Interferon stimulated gene 15 (ISG15): Molecular characterization and expression profile in endometrium of buffalo (Bubalus bubalis)

A sequential medium was evaluated on the survival, activation and growth rates of caprine preantral follicles submitted to a long-term culture period, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM(+) alone or supplemented with hormones (GH and/or FSH) added sequentially on different days of culture. Ovarian fragments were cultured in the first (days 0-8) and second (days 8-16) halves of the culture period, generating 10 treatments: α-MEM(+)/α-MEM(+), FSH/FSH, FSH/GH, FSH/FSH+GH, GH/GH, GH/FSH, GH/FSH+GH, FSH+GH/FSH+GH, FSH+GH/FSH and FSH+GH/GH. Follicle morphology, viability and ultrastructure were analyzed. After day 1 of culture, FSH treatments maintained the percentage of normal follicles similar to the fresh control. At day 16 of culture, the treatment FSH/GH showed the highest (P<0.05) percentage of normal follicles. The ultrastructure of follicles was preserved in the fresh control and FSH/GH treatment. Follicles cultured with FSH/GH had a higher (P<0.05) viability than α-MEM(+); however the viability was lower (P<0.05) when compared to the fresh control. The FSH/GH treatment showed the highest (P<0.05) percentage of follicular activation and secondary follicle formation and produced the largest (P<0.05) mean follicular diameter after 16 days of culture. In conclusion, a sequential medium supplemented with FSH followed by GH during a long-term culture maintains the survival, viability and ultrastructure of goat preantral follicles, and promotes activation and secondary follicles.     

Boosting the globalization of plant proteomics through INPPO: current developments and future prospects

The International Plant Proteomics Organization (INPPO) is a non-profit-organization consisting of people who are involved or interested in plant proteomics. INPPO is constantly growing in volume and activity, which is mostly due to the realization among plant proteomics researchers worldwide for the need of such a global platform. Their active participation resulted in the rapid growth within the first year of INPPO's official launch in 2011 via its website (www.inppo.com) and publication of the 'Viewpoint paper' in a special issue of PROTEOMICS (May 2011). Here, we will be highlighting the progress achieved in the year 2011 and the future targets for the year 2012 and onwards. INPPO has achieved a successful administrative structure, the Core Committee (CC; composed of President, Vice-President, and General Secretaries), Executive Council (EC), and General Body (GB) to achieve INPPO objectives. Various committees and subcommittees are in the process of being functionalized via discussion amongst scientists around the globe. INPPO's primary aim to popularize the plant proteomics research in biological sciences has also been recognized by PROTEOMICS where a section dedicated to plant proteomics has been introduced starting January 2012, following the very first issue of this journal devoted to plant proteomics in May 2011. To disseminate organizational activities to the scientific community, INPPO has launched a biannual (in January and July) newsletter entitled 'INPPO Express: News & Views' with the first issue published in January 2012. INPPO is also planning to have several activities in 2012, including programs within the Education Outreach committee in different countries, and the development of research ideas and proposals with priority on crop and horticultural plants, while keeping tight interactions with proteomics programs on model plants such as Arabidopsis thaliana, rice, and Medicago truncatula. Altogether, the INPPO progress and upcoming activities are because of immense support, dedication, and hard work of all members of the INPPO community, and also due to the wide encouragement and support from the communities (scientific and non-scientific).     

Statistical optimization of physical parameters for enhanced bacteriocin production by L. casei

Antimicrobial proteinaceous compounds such as bacteriocins produced from Lactobacillus sp. are widely known. They have potential antimicrobial activities towards closely related bacteria and several pathogens associated with food spoilage and hence can be a potential food bio-preservative agent. Bacteriocin production requires optimized process, complex media and well-controlled physical conditions including pH and temperature. A probiotic strain of L. casei LA-1 isolated from mango pickle was used in the present study. The influence of physical parameters viz. temperature (15 ?? 45°C), pH (4.0 ?? 7.0), incubation time (up to 48 h) and inoculum size (0.7 ?? 2.0 O.D) on bacteriocin production was analyzed. The effect of all the parameters was first investigated using the one-factor-at-a-time method (OFAT) to see the significance of these parameters on bacteriocin production and then further optimized by response surface methodology (RSM). Following OFAT analysis, all factors were found to have a significant effect on bacteriocin production. Bacteriocin production of 2,844 AU/mL was obtained at temperature 37°C, pH 6.7 and inoculum size 1.8 O.D at an incubation time of 20 h and it was produced during the stationary phase of growth. Statistical analysis showed that three variables-pH, temperature and incubation time have significant effects on bacteriocin production. RSM proved to be a powerful tool in the optimization of bacteriocin production by L. casei LA-1 with a two-fold increase, giving a production of 4652.15 AU/mL at pH 7.19, temperature 33.3°C and incubation time of 22.2 h.     

Using the “reverse Warburg effect” to identify high-risk breast cancer patients: Stromal MCT4 predicts poor clinical outcome in triple-negative breast cancers

We have recently proposed a new model of cancer metabolism to explain the role of aerobic glycolysis and L-lactate production in fueling tumor growth and metastasis. In this model, cancer cells secrete hydrogen peroxide (H₂O₂), initiating oxidative stress and aerobic glycolysis in the tumor stroma. This, in turn, drives L-lactate secretion from cancer-associated fibroblasts. Secreted L-lactate then fuels oxidative mitochondrial metabolism (OXPHOS) in epithelial cancer cells, by acting as a paracrine onco-metabolite. We have previously termed this type of two-compartment tumor metabolism the “reverse Warburg effect,” as aerobic glycolysis takes place in stromal fibroblasts, rather than epithelial cancer cells. Here, we used MCT4 immunostaining of human breast cancer tissue microarrays (TMAs; >180 triple-negative patients) to directly assess the prognostic value of the “reverse Warburg effect.” MCT4 expression is a functional marker of hypoxia, oxidative stress, aerobic glycolysis and L-lactate efflux. Remarkably, high stromal MCT4 levels (score = 2) were specifically associated with decreased overall survival (<18% survival at 10 years post-diagnosis). In contrast, patients with absent stromal MCT4 expression (score = 0), had 10-year survival rates of ∼97% (p-value < 10⁻³²). High stromal levels of MCT4 were strictly correlated with a loss of stromal Cav-1 (p-value < 10⁻¹⁴), a known marker of early tumor recurrence and metastasis. In fact, the combined use of stromal Cav-1 and stromal MCT4 allowed us to more precisely identify high-risk triple-negative breast cancer patients, consistent with the goal of individualized risk-assessment and personalized cancer treatment. However, epithelial MCT4 staining had no prognostic value, indicating that the “conventional” Warburg effect does not predict clinical outcome. Thus, the “reverse Warburg effect” or “parasitic” energy-transfer is a key determinant of poor overall patient survival. As MCT4 is a druggable target, MCT4 inhibitors should be developed for the treatment of aggressive breast cancers, and possibly other types of human cancers. Similarly, we discuss how stromal MCT4 could be used as a biomarker for identifying high-risk cancer patients that could likely benefit from treatment with FDA-approved drugs or existing MCT-inhibitors (such as, AR-C155858, AR-C117977 and AZD-3965).Key words: caveolin-1, oxidative stress, pseudohypoxia, lactate shuttle, MCT4, metabolic coupling, tumor stroma, predictive biomarker, SLC16A3, monocarboxylic acid transporter, two-compartment tumor metabolism     

Reporter gene imaging of protein-protein interactions in living subjects

In the past few years there has been a veritable explosion in the field of reporter gene imaging, with the aim of determining the location, duration and extent of gene expression within living subjects. An important application of this approach is the molecular imaging of interacting protein partners, which could pave the way to functional proteomics in living animals and might provide a tool for the whole-body evaluation of new pharmaceuticals targeted to modulate protein-protein interactions. Three general methods are currently available for imaging protein-protein interactions in living subjects using reporter genes: a modified mammalian two-hybrid system, a bioluminescence resonance energy transfer (BRET) system, and split reporter protein complementation and reconstitution strategies. In the future, these innovative approaches are likely to enhance our appreciation of entire biological pathway systems and their pharmacological regulation.     

A reporter gene assay for screening of PDE4 subtype selective inhibitors

Phosphodiesterase (PDE) constitutes a superfamily of enzymes that catalyze the hydrolysis of cAMP and cGMP into their corresponding monophosphates and play an important role in diverse physiological functions. The present study provides a process for identifying PDE4 subtypes selective inhibitors using a reporter gene assay. Stable recombinant HEK-293 cell lines expressing high levels of PDE4A4B, PDE4B2A, and PDE4D3 subtypes individually were generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene under the control of cAMP response element (CRE)-binding sequence, into these stable recombinant cell lines followed by treatment with PDE4 inhibitor, resulted in a dose dependent increase in luciferase activity. This methods provide a novel, simple and sensitive assay for high throughput screening of PDE4 subtype selective inhibitors for treatment of asthma and COPD.     

Precursor protein is readily degraded in mitochondrial matrix space if the leader is not processed by mitochondrial processing peptidase

It is not known why leader peptides are removed by the mitochondrial processing peptidase after import into the matrix space. The leaders of yeast aldehyde dehydrogenase (pALDH) and malate dehydrogenase were mutated so that they would not be processed after import. The recombinant nonprocessed precursor of yeast pALDH possessed a similar specific activity as the corresponding mature form but was much less stable. The nonprocessed pALDH was transformed into a yeast strain missing ALDHs. The transformed yeast grew slowly on ethanol as the sole carbon source showing that the nonprocessed precursor was functional in vivo. Western blot analysis showed that the amount of precursor was 15-20% of that found in cells transformed with the native enzyme. Pulse-chase experiments revealed that the turnover rate for the nonprocessed precursor was greater than that of the mature protein indicating that the nonprocessed precursor could have been degraded. By using carbonyl cyanide m-chlorophenylhydrazone, we showed that the nonprocessed precursor was degraded in the matrix space. The nonprocessed precursor forms of precursor yeast malate dehydrogenase and rat liver pALDH also were degraded in the matrix space of HeLa cell mitochondria faster than their corresponding mature forms. In the presence of o-phenanthroline, an inhibitor of mitochondrial processing peptidase, the wild type precursor was readily degraded in the matrix space. Collectively, this study showed that the precursor form is less stable in the matrix space than is the mature form and provides an explanation for why the leader peptide is removed from the precursors.     

A simple visualization technique to understand the system dynamics in bioreactors

In this article, we present a graph theoretic method to visualize and analyze the system behavior under different operating conditions. The system attributes (or variables) are the nodes in the graphs, and partial correlation between a pair of attributes defines the distance between corresponding nodes, resulting in a fully connected graph. Then, the redundant links are reduced using Pathfinder Network Scaling technique to uncover the latent network structure. We use a simulated biological reactor dataset in normal and faulty operation to validate our method. The method is general and can be used to analyze several different systems.