A region within the third extracellular loop of rat Aquaporin 6 precludes trafficking to plasma membrane in a heterologous cell line.

The inability to over-express Aquaporin 6 (AQP6) in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail using crystallographic approaches. Using an Aquaporin 3-tGFP Reporter (AGR) system we have identified a region within loop C of AQP6 that is responsible for severely hampering plasma membrane expression. Serine substitution corroborated that amino acids present within AQP6194-213 of AQP6 loop C contribute to intracellular endoplasmic reticulum (ER) retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous expression systems.

Consistency for the tree bootstrap in respondent-driven sampling.

Respondent-driven sampling is an approach for estimating features of populations that are difficult to access using standard survey tools, e.g., the fraction of injection drug users who are HIV positive. Baraff et al. (2016) introduced an approach to estimating uncertainty in population proportion estimates from respondent-driven sampling using the tree bootstrap method. In this paper we establish the consistency of this tree bootstrap approach in the case of [Formula: see text]-trees.

An uncharacterized region within the N-terminus of mouse TMC1 precludes trafficking to plasma membrane in a heterologous cell line.

Mechanotransduction by hair cell stereocilia lies at the heart of sound detection in vertebrates. Considerable effort has been put forth to identify proteins that comprise the hair cell mechanotransduction apparatus. TMC1, a member of the transmembrane channel-like (TMC) family, was identified as a core protein of the mechanotransduction complex in hair cells. However, the inability of TMC1 to traffic through the endoplasmic reticulum in heterologous cellular systems has hindered efforts to characterize its function and fully identify its role in mechanotransduction. We developed a novel approach that allowed for the detection of uncharacterized protein regions, which preclude trafficking to the plasma membrane (PM) in heterologous cells. Tagging N-terminal fragments of TMC1 with Aquaporin 3 (AQP3) and GFP fusion reporter, which intrinsically label PM in HEK293 cells, indicated that residues at the edges of amino acid sequence 138-168 invoke intracellular localization and/or degradation. This signal is able to preclude surface localization of PM protein AQP3 in HEK293 cells. Substitutions of the residues by alanine or serine corroborated that the information determining the intracellular retention is present within amino acid sequence 138-168 of TMC1 N-terminus. This novel signal may preclude the proper trafficking of TMC1 to the PM in heterologous cells.

Beyond prediction: A framework for inference with variational approximations in mixture models.

Variational inference is a popular method for estimating model parameters and conditional distributions in hierarchical and mixed models, which arise frequently in many settings in the health, social, and biological sciences. Variational inference in a frequentist context works by approximating intractable conditional distributions with a tractable family and optimizing the resulting lower bound on the log-likelihood. The variational objective function is typically less computationally intensive to optimize than the true likelihood, enabling scientists to fit rich models even with extremely large datasets. Despite widespread use, little is known about the general theoretical properties of estimators arising from variational approximations to the log-likelihood, which hinders their use in inferential statistics. In this paper we connect such estimators to profile M-estimation, which enables us to provide regularity conditions for consistency and asymptotic normality of variational estimators. Our theory also motivates three methodological improvements to variational inference: estimation of the asymptotic model-robust covariance matrix, a one-step correction that improves estimator efficiency, and an empirical assessment of consistency. We evaluate the proposed results using simulation studies and data on marijuana use from the National Longitudinal Study of Youth.

Generation of Flp-intm-ready DG44 and Lec 3.2.8.1 CHO cell lines for quick and easy constitutive protein expression.

The well-characterized cell line Chinese hamster ovary (CHO) has been used to produce numerous biopharmaceuticals and is an important tool for basic research. However, introducing foreign DNA into specially modified CHO cells such as DG44 and Lec 3.2.8.1 can sometimes be an arduous process. Here we show that the Flp-intm plasmid can be modified to produce a fluorescent tracer protein tag (mCherrytm) as a fusion reporter, to allow for the rapid selection of single-cell sorted, isogenic Flp-intm-ready DG44 and Lec 3.2.8.1 cell lines. These two cell lines are stable and viable and may be useful for applications such as antibody production and crystallographic studies. Here we provide key details on how the modified pFRT/CherryZeo plasmid may be used to incorporate Flp-intm technology into virtually any desired target cell line in a fast, safe and reliable manner.

Hyak mortality monitoring system: innovative sampling and estimation methods - proof of concept by simulation.

Traditionally health statistics are derived from civil and/or vital registration. Civil registration in low- to middle-income countries varies from partial coverage to essentially nothing at all. Consequently the state of the art for public health information in low- to middle-income countries is efforts to combine or triangulate data from different sources to produce a more complete picture across both time and space - data amalgamation. Data sources amenable to this approach include sample surveys, sample registration systems, health and demographic surveillance systems, administrative records, census records, health facility records and others. We propose a new statistical framework for gathering health and population data - Hyak - that leverages the benefits of sampling and longitudinal, prospective surveillance to create a cheap, accurate, sustainable monitoring platform. Hyak has three fundamental components: Data amalgamation: A sampling and surveillance component that organizes two or more data collection systems to work together: (1) data from HDSS with frequent, intense, linked, prospective follow-up and (2) data from sample surveys conducted in large areas surrounding the Health and Demographic Surveillance System (HDSS) sites using informed sampling so as to capture as many events as possible;Cause of death: Verbal autopsy to characterize the distribution of deaths by cause at the population level; andSocioeconomic status (SES): Measurement of SES in order to characterize poverty and wealth. We conduct a simulation study of the informed sampling component of Hyak based on the Agincourt HDSS site in South Africa. Compared with traditional cluster sampling, Hyak's informed sampling captures more deaths, and when combined with an estimation model that includes spatial smoothing, produces estimates of both mortality counts and mortality rates that have lower variance and small bias.

Effect of Ligand Modification on the Mechanism of Electrocatalytic Hydrogen Production by Ni(pyridinethiolate)3- Derivatives.

The effects of ligand modification on the catalytic mechanism of hydrogen production by Ni(PyS)3- derivatives, made with electron-withdrawing and -donating substitutions to the pyridinethiolate (PyS)- ligands, are studied experimentally and computationally using density functional theory. Thermodynamic data, spin density maps, and frontier molecular orbital diagrams were generated for reaction intermediates. Comparison of computed values for E0 and p Ka with experimental values supports the proposed mechanisms. The rate of electrochemical hydrogen production is correlated with the effect of ligand modification. Notably, the presence of an electron-donating substituent favors an alternative mechanism for hydrogen production. Computationally it was determined that the electron-donating substituent causes deviation from the original chemical-electrochemical-chemical-electrochemical (CECE) mechanism of Ni(PyS)3- to a CCEE mechanism, while the CECE mechanism is maintained for all catalysts substituted with electron-withdrawing groups.

Reproducibility of clinical research in critical care: a scoping review.

BACKGROUND: The ability to reproduce experiments is a defining principle of science. Reproducibility of clinical research has received relatively little scientific attention. However, it is important as it may inform clinical practice, research agendas, and the design of future studies. METHODS: We used scoping review methods to examine reproducibility within a cohort of randomized trials examining clinical critical care research and published in the top general medical and critical care journals. To identify relevant clinical practices, we searched the New England Journal of Medicine, The Lancet, and JAMA for randomized trials published up to April 2016. To identify a comprehensive set of studies for these practices, included articles informed secondary searches within other high-impact medical and specialty journals. We included late-phase randomized controlled trials examining therapeutic clinical practices in adults admitted to general medical-surgical or specialty intensive care units (ICUs). Included articles were classified using a reproducibility framework. An original study was the first to evaluate a clinical practice. A reproduction attempt re-evaluated that practice in a new set of participants. RESULTS: Overall, 158 practices were examined in 275 included articles. A reproduction attempt was identified for 66 practices (42%, 95% CI 33-50%). Original studies reported larger effects than reproduction attempts (primary endpoint, risk difference 16.0%, 95% CI 11.6-20.5% vs. 8.4%, 95% CI 6.0-10.8%, P = 0.003). More than half of clinical practices with a reproduction attempt demonstrated effects that were inconsistent with the original study (56%, 95% CI 42-68%), among which a large number were reported to be efficacious in the original study and to lack efficacy in the reproduction attempt (34%, 95% CI 19-52%). Two practices reported to be efficacious in the original study were found to be harmful in the reproduction attempt. CONCLUSIONS: A minority of critical care practices with research published in high-profile journals were evaluated for reproducibility; less than half had reproducible effects.

Acquiring and maintaining point-of-care ultrasound (POCUS) competence for anesthesiologists.

PURPOSE: Point-of-care ultrasound (POCUS) involves the bedside use of ultrasound to answer specific diagnostic questions and to assess real-time physiologic responses to treatment. Although POCUS has become a well-established resource for emergency and critical care physicians, anesthesiologists are still working to obtain POCUS skills and to incorporate them into routine practice. This review defines the benefits of POCUS to anesthesia practice, identifies challenges to establishing POCUS in routine anesthesia care, and offers solutions to help guide its incorporation going forward. PRINCIPAL FINDINGS: Benefits to POCUS include improving the sensitivity and specificity of the physical examination and helping to guide patient treatment. The challenges to establishing POCUS as a standard in anesthesia practice include developing and maintaining competence. There is a need to develop standards of practice and a common language between specialties to facilitate training and create guidelines regarding patient management. CONCLUSIONS: Presently, our specialty requires consensus by expert stakeholders to address issues of competence, certification, development of standards and terminology, and the management of unexpected diagnoses. To promote POCUS competency in our discipline, we support its incorporation into anesthesiology curricula and training programs and the continuing professional development of POCUS-related activities at a national level.

Photocatalytic water reduction using a polymer coated carbon quantum dot sensitizer and a nickel nanoparticle catalyst.

Hydrogen gas is produced photocatalytically using 470 nm light, PVP-coated carbon quantum dots (CQDs) as the photosensitizer, and nickel nanoparticles (NiNPs) as the catalyst. The effect of the amount of polyvinylpyrrolidone (PVP) on the ability of the CQD/NiNP composites to catalyze proton reduction was studied. A maximum of 330 mmols H2/g CQD is produced using 68 µg ml-1 of CQDs and 6 µg ml-1 of NiNPs, with activity persisting for 4 h when 20 wt%-PVP-coated CQDs were used. The H2 production quantum yield under these conditions is 6%. It was found that composites having higher weight percent PVP had decreased rates of H2 production, but increased duration. Increasing the weight percent of PVP coating also increases the fluorescence quantum yield of CQDs. Fluorescence quenching titrations reveal that H2 production could occur by either a reductive or oxidative quenching mechanism. The nanomaterials, prepared using simple methods, are used as the photosensitizer and catalyst in the proton reduction system that operates using visible light.

Antibody and cytokine levels in humans fed on by the common bedbug, Cimex lectularius L.

Little is known about cimicosis, the resultant dermal reaction from feeding activity by the common bedbug, Cimex lectularius L. We fed C. lectularius on human study subjects four times over four weeks and measured serum cytokine and antibody levels, and subjects recorded any cimicosis. The average time for subjects to develop cimicosis decreased with each feeding from 8.4, to 2.1, 1.5 and 1.3 days, respectively. There were no significant changes in total IgG, IgG1, IgG2, IgG4 or IgE levels between the first and fourth bedbug feedings, but there was a significant decrease in total IgG3 levels (P<.001). IgG4 was not required for cimicosis. Higher IgG2 and IgG4 levels at study visit 4 were associated with an increased duration of cimicosis (P=.04) and lower pruritis (P=.03), respectively. There were no significant changes in serum TNF-α, IL-1ß, IL-4, IL-5, IL-6, IL-10, IFN-γ and IL-17A levels before and one hour after the C. lectularius feeding. Lower post-C. lectularius feeding IL-6 levels were associated with increased pruritis (P=.001) and the time to maximum pruritis (P=.04), respectively. Higher post-C. lectularius feeding IL-5 levels were associated with a longer duration of pruritis (P=.05).

Visualization of atherosclerosis as detected by coronary artery calcium and carotid intima-media thickness reveals significant atherosclerosis in a cross-sectional study of psoriasis patients in a tertiary care center.

BACKGROUND: Psoriasis is a chronic inflammatory disease of the skin and joints that may also have systemic inflammatory effects, including the development of cardiovascular disease (CVD). Multiple epidemiologic studies have demonstrated increased rates of CVD in psoriasis patients, although a causal link has not been established. A growing body of evidence suggests that sub-clinical systemic inflammation may develop in psoriasis patients, even from a young age. We aimed to evaluate the prevalence of atherosclerosis and identify specific clinical risk factors associated with early vascular inflammation. METHODS: We conducted a cross-sectional study of a tertiary care cohort of psoriasis patients using coronary artery calcium (CAC) score and carotid intima-media thickness (CIMT) to detect atherosclerosis, along with high sensitivity C-reactive protein (hsCRP) to measure inflammation. Psoriasis patients and controls were recruited from our tertiary care dermatology clinic. Presence of atherosclerosis was defined using validated numeric values within CAC and CIMT imaging. Descriptive data comparing groups was analyzed using Welch's t test and Pearson Chi square tests. Logistic regression was used to analyze clinical factors associated with atherosclerosis, and linear regression to evaluate the relationship between psoriasis and hsCRP. RESULTS: 296 patients were enrolled, with 283 (207 psoriatic and 76 controls) having all data for the hsCRP and atherosclerosis analysis. Atherosclerosis was found in 67.6 % of psoriasis subjects versus 52.6 % of controls; Psoriasis patients were found to have a 2.67-fold higher odds of having atherosclerosis compared to controls [95 % CI (1.2, 5.92); p = 0.016], after adjusting for age, gender, race, BMI, smoking, HDL and hsCRP. In addition, a non-significant trend was found between HsCRP and psoriasis severity, as measured by PASI, PGA, or BSA, again after adjusting for confounders. CONCLUSIONS: A tertiary care cohort of psoriasis patients have a high prevalence of early atherosclerosis, increased hsCRP, and psoriasis remains a risk factor for the presence of atherosclerosis even after adjustment of key confounding clinical factors. Psoriasis may contribute to an accelerated systemic inflammatory cascade resulting in increased risk of CVD and CV events.

Elevated Intracranial Pressure Diagnosis with Emergency Department Bedside Ocular Ultrasound.

Bedside sonographic measurement of optic nerve sheath diameter can aid in the diagnosis of elevated intracranial pressure in the emergency department. This case report describes a 21-year-old female presenting with 4 months of mild headache and 2 weeks of recurrent, transient binocular vision loss. Though limited by patient discomfort, fundoscopic examination suggested the presence of blurred optic disc margins. Bedside ocular ultrasound (BOUS) revealed wide optic nerve sheath diameters and bulging optic discs bilaterally. Lumbar puncture demonstrated a cerebrospinal fluid (CSF) opening pressure of 54 cm H2O supporting the suspected diagnosis of idiopathic intracranial hypertension. Accurate fundoscopy can be vital to the appropriate diagnosis and treatment of patients with suspected elevated intracranial pressure, but it is often technically difficult or poorly tolerated by the photophobic patient. BOUS is a quick and easily learned tool to supplement the emergency physician's fundoscopic examination and help identify patients with elevated intracranial pressure.

Identifying low-value clinical practices in critical care medicine: protocol for a scoping review.

INTRODUCTION: Reducing unnecessary, low-value clinical practice (ie, de-adoption) is key to improving value for money in healthcare, especially among patients admitted to intensive care units (ICUs) where resource consumption exceeds other medical and surgical populations. Research suggests that low-value clinical practices are common in medicine, however systematically and objectively identifying them is a widely cited barrier to de-adoption. We will conduct a scoping review to identify low-value clinical practices in adult critical care medicine that are candidates for de-adoption. METHODS AND ANALYSIS: We will systematically search the literature to identify all randomised controlled trials or systematic reviews that focus on diagnostic or therapeutic interventions in adult patients admitted to medical, surgical or specialty ICUs, and are published in 3 general medical journals with the highest impact factor (New England Journal of Medicine, The Lancet, Journal of the American Medical Association). 2 investigators will independently screen abstracts and full-text articles against inclusion criteria, and extract data from included citations. Included citations will be classified according to whether or not they represent a repeat examination of the given research question (ie, replication research), and whether the results are similar or contradictory to the original study. Studies with contradictory results will determine clinical practices that are candidates for de-adoption. ETHICS AND DISSEMINATION: Our scoping review will use robust methodology to systematically identify a list of clinical practices in adult critical care medicine with evidence supporting their de-adoption. In addition to adding to advancing the study of de-adoption, this review may also serve as the launching point for clinicians and researchers in critical care to begin reducing the number of low-value clinical practices. Dissemination of these results to relevant stakeholders will include tailored presentations at local, national and international meetings, and publication of a manuscript. Ethical approval is not required for this study.

The key role of aquaporin 3 and aquaporin 10 in the pathogenesis of pompholyx.

Pompholyx remains a chronic skin affliction without a compelling pathophysiological explanation. The disease is characterized by the sudden onset of vesicles exclusively in the palms and soles which generally resolves. However, the disease may progress and the vesicles may expand and fuse; with chronicity there is deep fissuring. Multiple therapeutic approaches are available, but the disease is often resistant to conventional treatments. Currently, oral alitretinoin is used for patients with resistant chronic disease; however, this therapy is only approved for use in the UK, Europe and Canada. In this paper we wish to put forward a hypothesis: exposure to water and the subsequent steep osmotic gradient imbalance are key factors driving skin dehydration seen in pompholyx patients once the disease becomes chronic. The mechanistic explanation for the epidermal fissuring might lie in the over-expression across the mid and upper epidermis, including the stratum corneum, of two water/glycerol channel proteins aquaporin 3 and aquaporin 10, expressed in the keratinocytes of afflicted pompholyx patients. The over-expression of these two aquaporins may bridge the abundantly hydrated dermis and basal epidermis to the outer environment allowing cutaneous water and glycerol to flow outward. The beneficial effects reported in alitretinoin-treated patients with chronic hand eczemas may be due potential regulation of aquaporin 3 and aquaporin 10 by alitretinoin.

Female soccer knee injury: observed knowledge gaps in injury prevention among players/parents/coaches and current evidence (the KNOW study).

This study sought to determine if knowledge regarding the risk for knee injuries and the potential for their prevention is being translated to female adolescent soccer players (13-18 years), their parents, and coaches. Eligible participants in the 2007 indoor soccer season were surveyed to determine their knowledge of the risk for and the potential to prevent knee injuries, and their knowledge of effective prevention strategies, if they felt that injury prevention was possible. Team selection was stratified to be representative of both competitive and recreational level play and age group distributions within the selected soccer association. Of the study subjects, 773/1396 (55.4%) responded to the survey: 408 (53%) players, 292 (38%) parents, and 73 (9%) coaches. Most respondents (538 [71%]) were aware of the risk for knee injury. Coaches and parents were more likely than players to view knee injuries as preventable; however, appropriate prevention strategies were often not identified. Four hundred eighty-four (63.8%) respondents reported that they had never received information on knee injuries. Substantial knowledge gaps regarding knee injury prevention and effective preventative strategies were identified. Given the predominance of knee injuries in female adolescent soccer players, there is an urgent need for knowledge translation of prevention strategies to decrease both incidence and long-term consequences of knee injuries.

Successful cutaneous delivery of the photosensitizer silicon phthalocyanine 4 for photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) has been shown to be effective in the treatment of malignancies of a variety of organ systems, including the lungs, bladder, gastrointestinal tract and skin. Cutaneous lesions serve as ideal targets of PDT because of the accessibility of the skin to light. To achieve optimum results, the photosensitizer must be delivered effectively into the target layers of the skin within a practical timeframe, via noninvasive methods. AIM: To determine whether topical application of a second-generation photosensitizer, silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2 (CH2)3 N(CH3)2)(OH)], results in effective penetration of the skin barrier. METHODS: Penetration of Pc 4 was evaluated using standard Franz-type vertical diffusion cell experiments on surrogate materials (silicone membranes) and laser-scanning confocal microscopy of normal skin biopsy samples from human volunteers. RESULTS: The Franz diffusion data indicate that Pc 4 formulated in an ethanol/propylene glycol solution (70/30%, v/v) can penetrate the membrane at a flux that is appreciable and relatively invariant. Using the same formulation, Pc 4 uptake could be detected in human skin via laser-scanning confocal microscopy. CONCLUSION: After topical application, Pc 4 is absorbed into the epidermis in as little as 1 h, and the absorption increased with increasing time and dose. Pc 4 can be effectively delivered into human skin via topical application. The data also suggest that the degree of penetration is time- and dose-dependent.

Depletion of antigen-presenting cells by clodronate liposomes reverses the psoriatic skin phenotype in KC-Tie2 mice.

BACKGROUND: There is ongoing debate regarding the initiation of psoriatic plaque as primarily arising from an anomaly in epidermal keratinocytes (KCs) or from abnormalities in immunocytes that secondarily activate otherwise normal KCs. In mice engineered to overexpress the angiopoietin receptor Tie2 in KCs, skin spontaneously develops the characteristic clinical, histological and immune cell phenotypes of psoriasis which can be reversed with either transgene repression or ciclosporin administration, suggesting key roles for both KCs and T cells in mediating the skin disease in this murine model. OBJECTIVES: To determine if antigen-presenting cells (APCs) and macrophages alone are sufficient to sustain psoriasiform inflammation in the KC-Tie2 murine model of psoriasis. METHODS: Clodronate liposomes were intradermally injected into involved dorsal skin of KC-Tie2 or control animals once a week for 6weeks and acanthosis, angiogenesis, immune cell infiltration and cytokine production were quantitated using immunohistochemistry and interactive image analyses, enzyme-linked immunosorbent assay (ELISAs) and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Clodronate liposome injection eliminated CD11c+, F4/80+ and CD11b+ cells in the skin and returned CD8+ T-cell numbers to control mouse levels. APC depletion in KC-Tie2 mouse skin resulted in resolution of the acanthotic skin phenotype, decreased dermal angiogenesis, and a return to control mouse levels for interleukin (IL)-1α, IL-6, IL-23 and tumour necrosis factor (TNF)-α expression and modest reductions in interferon-γ and IL-17. CONCLUSIONS: These findings suggest a critical role for APCs and myeloid cell-derived IL-23 and TNF-α and underscore the importance of Th1 and Th17 T cells in maintaining the psoriasiform skin phenotype in the KC-Tie2 mouse model.