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.

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.

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.