High-throughput and direct measurement of androgen levels using turbulent flow chromatography liquid chromatography-triple quadrupole mass spectrometry (TFC-LC-TQMS) to discover chemicals that modulate dihydrotestosterone production in human prostate cancer cells

Objectives

To develop a high-throughput screening system to measure the conversion of testosterone to dihydrotestosterone (DHT) in cultured human prostate cancer cells using turbulent flow chromatography liquid chromatography-triple quadrupole mass spectrometry (TFC-LC-TQMS).

Results

After optimizing the cell reaction system, this method demonstrated a screening capability of 103 samples, including 78 single compounds and 25 extracts, in less than 12 h without manual sample preparation. Consequently, fucoxanthin, phenethyl caffeate, and Curcuma longa L. extract were validated as bioactive chemicals that inhibited DHT production in cultured DU145 cells. In addition, naringenin boosted DHT production in DU145 cells.

Conclusion

The method can facilitate the discovery of bioactive chemicals that modulate the DHT production, and four phytochemicals are potential candidates of nutraceuticals to adjust DHT levels in male hormonal dysfunction.

     

PKM2 knockdown influences SREBP activation and lipid synthesis in bovine mammary-gland epithelial MAC-T cells

Objective

The purpose of the article is to evaluate the changes in lipid metabolism in bovine mammary-gland epithelial MAC-T cells after PKM2 knockdown.

Results

MAC-T cells stably expressing low levels of PKM2 were established with lentivirus-mediated small hairpin RNA. Although the knockdown of PKM2 had no effect on MAC-T cell growth, the reduced expression of PKM2 attenuated the mRNA and protein expression of key enzymes involved in sterol synthesis through the SREBP pathway.

Conclusions

The downregulation of PKM2 significantly influenced lipid synthesis in bovine mammary-gland epithelial MAC-T cells. These findings extend our understanding of the crosstalk between glycolysis and lipid metabolism in bovine mammary-gland epithelial cells.

     

Copper oxide nanoparticles induce anticancer activity in A549 lung cancer cells by inhibition of histone deacetylase

Objectives

Copper oxide nanoparticles (CuO NPs) promoting anticancer activity may be due to the regulation of various classes of histone deacetylases (HDACs).

Results

Green-synthesized CuO NPs significantly arrested total HDAC level and also suppressed class I, II and IV HDACs mRNA expression in A549 cells. A549 cells treated with CuO NPs downregulated oncogenes and upregulated tumor suppressor protein expression. CuO NPs positively regulated both mitochondrial and death receptor-mediated apoptosis caspase cascade pathway in A549 cells.

Conclusion

Green-synthesized CuO NPs inhibited HDAC and therefore shown apoptosis mediated anticancer activity in A549 lung cancer cell line.

     

Recombinant neuritin affects the senescence,apoptosis, proliferation,and migration of rat bone marrow-derived mesenchymal stem cells

Objective

To study the effects of recombinant neuritin expressed by Pichia pastoris GS115 on the senescence, apoptosis, proliferation, and migration associated with rat bone marrow-derived mesenchymal stem cells (BMSCs).

Results

Recombinant neuritin was purified by Ni-affinity chromatography and identified by western blot and MALDI-TOF spectrometry. The effects of recombinant neuritin on senescence, apoptosis, proliferation, and migration of rat BMSCs WERE investigated. β-Galactosidase staining indicated that recombinant neuritin administration significantly inhibited BMSCs senescence at 1 μg neuritin/ml. Additionally, recombinant neuritin reduced the number of apoptotic cells at the early stage according to Annexin V/propidium iodide staining and inhibited cell proliferation according to MTT assay results. Moreover wound healing assay results showed that recombinant neuritin promoted BMSCs migration in the neuritin-treatment group.

Conclusion

Recombinant neuritin affects the senescence, apoptosis, proliferation, migration of rat BMSCs. Our findings offer insight into neuritin function outside of the nervous system.

     

RNA binding protein Lin28A promotes osteocarcinoma cells progression by associating with the long noncoding RNA MALAT1

Objectives

To explore the effects of Lin28A on progression of osteocarcinoma (OS) cells.

Results

Lin28A mRNA and protein expressions were significantly increased in OS tissues compared with that in normal adjacent tissues. Expressions of Lin28A and long noncoding RNA MALAT1 were positively correlated. Patients with higher Lin28A expression had shorter overall survival. Moreover, Lin28A knockdown inhibited OS cells proliferation, migration, invasion and promoted cell apoptosis; Lin28A was found to harbor binding sites on MALAT1 sequences and associated with MALAT1, and increased MALAT1 stability and expression. Notably, the inhibition of Lin28A knockdown was attenuated or even reversed by MALAT1 overexpression.

Conclusions

RNA binding protein Lin28A could facilitate OS cells progression by associating with the long noncoding RNA MALAT1.

     

Bone marrow-derived stem cells ameliorate hepatic fibrosis by down-regulating interleukin-17

Background

Accumulating evidences have identified the immunoregulatory features of stem cells. In this study, the immunoregulation of bone marrow-derived stem cells (BMSCs) transplanted into patients with HBV-related decompensated cirrhosis and mouse model of liver injury induced by carbon tetrachloride (CCl₄) administration was observed.

Results

Compared with healthy controls, patients with HBV-related decompensated cirrhosis showed significantly higher levels of TNF-alpha, IL-12, TGF-beta1, IL-17, and IL-8. However, only IL-17 was markedly decreased after autologous BMSCs transplantation during their follow-up. The same results were found in the CCl₄-treated mice. Furthermore, we found that exogenous IL-17 partly abolished the therapeutic effect of BMSCs whereas IL-17-specific antibody promoted improvement of liver injury in CCl₄-treated mice, resembling the therapeutic effect of BMSCs transplantation.

Conclusions

These data suggested that BMSCs transplantation induces a decrease of IL-17 level, which at least in part delineates the mechanisms of stem cells-mediated therapeutic benefit on liver disease.

     

MicroRNA-190b confers radio-sensitivity through negative regulation of Bcl-2 in gastric cancer cells

Objectives

To determine the role of miR-190b in radio-sensitivity of gastric cancer (GC).

Results

In radio-resistant GC cells, down-regulation of miR-190b and up-regulation of Bcl-2 were observed. The protein expression of Bcl-2 was negatively regulated by miR-190b. Overexpression of miR-190b significantly decreased cell viability and enhanced radio-sensitivity of GC cells. Of note, these effects of miR-190b on GC cells radio-sensitivity were abolished by Bcl-2.

Conclusion

miR-190b confers radio-sensitivity of GC cells, possibly via negative regulation of Bcl-2.

     

Effect of everolimus on the glucose metabolic pathway in mouse skeletal muscle cells (C2C12)

Introduction

Everolimus selectively inhibits mammalian target of rapamycin complex 1 (mTORC1) and exerts an antineoplastic effect. Metabolic disturbance has emerged as a common and unique side effect of everolimus.

Objectives

We used targeted metabolomic analysis to investigate the effects of everolimus on the intracellular glycometabolic pathway.

Methods

Mouse skeletal muscle cells (C2C12) were exposed to everolimus for 48 h, and changes in intracellular metabolites were determined by capillary electrophoresis time-of-flight mass spectrometry. mRNA abundance, protein expression and activity were measured for enzymes involved in glycometabolism and related pathways.

Results

Both extracellular and intracellular glucose levels increased with exposure to everolimus. Most intracellular glycometabolites were decreased by everolimus, including those involved in glycolysis and the pentose phosphate pathway, whereas no changes were observed in the tricarboxylic acid cycle. Everolimus suppressed mRNA expression of enzymes related to glycolysis, downstream of mTOR signaling enzymes and adenosine 5′-monophosphate protein kinases. The activity of key enzymes involved in glycolysis and the pentose phosphate pathway were decreased by everolimus. These results show that everolimus impairs glucose utilization in intracellular metabolism.

Conclusions

The present metabolomic analysis indicates that everolimus impairs glucose metabolism in muscle cells by lowering the activities of glycolysis and the pentose phosphate pathway.

     

Upregulation of citrullination pathway: From Autoimmune to Idiopathic Lung Fibrosis

Background

Increased protein citrullination and peptidylarginine deiminases (PADIs), which catalyze the citrullination process, are central in Rheumatoid arthritis pathogenesis and probably involved in the initial steps towards autoimmunity. Approximately, 10% of RA patients develop clinically significantly ILD. A possible shared role of protein citrullination in rheumatoid arthritis associated interstitial lung disease (RA-ILD), and idiopathic pulmonary fibrosis (IPF) pathogenesis remains unclear.

Methods

We evaluated PADI2 and PADI4 mRNA expression in bronchoalveolar lavage fluid (BALF) cells of 59 patients with IPF, 27 patients RA-ILD and 10 healthy controls. PADI 2 and 4 expression was analyzed by western blot and immunohistochemistry. Citrullinated protein levels were also quantified.

Results

PADI4 mRNA and protein levels were higher in RA-ILD and IPF than controls. Furthermore, PADI4 mRNA levels showed an increase among smokers in RA-ILD. PADI4 expression was detected in granulocytes and macrophages in all groups, with the strongest cytoplasmic expression observed in granulocytes in RA-ILD and IPF. PADI2 mRNA and immunostaining of BAL cells, were similar in all groups among smokers. Overall, stronger staining was observed in current smokers. Citrullinated peptides were significantly increased in IPF compared to RA-ILD and controls. In RA-ILD, protein citrullination strongly correlated with PADI4 expression and anti-citrullinated protein antibodies (ACPAs).

Conclusions

These results suggest that the citrullination pathway is upregulated in IPF and in RA-ILD.

     

Nanoformulation of curcumin protects HUVEC endothelial cells against ionizing radiation and suppresses their adhesion to monocytes: potential in prevention of radiation-induced atherosclerosis

Objectives

To investigated the potential of a novel dendrosomal nanoformulation of curcumin (DNC) in blocking radiation-induced changes in irradiated human umbilical vein endothelial cells (HUVECs), and their adhesion to human THP-1 monocytoid cells.

Results

Co⁶⁰ gamma rays reduced viability, raised the expression of adhesion molecules, ICAM-1, VCAM-1 and E-selectin (mRNA and protein), augmented the adhesion of THP-1 cells to HUVECs, activated NF-κB binding, increased the release of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) and induced oxidative damage (reduced glutathione declined, while 8-OHdG and TBARS increased). 5 µM DNC significantly inhibited these radiation-induced changes, activated the Nrf-2 pathway, and effectively suppressed THP-1 adhesion to HUVECs, implicating p38 MAPK signaling.

Conclusion

DNC treatment is a potential preventive method against inflammation and vascular damage from ionizing radiation.

     

Expression and purification of classical swine fever virus E2 protein from Sf9 cells using a modified vector

Objective

To develop a simple method for efficient expression of classical swine fever virus (CSFV) E2 protein.

Results

The pFastBac HT B vector (pFastHTB-M1) was modified by adding a melittin signal peptide sequence. The E2 gene fragment without the transmembrane region was cloned into pFastHTB-M1. The modified vector has clear advantage over the original one, as evidenced by the purified recombinant E2 protein that was detected significantly by SDS-PAGE.

Conclusions

The modified vector has the potential for large-scale production and easy purification of the CSFV E2 protein or other proteins of interests.

     

Differentiation of human adipose-derived stem cells into cardiomyocyte-like cells in fibrin scaffold by a histone deacetylase inhibitor

Background

Human adipose-derived stem cells (hADSCs) are capable of differentiating into many cells such as cardiac cells. Different types of inducers are used for cardiac cell differentiation, but this question still remains to be investigated, which one is the best. The aim of this paper was to investigate the effect of combination of fibrin scaffold and trichostatin A (TSA), for differentiation of hADSCs into cardiomyocyte-like cells.

Methods

After approval of characteristics of hADSCs and fibrin scaffold, hADSCs were cultured in fibrin scaffold with 10 µM TSA for 72 h and kept in standard conditions for 4 weeks. QRT-PCR and immunostaining assay were performed for evaluating the expression pattern of special cardiac genes and proteins.

Results

In particular, our study showed that fibrin scaffold alongside TSA enhanced expression of the selected genes and proteins.

Conclusions

We concluded that the TSA alone or with fibrin scaffold can lead to the generation of cardiac like cells in a short period of time.

     

Dexamethasone downregulates SIRT1 and IL6 and upregulates EDN1 genes in stem cells derived from gingivae via the AGE/RAGE pathway

Objectives

To evaluate the effects of dexamethasone on the aging of mesenchymal stem cells from human gingiva using next-generation sequencing.

Results

Four mRNAs were upregulated and 12 were downregulated when the results of dexamethasone at 24 h were compared with the control at 24 h. Expressions of SIRT1 and IL6 were decreased in dexamethasone at 24 h but expression of EDN1 was increased.

Conclusions

Application of dexamethasone reduced the expression of SIRT1 and IL6 but enhanced the expression of EDN1 of stem cells.

     

A methodology for elucidating regulatory mechanisms leading to changes in lipid profiles

Introduction

It is difficult to elucidate the metabolic and regulatory factors causing lipidome perturbations.

Objectives

This work simplifies this process.

Methods

A method has been developed to query an online holistic lipid metabolic network (of 7923 metabolites) to extract the pathways that connect the input list of lipids.

Results

The output enables pathway visualisation and the querying of other databases to identify potential regulators. When used to a study a plasma lipidome dataset of polycystic ovary syndrome, 14 enzymes were identified, of which 3 are linked to ELAVL1—an mRNA stabiliser.

Conclusion

This method provides a simplified approach to identifying potential regulators causing lipid-profile perturbations.

     

MiR-9 enhances the sensitivity of A549 cells to cisplatin by inhibiting autophagy

Objectives

To demonstrate that miR-9 inhibits autophagy by down-regulating Beclin1 and thus enhances the sensitivity of A549 cells to cisplatin.

Results

MiR-9 inhibited Beclin1 expression by binding to its 3′UTR. The inhibition decreased the cisplatin-induced autophagy in A549 cells, evidenced by the decreased expression of LC3II and GFP-LC3 puncta and the increased expression of P62. Upregulation of miR-9 level enhanced the sensibility of A549 cells to cisplatin and increased the cisplatin-induced apoptosis. Overexpression of Beclin1 reversed above effects of miR-9 mimics, cisplatin-induced autophagy was increased and apoptosis was decreased.

Conclusions

MiR-9 inhibits autophagy via targeting Beclin1 3′UTR and thus enhances cisplatin sensitivity in A549 cells.

     

A lost opportunity for science: journals promote data sharing in metabolomics but do not enforce it

Introduction

Data sharing is being increasingly required by journals and has been heralded as a solution to the ‘replication crisis’.

Objectives

(i) Review data sharing policies of journals publishing the most metabolomics papers associated with open data and (ii) compare these journals’ policies to those that publish the most metabolomics papers.

Methods

A PubMed search was used to identify metabolomics papers. Metabolomics data repositories were manually searched for linked publications.

Results

Journals that support data sharing are not necessarily those with the most papers associated to open metabolomics data.

Conclusion

Further efforts are required to improve data sharing in metabolomics.

     

Human umbilical endothelial cells (HUVECs) have a sex: characterisation of the phenotype of male and female cells

Background

Human umbilical endothelial cells (HUVECs) are widely used to study the endothelial physiology and pathology that might be involved in sex and gender differences detected at the cardiovascular level. This study evaluated whether HUVECs are sexually dimorphic in their morphological, proliferative and migratory properties and in the gene and protein expression of oestrogen and androgen receptors and nitric oxide synthase 3 (NOS3). Moreover, because autophagy is influenced by sex, its degree was analysed in male and female HUVECs (MHUVECs and FHUVECs).

Methods

Umbilical cords from healthy, normal weight male and female neonates born to healthy non-obese and non-smoking women were studied. HUVEC morphology was analysed by electron microscopy, and their function was investigated by proliferation, viability, wound healing and chemotaxis assays. Gene and protein expression for oestrogen and androgen receptors and for NOS3 were evaluated by real-time PCR and Western blotting, respectively, and the expression of the primary molecules involved in autophagy regulation [protein kinase B (Akt), mammalian target of rapamycin (mTOR), beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] were detected by Western blotting.

Results

Cell proliferation, migration NOS3 mRNA and protein expression were significantly higher in FHUVECs than in MHUVECs. Conversely, beclin-1 and the LC3-II/LC3-I ratio were higher in MHUVECs than in FHUVECs, indicating that male cells are more autophagic than female cells. The expression of oestrogen and androgen receptor genes and proteins, the protein expression of Akt and mTOR and cellular size and shape were not influenced by sex. Body weights of male and female neonates were not significantly different, but the weight of male babies positively correlated with the weight of the mother, suggesting that the mother’s weight may exert a different influence on male and female babies.

Conclusions

The results indicate that sex differences exist in prenatal life and are parameter-specific, suggesting that HUVECs of both sexes should be used as an in vitro model to increase the quality and the translational value of research. The sex differences observed in HUVECs could be relevant in explaining the diseases of adulthood because endothelial dysfunction has a crucial role in the pathogenesis of cardiovascular diseases, diabetes mellitus, neurodegeneration and immune disease.