Sequential viral introductions and spread of BA.1 across Pakistan provinces during the Omicron wave.

BACKGROUND: COVID-19 waves caused by specific SARS-CoV-2 variants have occurred globally at different times. We focused on Omicron variants to understand the genomic diversity and phylogenetic relatedness of SARS-CoV-2 strains in various regions of Pakistan. METHODS: We studied 276,525 COVID-19 cases and 1,031 genomes sequenced from December 2021 to August 2022. Sequences were analyzed and visualized using phylogenetic trees. RESULTS: The highest case numbers and deaths were recorded in Sindh and Punjab, the most populous provinces in Pakistan. Omicron variants comprised 93% of all genomes, with BA.2 (32.6%) and BA.5 (38.4%) predominating. The first Omicron wave was associated with the sequential identification of BA.1 in Sindh, then Islamabad Capital Territory, Punjab, Khyber Pakhtunkhwa (KP), Azad Jammu Kashmir (AJK), Gilgit-Baltistan (GB) and Balochistan. Phylogenetic analysis revealed Sindh to be the source of BA.1 and BA.2 introductions into Punjab and Balochistan during early 2022. BA.4 was first introduced in AJK and BA.5 in Punjab. Most recent common ancestor (MRCA) analysis revealed relatedness between the earliest BA.1 genome from Sindh with Balochistan, AJK, Punjab and ICT, and that of first BA.1 from Punjab with strains from KPK and GB. CONCLUSIONS: Phylogenetic analysis provides insights into the introduction and transmission dynamics of the Omicron variant in Pakistan, identifying Sindh as a hotspot for viral dissemination. Such data linked with public health efforts can help limit surges of new infections.

Evolutionary history and introduction of SARS-CoV-2 Alpha VOC/B.1.1.7 in Pakistan through international travelers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge, and their identification is important for the public health response to coronavirus disease 2019 (COVID-19). Genomic sequencing provides robust information but may not always be accessible, and therefore, mutation-based polymerase chain reaction (PCR) approaches can be used for rapid identification of known variants. International travelers arriving in Karachi between December 2020 and February 2021 were tested for SARS-CoV-2 by PCR. A subset of positive samples was tested for S-gene target failure (SGTF) on TaqPathTM COVID-19 (Thermo Fisher Scientific) and for mutations using the GSD NovaType SARS-CoV-2 (Eurofins Technologies) assays. Sequencing was conducted on the MinION platform (Oxford Nanopore Technologies). Bayesian phylogeographic inference was performed integrating the patients' travel history information. Of the thirty-five COVID-19 cases screened, thirteen had isolates with SGTF. The travelers transmitted infection to sixty-eight contact cases. The B.1.1.7 lineage was confirmed through sequencing and PCR. The phylogenetic analysis of sequence data available for six cases included four B.1.1.7 strains and one B.1.36 and B.1.1.212 lineage isolate. Phylogeographic modeling estimated at least three independent B.1.1.7 introductions into Karachi, Pakistan, originating from the UK. B.1.1.212 and B.1.36 were inferred to be introduced either from the UK or the travelers' layover location. We report the introduction of SARS-CoV-2 B.1.1.7 and other lineages in Pakistan by international travelers arriving via different flight routes. This highlights SARS-CoV-2 transmission through travel, importance of testing, and quarantine post-travel to prevent transmission of new strains, as well as recording detailed patients' metadata. Such results help inform policies on restricting travel from destinations where new highly transmissible variants have emerged.

Programmed cell death ligand 1 expression in aggressive pediatric non-Hodgkin lymphomas: frequency, genetic mechanisms, and clinical significance.

Programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are immunomodulatory molecules overexpressed in lymphomas and are promising immunotherapy targets for hematologic malignancies. However, studies of PD-1/PD-L1 overexpression and their clinical significance in aggressive pediatric non-Hodgkin lymphomas (NHL) are limited. We assessed PD-1/PD-L1 overexpression using immunohistochemistry in 68 aggressive pediatric NHL: ALK-positive anaplastic large cell lymphoma (ALK+ ALCL, n=8), Burkitt lymphoma (BL, n=27), and large B-cell lymphoma (LBCL) de novo LBCL, n=22 and diffuse LBCL arising as monomorphic post-transplant lymphoproliferative disorder [PTLD-DLBCL], n=11. In LBCL, correlations between PD-L1 overexpression and Epstein-Barr virus (EBV) status, cell of origin, stage, nodal status, overall survival (OS), and event-free survival (EFS) were examined. The genetic mechanisms of PD-L1 overexpression were investigated using targeted next-generation sequencing (NGS) and cytogenetic data. All ALK+ ALCL samples, 50.0% of de novo LBCL (11/22), 72.7% of PTLD-DLBCL (8/11), and no BL overexpressed PD-L1. Overexpressed PD-L1 correlated with EBV positivity (P=0.033) in LBCL and lower EFS in de novo LBCL (P=0.017). NGS of select LBCL revealed distinct somatic mutations and an ultra-hypermutated PTLD-DLBCL. Most cases with 9p24.1 copy gains overexpressed PD-L1 although some cases had no discernible genetic drivers of PD-L1 overexpression. Overexpressed PD-L1 is common in pediatric LBCL, associated with EBV positivity and 9p24.1 gains, and may have prognostic significance in de novo LBCL. Furthermore, diverse molecular mechanisms for PD-L1 overexpression in aggressive pediatric NHL can occur. Thus, additional studies exploring the therapeutic and prognostic significance and molecular mechanisms of PD-L1 overexpression in aggressive pediatric NHL are warranted.

The META tool optimizes metagenomic analyses across sequencing platforms and classifiers.

A major challenge in the field of metagenomics is the selection of the correct combination of sequencing platform and downstream metagenomic analysis algorithm, or "classifier". Here, we present the Metagenomic Evaluation Tool Analyzer (META), which produces simulated data and facilitates platform and algorithm selection for any given metagenomic use case. META-generated in silico read data are modular, scalable, and reflect user-defined community profiles, while the downstream analysis is done using a variety of metagenomic classifiers. Reported results include information on resource utilization, time-to-answer, and performance. Real-world data can also be analyzed using selected classifiers and results benchmarked against simulations. To test the utility of the META software, simulated data was compared to real-world viral and bacterial metagenomic samples run on four different sequencers and analyzed using 12 metagenomic classifiers. Lastly, we introduce "META Score": a unified, quantitative value which rates an analytic classifier's ability to both identify and count taxa in a representative sample.

Genetic and evolutionary analysis of SARS-CoV-2 circulating in the region surrounding Islamabad, Pakistan.

Genomic epidemiology of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has provided global epidemiological insight into the COVID-19 pandemic since it began. Sequencing of the virus has been performed at scale, with many countries depositing data into open access repositories to enable in-depth global phylogenetic analysis. To contribute to these efforts, we established an Oxford Nanopore Technologies (ONT) sequencing capability at the National Institutes of Health (NIH), Pakistan. This study highlights multiple SARS-CoV-2 lineages co-circulating during the peak of a second COVID-19 wave in Pakistan (Nov 2020-Feb 2021), with virus origins traced to the United States of America and Saudi Arabia. Ten SARS-CoV-2 positive samples were used for ONT library preparation. Sequence and phylogenetic analysis determined that the patients were infected with lineage B.1.1.250, originally identified in the United Kingdom and Bangladesh during March and April of 2020, and in circulation until the time of this study in Europe, USA and Australia. Lineage B.1.261 was originally identified in Saudi Arabia with widespread local dissemination in Pakistan. One sample clustered with the parental B.1 lineage and the other with lineage B.6 originally from Singapore. In the future, monitoring the evolutionary dynamics of circulating lineages in Pakistan will enable improved tracing of the viral spread, changing trends of their expansion trajectories, persistence, changes in their demographic dynamics, and provide guidance for better implementation of control measures.

Genomic trajectory in leukemogenesis of myeloproliferative neoplasms: a case report.

BACKGROUND: We report a patient with Essential Thrombocythemia (ET), subsequently diagnosed with concurrent myeloid and lymphoid leukemia. Generally, the molecular mechanisms underlying leukemic transformation of Philadelphia-negative myeloproliferative neoplasms (Ph-MPN) are poorly understood. Risk of transformation to acute myelogenous leukemia (AML) is low; transformation to both AML and acute lymphoblastic leukemia (ALL) is extremely low. Genetic defects, including allele burden, order of mutation acquisition, clonal heterogeneity and epigenetic mechanisms are important contributors to disease acceleration. CASE PRESENTATION: A 78-year-old Caucasian female originally treated for stable ET, underwent disease acceleration and transition to myeloid sarcoma and B-cell ALL. Genomic reconstruction based on targeted sequencing revealed the presence of a large del(5q) in all three malignancies and somatic driver mutations: TET2, TP53, SF3B1, and ASXL1 at high allele frequency. We propose that the combination of genetic and molecular abnormalities led to hematopoietic stem cell (HSC) injury and disease progression through sub-clone branching. We hypothesize that ancestral reconstruction of genomic data is a useful tool to uncover subclonal events leading to transformation. CONCLUSIONS: The use of ancestral reconstruction of genomic data sheds light on the unique clinical scenario described in this case report. By determining the mutational profile of tumors at several timepoints and deducing the most parsimonious relationship between them, we propose a reconstruction of their origin. We propose that blast progression originated from subclonal events with malignant potential, which coexisted with but did not originate from JAK2 p.V617F-positive ET. We conclude that the application of genomic reconstruction enhances our understanding of leukemogenesis by identifying the timing of molecular events, potentially leading to better chemotherapy choices as well as the development of new targeted therapies.

Medical Degree Disparity Among Authors of Original Research in Pediatric Journals.

INTRODUCTION: Research and scholarly output are integral parts of residency training for both residents and faculty. With the transition to a single accreditation system, scholarly activity and output of osteopathic physicians have garnered significant interest. Previous research has shown that osteopathic physicians in emergency medicine and obstetrics and gynecology infrequently publish original research in high impact journals. OBJECTIVE: To determine whether there is a disparity between osteopathic and allopathic physicians among authors who publish original research manuscripts in three high-impact pediatric journals. METHODS: The medical degree designation of the first and senior author (last author) and any advanced degree either author may have obtained were retrieved from the Journal of Pediatrics (J Pediatr), Pediatrics, and JAMA Pediatrics (JAMA Pediatr) for the years 2000, 2005, 2010 and 2015. Data was analyzed using simple descriptive statistics and linear regression. RESULTS: In total, 2232 manuscripts and 4296 authors were reviewed with 0.58% (25/4296) of all authors being osteopathic physicians. A total of 0.81% (18/2232) of first authors and 0.34% (7/2064) of senior authors were osteopathic physicians. For those with a dual degree, a total of 0.64% (5/777) of first and 0.33% (3/904) of senior authors were osteopathic physicians. No statistical trend could be established for increased first (p=0.24), senior (p=0.16), dual degree first (p=0.08) or dual degree senior (p=0.06) osteopathic physician authorship. Likewise, no statistical trend for increased authorship could be established for any Doctor of Osteopathic Medicine (DO) authorship role in the three journals over the time period studied. CONCLUSION: Very few osteopathic physicians have served as either the first or senior author in published original research manuscripts for the Journal of Pediatrics, Pediatrics, or JAMA Pediatrics for the years studied. Also, no statistical trend could be established for increased osteopathic physician publication over the same years.

GRIBCG: a software for selection of sgRNAs in the design of balancer chromosomes.

BACKGROUND: Balancer chromosomes are tools used by fruit fly geneticists to prevent meiotic recombination. Recently, CRISPR/Cas9 genome editing has been shown capable of generating inversions similar to the chromosomal rearrangements present in balancer chromosomes. Extending the benefits of balancer chromosomes to other multicellular organisms could significantly accelerate biomedical and plant genetics research. RESULTS: Here, we present GRIBCG (Guide RNA Identifier for Balancer Chromosome Generation), a tool for the rational design of balancer chromosomes. GRIBCG identifies single guide RNAs (sgRNAs) for use with Streptococcus pyogenes Cas9 (SpCas9). These sgRNAs would efficiently cut a chromosome multiple times while minimizing off-target cutting in the rest of the genome. We describe the performance of this tool on six model organisms and compare our results to two routinely used fruit fly balancer chromosomes. CONCLUSION: GRIBCG is the first of its kind tool for the design of balancer chromosomes using CRISPR/Cas9. GRIBCG can accelerate genetics research by providing a fast, systematic and simple to use framework to induce chromosomal rearrangements.

Medical Degree Disparity Among Authors in Obstetrics and Gynecology Journals.

CONTEXT: With the transition to a single accreditation system for graduate medical education, the scholarly activity among core faculty in osteopathic and allopathic residency programs has come under scrutiny. Currently, major differences in scholarly activity requirements exist between core faculty in obstetrics and gynecology residencies accredited by the Accreditation Council for Graduate Medical Education and those accredited by the American Osteopathic Association. OBJECTIVE: To determine whether there is a disparity between osteopathic and allopathic physicians among authors with original research published in 4 high-impact obstetrics and gynecology journals during 4 select years. METHODS: The authors reviewed Obstetrics & Gynecology (Obstet Gynecol), the American Journal of Obstetrics and Gynecology (Am J Obstet Gynecol), Fertility and Sterility (Fertil Steril), and Menopause for the degree designation of the first and senior (last) author of each original manuscript for the years of 2000, 2005, 2010, and 2015. Data were analyzed using simple descriptive statistics and linear regression. RESULTS: In total, 3311 articles and 5909 authors were reviewed. Of these authors, 0.80% (47) had a DO degree. Of 1692 authors with dual advanced degrees, only 0.53% (9) had a DO degree. On subgroup analysis of each journal, 0.87% (13 of 1494) of identified authors in Obstet Gynecol, 1.03% (21 of 2038) in Am J Obstet Gynecol, 0.44% (9 of 2030) in Fertil Steril, and 2.20% (4 of 347) in Menopause were osteopathic physicians. During the years studied, no statistically significant trend could be established for first or senior author publication by osteopathic physicians over time, for all 4 journals or for any individual journal. CONCLUSION: Very few osteopathic physicians have served as either the first or the senior author in articles published in Obstet Gynecol, Am J Obstet Gynecol, Fertil Steril, or Menopause during the years studied, and no trend was seen for increased publication by osteopathic physicians in these journals over time.

Identification of developmental stage and anatomical fraction contributions to cell wall recalcitrance in switchgrass.

BACKGROUND: Heterogeneity within herbaceous biomass can present important challenges for processing feedstocks to cellulosic biofuels. Alterations to cell wall composition and organization during plant growth represent major contributions to heterogeneity within a single species or cultivar. To address this challenge, the focus of this study was to characterize the relationship between composition and properties of the plant cell wall and cell wall response to deconstruction by NaOH pretreatment and enzymatic hydrolysis for anatomical fractions (stem internodes, leaf sheaths, and leaf blades) within switchgrass at various tissue maturities as assessed by differing internode. RESULTS: Substantial differences in both cell wall composition and response to deconstruction were observed as a function of anatomical fraction and tissue maturity. Notably, lignin content increased with tissue maturity concurrently with decreasing ferulate content across all three anatomical fractions. Stem internodes exhibited the highest lignin content as well as the lowest hydrolysis yields, which were inversely correlated to lignin content. Confocal microscopy was used to demonstrate that removal of cell wall aromatics (i.e., lignins and hydroxycinnamates) by NaOH pretreatment was non-uniform across diverse cell types. Non-cellulosic polysaccharides were linked to differences in cell wall response to deconstruction in lower lignin fractions. Specifically, leaf sheath and leaf blade were found to have higher contents of substituted glucuronoarabinoxylans and pectic polysaccharides. Glycome profiling demonstrated that xylan and pectic polysaccharide extractability varied with stem internode maturity, with more mature internodes requiring harsher chemical extractions to remove comparable glycan abundances relative to less mature internodes. While enzymatic hydrolysis was performed on extractives-free biomass, extractible sugars (i.e., starch and sucrose) comprised a significant portion of total dry weight particularly in stem internodes, and may provide an opportunity for recovery during processing. CONCLUSIONS: Cell wall structural differences within a single plant can play a significant role in feedstock properties and have the potential to be exploited for improving biomass processability during a biorefining process. The results from this work demonstrate that cell wall lignin content, while generally exhibiting a negative correlation with enzymatic hydrolysis yields, is not the sole contributor to cell wall recalcitrance across diverse anatomical fractions within switchgrass.

Cell wall composition and penetration resistance against the fungal pathogen Colletotrichum higginsianum are affected by impaired starch turnover in Arabidopsis mutants.

Penetration resistance represents the first level of plant defense against phytopathogenic fungi. Here, we report that the starch-deficient Arabidopsis thaliana phosphoglucomutase (pgm) mutant has impaired penetration resistance against the hemibiotrophic fungus Colletotrichum higginsianum. We could not determine any changes in leaf cutin and epicuticular wax composition or indolic glucosinolate levels, but detected complex alterations in the cell wall monosaccharide composition of pgm. Notably, other mutants deficient in starch biosynthesis (adg1) or mobilization (sex1) had similarly affected cell wall composition and penetration resistance. Glycome profiling analysis showed that both overall cell wall polysaccharide extractability and relative extractability of specific pectin and xylan epitopes were affected in pgm, suggesting extensive structural changes in pgm cell walls. Screening of mutants with alterations in content or modification of specific cell wall monosaccharides indicated an important function of pectic polymers for penetration resistance and hyphal growth of C. higginsianum during the biotrophic interaction phase. While mutants with affected pectic rhamnogalacturonan-I (mur8) were hypersusceptible, penetration frequency and morphology of fungal hyphae were impaired on pmr5 pmr6 mutants with increased pectin levels. Our results reveal a strong impact of starch metabolism on cell wall composition and suggest a link between carbohydrate availability, cell wall pectin and penetration resistance.

Somatic mutations in children with GATA2-associated myelodysplastic syndrome who lack other features of GATA2 deficiency.

Approximately 10% of children with primary myelodysplastic syndrome (MDS) have germ line GATA2 mutations, leading to the proposal that all children with primary MDS and certain cytogenetic findings, including monosomy 7, be tested for germ line GATA2 mutations regardless of family history or other clinical features associated with GATA2 deficiency. In adults with familial GATA2-MDS, those with somatic mutations in ASXL1 experience rapid disease progression to acute myeloid leukemia (AML) and poor prognosis after stem cell transplantation; however, the prevalence of somatic mutations in primary pediatric GATA2-MDS is unclear. Here, we studied a cohort of 8 pediatric patients with MDS and lacking additional GATA2-associated clinical features or significant family history and identified heterozygous germ line GATA2 mutations in 5 patients, including 1 with a normal karyotype. For those with GATA2-MDS, we screened for somatic mutations in genes with prognostic relevance in AML/MDS, using a targeted next-generation sequencing panel. Although no somatic mutations in ASXL1 were observed, somatic mutations were found in RUNX1, SETBP1, IKZF1, and CRLF2. One subject with deleterious mutations in RUNX1, SETBP1, and IKZF1 rapidly progressed to AML with disease that was refractory to treatment. Our findings confirm the importance of GATA2 testing in primary pediatric MDS, even in the absence of other clinical features of GATA2 deficiency. Further, similar to what has been observed in adults with GATA2-MDS, somatic mutations with potential prognostic effect occur in children with MDS associated with mutations in GATA2.

Childhood nodal marginal zone lymphoma with unusual clinicopathologic and cytogenetic features for the pediatric variant: a case report.

Nodal marginal zone lymphoma (NMZL) is a B-cell lymphoma that shares morphologic and immunophenotypic features with extranodal and splenic marginal zone lymphomas but lacks extranodal or splenic involvement at presentation. NMZL occurs mostly in adults with no sex predilection, at advanced stage (III or IV), with frequent relapses and a high incidence of tumoral genetic abnormalities including trisomies 3 and 18 and gain of 7q. Pediatric NMZL, however, is a rare but distinct variant of NMZL with characteristic features including male predominance, asymptomatic and localized (stage I) disease, low relapse rates with excellent outcomes, and a lower incidence of essentially similar genetic aberrations compared to adult NMZL. Here we describe a unique case of childhood NMZL with unusual clinicopathologic features for the pediatric variant including generalized lymphadenopathy, high-stage disease with persistence after therapy, unusual immunophenotype (CD5, CD23, and BCL6 positive), and unique chromosomal abnormalities including monosomy 20 and add(10)(p11.2).

Hemophagocytic lymphohistiocytosis associated with influenza A (H1N1) infection in a patient with chronic lymphocytic leukemia: an autopsy case report and review of the literature.

H1N1 influenza A virus can trigger fatal hemophagocytic lymphohistiocytosis in immunocompromised patients and in immunocompetent hosts, usually children. We present a case of a 50-year-old man with low-burden chronic lymphocytic leukemia who had sudden reactivation of his leukemia triggered by influenza A (H1N1) infection with hemophagocytic lymphohistiocytosis during the 2009 H1N1 pandemic. His rapid course was complicated by acute respiratory distress syndrome with diffuse alveolar damage, a 6-fold rise in lymphocyte count, disseminated intravascular coagulation, and, ultimately, cardiac arrest. Major findings at autopsy included: bilateral H1N1 pneumonitis with diffuse alveolar damage, intra-alveolar pulmonary hemorrhage, pulmonary microthromboemboli, pulmonary hemorrhagic infarction, hemophagocytic lymphohistiocytosis in multiple locations, and diffuse chronic lymphocytic leukemia. Hemophagocytic lymphohistiocytosis is a serious and often fatal condition, which may be primary or secondary. It may be associated with high-grade lymphoproliferative malignancies, especially in patients with therapy-related leukocytopenia, but only rarely is it seen in uncomplicated chronic lymphocytic leukemia. Hemophagocytic lymphohistiocytosis may be triggered by a variety of infections (viral, fungal, bacterial and parasitic), but H1N1 influenza A-associated hemophagocytic lymphohistiocytosis is often rapidly fatal, especially in children. This adult patient's clinical presentation with low tumor burden and leukocytosis is thus unique. We review the recently published autopsy findings in fatal influenza A (H1N1) infection and the association with resultant secondary hemophagocytic lymphohistiocytosis.