Detection of ESKAPE bacterial pathogens at the point of care using isothermal DNA-based assays in a portable degas-actuated microfluidic diagnostic assay platform

An estimated 1.5 billion microbial infections occur globally each year and result in ~4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridgebased system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuumdegassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/ reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ~10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting

Influenza Vaccination of Healthcare Workers and Vaccine Allocation for Healthcare Workers During Vaccine Shortages

Abstract Influenza causes substantial morbidity and mortality annually, particularly in high-risk groups such as the elderly, young children, immunosuppressed individuals, and individuals with chronic illnesses. Healthcare-associated transmission of influenza contributes to this burden but is often under-recognized except in the setting of large outbreaks. The Centers for Disease Control and Prevention has recommended annual influenza vaccination for healthcare workers (HCWs) with direct patient contact since 1984 and for all HCWs since 1993. The rationale for these recommendations is to reduce the chance that HCWs serve as vectors for healthcare-associated influenza due to their close contact with high-risk patients and to enhance both HCW and patient safety. Despite these recommendations as well as the effectiveness of interventions designed to increase HCW vaccination rates, the percentage of HCWs vaccinated annually remains unacceptably low. Ironically, at the same time that campaigns have sought to increase HCW vaccination rates, vaccine shortages, such as the shortage during the 2004-2005 influenza season, present challenges regarding allocation of available vaccine supplies to both patients and HCWs. This two-part document outlines the position of the Society for Healthcare Epidemiology of America on influenza vaccination for HCWs and provides guidance for the allocation of influenza vaccine to HCWs during a vaccine shortage based on influenza transmission routes and the essential need for a practical and adaptive strategy for allocation. These recommendations apply to all types of healthcare facilities, including acute care hospitals, long-term-care facilities, and ambulatory care settings

The predictive and prognostic potential of plasma telomerase reverse transcriptase (TERT) RNA in rectal cancer patients

Background: Preoperative chemoradiotherapy (CRT) followed by surgery is the standard care for locally advanced rectal cancer, but tumour response to CRT and disease outcome are variable. The current study aimed to investigate the effectiveness of plasma telomerase reverse transcriptase (TERT) levels in predicting tumour response and clinical outcome. Methods: 176 rectal cancer patients were included. Plasma samples were collected at baseline (before CRT\ubcT0), 2 weeks after CRT was initiated (T1), post-CRT and before surgery (T2), and 4\u20138 months after surgery (T3) time points. Plasma TERT mRNA levels and total cell-free RNA were determined using real-time PCR. Results: Plasma levels of TERT were significantly lower at T2 (Po0.0001) in responders than in non-responders. Post-CRT TERT levels and the differences between pre- and post-CRT TERT levels independently predicted tumour response, and the prediction model had an area under curve of 0.80 (95% confidence interval (CI) 0.73\u20130.87). Multiple analysis demonstrated that patients with detectable TERT levels at T2 and T3 time points had a risk of disease progression 2.13 (95% CI 1.10\u20134.11)-fold and 4.55 (95% CI 1.48\u201313.95)-fold higher, respectively, than those with undetectable plasma TERT levels. Conclusions: Plasma TERT levels are independent markers of tumour response and are prognostic of disease progression in rectal cancer patients who undergo neoadjuvant therapy

Antimicrobial sensing coupled with cell membrane remodeling mediates antibiotic resistance and virulence in Enterococcus faecalis.

Bacteria have developed several evolutionary strategies to protect their cell membranes (CMs) from the attack of antibiotics and antimicrobial peptides (AMPs) produced by the innate immune system, including remodeling of phospholipid content and localization. Multidrug-resistant Enterococcus faecalis, an opportunistic human pathogen, evolves resistance to the lipopeptide daptomycin and AMPs by diverting the antibiotic away from critical septal targets using CM anionic phospholipid redistribution. The LiaFSR stress response system regulates this CM remodeling via the LiaR response regulator by a previously unknown mechanism. Here, we characterize a LiaR-regulated protein, LiaX, that senses daptomycin or AMPs and triggers protective CM remodeling. LiaX is surface exposed, and in daptomycin-resistant clinical strains, both LiaX and the N-terminal domain alone are released into the extracellular milieu. The N-terminal domain of LiaX binds daptomycin and AMPs (such as human LL-37) and functions as an extracellular sentinel that activates the cell envelope stress response. The C-terminal domain of LiaX plays a role in inhibiting the LiaFSR system, and when this domain is absent, it leads to activation of anionic phospholipid redistribution. Strains that exhibit LiaX-mediated CM remodeling and AMP resistance show enhanced virulence in the Caenorhabditis elegans model, an effect that is abolished in animals lacking an innate immune pathway crucial for producing AMPs. In conclusion, we report a mechanism of antibiotic and AMP resistance that couples bacterial stress sensing to major changes in CM architecture, ultimately also affecting host-pathogen interactions

PTPN11 mosaicism causes a spectrum of pigmentary and vascular neurocutaneous disorders and predisposes to melanoma

Phakomatosis pigmentovascularis (PPV) is a diagnosis which denotes the coexistence of pigmentary and vascular birthmarks of specific types, accompanied by variable multisystem involvement including central nervous system disease, asymmetrical growth and a predisposition to malignancy. Using a tightly phenotyped group and high depth next generation sequencing of affected tissues we discover here clonal mosaic variants in gene PTPN11 encoding SHP2 phosphatase as a cause of PPV type III or spilorosea. Within an individual the same variant is found in distinct pigmentary and vascular birthmarks and is undetectable in blood. We go on to demonstrate that the same variants can cause either the specific pigmentary or vascular phenotypes alone, as well as driving melanoma development within the pigmentary lesion. Protein conformational modelling highlights that while variants lead to loss of function at the level of the phosphatase domain, resultant conformational changes promote longer ligand binding. In vitro modelling of the missense variants confirms downstream MAPK pathway overactivation, and widespread disruption of human endothelial cell angiogenesis. Importantly, PTPN11-mosaic patients theoretically risk passing on the variant to their children as the germline RASopathy Noonan syndrome with lentigines. These findings improve our understanding of the pathogenesis and biology of naevus spilus and capillary malformation syndromes, paving the way for better clinical management

The state of the Martian climate

60°N was +2.0°C, relative to the 1981–2010 average value (Fig. 5.1). This marks a new high for the record. The average annual surface air temperature (SAT) anomaly for 2016 for land stations north of starting in 1900, and is a significant increase over the previous highest value of +1.2°C, which was observed in 2007, 2011, and 2015. Average global annual temperatures also showed record values in 2015 and 2016. Currently, the Arctic is warming at more than twice the rate of lower latitudes

BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration

BRF1 is a rate-limiting factor for RNA Polymerase III-mediated transcription and is elevated in numerous cancers. Here, we report that elevated levels of BRF1 associate with poor prognosis in human prostate cancer. In vitro studies in human prostate cancer cell lines demonstrated that transient overexpression of BRF1 increased cell proliferation whereas the transient downregulation of BRF1 reduced proliferation and mediated cell cycle arrest. Consistent with our clinical observations, BRF1 overexpression in a Pten-deficient mouse (Pten BRF1 ) prostate cancer model accelerated prostate carcinogenesis and shortened survival. In Pten BRF1 tumours, immune and inflammatory processes were altered, with reduced tumoral infiltration of neutrophils and CD4 positive T cells, which can be explained by decreased levels of complement factor D (CFD) and C7 components of the complement cascade, an innate immune pathway that influences the adaptive immune response. We tested if the secretome was involved in BRF1-driven tumorigenesis. Unbiased proteomic analysis on BRF1-overexpresing PC3 cells confirmed reduced levels of CFD in the secretome, implicating the complement system in prostate carcinogenesis. We further identify that expression of C7 significantly correlates with expression of CD4 and has the potential to alter clinical outcome in human prostate cancer, where low levels of C7 associate with poorer prognosis

Activation Status of Wnt/ß-Catenin Signaling in Normal and Neoplastic Breast Tissues: Relationship to HER2/neu Expression in Human and Mouse

Wnt/ß-catenin signaling is strongly implicated in neoplasia, but the role of this pathway in human breast cancer has been controversial. Here, we examined Wnt/ß-catenin pathway activation as a function of breast cancer progression, and tested for a relationship with HER2/neu expression, using a human tissue microarray comprising benign breast tissues, ductal carcinoma in situ (DCIS), and invasive carcinomas. Cores were scored for membranous ß-catenin, a key functional component of adherens junctions, and for nucleocytoplasmic ß-catenin, a hallmark of Wnt/ß-catenin pathway activation. Only 82% of benign samples exhibited membrane-associated ß-catenin, indicating a finite frequency of false-negative staining. The frequency of membrane positivity was similar in DCIS samples, but was significantly reduced in carcinomas (45%, P<0.001), consistent with loss of adherens junctions during acquisition of invasiveness. Negative membrane status in cancers correlated with higher grade (P = 0.04) and estrogen receptor-negative status (P = 0.03), both indices of poor prognosis. Unexpectedly, a substantial frequency of nucleocytoplasmic ß-catenin was observed in benign breast tissues (36%), similar to that in carcinomas (35%). Positive-staining basal nuclei observed in benign breast may identify putative stem cells. An increased frequency of nucleocytoplasmic ß-catenin was observed in DCIS tumors (56%), suggesting that pathway activation may be an early event in human breast neoplasia. A correlation was observed between HER2/neu expression and nucleocytoplasmic ß-catenin in node-positive carcinomas (P = 0.02). Furthermore, cytoplasmic ß-catenin was detected in HER2/neu-induced mouse mammary tumors. The Axin2NLSlacZ mouse strain, a previously validated reporter of mammary Wnt/ß-catenin signaling, was utilized to define in vivo transcriptional consequences of HER2/neu-induced ß-catenin accumulation. Discrete hyperplastic foci observed in mammary glands from bigenic MMTV/neu, Axin2NLSlacZ mice, highlighted by robust ß-catenin/TCF signaling, likely represent the earliest stage of mammary intraepithelial neoplasia in MMTV/neu mice. Our study thus provides provocative evidence for Wnt/ß-catenin signaling as an early, HER2/neu-inducible event in breast neoplasia

Can sleep and resting behaviours be used as indicators of welfare in shelter dogs (Canis lupusfamiliaris)?

Previous research on humans and animals suggests that the analysis of sleep patterns may reliably inform us about welfare status, but little research of this kind has been carried out for non-human animals in an applied context. This study explored the use of sleep and resting behaviour as indicators of welfare by describing the activity patterns of dogs (Canis lupus familiaris) housed in rescue shelters, and comparing their sleep patterns to other behavioural and cognitive measures of welfare. Sleep and activity patterns were observed over five non-consecutive days in a population of 15 dogs. Subsequently, the characteristics of sleep and resting behaviour were described and the impact of activity on patterns of sleep and resting behaviour analysed. Shelter dogs slept for 2.8% of the day, 14.3% less than previously reported and experienced less sleep fragmentation at night (32 sleep bouts). There were no statistically significant relationships between behaviours exhibited during the day and sleep behaviour. A higher proportion of daytime resting behaviour was significantly associated with a positive judgement bias, less repetitive behaviour and increased time spent coded as ‘relaxed’ across days by shelter staff. These results suggest that, in the context of a busy shelter environment, the ability to rest more during the day could be a sign of improved welfare. Considering the non-linear relationship between sleep and welfare in humans, the relationship between sleep and behavioural indicators of welfare, including judgement bias, in shelter dogs may be more complex than this study could detect