association of NAD(P)H quinone oxidoreductase (NQO1*2) polymorphism with NQO1 levels and risk of diabetic nephropathy.

NAD(P)H quinone oxidoreductase 1 (NQO1) catalyzes reactions having a cyto-protective effect against redox cycling and oxidative stress. A single base polymorphism (C/T) at nucleotide 609 of the NQO1 gene impairs the stability and function of its protein. Its role in the development of diabetic nephropathy (DN) has not been deciphered. Therefore, this study aimed to evaluate the association of NQO1*2 (rs1800566) polymorphism with plasma NQO1 levels and DN. This study screened 600 participants including healthy controls (HC), type 2 diabetes mellitus without complications (T2DM) and diabetic nephropathy (DN): 200 each for studying NQO1*2 gene polymorphism using the PCR-RFLP. Plasma NQO1 levels were measured by ELISA. Analysis of variance and logistic regression were used to evaluate the association of NQO1 polymorphism with plasma NQO1 levels and DN. The allelic frequencies of NQO1*1/NQO1*2 were 0.88/0.12 in HC, 0.765/0.235 in T2DM and 0.65/0.35 in DN. Carriers of the NQO1*2 allele had significantly lower plasma NQO1 levels (p<0.05) and revealed higher risk towards the development of DN (OR=1.717, p=0.010). NQO1*2 SNP is a functional polymorphism having a significant effect on NQO1 levels. Our results indicate that NQO1*2 genotype may increase susceptibility to DN in north Indian subjects with T2DM.

Idiopathic Fetal Growth Restriction: Repercussion of Modulation in Oxidative Stress.

Oxidative stress has been proposed as one of the causes involved in idiopathic fetal growth restriction (IFGR). However, the exact relationship between oxidative stress and IFGR is not understood. This study aimed at understanding the role of oxidative stress and antioxidant status in IFGR materno-fetal dyads and matched controls. 75 materno-fetal dyads with IFGR were enrolled with equal number of normal low risk controls. Malondialdehyde (MDA) levels were measured as marker of oxidative stress, while paraoxonase-1 (PON1) activity and total antioxidant capacity (TAC) of serum were measured as markers of antioxidant status. MDA levels were increased in both maternal and cord blood of IFGR neonates as compared to controls (p < 0.001). TAC of serum were found to be decreased in IFGR (both maternal and cord blood) as compared to controls (p < 0.001; p < 0.05, respectively). PON1 activity was found to be decreased in the IFGR mothers while it was found increased in IFGR cord blood (p < 0.01; p < 0.001)). IFGR is a state of increased oxidative stress. Decreased PON1 enzymatic activity in mothers is also associated with IFGR.

Mitochondrial Dysfunction, Oxidative Stress and Premature Aging in Children With Nutritional Rickets.

OBJECTIVE: To assess the mitochondrial dysfunction, oxidative stress and premature aging in children with nutritional rickets. METHODS: This cross-sectional study enrolled children between 6 months - 5 years of age with nutritional rickets attending a tertiary care hospital between between January 2021 and August 2022. Mitochondrial dysfunction, oxidative stress and premature aging were assessed by measuring the mitochondrial DNA (mtDNA) content, total antioxidant status (TAOS) and telomere length (TL) in 40 children with nutritional rickets and 40 age- and sex- matched healthy children without rickets (controls). RESULTS: The median (IQR) mtDNA content was significantly higher in children with rickets as compared to controls [152.27 (111.83,218.66) vs 93.7 (72.5,134.14); P < 0.001], implying mitochondrial dysfunction attributed to increased mitochondrial biogenesis in children with rickets. The median (IQR) TAOS was significantly lesser in children with rickets than controls [4.54 (3.93, 5.73) vs 7.86 (5.09, 9.58); P < 0.001)]. The median (IQR) TL in cases was significantly longer in children with rickets compared to controls [417.31 (111.83,218.66) vs 93.7 (72.5,134.14); P < 0.001] implying that children with rickets do not have premature aging. CONCLUSION: Children with rickets have high oxidative stress and mitochondrial dysfunction but no evidence of premature aging.

Evaluation of CRP/Albumin Ratio in Polycystic Ovarian Syndrome.

Background: Polycystic ovarian syndrome is a common endocrine disorder among women of reproductive age. It is characterized by menstrual abnormalities, hyperandrogenism and polycystic ovaries and can lead to many complications. Studies have postulated the role of inflammation in the pathophysiology of PCOS. As acute phase reactants often serve as markers of inflammation, this study aimed to evaluate the role of inflammatory markers in women with PCOS and healthy controls. Material and Methods: A total of 60 participants were enrolled; 30 cases of PCOS and 30 age matched healthy controls. Peripheral venous blood was collected for assessment of CRP, serum albumin, serum total testosterone, serum fasting insulin and fasting blood glucose, following which statistical analysis was done. Results: The CRP/albumin ratio was found to be significantly higher in women with PCOS as compared to healthy controls along with serum total testosterone and HOMA-IR. Correlation between CRP/albumin ratio and the levels of serum total testosterone and insulin resistance was found to be non-significant. Conclusion: An elevated CRP/albumin ratio in cases of PCOS compared to healthy controls supports the hypothesis of inflammation playing a key role in the pathophysiology of PCOS. CRP/albumin ratio can serve as a cheaper biochemical marker of the disease subject to further validation studies to establish its use in Indian population.

Effect of Preoperative Oral Carbohydrate Intake on Perioperative Hyperglycemia in Indian Patients Undergoing Hip Fracture Fixation.

Objective: Preoperative fasting leads to a catabolic state aggravated by surgical stress. This leads to poor patient outcomes. This study aimed to determine the effect of preoperative oral carbohydrate administration on perioperative hyperglycemia and patient comfort. Methods: This prospective, randomized study was conducted on 60 adult American Society of Anesthesiologist I/II patients undergoing hip fracture fixation after obtaining institutional ethical committee clearance. Patients were randomly kept conventionally fasted before surgery (group F, n = 30) or were given oral carbohydrate 2 h before surgery (group C, n = 30). Under all aseptic precautions, a combined spinal epidural block was administered, and surgery was allowed. The primary outcome was blood glucose, and secondary outcomes included incidence of postoperative hyperglycemia, insulin level, blood urea, hunger, thirst, and anxiety. Results: Blood glucose levels were not statistically different between the two groups at baseline (T0; P=0.400), immediately after surgery (T1; P=0.399) and 24h after surgery (T2; P=0.619). The incidence of postoperative hyperglycemia was significantly higher in group F than in group C (P=0.045) at T2. Insulin levels, blood urea levels, and hunger scores were also not statistically different between the groups. The thirst and anxiety scores were lower at T0 and T1 in group C. Conclusion: Preoperative oral carbohydrate administration does not prevent perioperative increases in blood glucose levels. However, it reduces the incidence of perioperative hyperglycemia and decreases perioperative thirst and anxiety, thereby improving the quality of perioperative patient care.

Maternal Serum Vitamin E Levels and its Association with Cord Blood Telomere Length and Mitochondrial DNA Copy Number in Preterm Premature Rupture of Membranes.

Background and Objective: Oxidative stress is one of the pathophysiological factors of pPROM and Vit. E being antioxidant may have preventive role. Study was conducted to estimate maternal serum vitamin E levels and cord blood oxidative stress markers in pPROM cases. Methods: This was a case-control study including 40 pPROM cases and 40 controls. Maternal serum vitamin E levels were measured at recruitment. Cord blood was collected at delivery for estimation of telomere length and mtDNA copy number as oxidative stress markers. Levels were compared using student's t test or Mann Whitney test. For correlation Pearson coefficient was used. Results: Maternal serum vitamin E levels were normal in pPROM cases. Cord blood telomere length was more in pPROM than controls (428.99 ± 290.65 vs 322.35 ± 180.33) (p value 0.05). Cord blood mtDNA copy number was more in pPROM than controls (516.46 ± 443.55 vs 384.77 ± 328.27) (p value 0.13) though it was not significant. mtDNA copy number had negative correlation with Vit. E levels but it was statistically not significant (p value 0.49). There was no association of vitamin E levels with telomere length (p value 0.95). Interpretation and Conclusion: pPROM was not associated with vitamin E deficiency. There was insignificant oxidative stress in cord blood as measured by mtDNA copy number but cord blood telomere length measurement did not detect any oxidative stress in pPPROM cases.

PINK1 and oxidative stress in lean and obese patients with type 2 diabetes mellitus.

AIM: To compare mRNA [messenger RNA] expression of PINK1 in whole blood and the levels of biomarkers of Oxidative Stress (mitochondrial DNA [mtDNA] content & Total Antioxidant status [TAS]) in newly diagnosed lean and obese patients with T2DM. METHODS: Newly diagnosed patients of T2DM were enrolled in this study. The patients were divided into two groups of 30 patients each, lean (BMI < 18.5 kg/m2) and obese (BMI > 25 kg/m2). mRNA expression of PINK1 & mtDNA content was measured by real time PCR. Serum TAS was measured using a commercially available kit. RESULTS: There was a 1.78-fold decrease in mRNA expression of PINK1 in obese group compared to the lean group. Mean mtDNA content was 300.82 ± 169.66 in the obese group and 332.78 ± 147.07 in the lean group (p = 0.06). Mean levels of TAS was 5.39 ± 2.28 µM Trolox Equivalents in the obese group and 3.85 ± 3.33 µM Trolox Equivalents in the lean group (p = 0.001). CONCLUSION: The T2DM patient with obesity had greater OS than the lean patients. Thus, there is a compensatory increase in antioxidants in obese patients with T2DM. Our findings also suggest that decreased levels of PINK1 in obese group are unable to protect the mitochondria against OS leading to decreased mtDNA content. Does it also result in beta cell dysfunction or contribute to insulin resistance in obese patients with T2DM needs to be explored.

Children with severe acute malnutrition have high rates of bone resorption: A cross-sectional comparative study.

Malnutrition in under-5 children (i.e., children younger than age 5 years) remains a major public health problem. Because of the reductive adaptation in children with severe acute malnutrition (SAM), changes in bone health are often subtle. We hypothesized that children with SAM have higher rates of bone resorption than bone formation, which can be assessed using bone turnover markers. In this cross-sectional comparative study, we evaluated the status of bone turnover markers, serum osteocalcin and serum tartrate-resistant acid phosphatase-5b (TRAP-5b) in under-5 children with SAM. Thirty children (6-59 months) with SAM (defined as per World Health Organization criteria) were enrolled as cases and another 30 children (age and sex matched) with normal nutritional status (weight for height -1 standard deviation [SD] to +1 SD) were enrolled for comparison of bone turnover markers. Serum TRAP-5b concentrations were significantly higher in children with SAM compared with children with normal nutritional status (mean [SD] 22.6 [15.3] vs. 11.3 [9.6], P = .009), whereas serum osteocalcin concentrations were comparable between the 2 groups (mean [SD] 40.6 [17.9] vs. 36.0 [12.5], P = .344). Frequency of hypocalcemia and vitamin D deficiency were also significantly high in children with SAM (P < .05). An inverse correlation was found between serum calcium and serum osteocalcin (r = -0.383, P < .05). Our results indicate that children with SAM have a higher bone resorption rate than children with normal nutrition status indicating compromised bone health.

Oxidative Stress, Mitochondrial Dysfunction, and Premature Ageing in Severe Acute Malnutrition in Under-Five Children.

OBJECTIVES: To assess oxidative stress, mitochondrial dysfunction, and premature ageing in children with severe acute malnutrition (SAM). METHODS: This cross-sectional study was conducted in children (1 mo-5 y) with SAM (defined as per WHO criteria) presenting to Pediatrics inpatient department. Oxidative stress, mitochondrial dysfunction, and premature ageing were assessed by measuring and comparing total antioxidant status (TAOS), mitochondrial DNA (mtDNA) content, and telomere length (TL), respectively in 40 under-five children with SAM and 40 age- and sex-matched non-malnourished controls. RESULTS: Oxidative stress was significantly increased in children with SAM, reflected by lower median (IQR) TAOS in cases as compared to controls [10.78 (9.08, 12.3) vs. 16.63 (15.20, 18.03) mM Trolox, p < 0.001]. Median (IQR) mtDNA content was significantly increased in children with SAM [188.7 (105.2, 398.9) vs. 116.2 (67.2, 154.6), p < 0.001]. There was no significant difference in telomere length between cases and controls [1184.5 (894, 1408) vs.1082.6 (823.3, 1479), p = 0.747]. CONCLUSION: Children with SAM had significantly increased oxidative stress that possibly caused mitochondrial dysfunction but no premature ageing.

Association of Paraoxonase-2 (C1053G) Gene Polymorphism with the Expression of Paraoxonase-2 Gene in Patients of Ischemic Stroke - A Pilot Study in Indian Population.

Background and Objective: Oxidative stress plays an important role in atherosclerosis and ischemic stroke. Due to antioxidant properties of Paraoxonase-2, we studied the implication of Paraoxonase-2 gene polymorphism (C1053G) on expression of Paraoxonase-2 gene at mRNA level in ischemic stroke patients. Material and Methods: 40 patients of ischemic stroke and 40 age and sex-matched controls were included. Paraoxonase-2 genotypes were evaluated by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism and expression of Paraoxonase-2 gene at mRNA level was determined by quantitative real time Polymerase Chain Reaction analysed as delta-CT (△CT). Result and Discussion: The observed allele frequencies in patients for C and G allele were 0.61 and 0.39 respectively, and were 0.72 and 0.28 in control group. No significant association was found in C allele of C1053G polymorphism and ischemic stroke. The average △ CT value is significantly (p = 0.0001) higher in patients group (7.68 ± 2.0) as compared to controls (5.70 ± 1.8). We found a significant difference in the average delta-CT value (p = 0.0001), wherein down-regulated paraoxonase-2 gene expression (approximately 0.25 fold) was observed in case of patients as compared to controls. Down-regulated expression of paraoxonase-2 gene was observed in patients with GG genotype as compared to CG and CC genotypes in patients with ischemic stroke (p = 0.0001). Conclusion: Down-regulated Paraoxonase-2 gene expression, as evidenced by low mRNA levels in GG genotype may be one of the contributory factors in the progression of ischemic stroke.

Differential Expression of Suppressor of Cytokine Signaling and Interferon Gamma in Lean and Obese Patients with Type 2 Diabetes Mellitus.

Background: The model of obesity-induced insulin resistance has long been used to explain the development of type 2 diabetes mellitus (T2DM) in obese individuals (body mass index (BMI) > 25 kg/m2), but this model failed to explain the development of the disease in lean individuals (BMI < 18.5 kg/m2). Defects in the insulin signaling pathway have been postulated to play a role in these patients, particularly in suppressors of cytokine signaling (SOCS) proteins, which are involved in the downregulation of insulin transduction. The expression of SOCS is also known to be induced by cytokines such as interferon gamma (IFN-γ). It is still not clear whether these pathways operate differently in lean versus obese patients with T2DM. Therefore, this pilot study was designed to study the expression of SOCS1, SOCS3, and IFN-γ in lean and obese patients with T2DM. Objectives: The levels of IFN-γ in serum and the messenger RNA (mRNA) expression of SOCS (SOCS1 and SOCS3) and IFN-γ genes in whole blood in lean and obese patients with T2DM. Methods: Sixty newly diagnosed T2DM patients (not on any pharmacotherapy) were enrolled and divided into 2 groups of lean (BMI < 18.5 kg/m2) and obese (BMI > 25 kg/m2) patients (n = 30 per group). Serum IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA), and mRNA expression of IFN-γ, SOCS1, and SOCS3 was measured by real-time polymerase chain reaction (PCR) using the ∆∆ Ct method. Results: Serum IFN-γ levels were 10.83 ± 5.81 pg/mL in the lean group and 9.35 ± 5.14 pg/mL in the obese group (P = 0.02). Fasting serum insulin levels were 16.07 ± 8.39 µIU/mL in the lean group and 27.11 ± 4 .91 µIU/mL in the obese group (P = 0.001). There was a 3.16-fold increase in mRNA expression of IFN-γ and a 1.3-fold increase in mRNA expression of SOCS1 in the lean group compared to the obese group. mRNA expression of SOCS3 was similar in both groups. Conclusions: The level of IFN-γ increased at both transcriptional and translational levels, and mRNA expression of SOCS1 was higher in the lean group than in the obese group. The SOCS protein is a known negative regulator in insulin signaling pathways. Thus, our findings and available scientific literature suggest that IFN-γ might impair the insulin signaling pathway to a greater extent in lean patients than in obese patients via induction of SOCS1. This signaling pathway could be a major contributing factor to hyperglycemia in lean patients with T2DM compared with obese counterparts. This suggests that different therapeutic approaches to these groups might be of greater benefit in the treatment of T2DM.

Reduced fetal urine production rate-An early marker of fetal inflammatory response syndrome in preterm premature rupture of membranes: Prospective cohort study.

OBJECTIVE: To find association between fetal urine production rate (FUPR) and fetal inflammatory response syndrome (FIRS) in preterm premature rupture of membranes (PPROM). METHODS: A prospective cohort study of 70 pregnant women with PPROM at 28-34 weeks of pregnancy was conducted. FUPR was calculated by performing serial fetal bladder volume measurements ultrasonographically and was repeated weekly until delivery. After delivery, cord blood interleukin-6 (IL-6) levels were measured. Placental tissue histopathology was performed and neonatal outcomes were noted. RESULTS: Out of 70 recruited patients with PPROM, 44 had evidence of FIRS (62.86%). Mean FUPR at the time of delivery was significantly reduced in neonates with evidence of FIRS compared with the Non-FIRS group (13.89 ± 8.06 ml/h vs. 25.89 ± 4.94 ml/h). Out of 41 patients with reduced FUPR, 39 neonates had FIRS whereas only five out of 29 neonates with normal FUPR had FIRS (P < 0.001). Severe neonatal morbidity was found in 24 out of 41 (58.54%) neonates with reduced FUPR prenatally. The occurrence of respiratory distress syndrome, necrotizing enterocolitis, and sepsis was significantly high in neonates with reduced FUPR. CONCLUSION: Reduced FUPR is strongly associated with FIRS in cases of PPROM and hence can be used as an early predictor of adverse neonatal outcomes.

CpG hypermethylation of the C-myc promoter by dsRNA results in growth suppression.

Deregulation of the c-myc proto-oncogene plays an important role in carcinogenesis. It is, therefore, commonly found to be overexpressed in various types of tumors. Downregulation of c-myc expression assumes great importance in tumor therapy because of its ability to promote and maintain cancer stem cells. Apart from post-transcriptional gene silencing (PTGS), siRNAs have also been shown to cause transcriptional gene silencing (TGS) through epigenetic modifications of a gene locus. This approach can potentially be used to silence genes for longer periods and at a much lesser dosage than PTGS. In this study, we have examined the effect of transfection of a novel siRNA directed against a CpG island encompassing the CT-I(2) region in the P2 promoter of c-myc in U87MG and other cell lines. Transient transfection with this siRNA resulted in c-myc promoter CpG hypermethylation and decreased expression of c-myc (both mRNA and protein) and its downstream targets. A decrease was also observed in the expression of some stemness markers (oct-4 and nanog). Stable transfection also confirmed the promoter CpG hypermethylation and reduced c-myc expression along with reduced cell proliferation and an increase in apoptosis and senescence. A significant decrease in c-myc levels was also observed in three other cancer cell lines after transient transfection under similar conditions. Thus this novel siRNA has the capability of becoming an effective therapeutic tool in malignancies with overexpression of c-myc and may be of particular use in the eradication of recalcitrant cancer stem cells.

Quality Indicators for Evaluating Errors in the Preanalytical Phase.

Objective The aim of this study was to study the incidence of preanalytical errors in the clinical chemistry laboratory attached to a tertiary care hospital. Design and Methods The study was conducted in a clinical chemistry laboratory using the samples and forms received for analysis. Five hundred random samples were analyzed using a predefined set of quality indicators (QIs) over a period of 3 months. The incidence of each preanalytical error was described as a percentage of the total samples analyzed in the study. Statistical Analysis Individual QIs were assigned values as 0 and 1 and were used to assess each sample; 0 if the error was present, and 1 if absent. The incidence of each preanalytical error was described as a percentage of the total samples analyzed in the study. Result Out of the 500 samples observed, 138 samples were error free, while 21 samples had the maximum number of errors, that is, 6. The error committed most often was the omission of provisional diagnosis being mentioned on the requisition form. No preanalytical error was observed for QIs: selecting the appropriate blood collection vial or storage of sample. Conclusion This study confirms that error rate in the preanalytical phase is high and vastly ignored. Errors committed here may be overlooked, given the large number of samples received in the clinical laboratory of a tertiary center. To reduce these errors, the laboratory should provide training to all workers involved in the preanalytical phase. Daily or weekly QI scores should be recorded to assess and rectify shortcomings, thereby improving patient care.