Proteomic Profiling of Small Extracellular Vesicles Isolated from the Plasma of Vietnamese Patients with Non-Small Cell Lung Cancer Reveals Some Potential Biomarkers

Background: Considering the poor prognosis of non-small cell lung cancer (NSCLC), the objective of this study was to examine the potential of plasma-derived vesicles as a source of lung cancer-specific proteins. Extracellular vesicle (EV) cargos are specific to the source cells, hence they have the potential of being a source of cancer-specific proteins.  Methods: The proteins differently expressed in cancer were determined and derived from EVs isolated from the plasma of NSCLC patients at the National Lung Hospital. To this end, purification was done using gel filtration chromatography and ultracentrifugation. In addition, nano liquid chromatography mass spectrometry (LC–MS/MS) was used for analyzing. Results: Fifty-seven EV-derived proteins related to NSCLC were highlighted in this research. Some of them have not been addressed before, such as EEF1A1 (elongation factor 1-α1), KPNB1 (Importin subunit beta 1), SRC (proto-oncogene tyrosine-protein kinase) and ACTC1 (actin, alpha cardiac muscle 1). This list was further confirmed through a comparison with ExoCarta and Vesiclepedia. Conclusion: This study is the first work to show the involvement of several novel proteins of small EV (EEF1A1, KPNB1, SRC, and ACTC1) in the progression of NSCLC. The results suggested that they could serve as novel biomarkers for non-small cell lung cancer in the future.     

Differential expression of inflammatory cytokines and chemokines in lipopolysaccharide‐stimulated melanocytes from lightly and darkly pigmented skin

Increasing evidence suggests that human epidermal melanocytes play an important role in the skin immune system; however, a role of their pigmentation in immune and inflammatory responses is poorly examined. In the study, the expression of Toll‐like receptor 4 (TLR4) and inflammatory cytokines and chemokines by cultured normal melanocytes derived from lightly and darkly pigmented skin was investigated after cell stimulation with lipopolysaccharide (LPS). The basal TLR4 mRNA level in heavily pigmented cells was higher as compared to their lightly pigmented counterparts. Melanocyte exposure to LPS upregulated the expression of TLR4 mRNA and enhanced the DNA‐binding activity of NF‐κB p50 and p65. We found substantial differences in the LPS‐stimulated expression of numerous genes encoding inflammatory cytokines and chemokines between the cells with various melanin contents. In lightly pigmented melanocytes, the most significantly upregulated genes were nicotinamide phosphoribosyltransferase (NAMPT/visfatin), the chemokines CCL2 and CCL20, and IL6, while the genes for CXCL12, IL‐16 and the chemokine receptor CCR4 were the most significantly upregulated in heavily pigmented cells. Moreover, the lightly pigmented melanocytes secreted much more NAMPT, CCL2 and IL‐6. The results of our study suggest modulatory effect of melanogenesis on the immune properties of normal epidermal melanocytes.     

Vers un rapprochement du syndrome de Klinefelter et de la psychopathie en psychiatrie légale

Klinefelter syndrome (KS) (47,XXY) is the most common aneuploidy (1/650) of sexual chromosome among male (0,1 à 0,2 % of male population) (Hong and Reiss, 2014). Because its large physical phenotypic variability (high tall, sparse hairiness, gynecomastia), this syndrome is largely underdiagnosed (less than 25 % of affected persons) (Samango-Sprouse et al., 2018). Nevertheless, cognitive variability is smaller. Normal to low average total IQ, low verbal IQ, social problems and high levels of psychiatric comorbidities including early aggressiveness are commonly described (Hong and Reiss, 2014). In Denmark, higher risks of committing sexual crime and arson (compared to criminal controls) was recently reported (Stochholm et al., 2012). Quite a few clinically relevant cases reports scattered in the literature, suggests the presence of a pattern of a specific subtype of KS inpatients among forensic population (Bénézech, 1975). However, very few studies provide quantitative or qualitative pertaining to robust results. KS well-documented neurobiological (van Rijn, 2018) (e.g. low levels of testosterone), neuropsychological (Bénézech, 1975; Hong and Reiss, 2014; Samango-Sprouse et al., 2018; Savic, 2012; Seara-Cardoso et al., 2016; Senon, 2005; Stochholm et al., 2012; van Rijn, 2018; van Rijn et al., 2008; van Rijn et al., 2018; van Rijn et al., 2014; van Rijn et al., 2012) [29] (e.g. alterations of both complex cerebral — attention, empathy — and behavioral regulation functions - inhibition, mental flexibility, emotional response modulation, control of own actions) and neuroanatomical (Hong and Reiss, 2014; Itti et al., 2003; Savic, 2012; van Rijn et al., 2008; van Rijn et al., 2012) [29] (e.g. limbic system and temporal lobe abnormal volume, hemispheric specialization shortcoming) features may be helpful to understand comorbid symptoms psychopathology. Numbers of recent studies conduct on KS pediatric or adult population provide interesting results on conduct, anxiety, psychotic and autism spectrum disorders. In addition, some authors use genetic and epigenetic specific features of sex chromosome aneuploidies (e.g. X genes neurodevelopmental role; imprinting) in order to clarify genotype-phenotype links of comorbid symptoms (Bruining et al., 2011; Zitzmann et al., 2004;). With Belgian colleagues from the Social Defense Research Center (CRDS, Tournai, Belgium), we are currently recruiting KS inpatients from security hospitals or psychiatric units in Belgium and France. We aim to assess psychopathic traits with the Psychopathy Checklist Revised (PCL-R, Hare) (Hare, 2003). Our first results concerning 3 KS males outline that PCL-R is useful for the characterization of clinical phenotype among KS forensic sample. While three of them present psychopathic traits, two of them present categorical double diagnose “psychopathy-KS” (total PCL-R score > = 30/40 (Delannoy et al., 2017)). Moreover, dimensional analysis support our hypothesis of a higher prevalence of “explosive profile” in comparison to other psychopathic profiles in our sample (Delannoy et al., 2017). The present article summarizes historic background (e.g. “psychopathy” disappearance of mental disorder reference classification schemes, “crime chromosome” (Bénézech, 1975)) and current context argues (e.g. French psychiatrists court experts widely refer to psychopathy concept despite a lack of consensual definition (Senon, 2005), weak knowledge and training of PCL-R and its related biopsychological recent findings (Blair, 2013; de Oliveira-Souza et al., 2008; Dotterer et al., 2017; Glenn and Raine, 2014; Hosking et al., 2017; Korponay et al., 2017; Pham, 1995; Pham, 2005; Raine, 2008; Raine et al., 2003); stigma and discrimination apprehensions of KS and psychopath) that motivate our research project. Finally, we discuss the advantages of our research protocol on KS participants assessed with PCL-R, such as tackling stigma and discrimination, better understanding psychopathology, and clarifying murky interactions of biological, psychological and social factors entangled in the development of these two fascinating troubles.     

Fruit and Vegetable Intake and Stomach Cancer among Male Adults: A Case-Control Study in Northern Viet Nam

Objective: This study investigated the association between fruit and vegetable intake and stomach cancer, with considering the impacts of Helicobacter pylori (H. pylori) infection and tobacco smoking. Methods: A case-control study featuring 80 male incident stomach-cancer cases and 126 male controls was conducted in a general hospital in Viet Nam. A semi-quantitative food frequency and demographic lifestyle questionnaire were used; and venous blood samples were collected to determine H. pylori status by IgG ELISA. The respective associations between fruit and vegetable intake and stomach cancer were examined using unconditional logistic regression analysis with adjustments for possible cofactors. Results: Fruit intake and stomach cancer showed a weak inverse association when this became non-significant after adjusting for H. pylori infection (OR = 0.50, 95%CI: 0.22–1.12, p trend = 0.094). Stratifying by H. pylori status returned a negative trend for fruit intake and stomach cancer among H. pylori-negative participants (OR = 0.21, 95%CI: 0.06–0.69, p trend = 0.010), but no significant interaction for H. pylori-positive participants (OR = 0.76, 95%CI: 0.21–2.68, p trend = 0.670). Vegetable intake and stomach cancer showed no association, regardless of H. pylori status. Compared to ever-smokers with low intake, never-smokers with high vegetable (OR = 0.25, 95% CI: 0.06–0.95) and fruit intake (OR = 0.20, 95%CI: 0.06–0.65) showed the lowest odds of stomach cancer. Conclusions: Fruit, but not vegetable, intake showed a weak inverse association with stomach cancer. H. pylori infection and tobacco-smoking status may influence the protective effects of fruit and vegetable intake on stomach cancer.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A

Myostatin (MSTN) is a transforming growth factor-β (TGF-β) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-β family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis.

Myostatin (MSTN) is a secreted signaling molecule that normally acts to limit skeletal muscle growth (for review, see ref. 1). Mice lacking MSTN exhibit dramatic increases in muscle mass throughout the body, with individual muscles growing to about twice the normal size (2). MSTN appears to play two distinct roles in regulating muscle size, one to regulate the number of muscle fibers that are formed during development and a second to regulate the growth of those fibers postnatally. The sequence of MSTN has been highly conserved through evolution, with the mature MSTN peptide being identical in species as divergent as humans and turkeys (3). The function of MSTN has also been conserved, and targeted or naturally occurring mutations in MSTN have been shown to cause increased muscling in numerous species, including cattle (3–5), sheep (6), dogs (7), rabbits (8), rats (9), swine (10), goats (11), and humans (12). Numerous pharmaceutical and biotechnology companies have developed biologic agents capable of blocking MSTN activity, and these have been tested in clinical trials for a wide range of indications, including Duchenne and facioscapulohumeral muscular dystrophy, inclusion body myositis, muscle atrophy following falls and hip fracture surgery, age-related sarcopenia, Charcot–Marie–Tooth disease, and cachexia due to chronic obstructive pulmonary disease, end-stage kidney disease, and cancer.The finding that certain inhibitors of MSTN signaling can increase muscle mass even in Mstn^−/− mice revealed that the function of MSTN as a negative regulator of muscle mass is partially redundant with at least one other TGF-β family member (13, 14), and subsequent studies have identified activin A as one of these cooperating ligands (15, 16). MSTN and activin A share many key regulatory and signaling components. For example, the activities of both MSTN and activin A can be modulated extracellularly by naturally occurring inhibitory binding proteins, including follistatin (17, 18) and the follistatin-related protein, FSTL-3 or FLRG (19, 20). Moreover, MSTN and activin A also appear to share receptor components. Based on in vitro studies, MSTN is capable of binding initially to the activin type II receptors, ACVR2 and ACVR2B (also called ActRIIA and ActRIIB) (18) followed by engagement of the type I receptors, ALK4 and ALK5 (21). In previous studies, we presented genetic evidence supporting a role for both ACVR2 and ACVR2B in mediating MSTN signaling and regulating muscle mass in vivo. Specifically, we showed that mice expressing a truncated, dominant-negative form of ACVR2B in skeletal muscle (18) or carrying deletion mutations in Acvr2 and/or Acvr2b (13) have significantly increased muscle mass. One limitation of the latter study, however, was that we could not examine the consequence of complete loss of both receptors using the deletion alleles, as double homozygous mutants die early during embryogenesis (22). Moreover, the roles that the two type I receptors, ALK4 and ALK5, play in regulating MSTN and activin A signaling in muscle in vivo have not yet been documented using genetic approaches. Here, we present the results of studies in which we used floxed alleles for each of the type II and type I receptor genes in order to target these receptors alone and in combination in muscle fibers. We show that these receptors are functionally redundant and that signaling through each of these receptors contributes to the overall control of muscle mass.     

Protein kinase Cδ mediates trimethyltin-induced neurotoxicity in mice in vivo via inhibition of glutathione defense mechanism

We investigated whether protein kinase C (PKC) is involved in trimethyltin (TMT)-induced neurotoxicity. TMT treatment (2.8 mg/kg, i.p.) significantly increased PKCδ expression out of PKC isozymes (i.e., α, βI, βII, δ, and ?) in the hippocampus of wild-type (WT) mice. Consistently, treatment with TMT resulted in significant increases in cleaved PKCδ expression. Genetic or pharmacological inhibition (PKCδ knockout or rottlerin) was less susceptible to TMT-induced seizures than WT mice. TMT treatment increased glutathione oxidation, lipid peroxidation, protein oxidation, and levels of reactive oxygen species. These effects were more pronounced in the WT mice than in PKCδ knockout mice. In addition, the ability of TMT to induce nuclear translocation of Nrf2, Nrf2 DNA-binding activity, and upregulation of γ-glutamylcysteine ligase was significantly increased in the PKCδ knockout mice and rottlerin (10 or 20 mg/kg, p.o. × 6)-treated WT mice. Furthermore, neuronal degeneration (as shown by nuclear chromatin clumping and TUNEL staining) in WT mice was most pronounced 2 days after TMT. At the same time, TMT-induced inhibition of phosphoinositol 3-kinase (PI3K)/Akt signaling was evident, thereby decreasing phospho-Bad, expression of Bcl-xL and Bcl-2, and the interaction between phospho-Bad and 14-3-3 protein, and increasing Bax expression and caspase-3 cleavage were observed. Rottlerin or PKCδ knockout significantly protected these changes in anti- and pro-apoptotic factors. Importantly, treatment of the PI3K inhibitor LY294002 (0.8 or 1.6 µg, i.c.v.) 4 h before TMT counteracted protective effects (i.e., Nrf-2-dependent glutathione induction and pro-survival phenomenon) of rottlerin. Therefore, our results suggest that down-regulation of PKCδ and up-regulations of Nrf2-dependent glutathione defense mechanism and PI3K/Akt signaling are critical for attenuating TMT neurotoxicity.     

Diagnostic Accuracy of Cervical Low-Grade Squamous Intraepithelial Lesions Is Improved With MIB-1 Immunostaining.

There is considerable interobserver variation in the diagnosis of low-grade squamous intraepithelial lesion that involves mature squamous epithelium. Our aim was to evaluate the utility of MIB-1 immunostaining as an adjunct test to increase diagnostic accuracy. Consecutive cervical biopsies originally diagnosed as normal (n = 26) or low-grade squamous intraepithelial lesion (n = 23) were reviewed by three pathologists to obtain a consensus diagnosis. MIB-1 immunostaining was performed, and positive staining was defined as a cluster of at least two stained nuclei in the upper two thirds of the epithelial thickness. Human papillomavirus (HPV) DNA detection was performed using a polymerase chain reaction assay. All cases were subsequently reclassified as low-grade squamous intraepithelial lesion (LSIL) or normal (NL) when two or three of three gold standard criteria were satisfied (LSIL gold standard criteria = consensus diagnosis of LSIL, HPV+, MIB-1+; NL gold standard criteria = consensus diagnosis of NL, HPV-, MIB-1-). Using the gold standard diagnoses, we have identified that 14 normal cases (36%) were originally overdiagnosed as LSIL, and one LSIL case (10%) was originally underdiagnosed as normal. All MIB-1-positive cases were HPV+ and identified as LSIL in the consensus review. All MIB-1-negative cases were NL by gold standard criteria. The sensitivity (1.0) and the specificity (1.0) of MIB-1 staining for identifying LSIL were superior to the sensitivity (0.9) and the specificity (0.8) of HPV testing. In conclusion, MIB-1 is a highly sensitive and specific marker for identifying low-grade squamous intraepithelial lesion and is helpful in verifying the diagnosis of equivocal cases.     

Ontogeny of serum insulin-like growth factor binding proteins in the rat

Insulin-like growth factors (IGF-I and -II) are peptide growth factors that may be important for neonatal development. Specific high affinity IGF binding proteins (BPs) have been characterized in serum and extracellular fluids. The major serum binding complex in the adult has an apparent Mr of 150 K, while the predominant BP in the neonate is approximately 30 K. In the rat, the transition from the neonatal BP to the adult form occurs during the third postnatal week, concomitant with an increase in serum IGF-I and a decrease in serum IGF-II concentrations. Using specific RIAs and Western ligand blot analyses we have characterized the changes in serum IGF and IGF BPs, respectively, during the early postnatal period. Seven BPs were identified in serum with apparent Mr values of 42, 41, 40, 38, 28, 26, and 22 K. After deglycosylation, the 42, 41, 40, and 38 K BPs were reduced to two bands with apparent Mr values of 35 and 32 K, while the 28, 26, and 22 K BP were unchanged. In the neonate, the 28, 26, and 22 K BPs were present, with the 28 K BP in highest concentration. With increasing age, the 28 K BP decreased and the 42, 41, 40, and 38 K BPs appeared at approximately 19 days of age. Comparison of Western ligand blots of neonatal serum, BRL-3A conditioned media, rat amniotic fluid, and rat cerebrospinal fluid (CSF) demonstrated that all contained a prominent 28 K BP. A polyclonal antibody (alpha Hec 1) developed against the 31 K human IGF-BP (hBP-31) immunoprecipitated the 28 K BP from neonatal rat serum, BRL-3A media, rat amniotic fluid, and rat CSF, but did not react with adult rat serum. These findings suggest that, in the rat, the predominant neonatal serum BP is structurally and immunologically similar to the major BRL-3A, amniotic fluid, and CSF BPs, but distinct from the predominant adult serum BP.     

Mechanical Testing of Seven Fixation Methods for Generation of Compression Across a Midtarsal Osteotomy: A Comparison of Internal and External Fixation Devices

The purpose of this study was to assess 7 methods of fixation for a midtarsal osteotomy. Polyurethane foam models (N = 6) and cadaver specimens (N = 4-7) were used to examine the force generated by the different constructs of fixation. A midtarsal osteotomy was performed on each specimen in the test groups. The osteotomies were fixated either with 2 parallel 0.062-in Kirschner wires and 40-mm-long, 4-mm partially threaded, cancellous, cannulated titanium screws, an external ring fixator (frame), a frame with wires tensioned (tension), a frame with wires tensioned and compressed toward the osteotomy (tension and compression), a frame with tension, compression, and parallel Kirschner wires, or a frame with tension, compression, and two 4.0 cannulated parallel screws, respectively. Each model was fixated, and the force generated by the construct across the osteotomy was recorded via the use of pressure-sensitive film. Statistical analysis of the data in the polyurethane foam group determined that the use of frame with tension, compression, and two 4.0 parallel cannulated screws was statistically superior to 1) frame, 2) frame with tension, 3) 2 parallel Kirschner wires, 4) two 4.0 cannulated parallel screws, and 5) frame with tension and compression. A cadaver study determined that the frame with tension, compression, and 2 parallel Kirschner wires was statistically superior to 1) frame and 2) two parallel Kirschner wires. These findings suggest that there is a difference in the force generated by the type of fixation construct across a midtarsal osteotomy.