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Background: In patients with left heart failure, micro-RNAs (miRNAs) have been shown to be of diagnostic and prognostic value. The present study aims to identify those miRNAs in patients with univentricular heart (UVH) disease that may be associated with overt heart failure. Methods: A large panel of human miRNA arrays were used to determine miRNA expression profiles in the blood of 48 UVH patients and 32 healthy controls. For further selection, the most abundantly expressed miRNA arrays were related to clinical measures of heart failure and selected miRNAs validated by polymerase chain reaction were used for the prediction of overt heart failure and all-cause mortality. Results: According to microarray analysis, 50 miRNAs were found to be significantly abundant in UVH patients of which miR-150-5p was best related to heart failure parameters. According to ROC analysis, NT-proBNP levels (AUC 0.940, 95% CI 0.873–1.000; p = 0.001), miR-150-5p (AUC 0.905, 95% CI 0.779–1.000; p = 0.001) and a higher NYHA class ≥ III (AUC 0.893, 95% CI 0.713–1.000; p = 0.002) were the 3 most significant predictors of overt heart failure. Using a combined biomarker model, AUC increased to 0.980 indicating an additive value of miR-150-5p. Moreover, in the multivariate analysis, a higher NYHA class ≥ III (p = 0.005) and miR-150-5p (p = 0.006) turned out to be independent predictors of overt heart failure. Conclusion: In patients with UVH, miR-150-5p is an independent predictor of overt heart failure and thus may be used in the risk assessment of these patients. [ABSTRACT FROM AUTHOR]

Aberrant activation of the JAK/STAT pathway is thought to be the critical event in the pathogenesis of the chronic myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia and primary myelofibrosis. The most frequent genetic alteration in these pathologies is the activating JAK2V617F mutation, and expression of the mutant gene in mouse models was shown to cause a phenotype resembling the human diseases. Given the body of genetic evidence, it has come as a sobering finding that JAK inhibitor therapy only modestly suppresses the JAK2V617F allele burden, despite showing clear benefits in terms of reducing splenomegaly and constitutional symptoms in patients. To gain a better understanding if JAK2V617F is required for maintenance of myeloproliferative disease once it has evolved, we generated a conditional inducible transgenic JAK2V617F mouse model using the SCL-tTA-2S tet-off system. Our model corroborates that expression of JAK2V617F in hematopoietic stem and progenitor cells recapitulates key hallmarks of human myeloproliferative neoplasms, and exhibits gender differences in disease manifestation. The disease was found to be transplantable, and importantly, reversible when transgenic JAK2V617F expression was switched off. Our results indicate that mutant JAK2V617F-specific inhibitors should result in profound disease modification by disabling the myeloproliferative clone bearing mutant JAK2. [ABSTRACT FROM AUTHOR]

The problem of handling non-ignorable non-response has been typically addressed under the design-based approach using the well-known sub-sampling technique introduced by Hansen and Hurwitz [1946, Journal of the American Statistical Association, Vol 41(236), Page 517- 529]. Alternatively, the model-based paradigm emphasizes on utilizing the underlying model relationship between the outcome variable and one or more covariate(s) whose population values are known prior to the survey. This article utilizes the model relationship between the study variable and covariate(s) for handling non-ignorable non-response and obtaining an unbiased estimator for the population total under the sub-sampling technique. The main idea is to combine the estimates obtained from the sample on first call and the sub-sample from second call using separate model relationships. The contribution of this paper helps us in providing unbiased estimates with an improved efficiency under model-based paradigm in presence of non-ignorable non-response. The provided method is more economical than the available estimators under callback methods as we are working sub-sampling and also increase response rate as a stronger mode of interview is employed for data collection. A numerical study using Monte Carlo is presented to illustrate the behavior of the proposed and the efficiency comparison. [ABSTRACT FROM AUTHOR]

Streptococcus suis is an important porcine bacterial pathogen and a zoonotic agent responsible for sudden death, septic shock and meningitis, of which serotype 2 is the most widespread, with serotype 14 also causing infections in humans in South-East Asia. Knowledge of its pathogenesis and virulence are almost exclusively based on these two serotypes. Though serotype 9 is responsible for the greatest number of porcine cases in Spain, the Netherlands and Germany, very little information is currently available regarding this serotype. Of the different virulence factors, the capsular polysaccharide (CPS) is required for S. suis virulence as it promotes resistance to phagocytosis and killing and masks surface components responsible for host cell activation. However, these roles have been described for serotypes 2 and 14, whose CPSs are structurally and compositionally similar, both containing sialic acid. Consequently, we evaluated herein the interactions of serotype 9 with host cells and the role of its CPS, which greatly differs from those of serotypes 2 and 14. Results demonstrated that serotype 9 adhesion to but not invasion of respiratory epithelial cells was greater than that of serotypes 2 and 14. Furthermore serotype 9 was more internalized by macrophages but equally resistant to whole blood killing. Though recognition of serotypes 2, 9 and 14 by DCs required MyD88-dependent signaling, in vitro pro-inflammatory mediator production induced by serotype 9 was much lower. In vivo, however, serotype 9 causes an exacerbated inflammatory response, which combined with persistent bacterial presence, is probably responsible for host death during the systemic infection. Though presence of the serotype 9 CPS masks surface components less efficiently than those of serotypes 2 and 14, the serotype 9 CPS remains critical for virulence as it is required for survival in blood and development of clinical disease, and this regardless of its unique composition and structure. [ABSTRACT FROM AUTHOR]

Cell-free DNA (cfDNA) in the human blood circulation has been under investigation since its initial observation in 1948. Plasma cfDNA is known to be significantly elevated in diseased people. Due to possible variation in the population, evaluating cfDNA as a non-invasive biomarker at disease onset alone may not be sensitive enough to accurately diagnose diseases, particularly early stage cancers on a personal level. To understand the factors that define the cfDNA levels on the personal level and for better use as a non-invasive biomarker, we isolated cfDNA from the plasma of healthy individuals with varying degrees of genetic and/or environmental similarities (monozygotic twins, dizygotic twins, sibling pairs, and unrelated individuals) as well as from patients with varying stages of breast and ovarian cancer undergoing treatment. Cell-free DNA levels were quantified by a fluorometer (ng/ml) and/or real-time PCR (copies/ml). The associations between individuals with various degrees of genetic and/or environmental similarities and their plasma cfDNA levels were evaluated. The ACE model (A = additive genetic, C = common environment, and E = specific environmental factors) was used to determine the proportion of each factor on the cfDNA levels. We found a high correlation (r = 0.77; p < 0.0001) in plasma cfDNA levels between monozygotic twins (n = 39). However, the correlation was gradually reduced to moderate (r = 0.47; p = 0.016) between dizygotic twins (n = 13) and low correlation (r = 0.28; p = 0.043) between sibling pairs (n = 26). The ACE model analysis showed that the plasma cfDNA level of a given healthy individual is influenced both by genetic and the environmental components in similar proportions (53% and 47%, respectively; A = 53%, C = 22.5%, E = 24.5%). Moreover, while age had no effect, gender significantly influenced the individual's plasma cfDNA level. As expected, cfDNA levels were significantly higher in both breast (n = 26) (p<0.0001) and ovarian (n = 64) (p<0.0001) cancer patients compared to the healthy individuals. Our study demonstrated that both genome and environmental factors modulate the individual's cfDNA level suggesting that its diagnostic sensitivity may be improved only if the person's cfDNA level is known prior to disease presentation. [ABSTRACT FROM AUTHOR]

Peripheral Blood gene expression is widely used in the discovery of biomarkers and development of therapeutics. Recently, a spate of commercial blood collection and preservation systems have been introduced with proprietary variations that may differentially impact the transcriptomic profiles. Comparative analysis of these collection platforms will help optimize protocols to detect, identify, and reproducibly validate true biological variance among subjects. In the current study, we tested two recently introduced whole blood collection methods, RNAgard® and PAXgene® RNA, in addition to the traditional method of peripheral blood mononuclear cells (PBMCs) separated from whole blood and preserved in Trizol reagent. Study results revealed striking differences in the transcriptomic profiles from the three different methods that imply ex vivo changes in gene expression occurred during the blood collection, preservation, and mRNA extraction processes. When comparing the ability of the three preservation methods to accurately capture individuals' expression differences, RNAgard® outperformed PAXgene® RNA, and both showed better individual separation of transcriptomic profiles than PBMCs. Hence, our study recommends using a single blood collection platform, and strongly cautions against combining methods during the course of a defined study. [ABSTRACT FROM AUTHOR]

Gastric emptying (GE) can be either delayed or accelerated in diabetes mellitus (DM). However, most research has focused on delayed GE mediated by a chronic hyperglycemic condition in DM. As such, the function of GE in the early stages of DM is not well understood. Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal tract. In the present study, we investigated changes in GE and ICC networks in the early stages of DM using a streptozotocin-induced type 1 diabetic mouse model. The changes in GE were measured by the 13C-octanoic acid breath test. ICC networks were immunohistochemically detected by an antibody for c-Kit, a specific marker for ICC. Our results showed that GE in type 1 DM was significantly accelerated in the early stages of DM (2–4 weeks after onset). In addition, acute normalization of blood glucose levels by a single administration of insulin did not recover normal GE. ICC networks of the gastric antrum were significantly increased in DM and were not affected by the acute normalization of blood glucose. In conclusion, our results suggest that GE is accelerated in the early stages of DM, and it is associated with increased ICC networks. This mechanism may help to clarify a link between the onset of DM and GE disorders. [ABSTRACT FROM AUTHOR]

Sepsis can be caused by malaria infection, but little is known about the utility of the quick Sequential (Sepsis-Related) Organ Failure Assessment (qSOFA) and SOFA score in malaria. We conducted a prospective observational study from March 2013 to February 2017 to examine adults admitted with community-acquired infection in a tertiary-care hospital in Ubon Ratchathani, Northeast Thailand (Ubon-sepsis). Subjects were classified as having sepsis if they had a modified SOFA score ≥2 within 24 hours of admission. Serum was stored and later tested for malaria parasites using a nested PCR assay. Presence of severe malaria was defined using modified World Health Organization criteria. Of 4,989 patients enrolled, 153 patients (3%) were PCR positive for either Plasmodium falciparum (74 [48%]), P. vivax (69 [45%]), or both organisms (10 [7%]). Of 153 malaria patients, 80 were severe malaria patients presenting with sepsis, 70 were non-severe malaria patients presenting with sepsis, and three were non-severe malaria patients presenting without sepsis. The modified SOFA score (median 5; IQR 4–6; range 1–18) was strongly correlated with malaria severity determined by the number of World Health Organization severity criteria satisfied by the patient (Spearman’s rho = 0.61, p<0.001). Of 80 severe malaria patients, 2 (2.5%), 11 (14%), 62 (77.5%) and 5 (6%), presented with qSOFA scores of 0, 1, 2 and 3, respectively. Twenty eight-day mortality was 1.3% (2/153). In conclusion, qSOFA and SOFA can serve as markers of disease severity in adults with malarial sepsis. Patients presenting with a qSOFA score of 1 may also require careful evaluation for sepsis; including diagnosis of cause of infection, initiation of medical intervention, and consideration for referral as appropriate. [ABSTRACT FROM AUTHOR]

The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated. [ABSTRACT FROM AUTHOR]

Echocardiography, as a noninvasive hemodynamic evaluation technique, is frequently used in critically ill patients. Different opinions exist regarding whether it can be interchanged with traditional invasive means, such as the pulmonary artery catheter thermodilution (TD) technique. This systematic review aimed to analyze the consistency and interchangeability of cardiac output measurements by ultrasound (US) and TD. Five electronic databases were searched for studies including clinical trials conducted up to June 2019 in which patients’ cardiac output was measured by ultrasound techniques (echocardiography) and TD. The methodological quality of the included studies was evaluated by two independent reviewers who used the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2), which was tailored according to our systematic review in Review Manager 5.3. A total of 68 studies with 1996 patients were identified as eligible. Meta-analysis and subgroup analysis were used to compare the cardiac output (CO) measured using the different types of echocardiography and different sites of Doppler use with TD. No significant differences were found between US and TD (random effects model: mean difference [MD], -0.14; 95% confidence interval, -0.30 to 0.02; P = 0.08). No significant differences were observed in the subgroup analyses using different types of echocardiography and different sites except for ascending aorta (AA) (random effects model: mean difference [MD], -0.37; 95% confidence interval, -0.74 to -0.01; P = 0.05) of Doppler use. The median of bias and limits of agreement were -0.12 and ±0.94 L/min, respectively; the median of correlation coefficient was 0.827 (range, 0.140–0.998). Although the difference in CO between echocardiography by different types or sites and TD was not entirely consistent, the overall effect of meta-analysis showed that no significant differences were observed between US and TD. The techniques may be interchangeable under certain conditions. [ABSTRACT FROM AUTHOR]

The need for a validated set of emotional clips to elicit emotions in more ecological experiments is increasing. Here we present the validation of a new database of emotional films, named E-MOVIE, which includes, in this first validation phase, 39 excerpts arranged in six categories, three negative (Fear, Sadness and Compassion), two positive (Erotic and Scenery) and a Neutral category. Notably, Compassion and Scenery are new in the field as they were not included in other databases. The clips in E-MOVIE are characterized by homogenous durations of approximately two minutes, which make them suitable for psychophysiological research. In order to study the affective profile prompted by each category 174 participants (112 women) rated the movies on multiple dimensions, namely valence and arousal, intensity and discreteness of the induction of one of the six basic emotions and, finally, intensity of the experience of the emotional states defined by a series of emotional adjectives. Erotic clips were effective in the elicitation of a positive emotional state, characterized by high levels of arousal and excitement. On the other hand, Fear clips (selected without blood to avoid disgust reaction) prompted an affect characterized by high arousal, low valence and high levels of reported fear and anxiety. Women reported greater unpleasantness, distress, anxiety and jittery than men to the three negative categories. Compassion clips, characterized by the depiction of crying characters, were able to induce an affective state dominated by sadness and feeling touched, consistent with an empathic reaction to emotional sufferance. Sadness clips, instead, elicited an affective state characterized by sadness together with distress and angst. We also demonstrated that clips depicting natural environments (i.e. Scenery) prompted in the viewer a surprised, inspired affective state, characterized by high valence and arousal (especially in males), a result which suggests that their past categorization as neutral stimuli was inaccurate and problematic. [ABSTRACT FROM AUTHOR]

Concentrations of free amino acids and [K+] in human sweat can be many times higher than in plasma. Conversely, [Na+] and [Cl-] in sweat are hypotonic to plasma. It was hypothesised that the amino acids and K+ were directly or indirectly associated with the resorption of Na+ and Cl- in the sweat duct. The implication would be that, as resources of these components became limiting during prolonged exercise then the capacity to resorb [Na+] and [Cl-] would diminish, resulting in progressively higher levels in sweat. If this were the case, then [Na+] and [Cl-] in sweat would have inverse relationships with [K+] and the amino acids during exercise. Forearm sweat was collected from 11 recreational athletes at regular intervals during a prolonged period of cycling exercise after 15, 25, 35, 45, 55 and 65 minutes. The subjects also provided passive sweat samples via 15 minutes of thermal stimulation. The sweat samples were analysed for concentrations of amino acids, Na+, Cl-, K+, Mg2+ and Ca2+. The exercise sweat had a total amino acid concentration of 6.4 ± 1.2mM after 15 minutes which was lower than the passive sweat concentration at 11.6 ± 0.8mM (p<0.05) and showed an altered array of electrolytes, indicating that exercise stimulated a change in sweat composition. During the exercise period, [Na+] in sweat increased from 23.3 ± 3.0mM to 34.6 ± 2.4mM (p<0.01) over 65 minutes whilst the total concentrations of amino acids in sweat decreased from 6.4 ± 1.2mM to 3.6 ± 0.5mM. [Na+] showed significant negative correlations with the concentrations of total amino acids (r = -0.97, p<0.05), K+ (r = -0.93, p<0.05) and Ca2+ (r = -0.83, p<0.05) in sweat. The results supported the hypothesis that amino acids and K+, as well as Ca2+, were associated with resorption of Na+ and Cl-. [ABSTRACT FROM AUTHOR]

The general population is exposed to bisphenol A (BPA) orally, parenterally, transdermally, and environmentally as a result of the use of BPA in food packaging, plastics, and personal care products. The majority of the population nowadays (91–99%) has detectable levels of BPA inside their body. In this study, we successfully performed an inexpensive, rapid, and simple protein precipitation procedure for extraction of BPA from human plasma, followed by analysis by LC-MS/MS. This method was specifically developed for handling large numbers of samples with minimum cost and volume of sample. The developed method was accurate, precise, and reproducible for quantification of BPA from human plasma samples in the concentration range of 10–2000 ng/mL. The method was performed on samples from 150 healthy volunteers who were enrolled in the study. The mean of observed BPA level was 2.22 ± 9.91 ng/mL. Higher BPA levels were observed for females compare to that of males (p-value = 0.002), the BPA levels were higher in participants 33 years of age and older compared to those less than 33 years of age (p-value = 0.000), then the BPA levels higher in subjects with tap water as source of drinking (p-value = 0.005). This method may be valuable for general risk assessment of BPA for a large and varied population because of its efficiency and economical aspects. [ABSTRACT FROM AUTHOR]

Background: Personalized medicine requires accurate molecular profiling for targeted therapy decisions. Insufficient tissue yield or tumor heterogeneity frequently limits the correct tissue biomarker determination. As a noninvasive complement to traditional tissue biopsies, liquid biopsies detect and track cancer driver mutations from biofluids (e.g., blood, urine). Here we present the analytical validation of Target Selector™ ctDNA assays capable of single mutant DNA copy detection. Methods: The Target Selector ctDNA assay applies a patented Switch-Blocker technology to suppress amplification of background (wild-type) WT alleles, while allowing specific amplification of very low frequency mutant alleles. In contrast to allele specific enrichment technologies like ddPCR, one Switch-Blocker inhibits amplification of a DNA target up to 15 bp in length (e.g., one Switch-Blocker covers all KRAS exon 2, codon 12 and 13 variants). Target enrichment is achieved through a quantitative PCR reaction; subsequent DNA sequencing confirms mutation identity. Analytical validation with cancer cell line DNA was conducted by three independent operators using five instruments across five days. Results: A total of 3086 samples were tested on EGFR, BRAF and KRAS Target Selector ctDNA assays, with EGFR WT as a reference. All assays showed >99% analytical sensitivity and specificity. Single mutant copy detection is confirmed by experimental data and theoretical estimates. In the presence of 14000 WT DNA copies, limits of detection were: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. Inter- and intra-assay analyses showed r2>0.94, suggesting consistent performance among operational variables. Healthy donor samples (100 tests) showed clinical specificity at >99%. Finally, Target Selector clinical experience data of >2200 patient samples is consistent with published tissue mutation prevalence. Conclusions: Highly sensitive Target Selector ctDNA assays with single mutant copy detection and limit of detection at 0.02% or better enable accurate molecular profiling vital for disease management. [ABSTRACT FROM AUTHOR]

Background: RNA sequencing has been proposed as a means of increasing diagnostic rates in studies of undiagnosed rare inherited disease. Recent studies have reported diagnostic improvements in the range of 7.5–35% by profiling splicing, gene expression quantification and allele specific expression. To-date however, no study has systematically assessed the presence of gene-fusion transcripts in cases of germline disease. Fusion transcripts are routinely identified in cancer studies and are increasingly recognized as having diagnostic, prognostic or therapeutic relevance. Isolated reports exist of fusion transcripts being detected in cases of developmental and neurological phenotypes, and thus, systematic application of fusion detection to germline conditions may further increase diagnostic rates. However, current fusion detection methods are unsuited to the investigation of germline disease due to performance biases arising from their development using tumor, cell-line or in-silico data. Methods: We describe a tailored approach to fusion candidate identification and prioritization in a cohort of 47 undiagnosed, suspected inherited disease patients. We modify an existing fusion transcript detection algorithm by eliminating its cell line-derived filtering steps, and instead, prioritize candidates using a custom workflow that integrates genomic and transcriptomic sequence alignment, biological and technical annotations, customized categorization logic, and phenotypic prioritization. Results: We demonstrate that our approach to fusion transcript identification and prioritization detects genuine fusion events excluded by standard analyses and efficiently removes phenotypically unimportant candidates and false positive events, resulting in a reduced candidate list enriched for events with potential phenotypic relevance. We describe the successful genetic resolution of two previously undiagnosed disease cases through the detection of pathogenic fusion transcripts. Furthermore, we report the experimental validation of five additional cases of fusion transcripts with potential phenotypic relevance. Conclusions: The approach we describe can be implemented to enable the detection of phenotypically relevant fusion transcripts in studies of rare inherited disease. Fusion transcript detection has the potential to increase diagnostic rates in rare inherited disease and should be included in RNA-based analytical pipelines aimed at genetic diagnosis. [ABSTRACT FROM AUTHOR]

Introduction: The prevalence of bacteremia caused by Gram negative non-fermentative (GNNF) bacteria has been increasing globally over the past decade. Many studies have investigated their epidemiology but focus on the common GNNF including Pseudomonas aeruginosa and Acinetobacter baumannii. Knowledge of the uncommon GNNF bacteremias is very limited. This study explores invasive bloodstream infection GNNF isolates that were initially unidentified after testing with standard microbiological techniques. All isolations were made during laboratory-based surveillance activities in two rural provinces of Thailand between 2006 and 2014. Methods: A subset of GNNF clinical isolates (204/947), not identified by standard manual biochemical methodologies were run on the BD Phoenix automated identification and susceptibility testing system. If an organism was not identified (12/204) DNA was extracted for whole genome sequencing (WGS) on a MiSeq platform and data analysis performed using 3 web-based platforms: Taxonomer, CGE KmerFinder and One Codex. Results: The BD Phoenix automated identification system recognized 92% (187/204) of the GNNF isolates, and because of their taxonomic complexity and high phenotypic similarity 37% (69/187) were only identified to the genus level. Five isolates grew too slowly for identification. Antimicrobial sensitivity (AST) data was not obtained for 93/187 (50%) identified isolates either because of their slow growth or their taxa were not in the AST database associated with the instrument. WGS identified the 12 remaining unknowns, four to genus level only. Conclusion: The GNNF bacteria are of increasing concern in the clinical setting, and our inability to identify these organisms and determine their AST profiles will impede treatment. Databases for automated identification systems and sequencing annotation need to be improved so that opportunistic organisms are better covered. [ABSTRACT FROM AUTHOR]

Background: The National Program for Chagas disease was implemented in Bolivia in 2006, and it greatly decreased the number of infections through vector control. Subsequently, a treatment regimen of benznidazole (BNZ) was started in seropositive school-age children living in certified vector control areas. Methods and findings: We conducted a 12-month follow-up study and seven blood samples were taken during and after the treatment. Serology, conventional diagnostic PCR (cPCR) and quantitative Real-time PCR (qPCR) were performed. Plasma Th1/Th2/Th17 cytokines levels were also determined. Approximately 73 of 103 seropositive children complied with BNZ, with three interruptions due to side effects. To evaluate each individual’s treatment efficacy, the cPCR and qPCR values during the final 6 months of the follow-up period were observed. Among 57 children who completed follow-up, 6 individuals (11%) showed both cPCR(+) and qPCR(+) (non reactive), 24 (42%) cPCR(-) but qPCR(+) (ambiguous) and 27 (47%) cPCR(-) and qPCR(-) (reactive). Within 14 Th1/Th2/Th17 cytokines, IL-17A showed significantly higher levels in seropositive children before the treatment compared to age-matched seronegative children and significantly decreased to the normal level one-year after. Moreover, throughout the follow-up study, IL-17A levels were positively co-related to parasite counts detected by qPCR. At the 12 months’ time point, IL-17A levels of non-reactive subjects were significantly higher than either those of reactive or ambiguous subjects suggesting that IL-17A might be useful to determine the reactivity to BNZ treatment. Conclusions: Plasma levels of IL-17A might be a bio-marker for detecting persistent infection of T. cruzi and its chronic inflammation. [ABSTRACT FROM AUTHOR]

Background: Lymphatic filariasis (LF) is a neglected tropical disease, and the Global Program to Eliminate LF delivers mass drug administration (MDA) to 500 million people every year. Adverse events (AEs) are common after LF treatment. Methodology/Principal findings: To better understand the pathogenesis of AEs, we studied LF-patients from a treatment trial. Plasma levels of many filarial antigens increased post-treatment in individuals with AEs, and this is consistent with parasite death. Circulating immune complexes were not elevated in these participants, and the classical complement cascade was not activated. Multiple cytokines increased after treatment in persons with AEs. A transcriptomic analysis was performed for nine individuals with moderate systemic AEs and nine matched controls. Differential gene expression analysis identified a significant transcriptional signature associated with post-treatment AEs; 744 genes were upregulated. The transcriptional signature was enriched for TLR and NF-κB signaling. Increased expression of seven out of the top eight genes upregulated in persons with AEs were validated by qRT-PCR, including TLR2. Conclusions/Significance: This is the first global study of changes in gene expression associated with AEs after treatment of lymphatic filariasis. Changes in cytokines were consistent with prior studies and with the RNAseq data. These results suggest that Wolbachia lipoprotein is involved in AE development, because it activates TLR2-TLR6 and downstream NF-κB. Additionally, LPS Binding Protein (LBP, which shuttles lipoproteins to TLR2) increased post-treatment in individuals with AEs. Improved understanding of the pathogenesis of AEs may lead to improved management, increased MDA compliance, and accelerated LF elimination. [ABSTRACT FROM AUTHOR]

Understanding cell differentiation—the process of generation of distinct cell-types—plays a pivotal role in developmental and evolutionary biology. Transcriptomic information and epigenetic marks are useful to elucidate hierarchical developmental relationships among cell-types. Standard phylogenetic approaches such as maximum parsimony, maximum likelihood and neighbor joining have previously been applied to ChIP-Seq histone modification data to infer cell-type trees, showing how diverse types of cells are related. In this study, we demonstrate the applicability and suitability of quartet-based phylogenetic tree estimation techniques for constructing cell-type trees. We propose two quartet-based pipelines for constructing cell phylogeny. Our methods were assessed for their validity in inferring hierarchical differentiation processes of various cell-types in H3K4me3, H3K27me3, H3K36me3, and H3K27ac histone mark data. We also propose a robust metric for evaluating cell-type trees. [ABSTRACT FROM AUTHOR]

Objectives: Bloodstream infections in critically ill require a speeded-up microbiological diagnosis to improve clinical outcomes. In this pre-post intervention study, we evaluated how a molecular identification test directly performed on positive blood cultures of critically ill improves patient’s therapeutic management. Methods: All adult patients staying at the intensive care unit (ICU) at the time of positive blood culture detection were study-eligible. In the 8-month pre-intervention period (P0), standard positive blood culture management was performed. In the 10-month intervention period (P1), a BioFire® FilmArray® blood culture identification (FA-BCID) test (bioMérieux) was additionally performed 24/7 at detection. The evaluated clinical outcome was time to optimal antimicrobial treatment of the bloodstream infection. FA-BCID microbiological test performances were also analysed. Results: 163 positive blood culture episodes were allocated to P0 and 166 to P1. After the withdrawal of episodes in accordance with defined exclusion criteria, outcome analysis was performed on 110 bloodstream infections both in P0 and P1. Time to optimal antimicrobial treatment in P0 was 14h41 compared to 4h39 in P1. FA-BCID test results led to a treatment adjustment in 35/110 (31.8%) P1 episodes including 26 where the adjustment was the optimal antimicrobial treatment. FA-BCID testing identified 96.2% of the on-panel microorganisms thereby covering 85.2% of our ICU-strain epidemiology. Time to identification with FA-BCID testing was calculated at 1h35. Resistance detection was in complete concordance with routine results. Considering 150 FA-BCID tests were initially performed in P1, 4,3 tests were required to have 1 test leading to an improved therapeutic outcome. Conclusions: FA-BCID testing drastically reduced time to optimal antimicrobial treatment in critically ill with bloodstream infections. [ABSTRACT FROM AUTHOR]

The blood flow through the major vessels holds great diagnostic potential for the identification of cardiovascular complications and is therefore routinely assessed with current diagnostic modalities. Heart valves are subject to high hydrodynamic loads which render them prone to premature degradation. Failing native aortic valves are routinely replaced with bioprosthetic heart valves. This type of prosthesis is limited by a durability that is often less than the patient’s life expectancy. Frequent assessment of valvular function can therefore help to ensure good long-term outcomes and to plan reinterventions. In this article, we describe how unsupervised novelty detection algorithms can be used to automate the interpretation of blood flow data to improve outcomes through early detection of adverse cardiovascular events without requiring repeated check-ups in a clinical environment. The proposed method was tested in an in-vitro flow loop which allowed simulating a failing aortic valve in a laboratory setting. Aortic regurgitation of increasing severity was deliberately introduced with tube-shaped inserts, preventing complete valve closure during diastole. Blood flow recordings from a flow meter at the location of the ascending aorta were analyzed with the algorithms introduced in this article and a diagnostic index was defined that reflects the severity of valvular degradation. The results indicate that the proposed methodology offers a high sensitivity towards pathological changes of valvular function and that it is capable of automatically identifying valvular degradation. Such methods may be a step towards computer-assisted diagnostics and telemedicine that provide the clinician with novel tools to improve patient care. [ABSTRACT FROM AUTHOR]

Ookinetes, one of the motile and invasive forms of the malaria parasite, rely on gliding motility in order to establish an infection in the mosquito host. Here we characterize the protein PBANKA_0407300 which is conserved in the Plasmodium genus but lacks significant similarity to proteins of other eukaryotes. It is expressed in gametocytes and throughout the invasive mosquito stages of P. berghei, but is absent from asexual blood stages. Mutants lacking the protein developed morphologically normal ookinetes that were devoid of productive motility although some stretching movement could be detected. We therefore named the protein Ookinete Motility Deficient (OMD). Several key factors known to be involved in motility however were normally expressed and localized in the mutant. Importantly, the mutant failed to establish an infection in the mosquito which resulted in a total malaria transmission blockade. [ABSTRACT FROM AUTHOR]

We identified a mouse strain, HLB444, carrying an N-ethyl-N-nitrosourea (ENU)-induced mutation in a highly conserved C2H2 zinc-finger DNA binding motif of the transcriptional regulator KLF15 that exhibits resistance to diet-induced obesity. Characterization of the HLB444 mutant model on high-fat and chow diets revealed a number of phenotypic differences compared to wild-type controls. When fed a high fat diet, HLB444 had lower body fat, resistance to hepatosteatosis, lower circulating glucose and improved insulin sensitivity compared to C57BL/6J controls. Gut microbial profiles in HLB444 generated from 16S rRNA sequencing of fecal samples differed from controls under both chow and high fat diets. HLB444 shares similar phenotypic traits with engineered full- and adipose-specific Klf15 knockout strains; however, some phenotypic differences between this mutant and the other models suggest that the Klf15 mutation in HLB444 is a hypomorphic variant. The HLB444 model will inform further annotation of transcriptional functions of KLF15, especially with respect to the role of the first zinc-finger domain. [ABSTRACT FROM AUTHOR]

Acute appendicitis is one of the major causes for emergency surgery in childhood and adolescence. Appendectomy is still the therapy of choice, but conservative strategies are increasingly being studied for uncomplicated inflammation. Diagnosis of acute appendicitis remains challenging, especially due to the frequently unspecific clinical picture. Inflammatory blood markers and imaging methods like ultrasound are limited as they have to be interpreted by experts and still do not offer sufficient diagnostic certainty. This study presents a method for automatic diagnosis of appendicitis as well as the differentiation between complicated and uncomplicated inflammation using values/parameters which are routinely and unbiasedly obtained for each patient with suspected appendicitis. We analyzed full blood counts, c-reactive protein (CRP) and appendiceal diameters in ultrasound investigations corresponding to children and adolescents aged 0–17 years from a hospital based population in Berlin, Germany. A total of 590 patients (473 patients with appendicitis in histopathology and 117 with negative histopathological findings) were analyzed retrospectively with modern algorithms from machine learning (ML) and artificial intelligence (AI). The discovery of informative parameters (biomarker signatures) and training of the classification model were done with a maximum of 35% of the patients. The remaining minimum 65% of patients were used for validation. At clinical relevant cut-off points the accuracy of the biomarker signature for diagnosis of appendicitis was 90% (93% sensitivity, 67% specificity), while the accuracy to correctly identify complicated inflammation was 51% (95% sensitivity, 33% specificity) on validation data. Such a test would be capable to prevent two out of three patients without appendicitis from useless surgery as well as one out of three patients with uncomplicated appendicitis. The presented method has the potential to change today’s therapeutic approach for appendicitis and demonstrates the capability of algorithms from AI and ML to significantly improve diagnostics even based on routine diagnostic parameters. [ABSTRACT FROM AUTHOR]

Background: Positron emission tomography (PET) imaging of macrophages using the translocator protein (TSPO) tracer (R)-[11C]PK11195 has shown the promise to image rheumatoid arthritis (RA). To further improve TSPO PET for RA imaging, second generation TSPO tracers [11C]DPA-713 and [18F]DPA-714 have recently been evaluated pre-clinically showing better imaging characteristics. Objective: A clinical proof of concept study to evaluate [11C]DPA-713 and [18F]DPA-714 to visualize arthritis in RA patients. Methods: RA patients (n = 13) with at least two active hand joints were included. PET/CT scans of the hands were obtained after injection of [18F]DPA-714, [11C]DPA-713 and/or (R)-[11C]PK11195 (max. 2 tracers pp). Standardized uptake values (SUVs) and target-to-background (T/B) ratios were determined. Imaging data of the 3 different tracers were compared by pooled post-hoc testing, and by a head to head comparison. Results: Clinically active arthritis was present in 110 hand joints (2–17 pp). Arthritic joints were visualized with both [11C]DPA-713 and [18F]DPA-714. Visual tracer uptake corresponded with clinical signs of arthritis in 80% of the joints. Mean absolute uptake in PET-positive joints was significantly higher for [11C]DPA-713 than for [18F]DPA-714, the latter being not significantly different from (R)-[11C]PK11195 uptake. Background uptake was lower for both DPA tracers compared with that of (R)-[11C]PK11195. Higher absolute uptake and lower background resulted in two-fold higher T/B ratios for [11C]DPA-713. Conclusions: [11C]DPA-713 and [18F]DPA-714 visualize arthritic joints in active RA patients and most optimal arthritis imaging results were obtained for [11C]DPA-713. Second generation TSPO macrophage PET provides new opportunities for both early diagnosis and therapy monitoring of RA. [ABSTRACT FROM AUTHOR]

Perfusion-related information is reportedly embedded in the low-frequency component of a blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) signal. The blood-propagation pattern through the cerebral vascular tree is detected as an interregional lag variation of spontaneous low-frequency oscillations (sLFOs). Mapping of this lag, or phase, has been implicitly treated as a projection of the vascular tree structure onto real space. While accumulating evidence supports the biological significance of this signal component, the physiological basis of the “perfusion lag structure,” a requirement for an integrative resting-state fMRI-signal model, is lacking. In this study, we conducted analyses furthering the hypothesis that the sLFO is not only largely of systemic origin, but also essentially intrinsic to blood, and hence behaves as a virtual tracer. By summing the small fluctuations of instantaneous phase differences between adjacent vascular regions, a velocity response to respiratory challenges was detected. Regarding the relationship to neurovascular coupling, the removal of the whole lag structure, which can be considered as an optimized global-signal regression, resulted in a reduction of inter-individual variance while preserving the fMRI response. Examination of the T2* and S0, or non-BOLD, components of the fMRI signal revealed that the lag structure is deoxyhemoglobin dependent, while paradoxically presenting a signal-magnitude reduction in the venous side of the cerebral vasculature. These findings provide insight into the origin of BOLD sLFOs, suggesting that they are highly intrinsic to the circulating blood. [ABSTRACT FROM AUTHOR]

We previously reported that allogeneic, intraperitoneally administered “Neo-Islets,” composed of cultured pancreatic islet cells co-aggregated with high numbers of immunoprotective and cytoprotective Adipose-derived Stem Cells, reestablished, through omental engraftment, redifferentiation and splenic and omental up-regulation of regulatory T-cells, normoglycemia in autoimmune Type-1 Diabetic Non-Obese Diabetic (NOD) mice without the use of immunosuppressive agents or encapsulation devices. Based on these observations, we are currently testing this Neo-Islet technology in an FDA guided pilot study (INAD 012–776) in insulin-dependent, spontaneously diabetic pet dogs by ultrasound-guided, intraperitoneal administration of 2x10e5 Neo-Islets/kilogram body weight to metabolically controlled (blood glucose, triglycerides, thyroid and adrenal functions) and sedated animals. We report here interim observations on the first 4 canine Neo-Islet-treated, insulin-dependent pet dogs that are now in the early to intermediate-term follow-up phase of the planned 3 year study (> 6 months post treatment). Current results from this translational study indicate that in dogs, Neo-Islets appear to engraft, redifferentiate and physiologically produce insulin, and are rejected by neither auto- nor allo-immune responses, as evidenced by (a) an absent IgG response to the allogeneic cells contained in the administered Neo-Islets, and (b) progressively improved glycemic control that achieves up to a 50% reduction in daily insulin needs paralleled by a statistically significant decrease in serum glucose concentrations. This is accomplished without the use of anti-rejection drugs or encapsulation devices. No adverse or serious adverse events related to the Neo-Islet administration have been observed to date. We conclude that this minimally invasive therapy has significant translational relevance to veterinary and clinical Type 1 diabetes mellitus by achieving complete and at this point partial glycemic control in two species, i.e., diabetic mice and dogs, respectively. [ABSTRACT FROM AUTHOR]

The Duffy blood group plays a key role in Plasmodium knowlesi and Plasmodium vivax invasion into human erythrocytes. The geographical distribution of the Duffy alleles differs between regions with the FY*A allele having high frequencies in many Asian populations, the FY*B allele is found predominately in European populations and the FY*Bes allele found predominantly in African regions. A previous study in Peninsular Malaysia indicated high homogeneity of the dominant FY*A/FY*A genotype. However, the distribution of the Duffy genotypes in Malaysian Borneo is currently unknown. In the present study, the distribution of Duffy blood group genotypes and allelic frequencies among P. knowlesi infected patients as well as healthy individuals in Malaysian Borneo were determined. A total of 79 P. knowlesi patient blood samples and 76 healthy donor samples were genotyped using allele specific polymerase chain reaction (ASP-PCR). Subsequently a P. knowlesi invasion assay was carried out on FY*AB/ FY*A and FY*A/ FY*A Duffy genotype blood to investigate if either genotype conferred increased susceptibility to P. knowlesi invasion. Our results show almost equal distribution between the homozygous FY*A/FY*A and heterozygous FY*A/FY*B genotypes. This is in stark contrast to the Duffy distribution in Peninsular Malaysia and the surrounding Southeast Asian region which is dominantly FY*A/FY*A. The mean percent invasion of FY*A/FY*A and FY*A/FY*B blood was not significantly different indicating that neither blood group confers increased susceptibility to P. knowlesi invasion. [ABSTRACT FROM AUTHOR]

Objectives: This systematic review aimed to evaluate the therapeutic effects and safety of Chinese herbal medicine (CHM) formulae for managing menopausal hot flushes (MHF). Methods: Seven English and Chinese databases were searched for studies from respective inceptions to February 2019. Randomized controlled trials investigating the clinical effects and safety of CHM formulae on MHF were considered for inclusion. The outcomes of subjective feelings (MHF and quality of life), objective changes (hormones and peripheral blood flow) and safety were analyzed. The most frequently prescribed formulae and herbs were summarized. Results: Nineteen randomized clinical trials involving 2469 patients were included. When compared to menopausal hormone therapy, CHM had similar effects to menopausal hormone therapy on total effectiveness rate (OR 1.41, 95% CI 0.84 to 2.35) and total Kupperman index (KI) score (SMD -0.13, 95% CI -0.61 to 0.36), and could significantly reduce vasomotor symptom score (MD -0.43, 95% CI -0.55 to -0.31) and upper-body peripheral blood flow (MD -3.56, 95% CI -5.14 to -1.98 under the jaw, MD -7.10, 95% CI -11.01 to -3.19 in the fingertip). When compared to placebo, CHM could reduce MHF severity (MD -0.70, 95% CI-1.00 to -0.40) and improve total KI score (MD -12.61, 95% CI -15.21 to -10.01). However, no statistically significant changes to hormone levels were detected. Most commonly seen adverse events were mild gastrointestinal tract reactions. The most popularly studied formula was Kun Tai capsule and the most frequently prescribed herb was Bai shao (Paeoniae Radix Alba, Paeonia lactiflora Pall.). More than 50% included studies had low risks of bias in the domains of selection, performance, attrition and reporting. Conclusions: This review indicated that CHM formulae were safe to be applied in MHF females and able to improve MHF-related symptom scores as well as the peripheral blood flow. Further studies should focus on specific formulae. [ABSTRACT FROM AUTHOR]

Since their appearance, humanized mice carrying human immune system seemed promising tools to study the crosstalk between cancer and immunity. The NOD-scidIL2Rgammanull (NSG) mice engrafted with human cord blood (hCB)-CD34+ cells have been proposed to be a valuable tool to reproduce human immune system in mouse. However, the lack of solid evidences on the functionality of their human immune components limits their usage in immune-oncology. We report that (hCB)-CD34+ cells lose their ability to propagate and originate bone marrow-derived human immune cells after two serial transplantations in NSG mice. We demonstrate that transplants of bone marrow patient-derived acute myeloid leukemias (hAMLs) grow very similarly in the humanized (hCB)-CD34+ NSG and parental NSG mice. The similar extent of engraftment and development of leukemias in (hCB)-CD34+ NSG and controls suggests a poor human immune response against not compatible hAMLs. Our findings suggest that (hCB)-CD34+ NSG mice are transient and/or incomplete carriers of the human immune system and, therefore, represent a suboptimal tool to study the interaction between tumor and immune cells. [ABSTRACT FROM AUTHOR]

Objective: To explore clinical factors associated with bacterial translocation in Japanese patients with type 2 diabetes mellitus (T2DM). Methods: The data of 118 patients with T2DM were obtained from two previous clinical studies, and were retrospectively analyzed regarding the clinical parameters associated with bacterial translocation defined as detection of bacteremia and levels of plasma lipopolysaccharide binding protein (LBP), the latter of which is thought to reflect inflammation caused by endotoxemia. Results: LBP level was not significantly different between patients with and without bacteremia. No clinical factors were significantly correlated with the detection of bacteremia. On the other hand, plasma LBP level was significantly correlated with HbA1c (r = 0.312), fasting blood glucose (r = 0.279), fasting C-peptide (r = 0.265), body mass index (r = 0.371), high-density lipoprotein cholesterol (r = -0.241), and inflammatory markers (high-sensitivity C-reactive protein, r = 0.543; and interleukin-6, r = 0.456). Multiple regression analysis identified body mass index, HbA1c, high-sensitivity C-reactive protein, and interleukin-6 as independent determinants of plasma LBP level. Conclusion: The plasma LBP level was similar in patients with and without bacteremia. While both bacteremia and LBP are theoretically associated with bacterial translocation, the detection of bacteremia was not associated with LBP level in T2DM. [ABSTRACT FROM AUTHOR]

Second-generation mammalian target of rapamycin (mTOR) inhibitors such as CC-223 may have theoretical advantages over first-generation drugs by inhibiting TOR kinase in mTOR complex 1 (mTORC1) and 2 (mTORC2), potentially improving clinical efficacy for well-differentiated neuroendocrine tumors (NET).Enrolled patients had metastatic, well-differentiated NET of non-pancreatic gastrointestinal or unknown origin, with/without carcinoid symptoms, had failed ≥1 systemic chemotherapy, and were taking a somatostatin analog (SSA). Oral once-daily CC-223 was administered in 28-day cycles starting at 45 mg (n = 24), with a subsequent cohort starting at 30 mg (n = 23). Objectives were to evaluate tolerability, preliminary efficacy, and pharmacokinetic and biomarker profiles of CC-223. Forty-seven patients completed the study, with mean treatment duration of 378 days and mean dose of 26 mg; 26% of patients remained on the starting dose. Most frequent grade ≥3 toxicities were diarrhea (38%), fatigue (21%), and stomatitis (11%). By investigator, 3 of 41 evaluable patients (7%) showed partial response (PR) and 34 (83%) had stable disease (SD) for a disease control rate (DCR) of 90% (95% confidence interval [CI] 76.9–97.3%). Duration of PR was 125–401 days; median SD duration was 297 days (min–max, 50–1519 days). Median progression-free survival was 19.5 months (95% CI 10.4–28.5 months). Carcinoid symptoms of flushing, diarrhea, or both improved in 50%, 41%, and 39% of affected patients, respectively. For the first time, this study describes that a second-generation mTOR pathway inhibitor can result in highly durable tumor regression and control of NET carcinoid symptoms. The manageable safety profile, high DCR, and durable response, coupled with reduction in carcinoid symptoms refractory to SSA, make CC-223 a promising agent for further development. [ABSTRACT FROM AUTHOR]

This manuscript emerged from a larger third-party funded project investigating a new poly-trauma model and its influence upon secondary sepsis. The present sub-study compared selected leukocyte subpopulations in the circulation and bone marrow after polytrauma in BALB/c versus CD-1 mice. Animals underwent unilateral femur fracture, splenectomy and hemorrhagic shock. We collected blood and bone marrow for flow cytometry analysis at 24h and 48h post-trauma. Circulating granulocytes (Ly6G+CD11+) increased in both strains after trauma. Only in BALB/c mice circulating CD8+ T-lymphocytes decreased within 48h by 30%. Regulatory T-cells (Tregs, CD4+CD25+CD127low) increased in both strains by approx. 32%. Circulating Tregs and lymphocytes (CD11b-Ly6G-MHC-2+) were always at least 1.5-fold higher in BALB/c, while the bone marrow MHC-2 expression decreased in CD-1 mice (p<0.05). Overall, immune responses to polytrauma were similar in both strains. Additionally, BALB/c expressed higher level of circulating regulatory T-cells and MHC-2-positive lymphocytes compared to CD-1 mice. [ABSTRACT FROM AUTHOR]

Rhesus macaque is an important animal model in biomedical research, especially human disease, developmental, translational, and pre-clinical research. Blood physiological and biochemical parameters are important markers for physiology, pathology, and toxicology research. However, these parameters have not been systematically reported for Chinese rhesus macaques. To characterize the reference for these parameters, this study collected 1805 Chinese rhesus macaques living in Southwestern China. A total of 24 blood physiological indexes and 27 biochemical parameters were determined. Sex and age were found to affect these parameters. In conclusion, a comprehensive and systematic reference of hematological and biochemical parameters for Chinese rhesus macaque was established in this work on the basis of a large cohort. Such reference will benefit biomedical research employing rhesus macaques as animal models. [ABSTRACT FROM AUTHOR]

Congenital cytomegalovirus (CMV) infection is a major cause of birth defects ranging from developmental disorders to stillbirth. Most newborns affected by CMV do not present with symptoms at birth but are at risk of sequelae at later stages of their childhood. Stored dried blood spots (DBS) taken at birth can be used for retrospective diagnosis of hereditary diseases, but detection of pathogens is challenged by potentially low pathogen concentrations in the small blood volume available in a DBS. Here we test four different extraction methods for optimal recovery of CMV DNA from DBS at low to high CMV titers. The recovery efficiencies varied widely between the different extractions (from 3% to 100%) with the most efficient method extracting up to 113-fold more CMV DNA than the least efficient and 8-fold more than the reference protocol. Furthermore, we amplified four immunomodulatory CMV genes from the extracted DNA: the UL40 and UL111A genes which occur as functional knockouts in some circulating CMV strains, and the highly variable UL146 and US28 genes. The PCRs specifically amplified the CMV genes at all tested titers with sufficient quality for sequencing and genotyping. In summary, we here report an extraction method for optimal recovery of CMV DNA from DBSs that can be used for both detection of CMV and for genotyping of polymorphic CMV genes in congenital CMV infection. [ABSTRACT FROM AUTHOR]

Prions cause transmissible infectious diseases in humans and animals and have been found to be transmissible by blood transfusion even in the presymptomatic stage. However, the concentration of prions in body fluids such as blood and urine is extremely low; therefore, direct diagnostic tests on such specimens often yield false-negative results. Quantitative preanalytical prion enrichment may significantly improve the sensitivity of prion assays by concentrating trace amounts of prions from large volumes of body fluids. Here, we show that beads conjugated to positively charged peptoids not only captured PrP aggregates from plasma of prion-infected hamsters, but also adsorbed prion infectivity in both the symptomatic and preclinical stages of the disease. Bead absorbed prion infectivity efficiently transmitted disease to transgenic indicator mice. We found that the readout of the peptoid-based misfolded protein assay (MPA) correlates closely with prion infectivity in vivo, thereby validating the MPA as a simple, quantitative, and sensitive surrogate indicator of the presence of prions. The reliable and sensitive detection of prions in plasma will enable a wide variety of applications in basic prion research and diagnostics. [ABSTRACT FROM AUTHOR]

Background: Tyrosine kinase inhibitors targeted to anaplastic lymphoma kinase (ALK) have been demonstrated to be effective for lung cancer patients with an ALK fusion gene. Application of liquid biopsy, i.e., detection and quantitation of the fusion product in plasma cell-free DNA (cfDNA), could improve clinical practice. To detect ALK fusions, because fusion breakpoints occur somewhere in intron 19 of the ALK gene, sequencing of the entire intron is required to locate breakpoints. Results: We constructed a target sequencing system using an adapter and a set of primers that cover the entire ALK intron 19. This system can amplify fragments, including breakpoints, regardless of fusion partners. The data analysis pipeline firstly detected fusions by alignment to selected target sequences, and then quantitated the fusion alleles aligning to the identified breakpoint sequences. Performance was validated using 20 cfDNA samples from ALK-positive non-small cell lung cancer patients and samples from 10 healthy volunteers. Sensitivity and specificity were 50 and 100%, respectively. Conclusions: We demonstrated that PCR-based target sequencing using a tiling primer set and two-step mapping/alignment quantitatively detected ALK fusions in cfDNA from lung cancer patients. The system offers an alternative to existing approaches based on hybridization capture. [ABSTRACT FROM AUTHOR]

Bloodstream infections (BSI) are associated with high mortality. Therefore, reliable methods of detection are of paramount importance. Efficient strategies to improve diagnostic yield of bacteraemia within the emergency department (ED) are needed. We conducted a retrospective analysis of all ED encounters in a high-volume, city-centre university hospital within Germany during a five-year study period from October 2013 to September 2018. A time-series analysis was conducted for all ED encounters in which blood cultures (BCs) were collected. BC detection rates and diagnostic yield of community-onset bacteraemia were compared during the study period (which included 45 months prior to the start of a new diagnostic Antibiotic Stewardship (ABS) bundle and 15 months following its implementation). BCs were obtained from 5,191 out of 66,879 ED admissions (7.8%). Bacteraemia was detected in 1,013 encounters (19.5% of encounters where BCs were obtained). The overall yield of true bacteraemia (defined as yielding clinically relevant pathogens) was 14.4%. The new ABS-related diagnostic protocol resulted in an increased number of hospitalised patients with BCs collected in the ED (18% compared to 12.3%) and a significant increase in patients with two or more BC sets taken (59% compared to 25.4%), which resulted in an improved detection rate of true bacteraemia (2.5% versus 1.8% of hospital admissions) without any decrease in diagnostic yield. This simultaneous increase in BC rates without degradation of yield was a valuable finding that indicated success of this strategy. Thus, implementation of the new diagnostic ABS bundle within the ED, which included the presence of a skilled infectious disease (ID) team focused on obtaining BCs, appeared to be a valuable tool for the accurate and timely detection of community-onset bacteraemia. [ABSTRACT FROM AUTHOR]

Background: It is not clear whether the configuration of the posterior part of the circle of Willis (CW) depends on the proximal part of the vertebrobasilar system. Our aim is to evaluate the posterior part of CW in association with different size of vertebral arteries (VA) in healthy volunteers. Materials and methods: The present study was based on a sample of 923 healthy volunteers who were examined from 2013 through 2018. The duplex ultrasonographic examination of the extracranial vertebral (VA) and carotid arteries was performed. VA was defined as hypoplastic (VAH) when VA diameter in the entire course was less than 2.5 mm. All the participants underwent magnetic resonance angiography (MRA) examination. All the component vessels of the circle of Willis were assessed in each individual. We classified the posterior communicating artery (PCoA) as presence PCoA, absence/hypoplastic PCoA and fetal-type posterior circle of Willis (FCW) in which the major stem of the posterior cerebral artery (PCA) arises from ipsilateral internal carotid artery (ICA). The comparison of the posterior part of CW was made in subjects with normal VA and VAH of a different degree (communicating with basilar artery (VAH-BA) and not communicating with the basilar artery (VAH-PICA)). Results: FCW was found in 15.9% of subjects, bilaterally–in 2.3%. FCW was more frequent in individuals with VAH than in those with normal VA (accordingly, 28.8% vs. 13.5%, p<0.001. Moreover FCW was recorded in 50% of the subjects with VA—PICA in comparison with 13.5% of those with normal VA and 22.8% with VAH—BA, p<0.005. On the contrary, absence/hypoplasia of both PCoA was mostly found in the group with normal VA in comparison with VAH-BA and VAH-PICA (accordingly, 50.7%, 38.6% and 12.5%, p<0.01). Conclusion: Individuals with VAH have a different pattern of the posterior part of CW in comparison with those with normal VA. With the increasing degree of VAH, the proportion of FCW increases, while the proportion of absence/hypoplastic of both PCoA decreases. [ABSTRACT FROM AUTHOR]

Background: Identification of blood sources of hematophagous arthropods is crucial for understanding the transmission cycles of vector-borne diseases. Many different approaches towards host determination were proposed, including precipitin test, ELISA, DNA- and mass spectrometry-based methods; yet all face certain complications and limitations, mostly related to blood degradation. This study presents a novel method for blood meal identification, peptide mass mapping (PMM) analysis of host-specific hemoglobin peptides using MALDI-TOF mass spectrometry. Methodology/Principal findings: To identify blood meal source, proteins from abdomens of engorged sand fly females were extracted, cleaved by trypsin and peptide fragments of host hemoglobin were sequenced using MALDI-TOF MS. The method provided correct host identification of 100% experimentally fed sand flies until 36h post blood meal (PBM) and for 80% samples even 48h PBM. In females fed on two hosts, both blood meal sources were correctly assigned for 60% of specimens until 36h PBM. In a validation study on field-collected females, the method yielded unambiguous host determination for 96% of specimens. The suitability of PMM-based MALDI-TOF MS was proven experimentally also on lab-reared Culex mosquitoes. Conclusions/Significance: PMM-based MALDI-TOF MS analysis targeting host specific hemoglobin peptides represents a sensitive and cost-effective method with a fast and simple preparation protocol. As demonstrated here on phlebotomine sand flies and mosquitoes, it allows reliable and rapid blood source determination even 48h PBM with minimal material input and provides more robust and specific results than other currently used methods. This approach was also successfully tested on field-caught engorged females and proved to be a promising useful tool for large-scale screening of host preferences studies. Unlike other methods including MALDI-TOF protein profiling, it allows correct identification of mixed blood meals as was demonstrated on both experimentally fed and field-collected sand flies. [ABSTRACT FROM AUTHOR]

In the last decade a growing HIV/AIDS epidemic with increased incidence and AIDS-related mortality has been reported in Northern Brazil from which molecular data are scarce. Also, apparently healthy, adult blood donors, recently diagnosed with HIV-1 represent important sentinel populations for molecular studies. This cross-sectional study describes HIV-1 subtypes in blood donors from three reference public blood centers located in three States in Northern Brazil. HIV-1 pol sequencing (protease/PR, reverse transcriptase/RT) was performed on plasma samples of HIV-1 positive donors from HEMOAM, Manaus, Amazonas (n = 198), HEMERON, Porto Velho, Rondônia (n = 20) and HEMORAIMA, Boa Vista, Roraima (n = 9) collected from 2011–2017. HIV-1 subtypes were identified by REGA, phylogenetic inference; recombinant viruses were characterized by SIMPLOT. Young, single, males predominated, around half was first-time donors. Syphilis co-infection was detected in 17% (39 out of 227), 8% (18 out of 227) was anti-HBc positive. Subtype B represented ≥ 90% in Amazonas, Rondônia and Roraima, subtype C (3.1%) was found in Amazonas and Rondônia; subtype F1 (0.9%) and BF1 recombinants (5.3%) were only detected in Amazonas. Subtype B sequences from Amazonas (n = 179), Rondônia (n = 18) and Roraima (n = 9) were combined with viral strains representative of the BPANDEMIC (n = 300) and BCARIBBEAN/BCAR (n = 200) lineages. The BPANDEMIC lineage predominated (78%) although BCAR lineages were frequent in Roraima (56%) and Amazonas (22%). Subtype C and subtype F1 sequences identified here clustered within Brazilian CBR and F1BR lineages, respectively. Twelve BF1 mosaics showed 11 different recombination profiles: six were singleton unique-recombinant-forms/URFs, one displays a CRF28/29_BF-like recombinant pattern and the remaining four BF1 isolates branched with other Brazilian BF1 viruses previously described and may represent putative new CRF_BF1 from Northern Brazil. Our study shows a highly homogeneous molecular pattern with prevalent subtype B, followed by BF1, and sporadic subtype C and F1 in blood donors from the Northern region. Surveillance studies are important to monitor HIV-1 diversity which can reveal patterns of viral dissemination, especially in a highly endemic, remote and geographically isolated region as Northern Brazil. [ABSTRACT FROM AUTHOR]

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is the primary cause of acute, highly fatal, hemorrhagic diseases in young Asian elephants. Although monocytopenia is frequently observed in EEHV-HD cases, the role monocytes play in EEHV-disease pathogenesis is unknown. This study seeks to explain the responses of monocytes/macrophages in the pathogenesis of EEHV-HD. Samples of blood, frozen tissues, and formalin-fixed, paraffin-embedded (FFPE) tissues from EEHV1A-HD, EEHV4-HD, co-infected EEHV1A and 4-HD, and EEHV-negative calves were analyzed. Peripheral blood mononuclear cells (PBMCs) from the persistent EEHV4-infected and EEHV-negative calves were also studied. The results showed increased infiltration of Iba-1-positive macrophages in the inflamed tissues of the internal organs of elephant calves with EEHV-HD. In addition, cellular apoptosis also increased in the tissues of elephants with EEHV-HD, especially in the PBMCs, compared to the EEHV-negative control. In the PBMCs of persistent EEHV4-infected elephants, cytokine mRNA expression was high, particularly up-regulation of TNF-α and IFN-γ. Moreover, viral particles were observed in the cytoplasm of the persistent EEHV4-infected elephant monocytes. Our study demonstrated for the first time that apoptosis of the PBMCs increased in cases of EEHV-HD. Furthermore, this study showed that monocytes may serve as a vehicle for viral dissemination during EEHV infection in Asian elephants. [ABSTRACT FROM AUTHOR]

Inherited bleeding disorders including abnormalities of platelet number and function rarely occur in a variety of dog breeds, but are probably underdiagnosed. Genetically characterized canine forms of platelet disorders provide valuable large animal models for understanding similar platelet disorders in people. Breed-specific disease associated genetic variants in only eight different genes are known to cause intrinsic platelet disorders in dogs. However, the causative genetic variant in many dog breeds has until now remained unknown. Four cases of a mild to severe bleeding disorder in Cocker Spaniel dogs are herein presented. The affected dogs showed a platelet adhesion defect characterized by macrothrombocytopenia with variable platelet counts resembling human Bernard-Soulier syndrome (BSS). Furthermore, the lack of functional GPIb-IX-V was demonstrated by immunocytochemistry. Whole genome sequencing of one affected dog and visual inspection of the candidate genes identified a deletion in the glycoprotein IX platelet (GP9) gene. The GP9 gene encodes a subunit of a platelet surface membrane glycoprotein complex; this functions as a receptor for von Willebrand factor, which initiates the maintenance of hemostasis after injury. Variants in human GP9 are associated with Bernard-Soulier syndrome, type C. The deletion spanned 2460 bp, and included a significant part of the single coding exon of the canine GP9 gene on dog chromosome 20. The variant results in a frameshift and premature stop codon which is predicted to truncate almost two-thirds of the encoded protein. PCR-based genotyping confirmed recessive inheritance. The homozygous variant genotype seen in affected dogs did not occur in 98 control Cocker Spaniels. Thus, it was concluded that the structural variant identified in the GP9 gene was most likely causative for the BSS-phenotype in the dogs examined. These findings provide the first large animal GP9 model for this group of inherited platelet disorders and greatly facilitate the diagnosis and identification of affected and/or normal carriers in Cocker Spaniels. [ABSTRACT FROM AUTHOR]

Cord blood (CB) mononuclear cells (MNC) are being tested in clinical trials to treat hypoxic-ischemic (HI) brain injuries. Although early results are encouraging, mechanisms underlying potential clinical benefits are not well understood. To explore these mechanisms further, we exposed mouse brain organotypic slice cultures to oxygen and glucose deprivation (OGD) and then treated the brain slices with cells from CB or adult peripheral blood (PB). We found that CB-MNCs protect neurons from OGD-induced death and reduced both microglial and astrocyte activation. PB-MNC failed to affect either outcome. The protective activities were largely mediated by factors secreted by CB-MNC, as direct cell-to-cell contact between the injured brain slices and CB cells was not essential. To determine if a specific subpopulation of CB-MNC are responsible for these protective activities, we depleted CB-MNC of various cell types and found that only removal of CB CD14+ monocytes abolished neuroprotection. We also used positively selected subpopulations of CB-MNC and PB-MNC in this assay and demonstrated that purified CB-CD14+ cells, but not CB-PB CD14+ cells, efficiently protected neuronal cells from death and reduced glial activation following OGD. Gene expression microarray analysis demonstrated that compared to PB-CD14+ monocytes, CB-CD14+ monocytes over-expressed several secreted proteins with potential to protect neurons. Differential expression of five candidate effector molecules, chitinase 3-like protein-1, inhibin-A, interleukin-10, matrix metalloproteinase-9 and thrombospondin-1, were confirmed by western blotting, and immunofluorescence. These findings suggest that CD14+ monocytes are a critical cell-type when treating HI with CB-MNC. [ABSTRACT FROM AUTHOR]

Lung disease is regularly reported in human filarial infections but the molecular pathogenesis of pulmonary filariasis is poorly understood. We used Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity responsible for pleural inflammation, to model responses to human filarial infections and probe the mechanisms. Wild-type and Th2-deficient mice (ΔdblGata1 and Il-4receptor(r)a-/-/IL-5-/-) were infected with L. sigmodontis. Survival and growth of adult filariae and prevalence and density of microfilariae were evaluated. Cells and cytokines in the pleural cavity and bronchoalveolar space were characterized by imaging, flow cytometry and ELISA. Inflammatory pathways were evaluated by transcriptomic microarrays and lungs were isolated and analyzed for histopathological signatures. 40% of WT mice were amicrofilaremic whereas almost all mutant mice display blood microfilaremia. Microfilariae induced pleural, bronchoalveolar and lung-tissue inflammation associated with an increase in bronchoalveolar eosinophils and perivascular macrophages, production of mucus, visceral pleura alterations and fibrosis. Inflammation and pathology were decreased in Th2-deficient mice. An IL-4R-dependent increase of CD169 was observed on pleural and bronchoalveolar macrophages in microfilaremic mice. CD169+ tissue-resident macrophages were identified in the lungs with specific localizations. Strikingly, CD169+ macrophages increased significantly in the perivascular area in microfilaremic mice. These data describe lung inflammation and pathology in chronic filariasis and emphasize the role of Th2 responses according to the presence of microfilariae. It is also the first report implicating CD169+ lung macrophages in response to a Nematode infection. [ABSTRACT FROM AUTHOR]

Chronic atherosclerosis may cause cerebral hypoperfusion and inadequate brain oxygenation, contributing to the progression of cognitive decline. In this study, we exploited two-photon phosphorescence lifetime microscopy to measure the absolute partial pressure of oxygen (PO2) in cortical tissue in both young and old LDLR-/-, hApoB100+/+ mice, spontaneously developing atherosclerosis with age. Capillary red-blood-cell (RBC) speed, flux, hematocrit and capillary diameter were also measured by two-photon imaging of FITC-labelled blood plasma. Our results show positive correlations between RBC speed, flux, diameter and capillary-adjacent tissue PO2. When compared to the young mice, we observed lower tissue PO2, lower RBC speed and flux, and smaller capillary diameter in the old atherosclerotic mice. The old mice also exhibited a higher spatial heterogeneity of tissue PO2, and RBC speed and flux, suggesting a less efficient oxygen extraction. [ABSTRACT FROM AUTHOR]

Background: Large numbers of young people die yearly due to homicide, but little is known about homicide during adolescence. Research primarily focuses on youth violence among young men and masks important gender-related factors inherent in the adolescent age group. Although young women are less likely to be victims of homicide, violence against women is an important form of violence experienced during adolescence. In this paper, we describe the prevalence of and gender difference in adolescent homicide in South Africa in 2009. Methods: We conducted a retrospective national mortuary-based study to identify all child homicides (boys and girls < 18 years) in 2009 in a proportionate sample of mortuaries. Victim, perpetrator and crime data were collected in three phases: cases were identified from the mortuary register, the autopsy report and from police interviews. In this paper we focus on the adolescent group, aged 10 to 17 years. Findings: We identified 674 (95% CI: 520–823) adolescent homicides for 2009, with more male (520) than female (154) homicides. This gender disparity increased as children aged, with the male homicide rate 27.9/100 000 population (95% CI: 20.3–35.5), nearly 5 times the female rate (4.5:1) of 6.5/ 100 000 population (95% CI: 4.7–8.2) in older adolescents (15–17 year old). Adolescent males were significantly more likely (61.2%) to die in a public space compared to female adolescents (39.3%) but more adolescent females (48.4%) died at home compared to adolescent males (32.9%). Adolescent females (42.1%) were mainly killed by a family member or intimate partner while adolescent males were predominantly (58%) killed by an acquaintance. Conclusion: We found a distinct gender pattern for adolescent homicide in South Africa. This pattern appears to be driven by gender norms that support violence. South Africa requires an investment in developing evidence informed interventions to reduce violence. [ABSTRACT FROM AUTHOR]

For endovascular research pigs are an established animal model. However, experiences regarding analyses of platelet inhibition in pigs using the Multiplate® Analyzer are limited. The aims of the present study were to investigate if (1) the Multiplate® Analyzer is a suitable method for examination of porcine platelet function using manufacturers’ recommendations for human blood, and (2) platelet inhibition can be induced with acetylsalicylic acid (ASA) and clopidogrel in pigs reliably, and if (3) non-responders to one of the drug can be detected. Additionally we examined differences in (4) the effectiveness of ASA between oral administration and intravenous application, and (5) between domestic pigs (German Landrace; GL) and miniature pigs (MP). We investigated platelet function of 36 unmedicated pigs (GL n = 28; MP n = 8). In addition, 32 blood samples taken from medicated pigs (GL n = 15; MP n = 17) were analysed. Platelet inhibition was induced in four different ways: (1) 500 mg ASA intravenously (n = 11), (2) 500 mg ASA intravenously and 450 mg clopidogrel orally (n = 5), (3) 250 mg ASA orally (n = 11), (4) 250 mg ASA orally and 75 mg clopidogrel orally (n = 5). Results of the ASPI and ADP test of the Multiplate® Analyzer subtests in unmedicated and medicated pigs were in a comparable range to results known from humans. Application of ASA decreased the mean values of the ASPI test significantly regardless of the application method. Joined administration of ASA and clopidogrel also decreased the mean values of the ADP test significantly. Both, oral and intravenous administrations of ASA as well as oral administration of clopidogrel effectively inhibited platelet function in pigs. One pig did not respond to clopidogrel. We found no differences between domestic and miniature pigs regarding reference values in unmedicated pigs and the effectiveness of ASA and clopidogrel. [ABSTRACT FROM AUTHOR]

Intrarenal Doppler ultrasonography is a non-invasive method to evaluate the renal blood flow in patients with renal arterial stenosis as well as chronic kidney diseases (CKD). Until recently, the relationship between ultrasonography findings and CKD stage has not been fully understood. Overall, 162 patients with CKD without apparent renal arterial stenosis were included in this study, and the pulsed-wave Doppler ultrasonography findings were evaluated in terms of the following parameters: peak systolic velocity (PSV), end-diastolic velocity (EDV), and resistive index (RI) at the renal arterial trunk, hilum, segmental, and interlobar regions. Age showed a significant negative correlation with the estimated glomerular filtration rate (eGFR), kidney size, and aortic PSV. Additionally, age showed a significant positive correlation with RI in all 4 regions. The eGFR showed a positive correlation with the aortic PSV and kidney size, but a negative correlation with RI. Both age and eGFR were found to be independently associated with aortic blood flow. On the intrarenal ultrasound, EDV and RI showed stronger correlations with eGFR than PSV, suggesting that the former indices would be better markers of renal function. In particular, the interlobar EDV was found to be the best index that reflects renal function. Although the RI is also a good marker of renal function, it is confounded by age; thus, its utility would be weaker than that of the EDV. In conclusion, intrarenal pulsed-wave Doppler ultrasonography is a useful tool to estimate and evaluate the renal function; the interlobar EDV may be the best index to estimate the effective perfusion and filtration of the kidneys. [ABSTRACT FROM AUTHOR]

Background: Histoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly due to delayed diagnosis, caused by slow fungal growth in culture. Therefore, a fast, suitable and cost-effective assay is required for the diagnosis of histoplasmosis in resource-limited laboratories. This study aimed to develop and evaluate two new molecular approaches for a more cost-effective diagnosis of histoplasmosis. Methodology: Seeking a fast, suitable, sensitive, specific and low-cost molecular detection technique, we developed a new Loop-mediated Isothermal Amplification (LAMP) assay and nested PCR, both targeting the Internal Transcribed Spacer (ITS) multicopy region of Histoplasma capsulatum. The sensitivity was evaluated using 26 bone marrow and 1 whole blood specimens from patients suspected to have histoplasmosis and 5 whole blood samples from healthy subjects. All specimens were evaluated in culture, as a reference standard test, and Hcp100 nPCR, as a molecular reference test. A heparin-containing whole blood sample from a heathy subject was spiked with H. capsulatum cells and directly assayed with no previous DNA extraction. Results: Both assays were able to detect down to 1 fg/μL of H. capsulatum DNA, and ITS LAMP results could also be revealed to the naked-eye by adding SYBR green to the reaction tube. In addition, both assays were able to detect all clades of Histoplasma capsulatum cryptic species complex. No cross-reaction with other fungal pathogens was presented. In comparison with Hcp100 nPCR, both assays reached 83% sensitivity and 92% specificity. Furthermore, ITS LAMP assay showed no need for DNA extraction, since it could be directly applied to crude whole blood specimens, with a limit of detection of 10 yeasts/μL. Conclusion: ITS LAMP and nPCR assays have the potential to be used in conjunction with culture for early diagnosis of progressive disseminated histoplasmosis, allowing earlier, appropriate treatment of the patient. The possibility of applying ITS LAMP, as a direct assay, with no DNA extraction and purification steps, makes it suitable for resource-limited laboratories. However, more studies are necessary to validate ITS LAMP and nPCR as direct assay in other types of clinical specimens. [ABSTRACT FROM AUTHOR]