Metabolomics Suggests That Soil Inoculation with Arbuscular Mycorrhizal Fungi Decreased Free Amino Acid Content in Roots of Durum Wheat Grown under N-Limited,P-Rich Field Conditions

Arbuscular mycorrhizal fungi (AMF) have a major impact on plant nutrition, defence against pathogens, a plant’s reaction to stressful environments, soil fertility, and a plant’s relationship with other microorganisms. Such effects imply a broad reprogramming of the plant’s metabolic activity. However, little information is available regarding the role of AMF and their relation to other soil plant growth—promoting microorganisms in the plant metabolome, especially under realistic field conditions. In the present experiment, we evaluated the effects of inoculation with AMF, either alone or in combination with plant growth–promoting rhizobacteria (PGPR), on the metabolome and changes in metabolic pathways in the roots of durum wheat (Triticum durum Desf.) grown under N-limited agronomic conditions in a P-rich environment. These two treatments were compared to infection by the natural AMF population (NAT). Soil inoculation with AMF almost doubled wheat root colonization by AMF and decreased the root concentrations of most compounds in all metabolic pathways, especially amino acids (AA) and saturated fatty acids, whereas inoculation with AMF+PGPR increased the concentrations of such compounds compared to inoculation with AMF alone. Enrichment metabolomics analyses showed that AA metabolic pathways were mostly changed by the treatments, with reduced amination activity in roots most likely due to a shift from the biosynthesis of common AA to γ-amino butyric acid. The root metabolome differed between AMF and NAT but not AMF+PGPR and AMF or NAT. Because the PGPR used were potent mineralisers, and AMF can retain most nitrogen (N) taken as organic compounds for their own growth, it is likely that this result was due to an increased concentration of mineral N in soil inoculated with AMF+PGPR compared to AMF alone.     

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO₂, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.     

NGF Modulates trkANGFR/p75NTR in αSMA-Expressing Conjunctival Fibroblasts from Human Ocular Cicatricial Pemphigoid (OCP)

Objective

In a previous study, we reported the upregulation of Nerve Growth Factor (NGF) and trkA^NGFR expression in Ocular Cicatricial Pemphigoid (OCP), an inflammatory and remodeling eye disease. Herein, we hypothesize a potential NGF-driven mechanism on fibroblasts (FBs) during OCP remodeling events. To verify, human derived OCP-FBs were isolated and characterized either at baseline or after NGF exposure.

Materials and Methods

Conjunctival biopsies were obtained from 7 patients having OCP and 6 control subjects (cataract surgery). Both conjunctivas and primary FB cultures were characterised for αSMA, NGF and trkA^NGFR/p75^NTR expression. Subcultures were exposed to NGF and evaluated for αSMA, NGF, trkA^NGFR/p75^NTR expression as well as TGFβ1/IL4 release. For analysis, early and advanced subgroups were defined according to clinical parameters.

Results

OCP-conjunctivas showed αSMA-expressing FBs and high NGF levels. Advanced OCP-FBs showed higher αSMA expression associated with higher p75^NTR and lower trkA^NGFR expression, as compared to early counterparts. αSMA expression was in keeping with disease severity and correlated to p75^NTR. NGF exposure did not affect trkA^NGFR levels in early OCP-FBs while decreased both αSMA/p75^NTR expression and TGFβ1/IL4 release. These effects were not observed in advanced OCP-FBs.

Conclusions

Taken together, these data are suggestive for a NGF/p75^NTR task in the potential modulation of OCP fibrosis and encourages further studies to fully understand the underlying mechanism occurring in fibrosis. NGF/p75^NTR might be viewed as a potential therapeutic target.     

Cdx2 Polymorphism Affects the Activities of Vitamin D Receptor in Human Breast Cancer Cell Lines and Human Breast Carcinomas

Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D) and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954) human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative). These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression.     

A Stochastic Model for Detecting Overlapping and Hierarchical Community Structure

Community detection is a fundamental problem in the analysis of complex networks. Recently, many researchers have concentrated on the detection of overlapping communities, where a vertex may belong to more than one community. However, most current methods require the number (or the size) of the communities as a priori information, which is usually unavailable in real-world networks. Thus, a practical algorithm should not only find the overlapping community structure, but also automatically determine the number of communities. Furthermore, it is preferable if this method is able to reveal the hierarchical structure of networks as well. In this work, we firstly propose a generative model that employs a nonnegative matrix factorization (NMF) formulization with a l_2,1 norm regularization term, balanced by a resolution parameter. The NMF has the nature that provides overlapping community structure by assigning soft membership variables to each vertex; the l_2,1 regularization term is a technique of group sparsity which can automatically determine the number of communities by penalizing too many nonempty communities; and hence the resolution parameter enables us to explore the hierarchical structure of networks. Thereafter, we derive the multiplicative update rule to learn the model parameters, and offer the proof of its correctness. Finally, we test our approach on a variety of synthetic and real-world networks, and compare it with some state-of-the-art algorithms. The results validate the superior performance of our new method.     

Discovery of a Novel Immune Gene Signature with Profound Prognostic Value in Colorectal Cancer: A Model of Cooperativity Disorientation Created in the Process from Development to Cancer

Immune response-related genes play a major role in colorectal carcinogenesis by mediating inflammation or immune-surveillance evasion. Although remarkable progress has been made to investigate the underlying mechanism, the understanding of the complicated carcinogenesis process was enormously hindered by large-scale tumor heterogeneity. Development and carcinogenesis share striking similarities in their cellular behavior and underlying molecular mechanisms. The association between embryonic development and carcinogenesis makes embryonic development a viable reference model for studying cancer thereby circumventing the potentially misleading complexity of tumor heterogeneity. Here we proposed that the immune genes, responsible for intra-immune cooperativity disorientation (defined in this study as disruption of developmental expression correlation patterns during carcinogenesis), probably contain untapped prognostic resource of colorectal cancer. In this study, we determined the mRNA expression profile of 137 human biopsy samples, including samples from different stages of human colonic development, colorectal precancerous progression and colorectal cancer samples, among which 60 were also used to generate miRNA expression profile. We originally established Spearman correlation transition model to quantify the cooperativity disorientation associated with the transition from normal to precancerous to cancer tissue, in conjunction with miRNA-mRNA regulatory network and machine learning algorithm to identify genes with prognostic value. Finally, a 12-gene signature was extracted, whose prognostic value was evaluated using Kaplan–Meier survival analysis in five independent datasets. Using the log-rank test, the 12-gene signature was closely related to overall survival in four datasets (GSE17536, n = 177, p = 0.0054; GSE17537, n = 55, p = 0.0039; GSE39582, n = 562, p = 0.13; GSE39084, n = 70, p = 0.11), and significantly associated with disease-free survival in four datasets (GSE17536, n = 177, p = 0.0018; GSE17537, n = 55, p = 0.016; GSE39582, n = 557, p = 4.4e-05; GSE14333, n = 226, p = 0.032). Cox regression analysis confirmed that the 12-gene signature was an independent factor in predicting colorectal cancer patient’s overall survival (hazard ratio: 1.759; 95% confidence interval: 1.126–2.746; p = 0.013], as well as disease-free survival (hazard ratio: 2.116; 95% confidence interval: 1.324–3.380; p = 0.002).     

Alterations in mitochondrial dynamics with age‐related Sirtuin1/Sirtuin3 deficiency impair cardiomyocyte contractility

Sirtuin1 (SIRT1) and Sirtuin3 (SIRT3) protects cardiac function against ischemia/reperfusion (I/R) injury. Mitochondria are critical in response to myocardial I/R injury as disturbance of mitochondrial dynamics contributes to cardiac dysfunction. It is hypothesized that SIRT1 and SIRT3 are critical components to maintaining mitochondria homeostasis especially mitochondrial dynamics to exert cardioprotective actions under I/R stress. The results demonstrated that deficiency of SIRT1 and SIRT3 in aged (24–26 months) mice hearts led to the exacerbated cardiac dysfunction in terms of cardiac systolic dysfunction, cardiomyocytes contractile defection, and abnormal cardiomyocyte calcium flux during I/R stress. Moreover, the deletion of SIRT1 or SIRT3 in young (4–6 months) mice hearts impair cardiomyocyte contractility and shows aging‐like cardiac dysfunction upon I/R stress, indicating the crucial role of SIRT1 and SIRT3 in protecting myocardial contractility from I/R injury. The biochemical and seahorse analysis showed that the deficiency of SIRT1/SIRT3 leads to the inactivation of AMPK and alterations in mitochondrial oxidative phosphorylation (OXPHOS) that causes impaired mitochondrial respiration in response to I/R stress. Furthermore, the remodeling of the mitochondria network goes together with hypoxic stress, and mitochondria undergo the processes of fusion with the increasing elongated branches during hypoxia. The transmission electron microscope data showed that cardiac SIRT1/SIRT3 deficiency in aging alters mitochondrial morphology characterized by the impairment of mitochondria fusion under I/R stress. Thus, the age‐related deficiency of SIRT1/SIRT3 in the heart affects mitochondrial dynamics and respiration function that resulting in the impaired contractile function of cardiomyocytes in response to I/R.     

Hepatitis C virus core protein regulates NANOG expression via the stat3 pathway

HCV Core plays a role in the development of hepatocellular carcinoma. Aberrant expression of NANOG has been observed in many types of human malignancies. However, relationship between Core and NANOG has not been clarified. In this study, we found that Core is capable of up-regulating NANOG expression. Core-induced NANOG expression was accompanied by enforced expression of phosphorylated stat3 protein and was attenuated by inhibition of stat3 phosphorylation. ChIP showed that phosphorylated stat3 directly binds to the NANOG promoter. Core-induced NANOG expression resulted in enhanced cell growth and cell cycle progression. Knockdown of NANOG blocked the cell cycle at the G0/G1 phases and inhibited the cyclin D1 expression. Our findings provide a new insight into the mechanism of hepatocarcinogenesis by HCV infection.     

Identification of an elongation factor 1Bγ protein with glutathione transferase activity in both yeast and mycelial morphologies from human pathogenic Blastoschizomyces capitatus

Blastoschizomyces capitatus is an uncommon, opportunistic pathogenic fungus, which causes invasive and disseminated infections. This microorganism is normally present in both environmental and normal human flora. Within a host, B. capitatus is able to grow in both unicellular yeast and multicellular filamentous growth forms. In this study, we obtained in vitro morphological conversion of B. capitatus from yeast-to-mycelial phase to investigate the presence and expression of glutathione transferase (GST) enzymes in both cell forms. A protein with GST activity using the model substrate 1-chloro-2,4-dinitrobenzene was detected in both morphologies and identified by tandem mass spectrometry as a eukaryotic elongation factor 1Bγ (eEF1Bγ) protein, a member of the GST superfamily. No significant difference in GST-specific activity and kinetic constants were observed between mycelial and yeast forms, indicating that eEF1Bγ protein did not show differential expression between the two phases.