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Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a tumour target currently used for the development of novel therapeutic modalities, such as antibody-drug conjugates, chimeric antigen receptor T-cell therapies, and others. Success of these new drugs depends on the selection of relevant indications based on ROR1 tumour prevalence, staining heterogeneity, and subcellular localization, among other parameters. We investigated ROR1 immunophenotype using validated antibody clones for immunohistochemistry (IHC) and flow cytometry (FC), analyzing 292 tumour specimens from 7 haematological malignancies and triple negative breast cancer (TNBC) as a reference solid tumour indication. ROR1 prevalence varied significantly across distinct tumour types, showing 100% of ROR1 positivity in all chronic lymphocytic leukaemia (n = 48) and hairy cell leukaemia (n = 14) specimens analyzed via FC with ranges between 1.1–99.8% and 0.8–62.1%, respectively. Samples analysed via IHC showed ROR1 membrane/cytoplasmic positivity in 44% of mantle cell lymphoma tumour samples (n = 27; H-score range: 10–285 in positive cases); 30% in TNBC (n = 46; H-score range: 1–200); 15% in diffuse large B-cell lymphoma (n = 45; H-score: 40–250); and 11% in follicular lymphoma (n = 34; H-score: 2–300). Finally, all acute myeloid leukaemia (n = 52) and most T-cell non-Hodgkin lymphoma (n = 31/32) tested samples were negative for ROR1 via IHC. In conclusion, ROR1 shows a heterogeneous tumour cell expression profile across multiple leukaemias and lymphomas, making it a tumour target that would require different patient selection strategies to develop novel therapeutic modalities. [ABSTRACT FROM AUTHOR]

Gastric cancer (GC) remains one of the most common digestive cancers worldwide; however, most patients present at an advanced stage at initial diagnosis. Zic1 is a novel candidate tumor suppressor gene that is epigenetically silenced in GC. In this study, we investigated Zic1 promoter methylation in plasma DNA as a novel molecular marker for the early diagnosis and monitoring of GC. Methylation-specific polymerase chain reaction (MSP) assay was performed to detect Zic1 promoter methylation in plasma DNA from 20 healthy subjects, 50 gastric intraepithelial neoplasia patients, and 104 GC patients. The Zic1 promoter methylation rate in the plasma samples from the healthy control group was 0%, but it reached 54.0% in the intraepithelial neoplasia group and 60.6% in the GC group. The latter two values were significantly higher than that found in the healthy control group (p < 0.05), with a 100% specificity for intraepithelial neoplasia and GC diagnosis. The positive predictive value of plasma Zic1 promoter methylation for the diagnosis of intraepithelial neoplasia and GC was 100%. Methylation status in the GC group was not significantly associated with tumor size, tumor differentiation, lymph node metastasis, TNM staging, or tumor invasion (p > 0.05). Assessment of the significance of detection of the carcino-embryonic antigen (CEA) level and Zic1 promoter methylation rate for GC diagnosis revealed that the sensitivity of Zic1 promoter methylation was significantly higher than that of the CEA level as a marker and that the combined measurement of these two indices (parallel testing) improved sensitivity. Taken together, our results suggest that the Zic1 promoter methylation rate in plasma-derived DNA is of great significance for the early screening of GC and monitoring of tumorigenesis. Zic1 promoter methylation may serve as a novel non-invasive plasma biomarker for the early detection of GC and for risk assessment in high-risk populations. The combined measurement of the Zic1 promoter methylation rate and CEA level (parallel testing) may enhance the current guidelines for the early diagnosis of GC. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is an important and common cancer that constitutes a major public health problem, but early detection of small cell lung cancer can significantly improve the survival rate of cancer patients. A number of serum biomarkers have been used in the diagnosis of lung cancers; however, they exhibit low sensitivity and specificity. Methods: We used biochemical methods to measure blood levels of lactate dehydrogenase (LDH), C-reactive protein (CRP), Na+, Cl-, carcino-embryonic antigen (CEA), and neuron specific enolase (NSE) in 145 small cell lung cancer (SCLC) patients and 155 non-small cell lung cancer and 155 normal controls. A gene expression programming (GEP) model and Receiver Operating Characteristic (ROC) curves incorporating these biomarkers was developed for the auxiliary diagnosis of SCLC. Results: After appropriate modification of the parameters, the GEP model was initially set up based on a training set of 115 SCLC patients and 125 normal controls for GEP model generation. Then the GEP was applied to the remaining 60 subjects (the test set) for model validation. GEP successfully discriminated 281 out of 300 cases, showing a correct classification rate for lung cancer patients of 93.75% (225/240) and 93.33% (56/60) for the training and test sets, respectively. Another GEP model incorporating four biomarkers, including CEA, NSE, LDH, and CRP, exhibited slightly lower detection sensitivity than the GEP model, including six biomarkers. We repeat the models on artificial neural network (ANN), and our results showed that the accuracy of GEP models were higher than that in ANN. GEP model incorporating six serum biomarkers performed by NSCLC patients and normal controls showed low accuracy than SCLC patients and was enough to prove that the GEP model is suitable for the SCLC patients. Conclusion: We have developed a GEP model with high sensitivity and specificity for the auxiliary diagnosis of SCLC. This GEP model has the potential for the wide use for detection of SCLC in less developed regions. [ABSTRACT FROM AUTHOR]

Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC) opens up new avenues for basic research and regenerative medicine. However, the low efficiency of the procedure remains a major limitation. To identify iPSC, many studies to date relied on the activation of pluripotency-associated transcription factors. Such strategies are either retrospective or depend on genetically modified reporter cells. We aimed at identifying naturally occurring surface proteins in a systematic approach, focusing on antibody-targeted markers to enable live-cell identification and selective isolation. We tested 170 antibodies for differential expression between mouse embryonic fibroblasts (MEF) and mouse pluripotent stem cells (PSC). Differentially expressed markers were evaluated for their ability to identify and isolate iPSC in reprogramming cultures. Epithelial cell adhesion molecule (EPCAM) and stage-specific embryonic antigen 1 (SSEA1) were upregulated early during reprogramming and enabled enrichment of OCT4 expressing cells by magnetic cell sorting. Downregulation of somatic marker FAS was equally suitable to enrich OCT4 expressing cells, which has not been described so far. Furthermore, FAS downregulation correlated with viral transgene silencing. Finally, using the marker SSEA-1 we exemplified that magnetic separation enables the establishment of bona fide iPSC and propose strategies to enrich iPSC from a variety of human source tissues. [ABSTRACT FROM AUTHOR]

Background: Altered expression of miRNA expression contributes to human carcinogenesis. This study was designed to detect aberrant miRNA expressions as a potential biomarker for early detection and prognosis prediction of non-small cell lung cancer (NSCLC). Methods: miRNA array was used to profile differentially expressed miRNAs and Taqman-based quantitative RT-PCR assays were used to analyze levels of miR-29c, miR-93, and miR-429 expression in NSCLC tissue samples, corresponding normal tissue samples, and serum samples from 70 NSCLC patients as well as in serum samples from 48 healthy controls. Results: Levels of miR-29c and miR-93 expression were upregulated in NSCLC tissues, while serum levels of miR-29c were also upregulated, but levels of serum miR-429 were decreased in NSCLC. Moreover, the levels of miR-429 expression in NSCLC tissues were associated with those in serum samples. Receiver operating characteristic (ROC) curve analysis showed that at the optimal cut-off point, the areas under the ROC curve for serum levels of miR-29c and miR-429 were 0.723 and 0.727, respectively, levels which are higher than that of carcinoma embryonic antigen (0.534) in diagnosis of stage I NSCLC. In addition, serum levels of miR-429 were associated with poor overall survival of NSCLC patients. Both univariate and multivariate analyses showed that serum miR-429 level was an independent prognostic predictor for NSCLC. Conclusions: The results of the current study suggest that detection of serum miR-29c and miR-429 expression should be further evaluated as a novel, non-invasive biomarker for early stage NSCLC. [ABSTRACT FROM AUTHOR]

Replication-competent adenovirus type 5 (Ad5) vectors promise to be more efficient gene delivery vehicles than their replication-deficient counterparts, and chimeric Ad5 vectors that are capable of targeting CD46 are more effective than Ad5 vectors with native fibers. Although several strategies have been used to improve gene transduction and oncolysis, either by modifying their tropism or enhancing their replication capacity, some tumor cells are still relatively refractory to infection by chimeric Ad5. The oncolytic effects of the vectors are apparent in certain tumors but not in others. Here, we report the biological and oncolytic profiles of a replication-competent adenovirus 11p vector (RCAd11pGFP) in colon carcinoma cells. CD46 was abundantly expressed in all cells studied; however, the transduction efficiency of RCAd11pGFP varied. RCAd11pGFP efficiently transduced HT-29, HCT-8, and LS174T cells, but it transduced T84 cells, derived from a colon cancer metastasis in the lung, less efficiently. Interestingly, RCAd11p replicated more rapidly in the T84 cells than in HCT-8 and LS174T cells and as rapidly as in HT-29 cells. Cell toxicity and proliferation assays indicated that RCAd11pGFP had the highest cell-killing activities in HT29 and T84 cells, the latter of which also expressed the highest levels of glycoproteins of the carcinoma embryonic antigen (CEA) family. In vivo experiments showed significant growth inhibition of T84 and HT-29 tumors in xenograft mice treated with either RCAd11pGFP or Ad11pwt compared to untreated controls. Thus, RCAd11pGFP has a potent cytotoxic effect on colon carcinoma cells. [ABSTRACT FROM AUTHOR]

Background: Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells. Recently, selection of fully reprogrammed cells was achieved based on colony morphology reminiscent of embryonic stem (ES) cells. The maintenance of pluripotency was analysed. Methodology/Principal Findings: Clonal murine iPS cell line TiB7-4, which was derived from adult fibroblasts, was analysed for maintenance of pluripotency. Colony morphology, expression of pluripotency factors and stage specific embryonic antigen 1 (SSEA1) were analysed by real time PCR and flow cytometry. We found the iPS cell line TiB7-4 and its subclones to be rather diverse and exhibiting a tendency towards spontaneous differentiation and loss of pluripotency independent of their initial colony morphology. In contrast an undifferentiated transcription factor 1 (UTF1) promoter-driven G418 (Neo) resistance significantly improved the quality of these iPS cells. After selection with UTF-Neo for two weeks iPS subclones could be stably maintained for at least 40 passages in culture and differentiate into all three germ layers. As control, a construct expressing G418 resistance under the control of the ubiquitously active SV40 early promoter formed subclones with different colony morphology. Some of these subclones could be cultured for at least 12 passages without loosing their pluripotency, but loss of pluripotency eventually occured in an unpredictable manner and was independent of the subclones' initial morphology and SSEA1 expression. A UTF-Neo-based selection of a whole population of TiB7-4 without further subcloning resulted in the generation of cultures with up to 99% SSEA1 positive cells under stringent selection conditions. Conclusions: Our data indicate that temporary selection using a genetic UTF1-based system can generate homogenous pluripotent iPS cells that can be maintained without permanent selection pressure. [ABSTRACT FROM AUTHOR]

Background: The derivation of induced pluripotent stem cells (iPSCs) provides new possibilities for basic research and novel cell-based therapies. Limitations, however, include our current lack of understanding regarding the underlying mechanisms and the inefficiency of reprogramming. Methodology/Principal Findings: Here, we report identification and isolation of a subpopulation of human dermal fibroblasts that express the pluripotency marker stage specific embryonic antigen 3 (SSEA3). Fibroblasts that expressed SSEA3 demonstrated an enhanced iPSC generation efficiency, while no iPSC derivation was obtained from the fibroblasts that did not express SSEA3. Transcriptional analysis revealed NANOG expression was significantly increased in the SSEA3 expressing fibroblasts, suggesting a possible mechanistic explanation for the differential reprogramming. Conclusions/Significance: To our knowledge, this study is the first to identify a pluripotency marker in a heterogeneous population of human dermal fibroblasts, to isolate a subpopulation of cells that have a significantly increased propensity to reprogram to pluripotency and to identify a possible mechanism to explain this differential reprogramming. This discovery provides a method to significantly increase the efficiency of reprogramming, enhancing the feasibility of the potential applications based on this technology, and a tool for basic research studies to understand the underlying reprogramming mechanisms. [ABSTRACT FROM AUTHOR]

Background: Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. Methods and Findings: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. Conclusion: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates. [ABSTRACT FROM AUTHOR]

The rat is a reference animal model for physiological studies and for the analysis of multigenic human diseases such as hypertension, diabetes, neurological disorders, and cancer. The rats have long been used in extensive chemical carcinogenesis studies. Thus, the rat embryonic stem (rES) cell is an important resource for the study of disease models. Attempts to derive ES cells from various mammals, including the rat, have not succeeded. Here we have established two independent rES cells from Wister rat blastocysts that have undifferentiated characters such as Nanog and Oct3/4 genes expression and they have stage-specific embryonic antigen (SSEA) -1, -3, -4, and TRA-1-81 expression. The cells were successfully cultured in an undifferentiated state and can be possible over 18 passages with maintaining more than 40% of normal karyotype. Their pluripotent potential was confirmed by the differentiation into derivatives of the endoderm, mesoderm, and ectoderm. Most importantly, the rES cells are capable of producing chimera rats. Therefore, we established pluripotent rES cell lines that are widely used to produce genetically modified experimental rats for study of human diseases. [ABSTRACT FROM AUTHOR]

Purpose: This study investigated whether liver damage severity relates to the mobilization of multilineage-differentiating stress-enduring (Muse) cells, which are endogenous reparative pluripotent stem cells, into the peripheral blood (PB) and whether the degree of mobilization relates to the recovery of liver volume following human liver surgery. Methods: Forty-seven patients who underwent liver surgery were included in the present study. PB-Muse cells were counted before surgery, on postoperative days (PODs) 3 and on POD 7. Liver volume was measured using computed tomography before and after surgery. Results: The PB-Muse cell count increased after surgery. The number of PB-Muse cells before surgery was higher, but without statistical significance in the group with neoplasms than in the healthy group that included liver donors (p = 0.065). Forty-seven patients who underwent liver surgery were divided into major hepatic resection (MHR; hepatectomy of three or more segments according to the Couinaud classification, n = 22) and minor hepatic resection (mhr; hepatectomy of two segments or less according to the Couinaud classification, n = 25) groups. PB-Muse cells increased at high rates among MHR patients (p = 0.033). Except for complication cases, PB-Muse cells increased at higher rates in the group with advanced liver volume recovery (p = 0.043). The predictive impact of the rate of increase in PB-Muse cells on the recovery of liver volume was demonstrated by multivariate analysis (OR 11.0, p = 0.014). Conclusions: PB-Muse cell mobilization correlated with the volume of liver resection, suggesting that the PB-Muse cell number reflects the degree of liver injury. Given that the degree of PB-Muse cell mobilization was related to liver volume recovery, PB-Muse cells were suggested to contribute to liver regeneration, although this mechanism remains unclear. [ABSTRACT FROM AUTHOR]

Dermal wound healing is a complex process which requires the interaction of many cell types and mediators in a highly sophisticated temporal sequence. Myeloid cells which compose of a significant proportion of the inflammatory cells infiltrate to the to a wound site where they play important roles in clearance of damaged tissue and microorganisms. Myeloid cells have the capacity to be converted into fibroblast-like cells and endothelial cells during wound healing process. However, whether myeloid cells in wounds can convert into epithelial cells where they contribute to healing process is not clear. In this study, we performed double immunofluorescent staining with antibodies for hematopoietic cells and keratinocytes as well as cell tracing technique to investigate hematopoietic cell conversion. The result showed that during the healing process, some of the CD45-positive hematopoietic cells also expressed keratin 14, a marker for keratinocytes. Further, double immunofluorescent staining in dermal wounds, using CD11b and K14 antibodies indicated that CD11b-positive myeloid cells were the origin of newly generated epithelial cells. Through tracing injected labeled splenocyte-derived myeloid cells in skin, we confirmed that myeloid cells were able to convert into keratinocytes in repaired skin. Furthermore, our results from in vivo experiments provided new information on contribution of myeloid cells in hair follicle regeneration. In conclusion, this work highlights the myeloid cell contributions in wound repair and hair follicle regeneration through conversion of M-CSF-stimulated CD11b-positive myeloid cells into epithelial cells in a murine model. [ABSTRACT FROM AUTHOR]

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins. [ABSTRACT FROM AUTHOR]

Background: We recently reported that multilineage-differentiating stress enduring (Muse) cells intravenously administered after acute myocardial infarction (AMI), selectively engrafted to the infarct area, spontaneously differentiated into cardiomyocytes and vessels, reduced the infarct size, improved the left ventricular (LV) function and remodeling in rabbits. We aimed to clarify the efficiency of Muse cells in a larger animal AMI model of mini-pigs using a semi-clinical grade human Muse cell product. Method and result: Mini-pigs underwent 30 min of coronary artery occlusion followed by 2 weeks of reperfusion. Semi-clinical grade human Muse cell product (1x107, Muse group, n = 5) or saline (Vehicle group, n = 7) were intravenously administered at 24 h after reperfusion. The infarct size, LV function and remodeling were evaluated by echocardiography. Arrhythmias were evaluated by an implantable loop recorder. The infarct size was significantly smaller in the Muse group (10.5±3.3%) than in the Vehicle group (21.0±2.0%). Both the LV ejection fraction and fractional shortening were significantly greater in the Muse group than in the Vehicle group. The LV end-systolic and end-diastolic dimensions were significantly smaller in the Muse group than in the Vehicle group. Human Muse cells homed into the infarct border area and expressed cardiac troponin I and vascular endothelial CD31. No arrhythmias and no blood test abnormality were observed. Conclusion: Muse cell product might be promising for AMI therapy based on the efficiency and safety in a mini-pig AMI. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with a 5% 5-year survival rate for metastatic disease, yet with limited therapeutic advancements due to insufficient understanding of and inability to accurately capture high-risk CRC patients who are most likely to recur. We aimed to improve high-risk classification by identifying biological pathways associated with outcome in adjuvant stage II/III CRC. Methods and findings: We included 1062 patients with stage III or high-risk stage II colon carcinoma from the prospective three-arm randomized phase 3 AVANT trial, and performed expression profiling to identify a prognostic signature. Data from validation cohort GSE39582, The Cancer Genome Atlas, and cell lines were used to further validate the prognostic biology. Our retrospective analysis of the adjuvant AVANT trial uncovered a prognostic signature capturing three biological functions—stromal, proliferative and immune—that outperformed the Consensus Molecular Subtypes (CMS) and recurrence prediction signatures like Oncotype Dx in an independent cohort. Importantly, within the immune component, high granzyme B (GZMB) expression had a significant prognostic impact while other individual T-effector genes were less or not prognostic. In addition, we found GZMB to be endogenously expressed in CMS2 tumor cells and to be prognostic in a T cell independent fashion. A limitation of our study is that these results, although robust and derived from a large dataset, still need to be clinically validated in a prospective study. Conclusions: This work furthers our understanding of the underlying biology that propagates stage II/III CRC disease progression and provides scientific rationale for future high-risk stratification and targeted treatment evaluation in biomarker defined subpopulations of resectable high-risk CRC. Our results also shed light on an alternative GZMB source with context-specific implications on the disease's unique biology. [ABSTRACT FROM AUTHOR]

Background: Pancreatic adenocarcinoma (PDAC) is an incurable cancer without adequate tumor markers. Our previous study has showed a better diagnostic performance of Protein Induced by Vitamin K Absence II (PIVKA-II) compared to currently used PDAC biomarkers. To corroborate our previous data with a larger sample size and to assess a possible role of PIVKA-II in predicting surgical success. Additionally, to further evaluate the hypothesis of a direct PIVKA-II production by PDAC cells, we examined PIVKA-II tissue expression in a case of PDAC using immunofluorescence. Methods: We enrolled 76 newly diagnosed PDAC patients and selected 11 patients to determine PIVKA-II levels also after surgical resection. An immunofluorescence (IF) study of PIVKA-II tissue expression was carried out in one of them. PIVKA-II serum values were measured by chemiluminescent enzyme immunoassay method (CLEIA) on LUMIPULSE G1200 (Fujirebio-Europe, Belgium). Results: PIVKA-II serum levels were above the cut-off at baseline in 71 patients (94%) with a median value of 464 mAU/Ml (range 27–40783 mAU/mL); the sensitivity and specificity were 78.67% and 90.67% respectively. Patients with pre-operative PIVKA-II positivity showed a significant decrease (P < 0.015) of median PIVKA-II serum concentrations after surgery: 820 (91–40783) mAU/mL at diagnosis vs 123 (31–4666) mAU/mL post-operatively. IF assay on PDAC sections demonstrated PIVKA-II expression in cancer cells. Conclusion: These data are the first showing a decreased PIVKA-II serum levels after surgery in PDAC patients and reporting PIVKA-II expression in PDAC tissue. Further studies are needed to confirm these findings and to determine PIVKA-II usefulness in diagnosing and monitoring PDAC patients. [ABSTRACT FROM AUTHOR]

The medical applications of electrical biostimulation and silver ions have been evaluated in laboratory experiments and clinical studies for more than two decades. Their effects on preventing infection and promoting wound healing have been described. However, little is known about the role of electrical biostimulation and/or silver ion on changes in cellular transcriptome dynamics. To our knowledge, few studies have been conducted to investigate the potential of electrical biostimulation and silver ions in cell reprogramming. Besides, it is essential to assess any possible adverse effects or potential benefits of the silver ions on mammalian cells to address its safety concerns and to improve silver medical products. In this study, we investigated transcriptomic changes in porcine fibroblast cells in response to electrical biostimulation in the presence of silver ions. Exposed cells presented distinct morphological changes after treatment, which was mainly due to the exposure of silver ions rather than the electrical current itself. Gene expression analyses suggested that electrical biostimulation and silver ions did not increase the expression of pluripotency genes. Interestingly, a set of genes related to cellular metabolic processes were differentially expressed after cells were exposed to electrically generated silver ions for 21 hours. We found that 2.00 mg/L of electrically generated silver ion caused an increase of ATP generation and an increase of the total pool of NAD+ and NADH, while ROS production did not change. Aside from toxic effects, the results reported herein demonstrate the alternative effects of silver ions on mammalian cells, especially an oxidative phosphorylation burst. To our knowledge, this response of mammalian cells to silver ions has not been described previously. Although the function of this burst is not understood, it may lead to alterations in cellular activities such as metabolic remodeling and cell reprogramming, and/or serve an as-yet unknown function in neutralization or detoxification of the silver ions within the cells. [ABSTRACT FROM AUTHOR]

Natural antibodies are an innate-like subset of serum antibodies involved in host defense, tumor surveillance, homeostasis, and autoimmunity. Defining the natural antibody repertoire is critical for identifying biomarkers, developing vaccines, controlling and preventing autoimmunity, and understanding the development and organization of the immune system. While natural antibodies to protein antigens have been studied in depth, little is known about natural antibodies to carbohydrate antigens. To address this, we profiled IgM from umbilical cord blood and matched maternal sera on a glycan microarray. Since standard methods to detect maternal contamination in cord serum did not have sufficient sensitivity for our study, we developed a highly sensitive microarray-based assay. Using this method, we found that over 50% of the cord samples had unacceptable levels of maternal contamination. For the cord samples with high purity, anti-glycan IgM antibodies were prevalent and recognized a broad range of non-human and human glycans. Using principal component analysis and hierarchical clustering, cord IgM repertoires showed a high degree of similarity with each other but were distinct from maternal IgM repertoires. Our results demonstrate that many anti-glycan antibodies in human serum are natural antibodies and provide new insights into the development of anti-glycan antibody repertoires. [ABSTRACT FROM AUTHOR]

Background: Intensive follow-up after surgery for colorectal cancers is common in clinical practice, but evidence of a survival benefit is limited. Objective: To conduct a systematic review and meta-analysis on the effects of follow-up strategies for nonmetastatic colorectal cancer. Data sources: We searched Medline, Embase, and CENTRAL databases through May 30, 2018. Study selection: We included randomized clinical trials evaluating intensive follow-up versus less follow-up in patients with nonmetastatic colorectal cancer. Interventions: Intensive follow-up Main outcomes measures: Overall survival. Results: The analyses included 17 trials with a total of 8039 patients. Compared with less follow-up, intensive follow-up significantly improved overall survival in patients with nonmetastatic colorectal cancer after radical surgery (HR 0.85, 95% CI 0.74–0.97, P = 0.01; I2 = 30%; high quality). Subgroup analyses showed that differences between intensive-frequency and intensive-test follow-up (P = 0.04) and between short interval and long interval of follow-up (P = 0.02) in favor of the former one. Limitations: Clinical heterogeneity of interventions Conclusions: For patients with nonmetastatic colorectal cancer after curative resection, intensive follow-up strategy was associated with an improvement in overall survival compared with less follow-up strategy. Intensive-frequency follow-up strategy was associated with a greater reduction in mortality compared with intensive-test follow-up strategy. [ABSTRACT FROM AUTHOR]

Triple Negative Breast Cancer (TNBC), the most aggressive subtype of breast cancer, is characterized by the absence of hormone receptors usually targeted by hormone therapies like Tamoxifen. Because therapy success and survival rates for TNBC lag far behind other breast cancer subtypes, there is significant interest in developing novel anti-TNBC agents that can target TNBC specifically, with minimal effects on non-malignant tissue. To this aim, our study describes the anti-TNBC effect of strictinin, an ellagitanin previously isolated from Myrothamnus flabellifolius. Using various in silico and molecular techniques, we characterized the mechanism of action of strictinin in TNBC. Our results suggest strictinin interacts strongly with Receptor Tyrosine Kinase Orphan like 1 (ROR1). ROR1 is an oncofetal receptor highly expressed during development but not in normal adult tissue. It is highly expressed in several human malignancies however, owing to its numerous pro-tumor functions. Via its interaction and inhibition of ROR1, strictinin reduced AKT phosphorylation on ser-473, inhibiting downstream phosphorylation and inhibition of GSK3β. The reduction in AKT phosphorylation also correlated with decreased cell survival and activation of the caspase-mediated intrinsic apoptotic cascade. Strictinin treatment also repressed cell migration and invasion in a beta-catenin independent manner, presumably via the reactivated GSK3ß’s repressing effect on microtubule polymerization and focal adhesion turnover. This could be of potential therapeutic interest considering heightened interest in ROR1 and other receptor tyrosine kinases as targets for development of anti-cancer agents. Further studies are needed to validate these findings in other ROR1-expressing malignancies but also in more systemic models of TNBC. Our findings do however underline the potential of strictinin and other ROR1-targeting agents as therapeutic tools to reduce TNBC proliferation, survival and motility. [ABSTRACT FROM AUTHOR]

The use of Xenopus laevis as a model for vertebrate developmental biology is limited by a lack of antibodies specific for embryonic antigens. This study evaluated the use of immune and non-immune phage display libraries for the isolation of single domain antibodies, or nanobodies, with specificities for Xenopus embryonic antigens. The immune nanobody library was derived from peripheral blood lymphocyte RNA obtained from a llama immunized with Xenopus gastrula homogenates. Screening this library by immunostaining of embryonic tissues with pooled periplasmic material and sib-selection led to the isolation of several monoclonal phages reactive with the cytoplasm and nuclei of gastrula cells. One antigen recognized by a group of nanobodies was identified using a reverse proteomics approach as nucleoplasmin, an abundant histone chaperone. As an alternative strategy, a semi-synthetic non-immune llama nanobody phage display library was panned on highly purified Xenopus proteins. This proof-of-principle approach isolated monoclonal nanobodies that specifically bind uclear istribution lement-ike 1 (Ndel1) in multiple immunoassays. Our results suggest that immune and non-immune phage display screens on crude and purified embryonic antigens can efficiently identify nanobodies useful to the Xenopus developmental biology community. [ABSTRACT FROM AUTHOR]

Objective: Obesity is the result of white adipose tissue accumulation where excess of food energy is stored to form triglycerides. De novo lipogenesis (DNL) is the continuous process of new fat production and is driven by the transcription factor ChREBP. During adipogenesis, white adipocytes change their morphology and the entire cell volume is occupied by one large lipid droplet. Recent studies have implicated an essential role of autophagy in adipogenic differentiation, cytoplasmic remodelling and mitochondria reorganization. The phenolic monoterpenoid carvacrol (2-methyl-5-[1-methylethyl]phenol), produced by numerous aromatic plants, has been shown to reduce lipid accumulation in murine 3T3-L1 cells during adipogenic differentiation by modulating genes associated with adipogenesis and inflammation. Therefore, the aim of this study was to evaluate whether carvacrol could affect autophagy and ChREBP expression during adipogenic differentiation. Methods: The study was carried on by using the murine 3T3-L1 and the human WJ-MSCs (Wharton’s jelly-derived mesenchymal stem cells) cell lines. Cells undergoing adipogenic differentiation were untreated or treated with carvacrol. Adipogenic differentiation was assessed by analyzing cellular lipid accumulation with Oil-Red O staining and by ultrastructural examination with TEM. Autophagy was evaluated by western immunoblotting of autophagy markers LC3B and p62/SQSTM and by ultrastructural examination of autophagic bodies. Autophagic flux was evaluated by using autophagy inhibitor cloroquine (CQ). ChREBP expression levels was assessed by both western blotting and immunoelectron microscopy and ChREBP activity by analysis of adipogenic target genes expression. Results: We found that carvacrol reduced adipogenic differentiation of about 40% and 30% in, respectively, 3T3-L1 and in WJ-MSCs cells. The effect of carvacrol on adipogenic differentiation correlated with both reduction of autophagy and reduction of ChREBP expression. Conclusion: The results support the notion that carvacrol, through its effect on autophagy (essential for adipocyte maturation) and on ChREBP activity, could be used as a valuable adjuvant to reduce adipogenic differentiation. [ABSTRACT FROM AUTHOR]

The crosstalk between cells is important for differentiation of cells. Murine-derived feeder cells, SNL76/7 feeder cells (SNLs) or mouse primary embryonic fibroblast feeder cells (MEFs) are widely used for culturing undifferentiated human induced pluripotent stem cells (hiPSCs). It is still unclear whether different culture conditions affect the induction efficiency of definitive endoderm (DE) differentiation from hiPSCs. Here we show that the efficiency of DE differentiation from hiPSCs cultured on MEFs was higher than that of hiPSCs cultured on SNLs. The qPCR, immunofluorescent and flow cytometry analyses revealed that the expression levels of mRNA and/or proteins of the DE marker genes, SOX17, FOXA2 and CXCR4, in DE cells differentiated from hiPSCs cultured on MEFs were significantly higher than those cultured on SNLs. Comprehensive RNA sequencing and molecular network analyses showed the alteration of the gene expression and the signal transduction of hiPSCs cultured on SNLs and MEFs. Interestingly, the expression of non-coding hXIST exon 4 was up-regulated in hiPSCs cultured on MEFs, in comparison to that in hiPSCs cultured on SNLs. By qPCR analysis, the mRNA expression of undifferentiated stem cell markers KLF4, KLF5, OCT3/4, SOX2, NANOG, UTF1, and GRB7 were lower, while that of hXIST exon 4, LEFTY1, and LEFTY2 was higher in hiPSCs cultured on MEFs than in those cultured on SNLs. Taken together, our finding indicated that differences in murine-feeder cells used for maintenance of the undifferentiated state alter the expression of pluripotency-related genes in hiPSCs by the signaling pathways and affect DE differentiation from hiPSCs, suggesting that the feeder cells can potentiate hiPSCs for DE differentiation. [ABSTRACT FROM AUTHOR]

The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients. [ABSTRACT FROM AUTHOR]

There is a great unmet medical need in pancreatic carcinoma (PC) for novel drugs with other mechanisms of action than existing. PC cells express the onco-fetal RTK ROR1, absent on most normal post-partem cells. ROR1 is involved in proliferation, survival, EMT and metastasis of tumor cells in various malignancies. A small molecule inhibitor (KAN0439834) (530 Da) targeting the TK domain of ROR1 was developed and the activity in ROR1 expressing human PC cell lines (n = 8) evaluated. The effects were compared to a murine mAb against the external part of ROR1, gemcitabine, erlotinib and ibrutinib. KAN0439834 induced significant apoptosis of the tumor cells. EC50 values for KAN0439834 varied between 250–650 nM depending on the cell line. The corresponding values for erlotinib and ibrutinib were 10–40 folds higher. KAN0439834 was much more effective in inducing tumor cell death than the ROR1 mAb although both inhibited ROR1 phosphorylation and downstream non-canonical Wnt pathway molecules. Combination of KAN0439834 with erlotinib or ibrutinib had significant additive effects on tumor cell death. A first-in-class small molecule ROR1 inhibitor (KAN0439834) showed promising in vitro activity against a number of human PC cell lines. Interesting is the additive effects of erlotinib and ibrutinib which warrants further studies as both these agents are in clinical trials for pancreatic carcinoma. [ABSTRACT FROM AUTHOR]

The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model. [ABSTRACT FROM AUTHOR]

Stem cells derived from placental tissues are an attractive source of cells for regenerative medicine. Amniotic epithelial cells isolated from human amnion (hAECs) have desirable and competitive characteristics that make them stand out between other stem cells. They have the ability to differentiate toward all three germ layers, they are not tumorigenic and they have immunosuppressive properties. Although liver transplantation is the best way to treat acute and chronic hepatic failure patients, there are several obstacles. Recently, stem cells have been spotlighted as alternative source of hepatocytes because of their potential for hepatogenic differentiation. In this work, we aimed to study the proliferation and survival of the hAECs during their hepatic differentiation. We have also analyzed the changes in pluripotency and hepatic markers. We differentiated amniotic cells applying a specific hepatic differentiation (HD) protocol. We determined by qRT-PCR that hAECs express significant levels of SOX-2, OCT-4 and NANOG during at least 15 days in culture and these pluripotent markers diminish during HD. SSEA-4 expression was reduced during HD, measured by immunofluorescence. Morphological characteristics became more similar to hepatic ones in differentiated cells and representative hepatic markers significantly augmented their expression, measured by qRT-PCR and Western blot. Cells achieved a differentiation efficiency of 75%. We observed that HD induced proliferation and promoted survival of hAECs, during 30 days in culture, evaluated by 3H-thymidine incorporation and MTT assay. HD also promoted changes in hAECs cell cycle. Cyclin D1 expression increased, while p21 and p53 levels were reduced. Immunofluorescence analysis showed that Ki-67 expression was upregulated during HD. Finally, ERK 1/2 phosphorylation, which is intimately linked to proliferation and cell survival, augmented during all HD process and the inhibition of this signaling pathway affected not only proliferation but also differentiation. Our results suggest that HD promotes proliferation and survival of hAECs, providing important evidence about the mechanisms governing their hepatic differentiation. We bring new knowledge concerning some of the optimal transplantation conditions for these hepatic like cells. [ABSTRACT FROM AUTHOR]

Glioma is the most malignant type of primary central nervous system tumors, and has an extremely poor prognosis. One potential therapeutic approach is to induce the terminal differentiation of glioma through the forced expression of pro-neural factors. Our goal is to show the proof of concept of the neuronal conversion of C6 glioma through the combined action of small molecules. We investigated the various changes in gene expression, cell-specific marker expression, signaling pathways, physiological characteristics, and morphology in glioma after combination treatment with two small molecules (CHIR99021, a glycogen synthase kinase 3 [GSK3] inhibitor and forskolin, a cyclic adenosine monophosphate [cAMP] activator). Here, we show that the combined action of CHIR99021 and forskolin converted malignant glioma into fully differentiated neurons with no malignant characteristics; inhibited the proliferation of malignant glioma; and significantly down-regulated gene ontology and gene expression profiles related to cell division, gliogenesis, and angiogenesis in small molecule–induced neurons. In vivo, the combined action of CHIR99021 and forskolin markedly delayed neurological deficits and significantly reduced the tumor volume. We suggest that reprogramming technology may be a potential treatment strategy replacing the therapeutic paradigm of traditional treatment of malignant glioma, and a combination molecule comprising a GSK3 inhibitor and a cAMP inducer could be the next generation of anticancer drugs. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) plays an important role in abiotic stress response and physiological signal transduction resisting to the adverse environment. Therefore, it is very essential for the quantitative detection of abscisic acid (ABA) due to its indispensable role in plant physiological activities. Herein, a new detection method based on localized surface plasmon resonance (LSPR) using aptamer-functionalized gold nanoparticles (AuNPs) is developed without using expensive instrument and antibody. In the presence of ABA, ABA specifically bind with their aptamers to form the ABA-aptamer complexes with G-quadruplex-like structure and lose the ability to stabilize AuNPs against NaCl-induced aggregation. Meanwhile, the changes of the LSPR spectra of AuNP solution occur and therefore the detection of ABA achieved. Under optimized conditions, this method showed a good linear range covering from 5×10−7 M to 5×10−5 M with a detection limit of 0.33 μM. In practice, the usage of this novel method has been demonstrated by its application to detect ABA from fresh leaves of rice with the relative error of 6.59%-7.93% compared with ELISA bioassay. The experimental results confirmed that this LSPR-based biosensor is simple, selective and sensitive for the detection of ABA. The proposed LSPR method could offer a new analytical platform for the detection of other plant hormones by changing the corresponding aptamer. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most common primary bone tumor in children and young adults. The median survival of osteosarcoma patients has not significantly improved since 1990, despite administration of different classes of chemotherapy agents, such as methotrexate, cisplatin and doxorubicin. Cancer stem cells (CSCs) are responsible for the resistance of osteosarcoma to chemotherapy and OCT4, SOX2 and SSEA4 have been used to identify CSCs in osteosarcoma. Here, we used low-passage patient-derived osteosarcoma cells and osteosarcoma cells directly isolated from patients before and after chemotherapy treatments to evaluate the effects of chemotherapy on stem cell markers expression. We demonstrate that primary osteosarcoma cells are resistant to methotrexate treatment and sensitive to cisplatin and doxorubicin in vitro. We also verified that cisplatin and doxorubicin reduce the expression of SOX2 and OCT4 in primary osteosarcoma cells whereas methotrexate does not alter SOX2 and OCT4 expression, however it increases SSEA4 expression in primary osteosarcoma cells. Finally, we found that, although the combination treatment cisplatin plus doxorubicin inhibited the in vivo growth of osteosarcoma cells in NOD-SCID gamma mice subcutaneously injected with SaOs2, the combination treatment cisplatin plus doxorubicin plus methotrexate did not inhibit the in vivo growth of these cells. These observations may provide an explanation for the poor response of osteosarcomas to chemotherapy and point to the need of reevaluating the therapeutic strategies for human osteosarcomas. [ABSTRACT FROM AUTHOR]

Background: The aim of the study was to evaluate psychological effects of the state-of-art intensified follow-up protocol for colorectal cancer patients in the CEAwatch trial. Method: At two time points during the CEAwatch trial questionnaires regarding patients’ attitude towards follow-up, patients’ psychological functioning and patients’ experiences and expectations were sent to participants by post. Linear mixed models were fitted to assess the influences and secular trends of the intensified follow-up on patients’ attitude towards follow-up and psychological functioning. As secondary outcome, odds ratios were calculated using ordinal logistic mixed model to compare patients’ experiences to their expectations, as well as their experiences at two different time points. Results: No statistical significant effects of the intensified follow-up were found on patients’ attitude towards the follow-up and psychological functioning variables. Patients had high expectations of the intensified follow-up and their experiences at the second time point were more positive compared to the scores at the first time point. Conclusion: The intensified follow-up protocol posed no adverse effects on patients’ attitude towards follow-up and psychological functioning. In general, patients were more nervous and anxious at the start of the new follow-up protocol, had high expectations of the new follow-up protocol and were troubled by the nuisances of the blood sample testing. As they spent more time in the follow-up and became more adapted to it, the nervousness and anxiety decreased and the preference for the frequent blood test became high in replacement of conversations with the doctors. [ABSTRACT FROM AUTHOR]

Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD) did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane) and cholesteryl esters (stored in lipid droplets), revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs) among seasons and a dynamic organizational structure of cholesterol homeostasis within the COC. Modulation of membrane cholesterol by MβCD improved survival of bovine oocytes and preserved integrity of GM1-related rafts after vitrification. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate and compare the efficacy of B-mode, Doppler, contrast-enhanced ultrasonography (CEUS), and Acoustic Radiation Force Impulse (ARFI) elastography in predicting malignancy in canine mammary masses. This was a prospective cohort study from 2014 to 2016, which included 153 bitches with one or more mammary masses. A total of 300 masses were evaluated by ultrasonography (B-mode, Doppler, CEUS, and ARFI) and subsequently classified as benign or malignant by histopathology. Each ultrasound parameters studied were compared between benign and malignant masses by Chi-square or Student tests and differences were considered significant when P < 0.01. For the variables that proved significant differences were estimated the cut-off point, sensitivity, specificity, accuracy, and area under curve (AUC) by receiver-operating characteristic curve (ROC) analysis in a logistic regression model using histopathological classification as reference, to assess and compare diagnostic performance of each technique. Out of 300 mammary masses evaluated 246 were classified as malignant and 54 as benign. B-mode measurements showed sensitivity 67.9%, and specificity 67.6% as malignancy predictors on canine mammary masses; Doppler indexes systolic (>21.2 m/s) and diastolic velocity (>4.8 m/s) sensitivity 79.2% and specificity 70.8%; CEUS wash-out time (<80.5 s) sensitivity 80.2% and specificity 16.7%; and ARFI elastography shear velocity (SWV > 2.57 m/s) sensitivity 94.7% and specificity 97.2% In conclusion B-mode and Doppler ultrasound evaluations may assist in malignancy prediction of canine mammary masses with moderate sensitivity and specificity, already the SWV was an great accurate predictor. Therefore, ARFI elastography exam inclusion in veterinary clinic oncology and research is highly recommended, since it allows fast, non-invasive, and complication-free malignancy prediction of canine mammary masses. [ABSTRACT FROM AUTHOR]

The aims of the present study were to evaluate the predictive value of the platelet-to-lymphocyte ratio for peritoneal metastasis in patients with gastric cancer and to construct an available preoperative prediction system for peritoneal metastasis. A total of 1080 patients with gastric cancer were enrolled in our study. The preoperative platelet-to-lymphocyte ratio and other serum markers and objective clinical tumor characteristics were evaluated by receiver operating characteristic curves. A logistic analysis was performed to determine the independent predictive indicators of peritoneal metastasis. A prediction system that included the independent predictive indicators was constructed and evaluated by receiver operating characteristic curves. Based on the receiver operating characteristic curves, the ideal platelet-to-lymphocyte ratio cutoff value to predict peritoneal metastasis was 131.00. The logistic analysis showed that the platelet-to-lymphocyte ratio was an independent indicator to predict peritoneal metastasis. The area under the receiver operating characteristic curve was 0.599. When integrating all independent indicators (i.e., platelet-to-lymphocyte ratio, invasion depth, lymphatic invasion, pathological type), the prediction system more reliably predicted peritoneal metastasis with a higher area under the receiver operating characteristic curve (0.769). The preoperative platelet-to-lymphocyte ratio was an indicator that could be used to predict peritoneal metastasis. Our prediction system could be a reliable instrument to discriminate between patients with gastric cancer with and those without peritoneal metastasis. [ABSTRACT FROM AUTHOR]

The invasive larvae (cercariae) of schistosomes penetrate the skin of their definitive hosts. During the invasion, they undergo dramatic ultrastructural and physiological transitions. These changes result in the development of the subsequent stage, schistosomulum, which migrates through host tissues in close contact with host’s immune system. One of the striking changes in the transforming cercariae is the shedding of their thick tegumental glycocalyx, which represents an immunoattractive structure; therefore its removal helps cercariae to avoid immune attack. A set of commercial fluorescently labeled lectin probes, their saccharide inhibitors and monoclonal antibodies against the trisaccharide Lewis-X antigen (LeX, CD15) were used to characterize changes in the surface saccharide composition of the neuropathogenic avian schistosome Trichobilharzia regenti during the transformation of cercariae to schistosomula, both in vitro and in vivo. The effect of various lectins on glycocalyx shedding was evaluated microscopically. The involvement of peptidases and their inhibitors on the shedding of glycocalyx was investigated using T. regenti recombinant cathepsin B2 and a set of peptidase inhibitors. The surface glycocalyx of T. regenti cercariae was rich in fucose and mannose/glucose residues. After the transformation of cercariae in vitro or in vivo within their specific duck host, reduction and vanishing of these epitopes was observed, and galactose/N-acetylgalactosamine emerged. The presence of LeX was not observed on the cercariae, but the antigen was gradually expressed from the anterior part of the body in the developing schistosomula. Some lectins which bind to the cercarial surface also induced secretion from the acetabular penetration glands. Seven lectins induced the shedding of glycocalyx by cercariae, among which five bound strongly to cercarial surface; the effect could be blocked by saccharide inhibitors. Mannose-binding protein, part of the lectin pathway of the complement system, also bound to cercariae and schistosomula, but had little effect on glycocalyx shedding. Our study did not confirm the involvement of proteolysis in glycocalyx shedding. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the diagnostic accuracy of a single CEA (carcinoembryonic antigen) blood test in detecting colorectal cancer recurrence. Background: Patients who have undergone curative resection for primary colorectal cancer are typically followed up with scheduled CEA testing for 5 years. Decisions to investigate further (usually by CT imaging) are based on single test results, reflecting international guidelines. Methods: A secondary analysis was undertaken of data from the FACS trial (two arms included CEA testing). The composite reference standard applied included CT-CAP imaging, clinical assessment and colonoscopy. Accuracy in detecting recurrence was evaluated in terms of sensitivity, specificity, likelihood ratios, predictive values, time-dependent area under the ROC curves, and operational performance when used prospectively in clinical practice are reported. Results: Of 582 patients, 104 (17.9%) developed recurrence during the 5 year follow-up period. Applying the recommended threshold of 5μg/L achieves at best 50.0% sensitivity (95% CI: 40.1–59.9%); in prospective use in clinical practice it would lead to 56 missed recurrences (53.8%; 95% CI: 44.2–64.4%) and 89 false alarms (56.7% of 157 patients referred for investigation). Applying a lower threshold of 2.5μg/L would reduce the number of missed recurrences to 36.5% (95% CI: 26.5–46.5%) but would increase the false alarms to 84.2% (924/1097 referred). Some patients are more prone to false alarms than others—at the 5μg/L threshold, the 89 episodes of unnecessary investigation were clustered in 29 individuals. Conclusion: Our results demonstrated very low sensitivity for CEA, bringing to question whether it could ever be used as an independent triage test. It is not feasible to improve the diagnostic performance of a single test result by reducing the recommended action threshold because of the workload and false alarms generated. Current national and international guidelines merit re-evaluation and options to improve performance, such as making clinical decisions on the basis of CEA trend, should be further assessed. [ABSTRACT FROM AUTHOR]

Although there are many studies about pluripotent stem cells, little is known about pluripotent pathways and the difficulties of maintaining the pluripotency of bovine cells in vitro. Here, we investigated differently expressed genes (DEG) in bovine embryo-derived stem-like cells (eSLCs) from various origins to validate their distinct characteristics of pluripotency and differentiation. We identified core pluripotency markers and additional markers which were not determined as pluripotency markers yet in bovine eSLCs. Using the KEGG database, TGFβ, WNT, and LIF signaling were related to the maintenance of pluripotency. In contrast, some DEGs related to the LIF pathway were down-regulated, suggesting that reactivation of the pathway may be required for the establishment of true bovine embryonic stem cells (ESCs). Interestingly, oncogenes were co-down-regulated, while tumor suppressor genes were co-up-regulated in eSLCs, implying that this pattern may induce abnormal teratomas. These data analyses of signaling pathways provide essential information on authentic ESCs in addition to providing evidence for pluripotency in bovine eSLCs. [ABSTRACT FROM AUTHOR]

Introduction: Knowledge of ovine mesenchymal stromal cells (oMSCs) is currently expanding. Tissue engineering combining scaffolding with oMSCs provides promising therapies for the treatment of osteochondral diseases. Purpose: The aim was to isolate and characterize oMSCs from bone marrow aspirates (oBMSCs) and to assess their usefulness for osteochondral repair using β-tricalcium phosphate (bTCP) and type I collagen (Col I) scaffolds. Methods: Cells isolated from ovine bone marrow were characterized morphologically, phenotypically, and functionally. oBMSCs were cultured with osteogenic medium on bTCP and Col I scaffolds. The resulting constructs were evaluated by histology, immunohistochemistry and electron microscopy studies. Furthermore, oBMSCs were cultured on Col I scaffolds to develop an in vitro cartilage repair model that was assessed using a modified International Cartilage Research Society (ICRS) II scale. Results: oBMSCs presented morphology, surface marker pattern and multipotent capacities similar to those of human BMSCs. oBMSCs seeded on Col I gave rise to osteogenic neotissue. Assessment by the modified ICRS II scale revealed that fibrocartilage/hyaline cartilage was obtained in the in vitro repair model. Conclusions: The isolated ovine cells were demonstrated to be oBMSCs. oBMSCs cultured on Col I sponges successfully synthesized osteochondral tissue. The data suggest that oBMSCs have potential for use in preclinical models prior to human clinical studies. [ABSTRACT FROM AUTHOR]

Several inducers have been used to differentiate embryonic stem cells (ESCs) into male germ cells but the induction process has been inefficient. To solve the problem of low efficiency of inducer for ESCs differentiation into male germ cells, all-trans retinoic acid (ATRA), Am80(the retinoic acid receptor agonist), and estradiol (E2) was used to induce ESCs to differentiate into male germ cells in vitro. ESCs were cultured in media containing ATRA, Am80, or E2 respectively which can differentiate ESCs into a germ cell lineage. In process of ATRA and Am80 induction Group, germ cell-like cells can be observed in 10 days; but have no in E2 induction Group. The marker genes of germ cell: Dazl, Stra8, C-kit, Cvh, integrinα6, and integrinβ1 all showed a significant up-regulation in the expression level. The ATRA-induction group showed high expression of C-kit and Cvh around 4 days, and integrinα6 and integrinβ1 were activated on day 10, respectively, while the E2-,Am80- induction group showed a high expression of C-kit as early as 4 days immunocytochemistry results shown that, integrinα6 and integrinβ1 could be detected in the ATRA-, Am80-, and E2-induction group, Positive clones in the ATRA group were greater in number than those in the other two groups. we conclued that ATRA, Am80, and E2 can promote the expression of the corresponding genes of germ cells, and had different effect on the differentiation of ESCs into male germ cells. ATRA was the most effective inducer of germ cell differentiation. [ABSTRACT FROM AUTHOR]

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to -proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. [ABSTRACT FROM AUTHOR]

Purpose: Many studies have reported that carbohydrate antigen 153 (CA153) in breast secretions (BS) can discriminate breast cancer (BC) patients from healthy individuals, indicating CA153 in BS as a potential index for BC. This meta-analysis aimed to evaluate the actual diagnostic value of CA153 in BS. Methods: Related papers were obtained from Pubmed, Embase, Scopus, Ovid, Sciverse, the Cochrane library, Chinese Biomedical literature Database (CBM), Technology of Chongqing (VIP), Wan Fang Data, and Chinese National Knowledge Infrastructure (CNKI). Pooled sensitivity, specificity, and diagnostic odds ratio (DOR) of CA153 in BS for BC diagnosis were analyzed with the random effect model. SROC and the area under the curve (AUC) were applied to assess overall diagnostic efficiency. Results: This meta-analysis included five studies with a total of 329 BC patients and 381 healthy subjects. For CA153 in BS, the summary sensitivity, specificity, and DOR to diagnose BC were 0.63 (95% confidence interval (CI): 0.57∼0.68), 0.82 (95% CI: 0.78∼0.86), and 9.18 (95% CI: 4.22∼19.95), respectively. Furthermore, the AUC of BS CA153 in the diagnosis of BC was 0.8614. Conclusions: CA153 in BS is a valuable molecular marker in diagnosing BC and should be applied in standard clinical practices of BC screening upon confirmation of our findings in a larger prospective study. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor 5 (Fgf5) has been widely used as a marker for the epiblast in the postimplantation embryo and epiblast stem cells (mEpiSCs) in the mouse, making it valuable for study of differentiation of various tissues and epiblast cells in vivo and in vitro. Here, we report for the first time the generation of Fgf5-P2A-Venus BAC transgenic (Tg) mice and show that the BAC Tg can recapitulate endogenous Fgf5 expression in epiblast and visceral endodermal cells of E6.5 and 7.5 embryos. We also show that Fgf5-P2A-Venus BAC Tg mEpiSCs in the undifferentiated state expressed abundant Venus, and upon reprogramming into naïve state, Venus was suppressed. Furthermore, while most Tg mEpiSCs expressed Venus abundantly, surprisingly the Tg mEpiSCs contained a minor subpopulation of Venus-negative cells that were capable of conversion to Venus-positive cells, indicating that even Fgf5 expression shows dynamic heterogeneity in mEpiSCs. Taken together, Fgf5-P2A-Venus BAC Tg mice and mEpiSCs generated in this study will be useful for developmental biology as well as stem cell biology research. [ABSTRACT FROM AUTHOR]

One standard treatment option for advanced-stage cancer is surgical resection of malignant tumors following by adjuvant chemotherapy and chemoradiotherapy. Additionally, neoadjuvant chemotherapy may be applied if required. During the time course of treatments, patients are generally followed by computed tomography (CT) surveillance, and by tumor marker diagnosis. However, currently, early evidence of recurrence and/or metastasis of tumors with a clinically relevant biomarker remains a major therapeutic challenge. In particular, there has been no validated biomarker for predicting treatment outcomes in therapeutic settings. Recently, we have looked at glycoforms of serum α1-acid glycoprotein (AGP) by using a crossed affinoimmunoelectrophoresis with two lectins and an anti-AGP antibody. The primary glycan structures of AGP were also analyzed by a mass spectrometer and a novel software in a large number of patients with various cancers. Accordingly, the relative abundance of α1,3fucosylated glycans in AGP (FUCAGP) was found to be significantly high in cancer patients as compared with the healthy controls. Further, strikingly elevated levels of FUCAGP were found in patients with poor prognosis but not in patients with good prognosis. In the current study, levels of FUCAGP in serum samples from various cancer patients were analyzed and 17 patients including 13 who had undergone chemotherapy were followed for several years post operation. FUCAGP level determined diligently by using a mass spectrometer was found to change along with disease prognosis as well as with responses to treatments, in particular, to various chemotherapies. Therefore, FUCAGP levels measured during following-up of the patients after operation appeared to be clinically relevant biomarker of treatment intervention. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSC) have been proposed as suitable candidates for cell therapy for neurological disorderssince they exhibit good neuronal differentiation capacity. However, for better therapeutic outcomes, it is necessary to isolate MSC from a suitable tissue sourcethat posses high neuronal differentiation. In this context, we isolated MSC from extra ocular muscle (EOM) tissue and tested the in vitro neuronal differentiation potential. In the current study, EOM tissue derived MSC were characterized and compared with bone marrow derived MSC. We found that EOM derived MSC proliferated as a monolayer and showed similarities in morphology, growth properties and cell surface marker expression with bone marrow derived MSC and expressed high levels of NES, OCT4, NANOG and SOX2 in its undifferentiated state. They also expressed embryonic cell surface marker SSEA4 and their intracellular mitochondrial distribution pattern was similar to that of multipotent stem cells. Although EOM derived MSC differentiated readily into adipocytes, osteocytes and chondrocytes, they differentiated more efficiently into neuroectodermal cells. The differentiation into neuroectodermal cellswas confirmed by the expression of neuronal markers NGFR and MAP2B. Thus, EOM derived MSC might be good candidates for stem cell based therapies for treating neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the predictive value of the apparent diffusion coefficient (ADC) for pathologic complete response (pCR) to neoadjuvant chemoradiotherapy (NCRT) in locally advanced rectal cancer. Methods: A total of 265 patients with rectal adenocarcinoma, whole Diffusion-Weighted MRI (DWI-MRI) images, clinically stage II to III (cT3-4 and/or cN+) and treated with NCRT followed by TME were screened. Fifty patients with pCR and another 50 patients without pCR with similar clinical charcacters and treatment regimens were selected for statistical analysis. All the patients’ pre-CRT and post-CRT average ADC values were calculated from the coefficient maps created by DWI-MRI and recorded independently. The difference in the ADC values between the pCR and non-pCR was analyzed by the Mann-Whitney U test. The cut-off ADC value of the receiver operating characteristic (ROC) curve with pCR was then established. Results: The mean pre- and post-ADC values in all patients, and in pCR patients and non-pCR patients were 0.879±0.06 and 1.383±0.11, 0.859±0.04 and 1.440±0.10, 0.899±0.07 and 1.325±0.09 (×10-3mm2/s), respectively. The difference between the pre- and post-ADC values in all patients, pCR patients, and non-pCR patients were considered to be statistically significant. The pre-ADC value was significantly lower in the pCR patients than in the non-pCR patients (p = 0.003), whereas the post-ADC values were significantly higher in the pCR patients than in the non-pCR patients. The percentage increase of the ADC value (ΔADC%) in the pCR and non-pCR patients were 68% and 48% respectively (p<0.001). The ROC curves of the cut-off value of the pre-CRT patient ADC value was 0.866×10-3mm2/s. The AUC, sensitivity, specificity, PPV, NPV, and accuracy of diagnosing pCR were 0.670 (95% CI 0.563–0.777), 0.600, 0.640, 60%, 60%, and 60%, respectively. The cut-off value of ΔADC% was 58%. The corresponding AUC, sensitivity, specificity, PPV, NPV, and accuracy of diagnosing pCR were 0.856 (95% CI 0.783–0.930), 0.800, 0.760, 76.9%, 79.2%, and 78%, respectively. Conclusions: DWI-MRI technology can be efficient for predicting pCR for LARC after NCRT. Although the mean pre-CRT ADC value and the ΔADC% are moderate predictors for pCR, the latter would be more accurate. [ABSTRACT FROM AUTHOR]

To identify putative biomarkers of porcine spermatogonial stem cells (pSSCs), total RNA sequencing (RNA-seq) analysis was performed on 5- and 180-day-old porcine testes and on pSSC colonies that were established under low temperature culture conditions as reported previously. In total, 10,184 genes were selected using Cufflink software, followed by a logarithm and quantile normalization of the pairwise scatter plot. The correlation rates of pSSCs compared to 5- and 180-day-old testes were 0.869 and 0.529, respectively and that between 5- and 180-day-old testes was 0.580. Hierarchical clustering data revealed that gene expression patterns of pSSCs were similar to 5-day-old testis. By applying a differential expression filter of four fold or greater, 607 genes were identified between pSSCs and 5-day-old testis, and 2118 genes were identified between the 5- and 180-day-old testes. Among these differentially expressed genes, 293 genes were upregulated and 314 genes were downregulated in the 5-day-old testis compared to pSSCs, and 1106 genes were upregulated and 1012 genes were downregulated in the 180-day-old testis compared to the 5-day-old testis. The following genes upregulated in pSSCs compared to 5-day-old testes were selected for additional analysis: matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 1 (MMP1), glutathione peroxidase 1 (GPX1), chemokine receptor 1 (CCR1), insulin-like growth factor binding protein 3 (IGFBP3), CD14, CD209, and Kruppel-like factor 9 (KLF9). Expression levels of these genes were evaluated in pSSCs and in 5- and 180-day-old porcine testes. In addition, immunohistochemistry analysis confirmed their germ cell-specific expression in 5- and 180-day-old testes. These finding may not only be useful in facilitating the enrichment and sorting of porcine spermatogonia, but may also be useful in the study of the early stages of spermatogenic meiosis. [ABSTRACT FROM AUTHOR]

Background: The identification of surgical non-small cell lung cancer (NSCLC) patients with poor prognosis is a priority in clinical oncology because of their high 5-year mortality. This meta-analysis explored the prognostic value of maximal standardized uptake value (SUVmax), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) on disease-free survival (DFS) and overall survival (OS) in surgical NSCLC patients. Materials and Methods: MEDLINE, EMBASE and Cochrane Libraries were systematically searched until August 1, 2015. Prospective or retrospective studies that evaluated the prognostic roles of preoperative 18F-FDG PET/CT with complete DFS and OS data in surgical NSCLC patients were included. The impact of SUVmax, MTV or TLG on survival was measured using hazard ratios (HR). Sub-group analyses were performed based on disease stage, pathological classification, surgery only and cut-off values. Results: Thirty-six studies comprised of 5807 patients were included. The combined HRs for DFS were 2.74 (95%CI 2.33–3.24, unadjusted) and 2.43 (95%CI: 1.76–3.36, adjusted) for SUVmax, 2.27 (95%CI 1.77–2.90, unadjusted) and 2.49 (95%CI 1.23–5.04, adjusted) for MTV, and 2.46 (95%CI 1.91–3.17, unadjusted) and 2.97 (95%CI 1.68–5.28, adjusted) for TLG. The pooled HRs for OS were 2.54 (95%CI 1.86–3.49, unadjusted) and 1.52 (95%CI 1.16–2.00, adjusted) for SUVmax, 2.07 (95%CI 1.16–3.69, unadjusted) and 1.91 (95%CI 1.13–3.22, adjusted) for MTV, and 2.47 (95%CI 1.38–4.43, unadjusted) and 1.94 (95%CI 1.12–3.33, adjusted) for TLG. Begg’s test detected publication bias, the trim and fill procedure was performed, and similar HRs were obtained. The prognostic role of SUVmax, MTV and TLG remained similar in the sub-group analyses. Conclusions: High values of SUVmax, MTV and TLG predicted a higher risk of recurrence or death in patients with surgical NSCLC. We suggest the use of FDG PET/CT to select patients who are at high risk of disease recurrence or death and may benefit from aggressive treatments. [ABSTRACT FROM AUTHOR]

Background: Anaplastic lymphoma kinase (ALK) rearrangements define a subgroup of lung cancer which is eligible to targeted kinase inhibition. The aim of this study is to observe the incidence rate of ALK fusion in a large cohort of Chinese digestive tract cancer patients. Patients and Methods: Tissue microarray (TMA) was constructed from 808 digestive tract cancer cases, including 169 esophageal squamous cell carcinoma, 182 gastric cancer and 457 colorectal cancer (CRC) cases. We tested all cases for ALK expression via a fully automated immunohistochemistry (IHC) assay. The IHC-positive cases were subjected to fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (qRT-PCR), target gene enrichment and sequencing for confirmation of ALK gene rearrangement and discovery of novel fusion partner. Results: Among the tested cases, 2 (0.44%) CRC cases showed positive both by IHC and FISH. By qRT-PCR, EML4–ALK fusion was found in one IHC-positive CRC case. In another IHC-positive CRC case, target gene enrichment and sequencing revealed ALK was fused to a novel partner, spectrin beta non-erythrocytic 1 (SPTBN1). One gastric cancer case showed partially positive IHC result, but no fusion was found by FISH and gene sequencing. Conclusions: The incidence rate of ALK gene fusion in Chinese CRC patients was 0.44%,but not detectable in gastric and esophageal cancers. The novel SPTBN1 -ALK fusion, together with other ALK fusion genes, may become a potential target for anti-ALK therapy. [ABSTRACT FROM AUTHOR]

Compared with many induced pluripotent stem cell (iPSC) lines generated using retrovirus and other non-integrating methods, the utilization of human protein-induced iPSC (piPSC) lines may provide a safer alternative for the generation of retinal pigment epithelial (RPE) cells for transplantation in retinal degenerative diseases. Here we assess the ability of piPSCs to differentiate into RPE cells, and to perform native RPE cell behavior. piPSCs were seeded in 6-well low-attachment plates to allow embryoid body formation, and then analyzed for pluripotent stem cell markers NANOG, SSEA4 and TRA-1-60 by immunofluorescence. Following colony formation, piPSCs were assessed for confirmation of RPE cell differentiation by staining for zonula occludens (ZO-1), bestrophin, microphthalmia-associated transcription factor (MITF) and retinal pigment epithelium specific protein-65 (RPE65). To evaluate piPSC-RPE cell phagocytic ability, adult bovine photoreceptor rod outer segments (ROS) were fed to piPSC-RPE cells, which were analyzed by fluorescent microscopy and flow cytometry. Undifferentiated piPSCs expressed all pluripotent markers assessed and formed embryoid body aggregates after 7 days. Differentiated piPSC-RPE cells expressed ZO-1, bestrophin, MITF and RPE65, typical RPE cell markers. Flow cytometry revealed robust ingestion of fluorescently-labeled ROS by piPSC-RPE cells, which was over four-times greater than that of undifferentiated piPSCs and comparable to that of an immortalized RPE cell line. Phagocytosis activity by piPSC-RPE cells was significantly reduced after the addition of anti-integrin αVβ5. In conclusion, piPSCs can be differentiated toward an RPE cell fate, expressing RPE cell markers and resembling native RPE cells in behavior. These results demonstrate that piPSCs can be differentiated into RPE-like cells using a method that has an increased safety profile, a critical consideration for the development of better treatments for retinal degenerative diseases such as age-related macular degeneration (AMD). [ABSTRACT FROM AUTHOR]