Phytochemical Characterization, Antioxidant and Anti-Proliferative Properties of Rubia cordifolia L. Extracts Prepared with Improved Extraction Conditions.

Rubia cordifolia L. (Rubiaceae) is an important plant in Indian and Chinese medical systems. Extracts prepared from the root, stem and leaf have been used traditionally for the management of various diseases. Some of the known effects are anti-inflammation, neuroprotection, anti-proliferation, immunomodulation and anti-tumor. A comparative account of the extracts derived from different organs that lead to the identification of the most suitable solvent is lacking. We explored the presence of phytochemicals, antioxidant activity and anti-proliferative properties of a variety of solvent-based extracts of root, and methanol extracts of stem and leaf of R. cordifolia L. The antioxidant potential was determined by DPPH, hydrogen peroxide, nitric oxide and total antioxidant assays. The anti-proliferative nature was evaluated by MTT assay on HeLa, ME-180 and HepG2 cells. The composition of the extracts was determined by UPLC-UV-MS. We found that the root extracts had the presence of higher amounts of antioxidants over the stem and leaf extracts. The root extracts prepared in methanol exhibited the highest cytotoxicity in HepG2 cells. The main compounds identified through UPLC-UV-MS of the methanol extract give credibility to the previous results. Our comprehensive study corroborates the preference given to the root over the stem and leaf for extract preparation. In conclusion, we identified the methanol extract of the root to be the most suited to have bioactivity with anti-cancer potential.

Skeletal Muscle Atrophy in Simulated Microgravity Might Be Triggered by Immune-Related microRNAs.

Exposure to microgravity induces skeletal muscle disorders including atrophy, muscle force decrease, fiber-type shift. Microgravity also contributes to immune-function alterations and modifies microRNAs (miRs) expression. To understand the link between microgravity-induced skeletal muscle atrophy and immune function deregulation, a bioinformatics study was performed. The web platform MiRNet was used for miRs-targets interaction analysis from previous proteomic studies on human soleus (SOL) and vastus lateralis (VL) muscles. We predicted miRs targeting deregulated gene expression following bed rest as a model of microgravity exposure; namely, let-7a-5p, miR-125b-5p for over-expressed genes in SOL and VL; miR-1-3p, miR-125b-5p and miR-1-3p, miR-95-5p for down-expressed genes in VL and SOL. The predicted miRs have important immune functions, exhibiting a significant role on both inflammation and atrophy. Let-7a down-expression leads to proliferation pathways promotion and differentiation pathway inhibition, whereas miR-1-3p over-expression yields anti-proliferative effect, promoting early differentiation. Such conflicting signals could lead to impairment between proliferation and differentiation in skeletal muscles. Moreover, promotion of an M2-like macrophage phenotype (IL-13, IL-10) by let-7a down-regulation and simultaneous promotion of an M1-like macrophage (IL-6, TNF-α) phenotype through the over-expression of EEF2 lead to a deregulation between M1/M2 tuning, that is responsible for a first pro-inflammatory/proliferative phase followed by an anti-inflammatory pro-myogenic phase during skeletal muscle regeneration after injury. These observations are important to understand the mechanism by which inflammation may play a significant role in skeletal muscle dysfunction in spaceflights, providing new links between immune response and skeletal muscle deregulation, which may be useful to further investigate possible therapeutic intervention.

How Diet Intervention via Modulation of DNA Damage Response through MicroRNAs May Have an Effect on Cancer Prevention and Aging, an in Silico Study.

The DNA damage response (DDR) is a molecular mechanism that cells have evolved to sense DNA damage (DD) to promote DNA repair, or to lead to apoptosis, or cellular senescence if the damage is too extensive. Recent evidence indicates that microRNAs (miRs) play a critical role in the regulation of DDR. Dietary bioactive compounds through miRs may affect activity of numerous genes. Among the most studied bioactive compounds modulating expression of miRs are epi-gallocatechin-3-gallate, curcumin, resveratrol and n3-polyunsaturated fatty acids. To compare the impact of these dietary compounds on DD/DDR network modulation, we performed a literature search and an in silico analysis by the DIANA-mirPathv3 software. The in silico analysis allowed us to identify pathways shared by different miRs involved in DD/DDR vis-à-vis the specific compounds. The results demonstrate that certain miRs (e.g., -146, -21) play a central role in the interplay among DD/DDR and the bioactive compounds. Furthermore, some specific pathways, such as "fatty acids biosynthesis/metabolism", "extracellular matrix-receptor interaction" and "signaling regulating the pluripotency of stem cells", appear to be targeted by most miRs affected by the studied compounds. Since DD/DDR and these pathways are strongly related to aging and carcinogenesis, the present in silico results of our study suggest that monitoring the induction of specific miRs may provide the means to assess the antiaging and chemopreventive properties of particular dietary compounds.

Hormone replacement therapy enhances IGF-1 signaling in skeletal muscle by diminishing miR-182 and miR-223 expressions: a study on postmenopausal monozygotic twin pairs.

MiRNAs are fine-tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co-twin case-control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen-based hormone replacement therapy (HRT) to explore estrogen-dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54-62-years-old monozygotic female twin pairs discordant for HRT (median 7 years). MCF-7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen's causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR-182, miR-223 and miR-142-3p expressions in HRT using than in their nonusing co-twins. Insulin/IGF-1 signaling emerged one common pathway targeted by these miRNAs. IGF-1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR-182 and miR-223 on IGF-1R and FOXO3A mRNA as well as a dose-dependent miR-182 and miR-223 down-regulations concomitantly with up-regulation of FOXO3A and IGF-1R expression. Novel finding is the postmenopausal HRT-reduced miRs-182, miR-223 and miR-142-3p expression in female skeletal muscle. The observed miRNA-mediated enhancement of the target genes' IGF-1R and FOXO3A expression as well as the activation of insulin/IGF-1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.

Predicting microRNA modulation in human prostate cancer using a simple String IDentifier (SID1.0).

To make faster and efficient the identification of mRNA targets common to more than one miRNA, and to identify new miRNAs modulated in specific pathways, a computer program identified as SID1.0 (simple String IDentifier) was developed and successfully applied in the identification of deregulated miRNAs in prostate cancer cells. This computationally inexpensive Fortran program is based on the strategy of exhaustive search and specifically designed to screen shared data (target genes, miRNAs and pathways) available from PicTar and DIANA-MicroT 3.0 databases. As far as we know this is the first software designed to filter data retrieved from available miRNA databases. SID1.0 takes advantage of the standard Fortran intrinsic functions for manipulating text strings and requires ASCII input files. In order to demonstrate SID1.0 applicability, some miRNAs expected from the literature to associate with cancerogenesis (miR-125b, miR-148a and miR-141), were randomly identified as main entries for SID1.0 to explore matching sequences of mRNA targets and also to explore KEGG pathways for the presence of ID codes of targeted genes. Besides genes and pathways already described in the literature, SID1.0 has proven to useful for predicting other genes involved in prostate carcinoma. These latter were used to identify new deregulated miRNAs: miR-141, miR-148a, miR-19a and miR-19b. Prediction data were preliminary confirmed by expression analysis of the identified miRNAs in androgen-dependent (LNCaP) and independent (PC3) prostate carcinoma cell lines and in normal prostatic epithelial cells (PrEC).

Modulation of caspase activity regulates skeletal muscle regeneration and function in response to vasopressin and tumor necrosis factor.

Muscle homeostasis involves de novo myogenesis, as observed in conditions of acute or chronic muscle damage. Tumor Necrosis Factor (TNF) triggers skeletal muscle wasting in several pathological conditions and inhibits muscle regeneration. We show that intramuscular treatment with the myogenic factor Arg(8)-vasopressin (AVP) enhanced skeletal muscle regeneration and rescued the inhibitory effects of TNF on muscle regeneration. The functional analysis of regenerating muscle performance following TNF or AVP treatments revealed that these factors exerted opposite effects on muscle function. Principal component analysis showed that TNF and AVP mainly affect muscle tetanic force and fatigue. Importantly, AVP counteracted the effects of TNF on muscle function when delivered in combination with the latter. Muscle regeneration is, at least in part, regulated by caspase activation, and AVP abrogated TNF-dependent caspase activation. The contrasting effects of AVP and TNF in vivo are recapitulated in myogenic cell cultures, which express both PW1, a caspase activator, and Hsp70, a caspase inhibitor. We identified PW1 as a potential Hsp70 partner by screening for proteins interacting with PW1. Hsp70 and PW1 co-immunoprecipitated and co-localized in muscle cells. In vivo Hsp70 protein level was upregulated by AVP, and Hsp70 overexpression counteracted the TNF block of muscle regeneration. Our results show that AVP counteracts the effects of TNF through cross-talk at the Hsp70 level. Therefore, muscle regeneration, both in the absence and in the presence of cytokines may be enhanced by increasing Hsp70 expression.

Static magnetic fields enhance skeletal muscle differentiation in vitro by improving myoblast alignment.

Static magnetic field (SMF) interacts with mammal skeletal muscle; however, SMF effects on skeletal muscle cells are poorly investigated. The myogenic cell line L6, an in vitro model of muscle development, was used to investigate the effect of a 80 +/- mT SMF generated by a custom-made magnet. SMF promoted myogenic cell differentiation and hypertrophy, i.e., increased accumulation of actin and myosin and formation of large multinucleated myotubes. The elevated number of nuclei per myotube was derived from increased cell fusion efficiency, with no changes in cell proliferation upon SMF exposure. No alterations in myogenin expression, a modulator of myogenesis, occurred upon SMF exposure. SMF induced cells to align in parallel bundles, an orientation conserved throughout differentiation. SMF stimulated formation of actin stress-fiber like structures. SMF rescued muscle differentiation in the presence of TNF, a muscle differentiation inhibitor. We believe this is the first report showing that SMF promotes myogenic differentiation and cell alignment, in the absence of any invasive manipulation. SMF-enhanced parallel orientation of myotubes is relevant to tissue engineering of a highly organized tissue such as skeletal muscle. SMF rescue of muscle differentiation in the presence of TNF may have important therapeutic implications.

Static magnetic fields affect cell size, shape, orientation, and membrane surface of human glioblastoma cells, as demonstrated by electron, optic, and atomic force microscopy.

BACKGROUND: It is common knowledge that static magnetic fields (SMF) do not interact with living cells; thus, fewer studies of SMF compared with variable magnetic fields are carried out. However, evidence demonstrated that SMF affect cellular structures. To investigate the effect of exposure to increasing doses of SMF on cell morphology, human glioblastoma cells were exposed to SMF ranging between 80 and 3,000 G (8 and 300 mT). METHODS: Cell morphology of human glioblastoma cells, derived from a primary culture, was studied by electron and optic microscopy. FITC-phalloidin staining of actin filaments was also investigated. Finally, cell surface structure changes were detected by atomic force microscopy. RESULTS: Scanning electron microscopy demonstrated a dose-dependent cell shape modification, progressive cell detachment, loss of the long villi, and appearance of membrane roughness and blebs. FITC-phalloidin staining confirmed the villi retention and cell dimension decrease. At 3,000 G, the appearance of apoptotic morphology was also observed by transmission electron microscopy. Cell exposed to SMF showed different orientation and alignment when compared with nonexposed cells. The atomic force microscopy of the exposed cells' membrane surfaces demonstrated the disappearance of the ordered surface ripples and furrows typical of the unexposed cells, and the occurrence of surface membrane corrugation at increasing dose exposure CONCLUSIONS: Our experimental procedures demonstrated that exposure to SMF affects not only cell size, shape, and orientation but also human glioblastoma cells' membrane surfaces.

Automated analysis of morphometric parameters for accurate definition of erythrocyte cell shape.

BACKGROUND: Modification of erythrocyte morphology is clinically important in hematology and medicine. Its detection is routinely performed by subjective microscopic evaluation, which is difficult and strongly dependent on the operator's expertise. We developed an original automated methodology to analyze erythrocyte cell shape modification to support and improve the operator's capability and expedite measurements. METHODS: We used morphometric parameters derived from optical microscope images elaborated with an image processing software (NIH Scion Image) to construct a new application for statistical multivariate discriminant analysis. RESULTS: For each cell type the elaboration of the morphometric parameters allowed us to develop a chromogenic index, a dimension index, a biconcavity index, and a density profile. The measurements of these indexes were used to construct a statistical methodology that could discriminate among erythrocyte morphologies according to Bessis. When applied casewise, the model effectively differentiated between discocytes, target cells, ovalocytes, macrocytes, and microcytes, with an agreement of 70% between actual and predicted classifications. CONCLUSIONS: The results clearly demonstrated that a set of opportunely selected morphometric parameters derived from optical microscope images and statistically analyzed can effectively discriminate with a high degree of certainty among different shape modifications that red blood cells can undergo in various in vitro and in vivo conditions. This method represents the first attempt to automate the definition of erythrocyte morphology and may have important applications in cases in which the detection of erythrocyte cell shape changes is crucial.