Phenotypic characterization and genome analysis of a novel Salmonella Typhimurium phage having unique tail fiber genes.

Salmonella enterica serovar Typhimurium is a foodborne pathogen causing occasional outbreaks of enteric infections in humans. Salmonella has one of the largest pools of temperate phages in its genome that possess evolutionary significance for pathogen. In this study, we characterized a novel temperate phage Salmonella phage BIS20 (BIS20) with unique tail fiber genes. It belongs to the subfamily Peduovirinae genus Eganvirus and infects Salmonella Typhimurium strain (SE-BS17; Acc. NO MZ503545) of poultry origin. Phage BIS20 was viable only at biological pH and temperature ranges (pH7 and 37 °C). Despite being temperate BIS20 significantly slowed down the growth of host strain for 24 h as compared to control (P < 0.009). Phage BIS20 features 29,477-base pair (bp) linear DNA genome with 53% GC content and encodes for 37 putative ORFs. These ORFs have mosaic arrangement as indicated by its ORF similarity to various phages and prophages in NCBI. Genome analysis indicates its similarity to Salmonella enterica serovar Senftenberg prophage (SEStP) sequence (Nucleotide similarity 87.7%) and Escherichia virus 186 (~ 82.4% nucleotide similarity). Capsid genes were conserved however those associated with tail fiber formation and assembly were unique to all members of genus Eganvirus. We found strong evidence of recombination hotspot in tail fiber gene. Our study identifies BIS20 as a new species of genus Eganvirus temperate phages as its maximum nucleotide similarity is 82.4% with any phage in NCBI. Our findings may contribute to understanding of origin of new temperate phages.fals

Characterization of two novel lytic bacteriophages having lysis potential against MDR avian pathogenic Escherichia coli strains of zoonotic potential.

Avian pathogenic E. coli (APEC) is associated with local and systemic infections in poultry, ducks, turkeys, and many other avian species, leading to heavy economical losses. These APEC strains are presumed to possess zoonotic potential due to common virulence markers that can cause urinary tract infections in humans. The prophylactic use of antibiotics in the poultry sector has led to the rapid emergence of Multiple Drug Resistant (MDR) APEC strains that act as reservoirs and put human populations at risk. This calls for consideration of alternative strategies to decrease the bacterial load. Here, we report isolation, preliminary characterization, and genome analysis of two novel lytic phage species (Escherichia phage SKA49 and Escherichia phage SKA64) against MDR strain of APEC, QZJM25. Both phages were able to keep QZJM25 growth significantly less than the untreated bacterial control for approximately 18 h. The host range was tested against Escherichia coli strains of poultry and human UTI infections. SKA49 had a broader host range in contrast to SKA64. Both phages were stable at 37 °C only. Their genome analysis indicated their safety as no recombination, integration and host virulence genes were identified. Both these phages can be good candidates for control of APEC strains based on their lysis potential.fals

Immunoglobulin, glucocorticoid, or combination therapy for multisystem inflammatory syndrome in children: a propensity-weighted cohort study

Background Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory condition associated with SARS-CoV-2 infection, has emerged as a serious illness in children worldwide. Immunoglobulin or glucocorticoids, or both, are currently recommended treatments. Methods The Best Available Treatment Study evaluated immunomodulatory treatments for MIS-C in an international observational cohort. Analysis of the first 614 patients was previously reported. In this propensity-weighted cohort study, clinical and outcome data from children with suspected or proven MIS-C were collected onto a web-based Research Electronic Data Capture database. After excluding neonates and incomplete or duplicate records, inverse probability weighting was used to compare primary treatments with intravenous immunoglobulin, intravenous immunoglobulin plus glucocorticoids, or glucocorticoids alone, using intravenous immunoglobulin as the reference treatment. Primary outcomes were a composite of inotropic or ventilator support from the second day after treatment initiation, or death, and time to improvement on an ordinal clinical severity scale. Secondary outcomes included treatment escalation, clinical deterioration, fever, and coronary artery aneurysm occurrence and resolution. This study is registered with the ISRCTN registry, ISRCTN69546370. Findings We enrolled 2101 children (aged 0 months to 19 years) with clinically diagnosed MIS-C from 39 countries between June 14, 2020, and April 25, 2022, and, following exclusions, 2009 patients were included for analysis (median age 8·0 years [IQR 4·2–11·4], 1191 [59·3%] male and 818 [40·7%] female, and 825 [41·1%] White). 680 (33·8%) patients received primary treatment with intravenous immunoglobulin, 698 (34·7%) with intravenous immunoglobulin plus glucocorticoids, 487 (24·2%) with glucocorticoids alone; 59 (2·9%) patients received other combinations, including biologicals, and 85 (4·2%) patients received no immunomodulators. There were no significant differences between treatments for primary outcomes for the 1586 patients with complete baseline and outcome data that were considered for primary analysis. Adjusted odds ratios for ventilation, inotropic support, or death were 1·09 (95% CI 0·75–1·58; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids and 0·93 (0·58–1·47; corrected p value=1·00) for glucocorticoids alone, versus intravenous immunoglobulin alone. Adjusted average hazard ratios for time to improvement were 1·04 (95% CI 0·91–1·20; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids, and 0·84 (0·70–1·00; corrected p value=0·22) for glucocorticoids alone, versus intravenous immunoglobulin alone. Treatment escalation was less frequent for intravenous immunoglobulin plus glucocorticoids (OR 0·15 [95% CI 0·11–0·20]; p<0·0001) and glucocorticoids alone (0·68 [0·50–0·93]; p=0·014) versus intravenous immunoglobulin alone. Persistent fever (from day 2 onward) was less common with intravenous immunoglobulin plus glucocorticoids compared with either intravenous immunoglobulin alone (OR 0·50 [95% CI 0·38–0·67]; p<0·0001) or glucocorticoids alone (0·63 [0·45–0·88]; p=0·0058). Coronary artery aneurysm occurrence and resolution did not differ significantly between treatment groups. Interpretation Recovery rates, including occurrence and resolution of coronary artery aneurysms, were similar for primary treatment with intravenous immunoglobulin when compared to glucocorticoids or intravenous immunoglobulin plus glucocorticoids. Initial treatment with glucocorticoids appears to be a safe alternative to immunoglobulin or combined therapy, and might be advantageous in view of the cost and limited availability of intravenous immunoglobulin in many countries. Funding Imperial College London, the European Union's Horizon 2020, Wellcome Trust, the Medical Research Foundation, UK National Institute for Health and Care Research, and National Institutes of Health

Abstract P6-07-22: Association of Hedgehog signaling pathway with luminal B sub-type of breast cancer affected patients of Pakistan

Abstract Introduction Hedgehog pathway dysregulation is observed in different types of cancers including breast. In the present study, expression profiles of hedgehog pathway molecules in breast cancer cohort of Pakistan and their probable association with molecular sub-types were explored. Methods The study was preceded with ethical approval and informed consent from respective institutions and participants. During 2013-2015, a total of 150 cancer biopsies along with adjacent normal tissues were prospectively collected immediately after surgery and processed for RNA isolation. Transcriptional profiles of salient members including SHH, DHH, IHH, PTCH-1, SMO and GLI-1 were quantified using qRT-PCR. Cohort was categorized into molecular sub-types following St. Gallen International Expert Consensus System. Association of expression levels of these aforementioned molecules with various clinico-pathological parameters was explored. Result Both SHH (p=0.01) and DHH (p&amp;lt;0.001) showed elevated expression among tumors in comparison to their controls. Similarly, PTCH-1 (p&amp;lt;0.001) and GLI-1 (p=0.002) were also significantly up regulated in the cohort. Interestingly, strong positive correlations were observed among the pathway molecules (r-value ranging from 0.45 to 0.81) which highlight their interdependence towards tumor progression. A significant correlation of SHH, DHH, PTCH-1 and GLI-1 was observed with advanced tumor sizes, stages, grades and nodal involvement (p&amp;lt;0.05). Association of IHH, SMO and GLI-1 over expression with cancer metastasis was also established in the cohort. SHH, PTCH-1 and GLI-1 were significantly linked with laterality, age and menopausal status. Expression of SHH (p=0.002) was more related to younger age group (mean age &amp;lt; 45 yrs) patients in comparison to elderly women. Moreover all hedgehog molecules were strongly related to hormonal receptors (ER and PR) (r-value ranging from 0.51 to 0.86) while over-expression of HER-2 was not associated with any pathway component. Briefly, 53% (79) of the cohort was categorized as Luminal-B, 18% (27) triple negative, 15% (23) Luminal-A and 14% (21) HER-2 for sub-typing of breast cancer patients in the cohort. Expression of SHH was significantly associated with the molecular sub-types (p=0.02) and age (p=0.005) using Pearson Chi-Square test. Elevated expression of SHH was observed in 60% of the patients in Luminal B sub-type. Conclusion Hedgehog pathway plays a crucial role in breast cancer progression and is found to be activated in Luminal B sub-type in this cohort. As Luminal B is a more aggressive type of breast cancer having poor prognosis and early-onset, association of SHH with Luminal-B and younger age patients signify it importance as a biomarker for early diagnosis of young patient's. Hence therapeutic interventions for hedgehog pathway can improve the prognosis of patients categorized as Luminal B subtype of breast carcinogenesis. Citation Format: Riaz SK, Rashid R, Shah STA, Wang F, Malik MFA. Association of Hedgehog signaling pathway with luminal B sub-type of breast cancer affected patients of Pakistan [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-22.</jats:p

KIF1A novel frameshift variant p.(Ser887Profs*64) exhibits clinical heterogeneity in a Pakistani family with Hereditary Sensory and Autonomic Neuropathy Type IIC.

Hereditary sensory and autonomic neuropathies (HSANs) are rare heterogeneous group of neurological disorders caused by peripheral nerve deterioration. The HSANs sub-clinical classes have clinical and genetic overlap which often lead to misdiagnosis. In the present study a Pakistani family with five affected members suffering from severe neuropathy were genetically analyzed to identify the disease causative element in the family. Genome wide high-density single nucleotide polymorphism (SNP) microarray analysis was carried out followed by whole exome sequencing of the affected proband and another affected sibling. Shared homozygous regions in all severely affected members were identified through homozygosity mapping approach. The largest homozygous region of 14.1Mb shared by the five severely affected members of the family was identified on chromosome 2. Subsequent exome sequencing identified a novel single nucleotide deletion c.2658del; p.(Ser887Profs*64) in KIF1A. Segregation analysis revealed that this mutation was homozygous in all five affected individuals of the family with severe clinical manifestation, while members of the family that were heterozygous carriers shared abnormal skin features (scaly skin) only with the homozygous affected members. A novel frameshift mutation p.(Ser887Profs*64) in KIF1A is the potential cause of severe HSANIIC in a Pakistani family along with incomplete penetrance in mutation carriers. We demonstrate that using a combination of different techniques not only strengthens the gene finding approach but also helps in proper sub-clinical characterization along with identification of mutated alleles exhibiting incomplete penetrance leading to intrafamilial clinical variability in HSAN group of inherited diseases

Abstract CT009: Phase II trial of perioperative pembrolizumab plus capecitabine and oxaliplatin followed by adjuvant pembrolizumab for resectable gastric and gastroesophageal junction (GC/GEJ) adenocarcinoma

Abstract Background: Perioperative therapy for locally advanced (LA) GC/GEJ adenocarcinoma is standard of care. Although immune checkpoint blockade (ICB) following chemoradiotherapy and resection significantly improves disease free survival (DFS) for esophageal cancer, the effectiveness of ICB together with chemotherapy in LA GC/GEJ cancer is unknown. Methods: This is a multicenter, single-arm, phase II clinical trial of pembrolizumab 200 mg every 3 weeks with capecitabine 625 mg/m2 twice daily and oxaliplatin 130 mg/m2 every 3 weeks (CAPOX) in patients with resectable GC/GEJ adenocarcinoma. Subjects with ECOG PS of 0-1 received CAPOX with pembrolizumab for 3 cycles prior to and 3 cycles following surgery with an additional cycle of pembrolizumab just prior to surgery and 12 months of maintenance pembrolizumab following adjuvant chemoimmunotherapy. The primary endpoint was pathologic complete response (pCR) rate. The study had 80% power to detect an increase in pCR rate from 3% to 15% with a one-sided alpha of 0.05. Secondary endpoints included overall response rate, DFS, and overall survival (OS). This study was registered with ClinicalTrials.gov (NCT02918162). Results: Between 02/10/2017 and 06/17/2021, 36 patients were enrolled with 34 (21 gastric and 13 GEJ) evaluable for efficacy. The median age was 65 years and 17 (50%) patients had an ECOG PS of 1. In total, 29 (85%) patients underwent resection. Seven patients achieved a pCR (20.6% of evaluable patients and 24.1% of those who underwent resection). An additional 6 (17.6%) patients achieved a near CR and 8 (23.5%) demonstrated a significant treatment effect on pathologic review. One patient was deemed unfit for surgery, 2 expired prior to surgery, and 2 were found to have metastatic disease during surgery. At the time of data cut-off, the median follow-up was 19 mo. Of those who underwent resection, 4 (13.7%) experienced disease recurrence and 5 (17.2%) expired. The probability of survival at 1 and 2 years was 0.91 (0.82-1.0) and 0.80 (0.64-0.99), respectively. The median DFS and OS have not been reached. Of the 35 patients who received treatment, treatment related adverse events (AEs) of grade greater than or equal to 3 were reported in 18 (51%) patients. Grade greater than or equal to 3 immune-related AEs were reported in 10 (29%) patients. Three grade 5 AEs occurred, two possibly treatment-related (gastric hemorrhage and gastric perforation) and one unrelated to treatment (cardiac arrest). Conclusion: In LA GC/GEJ adenocarcinoma, the combination CAPOX and pembrolizumab resulted in a pCR rate of 20.6%. The combination was well tolerated and 85.3% of patients underwent surgical resection. This trial met its primary endpoint supporting further investigation of this regimen as an alternative for patients who are unlikely to tolerate triple combination chemotherapy. Correlative studies are in progress. Citation Format: Alexander Grenander Raufi, Shing Lee, Michael May, Armando Del Portillo, Naomi Sender, Sarah Sta Ana, Katarzyna Gautier, Emily Alouani, Haeseong Park, Paul Oberstein, Manish Shah, Gulam A. Manji. Phase II trial of perioperative pembrolizumab plus capecitabine and oxaliplatin followed by adjuvant pembrolizumab for resectable gastric and gastroesophageal junction (GC/GEJ) adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT009.</jats:p