Eight years of experience with molecular identification of human enteroviruses

We have successfully typed 1,121 human enterovirus (HEV) isolates during the last 8 years by adapting partial VP1 sequencing to routine identification of HEV isolated from diverse clinical and environmental specimens. The isolates include 48 of the 59 traditional nonpoliovirus HEV serotypes and members of 8 newly discovered types, which would have remained untypeable by neutralization using the conventional cross-sectional pools of antisera.     

Identification of lead compounds targeting the cathepsin B-like enzyme of Eimeria tenella

Cysteine peptidases have been implicated in the development and pathogenesis of Eimeria. We have identified a single-copy cathepsin B-like cysteine peptidase gene in the genome database of Eimeria tenella (EtCatB). Molecular modeling of the predicted protein suggested that it differs significantly from host enzymes and could be a good drug target. EtCatB was expressed and secreted as a soluble, active, glycosylated mature enzyme from Pichia pastoris. Biochemical characterization of the recombinant enzyme confirmed that it is cathepsin B-like. Screening of a focused library against the enzyme identified three inhibitors (a nitrile, a thiosemicarbazone, and an oxazolone) that can be used as leads for novel drug discovery against Eimeria. The oxazolone scaffold is a novel cysteine peptidase inhibitor; it may thus find widespread use.     

Directed HIV-1 evolution of protease inhibitor resistance by second-generation short hairpin RNAs

Despite the success of antiretroviral drugs in decreasing AIDS-related mortality, a substantial fraction of HIV-infected patients experience therapy failure due to the emergence of drug-resistant virus variants. For durable inhibition of HIV-1 replication, the emergence of such escape viruses must be controlled. In addition to antiretroviral drugs, RNA interference (RNAi)-based gene therapy can be used to inhibit HIV-1 replication by targeting the viral RNA genome. RNAi is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of the targeted mRNA. Here we investigated an alternative strategy combining the activity of a protease inhibitor (PI) with second-generation short hairpin RNAs (shRNAs) designed to specifically block the emergence of PI-resistant HIV-1 variants. We demonstrate that dominant viral escape routes can be effectively blocked by second-generation shRNAs and that virus evolution can be redirected toward less-fit variants. These results are of importance for a deeper understanding of HIV-1 evolution under combined drug and RNAi pressure and may be used to design future therapeutic approaches.     

How to implement monitoring tools for sedation, pain and delirium in the intensive care unit: an experimental cohort study

Purpose

Systematic monitoring of sedation, pain and delirium in the ICU is of paramount importance in delivering adequate patient care. While the use of systematic monitoring instruments is widely agreed upon, these tools are infrequently implemented into daily ICU care. The aim of this study is to compare the effectiveness of two different training strategies (training according to the local standard vs. modified extended method) on the implementation rate of scoring instruments on the ICU.

Methods

In this experimental cohort study we analyzed the frequency of scoring on three surgical ICUs before and after training, and in a 1?year follow-up. A modified extended training included establishing a local support team helping to resolve immediate problems. In addition we evaluated the impact on patients’ outcome.

Results

ICUs trained by the modified extended method showed increased documentation rates of all scores per patient and day. In a 1?year follow-up, increased scoring rates for all scores were maintained. Scoring rates with training according to the local standard training protocol did not increase significantly. Implementation of delirium and pain monitoring were associated with a decrease in mortality [odds ratio (OR) 0.451; 95?% confidence interval (CI): 0.22–0.924, and, respectively, OR 0.348; 95?% CI: 0.140–0.863]. Monitoring had no significant influence on ventilation time or ICU length of stay.

Conclusions

A modified extended training strategy for ICU monitoring tools (sedation, pain, delirium) leads to higher intermediate and long-term implementation rates and is associated with improved patient outcome. However, these findings may have been biased by unmeasured confounders.     

The association of ownership type with job insecurity and worry about job stability: the moderating effects of fair management, positive leadership, and employment type

This study examined whether the ownership type is associated with job insecurity and worry about job stability and whether the type of employment contract, positive leadership, and fair management moderated these associations. Survey data from 1249 Finnish female elderly care staff aged 18 to 69 years were used. Job insecurity and worry about job stability were highest in not-for-profit sheltered homes. However, positive leadership and fair management were able to mitigate this insecurity and worry. Job insecurity was highest among fixed-term employees in public sheltered homes or not-for-profit nursing homes. Thus, promoting good leadership and fair management would be of importance.     

Effects of Efflux Pump Inhibitors on Erythromycin, Ciprofloxacin, and Tetracycline Resistance in Campylobacter spp. Isolates

The aim was to assess the potency of the efflux pump inhibitors (EPIs) phenylalanine-arginine ?-naphthylamide (PA?N) and 1-(1-naphthylmethyl)-piperazine (NMP) and the putative natural EPI phenolic (-)-epigallocatechin gallate (EGCG) for the reversal of erythromycin, ciprofloxacin, and tetracycline resistance in Campylobacter jejuni and Campylobacter coli isolates. We investigated target mutations and resistant genes involved in erythromycin and tetracycline resistance and determined the roles of the bacterial drug efflux systems (cmeB, cmeF, and cmeR) in antimicrobial resistance. Our data show that most of the high-level erythromycin resistance and all of the tetracycline resistance can be explained through mutations in 23S rRNA and the presence of the tetO gene, respectively. The EPIs show the ability to partly reverse drug resistance in these Campylobacter isolates. Based on a fourfold or greater reduction in the erythromycin minimal inhibitory concentration (MIC), PA?N and NMP had clear effects in almost of all of the isolates tested. PA?N had a highly selective action on the ciprofloxacin and tetracycline MICs. Inactivation of cmeB increased susceptibility to all of the antimicrobials tested, whereas inactivation of cmeF and cmeR had no effects. A notable decrease in resistance to erythromycin and ciprofloxacin in the presence of subinhibitory concentrations of EGCG demonstrates the resistance-modifying activities of this natural EPI, and indicates its potential use in the control of Campylobacter spp. in the food chain.     

Molecular characterisation of a novel cassava associated circular ssDNA virus

The application of sequence non-specific rolling circle amplification of circular single stranded (ss) DNA molecules to viral metagenomics has facilitated the discovery in various ecosystems of what is probably a diverse array of novel ssDNA viruses. Here we describe a putative novel ssDNA virus (at a genome level), cassava associated circular DNA virus (CasCV), isolated from cassava leaf samples infected with the fungi Collectotrichum and Plectosphaerella. CasCV has a circular ambisense genome and shares significant genome similarities with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), Mosquito VEM virus SDBVL and Meles meles faecal virus (MmFV). The CasCV genome (2220 nt) has three large open reading frames. While it is probable that one of these encodes a capsid protein, the other two probably express a replication associated protein (Rep) following the removal of an intron such as that found in the Rep encoding genes of some geminiviruses. This Rep would contain four conserved rolling circle replication (RCR) related motifs that have previously been identified in geminivirus, circovirus and nanovirus Reps. Given both that the CasCV Rep and CP share 62.7% and 39.8% amino acid identity respectively with the Rep and CP of SsHADV-1, and that CasCV was discovered associated with cassava infecting fungi, we suggest that CasCV should be classified within the mycovirus taxonomic family. However, host range studies using infectious clones will be required to demonstrate the novel virus' likely origin and actual host species.     

Experimental infection of laboratory-bred bank voles (Myodes glareolus) with murid herpesvirus 4

MuHV-4 is a natural pathogen of rodents of the genus Apodemus (e.g., wood mice, yellow-necked mice) and Myodes glareolus (bank voles). We report experimental MuHV-4 infection of bank voles in comparison with infection of A. sylvaticus (wood mice) and BALB/c mice. Like in wood mice, the level of productive replication in the lungs of bank voles was significantly lower than in BALB/c mice. In contrast to other hosts, however, the level of latent infection in the lung and spleen of bank voles was extremely low. These findings, together with those of previous studies, suggest that bank voles are an occasional and inefficient host for MuHV-4.