Effect of donation time on platelet concentrates and fresh-frozen plasma. An in vitro study

To investigate the effect of donation time on the quality of blood components, we measured the platelet count and pH on platelet concentrates, and the factor V and VIII:C levels and fibrinopeptide A concentration on fresh-frozen plasma by duration of donation time. Platelet concentrates and fresh-frozen plasma were classified into three groups according to donation time: group 1, less than 10 min; group 2, 10-15 min, and group 3, longer than 15 min. Mean platelet counts of platelet concentrate were: group 1, 8.6 +/- 2.5 (in x 10(10], group 2, 8.1 +/- 2.6, and group 3, 6.5 +/- 3.2 (p less than 0.05). The same pH was maintained in all three groups. The fibrinopeptide A concentrations in groups 1 and 2 were 24 +/- 53 and 169 +/- 64 ng/ml, respectively, while in group 3 all were greater than 200 ng/ml, indicating correlation of a higher fibrinopeptide A level with longer donation time. Although a higher fibrinopeptide A level indicated a greater degree of thrombin generation, assays of factors V and VII:C did not show decreased activity in groups 2 and 3.     

Factors associated with intention to be tested for HIV among men who have sex with men in a country with a very low HIV prevalence

This study examined factors associated with the intention to take an HIV test among men who have sex with men (MSM) in South Korea. An internet website-based survey was conducted among users of the only and largest online MSM website between 20 July 2016, and 20 August 2016. A total of 2915 participants completed the survey and answered questions related to sociodemographic information, health behaviors, sexual behaviors, and HIV testing history. Of these, 2587 (88.7%) participants responded as having an intention to take an HIV test. A multivariable logistic regression analysis revealed the following as having reduced the intention to undergo HIV testing: very good subjective health status and no sexual interactions during the last 6 months (Adjusted odds ratios [AOR] 0.45 and 0.54, respectively). In contrast, increased intention to take an HIV test was associated with being 20–29 years old, 30–39 years old, not paying or receiving money for sex, having a history of HIV testing, and taking an HIV test once per 12 months (AOR 2.64, 2.13, 1.54, 1.81, and 2.17, respectively). In conclusion, HIV testing among MSM in this study was associated with age, subjective health status, sex(es) of one’s sexual partner(s) during the last 6 months, sexual risk behaviors, HIV testing history, and undergoing regular HIV testing.     

Fgf20 governs formation of primary and secondary dermal condensations in developing hair follicles

In hair follicle development, a placode-derived signal is believed to induce formation of the dermal condensation, an essential component of ectodermal organs. However, the identity of this signal is unknown. Furthermore, although induction and patterning of hair follicles are intimately linked, it is not known whether the mesenchymal condensation is necessary for inducing the initial epithelial pattern. Here, we show that fibroblast growth factor 20 (Fgf20) is expressed in hair placodes and is induced by and functions downstream from epithelial ectodysplasin (Eda)/Edar and Wnt/β-Catenin signaling to initiate formation of the underlying dermal condensation. Fgf20 governs formation of primary and secondary dermal condensations in developing hair follicles and subsequent formation of guard, awl, and auchene hairs. Although primary dermal condensations are absent in Fgf20 mutant mice, a regular array of hair placodes is formed, demonstrating that the epithelial patterning process is independent of known histological and molecular markers of underlying mesenchymal patterns during the initial stages of hair follicle development.     

Chemoradiation-induced changes in systemic inflammatory markers and their prognostic significance in oesophageal squamous cell carcinoma

Objective:Chemoradiation (CRT) may induce a change in systemic inflammatory state which could affect clinical outcomes in oesophageal cancer. We aimed to evaluate the changes and prognostic significance of systemic inflammatory markers following definitive CRT in oesophageal squamous cell carcinoma.Methods:A total of 53 patients treated with concurrent CRT were included in this retrospective analysis. We compared neutrophils, lymphocytes, platelets, neutrophil–lymphocyte ratio (NLR) and platelet–lymphocyte ratio (PLR) before and after CRT using Wilcoxon signed-rank test. Overall survival (OS) and progression-free survival (PFS) were calculated. Univariable and multivariable survival analysis were performed using Cox regression analysis. Clinical univariable survival prognostic factors with p < 0.1 were included in a multivariable cox regression analysis for backward stepwise model selection.Results:Both NLR (median ∆+2.8 [IQR −0.11, 8.62], p < 001) and PLR (median ∆+227 [81.3–523.5], p < 0.001) increased significantly after CRT. Higher levels of pre-CRT, post-CRT and change (∆) in NLR and PLR were associated with inferior OS and PFS. Post-CRT NLR (HR 1.04, 95% CI 1.02–1.07, p < 0.001), post-CRT platelets (HR 1.03, 95% CI 1.01–1.05, p = 0.005), cT-stage (HR 3.83, 95% CI 1.39–10.60, p = 0.01) and RT dose (HR 0.41, 95% CI 0.21–0.81, p = 0.01) were independent prognostic factors for OS in multivariable analysis. Change in NLR (HR 1.04, 95% CI 1.01–1.06, p = 0.001), post-CRT platelets (HR 1.03, 95% CI 1.01–1.05, p = 0.002), cT-stage (HR 3.98, 95% CI 1.55–10.25, p = 0.004) and RT dose (HR 0.41, 95% CI 0.21–0.80, p = 0.009) were independent prognostic factors for PFS.Conclusion:Both NLR and PLR increased following definitive CRT. Post-CRT NLR and ∆NLR were associated with adverse survival in oesophageal SCC.Advances in knowledge:We showed that CRT increased PLR and NLR, possibly reflecting a systemic inflammatory state which were associated with poor clinical outcomes in oesophageal SCC.     

Perceived Stress,Positive Resources and Their Interactions as Possible Related Factors for Depressive Symptoms

Objective The present study aimed to explore how the patterns of interaction between stress and positive resources differ according to the severity of depression and which resources play the most important role among the various positive resources. Methods The study included 1,806 people who had visited a health screening center for a mental health check-up to evaluate the levels of perceived stress, positive resources, and depressive symptoms. The participants were divided into a depressive group (n=1,642, mean age 50.60, female 68%) and a non-depressive group (n=164, mean age 48.42, female 66.6%). We conducted hierarchical regression analyses and simple slope analyses to examine the interaction between perceived stress and positive resources. Results The interaction between perceived stress and optimism was significantly associated with depression in non-depressive groups. In depressive groups, the interactions between five types of positive resources (optimism, purpose in life, self-control, social support and care) and perceived stress were all significantly related to depression. Conclusion Interventions that promote optimism can be helpful for preventing inevitable stress from leading to depression. A deficiency in positive resources may be a factor in aggravating depression in stressful situations for people reporting moderate to severe depressive symptoms.     

A Network Model of Positive Resources,Temperament, Childhood Trauma,and Comorbid Symptoms for Patient with Depressive Disorders

ObjectiveTemperament, positive resources, childhood trauma, and other clinical comorbid symptoms are related to depressive symptom severity. Here, we used network analysis to examine the interrelations between these clinical factors in patients with depressive disorders. MethodsPatients with depressive disorders (n=454) completed self-report questionnaires evaluating clinical symptoms, childhood trauma, temperament, and positive resources. To identify network pattern and the most central aspect, we performed network analysis and centrality analyses. First, we analyzed the network pattern in total participants. Second, we established two groups of those with severe depressive symptoms and those with mild depressive symptoms and compared their network patterns. ResultsDeficient optimism and depression were the central factors in the network of total participants. In the group with severe depressive symptoms, lack of social support and childhood emotional trauma showed high centrality. Deficient social support and other positive resources played central roles in the group with mild depressive symptoms. ConclusionNetwork pattern of psychological factors was different between those with mild or severe depression. Lack of positive resources is an important factor in psychological processes in both mild and severe depression. However, childhood emotional trauma may play a relatively important role in patients with severe depressive symptoms.     

Genome-wide bimolecular fluorescence complementation analysis of SUMO interactome in yeast

The definition of protein–protein interactions (PPIs) in the natural cellular context is essential for properly understanding various biological processes. So far, however, most large-scale PPI analyses have not been performed in the natural cellular context. Here, we describe the construction of a Saccharomyces cerevisiae fusion library in which each endogenous gene is C-terminally tagged with the N-terminal fragment of Venus (VN) for a genome-wide bimolecular fluorescence complementation assay, a powerful technique for identifying PPIs in living cells. We illustrate the utility of the VN fusion library by systematically analyzing the interactome of the small ubiquitin-related modifier (SUMO) and provide previously unavailable information on the subcellular localization, types, and protease dependence of SUMO interactions. Our data set is highly complementary to the existing data sets and represents a useful resource for expanding the understanding of the physiological roles of SUMO. In addition, the VN fusion library provides a useful research tool that makes it feasible to systematically analyze PPIs in the natural cellular context.Most biological processes are mediated by complicated networks of protein–protein interactions (PPIs). Thus, the identification of the occurrence and components of PPIs provides invaluable insights into the cellular functions of proteins. In addition to conventional coimmunoprecipitation techniques, several methods have been developed to study PPIs, including the yeast two-hybrid analysis (Fields and Song 1989), the split ubiquitin system (Johnsson and Varshavsky 1994), fluorescence resonance energy transfer (FRET) assays (Periasamy and Day 1999; Pollok and Heim 1999), tandem affinity purification (TAP) followed by mass spectrometry (MS) analysis (Rigaut et al. 1999), and protein fragment complementation (PFC) assays (Remy and Michnick 1999; Ghosh et al. 2000; Wehrman et al. 2002; Paulmurugan and Gambhir 2003). Recently, the bimolecular fluorescence complementation (BiFC) assay, a specialized form of the PFC assay that uses fluorescent proteins as a reporter, has been developed (Hu et al. 2002). The BiFC assay is based on the formation of a fluorescent complex when two proteins fused to nonfluorescent fragments of a fluorescent protein interact with each other. This approach enables direct visualization of the occurrence and subcellular localization of PPIs with simple equipment.The budding yeast Saccharomyces cerevisiae has been a valuable eukaryotic model system, not only for traditional molecular and cell biology but also for the fields of functional genomics and proteomics. In S. cerevisiae, several large-scale analyses of PPIs have been performed with the yeast two-hybrid method (Uetz et al. 2000; Ito et al. 2001) or TAP-MS analysis (Gavin et al. 2002, 2006; Ho et al. 2002; Krogan et al. 2006). However, these approaches do not measure PPI in the natural cellular context; the yeast two-hybrid method is not appropriate for analyzing the interactions between proteins that cannot be transported to the nucleus or that form interactions only in the presence of other stabilizing interactions, and TAP-MS analysis is not amenable to studying protein complexes that are weakly or transiently formed or that do not survive in vitro purification. Compared with the yeast two-hybrid method and TAP-MS analysis, the FRET, PFC, and BiFC assays have several advantages in that they can detect the interactions between proteins in their natural cellular environment. Recently, a large-scale PPI screen using the PFC assay based on reconstituted dihydrofolate reductase (DHFR) activity has been reported (Tarassov et al. 2008), which is the first example of genome-wide PPI analysis using the PFC assay. However, because positive PPIs are selected by methotrexate resistance in the DHFR PFC assay, it is possible that the addition of methotrexate to the assay medium may perturb cellular physiology and proper PPI networks. In this regard, large-scale PPI screens using the BiFC or FRET assay that do not require any exogenous reagents would provide more accurate information about the structural organization of PPI networks in cells. To date, however, there is no report describing the application of the BiFC or FRET assay to genome-wide PPI analyses, particularly using proteins expressed from their own native promoters.The small ubiquitin-related modifier (SUMO) proteins are ∼10 kDa in size and comprise a family of evolutionarily conserved polypeptides that are post-translationally attached to the lysine residues of target proteins to regulate their subcellular localization, stability, and activity (Kerscher et al. 2006; Geiss-Friedlander and Melchior 2007). SUMO conjugation plays a variety of important roles in diverse eukaryotic cellular processes. SUMO can also mediate the non-covalent interaction of substrate proteins with proteins containing SUMO-interacting motifs and modulate their function (Song et al. 2004). In this regard, the identification of SUMO target proteins is crucial for the elucidation of the function of SUMO. Recent genome-wide PPI screens have identified over 500 putative SUMO conjugates in S. cerevisiae (Panse et al. 2004; Wohlschlegel et al. 2004; Zhou et al. 2004; Denison et al. 2005; Hannich et al. 2005; Wykoff and O''Shea 2005). More recently, an elegant study integrating the information of PPIs and genetic interactions has been conducted and has uncovered novel functional relationships between the SUMO pathway and various biological processes (Makhnevych et al. 2009). These systematic analyses have expanded the pool of SUMO substrates and the understanding of the biological function of sumoylation. However, because SUMO substrates can undergo rapid cycles of modification and demodification, and most target proteins appear to be modified to a small percentage at steady state (Geiss-Friedlander and Melchior 2007), it is likely that many unknown SUMO substrates remain to be discovered. Moreover, previous systematic PPI screens to identify SUMO substrates have primarily been performed with the yeast two-hybrid method and TAP-MS, which do not measure PPIs in the natural cellular context. The low agreement between the data from previous systematic screens demonstrates the limitation of the experimental methods used in those analyses and indicates that no single screen has been comprehensive. For this reason, different systematic approaches, with methods that can detect the interactions between proteins in their natural cellular environment, would greatly contribute to the identification of novel SUMO substrates and thus to the understanding of the function of the SUMO pathway.In the present study, we generated a collection of yeast strains expressing full-length proteins tagged with the N-terminal fragment of Venus (VN), a yellow fluorescent protein variant, from their own native promoters. Through a systematic analysis with the VN fusion library, we identified the interactome of SUMO, comprising 367 proteins, and also obtained previously unavailable information on the subcellular localization, types, and protease dependence of SUMO interactions in living yeast cells. Our data not only highlight a novel relationship between sumoylation and various biological processes but also represent a valuable resource that can be used to study the functional roles of the SUMO pathway. This is the first report that describes the application of the BiFC assay to a genome-wide PPI analysis using proteins expressed from their own native promoters. As demonstrated here, the VN fusion library provides a useful research tool that makes it feasible to systematically analyze PPIs in the natural cellular context.