Gene co-opening network deciphers gene functional relationships.
Genome sequencing technology has generated a vast amount of genomic and epigenomic data, and has provided us a great opportunity to study gene functions on a global scale from an epigenomic view. In the last decade, network-based studies, such as those based on PPI networks and co-expression networks, have shown good performance in capturing functional relationships between genes. However, the functions of a gene and the mechanism of interaction of genes with each other to elucidate their functions are still not entirely clear. Here, we construct a gene co-opening network based on chromatin accessibility of genes. We show that genes related to a specific biological process or the same disease tend to be clustered in the co-opening network. This understanding allows us to detect functional clusters from the network and to predict new functions for genes. We further apply the network to prioritize disease genes for Psoriasis, and demonstrate the power of the joint analysis of the co-opening network and GWAS data in identifying disease genes. Taken together, the co-opening network provides a new viewpoint for the elucidation of gene associations and the interpretation of disease mechanisms.


Surface Plasmon Resonance Sensing on Naturally Derived Membranes: A Remyelination-Promoting Human Antibody Binds Myelin with Extraordinary Affinity.
rHIgM22 is a recombinant human monoclonal IgM designed to promote remyelination, and it is currently in Phase I clinical trials in patients with multiple sclerosis (MS). In animal models of demyelination, a single low dose of rHIgM22 stimulates oligodendrocyte maturation, induces remyelination, preserves axons, and slows the decline of locomotor deficits. Natural autoantibodies like rHIgM22 typically bind to multiple antigens with weak affinity. rHIgM22 binds to oligodendrocytes and myelin. Because the antigens for rHIgM22 is prevalent within and exclusive to central nervous system (CNS) myelin, we used CNS myelin particles in combination with surface plasmon resonance to determine the kinetic and affinity constants for the interaction of rHIgM22 to myelin. We found that both the serum and recombinant forms of the antibody bind to myelin with very small dissociation constants in the 100 pM range, which is highly unusual for natural autoantibodies. The extraordinary affinity between rHIgM22 and myelin may explain why such a low effective dose can stimulate CNS repair in animal models of demyelination and underlie the accumulation of rHIgM22 in the CSF in treated MS patients by targeting myelin.


The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the Rho GTPases Cdc42 and Rac1.
A mammalian homologue of the PDZ domain containing Caenorhabditis elegans protein PAR-6 was found in a yeast two-hybrid system screen as binding to the Rho family member Cdc42. PAR-6 contains a PDZ domain and in C. elegans it has been shown to be crucial for the asymmetric cleavage and establishment of cell polarity during the first cell divisions in the growing embryo. Mammalian PAR-6 interacted with Cdc42 and Rac1 both in the yeast two-hybrid system and in in vitro binding assays. Co-immunoprecipitation experiments, employing transiently transfected Cos-1 cells, further confirmed that Cdc42 and Rac1 are physiological binding partners for PAR-6. We found that, in epithelial Madin-Darby canine kidney cells (MDCK), endogenous PAR-6 was present in the tight junctions, as judged from its co-localisation with the tight junction protein ZO-1, however, PAR-6 was also detected in the cell nucleus. Stimulation of MDCK cells with scatter factor/hepatocyte growth factor induced a loss of PAR-6 from the areas of cell-cell contacts in conformity with their progressive breakdown. In C. elegans PAR-6 co-localises with PAR-3 and has been suggested to form a direct complex. In agreement with earlier studies, mammalian PAR-3 was found to be present in tight junctions of MDCK cells but, in contrast to PAR-6, the protein could not be detected in the nucleus. Furthermore, co-immunoprecipitation experiments, employing Cos-1 cells, demonstrated that mammalian PAR-6 and PAR-3 formed a direct complex. These findings, together with the reported roles of PAR-6 and PAR-3 in C. elegans, suggest that Cdc42 and Rac1 and PAR-6/PAR-3 are involved in the establishment of cell polarity in epithelial cells.


Characteristics of multi-institutional health sciences education research: a systematic review.
Multi-institutional research increases the generalizability of research findings. However, little is known about characteristics of collaborations across institutions in health sciences education research. Using a systematic review process, the authors describe characteristics of published, peer-reviewed multi-institutional health sciences education research to inform educators who are considering such projects. Two medical librarians searched MEDLINE, the Education Resources Information Center (ERIC), EMBASE, and CINAHL databases for English-language studies published between 2004 and 2013 using keyword terms related to multi-institutional systems and health sciences education. Teams of two authors reviewed each study and resolved coding discrepancies through consensus. Collected data points included funding, research network involvement, author characteristics, learner characteristics, and research methods. Data were analyzed using descriptive statistics. One hundred eighteen of 310 articles met inclusion criteria. Sixty-three (53%) studies received external and/or internal financial support (87% listed external funding, 37% listed internal funding). Forty-five funded studies involved graduate medical education programs. Twenty (17%) studies involved a research or education network. Eighty-five (89%) publications listed an author with a master's degree or doctoral degree. Ninety-two (78%) studies were descriptive, whereas 26 studies (22%) were experimental. The reported study outcomes were changes in student attitude (38%; n=44), knowledge (26%; n=31), or skill assessment (23%; n=27), as well as patient outcomes (9%; n=11). Multi-institutional descriptive studies reporting knowledge or attitude outcomes are highly published. Our findings indicate that funding resources are not essential to successfully undertake multi-institutional projects. Funded studies were more likely to originate from graduate medical or nursing programs.


The ARF tumor suppressor inhibits tumor cell colonization independent of p53 in a novel mouse model of pancreatic ductal adenocarcinoma metastasis.
Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2-invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies.


Folliculotropism Does Not Affect Overall Survival in Mycosis Fungoides: Results from a Single-Center Cohort and Meta-Analysis.
Folliculotropic mycosis fungoides (FMF) is a distinct subtype of mycosis fungoides (MF) with unique clinicopathological features. The medical literature suggests that FMF has a more aggressive course and worse survival than classic MF. Previous studies do not use standardized treatment, and no studies have reported an association between treatment response and overall survival (OS). To compare OS for MF, FMF, and Sézary syndrome (SS) patients. Data were collected retrospectively from 218 patients (171 MF, 15 SS, 32 FMF) treated in a single academic center between 1970 and 2016. Negative predictors of OS were age (OR = 1.07), male sex (OR = 1.63), and stage IIB, III, and IV (OR = 4.10, 5.42, and 7.54, respectively, vs. stage IA). Lack of initial PUVA response was strongly associated with negative OS (OR = 3.08). Kaplan-Meier analysis of age-, sex-, and stage-matched MF and FMF patients found similar OS between the 2 groups. The 5-year OS was 91% for FMF and 74% for MF. Meta-analysis of current data and 2 published studies where survival of FMF patients was compared to MF did not reveal statistically significant differences between these 2 diseases. When patients were matched for age, sex, and disease stage, folliculotropism did not affect OS in MF.


Characterization of the Snow Mountain agent of viral gastroenteritis.
Snow Mountain agent (SMA) is a 27- to 32-nm virus which is the etiologic agent of outbreaks of acute gastroenteritis in Colorado and Vermont. SMA is morphologically similar to but antigenically distinct from the Norwalk and Hawaii agents of viral gastroenteritis but, like those agents, has not been cultivated in vitro. We purified and characterized SMA directly from human stool specimens containing the virus. The density of the SMA virion was 1.29 g/cm3 and 1.21 to 1.22 g/cm3 on potassium tartrate-glycerol gradients and 1.33 to 1.34 g/cm3 on cesium chloride gradients. SMA had an S value of 170 to 183S on a sucrose velocity gradient. The purified virion was iodinated, immunoprecipitated with acute and convalescent sera from volunteers challenged with SMA, and analyzed on polyacrylamide gels. The virion contains one major structural protein of 62,000 molecular weight, which is similar in size to the 59,000-molecular-weight protein found in the Norwalk virion. The biophysical properties and single structural protein of SMA most closely resemble those of the calicivirus group.


Measuring enzymatic HIV-1 susceptibility to two reverse transcriptase inhibitors as a rapid and simple approach to HIV-1 drug-resistance testing.
Simple and cost-effective approaches for HIV drug-resistance testing are highly desirable for managing increasingly expanding HIV-1 infected populations who initiate antiretroviral therapy (ART), particularly in resource-limited settings. Non-nucleoside reverse trancriptase inhibitor (NNRTI)-based regimens with an NRTI backbone containing lamivudine (3TC) or emtricitabine (FTC) are preferred first ART regimens. Failure with these drug combinations typically involves the selection of NNRTI- and/or 3TC/FTC-resistant viruses. Therefore, the availability of simple assays to measure both types of drug resistance is critical. We have developed a high throughput screening test for assessing enzymatic resistance of the HIV-1 RT in plasma to 3TC/FTC and NNRTIs. The test uses the sensitive "Amp-RT" assay with a newly-developed real-time PCR format to screen biochemically for drug resistance in single reactions containing either 3TC-triphosphate (3TC-TP) or nevirapine (NVP). Assay cut-offs were defined based on testing a large panel of subtype B and non-subtype B clinical samples with known genotypic profiles. Enzymatic 3TC resistance correlated well with the presence of M184I/V, and reduced NVP susceptibility was strongly associated with the presence of K103N, Y181C/I, Y188L, and G190A/Q. The sensitivity and specificity for detecting resistance were 97.0% and 96.0% in samples with M184V, and 97.4% and 96.2% for samples with NNRTI mutations, respectively. We further demonstrate the utility of an HIV capture method in plasma by using magnetic beads coated with CD44 antibody that eliminates the need for ultracentifugation. Thus our results support the use of this simple approach for distinguishing WT from NNRTI- or 3TC/FTC-resistant viruses in clinical samples. This enzymatic testing is subtype-independent and can assist in the clinical management of diverse populations particularly in resource-limited settings.


The meaning of justice in safety incident reporting.
Safety experts contend that to make incident reporting work, healthcare organizations must establish a "just" culture-that is, an organizational context in which health professionals feel assured that they will receive fair treatment when they report safety incidents. Although healthcare leaders have expressed keen interest in establishing a just culture in their institutions, the patient safety literature offers little guidance as to what the term "just culture" really means or how one goes about creating a just culture. Moreover, the safety literature does not indicate what constitutes a just incident reporting process in the eyes of the health professionals who provide direct patient care. This gap is unfortunate, for knowing what constitutes a just incident reporting process in the eyes of front-line health professionals is essential for designing useful information systems to detect, monitor, and correct safety problems. In this article, we seek to clarify the conceptual meaning of just culture and identify the attributes of incident reporting processes that make such systems just in the eyes of health professionals. To accomplish these aims, we draw upon organizational justice theory and research to develop a conceptual model of perceived justice in incident reporting processes. This model could assist those healthcare leaders interested in creating a just culture by clarifying the multiple meanings, antecedents, and consequences of justice.


Laboratory Studies of Methane and Its Relationship to Prebiotic Chemistry.
To examine how prebiotic chemical evolution took place on Earth prior to the emergence of life, laboratory experiments have been conducted since the 1950s. Methane has been one of the key molecules in these investigations. In earlier studies, strongly reducing gas mixtures containing methane and ammonia were used to simulate possible reactions in the primitive atmosphere of Earth, producing amino acids and other organic compounds. Since Earth's early atmosphere is now considered to be less reducing, the contribution of extraterrestrial organics to chemical evolution has taken on an important role. Such organic molecules may have come from molecular clouds and regions of star formation that created protoplanetary disks, planets, asteroids, and comets. The interstellar origin of organics has been examined both experimentally and theoretically, including laboratory investigations that simulate interstellar molecular reactions. Endogenous and exogenous organics could also have been supplied to the primitive ocean, making submarine hydrothermal systems plausible sites of the generation of life. Experiments that simulate such hydrothermal systems where methane played an important role have consequently been conducted. Processes that occur in other Solar System bodies offer clues to the prebiotic chemistry of Earth. Titan and other icy bodies, where methane plays significant roles, are especially good targets. In the case of Titan, methane is both in the atmosphere and in liquidospheres that are composed of methane and other hydrocarbons, and these have been studied in simulation experiments. Here, we review the wide range of experimental work in which these various terrestrial and extraterrestrial environments have been modeled, and we examine the possible role of methane in chemical evolution. Key Words: Methane-Interstellar environments-Submarine hydrothermal systems-Titan-Origin of life. Astrobiology 17, 786-812.


A Decade of Progress Using Virtual Reality for Poststroke Lower Extremity Rehabilitation: Systematic Review of the Intervention Methods.
To develop a systematic review of the literature, to describe the different virtual reality (VR) interventions and interactive videogames applied to the lower extremity (LE) of stroke patients, and to analyse the results according to the most frequently used outcome measures. An electronic search of randomized trials between January 2004 and January 2014 in different databases (Medline, Cinahl, Web of Science, PEDro, and Cochrane) was carried out. Several terms (virtual reality, feedback, stroke, hemiplegia, brain injury, cerebrovascular accident, lower limb, leg, and gait) were combined, and finally 11 articles were included according to the established inclusion and exclusion criteria. The reviewed trials showed a high heterogeneity in terms of study design and assessment tools, which makes it difficult to compare and analyze the different types of interventions. However, most of them found a significant improvement on gait speed, balance and motor function, due to VR intervention. Although evidence is limited, it suggests that VR intervention (more than 10 sessions) in stroke patients may have a positive impact on balance, and gait recovery. Better results were obtained when a multimodal approach, combining VR and conventional physiotherapy, was used. Flexible software seems to adapt better to patients' requirements, allowing more specific and individual treatments.


SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV.
In a previous screen of putative interferon-stimulated genes, SUN2 was shown to inhibit HIV-1 infection in an uncharacterized manner. SUN2 is an inner nuclear membrane protein belonging to the linker of nucleoskeleton and cytoskeleton complex. We have analyzed here the role of SUN2 in HIV infection. We report that in contrast to what was initially thought, SUN2 is not induced by type I interferon, and that SUN2 silencing does not modulate HIV infection. However, SUN2 overexpression in cell lines and in primary monocyte-derived dendritic cells inhibits the replication of HIV but not murine leukemia virus or chikungunya virus. We identified HIV-1 and HIV-2 strains that are unaffected by SUN2, suggesting that the effect is specific to particular viral components or cofactors. Intriguingly, SUN2 overexpression induces a multilobular flower-like nuclear shape that does not impact cell viability and is similar to that of cells isolated from patients with HTLV-I-associated adult T-cell leukemia or with progeria. Nuclear shape changes and HIV inhibition both mapped to the nucleoplasmic domain of SUN2 that interacts with the nuclear lamina. This block to HIV replication occurs between reverse transcription and nuclear entry, and passaging experiments selected for a single-amino-acid change in capsid (CA) that leads to resistance to overexpressed SUN2. Furthermore, using chemical inhibition or silencing of cyclophilin A (CypA), as well as CA mutant viruses, we implicated CypA in the SUN2-imposed block to HIV infection. Our results demonstrate that SUN2 overexpression perturbs both nuclear shape and early events of HIV infection. Cells encode proteins that interfere with viral replication, a number of which have been identified in overexpression screens. SUN2 is a nuclear membrane protein that was shown to inhibit HIV infection in such a screen, but how it blocked HIV infection was not known. We show that SUN2 overexpression blocks the infection of certain strains of HIV before nuclear entry. Mutation of the viral capsid protein yielded SUN2-resistant HIV. Additionally, the inhibition of HIV infection by SUN2 involves cyclophilin A, a protein that binds the HIV capsid and directs subsequent steps of infection. We also found that SUN2 overexpression substantially changes the shape of the cell's nucleus, resulting in many flower-like nuclei. Both HIV inhibition and deformation of nuclear shape required the domain of SUN2 that interacts with the nuclear lamina. Our results demonstrate that SUN2 interferes with HIV infection and highlight novel links between nuclear shape and viral infection.


Comparison of crystal structures of human androgen receptor ligand-binding domain complexed with various agonists reveals molecular determinants responsible for binding affinity.
Androgens exert their effects by binding to the highly specific androgen receptor (AR). In addition to natural potent androgens, AR binds a variety of synthetic agonist or antagonist molecules with different affinities. To identify molecular determinants responsible for this selectivity, we have determined the crystal structure of the human androgen receptor ligand-binding domain (hARLBD) in complex with two natural androgens, testosterone (Testo) and dihydrotestosterone (DHT), and with an androgenic steroid used in sport doping, tetrahydrogestrinone (THG), at 1.64, 1.90, and 1.75 A resolution, respectively. Comparison of these structures first highlights the flexibility of several residues buried in the ligand-binding pocket that can accommodate a variety of ligand structures. As expected, the ligand structure itself (dimension, presence, and position of unsaturated bonds that influence the geometry of the steroidal nucleus or the electronic properties of the neighboring atoms, etc.) determines the number of interactions it can make with the hARLBD. Indeed, THG--which possesses the highest affinity--establishes more van der Waals contacts with the receptor than the other steroids, whereas the geometry of the atoms forming electrostatic interactions at both extremities of the steroid nucleus seems mainly responsible for the higher affinity measured experimentally for DHT over Testo. Moreover, estimation of the ligand-receptor interaction energy through modeling confirms that even minor modifications in ligand structure have a great impact on the strength of these interactions. Our crystallographic data combined with those obtained by modeling will be helpful in the design of novel molecules with stronger affinity for the AR.


Detection of statistically significant network changes in complex biological networks.
Biological networks contribute effectively to unveil the complex structure of molecular interactions and to discover driver genes especially in cancer context. It can happen that due to gene mutations, as for example when cancer progresses, the gene expression network undergoes some amount of localized re-wiring. The ability to detect statistical relevant changes in the interaction patterns induced by the progression of the disease can lead to the discovery of novel relevant signatures. Several procedures have been recently proposed to detect sub-network differences in pairwise labeled weighted networks. In this paper, we propose an improvement over the state-of-the-art based on the Generalized Hamming Distance adopted for evaluating the topological difference between two networks and estimating its statistical significance. The proposed procedure exploits a more effective model selection criteria to generate p-values for statistical significance and is more efficient in terms of computational time and prediction accuracy than literature methods. Moreover, the structure of the proposed algorithm allows for a faster parallelized implementation. In the case of dense random geometric networks the proposed approach is 10-15x faster and achieves 5-10% higher AUC, Precision/Recall, and Kappa value than the state-of-the-art. We also report the application of the method to dissect the difference between the regulatory networks of IDH-mutant versus IDH-wild-type glioma cancer. In such a case our method is able to identify some recently reported master regulators as well as novel important candidates. We show that our network differencing procedure can effectively and efficiently detect statistical significant network re-wirings in different conditions. When applied to detect the main differences between the networks of IDH-mutant and IDH-wild-type glioma tumors, it correctly selects sub-networks centered on important key regulators of these two different subtypes. In addition, its application highlights several novel candidates that cannot be detected by standard single network-based approaches.


Stroke-Associated Pneumonia: A Retrospective Study of Risk Factors and Outcomes.
Pneumonia is the most common complication after stroke, but our knowledge on risk factors and predictors of stroke-associated pneumonia (SAP) is limited. We sought to evaluate the predictors and outcomes of SAP among acute ischemic stroke (AIS) hospitalizations. This is a cross-sectional study of the Nationwide Inpatient Sample database from the year 2003 to 2014. We identified adult hospitalizations with AIS using International Classification of Diseases, ninth revision, clinical modification (ICD-9-CM) codes. The SAP was identified by the presence of a secondary diagnosis of hospital-acquired pneumonia and ventilator-associated pneumonia. Multivariable survey logistic regression models were utilized to evaluate the predictors of SAP. Overall, 4,224,924 AIS hospitalizations were identified, of which 149,169 (3.53%) had SAP. The prevalence of SAP decreased from 3.72% in 2003 to 3.17% in 2014 (P<0.0001). Mortality [17.12% vs. 4.77%; adjusted odds ratio (aOR): 1.71; P<0.0001] and morbidity (22.53% vs. 3.28%; aOR: 1.86; P<0.0001) were markedly elevated in SAP group compare to non-SAP group. The significant risk factors of pneumonia among AIS hospitalization were nasogastric tube (aOR: 1.21; P=0.0179), noninvasive mechanical ventilation (aOR: 1.65; P<0.0001), invasive mechanical ventilation (aOR: 4.09; P<0.0001), length of stay between 1 to 2 weeks (aOR: 1.99; P<0.0001), >2 weeks (aOR: 3.90; P<0.0001), hemorrhagic conversion (aOR: 1.17; P=0.0002), and epilepsy (aOR: 1.09; P=0.0009). Other concurrent comorbidities which increased the risk of SAP among AIS patients were acquired immune deficiency syndrome (aOR: 1.88; P<0.0001), alcohol abuse (aOR: 1.60; P=0.0006), deficiency anemia (aOR: 1.26; P<0.0001), heart failure (aOR: 1.62; P<0.0001), pulmonary disease (aOR: 1.73; P<0.0001), diabetes (aOR: 1.29; P=0.0288), electrolyte disorders (aOR: 1.50; P<0.0001), paralysis (aOR: 1.22; P<0.0001), pulmonary circulation disorders (aOR: 1.22; P<0.0001), renal failure (aOR: 1.12; P<0.0001), coagulopathy (aOR: 1.13; P=0.0006), and weight loss (aOR: 1.39; P<0.0001). Our data underline the considerable epidemiological and prognostic impact of SAP in patients with AIS leading to higher mortality, morbidity, length of stay, and hospital cost despite advancements in care.


Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins.
A subset of bacterial [NiFe]-hydrogenases have been shown to be capable of activating dihydrogen-catalysis under aerobic conditions; however, it remains relatively unclear how the assembly and activation of these enzymes is carried out in the presence of air. Acquiring this knowledge is important if a generic method for achieving production of O2-resistant [NiFe]-hydrogenases within heterologous hosts is to be developed. Salmonella enterica serovar Typhimurium synthesizes the [NiFe]-hydrogenase-5 (Hyd-5) enzyme under aerobic conditions. As well as structural genes, the Hyd-5 operon also contains several accessory genes that are predicted to be involved in different stages of biosynthesis of the enzyme. In this work, deletions in the hydF, hydG, and hydH genes have been constructed. The hydF gene encodes a protein related to Ralstonia eutropha HoxO, which is known to interact with the small subunit of a [NiFe]-hydrogenase. HydG is predicted to be a fusion of the R. eutropha HoxQ and HoxR proteins, both of which have been implicated in the biosynthesis of an O2-tolerant hydrogenase, and HydH is a homologue of R. eutropha HoxV, which is a scaffold for [NiFe] cofactor assembly. It is shown here that HydG and HydH play essential roles in Hyd-5 biosynthesis. Hyd-5 can be isolated and characterized from a ΔhydF strain, indicating that HydF may not play the same vital role as the orthologous HoxO. This study, therefore, emphasises differences that can be observed when comparing the function of hydrogenase maturases in different biological systems.


Direct Observation of Asphaltene Nanoparticles on Model Mineral Substrates.
The propensity for adherence to solid surfaces of asphaltenes, a complex solubility class of heteropolycyclic aromatic compounds from the heavy fraction of crude oil, has long been the root cause of scale deposition and remains an intractable problem in the petroleum industry. Although the adhesion is essential to understanding the process of asphaltene deposition, the relationship between the conformation of asphaltene molecules on mineral substrates and its impact on adhesion and mechanical properties of the deposits is not completely understood. To rationalize the primary processes in the process of organic scale deposition, here we use atomic force microscopy (AFM) to visualize the morphology of petroleum asphaltenes deposited on model mineral substrates. High imaging contrast was achieved by the differential adhesion of the tip between asphaltenes and the mineral substrate. While asphaltenes form smooth continuous films on all substrates at higher concentrations, they deposit as individual nanoparticles at lower concentrations. The size, shape, and spatial distribution of the nanoaggregates are strongly affected by the nature of the substrate; while uniformly distributed spherical particles are formed on highly polar and hydrophilic substrates (mica), irregular islands and thicker patches are observed with substrates of lower polarity (silica and calcite). Asphaltene nanoparticles flatten when adsorbed on highly oriented pyrolytic graphite due to π-π interactions with the polycyclic core. Force-distance profiles provide direct evidence of the conformational changes of asphaltene molecules on hydrophilic/hydrophobic substrates that result in dramatic changes in adhesion and mechanical properties of asphaltene deposits. Such an understanding of the nature of adhesion and mechanical properties tuned by surface properties, on the level of asphaltene nanoaggregates, would contribute to the design of efficient asphaltene inhibitors for preventing asphaltene fouling on targeted surfaces. Unlike flat surfaces, the AFM phase contrast images of defected calcite surfaces show that asphaltenes form continuous deposits to fill the recesses, and this process could trigger the onset for asphaltene deposition.


Mode-selective mapping and control of vectorial nonlinear-optical processes in multimode photonic-crystal fibers.
We demonstrate an experimental technique that allows a mapping of vectorial nonlinear-optical processes in multimode photonic-crystal fibers (PCFs). Spatial and polarization modes of PCFs are selectively excited in this technique by varying the tilt angle of the input beam and rotating the polarization of the input field. Intensity spectra of the PCF output plotted as a function of the input field power and polarization then yield mode-resolved maps of nonlinear-optical interactions in multimode PCFs, facilitating the analysis and control of nonlinear-optical transformations of ultrashort laser pulses in such fibers.


Image data sharing for biomedical research--meeting HIPAA requirements for De-identification.
Data sharing is increasingly recognized as critical to cross-disciplinary research and to assuring scientific validity. Despite National Institutes of Health and National Science Foundation policies encouraging data sharing by grantees, little data sharing of clinical data has in fact occurred. A principal reason often given is the potential of inadvertent violation of the Health Insurance Portability and Accountability Act privacy regulations. While regulations specify the components of private health information that should be protected, there are no commonly accepted methods to de-identify clinical data objects such as images. This leads institutions to take conservative risk-averse positions on data sharing. In imaging trials, where images are coded according to the Digital Imaging and Communications in Medicine (DICOM) standard, the complexity of the data objects and the flexibility of the DICOM standard have made it especially difficult to meet privacy protection objectives. The recent release of DICOM Supplement 142 on image de-identification has removed much of this impediment. This article describes the development of an open-source software suite that implements DICOM Supplement 142 as part of the National Biomedical Imaging Archive (NBIA). It also describes the lessons learned by the authors as NBIA has acquired more than 20 image collections encompassing over 30 million images.


Pleiotropy of segregating genetic variants that affect honey bee worker life expectancy.
In contrast to many other complex traits, the natural genetic architecture of life expectancy has not been intensely studied, particularly in non-model organisms, such as the honey bee (Apis mellifera L.). Multiple factors that determine honey bee worker lifespan have been identified and genetic analyses have been performed on some of those traits. Several of the traits are included in a suite of correlated traits that form the pollen hoarding syndrome, which was named after the behavior to store surplus pollen in the nest and is tied to social evolution. Here, seven quantitative trait loci that had previously been identified for their effects on different aspects of the pollen hoarding syndrome were studied for their genetic influence on the survival of adult honey bee workers. To gain a more comprehensive understanding of the genetic architecture of worker longevity, a panel of 280 additional SNP markers distributed across the genome was also tested. Allelic distributions were compared between young and old bees in two backcross populations of the bi-directionally selected high- and low-pollen hoarding strain. Our results suggest a pleiotropic effect of at least one of the behavioral quantitative trait loci on worker longevity and one significant and several other putative genetic effects in other genomic regions. At least one locus showed evidence for strong antagonistic pleiotropy and several others suggested genetic factors that influence pre-emergence survival of worker honey bees. Thus, the predicted association between worker lifespan and the pollen hoarding syndrome was supported at the genetic level and the magnitude of the identified effects also strengthened the view that naturally segregating genetic variation can have major effects on age-specific survival probability in the wild.


Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer.
The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion functions. S also contains the principal antigenic determinants and is the target of neutralizing antibodies. Here we present the structure of a mouse coronavirus S trimer ectodomain determined at 4.0 Å resolution by single particle cryo-electron microscopy. It reveals the metastable pre-fusion architecture of S and highlights key interactions stabilizing it. The structure shares a common core with paramyxovirus F proteins, implicating mechanistic similarities and an evolutionary connection between these viral fusion proteins. The accessibility of the highly conserved fusion peptide at the periphery of the trimer indicates potential vaccinology strategies to elicit broadly neutralizing antibodies against coronaviruses. Finally, comparison with crystal structures of human coronavirus S domains allows rationalization of the molecular basis for species specificity based on the use of spatially contiguous but distinct domains.


Similar Ratios of Introns to Intergenic Sequence across Animal Genomes.
One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here, we analyze the genomes of 68 species across 12 animal phyla and some single-cell eukaryotes for general trends in genome composition and transcription, taking into account problems of gene annotation. We show that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of nonmodel organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought.


Entrustable Professional Activities for Pathology: Recommendations From the College of American Pathologists Graduate Medical Education Committee.
Competency-based medical education has evolved over the past decades to include the Accreditation Council for Graduate Medical Education Accreditation System of resident evaluation based on the Milestones project. Entrustable professional activities represent another means to determine learner proficiency and evaluate educational outcomes in the workplace and training environment. The objective of this project was to develop entrustable professional activities for pathology graduate medical education encompassing primary anatomic and clinical pathology residency training. The Graduate Medical Education Committee of the College of American Pathologists met over the course of 2 years to identify and define entrustable professional activities for pathology graduate medical education. Nineteen entrustable professional activities were developed, including 7 for anatomic pathology, 4 for clinical pathology, and 8 that apply to both disciplines with 5 of these concerning laboratory management. The content defined for each entrustable professional activity includes the entrustable professional activity title, a description of the knowledge and skills required for competent performance, mapping to relevant Accreditation Council for Graduate Medical Education Milestone subcompetencies, and general assessment methods. Many critical activities that define the practice of pathology fit well within the entrustable professional activity model. The entrustable professional activities outlined by the Graduate Medical Education Committee are meant to provide an initial framework for the development of entrustable professional activity-related assessment and curricular tools for pathology residency training.


Micro-simulation of a smallpox outbreak using official register data.
To explore the efficacy of four vaccine-based policy strategies (ring vaccination, targeted vaccination, mass vaccination, and pre-vaccination of healthcare personnel combined with ring vaccination) for controlling smallpox outbreaks in Sweden, disease transmission on a spatially explicit social network was simulated. The mixing network was formed from high-coverage official register data of the entire Swedish population, building on the Swedish Total Population Register, the Swedish Employment Register, and the Geographic Database of Sweden. The largest reduction measured in the number of infections was achieved when combining ring vaccination with a pre-vaccination of healthcare personnel. In terms of per dose effectiveness, ring vaccination was by far the most effective strategy. The results can to some extent be adapted to other diseases and environments, including other countries, and the methods used can be analysed in their own right.


Vasculogenesis of the embryonic heart: origin of blood island-like structures.
The earliest vascular structures (blood island-like) in the embryonic heart are clusters of angioblasts and nucleated red blood cells (NRBCs), which differentiate into endothelial cells and erythrocytes, respectively. Our purpose was to define the area and chronology of NRBC appearance in the mouse embryonic heart at the stages before a patency between coronary vessels and peripheral circulation is established (10.5-13.5 dpc). Before and at the onset of vascularization, NBCs were not present within the proepicardium; however, Ter/119+ differentiating erythroblasts and single scattered CD45+ were found in the heart beginning from 10.5 dpc. The Ter/119+ cells were in close apposition to angioblasts (PECAM1+) and were recognized as components of blood island-like structures or vascular vesicles in transmission electron microscope and were located mostly in the subepicardium. Some of the NRBCs were not accompanied by angioblasts and located close to the endocardial endothelium or at the border of the endocardial endothelium or in the subepicardium. These erythroblasts were beginning to assemble with angioblasts. CD34+ NBCs as well as progenitor cells of erythroid lineage were not detected in the heart at these stages of development. The state of differentiation of NRBCs of blood islands was similar/the same as the morphology of circulating blood cells at the respective stages of embryo development. The presence of mature NRBCs in the subendocardial area and lack of progenitor cells of erythroid lineage within the heart indicate that erythroid commitment occurs outside the heart. We suggest that NRBCs enter the heart from the blood stream at 10.5-12 dpc independently from angioblasts.


Yeast DNA damage-inducible Rnr3 has a very low catalytic activity strongly stimulated after the formation of a cross-talking Rnr1/Rnr3 complex.
The ribonucleotide reductase system in Saccharomyces cerevisiae includes four genes (RNR1 and RNR3 encoding the large subunit and RNR2 and RNR4 encoding the small subunit). RNR3 expression, nearly undetectable during normal growth, is strongly induced by DNA damage. Yet an rnr3 null mutant has no obvious phenotype even under DNA damaging conditions, and the contribution of RNR3 to ribonucleotide reduction is not clear. To investigate the role of RNR3 we expressed and characterized the Rnr3 protein. The in vitro activity of Rnr3 was less than 1% of the Rnr1 activity. However, a strong synergism between Rnr3 and Rnr1 was observed, most clearly demonstrated in experiments with the catalytically inactive Rnr1-C428A mutant, which increased the endogenous activity of Rnr3 by at least 10-fold. In vivo, the levels of Rnr3 after DNA damage never reached more than one-tenth of the Rnr1 levels. We propose that heterodimerization of Rnr3 with Rnr1 facilitates the recruitment of Rnr3 to the ribonucleotide reductase holoenzyme, which may be important when Rnr1 is limiting for dNTP production. In complex with inactive Rnr1-C428A, the activity of Rnr3 is controlled by effector binding to Rnr1-C428A. This result indicates cross-talk between the Rnr1 and Rnr3 polypeptides of the large subunit.


The plasma-membrane component is the primary site of action of alloxan on ATP-driven Ca2+ transport in vascular-muscle microsomal fractions.
The direct effects of alloxan in vitro on the handling of Ca2+ by microsomal fractions from dog aortic smooth muscle were investigated. Preincubation of the vascular-muscle microsomal membranes with alloxan showed a suppression of both binding of Ca2+ (in the absence of ATP) and ATP-driven Ca2+ transport. Alloxan substantially inhibited the microsomal ATP-driven Ca2+ transport stimulated by Pi, but not that stimulated by oxalate. Studies using subfractions isolated from the microsomal membranes on a sucrose density gradient indicated that plasma membrane is the primary site of action of alloxan on the ATP-driven Ca2+ transport.


Decoupling Control of Micromachined Spinning-Rotor Gyroscope with Electrostatic Suspension.
A micromachined gyroscope in which a high-speed spinning rotor is suspended electrostatically in a vacuum cavity usually functions as a dual-axis angular rate sensor. An inherent coupling error between the two sensing axes exists owing to the angular motion of the spinning rotor being controlled by a torque-rebalance loop. In this paper, a decoupling compensation method is proposed and investigated experimentally based on an electrostatically suspended micromachined gyroscope. In order to eliminate the negative spring effect inherent in the gyroscope dynamics, a stiffness compensation scheme was utilized in design of the decoupled rebalance loop to ensure loop stability and increase suspension stiffness. The experimental results show an overall stiffness increase of 30.3% after compensation. A decoupling method comprised of inner- and outer-loop decoupling compensators is proposed to minimize the cross-axis coupling error. The inner-loop decoupling compensator aims to attenuate the angular position coupling. The experimental frequency response shows a position coupling attenuation by 14.36 dB at 1 Hz. Moreover, the cross-axis coupling between the two angular rate output signals can be attenuated theoretically from -56.2 dB down to -102 dB by further appending the outer-loop decoupling compensator. The proposed dual-loop decoupling compensation algorithm could be applied to other dual-axis spinning-rotor gyroscopes with various suspension solutions.


Down-regulation of microRNAs controlling tumourigenic factors in follicular thyroid carcinoma.
The molecular determinants of thyroid follicular nodules are incompletely understood and assessment of malignancy is a diagnostic challenge. Since microRNA (miRNA) analyses could provide new leads to malignant progression, we characterised the global miRNA expression in follicular adenoma (FA) and follicular carcinoma (FC). Comparison of carcinoma and adenoma with normal thyroid revealed 150 and 107 differentially expressed miRNAs respectively. Most miRNAs were down-regulated and especially miR-199b-5p and miR-144 which were essentially lost in the carcinomas. Integration of the changed miRNAs with differentially expressed mRNAs demonstrated an enrichment of seed sites among up-regulated transcripts encoding proteins implicated in thyroid tumourigenesis. This was substantiated by the demonstration that pre-miR-199b reduced proliferation when added to cultured follicular thyroid carcinoma cells. The down-regulated miRNAs in FC exhibited a substantial similarity with down-regulated miRNAs in anaplastic carcinoma (AC) and by gene set enrichment analysis, we observed a significant identity between target mRNAs in FC and transcripts up-regulated in AC. To examine the diagnostic potential of miRNA expression pattern in distinguishing malignant from benign nodules we employed a supervised learning algorithm and leave-one-out-cross-validation. By this procedure, FA and FC were identified with a negative predicted value of 83% (data generated by microarray platform) and of 92% (data generated by qRT-PCR platform). We conclude that follicular neoplasia is associated with major changes in miRNA expression that may promote malignant transformation by increasing the expression of transcripts encoding tumourigenic factors. Moreover, miRNA profiling may facilitate the diagnosis of carcinoma vs adenoma.


Vitamin E in the treatment of tardive dyskinesia.
In a double-blind placebo controlled trial, the efficacy of Vitamin E in the treatment of tardive dyskinesia (TD) was studied in 32 patients. After a two week wash-out phase a baseline (0 week) TD rating was assessed on the tardive dyskinesia rating scale (TDRS). Subsequently, the patients entered a four week treatment phase during which 17 patients received capsules of vitamin E (600 mg) and 15 patients received identical placebo capsules. In the first week the patients received 1 capsule daily which was then increased to two capsules per day from the second to the fourth week. All patients were rated on the TDRS at the end of each week. The baseline TDRS score in the vitamin E group was significantly higher than the placebo group. This was hence adjusted and the results were then subjected to analysis of co- variance. The TDRS score after four weeks treatment was significantly lower in the vitamin E group as compared to the placebo group (p = 0.03).


Prognostic implications of global LV dysfunction: a systematic review and meta-analysis of global longitudinal strain and ejection fraction.
Global longitudinal strain (GLS) is a robust, well validated and reproducible technique for the measurement of LV longitudinal deformation. We sought to assemble evidence that GLS is an accurate marker in predicting cardiovascular outcomes, compared to LVEF. We undertook a systematic review of the evidence from observational studies which compared GLS against LVEF in predicting major adverse cardiac events. The primary outcome was all-cause mortality. The secondary outcome was a composite of cardiac death, malignant arrhythmia, hospitalisation due to heart failure, urgent valve surgery or heart transplantation, and acute coronary ischaemic event. A random effects model was used to combine HR and 95% CIs. A meta-regression was undertaken to assess the impact of potential covariates. Data were collated from 16 published articles (n=5721 adults) comprising 15 prospective and 1 retrospective observational studies. The underlying cardiac conditions were heart failure, acute myocardial infarction, valvular heart disease, and miscellaneous cardiac diseases. Mortality was independently associated with each SD change in the absolute value of baseline GLS (HR 0.50, 95% CI 0.36 to 0.69; p<0.002) and less strongly with LVEF (HR 0.81, 95% CI 0.72 to 0.92; p=0.572). The HR per SD change in GLS was associated with a reduction in mortality 1.62 (95% CI 1.13 to 2.33; p=0.009) times greater than the HR per SD change in LVEF. There is strong evidence of the prognostic value of GLS, which appears to have superior prognostic value to EF for predicting major adverse cardiac events.


The great time series classification bake off: a review and experimental evaluation of recent algorithmic advances.
In the last 5 years there have been a large number of new time series classification algorithms proposed in the literature. These algorithms have been evaluated on subsets of the 47 data sets in the University of California, Riverside time series classification archive. The archive has recently been expanded to 85 data sets, over half of which have been donated by researchers at the University of East Anglia. Aspects of previous evaluations have made comparisons between algorithms difficult. For example, several different programming languages have been used, experiments involved a single train/test split and some used normalised data whilst others did not. The relaunch of the archive provides a timely opportunity to thoroughly evaluate algorithms on a larger number of datasets. We have implemented 18 recently proposed algorithms in a common Java framework and compared them against two standard benchmark classifiers (and each other) by performing 100 resampling experiments on each of the 85 datasets. We use these results to test several hypotheses relating to whether the algorithms are significantly more accurate than the benchmarks and each other. Our results indicate that only nine of these algorithms are significantly more accurate than both benchmarks and that one classifier, the collective of transformation ensembles, is significantly more accurate than all of the others. All of our experiments and results are reproducible: we release all of our code, results and experimental details and we hope these experiments form the basis for more robust testing of new algorithms in the future.


Cross-cultural adaptation and validation of the voice handicap index into Greek.
The objective was to culturally adapt and validate the Voice Handicap Index (VHI) to the Greek language. The study design used was a psychometric analysis. The VHI was translated into Greek with cultural adaptations to accommodate certain words. The translated version was then completed by 67 subjects with various voice disorders and by a control group of 79 subjects. All the participants also completed a self-rating scale regarding the severity of their voice disorder. Statistical analyses demonstrated high internal consistency and high test-retest reliability both for the overall VHI score and for the functional, physical, and emotional domains of the VHI. A moderate correlation was found between the VHI and the self-rating severity scale. The subjects in the control group had lower scores compared to the subjects with voice disorders for the overall VHI score and for the three domains. Based on the internal consistency values and the test-retest reliability, the Greek version of VHI is a valid and reliable measure for use by Greek subjects with voice disorders.


Genetic association studies in patients with traumatic brain injury.
Traumatic brain injury (TBI) constitutes a major cause of mortality and disability worldwide, especially among young individuals. It is estimated that despite all the recent advances in the management of TBI, approximately half of the patients suffering head injuries still have unfavorable outcomes, which represents a substantial health care, social, and economic burden to societies. Considerable variability exists in the clinical outcome after TBI, which is only partially explained by known factors. Accumulating evidence has implicated various genetic elements in the pathophysiology of brain trauma. The extent of brain injury after TBI seems to be modulated to some degree by genetic variants. The authors' current review focuses on the up-to-date state of knowledge regarding genetic association studies in patients sustaining TBI, with particular emphasis on the mechanisms underlying the implication of genes in the pathophysiology of TBI.


Darwin and the neural bases of emotion and affective style.
This article presents an overview of ways to think about the brain and emotion and consider the role of evolution and expression in shaping the neural circuitry of affective processing. Issues pertaining to whether there are separate unique neural modules hard-wired for emotion processing or whether affective processing uses more generalized circuitry are considered. Relations between affect and cognition--specifically, memory--are examined from the perspective of overlapping neural systems. The role of individual differences in neural function in affective style are discussed, and the concepts of affective chronometry, or the time course of emotional responding and emotion regulation, are introduced. Finally, the extent to which certain emotional traits can be viewed as trainable skills is considered, and the relevance of work on neural plasticity to the skill framework is addressed. Data from a variety of sources using different types of measures is brought to bear on these questions, including neuroimaging and psychophysiological measures, studies of individuals of different ages ranging from early childhood to old age, studies of nonhuman primates, and observations of patients with localized brain damage. Emotions are viewed as varying in both type and dimension. Honoring brain circuitry in parsing the domain of affects will result in distinctions and differentiations that are not currently incorporated in traditional classification schemes.


Locals, incomers and intra-national migration: place-identities and a Scottish island.
In a trans-national context, immigrants are often described as essentially different from existing residents. However these descriptions of group differences are occasioned constructions of immigrants, and talk about nations provides frameworks of history and space within which immigration is understood. Claimed group differences and the proposed commonality of nation together present a challenging context for immigrants to negotiate identities and to gain acceptance. Drawing on the concept of place-identity, we examined here whether similar issues arise in intra-national migration to a remote Scottish island. We conducted semi-structured interviews with individuals who had lived on the island for periods ranging from 14 months to 20 years. The interviewees described island residents as comprising different groups, in terms such as residence, motivation, place of birth, and connections to other locations. The interviewees negotiated place-identities that compared favourably with others with more transitory connections but unfavourably with residents of longer-standing. Findings show that spatial connections can be used to account for varying degrees of social status in such locations. But some issues relevant to trans-national immigration still arise in intra-national migration, even in the absence of racial, ethnic, religious, or language differences. In short, 'incomers' cannot readily do 'being local'.


Dynamic effects of antibody-dependent enhancement on the fitness of viruses.
Antibody-dependent enhancement (ADE), a phenomenon in which viral replication is increased rather than decreased by immune sera, has been observed in vitro for a large number of viruses of public health importance, including flaviviruses, coronaviruses, and retroviruses. The most striking in vivo example of ADE in humans is dengue hemorrhagic fever, a disease in which ADE is thought to increase the severity of clinical manifestations of dengue virus infection by increasing virus replication. We examine the epidemiological impact of ADE on the prevalence and persistence of viral serotypes. Using a dynamical system model of n cocirculating dengue serotypes, we find that ADE may provide a competitive advantage to those serotypes that undergo enhancement compared with those that do not, and that this advantage increases with increasing numbers of cocirculating serotypes. Paradoxically, there are limits to the selective advantage provided by increasing levels of ADE, because greater levels of enhancement induce large amplitude oscillations in incidence of all dengue virus infections, threatening the persistence of both the enhanced and nonenhanced serotypes. Although the models presented here are specifically designed for dengue, our results are applicable to any epidemiological system in which partial immunity increases pathogen replication rates. Our results suggest that enhancement is most advantageous in settings where multiple serotypes circulate and where a large host population is available to support pathogen persistence during the deep troughs of ADE-induced large amplitude oscillations of virus replication.


Predicting death or extended length of stay in infants with congenital diaphragmatic hernia.
To predict mortality or length of stay (LOS) >109 days (90th percentile) among infants with congenital diaphragmatic hernia (CDH). We conducted a retrospective analysis using the Children's Hospital Neonatal Database during 2010 to 2014. Infants born >34 weeks gestation with CDH admitted at 22 participating regional neonatal intensive care units were included; patients who were repaired or were at home before admission were excluded. The primary outcome was death before discharge or LOS >109 days. Factors associated with this outcome were used to develop a multivariable equation using 80% of the cohort. Validation was performed in the remaining 20% of infants. The median gestation and age at referral in this cohort (n=677) were 38 weeks and 6 h, respectively. The primary outcome occurred in 242 (35.7%) infants, and was distributed between mortality (n=180, 27%) and LOS >109 days (n=66, 10%). Regression analyses showed that small for gestational age (odds ratio (OR) 2.5, P=0.008), presence of major birth anomalies (OR 5.9, P<0.0001), 5- min Apgar score ⩽3 (OR 7.0, P=0.0002), gradient of acidosis at the time of referral (P<0.001), the receipt of extracorporeal support (OR 8.4, P<0.0001) and bloodstream infections (OR 2.2, P=0.004) were independently associated with death or LOS >109 days. This model performed well in the validation cohort (area under curve (AUC)=0.856, goodness-of-fit (GF) χ(2), P=0.16) and acted similarly even after omitting extracorporeal support (AUC=0.82, GF χ(2), P=0.05). Six variables predicted death or LOS ⩾109 days in this large, contemporary cohort with CDH. These results can assist in risk adjustment for comparative benchmarking and for counseling affected families.


SEC23B is required for the maintenance of murine professional secretory tissues.
In eukaryotic cells, newly synthesized secretory proteins require COPII (coat protein complex II) to exit the endoplasmic reticulum (ER). COPII contains five core components: SAR1, SEC23, SEC24, SEC13, and SEC31. SEC23 is a GTPase-activating protein that activates the SAR1 GTPase and also plays a role in cargo recognition. Missense mutations in the human COPII paralogues SEC23A and SEC23B result in craniolenticulosutural dysplasia and congenital dyserythropoietic anemia type II, respectively. We now report that mice completely deficient for SEC23B are born with no apparent anemia phenotype, but die shortly after birth, with degeneration of professional secretory tissues. In SEC23B-deficient embryonic pancreas, defects occur in exocrine and endocrine tissues shortly after differentiation. Pancreatic acini are completely devoid of zymogen granules, and the ER is severely distended. Similar ultrastructural alterations are also observed in salivary glands, but not in liver. Accumulation of proteins in the ER lumen activates the proapoptotic pathway of the unfolded protein response, suggesting a central role for apoptosis in the degeneration of these tissues in SEC23B-deficient embryos. Although maintenance of the secretory pathway should be required by all cells, our findings reveal a surprising tissue-specific dependence on SEC23B for the ER exit of highly abundant cargo, with high levels of SEC23B expression observed in professional secretory tissues. The disparate phenotypes in mouse and human could result from residual SEC23B function associated with the hypomorphic mutations observed in humans, or alternatively, might be explained by a species-specific shift in function between the closely related SEC23 paralogues.


Prevalence of dementia in an urban Indian population.
This article reports the findings of a 3-year epidemiological survey for dementia in an urban community-resident population in Mumbai (Bombay), India, wherein the prevalence of all types of dementia was determined. The study was conducted in three stages. Stage 1: From a potential pool of 30,000 subjects aged 40 years or more, 24,488 (male = 11,875; female = 12,613) persons completed self-report or interviewer-rated protocols based on the Sandoz Clinical Assessment Geriatric Scale, but 5,512 (18.37%) persons refused to participate. Scores on the protocol had a possible range from 0 through 34. Stage 2: Persons with a score +2 SD above the mean were selected in this stage where the persons were screened for cognitive functioning using a modified and translated version of the Mini-Mental State Examination. Individuals who scored below the 5th percentile were included in Stage 3 and underwent a detailed neurological, psychiatric, and neuropsychological evaluation as well as hematological, radiological, electrocardiographic, and electroencephalographic investigations. Diagnoses were made jointly by a neurologist, psychiatrist, and psychologist using the DSM-IV diagnostic criteria. Subjects were also rated on the Clinical Dementia Rating (CDR) scale and assessed for activities of daily living. One hundred five subjects with dementia (CDR > or = 0.5) were identified in this population of 24,488 persons. The prevalence rate for dementia in those aged 40 years and more was 0.43% and for persons aged 65 and above was 2.44%. Seventy-eight individuals had a CDR of > or = 1 yielding an overall prevalence rate of 0.32%, and a prevalence rate of 1.81% for those aged 65 years and older. The overall prevalence rate for Alzheimer's disease (AD) in the population was 0.25%, and 1.5% for those aged 65 years and above. AD (n = 62; 65%) was the most common cause of dementia followed byvascular dementia (n = 23; 22%). There were more women (n = 38) than men (n = 24) in the AD group. Increasing age was associated with a higher prevalence of the dementia syndrome in general as well as AD specifically. In the population surveyed, the prevalence of AD and other dementias is less than that reported from developed countries but similar to results of other studies in India. Prevalence of the dementia syndrome increased with age and was not related to gender. AD was the most common dementia and the prevalence was higher in women than in men. Results are discussed with respect to shorter life expectancy, relocation of affected persons, and differences in the risk factors as compared to developed countries.


Astrogliosis in a dish: substrate stiffness induces astrogliosis in primary rat astrocytes.
Astrogliosis due to brain injury or disease can lead to varying molecular and morphological changes in astrocytes. Magnetic resonance elastography and ultrasound have demonstrated that brain stiffness varies with age and disease state. However, there is a lack in understanding the role of varied stiffness on the progression of astrogliosis highlighting a critical need to engineer in vitro models that mimic disease stages. Such models need to incorporate the dynamic changes in the brain microenvironment including the stiffness changes. In this study we developed a polydimethyl siloxane (PDMS) based platform that modeled the physiologically relevant stiffness of brain in both a healthy (200 Pa) and diseased (8000 Pa) state to investigate the effect of stiffness on astrocyte function. We observed that astrocytes grown on soft substrates displayed a consistently more quiescent phenotype while those on stiff substrates displayed an astrogliosis-like morphology. In addition to morphological changes, astrocytes cultured on stiff substrates demonstrated significant increase in other astrogliosis hallmarks - cellular proliferation and glial fibrillary acidic protein (GFAP) protein expression. Furthermore, culturing astrocytes on a stiff surface resulted in increased reactive oxygen species (ROS) production, increased super oxide dismutase activity and decreased glutamate uptake. Our platform lends itself for study of potential therapeutic strategies for brain injury focusing on the intricate brain microenvironment-astrocytes signaling pathways.


The prevalence of major anxiety disorders in relatives of alcohol dependent men and women.
The relationship between alcohol dependence and lifelong major anxiety disorders is complex. The literature indicates a close association between anxiety symptoms and drinking behavior. However, it is difficult to determine whether the anxiety conditions are lifelong disorders or if they represent temporary organic conditions related to alcohol intoxication and withdrawal. One approach to understanding more about the relationships between alcohol dependence and major anxiety disorders is to observe the rate of anxiety-related diagnoses in close relatives of alcoholics. This approach evaluates whether alcoholism and major anxiety disorders might share a common genetic basis. The data presented here describe the rates of four major anxiety disorders in 591 interviewed first-degree relatives of alcohol dependent men and women. The data were gathered through face-to-face structured standardized interviews. The analyses reveal that after focusing on DSM-III-R anxiety disorders, controlling for the potential presence of temporary organic conditions in the subject and considering the impact of assortative mating in their parents, the life-time risk for panic disorder in close biological family members of alcoholics is 3.4%; for agoraphobia, 1.4%; for social phobia, 2.3%; and for obsessive-compulsive disease, 1.4%. These data do not indicate an exceptionally high rate of anxiety disorders among close relatives of alcoholics. While other mechanisms might contribute to relationship between alcoholism and major anxiety disorders, the results do not support evidence of a common genotype for the two disorders.


Reasons for failures of oral implants.
This study reviews the literature regarding the factors contributing to failures of dental implants. An electronic search was undertaken including papers from 2004 onwards. The titles and abstracts from these results were read to identify studies within the selection criteria. All reference lists of the selected studies were then hand-searched, this time without time restrictions. A narrative review discussed some findings from the first two parts where separate data from non-comparative studies may have indicated conclusions different from those possible to draw in the systematic analysis. It may be suggested that the following situations are correlated to increase the implant failure rate: a low insertion torque of implants that are planned to be immediately or early loaded, inexperienced surgeons inserting the implants, implant insertion in the maxilla, implant insertion in the posterior region of the jaws, implants in heavy smokers, implant insertion in bone qualities type III and IV, implant insertion in places with small bone volumes, use of shorter length implants, greater number of implants placed per patient, lack of initial implant stability, use of cylindrical (non-threaded) implants and prosthetic rehabilitation with implant-supported overdentures. Moreover, it may be suggested that the following situations may be correlated with an increase in the implant failure rate: use of the non-submerged technique, immediate loading, implant insertion in fresh extraction sockets, smaller diameter implants. Some recently published studies suggest that modern, moderately rough implants may present with similar results irrespective if placed in maxillas, in smoking patients or using only short implants.


Imaging technologies for preclinical models of bone and joint disorders.
Preclinical models for musculoskeletal disorders are critical for understanding the pathogenesis of bone and joint disorders in humans and the development of effective therapies. The assessment of these models primarily relies on morphological analysis which remains time consuming and costly, requiring large numbers of animals to be tested through different stages of the disease. The implementation of preclinical imaging represents a keystone in the refinement of animal models allowing longitudinal studies and enabling a powerful, non-invasive and clinically translatable way for monitoring disease progression in real time. Our aim is to highlight examples that demonstrate the advantages and limitations of different imaging modalities including magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT) and optical imaging. All of which are in current use in preclinical skeletal research. MRI can provide high resolution of soft tissue structures, but imaging requires comparatively long acquisition times; hence, animals require long-term anaesthesia. CT is extensively used in bone and joint disorders providing excellent spatial resolution and good contrast for bone imaging. Despite its excellent structural assessment of mineralized structures, CT does not provide in vivo functional information of ongoing biological processes. Nuclear medicine is a very promising tool for investigating functional and molecular processes in vivo with new tracers becoming available as biomarkers. The combined use of imaging modalities also holds significant potential for the assessment of disease pathogenesis in animal models of musculoskeletal disorders, minimising the use of conventional invasive methods and animal redundancy.


T cell-derived tumor necrosis factor induces cytotoxicity by activating RIPK1-dependent target cell death.
TNF ligation of TNF receptor 1 (TNFR1) promotes either inflammation and cell survival by (a) inhibiting RIPK1's death-signaling function and activating NF-κB or (b) causing RIPK1 to associate with the death-inducing signaling complex to initiate apoptosis or necroptosis. The cellular source of TNF that results in RIPK1-dependent cell death remains unclear. To address this, we employed in vitro systems and murine models of T cell-dependent transplant or tumor rejection in which target cell susceptibility to RIPK1-dependent cell death could be genetically altered. We show that TNF released by T cells is necessary and sufficient to activate RIPK1-dependent cell death in target cells and thereby mediate target cell cytolysis independently of T cell frequency. Activation of the RIPK1-dependent cell death program in target cells by T cell-derived TNF accelerates murine cardiac allograft rejection and synergizes with anti-PD1 administration to destroy checkpoint blockade-resistant murine melanoma. Together, the findings uncover a distinct immunological role for TNF released by cytotoxic effector T cells following cognate interactions with their antigenic targets. Manipulating T cell TNF and/or target cell susceptibility to RIPK1-dependent cell death can be exploited to either mitigate or augment T cell-dependent destruction of allografts and malignancies to improve outcomes.


COVID-19 and Pacific food system resilience: opportunities to build a robust response.
The unfolding COVID-19 pandemic has exposed the vulnerability of the Pacific food system to externalities and has had far-reaching impacts, despite the small number of COVID-19 cases recorded thus far. Measures adopted to mitigate risk from the pandemic have had severe impacts on tourism, remittances, and international trade, among other aspects of the political economy of the region, and are thus impacting on food systems, food security and livelihoods. Of particular concern will be the interplay between loss of incomes and the availability and affordability of local and imported foods. In this paper, we examine some of the key pathways of impact on food systems, and identify opportunities to strengthen Pacific food systems during these challenging times. The great diversity among Pacific Island Countries and Territories in their economies, societies, and agricultural potential will be an important guide to planning interventions and developing scenarios of alternative futures. Bolstering regional production and intraregional trade in a currently import-dependent region could strengthen the regional economy, and provide the health benefits of consuming locally produced and harvested fresh foods - as well as decreasing reliance on global supply chains. However, significant production, processing, and storage challenges remain and would need to be consistently overcome to influence a move away from shelf-stable foods, particularly during periods when human movement is restricted and during post-disaster recovery.


Therapeutic plants of Ayurveda: a review of selected clinical and other studies for 166 species.
This paper reports on the results of a literature survey involving 166 different species of plants used in the Ayurvedic pharmacopoeia, based on a sampling of the literature available to us. We found a wide range of clinical and other in vivo studies for many of the plant-based therapies utilized in the Ayurvedic system. Of the 166 plants investigated, 72 (43%) had at least one or more human studies and 103 (62%) had one or more animal studies. These results appear to contradict the generally held notion that herbal remedies used in non-Western systems of botanical medicine have not been evaluated in human or in vivo trials. Some of these studies are not always as large or methodologically rigorous as clinical studies reported in major medical journals. Indeed, a critical assessment of the research according to the standards of evidence-based medicine would eliminate many of these studies for lack of rigor according to criteria of randomization, sample size, adequacy of controls, etc. However, the studies do suggest which species might be appropriate for larger and better-controlled trials in the future. Accordingly, a synopsis of the plants, their therapeutic applications, and their clinical or experimental evaluations is presented.


On Biomineralization: Enzymes Switch on Mesocrystal Assembly.
Cellular machineries guide the bottom-up pathways toward crystal superstructures based on the transport of inorganic precursors and their precise integration with organic frameworks. The biosynthesis of mesocrystalline spines entails concerted interactions between biomolecules and inorganic precursors; however, the bioinorganic interactions and interfaces that regulate material form and growth as well as the selective emergence of structural complexity in the form of nanostructured crystals are not clear. By investigating mineral nucleation under the regulation of recombinant proteins, we show that SpSM50, a matrix protein of the sea urchin spine, stabilizes mineral precursors via vesicle-confinement, a function conferred by a low-complexity, disordered region. Site-specific proteolysis of this domain by a collagenase initiates phase transformation of the confined mineral phase. The residual C-type lectin domain molds the fluidic mineral precursor into hierarchical mesocrystals identical to structural crystal modules constituting the biogenic mineral. Thus, the regulatory functions of proteolytic enzymes can guide biomacromolecular domain constitutions and interfaces, in turn determining inorganic phase transformations toward hybrid materials as well as integrating organic and inorganic components across hierarchical length scales. Bearing striking resemblance to biogenic mineralization, these hybrid materials recruit bioinorganic interactions which elegantly intertwine nucleation and crystallization phenomena with biomolecular structural dynamics, hence elucidating a long-sought key of how nature can orchestrate complex biomineralization processes.


Sexual conflict and the evolution of female mate choice and male social dominance.
Conflicts between the sexes over control of reproduction are thought to lead to a cost of sexual selection through the evolution of male traits that manipulate female reproductive physiology and behaviour, and female traits that resist this manipulation. Although studies have begun to document negative fitness effects of sexual conflict, studies showing the expected association between sexual conflict and the specific behavioural mechanisms of sexual selection are lacking. Here we experimentally manipulated the opportunity for sexual conflict in the cockroach. Nauphoeta cinerea and showed that, for this species, odour cues in the social environment influence the behavioural strategies and fitness of males and females during sexual selection. Females provided with the opportunity for discriminating between males but not necessarily mating with preferred males produced fewer male offspring than females mated at random. The number of female offspring produced was not affected, nor was the viability of the offspring. Experimental modification of the composition of the males' pheromone showed that the fecundity effects were caused by exposure to the pheromone component that makes males attractive to females but also makes males less likely to be dominant. Female mate choice therefore carries a demographic cost but functions to avoid male manipulation and aggression. Male-male competition appears to function to circumvent mate choice rather than directly manipulating females, as the mate choice can be cryptic. The dynamic struggle between the sexes for control of mating opportunities and outcomes in N. cinerea therefore reveals a unique role for sexual conflict in the evolution of the behavioural components of sexual selection.


Implementation of a Pharmacist-Administered Influenza Vaccination Program in the Outpatient Setting at the Military Treatment Facility.
The goal of the Army's influenza prevention program is to protect all Soldiers, healthcare professionals, and beneficiaries from influenza and its complications to maximize force readiness. Commanders have the daunting and challenging task of ensuring 100% of Soldiers and Military Treatment Facility (MTF) assigned employees are vaccinated against influenza by a specified timeframe. Outpatient Pharmacy Services at the MTF are in the perfect position to provide enhanced accessibility to the influenza vaccination to assist in meeting this requirement. Irwin Army Community Hospital at Fort Riley, Kansas, became the first MTF to offer a pharmacistadministered influenza vaccination program in the outpatient setting, as a walk-in service to all Soldiers, MTF employees, and beneficiaries, in 2016. Since then over 1,500 flu shots have been administered with no documented adverse events, and a potential cost-savings between $23,205 to $61,880. Here we describe the planning and implementation of such a program, which can easily be reproduced at any MTF outpatient pharmacy with minimal efforts and resources, enabling outpatient pharmacists to provide a convenient and accessible alternative for seasonal flu vaccination, with the ultimate goal of maximizing force readiness.


Differential FSH Glycosylation Modulates FSHR Oligomerization and Subsequent cAMP Signaling.
Follicle-stimulating hormone (FSH) and its target G protein-coupled receptor (FSHR) are essential for reproduction. Recent studies have established that the hypo-glycosylated pituitary FSH glycoform (FSH21/18), is more bioactive in vitro and in vivo than the fully-glycosylated variant (FSH24). FSH21/18 predominates in women of reproductive prime and FSH24 in peri-post-menopausal women, suggesting distinct functional roles of these FSH glycoforms. The aim of this study was to determine if differential FSH glycosylation modulated FSHR oligomerization and resulting impact on cAMP signaling. Using a modified super-resolution imaging technique (PD-PALM) to assess FSHR complexes in HEK293 cells expressing FSHR, we observed time and concentration-dependent modulation of FSHR oligomerization by FSH glycoforms. High eFSH and FSH21/18 concentrations rapidly dissociated FSHR oligomers into monomers, whereas FSH24 displayed slower kinetics. The FSHR β-arrestin biased agonist, truncated eLHβ (Δ121-149) combined with asparagine56-deglycosylated eLHα (dg-eLHt), increased FSHR homomerization. In contrast, low FSH21/18 and FSH24 concentrations promoted FSHR association into oligomers. Dissociation of FSHR oligomers correlated with time points where higher cAMP production was observed. Taken together, these data suggest that FSH glycosylation may modulate the kinetics and amplitude of cAMP production, in part, by forming distinct FSHR complexes, highlighting potential avenues for novel therapeutic targeting of the FSHR to improve IVF outcomes.


Rate of first recorded diagnosis of autism and other pervasive developmental disorders in United Kingdom general practice, 1988 to 2001.
There has been concern that the incidence of autism and other pervasive developmental disorders (PDDs) is increasing. Previous studies have been smaller, restricted to autism (excluding other pervasive developmental disorders such as Asperger's syndrome), included boys only, or have not been based on a national sample. We investigated time trends in the rates of diagnosis of pervasive developmental disorders. We analysed the rates of first diagnosis of pervasive developmental disorders among people registered with a practice contributing to the United Kingdom General Practice Research Database during the period 1988 to 2001. We included 1410 cases from over 14 million person-years of observation. The main outcome measures were rates of diagnosis of pervasive developmental disorders by year of diagnosis, year of birth, gender and geographical region. The rate increased progressively from 0.40/10,000 person-years (95% CI 0.30 to 0.54) in 1991 to 2.98/10,000 (95% CI 2.56 to 3.47) in 2001. A similar change occurred in the age standardised incidence ratios, from 35 (95% CI: 26-47) in 1991 to 365 (95% CI: 314-425) in 2001. The temporal increase was not limited to children born during specific years nor to children diagnosed in a specific time period. The rate of diagnosis of PDDs other than autism rose from zero for the period 1988-1992 to 1.06/10,000 person-years in 2001. The rate of diagnosis of autism also increased but to a lesser extent. There was marked geographical variation in rates, with standardised incidence ratios varying from 66 for Wales to 141 for the South East of England. Better ascertainment of diagnosis is likely to have contributed to the observed temporal increase in rates of diagnosis of PDD, but we cannot exclude a real increase.


Voluntary exercise inhibits intestinal tumorigenesis in Apc(Min/+) mice and azoxymethane/dextran sulfate sodium-treated mice.
Epidemiological studies suggest that physical activity reduces the risk of colon cancer in humans. Results from animal studies, however, are inconclusive. The present study investigated the effects of voluntary exercise on intestinal tumor formation in two different animal models, Apc(Min/+) mice and azoxymethane (AOM)/dextran sulfate sodium (DSS)-treated mice. In Experiments 1 and 2, five-week old female Apc(Min/+) mice were either housed in regular cages or cages equipped with a running wheel for 6 weeks (for mice maintained on the AIN93G diet; Experiment 1) or 9 weeks (for mice on a high-fat diet; Experiment 2). In Experiment 3, male CF-1 mice at 6 weeks of age were given a dose of AOM (10 mg/kg body weight, i.p.) and, 12 days later, 1.5% DSS in drinking fluid for 1 week. The mice were then maintained on a high-fat diet and housed in regular cages or cages equipped with a running wheel for 16 weeks. In the Apc(Min/+) mice maintained on either the AIN93G or the high-fat diet, voluntary exercise decreased the number of small intestinal tumors. In the AOM/DSS-treated mice maintained on a high-fat diet, voluntary exercise also decreased the number of colon tumors. In Apc(Min/+) mice, voluntary exercise decreased the ratio of serum insulin like growth factor (IGF)-1 to IGF binding protein (BP)-3 levels. It also decreased prostaglandin E2 and nuclear beta-catenin levels, but increased E-cadherin levels in the tumors. These results indicate hat voluntary exercise inhibited intestinal tumorigenesis in Apc(Min/+) mice and AOM/DSS-treated mice, and the inhibitory effect is associated with decreased IGF-1/IGFBP-3 ratio, aberrant beta-catenin signaling, and arachidonic acid metabolism.


Adolescent sexuality and fertility in Kenya: a survey of knowledge, perceptions, and practices.
This article presents findings from a survey conducted in Kenya in 1985 of the reproductive health knowledge, attitudes, and practices among more than 3,000 unmarried Kenyan youth, students and nonstudents, between the ages of 12 and 19. The survey was designed to elicit information that would be useful in gauging the kinds of problems Kenyan adolescents face in order to design programs that meet their needs. The study shows that although a solid majority of adolescents appear to have received information on reproductive health, the quality of the information is generally low. Fewer than 8 percent could correctly identify the fertile period in a woman's menstrual cycle. A substantial proportion of the population surveyed, more than 50 percent, is sexually active, having initiated intercourse some time between 13 and 14 years of age, on average. In spite of a general disapproval of premarital sex (but approval of the use of contraceptives among the sexually active), most of the sexually active population--89 percent--have never used contraceptives. The many contradictions between attitudes and practices pose serious questions and demonstrate the need to reexamine the programs (and policies) that provide access to reproductive health services to adolescents in Kenya.


Plate size and children's appetite: effects of larger dishware on self-served portions and intake.
Dishware size is thought to influence eating behaviors, but effects on children's self-served portion sizes and intakes have not been studied. We aimed to evaluate whether larger dishware increased children's self-served portion sizes and intake during meals. A within-subjects experimental design was used to test the effects of dishware size (ie, plates and bowls) on children's self-served portion sizes and intakes in a naturalistic setting. Subjects were predominantly African American elementary school-aged children (n = 42) observed on repeated occasions during school lunch. Children served themselves an entree and side dishes using either child- or adult-size dishware, which represented a 100% increase in the surface area of plates and volume of bowls across conditions. Condition order was randomly assigned and counterbalanced across 2 first-grade classrooms. Entrées of amorphous and unit form were evaluated on separate days. Fruit and vegetable side dishes were evaluated at each meal. Fixed portions of milk and bread were provided at each meal. Children served more energy (mean = 90.1 kcal, SE = 29.4 kcal) when using adult-size dishware. Adult-size dishware promoted energy intake indirectly, where every additional calorie served resulted in a 0.43-kcal increase in total energy intakes at lunch (t = 7.72, P = .001). Children served themselves more with larger plates and bowls and consumed nearly 50% of the calories that they served. This provides new evidence that children's self-served portion sizes are influenced by size-related facets of their eating environments, which, in turn, may influence children's energy intake.


Peripheral circadian oscillators in mammals.
Although circadian rhythms in mammalian physiology and behavior are dependent upon a biological clock in the suprachiasmatic nuclei (SCN) of the hypothalamus, the molecular mechanism of this clock is in fact cell autonomous and conserved in nearly all cells of the body. Thus, the SCN serves in part as a "master clock," synchronizing "slave" clocks in peripheral tissues, and in part directly orchestrates circadian physiology. In this chapter, we first consider the detailed mechanism of peripheral clocks as compared to clocks in the SCN and how mechanistic differences facilitate their functions. Next, we discuss the different mechanisms by which peripheral tissues can be entrained to the SCN and to the environment. Finally, we look directly at how peripheral oscillators control circadian physiology in cells and tissues.


Development of thermo-responsive polycaprolactone macrocarriers conjugated with Poly(N-isopropyl acrylamide) for cell culture.
Poly(N-isopropyl acrylamide) (PNIPAAm) is a well-known 'smart' material responding to external stimuli such as temperature. PNIPAAm was successfully conjugated to polycaprolactone (PCL) bead surfaces through amidation reaction. Functionalization steps were characterized and confirmed by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and Energy Dispersion Spectroscopy. PNIPAAm-conjugated PCL allowed human dermal fibroblast cells (HDF) and mesenchymal stem cells (MSC) to adhere, spread, and grow successfully. By reducing the temperature to 30 °C, more than 70% of HDF were detached from PNIPAAm-conjugated PCL macrocarriers with 85% viability. The cell detachment ratio by trypsin treatment was slightly higher than that induced by reduced temperature, however, cell detachment from PNIPAAm-conjugated macrocarriers by lowering the temperature significantly reduced cell death and increased both cell viability and the recovery potential of the detached cells. HDF attachment and detachment were also observed by Live-Dead staining and phase contrast imaging. The expression of extracellular matrix proteins such as Laminin and Fibronectin was also affected by the trypsinization process but not by the reduced temperature process. Taken together, our results showed that thermo-responsive macrocarriers could be a promising alternative method for the non-invasive detachment of cells, in particular for tissue engineering, clinical applications and the use of bioreactors.


The molecular target of bicyclams, potent inhibitors of human immunodeficiency virus replication.
Bicyclams are a novel class of antiviral compounds which act as potent and selective inhibitors of the replication of human immunodeficiency virus type 1 (HIV-1) and HIV-2. They block an early step in the viral life cycle following adsorption to the CD4 receptor and preceding reverse transcription. To identify the molecular target of these compounds, we genetically analyzed variants of the HIV-1 molecular clone NL4-3, which developed resistance against two structurally related bicyclams, JM2763 and the more potent SID791. The resistant strains were obtained after long-term passaging in MT-4 cells in the presence of progressively increasing compound concentrations. Recombinants between selected genes of the resistant strains and the parental NL4-3 provirus were generated by adapting the marker rescue technique to MT-4 cells. The bicyclam-resistant phenotype was rescued by transferring the envelope gp120 gene of bicyclam-resistant virus into the NL4-3 parental genetic background. In the gp120 genes of the resistant strains, we identified several mutations leading to amino acid substitutions in the V3 loop. Furthermore, two substitutions of highly conserved amino acids in close proximity to the disulfide bridges of the V3 and V4 loops were found in both SID791- and JM2763-resistant strains. Additional mutations in regions encoding V3, C4, V5, and C5 were present in SID791-resistant viruses. Recombination experiments with overlapping parts of the envelope gene indicated that most, if not all, of the mutations were necessary to develop the fully SID791 resistant phenotype. The mutations in the C-terminal part of gp120 downstream of the V3 loop sequence conferred partial resistance to JM2763 but did not significantly decrease susceptibility to SID791. The genetic data and the biological properties of the resistant viruses point to inhibition of entry and fusion as the mode of action of the HIV-inhibitory bicyclams. A possible mechanism of binding of bicyclams to gp120 leading to inhibition of unfolding of gp120 and its shedding from the gp41 fusion domain is discussed.


In vitro and in vivo neuroprotection by gamma-glutamylcysteine ethyl ester against MPTP: relevance to the role of glutathione in Parkinson's disease.
Glutathione is an abundant intracellular thiol antioxidant whose levels are reduced both in Parkinson's disease itself and in a widely used animal model of the disorder, systemic MPTP administration. Previous in vitro work from our laboratory has suggested that glutathione depletion may be directly responsible for mitochondrial dysfunction, which ultimately leads to dopaminergic cell death associated with the disease. Here, we demonstrate the ability of gamma-glutamylcysteine ethyl ester, a lipid permeable derivative of the major substrate for scavenger glutathione synthesis, to counteract glutathione loss and neurodegeneration associated with in vitro and in vivo administration of MPTP or its derivatives. This data suggests that prevention of glutathione depletion is a likely therapeutic target for the disease.


The use of nonradioactive DNA probes for the characterization of Leishmania isolates from Peru.
We describe conditions for dot blot DNA hybridization studies using biotinylated kDNA probes from Leishmania. The sensitivity and specificity attained with biotinylated or 32P-labeled probes were equivalent. The lower level of detection obtained was 100 parasites that were blotted on nitrocellulose paper and then treated with Proteinase K. Studies were performed with 112 Leishmania isolates from Andean (uta) and sylvatic mucocutaneous (espundia) patients and all were determined to belong to the Leishmania braziliensis complex.


Development of a disease specific questionnaire to measure health related quality of life in patients with chronic liver disease.
To develop and assess a disease specific instrument for measuring health related quality of life (HRQL) in patients with chronic liver disease (CLD). Based on responses from 60 patients with chronic liver disease, from 20 liver experts, and from a Medline search of the literature, items potentially affecting the HRQL of these patients were identified. A separate sample of 75 patients identified which items they found problematic and rated their importance. Results were explored using factor analysis; domains were chosen and items placed within domains. Redundant questions were eliminated and the final questionnaire was pretested in 10 patients. Using this instrument, HRQL was assessed in a further 133 patients with various types and stages of liver disease. Patients, experts, and the literature search identified 156 items of potential importance. Of these, 35 proved important to over 50% of 75 respondents in the item reduction sample. The factor analysis suggested six domains. After eliminating redundancies, the Chronic Liver Disease Questionnaire (CLDQ) included 29 items in the following domains: fatigue, activity, emotional function, abdominal symptoms, systemic symptoms, and worry. In pretesting, patients found the CLDQ clear and easy to complete in 10 minutes. In another 133 patients, the CLDQ showed a gradient between patients without cirrhosis, Child's A cirrhosis, and those with Child's B or C cirrhosis. CLDQ has evidence for moderate reliability at six months and seems to be responsive. The CLDQ is short, easy to administer, produces both a summary score and domain scores, and correlates with the severity of liver disease.