Role of cytoskeleton-related proteins in the acrosome reaction of Eriocheir sinensis spermatozoa.

Cytoskeleton-related proteins are essential for cell shape maintenance and cytoskeleton remodeling. The spermatozoa of Eriocheir sinensis (Chinese mitten crab) have a unique cellular structure, and the mechanism of spermatozoal metamorphosis during the acrosome reaction is not well understood. In this study, the E. sinensis spermatozoa were induced using calcium ionophore A23187 to undergo the acrosome reaction in vitro, and the acrosome-reacting and fresh (non-reacting) spermatozoa were collected separately. The differential expression of cytoskeleton-related protein genes in acrosome-reacting and fresh spermatozoa of E. sinensis was analyzed by whole transcriptome sequencing and bioinformatics analysis, and PPI network and miRNA-mRNA regulation network were constructed to analyze their possible function and regulation mechanism. The results showed that numerous differentially expressed cytoskeleton-related protein genes, miRNAs and lncRNAs were found in acrosome-reacting and fresh spermatozoa of E. sinensis; 27 cytoskeleton-related protein genes were down regulated and 687 miRNAs were up regulated in acrosome-reacting spermatozoa; 147 miRNAs target these 27 cytoskeleton-related protein genes. In the PPI networks, RAC1, BCAR1, RDX, NCKAP1, EPS8, CDC42BPA, LIMK1, ELMO2, GNAI1 and OCRL were identified as hub proteins. These proteins are mainly involved in the regulation of cytoskeleton organization, actin cytoskeleton organization, microtubule skeleton organization and small GTPase-mediated signal transduction and other biological processes, and play roles in pathways such as actin cytoskeletal regulation and axon guidance. miR-9, miR-31 and two novel miRNAs in the miRNA-mRNA regulatory network are the core miRNAs targeting cytoskeleton-related protein genes. miR-9 targets and regulates OBSCN, CDC42BPA, ELMO2, BCAS3, TPR and OCRL; while miR-31 targets and regulates CDC42BPA and TPR. This study provides a theoretical basis for revealing the mechanism of acrosome reaction under the special spermatozoa morphology of E. sinensis.

Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl.

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell-cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.

Serum levels of cytoskeleton remodeling proteins and their mRNA expression in tumor tissue of metastatic laryngeal and hypopharyngeal cancers.

Actin-binding proteins (ABPs) and various signaling systems are involved in the process of squamous cell carcinoma of the larynx and hypopharynx (SCCLH) metastasis. The clinical significance of these proteins has not yet been determined. We analyzed the relationship between the mRNA levels of cofilin 1 (CFL1), profilin 1 (PFN1), adenylyl cyclase-associated protein 1 (CAP1), SNAI1 and RND3 and SCCLH metastasis. The serum levels of the above ABPs were estimated and the relationship between them and their mRNA expressions was analyzed. The expression levels of ABP mRNAs were measured by real-time RT-PCR in paired tissue samples taken from 54 patients with SCCLH (T1-4N0-1M0). Expression analysis was performed using the 2-ΔΔCT method. The levels of ABPs in the blood serum were measured by ELISA. Statistical analysis was carried out using the SPSS Statistica 20.0 software package. No significant difference in the mRNA gene expression in tumor tissue of patients with T1-3N0M0 SCCLH and patients with T2-4N1-2M0 SCCLH was found. High expression of RND3 mRNA was accompanied by an increase in mRNA expression of all studied ABPs. In the blood serum of T2-4N1-2M0 patients, the level of PFN1 was lower by 21% and the level of CAP1 was higher by 75% than those observed in T1-4N0M0 patients. The data obtained showed that RND3 is involved in the regulation of molecular cascades of SCCLH metastasis. PFN1 and CAP1 serum levels can be good classifiers of metastases in patients with SCCLH.

Ezrin is a prognostic biomarker in patients with clear cell metastatic renal cell carcinoma receiving sunitinib.

OBJECTIVE: Sunitinib is a novel oral multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities. This study evaluates ezrin expression in sunitinib-treated metastatic clear cell renal cell carcinoma (ccRCC) patients and elucidates its role as a possible marker for survival. MATERIALS AND METHODS: The expression of ezrin was measured by immunohistochemistry in 80 patients with ccRCC treated by first-line sunitinib between January 2007 and June 2012. Kaplan-Meier curves and log-rank tests were used for analysis of progression-free survival and overall survival (OS), and a multivariate Cox proportional hazard model was employed to identify factors with an independent effect on the survival. RESULTS: In multivariate analysis, liver metastasis (P = 0.018; hazard ratio [HR]: 3.707 (1.257-10.931) and overexpression of ezrin (P = 0.006; HR: 2.993 (1.373-6.523 95% confidence interval) were remained significant factors influencing OS. Overexpression of ezrin in the patients who had progressed in the first 3 months was higher than in the patients who had progressed after 3 months (P = 0.003). The median OS was longer in patients with low levels of ezrin expression (27 months) compared to patients overexpressing ezrin (12 months) (P = 0.001). CONCLUSION: This is the first study in the literature showing that ezrin status is related with prognosis in patients with metastatic ccRCC.

Changes in concentrations of NMDA receptor subunit GluN2B, Arc and syntaxin-1 in dorsal hippocampus Schaffer collateral synapses in a rat learned helplessness model of depression.

Major depressive disorder involves changes in synaptic structure and function, but the molecular underpinnings of these changes are still not established. In an initial pilot experiment, whole-brain synaptosome screening with quantitative western blotting was performed to identify synaptic proteins that may show concentration changes in a congenital rat learned helplessness model of depression. We found that the N-methyl-d-aspartate receptor (NMDAR) subunits GluN2A/GluN2B, activity-regulated cytoskeleton-associated protein (Arc) and syntaxin-1 showed significant concentration differences between congenitally learned helpless (LH) and nonlearned helpless (NLH) rats. Having identified these three proteins, we then performed more elaborate quantitative immunogold electron microscopic analyses of the proteins in a specific synapse type in the dorsal hippocampus: the Schaffer collateral synapse in the CA1 region. We expanded the setup to include also unstressed wild-type (WT) rats. The concentrations of the proteins in the LH and NLH groups were compared to WT animals. In this specific synapse, we found that the concentration of NMDARs was increased in postsynaptic spines in both LH and NLH rats. The concentration of Arc was significantly increased in postsynaptic densities in LH animals as well as in presynaptic cytoplasm of NLH rats. The concentration of syntaxin-1 was significantly increased in both presynaptic terminals and postsynaptic spines in LH animals, while pre- and postsynaptic syntaxin-1 concentrations were significantly decreased in NLH animals. These protein changes suggest pathways by which synaptic plasticity may be increased in dorsal hippocampal Schaffer collateral synapses during depression, corresponding to decreased synaptic stability.

A modified protein marker panel to identify four consensus molecular subtypes in colorectal cancer using immunohistochemistry.

Colorectal cancer (CRC) is a heterogeneous disease with different genetic and molecular backgrounds, leading to a diverse patient prognosis and treatment response. Four consensus molecular subtypes (CMS 1-4) have recently been proposed based on transcriptome profiling. A clinically practical immunohistochemistry (IHC) based CMS classifier consisting of the four markers FRMD6, ZEB1, HTR2B, and CDX2 was then demonstrated. However, the IHC-CMS classifier did not distinguish between CMS2 and CMS3 tumours. In this study, we have applied the proposed transcriptome based and IHC-based CMS classifiers in a CRC cohort of 65 patients and found a concordance of 77.5 %. Further, we modified the IHC-CMS classifier by analysing the differentially expressed genes between CMS2 and CMS3 tumours using RNA-sequencing data from the TCGA dataset. The result showed that WNT signalling was among the most upregulated pathways in CMS2 tumours, and the expression level of CTNNB1 (encoding ß-catenin), a WNT pathway hallmark, was significantly upregulated (P = 1.15 × 10-6). We therefore introduced nuclear ß-catenin staining to the IHC-CMS classifier. Using the modified classifier in our cohort, we found a 71.4 % concordance between the IHC and RNA-sequencing based CMS classifiers. Moreover, ß-catenin staining could classify 16 out of the 19 CMS2/3 tumours into CMS2 or CMS3, thereby showing an 84.2 % concordance with the RNA-sequencing-based classifier. In conclusion, we evaluated CMS classifiers based on transcriptome and IHC analysis. We present a modified IHC panel that categorizes CRC tumours into the four CMS groups. To our knowledge, this is the first study using IHC to identify all four CMS groups.

In situ Chelation of Monoazo Dyes in Human Hair Keratin Fibers Using Environmentally Benign Metal Ions.

The coloration of human hair keratin fibers has long involved the oxidative coupling of primarily aromatic amines and phenols inside the fibers with the aid of harsh agents such as H2O2 and NH4OH. Further, the traditional process has exposed millions of consumers and their hairstylists to toxic substances such as skin sensitizers. While alternative hair dyeing processes have been explored, they fail to be competitive with the traditional method, for reasons including impracticality and limited colors achievable. In the present study, we developed an approach to imparting color to human hair fibers that involves entrapping colorants inside hair fibers by forming chelated monoazo dyes in situ. Dyes employed were based on monoarylide, arylazopyrazolone, and arylazonaphthol families, which display yellow, orange, and magenta colors on dyed hair. The dyes were applied at 40 °C without the use of oxidants and alkali associated with current commercial hair dyes, with the best dye uptake observed when the arylazonaphthol dye was employed. The dyed hair fibers showed good durability to washing, and treatment of these fibers with Al3+ or Fe3+ ions at 40 °C led to the rapid in situ formation of 1:2 metal/dye structures. In addition, the dyed hair was soft, indicating that chelated dye occupies the interior of the fibers rather than the surface. Such an approach can be applied to the coloration of other materials, including textiles.

Analysis of Tumor Depth Invasion With Anti-Smoothelin Antibody in Equivocal Transurethral Resection of Urinary Bladder Tumor Surgical Specimens.

OBJECTIVE: To examine the expression and value of the smoothelin marker in control cases, to standardize the working method, and to analyze its application in pathologic staging process of problematic transurethral resection of bladder tumor (TURBT) cases. MATERIAL AND METHODS: Immunohistochemical (IHC) staining was performed on tumor-free bladder wall sections, tumor-free large bowel sections, TURBTs with unequivocal tumor stage, and TURBTs with equivocal stage. The IHC staining of muscularis mucosa (MM), muscularis propria (MP), and blood vessels was evaluated semiquantitatively. RESULTS: Smoothelin IHC staining pattern ranged from negative (30% to 67% cases) to 2+ (0% to 15% cases) in MM and from 1+ (10% to 50% cases) to 3+ (9% to 48% cases) in MP. When compared on the same slide, the smoothelin expression of MP showed a stronger staining intensity than the one of the MM in all the analyzed cases. Blood vessel muscle cells stained in a constant intensity as the MM (r = 0.9808; r = 0.9604). Smoothelin determined restaging of 33% of the problematic TURBT cases. CONCLUSION: Smoothelin is an IHC marker that shows differential staining between coexistent MM and MP; however, variations in staining intensity and pattern may occur, aspects that can be influenced by different technique variables. We recommend using this marker as a diagnostic tool in problematic TURBT cases only when there is sufficient experience in control cases with this antibody.

Microscopy of Intracellular Listeria monocytogenes in Epithelial Cells.

The pathogen Listeria monocytogenes is a facultative intracellular bacterium, which targets a large range of cell types. Following entry, bacteria disrupt the invasion vacuole and reach the cytoplasm where they replicate and use the actin cytoskeleton to propel themselves from cell to cell. Mammalian epithelial cells grown in vitro can be used to study the different steps of the intracellular life of Listeria. However, rapid multiplication and dissemination of bacteria can induce important cell death and detachment, resulting in the formation of lytic plaques. Thus, in vitro infections with L. monocytogenes are usually restricted to short time courses, from a few minutes to one day. Here, we present a method to study long-term L. monocytogenes infections in epithelial cells using epifluorescence microscopy. This protocol enables the observation of actin-based motility, intercellular dissemination foci, and entrapment of L. monocytogenes within vacuoles of persistence termed "Listeria-Containing Vacuoles" (LisCVs). We also describe a protocol to study the recruitment of cytoskeletal proteins at Listeria actin comet tails, as well as a method to assess the membrane integrity of intracellular bacteria using a LIVE/DEAD viability assay.

AHNAK2 is a novel prognostic marker and correlates with immune infiltration in papillary thyroid cancer: Evidence from integrated analysis.

Papillary thyroid cancer (PTC) is the most prevalent endocrine tumor, and its incidence is still increasing. The mechanisms of PTC dedifferentiation and malignant progression remain unclear. In this study, we identified AHNAK2 as a key gene in PTC by differential expression analysis among four GEO datasets and validated its overexpression profile by data from the Oncomine, TCGA, and HPA databases and IHC staining analysis. AHNAK2 upregulation significantly correlated with advanced grades, stages, and lymph node events. Survival analysis suggested that AHNAK2 overexpression was coupled with poor overall survival. The immune infiltration analysis by TIMER and CIBERSORT indicated that AHNAK2 expression tightly correlated with the infiltration of diverse immune cell types, especially T cell subtypes. In addition, AHNAK2 is correlated with the expression of other conventional key genes of TC, such as PIK3CA, MAPK1, CTNNB1, and SLC5A5. AHNAK2 may be a novel prognostic marker for PTC.

Anatomic characteristics of epididymis based on histology, proteomic, and 3D reconstruction.

BACKGROUND: The epididymis is a popular research topic in urology and reproduction. OBJECTIVES: To explore and identify the anatomical characteristics of the epididymis based on histology, proteomics, and 3D reconstruction of epididymal tubules. MATERIALS AND METHODS: A 3D reconstruction of epididymal tubules was generated based on 7-µm-thick transverse serial sections of an epididymis. The proteins in the subcompartments of the epididymis were obtained and analyzed by non-labeled sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS). Protein function, signaling pathways, protein expression, and the histology in different subcompartments were analyzed. RESULTS: The caput (Cap), corpus (Cor), and cauda (Cau) of the epididymis were divided into 6, 10, and 4 subcompartments, respectively, and the subcompartment between the Cap and Cor is mixed together. A total of 3411 proteins were identified, and 854 proteins were accurately quantified after screening. When the subcompartment Cap 5 transitioned to Cap 6 and Cap 6 to Cap 7, 87 and 52 proteins were upregulated and 14 and 7 proteins were downregulated, respectively. The Cor 9 transition to Cau 1 was marked by 230 proteins that were downregulated, while 74 proteins were upregulated. At the junction of the cauda and the vas deferens, 57 proteins were downregulated, and 410 proteins were upregulated. Cap 6 histology was consistent with that of Cor 1. DISCUSSION AND CONCLUSION: The epididymis contains distinct connective tissue septa that can be identified under a surgical tabletop microscope, enabling it to be divided into 20 subcompartments.

Ex Vivo Analysis of Axonal Degeneration Using Sciatic and Optic Nerve Preparations.

This chapter describes techniques associated to the study of axonal degeneration in the peripheral (PNS) and central nervous system (CNS) using in vitro cultured sciatic and optic nerves from mice, a technique commonly referred to as ex vivo nerve explant analysis. Degeneration of axons in this technique is induced by axotomy (or exeresis) upon dissection of nerves from the PNS or CNS. Nerves explants can be analyzed by different techniques hours or days after in vitro culture. This model has the advantage to represent an intermediate model between in vitro and in vivo. Importantly, it allows for easy administration of drugs, electrical stimulation, and is especially suited for biochemical and morphological analysis. In addition, nerve explants can be obtained from mice of different genetic backgrounds, including knockout and transgenic animals, and allows the study of Wallerian degeneration without interference from the inflammatory reaction and macrophage infiltration that takes place after nerve injury in vivo. The protocol presented here constitutes a valuable tool to analyze in vitro the mechanisms associated to axonal degeneration and the role of Schwann cells in this process.

Extremely diversified haplotypes observed among assemblage B population of Giardia intestinalis in Kenya.

In molecular epidemiological studies of Giardia intestinalis, an pathogenic intestinal flagellate, due to the presence of allelic sequence heterogeneity (ASH) on the tetraploid genome, the image of haplotype diversity in the field remains uncertain. Here we employed the nine assemblage B positive stool samples, which had previously reported from Kenyan children, for the clonal sequence analysis of multiple gene loci (glutamate dehydrogenase (GDH), triosephosphate isomerase (TPI), and beta-giardin (BG)). The diversified unique assemblage B haplotypes as GDH (n = 67), TPI (n = 84), and BG (n = 62), and the assemblage A haplotypes as GDH (n = 7), TPI (n = 14), and BG (n = 15), which were hidden in the previous direct-sequence results, were detected. Among the assemblage B haplotypes, Bayesian phylogeny revealed multiple statistically significant clusters (9, 7, and 7 clusters for GDH, TPI, and BG, respectively). A part of the clusters (2 for GDH and 1 for BG), which included >4 haplotypes from an individual sample, indicated the presence of co-transmission with multiple strains sharing a recent ancestor. Locus-dependent discrepancies, such as different compositions of derived samples in clusters and different genotyping results for the assemblages, were also observed and considered to be the traces of both intra- and inter-assemblage genetic recombination respectively. Our clonal sequence analysis for giardial population, which applied firstly in Kenya, could reveal the higher rates of ASH far beyond the levels reported in other areas and address the complex population structure. The clonal analysis is indispensable for the molecular field study of G. intestinalis.

Immunohistochemical negative expression of ezrin predicts poor prognosis in clear cell renal cell carcinoma.

PURPOSE: To analyze the immunohistochemical expression of ezrin and moesin in clear cell renal cell carcinoma (ccRCC). These proteins, as part of the ezrin-radixin-moesin complex link the cell membrane to the actin cytoskeleton, affecting such processes as cell adhesion, cell survival, cell motility, and signal transduction. Our aim was to examine the impact of their expression on clinical outcomes and survival rates. PATIENTS AND METHODS: Five hundred seventy-five consecutive patients who had been treated surgically for ccRCC in a single center between 1985 and 2016 were selected. A single pathologist reviewed all cases to perform a uniform reclassification and determined the most representative tumor areas for construction of a tissue microarray. RESULTS: Of all ccRCC specimens, 106 (18.3%) were negative for ezrin, and 469 (81.7%) had positive ezrin expression; 16 (2.8%) were negative and 559 (97.2%) were positive for moesin, respectively. Ezrin expression was associated with pT stage (P < 0.001), clinical stage (P = 0.012), synchronic metastasis (P < 0.001), incidental tumors (P = 0.007), and International Society of Urological Pathology histological grade (P = 0.025). There was a correlation between moesin expression and clinical stage (P = 0.027), pT stage (P = 0.025), and pN stage (P = 0.007). Ezrin expression significantly influenced tumor-related deaths. By multivariate analysis, negative ezrin expression was an independent risk factor for disease-specific survival (HR 1.89; 95% CI 1.11-3.20). CONCLUSIONS: Negativity for ezrin in ccRCC patients significantly impacts survival rates. We encourage further prospective studies to analyze ezrin analysis to evaluate its significance in the prognosis of ccRCC.

Perspectives for Ezrin and Radixin in Astrocytes: Kinases, Functions and Pathology.

Astrocytes are increasingly perceived as active partners in physiological brain function and behaviour. The structural correlations of the glia-synaptic interaction are the peripheral astrocyte processes (PAPs), where ezrin and radixin, the two astrocytic members of the ezrin-radixin-moesin (ERM) family of proteins are preferentially localised. While the molecular mechanisms of ERM (in)activation appear universal, at least in mammalian cells, and have been studied in great detail, the actual ezrin and radixin kinases, phosphatases and binding partners appear cell type specific and may be multiplexed within a cell. In astrocytes, ezrin is involved in process motility, which can be stimulated by the neurotransmitter glutamate, through activation of the glial metabotropic glutamate receptors (mGluRs) 3 or 5. However, it has remained open how this mGluR stimulus is transduced to ezrin activation. Knowing upstream signals of ezrin activation, ezrin kinase(s), and membrane-bound binding partners of ezrin in astrocytes might open new approaches to the glial role in brain function. Ezrin has also been implicated in invasive behaviour of astrocytomas, and glial activation. Here, we review data pertaining to potential molecular interaction partners of ezrin in astrocytes, with a focus on PKC and GRK2, and in gliomas and other diseases, to stimulate further research on their potential roles in glia-synaptic physiology and pathology.

Effects of proteome changes on the tenderness of yak rumen smooth muscle during postmortem storage based on the label-free mass spectrometry.

A label-free proteomics method was used to explore the effects of differentially expressed proteins on the tenderness of yak rumen smooth muscle during postmortem storage (0, 3 and 7 days) at 3 ±â€¯1 °C. The tenderness improved significantly during storage. A total of 212 differentially expressed proteins were identified by the following comparisons: Day 3 vs.0, day 7 vs.0, and day 7 vs.3. Twenty-eight proteins were correlated with the WBSF of yak rumen smooth muscle. Calpastatin, ADP/ATP translocase 1, zyxin, LMOD1 protein, tropomyosin α-3 chain, thrombospondin-4 and UQCRC1 protein are highly related to smooth muscle tenderness, and thus, they are candidates indicators of yak rumen smooth muscle tenderness during storage. Furthermore, bioinformatics analyses revealed that the identified proteins were related to focal adhesion, vascular smooth muscle contraction, cardiac muscle contraction and necroptosis. The present results could provide proteomic insights into changes in yak rumen smooth muscle tenderness during storage and may be a valuable resource for future investigations.

Ezrin, a Membrane Cytoskeleton Cross-Linker Protein, as a Marker of Epithelial Damage in Asthma.

RATIONALE: Bronchial epithelial cell damage occurs in patients with bronchial asthma. Ezrin, a membrane-cytoskeleton protein, maintains cellular morphology and intercellular adhesion and protects the barrier function of epithelial cells. OBJECTIVES: To study the role of ezrin in bronchial epithelial cells injury and correlate its expression with asthma severity. METHODS: Levels of ezrin were measured in exhaled breath condensate (EBC) and serum in patients with asthma and BAL fluid (BALF) from a mouse model of asthma by ELISA. The regulation of IL-13 on ezrin protein levels was studied in primary bronchial epithelial cells. Ezrin knockdown using shRNA was studied in human bronchial epithelial 16HBE cells. MEASUREMENTS AND MAIN RESULTS: Ezrin levels were decreased in asthmatic EBC (92.7 ± 34.99 vs. 150.5 ± 10.22 pg/ml, P < 0.0001) and serum (700.7 ± 55.59 vs. 279.2 ± 25.83 pg/ml, P < 0.0001) compared with normal subjects. Levels were much lower in uncontrolled (P < 0.001) and partly controlled patients (P < 0.01) compared with well-controlled subjects. EBC and serum ezrin levels correlated with lung function in patients with asthma and serum ezrin levels were negatively correlated with serum IL-13 and periostin. IL-13-induced downregulation of ezrin expression in primary bronchial epithelial cells was significantly attenuated by the Janus tyrosine kinase 2 inhibitor, TG101348. Ezrin knockdown changed 16HBE cell morphology, enlarged intercellular spaces, and increased their permeability. Ezrin expression was decreased in the lung tissue and BALF of "asthmatic" mice and negatively correlated with BALF IL-13 level. CONCLUSIONS: Ezrin downregulation is associated with IL-13-induced epithelial damage and might be a potential biomarker of asthma control.

Syntrophins entangled in cytoskeletal meshwork: Helping to hold it all together.

Syntrophins are a family of 59 kDa peripheral membrane-associated adapter proteins, containing multiple protein-protein and protein-lipid interaction domains. The syntrophin family consists of five isoforms that exhibit specific tissue distribution, distinct sub-cellular localization and unique expression patterns implying their diverse functional roles. These syntrophin isoforms form multiple functional protein complexes and ensure proper localization of signalling proteins and their binding partners to specific membrane domains and provide appropriate spatiotemporal regulation of signalling pathways. Syntrophins consist of two PH domains, a PDZ domain and a conserved SU domain. The PH1 domain is split by the PDZ domain. The PH2 and the SU domain are involved in the interaction between syntrophin and the dystrophin-glycoprotein complex (DGC). Syntrophins recruit various signalling proteins to DGC and link extracellular matrix to internal signalling apparatus via DGC. The different domains of the syntrophin isoforms are responsible for modulation of cytoskeleton. Syntrophins associate with cytoskeletal proteins and lead to various cellular responses by modulating the cytoskeleton. Syntrophins are involved in many physiological processes which involve cytoskeletal reorganization like insulin secretion, blood pressure regulation, myogenesis, cell migration, formation and retraction of focal adhesions. Syntrophins have been implicated in various pathologies like Alzheimer's disease, muscular dystrophy, cancer. Their role in cytoskeletal organization and modulation makes them perfect candidates for further studies in various cancers and other ailments that involve cytoskeletal modulation. The role of syntrophins in cytoskeletal organization and modulation has not yet been comprehensively reviewed till now. This review focuses on syntrophins and highlights their role in cytoskeletal organization, modulation and dynamics via its involvement in different cell signalling networks.

Methodological innovations for the study of irreplaceable samples reveal giardiasis in extinct animals (Nothrotherium maquinense and Palaeolama maior).

The use of diagnostic methods that prevent irreplaceable samples (from museum collections, archaeological and paleontological samples) of being consumed or that increase their yield is relevant. For museum collections, archaeological and paleontological samples it is essential to conserve samples, subsamples or portions for future research. We are addressing methods for conservation of irreplaceable samples that could be fully consumed. Innovations in methodologies that are used in studies of Paleoparasitology and Paleomicrobiology will contribute to the preservation of collections. Therefore, to the development of archaeology and paleontology in the future, we evaluated whether the discarded material of the immunochromatography test could be used for molecular diagnosis and vice versa. We used a genotyped experimental coprolite positive for Giardia duodenalis. The diagnosis was positive for giardiasis in both cases. This methodology can be corroborated with the coprolite of a Paleolama maior (extinct llama) previously diagnosed for G. duodenalis with an immunoenzymatic test. The residue of the pre-digestion step of the DNA extraction before adding Proteinase K was confirmed positive with the immunochromatographic test. Also, the DNA extraction residue from a coprolite of Nothrotherium maquinense (ground sloth) was tested positive with immunochromatographic test for G. duodenalis. These are the oldest findings for G. duodenalis confirming that this intestinal parasite occurred among Northeastern Brazilian Megafauna animals from the late Pleistocene period, correlated to human occupation. The relevance of these results will allow the study by different methodological approaches from a small amount of material, reusing discarded materials.

Ascending aortas from heart donors and CABG patients are not equivalent as control in aortopathy studies.

OBJECTIVES: A careful selection of reference samples in studies on the pathogenesis of thoracic ascending aorta (TAA) dilation is crucial for reliability, consistency and reproducibility of experimental results. Several studies include control TAA samples from heart donors. Others include samples harvested during coronary artery bypass graft (CABG) procedures or a mix of samples from heart donors and CABG patients. We verified the equivalence/homogeneity of TAA samples from heart donors and CABG patients in terms of basal gene expression and thus their reliability as reference groups in aortopathy studies. DESIGN: We analysed by RT-PCR and Western blot the differential expression of smoothelin, α-smooth muscle actin (α-SMA) and transforming growth factor-ß1 (TGF-ß1), selected as major players in smooth muscle cell and myofibroblast phenotype and remodelling. The mean age and comorbidities of subjects were consistent with data routinely seen in clinical practice. RESULTS: Data revealed the loss of smoothelin in samples from CABG patients, together with a significant increase of α-SMA, while TGF-ß1 dimer showed a marked increase in CABG patients versus heart donors, accompanied by a decrease of the corresponding mRNA. Differences in gene expression were maintained after adjustment for age. However, TGF-ß1 mRNA and CABG patients' age showed a positive correlation (ρ = 0.89, p < .05), while α-SMA mRNA and age showed a negative correlation (ρ = -0.85, p < .05). CONCLUSIONS: We revealed the non-equivalence of samples from heart donors and CABG patients, presumably for the presence of microscopic atherosclerotic lesions in CABG patients, suggesting the necessity of a careful selection of control groups in aortopathy studies.