Post-mortem culture of Balamuthia mandrillaris from the brain and cerebrospinal fluid of a case of granulomatous amoebic meningoencephalitis, using human brain microvascular endothelial cells

The first isolation in the UK of Balamuthia mandrillaris amoebae from a fatal case of granulomatous amoebic meningoencephalitis is reported. Using primary cultures of human brain microvascular endothelial cells (HBMECs), amoebae were isolated from the brain and cerebrospinal fluid (CSF). The cultures showed a cytopathic effect at 20–28 days, but morphologically identifiable B. mandrillaris amoebae were seen in cleared plaques in subcultures at 45 days. The identification of the organism was later confirmed using PCR on Chelex-treated extracts. Serum taken while the patient was still alive reacted strongly with slide antigen prepared from cultures of the post-mortem isolate, and also with those from a baboon B. mandrillaris strain at 1 : 10 000 in indirect immunofluorescence, but with Acanthamoeba castellanii (Neff) at 1 : 160, supporting B. mandrillaris to be the causative agent. If the presence of amoebae in the post-mortem CSF reflects the condition in life, PCR studies on CSF and on biopsies of cutaneous lesions may also be a valuable tool. The role of HBMECs in understanding the interactions of B. mandrillaris with the blood–brain barrier is discussed

Estimating parameters for probabilistic linkage of privacy-preserved datasets.

Background: Probabilistic record linkage is a process used to bring together person-based records from within the same dataset (de-duplication) or from disparate datasets using pairwise comparisons and matching probabilities. The linkage strategy and associated match probabilities are often estimated through investigations into data quality and manual inspection. However, as privacy-preserved datasets comprise encrypted data, such methods are not possible. In this paper, we present a method for estimating the probabilities and threshold values for probabilistic privacy-preserved record linkage using Bloom filters. Methods: Our method was tested through a simulation study using synthetic data, followed by an application using real-world administrative data. Synthetic datasets were generated with error rates from zero to 20% error. Our method was used to estimate parameters (probabilities and thresholds) for de-duplication linkages. Linkage quality was determined by F-measure. Each dataset was privacy-preserved using separate Bloom filters for each field. Match probabilities were estimated using the expectation-maximisation (EM) algorithm on the privacy-preserved data. Threshold cut-off values were determined by an extension to the EM algorithm allowing linkage quality to be estimated for each possible threshold. De-duplication linkages of each privacy-preserved dataset were performed using both estimated and calculated probabilities. Linkage quality using the F-measure at the estimated threshold values was also compared to the highest F-measure. Three large administrative datasets were used to demonstrate the applicability of the probability and threshold estimation technique on real-world data. Results: Linkage of the synthetic datasets using the estimated probabilities produced an F-measure that was comparable to the F-measure using calculated probabilities, even with up to 20% error. Linkage of the administrative datasets using estimated probabilities produced an F-measure that was higher than the F-measure using calculated probabilities. Further, the threshold estimation yielded results for F-measure that were only slightly below the highest possible for those probabilities. Conclusions: The method appears highly accurate across a spectrum of datasets with varying degrees of error. As there are few alternatives for parameter estimation, the approach is a major step towards providing a complete operational approach for probabilistic linkage of privacy-preserved datasets

A 160-Gb/s OTDM demultiplexer based on parametric wavelength exchange

Parametric wavelength exchange (PWE) has been demonstrated as a versatile device in providing different functionalities. In this paper, we will concentrate, numerically and experimentally, on one of these functionalities, namely, all-optical time demultiplexing of 160-Gb/s return-to-zero (RZ) signals based on a pulsed-pump PWE in a 400 m highly nonlinear dispersion-shifted fiber. Experimental results show power penalties < 2.7 dB at bit-error rate of 10-9 for all demultiplexed 10-Gb/s RZ signals. We also derive theoretical expressions for the conversion/residual efficiencies and investigate the impact of pump pulse width and phase mismatch on these efficiencies. Furthermore, the impacts of pulsed-pump wavelength and power level on the characteristics of the switching window are investigated numerically. As a result, the demultiplexer can be easily upgraded to an add-drop multiplexer because of the complete exchange nature of PWE, which is justified by the surviving channels' waveform performance. © 2009 IEEE.published_or_final_versio

Correlations of Behavioral Deficits with Brain Pathology Assessed through Longitudinal MRI and Histopathology in the R6/2 Mouse Model of HD

Huntington's disease (HD) is caused by the expansion of a CAG repeat in the huntingtin (HTT) gene. The R6/2 mouse model of HD expresses a mutant version of exon 1 HTT and develops motor and cognitive impairments, a widespread huntingtin (HTT) aggregate pathology and brain atrophy. Despite the vast number of studies that have been performed on this model, the association between the molecular and cellular neuropathology with brain atrophy, and with the development of behavioral phenotypes remains poorly understood. In an attempt to link these factors, we have performed longitudinal assessments of behavior (rotarod, open field, passive avoidance) and of regional brain abnormalities determined through magnetic resonance imaging (MRI) (whole brain, striatum, cortex, hippocampus, corpus callosum), as well as an end-stage histological assessment. Detailed correlative analyses of these three measures were then performed. We found a gender-dependent emergence of motor impairments that was associated with an age-related loss of regional brain volumes. MRI measurements further indicated that there was no striatal atrophy, but rather a lack of striatal growth beyond 8 weeks of age. T2 relaxivity further indicated tissue-level changes within brain regions. Despite these dramatic motor and neuroanatomical abnormalities, R6/2 mice did not exhibit neuronal loss in the striatum or motor cortex, although there was a significant increase in neuronal density due to tissue atrophy. The deposition of the mutant HTT (mHTT) protein, the hallmark of HD molecular pathology, was widely distributed throughout the brain. End-stage histopathological assessments were not found to be as robustly correlated with the longitudinal measures of brain atrophy or motor impairments. In conclusion, modeling pre-manifest and early progression of the disease in more slowly progressing animal models will be key to establishing which changes are causally related. © 2013 Rattray et al

Technical challenges of providing record linkage services for research

Background: Record linkage techniques are widely used to enable health researchers to gain event based longitudinal information for entire populations. The task of record linkage is increasingly being undertaken by specialised linkage units (SLUs). In addition to the complexity of undertaking probabilistic record linkage, these units face additional technical challenges in providing record linkage ‘as a service’ for research. The extent of this functionality, and approaches to solving these issues, has had little focus in the record linkage literature. Few, if any, of the record linkage packages or systems currently used by SLUs include the full range of functions required. Methods: This paper identifies and discusses some of the functions that are required or undertaken by SLUs in the provision of record linkage services. These include managing routine, on-going linkage; storing and handling changing data; handling different linkage scenarios; accommodating ever increasing datasets. Automated linkage processes are one way of ensuring consistency of results and scalability of service. Results: Alternative solutions to some of these challenges are presented. By maintaining a full history of links, and storing pairwise information, many of the challenges around handling ‘open’ records, and providing automated managed extractions are solved. A number of these solutions were implemented as part of the development of the National Linkage System (NLS) by the Centre for Data Linkage (part of the Population Health Research Network) in Australia.Conclusions: The demand for, and complexity of, linkage services are growing. This presents as a challenge to SLUs as they seek to service the varying needs of dozens of research projects annually. Linkage units need to be both flexible and scalable to meet this demand. It is hoped the solutions presented here can help mitigate these difficulties

Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises.Defense Threat Reduction Agency (DTRA) (HDTRA1-14-1-0006)United States. National Institutes of Health (NIH AI100190

Accuracy and completeness of patient pathways – the benefits of national data linkage in Australia

Background - The technical challenges associated with national data linkage, and the extent of cross-border population movements, are explored as part of a pioneering research project. The project involved linking state-based hospital admission records and death registrations across Australia for a national study of hospital related deaths. Methods - The project linked over 44 million morbidity and mortality records from four Australian states between 1st July 1999 and 31st December 2009 using probabilistic methods. The accuracy of the linkage was measured through a comparison with jurisdictional keys sourced from individual states. The extent of cross-border population movement between these states was also assessed. Results - Data matching identified almost twelve million individuals across the four Australian states. The percentage of individuals from one state with records found in another ranged from 3-5 %. Using jurisdictional keys to measure linkage quality, results indicate a high matching efficiency (F measure 97 to 99 %), with linkage processing taking only a matter of days. Conclusions - The results demonstrate the feasibility and accuracy of undertaking cross jurisdictional linkage for national research. The benefits are substantial, particularly in relation to capturing the full complement of records in patient pathways as a result of cross-border population movements. The project identified a sizeable ‘mobile’ population with hospital records in more than one state. Research studies that focus on a single jurisdiction will under-enumerate the extent of hospital usage by individuals in the population. It is important that researchers understand and are aware of the impact of this missing hospital activity on their studies. The project highlights the need for an efficient and accurate data linkage system to support national research across Australia

The role of PKCzeta in cord blood T-cell maturation towards Th1 cytokine profile and its epigenetic regulation by fish oil

While immunodeficiency of immaturity of the neonate has been considered important as the basis for unusual susceptibility to infection, it has also been recognized that the ability to progress from an immature Th2 cytokine predominance to a Th1 profile has relevance in determining whether children will develop allergy, providing an opportunity for epigenetic regulation through environmental pressures. However, this notion remains relatively unexplored. Here, we present evidence that there are two major control points to explain the immunodeficiency in cord blood (CB) T-cells, a deficiency in interleukin (IL)-12 (IL-12) producing and IL-10 overproducing accessory cells, leading to a decreased interferon γ (IFNγ) synthesis and the other, an intrinsic defect in T-cell protein kinase C (PKC) ζ (PKCζ) expression. An important finding was that human CB T-cells rendered deficient in PKCζ, by shRNA knockdown, develop into low tumour necrosis factor α (TNFα) and IFNγ but increased IL-13 producing cells. Interestingly, we found that the increase in PKCζ levels in CB T-cells caused by prenatal supplementation with fish oil correlated with modifications of histone acetylation at the PKCζ gene (PRKCZ) promoter. The data demonstrate that PKCζ expression regulates the maturation of neonatal T-cells into specific functional phenotypes and that environmental influences may work via PKCζ to regulate these phenotypes and disease susceptibility.Hani Harb, James Irvine, Manori Amarasekera, Charles S. Hii, Dörthe A. Kesper, YueFang Ma, Nina D′Vaz, Harald Renz, Daniel P. Potaczek, Susan L. Prescott and Antonio Ferrant

RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord

ALS is a rapidly progressive, devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression, and molecular insights into pathogenesis and progression are sorely needed. In that context, we used high-depth, next generation RNA sequencing (RNAseq, Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned \u3e50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2, DEseq2, EdgeR) for identification of differentially expressed genes (DEG’s). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples, with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNFα-induced protein 2 (TNFAIP2) as a major network “hub” gene (WGCNA). Using the oPOSSUM algorithm, we analyzed transcription factors (TF) controlling expression of the nine DEG/hub genes in the ALS samples and identified TF’s involved in inflammation (NFkB, REL, NFkB1) and macrophage function (NR1H2::RXRA heterodimer). Transient expression in human iPSC-derived motor neurons of TNFAIP2 (also a DEG identified by all three algorithms) reduced cell viability and induced caspase 3/7 activation. Using high-density RNAseq, multiple algorithms for DEG identification, and an unsupervised gene co-expression network approach, we identified significant elevation of inflammatory processes in ALS spinal cord with TNF as a major regulatory molecule. Overexpression of the DEG TNFAIP2 in human motor neurons, the population most vulnerable to die in ALS, increased cell death and caspase 3/7 activation. We propose that therapies targeted to reduce inflammatory TNFα signaling may be helpful in ALS patients