Mechanism of Nucleic Acid Unwinding by SARS-CoV Helicase

The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5′→3′ polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second. Therefore the net unwinding rate is ∼280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding. This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis

Long-term efficacy and safety of solifenacin in pediatric patients aged 6 months to 18 years with neurogenic detrusor overactivity: results from two phase 3 prospective open-label studies

Introduction The standard recommended treatment for neurogenic detrusor overactivity (NDO) is clean intermittent catheterization combined with an antimuscarinic agent. However, the adverse systemic side effects of oxybutynin, the most widely used agent, are of concern. Objective To evaluate the efficacy and safety of solifenacin in pediatric patients with NDO, aged 6 months–15 cmH2O), number of overactive detrusor contractions (>15 cmH2O), maximum catheterized volume (MCV)/24 h and incontinence episodes/24 h. Safety parameters were treatment-emergent adverse events (TEAEs), serious adverse events, laboratory variables, vital signs, electrocardiograms, and ocular accommodation and cognitive function assessments. Results After 24 weeks, MCC had significantly increased compared with baseline in patients aged 6 months–<5 years and 5–<18 years (37.0 ml and 57.2 ml, respectively; P < 0.001; Fig.). Improvement was also observed after 52 weeks’ treatment. Significant changes were observed from baseline to week 24 in all secondary endpoints in both age groups: increase in bladder compliance, increase in bladder volume to first detrusor contraction as a percentage of expected bladder capacity, reduction in the number of overactive detrusor contractions, increase in MCV, and decreased incontinence episodes. TEAEs were mostly mild or moderate and there were no new drug-related TEAEs compared with adult studies. Age-related improvements were noted in ocular accommodation and cognitive function. Discussion These long-term multicenter investigations demonstrated the efficacy and safety of solifenacin in pediatric patients with NDO. The observed increases in MCC were clinically relevant and demonstrated that an increase in fluid volume can be accommodated in the bladder prior to reaching intravesical pressures that endanger kidney function and/or are associated with leakage or discomfort. Solifenacin was well tolerated with low incidences of constipation and dry mouth (typically associated with antimuscarinics), central nervous system-related side effects and facial flushing. Conclusion Solifenacin was effective and well tolerated in pediatric patients with NDO, aged 6 months–<18 years, suggesting that it is a viable alternative to oxybutynin, the current standard of care

Numero XXXIII

<p><b>Cumulative Incidence of AUR (A) and UR (B) with FDC Soli 6 mg or 9 mg + TOCAS.</b> (A) Acute urinary retention. (B) Urinary retention. Black markers indicate 6 mg FDC dose at time of AUR/UR onset; orange markers indicate 9 mg FDC dose at time of AUR/UR onset. AUR cases were a subgroup of UR cases that required catheterization. Abbreviation: FDC, fixed-dose combination.</p

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

Recent human H3N2 influenza A viruses have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells, which are commonly employed to propagate influenza A virus, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase 1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAcs), would result in improved A/H3N2 propagation. Stable overexpression of β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase 1 in Madin-Darby Canine Kidney and "humanized" Madin-Darby Canine Kidney cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the β-1,3-N-acetylglucosaminyltransferase and/or β-1,4-galactosyltransferase 1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on "humanized" Madin-Darby Canine Kidney-β-1,3-N-acetylglucosaminyltransferase cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 influenza A viruses require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.</p

Contemporary human H3N2 influenza a viruses require a low threshold of suitable glycan receptors for efficient infection

Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells (MDCK and hCK) which are commonly employed to propagate IAV, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency

Sea ice working group (SIP)

The sea ice is a crucial component of the polar climate system, and has an impact on albedo, heat and gas ex- change, primary productivity and car- bon export, atmospheric and ocean circulation, freshwater budget, ocean stratification, and deep water mass for- mation. It is therefore critical that it is correctly specified as a forcing or pre- dicted as a feedback in modeling stud- ies

Emergence of Variability in Isogenic Escherichia coli Populations Infected by a Filamentous Virus

The spread of epidemics not only depends on the average number of parasites produced per host, but also on the existence of highly infectious individuals. It is widely accepted that infectiousness depends on genetic and environmental determinants. However, even in clonal populations of host and viruses growing in homogeneous conditions, high variability can exist. Here we show that Escherichia coli cells commonly display high differentials in viral burst size, and address the kinetics of emergence of such variability with the non-lytic filamentous virus M13. By single-cell imaging of a virally-encoded fluorescent reporter, we monitor the viral charge distribution in infected bacterial populations at different time following infection. A mathematical model assuming autocatalytic virus replication and inheritance of bacterial growth rates quantitatively reproduces the experimental distributions, demonstrating that deterministic amplification of small host inhomogeneities is a mechanism sufficient to explain large and highly skewed distributions. This mechanism of amplification is general and may occur whenever a parasite has an initial phase of exponential growth within its host. Moreover, it naturally reproduces the shift towards higher virulence when the host is experimenting poor conditions, as observed commonly in host-parasite systems

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by cells commonly employed to propagate these viruses (MDCK and hCK), resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency

The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation

The bacterial flagellum is a remarkable molecular motor, whose primary function in bacteria is to facilitate motility through the rotation of a filament protruding from the bacterial cell. A cap complex, consisting of an oligomer of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex, which reveals that FliD is pentameric, with the N-terminal region of the protomer forming an extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded, contrary to a previously published cryo-EM structure, and propose a molecular model for the filament-cap interaction