The Frequency of Refractory Status Epilepticus and Its Outcome in a Tertiary Care Hospital in Pakistan: A Retrospective Study.

Background Refractory status epilepticus (RSE) is a common neurologic emergency with refractory cases leading to increased rates of morbidity and mortality in patients. The lack of previous studies on the incidence, causes, and management of refractory status epilepticus in the pediatric population from our region prompted us to investigate further in this study. Methods We included retrospective data of all patients admitted to the pediatric intensive care unit (PICU) with a provisional diagnosis of RSE at a tertiary care hospital in Karachi from February 2019 to February 2021. No personal identification data was used, and confidentiality of the data was maintained throughout the analysis. The Statistical Package for the Social Sciences (SPSS) software version 22.0 (IBM SPSS Statistics, Armonk, NY, USA) was used to pool data and perform a descriptive analysis. Results Among the 687 patients who presented to the PICU with seizures, 50 (7.27%) patients were eventually diagnosed with RSE during the two-year period. The majority of the patients were male and less than one year of age. Infectious causes predominated our data cohort, and a four-drug regimen consisting of phenytoin, levetiracetam, valproic acid, and midazolam was able to terminate RSE in the majority of the patients in our setting (70%). The mortality rate was noted to be 22% among patients with RSE. Conclusion Morbidity and mortality among pediatric RSE patients are high in our settings. Urgent emergency services and timely cause-directed intervention could improve outcomes.

Unlocking the potential of co-applied biochar and plant growth-promoting rhizobacteria (PGPR) for sustainable agriculture under stress conditions.

Sustainable food security is a major challenge in today's world, particularly in developing countries. Among many factors, environmental stressors, i.e., drought, salinity and heavy metals are major impediments in achieving sustainable food security. This calls for finding environment-friendly and cheap solutions to address these stressors. Plant growth-promoting rhizobacteria (PGPR) have long been established as an environment-friendly means to enhance agricultural productivity in normal and stressed soils and are being applied at field scale. Similarly, pyrolyzing agro-wastes into biochar with the aim to amend soils is being proposed as a cheap additive for enhancement of soil quality and crop productivity. Many pot and some field-scale experiments have confirmed the potential of biochar for sustainable increase in agricultural productivity. Recently, many studies have combined the PGPR and biochar for improving soil quality and agricultural productivity, under normal and stressed conditions, with the assumption that both of these additives complement each other. Most of these studies have reported a significant increase in agricultural productivity in co-applied treatments than sole application of PGPR or biochar. This review presents synthesis of these studies in addition to providing insights into the mechanistic basis of the interaction of the PGPR and biochar. Moreover, this review highlights the future perspectives of the research in order to realize the potential of co-application of the PGPR and biochar at field scale.

Prophylactic Mesh Placement for the Prevention of Incisional Hernia in High-Risk Patients After Abdominal Surgery: A Systematic Review and Meta-Analysis.

Background and objectives In high-risk populations, the efficacy of mesh placement in incisional hernia (IH) prevention after elective abdominal surgeries has been supported by many published studies. This meta-analysis aimed at providing comprehensive and updated clinical implications of prophylactic mesh placement (PMP) for the prevention of IH as compared to primary suture closure (PSC). Materials and methods PubMed, Science Direct, Cochrane, and Google Scholar were systematically searched until March 3, 2020, for studies comparing the efficacy of PMP to PSC in abdominal surgeries. The main outcome of interest was the incidence of IH at different follow-up durations. All statistical analyses were carried out using Review Manager version 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration, 2014) and Stata 11.0 (Stata Corporation LP, College Station, TX). The data were pooled using the random-effects model, and odds ratio (OR) and weighted mean differences (WMD) were calculated with the corresponding 95% confidence interval (CI). Results A total of 3,330 were identified initially and after duplicate removal and exclusion based on title and abstract, 26 studies comprising 3,000 patients, were included. The incidence of IH was significantly reduced for PMP at follow-up periods of one year (OR= 0.16 [0.05, 0.51]; p=0.002; I2=77%), two years (OR= 0.23 [0.12, 0.45]; p<0.0001; I2=68%), three years (OR= 0.30 [0.16, 0.59]; p=0.0004; I2= 52%), and five years (OR=0.15 [0.03, 0.85]; p=0.03; I2=87%). However, PMP was associated with an increased risk of seroma (OR=1.67 [1.10, 2.55]; p= 0.02; I2=19%) and chronic wound pain (OR=1.71 [1.03, 2.83]; p= 0.04; I2= 0%). No significant difference between the PMP and PSC groups was noted for postoperative hematoma (OR= 1.04 [0.43, 2.50]; p=0.92; I2=0%), surgical site infection (OR=1.09 [0.78, 1.52]; p= 0.62; I2=12%), wound dehiscence (OR=0.69 [0.30, 1.62]; p=0.40; I2= 0%), gastrointestinal complications (OR= 1.40 [0.76, 2.58]; p=0.28; I2= 0%), length of hospital stay (WMD= -0.49 [-1.45, 0.48]; p=0.32; I2=0%), and operating time (WMD=9.18 [-7.17, 25.54]; p= 0.27; I2=80%). Conclusions PMP has been effective in reducing the rate of IH in the high-risk population at all time intervals, but it is associated with an increased risk of seroma and chronic wound pain. The benefits of mesh largely outweigh the risk, and it is linked with positive outcomes in high-risk patients.

Alignment and mapping methodology influence transcript abundance estimation.

BACKGROUND: The accuracy of transcript quantification using RNA-seq data depends on many factors, such as the choice of alignment or mapping method and the quantification model being adopted. While the choice of quantification model has been shown to be important, considerably less attention has been given to comparing the effect of various read alignment approaches on quantification accuracy. RESULTS: We investigate the influence of mapping and alignment on the accuracy of transcript quantification in both simulated and experimental data, as well as the effect on subsequent differential expression analysis. We observe that, even when the quantification model itself is held fixed, the effect of choosing a different alignment methodology, or aligning reads using different parameters, on quantification estimates can sometimes be large and can affect downstream differential expression analyses as well. These effects can go unnoticed when assessment is focused too heavily on simulated data, where the alignment task is often simpler than in experimentally acquired samples. We also introduce a new alignment methodology, called selective alignment, to overcome the shortcomings of lightweight approaches without incurring the computational cost of traditional alignment. CONCLUSION: We observe that, on experimental datasets, the performance of lightweight mapping and alignment-based approaches varies significantly, and highlight some of the underlying factors. We show this variation both in terms of quantification and downstream differential expression analysis. In all comparisons, we also show the improved performance of our proposed selective alignment method and suggest best practices for performing RNA-seq quantification.

A Bayesian framework for inter-cellular information sharing improves dscRNA-seq quantification.

MOTIVATION: Droplet-based single-cell RNA-seq (dscRNA-seq) data are being generated at an unprecedented pace, and the accurate estimation of gene-level abundances for each cell is a crucial first step in most dscRNA-seq analyses. When pre-processing the raw dscRNA-seq data to generate a count matrix, care must be taken to account for the potentially large number of multi-mapping locations per read. The sparsity of dscRNA-seq data, and the strong 3' sampling bias, makes it difficult to disambiguate cases where there is no uniquely mapping read to any of the candidate target genes. RESULTS: We introduce a Bayesian framework for information sharing across cells within a sample, or across multiple modalities of data using the same sample, to improve gene quantification estimates for dscRNA-seq data. We use an anchor-based approach to connect cells with similar gene-expression patterns, and learn informative, empirical priors which we provide to alevin's gene multi-mapping resolution algorithm. This improves the quantification estimates for genes with no uniquely mapping reads (i.e. when there is no unique intra-cellular information). We show our new model improves the per cell gene-level estimates and provides a principled framework for information sharing across multiple modalities. We test our method on a combination of simulated and real datasets under various setups. AVAILABILITY AND IMPLEMENTATION: The information sharing model is included in alevin and is implemented in C++14. It is available as open-source software, under GPL v3, at https://github.com/COMBINE-lab/salmon as of version 1.1.0.

Biotinidase Deficiency With Suspected Sotos Syndrome: A Rare Entity.

Biotinidase deficiency (BTD) is a rare yet treatable metabolic autosomal recessive (AR) disorder in which the body is unable to recycle the vitamin biotin. Early diagnosis and treatment can be life-saving, but some symptoms of the disease are irreversible, and the condition can even prove to be fatal if not correctly diagnosed and managed. Here we present a case of a six-month-old child who presented with cough, fever, and difficulty in breathing. Respiratory examination revealed deep subcostal and intercostal recessions, bilateral crepitations, and wheezes. On central nervous system (CNS) examination, the baby had a low Glasgow Coma Scale (GCS) score of 10 while the tone was decreased, and bulk was increased in all four limbs. Chest X-ray revealed haziness at the right middle and lower lobes. Antibiotics were started keeping pneumonia, bronchiolitis, and sepsis in mind along with an initial diagnosis of inborn error of metabolism (IEM). As the patient's condition deteriorated, nasal bubble continuous positive airway pressure (CPAP) and nebulization were provided and later put on a ventilator. Arterial blood gases (ABGs) showed severe metabolic acidosis and compensatory respiratory alkalosis with an anion gap of 15. Urine profile for organic acid was performed, and the diagnosis of sepsis with BTD was made. Unfortunately, our patient expired on the fourth day of admission before a biotin injection could be searched and administered. Moreover, our patient was also suspected of a possible Sotos syndrome, which is a rare genetic disorder characterized by excessive growth in the initial years of life. The case highlights the significance of the diagnosis of such metabolic disorders in the natal period of life and their immediate management.

Accuracy of Pediatric Risk of Mortality (PRISM) III Score in Predicting Mortality Outcomes in a Pediatric Intensive Care Unit in Karachi.

Background With the advancements in medicine and increasing access to modern technology, pediatric intensive care units (PICU) are becoming a vital part of any health care setting. PICUs play a key role in saving the life of young patients. Various scales have been designed by researchers to aid in predicting the mortality of a patient admitted in PICU. Pediatric Risk of Mortality (PRISM) and Pediatric Index of Mortality (PIM) are among the most commonly used scales. Calculating the risk of mortality enables the physicians to classify the patients and helps in identifying which patients require more urgent care and resources.  Methods A hospital-based prospective study was carried out at PICU in a tertiary care hospital in Karachi from December 2017 to June 2019. All patients between the age of one month and 12 years were included in our study after informed consent from parents/guardians. A standard questionnaire was used and the PRISM III score was calculated at 24 hours of admission. All necessary investigations were carried out, and all statistical analyses were carried out using SPSS v.23 (IBM, Armonk, NY). Results A total of 407 patients were included in our study with the majority being males (54.5%). The mean age was 27±33 months. The mean duration of stay of patients in PICU was 80.15±36.58 hours. The mortality rate in our study was 37.35 % (n=152). The need for mechanical ventilation, use of inotropic drugs, higher temperatures, and low Glasgow Coma Scale scores were associated with poor survival. It was noted that as the PRISM III score increased, the mortality rate also increased. In our study, we found that PRISM III had good predictive power in our population. The area under the curve was 0.903±0.016 (p<0.001, 95% confidence interval: 0.872-0.934). Conclusions PRISM III score showed excellent accuracy and predictive ability in our population. There was no significant difference in observed and expected mortality rates in our study. In a resource-limited setting, the prediction models highlight the cases where more medical attention is required and also enable the physicians to assess the prognosis of the patient so adequate measures can be taken beforehand.

Michelin Tire Baby Syndrome: A Rare Case with Review of Literature.

Michelin tire baby syndrome (MTBS) is a benign hamartomatous condition with ring-like lesions present on the limbs and trunk. MTBS is a rare genodermatosis. According to our search, only 20 cases have been reported. We present a case of a six-month-old female child, with complaints of fever and seizures. Since birth, she had asymptomatic multiple, asymmetric skin folds on all four limbs, resembling "Michelin Man" logo of the French tire manufacturer. She had microcephaly with characteristic round face hypertelorism, depressed nasal bridge, hypertrichosis with low set ears, a thin down-turned vermillion border of the upper lip, and a short neck. MRI was normal. Clinically, the diagnosis of MTBS was made. In addition, the parents were counseled about the self-limiting course of this disorder. MTBS itself might not be a single disorder but may manifest as a clinical finding associated with other disorders; therefore, a regular follow up of these patients is usually advised.

Alevin efficiently estimates accurate gene abundances from dscRNA-seq data.

We introduce alevin, a fast end-to-end pipeline to process droplet-based single-cell RNA sequencing data, performing cell barcode detection, read mapping, unique molecular identifier (UMI) deduplication, gene count estimation, and cell barcode whitelisting. Alevin's approach to UMI deduplication considers transcript-level constraints on the molecules from which UMIs may have arisen and accounts for both gene-unique reads and reads that multimap between genes. This addresses the inherent bias in existing tools which discard gene-ambiguous reads and improves the accuracy of gene abundance estimates. Alevin is considerably faster, typically eight times, than existing gene quantification approaches, while also using less memory.

Rich Chromatin Structure Prediction from Hi-C Data.

Recent studies involving the 3-dimensional conformation of chromatin have revealed the important role it has to play in different processes within the cell. These studies have also led to the discovery of densely interacting segments of the chromosome, called topologically associating domains. The accurate identification of these domains from Hi-C interaction data is an interesting and important computational problem for which numerous methods have been proposed. Unfortunately, most existing algorithms designed to identify these domains assume that they are non-overlapping whereas there is substantial evidence to believe a nested structure exists. We present a methodology to predict hierarchical chromatin domains using chromatin conformation capture data. Our method predicts domains at different resolutions, calculated using intrinsic properties of the chromatin data, and effectively clusters these to construct the hierarchy. At each individual level, the domains are non-overlapping in such a way that the intra-domain interaction frequencies are maximized. We show that our predicted structure is highly enriched for actively transcribing housekeeping genes and various chromatin markers, including CTCF, around the domain boundaries. We also show that large-scale domains, at multiple resolutions within our hierarchy, are conserved across cell types and species. We also provide comparisons against existing tools for extracting hierarchical domains. Our software, Matryoshka, is written in C++11 and licensed under GPL v3; it is available at https://github.com/COMBINE-lab/matryoshka.

Emergence of Multi-Resistant Enteric Infection In A Paediatric Unit Of Karachi, Pakistan.

From June 2018, onwards, there has been an upsurge of multi-resistant enteric infections in children admitted from various catchment areas of Abbasi Shaheed Hospital (ASH). This is a serious concern as very few antibiotics are available to treat the children. Children from June 2018 to September 2018 of age groups 5.7 ± 2.84 (range 1.6 to 11 years), referred to ASH, for admission, with clinical suspicion of enteric fever and having received a third generation injectable cephalosporin by a general practitioner, for 5 days or more, with no response, and continuation of fever, were included. A total number of 137 patients had culture proven salmonella typhi, of whom 61(44.52%) showed sensitivity only to meropenem, 45 (32.8%) to azithromycin,13(9.4%)to fosfomycin, 11(8.02%) to Amoxicillin/clavulanic acid, 5 patients showed sensitivity to ceftriaxone(3.64%) and one had sensitivity to amikacin. All patients were treated successfully for 10 days and discharged home. There were no reported complications at follow-up. Multi-Drug Resistance (MDR) enteric fever appears to be a major health concern in Karachi. Mass immunization with oral live attenuated Typhi 21a or injectable unconjugated Vi typhoid vaccine, rational use of antibiotics, improvement in public sanitation facilities, availability of clean drinking water, promotion of safe food handling practices and public health education are vital in the prevention of MDR enteric fever.

Combinatorial Loss of the Enzymatic Activities of Viral Uracil-DNA Glycosylase and Viral dUTPase Impairs Murine Gammaherpesvirus Pathogenesis and Leads to Increased Recombination-Based Deletion in the Viral Genome.

Misincorporation of uracil or spontaneous cytidine deamination is a common mutagenic insult to DNA. Herpesviruses encode a viral uracil-DNA glycosylase (vUNG) and a viral dUTPase (vDUT), each with enzymatic and nonenzymatic functions. However, the coordinated roles of these enzymatic activities in gammaherpesvirus pathogenesis and viral genomic stability have not been defined. In addition, potential compensation by the host UNG has not been examined in vivo The genetic tractability of the murine gammaherpesvirus 68 (MHV68) system enabled us to delineate the contribution of host and viral factors that prevent uracilated DNA. Recombinant MHV68 lacking vUNG (ORF46.stop) was not further impaired for acute replication in the lungs of UNG-/- mice compared to wild-type (WT) mice, indicating host UNG does not compensate for the absence of vUNG. Next, we investigated the separate and combinatorial consequences of mutating the catalytic residues of the vUNG (ORF46.CM) and vDUT (ORF54.CM). ORF46.CM was not impaired for replication, while ORF54.CM had a slight transient defect in replication in the lungs. However, disabling both vUNG and vDUT led to a significant defect in acute expansion in the lungs, followed by impaired establishment of latency in the splenic reservoir. Upon serial passage of the ORF46.CM/ORF54.CM mutant in either fibroblasts or the lungs of mice, we noted rapid loss of the nonessential yellow fluorescent protein (YFP) reporter gene from the viral genome, due to recombination at repetitive elements. Taken together, our data indicate that the vUNG and vDUT coordinate to promote viral genomic stability and enable viral expansion prior to colonization of latent reservoirs.IMPORTANCE Unrepaired uracils in DNA can lead to mutations and compromise genomic stability. Herpesviruses have hijacked host processes of DNA repair and nucleotide metabolism by encoding a viral UNG that excises uracils and a viral dUTPase that initiates conversion of dUTP to dTTP. To better understand the impact of these processes on gammaherpesvirus pathogenesis, we examined the separate and collaborative roles of vUNG and vDUT upon MHV68 infection of mice. Simultaneous disruption of the enzymatic activities of both vUNG and vDUT led to a severe defect in acute replication and establishment of latency, while also revealing a novel, combinatorial function in promoting viral genomic stability. We propose that herpesviruses require these enzymatic processes to protect the viral genome from damage, possibly triggered by misincorporated uracil. This reveals a novel point of therapeutic intervention to potentially block viral replication and reduce the fitness of multiple herpesviruses.

Grouper: graph-based clustering and annotation for improved de novo transcriptome analysis.

Motivation: De novo transcriptome analysis using RNA-seq offers a promising means to study gene expression in non-model organisms. Yet, the difficulty of transcriptome assembly means that the contigs provided by the assembler often represent a fractured and incomplete view of the transcriptome, complicating downstream analysis. We introduce Grouper, a new method for clustering contigs from de novo assemblies that are likely to belong to the same transcripts and genes; these groups can subsequently be analyzed more robustly. When provided with access to the genome of a related organism, Grouper can transfer annotations to the de novo assembly, further improving the clustering. Results: On de novo assemblies from four different species, we show that Grouper is able to accurately cluster a larger number of contigs than the existing state-of-the-art method. The Grouper pipeline is able to map greater than 10% more reads against the contigs, leading to accurate downstream differential expression analyses. The labeling module, in the presence of a closely related annotated genome, can efficiently transfer annotations to the contigs and use this information to further improve clustering. Overall, Grouper provides a complete and efficient pipeline for processing de novo transcriptomic assemblies. Availability and implementation: The Grouper software is freely available at https://github.com/COMBINE-lab/grouper under the 2-clause BSD license. Supplementary information: Supplementary data are available at Bioinformatics online.

RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency.

RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB) signaling during murine gammaherpesvirus 68 (MHV68) infection: a latent B cell line (HE-RIT) inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio) that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R) that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA.

Relationship between stress and coronary heart disease.

BACKGROUND: Coronary heart disease is the leading cause of death worldwide and accounts for 13.7% of deaths in countries like Pakistan. Its association with stress has not been well considered in our setup. Patients with coronary artery disease admitted for coronary artery bypass grafting may have a high prevalence of stress that might increase the risk of adverse outcomes. METHODS: 60 patients with coronary artery disease admitted to the Civil Hospital Karachi for coronary artery bypass graft surgery from January 1 to March 31, 2012, were evaluated using a stress evaluation scale. RESULTS: Stress of varying degrees was found to be a significant independent risk factor in patients with coronary heart disease. Analysis of our collected sample of patients with stress showed 60% with high stress (p = 0.025) and 36.7% moderate stress (p = 0.0025). An appreciable relationship was found between stress and patient age, sex, body mass index, blood group, and the incidence of myocardial infarction. CONCLUSION: Our study found evidence of an independent causative association between psychological stress and coronary heart disease, of a similar order to the more conventional coronary heart disease risk factors.