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The present study aimed to elucidate the genetic features of multiple lung cancer (MLC) and identify effective molecular markers for diagnosis using next generation sequencing (NGS). The present data may also inform patient treatment and prognosis. A total of 35 lesions were obtained from 17 patients with MLC. Based on lesion histology and NGS, 13 cases of multiple primary lung cancer (MPLC) were identified and 4 cases were classified as intrapulmonary metastasis (IPM). All 4 patients with IPM exhibited an epidermal growth factor receptor (EGFR) mutation and synchronous mutation of at least one tumor suppressor gene. The frequency and percentage of EGFR mutations, accompanied with tumor suppressor genes, were significantly higher in patients with IPM compared with MPLC. Furthermore, a high EGFR-heterogeneity score and male sex were risk factors of IPM occurrence. There were significant differences in mean EGFR mutation abundance alone, mutations of tumor suppressor genes and mutations of EGFR combined with tumor suppressor genes between patients with adenocarcinoma (ADC) and adenocarcinoma in situ (AIS). In conclusion, histological characteristics combined with genetic alterations may be an effective method for the diagnosis of MPLC and IPM, and NGS may serve as a useful diagnostic tool. MLC exhibited unique molecular characteristics, including higher rates of EGFR mutations, EGFR driver mutations accompanied with tumor suppressor gene mutations and the absence of anaplastic lymphoma kinase mutations, which may help distinguish between patients with MPLC or IPM. The present study hypothesized that the mean frequency of EGFR mutations, mutations of tumor suppressor genes and mutations of both EGFR and tumor suppressor genes may serve an important role in the development of AIS to ADC. The results of the present study highlight the potential underlying mechanisms of lung ADC development, which may assist with future elucidation of effective treatments to prevent the progression of lung cancer.

Glucoside xylosyltransferase2 (GXYLT2), a member of the human α-1,3- D -xylosyltransferases, functions to modify the first xylose to the O-Glucose residue on epidermal growth factor (EGF) repeats of Notch receptors. It is well-established that the Notch signaling pathway plays a critical role in proper development and homeostasis. However, the regulatory role of EGF xylosylation in Notch signaling and different cell activities in human cells remains unknown. In this study, we showed that knockdown of GXYLT2 suppressed human cell proliferation and induced G1/S phase cell cycle arrest. GXYLT2 downregulation also inhibited cell migration and invasion, whereas the overexpression of GXYLT2 had the opposite effects. Additionally, GXYLT2 activated Notch signaling and promoted the phosphorylation of MAPKs but not PI3K and Akt. Taken together, our findings indicated that GXYLT2 plays an important role in cell activities via regulation of the Notch signaling.

The aim of the current study was to investigate the effect of adipocytes on the differentiation of osteoblasts at different stages of adipocyte development.BMSCs were isolated from 4-week-old male wistar rat femurs and tibias, and flow cytometry was performed. Adipocytes were derived from BMSCs, cell morphology was continually observed from day 21 to day 50. Adipocyte medium was collected once every 2days (d) and ELISA kits were used for detection of triglycerides (TG), tumor necrosis factor-α (TNF-α), and interleukin-6(IL-6) expression level. 21d and 40d old adipocyte and osteoblast cells were co-cultured, and alizarin red staining was performed after 21d. After co-culture, the adherent cells were collected, and the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) was detected by real time PCR.Results of cell characterisation showed that the cells had positive expression of CD29 (97.92%) and CD44 (89.32%). With the increase of the induction time of mature adipocytes, the number of adipocyte on 21thd was significantly higher than 40thd, while the volume of adipocyte was significantly lower than 40thd (P0.05). The levels of TG(2.6±0.83mmol/l VS 3.8±0.66mmol/l), TNF-α(30.5±2.53pg/ml VS 57.6±5.1pg/ml), and IL-6(32.5±1.42pg/ml VS 55.1±5.97pg/ml) secreted by adipocytes increased with induction time: 40thd was significantly higher than 21thd (P0.01). When 21thd adipocytes and osteoblasts were co-cultured, the number of calcium nodules significantly increased over that of the positive control group, When 40thd adipocytes and osteoblasts were co-cultured, the number of calcium nodules significantly decreased over that of the positive control group (P0.05). The OPG(68.9±5.39 VS 1.00±0.36) expression was significantly increased, and the expression of RANKL (2.0±0.84 VS 34.4±2.01) was significantly decreased from the 21thd adipocytes co-cultured group compared with the 40thd adipocytes co-cultured group (P0.001).The differential size of adipocytes in the bone marrow can affect bone metabolism by regulating the expression of OPG/RANKL.

The aim of the current study was to investigate the effects of obesity, induced via a high-fat diet, on bone metabolism in rats.Two hundred healthy Wistar male rats aged 4 weeks were fed a standard diet and a high-fat diet. At specific time points (week 0, 4, 6, 8, and 10), plasma was collected to determine the levels of glucose and lipid metabolism. Additionally, enzyme-linked immunoassays were performed to determine the plasma levels of adipocyte and bone metabolism factors. Micro-CT imaging was used to determine the parameters of bone metabolism. At 10th week, immunohistochemistry evaluation of femoral bone samples was performed to determine the expression of adipocyte factors.Receptor activator of nuclear factor kB ligand (RANKL) was positively correlated with levels of triglyceride (TG), free fatty acids (FFA), and tumor necrosis factor alpha (TNF-α) (P0.05), while receptor activator of the NF-κB (RANK) showed a positive correlation with TG, FFA, TNF-α and leptin (LPT) (P0.05). CT imaging demonstrated that bone mineral density and trabecular thickness were elevated compared to controls before 6 weeks, but these values were found to be lower in rats fed a high fat diet in the following weeks (P0.05). Immunohistochemistry showed that the expression of TNF-α, Interleukin- 6 (IL-6) and peroxisome proliferator activated receptor-γ (PPAR-γ) were increased and the expression of adiponectin (APN) were diminished in rats fed a high-fat diet compared to controls at 10 weeks (P0.05).With obesity intensifies, the release of FFA cause inflammation factor increase, resulting in bone parameters decreased.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates collagen-mediated platelet activation through its cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). However, the function of CEACAM1’s extracellular cleavage fragments is currently unknown. In the present study, we used mass spectrometry (MS) to identify 9 cleavage fragments shed by matrix metallopeptidase 12 (MMP-12), and then we synthesized peptides with sequences corresponding to the fragments. QLSNGNRTLT (QLSN), a peptide from the A1-domain of CEACAM1, significantly attenuated collagen-induced platelet aggregation. QLSN also attenuated platelet static adhesion to collagen. Additionally, QLSN reduced human platelet secretion and integrin αIIbβ3 activation in response to glycoprotein VI (GPVI)–selective agonist, convulxin. Correspondingly, QLSN treatment significantly decreased convulxin-mediated phosphorylation of Src, protein kinase B (Akt), spleen tyrosine kinase (Syk) and phospholipase Cγ2 (PLCγ2) in human platelets. These data indicate that the CEACAM1-derived peptide QLSN inhibits GPVI-mediated human platelet activation. QLSN could potentially be developed as a novel antiplatelet agent. QLSN inhibit collagen-induced human platelet activation through glycoprotein VI.

Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.

Objective Recent studies have revealed a link between toll-like receptors (TLRs), Kruppel-like factors (KLFs), and the adipose tissue inflammation associated with obesity. TLR4 is associated with chronic inflammation in obesity. KLF7 is known to play an important role in the differentiation of adipocytes, but its role in visceral adipose tissue inflammation has not yet been investigated. Thus, the objective of this study was to determine the correlation of TLR4 and KLF7 in inflammation induced by obesity. Methods A total of 32 Wistar male rat subjects were fed in the center for experimental animals of Shihezi University. The rats were divided into normal control (NC) and high-fat diet (HFD) group. Surgical instruments were used to collect rats’ visceral adipose tissue samples in the 10th week after HFD feeding. Ninety-five Uygur subjects between 20 and 90 years old were enrolled in the present study. The subjects were divided into two groups: the normal control group (NC, 18.0 kg/m2 ≤ BMI ≤ 23.9 kg/m2, n = 50) and the obesity group (OB, BMI ≥ 28 kg/m2, n = 45), and visceral adipose tissue was collected from the subjects. Anthropometric and clinical parameters were measured using standard procedures; biochemical indices were detected using the glucose oxidase-peroxidase method and a standardized automatic biochemistry analyzer; the plasma levels of inflammatory factors and adipocytokines were measured by enzyme-linked immunosorbent assay (ELISA); the mRNA and protein expression levels of key genes involved in the inflammatory signaling pathway were measured by real-time PCR and Western blot. Results In rats, compared with the NC group, the weight, Lee’s index, waist circumference, visceral fat mass, and the plasma level of Glu, TG, FFA, and TNF-α were higher in the HFD group, while the plasma levels of LPT and APN were significantly lower in the HFD group in the 10th week. Furthermore, compared with the NC group, visceral adipose tissue’s mRNA expression levels of TLR4, KLF7, and SRC were higher in the HFD group, and KLF7 was significantly positively correlated with LDL, TLR4, SRC, and IL-6 (P

Guillain–Barre syndrome (GBS) is an autoimmune-mediated disease triggered by a preceding infection. A substantial body of evidence implicates antibodies to various gangliosides in subtypes of GBS. A significant proportion of patients with acute demyelinating subset of GBS have IgG antibodies against peripheral nervous system myelin specific neolactogangliosides such as LM1 and Hex-LM1. Although anti-neolactoganglioside antibodies in GBS were described more than two decades ago, their pathogenic role in neuropathy remains unknown due to the lack of suitable experimental models. In this study, we immunized ten guinea pigs with purified LM1 ganglioside mixed with keyhole limpet hemocyanin (KLH) and emulsified in complete Freund's adjuvant (CFA). Control guinea pigs were injected with KLH emulsified in CFA only. The animals were bled every four week intervals. The animals were boosted 3 times every four weeks. Experiments were terminated four months after initial immunization. Nine of 10 guinea pigs immunized with LM1 exhibited antibody responses to LM1. Anti-LM1 IgG titers in nine guinea pigs ranged from 1:400 to 1:12,800 at 16-weeks after initial immunization. Anti-LM1 antibodies were predominantly of IgG2 subclass. One guinea pig with the highest levels of IgG antibodies exhibited mild signs of neuropathy. There was no evidence of demyelination or inflammation in the sciatic nerves of LM1-immunized guinea pigs. Anti-LM1 antibodies bound to rat sciatic nerve myelin and to isolated rat Schwann cells. In summary, our findings suggest that relatively high levels of anti-LM1 IgG antibodies can be induced in guinea pigs and that LM1 is localized in peripheral nerve myelin and in Schwann cells. Further studies are needed to determine the pathogenic potential of anti-neolactoganglioside antibodies in neuropathy.

Adenylate kinases participate in maintaining the homeostasis of cellular nucleotides. Depending on the yeast strains, the GTP:AMP phosphotransferase is encoded by the nuclear gene ADK2 with or without a single base pair deletion/insertion near the 3′ end of the open reading frame, and the corresponding protein exists as either Adk2p (short) or Adk2p (long) in the mitochondrial matrix. These two forms are identical except that the three C-terminal residues of Adk2p (short) are changed in Adk2p (long), and the latter contains an additional nine amino acids at the C terminus of the protein. The short form of Adk2p has so far been considered to be inactive (Schricker, R., Magdolen, V., Strobel, G., Bogengruber, E., Breitenbach, M., and Bandlow, W. (1995) J. Biol. Chem. 270, 31103–31110). Using purified proteins, we show that at the physiological temperature for yeast growth (30 °C), both short and long forms of Adk2p are enzymatically active. However, in contrast to the short form, Adk2p (long) is quite resistant to thermal inactivation, urea denaturation, and degradation by trypsin. Unfolding of the long form by high concentrations of urea greatly stimulated its import into isolated mitochondria. Using an integration-based gene-swapping approach, we found that regardless of the yeast strains used, the steady state levels of endogenous Adk2p (long) in mitochondria were 5–10-fold lower compared with those of Adk2p (short). Together, these results suggest that the modified C-terminal domain in Adk2p (long) is not essential for enzyme activity, but it contributes to and strengthens protein folding and/or stability and is particularly important for maintaining enzyme activity under stress conditions.

Iron-sulfur (Fe-S) cluster biogenesis in mitochondria is an essential process and is conserved from yeast to humans. Several proteins with Fe-S cluster cofactors reside in mitochondria, including aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. We found that mitochondria isolated from wild-type yeast contain a pool of apoaconitase and machinery capable of forming new clusters and inserting them into this endogenous apoprotein pool. These observations allowed us to develop assays to assess the role of nucleotides (GTP and ATP) in cluster biogenesis in mitochondria. We show that Fe-S cluster biogenesis in isolated mitochondria is enhanced by the addition of GTP and ATP. Hydrolysis of both GTP and ATP is necessary, and the addition of ATP cannot circumvent processes that require GTP hydrolysis. Both in vivo and in vitro experiments suggest that GTP must enter into the matrix to exert its effects on cluster biogenesis. Upon import into isolated mitochondria, purified apoferredoxin can also be used as a substrate by the Fe-S cluster machinery in a GTP-dependent manner. GTP is likely required for a common step involved in the cluster biogenesis of aconitase and ferredoxin. To our knowledge this is the first report demonstrating a role of GTP in mitochondrial Fe-S cluster biogenesis.

IgM paraproteins in about 50% of the patients with neuropathy associated with IgM gammopathy react with carbohydrate moieties in myelin-associated glycoprotein (MAG) and in sulfated glucuronic glycolipids (SGGLs) in human peripheral nerves. However, the role of anti-MAG/SGGL antibodies in the pathogenesis of neuropathy remains unclear. In order to induce an animal model of neuropathy associated with anti-MAG/SGGL antibodies, cats were immunized with sulfoglucuronyl paragloboside (SGPG). All four cats immunized with SGPG developed clinical signs of sensory neuronopathy within 11 months after initial immunization, characterized by unsteadiness, falling, hind limb weakness and ataxia. In two cats the ataxia and hind limb paralysis were so severe that the animals had to be euthanized. Pathological examination revealed sensory ganglionitis with inflammatory infiltrates in the dorsal root ganglia. No overt signs of pathology were noted in the examined roots or nerves. High titer anti-SGPG/MAG antibodies were detected in all 4 cats immunized with SGPG but not in 3 control cats. Our data demonstrate that immunization of cats with SGPG induced anti-SGPG antibodies and sensory neuronopathy clinically resembling the sensory ataxia of patients with monoclonal IgM anti-MAG/SGPG antibodies. This study suggests that these anti-MAG/SGPG antibodies play a role in the pathogenesis of this neuropathy.

A 38-year-old man developed the Guillain–Barre´ syndrome (GBS) associated with untreated end-stage AIDS and CD4+ lymphocyte count of 3 cells/mm3. The patient had serum high titer anti-sulfatide antibodies and responded well to infusion of immunoglobulin. The data suggest that elevated levels of anti-sulfatide antibodies may play a role in the pathogenesis of GBS in this patient, although a direct neurotropic effect of HIV virus cannot be excluded.

In Saccharomyces cerevisiae, two mitochondrial inner-membrane proteins play critical roles in organellar morphology. One is a dynamin-related GTPase, Mgm1p, which participates in mitochondrial fusion. Another is Tim11p, which is required for oligomeric assembly of F1Fo-ATP synthase, which generates ATP through oxidative phosphorylation. Our data bring these findings together and define a novel role for Mgm1p in the formation and maintenance of mitochondrial cristae. We show that Mgm1p serves as an upstream regulator of Tim11p protein stability, ATP synthase assembly, cristae morphology and cytochrome c storage within cristae.