Intracytoplasmic trapping of influenza virus by a lipophilic derivative of aglycoristocetin

We report on a new anti-influenza virus agent, SA-19, a lipophilic glycopeptide derivative consisting of aglycoristocetin coupled to a phenylbenzyl-substituted cyclobutenedione. In Madin-Darby canine kidney cells infected with influenza A/H1N1, A/H3N2, or B virus, SA-19 displayed a 50% antivirally effective concentration of 0.60 μM and a selectivity index (ratio of cytotoxic versus antiviral concentration) of 112. SA-19 was 11-fold more potent than unsubstituted aglycoristocetin and was active in human and nonhuman cell lines. Virus yield at 72 h p.i. was reduced by 3.6 logs at 0.8 μM SA-19. In contrast to amantadine and oseltamivir, SA-19 did not select for resistance upon prolonged virus exposure. SA-19 was shown to inhibit an early postbinding step in virus replication. The compound had no effect on hemagglutinin (HA)-mediated membrane fusion in an HA-polykaryon assay and did not inhibit the low-pH-induced refolding of the HA in a tryptic digestion assay. However, a marked inhibitory effect on the transduction exerted by retroviral pseudoparticles carrying an HA or vesicular stomatitis virus glycoprotein (VSV-G) fusion protein was noted, suggesting that SA-19 targets a cellular factor with a role in influenza virus and VSV entry. Using confocal microscopy with antinucleoprotein staining, SA-19 was proven to completely prevent the influenza virus nuclear entry. This virus arrest was characterized by the formation of cytoplasmic aggregates. SA-19 appeared to disturb the endocytic uptake and trap the influenza virus in vesicles distinct from early, late, or recycling endosomes. The aglycoristocetin derivative SA-19 represents a new class of potent and broad-acting influenza virus inhibitors with potential clinical relevance.     

'Because we see them naked' - nurses' experiences in caring for hospitalized patients with dementia: considering artificial nutrition or hydration (ANH)

The aim of this study was to explore and describe how Flemish nurses experience their involvement in the care of hospitalized patients with dementia, particularly in relation to artificial nutrition or hydration (ANH). We interviewed 21 hospital nurses who were carefully selected from nine hospitals in different regions of Flanders. 'Being touched by the vulnerability of the demented patient' was the central experience of the nurses, having great impact on them professionally as well as personally. This feeling can be described as encompassing the various stages of the care process: the nurses' initial meeting with the vulnerable patient; the intense decision-making process, during which the nurses experienced several intense emotions influenced by supporting or hindering contextual factors; and the final coping process, a time when nurses came to terms with this challenging experience. From our examination of this care process, it is obvious that nurses' involvement in ANH decision-making processes that concern patients with dementia is a difficult and ethically sensitive experience. On the one hand, the feeling of 'being touched' can imply strength, as it demonstrates that nurses are willing to provide good care. On the other hand, the feeling of 'being touched' can also imply weakness, as it makes nurses vulnerable to moral distress stemming from contextual influences. Therefore, nurses have to be supported as they carry out this ethically sensitive assignment. Practical implications are given.     

TMEM165 deficiency causes a congenital disorder of glycosylation

Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.     

<Emphasis Type="Italic">Drosophila</Emphasis> as a platform to predict the pathogenicity of novel aminoacyl-tRNA synthetase mutations in CMT

Charcot-Marie-Tooth disease (CMT) is the major form of inherited peripheral neuropathy in humans. CMT is clinically and genetically heterogeneous and four aminoacyl-tRNA synthetases have been implicated in disease etiology. Mutations in the YARS gene encoding a tyrosyl-tRNA synthetase (TyrRS) lead to Dominant Intermediate CMT type C (DI-CMTC). Three dominant YARS mutations were so far associated with DI-CMTC. To further expand the spectrum of CMT causing genetic defects in this tRNA synthetase, we performed DNA sequencing of YARS coding regions in a cohort of 181 patients with various types of peripheral neuropathy. We identified a novel K265N substitution that in contrast to all previously described mutations is located at the anticodon recognition domain of the enzyme. Further genetic analysis revealed that this variant represents a benign substitution. Using our recently developed DI-CMTC Drosophila model, we tested in vivo the pathogenicity of this new YARS variant. We demonstrated that the developmental and behavioral defects induced by all DI-CMTC causing mutations were not present upon ubiquitous or panneuronal TyrRS K265N expression. Thus, in line with our genetic studies, functional analysis confirmed that the K265N substitution does not induce toxicity signs in Drosophila. The consistency observed throughout this work underscores the robustness of our DI-CMTC animal model and identifies Drosophila as a valid read-out platform to ascertain the pathogenicity of novel mutations to be identified in the future.     

Finger loop inhibitors of the HCV NS5b polymerase. Part II. Optimization of tetracyclic indole-based macrocycle leading to the discovery of TMC647055

Optimization of a novel series of macrocyclic indole-based inhibitors of the HCV NS5b polymerase targeting the finger loop domain led to the discovery of lead compounds exhibiting improved potency in cellular assays and superior pharmacokinetic profile. Further lead optimization performed on the most promising unsaturated-bridged subseries provided the clinical candidate 27-cyclohexyl-12,13,16,17-tetrahydro-22-methoxy-11,17-dimethyl-10,10-dioxide-2,19-methano-3,7:4,1-dimetheno-1H,11H-14,10,2,9,11,17-benzoxathiatetraazacyclo docosine-8,18(9H,15H)-dione, TMC647055 (compound 18a). This non-zwitterionic 17-membered ring macrocycle combines nanomolar cellular potency (EC(50) of 82 nM) with minimal associated cell toxicity (CC(50)>20 μM) and promising pharmacokinetic profiles in rats and dogs. TMC647055 is currently being evaluated in the clinic.     

Job strain and tobacco smoking: an individual-participant data meta-analysis of 166,130 adults in 15 European studies

Background

Tobacco smoking is a major contributor to the public health burden and healthcare costs worldwide, but the determinants of smoking behaviours are poorly understood. We conducted a large individual-participant meta-analysis to examine the extent to which work-related stress, operationalised as job strain, is associated with tobacco smoking in working adults.

Methodology and Principal Findings

We analysed cross-sectional data from 15 European studies comprising 166 130 participants. Longitudinal data from six studies were used. Job strain and smoking were self-reported. Smoking was harmonised into three categories never, ex- and current. We modelled the cross-sectional associations using logistic regression and the results pooled in random effects meta-analyses. Mixed effects logistic regression was used to examine longitudinal associations. Of the 166 130 participants, 17% reported job strain, 42% were never smokers, 33% ex-smokers and 25% current smokers. In the analyses of the cross-sectional data, current smokers had higher odds of job strain than never-smokers (age, sex and socioeconomic position-adjusted odds ratio: 1.11, 95% confidence interval: 1.03, 1.18). Current smokers with job strain smoked, on average, three cigarettes per week more than current smokers without job strain. In the analyses of longitudinal data (1 to 9 years of follow-up), there was no clear evidence for longitudinal associations between job strain and taking up or quitting smoking.

Conclusions

Our findings show that smokers are slightly more likely than non-smokers to report work-related stress. In addition, smokers who reported work stress smoked, on average, slightly more cigarettes than stress-free smokers.     

Longitudinal study on transmission of MRSA CC398 within pig herds

ABSTRACT: BACKGROUND: Since the detection of MRSA CC398 in pigs in 2004, it has emerged in livestock worldwide. MRSA CC398 has been found in people in contact with livestock and thus has become a public health issue. Data from a large-scale longitudinal study in two Danish and four Dutch pig herds were used to quantify MRSA CC398 transmission rates within pig herds and to identify factors affecting transmission between pigs. RESULTS: Sows and their offspring were sampled at varying intervals during a production cycle. Overall MRSA prevalence of sows increased from 33% before farrowing to 77% before weaning. Overall MRSA prevalence of piglets was > 60% during the entire study period. The recurrent finding of MRSA in the majority of individuals indicates true colonization or might be the result of contamination. Transmission rates were estimated using a Susceptible-Infectious-Susceptible (SIS-)model, which resulted in values of the reproduction ratio (R0) varying from 0.24 to 8.08. Transmission rates were higher in pigs treated with tetracyclins and beta-lactams compared to untreated pigs implying a selective advantage of MRSA CC398 when these antimicrobials are used. Furthermore, transmission rates were higher in pre-weaning pigs compared to post-weaning pigs which might be explained by an age-related susceptibility or the presence of the sow as a primary source of MRSA CC398. Finally, transmission rates increased with the relative increase of the infection pressure within the pen compared to the total infection pressure, implying that within-pen transmission is a more important route compared to between-pen transmission and transmission through environmental exposure. CONCLUSIONS: Our results indicate that MRSA CC398 is able to spread and persist in pig herds, resulting in an endemic situation. Transmission rates are affected by the use of selective antimicrobials and by the age of pigs.     

Characterization of anticoagulant heparinoids by immunoprofiling

Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and–when additional saccharides are present–inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics.