Metabarcoding analysis is an effective technique for monitoring the domoic acid-producing Pseudo-nitzschia species in marine environments, uncovering high-levels of molecular diversity. However, such efforts may result in the overinterpretation of Pseudo-nitzschia species diversity, as molecular diversity not only encompasses interspecies and intraspecies diversities but also exhibits extensive intragenomic variations (IGVs). In this study, we analyzed the V4 region of the 18S rDNA of 30 strains of Pseudo-nitzschia multistriata collected from the coasts of China. The results showed that each P. multistriata strain harbored about a hundred of unique 18S rDNA V4 sequence varieties, of which each represented by a unique amplicon sequence variant (ASV). This study demonstrated the extensive degree of IGVs in P. multistriata strains, suggesting that IGVs may also present in other Pseudo-nitzschia species and other phytoplankton species. Understanding the scope and levels of IGVs is crucial for accurately interpreting the results of metabarcoding analysis.

While canonical Wnt signaling is well recognized for its crucial regulatory functions in cell fate decisions, the role of non-canonical Wnt signaling in adult stem cells remains elusive and contradictory. Here, we identified Mcam , a potential member of the non-canonical Wnt signaling, as an important negative regulator of mammary gland epithelial cells (MECs) by genome-scale CRISPR-Cas9 knockout (GeCKO) library screening. Loss of Mcam increases the clonogenicity and regenerative capacity of MECs, and promotes the proliferation, differentiation, and ductal morphogenesis of mammary epithelial in knockout mice. Mechanically, Mcam knockout recruits and polarizes macrophages through the Il4-Stat6 axis, thereby promoting secretion of the non-canonical Wnt ligand Wnt5a and its binding to the non-canonical Wnt signaling receptor Ryk to induce the above phenotypes. These findings reveal Mcam roles in mammary gland development by orchestrating communications between MECs and macrophages via a Wnt5a/Ryk axis, providing evidences for non-canonical Wnt signaling in mammary development.

Efficient milk production in mammals confers evolutionary advantages by facilitating the transmission of energy from mother to offspring. However, the regulatory mechanism responsible for the gradual establishment of milk production efficiency in mammals, from marsupials to eutherians, remains elusive. Here, we find that mammary gland of the marsupial sugar glider contained milk components during adolescence, and that mammary gland development is less dynamically cyclic compared to that in placental mammals. Furthermore, fused in sarcoma (FUS) is found to be partially responsible for this establishment of low efficiency. In mouse model, FUS inhibit mammary epithelial cell differentiation through the cyclin-dependent kinase inhibitor p57Kip2, leading to lactation failure and pup starvation. Clinically, FUS levels are negatively correlated with milk production in lactating women. Overall, our results shed light on FUS as a negative regulator of milk production, providing a potential mechanism for the establishment of milk production from marsupial to eutherian mammals. Efficient milk production in mammals confers advantages by facilitating the transmission of energy from mother to offspring. However, the factors for establishing the efficiency in mammals are unknown. Here, the authors identify FUS as a regulator of efficient milk production in mammals.

Background: Peripheral blood inflammation indices, including the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and systemic immune-inflammation index (SII), have become research hotspots in the diagnosis, treatment, and prognosis prediction of breast cancer, whereas existing research findings remain controversial.Methods: Data pertaining to 1808 breast cancer patients were collected retrospectively to analyze the predictive value of NLR/PLR/ SII for breast cancer clinicopathological characteristics, chemotherapy response, and relapse. 1489, 258, and 53 eligible breast cancer patients entered into the three analyses, respectively. Logistic regression analyses were used to assess the correlation between these indices and poor response to chemotherapy. A predictive scoring model was established to predict chemotherapeutic responses based upon the odds ratio values of significant variables identified in logistic regression analyses. Results: Higher pretherapeutic NLR/PLR/SII values were significantly correlated with higher tumor stage, triple-negative breast cancer, premenopausal status, and younger age. Logistic regression analyses indicated that pretherapeutic high SII (as a continuous variable or with a cut-off value of 586.40) and HER2-negative status were independent predictors of poor response to neoadjuvant chemotherapy. A first-in-class SII-based predictive scoring model well distinguished patients who might not benefit from neoadjuvant chemotherapy, with an area under the curve of 0.751. In HR-positive cancers, SII was more strongly associated with clinicopathological features and chemotherapy response. In addition, a receiver operating characteristic curve analysis indicated that the specificity of follow-up SII in identifying cancer relapse was greater than 98.0% at a cut-off value of 900.Conclusion: As a predictor of breast cancer, especially in the HR-positive subtype, SII may eclipse NLR/PLR. SII-high patients are more likely to have a worse chemotherapy response and a higher risk of recurrence.

Objective: To analyze the role of circMYC in cervical cancer. Methods: Protein and RNA expression was detected by RT-qPCR and western blotting. Transwell, CCK8, and colony formation assays were used for measuring metastasis, cell viability, and proliferation, respectively. Lactate production, glucose uptake, and ATP generation were examined to evaluate cell glycolysis. Interactions between circMYC, miR-577, and MET were determined by RNA pull-down and immunoprecipitation, and dual-luciferase reporter assays. Xenografts were established in mice to evaluate the functions of circMYC in vivo. Results: circMYC was overexpressed in tumor tissue, which was related to poor prognosis. CircMYC knockdown reduced proliferation, colony formation, metastasis, and glycolysis in cervical cancer cells as well as inhibiting tumor growth in vivo. Mechanistically, circMYC targeted miR-577, and the effects of circMYC knockdown could be reversed by miR-577 inhibition. Moreover, miR-577 downregulated the expression of MET. Therefore, the oncogenic role of circMYC in cervical cancer was achieved by sponging miR-577 and maintaining MET expression. Conclusion: circMYC promotes cervical cancer progression through regulation of the miR-577/ MET axis. circMYC may thus be a potential target for diagnosing and treating cervical cancer.

The present study aimed to investigate the protective effects of ghrelin against oxidative stress, inducible nitric oxide synthase (iNOS) and inflammation in a mouse model of myocardial ischemia/reperfusion injury (MIRI). In addition, the study aimed to determine its underlying mechanisms. A mouse model of MIRI was used in vivo, in order to ascertain the protective effects of ghrelin on MIRI. Commercial kits were used to measure the serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH) in MIRI mice. Furthermore, Evan's Blue-triphenyltetrazolium chloride solution was used to analyze the protective effects of ghrelin against infarct size in MIRI mice. The underlying mechanisms were determined by measuring MIRI-induced tumor necrosis factor (TNF)‑α, interleukin (IL)‑6, superoxide dismutase (SOD), glutathione (GSH), GSH-peroxidase (GSH‑PX), malondialdehyde (MDA) and caspase‑3/caspase‑9 activities, and iNOS, high mobility group box 1 (HMGB1), Toll‑like receptor 4 (TLR4) and nuclear factor (NF)‑κB protein expression in MIRI mice. The results demonstrated that MIRI led to an increase in infarct size; CK, LDH, TNF‑α, IL‑6, MDA, caspase‑3 and caspase-9 serum levels; and iNOS protein expression. MIRI resulted in inhibition of SOD, FSH and GSH‑PX levels. Conversely, these alterations were significantly inhibited following treatment with ghrelin. In addition, the protective effects of ghrelin against MIRI suppressed HMGB1, TLR4 and NF‑κB protein expression in MIRI mice. The present study revealed that ghrelin exerted protective effects against oxidative stress, iNOS and inflammation in MIRI mice via the HMGB1/TLR4/NF-κB pathway.

Melatonin can modulate neural stem cell ( NSC ) functions such as proliferation and differentiation into NSC ‐derived pluripotent stem cells (N‐ iPS ) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSC s isolated from the retinal neural layer could transform into N‐ iPS cell. NSC s were exposed to 0.01, 0.1, 1, 10, or 100 μ m melatonin, and cell viability studies indicated that 10 μ m melatonin can significantly increase cell viability and promote cell proliferation in NSC s in vitro. Thus, 10 μ m melatonin was used to study miR‐302/367‐mediated cell reprogramming of NSC s. We noted that this concentration of melatonin increased reprogramming efficiency of N‐ iPS cell generation from primary cultured bovine NSC s and that this was mediated by downregulation of apoptosis‐related genes p53 and p21. Then, N‐ iPS cells were treated with 1, 10, 100, or 500 μ m melatonin, and N‐ iPS (M‐N‐ iPS ) cell proliferation was measured. We noted that 100 μ m melatonin increased proliferation of N‐ iPS cells via increased phosphorylation of intracellular ERK 1/2 via activation of its pathway in M‐N‐ iPS via melatonin receptors 1 ( MT 1). Finally, we verified that N‐ iPS cells and M‐N‐ iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo , and the capacity to differentiate into all three embryonic germ layers.

Limited microRNAs (miRNAs, miRs) have been reported to be necessary for exercise-induced cardiac growth and essential for protection against pathological cardiac remodeling. Here we determined members of the miR-17-92 cluster and their passenger miRNAs expressions in two distinct murine exercise models and found that miR-17-3p was increased in both. miR-17-3p promoted cardiomyocyte hypertrophy, proliferation, and survival. TIMP-3 was identified as a direct target gene of miR-17-3p whereas PTEN was indirectly inhibited by miR-17-3p. Inhibition of miR-17-3p in vivo attenuated exercise-induced cardiac growth including cardiomyocyte hypertrophy and expression of markers of myocyte proliferation. Importantly, mice injected with miR-17-3p agomir were protected from adverse remodeling after cardiac ischemia/reperfusion injury. Collectively, these data suggest that miR-17-3p contributes to exercise-induced cardiac growth and protects against adverse ventricular remodeling. miR-17-3p may represent a novel therapeutic target to promote functional recovery after cardiac ischemia/reperfusion.

Improving the dysfunction of endothelial progenitor cell (EPCs) in patients with diabetes mellitus is important for preventing vascular complication. Vaspin, an adipocytokine, has the anti-atherogenic properties rely on its positive effect on nitric oxide (NO) bioavailability. We hypothesis that vaspin may ameliorate high glucose induced dysfunction of EPCs. In rat bone marrow derived EPCs, glucose treatment results in a decrease in the proliferation and migration capacity in a dose dependent manner. These detrimental effects can be alleviated by vaspin. Furthermore, vaspin increased the production of NO and the effect of vaspin on EPCs can be diminished partly by the eNOS inhibitor (L-NAME). We assessed total eNOS protein expression and Ser(1177)-phospho-eNOS expression and found that vaspin not only induced eNOS protein expression but also up regulate the eNOS activation. Subsequently, we investigated protein kinase B (Akt) activation in the presence and absence of phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor (LY-2940002). Vaspin increased total Akt and Ser(473)-phospho-Akt expression and these effects can be blocked by LY-2940002. The results of our study indicate a novel effect of vaspin to regulate eNOS expression and function in EPCs via a PI3K/Akt/eNOS pathway; vaspin may have a protective effect in patients with diabetes to prevent the occurrence of vascular complication.

Although the mortality of cerebrovascular disease (CVD) has been steadily declined in the European Union (EU), CVD remains among the major causes of death in EU. As risk factors such asobesity and diabetes mellitus are increasing, the trends of European CVD mortality remains unknown. To understand the variation in CVD mortality of different EU countries, we studied the trends in CVD mortality in EU countries over the last three decades between males and females. Age- and sex-specific mortality rates between 1980 and 2011 were calculated by data from the WHO mortality database. Joinpoint software was used to calculate annual percentage changes and to characterize trends in mortality rates over time. Our study showed that between 1980 and 2011, CVD mortality significantly decreased in both men and women across all age groups. The specific mortality trends varied largely between EU countries. The plateau trend was observed in little regions at different age groups, however, the EU as a whole displayed declined trend CVD mortality. During the last three decades, CVD mortality decreased substantially in the entire population of EU. However, despite this overall decline in CVD mortality, several areas were identified as having no change in their CVD mortality rates at different period. The whole EU needs to establish strict prevention measures toreduce the incidence of CVD risk factors.

Bovine bone marrow-derived mesenchymal stem cells (bBMSCs) were isolated from the bone marrow of a 4-6-month-old fetal bovine and then characterized by immunofluorescence and reverse transcriptase polymerase chain reaction. We found that primary bBMSCs could be expanded for 46 passages; the total culture time in vitro was 125 days. The results of surface antigen detection showed that bBMSCs expressed CD29, CD44, and CD73 but did not express endothelial cells and hematopoietic cells-specific marker CD31, CD34, and CD45. The cells from four passages (passages 3, 9, 15, and 25) were successfully induced to differentiate into osteoblasts, adipocytes, hepatic, and islet-like cells. The results indicate the potential for multi-lineage differentiation of bBMSCs that may represent an ideal candidate for cellular transplantation therapy.

Aims: Endurance exercise has been proved to be a stimulus for cardiac growth and it can induce cardiac hypertrophy and cardiomyocyte proliferation. Although genes and transcriptional factors responsible for that has been well-established, little is known about the potential role of microRNAs (miRNAs, miRs) in this physiological process. The miR-17-92 cluster has been reported to promote cell proliferation and play anti-apoptotic roles in tumor cells. This study aims at investigating the role of miR-17-92 cluster or its member in exercise-induced cardiac growth. Methods: Endurance exercise was induced by swimming the mice for 3 weeks. Using quantitative reverse transcription polymerase chain reactions (RT-PCRs), members in miR-17-92 clusters were determined and miR-17-3p was found to be significantly up-regulated. The expression levels of miR-17-3p in ventricular tissue of mice subjected to transaortic constriction (TAC) and DCM patients were also determined using RT-PCRs. In cultured cardiomyocytes, the e...

Cetuximab, a monoclonal antibody (mAb) targeting the epidermal growth factor receptor (EGFR), has been intensively investigated as a promising cancer treatment strategy. The specific mechanism of cetuximab endocytosis and its influence on cetuximab uptake, biodistribution and efficacy still remain elusive. Recently, statins have been reported to synergize with EGFR-targeting agents. Our prior work established that nystatin, a cholesterol-sequestering antifungal drug, facilitates endocytosis via the clathrin-dependent pathway. This study aimed to investigate whether nystatin regulates the uptake and efficacy of cetuximab and cetuximab-based antibody-drug conjugates (cetuximab-ADCs). In vitro and in vivo efficacies of nystatin on the uptake and activity of cetuximab/cetuximab-ADCs were studied in multiple human carcinoma cell lines and xenograft models, respectively. We identified that cholesterol sequestration by nystatin enhanced cetuximab internalization in EGFR-positive carcinoma cells by regulating EGFR trafficking/turnover and facilitating a switch from lipid rafts to clathrin-mediated endocytosis. Combination treatment with cetuximab and nystatin selectively increased cetuximab uptake by tumor tissues, translating into potentiated antitumor efficacy of cetuximab in vivo (A431 and A549 tumors). Nystatin-enhanced internalization of cetuximab further improved the uptake and potency of cetuximab-doxorubicin and cetuximab-methotrexate conjugates in EGFR-positive cetuximab-resistant tumors. Combination therapy with nystatin plus either cetuximab or cetuximab-ADC further prolonged animal survival and significantly suppressed tumor growth, as compared with single-agent cetuximab or cetuximab-ADC. In summary, our results identify a novel mechanism whereby cholesterol sequestration enhances the uptake of EGFR-targeting mAb and ADCs, therefore providing preclinical proof-of-concept that combination with nystatin can potentiate the delivery and efficacy of these EGFR-targeted agents.

1. Cardiac troponin I-interacting kinase (TNNI3K) is a novel cardiac-specific kinase gene. Quantitative real-time reverse transcription polymerase chain reaction analysis showed a significant increase in TNNI3K mRNA expression in hypertrophic cardiomyocytes induced by endothelin-1 (ET-1). The aim of the present study was to investigate the effects of TNNI3K on neonate rat cardiomyocyte hypertrophy induced by ET-1. 2. Adenoviruses were amplified in 293A cells. To determine a reasonable adenovirus infection dose cardiomyocytes were infected with an adenovirus carrying human TNNI3K (Ad-TNNI3K) at varying multiplicity of infection (MOI) and the expression of TNNI3K was analysed by western blot. 3. Cardiomyocytes were infected with either a control adenovirus carrying green fluorescent protein (Ad-GFP) or Ad-TNNI3K. Compared with Ad-GFP, the Ad-TNNI3K induced an increase in sarcomere organization, cell surface area, (3) H-leucine incorporation and β-MHC re-expression. This type of hypertrophic phenomenon is similar to that observed in Ad-GFP-infected hypertrophic cardiomyocytes induced by ET-1. To determine the functional role of TNNI3K in ET-1-induced hypertrophic cardiomyocytes, the cells were infected with Ad-GFP or Ad-TNNI3K. Ad-TNNI3K induced an increase in sarcomere organization, cell surface area and (3) H-leucine incorporation compared with Ad-GFP. 4. These results suggest that TNNI3K overexpression induces cardiomyocytes hypertrophy and accelerates hypertrophy in hypertrophic cardiomyocytes. Therefore, TNNI3K might be an interesting target for the clinical treatment of hypertrophy.

A fibroblast line was successfully established from ear marginal tissues of Piedmontese cattle by direct culturing of explants. Biological analysis showed that the population doubling time (PDT) for reviving cells was approximately 24 h. The average viability of the cells was 96.6% before freezing and 92.7% after thawing. Isoenzyme polymorphorism of lactic dehydrogenase (LDH) and malic dehydrogenase (MDH) ruled out cross-contamination among cell lines. Karyotyping showed that the proportion of cells with chromosome number 2n = 60 was above 92%. Tests for bacteria, fungi, viruses and mycoplasmas were negative. The transfection efficiencies of pEGFP-N3, pEYFP-N1 and pDsRed1-N1 were between 7.1 and 30.8%; fluorescences were homogenously distributed throughout cytoplasm and nucleus except in some cryptomeric vesicles. Every index of the Piedmontese cattle cell line met the quality control standards of the American Type Culture Collection (ATCC). Key words : Piedmontese cattle, fibroblast line, biological characterization.

BACKGROUND:Mutations in the lamin A/C gene (LMNA) may cause familial dilated cardiomyopathy (dilated cardiomyopathy) characterized by early onset atrio-ventricular block (A-V block) before the manifestation of dilated cardiomyopathy and high risk of sudden death due to ventricular arrhythmia, which is very similar to the phenotype of gap junction related heart disease. This study aimed to determine the expression and localization of connexins in neonatal myocytes transfected with wild-type (WT) or mutant LMNA to elucidate how these mutations cause heart diseases.METHODS:We studied the connexin 43 (Cx43) and connexin 40 (Cx40) expression in cultured neonatal myocytes transfected with wild-type (WT) or mutant LMNA (Glu82Lys (E82K) and Arg644Cys (R644C)) using confocal imaging and Western blotting analysis.RESULTS:Cx43 protein expression was reduced by 40% in cells transfected with LMNA E82K than that in cells transfected with WT LMNA cDNA. Confocal imaging showed that the Cx43 located inside the cells by LMNA E82K. By contrast, LMNA E82K mutation had no effect on expression and localization of Cx40. LMNA R644C transfection did not show any significant effects on gap junctions at all.CONCLUSIONS:Our findings suggest that LMNA E82K significantly reduced the Cx43 expression and altered its localization which may be one of the pathological mechanisms underlying LMNA-related heart disease.