CRISPR/Cas genome editing revealed non-angiogenic role of VEGFA gene in porcine luteal cells: a preliminary report.

BACKGROUND: The angiogenic cytokine vascular endothelial growth factor A (VEGFA) also exerts non-angiogenic effects on endocrine functionality of porcine luteal cells critical for progesterone (P4) production. METHOD AND RESULTS: The expression dynamics of VEGFA-FLT/KDR system were investigated using RT-qPCR during luteal stages and VEGFA gene knock out (KO) porcine luteal cells were generated using CRISPR/Cas9 technology. The downstream effects of VEGFA ablation were studied using RT-qPCR, Annexin V, MTT, ELISA for P4 estimation and scratch wound assay. Bioinformatics analysis of RNA-Seq data of porcine mid-luteal stage was conducted for exploring protein-protein interaction network, KEGG pathways, transcription factors and kinase mapping for VEGFA-FLT/KDR interactomes. The VEGFA-FLT/KDR system expressed throughout the luteal stages with highest expression during mid- luteal stage. Cellular morphology, structure and oil-red-o staining for lipid droplets did not differ significantly between VEGFA KO and wild type cells, however, VEGFA KO significantly decreased (p < 0.05) viability and proliferation efficiency of edited cells on subsequent passages. Expression of apoptotic gene, CASP3 and hypoxia related gene, HIF1A were significantly (p < 0.05) upregulated in KO cells. The relative mRNA expression of VEGFA and steroidogenic genes STAR, CYP11A1 and HSD3B1 decreased significantly (p < 0.05) upon KO, which was further validated by the significant (p < 0.05) decrease in P4 output from KO cells. Bioinformatics analysis mapped VEGFA-FLT/KDR system to signalling pathways associated with steroidogenic cell functionality and survival, which complemented the findings of the study. CONCLUSION: The ablation of VEGFA gene resulted in decreased steroidogenic capability of luteal cells, which suggests that VEGFA exerts additional non-angiogenic regulatory effects in luteal cell functionality.

Androgen receptor gene deficiency results in the reduction of steroidogenic potential in porcine luteal cells.

Luteal steroidogenesis is critical to implantation and pregnancy maintenance in mammals. The role of androgen receptors (AR) in the progesterone (P4) producing luteal cells of porcine corpus luteum (CL) remains unexplored. The aim of the present study was to establish AR gene knock out (KO) porcine luteal cell culture system model by CRISPR/Cas9 genome editing technology and to study the downstream effects of AR gene deficiency on steroidogenic potential and viability of luteal cells. For this purpose, genomic cleavage detection assay, microscopy, RT-qPCR, ELISA, annexin, MTT, and viability assay complemented by bioinformatics analysis were employed. There was significant downregulation (p < 0.05) in the relative mRNA expression of steroidogenic marker genes STAR, CYP11A1, HSD3B1 in AR KO luteal cells as compared to the control group, which was further validated by the significant (p < 0.05) decrease in the P4 production. Significant decrease (p < 0.05) in relative viability on third passage were also observed. The relative mRNA expression of hypoxia related gene HIF1A was significantly (p < 0.05) downregulated in AR KO luteal cells. Protein-protein interaction analysis mapped AR to signaling pathways associated with luteal cell functionality. These findings suggests that AR gene functionality is critical to luteal cell steroidogenesis in porcine.

A Study to Compare Ultrasound-guided and Clinically Guided Fluid Management in Children with Septic Shock.

Background: To evaluate the role of ultrasound during initial fluid resuscitation along with clinical guidance in reducing the incidence of fluid overload on day 3 in children with septic shock. Materials and methods: It was a prospective, parallel limb open-labeled randomized controlled superiority trial done in the PICU of a government-aided tertiary care hospital in Eastern India. Patient enrolment took place between June 2021 and March 2022. Fifty-six children aged between 1 month and 12 years, with proven or suspected septic shock, were randomized to receive either ultrasound-guided or clinically guided fluid boluses (1:1 ratio) and subsequently followed up for various outcomes. The primary outcome was frequency of fluid overload on day 3 of admission. The treatment group received ultrasound-guided fluid boluses along with the clinical guidance and the control group received the same but without ultrasound guidance upto 60 mL/kg of fluid boluses. Results: The frequency of fluid overload on day 3 of admission was significantly lower in the ultrasound group (25% vs 62%, p = 0.012) as was the median (IQR) cumulative fluid balance percentage on day 3 [6.5 (3.3-10.3) vs 11.3 (5.4-17.5), p = 0.02]. The amount of fluid bolus administered was also significantly lower by ultrasound [median 40 (30-50) vs 50 (40-80) mL/kg, p = 0.003]. Resuscitation time was shorter in the ultrasound group (13.4 ± 5.6 vs 20.5 ± 8 h, p = 0.002). Conclusion: Ultrasound-guided fluid boluses were found to be significantly better than clinically guided therapy, in preventing fluid overload and its associated complications in children with septic shock. These factors make ultrasound a potentially useful tool for resuscitation of children with septic shock in the PICU. How to cite this article: Kaiser RS, Sarkar M, Raut SK, Mahapatra MK, Uz Zaman MA, Roy O, et al. A Study to Compare Ultrasound-guided and Clinically Guided Fluid Management in Children with Septic Shock. Indian J Crit Care Med 2023;27(2):139-146.

Transcriptome profiling of different developmental stages of corpus luteum during the estrous cycle in pigs.

To better understand the molecular basis of corpus luteum (CL) development and function RNA-Seq was utilized to identify differentially expressed genes (DEGs) in porcine CL during different physiological stages of the estrous cycle viz. early (EL), mid (ML), late (LL) and regressed (R) luteal. Stage wise comparisons obtained 717 (EL vs. ML), 568 (EL vs. LL), 527 (EL vs. R), 786 (ML vs. LL), 474 (ML vs. R) and 534 (LL vs. R) DEGs with log2(FC) ≥1 and p < 0.05. The process of angiogenesis, steroidogenesis, signal transduction, translation, cell proliferation and tissue remodelling were significantly (p < 0.05) enriched in EL, ML and LL stages, where as apoptosis was most active in regressed stage. Pathway analysis revealed that most annotated genes were associated with lipid metabolism, translation, immune and endocrine system pathways depicting intra-luteal control of diverse CL function. The network analysis identified genes AR, FOS, CDKN1A, which were likely the novel hub genes regulating CL physiology.

Blood Pressure Variation with Altitudes in Children: A Cross-Sectional Observational Study from Himalayan Hills.

OBJECTIVES: We aimed to evaluate blood pressure (BP) in Indian children who had similar demographic characteristics but hailed from different altitudes. METHODS: BP of school going children, aged 5 to 12 years, at five different locations varying in altitude (near sea level: n = 425; 2000 feet: n = 244; 4000 feet: n = 248; 6000 feet: n = 242 and 8000 feet: n = 250) was measured in a mountainous district in Himalaya. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded by aneroid sphygmomanometer in the sitting posture using a calibrated instrument and four trained resident doctors. The average of three readings was taken. RESULTS: Of the 1229 children whose data were analyzed, 50.4% were boys. SBP showed a progressive rise from 99.5 (4.00) mmHg [mean (standard deviation)] at near sea level to 106.7 (4.17) mmHg at 8000 feet. Similarly, DBP showed a rise from 60.1 (3.67) mmHg to 66.8 (4.58) mmHg over the same altitude range. Analysis of covariance for BP variation with altitude, with age as covariate, indicated a modest but statistically significant rise in both SBP and DBP with altitude. Higher BP values was noted among children residing at higher than at lower altitude (0.8% at sea level to 18.8% at 8000 feet). Correlations between SBP and DBP values and height and weight, though positive and significant at p < 0.01 level, were weak. CONCLUSIONS: These data will help in correct interpretation of BP readings in children residing at high altitude.

Evaluation of thermo-adaptability between Tharparkar (Bos indicus) and crossbred (Bos indicus X Bos taurus) calves in a controlled environment.

The global warming driven climate change has increased the susceptibility of livestock around the globe to heat stress (HS), which reduces animal productivity and threatens the sustainability of marginal farmers. The objective of this investigation was to evaluate thermo-adaptability between Tharparkar calves (TC), an indigenous milch breed of India and crossbred calves (CC) during induced heat stress in controlled environment. For this purpose, 12 apparently healthy male calves (six in each group) aged 5-6 months, were selected. The experiment was conducted at physiologically comfortable temperature (25 °C), moderate HS (31 °C) and severe HS (37 °C) for 21 days each in a psychrometric chamber. In each experimental day, the calves were exposed to 6 h of heat. There were 7 days of acclimatization period before experiment and 10 days of recovery period at ambient temperature between each 21 day exposure period. During experimental period, the blood was collected at 1st, 6th, 11th, 16th, 21st day and among ten-day recovery period the blood was collected at 5th day. Physiological responses, serum electrolytes, metabolic enzymes profiles, antioxidant capacity, oxidative stress status and general endocrine milieu were studied. Relative mRNA expression study of Heat Shock Protein (HSP) 70, HSP90, induced Nitric Oxide Synthase (iNOS) and endothelial NOS (eNOS) were carried out by qPCR. There was significant (p < 0.05) change in the displacement in rectal temperature, respiration rate, serum alanine aminotransferase level between two breeds at moderate and severe HS. Similar change was observed in total antioxidant capacity, superoxide dismutase, and endocrinological parameters. The comparatively lower mRNA expression of HSP70 and higher expression of HSP90 in TC than CC point the better thermo-adaptability of the same. The results of the experiment indicated that TC are more thermo-adaptable than CC at different modality of stress in controlled temperature conditions.

A Retrospective Analysis of Clinical Manifestations, Management and Outcome of Acute Respiratory Distress Syndrome Associated with Coronavirus Disease-2019 Infection in Children.

Background: Acute respiratory distress syndrome (ARDS) associated with COVID-19 in children is not well described in the literature, so this study was designed to assess the severity, clinical course, different treatment measures, and outcome of this group of patients. Patients and methods: This descriptive study was performed by retrospective chart review of children admitted in pediatric intensive care unit (PICU) in the age-group of 1 month to 12 years over the period of 6 months (July-December 2020) in a tertiary care pediatric COVID facility in eastern India. Severity of ARDS, ventilator settings, oxygenation and laboratory parameters, and outcomes were documented. Predictors associated with severe ARDS were evaluated. Results: Among 128 laboratory-confirmed pediatric COVID-19 cases admitted in PICU, 18 (14%) developed ARDS, 6 (33.3%) had severe ARDS, and 3 (16.6%) succumbed to death. Outcome was measured by median hospital stay [20 days (IQR 19, 21)], PICU stay [13 days (IQR 10, 16)], and 28-day ventilator-free days [14 days (IQR 13, 22)]. Half (n = 9) of our study cohort had different comorbidities and congenital heart disease being the most common (4, 22.2%). Median positive end-expiratory pressure requirement was 10 cm H2O (9, 11) for invasively ventilated children (n = 13, 72.2%) along with peak inspiratory pressure of 24 cm H2O (20, 29) and mean airway pressure of 17 cm H2O (14, 20). Median oxygenation index was 13.3 (10.5, 18.6). Nine (69.2%) out of 13 intubated children had undergone prone ventilation. C-reactive protein (CRP) and D-dimer levels were significantly high in children with severe ARDS alongside pSOFA and lung USG score. Conclusion: Incidence of ARDS in pediatric COVID-19 though less but is not rare. Elevated CRP, D-dimer values, and high lung USG scores were associated with severe ARDS. Those who died had significant comorbidity. How to cite this article: Sarkar M, Das B, Mahapatra MK, Roychowdhoury S, Das S, Konar MC. A Retrospective Analysis of Clinical Manifestations, Management and Outcome of Acute Respiratory Distress Syndrome Associated with Coronavirus Disease-2019 Infection in Children. Indian J Crit Care Med 2022;26(3):331-338.

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo.

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.

A Case Study of Dual Infection of Dengue and COVID-19: Presenting as Multiorgan Dysfunction in an Infant.

Dengue is a major health concern in South Asian countries transmitted by bite of day breeder mosquitoes Aedes aegypti and Aedes albopictus. Severity of plasma leak, shock, bleeding tendency and other organ dysfunction can be more pronounced in infants. The management becomes further complicated in the presence of a co-existing COVID-19 infection. Although COVID-19 infection is usually asymptomatic or has mild manifestations in children, however in presence of serious co-infection like dengue it can modify the course of the illness and lead to drastic consequences. Here, we present one such case of a 9-month-old female child who tested positive for dengue as well as COVID-19 during the ongoing corona pandemic and went on to develop shock, encephalopathy with deranged liver enzymes but managed to overcome all odds and recover from the disease by day 14 of illness.

Incomplete Kawasaki Disease as Presentation of COVID-19 Infection in an Infant: A Case Report.

BACKGROUND: Recently a severe form of COVID-19 infection has been described in a cluster of children presenting as multisystem inflammatory condition. One of the important spectrum of this condition is incomplete Kawasaki disease (KD). CASE REPORT: A 5-month-old male child presented with high-spiking fever for 5 days with skin rash, bilateral non-purulent conjunctivitis and irritability. His C-reactive protein was markedly elevated (215.4 mg/l). Echocardiography revealed dilated left main coronary artery (3.0 mm, Z score +4.30) and left anterior descending artery (2.37 mm, Z score +3.76). Concomitantly Reverse Transcription- Polymerase Chain Reaction for COVID-19 was positive on fifth day sample. He was diagnosed as incomplete KD with COVID-19 infection and treated with intravenous immunoglobulin (IVIG) (2 g/kg), oral aspirin and azithromycin. Patient improved after 48 h and was discharged on oral aspirin. CONCLUSION: Incomplete KD may co-exist with COVID-19 infection in infant. Early institution of IVIG may lead to better outcome.

COVID-19 and Co-infection in Children: The Indian Perspectives.

BACKGROUND AND OBJECTIVES: Assessing the co-infections with COVID-19 is crucial to delineate its true clinical impact. Pediatric information in this aspect is limited. Our study aims to analyze the spectrum of co-infections in pediatric COVID-19 patients and determine the clinical as well as laboratory parameters predicting co-infection. METHODOLOGY: In this prospective observational study conducted from June to December 2020 in a single tertiary care institution, data pertaining to demographic, illness and treatment-related variables were analyzed among two subsets of pediatric patients of age 1 month-12 years with RT-PCR-confirmed COVID-19 infection-Group A: those with confirmed co-infection and Group B: moderate to severe disease without co-infection. Among Group A, etiology of co-infection was characterized through relevant microbiological examination within 48 h admission. RESULT: Among our study population, 15.03% and 20.6% had co-infections and moderate to severe disease respectively. Among those with confirmed co-infection, 32.5%, 11.6% and 6.97% recorded blood culture, respiratory secretion and CSF growth, respectively, the picture being dominated by Methicillin resistant and sensitive Staphylococcus aureus. Serum serology demonstrated Scrub typhus infection to be most prevalent. Concurrent respiratory viral infections were seen in 11.6%. Children with co-infection had significantly higher morbidity and need for supportive therapy. Predictors of co-infection were localization of infection, Neutrophil count ≥10×109, age-specific lymphopenia, CRP > 100 mg/dl and hyperferritinemia. CONCLUSION: Co-infections are an important factor prognosticating pediatric COVID infection. Their early detection, prompt and appropriate treatment is of paramount importance.

Scrub Typhus-Associated Hemophagocytic Lymphohistiocytosis: Not a Rare Entity in Pediatric Age Group.

BACKGROUND: Our goal was to study the demographic, clinical and laboratory profile and outcome of scrub typhus-associated hemophagocytic lymphohistiocytosis (HLH) in the pediatric age group. METHODS: We conducted a prospective observational study in a tertiary care teaching hospital over a period of 1 year. Children in the age group of 1 month to 12 years with IgM ELISA positive for scrub typhus were included in the study. HLH was diagnosed using HLH-2004 criteria. Demographic, clinical and laboratory profile, treatment and outcome of HLH patients were noted and also compared with non-HLH scrub typhus patients. RESULTS: Among 58 children with scrub typhus infection, 18 had HLH. The mean age of patients with HLH was 35.3 ± 44.8 months and 61% were male. Anemia, thrombocytopenia and hyperferritinemia were seen in all the patients. Hypertriglyceridemia, hypofibrinogenemia and coagulopathy were noted in 78%, 56% and 44%, respectively. All the patients were treated with intravenous doxycycline for an average duration of 9.5 days. Intravenous immunoglobulin and methylprednisolone were given in 33% and 22% cases, respectively. Complications like acute respiratory distress syndrome (ARDS) (p = 0.001) and MODS (p = 0.004) were significantly high in the HLH group. Younger age (<3 years), fever > 7 days, presence of convulsion, ARDS and MODS were the clinical predictors of scrub typhus-associated HLH. CONCLUSION: HLH in scrub typhus infected children is being increasingly recognized. Younger age, prolonged fever, presence of convulsion, ARDS and MODS should alert clinicians of the risk of HLH. Treating the primary cause usually cures the disease and immunomodulator therapy need not be routinely administered.

Leave against Medical Advice in Children: Rural Indian Perspective.

OBJECTIVES: To determine the burden and etiological factors of leave against medical advice (LAMA) in Indian children. METHODOLOGY: In this prospective study, legal guardians of 528 patients who took the decision of LAMA were interviewed (using structured question answers based multi-option) and data were captured over a period of 16 months. The resultant database was analyzed using standard statistical methods. RESULTS: About 6.12% of childhood LAMA cases were dealt out of total pediatric admission including newborns. Neonatal preponderance noted in cases of LAMA from intensive care unit (ICU; 57.14%, p < 0.05). The overall male (n = 293) to female (n = 235) ratio in this study was 1.25:1. Number of LAMA patients was higher from rural area (83.33%), mostly admitted in ICU (93.65%, Pearson's chi-squared test, p < 0.05). Higher proportion (29.47%) of patients with infection availed LAMA from neonatal age group but overall LAMA patients fall under category of respiratory disorders (22.35%). Interest of the domestics issues other than suffering child was considered primary during LAMA for those admitted in ward as compared with ICU patients [odds ratio (OR): 1.73, CI: 1.02-2.94, p < 0.05]. ICU patients were reportedly to be taken to private health care facility (OR: 1.93, CI: 1.06-3.49, p < 0.05). Duration of stay before taking LAMA from ward was <7 days (85.59%, OR: 0.19, CI: 0.11-0.35, p < 0.05). Upper-lower socio-economic class attributed financial constraint as the main reason for LAMA (Pearson's chi-squared test, Chi-square value: 152.23, p < 0.05). CONCLUSIONS: This study tried to elucidate the determinants of childhood LAMA in rural Indian setting.

Tracheostomy before 14 Days: Is It Associated with Better Outcomes in Pediatric Patients on Prolonged Mechanical Ventilation?

INTRODUCTION: With the advancement of pediatric critical care services across India, many children require prolonged mechanical ventilation (MV), and tracheostomy is recommended to them. However, many pediatric intensivists have concerns regarding the safety, feasibility, and outcome of tracheostomy.We aimed to analyze clinical characteristics, indication, duration, and outcome of tracheostomized children with respect to timing of tracheostomy. MATERIALS AND METHODS: We conducted a retrospective study from the hospital clinical database of consecutive patients below 12 years who had undergone tracheostomy after admission into the pediatric intensive care unit (PICU) for prolonged ventilation (≥96 hours) from January 2015 to December 2019. The study was approved by the Institutional Ethics Committee. Patients were divided into two groups: tracheostomies done within 14 days of MV (early tracheostomy) and patients with tracheostomies performed after 14 days (late tracheostomy). Patients' age, sex, indications, complications, decannulation rate, length of MV, PICU, and hospital stay were analyzed. RESULTS: Of the 1,425 patients on invasive MV, 87 (6.1%) patients required tracheostomy after a mean 13.37 days of MV. The most common indication was encephalopathy 32 (36.7%) followed by acute respiratory distress syndrome 20 (22.9%). Factors like higher pediatric logistic organ dysfunction score, vasoactive inotrop score, incidence of pretracheostomy ventilator-associated pneumonia, and difficulty in obtaining parental consent were associated with late tracheostomy. The early tracheostomy group had a higher decannulation rate (odds ratio, 5.17; p, 0.01) and weaning rate (odds ratio, 5.94; p, 0.032). The late tracheostomy group needed a longer duration of MV, PICU, and hospital stay. Complications of tracheostomy were less in the early tracheostomy patients (odds ratio, 2.95; p, 0.03). CONCLUSION: Early tracheostomy was associated with lower complications, higher successful weaning rates, and less utilization of intensive care facilities in patients receiving prolonged MV. CLINICAL SIGNIFICANCE: In the context of scarcity of data on the timing of tracheostomy in children with prolonged ventilation (≥96 hours) the study shows that early (<14 days) tracheostomy is associated with a better outcome. HOW TO CITE THIS ARTICLE: Sarkar M, Roychowdhoury S, Bhakta S, Raut S, Nandi M. Tracheostomy before 14 Days: Is It Associated with Better Outcomes in Pediatric Patients on Prolonged Mechanical Ventilation? Indian J Crit Care Med 2021;25(4):435-440.

Marker-less deletion of cctA gene of Clostridium chauvoei.

Clostridium chauvoei causes blackleg disease in domestic animals, especially cattle and sheep. The pathogen produces several toxins including CctA - a hemolysin and protective antigen. Molecular pathogenesis of the disease is poorly understood, possibly due to lack of genetic manipulation tools for C. chauvoei. In the present study, we report the marker-less deletion of cctA gene using the CRISPR-Cas9 system. The C. chauvoei cctA deletion mutant had negligible hemolytic and significantly reduced cytotoxic activities. To the best of our knowledge, this is the first report of genetic manipulation of C. chauvoei. The method we used in this study can be applied for genetic manipulation of C. chauvoei to better understand the pathogenesis and genetics of the pathogen.

The unusual glycine-rich C terminus of the Acinetobacter baumannii RNA chaperone Hfq plays an important role in bacterial physiology.

Acinetobacter baumannii is a Gram-negative nosocomial pathogen that causes soft tissue infections in patients who spend a long time in intensive care units. This recalcitrant bacterium is very well known for developing rapid drug resistance, which is a combined outcome of its natural competence and mobile genetic elements. Successful efforts to treat these infections would be aided by additional information on the physiology of A. baumannii Toward that end, we recently reported on a small RNA (sRNA), AbsR25, in this bacterium that regulates the genes of several efflux pumps. Because sRNAs often require the RNA chaperone Hfq for assistance in binding to their cognate mRNA targets, we identified and characterized this protein in A. baumannii The homolog in A. baumannii is a large protein with an extended C terminus unlike Hfqs in other Gram-negative pathogens. The extension has a compositional bias toward glycine and, to a lower extent, phenylalanine and glutamine, suggestive of an intrinsically disordered region. We studied the importance of this glycine-rich tail using truncated versions of Hfq in biophysical assays and complementation of an hfq deletion mutant, finding that the tail was necessary for high-affinity RNA binding. Further tests implicate Hfq in important cellular processes of A. baumannii like metabolism, drug resistance, stress tolerance, and virulence. Our findings underline the importance of the glycine-rich C terminus in RNA binding, ribo-regulation, and auto-regulation of Hfq, demonstrating this hitherto overlooked protein motif to be an indispensable part of the A. baumannii Hfq.

Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

BACKGROUND/AIMS: Thrombospondins (TSPs) are large multi-modular proteins, identified as natural angiogenesis inhibitors that exert their activity by binding to CD36 and CD47 receptors. The anti-angiogenic effect of TSPs in luteal regression of water buffalo has not been addressed. The present study characterized the expression pattern and localization of TSPs and their receptors in ovarian corpus luteum during different stages of development in buffalo. This study also elucidated the effect of exogenous Thrombospondin1 (TSP1) or the knocking out of the endogenous protein on luteal cell viability and function. Further, the in vitro transcriptional interaction of TSP1 with hormones, LH, PGF2α and angiogenic growth factors, VEGF and FGF2 were also evaluated. METHODS: First, the CLs were classified into four groups based on macroscopic observation and progesterone concentration. mRNA expression of examined factors was measured by qPCR, localization by immunoblotting and immunohistochemistry. TSP1 was knocked out (KO) in cultured luteal cells isolated from late luteal stage CLs (day 1116) by CRISPR/Cas9 mediated gene editing technology in order to functionally validate the TSP1 gene. Isolated cells from late stage CLs were also stimulated with different doses of TSP1, LH, PGF2α, VEGF and FGF2 for various time intervals to determine transcriptional regulation of thrombospondins. RESULTS: mRNA expression of TSPs and their receptors were found to be significantly higher in late and regressed stage of CL as compared to other groups which was consistent with the findings of immunoblotting and immunolocalization experiments. It was observed that TSP1 induced apoptosis, down regulated angiogenic growth factors, VEGF and FGF2 and attenuated progesterone production in cultured luteal cells. However, knocking out of endogenous TSP1 with CRISPR/Cas9 system improved the viability of luteal cells, progesterone synthesis and upregulated the expression of VEGF and FGF2 in the KO luteal cells. PGF2α induced the upregulation of TSPs and Caspase 3 transcripts, whereas treatment with LH and angiogenic growth factors (VEGF and FGF2) down regulated the TSP system in luteal cells. CONCLUSION: Collectively, these data provide evidence that thrombospondins along with their receptors are expressed at varying levels in different stages of CL progression with maximum expression during the late and regressing stages. These results are consistent with the hypothesis that thrombospondins stimulated by PGF2α plays an essential modulatory role in bringing about structural and functional luteolysis in buffalo.

Effect of dietary n-3 polyunsaturated fatty acid rich fish oil on the endometrial prostaglandin production in the doe (Capra hircus).

Recently, we showed that dietary supplementation of n-3 PUFA rich fish oil (FO) decreased the metabolites of serum prostaglandin (PG) F2α and E2 during the window of pregnancy recognition in the doe. In this study, we investigated its effect on the changes on endometrial PG production in vitro. Cycling does (n = 12) of Rohilkhand region were divided into two equal groups and fed a concentrate diet supplemented with either FO containing 26% n-3 PUFA (TRT; n = 6) or palm oil (CON; n = 6) @ 0.6 mL/kg body weight for 57 days. Estrus was synchronized by two injections of PGF2α analogue viz, on day 25 and 36 of supplementation and laparo-hysterotomy was performed to obtain endometrial tissue on day 16 of the synchronized estrus. Endometrial explant culture was done using a defined medium.The basal PG production was assayed at 6 and 12 h. Endometrial explant was stimulated with oxytocin (OXT) and/or recombinant ovine interferon tau (roIFN-τ) and PGs were assayed at 3 and 12 h post-treatment. The relative expression of genes related to PG metabolism in the endometrium was done by Quantitative Real Time PCR technique (qRT-PCR). There was a significant (P < 0.05) decline in the basal production of PGF2α and PGE2 in the TRT as compared to the CON group. The cultured endometrial tissue produced PGF2α in a time- dependent fashion in both the groups (P < 0.05). Neither OXT nor roIFN-τ had a significant (P > 0.05) effect on the PGF2α and PGE2 production in the TRT group. Similarly, the PG production in the OXT and roIFN-τ was comparable with the control in TRT. Expression of mRNA for cyclooxygenase-2 (COX-2), cytosolic phospholipase A2 (cPLA2) and PGF synthase (PGFS) was lower (P < 0.05) whereas, PGE synthase and peroxisome proliferator-activated receptors such as PPAR-γ and δ was increased (P < 0.05) in n-3 PUFA fed doe. In conclusion, dietary supplementation of FO decreased the endometrial production of PGF2α and PGE2 by downregulating the COX-2, cPLA2 and PGFS transcripts in the doe. The findings suggest that n-3 PUFA influence embryo survival by modulating the endometrial PG.

Expression Profiling and Identification of Novel SNPs in CatSper2 Gene and Their Influence on Sperm Motility Parameters in Bovines.

122 randomly selected Vrindavani cattle were studied to detect polymorphism in four fragments of the CatSper2 gene that were comprised of exon 2, 4, 5, and 6 with flanking regions. Using PCR-SSCP and sequencing analysis, three SNPs (T157C, C273A, and A274C) in the first fragment, one SNP (C30G) in the second fragment, and two SNPs (T86G and T292C) in the fourth fragment were identified. The third fragment did not reveal any polymorphism. The SNPs were used for construction of haplotypes and three haplotypes were found. The least square analysis of variance revealed a significant (P < 0.01) effect of haplotype on all three motility parameters. The haplotype II and III were nonsignificantly different from each other while being significantly (P < 0.01) different from haplotype I. The nonsignificant difference of haplotype II with III can lead to a hypothesis that T>G or C>T SNPs may not play a role in sperm motility. However, when the comparison was made between haplotype I and II, it can be inferred that C>T SNP may have a role in sperm motility, as haplotype II has better motility parameters. Expression profiling of Catper2 gene revealed nonsignificant down regulation of CatSper2 gene in poor motility sperm compared to good motility sperm.

Comparative Study between Noninvasive Continuous Positive Airway Pressure and Hot Humidified High-flow Nasal Cannulae as a Mode of Respiratory Support in Infants with Acute Bronchiolitis in Pediatric Intensive Care Unit of a Tertiary Care Hospital.

BACKGROUND: Early initiation of appropriate noninvasive respiratory support is utmost important intervention to avoid mechanical ventilation in severe bronchiolitis. AIM: This study aims to compare noninvasive continuous positive airway pressure (nCPAP) and hot humidified high-flow nasal cannulae (HHHFNC) as modes of respiratory support in infants with severe bronchiolitis. METHODS: Prospective, randomized, open-label pilot study done in a tertiary-care hospital Pediatric Intensive Care Unit (PICU). Participants: 31 infants (excluding neonates) clinically diagnosed with acute bronchiolitis having peripheral capillary oxygen saturation (SpO2) <92% (with room air oxygen); Respiratory Distress Assessment Index (RDAI) ≥11. Intervention: nCPAP (n = 16) or HHHFNC (n = 15), initiated at enrollment. Primary outcome: Reduction of need of mechanical ventilation assessed by improvements in (i) SpO2% (ii) heart rate (HR); respiratory rate; (iii) partial pressure of carbon dioxide; (iv) partial pressure of oxygen; (v) COMFORT Score; (vi) RDAI from preintervention value. Secondary outcome: (i) total duration of noninvasive ventilation support; (ii) PICU length of stay; and (iii) incidence of nasal injury (NI). RESULTS: Mean age was 3.41 ± 1.11 months (95% confidence interval 2.58-4.23). Compared to nCPAP, HHHFNC was better tolerated as indicated by better normalization of HR (P < 0.001); better COMFORT Score (P < 0.003) and lower incidence of NI (46.66% vs. 75%; P = 0.21). Improvements in other outcome measures were comparable for both groups. For both methods, no major patient complications occurred. CONCLUSION: HHHFNC is an emerging alternative to nCPAP in the management of infants with acute bronchiolitis.