Sustained exposure to Helicobacter pylori induces immune tolerance by desensitizing TLR6.

Helicobacter pylori (H. pylori, Hp) has been designated a class I carcinogen and is closely associated with severe gastric diseases. During colonization in the gastric mucosa, H. pylori develops immune escape by inducing host immune tolerance. The gastric epithelium acts as the first line of defense against H. pylori, with Toll-like receptors (TLRs) in gastric epithelial cells being sensitive to H. pylori components and subsequently activating the innate immune system. However, the mechanism of immune tolerance induced by H. pylori through the TLR signalling pathway has not been fully elucidated. In this research, we detected the expression of TLRs and inflammatory cytokines in GES-1 cells upon sustained exposure to H. pylori or H. pylori lysate from 1 to 30 generations and in Mongolian gerbils infected with H. pylori for 5 to 90 weeks. We found that the levels of TLR6 and inflammatory cytokines first increased and then dropped during the course of H. pylori treatment in vitro and in vivo. The restoration of TLR6 potentiated the expression of IL-1ß and IL-8 in GES-1 cells, which recruited neutrophils and reduced the colonization of H. pylori in the gastric mucosa of gerbils. Mechanistically, we found that persistent infection with H. pylori reduces the sensitivity of TLR6 to bacterial components and regulates the expression of inflammatory cytokines in GES-1 cells through TLR6/JNK signaling. The TLR6 agonist obviously alleviated inflammation in vitro and in vivo. Promising results suggest that TLR6 may be a potential candidate immunotherapy drug for H. pylori infection.

Development of a new cerebral ischemia reperfusion model of Mongolian gerbils and standardized evaluation system.

BACKGROUND: The Mongolian gerbil is an excellent laboratory animal for preparing the cerebral ischemia model due to its inherent deficiency in the circle of Willis. However, the low incidence and unpredictability of symptoms are caused by numerous complex variant types of the circle. Additionally, the lack of an evaluation system for the cerebral ischemia/reperfusion (I/R) model of gerbils has shackled the application of this model. METHODS: We created a symptom-oriented principle and detailed neurobehavioral scoring criteria. At different time points of reperfusion, we analyzed the alteration in locomotion by rotarod test and grip force score, infarct volume by triphenyltetrazolium chloride (TTC) staining, neuron loss using Nissl staining, and histological characteristics using hematoxylin-eosin (H&E) straining. RESULTS: With a successful model rate of 56%, 32 of the 57 gerbils operated by our method harbored typical features of cerebral I/R injury, and the mortality rate in the male gerbils was significantly higher than that in the female gerbils. The successfully prepared I/R gerbils demonstrated a significant reduction in motility and grip strength at 1 day after reperfusion; formed obvious infarction; exhibited typical pathological features, such as tissue edema, neuronal atrophy and death, and vacuolated structures; and were partially recovered with the extension of reperfusion time. CONCLUSION: This study developed a new method for the unilateral common carotid artery ligation I/R model of gerbil and established a standardized evaluation system for this model, which could provide a new cerebral I/R model of gerbils with more practical applications.

ELAVL4 promotes the tumorigenesis of small cell lung cancer by stabilizing LncRNA LYPLAL1-DT and enhancing profilin 2 activation.

Small cell lung cancer (SCLC) is one of the most malignant tumors that has an extremely poor prognosis. RNA-binding protein (RBP) and long noncoding RNA (lncRNA) have been shown to be key regulators during tumorigenesis as well as lung tumor progression. However, the role of RBP ELAVL4 and lncRNA LYPLAL1-DT in SCLC remains unclear. In this study, we verified that lncRNA LYPLAL1-DT acts as an SCLC oncogenic lncRNA and was confirmed in vitro and in vivo. Mechanistically, LYPLAL1-DT negatively regulates the expression of miR-204-5p, leading to the upregulation of PFN2, thus, promoting SCLC cell proliferation, migration, and invasion. ELAVL4 has been shown to enhance the stability of LYPLAL1-DT and PFN2 mRNA. Our study reveals a regulatory pathway, where ELAVL4 stabilizes PFN2 and LYPLAL1-DT with the latter further increasing PFN2 expression by blocking the action of miR-204-5p. Upregulated PFN2 ultimately promotes tumorigenesis and invasion in SCLC. These findings provide novel prognostic indicators as well as promising new therapeutic targets for SCLC.

LncRNA LYPLAL1-DT screening from type 2 diabetes with macrovascular complication contributes protective effects on human umbilical vein endothelial cells via regulating the miR-204-5p/SIRT1 axis.

Long noncoding RNAs (lncRNAs) are involved in diabetes related diseases. However, the role of lncRNAs in the pathogenesis of type 2 diabetes with macrovascular complication (DMC) has seldomly been recognized. This study screened lncRNA profiles of leukocytes from DMC patients and explored protective role of lncRNA LYPLAL1-DT in endothelial cells (EC) under high glucose (HG) and inflammatory conditions (IS). Between DMC and healthy controls, 477 differential expression lncRNAs (DE-lncRNAs) were identified. The enrichment and pathway analysis showed that most of the DE-lncRNAs belonged to inflammatory, metabolic, and vascular diseases. A total of 12 lncRNAs was validated as significant DE-lncRNAs in expanding cohorts. Furthermore, these DE-lncRNAs were shown to be significantly related to hypoxia, HG, and IS in EC, especially lncRNA LYPLAL1-DT. LYPLAL1-DT overexpression results in the promotion of the proliferation, and migration of EC, as well as an elevation of autophagy. Overexpressed LYPLAL1-DT reduces the adhesion of monocytes to EC, boosts anti-inflammation, and suppresses inflammatory molecules secreted in the medium. Mechanistically, LYPLAL1-DT acts as competing endogenous RNA (ceRNA) by downregulating miR-204-5p, therefore enhancing SIRT1 and protecting EC autophagy function; thus, alleviating apoptosis. Finally, exosome sequencing revealed LYPLAL1-DT expression was 4 times lower in DMC cells than in healthy samples. In general, we identified LYPLAL1-DT having protective effects on EC as ceRNA mediated through the miR-204-5p/SIRT1 pathway. Therefore, it inhibits the autophagy of EC as well as modulating systemic inflammation. This approach could be regarded as a new potential therapeutic target in DMC.

FAK PROTAC Inhibits Ovarian Tumor Growth and Metastasis by Disrupting Kinase Dependent and Independent Pathways.

Focal adhesion kinase (FAK) is highly expressed in a variety of human cancers and is a target for cancer therapy. Since FAK kinase inhibitors only block the kinase activity of FAK, they are not highly effective in clinical trials. FAK also functions as a scaffold protein in a kinase-independent pathway. To effectively target FAK, it is required to block both FAK kinase-dependent and FAK-independent pathways. Thus, we tested a new generation drug FAK PROTAC for ovarian cancer therapy, which blocks both kinase and scaffold activity. We tested the efficacy of FAK PROTAC and its parent kinase inhibitor (VS-6063) in ovarian cancer cell lines in vitro by performing cell functional assays including cell proliferation, migration, invasion. We also tested in vivo activity in orthotopic ovarian cancer mouse models. In addition, we assessed whether FAK PROTAC disrupts kinase-dependent and kinase-independent pathways. We demonstrated that FAK PROTAC is highly effective as compared to its parent FAK kinase inhibitor VS-6063 in inhibiting cell proliferation, survival, migration, and invasion. FAK PROTAC not only inhibits the FAK kinase activity but also FAK scaffold function by disrupting the interaction between FAK and its interaction protein ASAP1. We further showed that FAK PROTAC effectively inhibits ovarian tumor growth and metastasis. Taken together, FAK PROTAC inhibits both FAK kinase activity and its scaffold protein activity by disrupting the interaction between FAK and ASAP1 and is highly effective in inhibiting ovarian tumor growth and metastasis.

Establishment of Noninvasive Methods for the Detection of Helicobacter pylori in Mongolian Gerbils and Application of Main Laboratory Gerbil Populations in China.

Identifying Helicobacter pylori (H. pylori, Hp) infection in animals before and after artificial infection influences the subsequent experiment. We established effective and noninvasive detection methods, including the gastric fluid nested polymerase chain reaction (PCR) method and the 13C-urea breath test, which can detect Hp before modeling Hp infection in Mongolian gerbils. We designed a gas collection equipment for gerbils. Hp nested PCR was also performed on gastric fluid, gastric mucosa, duodenal contents, and faeces of gerbils challenged with Hp. Conventional Hp detection methods, including rapid urease assay and immunohistochemistry, were compared. Moreover, we assessed the natural infection of Hp in 135 gerbils that had never been exposed to Hp artificially from the major laboratory gerbil groups in China. In 10 Hp infected gerbils, the positive detection results were 100%, 100%, 90%, and 10% in gastric fluid, gastric mucosa, duodenal contents, and faeces with nested PCR, respectively. A rapid urease test performed on gastric mucosa showed that all animals were infected with Hp. Immunohistochemical detection and bacteria culture of gastric mucosa samples that were positive by the nested PCR method also confirmed the presence of Hp. 9% (3/35) and 6% (2/31) natural infection rates were found in conventional gerbil groups from the Capital Medical University and Zhejiang Laboratory Animal Center. In conclusion, we established two noninvasive Hp detection methods that can be performed before modelingHp infection, including the gastric fluid nested PCR method and the 13C-urea breath test.

Profilin 2 and Endothelial Exosomal Profilin 2 Promote Angiogenesis and Myocardial Infarction Repair in Mice.

Cardiovascular diseases (CVD) are the leading cause of death worldwide, wherein myocardial infarction (MI) is the most dangerous one. Promoting angiogenesis is a prospective strategy to alleviate MI. Our previous study indicated that profilin 2 (PFN2) may be a novel target associated with angiogenesis. Further results showed higher levels of serum PFN2 and exosomal PFN2 in patients, mice, and pigs with MI. In this study, we explored whether PFN2 and endothelial cell (EC)-derived exosomal PFN2 could increase angiogenesis and be beneficial for the treatment of MI. Serum PFN2, exosomes, and exosomal PFN2 were elevated in rats with MI. PFN2 and exosomes from PFN2-overexpressing ECs (OE-exo) enhanced EC proliferation, migration, and tube formation ability. OE-exo also significantly increased the vessel number in zebrafish and protected the ECs from inflammatory injury. Moreover, OE-exo-treated mice with MI showed improvement in motor ability, ejection fraction, left ventricular shortening fraction, and left ventricular mass, as well as increased vessel numbers in the MI location, and decreased infarction volume. Mechanistically, PI3K might be the upstream regulator of PFN2, while ERK might be the downstream regulator in the PI3K-PFN2-ERK axis. Taken together, our findings demonstrate that PFN2 and exosomal PFN2 promote EC proliferation, migration, and tube formation through the PI3K-PFN2-ERK axis. Exosomal PFN2 may be a valuable target in the repair of MI injury via angiogenesis.

Characterization and clinical evaluation of microsatellite instability and loss of heterozygosity within tumor-related genes in colorectal cancer.

BACKGROUND: Microsatellite instability (MSI) is a biomarker for better outcomes in colorectal cancer (CRC). However, this conclusion is controversial. In addition, MSs can be a useful marker for loss of heterozygosity (LOH) of genes, but this finding has not been well studied. Here, we aimed to clarify the predictive value of MSI/LOH within tumor-related genes in CRC. METHODS: We detected MSI/LOH of MSs in tumor-related genes and the Bethesda (B5) panel by STR scanning and cloning/sequencing. We further analyzed the relationship between MSI/LOH status and clinical features or outcomes by Pearson's Chi-square test, Fisher's exact test and the Kaplan-Meier method. RESULTS: The findings indicated that the MSI rates of B5 loci were all higher than those of loci in tumor-related genes. Interestingly, MSI/LOH of 2 loci in the B5 panel and 12 loci in tumor-related genes were associated with poorer outcomes, while MSI/LOH of the B5 panel failed to predict outcomes in CRC. MSI of BAT25, MSI/LOH of BAT26 and MSI of the B5 panel showed closer relationships with mucinous carcinoma. In addition, LOH-H of the B5 panel was associated with increased lymphatic metastasis. CONCLUSIONS: In summary, MSI/LOH of certain loci or the whole panel of B5 is related to clinical features, and several loci within tumor-related genes showed prognostic value in the outcomes of CRC.

Characterization and clinical evaluation of microsatellite instability and loss of heterozygosity in tumor-related genes in gastric cancer.

Microsatellite instability (MSI) detection is widely used in the diagnosis and prognosis evaluation of colorectal cancer. However, for gastric cancer (GC), there is no standard panel of microsatellites (MSs) used in clinical guidance. The present study aimed to identify useful predictors of the clinical features and for the prognosis of GC, based on an investigation of MSI and loss of heterozygosity (LOH) in tumor-related genes. First, from 20 tumor-related genes which were proven to be important to the development of GC, 91 MSs were identified, and PCR amplification, short tandem repeat scanning analysis and TA clone sequencing were used to analyze MSI and LOH in the first set of 90 GC samples. Subsequently, the same method was used to detect the MSI/LOH of the optimized loci in the second set of 136 GC samples. MSI/LOH in the mismatch repair genes was highly consistent with that in oncogenes and tumor suppressor genes, respectively. The length of the core sequence was a main factor for the MSI/LOH rate. The MSI of 12 single loci was significantly associated with lymph node metastasis. The MSI in TP53-1 and the LOH in MGMT-10 were significantly associated with early stages of tumor infiltration depth. The LOH in MGMT-10, PTN-2 and MCC-17 was significantly associated with TNM stage. The LOH in TP53-1 and ERBB2-12 was associated with adenocarcinoma. The MSI/LOH in 6 single loci of 5 tumor-related genes was associated with poor prognosis of GC. The present study demonstrated that the MSI/LOH of loci in tumor-associated genes was associated with 4 clinicopathological characteristics and outcomes of GC. These results may provide potential specific biomarkers for the clinical prediction and treatment of GC.

Development of Microsatellite Marker System to Determine the Genetic Diversity of Experimental Chicken, Duck, Goose, and Pigeon Populations.

Poultries including chickens, ducks, geese, and pigeons are widely used in the biological and medical research in many aspects. The genetic quality of experimental poultries directly affects the results of the research. In this study, following electrophoresis analysis and short tandem repeat (STR) scanning, we screened out the microsatellite loci for determining the genetic characteristics of Chinese experimental chickens, ducks, geese, and pigeons. The panels of loci selected in our research provide a good choice for genetic monitoring of the population genetic diversity of Chinese native experimental chickens, ducks, geese, and ducks.

Adipose-Derived Stem Cells Facilitate Ovarian Tumor Growth and Metastasis by Promoting Epithelial to Mesenchymal Transition Through Activating the TGF-ß Pathway.

Adipose-derived stem cells (ADSC) are multipotent mesenchymal stem cells derived from adipose tissues and are capable of differentiating into multiple cell types in the tumor microenvironment (TME). The roles of ADSC in ovarian cancer (OC) metastasis are still not well defined. To understand whether ADSC contributes to ovarian tumor metastasis, we examined epithelial to mesenchymal transition (EMT) markers in OC cells following the treatment of the ADSC-conditioned medium (ADSC-CM). ADSC-CM promotes EMT in OC cells. Functionally, ADSC-CM promotes OC cell proliferation, survival, migration, and invasion. We further demonstrated that ADSC-CM induced EMT via TGF-ß growth factor secretion from ADSC and the ensuing activation of the TGF-ß pathway. ADSC-CM-induced EMT in OC cells was reversible by the TGF-ß inhibitor SB431542 treatment. Using an orthotopic OC mouse model, we also provide the experimental evidence that ADSC contributes to ovarian tumor growth and metastasis by promoting EMT through activating the TGF-ß pathway. Taken together, our data indicate that targeting ADSC using the TGF-ß inhibitor has the therapeutic potential in blocking the EMT and OC metastasis.

Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of triple negative breast cancer cells.

BACKGROUND: Triple negative breast cancer (TNBC) is the most aggressive subtype with the worst prognosis. The role of profilin 2 (PFN2) in TNBC is very controversial. The current study is to explore the role of PFN2 in TNBC. METHODS: PFN2 expression in TNBC and normal breast tissues were evaluated by immunohistochemical analysis. The association between PFN2 expression and prognosis in TNBC patients was analyzed from the TCGA database. A cell counting kit-8 (CCK8) assay was employed to investigate the effects of PFN2 in TNBC cell proliferations. The migration and invasion capability of TNBC cells was evaluated by transwell assays. Western blot was performed to assess the related protein expression of TGF-ß/Smad signaling and epithelial to mesenchymal transition. Finally, TNBC xenografts were established to determine the tumorigenicity in vivo using female Nod/Scid mice. RESULTS: PFN2 is upregulated in TNBC and the higher expression was associated with worse survival. CCK8 assays and Transwell assays demonstrated that PFN2 promoted the proliferation, migration and invasion of TNBC cells. Smad2 and Smad3 were upregulated in PFN2 overexpressing TNBC cells, which further induced the process of epithelial­to­mesenchymal transition. Similarly, the overexpressing PFN2 TNBC cells exhibited stronger tumorigenicity in vivo. CONCLUSIONS: Higher PFN2 expression is associated with a worse 10-year overall survival and relapse-free survival in breast cancer patients, as well as worse 10-year relapse-free survival in TNBC patients. PFN2 promotes the proliferation, migration and invasion of TNBC cells by regulating epithelial-to-mesenchymal transition.

Sustained Exposure to Helicobacter pylori Lysate Inhibits Apoptosis and Autophagy of Gastric Epithelial Cells.

Helicobacter pylori is designated as a class I carcinogen of human gastric cancer following long-term infection. During this process, H. pylori bacteria persist in proliferation and death, and release bacterial components that come into contact with gastric epithelial cells and regulate host cell function. However, the impact of long-term exposure to H. pylori lysate on the pathological changes of gastric cells is not clear. In this study, we aimed to investigate the regulation and mechanisms involved in gastric cell dysfunction following continuous exposure to H. pylori lysate. We co-cultured gastric cell lines GES-1 and MKN-45 with H. pylori lysate for 30 generations, and we found that sustained exposure to H. pylori lysate inhibited GES-1 cell invasion, migration, autophagy, and apoptosis, while it did not inhibit MKN-45 cell invasion or migration. Furthermore, Mongolian gerbils infected with H. pylori ATCC 43504 strains for 90 weeks confirmed the in vitro results. The clinical and in vitro data indicated that sustained exposure to H. pylori lysate inhibited cell apoptosis and autophagy through the Nod1-NF-κB/MAPK-ERK/FOXO4 signaling pathway. In conclusion, sustained exposure to H. pylori lysate promoted proliferation of gastric epithelial cells and inhibited autophagy and apoptosis via Nod1-NF-κB/MAPK-ERK/FOXO4 signaling pathway. In the process of H. pylori-induced gastric lesions, H. pylori lysate plays as an "accomplice" to carcinogenesis.

Profilin 2 promotes growth, metastasis, and angiogenesis of small cell lung cancer through cancer-derived exosomes.

Small cell lung cancer (SCLC) is highly aggressive and prone to hypervascular metastases. Recently, we found profilin 2 (PFN2) expression in SCLC but not in normal tissues. Furthermore, PFN2 expression had been shown to promote angiogenesis through exosomes. However, it remains unclear whether PFN2 contributes to the progression and metastasis of SCLC through angiogenesis. We report here that overexpression (OE) of PFN2 increased, whereas its knockdown (KD) decreased the proliferation, migration, and invasion of SCLC cell H446. The exosomes from OE-H446 (SCLC-OE-exo) exhibited similar effects on H446 properties. Culturing of endothelial cells (ECs) in SCLC-OE conditioned medium (CM) or SCLC-OE-exo increased the migration and tube formation ability of ECs, whereas SCLC-KD-CM and SCLC-KD-exo had inhibitory effects. Interestingly, both SCLC- and EC-derived exosomes were internalized in H446 more rapidly than in ECs. More importantly, OE-PFN2 dramatically elevated SCLC growth and vasculature formation as well as lung metastasis in tumor xenograft models. Finally, we found that PFN2 activated Smad2/3 in H446 and pERK in ECs, respectively. Taken together, our study revealed the role of PFN2 in SCLC development and metastasis, as well as tumor angiogenesis through exosomes, providing a new molecular target for SCLC treatment.

Exploring the mechanism of cisplatin resistance by transcriptome sequencing and reversing the chemoresistance by autophagy inhibition in small cell lung cancer.

Cisplatin plays a key role in treating small cell lung cancer (SCLC); however, the rapid development of cisplatin resistance limits its treatment effect. The detailed mechanisms of cisplatin-resistance, particularly in SCLC, remain unclear. We analyzed the differentially expressed genes (DEGs) between cisplatin-resistant small cell lung cancer cell line H446/CDDP and its parental cell line H446, using the transcriptome sequencing technique. Gene ontology (GO) analysis and the subsequent tests demonstrated that the functions of protein ubiquitination and autophagy are more active in the H446/CDDP cells. Autophagy plays a protective role in the H446/CDDP cells by using the autophagy inhibitors, 3-methyladenine and bafilomycin A1. Moreover, antimalarial drugs that inhibit autophagy by increasing the pH of lysosomes can also enhance cisplatin-induced cell death.

Generation of Gene-Knockout Mongolian Gerbils via CRISPR/Cas9 System.

The Mongolian gerbil (Meriones unguiculatus), a well-known "multifunctional" experimental animal, plays a crucial role in the research of hearing, cerebrovascular diseases and Helicobacter pylori infection. Although the whole-genome sequencing of Mongolian gerbils has been recently completed, lack of valid gene-editing systems for gerbils largely limited the further usage of Mongolian gerbils in biomedical research. Here, efficient targeted mutagenesis in Mongolian gerbils was successfully conducted by pronuclear injection with Cas9 protein and single-guide RNAs (sgRNAs) targeting Cystatin C (Cst3) or Apolipoprotein A-II (Apoa2). We found that 22 h after human chorionic gonadotropin (hCG) injection, zygote microinjection was conducted, and the injected zygotes were transferred into the pseudopregnant gerbils, which were induced by injecting equine chorionic gonadotropin (eCG) and hCG at a 70 h interval and being caged with ligated male gerbils. We successfully obtained Cst3 and Apoa2 gene knockout gerbils with the knockout efficiencies of 55 and 30.9%, respectively. No off-target effects were detected in all knockout gerbils and the mutations can be germline-transmitted. The absence of CST3 protein was observed in the tissues of homozygous Cst3 knockout (Cst3-KO) gerbils. Interestingly, we found that disruption of the Cst3 gene led to more severe brain damage and neurological deficits after unilateral carotid artery ligation, thereby indicating that the gene modifications happened at both genetic and functional levels. In conclusion, we successfully generated a CRISPR/Cas9 system based genome editing platform for Mongolian gerbils, which provided a foundation for obtaining other genetically modified gerbil models for biomedical research.

Impact of age on N-methyl-N-nitrosourea induced microsatellite instability in young and old C57BL/6J mice.

Age is an important factor in the evaluation of chemical toxicology. Chemical carcinogenic compounds can induce genomic mutations. However, few studies have been conducted on the association between genomic mutation frequency, such as microsatellite instability (MSI), and the age of mice treated with a nitrosourea mutagen. In the current work, we treated young (6 weeks) and old (10 months) mice with N-methyl-N-nitrosourea (MNU) for 4 months; the MSI frequency was then measured using polymerase chain reaction (PCR) and short tandem repeat (STR) scanning. The percentage of animals with MSI in the old group was significantly higher than that in the young group (100% and 75%). The frequency of MSI events was significantly different between the two groups as well (15.8% for old and 9.4% for young). The ratio of MSI loci displayed no obvious difference between the two groups. In addition, a few loci, including D15Mit5 and D8Mit14 exhibited the highest frequency of MSI events. Since specific loci showed increased MSI in the present study and a higher frequency in previous studies, these loci could be regarded as "hot spot". These results suggested that old mice would be more susceptible to this mutagen, and prone to accrue MSI. The hot spot microsatellite loci are potentially useful markers for genomic instability analysis.

Development of an effective microsatellite marker system to determine the genetic structure of Meriones meridianus populations.

Understanding the genetic quality of the gerbil, Meriones meridianus, plays an important role in the study of medical biology. However, no effective system has been established for evaluating a population's genetic diversity to date. In the present study, we established a set of reasonable evaluative systems based on microsatellite markers of the Mongolian gerbil by using the method of cross-amplification of species. Following electrophoresis analysis, short tandem repeat (STR) scanning, and sequencing, 11 microsatellite loci were identified by matching the criteria characteristics and were used to evaluate the genetic diversity of two stocks of Meriones meridianus: Meriones meridianus jei Wang, 1964 (M. m. jei) and Meriones meridianus cryptorhinus Blanford, 1875 (M. m. cryptorhinus) from Xinjiang, China. The microsatellite loci screened were highly polymorphic and were suitable for genetic quality control of Meriones meridianus. In addition, the quality of the non-bred M. m. jei and M. m. cryptorhinus strains in our study is sufficient for them to be promising stocks in the future for the farmed animal industry.

Association of intron microsatellite status and exon mutational profiles of TP53 in human colorectal cancer.

Microsatellite instability (MSI) and loss of heterozygosity (LOH), which cause genomic instability, contribute to cancer pathogenesis. However, only few studies have evaluated the association of a single microsatellite locus of the TP53 gene with the mutation spectra of TP53 exons. A total of 256 patients with colorectal cancer were enrolled in the present study. MSI/LOH alterations of a microsatellite in the TP53 intron (TP53ALU) were assessed via short tandem repeat scanning. The exon mutation profile was evaluated by direct sequencing. The mutation rate of TP53 exons was significantly higher in tumors with LOH alterations of TP53 introns compared with those in tumors with a microsatellite-stable status in the TP53 intron (P=0.0047). TNM stage II was significantly more frequent in MSI vs. LOH or MSS of the TP53 intron (P=0.027 and P=0.048, respectively). Thus, microsatellite alterations may be valuable predictors of TP53 exon mutation and the TNM stage of colorectal cancers.

Cystatin C Expression is Promoted by VEGFA Blocking, With Inhibitory Effects on Endothelial Cell Angiogenic Functions Including Proliferation, Migration, and Chorioallantoic Membrane Angiogenesis.

Background Vascular development, including vasculogenesis and angiogenesis, is involved in many diseases. Cystatin C ( CST 3) is a commonly used marker of renal dysfunction, and we have previously reported that its expression level is associated with variations in the gerbil circle of Willis. Thus, we hypothesized that CST 3 may affect endothelial function and angiogenic capacity. In the current study, we sought to determine the influence of CST 3 on endothelial function and explore its potential regulatory pathway. Methods and Results We analyzed CST 3 and vascular endothelial growth factor A ( VEGFA) levels in different developmental stages of gerbils using ELISA s and immunofluorescence (to examine the relationship between CST 3 and VEGFA . We used a real-time cell analyzer, cytotoxicity assays, and the chorioallantoic membrane assay to investigate the function of CST 3 in endothelial cells and the chorioallantoic membrane. Additionally, we used Western blotting to explore the downstream targets of CST 3. The expression levels of both CST 3 and VEGFA were at their highest on day 10 of the embryonic stage. CST 3 inhibited endothelial cell proliferation, migration, tube formation, and permeability, as well as vascular development in the chorioallantoic membrane. Blocking of VEGFA dose-dependently increased CST 3 expression in arterial and venous endothelial cells. Furthermore, overexpression and knockdown of CST 3 significantly affected the protein levels of p53 and CAPN10 (calpain 10), suggesting that CST 3 might play a role in vascular development through these proteins. Conclusions CST 3 may be associated with vascular development and angiogenesis, and this effect could be promoted by blocking VEGFA .