Determination of Neuraminidase Kinetic Constants Using Whole Influenza Virus Preparations and Correction for Spectroscopic Interference by a Fluorogenic Substrate

The influenza neuraminidase (NA) enzyme cleaves terminal sialic acid residues from cellular receptors, a process required for the release of newly synthesized virions. A balance of NA activity with sialic acid binding affinity of hemagglutinin (HA) is important for optimal virus replication. NA sequence evolution through genetic shift and drift contributes to the continuous modulation of influenza virus fitness and pathogenicity. A simple and reliable method for the determination of kinetic parameters of NA activity could add significant value to global influenza surveillance and provide parameters for the projection of fitness and pathogenicity of emerging virus variants. The use of fluorogenic substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) and cell- or egg-grown whole influenza virus preparations have been attractive components of NA enzyme activity investigations. We describe important criteria to be addressed when determining K_m and V_max kinetic parameters using this method: (1) determination of the dynamic range of MUNANA and 4-methylumbelliferone product (4-MU) fluorescence for the instrument used; (2) adjustment of reaction conditions to approximate initial rate conditions, i.e. ≤15% of substrate converted during the reaction, with signal-to-noise ratio ≥10; (3) correction for optical interference and inner filter effect caused by increasing concentrations of MUNANA substrate. The results indicate a significant interference of MUNANA with 4-MU fluorescence determination. The criteria proposed enable an improved rapid estimation of NA kinetic parameters and facilitate comparison of data between laboratories.     

A Treatment Plant Receiving Waste Water from Multiple Bulk Drug Manufacturers Is a Reservoir for Highly Multi-Drug Resistant Integron-Bearing Bacteria

The arenas and detailed mechanisms for transfer of antibiotic resistance genes between environmental bacteria and pathogens are largely unclear. Selection pressures from antibiotics in situations where environmental bacteria and human pathogens meet are expected to increase the risks for such gene transfer events. We hypothesize that waste-water treatment plants (WWTPs) serving antibiotic manufacturing industries may provide such spawning grounds, given the high bacterial densities present there together with exceptionally strong and persistent selection pressures from the antibiotic-contaminated waste. Previous analyses of effluent from an Indian industrial WWTP that processes waste from bulk drug production revealed the presence of a range of drugs, including broad spectrum antibiotics at extremely high concentrations (mg/L range). In this study, we have characterized the antibiotic resistance profiles of 93 bacterial strains sampled at different stages of the treatment process from the WWTP against 39 antibiotics belonging to 12 different classes. A large majority (86%) of the strains were resistant to 20 or more antibiotics. Although there were no classically-recognized human pathogens among the 93 isolated strains, opportunistic pathogens such as Ochrobactrum intermedium, Providencia rettgeri, vancomycin resistant Enterococci (VRE), Aerococcus sp. and Citrobacter freundii were found to be highly resistant. One of the O. intermedium strains (ER1) was resistant to 36 antibiotics, while P. rettgeri (OSR3) was resistant to 35 antibiotics. Class 1 and 2 integrons were detected in 74/93 (80%) strains each, and 88/93 (95%) strains harbored at least one type of integron. The qPCR analysis of community DNA also showed an unprecedented high prevalence of integrons, suggesting that the bacteria living under such high selective pressure have an appreciable potential for genetic exchange of resistance genes via mobile gene cassettes. The present study provides insight into the mechanisms behind and the extent of multi-drug resistance among bacteria living under an extreme antibiotic selection pressure.     

Safety,acceptability, and pharmacokinetics of a monoclonal antibody-based vaginal multipurpose prevention film (MB66): A Phase I randomized trial

BackgroundMB66 film is a multipurpose prevention technology (MPT) product with monoclonal antibodies (mAbs) against HIV-1 (VRC01-N) and HSV-1 and 2 (HSV8-N). The mAbs were produced by transient expression in Nicotiana benthamiana (N). We conducted a Phase I clinical trial to assess the safety, pharmacokinetics (PK), and ex vivo efficacy of single and repeated doses of MB66 when used intravaginally.Methods and findingsThe clinical trial enrolled healthy reproductive-aged, sexually abstinent women. In Segment A, 9 women received a single MB66 film which was inserted into the vaginal posterior fornix by a clinician. In Segment B, 29 women were randomly assigned to MB66 (Active) or Placebo film groups and were instructed to insert 1 film vaginally for 7 consecutive days. Visits and clinical sampling occurred predose and at various time points after single and repeated film doses. The primary endpoint was number of adverse events (AEs) Grade 2 or higher related to product use. Secondary endpoints included film dissolution rate, Nugent score (a Gram stain scoring system to diagnose bacterial vaginosis), vaginal pH, post-use survey results, cytokine concentrations in cervicovaginal lavage (CVL) specimens (assessed by Luminex assay), mAb concentrations in vaginal fluid collected from 4 sites (assessed by ELISA), and HIV and HSV neutralization activity of CVL samples ex vivo (assessed by TZM-bl and plaque reduction assay, respectively).The product was generally safe and well tolerated, with no serious AEs recorded in either segment. The AEs in this study were primarily genitourinary in nature with the most commonly reported AE being asymptomatic microscopic hematuria. There were no differences in vaginal pH or Nugent scores or significant increases in levels of proinflammatory cytokines for up to 7 days after film insertion in either segment or between Active and Placebo groups. Acceptability and willingness to use the product were judged to be high by post-use surveys.Concentrations of VRC01-N and HSV8-N in vaginal secretions were assessed over time to generate pharmacokinetic curves. Antibody levels peaked 1 hour postdosing with Active film (median: 35 μg/mL) and remained significantly elevated at 24 hours post first and seventh film (median: 1.8 μg/mL). Correcting for sample dilution (1:20), VRC01-N concentrations ranged from 36 to 700 μg/mL at the 24-hour time point, greater than 100-fold the IC₅₀ for VRC01 (0.32 μg/mL); HSV8-N concentrations ranged from 80 to 601 μg/mL, well above the IC₅₀ of 0.1 μg/m. CVL samples collected 24 hours after MB66 insertion significantly neutralized both HIV-1 and HSV-2 ex vivo. Study limitations include the small size of the study cohort, and the fact that no samples were collected between 24 hours and 7 days for pharmacokinetic evaluation.ConclusionsSingle and repeated intravaginal applications of MB66 film were safe, well tolerated, and acceptable. Concentrations and ex vivo bioactivity of both mAbs in vaginal secretions were significantly elevated and thus could provide protection for at least 24 hours postdose. However, further research is needed to evaluate the efficacy of MB66 film in women at risk for HIV and HSV infection. Additional antibodies could be added to this platform to provide protection against other sexually transmitted infections (STIs) and contraception.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02579083","term_id":"NCT02579083"}}NCT02579083.

Joseph Politch and co-workers report on a first-in-human evaluation of a vaginal film deploying monoclonal antibodies to target viral infections.     

Lateral gene transfer between prokaryotes and multicellular eukaryotes: ongoing and significant?

The expansion of genome sequencing projects has produced accumulating evidence for lateral transfer of genes between prokaryotic and eukaryotic genomes. However, it remains controversial whether these genes are of functional importance in their recipient host. Nikoh and Nakabachi, in a recent paper in BMC Biology, take a first step and show that two genes of bacterial origin are highly expressed in the pea aphid Acyrthosiphon pisum. Active gene expression of transferred genes is supported by three other recent studies. Future studies should reveal whether functional proteins are produced and whether and how these are targeted to the appropriate compartment. We argue that the transfer of genes between host and symbiont may occasionally be of great evolutionary importance, particularly in the evolution of the symbiotic interaction itself.     

Pre-clinical toxicity of IDPH-791: a new centrally acting muscle relaxant in rats.

IDPH-791, a novel centrally acting muscle relaxant, in doses up to 500 mg/kg (po) for 14 days did not result in any appreciable adverse effect on body weight gain, food or water consumption including biochemical and haematologica parameters in rats. Variations observed in the biochemistry and haematology were either comparable to controls or were within normal limits.     

An ensemble of B-DNA dinucleotide geometries lead to characteristic nucleosomal DNA structure and provide plasticity required for gene expression

Background  

A nucleosome is the fundamental repeating unit of the eukaryotic chromosome. It has been shown that the positioning of a majority of nucleosomes is primarily controlled by factors other than the intrinsic preference of the DNA sequence. One of the key questions in this context is the role, if any, that can be played by the variability of nucleosomal DNA structure.     

Noncontact dipole effects on channel permeation. III. Anomalous proton conductance effects in gramicidin

Proton transport on water wires, of interest for many problems in membrane biology, is analyzed in side-chain analogs of gramicidin A channels. In symmetrical 0.1 N HCl solutions, fluorination of channel Trp(11), Trp-(13), or Trp(15) side chains is found to inhibit proton transport, and replacement of one or more Trps with Phe enhances proton transport, the opposite of the effects on K(+) transport in lecithin bilayers. The current-voltage relations are superlinear, indicating that some membrane field-dependent process is rate limiting. The interfacial dipole effects are usually assumed to affect the rate of cation translocation across the channel. For proton conductance, however, water reorientation after proton translocation is anticipated to be rate limiting. We propose that the findings reported here are most readily interpreted as the result of dipole-dipole interactions between channel waters and polar side chains or lipid headgroups. In particular, if reorientation of the water column begins with the water nearest the channel exit, this hypothesis explains the negative impact of fluorination and the positive impact of headgroup dipole on proton conductance.