Validation of Multiplex Serology detecting human herpesviruses 1-5

Human herpesviruses (HHV) cause a variety of clinically relevant conditions upon primary infection of typically young and immunocompetent hosts. Both primary infection and reactivation after latency can lead to more severe disease, such as encephalitis, congenital defects and cancer. Infections with HHV are also associated with cardiovascular and neurodegenerative disease. However, most of the associations are based on retrospective casecontrol analyses and well-powered prospective cohort studies are needed for assessing temporality and causality. To enable comprehensive investigations of HHV-related disease etiology in large prospective population-based cohort studies, we developed HHV Multiplex Serology. This methodology represents a low-cost, high-throughput technology that allows simultaneous measurement of specific antibodies against five HHV species: Herpes simplex viruses 1 and 2, Varicella zoster virus, Epstein-Barr virus, and Cytomegalovirus. The newly developed HHV species-specific (‘Monoplex’) assays were validated against established gold-standard reference assays. The specificity and sensitivity of the HHV speciesspecific Monoplex Serology assays ranged from 92.3% to 100.0% (median 97.4%) and 91.8% to 98.7% (median 96.6%), respectively. Concordance with reference assays was very high with kappa values ranging from 0.86 to 0.96 (median kappa 0.93). Multiplexing the Monoplex Serology assays resulted in no loss of performance and allows simultaneous detection of antibodies against the 5 HHV species in a high-throughput manner

Universal Alcohol/Drug Screening in Prenatal Care: A Strategy for Reducing Racial Disparities? Questioning the Assumptions

Agencies and organizations promoting universal screening for alcohol and drug use in prenatal care argue that universal screening will reduce White versus Black racial disparities in reporting to Child Protective Services (CPS) at delivery. Yet, no published research has assessed the impact of universal screening on reporting disparities or explored plausible mechanisms. This review defines two potential mechanisms: Equitable Surveillance and Effective Treatment and identifies assumptions underlying each mechanism. It reviews published literature relating to each assumption. Research relating to assumptions underlying each mechanism is primarily inconclusive or contradictory. Thus, available research does not support the claim that universal screening for alcohol and drug use in prenatal care reduces racial disparities in CPS reporting at delivery. Reducing these reporting disparities requires more than universal screening

Gamma-Secretase-Dependent and -Independent Effects of Presenilin1 on β-Catenin·Tcf-4 Transcriptional Activity

Presenilin1 (PS1) is a component of the γ-secretase complex mutated in cases of Familial Alzheimer's disease (FAD). PS1 is synthesized as a 50 kDa peptide subsequently processed to two 29 and 20 kDa subunits that remain associated. Processing of PS1 is inhibited by several mutations detected in FAD patients. PS1 acts as negative modulator of β-catenin·Tcf-4 transcriptional activity. In this article we show that in murine embryonic fibroblasts (MEFs) the mechanisms of action of the processed and non-processed forms of PS1 on β-catenin·Tcf-4 transcription are different. Whereas non-processed PS1 inhibits β-catenin·Tcf-4 activity through a mechanism independent of γ-secretase and associated with the interaction of this protein with plakoglobin and Tcf-4, the effect of processed PS1 is prevented by γ-secretase inhibitors, and requires its interaction with E- or N-cadherin and the generation of cytosolic terminal fragments of these two cadherins, which in turn destabilize the β-catenin transcriptional cofactor CBP. Accordingly, the two forms of PS1 interact differently with E-cadherin or β-catenin and plakoglobin: whereas processed PS1 binds E-cadherin with high affinity and β-catenin or plakoglobin weakly, the non-processed form behaves inversely. Moreover, contrarily to processed PS1, that decreases the levels of c-fos RNA, non-processed PS1 inhibits the expression c-myc, a known target of β-catenin·Tcf-4, and does not block the activity of other transcriptional factors requiring CBP. These results indicate that prevention of PS1 processing in FAD affects the mechanism of repression of the transcriptional activity dependent on β-catenin

Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica

Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents

Tigers of Sundarbans in India: Is the Population a Separate Conservation Unit?

The Sundarbans tiger inhabits a unique mangrove habitat and are morphologically distinct from the recognized tiger subspecies in terms of skull morphometrics and body size. Thus, there is an urgent need to assess their ecological and genetic distinctiveness and determine if Sundarbans tigers should be defined and managed as separate conservation unit. We utilized nine microsatellites and 3 kb from four mitochondrial DNA (mtDNA) genes to estimate genetic variability, population structure, demographic parameters and visualize historic and contemporary connectivity among tiger populations from Sundarbans and mainland India. We also evaluated the traits that determine exchangeability or adaptive differences among tiger populations. Data from both markers suggest that Sundarbans tiger is not a separate tiger subspecies and should be regarded as Bengal tiger (P. t. tigris) subspecies. Maximum likelihood phylogenetic analyses of the mtDNA data revealed reciprocal monophyly. Genetic differentiation was found stronger for mtDNA than nuclear DNA. Microsatellite markers indicated low genetic variation in Sundarbans tigers (He= 0.58) as compared to other mainland populations, such as northern and Peninsular (Hebetween 0.67- 0.70). Molecular data supports migration between mainland and Sundarbans populations until very recent times. We attribute this reduction in gene flow to accelerated fragmentation and habitat alteration in the landscape over the past few centuries. Demographic analyses suggest that Sundarbans tigers have diverged recently from peninsular tiger population within last 2000 years. Sundarbans tigers are the most divergent group of Bengal tigers, and ecologically non-exchangeable with other tiger populations, and thus should be managed as a separate "evolutionarily significant unit" (ESU) following the adaptive evolutionary conservation (AEC) concept.Wildlife Institute of India, Dehra Dun (India)

A supermatrix analysis of genomic, morphological, and paleontological data from crown Cetacea

<p>Abstract</p> <p>Background</p> <p>Cetacea (dolphins, porpoises, and whales) is a clade of aquatic species that includes the most massive, deepest diving, and largest brained mammals. Understanding the temporal pattern of diversification in the group as well as the evolution of cetacean anatomy and behavior requires a robust and well-resolved phylogenetic hypothesis. Although a large body of molecular data has accumulated over the past 20 years, DNA sequences of cetaceans have not been directly integrated with the rich, cetacean fossil record to reconcile discrepancies among molecular and morphological characters.</p> <p>Results</p> <p>We combined new nuclear DNA sequences, including segments of six genes (~2800 basepairs) from the functionally extinct Yangtze River dolphin, with an expanded morphological matrix and published genomic data. Diverse analyses of these data resolved the relationships of 74 taxa that represent all extant families and 11 extinct families of Cetacea. The resulting supermatrix (61,155 characters) and its sub-partitions were analyzed using parsimony methods. Bayesian and maximum likelihood (ML) searches were conducted on the molecular partition, and a molecular scaffold obtained from these searches was used to constrain a parsimony search of the morphological partition. Based on analysis of the supermatrix and model-based analyses of the molecular partition, we found overwhelming support for 15 extant clades. When extinct taxa are included, we recovered trees that are significantly correlated with the fossil record. These trees were used to reconstruct the timing of cetacean diversification and the evolution of characters shared by "river dolphins," a non-monophyletic set of species according to all of our phylogenetic analyses.</p> <p>Conclusions</p> <p>The parsimony analysis of the supermatrix and the analysis of morphology constrained to fit the ML/Bayesian molecular tree yielded broadly congruent phylogenetic hypotheses. In trees from both analyses, all Oligocene taxa included in our study fell outside crown Mysticeti and crown Odontoceti, suggesting that these two clades radiated in the late Oligocene or later, contra some recent molecular clock studies. Our trees also imply that many character states shared by river dolphins evolved in their oceanic ancestors, contradicting the hypothesis that these characters are convergent adaptations to fluvial habitats.</p

Impact of Intraoperative Parathyroid Hormone Monitoring on the Prediction of Multiglandular Parathyroid Disease

Optimal interpretation of the results of intraoperative parathyroid hormone (IOPTH) monitoring during neck exploration for primary hyperparathyroidism (pHPT) is still controversial. The reliability of the “50% rule” in multiglandular disease (MGD) is often disputed, mostly because of competing pathophysiologic paradigms. The aim of this study was to ascertain and corroborate the ability of IOPTH monitoring to detect MGD in a practice, combining conventional and alternative parathyroidectomy techniques. This is a retrospective single institution analysis of 69 consecutive patients undergoing cervical exploration for pHPT by various approaches. The IOPTH measurements were performed after induction of anesthesia but prior to skin incision and 10 minutes after excision of the first visualized enlarged parathyroid gland. In this series, 55 patients (80%) had single adenomas, and 14 patients (20%) had MGD. In 8 of the 14 patients with MGD, IOPTH levels were obtained sequentially after removal of every enlarged gland. Of these 8 patients, 6 (75%) had a false-positive decrease (decrease below 50% of baseline value in presence of another enlarged gland) failing to predict the presence of a second enlarged gland. In 2 cases IOPTH monitoring provided a true-negative result, correctly predicting MGD. If MGD is defined by gross morphologic criteria, IOPTH monitoring fails to predict the presence of MGD reliably. However, if MGD is defined by functional criteria, the course of these patients does not seem significantly affected. The importance of these findings must be further investigated, especially with regard to the outcome of minimally invasive parathyroid procedures.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41301/1/268_2003_Article_7255.pd

The Immune System in Stroke

Stroke represents an unresolved challenge for both developed and developing countries and has a huge socio-economic impact. Although considerable effort has been made to limit stroke incidence and improve outcome, strategies aimed at protecting injured neurons in the brain have all failed. This failure is likely to be due to both the incompleteness of modelling the disease and its causes in experimental research, and also the lack of understanding of how systemic mechanisms lead to an acute cerebrovascular event or contribute to outcome. Inflammation has been implicated in all forms of brain injury and it is now clear that immune mechanisms profoundly influence (and are responsible for the development of) risk and causation of stroke, and the outcome following the onset of cerebral ischemia. Until very recently, systemic inflammatory mechanisms, with respect to common comorbidities in stroke, have largely been ignored in experimental studies. The main aim is therefore to understand interactions between the immune system and brain injury in order to develop novel therapeutic approaches. Recent data from clinical and experimental research clearly show that systemic inflammatory diseases -such as atherosclerosis, obesity, diabetes or infection - similar to stress and advanced age, are associated with dysregulated immune responses which can profoundly contribute to cerebrovascular inflammation and injury in the central nervous system. In this review, we summarize recent advances in the field of inflammation and stroke, focusing on the challenges of translation between pre-clinical and clinical studies, and potential anti-inflammatory/immunomodulatory therapeutic approaches

miRNA expression profiling of 51 human breast cancer cell lines reveals subtype and driver mutation-specific miRNAs

INTRODUCTION: Breast cancer is a genetically and phenotypically complex disease. To understand the role of miRNAs in this molecular complexity, we performed miRNA expression analysis in a cohort of molecularly well-characterized human breast cancer cell lines to identify miRNAs associated with the most common molecular subtypes and the most frequent genetic aberrations. METHODS: Using a microarray carrying LNA™ modified oligonucleotide capture probes), expression levels of 725 human miRNAs were measured in 51 breast cancer cell lines. Differential miRNA expression was explored by unsupervised cluster analysis and was then associated with the molecular subtypes and genetic aberrations commonly present in breast cancer. RESULTS: Unsupervised cluster analysis using the most variably expressed miRNAs divided the 51 breast cancer cell lines into a major and a minor cluster predominantly mirroring the luminal and basal intrinsic subdivision of breast cancer cell lines. One hundred and thirteen miRNAs were differentially expressed between these two main clusters. Forty miRNAs were differentially expressed between basal-like and normal-like/claudin-low cell lines. Within the luminal-group, 39 miRNAs were associated with ERBB2 overexpression and 24 with E-cadherin gene mutations, which are frequent in this subtype of breast cancer cell lines. In contrast, 31 miRNAs were associated with E-cadherin promoter hypermethylation, which, contrary to E-cadherin mutation, is exclusively observed in breast cancer cell lines that are not of luminal origin. Thirty miRNAs were associated with p16(INK4 )status while only a few miRNAs were associated with BRCA1, PIK3CA/PTEN and TP53 mutation status. Twelve miRNAs were associated with DNA copy number variation of the respective locus. CONCLUSION: Luminal-basal and epithelial-mesenchymal associated miRNAs determine the subdivision of miRNA transcriptome of breast cancer cell lines. Specific sets of miRNAs were associated with ERBB2 overexpression, p16(INK4a )or E-cadherin mutation or E-cadherin methylation status, which implies that these miRNAs may contribute to the driver role of these genetic aberrations. Additionally, miRNAs, which are located in a genomic region showing recurrent genetic aberrations, may themselves play a driver role in breast carcinogenesis or contribute to a driver gene in their vicinity. In short, our study provides detailed molecular miRNA portraits of breast cancer cell lines, which can be exploited for functional studies of clinically important miRNAs