Neurogenic potential of mesenchymal-like stem cells from human amniotic fluid: the influence of extracellular growth factors

Amniotic fluids contain human stem cells, among which mesenchymal stem cells could be isolated. These cells have multipotent differentiation ability and no tumorigenic potential after transplantation in mice. These features make them good candidates for in vitro studies and for therapeutic purposes. The aim of this study was to isolate mesenchymal stem cell-like cultures from different amniotic fluids in order to study in vitro their neurogenic potential and assess if this process could be reproducible and standardized. We focused attention on the possible differential effects of soluble growth factors. Immunophenotypical and molecular characterization showed that the 31 amniotic fluid-derived cultures expressed mesenchymal markers as well as some stemness properties. These cells also appeared to be responsive to purines or acetylcholine showing an intracellular calcium increase, also reported for mesenchymal stem cells derived from other sources. Interestingly, in the presence of retinoic acid, these cells assumed a neuronal-like morphology. In addition, functional and molecular analyses revealed that retinoic acid-treated cells showed immature electric functional properties, the expression of neuronal markers and stemness genes. In conclusion, even if further investigations are required, the results presented here contribute to support the finding that amniotic fluid contains cells able to differentiate in vitro towards neural-like lineage in the presence of retinoic acid. The ability of retinoic acid to induce a possible neuronal progenitor culture makes the model useful to study a possible in vivo transplantation of these cells and to contribute to define the protocols for cell therapy.     

An early fish oil-enriched diet reverses biochemical, liver and adipose tissue alterations in male offspring from maternal protein restriction in mice

Fetal programming is linked to adulthood metabolic and chronic diseases. We hypothesized that early fish oil (FO) intake would revert the programming responses in adult offspring. Pregnant mice were fed either standard chow (SC) or a low-protein diet (LP) throughout pregnancy/lactation. At weaning, the following groups were formed: SC and SC-FO, LP and LP-FO, which were fed SC or SC+FO, respectively. The LP offspring are predisposed to becoming fat, hypercholesterolemic and hyperglycemic. In addition, during adulthood, they become hypertensive with hepatic steatosis and have a high level of sterol regulatory element binding protein (SREBP-1). However, LP offspring that were fed an FO-enriched diet have decreased body mass (BM) gain and lower final BM. In addition, with this diet, these mice have improved lipid metabolism with a decrease in total cholesterol (TC) and triacylglyceride (TG) levels, reduced fat pad masses and reduced adipocyte size. Furthermore, these LP offspring show reduced liver structural damage of alanine aminotransferase (ALT), liver steatosis with low SREBP-1 protein expression and high peroxisome proliferator activity receptor-alpha expression, and improvement of blood pressure (BP) and tumor necrosis factor (TNF)-alpha level. Early fish oil intake has beneficial effects on the programming responses that control body fat pad, glucose and lipid metabolism, and liver and adipose tissue structure in adult programmed offspring.     

Nicotinamide N-methyltransferase upregulation inversely correlates with lymph node metastasis in oral squamous cell carcinoma

We investigated expression levels of Nicotinamide N-Methyltransferase (NNMT), an enzyme involved in the biotransformation of many drugs and xenobiotic compounds, in oral squamous cell carcinoma (OSCC). Measurements were performed by semi-quantitative RT-PCR and quantitative real-time PCR in tumor and matched adjacent healthy tissue. Interestingly, NNMT was up-regulated in most of the favorable OSCCs, while no marked NNMT expression alterations between tumor and normal mucosa were detected in most of the unfavorable OSCCs. Western blot and immunohistochemical analyses also were performed and the relationship between tumor characteristics and NNMT levels in OSCC were studied to evaluate the effectiveness of NNMT as a prognostic marker in the squamous cell carcinoma of the oral cavity. In summary, the present study suggests that NNMT may have potential as a biomarker and a therapeutic target for OSCC.     

Molecular Identification and Genetic Relationships of Palestinian Grapevine Cultivars

Palestine has a wide range of agro-ecological concerns and hosts a large variety of plants. Grapes are part of the cultural heritage and provide an indispensable food ingredient. Local cultivars have been traditionally identified on the basis of morphological traits, geographical origin, or names of the vineyard owner; therefore, the occurrence of homonymy, synonymy, and misnaming significantly prevents their valorization. DNA profiling by 22 common SSR markers was used to characterize 43 putative cultivars grown mainly for local table grape consumption at the southern highland regions of West-Bank, to further evaluate genetic diversity and relationships of the population. Consistent matching of SSR markers with grapevines cultivated in neighboring countries or maintained in European germplasm collections was found for 8 of the 21 different non-redundant genotypes discovered, suggesting possible synonyms as well as the occurrence of breeding selections formerly developed in the USA. Genetic relationships inferred from SSR markers clearly assigned Palestinian cultivars to the Proles orientalis subpr. Antasiatica ancestral population, and they even remarked the connection between local resources and cultivars generated from international table grape breeding. This study supports the value of collection and conservation of vines endemic to a region of immense historical importance for viticulture.     

Comparative Salivary Proteome of Hepatitis B- and C-Infected Patients

Hepatitis B and C virus (HBV and HCV) infections are an important cause of cirrhosis and hepatocellular carcinoma. The natural history has a prominent latent phase, and infected patients may remain undiagnosed; this situation may lead to the continuing spread of these infections in the community. Compelling reasons exist for using saliva as a diagnostic fluid because it meets the demands of being an inexpensive, noninvasive and easy-to-use diagnostic method. Indeed, comparative analysis of the salivary proteome using mass spectrometry is a promising new strategy for identifying biomarkers. Our goal is to apply an Orbitrap-based quantitative approach to explore the salivary proteome profile in HBV- and HCV-infected patients. In the present study, whole saliva was obtained from 20 healthy, (control) 20 HBV-infected and 20 HCV-infected subjects. Two distinct pools containing saliva from 10 subjects of each group were obtained. The samples were ultracentrifuged and fractionated, and all fractions were hydrolyzed (trypsin) and injected into an LTQ-VELOS ORBITRAP. The identification and analyses of peptides were performed using Proteome Discoverer1.3 and ScaffoldQ + v.3.3.1. From a total of 362 distinct proteins identified, 344 proteins were identified in the HBV, 326 in the HCV and 303 in the control groups. Some blood proteins, such as flavin reductase (which converts biliverdin to bilirubin), were detected only in the HCV group. The data showed a reduced presence of complement C3, ceruloplasmin, alpha(1)-acid glycoprotein and alpha(2)-acid glycoprotein in the hepatitis-infected patients. Peptides of serotransferrin and haptoglobin were less detected in the HCV group. This study provides an integrated perspective of the salivary proteome, which should be further explored in future studies targeting specific disease markers for HBV and HCV infection.     

In vitro pathogenicity of different Metarhizium anisopliae s.l. isolates in oil formulations against Rhipicephalus microplus

The present study aimed to evaluate the in vitro pathogenicity of the Metarhizium anisopliae s.l. CG 112, CG 347, CG 32, CG 148 and CG 629 isolates formulated in vegetable or mineral oil against Rhipicephalus microplus. The bioassays were performed with R. microplus engorged females. The ticks were immersed for three minutes in oil-based formulations containing 10% oil (mineral or vegetable). The effects of the different fungal isolates were evaluated analyzing the females’ reproductive parameters (the total weight of the egg mass, hatching percentage, egg production index and nutritional index). The present study showed that all of the tested isolates and both oil formulations (vegetable or mineral oil) changed the biological parameters of the R. microplus females. However, the mineral oil formulation was more effective than the vegetable oil formulation, as the former showed a higher tick control percentage. It was concluded that there was variation in the virulence among the different M. anisopliae s.l. isolates and between the different types of oil. Moreover, that either of the isolates GC 148 and GC 629 formulated in oil confers the good potential for controlling R. microplus engorged females.     

In vitro contractile effect of platelet-activating factor on guinea-pig myometrium

Platelet-activating factor (PAF) evoked myometrial contractions in two different patterns, depending on whether spontaneous activity was present. In spontaneously active myometrial strips (58%), both PAF and oxytocin enhanced the amplitude of myometrial contractions. In quiescent myometrial strips, PAF induced contractions characterized by a prompt development of tension, a plateau, and a final, rapid relaxation. In 54% of these strips, PAF-induced contraction was followed by rhythmic activity. PAF contractile response was dependent upon the concentration (0.1–100 nM); the minimal effective concentration of PAF was 0.1 nM and the EC₅₀ was 1 nM. The response to oxytocin (0.01–10 mU/ml), assumed as reference stimulus, was characterized by a prompt development of tension, which was followed by a sustained, slow contraction and relaxation. PAF response was almost completely dependent on cyclooxygenase and partially on lipoxygenase pathways, as inferred from studies with indomethacin and FPL 55712, respectively. A receptor mediated mechanism of PAF action was suggested by specific desentization of the myometrium to a second challenge with an equimolar concentration of PAF (but not with oxytocin) and the blocking effect of CV 3988, a specific PAF receptor antagonist.     

Development of user-friendly functional molecular markers for VvDXS gene conferring muscat flavor in grapevine

High fruit and wine quality combined with good climatic adaptation and disease resistance are essential objectives of grape breeding. While several molecular markers are available for pyramiding resistance to fungal pathogens, molecular tools for predicting fruit composition are still scarce. Muscat flavor, caused by the accumulation of monoterpenoids in the berry, is an important target trait for breeding, sought after in both table grapes and wine. Four missense mutations in the VvDXS gene in grape germplasm have been shown to be tightly linked to muscat flavor. Here we present highly reproducible and breeder-friendly functional markers for each of the targeted polymorphisms developed by using either the multiplexed minisequencing SNaPshot? method, the high-resolution melting (HRM) assay or the cleaved amplified polymorphic sequence system. A total of 242 grapevine accessions were analyzed to optimize these different genotyping methods and to provide allele-specific markers for accurate selection of muscat flavor at early stages of grape breeding programs. The HRM and the minisequencing SNaPshot multiplex assays allow for high-throughput automated screening and are suitable for large-scale breeding programs and germplasm characterization.     

SOCS-3 inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of obese mice

SOCS (suppressor of cytokine signaling) proteins are inhibitors of cytokine signaling involved in negative feedback loops. We have recently shown that insulin increases SOCS-3 mRNA expression in 3T3-L1 adipocytes. When expressed, SOCS-3 binds to phosphorylated Tyr(960) of the insulin receptor and prevents Stat 5B activation by insulin. Here we show that in COS-7 cells SOCS-3 decreases insulin-induced insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with p85, a regulatory subunit of phosphatidylinositol-3 kinase. This mechanism points to a function of SOCS-3 in insulin resistance. Interestingly, SOCS-3 expression was found to be increased in the adipose tissue of obese mice, but not in the liver and muscle of these animals. Two polypeptides known to be elevated during obesity, insulin and tumor necrosis factor-alpha (TNF-alpha), induce SOCS-3 mRNA expression in mice. Insulin induces a transient expression of SOCS-3 in the liver, muscle, and the white adipose tissue (WAT). Strikingly, TNF-alpha induced a sustained SOCS-3 expression, essentially in the WAT. Moreover, transgenic ob/ob mice lacking both TNF receptors have a pronounced decrease in SOCS-3 expression in the WAT compared with ob/ob mice, providing genetic evidence for a function of this cytokine in obesity-induced SOCS-3 expression. As SOCS-3 appears as a TNF-alpha target gene that is elevated during obesity, and as SOCS-3 antagonizes insulin-induced IRS-1 tyrosine phosphorylation, we suggest that it is a player in the development of insulin resistance.