Glycodelin protein and mRNA is downregulated in human first trimester abortion and partially upregulated in mole pregnancy.

Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy.     

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.Biological membranes are conceptually simple structures that may be generated in vitro according to simple physicochemical principles. In vivo, however, membranes are highly complex and host a plethora of proteins that mediate the transfer of molecules and communication across the membrane. Proteins may be trapped in membrane by their transmembrane domains, anchored by lipid tails, or attach to membrane-integral proteins. A further level of complexity is seen when membrane proteins are not equally distributed but occupy only a limited fraction of the available surface (i.e. when they are polarly localized or when they form small membrane subdomains in the micrometer range). The question of how membrane proteins are retained locally and prevented from diffusing freely is of high importance to cell biology. Polarly localized proteins may be retained in their respective domains by membrane fences; in such a situation, polarly localized proteins are mobile in their domains but cannot diffuse through tightly packed scaffold proteins forming a molecular fence within the membrane. Membrane fences delimiting polar domains have been described in different organisms. For example, diffusion between membrane compartments is prevented in budding yeast (Saccharomyces cerevisiae) at the level of the bud neck (Barral et al., 2000; Takizawa et al., 2000); in ciliated vertebrate cells, between ciliary and periciliary membranes (Hu et al., 2010); in epithelial cells, between apical and basolateral membranes (van Meer and Simons, 1986); in neurons, between axon and soma (Kobayashi et al., 1992; Winckler et al., 1999; Nakada et al., 2003); and in spermatozoa, at the level of the annulus (Myles et al., 1984; Nehme et al., 1993). The existence of membrane scaffolds that prevent free protein diffusion has also been described in bacteria (Baldi and Barral, 2012; Schlimpert et al., 2012). In plants, we have shown the existence of a strict membrane fence in the root endodermis, where a median domain splits the cell in two lateral halves occupied by different sets of proteins (Alassimone et al., 2010). The situation in the plant endodermis is analogous to the separation of animal epithelia into apical and basolateral domains; indeed, a parallel between epithelia and endodermal cells has been drawn, despite the different origin of multicellularity in plants and animals (Grebe, 2011).The protein complexes responsible for the formation of membrane fences have been identified. Septins are a family of proteins able to oligomerize and form filaments (Saarikangas and Barral, 2011); their role in the formation of membrane fences has been demonstrated in several organisms and cellular situations, including the yeast bud neck (Barral et al., 2000; Takizawa et al., 2000), animal cilia (Hu et al., 2010), and mammalian spermatozoa (Ihara et al., 2005; Kissel et al., 2005; Kwitny et al., 2010). At the axonal initial segment of neurons, AnkyrinG is necessary to establish and maintain a membrane scaffold where different membrane proteins are immobilized and stabilized (Hedstrom et al., 2008; Sobotzik et al., 2009). In Caulobacter crescentus, the stalk protein Stp forms a complex that prevents diffusion between the cell body and stalk and between stalk compartments. Claudins and occludin are the main components of epithelial tight junctions (Furuse et al., 1993, 1998). Occludins are four-membrane-span proteins and belong to the MARVEL protein family (Sánchez-Pulido et al., 2002), as do Tricellulin and MARVELD3, which are also tight junction-associated proteins (Furuse et al., 1993; Ikenouchi et al., 2005; Steed et al., 2009).In Arabidopsis (Arabidopsis thaliana), our group identified a family of proteins that form a membrane fence in the endodermis (Roppolo et al., 2011). These CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASP1 to CASP5) are four-transmembrane proteins that form a median domain referred to as the Casparian strip membrane domain (CSD). CASPs are initially targeted to the whole plasma membrane, then they are quickly removed from lateral plasma membranes and remain localized exclusively at the CSD; there, they show an extremely low turnover, although they are eventually removed (Roppolo et al., 2011). The membrane proteins NOD26-LIKE INTRINSIC PROTEIN5;1 and BORON TRANSPORTER1 are restricted from diffusing through the CSD and remain polarly localized in the outer and inner lateral membranes, respectively; a fluorescent lipophilic molecule, when integrated in the outer endodermal membrane, was blocked at the level of the CSD and could not diffuse into the inner membrane (Roppolo et al., 2011). Besides making a plasma membrane diffusion barrier, CASPs have an important role in directing the modification of the cell wall juxtaposing their membrane domain: by interacting with secreted peroxidases, they mediate the deposition of lignin and the building up of the Casparian strips (Roppolo et al., 2011; Naseer et al., 2012; Lee et al., 2013). The two CASP activities, making membrane scaffolds and directing a modification of the cell wall, can be uncoupled: indeed, (1) formation of the CASP domain is independent from the deposition of lignin, and (2) interaction between CASPs and peroxidases can take place outside the CSD when CASPs are ectopically expressed (Lee et al., 2013).As CASPs are currently the only known proteins forming membrane fences in plants and because of their essential role in directing a local cell wall modification, we were interested in characterizing the repertoire of a large number of CASP-like (CASPL) proteins in the plant kingdom. Our aim was to provide the molecular basis for the discovery of additional membrane domains in plants and for the identification of proteins involved in local cell wall modifications. We extended our phylogenetic analysis outside of the plant kingdom and found conservation between CASPLs and the MARVEL protein family. Conserved residues are located in transmembrane domains, and we provide evidence suggesting that these domains are involved in CASP localization. We explored the potential use of the CASPL module in plants by investigating CASPL expression patterns and their ability to form membrane domains in the endodermis. Moreover, we related the appearance of the Casparian strips in the plant kingdom to the emergence of a CASP-specific signature that was not found in the genomes of plants lacking Casparian strips.     

Quinidine: an “Endangered Species” Drug Appropriate for Management of Electrical Storm in Brugada Syndrome

Brugada syndrome is an inherited channelopathy associated with an increased risk of syncope and sudden cardiac death. In rare cases it can be manifested with electrical storm. We report two cases of Brugada syndrome that presented with electrical storm and were treated successfully with oral quinidine, an "endangered species" drug.     

An Extended,Boolean Model of the Septation Initiation Network in S.Pombe Provides Insights into Its Regulation

Cytokinesis in fission yeast is controlled by the Septation Initiation Network (SIN), a protein kinase signaling network using the spindle pole body as scaffold. In order to describe the qualitative behavior of the system and predict unknown mutant behaviors we decided to adopt a Boolean modeling approach. In this paper, we report the construction of an extended, Boolean model of the SIN, comprising most SIN components and regulators as individual, experimentally testable nodes. The model uses CDK activity levels as control nodes for the simulation of SIN related events in different stages of the cell cycle. The model was optimized using single knock-out experiments of known phenotypic effect as a training set, and was able to correctly predict a double knock-out test set. Moreover, the model has made in silico predictions that have been validated in vivo, providing new insights into the regulation and hierarchical organization of the SIN.     

Impact of definitive therapy with beta-lactam monotherapy or combination with an aminoglycoside or a quinolone for Pseudomonas aeruginosa bacteremia

Background

Bacteremia by Pseudomonas aeruginosa represents one severe infection. It is not clear whether beta-lactam monotherapy leads to similar rates of treatment success compared to combinations of beta-lactams with aminoglycosides or quinolones.

Methods

Retrospective cohort study from 3 tertiary hospitals (2 in Greece and 1 in Italy). Pseudomonas aeruginosa isolates were susceptible to a beta-lactam and an aminoglycoside or a quinolone. Patients received appropriate therapy for at least 48 hours. Primary outcome of interest was treatment success in patients with definitive beta-lactam combination therapy compared to monotherapy. Secondary outcomes were treatment success keeping the same empirical and definitive regimen, mortality, and toxicity.

Results

Out of 92 bacteremias there were 54 evaluable episodes for the primary outcome (20 received monotherapy). Treatment success was higher with combination therapy (85%) compared to beta-lactam monotherapy (65%), however not statistically significantly [Odds ratio (OR) 3.1; 95% Confidence Interval (CI) 0.69–14.7, p = 0.1]. Very long (>2 months) hospitalisation before bacteremia was the only factor independently associated with treatment success (OR 0.73; 95% CI 0.01–0.95, p = 0.046), however this result entailed few episodes. All-cause mortality did not differ significantly between combination therapy [6/31 (19%)] and monotherapy [8/19 (42%)], p = 0.11. Only Charlson comorbidity index was associated with excess mortality (p = 0.03).

Conclusion

Our study, in accordance with previous ones, indicates that the choice between monotherapy and combination therapy may not affect treatment success significantly. However, our study does not have statistical power to identify small or moderate differences. A large randomized controlled trial evaluating this issue is justified.     

Follicular atresia during Dacus oleae oogenesis

Programmed cell death, constitutes a common fundamental incident that occurs during oogenesis in a variety of different animals. It plays a significant role in the maturation process of the female gamete and also in the removal of abnormal and superfluous cells at certain checkpoints of development. In the present study, we demonstrate the existence of follicular atresia during mid-oogenesis in the olive fruit fly Dacus oleae (Tephritidae). The number of atretic follicles increases following the age of the fly, suggesting for the presence of an age-susceptible process. The atretic follicles contain nurse cells that exhibit chromatin condensation, DNA fragmentation and actin cytoskeleton alterations, as revealed by propidium iodide staining, TUNEL labeling and phalloidin-FITC staining. Conventional light and electron microscopy disclose that the nurse cell remnants are phagocytosed by the adjacent follicle cells. The follicular epithelium also eliminates the oocyte through phagocytosis, resulting to an egg chamber with no compartmentalized organization. The data presented herein are very similar compared to previous reported results in other Diptera species, strongly suggesting the occurrence of a phylogenetically conserved mechanism of follicular atresia. All these observations also support the notion that mid-oogenesis in D. oleae may be the critical regulation point at which superfluous and defective egg chambers are selectively eliminated before they reach maturity.     

Leaf anatomy and chloroplast ultrastructure of Mn-deficient orange plants

Leaf samples of Mn-deficient and Mn-sufficient (control) ‘Navelate’ orange plants grown in a greenhouse were taken to investigate the effects of Mn deficiency in leaf structure and chloroplast ultrastructure. Total leaf chlorophyll concentration was significantly lower in Mn-deficient plants than in control ones. Entire lamina thickness was not altered due to Mn deficiency. However, Mn deficiency resulted in disorganization of mesophyll cells, mainly of palisade parenchyma cells. The number of mesophyll chloroplasts per cellular area and their length were both affected negatively. The membranous system of chloroplasts was also disorganized. The percentages of starch grains and plastoglobuli per chloroplast of Mn-deficient leaves were significantly greater than those of control leaves.     

Elevated serum triglycerides is the strongest single indicator for the presence of metabolic syndrome in patients with type 2 diabetes

Objectives

South-Asians have lower adiponectin levels compared to Caucasians. It was not clear however, if this intrinsic feature is related to aspects of glucose metabolism. This study aims to determine the relationship between body fat distribution and adipocytokine in South-Asian subjects by measuring serum adipocytokines, adiposity, insulinemia, and glucose tolerance levels.

Methods

In this cross-sectional study, 150 South-Asians (80 males, 70 females) were included, 60 had NGT (Control group, Age 51.33 ± 11.5, BMI 27 ± 2.3), 60 had IGT (Age 57.7 ± 12.5, BMI 27.2 ± 2.7), 30 had type 2 DM (Age 49.5 ± 10.9, BMI 28 ± 1.7). Measures of adiposity, adipocytokines and other metabolic parameters were determined. Parameters were measured using the following: a) Plasma glucose by glucose oxidase method b) CRP by immunoturbidimetric method (Roche/Hitachi analyser) c) insulin by Medgenix INS-ELISA immunoenzymetric assay by Biosource (Belgium) d) Leptin, Adiponectin by radioimmunoassay kits by Linco Research (St. Charles MO) e) Resistin by immunoassay kits by Phoenix Pharmaceuticals INC (530 Harbor Boulevard, Belmont CA 94002, USA).

Results

Adiponectin concentrations were highest in NGT, decreased in IGT and lowest in DMT2, (both p < 0.01). Leptin was significantly higher in DMT2 than IGT and NGT p = 0.02 and 0.04 respectively. There was a significant positive relationships between log adiponectin and 2-hr insulin values, p = 0.028 and history of hypertensions and a ischemic heart disease p = 0.008 with R = 0.65. There was a significant inverse correlation between log adiponectin and resistin, p < 0.01.

Conclusion

Resistin levels had an inverse correlation with adiponectin levels, indicating an inverse relationship between pro-inflammatory cytokines and adiponectin. Adiponectin levels were related to glucose tolerance.     

Immune Response Gene Expression in Colorectal Cancer Carries Distinct Prognostic Implications According to Tissue,Stage and Site: A Prospective Retrospective Translational Study in the Context of a Hellenic Cooperative Oncology Group Randomised Trial

Background

Although host immune response is an emerging prognostic factor for colorectal cancer, there is no consensus on the optimal methodology, surrogate markers or tissue for study.

Patients and Methods

Tumour blocks were prospectively collected from 344 patients with stage II/III colorectal cancer (CRC) treated with adjuvant chemotherapy. Whole section lymphocytic infiltration was studied along with mRNA expression of CD3Z, CD8, CD4, CXCL9, CXCL13, IGHM, FOXP3, SNAI2 and ESR1 by qRT-qPCR in tissue microarray (TMA) cores from the centre of tumour, invasive margin and adjacent normal mucosa.

Results

Lymphocytic infiltration, deficient MMR (10.9%), KRAS (40.7%) and BRAF (4.9%) mutations or single mRNA gene expression were not prognostic. Tumour ESR1 gene expression (Hazard Ratio [HR] for relapse 2.33, 95% CI 1.35-4.02; HR for death 1.74, 95% CI 1.02-2.97) and absence of necrosis (HR for relapse 1.71, 95% CI 1.05-2.71; HR for death 1.98, 95% CI 1.14-3.43) were adverse prognostic features. We used CD3Z and CD8 expression in order to devise the mRNA-based Immune Score (mIS) and proceeded to partitioning analysis in 267 patients, with age, stage, tumour site (Right vs Left CRC), KRAS mutation and tumour mIS as input factors. Only in patients with stage III right-sided colon cancer, a low immune response was associated with inferior disease-free survival (mIS-low, HR for relapse 2.28, 95% CI 1.05-8.02). No prognostic significance was seen for tumour mIS in any other stage or site of CRC, or for a similar mIS score derived from adjacent normal mucosa. Independent adverse prognostic significance was retained in multivariable analysis for absence of necrosis, tumour ESR1 expression in all patients and low tumour mIS in stage III right-sided CRC.

Conclusions

In localised CRC, mRNA-based CD3Z/CD8 profiling of tumour immune response may have stage, site and tissue-specific prognostic significance, along with ESR1 expression.

Trial Registration

ANZCTR.org.au ACTRN12610000509066