[Methylation of p16 gene and reduced expression of p16 protein in insulinoma associated with clinicopathological features].

Objective: To study the alterations of p16 gene and its expression in insulinoma and to correlate the findings with clinicopathological characteristics. Methods: Expression of p16 protein was detected in 72 insulinomas and 49 para-tumoral or normal pancreatic tissues by immunohistochemical staining. Genomic DNA was isolated from 32 tumor tissue and 17 paired pancreatic tissues and bisulfite-modified. Promoter methylation status of p16 gene was detected in 32 tumor tissue and 17 paired pancreatic tissues by methylation specific PCR. The findings were correlated with the clinicopathological features. Results: There were 30 males and 42 females in all 72 patients, aged (46.5±14.0) years. Loss or reduced expression of p16 protein was found in 42 of 72 insulinomas (58.3%) while loss or reduced expression of p16 was seen in only 34.7% (17/49) of para-tumoral or normal pancreatic tissues (χ²=6.52, P=0.011). Promoter methylation of p16 gene was found in 13 of 32 insulinomas (40.6%) and only 2 of 17 (11.8%) para-tumoral tissues (χ²=4.35, P=0.037). The expression of p16 protein in insulinoma was not associated with clinicopathological features such as gender, age, tumor size and tumor grade. Conclusions: Loss or reduced expression of p16 protein was found in insulinomas, and associated with p16 gene promoter methylation.

[Application of posterior gastric mesentery in laparoscopic gastric surgery].

The posterior gastric mesentery is one of the six mesenteries of the stomach in the membrane anatomy theory. It locates in the upper area of the pancreas, surrounds the posterior gastric vessels, and is adjacent to the short gastric mesentery by the left side, and is adjacent to the left gastric mesentery by the right side, which fixes the fundus body to the posterior abdominal wall of the upper area of pancreas. Due to its anatomical structure, in complete mesentery excision (CME)+D2 surgery, it is a surgical approach to deal with gastric mesentery in the upper area of pancreas; the second step of the "Huang's three-step method" corresponds to the posterior gastric mesentery in the theory of membrane anatomy. In the surgery of benign diseases of the stomach, laparoscopic sleeve gastrectomy (LSG) and laparoscopic Nissen fundoplication, if the short gastric vessels are difficult to be exposed and safely divided, we can dissect the posterior gastric mesentery firstly, and then hoist the fundus of the stomach in order to help dissection of the short gastric vessels. The membrane anatomy theory, as a frontier theory, provides us the new surgical perspectives and paths in gastric surgery.

ST8SIA1 inhibition sensitizes triple negative breast cancer to chemotherapy via suppressing Wnt/ß-catenin and FAK/Akt/mTOR.

BACKGROUND: Chemoresistance is the major cause of therapeutic failure in triple negative breast cancer (TNBC). In this work, we investigated the molecular mechanism for the development of TNBC chemoresistance. METHODS: mRNA and protein levels of ST8SIA1 were analyzed in chemosensitive and chemoresistant TNBC cells and tissues. Proliferation and survival assays were performed to determine the role of ST8SIA1 in TNBC chemoresistance. RESULTS: We found that ST8SIA1 mRNA and protein levels were increased in multiple TNBC cell lines after prolonged exposure to chemotherapeutic drugs. Consistently, retrospective study demonstrated that the majority of TNBC patients who developed chemoresistance displayed upregulation of ST8SIA1. We further found that chemoresistant TNBC cells were more sensitive than chemosensitive cells to ST8SIA1 inhibition in decreasing growth and viability. Consistently, ST8SIA1 inhibition augmented the efficacy of chemotherapy in TNBC cells. Mechanism studies demonstrated that ST8SIA1 inhibition led to suppression of FAK/Akt/mTOR and Wnt/ß-catenin signalling pathways. CONCLUSIONS: These findings provide an explanation for the heterogeneity of chemotherapy responses across TNBC individuals and reveal the supportive roles of ST8SIA1in TNBC chemoresistance.

Identification and characterisation of a novel FT orthologous gene in London plane with a distinct expression response to environmental stimuli compared to PaFT.

FLOWERING LOCUS T (FT) is a key integrator of environmental signals and internal cues, and codes for florigen-like activity which regulates the transition from vegetative to reproductive growth in flowering plants. Unlike annual plants, perennial tree species undergo several years of vegetative growth prior to the transition to the reproductive stage, as characterised by the ability to form flower buds. Thereafter, trees in temperate regions typically display an annual growth cycle involving distinct vegetative growth, flowering and dormancy stages. In London plane (Platanus acerifolia Willd.), a FT-like gene has previously been identified. Here, we report the isolation of a novel FT orthologous gene, PaFTL, and investigate the functions of PaFT and PaFTL through the analysis of expression profiles and transgenic phenotypes. PaFT displayed the highest levels of expression during tree dormancy, and similarly elevated expression levels were seen under conditions of low temperature and short days (LT/SD). In contrast, PaFTL transcripts were up-regulated during the floral transition phase, the early stages of inflorescence development and throughout the main flowering period, whereas expression levels were low and variable during dormancy and in response to LT/SD treatments. Ectopic expression of 35s::PaFTL in tobacco produced a phenotype similar to that with PaFT, namely, advanced floral initiation. Overall, the results suggest that PaFT and PaFTL have both conserved and diverse functions in floral initiation, floral development and dormancy regulation.

Oxcarbazepine causes neurocyte apoptosis and developing brain damage by triggering Bax/Bcl-2 signaling pathway mediated caspase 3 activation in neonatal rats.

OBJECTIVE: Anti-epileptic drugs (AEDs) are the main methods for treatment of neonatal seizures; however, a few AEDs may cause developing brain damage of neonate. This study aims to investigate effects of oxcarbazepine (OXC) on developing brain damage of neonatal rats. MATERIALS AND METHODS: Both of neonatal and adult rats were divided into 6 groups, including Control, OXC 187.5 mg/kg, OXC 281.25 mg/kg, OXC 375 mg/kg group, LEV and PHT group. Body weight and brain weight were evaluated. Hematoxylin and eosin (HE) and Nissl staining were used to observe neurocyte morphology and Nissl bodies, respectively. Apoptosis was examined using TUNEL assay, and caspase 8 activity was evaluated using spectrophotometer method. Cytochrome C-release was evaluated using flow cytometry. Western blot was used to examine Bax and Bcl-2 expression. RESULTS: OXC 375 mg/kg treatment significantly decreased brain weight compared to Control group in neonatal rats (P5 rats) (p<0.05). OXC administration causes histological changes of neurocytes. OXC 281.25 mg/kg or more concentration significantly decreased neurocytes counts and increased TUNEL-staining positive neurocytes compared to Control group (p<0.05). OXC 281.25 mg/kg and OXC 375 mg/kg significantly increased caspase 3 activity compared to Control group in P5 rats (p<0.05). OXC 281.25 mg/kg and OXC 375 mg/kg significantly increased Bax, Bax/Bcl-2 ratio and cytochrome C release in frontal lobes compared to Control group in P5 rats (p<0.05). CONCLUSIONS: Oxcarbazepine at a concentration of 281.25 mg/kg or more causes neurocyte apoptosis and developing brain damage by triggering Bax/Bcl-2 signaling pathway mediated caspase 3 activation in neonatal rats.

[Clinical observation of visual quality after the individual implantation of intraocular lens guided by corneal Q value].

Objective: To describe the feasibility of individual IOL implantation guided by corneal Q value and observe patients postoperative visual quality under different residual spherical aberration. Methods: Prospective study. One hundred and twenty cases (171 eyes)cataract patients in our hospital were selected for the individual implantation of intraocular lens guided by corneal Q value which obtained by Oberscan before operation. Based on spherical aberration calculated by corneal Q value, choose appropriate IOL personalitily to make postoperative whole eye surface aberration +0.1 µm (group of positive spherical aberration) or 0 µm (group of zero spherical aberration). To observe spherical aberration, the uncorrected visual acuity, best corrected visual acuity and contrast sensitivity (including no glare and glare) 1 month and 3 months after surgery. Dates were analyzed with one-way ANOVA and LSD method for multiple comparisons between groups. Results: Spherical aberration after operation: group of positive spherical aberration: (0.111±0.023)µm, group of zero spherical aberration: (0.020±0.019)µm, control group: (0.299±0.073)µm. At 1 months and 3 months, uncorrected visual acuity, and corrected visual acuity were not statistically different between groups (t=0.474, 1.607, P>0.05). Contrast sensitivity (including no glare and glare) 3 months after surgery display: at whole space frequency, the group of positive spherical aberration(reserved +0.1 µm spherical aberration) contrast sensitivity is better than that of the group of zero spherical aberration(reserved 0.0 µm spherical aberration) and the control group(F=32.885, 35.493, 19.969, 20.572,P<0.05). The group of zero spherical aberration is better than control group at space frequency of 3 and 6 c/d(F=6.506, 7.521, P<0.05). Conclusions: The individual implantation of introcular lens guided by corneal Q value is feasible. + 0.1 µm spherical aberration after surgery can achieve the best contrast sensitivity and stereo vision, and 0 spherical aberration after surgery can improve the postoperative contrast sensitivity and stereo vision than a traditional method, but its advantage mainly embodies in the middle and lower spatial frequency. (Chin J Ophthalmol, 2017, 53: 814-820).

Aging and age related stresses: a senescence mechanism of intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration is a complicated process that involves both age-related change and tissue damage caused by multiple stresses. In a degenerative IVD, cellular senescence accumulates and is associated with reduced proliferation, compromised self-repair, increased inflammatory response, and enhanced catabolic metabolism. In this review, we decipher the senescence mechanism of IVD degeneration (IVDD) by interpreting how aging coordinates with age-related, microenvironment-derived stresses in promoting disc cell senescence and accelerating IVDD. After chronic and prolonged replication, cell senescence may occur as a natural part of the disc aging process, but can potentially be accelerated by growth factor deficiency, oxidative accumulation, and inflammatory irritation. While acute disc injury, excessive mechanical overloading, diabetes, and chronic tobacco smoking contribute to the amplification of senescence-inducing stresses, the avascular nature of IVD impairs the immune-clearance of the senescent disc cells, which accumulate in cell clusters, demonstrate inflammatory and catabolic phenotypes, deteriorate disc microenvironment, and accelerate IVDD. Anti-senescence strategies, including telomerase transduction, supply of growth factors, and blocking cell cycle inhibitors, have been shown to be feasible in rescuing disc cells from early senescence, but their efficiency for disc regeneration requires more in vivo validations. Guidelines dedicated to avoiding or alleviating senescence-inducing stresses might decelerate cellular senescence and benefit patients with IVD degenerative diseases.

Brief Exercises Affect Gene Expression in Circulating Monocytes.

We aimed to give a systematic hypothesis on the functions of exercise on circulating monocytes by identifying a discrete set of genes in circulating monocytes that were altered by exercise. The microarray expression profile of GSE51835 was downloaded from gene expression omnibus (GEO) database for the identification of differentially expressed genes (DEGs) using limma and affy packages in R language. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for DEGs, followed by the construction of co-expression network and protein-protein interaction (PPI) network. The top 10 nodes in PPI network were screened, and subnetwork was constructed for the key genes identification. Totally, 35 DEGs, including 2 upregulated genes and 33 downregulated genes, were identified. The enriched GO terms were mainly linked to immune response and defence response, and the enriched KEGG pathways were mainly associated with natural killer cell-mediated cytotoxicity and graft-versus-host disease. Dual-specificity phosphatase 2 (DUSP2) was identified as a key node in the co-expression network. In the PPI network, CD247 module (CD247), chemokine (C-X-C motif) receptor 4 (CXCR4), granzyme B (GZMB) and perforin 1 (PRF1) were identified as key nodes. An important interaction, GZMB/PRF1, was detected. Five key genes, including DUSP2, CD247, CXCR4, GZMB and PRF1, and an interaction of GZMB/PRF1, were significant factors in the immune processes of circulating monocytes, which might be regulated by brief exercises, leading to the enhancement of immune function.

Long interspersed nucleotide acid element-1 ORF-1 protein promotes proliferation and invasion of human colorectal cancer LoVo cells through enhancing ETS-1 activity.

The human proto-oncogene long interspersed nucleotide acid element-1 (LINE-1) open reading frame-1 protein (ORF-1p) is involved in the progress of several cancers. The transcription factor ETS-1 can mediate the transcription of some downstream genes that play specific roles in the regulation of cancerous cell invasion and metastasis. In this study, the effects of LINE-1 ORF-1p on ETS-1 activity and on the proliferation and invasion of human colorectal cancer LoVo cells were investigated. Results showed that the overexpression of LINE-1 ORF-1p enhanced the transcription of ETS-1 downstream genes and increased their protein levels, and downregulation of the LINE-1 ORF-1p level by small interfering RNA (siRNA) reduced the transcriptional activation of ETS-1. In addition, overexpression of LINE-1 ORF-1p promoted LoVo cell proliferation and anchor-independent growth, and a knockdown of the LINE-1 protein level by siRNA reduced the proliferation and anchor-independent growth ability of LoVo cells. In vivo data revealed that LINE-1 ORF-1p overexpression increased LoVo tumor growth in nude mice, whereas the siRNA knockdown of endogenous LINE-1 ORF-1p expression decreased LoVo cell growth in nude mice. Therefore, LINE- 1 ORF-1p could promote LoVo cell proliferation and invasion both in vitro and in vivo, indicating that it might be a useful molecular target for the treatment of human colorectal cancer.

Protective effect of Puerarin on ß-amyloid-induced neurotoxicity in rat hippocampal neurons.

Puerarin (CAS Number 3681-99-0), a major isoflavone glycoside purified from Pueraria lobata, was reported to possess antioxidative and estrogen-like biological activities. Recent studies showed that puerarin protects different cell types from damage caused by a variety of toxic stimuli. In the present study, we investigated the neuroprotective effect of puerarin against Aß25-35-induced neurotoxicity in cultured hippocampal neurons, as well as the underlying mechanism(s). Following exposure of cells to Aß25-35, cell survival and glutathione peroxidase (GSH-Px) and catalase (CAT) activities were reduced while production of reactive oxygen species (ROS) was increased. Preincubation of the cells with puerarin prior to Aß25-35 exposure increased cell survival and GSH-Px and CAT activities and decreased ROS production. It was previously shown that overactivation of glycogen synthase kinase-3ß (GSK-3ß) is implicated in Aß-induced cell death. In this study, Aß25-35 treatment is found to increase GSK-3ß activity and pretreatment with puerarin preventesAß-induced activation of GSK-3ß based on Western blot analysis. In addition, puerarin is shown to activate protein kinase B (PKB)/Akt, an important upstream kinase of GSK-3ß, possibly promoting subsequent GSK-3ß inhibition. Our data suggest that puerarin attenuates cell death induced by Aß25-35 via various mechanisms, which might be beneficial for the treatment of Alzheimer's disease.

The physiology functions of estrogen receptor α (ERα) in reproduction cycle of ovoviviparous black rockfish, Sebastes schlegeli Hilgendorf.

This paper revealed the expression pattern of ERα in the ovoviviparous teleost, Sebastes schlegeli. In this paper, we isolated the cDNA encoding for estrogen receptor alpha of black rockfish (S. schlegeli) from its ovary, named as black rockfish ERα (brfERα). The cDNA sequence of brfERα consists of 2972bp with an open reading frame encoding a 624 amino acid putative protein which exhibits high identities with other teleosts'. The tissue distribution of brfERα mRNA was examined using RT-PCR. BrfERα showed generally expressions in most tissues of female black rockfish, besides, the higher degree of expressions were seen in ovary, liver, duodenum and fat, whereas it had a more restricted distribution in male fish. In ovary, the expression level of brfERα was as similar as the serum levels of E2 and P in female. However, it was a different situation in male, where the serum concentration of E2 showed higher levels after spermiation and Serum concentration of P did not show any significant changes during a year. Based on the present study, it is supposed that brfERα plays an important role in ovary and other target organs during the reproductive cycle, Further studies will focus on the transcriptional regulation and localization of brfERα in gonad in order to get a better understand of the physiological function of brfERα in ovoviviparous teleost. This study indicates that the black rockfish may be a good candidate for understanding the mechanism of estrogen in ovoviviparous fish.

Cloning and expression of P450c17-I (17α-hydroxylase/17,20-lyase) in brain and ovary during gonad development in Cynoglossus semilaevis.

Cytochrome P450c17 (CYP17, 17a-hydroxylase/17,20-lyase) is a critical enzyme in the production of androgens and estrogens in vertebrates. A 2,469 bp full length cDNA of P450c17-I (CYP17A1) has been isolated from the ovary of half-smooth tongue sole, Cynoglossus semilaevis which encodes 509 amino acids. Additionally, a relatively shorter cDNA (1,742 bp), a likely result of polyadenylation, was also found. The putative P450c17-I enzyme shares high sequence identity with that of the fathead minnow (73%), zebrafish (71%), the Japanese eel (70%), catfish (70%), tilapia (79%), three-spined stickleback (81%), medaka (79%), dogfish (60%), chicken (65%), rat (47%), and human (49%). Semi-quantitative RT-PCR analysis of spatial expression showed the enzyme was predominantly expressed in the ovaries and the brain. P450c17-I was also detected in the stomach, intestine, gill, spleen, kidney, and head kidney, albeit weakly. Further examination of temporal expression pattern of P450c17-I in ovary and brain revealed developmental stage-dependency. In addition to this our data on T and E2 levels further endorse the critical role of P450c17-I during shift in steroidogenesis. Based on the present study we indicate an important role for P450c17-I during ovarian development. However, further studies are needed at transcriptional regulation level for deeper insights into the physiological functions of P450c17-I.

Pesticide multiresidue analysis of peanuts using automated gel permeation chromatography clean-up/gas chromatography-mass spectrometry.

A method based on automated gel permeation chromatography (GPC) and on-line quantitative concentration was developed for the determination of residues of 38 typically used pesticides in high-oil peanuts. Pesticides were extracted using acetonitrile into an oil fraction containing little of the peanut matrix. After changing the solvent to the mobile phase (1 : 1 cyclohexane : ethyl acetate), clean-up was carried out using GPC, the final collected solution being automatically concentrated to a fixed volume and transferred into vials for gas chromatography (GC) injection. The pesticides were analysed by gas chromatography-mass spectrometry (GC/MS) in the selected ion monitoring (SIM) mode. Average recoveries (spiked levels of 0.02, 0.05, and 0.1 microg g(-1)) were between 70 and 117%. The relative standard deviation of the method was 3.5-21% (n = 6). This approach gave comparable results with a previously published method.

Association of reproductive performance with SNPs of FOXL2 gene by SSCP in Japanese flounder (Paralichthys olivaceus).

FOXL2 which is a putative winged helix/forkhead transcription factor gene and a sexually dimorphic marker of ovarian differentiation plays an important role in ovarian development, granulosa cell differentiation, and thus the proper maintenance of ovarian function. The aims of this study were to characterize polymorphisms within the FOXL2 gene in a population of 52 female Japanese flounder and analyze the association of FOXL2 polymorphisms with reproductive performance by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP). Results indicated that five single nucleotide polymorphisms (SNPs), which were SNP1 [c.540A>C (p.Asn102His) and c.591A>G (p.Asn119Asp)], SNP2 [c.864G>A (p.Lys210Glu)and c.875G>A] and SNP3 (c.1169C>A), were identified in the FOXL2 gene. General Linear Model (GLM) analysis showed that SNP1 in the forkhead domain was significantly associated with gonadosomatic index (GSI) (P<0.05). SNP2 in the downstream of forkhead domain was significantly associated with serum 17beta-estradiol (E(2)) level (P<0.05). And SNP3 in the 3'-UTR was significantly associated with hepatosomatic index (HSI) (P<0.05). Moreover, the evaluation of the genetic effects for both Testosterone (T) level of diplotype D3 and GSI of diplotype D5 suggested they were significantly higher than those of other four diplotypes (P<0.05), respectively. These results implied that these SNPs could influence reproductive endocrinology of female Japanese flounder and be also used in marker-assisted selection (MAS) program to reproductive performance in female Japanese flounder in the future.

Hypothalamic preproghrelin gene expression is repressed by insulin via both PI3-K/Akt and ERK1/2 MAPK pathways in immortalized, hypothalamic neurons.

The gut peptide ghrelin is expressed within neurons of the hypothalamus. Using a hypothalamic cell line, mHypoE-38 neurons, the effect of insulin on preproghrelin gene expression was assayed. These cells contain neuron-specific markers, preproghrelin and the insulin receptor. We determined that insulin has direct effects on preproghrelin gene expression. Insulin (10 nM) stimulated protein kinase B (Akt) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation from 5 to 60 min and 5 min, respectively, and led to repression of preproghrelin gene expression at 2 h. Pharmacological inhibitors to phosphoinositide-3-kinase (PI3-K; LY294002) and MEK (PD98059) demonstrated that basal ghrelin gene expression is regulated by the PI3-K pathway and requires the mitogen-activated protein kinase pathway for insulin-stimulated preproghrelin repression. These results demonstrate that insulin has a direct effect on hypothalamic neurons to decrease preproghrelin gene expression through classic insulin pathways.

Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies.

Mouse PNAS-4 (mPNAS-4) has 96% identity with human PNAS-4 (hPNAS-4) in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data), it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2) and colon carcinoma cells (CT26) of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3). The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4) was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37 degrees C, while it was almost exclusively expressed in soluble form at 20 degrees C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28%. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.

Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies

Mouse PNAS-4 (mPNAS-4) has 96 percent identity with human PNAS-4 (hPNAS-4) in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data), it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2) and colon carcinoma cells (CT26) of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3). The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4) was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37°C, while it was almost exclusively expressed in soluble form at 20°C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28 percent. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.