Department of Structural Biology, Weizmann Institute, Rehovot 76100
Ribosomes, the universal cellular machines for translation of the genetic code into proteins, possess spectacular architecture accompanied by inherent mobility, allowing for their smooth performance as polymerases that translate the genetic code into proteins. The site for peptide bond formation is located within a universal internal semi-symmetrical region. The high conservation of this region implies its existence irrespective of environmental conditions and indicates that it may represent an ancient RNA machine. Hence, it could be the kernel around which life originated. The mechanistic and genetic applications of this finding will be discussed
Owing to the key role played by ribosomes in life cycles, almost half of the clinically useful antibiotics paralyze ribosomes by binding to their functional sites. By investigating the three dimensional structures of ribosomes from nonpathogenic bacteria as models for genuine pathogens, common features were identified. Thus, the antibiotics binding modes, inhibitory actions and synergism pathways have been determined for almost all ribosomal antibiotics. These indicated the principles of differentiation between patients and pathogens and suggested common principles of mechanisms leading to bacterial resistance.
The incredible global increase in resistance to antibiotics that we are witnessing recently is a serious medical threat. It seems that the world is approaching a post-antibiotic era, in which common infections and minor injuries that have been treatable for decades could become fatal once again.
As species specific diversity was detected in susceptibility to infectious diseases and in developing specific resistance mechanisms, our structural studies have been extended to ribosomes from genuine pathogens. By determining the high resolution structure of the first and only ribosomal particle from a genuine pathogen with several antibiotics, we identified subtle, albeit highly significant structural elements that can account for the species specificity in resistance, thus could paved ways for improvement of existing antibiotics as well as for the design of advanced therapeutics capable of minimizing antibiotics resistance.
Program for Evolutionary Dynamics, Harvard University, Cambridge, MA 02138
Cancer is an evolutionary process. Cancer initiation and progression are caused by somatic mutation and selection of dividing cells. The mathematical theory of evolution can therefore provide quantitative insights into human cancer. I will discuss the role of chromosomal instability (CIN) and the accumulation of drivers and passengers in growing tumors. I will study success and failure of targeted therapy including combination of different drugs and evolution of resistance. A simple conclusion is that combination treatment can succeed, if the cancer requires at least two point mutations to gain resistance. From the perspective of preventing resistance, simultaneous therapy is highly recommended whereas sequential therapy is a recipe for almost certain treatment failure.
Bozic et al. (2010). Accumulation of driver and passenger mutations during tumor progression. Proc Natl Acad Sci USA 107: 18545- 18550.
Diaz et al. (2012) The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 486: 537- 540.
Bozic et al. (2013). Evolutionary dynamics of cancer in response to targeted combination therapy. eLife 2: e00747.
Charles N. Serhan
Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Harvard Institutes of Medicine, Brigham and Women’s Hospital & Harvard Medical School, Boston, Massachusetts, 02115
Endogenous mechanisms controlling inflammation are of paramount importance because persistent and chronic inflammation can impact all organs and tissues throughout the body and are involved in many widely occurring diseases. Recent advances in our appreciation of the molecular mechanisms in resolution of acute inflammation (RoI) and ischemia-reperfusion injury systematically uncovered a novel genus of potent pro-resolving autacoid families, each biosynthesized from essential polyunsaturated fatty acids (PUFA) to activate potent responses not shared by the substrate. These include the resolvins (Rv), protectins (PD) and maresins (MaR), collectively termedspecialized proresolving mediators (SPM) that act in pico-nanogram range. SPM are temporally and spatially biosynthesized by resolving-inflammatory exudates, which proved to evoke potent antiinflammatory and pro-resolving actions as well as enhance microbial clearance. The potent SPM actions and complete structures are confirmed, which also permitted use of LC-MS-MS-based metabololipidomics to identify SPM in human and murine tissues (i.e. peripheral blood, breast milk, adipose, lymphoid, placenta), isolated human cells types (e.g. apoptotic human neutrophils, microparticles and macrophage phenotypes M1, M2), fish and diminished SPM in human pathologies e.g. breath condensates, Alzheimer brain, and synovial fluids from rheumatoid patients (CN Serhan Nature June vol 510, 2014 doi:10.1038/nature13479). Specific SPM demonstrate potent and stereoselective actions that involve specific G-protein-coupled receptors and are not immunosuppressive. Lipid mediator-metabololipidomics with selflimited resolving inflammatory exudates and human tissues demonstrated temporal orchestration of the SPM, i.e. RvD1 and RvD2 antecede RvD3, and MaR1 in mice and human tissues (Colas et al. AJP 2014). Many of the SPM born in inflammation-resolution are now shown to have potent actions and roles within host defense against bacteria and virus, pain, organ protection, tissue regeneration, exercise and neurobiology/cognitive function. This Plenary Lecture will update advances in SPM mechanisms in RoI, their new sites of formation and novel actions that opened the door for their role(s) in resolution physiology and pharmacology. Together, these new SPM families provide opportunities for resolution-based pharmacology and resolution physiology.
Department of Systems Biology, Harvard Medical School and The Wyss Institute for Biologically Inspired Engineering, Harvard University
The engineering of Biology presents infinite opportunities for therapeutic design, diagnosis, and prevention of disease. Towards these goals, we seek to make the engineering of Biology faster, more predictable and cheaper. This ‘Synthetic Biology’ has deep practical and social consequences for the pharmaceutical as well as the commodity industry. Here, I will present concepts and experiments that begin to address how we approach these problems in a systematic way.
By one strategy, we seek to predictably engineer mammalian cells to produce novel compounds that could potentially act as new therapeutics. For example, we have developed an algorithm for biosynthesis of new steroids that could have increased specificity towards their respective targets. This has implications in treatment of inflammation and clean production of other chemicals of interest.
By a second strategy, we design chimeric proteins to act as specific therapeutics. Specificity in biologics remains one of the outstanding issues in their use. We have again developed an algorithm based on coarse grain modeling for the predictable design on new proteins. Some have been tested in animals and show the predicted effects.
Lastly, we engineer components of the microbiome to act as both diagnostics and therapeutics. In one example, we have engineered natural gut bacteria to record the exposure of animals to antibiotics and to count the number of cell divisions as the bacteria passes through the gut. We can engineer the same bacteria to secrete toxins that could result in localized killing of pathogens and to act in a communal manner. Taken together, these experiments have far-reaching implications for the use of biology to prevent and treat disease in the future.
Departments of Bioengineering and Pediatrics University of California, San Diego, San Diego, USA
Following the availability of full genome sequences in the mid 1990s, an effort was initiated to reconstruct, on a genome-scale, the biochemical reaction networks that underlie cellular functions. After 15 years of intense efforts, we now have highly curated network reconstructions, their experimental validation, and the generation of mathematical and modeling procedures available that allow the computation of cellular functions from genome- and bibliome-wide data sets. This effort has put a mechanistic basis into the most fundamental relationship in the life sciences; the genotypephenotype relationship. This effort has started with simple organisms and the best characterized cellular functions and it is steadily growing in scope and biological complexity.
Center for Genetic Medicine Research, Children’s National Medical Center and Department of Integrative Systems Biology, George Washington University, Washington, DC 20010, USA
Drug development is a lengthy and time consuming process, where it has been suggested that it typically costs $500 million and takes 15 years to bring a drug to market. The high costs become problematic with orphan drugs, where there may be very few patients to prescribe the new drug and distribute costs. Also, there are increasing opportunities for highly targeted drugs, but often in ever more stratified patient populations, so the proportion of orphan drugs is expected to increase dramatically. Methods to decrease the costs and time associated with orphan drug development include early de-risking (reducing late stage expensive failures), and accelerated approval (moving larger efficacy trials to the post-marketing space). VBP15 is a first in human drug developed for Duchenne muscular dystrophy (DMD). Intellectual property for the program was transferred from Children’s National Medical Center to ReveraGen Biopharma. Initial seed funding for lead compound selection was accomplished through support of the Department of Defense CDMRP, and early de-risking of the lead (VBP15) was done in partnership with the National Institutes of Health TRND program. Pre-clinical studies and Phase 1 trials were funded by venture philanthropy support of seven DMD foundations through a risk sharing and profit sharing model based on later drug sales. Phase 2a and 2b trials in DMD patients are to run by international academic networks, with the possibility of accelerated approval after a short-term (3-6 month) trial with a measure of strength as the primary outcome measure. VBP15 is a steroidal compound built on a delta 9,11 backbone, where the drug shows a high affinity for the glucocorticoid receptor, but is effective in dissociating anti-inflammatory transrepression subactivities (retention of efficacy), from the transactivation subproperties associated with side effects (growth stunting, adrenal suppression, immune suppression). VBP15 has shown efficacy in pre-clinical models of multiple chronic inflammatory states, including allergic lung disease, arthritis, multiple sclerosis, inflammatory bowel disease, and sickle cell anemia. Thus, VBP15 holds potential for replacing traditional glucocorticoids in multiple indications, including DMD.
Mark G. Moloney
University of Oxford, Department of Chemistry, Chemistry Research Laboratory, Mansfield Rd., UK
The discovery of new antibiotics has become urgent as a result of the emergence of resistance and new pathogenic bacterial strains. However, this need has coincided with unprecedented lowering of levels of productivity in the drug discovery process and consequent reduced investment from large pharma. New strategies for antibacterial drug discovery are required, and a renewed understanding of the value of a natural products’ guided approach has emerged. Our focus has been on the use of antibacterial natural products containing tetramate core structures, and using equisetin and reutericyclin as inspiration, we have developed novel chemistry that uses suitable serine, threonine and cysteine-derived oxazolidine templates for highly chemo- and diasteroselective ring closure reactions leading to tetramic acid derivatives. Although simple unsubstituted pyrrolidines and tetramates appear to be intrinsically devoid of activity, application of these templates for fragment-based synthesis, has permitted access to several compound series which possess high levels of antibacterial activity, SAR analysis has permitted some optimization of the initial activity and MOA and other pharmacokinetic data been obtained.
This lecture will illustrate the potential of natural products to guide antibacterial drug discovery, the role of synthetic organic chemistry in the construction of libraries which mimic these natural products, and suggest a possible way forward for more efficient drug discovery strategies.
“Natural product inspired antibacterial tetramic acid libraries with dual enzyme target activity”, Y.-C. Jeong, M. Anwar, M. G. Moloney, Z. Bikadi and E. Hazai, Chem. Sci., 2013, 4, 1008-1015; “Chiral Bicyclic Tetramates as Non-Planar Templates for Chemical Library Synthesis”, M. Anwar and M.G. Moloney, Chem Biol Drug Des, 2013, 81, 645-649; “Synthesis and antibacterial activity of monocyclic 3-carboxamidotetramic acids”, Y.-C. Jeong, M. G. Moloney, Beilstein J. Org. Chem., 2013, 9, 1899-1906; “Synthesis of 3- Acyltetramates by Side Chain Manipulation and their Antibacterial Activity”, Song Wei Benjamin Tan, Christina Chai and Mark G. Moloney, Org. Biomol. Chem., 2014, 12 (11), 1711-1716; “A detailed study of antibiotic 3-acyltetramic acids and 3-acylpiperidine-2,4- diones”, Y.-C. Jeong, M. Anwar, M. G. Moloney, Z. Bikadi and E. Hazai, ChemMedChem, 2014, 9, 1826-1837; “Antibiotic activity and structure-activity relationship study of some 3-enaminetetramic acids”, Y.-C. Jeong, M. Anwar, M. G. Moloney, Biorg. Med. Chem. Lett., 2014, 24, 1901-1906.
Harvard-MIT Division of Health Sciences and Technology, Brigham and Women’s Hospital and Harvard Medical School, Cambridge MA 02139, USA
Engineered materials that integrate advances in polymer chemistry, nanotechnology, and biological sciences have the potential to create powerful medical therapies. Our group aims to engineer tissue regenerative therapies using water-containing polymer networks, called hydrogels, that can regulate cell behavior. Specifically, we have developed photocrosslinkable hybrid hydrogels that combine natural biomolecules with nanoparticles to regulate the chemical, biological, mechanical and electrical properties of gels. These functional scaffolds induce the differentiation of stem cells to desired cell types and direct the formation of vascularized heart or bone tissues. Since tissue function is highly dependent on architecture, we have also used microfabrication methods, such as microfluidics, photolithography, bioprinting, and molding, to regulate the architecture of these materials. We have employed these strategies to generate miniaturized tissues. To create tissue complexity, we have also developed directed assembly techniques to compile small tissue modules into larger constructs. It is anticipated that such approaches will lead to the development of next-generation regenerative therapeutics and biomedical devices.
Attilio Di Pietro
BMSSI UMR5086 CNRS-University of Lyon, Institute of Protein Biology and Chemistry, Passage du Vercors 7, 69367 Lyon, France
Multidrug ABC (“ATP-binding cassette”) transporters are involved, upon overexpression, in chemoresistant tumors by pumping anticancer drugs out of the cells. For early discovered ABCB1/ “P-glycoprotein”, third-generation drug-efflux inhibitors are under clinical development. For more recently identified ABCG2/“breast cancer resistance protein”, we have screened different series of flavonoids and derivatives, such as flavones, rotenoids and acridones, and more recently chalcones [1, 2], chromones [3, 4], and indenoindoles , as inhibitors of mitoxantrone efflux from transfected HEK293 human cells and chemosensitizers of cell proliferation, to establish 3D-Quantitative Structure-Activity Relationships. Two types of selective, non-competitive, inhibitors have been characterized, either inhibiting or stimulating the basal ATPase activity. The most potent one is indeed efficient in vivo on SCID mice, xenografted with human ABCG2-transfected cells, by chemosensitizing tumor growth to the drug-substrate irinotecan . These selective inhibitors constitute good drug candidates, with low intrinsic toxicity, as sensitizers of cell proliferation to conventional chemotherapeutics.
The “Multidrug Resistance Protein 1” ABCC1 is able to catalyze the efflux of either glutathione conjugates or free glutathione together with hydrophobic substrate drugs. We have identified modulators such as verapamil [7, 8] mimicking substrates and inducing a fast and massive efflux of intracellular glutathione from ABCC1-overexpressing cells, leading to a selective cell death through apoptosis, due to “collateral sensitivity”, or hypersensitivity. The overexpressed transporter then constitutes the Achilles’heel of such resistant cancer cells. Since verapamil is known for its cadiotoxic effects, we investigated other types of modulators such as xanthones, flavones  and flavonoid dimers. Glutathione efflux appeared to be necessary, but not sufficient alone, to trigger apoptosis, indicating the contribution of other partner(s) or signaling pathway(s). Such apoptosis inducers may constitute a new type of anticancer drugs operating through an original strategy aimed at selectively targeting and eliminating multidrug-resistant tumors overexpressing the ABCC1 transporter .
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Center for the Study of Systems Biology, Georgia Institute of Technology, 250 14th Street NW Atlanta, GA 30318, USA
The intrinsic ability of protein structures to exhibit the geometric and sequence properties required for ligand binding without evolutionary selection is shown by the coincidence of the properties of pockets in native, single domain proteins with those in computationally generated, compact homopolypeptide, artificial structures, ART. The library of native pockets is covered by a remarkably small number of representative pockets (~400), with virtually every native pocket having a statistically significant match in the ART library, suggesting that the library is complete. When sequences are selected for ART structures based on fold stability, pocket sequence conservation is coincident to native. The fact that structurally and sequentially similar pockets occur across fold classes combined with the small number of representative pockets in native proteins implies that promiscuous interactions are inherent to proteins. Based on comparison of PDB and ART structures and pockets, the widespread assumption that the co-occurrence of global structure, pocket similarity, and amino acid conservation demands an evolutionary relationship between proteins is shown to significantly underestimate the random background probability. Indeed, many features of biochemical function arise from the physical properties of proteins which evolution likely fine-tunes to achieve specificity. This study suggests that a repertoire of thermodynamically (marginally) stable proteins could engage in many of the biochemical reactions needed for living systems without selection for function, a conclusion with significant implications for the origin of life. Finally, examples of experimental validation of promising small molecule hits that exploit the degeneracy of ligand binding pockets are presented.
Diplom-Physiker, Scientific Director, RI-B-NT Research Institute of Bioinformatics and Nanotechnology, Franziusallee 177, D-24148 Kiel, Germany
The strategic combination of various biophysical and biochemical methods such as NMR, X-ray crystallography, Atomic Force Microscopy, Accelerated Mass Spectrometry and Surface Plasmon Resonance techniques with cell biological approaches and patient studies leads to new perspectives in the fields of nanomedicine and nanopharmacology concerning innovative routes in applied health care. These routes show solutions for different so-far unsolved medical and pharmacological problems in oncology, antiinfection, tissue-engineering and pathobiochemistry of the endocrine system [1-7]. It was essential for this purpose to decipher the three alphabets of life completely (1: nucleic acid code, 2: amino acid code, 3: sugar code ) and figure out how they work together on a sub-molecular level. It turned out in our molecular dynamics simulations that ab initio calculations play an important role in order to fit all the results from the different approaches together in a convincing way.
 V. B. Krylov, D. A. Argunov, D. Z. Vinnitskiy, S. A. Verkhnyatskaya, A. G. Gerbst, N. E. Ustyuzhanina, A. S. Dmitrenok, J. Huebner, O. Holst, H.-C. Siebert, N. E. Nifantiev (2014) Pyranoside into furanoside rearrangement: new reaction in carbohydrate chemistry and its application in oligosaccharide synthesis. Chem – Eur J: in press.
 R. Zhang, A. K. Petridis, G. Scheiner-Bobis, M. Burg-Roderfeld, T. Eckert, A. Wehrend, M. Bergmann, L. Wu, B. Norden, M. Billeter, A. Scheidig, R. Schauer, S. Fraune, T. C. G. Bosch, H. Wienk, R. Boelens, B. K. Chatterjee, H-C. Siebert (2014) How biophysical and nanomedical tools support therapeutical improvements in neurooncology. Physics of Cancer Symposium. Open Access: https://www.dropbox.com/s/euhdlyzhdaw633e/Poster-Leipzig-Sept-2014.pdf?dl=0
 S. Schadow, H.-C. Siebert, G. Lochnit, J. Kordelle, M. Rickert, J. Steinmeyer (2013) Collagen metabolism of human osteoarthritic articular cartilage as modulated by bovine collagen hydrolysates. PLOS ONE 8, 1, e53955.
 H.-C. Siebert (2013) What can we expect from new therapeutic strategies in nanopharmacology and nanomedicine? Medicinal chemistry and computer aided drug designing. Open Access: https://www.dropbox.com/s/tu95nji7liv7mz7/lecture- LasVegas.mp4
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 T. Eckert, S. Stötzel, M. Burg-Roderfeld, J. Sewing, T. Lütteke, N. E. Nifantiev, J. F. G. Vliegenthart, H.-C. Siebert (2012) In silico study on sulfated and non-sulfated carbohydrate chains from proteoglycans in Cnidaria and their interaction with collagen. O. J. Phys. Chem. 2, 123-133.
 Y. E. Tsvetkov, M. Burg-Roderfeld, G. Loers, A. Ardá, E. V. Sukhova, E. A. Khatuntseva, A. A. Grachev, A. O. Chizhov, H.- C. Siebert, M. Schachner, J. Jiménez-Barbero, N. E. Nifantiev (2012) Synthesis and molecular recognition studies of the HNK- 1 trisaccharide and related oligo-saccharides. The specificity of monoclonal anti-HNK-1 antibodies as assessed by surface plasmon resonance and STD NMR. J. Am. Chem. Soc. 134, 426-435.
 H.-J. Gabius, H.-C. Siebert, S. André, J. Jiménez-Barbero, H. Rüdiger (2004) Chemical biology of the sugar code. ChemBioChem 5, 740-764.
Southwest National Primate Research Center, Texas Biomedical Research Institute, 7620 Northwest Loop 410, San Antonio, Texas, 78227-5301, USA
Traditionally, CD34 positive cells are predominantly found in the umbilical cord and bone marrow, thus are considered as hematopoietic progenitors. Increasing evidence has suggested that the CD34+ cells represent a distinct subset of cells with enhanced progenitor activity; CD34 is a general marker of progenitor cells in a variety of cell types. Because the CD34 protein shows expression early on in hematopoietic and vascular-associated tissues, the CD34+ cells have enormous potential as cellular agents for research and for clinical cell transplantation. To date, the clinical trials using CD34+ cells cover various diseases including acute and chronic ischemic heart failure, spinal cord injury, liver cirrhosis, and peripheral vascular diseases. Current clinical use of CD34+ cells are wither autologous or allogeneic transplantation from adult tissues, which might not be practical in future application. Differentiation of the embryonic stem cells will give rise to inexhaustible CD34+ cells, which becomes an exciting approach for biomedical research and for regenerative medicine. Here, we describe the main methods that have been published for the derivation of CD34+ cells by embryonic stem cells; specifically those approaches using the human and non-human primate stem cells. Therefore, it enables us to oversee the current status of this field, to compare the methods used for this purpose, and to consider the issues in translating the bench science to bedside therapy.
Keywords: CD34+ progenitor cells, Embryonic stem cells, Differentiation, Cell therapy, Non-human primate model, Bioassay.
William J. Rowe
M.D. FBIS FACN, Fellow of the British Interplanetary Society, Former Assistant Clinical Professor of Medicine, Medical U. of Ohio at Toledo, 1485 Bremerton La., Keswick, Virginia, 22947, USA
In 2002, the author presented and subsequently published that with space flight endothelial dysfunction; there is the potential for congestive heart failure despite invariable dehydration. There is decreased thirst, inappropriate diuresis, atrophy of water storage sites in skeletal muscles and reduced plasma volume; this may be triggered by endothelial intercellular gaps with inflammation in post -capillary venules. These leaks may be responsible for an invariable 10% loss of plasma volume in microgravity. Furthermore, the invariable significant reductions of the serum magnesium (p<0.0001) in large groups of astronauts and cosmonauts, despite very poor serum sensitivity, along with catecholamine elevations to levels twice those in the supine position on Earth with ischemia and multiple vicious cycles, are conducive to further endothelial injuries and in turn, to catecholamine cardiomyopathy ( acute temporary heart failure.)
During his lunar last 20 minutes, Neil Armstrong notified Houston twice at 4 minute intervals that he was “short of breath” at 111 hours and 32 minutes and “still short of breath” at 111: 36. This symptom occurred prior to the potential confounder of inhalation of highly toxic iron-laden dust, brought into the habitat on space suits. Whereas his heart rate on the moon was 130-160/ minute, just prior to splashdown in the Pacific, after his 3 day journey back to Earth, his heart rate was down to 61. The explanation for this correction of tachycardia, could only be that by quenching his severe thirst, the very high adrenaline levels were reduced because of expansion of the left ventricle by replenished plasma volume with in turn reduction in the gradient; this could have been precipitated by protrusion of the septum into the left ventricle as postulated by Merli et al.
Neil Armstrong Space Syndrome: Armstrong developed severe shortness of breath and very rapid heart rate on the moon BEFORE inhalation of toxic iron-laden dust, brought into the habitat on the space suit.
1. SEVERE DYSPNEA, manifested by Armstrong’s notifying Houston twice during a 4 minute interval that he was short of breath, consistent with acute congestive heart failure.
2. SEVERE THIRST; with microgravity, there is invariably decreased thirst, but in Armstrong’s case, despite the 3 day trip back to Earth in microgravity, he apparently quenched his severe thirst.
3. SEVERE TACHYCARDIA CORRECTED BY FLUID REPLENISHMENT; with decreased plasma volume and in turn increased adrenaline with tachycardia, restoring his plasma volume, would in turn, markedly reduce a high heart rate.
Keywords: Neil Armstrong, dyspnea, tachycardia
Department of HumanBiology, Faculty of Natural Sciences, University of Haifa, and Department of Molecular Genetics, Carmel Medical Center, Haifa, Israel
One major issue regarding the clinical use of many peptides is their short half-life due to the rapid clearance from the circulation. To overcome this problem, we used overlapping PCR technique to add the signal sequence of O-linked oligosaccharides to the coding sequence of glycoprotein hormones. The used cassette gene contains the sequence of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin β (hCGβ) subunit. It was postulated that the O-linked oligosaccharides add flexibility, hydrophilicity, stability and prevent plasma clearance and thus increasing the half-life of the protein in circulation. Using this strategy we succeeded to ligate the CTP to the coding sequence of follitropin (FSH), thyrotropin (TSH), erythropoietin (EPO) growth hormone (GH) and thus to increase the longevity and bioactivity of these proteins in-vivo. Interestingly, the new analog of FSH was found not immunogenic in humans and it was approved by the European Commission (EC) for treatment of fertility. In addition, our results indicated that long acting GH is not toxic in monkeys and the results from clinical trials phases I and II seem to be promising. Designing long acting peptides will diminish the cost of these drugs and perhaps reduce the number of injections in the clinical protocols.
Deletion of the N-linked oligosaccharides from hTSH significantly reduced its activity in vitro and in vivo. Moreover, deglycosylated TSH compete with hTSH and human Thyroid Stimulating immunoglobulin (TSI) in a dose dependent manner. These variants may offer a novel therapeutic strategy in the treatment of hyperthyroidism and Grave’s disease.
Keywords: Glycoprotein,follitropin,thyrotropin and erythropoietin.
The Jerome Lipper Multiple Myeloma Center and LeBow Institute for Myeloma Therapeutics, Dana- Farber Cancer Institute, Boston, MA, USA
Targeted immunotherapy with monoclonal antibodies (mAbs) has become critical for the successful treatment of many cancers but not yet for human multiple myeloma (MM). Recently, encouraging activity was reported in ongoing clinical trials of anti-SLAMF7 mAb elotuzumab, when combined with lenalidomide/dexamethasone or bortezomib. In addition, two clinical grade anti-CD38 mAbs continue to show acceptable safety profiles with signs of single agent activity in refractory MM. However, these antigens still lack specificity which could limit their clinical utility. B cell maturation antigen (BCMA) is expressed exclusively on malignant plasma cells in MM but has not been effectively targeted with therapeutic mAbs. Here, a novel anti-BCMA mAb was developed and aimed to achieve improved MM selectivity and simultaneously targeting cytotoxic drugs to MM cells. We demonstrate selective anti-MM activity of a novel humanized and afucosylated antagonistic anti- BCMA antibody-drug conjugate (ADC) via a noncleavable linker. This ADC specifically blocks cell growth via G2/M arrest and induces caspase 3-dependent apoptosis in MM cells, alone and in coculture with bone marrow stromal cells (BMSC) or various effector cells. It strongly inhibits colony formation by MM cells while sparing surrounding BCMAnegative normal cells (BMSC, NK, monocytes, PBMCs). This MM-specific ADC significantly induces effector cellmediated antibody-dependent cellular cytotoxicity (ADCC) against allogeneic or autologous patient MM cells, with an increased potency of >10-fold and maximum MM cell lysis up to 9-fold compared to the wild-type anti-BCMA antibody without Fc enhancement. The ADCC and apoptotic activity of this MM specific ADC is further enhanced by immunomodulatory drug lenalidomide. Importantly, this ADC rapidly eradicates detectable myeloma cells in subcutaneous and disseminated mouse models, and mice remain tumor-free and healthy up to 3.5 months. Furthermore, it recruits macrophages and mediates antibody-dependent cellular phagocytosis (ADCP) of MM cells. Together, these results demonstrate that this MM specific ADC has distinct and selective anti-MM activities via multiple cytotoxic mechanisms, providing a promising next-generation immunotherapeutic in this cancer.
Keywords: GSK2857916, monoclonal antibodies, multiple myeloma.
Ekram W. Abd El- Wahab, Redmond P. Smyth, Elodie Mailler, Serena Bernacchi, Valérie Vivet-Boudou, Marcel Hijnen, Fabrice Jossinet, Johnson Mak, Jean-Christophe Paillart and Roland Marquet
Tropical Health Department, High Institute of Public Health, Alexandria University, Egypt
During assembly, HIV-1 must select its genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. Despite a large number of studies, there is no consensus on how the Pr55Gag precursor achieves this selection. These studies were limited by the expression and purification of intact full-length Pr55Gag protein. Here, we purified soluble full-length Pr55Gag and we investigated the specific determinants of the selective binding of Pr55Gag to HIV-1 gRNA using RNA binding and footprinting assays. Our results revealed that Pr55Gag exhibits a higher binding affinity for gRNA than for spliced vRNA species. Importantly, we demonstrate that the primary Pr55Gag binding site consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. Further analyses on viral RNA fragments of different length spanning the Psi and/or its flanking regions are in favor of a long-distance tertiary interaction involving sequences upstream of SL1 and downstream of SL4, which promotes the optimal binding of Pr55Gag to gRNA. Altogether our data shed light on the importance of a proper gRNA conformation that regulates its specific binding to Pr55Gag, and could result in the competent selection and packaging of the genome. We propose a new model to explain how Pr55Gag discriminates and specifically selects gRNA from cellular RNAs and viral spliced vRNAs that also harbor functional SL1 in their first common exon. A double regulation ensures specific binding of Pr55Gag to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all viral RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA.
Keywords: HIV-1, genomic RNA, specific recognition, Gag precursor.
This research was published in nature communications, 2014.
Ekram W. Abd El-Wahab, Redmond P. Smyth, Serena Bernacchi, Marcel Hijnen, Fabrice Jossinet, Johnson Mak, Jean- Christophe Paillart & Roland Marquet. Specific Recognition of the HIV-1 genomic RNA by the Gag precursor. Nat Commun. 2014 Jul 2;5:4304. doi: 10.1038/ncomms5304.
Sandeep Bisht, Shivesh Sharma, V. Kumar, S.S. Bisht and B.P Nautiyal
Department of Molecular Biology & Biotechnology, VCSG College of Horticulture, Bharsar campus, Uttarakhand University of Horticulture & Forestry, Bharsar, Pauri, Uttarakhand, India
Keywords: PAH, Bacillus sp., Populus deltoides, pgp, rhizoremediation, bioinoculant.
Hala A. Amin, Mostafa M. Abo Elsoud, Ahmed. F. Sahab
Chemistry of Natural and Microbial products Department, National Research Center, Egypt
Soyasapogenol B, aglycone of soybean saponin, is known to have hepatoprotective, antimutagenic, antivirus, and anti-inflammatory activities. This research examined the use of whole-cell biocatalyst to produce soyasapogenol B from soybean saponin. It was found that Aspergillus flavus, a fungus isolated from peanut pods, was capable of expressing extracellular and intracellular saponin hydrolase enzyme. However, the total enzyme activity produced using fungal whole cells (37U) in the reaction mixture was about 3times that produced using the extracellular (12.4U) or intracellular (11.5U) enzyme. Cells with maximum hydrolytic activity for production of soyasapogenol B (12.2 U/g) was obtained using production medium supplemented by 2% soybean saponin, as inducer for enzyme production, adjusted at pH 9 and incubated at 30°C for 2 days. The highest yield of soyasapogenol B was achieved when the reaction mixture was incubated at pH 5.5 and 45°C for 48h; using 20 g wet cells (corresponding to 4% cell dry weight) and soybean saponin (2%, w/v) as a substrate. Under these optimal conditions, the cells bioconversion efficiency (soysapogenole B yield) increased from 5.3 to 60%. Whole cell biocatalyst has several advantages with regard to industrial applications: a consistent quality, easy to be prepared and a very low price compared with purified enzyme. Consequently, this study is significant for production of soyasapogenol B from soybean saponin on an industrial scale.
The authors would like to thank for financial support via the tenth research grant (2013-2016) of the National Research Center of Egypt.
Keywords: Whole cell biocatalyst, Soybean saponin, Soyasapogenol B, Aspergillus flavus.
Syahriel Abdullah, Januarius Gobilik and Khim Phin Chong
Sustainable Palm Oil Research unit (SPOR), Faculty of Science and Natural Resources, Universiti Malaysia Sabah, 88400, Kota Kinabalu, Sabah, Malaysia
Cynodon dactylon (L.) Pers. is belonging to Poaceae family which used as folk medicine to treat many diseases and infections. The present study reports the antibacterial activity and mechanism of action of C. dactylon Solid Phase Extract (SPE) against Bacillus cereus, Bacillus subtilis, Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumonia along with the possible antibacterial compounds. Antibacterial activity and Minimum Inhibitory Concentrations (MICs) were evaluated using disc-diffusion and micro-broth dilution bioassays respectively. Mode of action study was done via assessment on the leakage of 260nm-absorbing materials, fluorometric assay and Scanning Electron Microscope (SEM) observation for membrane disintegration effect while luminometric assay was used for metabolic inhibition assessment. Remarkable antibacterial activity was observed from flush fraction of C. dactylon SPE against the tested bacterial pathogens (MICs=10.00 mgmL-1). Assessment on membrane degradation based on 260nm-absorbing material leakage and fluorometric assay showed membrane disruption on B. cereus, B. subtilis and E. coli after treated with the plant extract. SEM observation further confirmed the membrane disruption. Luminometric assay based on ATP quantification suggests the bacterial death was probably due to other metabolic factor. Liquid Chromatography-Mass Spectrometry (LCMS) analysis revealed some possible antibacterial compounds including peptides, polyketides, triterpenoid, cardenolide glycoside and some steroidal compounds.
Keywords: Cynodon dactylon, antibacterial, mode of action, phytochemical.
Jianmin Xing, Yilan Liu, Maohua Yang, Qinhong Wang and Yanhe Ma
National Key Lab of Biochemical Engineering, Institute of Process Engineering, CAS, China
Microbial synthesis of fatty alcohols from renewable resources has attracted increasing attentions. However, the low tilters of fatty alcohols hampered its industrialization. Here we designed a novel strategy for fatty alcohol production based on fatty acid starvation. For the first time, all three acyl- ACP thioesterases related to formation of fatty acids were knocked out to enhance fatty alcohol production. The fatty alcohol titer increased about 58%, while fatty acids concentration dropped 73%. Transcriptome analysis showed that expression levels of genes for fatty acid synthesis and glycolysis pathway were upregulated, while fatty acid degradation and TCA pathway were downregulated. Furthermore, Fatty alcohol production was enhanced by deleting the genes responsible for lactate and acetate formation. The optimized strain, E. coli MGL2 with exogenous fatty acyl-ACP reductase, accumulated fatty alcohols at a remarkable level of 6.33 g/L under fed-batch fermentation. This is the highest reported titer of fatty alcohols produced by microorganisms.
Keywords: E. coli, Fatty alcohols, Acyl-ACP thioesterases, Fatty acid starvation.
Health Care Valuation, Amsterdam, The Netherlands
Registration is not the sole success factor anymore for future sales and valuation of the share of company. Reimbursement procedures and the introduction of new business models like value-based pricing and risk sharing agreements, have financial consequences for biotech companies. As the future financial performance of a biotech company is directly related to the revenues of new products, an appropriate assessment of the potential sales forecast of the portfolio of forthcoming new products is an important predictor of the financial value of a pharmaceutical company. Therefore information on positive clinical trials results of a new product should be followed by a reimbursement scan and a sales forecast model including the key global markets.
The objective of this lecture is to present a financial valuation algorithm for the assessment of the futures sales of a new, innovative medicinal product based on the current reimbursement policies and future business models for reimbursement. The algorithm consists of a number pathways leading to an assessment of the market potential of the new drug: no potential, limited potential, moderate potential and expected potential. The algorithm is applied to possible new innovative products for treatment of depression and multiple sclerosis.
Keywords: valuation, reimbursement, algorithm.
Associate Professor, Harvard Medical School, Head, Molecular Neurotherapy and Imaging Laboratory,Director, Stem Cell Therapeutics and Imaging Program, Department of Radiology and Neurology, Massachusetts General Hospital, Principal Faculty, Harvard Stem Cell Institute, 149 13th Street, Charlestown, MA 02129, USA
The recognition that different stem cell types can home to tumors following transplantation has unveiled new possibilities for their use in cancer therapy. Our research is based on simultaneously targeting cell death and proliferation pathways in tumor cells in an effort to eradicate both primary and metastatic tumors in the brain using therapeutically engineered stem cells. We have engineered different adult stem cells types to release therapeutic: (i) pro-apoptotic protein, S-TRAIL (secreted tumor necrosis factor receptor-apoptosis inducing ligand) to specifically induce apoptosis in tumor cells; (ii) anti-proliferative anti-epidermal growth factor (EGFR) nanobodies (ENb) to inhibit tumor cell proliferation; (iii) anti-angiogenic thrombospondin (TSP)-1 to target blood vessels supplying the tumor; and (iv) bimodal anti-proliferative and pro-apoptotic proteins that target both cell proliferation and death pathways in tumors cells. We have extensively demonstrated the therapeutic efficacy of these engineered stem cells both in culture and in mouse tumor models. Recently, we have also explored the potential of stem cell loaded oncolytic viruses for cancer therapy and shown that mesenchymal stem cell (MSC) loaded with different variants of oncolytic herpes viruses (oHSV) eradicate tumors in mice brains. In an effort to translate these therapeutics into clinical settings, we have utilized different mouse tumor models generated from our extensively characterized patient derived brain tumor cells. These mouse models mimic clinical settings of primary tumors and their secondary micro-invasive deposits in the brain. Inherently linked to the brain tumor therapy paradigm, we have employed imaging markers and optical imaging, MRI and PET techniques to track stem cells, image apoptosis and changes in tumor volumes in real time in vivo. Within next 2-5 years, we expect to initiate a clinical study using therapeutic stem cells to treat brain tumor patients and hope to have a major impact in saving the lives of these patients.
Keywords: Stem cell, metastatic tumors, brain tumor, tumor cells.
Zhang Yan, Xu Li, Huang Zhengnan and Yan Yunjun
Key Laboratory of Molecular Biophysics, The Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P. R. China
Aimed to major bottlenecks of high cost and low operation stability of free lipases, Candida rugosa lipase (CRL) and Rhizopus oryzae lipase (ROL) were co-displayed on the cell surface of Pichia pastoris and used as a whole-cell catalyst to produce biodiesel from tallow seed oil in this study. After screened by double resistance and tributyrin medium, the resultant co-displayed recombinant GS115/pRCS with the maximum activity of 470.59 U/g dry cells, being 3.9- and 1.3- fold compared with that of the single displayed ROL and CRL1, respectively. The analysis of fluorescence microscope and Flow Cytometer demonstrated that ROL and CRL1 were successfully co-displayed on the surface of recombinant P. pastoris GS115. When the self-immobilized lipases were utilized as whole cell catalysts, the rate of methyl ester from co-displayed recombinants strain GS115/pRCS harboring ROL and CRL1 is 1.4-fold compared with that of single displayed ROL. All these results indicate that biodiesel catalyzed by the co-displayed enzymes with synergetic effect could be an alternative strategy for producing biodiesel in low cost.
Keywords: Rhizopus oryzae lipase; Candida rugosa lipase 1; co-display; Pichia pastoris; whole-cell catalysts
Harilal U. Gangar
Ex-Head, Engineering & Workshop, Central Institute for Research on Cotton Technology, (Indian Council of Agricultural Research), Matunga, Mumbai-400019, India
Study carried out on plant bodies revealed that genetic activity within respective body can be increased, decreased, started or stopped by use of water containing small amount of appropriate homoeopathic drugs. Investigation of this medicated water has shown that it emits electromagnetic waves (signals) oscillating at very high frequencies in the range of GHz. It was noticed that spectrums of signals were different for different drugs. Even for different potencies of same drug, also, they were different. Addition of another drug in medicated water increased the spread and intensity of spectrum of original signal.
From further study it was found that during germination of seed, also, such signals are emitted due to the relevant genetic activity. Again spectrums of signals emitted by seeds of cotton were different than those emitted by mung beans during their respective germination process.
However, when medicated water was used for germination of cotton and mung bean seeds from same variety, the electro-magnetic properties of said signals were altered significantly. In case of cotton seeds, the intensity and spread of spectrum of signal increased. It resulted in significant increase in rate of genetic activity resulting in enhanced rate of germination. In case of mung bean seeds, intensity and spread of spectrum of signals decreased. This resulted in reduction in genetic activity and corresponding decrease in rate of germination. It clearly indicated heterodyning of electro-magnetic waves from medicated water with those originating from genes during relevant genetic activity. This heterodyning process resulted in modifying the genetic signal and thereby affecting the end result.
The findings of study can be used for 1) Replacement of diseased tissues of human body by redevelopment through activation of respective stem cell genes 2) Increasing the yield of crops by increasing the rate of activity of respective stem cell genes in the field of agriculture 3) Speedy recovery from diseases caused by attacks from microbes by increasing the rate of activity of defense response genes in humans, animals and plants. Few such cases of application of findings are listed here.
Keywords: Genetic activity, spectrum and electro-magnetic waves.
Nawrot I, Bogdan Marai Woźniewicz, Maruszewski B, Janas R, Wozniewicz M and Nazarewskis
Department of Pathology, Childrens'' Memorial Health Institute, Warszawa, Poland
Thymus is important organ of the immune system. From discovery of thymosine alpha 1 play essential role in maturation of peripheral T lymphocytes. Many factors was discovered that cause precocious thymus atrophy and developing of autoimmune diseases. Many methods are described to stop autoimmune diseases including revolution in oncology (Ipilimumab), and stem cell therapy. This paper describes a new method to improve natural thymus defence system by ectopic thymus derived stem cell implantation. Material and Methods. Thymus explants were cultured for 34 days to observe secretion of thymosine alpha 1 in vitro and after implantation into volunteers. Cells were implanted into the subcutaneous adipose tissue and Tha1 level was observed during 12 years. Number of implanted cells was adequate to the size of 3,0 thymus remnants of adult man. Results. Thymus derived progenitor/stem cells secrete thymosin alpha 1 and may be used as natural method improving immune system, Conclusion. Thymus progenitor/stem cells may be used in future as a new additional toolls in case of thymus deficiency and in autoimmune diseases. Instead of very expensive pharmaco-therapy using synthetic thymosine (Zadaxin) and other pharmaceuthicals.
Keywords: Stem cell, thymus, human, immunotherapy, autoimmune.
Om Prakash Yadav
Chemistry and Bio-Chemistry, CCS Haryana Agricultural University, India
In the recent years, due to the development of new strains, resistance of bacteria to the commonly used antibiotics drug has increased. Most of the Antibiotics being used, today, are of organic nature. Some of these are extremely irritant and even toxic, therefore, there is much interest in finding ways to formulate new types of safe and cost-effective biocidal materials. Nanosize inorganic material such as metals and their salts during their antibiotic action, can cause denaturation of proteins present in bacterial cell walls. This paper will highlight the synthesis, characterization and antimicrobial activity of as-synthesized inorganic antibiotics against gram positive (Staphylococcus aureus and Streptococcus) as well as gram negative (Pseudomonas aeruginosa and Escherichia coli) bacteria using paper disc diffusion technique. Effects of doping silver and Sulphur in ZnS nanoparticles on their antimicrobial activity will be reported and discussed. The antimicrobial performance of assynthesized nanomaterial will be compared with the commonly used antibiotic drugs.
Keywords: Antibiotics, inorganic, Escherichia coli, nanomaterial, Streptococcus.
Abbas Teimouri, Leila. Ghorbanianb and Alireza Najafi Chermahinic
Department of Chemistry, Payame Noor University (PNU), Isfahan, P.O.Box: 81395-671, Iran
Tissue engineering uses a scaffold inhabited with signifying molecules and cells to encourage the regeneration of host tissue . Silk fibroin (SF) has attracted important interest in the generation of new materials, because SF is a biocompatible and biodegradable natural polymer with excellent mechanical properties and high chemical reactivity . Chitosan (CS) is a deacetylated product of chitin that can be extracted from crustaceans. Due to its excellent bioactivity and degradability, diopside has been proposed as a potential material for the bone tissue regeneration. In continuation of our recent study on the construction of composite scaffolds [3, 4] In the present study, Silk (SF), Chitosan (CS) and Nano Diopside were all combined using the freeze drying technique to fabricate a bio-compositescaffold. The composite scaffold (SF/CS/ Nano Diopside) was characterized by SEM, XRD, TGA, BET and FT-IR studies. The scaffold possessed a porous nature with pore dimensions suitable for cell infiltration and colonization. The presence of Diopside in the SF/CS/ Nano Diopside scaffold increased compressive strength and water uptake capacity and decreased porosity. Cytocompatibility of the SF/CS/ Nano Diopside scaffold was assessed by MTT assay revealed non-toxicity to the Human Gingival Fibroblast (HGF, NCBI: C-131(. Thus, we suggest that SF/CS/N Nano Diopside composite scaffold is a potential candidate to be used for tissue engineering.
Keywords: Silk fibroin; Chitosan; tissue engineering; microporous materials.
 J.P. Vacanti, C.A. Vacanti, R.P. Lanza, R. Langer, J. Vacanti, Principles of Tissue Engineering, 2nd ed., Academic Press, CA, 2000, pp. 3-9.
 C. Vepari, D.L. Kaplan,Progress in Polymer Science 32; (2007), 991-1007.
 L. Ghorbanian, R. Emadi, S.M Razavi, H. Shin, A. Teimouri, International Journal of Biological Macromolecules 58; (2013), 275-280.
 A. Teimouri, L. Ghorbanian, A.R. NajafiChermahini, R. Emadi, Ceramics International, 40; (2014), 6405-6411.
Mohamed Mustafa Ibrahim, Hapipah Mohd Ali, and Mahmood Ameen Abdullah
Department of Chemistry, University of Al-Baha, Saudi Arabia
Tryptamine Schiff bases were prepared by the condensation reaction of ethanolic solution of tryptamine with 5-chlorosalicylaldehyde, 5-nitosalicylaldehyde and 3,5 ditertiarybutyl salicylalehyde. The ligands were coordinated to Cu(II), Ni(II), and Zn(II). All the ligands and complexes prepared have been characterized by spectroscopic methods such as IR spectra, 1H and 13C NMR, UV-Vis, mass spectra, elemental C H N analysis, X-ray crystallography and magnetic susceptibility measurements, as well as atomic absorption spectroscopy for metal analysis. Spectroscopic data shows that the ligands are bonded to Zn and Cu metal centers via N and O atoms in a distorted tetrahedral geometry, whereas square planar geometry is proposed for Ni complexes.
The biological activities of the ligands and complexes as anti ulcer agents have been investigated using Cimetidine as a standard drug. The screening was done on high and low doses (60mg/kg and 30mg/kg body weight respectively) on Sprague-Dawley rats which had been ulcer-induced by absolute ethanol. The ulcer lesion of the stomachs were identified and counted and then correlations between the effects of the different compound have been made. The results revealed very effective inhibition of gastric ulcer for ligands and complexes.
Keywords: Schiff bases, ligands, transition metal complexes, gastric ulcer.
Rana A. Hameed Nidhal N. Hussain and Abed Aljasim M. Jasim Almanssoori
Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq
Soil bacteria Sinorhizobium meliloti had enormous agricultural value,due to their ability in fixing nitrogen symbiotically with an important forage crop legume- alfalfa.The aim of this study (i)isolate indigenous S.meliloti from different field sites in Iraq,(ii) evaluate the isolates tolerance to induced drought using polyethylene glycol-6000, (iii)assessing genetic diversity and genetic relationships among isolates of natural population with drought tolerant abilities. Drought tolerance study revealed vast variations between Sinorhizobium isolates, the highest tolerant isolates to drought were twelve from total thirty (40%),tolerated from -3 up to -4 Mpa(mega pascal),while the drought sensitive isolates tolerated up to – 1.5 Mpa,except isolate Bs58 which tolerated up to -1 Mpa water potential.The growth declined with the increase of drought stress. Cluster analysis based on RAPD-PCR showed significant differences among S. meliloti isolates, and the results gave almost identical grouping of isolates in regards to drought experiment. Among indigenous isolates two divergent groups could be determined,the first major group included drought tolerant isolates and the second major group comprised all drought moderate and sensitive isolates with 40% similarity between the two major groups.
Essam A. Makky, Chan Cai Wen, Muna Jalal, Mashitah M. Yusoff
Department of Biotechnology, University Malaysia Pahang, Kuantan, Lebuhraya Tun Razak, 26300 Gambang, Pahang, Malaysia
Termites are considered as important pests that could cause severe wood damage and economic loses in urban, agriculture and forest of Malaysia. The ability of termites to degrade cellulose depends on association of gut cellulolytic microflora or better known as mutual symbionts. With the idea of disrupting the mutual symbiotic association, better pest control practices can be attained. This study is aimed to isolate cellulolytic bacteria from the gut of termites and carry out antibacterial studies for termite. Confirmation of cellulase activity is done by qualitative and quantitative methods. Impacts of antibiotics and their combinations as well as heavy metals and disinfectants are conducted by using disc diffusion method. Effective antibacterial agents are then subjected for termite treatment to study the effectiveness of the agents as termiticides. 24 cellulolytic bacteria are isolated, purified and screened from the gut of termites. All isolates were identified as Gram-negative with either rod or cocci in shape. For antibacterial studies result, isolates were found to be 100% sensitive to 4 antibiotics (rifampicin, tetracycline, gentamycin and neomycin), 2 heavy metals (cadmium and mercury) and 3 disinfectants (lactic acid, formalin and hydrogen peroxide). 22 out of 36 antibiotic combinations showed synergistic effect, while 15 antibiotic combinations showed antagonistic effect on isolates. The 2 heavy metals and 3 disinfectants that showed 100% effectiveness as well as 22 antibiotic combinations that showed synergistic effect were used for termite control. Among the 27 selected antibacterial agents, 12 of them were found to be effective to kill all the termites within 1 to 6 days. Mercury, lactic acid, formalin and hydrogen peroxide were found to be the most effective termiticides in which all termites were killed within 1 day only. These effective antibacterial agents possess a great potential to be a new application to control the termite pest species in the future.
Keywords: Antibacterial, Cellulose, Termiticide, Termites.
Sultan Ayoub Meo
Physiology, College of Medicine, King Saud University, Riyadh, KSA
Diabetes mellitus is a life-long disease and is swiftly increasing in all age groups and both genders. It involves various physiological functions, organs and multiple systems, and is associated with wide ranging and devastating health complications. New figures have broken all the previous prevalence records, global prevalence of diabetes is 8.3% which means that 382 million adults are diabetic, and the number is expected to rise to 592 million by 2035. Currently, six countries in the Middle East including Saudi Arabia, Bahrain, United Arab Emirates, Kuwait, Oman and Egypt are among the world's highest for the prevalence of diabetes. Management of Diabetes Mellitus presents challenges to the health care providers. In the 21st century, there has been constant progress in the management of diabetes mellitus. The introduction of new insulin delivery systems and glucose monitoring devices has enhanced the ability of both patients and medical teams to better define and develop the therapeutic strategies. Recent advances in devices for insulin administration and glucose monitoring having a profound effect on the lives of diabetic patients. It is strongly suggested that, the science community, physicians must adopt the new bio-technology tools in the management of diabetes mellitus to defeat this debilitating disease.
Keywords: Diabetes Mellitus, Bio-technology Tools.
Catherine Michaux and Eric A Perpete
Unite de Chimie Physique Theorique et Structurale, University of Namur, rue de Bruxelles, 61, 5000 Namur, Belgium
Escherichia coli is one of the most widely used hosts for the production of recombinant proteins of therapeutic or commercial interest dedicated to structural and functional analysis. However, this expression system is often hampered by the formation of insoluble protein aggregates (inclusion bodies). In vitro refolding of such proteins into their native states requires screening of numerous experimental parameters specifically optimized for each system. Hence, there is currently no reliable straightforward and universal experimental solution providing the optimal refolding of proteins. The development of new original techniques in this field is therefore crucial.
In that context, we have successfully demonstrated the reliability of a new procedure for protein refolding [1-3]. This peculiar protocol is based on the association of an ionic detergent with a cosolvent. Indeed, though being known to feature denaturing abilities, some detergents appear to have their properties strongly altered when interacting with a cosolvent, and strikingly an appropriate combination of both even turns to a refolding of the protein. This remarkable procedure has successfully been applied to soluble α-helix and β-sheet peptides, as well as soluble and membrane proteins with several types of structures and properties.
In this contribution, we summarize our progress in the understanding by experimental (spectroscopy) and theoretical methods (molecular dynamics), of our recently reported approach.
Keywords: Protein refolding, detergent, cosolvent, spectroscopy, molecular dynamics.
 Michaux, C., Pomroy, N. C. & Privé, G. G. J. Mol. Biol. (2008) 375, 1477-1488.
 Roussel, G., Perpète, E. A., Matagne, A., Tinti, E. & Michaux, C. Biotechnol. Bioeng. (2013) 110, 417-423.
 Roussel, G., Rouse, S. L., Sansom, M. S. P., Michaux, C. & Perpète, E. A. Colloids Surfaces B Biointerfaces (2014) 114, 357- 362.
Mian Zhou, Jinhu Guo, Joonseok Cha, Michael Chae, She Chen, Jose M. Barral, Matthew S. Sachs and Yi Liu
School of Biotechnology, East China University of Science and Technology, Shanghai, China
During heterologous protein expression, the aim is to increase expression levels without altering native activities. Gene expression is mostly known to be regulated at the transcriptional level, while protein function can be regulated post-translationally. However, as a co-translational mechanism, the role of codon usage in regulating protein expression and function is less well studied.
In many organisms, there is a bias for preferred codons and it is thought to be a mechanism to enhance expression for highly expressed genes. For example, Neurospora crassa prefers to use G/C rather than A/T at the 3rd position of codons in highly expressed genes. FREQUENCY (FRQ) is a key component in the negative feedback loop of Neurospora crassa circadian clock. Compared with other genes, frequency (frq) exhibits a very non-optimal codon usage. It's not clear whether this "poor" codon usage has any biological significance.
To test the role of codon usage in frq, here we made a series of codon optimized frq constructs, and introduced them into a frq null strain. We found that strains carrying the N-terminal optimized frq exhibited severe circadian clock phenotypes, with higher FRQ protein levels but similar frq mRNA levels compared with wild-type. Surprisingly, shown by both trypsin sensitivity assay and freeze-thaw assay, FRQ proteins in these strains were less stable. Besides, they were defective in binding their WCC partners, and optimized FRQ had a compromised function to support WC-1 and WC-2 levels in the positive feedback loop, suggesting that codon optimization also influences protein function even though its sequence is unchanged. Taken together, codon usage of frq regulates its protein expression, folding and function. Therefore a scientific analysis is needed during codon optimization design when expressing heterologous proteins.
Keywords: Codon usage, expression, folding.
Sin Hang Lee and Guofan Hong
Milford Hospital and Milford Molecular Diagnostics Laboratory, 2044 Bridgeport Avenue, Milford, CT 06460, USA
 Hong G et al. Inter J Mol Sci. 2013; 14:12853-12862.
 Lee SH. Methods Mol Biol 2012; 903:65-101.
 Lee SH et al. Am J Clin Pathol. 2008; 129:852-859.
 Lee SH et al. Am J Clin Path. 2010; 133:569-576.
 Lee SH et al. Inter J Mol Sci. 2014; 15:4284-4298.
 Lee SH et al. Inter J Mol Sci. 2014; 15:11364-11386.
Yael Hacham, Jong Yoon, Ifat Matityahu, Alex Kaplan, Lilach Kachan, David Oliver and Rachel Amir
Chairperson and Research Scientist, Laboratory of Plant Science, Migal Galilee Technology Center, P.O. Box 831, Kiryat Shmona 11016, Israel
Cysteine (Cys), the first organic sulfur-containing metabolite, serves as a precursor for the synthesis of glutathione and methionine (Met), two metabolites that are central to plant growth and survival. Glutathione plays a crucial role in the defence against a wide variety of environmental stresses, while Met is a protein constituent, and through its first metabolite, S-adenosylMet (SAM), regulates essential processes required for plant growth. To reveal the relations between glutathione and Met, we used tobacco plants overexpressing the regulatory enzyme of Met biosynthesis pathway, cystathionine γ-snthase (CGS), and those overexpressing the yeast gene encoding a feedback-insensitive O-acetylserine (thiol)lyase (OASTL) in the plastids and in the cytosol that regulate the levels of Cys and glutathione. We crossed between the two transgenic lines to determine that the level of Met can significantly increase in plants overexpressing the plastidic OASTL with AtCGS, accompanied by a reduction in glutathione. The results strongly suggest that the flux towards Met is relatively high, and thus Met can be considered as an intermediate metabolite in the pathways leading to its various associated metabolites. In addition, flux and metabolic profiling analyses indicated the existence of metabolic competition between the biosynthesis pathways of Met and glutathione on their common precursor, Cys, and that this competition is more crucial under oxidative conditions when more Cys is required for the synthesis of glutathione. Plants overexpressing AtCGS with or without the yeast enzyme were significantly more sensitive to oxidative stress, indicating the reason why the levels of Met remained low during the evolution.
Muhammad Saeed, Md. Shah Kamal and Javed Iqbal
Department of Botany, Government College University, Faisalabad, Pakistan
Climate change is adversely affecting World crop production. Climate change has intensified already existing menace of soil salinity in most of the countries. Na+ uptake is an important attribute with respect to salinity tolerance in plants. Plants which manage to keep low concentrations of Na+ in their internal environment, relative to the external environment surrounding their roots, perform better under saline conditions. Quantitative trait loci (QTL) involved in salinity tolerance in rice (Oryza sativa) were identified by using an F2 population derived from the cross of IR - 36 (salt sensitive) and Pokkali (salt tolerant). Plant material was evaluated under normal and saline conditions and data for biochemical traits and grain yield were collected at the maturity stage. A major locus controlling Na+ uptake was identified on chromosome 7 with a phenotypic variance explained (R2) value of 72.57%. This was a novel QTL not reported previously. Associated marker with this QTL was RM248. Allele from salt tolerant parent, Pokkali, was responsible for an increase in Na+ uptake. This QTL may be a good candidate for marker-assisted selection (MAS) to develop salt tolerant rice cultivars with potential to give stable yield under climate change scenario.
Keywords: Salinity, quantitative trait loci, rice, Na+ uptake.
Naima BOU M’HANDI
National Research Institute of Marine/Fisheries (INRH), Seafood Processing Technology Center (CSVTPM), BP: 1050 Poste principale, Agadir- Morocco
In Morocco, fisheries are very diversified. However, the sources used are dominated by small pelagics. According to FAO, Morocco's fisheries production in 2014 amounted to one million tonnes. This record is due to a single species, sardine (Sardina pilchardus), which Morocco is both the largest producer and exporter worldwide.
The establishment of a regulatory environmental policy increasingly strict encourages Moroccan manufacturers to consider waste / by-products resulting from the processing of fish. The search for biologically active molecule extracted from these co-products begins to generate interest. It seems interesting, as part of the new strategy of development of the fisheries sector in Morocco called "Halieutis Plan" and the development of a National Charter for Environment and Sustainable Development, to develop a new technique to guide enhancement of marine by-products into high value products rather than considering them as waste low interest.
It is in this context that the present project is part of the value scales of sardine (Sardina pilchardus) to reduce the pollution load discharged and recover valuable biomolecules with high added value, the collagen in this case.
The preliminary results of this study demonstrated the ability to produce, in the pre industrial scale, collagen powder form sardine scales with a yield of 26%. This collagen contains a large amount of 78% protein and a medium amount of ashes 16.8%. Regarding the amount of water, it represents only 9.5%. Biomass scales representing 2% of the weight of the fish and is not even valued, can become raw material for the production of collagen used for health nutrition, pharmaceutical, cosmetic and biomedical.
Kasim Sakran Abass
School of Public Health, Faculty of Veterinary Medicine, University of Kirkuk, Kirkuk, Iraq
Organophosphate inhibits carboxylesterase thus regular treatment includes carboxylesterase oxime reactivator in combination with anti-muscarinic agents. In order to progress the understanding of species differences and to enable an additional reliable extrapolation of animal result to quail a study was initiated to inspect the effect of insecticide compounds, i.e. mevinphos, ethoprop and fenthion, with quail’s and chick’s serum carboxylesterase. The rate constants for the inhibition of carboxylesterase by these organophosphate and for the aging and spontaneous reactivation of organophosphate-inhibited carboxylesterase enzyme as well as for the oxime-induced reactivation of organophosphate-inhibited carboxylesterase enzyme by the oximes trimedoxime, methoxime, N,N'-(ethano)bis(4-hydroxyiminomethyl)pyridinium methanosulphonate, HI-6 and HLö-7 were determined in this study. Compared to serum quail carboxylesterase and chicks carboxylesterase displayed a lower sensitivity towards the inspected organophosphate. Furthermore, a slower aging and spontaneous reactivation of serum quail carboxylesterase enzyme was recorded. The potency of the detected oxime reactivator was remarkably lower with organophosphate -inhibited serum quail carboxylesterase.
Keywords: Carboxylesterase, Quail's, Chick's, Organophosphate; Oximes.
V. Barbakadze, L. Gogilashvili, L. Amiranashvili, M. Merlani and K. Mulkijanyan
Plant Biopolymers, Tbilisi Medical University Institute of Pharmacochemistry, Georgia
The high-molecular fractions from the species of two genera (Symphytum and Anchusa) of Boraginaceae family Symphytum asperum, S. caucasicum, S. officinale and Anchusa italica were isolated. According to 13C, 1H NMR and 2D heteronuclear 1H/13C HSQC spectral data the main structural element of these preparations was found to be a regularly substituted polyoxyethylene, namely poly[3-(3,4-dihydroxyphenyl)glyceric acid] (PDPGA) or poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene]. PDPGA represents a new class of natural polyethers with a residue of 3-(3,4-dihydroxyphenyl)glyceric acid as the repeating unit. Most of the carboxylic groups of PDPGA from A. italica unlike the polymer of Symphytum species are methylated. Such caffeic acid-derived biopolymer to our knowledge has not been known and has been identified for the first time. According to results of in vitro and in vivo experiments PDPGA could be considered as potential antiinflammatory, wound healing and anti-cancer therapeutic agent.
PDPGA exerted anti-cancer efficacy in vitro and in vivo against androgen-dependent and -independent PCA cells via targeting androgen receptor, cell cycle arrest and apoptosis without any toxicity, together with a strong decrease in PSA level in plasma.
Overall, this study identifies PDPGA as a potent agent against PCA without any toxicity, and supports its clinical application.
Mohamed Hamdy Doweidar, Seyed Jamaleddin Mousavi
Group of Structural Mechanics and Materials Modelling (GEMM), Aragón Institute of Engineering Research (I3A), University of Zaragoza, Spain.
Mechanical Engineering Department, School of Engineering and Architecture (EINA), University of Zaragoza, Spain.
Cell morphology is a key aspect in many biological processes such as morphogenesis, tumor growth and wound healing. Among other cues, mechanical characteristic of the surrounding micro-environment can control the cell morphology. It is well known that traction forces transmitted to the extracellular matrix (ECM) through cell focal adhesions and integrins play a fundamental role in this process by rearranging the cell cytoskeleton (CSK). In this work we have developed a novel 3D computational model to comprehensively predict the evolution of cell morphology during migration due to mechanotaxis.
A discrete methodology is here chosen by which the cell is represented by a group of finite elements. Therefore, during migration, the cell shape can be efficiently remodeled in a free mode. The present model is developed based on equilibrium of the effective forces over the cell body; the traction force, the protrusion force and the drag force. The cell traction force is governed by the cell internal deformation. The random protrusion force is generated by actin polymerization. The drag force is the substrate viscous resistance.
Correlated with experimental observations, the present model illustrates that the morphology of an adherent cell can be controlled by substrate stiffness and boundary conditions. Our findings indicate that within an unconstrained substrate with a soft (several kPa) and hard (>200 kPa) stiffnesses, the cell is unable to adhere or penetrate into the substrate so that the cell remains mainly rounded without any specific preference of migration direction. In contrast, when a cell is located within a substrate with an intermediate (10 kPa) and rigid (100 kPa) stiffnesses the cell can actively adhere to the substrate migrating towards the constrained surfaces. It can be concluded that in the intermediate and rigid substrates the higher the traction force, the greater the cell elongation, the larger the cell membrane area, and the less random the cell alignment.
Keywords: Finite Element Method, Cell Morphology, Cell migration, Mechanotaxis.
Acknowledgements: The authors gratefully acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (MINECO MAT2013-46467-C4-3-R) and the CIBER-BBN initiative. CIBER-BBN is financed by the Instituto de SaludCarlos III with assistance from the European Regional Development Fund.
Mufeed J. Ewadh, Intisar R. Alsultanee and Muna M. Ewadh
Babylon University, College Of Medicine, Clinical Biochemistry Dept., IRAQ
The present study aims to investigate the methanolic extracts effect of Citrullus colocynthis cortex on xanthine oxidase. It is also known for its highly bitter taste such as 'bitter cucumber'. Cortex of such herb tested in vitro at concentration (100 µg/ml) for their inhibition potencies expressed as % inhibition of XO activity. The test plant inhibition activity of xanthine oxidase found to be (78.30±0.92 %) compared with Allopurinol (positive inhibitor) (p < 0.05). The in vivo hypouricaemic activity of such extract (dose of 0.5 mg/kg b.w.) using oxonate-induced hyperuricaemic mice were tested for their ability to reduce the serum urate level and inhibitory actions on the XO enzyme activities in the mouse liver. Potassium oxonate (150 mg/kg, i.p.), an uricase inhibitor was used to induce hyperuricaemia. Allopurinol (10 mg/kg, p.o.) was used as the positive control. The fractions when administered to hyperuricaemic mice produced a significant reduction in serum urate levels due to inhibitory actions on the XO enzyme activities in the mouse liver. Phytochemical screening of the Citrullus colocynthis cortex revealed the presence of tannins, flavonoids, alkaloids and terpenoids which may be partly responsible for the beneficial effect on hyperuricaemia and gout.These results suggest that fractions of Citrullus colocynthis cortex could be used as a potential source to treat gout and other inflammatory disorders.
Azza EL-Medany1 and Jamila EL-Medany2
1Department of Pharmacology, College of Medicine and KHUH, King Saud University, Kingdom of Saudi Arabia 2Department of Anatomy & Histology, College of Medicine and KHUH, King Saud University
Lung fibrosis is a common side effect of the chemotherapeutic agent, bleomycin. Current evidence suggests that reactive oxygen species may play a key role in the development of lung fibrosis. The present work studied the effect of green tea extract on bleomycin–induced lung fibrosis in rats. Animals were divided into three groups: (1) Saline control group; (2) bleomycin group in which rats were injected with bleomycin (15mg/kg,i.p.) three times a week for four weeks; (3) bleomycin and green tea group in which green tea extract was given to rats (100mg/kg/day, p.o) a week prior to bleomycin and daily during bleomycin injections for 4 weeks until the end of the experiment. Bleomycin–induced pulmonary injury and lung fibrosis that was indicated by increased lung hydroxyproline content, elevated nitric oxide synthase, myeoloperoxidase (MPO), platelet activating factor (PAF), tumor necrosis factor α (TNF_α ), transforming growth factor 1ß ( TGF1ß ) and angiotensin converting enzyme (ACE) activity in lung tissues. On the other hand, bleomycin induced a reduction in reduced glutathione concentration (GSH). Moreover, bleomycin resulted in severe histological changes in lung tissues revealed as lymphocytes and neutrophils infiltration, increased collagen deposition and fibrosis. Co-administration of bleomycin and green tea extract reduced bleomycin–induced lung injury as evaluated by the significant reduction in hydroxyproline content, nitric oxide synthase activity, levels of MPO, PAF & TNF-α & ACE in lung tissues. Furthermore, green tea extract ameliorated bleomycin– induced reduction in GSH concentration. Finally, histological evidences supported the ability of green tea extract to attenuate bleomycin–induced lung fibrosis and consolidation. Thus, the finding of the present study provides that green tea may serve as a novel target for potential therapeutic treatment of lung fibrosis.
Keywords: Bleomycin, Lung fibrosis, Green tea, Oxygen species.
El- Medany Azza1 and EL-Medany Jamila2
1Department of Pharmacology, Faculty of Medicine KHUH, King Saud University, P.O. Box 2925, Riyadh 11461, Saudi Arabia
2Department of Anatomy, Faculty of Medicine, King Saud University, Saudi Arabia
The present study investigated the analgesic and anti-inflammatory effects of chlorogenic acid (CGA), a polyphenolic compound present in many foods and beverages using carrageenan (Carr)-induced paw edema in rats and formalin-induced algesia in mice. The molecular mechanisms of CGA were studied through induction of endogenous & exogenous superoxide anion stress in the aforementioned rodent inflammation & algesia models. The results revealed that treatment of rats with CGA (50, 100 or 150 mg/kg) significantly reduced the rats paw edema induced by carrageenan and the formalin- induced pain in mice (P<.05) as compared to control groups. A significant reduction in rat paw volume in nitric oxide induced edema was observed (P<.05), also CGA produced a significant reduction in malondialdehyde (MDA) and a significant increase in reduced glutathione (GSH) in paw tissues (P<.05). These results confirm that CGA has both analgesic and anti-inflammatory properties which may be related to the ability of this polyphenol to reduce the levels of superoxide and peroxynitrite anion radicals. The results of the present work showed that CGA represents a promising potential drug of natural anti-inflammatory property for the development of new drugs that may help to control oxidative stress and consequently the inflammatory response.
Sarman Singh, Beenish Syed Rufai, Parveen Kumar and Jitender Singh
Division of Clinical Microbiology & Molecular Medicine, All India Institute of Medical Sciences, New Delhi, India
Tuberculosis is global health problem, specially after AIDS epidemic. India is having largest pool of drug resistant tuberculosis. The most important concern is its early diagnosis and drug resistance. The conventional methods of diagnosis and drug resistance detection are cumbersome, takes several weeks and lack reproducibility. Therefore in recent years several molecular methods such as conventional monoplex and multiplex polymerase chain reactions, real time PCR assays, Line probe assay and Xpert MTB/RIF have been made commercially available. The MTBDRplus line probe assay (LPA) and Xpert MTB/RIF have been endorsed by World Health Organization and both these tests can be used for detecting Tuberculosis as well as for drug resistant detection in Mycobacterium tuberculosis. However, there is no clarity regarding the superiority of one over the other. Therefore, for the first time from India, we carried out a prospective study, to evaluated the efficacy of Xpert MTB/RIF and LPA on culture confirmed samples. A total of 405 sputa of suspected drug resistant tuberculosis patients were included. Of these, 285 samples were smear positive and all these were subjected to LPA. Seventy-two (25.8%) samples showed multi-drug resistance, 62 (22.2%) showed rifampicin monoresistance, 29 (10.3%) isoniazid monoresistance and 116 (41.5%) were pan-susceptible. All 62 rifampicin monoresistant samples detected by LPA were tested by Xpert MTB/RIF using cartridge version G4. Of these, 38 (61.4%) showed concordance with LPA showing rifampicin resistance while 21 (33.8%) were found discordant susceptible to rifampicin by Xpert MTB/RIF using cartridge version G4. Of the 116 pan-susceptible samples, only 83 were available for Xpert MTB/RIF testing; of which 4 (5.1%) were found rifampicin resistant, 74 (94.8%) were susceptible. The 25 discrepant samples were further subjected to MGIT960 drug-susceptibility testing. The MGIT960 results showed 100% agreement with LPA results but only 64.4% agreement with Xpert MTB/RIF results. Sequencing analysis of discrepant samples showed 91.3% concordance with LPA but only 8.7% concordance with Xpert MTB/RIF assay. These isolates were characterized by spoligotyping and mycobacterial interspersed repetitive-unit–variable-number-tandem-repeat (MIRU-VNTR) analysis. Interestingly, most of these strains were CAS (CAS-1_Delhi [SIT 26] and 2 of SIT 846), followed by Orphan (SIT 27), and only 1 belonged to the MANU2 (SIT 1976) genotype. NO isolate was Beijing type.
This study shows that Xpert MTB/RIF may not be the first choice of molecular test to be used as point of care as primary screening test.
Rasha F. Mady, Radwa G. Diab and Doaa A. Abdel Monsef
Department of Medical Parasitology and Medical Biochemistry, Faculty of Medicine, Alexandria University, Egypt
Microsporidia, of the genus Enterocytozoon, are an important cause of life-threatening diarrhea especially in immunocompromised hosts. There are controversies on the use of albendazole in treatment, whereas, fumagillin was to be more effective but with undesirable side effects. Ginger has been used as an antimicrobial agent since ancient times. However its potential therapeutic effect against Enterocytozoon bieneusi has not been tested. This study was done to investigate the effect of ginger as a prospective therapy for microsporidia versus fumagillin in immunocompetent and immunosuppressed mice. Also, to report the synergistic effect of the two compounds together in a drug-combination regimen. Enterocytozoon bieneusi was the species identified in the stool samples collected from immunocompromised patients and was used to initiate the in vivo infection in albino mice. Animals were divided into three major groups. Group I: Normal, non-infected nontreated, control group; group II: infected, immunocompetent group; and group III: infected, immunosuppressed group. Each infected group was subdivided into four equal subgroups a, b, c and d which comprise non-treated, fumagillintreated, ginger-treated, and combined ginger/fumagillin treated mice respectively. Evaluation of the ginger efficacy in infected mice was achieved by assessment of fecal spore shedding, intestinal spore load, and biochemical assay which aimed at estimation of the malondialdehyde level and total antioxidant capacity. Spore count in both stool and intestinal sections and malondialdehyde level decreased significantly with ginger treatment. Best results were obtained when ginger is combined with fumagillin in all measured parameters. Ginger could be a good enhancer for fumagillin efficacy to eradicate infection. However, further studies on their its principles, mechanisms of action, toxicity evaluation are still needed.
Wei Li, Chun Chen, Zhiwei Jia, Xuedong Bai and Dike Ruan
Department of Orthopedics, Navy General Hospital, Beijing 100048, China
Introduction: The debilitating effects of lower back pain are a major health issue worldwide. A variety of factors contribute to this, and oftentimes intervertebral disk degeneration (IDD) is an underlying cause of this disorder. Inflammation contributes to IDD, and inflammatory cytokines play key roles in the pathology of IDD. This study characterized the potential to suppress inflammatory cytokine production in degenerative intervertebral disc (NP) cells by treatment with IL-10 and TGF-β in a canine model of IDD.
Methods: IDD was induced surgically in six male beagles, and degenerative NP cells were isolated and cultured for in vitro studies on cytokine production. Cultured degenerative NP cells were divided into four experimental treatment groups: untreated control, IL-10-treated, TGF-β-treated, and IL-10- plus TGF-β-treated cells. Cultured normal NP cells served as a control group. TNF-α expression was evaluated by FACS analysis and ELISA; moreover, ELISA and realtime PCR were also performed to evaluate the effect of IL-10 and TGF-β on NP cell cytokine expression in vitro.
Results: The major findings of these analysis are that after treatment with IL-10 and TGF-β, the expression of extracellular and intracellular TNF-α and IL-1β was suppressed, while the expression of inflammatory cytokines in untreated normal NP cells was significantly lower than that in untreated degenerative NP cells. Our results demonstrated that IL-10 and TGF-β treatment suppressed the expression of IL-1β and TNF-α and inhibited the development of inflammatory responses.
Discussion: We observed that either TGF-β or IL-10 alone suppressed the expression of inflammatory cytokines. Furthermore, their combined use produced a higher level of inhibition of TNF-α and IL-1β than either TGF-β or IL-10 alone. IL-10 and TGF-β should be evaluated as therapeutic approaches for the treatment of lower back pain mediated by IDD.
Manolov V, Atanasova B, Velizarova M, Vasilev V, Bogov B, Yonova D, Vazelov E, Paskaleva- Peycheva V, Hadjiev E, Hadjidekova S, Tzatchev K
Medical University - Sofia, Medical Faculty, Dept. of Medical Genetics, Bulgaria
Aim: Hepcidin is a key regulator of the iron homeostasis. The pathogenesis of the anemia of chronic disease (ACD) is coupled with hepcidin stimulated synthesis as a result of inflammation. Hereditary hemochromatosis covers genetic disorders in the metabolism of iron by the appearance of superimposition. Iron overload leads to organ damage such as liver fibrosis, cirrhosis, cardiomyopathia. In patients with iron-deficiency anemia (IDA) and those with low serum ferritin latent and pre-latent form of iron deficiency without concurrent anemia (due to blood loss or insufficient iron intake from food) sets very low hepcidin levels. The purpose of our study was to investigate serum hepcidin in patients with ACD and IDA in different diseases in order to help finding the right therapeutic choice.
Data: For a period 2012-2014 we study serum hepcidin levels using verified ELISA method in 50 healthy volunteers, 50 patients with chronic kidney disease (CKD, stages II to IV), 30 patients with CKD (stage V) and 30 patients with rheumatoid arthritis.
Results: We found significantly high serum hepcidin levels in patients with ACD [0.3 < r < 0.7; P < 0.001]: in CKD stages II to IV 81.52 ± 10.7 μg/L; in CKD stage V 219.14 ± 30.6 μg/L and in patients with RA 103.83 ± 18.4 μg/L compared to control group 13.67 ± 4.9 μg/L. In patients with RA and IDA we found significantly low serum hepcidin levels 0.78 ± 0.3 μg/L (P < 0.001).
Discussion: Reliable method for serum hepcidin is important for the proper therapeutic approach in patients with ACD and IDA in different diseases. The rate of rise may differentiate patients with functional iron deficiency than those with reticuloendothelial blockade. Monitoring of serum hepcidin concentrations in parenteral therapy with iron preparations reduces the risk of toxic overload of iron in the body. Hepcidin is a potential indicator of iron deficiency in RA patients with anemia and an active inflammatory process.
Shree Prakash Panday
Biological Sciences, Indian Institute of Science Education and Research Kolkata, India
Plants face a plethora of biotic stresses in their agro-ecological environments. Responses of plants tailored to these stresses involve perception, processing and integration of external information into cellular and physiological machinery. This involves elicitation of complex signaling networks. But hese signaling networks remain poorly characterized in crop species such as wheat. Moreover, other than the involvement of some transcription factors (TFs), how cellular signaling is modulated during attack of fungal pathogens and herbivores remains poorly understood even in model plants. On the other hand, small regulatory RNAs (smRNAs), such as microRNAs, have appeared as master regulators of cellular signaling events in processes such as development and differentiation. Our functional studies on components small RNA machinery suggest that smRNA biogenesis pathways have evolved in specialized manner during adaptation to specific stresses. Use the Argonaute (AGO) proteins (the central component of the smRNA pathways) as candidates for studying evolution of smRNA pathways; we conclude that the evolution of smRNA pathways has been a dynamic process that could generate signatures of their diversification of function in plants. We have extended our investigations to the wheat genomes to identify, annotate and understand signaling pathways and to determine phyloenetic linkages in other grass genomes. Our results have direct implications for biotechnological applications for crop improvement in cereals in general and wheat in particular.
Keywords: Phytohormone signaling, small RNA, miRNA, transcription factor, stress adaptation.
James Petrie, Thomas Vanhercke, Allan Green and Surinder Singh
CSIRO Food, Nutrition and Bioproducts Flagship, ACT, Australia
Supply of vegetable oils as a major commodity faces continuous pressure. Global demand is expected to double in the next two decades due to increasing world population and rising petroleum prices. Increasing limitations on arable land and agricultural inputs mean it will be difficult to meet this additional demand with current oilseed-based production systems. The concept of producing oil in the leaves and stems of high biomass species has attracted attention as a way to intensify oil production. The engineering of such a new oil production platform would not only yield greater amount of oil for a given land area but also provide a way to more easily segregate bioeconomy traits such as unusual fatty acids away from food production. We previously reported the accumulation of up to 17% triacylglycerol (TAG, dry weight) in leaf tissue of Nicotiana species. This was achieved by combinatorial metabolic engineering in which we increased fatty acid biosynthesis (‘Push’) by limited overexpression of the WRI1 transcription factor, increased TAG assembly (‘Pull’) by expressing DGAT1, and encouraged oil body formation (‘Packaging’) by expressing oleosin in plant leaves1,2.
In this presentation, we will describe some of our second generation construct designs which have more than doubled the previously reported TAG content. Oil content in leaves now matches elite oilseed crop seed levels of +40%. We will describe the implications that this technology has for global plant oil production from a yield and intensification perspective, as well as the challenges that remain for integration into the existing industry. We will also present data demonstrating that the newly produced fatty acids can be modified for industrial or nutritional applications as well as preliminary data of a transcriptome comparison between wild type and high oil leaf tissue, harvested at different stages during plant development.
 Vanhercke et al., 2012. Synergistic effect of WRI1 and DGAT1 coexpression on triacylglycerol biosynthesis in plants. FEBS Letters 587(4):364-9.
 Vanhercke et al., 2014. Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves. Plant Biotechnology Journal 12(2):231-9.
Munusamy Madhaiyan and Lianghui Ji
Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604
Jatropha curcas is promising new non-food crop for biodiesel production because of its ability to thrive on marginal land, with strong tolerance to draught and poor soil nutrient content [1-3]. Nevertheless, high fertilizer input remains essential in order to have commercially acceptable oil productivity. This reduces the green index of Jatropha biofuel. To address this issue, we studied the taxonomical distribution of 1017 cultivable endophytic bacterial strains isolated from different parts of Jatropha with an emphasis on nitrogen-fixing bacteria. Like other reports, we found strong tissue preferences of the bacteria in Jatropha. The16S rRNA gene sequences can be assigned to five major phyla and, surprisingly, 31.2% of them potentially represent new taxa. Nitrogen fixing isolates were found diverse and present in five classes belonging to α, β, γ- Proteobacteria, Actinobacteria and Firmicutes. The phylum Proteobacteria was the most dominant amongst strains that were positive for both nifH gene and endoglucanase activity. Methylobacterium species account for 69.1% of the leaf endophytic bacterial isolates. Notably, many Methylobacterium isolates were able to fix nitrogen.
We will present genomic, physiology and plant-bacteria interaction studies on two strong nitrogen-fixing isolates: Kosakonia sp. R4-368 (previously Enterobacter R4-368 [4,5]) that mainly colonizes in roots and stems and Methylobacterium radiotolerans L2-4 that mainly colonizes leaf tissues both as endophyte and epiphyte. Root treatment of R4-368 or foliar application of L2-4 significantly improved growth parameters, such as plant height, leaf number, relative chlorophyll content and stem volume. Importantly, strain L2-4 improved seed yield by 222.2% and 96.3% in plants potted in sterilized and non-sterilized soil pots respectively. Strain R4-368 improved seed set by approximately 177% and 49.0% in sterilized and non-sterilized soil respectively. The average single seed weight was increased approximately 10% by strain R4-368. Yield improvements were mainly attributed to an increase of female-male flower ratio, which led to a corresponding increase of fruit and seed sets. Furthermore, there was an additive effect for seed yield of root and leaf treatments were both performed. We will present the effects of application of the two isolates on other crops.
Key words: Nitrogen-fixation, biofuel, plant growth promoting bacteria, Jatropha curcas
 Fairless D (2007) Biofuel: The little shrub that could - maybe. Nature 449:652-655. doi:doi:10.1038/449652a
 Gaydou AM, Menet L, Ravelojaona G, Geneste P (1982) Vegetable energy sources in Madagascar: ethyl alcohol and oil seeds.
Oleagineux 37 ((3)):135–141
 Openshaw K (2000) A review of Jatropha curcas: an oil plant of unfulfilled promise. Biomass Bioenergy 19 (1):1-15
 Madhaiyan M, Peng N, Ji L (2013) Complete Genome Sequence of Enterobacter sp. Strain R4-368, an Endophytic N-Fixing Gammaproteobacterium Isolated from Surface-Sterilized Roots of Jatropha curcas L. Genome announcements 1 (4):e00544- 00513
 Yan H, Ji L (2013) Improvement of plant growth and seed yield in Jatropha curcas by a novel nitrogen-fixing root associated Enterobacter species.
Adel M. A. Assiri, Hala F. M. Kamel and Abeer A. EL-Refai
Biochemistry Department, Faculty of Medicine, Umm Al-Qura University, Makah, KSA and Medical Biochemistry Department, Faculty of Medicine,Ain Shams University, Cairo, Egypt
AGEs are a heterogeneous group of compounds derived from non-enzymatic glycation of proteins, lipids and nuclic acids through complex reaction known as the Maillard reaction. AGEs interact with the receptor for AGEs (RAGE) on the membrane and induce harmfull effects through activation of nuclear factor kappa-B, and increased oxidative stress and inflammatory mediators. AGEs combine with membrane receptors (RAGEs) also combine with circulating soluble receptors (sRAGE) and act as a decay agent. AGEs are thought to be involved in many complications of angiopathy, cardiovascular diseases (CVD) and nephropathy in diabetes as well as those of end‐stage renal failure. The aim of this study was to evaluate the changes of (AGEs), (sRAGE) and Oxidized Low-density Lipoproteins ( OxLDL) levels, in hemodialysis patients with different underlying causes and to evaluate them in relation to occurrence of CVD or with diabetic nephropathy as an underlying cause in relation to other glycemic, lipid profile and renal functions' biomarkers.Our study included 279 patients with end stage renal disease (ERD) who received maintenance hemodialysis (HD) (duration of HD, 6.7 ± 3.4 years.) and 112 sex and age matched healthy control subjects.The underlying casual disorders for them were as follows :diabetic nephropathy (56.9%) and non-diabetic nephropathy diseases (43.1%) including chronic hypertensive nephrosclerosis, glomerulonephritis, polycystic kidney disease, and rest of unknown etiology. We also categorized HD patients into 2 subgroups according to their positive medical history of CVD ( 52.09%) and another subgroups with no medical history of CVD (47,01%).We demonstrated significantly higher levels of oxLDL, AGES and sRAGE in HD than healthy control group ( P< 0.001). We found significant increase of OxLDL, AGEs and sRAGE in HD diabetic nephropathy subgroup when compared with non-diabetic subgroup as an underlying cause. Interestingly sRAGEs significantly decreased in HD patients who had positive history for CVD in comparison with those HD patients with no history of CVD (1970 ± 870, 2660 ± 908; P < 0.001). There was positive correlation between AGEs and sRAGE in whole HD patients (r =0.441;P = < 0.001, r = 0.395; P= < 0.05) while only in HD group with CVD There was negative correlation ( r = - 0.294 ; p = <0.01). We found that AGEs was an independent determine for diabetic nephropathy as an underlying cause for ESR (OR, 1.25; 95% CI, 1.04 to 1.49; P < 0.016). Another factor was the oxLDL level (OR, 1.10; 95% CI, 1.02 to 1.19, P < 0.018) and sRAGE (OR, 1.04; 95% CI, 1.01 to 1.06, P < 0.007). and we identified sRAGE as an independent factor associated with the prevalence of CVD(OR, -0. 49; 95% CI, -0.086 to 0.034; P < 0.001). The other factors were AGEs (OR, 2.81; 95% CI, 1.79 to 4.41; P < 0.001), ox LDL (OR, 1.04; 95% CI, 1.02 to 1.06; P < 0.001),and hs-CRP level (OR, 1.02; 95% CI, 1.00 to 1.05, P <0.046).Conclusions plasma OxLDL,AGEs and sRAGE levels are strongly associated with the prevalence of cardiovascular disease and diabetic nephropathy in hemodialysis patients and could be considered future markers for diabetic nephropathy and cardiovascular diseases in hemodialysis patients with end stage renal disease.
Orenburg State Medical Academy, 460000 Orenburg, Russia
Studies on computer simulation of the genome (see, e.g., ) lead to understanding that not only the characteristics of body elements (structure, weight, color, etc.), but also all interactions between them are directly determined at the genomic level. Tolerance of self-antigens should also be directly determined by the genome, through genomic or "smart" recognition, rather than through negative selection against self-reactive lymphocytes. The concept of linked functions  holds that the very presence of the genes of class I MHC self-antigens in the genome "automatically" precludes immune response to these antigens. Therefore, integration of certain class I MHC genes of the donor into the genome of the recipient's hematopoietic stem cells in the course of preoperative treatment should result in tolerance of the donor's MHC antigens. Conceivably, this approach to the formation of tolerance should also work for xenogenic grafts, which would considerably enhance the possibilities of tissue and organ transplantation. The technique for integrating foreign class I MHC genes into the genome of hematopoietic stem cells has not been developed thus far. Solution of the numerous problems involved requires experimental research. This research is going to take much effort, and I would appreciate any suggestions on collaboration and/or sponsorship.
 Galimov, E.M. Origins of Life and Evolution of the Biosphere. Springer, 2004.
 Malyshkin A.P. Adaptive Immunity: The Concept of Linked Functions. Immunology Innovation, 2013, 1:1. http://dx.doi.org/10.7243/2053-213X-1-1.
James Petrie, Malcolm Devine, Greg Buzza and Surinder Singh
CSIRO Food, Nutrition and Bioproducts Flagship, ACT, Australia and 2 Nuseed Pty Ltd, VIC, Australia
Omega-3 long chain polyunsaturated fatty acids like EPA and DHA have critical roles in human health and development with studies indicating that deficiencies in these fatty acids can increase the risk or severity of cardiovascular and inflammatory diseases in particular. These fatty acids are predominantly sourced from fish and algal oils. In order to meet the increasing demand for these oils there is an urgent need for an alternative and sustainable source of EPA and DHA. We have focused on maximising the production of DHA in seed. This talk will describe the transition of DHA production in seed of our model species Arabidopsis  through to Camelina , Brassica juncea and our target crop Brassica napus. DHA levels that exceed the amount typically found in bulk fish oil have now been achieved in all four species. This talk will describe gene characterisation, construct designs, transgenic plants and seed oil fatty acid profiles. We will describe the progress of the ongoing GM canola field trials conducted by Nuseed as well as the effects of growth temperature on the performance of transgenic and endogenous desaturases. We will also describe some of the intellectual property challenges this project faced and how we have solved them to enable the real-world deployment of this new, high value GM crop.
 Petrie et al., 2012. Metabolic engineering plant seeds with fish oil-like levels of DHA. PLoS One 7(11):e49165.
 Petrie et al., 2014. Metabolic engineering Camelina sativa with fish oil-like levels of DHA. PLoS One 9(1):e85061.
Reza Mohammad Emon, A. Y. M. Nevame, G. B. Gregorio, M. R. Islam and Fan Ye-Yang
State Key Laboratory of Rice Biology, China National Rice Research Institute, China
Selection of new rice (Oryza sativa L.) variety tolerant to soil salinity is one of the most important issues to avoid salt stress in rice production. In this study, the morphological characterization was performed on 5 rice genotypes under two salt stress treatments. Morphological characters and Na+/K+ uptake ratio were surveyed to evaluate salt stress effects. Under EC 12 dS/m of salt stress at seedling stage, NSIC Rc222 and IR64 were found sensitive to salt compared to BRRI dhan47, Binadhan-8, and Binadhan-10, which were moderately tolerant. Yet, at vegetative stage all the genotypes have survived. However, at reproductive stage all the genotypes were affected by salt stress except for the Binadhan-10 that survived the whole life cycle. A total of 160 SSR markers used have revealed 209 alleles among the 5 rice genotype. Interestingly, 4 SSR markers with highest PIC (0.67) value have scored the highest level of genetic diversity value (0.72). The present investigation on variant salt stress responsiveness of 5 promising rice genotype will be of great value in rice breeding.
Keywords: Salt stress; Na+/K+ ratio; genetic diversity; promising genotype; rice.
Mohamed F. Foda, K. Shao, J. Wang, L. Huang, L. Wu, K. Cai, J.W. Liu, Z.C. Lu, and H.Y. Han
State Key Laboratory of Agricultural Microbiology, College of Science, Huazhong Agricultural University, Wuhan, 430070, P.R. China
The detection of low-abundance biomarkers and targeted therapy of cancers using fabricated hybrid nano-bioprobe are particularly desirable but remain a great challenge. In current work, designing signal amplifying electrochemical biosensors by silicon carrier nanoparticles integrated with hybrid gold nanoparticle-graphene nanosheet matrix became one appropriate way to overcome the drawback of universal methods such as time consuming and insensitivity. By this manner, the combination of electrochemical and luminescent methods has been proved to be a powerful analytical technique for ultrasensitive immunosensor and early diagnosis with a detection limit up to 0.40 pg mL-1.
On the other hand, fabricating drug carrier with excellent performance, another remarkable coating strategy for I-III-VI semiconductor quantum dots, the most promising and challenging QDs, has been achieved. In this regard, we incorporated multiple hydrophobic CuInS2/ZnS and CdSe/CdS/ZnS directly into silica beads and further TEOS coating via Stöber method was carried out. Afterwards, a mesoporous nanostructure was established via CTAB/NaOH mixture with an overall size 45-50 nm in diameter, achieving a porous size 4 nm. More interesting, the addressed mesoporous QDs turned to be a promising drug delivery carrier in the biomedical application and cancer therapy.
Keywords: Biosensors, immunosensor, silicon nanoparticles, antibody, CuInS2/ZnS QDs.
Ayaz Mohammed, Aftab Aslam Parwaz Khan and Shaista Bano & K. S. Siddiqi
Applied Biotechnology Department, Sur College of Applied Science, Sur Sultanate of Oman
A simple, sensitive, and rapid method based on ion association, for the determination of FLD has been developed. Flutamide (FLD) can react with Cu(II) to form 1:1 cationic chelate at pH 2.2-7.0 Mclivaine buffer medium, which can further react with anionic surfactants (AS) such as sodium dodecyl sulfate (SDS), sodium lauryl sulfonate (SLS) and sodium dodecylbenzene sulfonate (SDBS) to form 1:1 ion association complexes. As a result, the resonance Rayleigh scattering (RRS), second-order scattering (SOS) and frequency doubling scattering (FDS) were enhanced to the highest degree. The maximum RRS, SOS and FDS wavelengths of three ion-association complexes were located at 345/345 nm, 610/305 nm and 430/ 860 nm, respectively. The increments of scattering intensity (ΔI) were directly proportional to the concentration of FLD in certain ranges. The detection limits (3σ) of FLD for SDS, SLS and SDBS systems were 1.9 ng ml−1, 2.1 ng ml−1 and 2.2 ng ml−1(RRS method), 2.4 ng ml−1, 2.7 ng ml−1 and 2.6 ng ml−1 (SOS method) and 2.3 ng ml−1, 2.4 ng ml−1 and 2.5 ng ml−1 (FDS method), separately. The sensitivity of RRS method was higher than those of FDS and SOS methods. The optimum conditions of RRS method and the influence factors, the composition and the reaction mechanism have been discussed. Since the method is highly selective, it does not interference concomitant substances. These methods were applied successfully for the determination of FLD in pharmaceutical formulation and urine.
Keywords: Flutamide (FLD), Cu(II), Resonance Rayleigh scattering, Anionic surfactants, Urine sample.
CSIRO Agriculture Flagship, Canberra Australia
Dietary fibre is largely derived from the cell walls of plants and in cereal grains, arabinoxylan and 1,3;1,4-β-D-glucan (betaglucan) are the major cell wall polysaccharides. Dietary fibre is an essential part of a healthy diet however fibre consumption in most Western countries is below target levels. Food products made from grains that have a high content of water soluble betaglucan such as oats and barley are allowed specific health claims in some countries related to the lowering of blood cholesterol. Wheat grain has only low levels of betaglucan, most of which is insoluble. The cellulose-synthase-like CslF6 gene is a major component of the betaglucan synthase and we will describe work to characterise the function of this protein from various cereals in Nicotiana benthamiana leaf expression system. Each CslF6 gene produces a betaglucan with a distinct structure and this affects water solubility. Overexpression of selected CslF6 genes in wheat grain increases levels of soluble betaglucan and dietary fibre.