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Kanthala S.,University of Louisiana at Monroe | Gauthier T.,Biotechnology Laboratory | Satyanarayanajois S.,University of Louisiana at Monroe
Biopolymers | Year: 2014

Human epidermal growth factor receptor-2 (HER2) is a tyrosine kinase family protein receptor that is known to undergo heterodimerization with other members of the family of epidermal growth factor receptors (EGFR) for cell signaling. Overexpression of HER2 and deregulation of signaling has implications in breast, ovarian, and lung cancers. We have designed several peptidomimetics to block the HER2-mediated dimerization, resulting in antiproliferative activity for cancer cells. In this work, we have investigated the structure-activity relationships of peptidomimetic analogs of Compound 5. Compound 5 was conformationally constrained by N- and C-terminal modification and cyclization as well as by substitution with d-amino acids at the N-and C-termini. Among the compounds studied in this work, a peptidomimetic Compound 21 with d-amino acid substitution and its N- and C-termini capped with acetyl and amide functional groups and a reversed sequence compared to that of Compound 5 exhibited better antiproliferative activity in HER2-overexpressed breast, ovarian, and lung cancer cell lines. Compound 21 was further evaluated for its protein-protein interaction (PPI) inhibition ability using enzyme fragment complementation assay, proximity ligation assay, and Western blot analysis. Results suggested that Compound 21 is able to block HER2:HER3 interaction and inhibit phosphorylation of the kinase domain of HER2. The mode of binding of Compound 21 to HER2 protein was modeled using a docking method. Compound 21 seems to bind to domain IV of HER2 near the PPI site of EGFR:HER2, and HER:HER3 and inhibit PPI. Copyright © 2013 Wiley Periodicals, Inc.

Albani M.C.,Max Planck Institute for Plant Breeding Research | Albani M.C.,University of California at San Diego | Vincent C.,Max Planck Institute for Plant Breeding Research | Bergonzi S.,Max Planck Institute for Plant Breeding Research | And 6 more authors.
Plant Cell | Year: 2011

Flowering of many plants is induced by environmental signals, but these responses can depend on the age of the plant. Exposure of Arabidopsis thaliana to vernalization (winter temperatures) at germination induces flowering, whereas a close perennial relative Arabis alpina only responds if exposed when at least 5 weeks old. We show that vernalization of these older A. alpina plants reduces expression of the floral repressor PEP1 and activates the orthologs of the Arabidopsis flowering genes SOC1 (Aa SOC1) and LFY (Aa LFY). By contrast, when younger plants are vernalized, PEP1 and Aa SOC1 mRNA levels change as in older plants, but Aa LFY is not expressed. We demonstrate that A. alpina TFL1 (Aa TFL1) blocks flowering and prevents Aa LFY expression when young plants are exposed to vernalization. In addition, in older plants, Aa TFL1 increases the duration of vernalization required for Aa LFY expression and flowering. Aa TFL1 has similar functions in axillary shoots, thus ensuring that following a flowering episode vegetative branches are maintained to continue the perennial life cycle. We propose that Aa TFL1 blocks flowering of young plants exposed to vernalization by setting a threshold for a flowering pathway that is increased in activity as the shoot ages, thus contributing to several perennial traits. © 2011 American Society of Plant Biologists.

Rathore M.S.,Jai Narain Vyas University | Rathore M.S.,Biotechnology Laboratory | Shekhawat N.S.,Jai Narain Vyas University
Environmental and Experimental Botany | Year: 2013

Leptadenia reticulata (Retz.) Wight& Arnis an important medicinal plant, belongs to the family AsclepiadaceaeThis plant is known for its medicinal uses since 4500 BCPresently this is an endangered species (Arya et al., 2003)Six shoots (2-4cm long) per node differentiated on MS medium+5.0mg/l of BAP+additivesIncorporation of additives in the culture medium promoted growth of culturesThe shoots differentiated per explant were repeatedly transferred on to fresh MS+1.0mg/l of BAP+0.1mg/l of NAA and additivesThe regenerated shoots were subcultured for further multiplication on MS+1.0mg/l BAP+0.5mg/l Kin+2-iP (0.5mg/l) and 0.1mg/l of NAA+additives regularly after an interval of 3 weeksAddition of ammonium sulphate in the medium resulted in increase in shoot number and promoted elongation also growth of cultures was sustained even if subculturing was delayed (26±2 days)Success was also achieved in defining protocol for in vitro regeneration of shoots from petiole derived callusShoots regenerated in vitro by both processes were rooted in vitro on 1/4 strength of MS medium+3.0mg/l of IBA after 15-20 daysCent percent of the shoots rooted ex vitro, if the in vitro regenerated shoots were treated with 200mg/l of IBAThe in vitro-ex vitro rooted plantlets were hardened under different regimes of temperature and humidity in a greenhouseThe hardened plantlets were transferred to soil in polybagsMore than 95% plants survived in field conditionsTotal dry biomass harvested per year was 2800kg/acre © 2010 Elsevier B.V.

Barbet-Massin E.,University Claude Bernard Lyon 1 | Ricagno S.,Biotechnology Laboratory | Ricagno S.,University of Pavia | Ricagno S.,University of Milan | And 7 more authors.
Journal of the American Chemical Society | Year: 2010

Elucidating the fine structure of amyloid fibrils as well as understanding their processes of nucleation and growth remains a difficult yet essential challenge, directly linked to our current poor insight into protein misfolding and aggregation diseases. Here we consider β-2-microglobulin (β2m), the MHC-1 light chain component responsible for dialysis-related amyloidosis, which can give rise to amyloid fibrils in vitro under various experimental conditions, including low and neutral pH. We have used solid-state NMR to probe the structural features of fibrils formed by full-length β2m (99 residues) at pH 2.5 and pH 7.4. A close comparison of 2D 13C-13C and 15N-13C correlation experiments performed on β2m, in both the crystalline and fibrillar states, suggests that, in spite of structural changes affecting the protein loops linking the protein β-strands, the protein chain retains a substantial share of its native secondary structure in the fibril assembly. Moreover, variations in the chemical shifts of the key Pro32 residue suggest the involvement of a cis-trans isomerization in the process of β2m fibril formation. Lastly, the analogy of the spectra recorded on β2m fibrils grown at different pH values hints at a conserved architecture of the amyloid species thus obtained. © 2010 American Chemical Society.

Shekhawat M.S.,Biotechnology Laboratory | Manokari M.,Biotechnology Laboratory
Physiology and Molecular Biology of Plants | Year: 2016

In vitro propagation methods using seeds and nodal segments of a 21-year old Couroupita guianensis - a medicinally important but threatened tree have been developed. Hundred percent of the seeds germinated on half strength Murashige and Skoog (MS) medium with 2.0 mg l−1 indole-3 butyric acid (IBA). Nodal segments were found most suitable for the establishment of cultures. About 90 % explants responded and 4.1 ± 0.23 shoots per node were induced after five weeks of inoculation on MS medium +4.0 mg l−1 6-benzylaminopurine (BAP). Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoots on fresh medium. Maximum number (8.2 ± 0.17) of shoots were regenerated on MS medium with 1.0 mg l−1 each of BAP and Kinetin (Kin) + 0.5 mg l−1 α-naphthalene acetic acid (NAA) with additives (50 mg l−1 of ascorbic acid and 25 mg l−1 each of adenine sulphate, L-arginine and citric acid). The multiplied shoots rooted (4.3 ± 0.26 roots/shoot) on half strength MS medium with 2.5 mg l−1 IBA. All the shoots were rooted ex vitro when pulse treated with 400 mg l−1 of IBA for five min with an average of 7.3 ± 0.23 roots per shoot. Nearly 86 % of these plantlets were acclimatized within 7–8 weeks and successfully transferred in the field. Biologically significant developmental changes were observed during acclimation particularly in leaf micromorphology in terms of changes in stomata, veins and vein-islets, and trichomes. This study helps in understanding the response by the plants towards outer environmental conditions during acclimatization. This is the first report on micropropagation of C. guianensis, which could be used for the large-scale multiplication, restoration and conservation of germplasm of this threatened and medicinally important tree. © 2016 Prof. H.S. Srivastava Foundation for Science and Society

Shekhawat M.S.,Biotechnology Laboratory | Kannan N.,Biotechnology Unit | Manokari M.,Biotechnology Laboratory
South African Journal of Botany | Year: 2015

An efficient micropropagation protocol has been developed successfully for Morinda coreia Buch.-Ham. by culturing nodal segments. The explants were washed, sterilized with HgCl2 and inoculated on semi-solid Murashige and Skoog (MS) medium containing various concentrations and combinations of plant growth regulators (PGRs). Shoot bud initiation was observed after one week and 8.6±0.32 shoots (per explant) harvested after five weeks on MS medium with 4.0mgl-1 concentration of 6-benzylaminopurine (BAP). The regenerated shoots were further multiplied on semi-solid MS medium augmented with 2.0mgl-1 BAP+1.0mgl-1 Kinetin (Kin). Maximum 24.5±0.34 shoots per explant was obtained after five weeks on this media combination. The long (4-5cm) and healthy shoots were rooted in vitro with 100% success rate on half strength MS medium+1.0mgl-1 indole-3 butyric acid (IBA). Rooting and acclimatization were achieved simultaneously by ex vitro rooting method using 200mgl-1 IBA for 5min with very good success rate (28.67±05.51 roots per shoot with 100% response). The rooted shoots were transferred to the greenhouse for acclimatization for 4-5weeks. The hardened plantlets were finally shifted to the field for further growth in the natural conditions after another five weeks. This is the first report on micropropagation of M. coreia, which can be successfully used for the large-scale multiplication and conservation of germplasm of this important medicinal plant. © 2015 South African Association of Botanists.

Shekhawat M.S.,Biotechnology Laboratory | Manokari M.,Biotechnology Laboratory
Indian Journal of Plant Physiology | Year: 2016

Hemidesmus indicus is an endangered medicinal plant of Peninsular India. A rapid micropropagation method using nodal segments of a 4 year old plant has been achieved in the present study. The nodal explants (2.0–3.0 cm long) were harvested from actively growing shoots of conventionally raised plants and cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg l−16-benzylaminopurine (BAP). Hundred percent explants responded with 5.2 ± 0.25 cm long multiple shoots (9.0 ± 0.53) from the nodal meristems. The shoots were further multiplied enormously (272 ± 4.12 shoots per explant) by repeated subculture of mother explants and freshly induced shoot clumps on MS medium with the reduced concentrations of BAP (1.0 mg l−1), kinetin (Kin; 0.5 mg l−1) and indole-3 acetic acid (IAA; 0.1 mg l−1) within 4 weeks. Highest root induction (62.0 ± 0.54 roots per shoot) was observed on 1/4th strength MS medium supplemented with 3.0 mg l−1indole-3 butyric acid (IBA). About 96 % shoots were rooted (45.0 ± 0.48 roots per shoot) by ex vitro methods when the cut ends of the shoots pulse treated with IBA (400 mg l−1) for 5 min. The in vitro as well as ex vitro rooted plantlets were acclimatized successfully in the greenhouse. Ex vitro rooted plantlets exhibited higher percentage (98 %) of survival as compared to the in vitro rooted plantlets (91 %) in the field conditions. There were not any observable differences between in vitro propagated and field transplanted plantlets. A comparative foliar micro-morphological study of H. indicus was conducted to understand the micro-morphological changes in the plants while shifting from in vitro to the in vivo conditions in terms of variations in stomata, venation pattern and vein density, and trichomes. The aforementioned protocol could be successfully used for the large-scale multiplication and conservation of germplasm of this endangered plant species. © 2016, Indian Society for Plant Physiology.

Shekhawat M.S.,Biotechnology Laboratory | Manokari M.,Biotechnology Laboratory
Physiology and Molecular Biology of Plants | Year: 2016

The optimum concentrations of the plant hormones for in vitro regeneration and subsequent effect of auxins on rooting (in vitro and ex vitro) of shoots of Basella alba L. have been investigated in present study. Nodal shoot segments were used as explants to initiate the cultures. The bud breaking from explants was observed within 1 week of incubation on agar gelled Murashige and Skoog’s (MS) medium. Multiple axillary shoots (7.30 ± 0.56 shoots per explant) were induced on MS medium supplemented with 2.0 mg/L 6-benzylaminopurine (BAP). The shoots were multiplied (maximum 17.10 ± 0.44 shoots per explant) on the same medium fortified with 0.5 mg/L each of BAP and Kin (Kinetin) +0.1 mg/L IAA. These shoots were excised and rooted in vitro (10.73 ± 0.92 roots per shoot) on half-strength MS medium augmented with 2.0 mg/L indole-3 butyric acid (IBA). Hundred percentage success rates have been achieved by ex vitro rooting of the in vitro regenerated shoots with IBA at 300 mg/L. The in vitro and ex vitro rooted shoots were acclimatized in greenhouse and subsequently transferred to the natural field conditions where 100 % survival rate was reported. The ex vitro rooting method was found more advantageous than in vitro rooting in terms of time, energy and survival percentage of B. alba. A comparative foliar micromorphological study of B. alba was conducted to understand the micromorphological changes in plants while shifting from in vitro to the in vivo conditions in terms of variations in stomatal index, venation pattern and vein density, and the arrangement of crystals. The study could help in understanding the response of in vitro raised plants towards in vivo environment. © 2016 Prof. H.S. Srivastava Foundation for Science and Society

Bantawa P.,Biotechnology Laboratory | Saha-Roy O.,Biotechnology Laboratory | Ghosh S.K.,Biotechnology Laboratory | Mondal T.K.,Biotechnology Laboratory
Biologia Plantarum | Year: 2011

A reproducible in vitro regeneration system for Nepalese kutki (Picrorhiza scrophulariiflora Pennell) was developed from in vitro leaf derived callus. Induction of more than seven shoot buds per explant was achieved on Woody plant medium (WPM) supplemented with 0.53 μM α-napthaleneacetic acid (NAA) and 0.23 μM kinetin (KIN). The shoots were elongated on WPM supplemented with 0.44 μM 6-benzylaminopurine (BAP) and rooted on WPM supplemented with 5.3 μM NAA within 2 weeks. The random amplified polymorphic DNA (RAPD) analysis indicated genetic uniformity of the micropropagated plants with its donor plants. © 2011 Springer Science+Business Media B.V.

News Article | December 1, 2016
Site: phys.org

A team of scientists from the Centre for Biological Research (CIB-CSIC) in Madrid has developed a system for producing PHA bioplastics, which are considered an alternative to plastics derived from petroleum. The system works by "using predatory bacteria to extract the bioproduct from inside other bacteria, which are killed in the predatory process," explains Virginia Martínez, lead author of the study. The findings of the study were published in Scientific Reports (Nature Group). Martínez is currently a researcher at the biotech firm Evolva in Copenhagen, having previously worked at the CIB's Polymer Biotechnology Laboratory, and specialist in developing bacterial cell factories. "The aim is to sustainably obtain the products we're interested in, such as bioplastics, a very interesting alternative where very large amounts of money are at stake." The issue, she adds, "is that bacteria can produce and accumulate up to 90 percent of their own weight as bioplastic. But the polymer is contained inside the cell and it is difficult to extract. Until now, different detergents and cell disruption systems have been developed and applied to lyse the producers and release the product. However, these processes are not environmentally friendly and also quite inefficient, which increases production costs." Aiming to reduce costs and improve the downstream process, the team chose a bacterium that preys on other bacteria, named Bdellovibrio bacteriovorus. Moreover, the predator was genetically redesigned, transforming it into a biological tool that enables the disruption of bioplastic-producing bacteria, facilitating the extraction and further purification of the bioproduct. The innovative extraction method is novel and has already been patented. "What we did was to use the predatory bacterium B. bacteriovorus as a lytic agent to kill other bacteria (in this particular case P. putida KT2440, a natural PHA producer) and recover the intracellular bioproduct. We also engineered the predator so that it doesn't degrade the bioplastic accumulated by the prey," the researcher explains. This lytic system enables the bioplastic to be recovered in a single step "with no need for complex equipment or toxic compounds," Martínez says. The method could also be used to obtain other added-value compounds, such as enzymes or proteins previously accumulated in other bacteria. This is due to the ability of B. bacteriovorus to attack a wide variety of bacterial strains, including those most used in industry and also at high cell density. In addition, "it is safe for use by humans, as it does not attack mammalian cells," the researcher says. With the system now patented, some companies have already shown interest in it. "We hope it will be used commercially to produce bioplastics or any other intracellular compound. This is an innovative process because it is the first time a predatory bacterium strategy has been used as an alternative method for the recovery of intracellular products of industrial interest," Martínez concludes. More information: Virginia Martínez et al. Engineering a predatory bacterium as a proficient killer agent for intracellular bio-products recovery: The case of the polyhydroxyalkanoates, Scientific Reports (2016). DOI: 10.1038/srep24381

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