A New Member of the AFAP Family, AFAP1L1, Binds to Cortactin and Localizes to Invadosomes

Cellular motility and invasion in normal cellular processes and disease states such as cancer are dependent upon the ability of a cell to efficiently interact with its microenvironment, rearrange its cytoskeleton and degrade tissue barriers for purposes of cell movement. The AFAP family of adaptor proteins, AFAP1, AFAP1L1 and AFAP1L2, integrates signals received from the microenvironment into coordinated cytoskeletal changes. While there have been many reports on the functions and binding partners of AFAP1 and AFAP1L2, this work aimed to determine the cellular location and function of newly discovered AFAP1L1. The overall amino acid and protein structures of AFAP family members were compared so as to determine similarities and differences as well as to propose an evolutionary link between all three family members. As AFAP1 and AFAP1L1 have been shown to be more closely related, studies focused on a detailed comparison of these two family members. AFAP1 and AFAP1L1 were shown to have similar cellular localization in the cell by associating with stress filaments and cortical actin and also showing localization to invadosomes. Immunohistochemistry demonstrated differential expression of AFAP1L1 in the brain, particularly surrounding the Purkinje neurons, granular cells and neurons of the dentate nucleus. Although AFAP1 is a well known cSrc binding partner and activator, AFAP1L1 was determined to be a binding partner for cortactin, possibly through the SH3 domain. As other AFAP family members have been shown to be increased in various cancers, AFAP1L1 expression levels are upregulated in a number of cancers, particularly neuroblastoma and glioblastoma. While the similar amino acid sequence and modular domain identifies AFAP1L1 as a previously undescribed member of the AFAP family, the ability of AFAP1L1 to interact with cortactin and localize to distinct areas of the brain implies that AFAP1L1 has unique functions separate from AFAP1 and AFAP1L2

Multi-walled carbon nanotubes induce human microvascular endothelial cellular effects in an alveolar-capillary co-culture with small airway epithelial cells

Background Nanotechnology, particularly the use of multi-walled carbon nanotubes (MWCNT), is a rapidly growing discipline with implications for advancement in a variety of fields. A major route of exposure to MWCNT during both occupational and environmental contact is inhalation. While many studies showed adverse effects to the vascular endothelium upon MWCNT exposure, in vitro results often do not correlate with in vivo effects. This study aimed to determine if an alveolar-capillary co-culture model could determine changes in the vascular endothelium after epithelial exposure to MWCNT. Methods A co-culture system in which both human small airway epithelial cells and human microvascular endothelial cells were separated by a Transwell membrane so as to resemble an alveolar-capillary interaction was used. Following exposure of the epithelial layer to MWCNT, the effects to the endothelial barrier were determined. Results Exposure of the epithelial layer to MWCNT induced multiple changes in the endothelial cell barrier, including an increase in reactive oxygen species, actin rearrangement, loss of VE-cadherin at the cell surface, and an increase in endothelial angiogenic ability. Overall increases in secreted VEGFA, sICAM-1, and sVCAM-1 protein levels, as well as increases in intracellular phospho-NF-κB, phospho-Stat3, and phospho-p38 MAPK, were also noted in HMVEC after epithelial exposure. Conclusion The co-culture system identified that alveolar-capillary exposure to MWCNT induced multiple changes to the underlying endothelium, potentially through cell signaling mediators derived from MWCNT-exposed epithelial cells. Therefore, the co-culture system appears to be a relevant in vitromethod to study the pulmonary toxicity of MWCNT

Multi-Walled Carbon Nanotube-Induced Gene Expression Biomarkers for Medical and Occupational Surveillance

As the demand for multi-walled carbon nanotube (MWCNT) incorporation into industrial and biomedical applications increases, so does the potential for unintentional pulmonary MWCNT exposure, particularly among workers during manufacturing. Pulmonary exposure to MWCNTs raises the potential for development of lung inflammation, fibrosis, and cancer among those exposed; however, there are currently no effective biomarkers for detecting lung fibrosis or predicting the risk of lung cancer resulting from MWCNT exposure. To uncover potential mRNAs and miRNAs that could be used as markers of exposure, this study compared in vivo mRNA and miRNA expression in lung tissue and blood of mice exposed to MWCNTs with in vitro mRNA and miRNA expression from a co-culture model of human lung epithelial and microvascular cells, a system previously shown to have a higher overall genome-scale correlation with mRNA expression in mouse lungs than either cell type grown separately. Concordant mRNAs and miRNAs identified by this study could be used to drive future studies confirming human biomarkers of MWCNT exposure. These potential biomarkers could be used to assess overall worker health and predict the occurrence of MWCNT-induced diseases

A Polymorphic Variant of AFAP-110 Enhances cSrc Activity12

Enhanced expression and activity of cSrc are associated with ovarian cancer progression. Generally, cSrc does not contain acti- vating mutations; rather, its activity is increased in response to signals that affect a conformational change that releases its auto- inhibition. In this report, we analyzed ovarian cancer tissues for the expression of a cSrc-activating protein, AFAP-110. AFAP-110 activates cSrc through a direct interaction that releases it from its autoinhibited conformation. Immunohistochemical analysis re- vealed a concomitant increase of AFAP-110 and cSrc in ovarian cancer tissues. An analysis of the AFAP-110 coding sequence revealed the presence of a nonsynonymous, single-nucleotide polymorphism that resulted in a change of Ser403 to Cys403. In cells that express enhanced levels of cSrc, AFAP-110403C directed the activation of cSrc and the formation of podosomes indepen- dently of input signals, in contrast to wild-type AFAP-110. We therefore propose that, under conditions of cSrc overexpression, the polymorphic variant of AFAP-110 promotes cSrc activation. Further, these data indicate a mechanism by which an inherited genetic variation could influence ovarian cancer progression and could be used to predict the response to targeted therapy

Evidence for a functional specialization of ventral anterior temporal lobe for language.

The controlled semantic cognition framework proposes that the ventral anterior temporal lobes (vATL) in the left and right hemisphere function as an integrated hub region supporting transmodal semantic representations. The clinical evidence for the transmodal function of vATL is largely based on studies of semantic dementia patients with severe anomia, who also show impaired performance on nonverbal tasks that involve the retrieval of knowledge about objects and their prototypical use, such as the production of tool use pantomimes. Yet, evidence from patients with apraxia and functional neuroimaging studies in healthy adults does not implicate vATL in pantomime production. We, therefore, compared semantic retrieval of object-action associations for overt verb and pantomime production from picture and word stimuli. Our results show that, independent of stimulus modality, the retrieval of object-action associations for verb, but not pantomime, production is related to activity in bilateral vATL. Bilateral vATL activation was also observed for meaningless verbal responses that did not require the retrieval of object-action associations. Taken together, our results suggest that bilateral vATL is not engaged in the retrieval of object-action associations per se, but rather supports semantic representations that are functionally specialized for language. These findings have implications for the semantic cognition framework and our understanding of the dependence of conceptual knowledge on language

Non-invasive diagnostic tests for Helicobacter pylori infection

BACKGROUND: Helicobacter pylori (H pylori) infection has been implicated in a number of malignancies and non-malignant conditions including peptic ulcers, non-ulcer dyspepsia, recurrent peptic ulcer bleeding, unexplained iron deficiency anaemia, idiopathic thrombocytopaenia purpura, and colorectal adenomas. The confirmatory diagnosis of H pylori is by endoscopic biopsy, followed by histopathological examination using haemotoxylin and eosin (H & E) stain or special stains such as Giemsa stain and Warthin-Starry stain. Special stains are more accurate than H & E stain. There is significant uncertainty about the diagnostic accuracy of non-invasive tests for diagnosis of H pylori. OBJECTIVES: To compare the diagnostic accuracy of urea breath test, serology, and stool antigen test, used alone or in combination, for diagnosis of H pylori infection in symptomatic and asymptomatic people, so that eradication therapy for H pylori can be started. SEARCH METHODS: We searched MEDLINE, Embase, the Science Citation Index and the National Institute for Health Research Health Technology Assessment Database on 4 March 2016. We screened references in the included studies to identify additional studies. We also conducted citation searches of relevant studies, most recently on 4 December 2016. We did not restrict studies by language or publication status, or whether data were collected prospectively or retrospectively. SELECTION CRITERIA: We included diagnostic accuracy studies that evaluated at least one of the index tests (urea breath test using isotopes such as13C or14C, serology and stool antigen test) against the reference standard (histopathological examination using H & E stain, special stains or immunohistochemical stain) in people suspected of having H pylori infection. DATA COLLECTION AND ANALYSIS: Two review authors independently screened the references to identify relevant studies and independently extracted data. We assessed the methodological quality of studies using the QUADAS-2 tool. We performed meta-analysis by using the hierarchical summary receiver operating characteristic (HSROC) model to estimate and compare SROC curves. Where appropriate, we used bivariate or univariate logistic regression models to estimate summary sensitivities and specificities. MAIN RESULTS: We included 101 studies involving 11,003 participants, of which 5839 participants (53.1%) had H pylori infection. The prevalence of H pylori infection in the studies ranged from 15.2% to 94.7%, with a median prevalence of 53.7% (interquartile range 42.0% to 66.5%). Most of the studies (57%) included participants with dyspepsia and 53 studies excluded participants who recently had proton pump inhibitors or antibiotics.There was at least an unclear risk of bias or unclear applicability concern for each study.Of the 101 studies, 15 compared the accuracy of two index tests and two studies compared the accuracy of three index tests. Thirty-four studies (4242 participants) evaluated serology; 29 studies (2988 participants) evaluated stool antigen test; 34 studies (3139 participants) evaluated urea breath test-13C; 21 studies (1810 participants) evaluated urea breath test-14C; and two studies (127 participants) evaluated urea breath test but did not report the isotope used. The thresholds used to define test positivity and the staining techniques used for histopathological examination (reference standard) varied between studies. Due to sparse data for each threshold reported, it was not possible to identify the best threshold for each test.Using data from 99 studies in an indirect test comparison, there was statistical evidence of a difference in diagnostic accuracy between urea breath test-13C, urea breath test-14C, serology and stool antigen test (P = 0.024). The diagnostic odds ratios for urea breath test-13C, urea breath test-14C, serology, and stool antigen test were 153 (95% confidence interval (CI) 73.7 to 316), 105 (95% CI 74.0 to 150), 47.4 (95% CI 25.5 to 88.1) and 45.1 (95% CI 24.2 to 84.1). The sensitivity (95% CI) estimated at a fixed specificity of 0.90 (median from studies across the four tests), was 0.94 (95% CI 0.89 to 0.97) for urea breath test-13C, 0.92 (95% CI 0.89 to 0.94) for urea breath test-14C, 0.84 (95% CI 0.74 to 0.91) for serology, and 0.83 (95% CI 0.73 to 0.90) for stool antigen test. This implies that on average, given a specificity of 0.90 and prevalence of 53.7% (median specificity and prevalence in the studies), out of 1000 people tested for H pylori infection, there will be 46 false positives (people without H pylori infection who will be diagnosed as having H pylori infection). In this hypothetical cohort, urea breath test-13C, urea breath test-14C, serology, and stool antigen test will give 30 (95% CI 15 to 58), 42 (95% CI 30 to 58), 86 (95% CI 50 to 140), and 89 (95% CI 52 to 146) false negatives respectively (people with H pylori infection for whom the diagnosis of H pylori will be missed).Direct comparisons were based on few head-to-head studies. The ratios of diagnostic odds ratios (DORs) were 0.68 (95% CI 0.12 to 3.70; P = 0.56) for urea breath test-13C versus serology (seven studies), and 0.88 (95% CI 0.14 to 5.56; P = 0.84) for urea breath test-13C versus stool antigen test (seven studies). The 95% CIs of these estimates overlap with those of the ratios of DORs from the indirect comparison. Data were limited or unavailable for meta-analysis of other direct comparisons. AUTHORS' CONCLUSIONS: In people without a history of gastrectomy and those who have not recently had antibiotics or proton ,pump inhibitors, urea breath tests had high diagnostic accuracy while serology and stool antigen tests were less accurate for diagnosis of Helicobacter pylori infection.This is based on an indirect test comparison (with potential for bias due to confounding), as evidence from direct comparisons was limited or unavailable. The thresholds used for these tests were highly variable and we were unable to identify specific thresholds that might be useful in clinical practice.We need further comparative studies of high methodological quality to obtain more reliable evidence of relative accuracy between the tests. Such studies should be conducted prospectively in a representative spectrum of participants and clearly reported to ensure low risk of bias. Most importantly, studies should prespecify and clearly report thresholds used, and should avoid inappropriate exclusions

Multi-Walled Carbon Nanotube-Induced Gene Expression Biomarkers for Medical and Occupational Surveillance

As the demand for multi-walled carbon nanotube (MWCNT) incorporation into industrial and biomedical applications increases, so does the potential for unintentional pulmonary MWCNT exposure, particularly among workers during manufacturing. Pulmonary exposure to MWCNTs raises the potential for development of lung inflammation, fibrosis, and cancer among those exposed; however, there are currently no effective biomarkers for detecting lung fibrosis or predicting the risk of lung cancer resulting from MWCNT exposure. To uncover potential mRNAs and miRNAs that could be used as markers of exposure, this study compared in vivo mRNA and miRNA expression in lung tissue and blood of mice exposed to MWCNTs with in vitro mRNA and miRNA expression from a co-culture model of human lung epithelial and microvascular cells, a system previously shown to have a higher overall genome-scale correlation with mRNA expression in mouse lungs than either cell type grown separately. Concordant mRNAs and miRNAs identified by this study could be used to drive future studies confirming human biomarkers of MWCNT exposure. These potential biomarkers could be used to assess overall worker health and predict the occurrence of MWCNT-induced diseases

Differential gene regulation in human small airway epithelial cells grown in monoculture versus coculture with human microvascular endothelial cells following multiwalled carbon nanotube exposure

Concurrent with rising production of carbon-based engineered nanomaterials is a potential increase in respiratory and cardiovascular diseases due to exposure to nanomaterials in the workplace atmosphere. While single-cell models of pulmonary exposure are often used to determine the potential toxicity of nanomaterials in vitro, previous studies have shown that coculture cell models better represent the cellular response and crosstalk that occurs in vivo. This study identified differential gene regulation in human small airway epithelial cells (SAECs) grown either in monoculture or in coculture with human microvascular endothelial cells following exposure of the SAECs to multiwalled carbon nanotubes (MWCNTs). SAEC genes that either changed their regulation direction from upregulated in monoculture to downregulated in coculture (or vice versa) or had a more than a two-fold changed in the same regulation direction were identified. Genes that changed regulation direction were most often involved in the processes of cellular growth and proliferation and cellular immune response and inflammation. Genes that had a more than a two-fold change in regulation in the same direction were most often involved in the inflammatory response. The direction and fold-change of this differential gene regulation suggests that toxicity testing in monoculture may exaggerate cellular responses to MWCNTs, and coculture of cells may provide a more in-depth assessment of toxicological responses

Multi-walled carbon nanotubes induce human microvascular endothelial cellular effects in an alveolar-capillary co-culture with small airway epithelial cells

Background: Nanotechnology, particularly the use of multi-walled carbon nanotubes (MWCNT), is a rapidly growing discipline with implications for advancement in a variety of fields. A major route of exposure to MWCNT during both occupational and environmental contact is inhalation. While many studies showed adverse effects to the vascular endothelium upon MWCNT exposure, in vitro results often do not correlate with in vivo effects. This study aimed to determine if an alveolar-capillary co-culture model could determine changes in the vascular endothelium after epithelial exposure to MWCNT. Methods: A co-culture system in which both human small airway epithelial cells and human microvascular endothelial cells were separated by a Transwell membrane so as to resemble an alveolar-capillary interaction was used. Following exposure of the epithelial layer to MWCNT, the effects to the endothelial barrier were determined. Results: Exposure of the epithelial layer to MWCNT induced multiple changes in the endothelial cell barrier, including an increase in reactive oxygen species, actin rearrangement, loss of VE-cadherin at the cell surface, and an increase in endothelial angiogenic ability. Overall increases in secreted VEGFA, sICAM-1, and sVCAM-1 protein levels, as well as increases in intracellular phospho-NF-κB, phospho-Stat3, and phospho-p38 MAPK, were also noted in HMVEC after epithelial exposure. Conclusion: The co-culture system identified that alveolar-capillary exposure to MWCNT induced multiple changes to the underlying endothelium, potentially through cell signaling mediators derived from MWCNT-exposed epithelial cells. Therefore, the co-culture system appears to be a relevant in vitro method to study the pulmonary toxicity of MWCN