The pyrin inflammasome and the Yersinia effector interaction

Pyrin is a cytosolic pattern-recognition receptor that normally functions as a guard to trigger capase-1 inflammasome assembly in response to bacterial toxins and effectors that inactivate RhoA. The MEFV gene encoding human pyrin is preferentially expressed in phagocytes. Key domains in pyrin include a pyrin domain (PYD), a linker region, and a B30.2 domain. Binding of ASC to pyrin by a PYD-PYD interaction triggers inflammasome assembly. Pyrin is held in an inactive conformation by negative regulation mechanisms to avoid premature inflammasome assembly. One mechanism of negative regulation involves phosphorylation of the linker by PRK kinase which in turn is positively regulated by active RhoA. The B30.2 domain also negatively regulates pyrin. Gain of function mutations in MEFV responsible for the autoinflammatory disease Familial Mediterranean Fever (FMF) map to exon 10 encoding the B30.2 domain. Insights into pyrin regulation have come from studies of several Yersinia effectors, which are injected into phagocytes and interact with the RhoA-PRK-pyrin axis during infection. Two effectors, YopE and YopT, inactivate RhoA to disrupt phagocytic signaling. To counteract an effector-triggered immune response, a third effector, YopM, binds to and inhibits pyrin by hijacking PRK and RSK and directing linker phosphorylation. Inhibition of pyrin by YopM is required for virulence of Yersinia pestis, the agent of plague. Recent results from infection studies with human phagocytes and mice producing pyrin B30.2 FMF variants show that gain of function MEFV mutations bypass inhibition by YopM. Population genetic data suggest that MEFV mutations were selected for in individuals of Mediterranean decent during historic plague pandemics. This review discusses current concepts of pyrin regulation and its interaction with Yersinia effectors.     

Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel V_HHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN). Human hepatic (Huh7) cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies) had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-V_HH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β), indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the V_HHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents.     

Analytical strategy for the detection of ecdysterone and its metabolites in vivo in uPA(+/+)-SCID mice with humanized liver,human urine samples,and estimation of prevalence of its use in anti-doping samples

Ecdysteroids are of interest as potential sport performance enhancers, due to their anabolic effects. The current study aimed to analyze levels of the most abundant ecdysteroid, ecdysterone (20-hydroxyecdysone, 20-OHE) in easily available dietary supplements, and, outline an analytical strategy for its detection, and that, of its metabolites, (1) following administration of pure 20-OHE to uPA(+/+)-SCID mice with humanized liver, (2) in a human volunteer after ingestion of two supplements, one with a relatively low, and the other a high, concentration of 20-OHE, and, (3) to estimate the prevalence of use of 20-OHE in elite athletes (n = 1000). Of the 16 supplements tested, only five showed detectable levels of 20-OHE, with concentrations ranging from undetectable up to 2.3 mg per capsule. Urine of uPA(+/+)-SCID urine showed the presence of 20-OHE and its metabolite, 14 deoxy ecdysterone, within 24 hours (hr) of ingestion. In humans, both the parent and the metabolite were detectable within 2 to 5 hr of ingestion, with the metabolite being detectable for longer than the parent. After ingestion of a low dose supplement, the parent and metabolite were detectable for 70 and 48 hr, while following the higher dose it was 96 and 48 hr, respectively. Analysis of urines from athletes (n = 1000) confirmed four positives for 20-OHE, suggesting a prevalence of use of 0.4%. Prevalence of its use by elite athletes was relatively low, however, this needs to be confirmed in other populations, and with other related ecdysteroids.     

Phylogenetic Analysis of MERS-CoV in a Camel Abattoir,Saudi Arabia, 2016–2018

We detected Middle East respiratory syndrome coronavirus (MERS-CoV) RNA in 305/1,131 (27%) camels tested at an abattoir in Al Hasa, Eastern Province, Saudi Arabia, during January 2016–March 2018. We characterized 48 full-length MERS-CoV genomes and noted the viruses clustered in MERS-CoV lineage 5 clade B.     

Why is Pakistan vulnerable to COVID-19 associated morbidity and mortality? A scoping review

The scoping review was undertaken to outline the vulnerabilities of Pakistan's public health and healthcare system, which put the population at increased risk of coronavirus disease 2019 (COVID-19) associated morbidity and mortality. The major electronic databases were searched using both “text words” and “thesaurus terms,” focusing on viral infections, COVID-19 and healthcare systems in Pakistan. The content of the selected articles was analyzed by using thematic approach. Out of the total 171 potentially relevant citations, 24 articles were included in the data synthesis. We found that the recent COVID-19 outbreak is a major threat to Pakistan's public health and healthcare system, and the country is not in a position to control spread of disease and provide required standards of care deemed necessary by the World Health Organization. A number of intertwined reasons that expose the Pakistani population at increased risk of COVID-19 associated morbidity and mortality, include public related demurrals, healthcare workforce related demurrals, organizational and regulatory voids, and travel patterns. To cope with the upsurge of COVID-19 in Pakistan, the regulators need to re-examine and recognize deficiencies in the healthcare system, and thereafter reinforce core capacities in workforce and monetary resources, surveillance, laboratory services, and hospital preparedness for isolation and ventilation of patients.     

Brainstem auditory evoked response in newborns with hyperbilirubinemia

OBJECTIVE: To determine the initial Brainstem Auditory Evoked Response (BAER) abnormalities in neonates with hyperbilirubinemia and the possible reversibility of abnormal BAER after therapy. DESIGN: Prospective cohort study. SETTING: Tertiary care hospital. SUBJECTS: 30 term neonates with hyperbilirubinemia (S. bilirubin < 15 mg/dl) as cases and 25 normal term neonates as controls. METHODS: Duration of study was from August 1995 to August 1996. BAER were recorded before therapy at peak hyperbilirubinemia, after therapy, and the age of 2-4 months using electric response audiometer (Nihon Neuropack Four Machine). Denver Development Screening Test (Denver II) was performed at 1 year of age. RESULTS: Seventeen out of thirty (56.7%) neonates with hyperbilirubinemia showed abnormalities on initial BAER. Commonest abnormality seen was raised threshold of wave V in 12 neonates (40%). Other abnormalities observed were absence of all waves at 90 dB (23.3%), prolongation of latencies of various waves (26.7%) and prolongation of various intervals (26.7%). Abnormalities in BAER correlated significantly with bilirubin level. After therapy abnormalities reverted back to normal in 10 cases but persisted in 7 out of 17 (41.17%) cases with initial abnormal BAER. Development screening at 1 yr was abnormal in 3 infants all of whom had persistent abnormalities in BAER. CONCLUSION: Serial BAER is a useful, non invasive tool to detect neurodevelopment delay secondary to neonatal hyperbilirubinemia.