Development of Lateral-flow Immunoassay for WSSV with Polyclonal Antibodies Raised against Recombinant VP （19＋28） Fusion Protein

We have developed a sensitive and rapid lateral-flow immunoassay (LFIA) for WSSV, using colloidal gold as an indicator. The fusion protein, VP (19 28), was expressed in E. coli, purified and used to prepare polyclonal antibodies. The purified anti-VP (19 28) IgG were conjugated with colloidal gold. Unconjugated anti-VP (19 28) IgG and goat anti-rabbit IgG were immobilized on nitrocellulose membranes. After assembly, three groups (5 individual animals in each group) of shrimp samples were tested which included healthy, moribund and dead shrimps. For each group, three different tissues (body juices, gills and hepatopancreas) were tested at the same time. In parallel, all the samples were also analyzed using PCR for comparison. Out of 45 samples tested, 30 were detected as positive while 15 were classified as negative. The results of LFIA correlate with those obtained by the PCR analysis, indicating that these two detection methods have the same efficacy in the limited number of samples tested in this preliminary study.     

Clinical Performance of a New Soluble CD14-Subtype Immunochromatographic Test for Whole Blood Compared with Chemiluminescent Enzyme Immunoassay: Use of Quantitative Soluble CD14-Subtype Immunochromatographic Tests for the Diagnosis of Sepsis

We previously reported that a soluble CD14-subtype (sCD14-ST) immunochromatographic test (ICT) for plasma is more convenient than chemiluminescent enzyme immunoassay (CLEIA), but plasma separation makes bedside measurements difficult. We developed a new sCD14-ST ICT for whole blood and investigated whether quantitative determinations of sCD14-ST by ICT were useful for diagnosing sepsis and severe sepsis/septic shock. We studied 20 patients who fulfilled two or more systemic inflammatory response syndrome (SIRS) criteria and 32 patients who had been diagnosed with sepsis or severe sepsis/septic shock. Whole blood was collected on day 0 (on admission) and day 7, and the sCD14-ST concentration was quantitatively measured by CLEIA and ICT for whole blood. The patients’ Acute Physiology and Chronic Health Evaluation (APACHE) II, Sequential Organ Failure Assessment (SOFA), and Mortality in Emergency Department Sepsis (MEDS) scores were also calculated. The cut-off values obtained by the quantitative measurements made by ICT were 464.5 pg/mL for sepsis and 762.7 pg/mL for severe sepsis/septic shock (P < 0.0001). A Bland–Altman plot showed that no fixed bias or proportional bias was detected between CLEIA and quantitative ICT for whole blood. sCD14-ST concentrations were significantly correlated with APACHE II, SOFA, and MEDS scores (P < 0.0001). These results suggest that the new sCD14-ST ICT for whole blood may be a useful tool for the convenient, rapid bedside diagnosis and treatment of sepsis.     

Historical Invasion Records Can Be Misleading: Genetic Evidence for Multiple Introductions of Invasive Raccoons (Procyon lotor) in Germany

Biological invasions provide excellent study systems to understand evolutionary, genetic and ecological processes during range expansions. There is strong evidence for positive effects of high propagule pressure and the associated higher genetic diversity on invasion success, but some species have become invasive despite small founder numbers. The raccoon (Procyon lotor) is often considered as a typical example for such a successful invasion resulting from a small number of founders. The species’ largest non-native population in Germany is commonly assumed to stem from a small number of founders and two separate founding events in the 1930s and 1940s. In the present study we analyzed 407 raccoons at 20 microsatellite loci sampled from the invasive range in Western Europe to test if these assumptions are correct. Contrary to the expectations, different genetic clustering methods detected evidence for at least four independent introduction events that gave rise to genetically differentiated subpopulations. Further smaller clusters were either artifacts or resulted from founder events at the range margin and recent release of captive individuals. We also found genetic evidence for on-going introductions of individuals. Furthermore a novel randomization process was used to determine the potential range of founder population size that would suffice to capture all the alleles present in a cluster. Our results falsify the assumption that this species has become widespread and abundant despite being genetically depauperate and show that historical records of species introductions may be misleading.     

Blood Levels of Glial Fibrillary Acidic Protein (GFAP) in Patients with Neurological Diseases

Background and Purpose

The brain-specific astroglial protein GFAP is a blood biomarker candidate indicative of intracerebral hemorrhage in patients with symptoms suspicious of acute stroke. Comparably little, however, is known about GFAP release in other neurological disorders. In order to identify potential “specificity gaps” of a future GFAP test used to diagnose intracerebral hemorrhage, we measured GFAP in the blood of a large and rather unselected collective of patients with neurological diseases.

Methods

Within a one-year period, we randomly selected in-patients of our university hospital for study inclusion. Patients with ischemic stroke, transient ischemic attack and intracerebral hemorrhage were excluded. Primary endpoint was the ICD-10 coded diagnosis reached at discharge. During hospital stay, blood was collected, and GFAP plasma levels were determined using an advanced prototype immunoassay at Roche Diagnostics.

Results

A total of 331 patients were included, covering a broad spectrum of neurological diseases. GFAP levels were low in the vast majority of patients, with 98.5% of cases lying below the cut-off that was previously defined for the differentiation of intracerebral hemorrhage and ischemic stroke. No diagnosis or group of diagnoses was identified that showed consistently increased GFAP values. No association with age and sex was found.

Conclusion

Most acute and chronic neurological diseases, including typical stroke mimics, are not associated with detectable GFAP levels in the bloodstream. Our findings underline the hypothesis that rapid astroglial destruction as in acute intracerebral hemorrhage is mandatory for GFAP increase. A future GFAP blood test applied to identify patients with intracerebral hemorrhage is likely to have a high specificity.     

Potential Cost-Effectiveness of a New Infant Tuberculosis Vaccine in South Africa - Implications for Clinical Trials: A Decision Analysis

Novel tuberculosis vaccines are in varying stages of pre-clinical and clinical development. This study seeks to estimate the potential cost-effectiveness of a BCG booster vaccine, while accounting for costs of large-scale clinical trials, using the MVA85A vaccine as a case study for estimating potential costs. We conducted a decision analysis from the societal perspective, using a 10-year time frame and a 3% discount rate. We predicted active tuberculosis cases and tuberculosis-related costs for a hypothetical cohort of 960,763 South African newborns (total born in 2009). We compared neonatal vaccination with bacille Calmette-Guérin alone to vaccination with bacille Calmette-Guérin plus a booster vaccine at 4 months. We considered booster efficacy estimates ranging from 40% to 70%, relative to bacille Calmette-Guérin alone. We accounted for the costs of Phase III clinical trials. The booster vaccine was assumed to prevent progression to active tuberculosis after childhood infection, with protection decreasing linearly over 10 years. Trial costs were prorated to South Africa''s global share of bacille Calmette-Guérin vaccination. Vaccination with bacille Calmette-Guérin alone resulted in estimated tuberculosis-related costs of $89.91 million 2012 USD, and 13,610 tuberculosis cases in the birth cohort, over the 10 years. Addition of the booster resulted in estimated cost savings of $7.69–$16.68 million USD, and 2,800–4,160 cases averted, for assumed efficacy values ranging from 40%–70%. A booster tuberculosis vaccine in infancy may result in net societal cost savings as well as fewer active tuberculosis cases, even if efficacy is relatively modest and large scale Phase III studies are required.     

Patient-Reported Outcome (PRO) Assessment in Clinical Trials: A Systematic Review of Guidance for Trial Protocol Writers

Background

Evidence suggests there are inconsistencies in patient-reported outcome (PRO) assessment and reporting in clinical trials, which may limit the use of these data to inform patient care. For trials with a PRO endpoint, routine inclusion of key PRO information in the protocol may help improve trial conduct and the reporting and appraisal of PRO results; however, it is currently unclear exactly what PRO-specific information should be included. The aim of this review was to summarize the current PRO-specific guidance for clinical trial protocol developers.

Methods and Findings

We searched the MEDLINE, EMBASE, CINHAL and Cochrane Library databases (inception to February 2013) for PRO-specific guidance regarding trial protocol development. Further guidance documents were identified via Google, Google scholar, requests to members of the UK Clinical Research Collaboration registered clinical trials units and international experts. Two independent investigators undertook title/abstract screening, full text review and data extraction, with a third involved in the event of disagreement. 21,175 citations were screened and 54 met the inclusion criteria. Guidance documents were difficult to access: electronic database searches identified just 8 documents, with the remaining 46 sourced elsewhere (5 from citation tracking, 27 from hand searching, 7 from the grey literature review and 7 from experts). 162 unique PRO-specific protocol recommendations were extracted from included documents. A further 10 PRO recommendations were identified relating to supporting trial documentation. Only 5/162 (3%) recommendations appeared in ≥50% of guidance documents reviewed, indicating a lack of consistency.

Conclusions

PRO-specific protocol guidelines were difficult to access, lacked consistency and may be challenging to implement in practice. There is a need to develop easily accessible consensus-driven PRO protocol guidance. Guidance should be aimed at ensuring key PRO information is routinely included in appropriate trial protocols, in order to facilitate rigorous collection/reporting of PRO data, to effectively inform patient care.     

Phage Shock Protein C (PspC) of Yersinia enterocolitica Is a Polytopic Membrane Protein with Implications for Regulation of the Psp Stress Response

Phage shock proteins B (PspB) and C (PspC) are integral cytoplasmic membrane proteins involved in inducing the Yersinia enterocolitica Psp stress response. A fundamental aspect of these proteins that has not been studied in depth is their membrane topologies. Various in silico analyses universally predict that PspB is a bitopic membrane protein with the C terminus inside. However, similar analyses yield conflicting predictions for PspC: a bitopic membrane protein with the C terminus inside, a bitopic membrane protein with the C terminus outside, or a polytopic protein with both termini inside. Previous studies of Escherichia coli PspB-LacZ and PspC-PhoA fusion proteins supported bitopic topologies, with the PspB C terminus inside and the PspC C terminus outside. Here we have used a series of independent approaches to determine the membrane topologies of PspB and PspC in Y. enterocolitica. Our data support the predicted arrangement of PspB, with its C terminus in the cytoplasm. In contrast, data from multiple independent approaches revealed that both termini of PspC are located in the cytoplasm. Additional experiments suggested that the C terminus of PspC might be the recognition site for the FtsH protease and an interaction interface with PspA, both of which would be compatible with its newly proposed cytoplasmic location. This unexpected arrangement of PspC allows a new model for events underlying activation of the Psp response, which is an excellent fit with observations from various previous studies.     

A Genetic Association Study of Serum Acute-Phase C-Reactive Protein Levels in Rheumatoid Arthritis: Implications for Clinical Interpretation

Background

The acute-phase increase in serum C-reactive protein (CRP) is used to diagnose and monitor infectious and inflammatory diseases. Little is known about the influence of genetics on acute-phase CRP, particularly in patients with chronic inflammation.

Methods and Findings

We studied two independent sets of patients with chronic inflammation due to rheumatoid arthritis (total 695 patients). A tagSNP approach captured common variation at the CRP locus and the relationship between genotype and serum CRP was explored by linear modelling. Erythrocyte sedimentation rate (ESR) was incorporated as an independent marker of inflammation to adjust for the varying levels of inflammatory disease activity between patients. Common genetic variants at the CRP locus were associated with acute-phase serum CRP (for the most associated haplotype: p = 0.002, p<0.0005, p<0.0005 in patient sets 1, 2, and the combined sets, respectively), translating into an approximately 3.5-fold change in expected serum CRP concentrations between carriers of two common CRP haplotypes. For example, when ESR = 50 mm/h the expected geometric mean CRP (95% confidence interval) concentration was 43.1 mg/l (32.1–50.0) for haplotype 1 and 14.2 mg/l (9.5–23.2) for haplotype 4.

Conclusions

Our findings raise questions about the interpretation of acute-phase serum CRP. In particular, failure to take into account the potential for genetic effects may result in the inappropriate reassurance or suboptimal treatment of patients simply because they carry low-CRP–associated genetic variants. CRP is increasingly being incorporated into clinical algorithms to compare disease activity between patients and to predict future clinical events: our findings impact on the use of these algorithms. For example, where access to effective, but expensive, biological therapies in rheumatoid arthritis is rationed on the basis of a DAS28-CRP clinical activity score, then two patients with identical underlying disease severity could be given, or denied, treatment on the basis of CRP genotype alone. The accuracy and utility of these algorithms might be improved by using a genetically adjusted CRP measurement. Please see later in the article for the Editors'' Summary     

The Impacts of Inclusion in Clinical Trials on Outcomes among Patients with Metastatic Breast Cancer (MBC)

Background

Metastatic breast cancer (MBC) remains a devastating and incurable disease. Over the past decade, the implementation of clinical trials both with and without molecular targeted therapeutics has impacted the daily clinical treatment of patients with MBC. In this study, we determine whether including MBC patients in clinical trials affects clinical outcomes.

Methods

We retrospectively reviewed data for a total of 863 patients diagnosed with initial or recurrent (after receiving adjuvant systemic treatments following surgery) metastatic disease between January 2000 and December 2013. Data were obtained from the breast cancer database of Samsung Medical Center.

Results

Among the 806 patients selected for inclusion, 188 (23%) had participated in clinical trials. A total of 185 clinical trials were conducted from 2000 to 2014. When compared with earlier periods (n = 10 for 2000–2004), clinical trial enrollment significantly increased over time (n = 103 for 2005–2009, P = 0.024; n = 110 for 2010–2014, P = 0.046). Multivariate analyses revealed that biologic subtype, distant recurrence free interval (DRFI), and clinical trial enrollment were independent predictors of overall survival. Patients who participated in clinical trials showed improved survival, with a hazard ratio of 0.75 (95% CI, 0.59–0.95), which was associated with a 25% reduction in the risk of death. However, subgroup analysis showed that this improved survival benefit was not maintained in patients with triple negative breast cancer (TNBC).

Conclusions

Although not conclusive, we could speculate that there were differences in the use of newer agents or regimens over time, and these differences appear to be associated with improved survival.     

Effect of Seasonal Variation on Adult Clinical Laboratory Parameters in Rwanda,Zambia, and Uganda: Implications for HIV Biomedical Prevention Trials

Objectives

To investigate the effect of seasonal variation on adult clinical laboratory parameters in Rwanda, Zambia, and Uganda and determine its implications for HIV prevention and other clinical trials.

Methods

Volunteers in a cross-sectional study to establish laboratory reference intervals were asked to return for a seasonal visit after the local season had changed from dry to rainy or vice versa. Volunteers had to be clinically healthy, not pregnant and negative for HIV, Hepatitis B and C, and syphilis infection at both visits. At each visit, blood was taken for measurement of hemoglobin, haematocrit, mean corpuscular volume, red blood cells, platelets, total white blood cells (WBC), neutrophils, lymphocytes, monocytes, eosinophils, basophils, CD4/CD8 T cells, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, direct bilirubin, total bilirubin, total immunoglobulin gamma, total protein, creatinine, total amylase, creatine phosphokinase and lactate dehydrogenase (LDH). Consensus dry season reference intervals were applied to rainy season values (and vice versa) and the proportion of ‘out-of-range’ values determined. Percentage differences between dry and rainy season parameter mean values were estimated.

Results

In this cohort of 903 volunteers, less than 10.0% of consensus parameter (except LDH) values in one season were “out-of-range” in the other. Twenty-two (22) percent of rainy season LDH values fell outside of the consensus dry season interval with the higher values observed in the rainy season. Variability between consensus seasonal means ranged from 0.0% (total WBC, neutrophils, monocytes, basophils, and direct bilirubin) to 40.0% (eosinophils). Within sites, the largest seasonal variations were observed for monocytes (Masaka, 11.5%), LDH (Lusaka, 21.7%), and basophils (Kigali, 22.2%).

Conclusions

Seasonality had minimal impact on adult clinical laboratory parameter values in Rwanda, Zambia, and Uganda. Seasonal variation may not be an important factor in the evaluation of adult clinical laboratory parameters in HIV prevention and other clinical trials in these countries.     

Extracellular HCV-Core Protein Induces an Immature Regulatory Phenotype in NK Cells: Implications for Outcome of Acute Infection

Background

Hepatitis C viral (HCV) proteins, including core, demonstrate immuno-modulatory properties; however, the effect of extracellular core on natural killer (NK) cells has not previously been investigated.

Aims

To characterise NKs in acute HCV infection over time, and, to examine the effect of exogenous HCV-core protein on NK cell phenotype and function.

Methods

Acute HCV patients (n = 22), including 10 subjects who spontaneously recovered, were prospectively studied. Flow-cytometry was used to measure natural cytotoxicity and to phenotype NKs directly ex vivo and after culture with HCV-core protein. Microarray analysis was used to identify pathways involved in the NK cell response to exogenous HCV-core.

Results

Direct ex vivo analysis demonstrated an increased frequency of immature/regulatory CD56^bright NKs early in acute HCV infection per se which normalized with viral clearance. Natural cytotoxicity was reduced and did not recover after viral clearance. There was a statistically significant correlation between the frequency of CD56^bright NKs and circulating serum levels of HCV core protein. In vitro culture of purified CD56^bright NK cells with HCV-core protein in the presence of IL-15 maintained a significant proportion of NKs in the CD56^bright state. The in vitro effect of core closely correlates with NK characteristics measured directly ex vivo in acute HCV infection. Pathway analysis suggests that HCV-core protein attenuates NK interferon type I responses.

Conclusions

Our data suggest that HCV-core protein alters NK cell maturation and may influence the outcome of acute infection.     

Failure of Hairpin-Ended and Nicked DNA To Activate DNA-Dependent Protein Kinase: Implications for V(D)J Recombination

V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PK_CS or p460). Ku directs DNA-PK_CS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PK_CS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PK_CS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.     

Post-synaptic Density-95 (PSD-95) Binding Capacity of G-protein-coupled Receptor 30 (GPR30), an Estrogen Receptor That Can Be Identified in Hippocampal Dendritic Spines

The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity.     

The First Mitochondrial Genome for Caddisfly (Insecta: Trichoptera) with Phylogenetic Implications

The Trichoptera (caddisflies) is a holometabolous insect order with 14,300 described species forming the second most species-rich monophyletic group of animals in freshwater. Hitherto, there is no mitochondrial genome reported of this order. Herein, we describe the complete mitochondrial (mt) genome of a caddisfly species, Eubasilissa regina (McLachlan, 1871). A phylogenomic analysis was carried out based on the mt genomic sequences of 13 mt protein coding genes (PCGs) and two rRNA genes of 24 species belonging to eight holometabolous orders. Both maximum likelihood and Bayesian inference analyses highly support the sister relationship between Trichoptera and Lepidoptera.     

Sex differences in mitochondrial (dys)function: Implications for neuroprotection

Journal of Bioenergetics and Biomembranes - Decades of research have revealed numerous differences in brain structure size, connectivity and metabolism between males and females. Sex differences in...     

Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV

Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for viral propagation. Using protein microarray analysis, we identified 90 cellular proteins as HCV nonstructural 5A (NS5A) interacting partners, and selected telomere length regulation protein (TEN1) for further study. TEN1 forms a heterotrimeric complex with CTC and STN1, which is essential for telomere protection and maintenance. Telomere length decreases in patients with active HCV, chronic liver disease, and hepatocellular carcinoma. However, the molecular mechanism of telomere length shortening in HCV-associated disease is largely unknown. In the present study, protein interactions between NS5A and TEN1 were confirmed by immunoprecipitation assays. Silencing of TEN1 reduced both viral RNA and protein expression levels of HCV, while ectopic expression of the siRNA-resistant TEN1 recovered the viral protein level, suggesting that TEN1 was specifically required for HCV propagation. Importantly, we found that TEN1 is re-localized from the nucleus to the cytoplasm in HCV-infected cells. These data suggest that HCV exploits TEN1 to promote viral propagation and that telomere protection is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the telomere shortening in HCV-infected cells.