[The distribution and clinical significance of IgG subclasses of anti-glomerular basement membrane antibodies].
It was reported that major subclasses of anti-glomerular basement membrane (GBM) antibody were IgG1 and IgG4. The IgG1 subclass was mainly found in male patients and IgG4 subclass in female patients with anti-GBM disease. This study investigates the distribution of anti-GBM IgG subclasses and their association with clinical characteristics. Sera from 50 patients diagnosed as anti-GBM disease during 1991-2003 were collected. The anti-GBM antibodies with IgG1, IgG2, IgG3, and IgG4 subclasses were detected with purified bovine alpha (IV) NC1 as solid phase ligand in ELISA. The association with clinical manifestations was further investigated. The positive rates of IgG anti-GBM subclasses were IgG1 94%, IgG2 56%, IgG3 12%, IgG4 88%, respectively. IgG1 and IgG4 were the main subclasses in anti-GBM IgG subclasses (P<0.01). The positive rates of the four subclasses had no significant difference between male and female, whereas the average positive percentage of IgG4 anti-GBM antibody was much higher in males than that in females (P<0.01). The average ages of patients with each subclass had no difference. The distribution of anti-GBM subclasses had no association with antineutrophil cytoplasmic antibody (ANCA), hematuria, proteinuria,or hemoptysis. The average positive percentage of IgG1 in patients with serum creatinine over 600 micromol/L was significantly higher than that in patients with serum creatinine less than 600 micromol/L (P<0.05), which suggested IgG1 might play an important role in the evolution and development of the disease. In the four patients with mild clinical presentations, three had IgG4 subclass, and only one patient who retained normal renal function in the follow-up exhibited pure IgG4 subclass. This suggested that IgG4 might not be inclined to cause severe damage. The average positive percentage of IgG4 subclass was higher in smoking patients than that in non-smoking patients (P<0.01). There is a restrictive distribution among the subclasses of anti-GBM antibody, in which IgG1 and IgG4 are the main subclasses. The distribution of anti-GBM IgG subclasses was not associated with gender.


Sports activity and the use of cigarettes and snus among young males in Finland in 1999-2010.
Studies of the relationship between sports activity and smoking among adolescents and young adults report contradictory results. We examined the association between sports activity (intensity and type of sport) and the current use of snus (Swedish snuff), cigarette smoking, and the combined use of cigarettes and snus (dual use) among young males in Finland. Data were collected from 16,746 male conscripts who completed a survey during the first days of their conscription during the years 1999-2010 (median age 19 years, response rate 95%). Main outcome measures were self-reported daily/occasional use of snus, cigarette smoking, and dual use. The association between sports activity, type of sport, and several sociodemographic background variables was assessed using logistic regression analysis. Over the study period (1999-2010), the prevalence of cigarette smoking decreased from 42% to 34%, while snus use increased from 5% to 12%, and dual use increased from 7% to 13% (p < 0.001). Compared with no physical activity, regular competitive sports activity (defined as high-intensity sports activity) was positively associated with use of snus (odds ratio [OR] 10.2; 95% confidence interval [CI]: 7.8-13.5) and negatively with cigarette smoking (OR 0.2; 95% CI: 0.1-0.3). When stratified by type of sport in multivariate models, ice hockey was most strongly associated with snus use (OR 1.6; 95% CI: 1.4-1.9) and dual use (OR 2.0; 95% CI 1.8-2.3) compared with those not playing ice-hockey, followed by other team sports for snus use (OR 1.5; 95% CI: 1.3-1.8) and dual use (OR 1.8; 95% CI: 1.6-2.0) compared with those not participating in other team-sports. Our results show a clear association between snus use and intensity and type of training. Team sports were associated with increased use of snus and dual use compared with no participation in team sports. These findings should be acknowledged when planning and implementing preventive strategies.


Immunoglobulin therapy for autoimmune thrombocytopenia purpura during pregnancy. A report of two cases.
High-dose, intravenous immunoglobulin therapy may be effective in elevating low platelet counts in nonpregnant patients with autoimmune thrombocytopenia purpura (ATP). We used immunoglobulin successfully on two pregnant women with ATP who had been refractory to high-dose corticosteroid therapy. The 5-day infusions were started at 12 and 29 weeks' gestation, and the subsequent uncomplicated vaginal deliveries occurred after the shortest reported interval (8 days) and longest (28 weeks) following a single immunoglobulin infusion. A splenectomy was avoided, and no adverse maternal or neonatal effects were apparent.


Two dietary polyphenols, fisetin and luteolin, reduce inflammation but augment DNA damage-induced toxicity in human RPE cells.
Plant-derived polyphenols are known to possess anti-inflammatory and antioxidant effects. In recent years, several studies have investigated their potential benefits for treating chronic diseases associated with prolonged inflammation and excessive oxidative stress, such as age-related macular degeneration (AMD). Previously, two polyphenols, fisetin and luteolin, have been reported to increase the survival of retinal pigment epithelial (RPE) cells suffering from oxidative stress as well as decreasing inflammation but the benefits of polyphenol therapy seem to depend on the model system used. Our aim was to analyze the effects of fisetin and luteolin on inflammation and cellular viability in a model of nonoxidative DNA damage-induced cell death in human RPE (hRPE) cells. Pretreatment of ARPE-19 or primary hRPE cells with the polyphenols augmented etoposide-induced cell death as measured by the lactate dehydrogenase and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. However, the treatment was able to reduce the release of two proinflammatory cytokines, IL-6 and IL-8, which were determined by enzyme-linked Immunosorbent assay. Analyses of caspase 3 activity, p53 acetylation and SIRT1 protein levels revealed the apoptotic nature of etoposide-evoked cell death and that fisetin and luteolin augmented the etoposide-induced acetylation of p53 and decreased SIRT1 levels. Taken together, our findings suggest that the cytoprotective effects of fisetin and luteolin depend on the stressor they need to combat, whereas their anti-inflammatory potential is sustained over a variety of model systems. Careful consideration of disease pathways will be necessary before fisetin or luteolin can be recommended as therapeutic agents for inflammatory diseases in general and specifically AMD.


Colorimetric Properties of Bombyx mori Silk Fibroin.
Recent reports highlighted several novel applications for the Bombyx mori silk fibroin (SF), as edible coatings for the preservation of food freshness, smart labels, or packaging materials. This study complements these reports and additionally describes the colorimetric sensing properties of the natural protein that could be explored to enhance the practical value of such applications. Our data show that in response to pH changes, reconstituted SF is able to undergo visible color changes that correlate with the intensity of the stimuli, regardless of its physical format or physical cross-linking state. The intensity of the developed color was proportional to the extent of the protein's hydrolytic degradation. We also found that these pH-driven color changes were reversible and interchangeable, with colorless samples at neutral pH, purple in acidic environments, and yellow under basic conditions. Our mechanistic studies identified tryptophan as being responsible for these colorimetric responses, which could be further intensified by the presence of ionized tyrosine functionalities. In addition, we determined that SF's sensing properties also applied to ultraviolet light exposure. Finally, we showed that the innate sensing capabilities of activated SF can be enhanced via the covalent incorporation of additional tryptophan into the protein. Overall, our results further support the utility of SF for sensing applications.


Structural symmetry and protein function.
The majority of soluble and membrane-bound proteins in modern cells are symmetrical oligomeric complexes with two or more subunits. The evolutionary selection of symmetrical oligomeric complexes is driven by functional, genetic, and physicochemical needs. Large proteins are selected for specific morphological functions, such as formation of rings, containers, and filaments, and for cooperative functions, such as allosteric regulation and multivalent binding. Large proteins are also more stable against denaturation and have a reduced surface area exposed to solvent when compared with many individual, smaller proteins. Large proteins are constructed as oligomers for reasons of error control in synthesis, coding efficiency, and regulation of assembly. Symmetrical oligomers are favored because of stability and finite control of assembly. Several functions limit symmetry, such as interaction with DNA or membranes, and directional motion. Symmetry is broken or modified in many forms: quasisymmetry, in which identical subunits adopt similar but different conformations; pleomorphism, in which identical subunits form different complexes; pseudosymmetry, in which different molecules form approximately symmetrical complexes; and symmetry mismatch, in which oligomers of different symmetries interact along their respective symmetry axes. Asymmetry is also observed at several levels. Nearly all complexes show local asymmetry at the level of side chain conformation. Several complexes have reciprocating mechanisms in which the complex is asymmetric, but, over time, all subunits cycle through the same set of conformations. Global asymmetry is only rarely observed. Evolution of oligomeric complexes may favor the formation of dimers over complexes with higher cyclic symmetry, through a mechanism of prepositioned pairs of interacting residues. However, examples have been found for all of the crystallographic point groups, demonstrating that functional need can drive the evolution of any symmetry.


ZAKα Recognizes Stalled Ribosomes through Partially Redundant Sensor Domains.
Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) remains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix 14 of 18S ribosomal RNA (rRNA). An adjacent domain in ZAKα also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxin action. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.


Role of dietary fiber in diverticular disease and colon cancer.
This paper reviews recent research on the characterization, properties, and definition of dietary fiber as well as its possible role in colonic carcinogenesis and diverticulosis. Despite progress in analytic methods and characterization, an accepted definition and terminology for fiber are lacking as is an accurate, rapid method for measurement of total dietary fiber or fiber in foods. Mechanisms of effects of fiber in the gut and the significant of interactions between fiber, nutrients gut flora and associated metabolites, and enteric secretions are unclear. Epidemiologic and experimental data indicate an increased risk of diverticular disease and colonic cancer with low-fiber intakes; however, genetic, environmental, cultural, dietary, and other variables were often uncontrolled in the epidemiologic studies. Thus, conclusive evidence for a causal relationship between low intake of fiber and diverticulosis or colonic cancer is not available, and the question whether first protects against human colonic cancer and/or diverticulosis is not completely resolved. Clinical trials in which symptomatic diverticular disease was treated with supplementary dietary fiber have generally had favorable results. Numerous specific questions require additional study before a role for dietary fiber in the prevention of human colonic diverticulosis and cancer of the colon can be established. Suggestions for possible future investigation are provided.


Water diffusion measurements in perfused human hippocampal slices undergoing tonicity changes.
Diffusion MRI has the potential to probe the compartmental origins of MR signals acquired from human nervous tissue. However, current experiments in human subjects require long diffusion times, which may confound data interpretation due to the effects of compartmental exchange. To investigate human nervous tissue at shorter diffusion times, and to determine the relevance of previous diffusion studies in rat hippocampal slices, water diffusion in 20 perfused human hippocampal slices was measured using a wide-bore 17.6-T magnet equipped with 1000-mT/m gradients. These slices were procured from five patients undergoing temporal lobectomy for epilepsy. Tissue viability was confirmed with electrophysiological measurements. Diffusion-weighted water signal attenuation in the slices was well-described by a biexponential function (R(2) > 0.99). The mean diffusion parameters for slices before osmotic perturbation were 0.686 +/- 0.082 for the fraction of fast diffusing water (F(fast)), 1.22 +/- 0.22 x 10(-3) mm(2)/s for the fast apparent diffusion coefficient (ADC), and 0.06 +/- 0.02 x 10(-3) mm(2)/s for the slow ADC. Slice perturbations with 20% hypotonic and 20% hypertonic artificial cerebrospinal fluid led to changes in F(fast) of -8.2% and +10.1%, respectively (ANOVA, P < 0.001). These data agree with previous diffusion studies of rat brain slices and human brain in vivo, and should aid the development of working models of water diffusion in nervous tissue, and thus increase the clinical utility of diffusion MRI.


Resident perceptions of opportunity for communication and contribution to care planning in residential aged care.
Irrespective of age, communication is a tool of expression and a key daily activity meeting the human need for social interaction and connection. The introduction of consumer-directed care (CDC) emphasises the importance of communication to provide consumers with the opportunity to exercise choice over the care they receive. As consumer-directed care progresses, it is hypothesised that the feasibility of shared decision-making and care planning in residential aged care will be largely determined by the communication opportunities afforded to the residents. Therefore, the aim of this study was to explore resident perceptions of the opportunities they have to communicate, including the opportunity to express their care preferences and contribute opinions about their care. A qualitative inductive design was adopted. An individual interview format was used to gather the perspectives of 102 residents. Data were analysed using qualitative content analysis to generate themes illustrating patterns in participant views. Overall, residents desired increased involvement in their care planning and increased opportunity for more meaningful communication and social opportunities. Residents described the negative impact of the communication difficulties they face on their communication and the need for support and activities to be tailored to residents' individual communication needs. To facilitate resident participation in CDC and meet resident desire for increased social communication, further investment in resources to support resident-staff communication and accommodate residents' individual communication needs is required. By highlighting communication as a stand-alone activity and a priority of residents, the findings of this study raise the profile of communication and demonstrate the need for explicit allocation of care time and specialist services to support resident-staff communication and social communication in residential aged care. Such support must be tailored to meet residents' individual communication needs and be coupled with increased staff training in providing communication support. Without facilitating resident communication and increasing the opportunity to communicate, shared decision-making and care planning in residential aged care consistent with person-centred and consumer-directed models of care will be limited.


Gas sensing properties of columnar CeO2 nanostructures prepared by chemical vapor deposition.
Columnar CeO2 nanostructures are grown on alumina substrates by a template- and catalyst-free Chemical Vapor Deposition (CVD) approach and subsequently tested as resistive gas sensors of CH3COCH3, H2, NO2. The sensor response is stable and reproducible throughout the whole working temperature range (200-500 degrees C) and directly dependent on the analyte gas and the adopted operating conditions. The higher sensitivity with respect to that displayed by continuous CeO2 thin films demonstrates the potential of fabricating nanostructured sensing devices characterized by improved functional performances.


Cytotoxic hepatosplenic gammadelta T-cell lymphoma following acute myeloid leukemia bearing two distinct gamma chains of the T-cell receptor. Biologic and clinical features.
Hepatosplenic gd T-cell lymphoma is a rare entity of peripheral T-cell lymphomas. We characterized in detail the first case of hepatosplenic gd -T-cell lymphoma following acute myeloid leukemia. Hepatosplenic gd -T-cell lymphoma was diagnosed in a woman who had been in complete remission (CR) of acute myeloid leukemia (AML) for two years. Improvement but no objective response of the disease was observed after various types of chemotherapy. CR was achieved after related donor stem cell transplantation. Thirteen months later relapse of hepatosplenic gd T-cell lymphoma was diagnosed. While being prepared for a second transplantation the patient developed meningeal lymphoma and died. The patient's lymphoma cells were studied by immunologic, functional and molecular techniques. Lymphoma cells expressed the gd T-cell receptor (TCR), CD2, CD3, CD5, CD7, CD38, CD45, CD161 (NKR-P1), TIA and Ki67. Further analysis revealed expression of Vd1 and two distinct TCRg chains, Vg3 and Vg9, by the malignant cell clone. The clonality of the T-cells was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) followed by sequencing of TCR Vg3, Vg9 and Vd1 junctional regions. Clone-specific PCR was negative at diagnosis of AML and was positive at all times during follow-up of the hepatosplenic gd T-cell lymphoma. The lymphoma cells mediated strong natural killer cell-like cytotoxic activity, possibly explained by expression of CD161 and a lack of killer inhibitory receptor. Several so far undescribed features were observed in this case of hepatosplenic gd T-cell lymphoma, such as T-cell lymphoma following AML, expression of two distinct T-cell receptor g-chains, and an unexpected cytotoxic phenotype.


Neurotensin and neuromedin N stimulate mucin output from human goblet cells (Cl.16E) via neurotensin receptors.
The stably differentiated human intestinal goblet cell line Cl.16E was used to study the effects of two structurally related regulatory peptides, neurotensin (NT) and neuromedin N (NN), on mucus secretion. NT and NN stimulated rapid release of mucins from filter-grown Cl.16E cells, this effect being dose related with a mean effective dose of 36 nM for NT and 422 nM for NN. The order of potency of NT, three NT fragments corresponding to the NH2-terminal part [NT-(1-11)] or to the COOH-terminal part [NT-(8-13) and NT-(9-13)], and NN in promoting mucin release and in inhibiting 125I-labeled NT binding to Cl.16E cell membranes was identical with NT greater than or equal to NT-(8-13) greater than NN greater than NT-(9-13) much greater than NT-(1-11) supporting the hypothesis that NT and NN stimulate mucin output through interaction with a common NT-preferring receptor. Scatchard analysis of equilibrium binding data showed one population of NT binding sites in Cl.16E cell membranes with the following characteristics: binding capacity (Bmax) was 141 fmol/mg of protein and dissociation constant (Kd) was 1.00 nM.(ABSTRACT TRUNCATED AT 250 WORDS)


Interleukin-12p70 expression by dendritic cells of HIV-1-infected patients fails to stimulate gag-specific immune responses.
A variety of immune-based therapies has been developed in order to boost or induce protective CD8(+) T cell responses in order to control HIV replication. Since dendritic cells (DCs) are professional antigen-presenting cells (APCs) with the unique capability to stimulate naïve T cells into effector T cells, their use for the induction of HIV-specific immune responses has been studied intensively. In the present study we investigated whether modulation of the activation state of DCs electroporated with consensus codon-optimized HxB2 gag mRNA enhances their capacity to induce HIV gag-specific T cell responses. To this end, mature DCs were (i) co-electroporated with mRNA encoding interleukin (IL)-12p70 mRNA, or (ii) activated with a cytokine cocktail consisting of R848 and interferon (IFN)-γ. Our results confirm the ability of HxB2 gag-expressing DCs to expand functional HIV-specific CD8(+) T cells. However, although most of the patients had detectable gag-specific CD8(+) T cell responses, no significant differences in the level of expansion of functional CD8(+) T cells could be demonstrated when comparing conventional or immune-modulated DCs expressing IL-12p70. This result which goes against expectation may lead to a re-evaluation of the need for IL-12 expression by DCs in order to improve T-cell responses in HIV-1-infected individuals.


The QUTA activator and QUTR repressor proteins of Aspergillus nidulans interact to regulate transcription of the quinate utilization pathway genese.
Genetic evidence suggests that the activity of the native QUTA transcription activator protein is negated by the action of the QUTR transcription repressor protein. When Aspergillus nidulans was transformed with plasmids containing the wild-type qutA gene, transformants that constitutively expressed the quinate pathway enzymes were isolated. The constitutive phenotype of these transformants was associated with an increased copy number of the transforming qutA gene and elevated qutA mRNA levels. Conversely, when A. nidulans was transformed with plasmids containing the qutR gene under the control of the constitutive pgk promoter, transformants with a super-repressed phenotype (unable to utilize quinate as a carbon source) were isolated. The super-repressed phenotype of these transformants was associated with an increased copy number of the transforming qutR gene and elevated qutR mRNA levels. These copy-number-dependent phenotypes argue that the levels of the QUTA and QUTR proteins were elevated in the high-copy-number transformants. When diploid strains were formed by combining haploid strains that contained high copy numbers of either the qutA gene (constitutive phenotype) or the qutR gene (super-repressing; non-inducible phenotype), the resulting diploid phenotype was one of quinate-inducible production of the quinate pathway enzymes, in a manner similar to wild-type. The simplest interpretation of these observations is that the QUTR repressor protein mediates its repressing activity through a direct interaction with the QUTA activator protein. Other possible interpretations are discussed in the text. Experiments in which truncated versions of the QUTA protein were produced in the presence of a wild-type QUTA protein indicate that the QUTR repressor protein recognizes and binds to the C-terminal half of the QUTA activator protein.


HIV prevention research and COVID-19: putting ethics guidance to the test.
Critical public health measures implemented to mitigate the spread of the novel coronavirus disease (COVID-19) pandemic have disrupted health research worldwide, including HIV prevention research. While general guidance has been issued for the responsible conduct of research in these challenging circumstances, the contours of the dueling COVID-19 and HIV/AIDS pandemics raise some critical ethical issues for HIV prevention research. In this paper, we use the recently updated HIV Prevention Trials Network (HPTN) Ethics Guidance Document (EGD) to situate and analyze key ethical challenges related to the conduct of HIV prevention research during the COVID-19 pandemic as well as identify potential areas for refinement of the guidance document based on this unprecedented state of affairs. Necessary actions taken for HIV prevention research studies due to the COVID-19 pandemic involve an array of ethical issues including those related to: (1) risk mitigation; (2) behavior change; (3) compounding vulnerability; (4) community engagement; (5) trial reopening; and 6) shifting research priorities. In the context of the dueling HIV and COVID-19 global pandemics, research teams and sponsors must be nimble in responding to the rapidly changing environment by being sensitive to the associated ethical issues. The HTPN EGD provides a rich set of tools to help identify, analyze and address many of these issues. At the same time, future refinements of the HPTN EGD and other research ethics guidance could be strengthened by providing explicit advice regarding the ethical issues associated with disrupted research and the reopening of studies. In addition, additional consideration should be given to appropriately balancing domains of risk (e.g., physical versus social), addressing the vulnerability of research staff and community partners, and responding to un-anticipatable ancillary care needs of participants and communities. Appropriately addressing these issues will necessitate conceptual work, which would benefit from the careful documentation of the actual ethical issues encountered in research, the strategies implemented to overcome them, and their success in doing so. Throughout all of these efforts, it is critical to remember that the HIV pandemic not be forgotten in the rush to deal with the COVID-19 pandemic.


Infections acquired in the fields and forests of the United States.
Sportsmen, backpackers, and outdoor workers may present with unusual infections acquired in the fields and forests of the United States. Infections to be considered in such persons with a febrile illness include Rocky Mountain spotted fever, Colorado spotted tick fever, babesiosis, borreliosis, and Lyme disease. The differential diagnoses for clinical presentations of pulmonary and gastrointestinal disease are also discussed.


Dependence of DNA looping on Escherichia coli culture density.
Repression of a promoter by entrapment within a tightly bent DNA loop is a common mechanism of gene regulation in bacteria. Besides the mechanical properties of the looped DNA and affinity of the protein that anchors the loop, cellular energetics and DNA negative supercoiling are likely factors determining the stability of the repression loop. E. coli cells undergo numerous highly regulated and dynamic transitions as resources are depleted during bacterial growth. We hypothesized that the probability of DNA looping depends on the growth status of the E. coli culture. We utilized a well-characterized repression loop model assembled from elements of the lac operon to measure loop length-dependent repression at three different culture densities. Remarkably, even with changes in supercoiling, there exists a dynamic compensation in which the contribution of DNA looping to gene repression remains essentially constant.


Tanshinone IIA attenuates bleomycin-induced pulmonary fibrosis in rats.
Idiopathic pulmonary fibrosis is a chronic and progressive fibrotic lung disorder with unknown etiology and a high mortality rate. Tanshinone ⅡA (Tan ⅡA) is a lipophilic diterpene extracted from the Chinese herb Salvia miltiorrhiza Bunge with diverse biological functions. The present study was conducted to evaluate the effects of Tan ⅡA on bleomycin (BLM)‑induced pulmonary fibrosis in rats. Rats received an intraperitoneal injection of Tan ⅡA and normal rats were used as controls. Severe pulmonary edema, inflammation and fibrosis were observed in the BLM‑treated rats and the counts of total cells, neutrophils and lymphocytes were significantly increased in the bronchoalveolar lavage fluids of those rats. These pathological changes were markedly attenuated by subsequent treatment with Tan ⅡA. In addition, BLM‑induced increased expression of tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6, cyclooxygenase‑2, prostaglandin E2, malondialdehyde, inducible nitric oxide synthase and nitric oxide in rats, which was also suppressed by Tan ⅡA injection. The present findings suggest therapeutic potential of Tan ⅡA for pulmonary fibrosis.


Solution-state (15)N NMR spectroscopic study of alpha-C-phycocyanin: implications for the structure of the chromophore-binding pocket of the cyanobacterial phytochrome Cph1.
The detailed structure of the chromophore-binding pocket in phytochrome proteins and the structural changes associated with its photocycle are still matters of debate. Insight into the structure and dynamics of the binding pocket has been gained through the comparison of a (15)N NMR spectrum of alpha-C-phycocyanin, which is often used as a model system for the study of phytochromes, with the previously described (15)N NMR spectrum of the cyanobacterial phytochrome Cph1. The former spectrum supports the hypothesis that all four nitrogen atoms of the alpha-C-phycocyanin chromophore are protonated, in analogy with the proposed protonation state for the P(r) and P(fr) forms of Cph1. The spectra show that the chromophores in both proteins exhibit a distinct dynamic behavior, as also indicated by a NOESY spectrum of Cph1. Finally, stereochemical arguments and a Cph1 homology model support the hypothesis that the chromophore in Cph1 is most likely in the ZZZssa conformation in the P(r) form of the protein.


Apoptosis in Drosophila: neither fish nor fowl (nor man, nor worm).
Studies in a wide variety of organisms have produced a general model for the induction of apoptosis in which multiple signaling pathways lead ultimately to activation of the caspase family of proteases. Once activated, these enzymes cleave key cellular substrates to promote the orderly dismantling of dying cells. A broad similarity exists in the cell death pathways operating in different organisms and there is a clear evolutionary conservation of apoptotic regulators such as caspases, Bcl-2 family members, inhibitor of apoptosis (IAP) proteins, IAP antagonists and caspase activators. Despite this, studies in Caenorhabditis elegans, Drosophila and vertebrates have revealed some apparent differences both in the way apoptosis is regulated and in the way individual molecules contribute to the propagation of the death signal. For example, whereas cytochrome c released from mitochondria clearly promotes caspase activation in vertebrates, there is no documented role for cytochrome c in C. elegans apoptosis and its role in Drosophila is highly controversial. In addition, the apoptotic potency of IAP antagonists appears to be greater in Drosophila than in vertebrates, indicating that IAPs may be of different relative importance in different organisms. Thus, although Drosophila, worms and humans share a host of apoptotic regulators, the way in which they function may not be identical.


Effects of fluoride on growth, body composition, and serum biochemical profile in a freshwater teleost, Cyprinus carpio.
Fluoride is ubiquitously distributed in natural waters. Elevated fluoride may cause histopathological changes and induce oxidative stress in the gills of the common carp (Cyprinus carpio). The present study further evaluates the effects of fluoride on growth performance, body composition, and biochemical measurements of C. carpio. The results showed that food intake, growth, serum osmolality, body composition, and biochemical measures in the blood were affected by fluoride. Weight gain rate and specific growth rate in the exposed fish decreased compared with those of the control fish. Levels of crude protein and crude lipids were reduced in the fluoride-exposed fish. The major ion levels in the sera of fluoride-exposed fish were severely disturbed, resulting in a lower osmolality. All the biochemical parameters measured in the blood were affected by the exposure to fluoride. Total protein, albumin, globulin, and glucose in fish exposed to 63.6 mg/L, 77.7 mg/L, and 124.4 mg/L were lower than those in the control fish. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were markedly increased in exposed fish compared with control. Taken together, the data showed that exposure to fluoride caused a suite of detrimental effects in C. carpio, which might lead to a decrease in growth and food utilization efficiency. The results of the present study indicated that high levels of fluoride could pose a threat to carp in the field.


Prediction of ductus closure and development of adverse clinical outcome by functional echocardiography in very low birth weight newborn.
To evaluate the role of functional echocardiography for prediction of patent ductus arteriosus (PDA) closure and development of adverse outcome in very low birth weight newborn. Functional echocardiography was done in 143 neonates with birth weight less than 1250 g on day 3. Nine echocardiographic parameters were analyzed for babies with PDA for prediction of spontaneous closure and development of one or more adverse outcomes (bronchopulmonary dysplasia, necrotizing enterocolitis≥stage 2 and intraventricular hemorrhage > grade 2) during hospital stay. The mean (±SD) birthweight and gestational age of the cohort were 1017 (±17) g and 30.8 (±2.7) weeks. PDA was found in 60 (41.9%) babies on day 3 among the recruited babies. Binary logistic regression model showed strong prediction ability of early (day 3) functional echocardiography markers in predicting future spontaneous closure of PDA and development of one or more adverse clinical outcome. In multivariate analysis, abnormal flow pattern (growing or pulsatile) was found to have increased risk for persistence of PDA (adjusted OR 22.9, CI 1.97-269) in comparison to PDA with closing/bidirectional pattern. Early functional echocardiography, especially the flow pattern, is useful for prediction of PDA closure. Adverse clinical outcome prediction is possible with functional echocardiography markers when combined with gestational age and PDA closure status.


Treatment of excessive mediastinal bleeding after cardiopulmonary bypass.
Excessive mediastinal bleeding after cardiopulmonary bypass is one of the most frequently reported complications of cardiac operations. Appropriate treatment requires a rapid and effective diagnostic work-up, based on the knowledge of the pathophysiology induced by cardiopulmonary bypass. Possible causes, diagnostic methods available, and therapeutic approaches are reviewed in the light of the literature published on excessive bleeding after cardiac operations. When bleeding is massive (> 250 to 300 mL/h for the first 2 hours, > 150 mL/h thereafter), immediate surgical reexploration is mandatory. When bleeding is less important (50 to 150 mL/h), the decision to reoperate should be based on the presence of hemodynamic compromise or a suspected surgical cause. Otherwise, coagulation testing should allow the correction of hemostatic defects as appropriate with protamine, platelet concentrates, fresh frozen plasma, desmopressin, or antifibrinolytics. Hypothermia and hypotension should be corrected and a trial of positive end-expiratory pressure may be considered if diffuse mediastinal oozing (especially from the bed of the mammary artery) is suspected. A protocol is suggested to guide treatment, taking into account the rapidity of blood loss and the suspected underlying cause.


Splicing landscape of the eight collaborative cross founder strains.
The Collaborative Cross (CC) is a large panel of genetically diverse recombinant inbred mouse strains specifically designed to provide a systems genetics resource for the study of complex traits. In part, the utility of the CC stems from the extensive genome-wide annotations of founder strain sequence and structural variation. Still missing, however, are transcriptome-specific annotations of the CC founder strains that could further enhance the utility of this resource. We provide a comprehensive survey of the splicing landscape of the 8 CC founder strains by leveraging the high level of alternative splicing within the brain. Using deep transcriptome sequencing, we found that a majority of the splicing landscape is conserved among the 8 strains, with ~65% of junctions being shared by at least 2 strains. We, however, found a large number of potential strain-specific splicing events as well, with an average of ~3000 and ~500 with ≥3 and ≥10 sequence read coverage, respectively, within each strain. To better understand strain-specific splicing within the CC founder strains, we defined criteria for and identified high-confidence strain-specific splicing events. These splicing events were defined as exon-exon junctions 1) found within only one strain, 2) with a read coverage ≥10, and 3) defined by a canonical splice site. With these criteria, a total of 1509 high-confidence strain-specific splicing events were identified, with the majority found within two of the wild-derived strains, CAST and PWK. Strikingly, the overwhelming majority, 94%, of these strain-specific splicing events are not yet annotated. Strain-specific splicing was also located within genomic regions recently reported to be over- and under-represented within CC populations. Phenotypic characterization of CC populations is increasing; thus these results will not only aid in further elucidating the transcriptomic architecture of the individual CC founder strains, but they will also help in guiding the utilization of the CC populations in the study of complex traits. This report is also the first to establish guidelines in defining and identifying strain-specific splicing across different mouse strains.


Defense mechanisms in Peyer's patches and mesenteric lymph nodes against Yersinia enterocolitica involve integrins and cytokines.
Adhesion molecules and cytokines are involved in regulation of cellular host responses in infection processes. In this study the roles of the integrins Mac-1 and VLA-4, as well as those of the cytokines tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma), in defense mechanisms against Yersinia enterocolitica in Peyer's patches (PP) and mesenteric lymph nodes (MLN) were investigated by blocking these molecules with antibodies in vivo prior to orogastric Yersinia infection. Intestinal Yersinia infection caused abscesses composed of polymorphonuclear (Mac-1+ VLA-4+ Pgp-1+ ICAM-1-) and mononuclear (Mac-1+ VLA-4+ Pgp-1+ ICAM-inhibited phagocytosis of yersiniae by macrophages, (ii) reduced Yersinia-specific proliferation and IFN-gamma production of T cells from PP and MLN, and (iii) caused increased bacterial growth in PP and MLN followed by profound tissue destruction. Neutralization of TNF-alpha or IFN-gamma had comparable effects, suggesting that cell-mediated host responses including activated macrophages are required for control of yersiniae in intestinal tissues. The number of Mac-1+ cells in PP and MLN increased after yersinia infection, and recruitment of these cells was not blocked by administration of anticytokine or anti-integrin antibodies. While anti-VLA-4, -TNF-alpha, or -IFN-gamma antibody treatment caused an increased dissemination of yersiniae from PP to the spleen systemic dissemination was reduced by anti-Mac-1 antibodies. The results of this study suggest that the cytokines IFN-gamma and TNF-alpha as well as the integrins Mac-1 and VLA-4 are involved in protective cellular host defense mechanisms in PP and MLN against Y. enterocolitica, the latter probably being involved in both cell-cell and cell-pathogen interactions.


[Reconstruction of defects at the neuromuscular junction].
Loss of muscle tissue at the area of the neuromuscular junction after tumor resection or after trauma precludes the reconstruction with conventional nerve grafts, because the distal nerve stump is absent. For these cases, we recommend direct insertion of the nerve grafts into the muscle. We describe a standardized technique, which has been performed in 19 patients and led to a mean motor recovery of grade M4 after Highet. The key procedure of this technique is the interfascicular dissection of the nerve grafts, which allows a wide distribution of the grafts into the muscle tissue.


A Multimodal A* Algorithm to Solve the Two-Dimensional Optimization Problem of Accompanying a Person for an Intelligent Wheelchair.
Impaired mobility have far-reaching consequences for handicapped persons and their relatives. Mobile robotic technologies enable intelligent wheelchairs to regain mobility for those affected. Multiple research projects address human aware navigation and the task of following a person for assistive robots. But just a few projects focus on accompanying a person to enable social interaction. Therefore, we present a navigation system for indoor navigation in dynamic and cluttered environments as well as a novel algorithm for accompanying a person.First, we developed an autonomous driving wheelchair for indoor navigation based on the robot operating system (ROS). Thereby, a multi sensor setup using cameras and laser scanner enables localization within a map. People are detected by the same sensors and tracked by a Kalman filter. Afterwards we propose a novel algorithm to achieve a dynamic accompanying behaviour. An attractiveness distribution is introduced to evaluate the possible accompanying positions next to the manually selected target person regarding social interaction. The resulting two-dimensional optimization problem is solved by a novel multimodal extension of the A* algorithm.The proposed intelligent wheelchair is able to navigate in indoor environments and to accompany any person. In addition it allows social interaction while walking to relieve relatives or nursing staff, which otherwise need to push the wheelchair. The aim is to increase participation in everyday life for those affected.


The Case for Increased Physical Activity in Chronic Inflammatory Bowel Disease: A Brief Review.
Regular physical activity reduces the risk of colon cancer, but there is little information on the merits of such activity in the prevention and management of chronic inflammatory bowel disease (CIBD). The present systematic review thus documents current levels of habitual physical activity and aerobic and muscular function in CIBD, and examines the safety, practicality and efficacy of exercise programmes in countering the disease process, correcting functional deficits and enhancing quality of life. A systematic search of the Ovid/Medline database from January 1996 to May 2015 linked the terms physical activity/motor activity/physical fitness/physical training/physical education/training/exercise/exercise therapy with Crohn's disease/colitis/ulcerative colitis/inflammatory bowel disease, supplementing this information by a scanning of reference lists and personal files.12 of 16 published studies show a low level of habitual physical activity in CIBD, with sub-normal values for aerobic power, lean tissue mass and muscular strength. 3 of 4 studies suggest physical activity may reduce the risk of developing IBD, and 11 interventions all note that exercise programmes are well tolerated with some decreases of disease activity, and functional gains leading to an increased health-related quality of life. Moreover, programme compliance rates compare favourably with those seen in the treatment of other chronic conditions. More information on mechanisms is needed, but regular moderate aerobic and/or resistance exercise improves the health status of patients with CIBD both by modulating immune function and by improving physical function. A regular exercise programme should thus become an important component in the management of CIBD.


Characterization and spatial distribution of methanogens and methanogenic biosignatures in hypersaline microbial mats of Baja California.
Well-developed hypersaline cyanobacterial mats from Guerrero Negro, Baja California Sur, sustain active methanogenesis in the presence of high rates of sulfate reduction. Very little is known about the diversity and distribution of the microorganisms responsible for methane production in these unique ecosystems. Applying a combination of 16S rRNA and metabolic gene surveys, fluorescence in situ hybridization, and lipid biomarker analysis, we characterized the diversity and spatial relationships of methanogens and other archaea in the mat incubation experiments stimulated with methanogenic substrates. The phylogenetic and chemotaxonomic diversity established within mat microcosms was compared with the archaeal diversity and lipid biomarker profiles associated with different depth horizons in the in situ mat. Both archaeal 16S rRNA and methyl coenzyme M reductase gene (mcrA) analysis revealed an enrichment of diverse methanogens belonging to the Methanosarcinales in response to trimethylamine addition. Corresponding with DNA-based detection methods, an increase in lipid biomarkers commonly synthesized by methanogenic archaea was observed, including archaeol and sn-2-hydroxyarchaeol polar lipids, and the free, irregular acyclic isoprenoids, 2,6,10,15,19-pentamethylicosene (PMI) and 2,6,11,15-tetramethylhexadecane (crocetane). Hydrogen enrichment of a novel putative archaeal polar C(30) isoprenoid, a dehydrosqualane, was also documented. Both DNA and lipid biomarker evidence indicate a shift in the dominant methanogenic genera corresponding with depth in the mat. Specifically, incubations of surface layers near the photic zone predominantly supported Methanolobus spp. and PMI, while Methanococcoides and hydroxyarchaeol were preferentially recovered from microcosms of unconsolidated sediments underlying the mat. Together, this work supports the existence of small but robust methylotrophic methanogen assemblages that are vertically stratified within the benthic hypersaline mat and can be distinguished by both their DNA signatures and unique isoprenoid biomarkers.


Implementing School-Based Policies to Prevent Obesity: Cluster Randomized Trial.
Healthier school environments can benefit students, and school wellness policies may result in meaningful enhancements. Schools participating in federal child nutrition programs must implement wellness policies as mandated by law. The primary study objective is to assess effectiveness of implementing school-based nutrition and physical activity policies on student BMI trajectories. Cluster randomized trial using 2 × 2 factorial design. Twelve randomly selected schools in an urban district. Students were followed for 3 years through middle school, fifth to eighth grades (2011-2015, n=595 students, 92.3% participation, 85.2% retention). Specific to randomized condition, support was provided for implementation of nutrition policies (e.g., alternatives to food-based rewards/celebrations) and physical activity policies (e.g., opportunities for physical activity during/after school). Sex-/age-adjusted BMI percentile and BMI z-score; behavioral indicators. Data collected via standardized protocols. Analyses followed intention-to-treat principles, with planned secondary analyses (conducted 2016-2018). Students at schools randomized to receive support for nutrition policy implementation had healthier BMI trajectories over time (F=3.20, p=0.02), with a greater magnitude over time and cumulatively significant effects 3 years post-intervention (β=-2.40, p=0.04). Overall, students at schools randomized to receive the nutrition intervention had an increase in BMI percentile of <1%, compared with students in other conditions, whereas BMI percentile increased 3%-4%. There was no difference in student BMI between those in schools with and without physical activity policy implementation. Examining behavioral correlates in eighth grade, students at schools randomized to the nutrition condition consumed fewer unhealthy foods and sugar-sweetened beverages, and ate less frequently at fast-food restaurants (all p<0.03). This cluster randomized trial demonstrated effectiveness of providing support for implementation of school-based nutrition policies, but not physical activity policies, to limit BMI increases among middle school students. Results can guide future school interventions. This study is registered at www.clinicaltrials.gov NCT02043626.


Does It Help? The Contribution of Wives' Ways of Giving Support to Their Veteran Husbands' Posttraumatic Stress Symptoms and Functional Impairment.
In this study, we aimed to understand female partners' ways of giving support to their male military veteran partners' adjustment. Specifically, we examined the direct and moderating contributions female partners' ways of giving support-active engagement, protective buffering, or overprotection-make on their male partners' posttraumatic stress symptoms (PTSS) and functional impairment. Our hypotheses were that (a) female partners' active engagement would be negatively correlated with male veterans' PTSS and positively associated with veterans' functioning, (b) female partners' protective buffering and overprotection would be positively correlated with veterans' PTSS and negatively associated with veterans' functioning, and (c) female partners' ways of giving support would moderate the association between their secondary PTSS and male partners' adjustment. Participants were 300 male Israeli veterans of the 2006 Israel-Lebanon War and their female partners, all of whom completed self-report questionnaires. Active engagement did not contribute to female partners' or veterans' adjustment. In addition, whereas the correlations showed both female partners' protective buffering and overprotection were associated with male veterans' adjustment, the regression analysis showed only protective buffering made a direct, η2 = .040 and .053, and moderating contribution to veterans' adjustment, η2 = .019 and .016. Results revealed that when the level of protective buffering was high, female partners' secondary PTSS was associated more positively and strongly with veterans' PTSS than when protective buffering was low. The discussion reviews the complexity of giving support in couples when the veteran has PTSS.


Neurodevelopment at 3 Years in Neonates Born by Vaginal Delivery versus Cesarean Section at <26 Weeks of Gestation: Retrospective Analysis of a Nationwide Registry in Japan.
A high proportion of extremely preterm (EPT) infants are born by cesarean section (CS). However, whether the mode of delivery is related to long-term neurodevelopment in these infants is unclear. This study aimed to determine whether the mode of delivery is associated with mortality and long-term outcomes in EPT infants. We analyzed data of the Neonatal Research Network in Japan (NRNJ), a population-based, nationwide registry. Inclusion criteria were neonates who were born between 2003 and 2012 with a gestational age <26 weeks. The primary composite outcome was death before 3 years or neurodevelopmental impairment (NDI) at 3 years. Confounder-adjusted odds ratios (OR) were estimated by logistic generalized linear mixed models, which accounted for clustering within hospitals. 2,138 eligible infants (703 by vaginal delivery [VD] and 1,435 by CS) were identified for primary analysis. The composite outcome of death or NDI was not different between both groups (66.7% by VD and 62.7% by CS, p = 0.075). After multivariate analysis adjusting for confounders, we found that CS did not improve the composite outcome of death or NDI (OR = 0.839, 95% confidence interval = 0.816-1.328, p = 0.742). For secondary outcomes, mortality (OR = 0.824, p = 0.150), NDI (OR = 1.237, p = 0.165), and other neurodevelopmental outcomes were not different between the groups. Among neonates born at <26 weeks, CS does not improve mortality and neurodevelopmental outcomes at 3 years in the NRNJ cohort. However, because of several potential biases such as high rates of infants lost to follow-up, further evidence may be required.


Comparing five diagnostic criteria for multiple myeloma: a retrospective study of 227 cases.
Several diagnostic criteria for multiple myeloma are used in clinical practice, and it can be difficult to reach a diagnosis when a patient's clinical presentation is consistent with one criterion but not with another. However, no study to date has compared the superiority of the different diagnostic criteria. The aim of this research is to compare the efficacy of five diagnostic criteria for multiple myeloma and to find the reasons for misdiagnosis of atypical multiple myeloma cases. A total of 227 multiple myeloma cases were retrospectively studied. The clinical data (including plasma cell morphology, flow cytometry, immunofixation electrophoresis, imaging information and clinical manifestations) were scrutinized and the reasons underlying the misdiagnoses analyzed. The Traditional Domestic criteria had the highest misdiagnosis rate due to the high fixed bone marrow plasma cell percentage and serum M-protein thresholds. The WHO criteria and the International Myeloma Working Group 2009 criteria exhibited relatively low misdiagnosis rates due to their lower bone marrow plasma cell percentage thresholds, flexible criteria and detailed end-organ damage descriptions. The 2003 International Myeloma Working Group criteria and the 2011 Chinese Myeloma Working Group criteria exhibited perfect performance, as each focused on monoclonal plasma cell proliferation and not on fixed bone marrow plasma cell percentage and serum M-protein thresholds. The 2003 International Myeloma Working Group criteria and the 2011 Chinese Myeloma Working Group criteria have advantages in diagnosing early or atypical multiple myeloma cases. To avoid misdiagnosing some atypical cases of multiple myeloma, attention should be paid to evidence of monoclonal plasma cell proliferation, and flow cytometry may be a useful tool for discovering monoclonal plasma cell proliferation. Advanced imaging techniques should be used to confirm any suspected or atypical findings on metastatic bone survey.


Model-driven discovery of calcium-related protein-phosphatase inhibition in plant guard cell signaling.
The plant hormone abscisic acid (ABA) promotes stomatal closure via multifarious cellular signaling cascades. Our previous comprehensive reconstruction of the stomatal closure network resulted in an 81-node network with 153 edges. Discrete dynamic modeling utilizing this network reproduced over 75% of experimental observations but a few experimentally supported results were not recapitulated. Here we identify predictions that improve the agreement between model and experiment. We performed dynamics-preserving network reduction, resulting in a condensed 49 node and 113 edge stomatal closure network that preserved all dynamics-determining network motifs and reproduced the predictions of the original model. We then utilized the reduced network to explore cases in which experimental activation of internal nodes in the absence of ABA elicited stomatal closure in wet bench experiments, but not in our in silico model. Our simulations revealed that addition of a single edge, which allows indirect inhibition of any one of three PP2C protein phosphatases (ABI2, PP2CA, HAB1) by cytosolic Ca2+ elevation, resolves the majority of the discrepancies. Consistent with this hypothesis, we experimentally show that Ca2+ application to cellular lysates at physiological concentrations inhibits PP2C activity. The model augmented with this new edge provides new insights into the role of cytosolic Ca2+ oscillations in stomatal closure, revealing a mutual reinforcement between repeated increases in cytosolic Ca2+ concentration and a self-sustaining feedback circuit inside the signaling network. These results illustrate how iteration between model and experiment can improve predictions of highly complex cellular dynamics.


The role of microparticles in inflammation and thrombosis.
Microparticles (MP) are small membrane-bound vesicles that circulate in the peripheral blood and play active roles in thrombosis, inflammation and vascular reactivity. While MP can be released from nearly every cell type, most investigation has focused on MP of platelet, leucocyte and endothelial cell origin. Cells can release MP during activation or death. Flow cytometry is the usual method to quantify MP; the small size of these structures and lack of standardization in methodology complicate measurement. As MP contain surface and cytoplasmic contents of the parent cells and bear phosphatidylserine, antibodies to specific cell surface markers and annexin V can be used for identification. Through various mechanisms, MP participate in haemostasis and have procoagulant potential in disease. MP contribute to inflammation via their influence on cell-cell interactions and cytokine release, and MP also function in mediating vascular tone. In several disease states characterized by inflammation and vascular dysfunction, MP subpopulations are elevated, correlate with clinical events, and may have important roles in pathogenesis. In the rheumatic conditions such as rheumatoid arthritis and systemic lupus erythematosus, MP are potentially important markers of disease activity and have an increasingly recognized role in immunopathogenesis. It is clear that MP play an important role in atherosclerosis, and study of these structures may provide insight into the link between chronic inflammatory conditions and accelerated atherosclerosis. As biomarkers, MP allow access to usually inaccessible tissues such as the endothelium. Further research will hopefully lead to interventions targeting MP release and function.


Unusual occurrence of ankylosing spondylitis and multiple sclerosis in a black patient.
A possible association between ankylosing spondylitis (AS) and multiple sclerosis (MS) has been suggested in whites. The authors describe the first report of the coexistence of AS and MS in a black patient. The clinical diagnosis of MS was further substantiated by the results of magnetic resonance imaging of the brain. The diagnosis of AS met the Rome as well as the New York criteria. The patient possessed HLA-B27; his complete HLA phenotype was Aw66, B27, Cw2, DR3, and DR5.


Long-term infliximab maintenance therapy for ulcerative colitis: the ACT-1 and -2 extension studies.
The aim was to evaluate long-term efficacy, quality of life, and safety in ulcerative colitis patients who received infliximab during the ACT-1 and -2 extension studies. Adults with moderate-to-severely active ulcerative colitis in the 54-week ACT-1 and 30-week ACT-2 studies who achieved benefit from infliximab were eligible to participate in extension studies and receive up to 3 additional years of therapy. Patients received randomized study medication until all sites were unblinded; placebo-treated patients were discontinued. Patients receiving 5 or 10 mg/kg infliximab continued to receive open-label infliximab every 8 weeks. Patients receiving infliximab 10 mg/kg could decrease to 5 mg/kg; patients receiving infliximab 5 mg/kg could increase to 10 mg/kg if response was lost. A total of 229 of 484 infliximab-treated patients from the ACT-1 and ACT-2 main studies entered the long-term extensions. Overall, 70 (30.6%) patients discontinued infliximab infusions for adverse events (24 [10.5%]), lack of efficacy (11 [4.8%]), required a colectomy (1 [0.4%]), or for other reasons (34 [14.8%]). Proportions of patients whose Physician's Global Assessment scores were indicative of no or mild disease (score = 0 or 1) were maintained during the extension studies; 76.5% at Extension week 0 and ranged between 90.0% and 94.3% through Extension week 152. Improvement in Inflammatory Bowel Disease Questionnaire scores observed in the main studies was maintained. During the long-term extension, the infliximab safety profile was consistent with that of the main studies; no new or unexpected safety signals were observed. Long-term treatment with infliximab for up to 3 additional years was effective and well tolerated.


Glucose increases rat plasma norepinephrine levels by direct action on the brain.
The hypothesis that glucose can selectively activate the sympathetic nervous system (SNS) by direct action on the brain was tested using plasma norepinephrine (NE) and epinephrine (Epi) responses to intracarotid and intravenous glucose injections as indexes of SNS and adrenal medullary responses, respectively. Intracarotid glucose bolus injections (0.1 g/kg) transiently raised plasma glucose (22%) and insulin (98%) levels at 2 min and increased plasma NE, but not Epi, levels from 2 to 60 min. Areas under the NE curve were 700% higher than equiosmolar doses of mannitol. An intravenous glucose bolus (1 g/kg) gave quantitatively similar but delayed (30 min) NE responses to the 0.1 g/kg intracarotid dose but raised plasma glucose 500% and insulin 1,700% above baseline at 2 min postinjection. Slow intracarotid glucose infusions for 60 min at 4 mg.kg-1.min-1 raised plasma NE levels from 30 to 60 min with 250% higher areas under the NE curve than the intracarotid and intravenous bolus doses but without a change in plasma glucose, insulin, or Epi levels. Infusions at 6 mg.kg-1.min-1 transiently raised plasma NE levels at 30 min without altering glucose, insulin, or Epi levels. These results suggest that glucose alone can produce a selective, delayed SNS activation by a direct action on the brain.


Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources.
Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC.


Preoperative progressive pneumoperitoneum for giant inguinal hernias.
Reduction of giant hernia contents into the abdominal cavity may cause intraoperative and postoperative problems such as abdominal compartment syndrome. Preoperative progressive pneumoperitoneum expands the abdominal cavity, increases the patient's tolerability to operation, and can diminish intraoperative and postoperative complications. Preoperative progressive pneumoperitoneum is recommended for giant ventral hernias, but rarely for giant inguinal hernias. We present two giant inguinal hernia patients who were prepared for hernia repair with preoperative progressive pneumoperitoneum and then treated successfully by graft hernioplasty. We observed that abdominal expansion correlated with the inflated volume and pressure during the first four days of pneumperitoneum. Although insufflated gas volume can be different among patients, we observed that the duration of insufflation may be the same for similar patients.


Effect of Togu-exercise on Lumbar Back Strength of Women with Chronic Low Back Pain.
[Purpose] The present study analyzed the effect of the Togu exercise program on the lumbar back strength of middle-aged women who did not perform regular exercise, and presents an effective exercise method for middle-aged women with chronic low back pain symptom. [Subjects] The subjects were 16 women with chronic low back pain attending N University, Chungcheong-do, Korea. [Methods] Height, weight, body fat, and body mass index (BMI) were measured using a body composition analyzer (Inbody 720, Biospace, Korea), which utilizes bioelectrical impedance analysis. Using isokinetic lumbar muscle strength measurement equipment [Isomed 2000, Back system, (Germany)], peak torque/body weight, total work and average power of flexion and extension of the lumbar region were measured. For lumbar stabilization exercises, an air cushion (Germany), jumper (Germany), and aero step (Germany) were employed. First, warm-up exercise was conducted for 10 minutes, followed by 10 sets of the 3 main exercises using the above tools with 10-second rest intervals. The main exercise was done for 40 minutes in total. [Results] The paired sample t-test showed significant in the Togu exercise group peak torque of flexion, peak torque of extension, total work of flexion, total work of extension, average power of flexion, average power of extension, after the exercise showed a significant difference in the Togu exercise group. [Conclusion] The trunk muscle actiation program for middle-aged women had a significant effect on muscle strength and low back pain. Therefore the trunk muscle activation program is effective at increasing muscular strength of middle-aged women, and the results suggest improve muscle strength is effectively stabilizaties the lumbar region.


[Cholinergic neurons of the human motor cortex].
Cytoarchitectonic area 4 of human cerebral cortex was studied by histochemical demonstration of choline acetyltransferase (CAT). In layer I CAT reaction demonstrated the presence of tangential nerve fibers and synaptic endings on Cajal-Retzius neuron. High activity of the enzyme was detected in non-pyramidal cell with the axon forming the arcades and establishing contacts on bodies and apical dendrites of the layer III pyramidal neurons. Pyramidal neurons were divided into cholinergic-non-cholinoceptive, cholinergic-cholinoceptive and non-cholinergic-cholinoceptive types.


Effect of prior treatment with antipsychotic long-acting injection on randomised clinical trial treatment outcomes.
It is uncertain whether antipsychotic long-acting injection (LAI) medication in schizophrenia is associated with better clinical outcomes than oral preparations. To examine the impact of prior treatment delivery route on treatment outcomes and whether any differences are moderated by adherence. Analysis of data from two pragmatic 1-year clinical trials in which patients with schizophrenia were randomised to either an oral first-generation antipsychotic (FGA), or a non-clozapine second-generation antipsychotic (SGA, CUtLASS 1 study), or a non-clozapine SGA or clozapine (CUtLASS 2 study). Across both trials, 43% (n = 155) of participants were prescribed an FGA-LAI before randomisation. At 1-year follow-up they showed less improvement in quality of life, symptoms and global functioning than those randomised from oral medication. This difference was confined to patients rated as less than consistently adherent pre-randomisation. The relatively poor improvement in the patients prescribed an LAI pre-randomisation was ameliorated if they had been randomised to clozapine rather than another SGA. There was no advantage to being randomly assigned from an LAI at baseline to a non-clozapine oral SGA rather than an oral FGA. A switch at randomisation from an LAI to an oral antipsychotic was associated with poorer clinical and functional outcomes at 1-year follow-up compared with switching from one oral antipsychotic to another. This effect appears to be moderated by adherence, and may not extend to switching to clozapine. This has implications for clinical trial design: the drug from which a participant is randomised may have a greater effect than the drug to which they are randomised.


Delayed versus Delayed-Immediate Autologous Breast Reconstruction: A Blinded Evaluation of Aesthetic Outcomes.
The technique of delayed-immediate breast reconstruction includes immediate insertion of a tissue expander, post-mastectomy radiation, followed by reconstruction. The aesthetic benefits of delayed-immediate reconstruction compared to delayed reconstruction are postulated but remain unproven. The purpose of this study was to compare aesthetic outcomes in patients following delayed and delayed-immediate autologous breast reconstruction. A retrospective analysis was performed of all patients who underwent delayed or delayed-immediate autologous breast reconstruction by the senior author from 2005 to 2011. Postoperative photographs were used to evaluate aesthetic outcomes: skin quality, scar formation, superior pole contour, inferior pole contour, and overall aesthetic outcome. Ten non-biased reviewers assessed outcomes using a 5-point Likert scale. Fisher's Exact and Wilcoxon-Mann-Whitney tests were used for comparative analysis. Patient age and body mass index were similar between delayed (n=20) and delayed-immediate (n=20) cohorts (P>0.05). Skin and scar quality was rated significantly higher in the delayed-immediate cohort (3.74 vs. 3.05, P<0.001 and 3.41 vs. 2.79, P<0.001; respectively). Assessment of contour-related parameters, superior pole and inferior pole, found significantly improved outcomes in the delayed-immediate cohort (3.67 vs. 2.96, P<0.001 and 3.84 vs. 3.06, P<0.001; respectively). Delayed-immediate breast reconstruction had a significantly higher overall score compared to delayed breast reconstructions (3.84 vs. 2.94, P<0.001). Smoking and the time interval from radiation to reconstruction were found to affect aesthetic outcomes (P<0.05). Preservation of native mastectomy skin may allow for improved skin/scar quality, breast contour, and overall aesthetic outcomes following a delayed-immediate reconstructive algorithm as compared to delayed breast reconstruction.


An Objective Approach to Deriving the Clinical Performance of Autoverification Limits.
This study describes an objective approach to deriving the clinical performance of autoverification rules to inform laboratory practice when implementing them. Anonymized historical laboratory data for 12 biochemistry measurands were collected and Box-Cox-transformed to approximate a Gaussian distribution. The historical laboratory data were assumed to be error-free. Using the probability theory, the clinical specificity of a set of autoverification limits can be derived by calculating the percentile values of the overall distribution of a measurand. The 5th and 95th percentile values of the laboratory data were calculated to achieve a 90% clinical specificity. Next, a predefined tolerable total error adopted from the Royal College of Pathologists of Australasia Quality Assurance Program was applied to the extracted data before subjecting to Box-Cox transformation. Using a standard normal distribution, the clinical sensitivity can be derived from the probability of the Z-value to the right of the autoverification limit for a one-tailed probability and multiplied by two for a two-tailed probability. The clinical sensitivity showed an inverse relationship with between-subject biological variation. The laboratory can set and assess the clinical performance of its autoverification rules that conforms to its desired risk profile.


Accuracy of optical coherence tomography for diagnosing glaucoma: an overview of systematic reviews.
To assess the diagnostic accuracy (DTA) of optical coherence tomography (OCT) for detecting glaucoma by systematically searching and appraising systematic reviews (SRs) on this issue. We searched a database of SRs in eyes and vision maintained by the Cochrane Eyes and Vision United States on the DTA of OCT for detecting glaucoma. Two authors working independently screened the records, abstracted data and assessed the risk of bias using the Risk of Bias in Systematic Reviews checklist. We extracted quantitative DTA estimates as well as qualitative statements on their relevance to practice. We included four SRs published between 2015 and 2018. These SRs included between 17 and 113 studies on OCT for glaucoma diagnosis. Two reviews were at low risk of bias and the other two had two to four domains at high or unclear risk of bias with concerns on applicability. The two reliable SRs reported the accuracy of average retinal nerve fibre layer (RNFL) thickness and found a sensitivity of 0.69 (0.63 to 0.73) and 0.78 (0.74 to 0.83) and a specificity of 0.94 (0.93 to 0.95) and 0.93 (0.92 to 0.95) in 57 and 50 studies, respectively. Only one review included a clear specification of the clinical pathway. Both reviews highlighted the limitations of primary DTA studies on this topic. The quality of published DTA reviews on OCT for diagnosing glaucoma was mixed. Two reliable SRs found moderate sensitivity at high specificity for average RNFL thickness in diagnosing manifest glaucoma. Our overview suggests that the methodological quality of both primary and secondary DTA research on glaucoma is in need of improvement.


Autoregulation of human retinal blood flow. An investigation with laser Doppler velocimetry.
The effect of acute changes in mean retinal perfusion pressure, P (2/3 of mean brachial artery blood pressure minus IOP), on retinal volumetric blood flow rate, Q, was investigated in normal volunteers. Changes in Q were determined from Q = k X Vmax X D2, where Vmax is the center line red blood cell velocity measured from temporal veins by laser Doppler velocimetry, D is the vessel diameter obtained by monochromatic fundus photography, and k is a constant of proportionality. A suction cup was used to induce step changes in IOP and, consequently, in P. The magnitude of the steps ranged from 10-32 mmHg. During the first 30 sec after a step decrease in P, Vmax and Q were significantly smaller than at rest by an amount proportional to the decrease in P. Thereafter, Vmax and Q increased markedly towards their values at rest, although P changed comparatively little during this period of time. Time constant of the corresponding decrease in vascular resistance, R(t) = P(t)/Q(t), was approximately 45 sec. There was no significant change in D during elevated IOP. Removal of the cup induced an immediate step increase in P, Vmax, D, Q, and R. Thereafter, Vmax, D, Q, and R returned to their values at rest (time constant of the change in R was about 30 sec), while P remained nearly constant. The rapid change in vascular resistance following a step decrease and increase in P can be attributed to an active process that attempts to maintain blood flow close to normal, in spite of changes in perfusion pressure (autoregulation).(ABSTRACT TRUNCATED AT 250 WORDS)


Freshwater salinization syndrome limits management efforts to improve water quality.
Freshwater Salinization Syndrome (FSS) refers to groups of biological, physical, and chemical impacts which commonly occur together in response to salinization. FSS can be assessed by the mobilization of chemical mixtures, termed "chemical cocktails", in watersheds. Currently, we do not know if salinization and mobilization of chemical cocktails along streams can be mitigated or reversed using restoration and conservation strategies. We investigated 1) the formation of chemical cocktails temporally and spatially along streams experiencing different levels of restoration and riparian forest conservation and 2) the potential for attenuation of chemical cocktails and salt ions along flowpaths through conservation and restoration areas. We monitored high-frequency temporal and longitudinal changes in streamwater chemistry in response to different pollution events (i.e., road salt, stormwater runoff, wastewater effluent, and baseflow conditions) and several types of watershed management or conservation efforts in six urban watersheds in the Chesapeake Bay watershed. Principal component analysis (PCA) indicates that chemical cocktails which formed along flowpaths (i.e., permanent reaches of a stream) varied due to pollution events. In response to winter road salt applications, the chemical cocktails were enriched in salts and metals (e.g., Na+, Mn, and Cu). During most baseflow and stormflow conditions, chemical cocktails were less enriched in salt ions and trace metals. Downstream attenuation of salt ions occurred during baseflow and stormflow conditions along flowpaths through regional parks, stream-floodplain restorations, and a national park. Conversely, chemical mixtures of salt ions and metals, which formed in response to multiple road salt applications or prolonged road salt exposure, did not show patterns of rapid attenuation downstream. Multiple linear regression was used to investigate variables that influence changes in chemical cocktails along flowpaths. Attenuation and dilution of salt ions and chemical cocktails along stream flowpaths was significantly related to riparian forest buffer width, types of salt pollution, and distance downstream. Although salt ions and chemical cocktails can be attenuated and diluted in response to conservation and restoration efforts at lower concentration ranges, there can be limitations in attenuation during road salt events, particularly if storm drains bypass riparian buffers.


Control analysis applied to single enzymes: can an isolated enzyme have a unique rate-limiting step?
Control analysis is used to analyse and quantify the concept of a rate-limiting step within an enzyme. The extent to which each rate constant within the enzyme limits the steady-state rate of the enzyme and the levels of enzyme intermediate species are quantified as flux and concentration control coefficients. These coefficients are additive and obey summation theorems. The control coefficients of triose phosphate isomerase, carbamate kinase and lactate dehydrogenase are calculated from literature values of the rate constants. It is shown that, contrary to previous assumption, these enzymes do not have a unique rate-limiting step, but rather flux control is shared by several rate constants and varies with substrate, product and effector concentrations, and with the direction of the reaction. Thus the general assumption that an enzyme will have a unique rate-limiting step is unjustified.


Role of ferrocyanides in the prebiotic synthesis of α-amino acids.
We investigated the synthesis of α-amino acids under possible prebiotic terrestrial conditions in the presence of dissolved iron (II) in a simulated prebiotic ocean. An aerosol-liquid cycle with a prebiotic atmosphere is shown to produce amino acids via Strecker synthesis with relatively high yields. However, in the presence of iron, the HCN was captured in the form of a ferrocyanide, partially inhibiting the formation of amino acids. We showed how HCN captured as Prussian Blue (or another complex compound) may, in turn, have served as the HCN source when exposed to UV radiation, allowing for the sustained production of amino acids in conjunction with the production of oxyhydroxides that precipitate as by-products. We conclude that ferrocyanides and related compounds may have played a significant role as intermediate products in the prebiotic formation of amino acids and oxyhydroxides, such as those that are found in iron-containing soils and that the aerosol cycle of the primitive ocean may have enhanced the yield of the amino acid production.


Hsf1 and the molecular chaperone Hsp90 support a 'rewiring stress response' leading to an adaptive cell size increase in chronic stress.
Cells are exposed to a wide variety of internal and external stresses. Although many studies have focused on cellular responses to acute and severe stresses, little is known about how cellular systems adapt to sublethal chronic stresses. Using mammalian cells in culture, we discovered that they adapt to chronic mild stresses of up to two weeks, notably proteotoxic stresses such as heat, by increasing their size and translation, thereby scaling the amount of total protein. These adaptations render them more resilient to persistent and subsequent stresses. We demonstrate that Hsf1, well known for its role in acute stress responses, is required for the cell size increase, and that the molecular chaperone Hsp90 is essential for coupling the cell size increase to augmented translation. We term this translational reprogramming the 'rewiring stress response', and propose that this protective process of chronic stress adaptation contributes to the increase in size as cells get older, and that its failure promotes aging.


Buddhist philosophy for the treatment of problem gambling.
Purpose In the last five years, scientific interest into the potential applications of Buddhist-derived interventions (BDIs) for the treatment of problem gambling has been growing. This paper reviews current directions, proposes conceptual applications, and discusses integration issues relating to the utilisation of BDIs as problem gambling treatments. Method Aliterature search and evaluation of the empirical literature for BDIs as problem gambling treatments was undertaken. Results To date, research has been limited to cross-sectional studies and clinical case studies and findings indicate that Buddhist-derived mindfulness practices have the potential to play an important role in ameliorating problem gambling symptomatology. As an adjunct to mindfulness, other Buddhist-derived practices are also of interest including: (i) insight meditation techniques (e.g., meditation on 'emptiness') to overcome avoidance and dissociation strategies, (ii) 'antidotes' (e.g., patience, impermanence, etc.) to attenuate impulsivity and salience-related issues, (iii) loving-kindness and compassion meditation to foster positive thinking and reduce conflict, and (iv) 'middle-way' principles and 'bliss-substitution' to reduce relapse and temper withdrawal symptoms. In addition to an absence of controlled treatment studies, the successful operationalisation of BDIs as effective treatments for problem gambling may be impeded by issues such as a deficiency of suitably experienced BDI clinicians, and the poor provision by service providers of both BDIs and dedicated gambling interventions. Conclusions Preliminary findings for BDIs as problem gambling treatments are promising, however, further research is required.


One-year results of a community-based translation of the Diabetes Prevention Program: Healthy-Living Partnerships to Prevent Diabetes (HELP PD) Project.
Although the Diabetes Prevention Program (DPP) and the Finnish Diabetes Prevention Study (FDPS) demonstrated that weight loss from lifestyle change reduces type 2 diabetes incidence in patients with prediabetes, the translation into community settings has been difficult. The objective of this study is to report the first-year results of a community-based translation of the DPP lifestyle weight loss (LWL) intervention on fasting glucose, insulin resistance, and adiposity. We randomly assigned 301 overweight and obese volunteers (BMI 25-40 kg/m(2)) with fasting blood glucose values between 95 and 125 mg/dL to a group-based translation of the DPP LWL intervention administered through a diabetes education program (DEP) and delivered by community health workers (CHWs) or to an enhanced usual-care condition. CHWs were volunteers with well-controlled type 2 diabetes. A total of 42.5% of participants were male, mean age was 57.9 years, 26% were of a race/ethnicity other than white, and 80% reported having an education beyond high school. The primary outcome is mean fasting glucose over 12 months of follow-up, adjusting for baseline glucose. Compared with usual-care participants, LWL intervention participants experienced significantly greater decreases in blood glucose (-4.3 vs. -0.4 mg/dL; P<0.001), insulin (-6.5 vs. -2.7 μU/mL; P<0.001), homeostasis model assessment of insulin resistance (-1.9 vs. -0.8; P<0.001), weight (-7.1 vs. -1.4 kg; P<0.001), BMI (-2.1 vs. -0.3 kg/m2; P<0.001), and waist circumference (-5.9 vs. -0.8 cm; P<0.001). This translation of the DPP intervention conducted in community settings, administered through a DEP, and delivered by CHWs holds great promise for the prevention of diabetes by significantly decreasing glucose, insulin, and adiposity.


Effect of levamisole as a surgical adjuvant therapy for malignant melanoma.
The preliminary results of a trial in progress are reported. Two hundred patients have been enrolled in a randomized, double-blind, placebo-controlled trial of levamisole in malignant melanoma. A median followup of 20 months has been reached. Endpoints of the study include disease-free interval, interval free of visceral disease, and survival. Overall, there is a trend in favor of the levamisole group with regard to all three endpoints, but the difference is not significant. Significant differences between the control and levamisole groups are found at some, but not all, time intervals when the analysis includes only patients with stage II disease (positive lymph nodes at the initiation of the study). Although these preliminary results are encouraging, at the present time the overall curve for patients with stage II disease is not statistically significantly different when analyzed by the method of Mantel.


Biomarkers in Lysosomal Storage Diseases.
A biomarker is generally an analyte that indicates the presence and/or extent of a biological process, which is in itself usually directly linked to the clinical manifestations and outcome of a particular disease. The biomarkers in the field of lysosomal storage diseases (LSDs) have particular relevance where spectacular therapeutic initiatives have been achieved, most notably with the introduction of enzyme replacement therapy (ERT). There are two main types of biomarkers. The first group is comprised of those molecules whose accumulation is directly enhanced as a result of defective lysosomal function. These molecules represent the storage of the principal macro-molecular substrate(s) of a specific enzyme or protein, whose function is deficient in the given disease. In the second group of biomarkers, the relationship between the lysosomal defect and the biomarker is indirect. In this group, the biomarker reflects the effects of the primary lysosomal defect on cell, tissue, or organ functions. There is no "gold standard" among biomarkers used to diagnosis and/or monitor LSDs, but there are a number that exist that can be used to reasonably assess and monitor the state of certain organs or functions. A number of biomarkers have been proposed for the analysis of the most important LSDs. In this review, we will summarize the most promising biomarkers in major LSDs and discuss why these are the most promising candidates for screening systems.


Modeling cost-effectiveness of pharmaceuticals in Alzheimer's disease.
Alzheimer's disease is a neurodegenerative disorder characterized by cognitive and behavioral deficits. Donepezil and memantine are two medications used to treat the symptoms of cognitive decline in Alzheimer's disease. A modeled cost-effectiveness analysis found that treatment with donepezil to be less costly and more effective than no treatment or treatment with memantine in Germany. However, the model drew on non-German population data and could have been more transparent with regard to 'back-end' processes. In addition, the authors would question the extent to which the differences in effectiveness between treatments would be considered clinically significant. Identifying sources of cost savings for donepezil remains an important issue for future consideration.


Discovery of high-affinity ligands of sigma1 receptor, ERG2, and emopamil binding protein by pharmacophore modeling and virtual screening.
ERG2, emopamil binding protein (EBP), and sigma-1 receptor (sigma(1)) are enzymes of sterol metabolism and an enzyme-related protein, respectively, that share high affinity for various structurally diverse compounds. To discover novel high-affinity ligands, pharmacophore models were built with Catalyst based upon a series of 23 structurally diverse chemicals exhibiting K(i) values from 10 pM to 100 microM for all three proteins. In virtual screening experiments, we retrieved drugs that were previously reported to bind to one or several of these proteins and also tested 11 new hits experimentally, of which three, among them raloxifene, had affinities for sigma(1) or EBP of <60 nM. When used to search a database of 3525 biochemicals of intermediary metabolism, a slightly modified ERG2 pharmacophore model successfully retrieved 10 substrate candidates among the top 28 hits. Our results indicate that inhibitor-based pharmacophore models for sigma(1), ERG2, and EBP can be used to screen drug and metabolite databases for chemically diverse compounds and putative endogenous ligands.


Activation of the regulator of G protein signaling 14-Gαi1-GDP signaling complex is regulated by resistance to inhibitors of cholinesterase-8A.
RGS14 is a brain scaffolding protein that integrates G protein and MAP kinase signaling pathways. Like other RGS proteins, RGS14 is a GTPase activating protein (GAP) that terminates Gαi/o signaling. Unlike other RGS proteins, RGS14 also contains a G protein regulatory (also known as GoLoco) domain that binds Gαi1/3-GDP in cells and in vitro. Here we report that Ric-8A, a nonreceptor guanine nucleotide exchange factor (GEF), functionally interacts with the RGS14-Gαi1-GDP signaling complex to regulate its activation state. RGS14 and Ric-8A are recruited from the cytosol to the plasma membrane in the presence of coexpressed Gαi1 in cells, suggesting formation of a functional protein complex with Gαi1. Consistent with this idea, Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex in cells and in vitro using purified proteins. Purified Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex to form a stable Ric-8A-Gαi complex in the absence of GTP. In the presence of an activating nucleotide, Ric-8A interacts with the RGS14-Gαi1-GDP complex to stimulate both the steady-state GTPase activity of Gαi1 and binding of GTP to Gαi1. However, sufficiently high concentrations of RGS14 competitively reverse these stimulatory effects of Ric-8A on Gαi1 nucleotide binding and GTPase activity. This observation correlates with findings that show RGS14 and Ric-8A share an overlapping binding region within the last 11 amino acids of Gαi1. As further evidence that these proteins are functionally linked, native RGS14 and Ric-8A coexist within the same hippocampal neurons. These findings demonstrate that RGS14 is a newly appreciated integrator of unconventional Ric-8A and Gαi1 signaling.


Structural mechanism for regulation of the AAA-ATPases RUVBL1-RUVBL2 in the R2TP co-chaperone revealed by cryo-EM.
The human R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) is an HSP90 co-chaperone required for the maturation of several essential multiprotein complexes, including RNA polymerase II, small nucleolar ribonucleoproteins, and PIKK complexes such as mTORC1 and ATR-ATRIP. RUVBL1-RUVBL2 AAA-ATPases are also primary components of other essential complexes such as INO80 and Tip60 remodelers. Despite recent efforts, the molecular mechanisms regulating RUVBL1-RUVBL2 in these complexes remain elusive. Here, we report cryo-EM structures of R2TP and show how access to the nucleotide-binding site of RUVBL2 is coupled to binding of the client recruitment component of R2TP (PIH1D1) to its DII domain. This interaction induces conformational rearrangements that lead to the destabilization of an N-terminal segment of RUVBL2 that acts as a gatekeeper to nucleotide exchange. This mechanism couples protein-induced motions of the DII domains with accessibility of the nucleotide-binding site in RUVBL1-RUVBL2, and it is likely a general mechanism shared with other RUVBL1-RUVBL2-containing complexes.


Cancer risks in BRCA2 families: estimates for sites other than breast and ovary.
In BRCA2 mutation carriers, increased risks have been reported for several cancer sites besides breast and ovary. As most of the families included in earlier reports were selected on the basis of multiple breast/ovarian cancer cases, it is possible that risk estimates may differ in mutation carriers with a less striking family history. In the Netherlands, 139 BRCA2 families with 66 different pathogenic mutations were included in a nationwide study. To avoid testing bias, we chose not to estimate risk in typed carriers, but rather in male and female family members with a 50% prior probability of being a carrier (n = 1811). The relative risk (RR) for each cancer site with the exception of breast and ovarian cancer was determined by comparing observed numbers with those expected, based on Dutch cancer incidence rates. We observed an excess risk for four cancer sites: pancreas (RR 5.9; 95% confidence interval (CI) 3.2 to 10.0), prostate (2.5; 1.6 to 3.8), bone (14.4; 2.9 to 42.1) and pharynx (7.3; 2.0 to 18.6). A small increase was observed for cancer of the digestive tract (1.5; 1.1 to 1.9). Histological verification was available for 46% of the tumours. Nearly all increased risks reached statistical significance for men only. Cancer risks tended to be higher for people before the age of 65 years. Moreover, families with mutations outside the previously defined ovarian cancer cluster region tended to have a higher cancer risk. We found that BRCA2 carriers are at increased risk for cancers of the prostate and pancreas, and possibly bone and pharynx. Larger databases with extended follow up are needed to provide insight into mutation specific risks of selected carriers in BRCA2 families.


Dakin-West synthesis of beta-aryl ketones.
Triethylamine and 1-methylimidazole were found to be selective catalysts for the Dakin-West synthesis of diaryl ketones and aryl methyl ketones, respectively. In the 1-methylimidazole-catalyzed reaction, catalysis is due to the simultaneous formation of both an effective acylating agent, 1-acyl-3-methylimidazolium, and a base, carboxylate anion. Hydrocinnamic acid, a compound previously reported to be unreactive under Dakin-West conditions, forms 4-phenyl-2-butanone when the reaction is catalyzed by 1-methylimidazole.


Protein Glycation by Glyoxal Promotes Amyloid Formation by Islet Amyloid Polypeptide.
Protein glycation, also known as nonenzymatic glycosylation, is a spontaneous post-translational modification that would change the structure and stability of proteins or hormone peptides. Recent studies have indicated that glycation plays a role in type 2 diabetes (T2D) and neurodegenerative diseases. Over the last two decades, many types of advanced glycation end products (AGEs), formed through the reactions of an amino group of proteins with reducing sugars, have been identified and detected in vivo. However, the effect of glycation on protein aggregation has not been fully investigated. In this study, we aim to elucidate the impact of protein glycation on islet amyloid polypeptide (IAPP, also known as amylin) aggregation, which was strongly associated with T2D. We chemically synthesized glycated IAPP (AGE-IAPP) to mimic the consequence of this hormone peptide in a hyperglycemia (high blood sugar) environment. Our data revealed that AGE-IAPP formed amyloid faster than normal IAPP, and higher-molecular-weight AGE-IAPP oligomers were also observed in the early stage of aggregation. Circular dichroism spectra also indicated that AGE-IAPP exhibited faster conformational changes from random coil to its β-sheet fibrillar states. Moreover, AGE-IAPP can induce normal IAPP to expedite its aggregation process, and its fibrils can also act as templates to promote IAPP aggregation. AGE-IAPP, like normal IAPP, is capable of interacting with synthetic membranes and also exhibits cytotoxicity. Our studies demonstrated that glycation modification of IAPP promotes the amyloidogenic properties of IAPP, and it may play a role in accumulating additional amyloid during T2D progression.


The effect of leptin on mouse trophoblast cell invasion.
The hormone leptin is produced by adipose tissue and can function as a signal of nutritional status to the reproductive system. The expression of leptin receptor and, in some species, leptin, in the placenta suggests a role for leptin in placental development, but this role has not been elucidated. Leptin is required at the time of embryo implantation in the leptin-deficient ob/ ob mouse and has been shown to upregulate expression of matrix metalloproteinases (MMPs), enzymes involved in trophoblast invasion, in cultured human trophoblast cells. This led us to the hypothesis that leptin promotes the invasiveness of trophoblast cells crucial to placental development. We found that leptin stimulated mouse trophoblast cell invasion through a matrigel-coated insert on Day 10, but not Day 18 of pregnancy. Optimal stimulation occurred at a concentration of 50 ng/ml leptin, similar to the peak plasma leptin concentration during pregnancy in the mouse. Leptin treatment did not stimulate proliferation of mouse trophoblast cells in primary culture. Leptin stimulation of invasion was prevented by 25 muM GM6001, an inhibitor of MMP activity. Our results suggest that leptin may play a role in the establishment of the placenta during early pregnancy and that this function is dependent on MMP activity. This effect of leptin may represent one mechanism by which body condition affects placental development.