Lung cancer is a malignant tumor with high mortality in both women and men. To study the mechanisms of smoking-induced lung cancer, we analyzed microarray of GSE4115. GSE4115 was downloaded from Gene Expression Omnibus including 78 and 85 bronchial epithelium tissue samples separately from smokers with and without lung cancer. Limma package in R was used to screen differentially expressed genes (DEGs). Hierarchical cluster analysis for DEGs was conducted using orange software and visualized by distance map. Using DAVID software, functional and pathway enrichment analyses separately were conducted for the DEGs. And protein-protein interaction (PPI) network was constructed using Cytoscape software. Then, the pathscores of enriched pathways were calculated. Besides, functional features were screened and optimized using the recursive feature elimination (RFE) method. Additionally, the support vector machine (SVM) method was used to train model. Total 1923 DEGs were identified between the two groups. Hierarchical cluster analysis indicated that there were differences in gene level between the two groups. And SVM analysis indicated that the five features had potential diagnostic value. Importantly, MAPK1 (degree=30), SRC (degree=29), SMAD4 (degree=23), EEF1A1 (degree=21), TRAF2 (degree=21) and PLCG1 (degree=20) had higher degrees in the PPI network of the DEGs. They might be involved in smoking-induced lung cancer by interacting with each other (e.g. MAPK1-SMAD4, SMAD4-EEF1A1 and SRC-PLCG1). MAPK1, SRC, SMAD4, EEF1A1, TRAF2 and PLCG1 might be responsible for the development of smoking-induced lung cancer.

High blood sugar is a symptom of diabetes mellitus (DM). Vascular endothelial cells (VECs) directly contact the blood and are damaged when blood sugar levels are high. However, the molecular mechanism underlying this process remains elusive. To analyze the effects of DM on migration, we simulated DM by applying high glucose (HG) to the human VEC. HG delayed cell migration and induced phosphorylation of MAPKs (JNK and ERK). By contrast, in presence of bFGF, cell migration was promoted and MAPK phosphorylation levels were reduced. Furthermore, treatment with JNK and ERK inhibitors rescued HG-mediated delay of cell migration. Molecular and cell biological studies demonstrated that HG increased ROS production, whereas treatment with bFGF or JNK/ERK inhibitors blocked HG-induced ROS accumulation. Addition of MnTMPyP, a ROS scavenger, reduced HG-induced ROS production and accelerated cell migration, suggesting that the influence of HG on bFGF-MAPK signaling causes accumulation of ROS, which in turn regulate cell migration. This is the first study to elucidate the molecular mechanism of HG-mediated VEC migration; these findings could facilitate the development of novel therapies for DM.

Endocytosis of G-protein-coupled receptors (GPCRs) and associated channels contributes to desensitization and adaptation of a variety of signaling cascades. In Drosophila melanogaster, the main light-sensing rhodopsin (Rh1; encoded by ninaE) and the downstream ion channel, transient receptor potential like (TRPL), are endocytosed in response to light, but the mechanism is unclear. By using an RNA-Sequencing (RNA-Seq) approach, we discovered a protein we named CULD, a photoreceptor-cell enriched CUB- and LDLa-domain transmembrane protein, that is required for endocytic trafficking of Rh1 and TRPL. CULD localized to endocytic Rh1-positive or TRPL-positive vesicles. Mutations in culd resulted in the accumulation of Rh1 and TRPL within endocytic vesicles, and disrupted the regular turnover of endocytic Rh1 and TRPL. In addition, loss of CULD induced light- and age-dependent retinal degeneration, and reduced levels of Rh1, but not of TRPL, suppressed retinal degeneration in culd-null mutant flies. Our data demonstrate that CULD plays an important role in the endocytic turnover of Rh1 and TRPL, and suggest that CULD-dependent rhodopsin endocytic trafficking is required for maintaining photoreceptor integrity.

Osteocytes comprising over 90% of the bone cell population are highly susceptible to the adverse effects of glucocorticoids (GC) administration. Here we observed that Dexamethasone (Dex) induces a robust cytoskeleton rearrangement and decreases Cx43 protein expression in osteocyte-like MLO-Y4 cells. Using a Dmp1Cre-mT/mG osteocyte ex vivo culture system, we found significant shortening of dendritic processes in primary osteocytes following Dex administration. Loss of dendritic processes is a consequence of reduced Cx43 connectivity upon Dex induced autophagy in both RFP-GFP-LC3B transfected MLO-Y4 cells and primary calvarial osteocytes from LC3GFP transgenic mice. Upon the induction of autophagy by Dex, Cx43 was internalized into autophagosome/autolysosomes and degraded by autophagy. The degradation was attenuated following lysosomal inhibition using chloroquine (CLQ) and suppression of autophagy by Atg5 silencing. Inhibition Akt-mTORC1 signaling by Dex induces autophagy subsequently resulting in Cx43 degradation.Activation of Akt phosphorylation by IGF-1 attenuated Dex induced autophagy and degradation of Cx43. Together, we demonstrated that GC impair osteocyte cell-cell connectivity via autophagy mediated degradation of Cx43 through inhibition of the Akt-mTORC1 signaling. This may account for the deleterious effect of GC-induced bone loss.

Immune checkpoint blockade of the inhibitory immune receptors PD-L1, PD-1 and CTLA-4 has emerged as a successful treatment strategy for several advanced cancers. Here we demonstrate that miR-424(322) regulates the PD-L1/PD-1 and CD80/CTLA-4 pathways in chemoresistant ovarian cancer. miR-424(322) is inversely correlated with PD-L1, PD-1, CD80 and CTLA-4 expression. High levels of miR-424(322) in the tumours are positively correlated with the progression-free survival of ovarian cancer patients. Mechanistic investigations demonstrated that miR-424(322) inhibited PD-L1 and CD80 expression through direct binding to the 3'-untranslated region. Restoration of miR-424(322) expression reverses chemoresistance, which is accompanied by blockage of the PD-L1 immune checkpoint. The synergistic effect of chemotherapy and immunotherapy is associated with the proliferation of functional cytotoxic CD8+ T cells and the inhibition of myeloid-derived suppressive cells and regulatory T cells. Collectively, our data suggest a biological and functional interaction between PD-L1 and chemoresistance through the microRNA regulatory cascade.

Copy number variants (CNVs) at the Breakpoint 1 to Breakpoint 2 region at 15q11.2 (BP1-2) are associated with language-related difficulties and increased risk for developmental disorders in which language is compromised. Towards underlying mechanisms, we investigated relationships between single nucleotide polymorphisms (SNPs) across the region and quantitative measures of human brain structure obtained by magnetic resonance imaging of healthy subjects. We report an association between rs4778298, a common variant at CYFIP1, and inter-individual variation in surface area across the left supramarginal gyrus (lh.SMG), a cortical structure implicated in speech and language in independent discovery (n = 100) and validation cohorts (n = 2621). In silico analyses determined that this same variant, and others nearby, is also associated with differences in levels of CYFIP1 mRNA in human brain. One of these nearby polymorphisms is predicted to disrupt a consensus binding site for FOXP2, a transcription factor implicated in speech and language. Consistent with a model where FOXP2 regulates CYFIP1 levels and in turn influences lh.SMG surface area, analysis of publically available expression data identified a relationship between expression of FOXP2 and CYFIP1 mRNA in human brain. We propose that altered CYFIP1 dosage, through aberrant patterning of the lh.SMG, may contribute to language-related difficulties associated with BP1-2 CNVs. More generally, this approach may be useful in clarifying the contribution of individual genes at CNV risk loci.

Rheumatoid arthritis (RA) affects millions world-wide. While anti-TNF treatment is widely used to reduce disease progression, treatment fails in ∼one-third of patients. No biomarker currently exists that identifies non-responders before treatment. A rigorous community-based assessment of the utility of SNP data for predicting anti-TNF treatment efficacy in RA patients was performed in the context of a DREAM Challenge (http://www.synapse.org/RA_Challenge). An open challenge framework enabled the comparative evaluation of predictions developed by 73 research groups using the most comprehensive available data and covering a wide range of state-of-the-art modelling methodologies. Despite a significant genetic heritability estimate of treatment non-response trait (h(2)=0.18, P value=0.02), no significant genetic contribution to prediction accuracy is observed. Results formally confirm the expectations of the rheumatology community that SNP information does not significantly improve predictive performance relative to standard clinical traits, thereby justifying a refocusing of future efforts on collection of other data.

Invasive progression is the major lethal cause of prostate cancer. In this study, we aimed to investigate the role of kindlin-2, an integrin-binding focal adhesion protein, in the regulation of invasiveness of prostate cancer. We found that downregulation of kindlin-2 using small interfering RNA (siRNA) technology significantly inhibited the invasion of PC-3 and DU-145 prostate cancer cells in a Matrigel Transwell assay. Conversely, overexpression of kindlin-2 promoted the invasiveness of prostate cancer cells. Kindlin-2 overexpression was found to activate nuclear factor (NF)-κB-dependent signaling and upregulate the expression of matrix metalloproteinase-9 (MMP-9) and MMP-2, whereas kindlin-2 silencing led to opposing effects on the expression of NF-κB and MMPs. Most importantly, kindlin-2-induced invasiveness was almost completely abolished by pretreatment with pyrrolidine dithiocarbamate (an inhibitor of NF-κB signaling) or co-transfection with MMP-9 or MMP-2 siRNA. Taken together, our data indicate that kindlin-2 promotes the invasiveness of prostate cancer cells largely through NF-κB-dependent upregulation of MMP-9 and MMP-2. Further studies are warranted to evaluate the significance of kindlin-2 as a therapeutic target for metastatic prostate cancer.

Detection and quantification of cell viability and growth in two-dimensional (2D) and three-dimensional (3D) cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up of several replicates for time-lapse or dose-response studies. Thus, developing a non-invasive and touch-free detection of cell growth in longitudinal studies of 3D tumor spheroid cultures or of stem cell regeneration remains a major unmet need. Since surface acoustic waves (SAWs) permit mass loading-based biosensing and have been touted due to their many advantages including low cost, small size and ease of assembly, we examined the potential of SAW-biosensing to detect and quantify cell growth. Herein, we demonstrate that a shear horizontal-surface acoustic waves (SH-SAW) device comprising two pairs of resonators consisting of interdigital transducers and reflecting fingers can be used to quantify mass loading by the cells in suspension as well as within a 3D cell culture platform. A 3D COMSOL model was built to simulate the mass loading response of increasing concentrations of cells in suspension in the polydimethylsiloxane (PDMS) well in order to predict the characteristics and optimize the design of the SH-SAW biosensor. The simulated relative frequency shift from the two oscillatory circuit systems (one of which functions as control) were found to be concordant to experimental data generated with RAW264.7 macrophage and A549 cancer cells. In addition, results showed that SAW measurements per se did not affect viability of cells. Further, SH-SAW biosensing was applied to A549 cells cultured on a 3D electrospun nanofiber scaffold that generate tumor spheroids (tumoroids) and the results showed the device's ability to detect changes in tumor spheroid growth over the course of eight days. Taken together, these results demonstrate the use of SH-SAW device for detection and quantification of cell growth changes over time in 2D suspension cultures and in 3D cell culture models, which may have potential applications in both longitudinal 3D cell cultures in cancer biology and in regenerative medicine.

Osteoporosis is a debilitating skeletal disorder with an increased risk of low-energy fracture, which commonly occurs among postmenopausal women. Andrographolide (AP), a natural product isolated from Andrographis paniculata, has been found to have anti-inflammatory, anti-cancer, anti-asthmatic, and neuro-protective properties. However, its therapeutic effect on osteoporosis is unknown. In this study, an ovariectomy (OVX) mouse model was used to evaluate the therapeutic effects of AP on post-menopausal osteoporosis by using micro-computed tomography (micro-CT). Bone marrow-derived osteoclast culture was used to examine the inhibitory effect of AP on osteoclastogenesis. Real time PCR was employed to examine the effect of AP on the expression of osteoclast marker genes. The activities of transcriptional factors NF-κB and NFATc1 were evaluated using a luciferase reporter assay, and the IκBα protein level was analyzed by Western blot. We found that OVX mice treated with AP have greater bone volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) compared to vehicle-treated OVX mice. AP inhibited RANKL-induced osteoclastogenesis, the expression of osteoclast marker genes including cathepsin K (Ctsk), TRACP (Acp5), and NFATc1, as well as the transcriptional activities of NF-κB and NFATc1. In conclusion, our results suggest that AP inhibits estrogen deficiency-induced bone loss in mice via the suppression of RANKL-induced osteoclastogensis and NF-κB and NFATc1 activities and, thus, might have therapeutic potential for osteoporosis.

Stem cell transplantation and low-energy shock-wave therapy (LESWT) have emerged as potential and effective treatment protocols for diabetic erectile dysfunction. During the tracking of transplanted stem cells in diabetic erectile dysfunction models, the number of visible stem cells was rather low and decreased quickly. LESWT could recruit endogenous stem cells to the cavernous body and improve the microenvironment in diabetic cavernous tissue. Thus, we deduced that LESWT might benefit transplanted stem cell survival and improve the effects of stem cell transplantation. In this research, 42 streptozotocin-induced diabetic rats were randomized into four groups: the diabetic group (n = 6), the LESWT group (n = 6), the bone marrow-derived mesenchymal stem cell (BMSC) transplantation group (n = 15), and the combination of LESWT and BMSC transplantation group (n = 15). One and three days after BMSC transplantation, three rats were randomly chosen to observe the survival numbers of BMSCs in the cavernous body. Four weeks after BMSC transplantation, the following parameters were assessed: the surviving number of transplanted BMSCs in the cavernous tissue, erectile function, real-time polymerase chain reaction, and penile immunohistochemical assessment. Our research found that LESWT favored the survival of transplanted BMSCs in the cavernous body, which might be related to increased stromal cell-derived factor-1 expression and the enhancement of angiogenesis in the diabetic cavernous tissue. The combination of LESWT and BMSC transplantation could improve the erectile function of diabetic erectile function rats more effectively than LESWT or BMSC transplantation performed alone.

Epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors (TKIs) have emerged as first-line drugs for non-small cell lung cancers (NSCLCs). However, the resistance to TKIs represents the key limitation for their therapeutic efficacy. We found that miR-26a was upregulated in gefitinib-refractory NSCLCs; miR-26a is downstream of EGFR signaling and directly targets and silences protein tyrosine phosphatase non-receptor type 13 (PTPN13) to maintain the activation of Src, a dephosphorylation substrate of PTPN13, thus reinforcing EGFR pathway in a regulatory circuit. miR-26a inhibition significantly improved NSCLC responses to gefitinib. These data revealed a novel mechanism of NSCLC resistance to TKI treatment.

Growing evidence indicates that the deregulation of microRNAs (miRNAs) contributes to the tumorigenesis. We previously revealed that microRNA-520b (miR-520b) was involved in the complement attack and migration of breast cancer cells. In this report, we show that miR-520b is an important miRNA in the development of hepatocellular carcinoma (HCC). Our data showed that the expression levels of miR-520b were significantly reduced in clinical HCC tissues and hepatoma cell lines. We observed that the introduction of miR-520b dramatically suppressed the growth of hepatoma cells by colony formation assays, 5-ethynyl-2-deoxyuridine (EdU) incorporation assays and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Moreover, ectopic expression of miR-520b was able to inhibit the growth of hepatoma cells in nude mice. Further studies revealed that the mitogen-activated protein kinase kinase kinase 2 (MEKK2) and cyclin D1 were two of direct target genes of miR-520b. Silencing of MEKK2 or cyclin D1 was able to inhibit the growth of hepatoma cells in vitro and in vivo, which is consistent with the effect of miR-520b overexpression on the growth of hepatoma cells. In addition, miR-520b significantly decreased the phosphorylation levels of c-Jun N-terminal kinase (p-JNK, a downstream effector of MEKK2) or retinoblastoma (p-Rb, a downstream effector of cyclin D1). In conclusion, miR-520b is able to inhibit the growth of hepatoma cells by targeting MEKK2 or cyclin D1 in vitro and in vivo. Our findings provide new insights into the role of miR-520b in the development of HCC, and implicate the potential application of miR-520b in cancer therapy.

APSL (active peptide from shark liver) is a hepatic stimulator cytokine from the liver of Chiloscyllium. It can effectively protect islet cells and improve complications in mice with alloxan-induced diabetes. Here, we demonstrate that the APSL sequence is present in the N-terminus of novel TBC (Tre-2, Bub2 and Cdc16) domain family, member 15 (TBC1D15) from Chiloscyllium plagiosum. This shark TBC1D15 gene, which contains an ORF of 2088 bp, was identified from a cDNA library of regenerating shark liver. Bioinformatic analysis showed that the gene is highly homologous to TBC1D15 genes from other species. Moreover, the N-terminus of shark TBC1D15 contains a motif of unknown function (DUF3548), which encompasses the APSL fragment. Rab-GAP activity analysis showed that shark TBC1D15 is a new member of the TBC1D15 family. These results demonstrated that shark TBC1D15 possesses Rab-GAP activity using Rab7 as a substrate, which is a common property of the TBC1D15 family. The involvement of APSL at the N-terminus of TBC1D15 also demonstrates that this protein might be involved in insulin signaling and may be associated with the development of type 2 diabetes. The current findings pave the way for further functional and clinical studies of these proteins from marine sources.

B cell-mediated antibody response plays critical roles in protective immunity, as well as in the pathogenesis of allergic and autoimmune diseases. Epigenetic histone and DNA modifications regulate gene transcription and immunity; however, so far, little is known about the role of epigenetic regulation in antibody responses. In this study, we found that mice deficient in the histone H2A deubiquitinase MYSM1, despite their severe defect in B cell development, exhibit an enhanced antibody response against both T cell-dependent and independent antigens. We revealed that MYSM1 intrinsically represses plasma cell differentiation and antibody production. Mechanistic studies demonstrated that MYSM1 is a transcriptional activator of Pax5, the repressors of plasma cell differentiation, by facilitating key transcriptional factor recruitment and coordinating histone modifications at the Pax5 loci. Hence, this study uncovers a critical role for MYSM1 in epigenetically repressing plasma cell differentiation and antibody production, in addition to its opposing, active role in B cell development. Importantly, this study further provides a new target and strategy to modulate antibody production and responses with profound therapeutic implications.

Koumiss is a traditionally fermented mare's milk described with health-promoting potentials for decades. However, only a few studies focused on the probiotic strains isolated from koumiss. In this study, we collected koumiss samples from Inner Mongolian pasturing area of China and selected a promising strain of Lactobacillus helveticus, isolate NS8, based on the survival abilities in gastrointestinal tract (GIT) and adhesion to intestinal endothelial cells in vitro. As the ability to positively modulate host immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain, our study mainly focus on the immunomodulatory properties of L. helveticus NS8 by using in vivo and ex vivo analyses.

RNA sequencing (RNA-Seq) is a powerful tool for analyzing the identity of cellular RNAs but is often limited by the amount of material available for analysis. In spite of extensive efforts employing existing protocols, we observed that it was not possible to obtain useful sequencing libraries from nuclear RNA derived from cultured human cells after crosslinking and immunoprecipitation (CLIP). Here, we report a method for obtaining strand-specific small RNA libraries for RNA sequencing that requires picograms of RNA. We employ an intramolecular circularization step that increases the efficiency of library preparation and avoids the need for intermolecular ligations of adaptor sequences. Other key features include random priming for full-length cDNA synthesis and gel-free library purification. Using our method, we generated CLIP-Seq libraries from nuclear RNA that had been UV-crosslinked and immunoprecipitated with anti-Argonaute 2 (Ago2) antibody. Computational protocols were developed to enable analysis of raw sequencing data and we observe substantial differences between recognition by Ago2 of RNA species in the nucleus relative to the cytoplasm. This RNA self-circularization approach to RNA sequencing (RC-Seq) allows data to be obtained using small amounts of input RNA that cannot be sequenced by standard methods.

Although the medial subdivision of the central nucleus of the amygdala (CeM) and serotonin-1A (5-HT1A) receptors are involved in the regulation of anxiety, their roles in Parkinson's disease (PD)-associated anxiety are still unknown. Here we assessed the importance of CeM 5-HT1A receptors for anxiety in rats with unilateral 6-hydroxydopamine (6-OHDA) lesion of the medial forebrain bundle (MFB). The lesion induced anxiety-like behaviors, increased the firing rate and burst-firing pattern of CeM γ-aminobutyric acid (GABA) neurons, as well as decreased dopamine (DA) levels in the striatum, medial prefrontal cortex (mPFC), amygdala and ventral part of hippocampus (vHip). Intra-CeM injection of the selective 5-HT1A receptor agonist 8-OH-DPAT produced anxiolytic effects in the lesioned rats, and decreased the firing rate of CeM GABAergic neurons in two groups of rats. Compared to sham-operated rats, the duration of the inhibitory effect on the firing rate of GABAergic neurons was shortened in the lesioned rats. The injection increased DA levels in the mPFC and amygdala in two groups of rats and the vHip in the lesioned rats, and increased 5-HT level in the lesioned rats, whereas it decreased NA levels in the mPFC in two groups of rats and the vHip in the lesioned rats. Moreover, the mean density of 5-HT1A receptor and GABA double-labeled neurons in the CeM was reduced after the lesioning. These results suggest that activation of CeM 5-HT1A receptor produces anxiolytic effects in the 6-OHDA-lesioned rats, which involves decreased firing rate of the GABAergic neurons, and changed monoamine levels in the limbic and limbic-related brain regions.

Although microRNA-1 (miR-1) is a known liver cancer suppressor, the role of miR-1 in apoptosis of hepatoma cells has remained largely unknown. Our study shows that ectopic miR-1 overexpression induced apoptosis of liver hepatocellular carcinoma (HepG2) cells. Apoptosis inhibitor 5 (API-5) was found to be a potential regulator of miR-1 induced apoptosis, using a bioinformatics approach. Furthermore, an inverse relationship between miR-1 and API-5 expression was observed in human liver cancer tissues and adjacent normal liver tissues. Negative regulation of API-5 expression by miR-1 was demonstrated to promote apoptosis of HepG2 cells. Our study provides a novel regulatory mechanism of miR-1 in the apoptosis of hepatoma cells.

The widely recognized neuroprotective effect of iron chelators is contributed by their ability to prevent reactive oxygen species (ROS) generation via the Fenton reaction, which sequesters redox-active Fe. An additional neuroprotective mechanism of iron-chelating compounds is to regulate the transcriptional activator hypoxia-inducible factor 1α (HIF-1α). In the present study, we observed that intranasal administration of deferoxamine decreased beta-amyloid (Aβ) deposition and rescued synapse loss in the brain of Aβ precursor protein and presenilin-1 (APP/PS1) double transgenic mice. We found that deferoxamine (DFO) up-regulated HIF-1α mRNA expression and its protein level, and further induced the proteins that are encoded from HIF-1-adaptive genes, including transferrin receptor (TFR), divalent metal transporter 1 (DMT1), and brain-derived neurotrophic factor (BDNF). The effects of DFO on the induction and stabilization of HIF-1α were further confirmed in vitro. This was accompanied by a decrease of Fe in the CA3 region of the hippocampus. Western blotting studies revealed that DFO differentially enhanced the phosphorylation of mitogen-activated protein kinase (MAPK)/P38 kinase in vitro and in vivo. The results suggest that the DFO may up-regulate several HIF-1-dependent neuroprotective-adaptive genes in AD via activating P38/HIF-1α pathway, which may serve as important therapeutic targets to the disease.