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N6‐Methyladenosine (m6A) is the most common internal RNA modification in the consensus sequence of 5′‐RRACH‐3′. The methyl mark is added by writer proteins (METTL3/METTL14 metyltransferase complex) and removed by eraser proteins (m6A demethylases; FTO and ALKBH5). Recognition of this methyl mark by m6A reader proteins leads to changes in RNA metabolism. How the writer and eraser proteins determine their targets is not well‐understood, despite the importance of this information in understanding the regulatory mechanisms and physiological roles of m6A. However, approaches for targeted manipulation of the methylation state at specific sites are being developed. In this review, I summarize the recent findings on the mechanisms of target identification of m6A regulatory proteins, as well as recent approaches for targeted m6A modifications. [ABSTRACT FROM AUTHOR]

A general framework by which dynamic interactions within a protein will promote the necessary series of structural changes, or "conformational cycle," required for function is proposed. It is suggested that the free‐energy landscape of a protein is biased toward this conformational cycle. Fluctuations into higher energy, although thermally accessible, conformations drive the conformational cycle forward. The amino acid interaction network is defined as those intraprotein interactions that contribute most to the free‐energy landscape. Some network connections are consistent in every structural state, while others periodically change their interaction strength according to the conformational cycle. It is reviewed here that structural transitions change these periodic network connections, which then predisposes the protein toward the next set of network changes, and hence the next structural change. These concepts are illustrated by recent work on tryptophan synthase. Disruption of these dynamic connections may lead to aberrant protein function and disease states. [ABSTRACT FROM AUTHOR]

The endo‐lysosome system is involved in endocytosis, protein sorting, and degradation as well as autophagy. Numerous toxins and pathogens exploit this system to enter host cells and exert their deleterious effects. Modulation of host endo‐lysosome pathway may restrict multiple toxins intoxication as well as pathogen infection. ABMA, selected from a high‐throughput screening against the cytotoxicity of ricin toxin, exhibits a broad‐spectrum antitoxin and antipathogen activity. Here, we show that ABMA selectively retains endocytosed protein and toxin to late endosomes and thus delaying their intracellular trafficking. It also impairs the autophagic flux by excessive fusion of late endosomes and autophagosomes. Its exclusive action on late endosomes and corresponding consequences on the endo‐lysosomal pathway and autophagic flux are distinct from known inhibitors such as bafilomycin A1, EGA, or chloroquine. Hence, besides being a broad‐spectrum inhibitor of endocytosed toxins and pathogens, ABMA may serve as a molecular tool to dissect endo‐lysosome system‐related cellular physiology and mechanisms of pathogenesis. [ABSTRACT FROM AUTHOR]

Background: The molecular mechanisms involved in the acquisition of mammalian sperm fertilizing ability are still poorly understood, reflecting the complexity of this process. Objectives: In this review, we describe the role of Cysteine RIch Secretory Proteins (CRISP1–4) in different steps of the sperm journey to the egg as well as their relevance for fertilization and fertility. Materials and Methods: We analyze bibliography reporting the phenotypes of CRISP KO mice models and combine this search with recent findings from our team. Results: Generation of individual KO for CRISP proteins reveals they are key mediators in different stages of the fertilization process. However, in spite of their important functional roles, KO males for each of these proteins remain fertile, supporting the existence of compensatory mechanisms between homologous CRISP family members. The development of mice lacking epididymal CRISP1 and CRISP4 simultaneously (DKO) revealed that mutant males exhibit an impaired fertility due to deficiencies in the sperm ability to fertilize the eggs in vivo, consistent with the proposed roles of the two proteins in fertilization. Interestingly, DKO males show clear defects in both epididymal epithelium differentiation and luminal acidification known to be critical for sperm maturation and storage. Whereas in most of the cases, these epithelium defects seem to specifically affect the sperm fertilizing ability, some animals exhibit a disruption of the characteristic immune tolerance of the organ with clear signs of inflammation and sperm viability defects. Discussion and Conclusion: Altogether, these observations confirm the relevance of CRISP proteins for male fertility and contribute to a better understanding of the fine‐tuning mechanisms underlying sperm maturation and immune tolerance within the epididymis. Moreover, considering the existence of a human epididymal protein functionally equivalent to rodent CRISP1 and CRISP4, DKO mice may represent an excellent model for studying human epididymal physiology and pathology. [ABSTRACT FROM AUTHOR]

Objective: Previously, we have shown that miRNA‐342‐3p was increased during osteogenic differentiation of human umbilical mesenchymal stem cells (hUCMSCs) via regulating the sonic hedgehog (Shh) pathway. In this study, our objective is to further investigate the role of miRNA‐342‐3p in activation of Shh pathway by targeting suppressor of fused protein (Sufu), a suppressor of transcriptional factor Gli, as well as the potential interaction with transforming growth factor beta (TGF‐β) signaling pathway during osteogenic induction of hUCMSCs. Materials and methods: HUCMSCs that stable overexpression or knockdown of miRNA‐342‐3p were established by infection with lentiviral vectors. mRNA and protein levels of Hedgehog signaling pathway and osteogenic genes were measured by RT‐qPCR and western blot assays. Luciferase reporter assay was performed to test the direct binding site of Sufu 5′UTR targeted by miRNA‐342‐3p. Results: Overexpression of miRNA‐342‐3p in hUCMSCs enhanced the expression of osteogenic genes by targeting Sufu. And the potential of osteogenic differentiation of hUCMSCs was inhibited while knocking down miRNA‐342‐3p. Meanwhile, induced the TGF‐β expression level was also observed upon overexpressing miRNA‐342‐3p, suggesting activation of TGF‐β signaling pathway was a potential mechanism of miRNA‐342‐3p‐mediated osteogenesis in hUCMSCs. Conclusions: Our findings provide new mechanistic evidence that miRNA‐342‐3p might be a valuable therapeutic target in bone regeneration. [ABSTRACT FROM AUTHOR]

Objectives: We aimed to accelerate angiogenesis in pulp regeneration by modulating ephrinB2 expression in stem cells from apical papilla (SCAPs). Materials and Methods: Stem cells from apical papilla were transducted with ephrinB2‐lentiviral expression vector (ephrinB2‐SCAPs) in experimental group and green fluorescent protein (GFP‐SCAPs) in control group. The transduction efficiency was confirmed by real‐time PCR and Western blot assays. MTT assay was performed to detect the proliferative capacity of SCAPs after transduction. In vitro Matrigel assay and in vivo Matrigel plug assay were carried out to evaluate the angiogenic capacity. Results: Results showed that ephrinB2‐SCAPs had significantly higher ephrinB2 expression than GFP‐SCAPs. EphrinB2‐SCAPs upregulated vascular endothelial growth factor (VEGF) secretion under hypoxia. In vitro Matrigel assay demonstrated that human umbilical vein endothelial cells (HUVECs) cocultured with ephrinB2‐SCAPs under hypoxia formed vascular‐like structures earlier than GFP‐SCAPs. Animal experiments confirmed that SCAPs co‐transplanted with HUVECs enabled to generate greater amount of blood vessels than SCAPs alone. EphrinB2‐SCAPs produced increased number of blood vessels with references to GFP‐SCAPs, and those co‐transplanted with HUVECs generated vessels with larger and functional tubule volumes. Conclusions: Regulating ephrinB2 expression in SCAPs may act as a new avenue for enhancing angiogenesis in dental pulp regeneration. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the nutritional properties of fresh (F), boiled (B), ambient‐dried (Holothuria tubulosa) (DA, 23 ± 2°C) and oven‐dried sea cucumber (DO, 45 ± 1°C) were compared in terms of proximate composition and fatty acid profiles. The results of the proximate analyses showed that the highest moisture content (86.76%) was determined in fresh samples, whereas the lowest moisture content (9.35%) was obtained in the oven‐dried group (DO). The crude fat and protein contents were in the range of 0.19% (B) to 0.87% (DA) and 12.30% (F) to 62.13% (DA), respectively. The highest ash content (30.30%) was obtained in group DO, while the lowest ash content (0.61%) was observed in the boiled samples (B). According to fatty acid analyses, there were no significant differences (p > 0.05) between the two drying methods. The monounsaturated fatty acid (MUFA) (21.405%) and polyunsaturated fatty acid (PUFA) (36.018%) contents of H. tubulosa were high. Holothuria tubulosa can be used as protein and PUFA sources. Comparing the two methods, oven‐drying is better in terms of preservation, whereas drying at the ambient temperature is better in terms of nutrient value. [ABSTRACT FROM AUTHOR]

Objective: Research has highlighted the importance of reward‐based processes in binge eating (BE). However, both increased and decreased activation have been observed in reward related brain regions for BE. Differences may be similar to addiction research, where the reward system is initially hyper‐responsive at early stages of use, but becomes hypo‐responsive with prolonged drug/alcohol use. This study was the first to examine differences in reward system responsivity at early versus chronic BE stages. Method: Using an animal model, Sprague–Dawley female rats were identified as BE prone (BEP) or BE resistant (BER) and randomly assigned to an early or chronic stage group. Neural activation (via Fos protein) was quantified in the nucleus accumbens core (NAC) and shell (NAS). Results: Early stage BEP rats had the highest levels of Fos expression of all of the study groups. By contrast, chronic stage BEP rats exhibited decreased activation in the NAS and NAC that was similar to the activation in chronic stage BER rats. Discussion: Findings are significant in suggesting hyper‐neural activation to reward in the early stages of BE and decreased activation in later stages of BE. Additional studies are needed to elucidate how these differences may impact risk for and maintenance of BE. [ABSTRACT FROM AUTHOR]

We report the whole genome sequence of the serotype e Cbm+ strain LAR01 of Streptococcus mutans, a dental pathogen frequently associated with extra-oral infections. The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. The genome contains 15 phosphotransferase system gene clusters, seven cell wall-anchored (LPxTG) proteins, all genes required for the development of natural competence and genes coding for mutacins VI and K8. Interestingly, the cbm gene is genetically linked to a putative type VII secretion system that has been found in Mycobacteria and few other Gram-positive bacteria. When compared with the UA159 type strain, phenotypic characterization of LAR01 revealed increased biofilm formation in the presence of either glucose or sucrose but similar abilities to withstand acid and oxidative stresses. LAR01 was unable to inhibit the growth of Strpetococcus gordonii, which is consistent with the genomic data that indicate absence of mutacins that can kill mitis streptococci. On the other hand, LAR01 effectively inhibited growth of other S. mutans strains, suggesting that it may be specialized to outcompete strains from its own species. In vitro and in vivo studies using mutational and heterologous expression approaches revealed that Cbm is a virulence factor of S. mutans by mediating binding to extracellular matrix proteins and intracellular invasion. Collectively, the whole genome sequence analysis and phenotypic characterization of LAR01 provides new insights on the virulence properties of S. mutans and grants further opportunities to understand the genomic fluidity of this important human pathogen. [ABSTRACT FROM AUTHOR]

Summary: Leptospires and other members of the evolutionarily ancient phylum of Spirochaetes are bacteria often characterized by long, highly motile spiral‐ or wave‐shaped cells. Morphology and motility are critical factors in spirochete physiology, contributing to the ability of these bacteria to successfully colonize diverse environments. However, the mechanisms conferring the helical structure of Leptospira spp. have yet to be fully elucidated. We have identified five Leptospira biflexa bactofilin proteins, a recently characterized protein family with cytoskeletal properties. These five bactofilins are conserved in all species of the Leptospiraceae, indicating that these proteins arose early in the evolution of this family. One member of this protein family, LbbD, confers the optimal pitch distance in the helical structure of L. biflexa. Mutants lacking lbbD display a unique compressed helical morphology, a reduced motility and a decreased ability to tolerate cell wall stressors. The change in the helical spacing, combined with the motility and cell wall integrity defects, showcases the intimate relationship and coevolution between shape and motility in these spirochetes. [ABSTRACT FROM AUTHOR]

Background and Purpose: Rescue of F508del-cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the most common CF mutation, requires small molecules that overcome protein processing, stability and channel gating defects. Here, we investigate F508del-CFTR rescue by CFFT-004, a small molecule designed to independently correct protein processing and channel gating defects.Experimental Approach: Using CFTR-expressing recombinant cells and CF patient-derived bronchial epithelial cells, we studied CFTR expression by Western blotting and channel gating and stability with the patch-clamp and Ussing chamber techniques.Key Results: Chronic treatment with CFFT-004 improved modestly F508del-CFTR processing, but not its plasma membrane stability. By contrast, CFFT-004 rescued F508del-CFTR channel gating better than C18, an analogue of the clinically used CFTR corrector lumacaftor. Subsequent acute addition of CFFT-004, but not C18, potentiated F508del-CFTR channel gating. However, CFFT-004 was without effect on A561E-CFTR, a CF mutation with a comparable mechanism of CFTR dysfunction as F508del-CFTR. To investigate the mechanism of action of CFFT-004, we used F508del-CFTR revertant mutations. Potentiation by CFFT-004 was unaffected by revertant mutations, but correction was abolished by the revertant mutation G550E. These data suggest that correction, but not potentiation, by CFFT-004 might involve nucleotide-binding domain 1 of CFTR.Conclusions and Implications: CFFT-004 is a dual-acting small molecule with independent corrector and potentiator activities that partially rescues F508del-CFTR in recombinant cells and native airway epithelia. The limited efficacy and potency of CFFT-004 suggests that combinations of small molecules targeting different defects in F508del-CFTR might be a more effective therapeutic strategy than a single agent. [ABSTRACT FROM AUTHOR]

Background: Nonfunctioning pancreatic neuroendocrine tumors (NFPanNETs) may be sporadic or inherited because of germline mutations associated with von Hippel-Lindau disease (VHL) or multiple endocrine neoplasia type 1 (MEN1). The clinical behavior of NFPanNETs is difficult to predict, even in tumors of the same stage and grade. The authors analyzed genotype-specific patterns of transcriptional messenger RNA (mRNA) levels of NFPanNETs to understand the molecular features that determine PanNET phenotype.Methods: Thirty-two samples were included for genome-wide mRNA gene expression analysis (9 VHL-associated, 10 MEN1-associated, and 9 sporadic NFPanNETs and 4 purified normal islet cell [NIC] samples). Validation of genes was performed by real-time polymerase chain reaction analysis and immunohistochemistry. Gene expression profiles were analyzed by tumor genotype, and pathway analysis was curated.Results: Consensus clustering of mRNA expression revealed separate clustering of NICs, VHL-associated NFPanNETs, and MEN1-associated NFPanNETs; whereas some sporadic tumors clustered with MEN1. Four of 5 MEN1-like sporadic PanNET subtypes had loss of heterozygosity at the MEN1 gene locus. Pathway analysis demonstrated subtype-specific pathway activation, comprising angiogenesis and immune response in VHL; neuronal development in MEN1; protein ubiquitination in the new MEN1/sporadic subtype; and cytokinesis and cilium/microtubule development in sporadic NFPanNETs. Among many genes, platelet-derived growth factor receptor β (PDGFRB), lymphoid enhancer-binding factor-1 (Lef-1), cyclin-dependent kinase 4 (CDK4), and CDK6 were upregulated in VHL or MEN1 NFPanNETs, providing potential subtype-specific treatment targets.Conclusions: Distinct mRNA expression patterns were identified in sporadic-associated, VHL-associated, and MEN1-associated NFPanNETs. The current results uncover new pathways involved in NFPanNETs that are subtype-specific and provide potential new diagnostic or therapeutic targets based on tumor subtype. Cancer 2018;124:636-47. © 2017 American Cancer Society. [ABSTRACT FROM AUTHOR]

Purpose: To demonstrate that sonic hedgehog (Shh) is present in human parotid saliva and is decreased in human taste dysfunction.Methods: Shh was measured in parotid saliva of 27 normal subjects and 81 patients with taste dysfunction of multiple etiologies using a sensitive spectrophotometric ELISA assay. Taste dysfunction was defined clinically both by subjective decreases of taste acuity and flavor perception and by impaired gustometry.Results: Shh was found in parotid saliva of both normal subjects and patients with taste dysfunction. Levels were significantly lower in patients than in normal subjects. Both subjective loss of taste acuity and flavor perception and impaired gustometry was measured in untreated patients.Conclusions: This is the first demonstration of Shh in human saliva. As Shh has been related to taste bud growth and development, its presence in saliva is consistent with its role as a cell signaling moiety involved with stimulation of taste bud stem cells to generate taste receptors. Decreased saliva Shh secretion can be considered a marker of taste dysfunction in patients with multiple pathologies for their dysfunction. [ABSTRACT FROM AUTHOR]

Objective: Autosomal recessive primary microcephaly (MCPH) is a rare condition characterized by a reduced cerebral cortex accompanied with intellectual disability. Mutations in 17 genes have been shown to cause this phenotype. Recently, mutations in CIT, encoding CRIK (citron rho-interacting kinase)-a component of the central spindle matrix-were added. We aimed at identifying novel MCPH-associated genes and exploring their functional role in pathogenesis.Methods: Linkage analysis and whole exome sequencing were performed in consanguineous and nonconsanguineous MCPH families to identify disease-causing variants. Functional consequences were investigated by RNA studies and on the cellular level using immunofluorescence and microscopy.Results: We identified homozygous mutations in KIF14 (NM_014875.2;c.263T>A;pLeu88*, c.2480_2482delTTG; p.Val827del, and c.4071G>A;p.Gln1357=) as the likely cause in 3 MCPH families. Furthermore, in a patient presenting with a severe form of primary microcephaly and short stature, we identified compound heterozygous missense mutations in KIF14 (NM_014875.2;c.2545C>G;p.His849Asp and c.3662G>T;p.Gly1221Val). Three of the 5 identified mutations impaired splicing, and 2 resulted in a truncated protein. Intriguingly, Kif14 knockout mice also showed primary microcephaly. Human kinesin-like protein KIF14, a microtubule motor protein, localizes at the midbody to finalize cytokinesis by interacting with CRIK. We found impaired localization of both KIF14 and CRIK at the midbody in patient-derived fibroblasts. Furthermore, we observed a large number of binucleated and apoptotic cells-signs of failed cytokinesis that we also observed in experimentally KIF14-depleted cells.Interpretation: Our data corroborate the role of an impaired cytokinesis in the etiology of primary and syndromic microcephaly, as has been proposed by recent findings on CIT mutations. Ann Neurol 2017;82:562-577. [ABSTRACT FROM AUTHOR]

The complement system is an arm of innate immunity that aids in the removal of pathogens and dying cells. Due to its harmful, pro-inflammatory potential, complement is controlled by several soluble and membrane-bound inhibitors. This family of complement regulators has been recently extended by the discovery of several new members, and it is becoming apparent that these proteins harbour additional functions. In this review, the current state of knowledge of the physiological functions of four complement regulators will be described: cartilage oligomeric matrix protein (COMP), CUB and sushi multiple domains 1 (CSMD1), sushi domain-containing protein 4 (SUSD4) and CD59. Complement activation is involved in both the development of and defence against cancer. COMP expression is pro-oncogenic, whereas CSMD1 and SUSD4 act as tumour suppressors. These effects may be related in part to the complex influence of complement on cancer but also depend on unrelated functions such as the protection of cells from endoplasmic reticulum stress conveyed by intracellular COMP. CD59 is the main inhibitor of the membrane attack complex, and its deficiency leads to complement attack on erythrocytes and severe haemolytic anaemia, which is now amenable to treatment with an inhibitor of C5 cleavage. Unexpectedly, the intracellular pool of CD59 is crucial for insulin secretion from pancreatic β-cells. This finding is one of several relating to the intracellular functions of complement proteins, which until recently were only considered to be present in the extracellular space. Understanding the alternative functions of complement inhibitors may unravel unexpected links between complement and other physiological systems, but is also important for better design of therapeutic complement inhibition. [ABSTRACT FROM AUTHOR]

This clinical study aimed to determine whether periodontal disease is associated with expression of developmental endothelial locus-1 (Del-1) and pentraxin-3 ( PTX-3), endogenous inhibitors of leukocyte extravasation in humans. Expression of DEL1, PTX3, interleukin-17A ( IL17A), and lymphocyte function-associated antigen-1 ( LFA1) was determined, using RT-PCR and melting curve analysis, in biopsies of gingival tissues from 95 patients: 42 with moderate periodontitis; 40 with severe periodontitis; and 13 healthy controls. Relative expression of DEL1 and PTX3 was statistically significantly weaker in patients with periodontitis than in the control subjects. On the contrary, both IL17A and LFA1 showed statistically significant stronger expression in patients with periodontitis than in healthy controls. Correlation analysis, performed using Spearman's test, showed that expression of DEL1 was statistically significantly linked to periodontitis ( ρ = −0.103) and to age ( ρ = −0.134), but not to the gender of the patient, and that expression of PTX3 was significantly correlated with periodontitis ( ρ = −0.354). Expression of neutrophil extravasation inhibitors DEL1 and PTX3 show significant, but weak, association with the clinical manifestation of chronic periodontitis. [ABSTRACT FROM AUTHOR]

Background: Periodontal disease is chronic inflammatory process that affects the attachment structures of the teeth and constitutes a significant cause of tooth loss in adults. Although different bacteria play an important role in the triggering of this condition, the progression and severity of the disease are strongly affected by the host immune response, which is under the control of different immune regulatory mechanisms, including T regulatory (Treg) cells. The aim of this study was to assess the frequency and function of CD69+ Treg lymphocytes in patients with chronic periodontal disease.Methods: Peripheral blood samples (n = 33) and gingival tissue (n = 9) were obtained from patients with chronic periodontal disease. Blood samples from 25 healthy individuals were also studied. Levels of CD69+ Treg lymphocytes in peripheral blood and gingival tissue were determined by six-color multiparametric flow cytometry, immunofluorescence, and immunohistochemistry. The immune regulatory function of CD69+ Treg cells was tested by an in vitro assay of inhibition of lymphocyte activation.Results: Percentages of CD69+ Treg cells were significantly higher in the peripheral blood from patients with active periodontal disease compared to healthy controls, and these percentages inversely correlated with the periodontal attachment loss. Increased numbers of these Treg cells were detected in the gingival tissue from active PD patients compared to their peripheral blood. However, the suppressive function of CD69+ Treg cells was significantly diminished in patients with periodontal disease compared to healthy controls.Conclusions: Our data suggest that CD69+ Treg cells seem to be another important piece in the complex immunopathogenesis of periodontal disease. [ABSTRACT FROM AUTHOR]

Introduction: Muscles add sarcomeres in response to stretch, presumably to maintain optimal sarcomere length. Clinical evidence from patients with cerebral palsy, who have both decreased serial sarcomere number and reduced satellite cells (SCs), suggests a hypothesis that SCs may be involved in sarcomere addition.Methods: A transgenic Pax7-DTA mouse model underwent conditional SC depletion, and their soleii were then stretch-immobilized to assess the capacity for sarcomere addition. Muscle architecture, morphology, and extracellular matrix (ECM) changes were also evaluated.Results: Mice in the SC-reduced group achieved normal serial sarcomere addition in response to stretch. However, muscle fiber cross-sectional area was significantly smaller and was associated with hypertrophic ECM changes, consistent with fibrosis.Conclusions: While a reduced SC population does not hinder serial sarcomere addition, SCs play a role in muscle adaptation to chronic stretch that involves maintenance of both fiber cross-sectional area and ECM structure. Muscle Nerve 55: 384-392, 2017. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis is a bacterium associated with chronic periodontitis that possesses a family of genes encoding hemagglutinins required for heme acquisition. In this study we generated ΔhagB and ΔhagC mutants in strain W83 and demonstrate that both hagB and hagC are required for adherence to oral epithelial cells. Unexpectedly, a double ΔhagB/ΔhagC mutant had less severe adherence defects than either of the single mutants, but was found to exhibit increased expression of the gingipain-encoding genes rgpA and kgp, suggesting that a ΔhagB/ΔhagC mutant is only viable in populations of cells that exhibit increased expression of genes involved in heme acquisition. Disruption of hagB in the fimbriated strain ATCC33277 demonstrated that HagB is also required for stable attachment of fimbriated bacteria to oral epithelial cells. Mutants of hagC were also found to form defective single and multi-species biofilms that had reduced biomass relative to biofilms formed by the wild-type strain. This study highlights the hitherto unappreciated importance of these genes in oral colonization and biofilm formation. [ABSTRACT FROM AUTHOR]

The brain extracellular matrix (ECM) occupies the space between cells and is involved in cell-matrix and cell-cell adhesion. However, in addition to providing structural support to brain tissue, the ECM activates cell signaling and controls synaptic transmission. The expression and activity of brain ECM components are regulated by alcohol exposure. This review will discuss what is currently known about the effects of alcohol on the activity and expression of brain ECM components. An interpretation of how these changes might promote alcohol use disorder (AUD) will be also provided. Ethanol (EtOH) exposure decreases levels of structural proteins involved in the interstitial matrix and basement membrane, with a concomitant increase in proteolytic enzymes that degrade these components. In contrast, EtOH exposure generally increases perineuronal net components. Because the ECM has been shown to regulate both synaptic plasticity and behavioral responses to drugs of abuse, regulation of the brain ECM by alcohol may be relevant to the development of alcoholism. Although investigation of the function of brain ECM in alcohol abuse is still in early stages, a greater understanding of the interplay between ECM and alcohol might lead to novel therapeutic strategies for treating AUD. [ABSTRACT FROM AUTHOR]

Objective The shared epitope hypothesis posits that amino acids QR/KRAA in positions 70-74 of the DRΒ1 chain are responsible for rheumatoid arthritis susceptibility. However, even DRB1*04 alleles containing the shared epitope vary greatly with respect to degrees of susceptibility. This study was undertaken to conduct a molecular examination of the shared epitope hypothesis by measuring binding of arthritogenic peptides to susceptibility and resistance alleles. Methods We measured binding of native and citrullinated forms of vimentin66-78 and α-enolase11-25 and noncitrullinated type II collagen258-272 to 88 class II alleles on Luminex beads (which includes alleles of many varying degrees of susceptibility and resistance). We expressed DRΒ1*04:01, *04:02, and *08:01 in T2 cells and mutated DRΒ1*04:01 at positions 67, 70, 71, 74, and 86 to corresponding residues in DRB1*04:02, *04:03, *04:04, *04:05, and *08:01. Finally, we measured responses of 4 DRΒ1*04:01 restricted collagen258-272 T cell hybridomas against wild-type DRΒ1*04:01, *04:02, and all mutated alleles. Results The most susceptible allele, DRΒ1*04:01, preferentially bound citrullinated vimentin66-78 and citrullinated α-enolase11-25 over the native forms. DRΒ1*04:02 exhibited no preference for citrullinated peptides, and *08:01 preferred native peptides. Similarly, DRB1*04:01 bound collagen258-272, but *04:02 and *08:01 did not. Mutating DRΒ1*04:01 at positions 70, 71, 74, and 86 to the corresponding residues in DRΒ1*04:02 or *08:01 dramatically reduced the specificity for citrullinated peptides and collagen258-272 binding. Conclusion These observations demonstrate that while amino acids at positions 70, 71, and 74 within the shared epitope in DRΒ1 mediate binding and T cell responses of arthritogenic peptides, position 86 outside the shared epitope also plays a critical role. [ABSTRACT FROM AUTHOR]

Background: The normal red blood cell (RBC) life span may be significantly reduced when RBCs are stored under blood bank conditions, resulting in a reduced 24-hour survival after transfusion. The damage of stored RBCs is probably multifactorial as stored RBCs share features of both senescence and suicidal RBC death (eryptosis). Since an increased intracellular Ca(2+) concentration ([Ca(2+) ]i ) is one key feature of eryptosis, we here investigated if stored human RBCs had increased [Ca(2+) ]i and the mechanisms behind uptake of such RBCs in a murine model.Study Design and Methods: The intracellular Ca(2+) content of RBCs was determined using the Ca(2+) probe Fluo-3 and flow cytometry. In vivo uptake of Ca(2+) ionophore-treated murine RBCs (Ca(2+) -RBCs) was investigated in recipient mice, using flow cytometry and immunohistochemical analysis.Results: A small fraction of human RBCs accumulated [Ca(2+) ]i during storage for up to 42 days under blood bank conditions. In a murine model, where fresh or Ca(2+) -RBCs were transfused, Ca(2+) -RBCs were mainly trapped by MARCO+ splenic marginal zone macrophages and CD11c+ CD207+ dendritic cells (DCs) within 1 hour after transfusion. In marked contrast, freshly transfused RBCs aging normally in circulation were cleared much slower and preferentially by F4/80+ red pulp macrophages. CD47 on the Ca(2+) -RBCs did not affect their clearance by splenic phagocytic cells.Conclusions: A small fraction of RBCs accumulate [Ca(2+) ]i during storage, and in a murine model such RBCs are recognized by splenic macrophages and DCs in ways similar to what has been reported for nucleated apoptotic cells. [ABSTRACT FROM AUTHOR]

Background and Purpose: The NMDA receptor is an important target of alcohol action in the brain. Recent studies in this laboratory have demonstrated that alcohol-sensitive positions in the intersubunit interfaces of the M3 and M4 domains of GluN1 and GluN2A subunits interact with respect to ethanol sensitivity and receptor kinetics and that alcohol-sensitive positions in the M domains of GluN2A and GluN2B subunits differ. In this study, we tested for interactions among alcohol-sensitive positions at the M domain intersubunit interfaces in GluN1/GluN2B NMDA receptors.Experimental Approach: We used whole-cell patch-clamp recording in tsA201 cells expressing tryptophan substitution mutants at ethanol-sensitive positions in the GluN1 and GluN2B NMDA receptor subunits to test for interactions among positions.Key Results: Six pairs of positions in GluN1/GluN2B significantly interacted to regulate ethanol inhibition: Gly(638) /Met(824) , Gly(638) /Leu(825) , Phe(639) /Leu(825) , Phe(639) /Gly(826) , Met(818) /Phe(637) and Val(820) /Phe(637) . Tryptophan substitution at Met(824) or Leu(825) in GluN2B did not alter ethanol sensitivity but interacted with positions in the GluN1 M3 domain to regulate ethanol action, whereas tryptophan substitution at Gly(638) , which is the cognate of an ethanol-sensitive position in GluN2A, did not alter ethanol sensitivity or interact with positions in GluN1. Two and three pairs of positions interacted to regulate glutamate steady-state and peak current EC50 , respectively, and one pair interacted with respect to macroscopic desensitization.Conclusions: Despite highly-conserved M domain sequences and similar ethanol sensitivity in the GluN2A and GluN2B subunits, the manner in which these subunits interact with the GluN1 subunit to regulate ethanol sensitivity and receptor kinetics differs. [ABSTRACT FROM AUTHOR]

PG0162, annotated as an extracytoplasmic function (ECF) sigma factor in Porphyromonas gingivalis, is composed of 193 amino acids. As previously reported, the PG0162-deficient mutant, P. gingivalis FLL350 showed significant reduction in gingipain activity compared with the parental strain. Because this ECF sigma factor could be involved in the virulence regulation in P. gingivalis, its genetic properties were further characterized. A 5'-RACE analysis showed that the start of transcription of the PG0162 gene occurred from a guanine (G) residue 69 nucleotides upstream of the ATG translation initiation codon. The function of PG0162 as a sigma factor was confirmed in a run-off in vitro transcription assay using the purified rPG0162 and RNAP core enzyme from Escherichia coli with the PG0162 promoter as template. As an appropriate PG0162 inducing environmental signal is unknown, a strain overexpressing the PG0162 gene designated P. gingivalis FLL391 was created. Compared with the wild-type strain, transcriptome analysis of P. gingivalis FLL391 showed that approximately 24% of the genome displayed altered gene expression (260 upregulated genes; 286 downregulated genes). Two other ECF sigma factors (PG0985 and PG1660) were upregulated more than two-fold. The autoregulation of PG0162 was confirmed with the binding of the rPG0162 protein to the PG0162 promoter in electrophoretic mobility shift assay. In addition, the rPG0162 protein also showed the ability to bind to the promoter region of two genes (PG0521 and PG1167) that were most upregulated in P. gingivalis FLL391. Taken together, our data suggest that PG0162 is a sigma factor that may play an important role in the virulence regulatory network in P. gingivalis. [ABSTRACT FROM AUTHOR]

Aim To determine whether preadipocyte factor 1 could be a predictive marker for the development of diabetes in people without diabetes at baseline. Methods We conducted a population-based, nested case-control study of individuals who progressed to diabetes ( n = 43) or prediabetes ( n = 345) and control participants matched on age, sex and fasting plasma glucose concentration, who maintained normal glucose tolerance ( n = 389) during a 4-year follow-up using data from the Chungju Metabolic disease Cohort Study. Circulating levels of preadipocyte factor 1 were measured using an enzyme-linked immunosorbent assay. Results Baseline serum preadipocyte factor 1 levels showed a stepwise decrease across the glucose tolerance status groups at follow-up (normal glucose tolerance: 10.02 ± 3.02 ng/ml; prediabetes: 9.48 ± 3.35 ng/ml; diabetes: 8.66 ± 3.29 ng/ml; P for trend, 0.0151). Individuals whose fasting plasma glucose level had increased or whose homeostasis model assessment of β-cell function had decreased at follow-up showed significantly lower levels of preadipocyte factor 1 compared with their control group counterparts. After adjusting for age, BMI, fasting plasma glucose, serum insulin levels, systolic blood pressure and triglycerides, the incidence of diabetes was nearly threefold higher in the lowest vs. the upper three quartiles of circulating preadipocyte factor 1 (relative risk 2.794; 95% CI 1.188-6.571; P = 0.0185). Notably, these findings were significant in women but not in men. Conclusions Levels of circulating preadipocyte factor 1 may be a useful biomarker for identifying women at high risk of developing diabetes. [ABSTRACT FROM AUTHOR]

Background: In vitro generation of red blood cells (RBCs) from hematopoietic stem cells (HSCs) has been reported, but the collection of 1 × 10(5) to 1 × 10(6) CD34+ cells present in cord and peripheral blood is too small for expansion to 1 × 10(12) cells in 1 unit of RBCs. We transduced JAK2V617F gene, the most common mutation with polycythemia vera (PV), into cord blood-derived CD34+ cells. This PV model was expected to increase cell proliferation without the addition of erythropoietin (EPO) in early phase of differentiation.Study Design and Methods: Empty vector (control), wild-type JAK2 (wJAK2), and mutant JAK2V617F (mJAK2) were transduced into CD34+ cells using a lentivirus system. The CD34+ cells were then differentiated to the RBCs in a culture system. The cells were analyzed for cell number, differential count, and morphologic changes. Cultured RBCs were tested for oxygen equilibrium.Results: wJAK2- and mJAK2-transduced cells showed higher proliferation capacity until Day 21 than control cells; interestingly, only mJAK2-transduced cells were highly increased on Day 7 during EPO-free culture. However, both wJAK2- and mJAK2-tranduced cells had more delayed differentiation than control, but they had a higher portion of completely matured RBCs and orthochromatic erythroblasts. Furthermore, mJAK2-tranduced cells showed more differentiation into RBCs than wJAK2-transduced cells and they had a normal hemoglobin dissociation curve.Conclusion: This is the first trial to use a PV erythropoiesis model for RBC differentiation from stem cells. The transduction of HSCs with mJAK2 increased their proliferation capacity in EPO-free culture conditions. This model may also be useful for investigating the pathogenesis of PV. [ABSTRACT FROM AUTHOR]

Recent studies have shown that the transcriptional landscape of the pleiomorphic fungus Candida albicans is highly dependent upon growth conditions. Here using a dual RNA-seq approach we identified 299 C. albicans and 72 Streptococcus gordonii genes that were either upregulated or downregulated specifically as a result of co-culturing these human oral cavity microorganisms. Seventy-five C. albicans genes involved in responses to chemical stimuli, regulation, homeostasis, protein modification and cell cycle were significantly (P ≤ 0.05) upregulated, whereas 36 genes mainly involved in transport and translation were downregulated. Upregulation of filamentation-associated TEC1 and FGR42 genes, and of ALS1 adhesin gene, concurred with previous evidence that the C. albicans yeast to hypha transition is promoted by S. gordonii. Increased expression of genes required for arginine biosynthesis in C. albicans was potentially indicative of a novel oxidative stress response. The transcriptional response of S. gordonii to C. albicans was less dramatic, with only eight S. gordonii genes significantly (P ≤ 0.05) upregulated at least two-fold (glpK, rplO, celB, rplN, rplB, rpsE, ciaR and gat). The expression patterns suggest that signals from S. gordonii cause a positive filamentation response in C. albicans, whereas S. gordonii appears to be transcriptionally less influenced by C. albicans. [ABSTRACT FROM AUTHOR]

The respective contributions of components of the protein translocation/maturation machinery to cell surface biogenesis in Streptococcus mutans are not fully understood. Here we used a genetic approach to characterize the effects of deletion of genes encoding the ribosome-associated chaperone RopA (Trigger Factor), the surface-localized foldase PrsA, and the membrane-localized chaperone insertases YidC1 and YidC2, both singly and in combination, on bacterial growth, chain length, self-aggregation, cell surface hydrophobicity, autolysis, and antigenicity of surface proteins P1 (AgI/II, PAc), WapA, GbpC, and GtfD. The single and double deletion mutants, as well as additional mutant strains lacking components of the signal recognition particle pathway, were also evaluated for their effects on mutacin production and genetic competence. [ABSTRACT FROM AUTHOR]

Background and Purpose: There is emerging evidence suggesting that abnormal transport of amyloid-β (Aβ) across the blood-brain barrier (BBB) is involved in diabetes-associated cognitive decline. We investigated whether PPARγ agonists restore Aβ transport across the BBB and hippocampal plasticity in db/db mice.Experimental Approach: Efflux and influx of Aβ across the BBB were determined by stereotaxic intra-cerebral or i.a. infusion of [(125) I]-Aβ1-40 respectively. Receptor for advanced glycation end products (RAGE) and low-density lipoprotein receptor-related protein 1 (LRP1), which are involved in Aβ influx and efflux, PPARγ and NF-κB p65 at the BBB, as well as hippocampal Aβ, caspase-3, Bax and Bcl-2 were assayed by Western blot, immunohistochemistry and RT-PCR. In vivo, hippocampal LTP was recorded, and Morris water maze and Y-maze tasks were performed.Key Results: Treatment with PPARγ agonists, rosiglitazone (0.8 mg·kg(-1) ) and pioglitazone (9.0 mg·kg(-1) ), for 6 weeks significantly increased Aβ efflux and decreased Aβ influx across the BBB in db/db mice. Concomitantly, they decreased hippocampal Aβ1-40 and Aβ1-42 , suppressed neuronal apoptosis, as indicated by decreased caspase-3 activity and increased ratio of Bcl-2/Bax, and increased hippocampal plasticity, characterized by an enhanced in vivo LTP and better performance in behavioural tests. Furthermore, the PPARγ agonists induced the expression of LRP1 gene by activation of PPARγ and suppressed RAGE gene expression by inactivation of NF-κB signalling at the BBB of db/db mice.Conclusions and Implications: PPARγ agonists modify abnormal Aβ transport across the BBB and this is accompanied by amelioration of β-amyloidosis and an improvement in hippocampal plasticity in diabetic mice. [ABSTRACT FROM AUTHOR]

Background: The reverse Warburg effect describes the phenomenon that epithelial cancer cells take advantage of the metabolic machinery from nearby cancer-associated fibroblast, inducing them to produce lactate and ketones to fuel the high metabolic demands of the epithelial tumour tissues. This is in breast cancer observed as a lack of stromal caveolin-1 (CAV-1) and an increased expression of monocarboxylate transporter 4 (MCT-4) in the tumour stroma, with a concomitant increase in the expression of monocarboxylate transporter 1 (MCT-1) in the epithelial, tumour compartment. The lack of CAV-1 and increased expression of MCT-4 have been shown to have prognostic importance, primarily in patients with breast cancer. However, this phenomenon has only scarcely been described in oral squamous cell carcinoma (OSCC). Given the prognostic importance of myofibroblasts in OSCC, we also examined a potential relationship between the expression of MCT-4 and the presence of myofibroblasts.Methods: Paraffin-embedded tissues from 30 patients with OSCC were immunostained with antibodies towards MCT-1, MCT-4, Cav-1, GLUT-1, α-SMA, TOMM20 and KI-67, and evaluated for their specific epithelial and stromal expression.Results and Conclusions: In patients with OSCC, we find an increased expression of MCT-1 and MCT-4 in both the epithelial and stromal compartment, with almost no overlap in their spatial expression. We found a large spatial overlap between α-SMA and MCT-1 in the stroma compartment, but no relationship between MCT-4 and myofibroblasts. Interestingly, we did not observe any relationship between the absence of CAV-1 and the presence of MCT-4 as has been shown in breast carcinomas. [ABSTRACT FROM AUTHOR]

Background: Oral submucous fibrosis (OSF) is regarded as a pre-cancerous condition with fibrosis in oral subepithelial connective tissue. Hypoxia-inducible factor (HIF)-1α regulates a wide variety of profibrogenic genes, which are closely associated with tissue fibrosis. The aim of this study was to compare HIF-1α expression in normal buccal mucosa tissues and OSF specimens and further explore the potential mechanisms that may lead to the induction of HIF-1α expression.Method: Twenty-five OSF specimens and six normal buccal mucosa were examined by immunohistochemistry. The expression of HIF-1α from fibroblasts cultured from OSF and normal buccal mucosa was measured by Western blot. Arecoline, a major areca nut alkaloid, was challenged to normal buccal mucosa fibroblasts (BMFs) to elucidate whether HIF-1α expression could affect by arecoline. In addition, the effects of arecoline on plasminogen activator inhibitor (PAI)-1 expression were evaluated in environmental hypoxia.Results: HIF-1α expression was significantly higher in OSF specimens and expressed mainly by fibroblasts, epithelial cells, and inflammatory cells. Fibroblasts derived from OSF were found to exhibit higher HIF-1α protein expression than BMFs (P < 0.05). Arecoline was found to upregulate HIF-1α protein in a dose-dependent manner (P < 0.05). Hypoxia increased arecoline-induced PAI-1 protein expression than normoxic conditions (P < 0.05).Conclusion: These results suggest that HIF-1α expression is significantly upregulated in OSF tissues from areca quid chewers, implying a potential role as a biomarker for local tissue hypoxia. The activation of HIF-1α may promote fibrogenesis by an increase of PAI-1 expression and a subsequent elevation of extracellular matrix production in oral submucosa leading to fibrosis. [ABSTRACT FROM AUTHOR]

Plant growth-promoting rhizobacteria (PGPR) are used in agriculture to improve crop yield. Crude smoke-water (made by bubbling plant-derived smoke through water) stimulates germination and improves seedling growth. Some active compounds have been isolated from smoke with karrikinolide (KAR1) stimulating plant growth and trimethylbutenolide (TMB) being inhibitory. These smoke compounds have great potential in agriculture but their interaction with PGPR is unknown. In the present study, a two-factorial pot trial with three replicates per treatment was designed to investigate the interactions between Bacillus licheniformis and two concentrations each of smoke-water, KAR1, and TMB on okra ( Abelmoschus esculentus). Growth and physiological parameters (chlorophyll, carotenoid, protein, sugar and α-amylase) of okra as well as bacterial abundance in the rhizosphere were measured after 5 weeks. Application of B. licheniformis and 10−7 M KAR1 significantly improved the shoot biomass and 10−7 M KAR1 also significantly improved leaf area of okra. However, when 10−7 M KAR1 was applied in combination with B. licheniformis, there was an antagonistic effect on plant growth. While TMB had a negative effect on plant growth, a combination treatment of TMB and B. licheniformis overcame the inhibitory effect of TMB resulting in plant growth similar to the control plants. All treatments had no effect on chlorophyll, carotenoid, protein and sugar concentrations, while α-amylase activity was significantly elevated in okra root treated with 1:500 (v/v) smoke-water. Determining the rhizobacteria populations at harvest showed that all treatments had no significant effect on the rhizosphere microbial abundance. The modes of interaction between PGPR and smoke-derived compounds need to be further elucidated. [ABSTRACT FROM AUTHOR]

The expression of filaggrin in differentiated keratinocytes and the association of filaggrin mutations with ichthyosis vulgaris and atopic dermatitis suggest that this prototypical member of the S100 fused-type protein ( SFTP) family plays a key role in the epidermal barrier to the environment. Here, we report that SFTP genes are present not only in amniotes but also in amphibians. Four SFTPs are expressed in the skin of the frog Xenopus laevis. The results of this study indicate that filaggrin has evolved from an ancestral SFTP that may have contributed to skin modifications during the evolutionary transition to terrestrial life. [ABSTRACT FROM AUTHOR]

This study aimed to compare the biochemical composition of saliva from patients with eating disorders (EDs) with saliva from control subjects with no ED. All patients who initiated outpatient treatment in an ED clinic during a 12-month period were invited to participate. Of the 65 patients who started treatment during the period, 54 (50 female patients/four male patients; mean age: 21.5 yr) agreed to participate. The controls were 54 sex- and age-matched patients from a dental health clinic. All participants completed a questionnaire and underwent dental clinical examinations, including laboratory analyses of saliva. The proportion of subjects with unstimulated salivary hyposalivation was lower in the ED group and not correlated with intake of xerogenic drugs. Significant differences in the biochemical composition of saliva were found almost exclusively in the unstimulated state, with albumin, inorganic phosphate, aspartate aminotransferase (ASAT), chloride, magnesium, and total protein all being significantly higher in the ED group. Conditional logistic regression showed that higher ASAT and total protein concentrations were relatively good predictors of ED, with sensitivity and specificity of 65% and 67%, respectively. In conclusion, elevated salivary concentrations of ASAT and total protein may serve as indicators of ED as well as of disease severity. Future studies are needed to corroborate these initial findings. [ABSTRACT FROM AUTHOR]

Aim This experimental in vitro study examined differences in the expression and activity of calcium release-activated calcium ( CRAC) channels of human term-pregnant and non-pregnant myometrium. Material and Methods The tissue samples were obtained from term-pregnant myometrium in labor of women undergoing cesarean section and from non-pregnant myometrium of women undergoing total hysterectomy due to uterine myoma. The expression of Orai1 protein, a pore-forming subunit of CRAC channels, in human myometrium was examined using immunohistochemistry. CRAC channel involvement in the amplitude and frequency of myometrial contractions was evaluated in vitro using a tissue bath method with a CRAC ion channel blocker 3-fluropyridine-4-carboxylic acid ( FPCA). Results Decreased Orai1 expression was observed in human term-pregnant laboring myometrium compared with non-pregnant myometrium. However, the initial oxytocin-induced contraction of myometrium was significantly suppressed at different doses of FPCA in both non-pregnant human isolated myometrium and non-pregnant myometrium. The frequency of contractions was the most significantly reduced at the lowest dose of FPCA in non-pregnant myometrium and remained suppressed at all doses of FPCA in term-pregnant myometrium. Salbutamol was shown as more effective in suppression of amplitude in term-pregnant isolated myometrium. Conclusion Our results provide the first information about the changes in the Orai1 protein expression and activity of human myometrial CRAC channels in term-pregnant laboring myometrium. [ABSTRACT FROM AUTHOR]

Objective Injection of agonistic anti-Fas antibody has been shown to decrease disease symptoms in mouse models of arthritis. Additionally, membrane-bound FasL (mFasL) has been shown to induce cell death in fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. However, levels of soluble FasL (sFasL) are increased in the joints of RA patients and have been associated with disease severity, indicating that mFasL and sFasL play opposing roles in RA. The purpose of this study was to analyze the effects of FasL on RA FLS responses. Methods The responses of FLS from RA and osteoarthritis (OA) patients to soluble and oligomeric FasL, the latter mimicking mFasL, were analyzed by fluorescence-activated cell sorting and proliferation assays, using 3 different FasL variants. The signaling pathways that trigger FasL responses were characterized by Western blotting. Results We found that mFasL and sFasL have distinct roles in RA FLS. Crosslinked FasL preferentially induced apoptosis, whereas sFasL stimulated proliferation. Moreover, sFasL activated several signaling pathways in RA FLS, such as ERK-1/2, phosphatidylinositol 3-kinase, caspase 8, and JNK, with a prominent role of JNK, since only the blockade of this pathway rendered FLS more susceptible to FasL-induced apoptosis. Crosslinked FasL induced apoptosis in FLS from OA patients, but sFasL failed to stimulate their proliferation. Conclusion Our findings suggest that sFasL is a disease promoter in RA, a finding consistent with previous reports describing a tumor-promoting role of FasL. Therefore, blocking of sFasL could be a therapeutic strategy for RA. [ABSTRACT FROM AUTHOR]

Objective To evaluate whether olfactory 1/early B cell factor-associated zinc-finger protein (OAZ), a candidate lupus susceptibility gene involved in antinuclear antibody (ANA) production, plays a role in the regulation of B cells by mesenchymal stem cells (MSCs). Methods MSCs derived from the bone marrow of patients with systemic lupus erythematosus (SLE) and healthy control subjects were expanded and incubated with small interfering RNAs specific for OAZ or a nontargeting sequence. Knockdown of messenger RNA levels of OAZ and its downstream genes was measured using real-time polymerase chain reaction, and protein levels of chemokine/cytokine and immunoglobulins were determined by enzyme-linked immunosorbent assay or Western blotting. The effects of modulating the OAZ levels in MSCs, by either silencing or overexpression, on B cell proliferation and terminal differentiation were assessed by coculturing MSCs with mouse spleen cells. Results OAZ gene expression was highly enriched in MSCs compared with peripheral blood leukocytes and was increased in patients with SLE compared with control subjects. After the silencing of OAZ expression, SLE MSCs could regain the ability to inhibit B cell proliferation and terminal differentiation, as indicated by decreased percentages of bromodeoxyuridine-positive cells and CD138+ cells as well as decreased levels of IgG, IgM, and ANAs. The level of CCL2 was increased after OAZ knockdown, while anti-CCL2 antibodies completely counteracted the effect of OAZ silencing. Umbilical cord-derived normal MSCs that overexpressed OAZ had a diminished ability to inhibit B cell proliferation and terminal differentiation. Conclusion OAZ down-regulation could restore the impaired function of SLE MSCs in the immune regulation of B cells, contributing to a reduction in ANA levels. OAZ might represent a new target for therapy in patients with SLE. [ABSTRACT FROM AUTHOR]

Background A high expression of squalene epoxidase ( SQLE) is related to tumor occurrence, development, and prognosis in a variety of cancers. In this study, the expression and significance of SQLE was analyzed in patients with squamous lung cancer and pericarcinoma tissues. Methods The SQLE mRNA and protein expression were separately examined by semi-quantitative reverse transcription polymerase chain reaction, fluorescence quantitative polymerase chain reaction and Western blot in 65 cases of squamous cell lung carcinoma tissues and adjacent non-cancerous lung tissues. Results The expression of SQLE mRNA and protein in lung squamous cancerous tissues was significantly higher than in pericarcinoma tissues (63.07% vs. 44.61%, P = 0.0348; 67.69% vs. 38.46%, P = 0.0008). The positive expression rate of SQLE mRNA was not associated with gender, age, smoking, or tumor size ( P > 0.05). The expression of SQLE mRNA was closely correlated with poor differentiation, clinical stages, and lymphatic metastasis ( P < 0.05). The expression of SQLE mRNA was negatively associated with overall survival rate ( P < 0.05). Conclusion A high expression of SQLE was found in squamous lung cancer tissues. The high expression of the SQLE gene may be closely related to the occurrence and development of squamous cell lung carcinoma. SQLE expression predicts a poor prognosis and may serve as a novel lung carcinoma molecule marker. [ABSTRACT FROM AUTHOR]

Objective Nuclear protein 1 (Nupr1) is a stress-inducible protein that is involved in gene transcription. The present study was undertaken to determine whether chondrocytes express Nupr1 and whether Nupr1 regulates matrix metalloproteinase 13 (MMP-13) expression. Methods Paraffin-embedded cartilage sections from normal human and osteoarthritic (OA) cartilage were immunostained using anti-Nupr1 antibody. To measure Nupr1 expression, total RNA was isolated from joint tissue obtained 8 weeks after surgery from young (12-week-old) and older (12-month-old) mice that underwent destabilization of the medial meniscus (DMM) to induce OA. Human chondrocytes were stimulated with 1-10 ng/ml interleukin-1β (IL-1β), 25 μ M tert-butyl-hydroperoxide (tBHP), or 2 μ M thapsigargin, and Nupr1 expression was analyzed by quantitative polymerase chain reaction. In addition, chondrocytes were transfected with small interfering RNA to knock down Nupr1 expression and then stimulated overnight with IL-1β. After incubation, the conditioned medium was collected and MMP levels measured. Results Increased Nupr1 immunostaining was noted in human OA cartilage compared to normal cartilage. Expression was also increased in joint tissue from 12-month-old mice that underwent DMM surgery compared to sham-operated controls. Stimulation of chondrocytes with IL-1β induced a 2-fold increase in Nupr1 messenger RNA (mRNA) within 1 hour, with the increase peaking to 4-fold at 6 hours. Treatment of chondrocytes with tBHP to induce oxidative stress increased Nupr1 mRNA expression by >2-fold; treatment with thapsigargin to induce endoplasmic reticulum stress did not produce a similar effect. Knockdown of Nupr1 inhibited IL-1β-mediated induction of MMP-13. Conclusion Nupr1 is expressed in cartilage, and its levels are increased in OA. Nupr1 expression is required for IL-1β-mediated expression of MMP-13. These findings provide evidence of a novel pathway for regulation of IL-1β-mediated production of MMPs in chondrocytes. [ABSTRACT FROM AUTHOR]

Aims To examine single nucleotide polymorphisms in the protein tyrosine phosphatase N22 gene ( PTPN22) and to study their association with Type 1 diabetes in a Chinese cohort. Methods Three hundred and sixty-four young patients with Type 1 diabetes and 719 healthy children were included in this case-controlled study. The genotypes of rs1217385, rs2488457 (-1123C>G), rs1217414, rs1217419, rs3765598 and rs2476601 (1858C>T) in the PTPN22 gene were determined using the SNaPshot method. Alleles, genotypes and haplotype frequencies were compared between patients with Type 1 diabetes and healthy control subjects. The association between single nucleotide polymorphisms and clinical traits/autoantibody status was also analysed. Results The single nucleotide polymorphism, rs1217419, located in the second intron of the PTPN22 gene was associated with Type 1 diabetes (odds ratio 1.5, 95% CI 1.14-1.97, P = 0.003). An additional single nucleotide polymorphism, rs1217385, was also associated with Type 1 diabetes; however, the association was secondary to that of rs1217419. The previously reported single nucleotide polymorphism that is associated with Type 1 diabetes (-1123G>C) had only marginal association with Type 1 diabetes in our study. A marginal association was also identified between -1123G>C and glutamic acid decarboxylase autoantibody positivity in patients with Type 1 diabetes. There was no association between the single nucleotide polymorphism 1858C>T and Type 1 diabetes in our studied cohort. Conclusions Our study confirmed that PTPN22 is a gene that contributes to Type 1 diabetes susceptibility. The primary association occurs with single nucleotide polymorphism rs1217419 and there is clear heterogeneity of the association between PTPTN22 polymorphisms and Type 1 diabetes in a Chinese population compared with other populations. [ABSTRACT FROM AUTHOR]

Objective We previously described a messenger RNA variant of ADAMTS4 ( ADAMTS4_v1) in human synovial cell cocultures obtained from patients with osteoarthritis (OA). This RNA message has been found only in OA synovium and, if translated, would result in a protein identical to ADAMTS-4, except that the C-terminal spacer domain would be different. The purpose of this study was to determine whether ADAMTS4_v1 is translated into a protein, is expressed in vivo, and acts as a functional aggrecanase. Methods Polyclonal antibodies were raised against unique C-terminal sequences of ADAMTS-4_v1. An immunohistochemical study of human OA synovium was performed. A mammalian expression vector coding for FLAG-tagged human ADAMTS4 was mutated to contain the different sequences of ADAMTS4_v1, and the resultant plasmid was used to transfect HEK 293 cells. ADAMTS-4_v1 produced by these cells was purified via the FLAG epitope, and the ability of this recombinant protein to cleave aggrecan, biglycan, and decorin was investigated. Results An antibody specific for ADAMTS-4_v1 was found to bind to the synovial membrane surface on cryosections, and the protein was detected in cell lysates from synovium obtained from OA patients. The recombinant ADAMTS-4_v1 demonstrated enzyme activity toward the target substrate in a commercial aggrecanase 1 enzyme-linked immunosorbent assay and was also found to cleave aggrecan at the pathologically important Glu373↓374Ala aggrecanase site. Conclusion ADAMTS-4_v1 is expressed as a protein in vivo in human OA synovium, functions as an aggrecanase, and cleaves other proteoglycan substrates. This splice variant may be a major contributor to loss of aggrecan from the superficial zone of OA cartilage. [ABSTRACT FROM AUTHOR]

Objective: Innate and adaptive immunity may contribute to gland dysfunction in patients with primary Sjögren's syndrome (pSS). The P2X7 receptor (P2X7 R)-NLRP3 inflammasome complex modulates the release of the inflammatory cytokines IL-1β and IL-18. The presence of P2X7 R in salivary glands suggests an interesting scenario for the initiation and amplification of the innate immune response in pSS. Therefore, the aim of this study was to assess the role of the P2X7 R-NLRP3 inflammasome in pSS. Subjects and Methods: Twenty-one consecutive patients with pSS according to the American-European Consensus Group criteria and 15 patients with sicca syndrome (i.e. without Sjögren's syndrome, non-SS) were enrolled in this study, together with six control (CTL) subjects. Expression of the P2X7R-NLRP3 platform and IL-18 was determined by real-time PCR and western blotting in gland specimens and peripheral lymphomonocytes; data were related to patients\x92 clinical, serological and histopathological characteristics. The presence of IL-18 was determined in gland and saliva samples. Results: P2X7 R expression was significantly higher in salivary glands from individuals with pSS than in those from non-SS and CTL subjects. Accordingly, the gene expression levels of the inflammasome components NLRP3, ASC and caspase-1 were significantly higher in pSS gland specimens, and this was paralleled by an increased expression of mature IL-18 in pSS saliva samples. The expression of both the P2X7 R and the inflammasome components was a marker of disease-related glandular involvement, being increased in patients with anti-Ro/SSA positivity and correlated with focus score. Conclusion: The results of this study suggest an involvement of the P2X7 R-inflammasome-caspase-1-IL-18 axis in the development of pSS exocrinopathy. This finding provides the basis for studying the complex mechanisms underlying pSS, as well as for developing novel potential therapeutic strategies. [ABSTRACT FROM AUTHOR]

The homeobox, msh-like 1 (MSX1) protein is essential for cell proliferation and differentiation. Tooth germ development of Msx1 knockout mouse is arrested at the bud stage, impeding an understanding of its role beyond this stage of tooth development. The aims of this study were to investigate the potential role of MSX1 in the regulation of proliferation and differentiation of dental mesenchymal cells in culture, and to preliminarily explore its underlying mechanism of action. Tooth germs were isolated from embryonic day (E)15.5 mice. The mesenchyme was separated and digested into a single-cell suspension, and then cultured in vitro. Isolated dental mesenchymal cells were transfected with MSX1 small interfering RNA, and the effects on cell proliferation, cell cycle distribution, and the expression of bone morphogenetic protein 2 ( Bmp2) and bone morphogenetic protein 4 ( Bmp4) were studied. We also compared the expression levels of alkaline phosphatase ( Alp), type I collagen ( Col1A), osteocalcin ( Ocn), runt-related transcription factor 2 ( Runx2), dentin sialophosphoprotein ( Dspp) and dentin matrix protein 1 ( Dmp1), and mineralized nodule formation, between control and MSX1 siRNA-transfected groups after the induction of odontoblast differentiation. Knockdown of Msx1 expression was associated with decreased cell proliferation, prolonged time in the S phase of the cell cycle, enhanced odontoblast differentiation, and elevated Bmp2 and Bmp4 expression. We conclude that MSX1 may promote proliferation and prevent the differentiation of dental mesenchymal cells by the inhibition of Bmp2 and Bmp4 expression. [ABSTRACT FROM AUTHOR]

Saurochory (seed dispersal by reptiles) among crocodilians has largely been ignored, probably because these reptiles are generally assumed to be obligate carnivores incapable of digesting vegetable proteins and polysaccharides. Herein we review the literature on crocodilian diet, foraging ecology, digestive physiology and movement patterns, and provide additional empirical data from recent dietary studies of Alligator mississippiensis. We found evidence of frugivory in 13 of 18 (72.2%) species for which dietary information was available, indicating this behavior is widespread among the Crocodylia. Thirty-four families and 46 genera of plants were consumed by crocodilians. Fruit types consumed by crocodilians varied widely; over half (52.1%) were fleshy fruits. Some fruits are consumed as gastroliths or ingested incidental to prey capture; however, there is little doubt that on occasion, fruit is deliberately consumed, often in large quantities. Sensory cues involved in crocodilian frugivory are poorly understood, although airborne and waterborne cues as well as surface disturbances seem important. Crocodilians likely accrue nutritional benefits from frugivory and there are no a priori reasons to assume otherwise. Ingested seeds are regurgitated, retained in the stomach for indefinite and often lengthy periods, or passed through the digestive tract and excreted in feces. Chemical and mechanical scarification of seeds probably occurs in the stomach, but what effects these processes have on seed viability remain unknown. Because crocodilians have large territories and undertake lengthy movements, seeds are likely transported well beyond the parent plant before being voided. Little is known about the ultimate fate of seeds ingested by crocodilians; however, deposition sites could prove suitable for seed germination. Although there is no evidence for a crocodilian-specific dispersal syndrome similar to that described for other reptiles, our review strongly suggests that crocodilians function as effective agents of seed dispersal. Crocodilian saurochory offers a fertile ground for future research. [ABSTRACT FROM AUTHOR]

The molecular circuitries controlling the process of skin wound healing have gained new significant insights in recent years. This knowledge is built on landmark studies on skin embryogenesis, maturation, and differentiation. Furthermore, the identification, characterization, and elucidation of the biological roles of adult skin epithelial stem cells and their influence in tissue homeostasis have provided the foundation for the overall understanding of the process of skin wound healing and tissue repair. Among numerous signaling pathways associated with epithelial functions, the PI3 K/ Akt/m TOR signaling route has gained substantial attention with the generation of animal models capable of dissecting individual components of the pathway, thereby providing a novel insight into the molecular framework underlying skin homeostasis and tissue regeneration. In this review, we focus on recent findings regarding the mechanisms involved in wound healing associated with the upregulation of the activity of the PI3 K/ Akt/m TOR circuitry. This review highlights critical findings on the molecular mechanisms controlling the activation of mTOR, a downstream component of the PI3 K- PTEN pathway, which is directly involved in epithelial migration and proliferation. We discuss how this emerging information can be exploited for the development of novel pharmacological intervention strategies to accelerate the healing of critical size wounds. [ABSTRACT FROM AUTHOR]

The aim of the present study was to clarify an involvement of growth-associated protein-43 (GAP-43) in the regeneration of primary afferent trigeminal ganglion (TG) neurons following inferior alveolar nerve transection (IANX). A larger number of GAP-43 immunoreactive (GAP-43 IR) TG neurons was observed in rats 3 d after IANX compared with sham rats. Growth-associated protein-43 IR TG neurons were also detected for 30 d after IANX, and the number of GAP-43 IR TG neurons was significantly higher in the IANX model until day 30. The relative number of large (>600 μm2) GAP-43 IR TG neurons was significantly lower, whereas the relative number of small (<400 /μm2) GAP-43 IR TG neurons was significantly higher than that at day 0 until 30 d after IANX. To evaluate the functional recovery of damaged IAN, the jaw opening reflex (JOR), elicited by the electrical stimulation of the IAN, was measured before and after IANX. Jaw opening reflex occurrence was gradually increased and the relative threshold of electrical stimulation eliciting JOR was gradually decreased over the 30-d duration of the study. On day 30 after IANX, the JOR occurrence and relative JOR threshold were similar to those in sham rats. The present findings suggest that changes in the expression of GAP-43 in TG neurons after IANX are involved in regeneration and functional recovery of the transected IAN. [ABSTRACT FROM AUTHOR]

The contractile phenotype and function of myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized that smooth muscle α-actin expressed in myofibroblasts is critical for its formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin-null and wild-type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a myofibroblast-like distribution and a stress fiber-like pattern, showing that these cells acquired the myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild-type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-β1. Smooth muscle γ-actin and skeletal muscle α-actin were expressed in smooth muscle α-actin-null myofibroblasts, as shown by immunostaining, real-time polymerase chain reaction, and mass spectrometry. These results show that smooth muscle α-actin is not necessary for myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in myofibroblasts. [ABSTRACT FROM AUTHOR]

The formation of dentin and enamel is initiated by the differentiation of odontogenic precursor cells into odontoblasts and ameloblasts, respectively. This study was performed to identify new molecules involved in the differentiation of odontogenic cells. The genes expressed differentially between the root stage (after the differentiation of odontogenic cells and dental hard-tissue formation) and the cap stage (before the differentiation of odontogenic cells and dental hard-tissue formation) were searched using differential display PCR. For the first time, synaptic vesicle protein ( SV) 2b, an important transmembrane transporter of Ca2+-stimulated vesicle exocytosis, was identified as a differentially expressed molecule. Real-time PCR and western blotting revealed an increase in the transcriptional and translational levels of SV2b during or after the differentiation of odontogenic cells. Immunofluorescence revealed this molecule to be localized in not only fully differentiated odontoblasts but also in pre-odontoblasts before dentin matrix secretion. The expression pattern of the SV2a isoform was similar to that of the SV2b isoform, whereas the SV2c isoform showed a contrasting pattern of expression. After treatment with alendronate, an inhibitor of protein isoprenylation for the transport of secretory vesicles, the expression of SV2a and SV2b decreased, whereas that of SV2c increased. These results suggest that the SV2 isoforms are functional molecules of (pre)odontoblasts which may be involved in vesicle transport. [ABSTRACT FROM AUTHOR]

Key points The CXC chemokine ligand 12 (CXCL12) modulates spontaneous firing of Cajal-Retzius cells via the CXC chemokine receptor 4 (CXCR4). However, the underlying mechanism(s) are poorly understood. CXCR4 also binds the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120), but the functional effects of this interaction on Cajal-Retzius cell excitability remain unknown., We show that CXCL12 reduces spontaneous firing in Cajal-Retzius cells by opening a BK-type calcium-activated potassium conductance, whereas gp120 increases their excitability via a calcium- and chloride-dependent mechanism., Our data suggest that, depending on the use of CXCL12 or gp120 as ligands, partial agonism at the CXCR4 receptor generates calcium responses of different strengths, which lead to the recruitment of either calcium-activated potassium or chloride channels., We propose that HIV infection disrupts a signalling pathway important for the regulation of the excitability of Cajal-Retzius cells, and alters their functions., Abstract Activation of the CXC chemokine receptor 4 (CXCR4) in Cajal-Retzius cells by CXC chemokine ligand 12 (CXCL12) is important for controlling their excitability. CXCR4 is also a co-receptor for the glycoprotein 120 (gp120) of the envelope of the human immunodeficiency virus type 1 (HIV-1), and binding of gp120 to CXCR4 may produce pathological effects. In order to study CXCR4-dependent modulation of membrane excitability, we recorded in cell-attached configuration spontaneous action currents from hippocampal stratum lacunosum-moleculare Cajal-Retzius cells of the CXCR4-EGFP mouse. CXCL12 (50 n m) powerfully inhibited firing independently of synaptic transmission, suggesting that CXCR4 regulates an intrinsic conductance. This effect was prevented by conditioning slices with BAPTA-AM (200 μ m), and by blockers of the BK calcium-dependent potassium channels (TEA (1 m m), paxilline (10 μ m) and iberiotoxin (100 n m)). In contrast, exposure to gp120 (pico- to nanomolar range, alone or in combination with soluble cluster of differentiation 4 (CD4)), enhanced spontaneous firing frequency. This effect was prevented by the CXCR4 antagonist AMD3100 (1 μ m) and was absent in EGFP-negative stratum lacunosum-moleculare interneurons. Increased excitability was prevented by treating slices with BAPTA-AM or bumetanide, suggesting that gp120 activates a mechanism that is both calcium- and chloride-dependent. In conclusion, our results demonstrate that CXCL12 and gp120 modulate the excitability of Cajal-Retzius cells in opposite directions. We propose that CXCL12 and gp120 either generate calcium responses of different strength or activate distinct pools of intracellular calcium, leading to agonist-specific responses, mediated by BK channels in the case of CXCL12, and by a chloride-dependent mechanism in the case of gp120. [ABSTRACT FROM AUTHOR]