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.

Infant COVID-19 Infection: An Experience from Pediatric Intensive Care Unit of a Tertiary Care Dedicated Pediatric COVID Hospital.

This study aimed to assess different clinical, disease severity, laboratory, treatment, and outcome-related factors of COVID-19 positive infants admitted to a pediatric intensive care unit (PICU) and to compare these parameters with COVID-19 positive noninfants (1-12 years of age) who also required intensive care admission. This retrospective observational study was conducted in a PICU of a tertiary care, dedicated pediatric COVID facility. The clinical, epidemiological, laboratory parameters, and treatment outcomes of COVID-19 infected infants admitted to the PICU were recorded and analyzed. During comparison with the noninfant group, malignancy and coinfection with dengue and scrub typhus were excluded from both groups. A total 313 COVID-19 positive children aged from 1 month to 12 years old were admitted, of which 115 (36.7%) children required PICU admission. Infants constituted 37.4% of total PICU admissions. Most common symptoms were respiratory (83.7%) followed by fever (60.5%). Fifteen (34.9%) infants presented with shock. Ten infants (23.3%) had myocardial dysfunction. C-reactive protein (CRP) and ferritin were high in 60.5 and 16.7% infants, respectively. Fourteen infants needed invasive mechanical ventilation. Nine patients had acute respiratory distress syndrome (ARDS) and five had MIS-C. However, 53.5% infants had different comorbidities. Four infants died and all of them had severe comorbidities. Respiratory distress ( p = 0.009), pediatric sequential organ failure assessment score ( p = 0.032) and number of ARDS cases ( p = 0.044) were significantly higher in infants than noninfants. Infants are one of the most vulnerable groups of children suffering from serious illness from COVID-19 infection requiring PICU admission due to predominantly respiratory involvement. Overall outcome was good among infants without significant comorbidity.

Retrospective analysis of clinico-laboratory parameters, outcome and predictors of scrub typhus co- infection in covid-19 affected children.

The objective of this study was to describe clinico-laboratory parameters, outcome and predictors of scrub typhus co- infection in COVID-19 affected children. Ours was a retrospective analysis done in a tertiary care pediatric COVID facility. COVID-19 positive children in the age group of 1 month to 12 years who were tested for scrub typhus co-infection between May to August 2020 were included in the study. In all positive cases, clinical, laboratory, treatment and outcome data were analyzed. Relevant data were compared between scrub typhus confirmed, and suspected but negative patients. All six patients with co-infection had fever for >5 days and four had features of cerebral involvement. These had lymphocytic pleocytosis in cerebrospinal fluid (CSF) analysis and two had eschar. Screening for scrub typhus in persistently febrile children residing in highly endemic areas is highly recommended.

CRISPR/Cas mediated disruption of BMPR-1B gene and introduction of FecB mutation into the Caprine embryos using Easi-CRISPR strategy.

Bone Morphogenetic Proteins play a significant role in ovarian physiology and contribute to the reproductive fitness of mammals. The BMPR-1B/FecB mutation, a loss of function mutation increases litter size by 1-2 with each number of mutated alleles in sheep. Considering demand-supply gap of the meat industry, and low replacement rate of indigenous caprine species, the conservative BMPR-1B locus can be explored, and FecB mutated goats can be produced. The experiment one produced CRISPR/Cas mediated KO transferable caprine embryos, and experiment two generated caprine embryos with desired FecB mutation using Easi-CRISPR strategy. In the KO experiment, Cas9 and BMPR-1B guide RNA (100:100ng/ul) were electroporated into single stage caprine zygotes at 750V, 10 ms and 1pulse using Neon transfection system. In the second experiment, phosphorothioate (PS) modified single-stranded oligodeoxynucleotide (ssODN) was used as an HDR template along with CRISPR components (100:100ng/ul, ssODN 100ng/ul). The precise time and method of electroporation, RNP format of CRISPR components and PS modified asymmetric ssODN were the factors that affected the production of mosaicism free BMPR-1B edited caprine embryos. The editing efficiency of KO and KI experiments was 68.52 and 63.16% respectively, and successful production of goats with higher mean ovulation rate can be realized with addition of embryo transfer technology to these experiments.

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.

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.

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.

Characteristics and predictors of outcomes of critically Ill children with SARS-CoV-2 infection - the PICU experience

Abstract Objective To describe the clinical characteristics, laboratory parameters, treatment, and predictors of an unfavorable outcome of critically ill children with SARS-CoV-2 infection. Method This was a prospective observational study performed in a pediatric intensive care unit (PICU) of a tertiary care COVID referral hospital among critically ill children in the age group 1 month - 12 years admitted due to SARS-CoV-2 infection from June to December 2020. Demographic, clinical profile, pSOFA and PRISM III scores, laboratory parameters, treatment, and outcomes of the patients were recorded. Children who had a prolonged PICU stay (>14 days) or died were compared with those who were discharged from PICU within 14 days to assess predictors of unfavorable outcomes. Results PICU admission rate among hospitalized SARS-CoV-2 infected children was 22.1% (92/416). Infants comprised the majority of the ICU population. Invasive mechanical ventilation and inotropic support were required for 28.3% and 37% of patients, respectively. Remdesivir, IVIg, and steroids were administered to 15.2%, 26.1%, and 54.3% of the subjects, respectively. The mortality rate was 7.6 %. MIS-C patients were older, less comorbid, and required less ventilator support but more inotrope support than acute severe COVID-19 patients. Predictors of unfavorable outcomes were age < 1 year, fever duration > 5 days, respiratory distress, shock, comorbidity, elevated CRP (> 50 mg/L), procalcitonin (> 6 ng/L), D-dimer (> 6 µg/L) and arterial lactate (> 2 mmol/L). Conclusion Critically ill children with unfavorable outcomes were predominantly infants, comorbid, prolonged fever, respiratory distress, shock and elevated inflammatory markers, D-dimer and lactate. These factors may be useful for watchful monitoring and early intervention.

Predictors of Severity of Scrub Typhus in Children Requiring Pediatric Intensive Care Admission.

Objective of our study was to determine the clinical characteristics and laboratory profile of scrub typhus patients requiring pediatric intensive care admission and to find out risk factors for the severity of illness. This was a cross-sectional observational study conducted on 1-month to 12-year-old children admitted with scrub typhus in a tertiary care pediatric intensive care unit (PICU). Relevant demographic, clinical, laboratory, treatment, and outcome-related data were documented. The severity of the disease was measured in the form of multiple organ dysfunction syndrome (MODS). With further correlation, and univariate and multivariate analyses, factors associated with severe disease were identified. During the study period, out of 586 PICU admission, 62 patients (10.6%) were diagnosed with scrub typhus. The mean age was 63.85 ± 52.78 months, where infants constituted 32.3% of the total population. Fever was present in 100% of the cases. Common indications of PICU admission were: respiratory distress 42 (67.7%), altered sensorium 41 (66.1%), convulsion 37 (59.7%), and shock 31 (50%). Total number of patients with MODS was 40 (64.5%). The case fatality rate was 8%. On multivariate analysis, infant age group ( p = 0.02), altered sensorium ( p = 0.001), reduced urine output ( p = 0.02), thrombocytopenia ( p = 0.001), raised C-reactive protein ( p = 0.004), hyponatremia ( p = 0.005), hypoalbuminemia ( p = 0.01), deranged international normalized ratio ( p = 0.02), and hyperferritinemia ( p = 0.02) came out to be independent factors in predictability for development of MODS. Multiorgan dysfunction is a life-threatening manifestation of scrub typus in children, which necessitates PICU admission. Infant age group, presence of altered sensorium, reduced urine output, thrombocytopenia, elevated inflammatory markers, coagulopathy, hypoalbuminemia, and hyponatremia predict risk for MODS.

Clinical Characteristics of Children With SARS-CoV-2 Infection During the Third Wave of the Pandemic: Single Center Experience.

OBJECTIVE: To evaluate pulmonary functions in children with transfusion-dependent thalassemia, and its reversal (lung dysfunction) using intensive intravenous chelation with desferrioxamine (DFO) (4 weeks). METHODS: This descriptive study enrolled 77 children with transfusion-dependent thalassemia. Pulmonary function test (PFT) and iron load (serum ferritin (SF) and T2* MRI of heart and liver) were done. PFT included spirometry, total lung capacity (TLC) by helium dilution test and diffusion capacity by carbon monoxide (DLCO). Follow-up PFT was available for 13 children with moderate to severe lung dysfunction given intravenous DFO. RESULTS: 50 (68.8%) patients had lung dysfunction, most commonly diffusional impairment (48; 96%), and reduced TLC (11; 22%); and none had obstructive pattern. 9 (81.8%) patients with restrictive defect had moderate to severely deranged DLCO. PFT and T2* MRI values were inversely correlated with serum ferritin. Among 13 patients receiving intensive chelation for 4 weeks, significant improvement was noticed in forced expiratory volume in one minute/ forced vital capacity ratio (DFEV1/FVC) (P=0.009), DDLCO (P=0.006) and DSF (P=0.01). CONCLUSIONS: Pulmonary dysfunction is common in children with multi-transfused thalassemia, and routine screening by PFT needs to be part of the management guidelines.

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.

Role of lung ultrasound patterns in monitoring coronavirus disease 2019 pneumonia and acute respiratory distress syndrome in children.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, lung ultrasonography (US) has been gaining importance in pediatric intensive care and emergency settings for the screening, diagnosis, and monitoring of pulmonary pathology. PURPOSE: To describe the pattern of lung US changes in patients with COVID-19 pneumonia and its potential role in monitoring ventilated patients. METHODS: This prospective observational study included children aged 1 month to 12 years with a confirmed diagnosis of COVID-19. Lung US was performed using a high-frequency linear probe (5-12 MHz) in all children with moderate/severe respiratory symptoms within 24 hours of admission and then daily until the patient required oxygen therapy. Lung involvement severity was assessed using lung US scores, while lung aeration improvement or deterioration was measured using lung ultrasound reaeration scores (LUSReS). RESULTS: Of 85 children with moderate to severe disease, 54 with pulmonary disease were included. Of them, 50 (92.5%) had an interstitial pattern, followed by pleural line abnormalities in 44 (81.5%), reduced or absent lung sliding in 31 (57.4%), and consolidation in 28 (51.8%). A significantly higher lung US score (median, 18; interquartile range [IQR], 11-22) was observed in ventilated versus nonventilated patients (median, 9; IQR, 6-11). LUSReS improvement after positive end-expiratory pressure titration was positively correlated with improved dynamic lung compliance and oxygenation indices and negatively correlated with the requirement for driving pressure. Successful weaning could be predicted with 100% specificity if loss of LUSReS ≤ 5. CONCLUSION: Interstitial syndrome, fragmented pleural line, and subpleural microconsolidation were the most prevalent lung US findings in children with COVID-19 pneumonia. Thus, lung US may have the ability to monitor changes in lung aeration caused by mechanical ventilation and predict its successful weaning in children with COVID-19.

Smad4 and γ-secretase knock-down effect on osteogenic differentiation mediated via Runx2 in canine mesenchymal stem cells.

Cell lineage determination during mesenchymal stem cell (MSCs) differentiation is a highly orchestrated process involving diverse signaling pathways and distinct classes of regulatory molecules. Bone morphogenetic protein (BMP) signaling positively influence the osteoblast lineage determination, whereas the Notch signaling may have a dimorphic action. Effective regenerative therapy for repairing bone defects requires ample knowledge of the signaling pathways responsible for the differentiation of MSCs. To elucidate the signaling pathways that drives canine bone-marrow derived MSCs towards osteogenic lineage, the current work was focused on BMP and Notch signaling. Target genes of Runx2, Smad4 and γ-secretase were silenced by short hairpin RNA (shRNA) in canine MSCs. Evaluation of the effect of gene silencing on in-vitro osteogenic differentiation potential was done by quantitative polymerase chain reaction (qPCR) for osteoblastic markers (Osteocalcin and Osteopontin) and Alizarin red S staining for the extracellular deposition of calcium. Silencing of Runx2 significantly reduced the osteocalcin and osteopontin gene expression while a similar trend was observed in the case of smad 4 silencing and their combination groups, but there was no difference found in Hey 1 expression. Runx2 and Smad4 silencing groups showed very less positive staining with Alizarin red S staining, whereas knockdown of γ-secretase and its combination groups showed reverse results as that of Runx2 and Smad4. Runx2 plays an indispensable part in directing the canine mesenchymal stem cells towards osteogenic lineage. Also, Smad-mediated BMP signaling induced the osteoblast-specific gene expression, whereas the notch pathway negatively regulated the osteogenic differentiation of canine MSCs.

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.

Characteristics and predictors of outcomes of critically Ill children with SARS-CoV-2 infection - the PICU experience.

OBJECTIVE: To describe the clinical characteristics, laboratory parameters, treatment, and predictors of an unfavorable outcome of critically ill children with SARS-CoV-2 infection. METHOD: This was a prospective observational study performed in a pediatric intensive care unit (PICU) of a tertiary care COVID referral hospital among critically ill children in the age group 1 month - 12 years admitted due to SARS-CoV-2 infection from June to December 2020. Demographic, clinical profile, pSOFA and PRISM III scores, laboratory parameters, treatment, and outcomes of the patients were recorded. Children who had a prolonged PICU stay (>14 days) or died were compared with those who were discharged from PICU within 14 days to assess predictors of unfavorable outcomes. RESULTS: PICU admission rate among hospitalized SARS-CoV-2 infected children was 22.1% (92/416). Infants comprised the majority of the ICU population. Invasive mechanical ventilation and inotropic support were required for 28.3% and 37% of patients, respectively. Remdesivir, IVIg, and steroids were administered to 15.2%, 26.1%, and 54.3% of the subjects, respectively. The mortality rate was 7.6 %. MIS-C patients were older, less comorbid, and required less ventilator support but more inotrope support than acute severe COVID-19 patients. Predictors of unfavorable outcomes were age < 1 year, fever duration > 5 days, respiratory distress, shock, comorbidity, elevated CRP (> 50 mg/L), procalcitonin (> 6 ng/L), D-dimer (> 6 µg/L) and arterial lactate (> 2 mmol/L). CONCLUSION: Critically ill children with unfavorable outcomes were predominantly infants, comorbid, prolonged fever, respiratory distress, shock and elevated inflammatory markers, D-dimer and lactate. These factors may be useful for watchful monitoring and early intervention.

Genomic Diversity Profiling and Breed-Specific Evolutionary Signatures of Selection in Arunachali Yak.

Arunachali yak, the only registered yak breed of India, is crucial for the economic sustainability of pastoralist Monpa community. This study intended to determine the genomic diversity and to identify signatures of selection in the breed. Previously available double digest restriction-site associated DNA (ddRAD) sequencing data of Arunachali yak animals was processed and 99,919 SNPs were considered for further analysis. The genomic diversity profiled based on nucleotide diversity, π (π = 0.041 in 200 bp windows), effective population size, Ne (Ne = 83) and Runs of homozygosity (ROH) (predominance of shorter length ROHs) was found to be optimum. Subsequently, 207 regions were identified to be under selective sweeps through de-correlated composite of multiple signals (DCMS) statistic which combined three individual test statistics viz. π, Tajima's D and |iHS| in non-overlapping 100 kb windows. Mapping of these regions revealed 611 protein-coding genes including KIT, KITLG, CDH12, FGG, FGA, FGB, PDGFRA, PEAR1, STXBP3, olfactory receptor genes (OR5K3, OR5H6 and OR1E1) and taste receptor genes (TAS2R1, TAS2R3 and TAS2R4). Functional annotation highlighted that biological processes like platelet aggregation and sensory perception were the most overrepresented and the associated regions could be considered as breed-specific signatures of selection in Arunachali yak. These findings point towards evolutionary role of natural selection in environmental adaptation of Arunachali yak population and provide useful insights for pursuing genome-wide association studies in future.

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.

Improved quality and fertilizability of cryopreserved buffalo spermatozoa with the supplementation of methionine sulfoxide reductase A.

BACKGROUND: The excessive reactive oxygen species produced during semen-freezing and -thawing damage the macromolecules resulting in impairment of cellular functions. Proteins are the primary targets of oxidative damage, wherein methionine residues are more prone to oxidation and get converted into methionine sulfoxide, thus affecting the protein function. The methionine sulfoxide reductase A (MsrA) catalyzes the conversion of methionine sulfoxide to methionine and restores the functionality of defective proteins. OBJECTIVES: To establish the expression of MsrA in male reproductive organs, including semen and its effect on quality of cryopreserved semen upon exogenous supplementation, taking buffalo semen as a model. MATERIALS AND METHODS: The expression of MsrA was established by immunohistochemistry, PCR, and Western blots. Further, the effect of recombinant MsrA (rMsrA) supplementation on the quality of cryopreserved spermatozoa was assessed in three treatment groups containing 1.0, 1.5, and 2.0 µg of rMsrA/50 million spermatozoa in egg yolk glycerol extender along with a control group; wherein the post-thaw progressive motility, viability, membrane integrity, and zona binding ability of cryopreserved spermatozoa were studied. RESULTS: The MsrA was expressed in buffalo testis, epididymis, accessory sex glands, and spermatozoa except in seminal plasma. In group 2, the supplementation has resulted in a significant (p < 0.05) improvement as compared to the control group in mean progressive motility (47.50 ± 2.50 vs. 36.25 ± 2.63), viability (56.47 ± 1.85 vs. 48.05 ± 2.42), HOST (50.76 ± 1.73 vs. 44.29 ± 1.29), and zona binding ability of spermatozoa (149.50 ± 8.39 vs. 29.50 ± 2.85). DISCUSSION AND CONCLUSION: In the absence of native MsrA of seminal plasma, the supplementations of rMsrA may repair the oxidatively damaged seminal plasma proteins and exposed sperm plasma membrane proteins resulting in better quality with a fivefold increase in fertilizability of frozen-thawed spermatozoa. The findings can be extended to other species to improve the semen quality with the variation in the amounts of rMsrA supplementation.

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.