3D Cell Printing of Advanced Vascularized Proximal Tubule-on-a-Chip for Drug Induced Nephrotoxicity Advancement.

Renal dysfunction due to drug-induced nephrotoxicity (DIN) affects >20% of the adult population worldwide. The vascularized proximal tubule is a complex structure that is often the primary site of drug-induced kidney injury. Herein, a vascularized proximal tubule-on-a-chip (Vas-POAC) was fabricated, demonstrating improved physiological emulation over earlier single-cell proximal tubule models. A perfusable model of vascularized proximal tubules permits the growth and proliferation of renal proximal tubule cells and adjacent endothelial cells under various conditions. An in vitro Vas-POAC showed mature expressions of the tubule and endothelial cell markers in the mature epithelium and endothelium lumens after 7 days of culture. Expression in the mature proximal tubule epithelium resembled the polarized expression of sodium-glucose cotransporter-2 and the de novo synthesis of ECM proteins. These perfusable Vas-POACs display significantly improved functional properties relative to the proximal tubules-on-a-chip (POAC), which lacks vascular components. Furthermore, the developed Vas-POAC model evaluated the cisplatin-induced nephrotoxicity and revealed enhanced drug receptivity compared to POAC. We further evaluated the capability of the developed proximal tubule model to act as a functional platform that targets screening drug doses that can cause renal proximal tubule injury in adults. Thus, our cell-printed models may prove valuable for screening, thoughtful mechanistic investigations of DIN, and discovery of drugs that interfere with tubule formation.

Coaxial cell printing of a human glomerular model: anin vitroglomerular filtration barrier and its pathophysiology.

Much effort has been expended in emulating the kidney's glomerular unit because of its limitless potential in the field of drug screening and nephrotoxicity testing in clinics. Herein, we fabricate a functional bilayer glomerular microvessel-on-a-chip that recapitulates the specific arrangement of the glomerular endothelial cell, podocyte layers, and the intervening glomerular basement membrane (GBM) in a single step. Our perfusable chip allows for the co-culture of monolayer glomerular endothelium and podocyte epithelium, which display mature functional markers of glomerular cells, and their proper interactions produce GBM proteins, which are the major components of the GBMin vivo. Furthermore, we test the selective permeability capacity, a representative hallmark function of the glomerular filtration barrier. Lastly, we evaluate the response of our glomerular model to Adriamycin- and hyperglycemia-induced injury to evaluate its applicability for drug screening and glomerular disease modeling.

Three-dimensional cell-printing of advanced renal tubular tissue analogue.

Despite significant progress in the development of renal tissue, recapitulation of perfusable complex renal tubular tissue with clinically relevant cellular heterogeneity is still remaining a challenge. In this study, using coaxial 3D cell-printing technique, we present microfluidic hollow tubes to realize tubular/vascular renal parenchyma composed of renal tubular epithelial and endothelial cells, respectively. We developed a functional hybrid bioink that inherits microenvironments for vascularized native kidney tissue with rapidly crosslinkable character to optimize cell functionality and retain the predefined hollow tubular structure. In addition, the novel bioink and 3D coaxial cell-printing technique provided a complex tube with tunable feature of monolayer and bilayer structure across the length of printed tube. Through prototyping a vascularized renal proximal tubule-on-a-chip, we showed its applicability to novel microfluidic renal tissue models. The renal subcapsular transplantation of the hollow tubes showed a long-term graft survival with the therapeutic capability of the tubular constructs in in vivo model of renal disease, which serves their applicability in regenerative medicine.

Directed differential behaviors of multipotent adult stem cells from decellularized tissue/organ extracellular matrix bioinks.

The decellularized tissue/organ extracellular matrix (dECM) is a naturally derived biomaterial that inherits various functional components from the native tissue or organ. Recently, various kinds of tissue/organ dECM bioinks capable of encapsulating cells, combined with 3D cell printing, have enabled remarkable progress in tissue engineering and regenerative medicine. However, the way in which the dECM component compositions of each tissue of different origins interact with cells and dictate tissue-specific cell behavior in the 3D microenvironment remains mostly unknown. To address this issue, in-depth differential proteomic analyses of four porcine dECMs were performed. Specifically, the differential variations of matrisome protein composition in each decellularized tissue type were also uncovered, which can play a significant role by affecting the resident cells in specific tissues. Furthermore, microarray analyses of human bone marrow mesenchymal stem cells (hBMMSCs) printed with various dECM bioinks were conducted to reveal the effect of compositional variations in a tissue-specific manner at the cellular level depending on the multipotency of MSCs. Through whole transcriptome analysis, differential expression patterns of genes were observed in a tissue-specific manner, and this research provides strong evidence of the tissue-specific functionalities of dECM bioinks.

Wound healing activity of the ethanol root extract and polyphenolic rich fraction from Potentilla fulgens.

CONTEXT: Potentilla fulgens Wall. ex Hook (Rosaceae) is a potent medicinal plant of the Western Himalayas, where its roots are traditionally used by the local people of Uttaranchal (India) to treat wounds and tiger bites. OBJECTIVE: The present study scientifically evaluates the wound healing activity of P. fulgens ethanol root extract (EPF) and its ethyl acetate fraction (PFEA) on experimental rats. MATERIALS AND METHODS: Wounds were inflicted on animals by using both excision and incision models. The wounded animals were treated for 16 days with EPF (oral: 200-400 mg/kg and topical: 5-10% w/w) and PFEA (oral: 75 mg/kg; topical: 1.75% w/w). Various physical (wound contraction, epithelialization rate, tensile strength) and biochemical parameters (hydroxyproline, hexosamine, proteins, DNA) were examined during the study. Oxidant product (lipidperoxidase), antioxidant enzymes (catalase, superoxide-dismutase) and reduced glutathione were determined. Morphological and histopathological studies of the skin tissues were monitored. RESULTS: A significant (p < 0.05) wound healing property was observed when the animals were treated topically with EPF (10% w/w) and PFEA (1.75% w/w). A significantly (p < 0.05) increased in the levels of hydroxyproline, hexosamine, protein and DNA up to 59.22, 70.42, 61.01 and 60.00% was observed, respectively. This effect was further demonstrated by the morphological and histopathological representation, thus showing significant (p < 0.05) re-epethelialization on the healing area. EPF and PFEA also showed significant (p < 0.05) antioxidant activity. CONCLUSIONS: The present study provided the scientific evidence, where P. fulgens rich in polyphenolic components possess remarkable wound healing activities, thereby supporting the traditional claims.

Transcriptome Analysis of Cotton (Gossypium hirsutum L.) Genotypes That Are Susceptible, Resistant, and Hypersensitive to Reniform Nematode (Rotylenchulus reniformis).

Reniform nematode is a semi-endoparasitic nematode species causing significant yield loss in numerous crops, including cotton (Gossypium hirsutum L.). An RNA-sequencing analysis was conducted to measure transcript abundance in reniform nematode susceptible (DP90 & SG747), resistant (BARBREN-713), and hypersensitive (LONREN-1) genotypes of cotton (Gossypium hirsutum L.) with and without reniform nematode infestation. Over 90 million trimmed high quality reads were assembled into 84,711 and 80, 353 transcripts using the G. arboreum and the G. raimondii genomes as references. Many transcripts were significantly differentially expressed between the three different genotypes both prior to and during nematode pathogenesis, including transcripts corresponding to the gene ontology categories of cell wall, hormone metabolism and signaling, redox reactions, secondary metabolism, transcriptional regulation, stress responses, and signaling. Further analysis revealed that a number of these differentially expressed transcripts mapped to the G. raimondii and/or the G. arboreum genomes within 1 megabase of quantitative trait loci that had previously been linked to reniform nematode resistance. Several resistance genes encoding proteins known to be strongly linked to pathogen perception and resistance, including LRR-like and NBS-LRR domain-containing proteins, were among the differentially expressed transcripts mapping near these quantitative trait loci. Further investigation is required to confirm a role for these transcripts in reniform nematode susceptibility, hypersensitivity, and/or resistance. This study presents the first systemic investigation of reniform nematode resistance-associated genes using different genotypes of cotton. The candidate reniform nematode resistance-associated genes identified in this study can serve as the basis for further functional analysis and aid in further development of reniform a nematode resistant cotton germplasm.

The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs.

MicroRNAs (miRNAs) and secondary small interfering RNAs (principally phased siRNAs or trans-acting siRNAs) are two distinct subfamilies of small RNAs (sRNAs) that are emerging as key regulators of posttranscriptional gene expression in plants. Both miRNAs and secondary-siRNAs (sec-siRNAs) are processed from longer RNA precursors by DICER-LIKE proteins (DCLs). Gossypium arboreum L., also known as tree cotton or Asian cotton, is a diploid, possibly ancestral relative of tetraploid Gossypium hirsutum L., the predominant type of commercially grown cotton worldwide known as upland cotton. To understand the biological significance of these gene regulators in G. arboreum, a bioinformatics analysis was performed on G. arboreum small RNAs produced from G. arboreum leaf, flower, and boll tissues. Consequently, 263 miRNAs derived from 353 precursors, including 155 conserved miRNAs (cs-miRNAs) and 108 novel lineage-specific miRNAs (ls-miRNAs). Along with miRNAs, 2,033 miRNA variants (isomiRNAs) were identified as well. Those isomiRNAs with variation at the 3'-miRNA end were expressed at the highest levels, compared to other types of variants. In addition, 755 pha-siRNAs derived 319 pha-siRNA gene transcripts (PGTs) were identified, and the potential pha-siRNA initiators were predicted. Also, 2,251 non-phased siRNAs were found as well, of which 1,088 appeared to be produced by so-called cis- or trans-cleavage of the PGTs observed at positions differing from pha-siRNAs. Of those sRNAs, 148 miRNAs/isomiRNAs and 274 phased/non-phased siRNAs were differentially expressed in one or more pairs of tissues examined. Target analysis revealed that target genes for both miRNAs and pha-siRNAs are involved a broad range of metabolic and enzymatic activities. We demonstrate that secondary siRNA production could result from initial cleavage of precursors by both miRNAs or isomiRNAs, and that subsequently produced phased and unphased siRNAs could result that also serve as triggers of a second round of both cis- and trans-cleavage of additional siRNAs, leading to the formation of complex sRNA regulatory networks mediating posttranscriptional gene silencing. Results from this study extended our knowledge on G. arboreum sRNAs and their biological importance, which would facilitate future studies on regulatory mechanism of tissue development in cotton and other plant species.

Nanostructure controlled sustained delivery of human growth hormone using injectable, biodegradable, pH/temperature responsive nanobiohybrid hydrogel.

The clinical efficacy of a therapeutic protein, the human growth hormone (hGH), is limited by its short plasma half-life and premature degradation. To overcome this limitation, we proposed a new protein delivery system by the self-assembly and intercalation of a negatively charged hGH onto a positively charged 2D-layered double hydroxide nanoparticle (LDH). The LDH-hGH ionic complex, with an average particle size of approximately 100 nm, retards hGH diffusion. Nanobiohybrid hydrogels (PAEU/LDH-hGH) were prepared by dispersing the LDH-hGH complex into a cationic pH- and temperature-sensitive injectable PAEU copolymer hydrogel to enhance sustained hGH release by dual ionic interactions. Biodegradable copolymer hydrogels comprising poly(ß-amino ester urethane) and triblock poly(ε-caprolactone-lactide)-poly(ethylene glycol)-poly-(ε-caprolactone-lactide) (PCLA-PEG-PCLA) were synthesized and characterized. hGH was self-assembled and intercalated onto layered LDH nanoparticles through an anion exchange technique. X-ray diffraction and zeta potential results showed that the LDH-hGH complex was prepared successfully and that the PAEU/LDH-hGH nanobiohybrid hydrogel had a disordered intercalated nanostructure. The biocompatibility of the nanobiohybrid hydrogel was confirmed by an in vitro cytotoxicity test. The in vivo degradation of pure PAEU and its nanobiohybrid hydrogels was investigated and it showed a controlled degradation of the PAEU/LDH nanobiohybrids compared with the pristine PAEU copolymer hydrogel. The LDH-hGH loaded injectable hydrogels suppressed the initial burst release of hGH and extended the release period for 13 days in vitro and 5 days in vivo. The developed nanohybrid hydrogel has the potential for application as a protein carrier to improve patient compliance.

Integrated signaling networks in plant responses to sedentary endoparasitic nematodes: a perspective.

Sedentary plant endoparasitic nematodes can cause detrimental yield losses in crop plants making the study of detailed cellular, molecular, and whole plant responses to them a subject of importance. In response to invading nematodes and nematode-secreted effectors, plant susceptibility/resistance is mainly determined by the coordination of different signaling pathways including specific plant resistance genes or proteins, plant hormone synthesis and signaling pathways, as well as reactive oxygen signals that are generated in response to nematode attack. Crosstalk between various nematode resistance-related elements can be seen as an integrated signaling network regulated by transcription factors and small RNAs at the transcriptional, posttranscriptional, and/or translational levels. Ultimately, the outcome of this highly controlled signaling network determines the host plant susceptibility/resistance to nematodes.

Characterization of the recombinant succinic semi-aldehyde dehydrogenase from Saccharomyces cerevisiae.

The yeast succinic semi-aldehyde dehydrogenase gene (SSADH; EC 1.2.1.16) was cloned and overexpressed in Escherichia coli. Based on SDS-PAGE, the molecular mass of the subunit was around 54 kDa, and the purified recombinant enzyme had a tetrameric molecular mass of ca. 200 kDa. The specific activity of the recombinant enzyme was 1.90 µM NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi-aldehyde (Km = 15.48 ± 0.14 µM) and could use both NAD(+) and NADP(+) as co-factors, with Km values of 579.06 ± 30.1 µM and 1.017 ± 0.46 mM, respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD(+) (Ki = 129.5 µM) but a non-competitive inhibitor with respect to succinate semi-aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with Ki = 1.13-10.2 mM, and showed competitive inhibition with respect to NAD(+) and mixed-competitive, non-competitive and non-competitive inhibition, respectively, with respect to succinate semi-aldehyde. The kinetic data suggest a 'ping-pong' mechanism.

In situ gelling pH- and temperature-sensitive biodegradable block copolymer hydrogels for drug delivery.

Stimuli-sensitive injectable polymeric hydrogels have been extensively investigated during the past decade as bioactive agent delivery vehicles and for tissue engineering applications. An aqueous solution of these polymers undergoes a sol-to-gel phase transition in response to external stimuli such as pH, temperature, salt, light, biomolecules, electromagnetic field, etc. Bioactive molecules or cells can be mixed into the low-viscosity state of the polymer solution and injected into the body at a target site, forming an in situ hydrogel depot, which can then serve as bioactive-molecule-releasing carriers or a cell-growing microenvironment. This review systematically summarizes the recent progress in biodegradable and injectable block copolymer hydrogels, giving special attention to the novel and promising pH- and temperature-sensitive injectable block copolymer hydrogels for biomedical applications. The gelation mechanism, formation of ionic complexes, and biodegradation are highlighted as key factors responsible for controlled protein/drug delivery. The advantages and perspectives of pH- and temperature-sensitive injectable block copolymer hydrogels are also highlighted.

GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production.

The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (α-ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45°C. Double and triple mutation constructs were compared for thermotolerance with the wild-type and single mutant strains. Although wild-type and all mutant strains were highly susceptible to brief heat stress at 50°C, a non-lethal 30 min at 40°C temperature pretreatment induced tolerance of the wild-type and all of the mutants to 50°C. The mutant strains collectively exhibited similar susceptibility at 45°C to the induced 50°C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild-type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N-tert-butyl-α-phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild-type. Measurement of intracellular GABA, glutamate and α-ketoglutarate during lethal heat exposure at 45°C showed higher levels of accumulation of GABA and α-ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from α-ketoglutarate to succinate during heat stress.

Overexpression of tobacco osmotin (Tbosm) in soybean conferred resistance to salinity stress and fungal infections.

Salinity and fungal diseases are the two significant constraints limiting soybean productivity. In order to address these problems, we have transformed soybean cv. Pusa 16 via somatic embryogenesis with salinity induced and apoplastically secreted pathogenesis-related tobacco osmotin (Tbosm) gene using Agrobacterium-mediated genetic transformation. Integration of Tbosm in randomly selected five GUS assay-positive independently transformed soybean plants was confirmed by polymerase chain reaction (PCR) and Southern hybridization. Reverse transcriptase-PCR (RT-PCR) and Western blotting confirmed that the Tbosm was expressed in three of the five transformed soybean plants. Further the Western blotting revealed that the truncated osmotin protein accumulated more in apoplastic fluid. The transformed (T(1)) soybean plants survived up to 200 mM NaCl, whereas non-transformed (NT) plants could withstand till 100 mM and perished at 150 mM NaCl. The biochemical analysis revealed the T(1) soybean plants accumulated higher amount of proline, chlorophyll, APX, CAT, SOD, DHAR, MDHAR, and RWC than NT plants. Leaf gas exchange measurements revealed that T(1) soybean plants maintained higher net photosynthetic rate, CO(2) assimilation, and stomatal conductance than NT plants. The three T(1) soybean plants expressing the osmotin gene also showed resistance against three important fungal pathogens of soybean--Microsphaera diffusa, Septoria glycines and Phakopsora pachyrhizi. The T(1) soybean plants produced 32-35 soybean pods/plant containing 10.3-12.0 g of seeds at 200 mM NaCl, whereas NT plant produced 28.6 soybean pods containing 9.6 g of seeds at 100 mM NaCl. The present investigation clearly shows that expression of Tbosm enhances salinity tolerance and fungal disease resistance in transformed soybean plants.

Polycaprolactone composites with TiO2 for potential nanobiomaterials: tunable properties using different phases.

TiO(2) nanoparticles of different phases play a key role in property alteration of nanocomposite fibers. Polycaprolactone (PCL)/TiO(2) composite fibers were prepared using the electrospinning method. Pure anatase and rutile phases were synthesized using the sol-gel route for nanocomposite synthesis. The Effect of nanoparticle phases on crystallinity of fibers and interaction with polymer molecules have been studied using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, morphology through SEM, surface properties using BET method and wetting property of fibers commencing from contact angle measurement. Biocompatibility and biodegradation of hybrid materials have been studied in simulated body fluid (SBF) and phosphate buffer (PBS), respectively. The anatase phase with smaller particle dimensions exhibited significant improvement of most of the properties as compared to composites made of the rutile phase. Better interaction between polymer chain and anatase particle PCL-A nanocomposite fibers leads to better mechanical property and biocompatibility vis-à-vis PCL-R and pristine PCL fibers. Biocompatibility of PCL nanocomposite has been testified through proliferation of fibroblast cell and its adhesion; MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay demonstrates good proliferation rate for cells on PCL-A nanocomposite fibres.

Antioxidant and hepatoprotective effects of ethanol extract of Vitex glabrata on carbon tetrachloride-induced liver damage in rats.

This study was aimed at evaluating the antioxidant and hepatoprotective effects of the ethanol extract of Vitex glabrata (EEVG) in a CCl(4)-induced liver damage model in rats; and to isolate and characterise the bioactive constituent from EEVG. Hepatoprotective activity was evaluated by changes in the levels of the serum enzymes viz. AST, ALT, ALP and total bilirubin, and further by histopathological examinations of liver tissues. Antioxidant activity was measured in terms of superoxide dismutase, GSH, lipid peroxidation (LPO), catalase and peroxidase levels in liver homogenate. The pentamethoxy flavonoid artemetin was isolated and characterised from EEVG. Artemetin and EEVG pre-treatment significantly (p < 0.001) inhibited LPO in CCl(4)-intoxicated livers and reduced the elevated serum levels of AST, ALT, ALP and bilirubin to normal; it also brought back the normal architecture of liver tissues. Antioxidant activity of EEVG was found to be comparable with silymarin (p < 0.05). In conclusion, EEVG possesses significant hepatoprotective activity, which may be mediated by the antioxidant mechanisms of its components, predominantly artemetin.

Expression, in vivo localization and phylogenetic analysis of a pyridoxine 5'-phosphate oxidase in Arabidopsis thaliana.

Pyridoxal phosphate (PLP), a vitamin B(6) vitamer, is an essential cofactor for numerous enzymes. Pyridoxine/pyridoxamine phosphate oxidase (PPOX) catalyzes the synthesis of pyridoxal phosphate from pyridoxine phosphate (PNP) and/or pyridoxamine phosphate (PMP). The At5g49970 locus in Arabidopsis thaliana encodes an AtPPOX, a PNP/PMP oxidase. The expression of the AtPPOX gene varied in different tissues of Arabidopsis examined, being up-regulated by light, heat shock, ABA, and ethylene treatments, and down-regulated by exposure to drought and NaCl. Monoclonal antibodies raised against two different domains of AtPPOX recognized different sizes of AtPPOX, suggesting that AtPPOX proteins are produced as splice variants of the AtPPOX gene in Arabidopsis. Phylogenetic analysis of AtPPOX across all domains of life demonstrated that plant AtPPOX homologs have an additional Yjef_N domain preceding the Pyridox_Oxidase domain at the C-terminal end of the protein, while AtPPOX homologs from bacteria, fungi and animals have only Pyridox_Oxidase domain. The presence of the Yjef_N domain in plant AtPPOX homologs suggests that acquisition of this domain, and its fusion with the pyridox_oxidase domain began with the endosymbiotic acquisition of the chloroplast. Bioinformatic analysis suggested that AtPPOX is localized in chloroplast, but the monoclonal antibody could not be used for subcellular localization of this protein. A GFP-AtPPOX fusion construct introduced into the Arabidopsis protoplast confirmed localization of AtPPOX into the chloroplast. An RNAi mutant of AtPPOX showed sensitivity to high light suggesting a role for PPOX in resistance to photooxidative damage, and alteration in root growth in the presence of sucrose suggests a role for PPOX in root development.

Expression of truncated tobacco osmotin in Escherichia coli: purification and antifungal activity.

PURPOSE OF WORK: Tobacco osmotin is a functional homolog of mammalian adiponectin, and has antifungal activity. This work was undertaken to produce recombinant osmotin that has previously been unsuccessful because of its toxicity. Expression of recombinant tobacco osmotin (rOSM) in Escherichia coli inclusion bodies has been achieved. The optimal pH for rOSM expression in ZYM 505 medium is 7.0 at OD(650) of 1.5 of culture growth. The rOSM from the inclusion body was extracted with 8 M urea, and purified using CM-cellulose and cobalt-agarose bead affinity chromatography to a high purity. Approximately 80% of the rOSM remained bound to CM-cellulose and Cobalt-agarose beads after initial elution. The yield of purified rOSM was between 40 and 50 mg from 2 l of culture. Repeated elution of protein from CM-cellulose and Co-agarose increased the yield of rOSM to 200 mg from 2 l culture. The purified rOSM showed variable antifungal activities against two pathogenic yeast strains; Cryptococcus neoformans, Candida albicans, and non-pathogenic strains; Saccharomyces cerevisiae and Pichia methanolica.

Expression of calmodulin genes in wild type and calmodulin mutants of Arabidopsis thaliana under heat stress.

Calmodulin (CaM), a calcium-regulated protein, regulates the activity of a number of key enzymes and plays important roles in cellular responses to environmental changes. The Arabidopsis thaliana genome contains nine calmodulin (CAM) genes. To understand the role of specific CAM genes in heat stress, the steady-state level of mRNA for the nine CAM genes in root and shoot tissues of seedlings grown at normal growth temperature (25 degrees C) and during heat stress at 42 degrees C for 2h was compared in T-DNA insertional mutant lines of 7 CAM genes and the wild type using gene specific primers and RT-PCR. Compared to growth at 25 degrees C, the mRNA levels of all CAM genes were up-regulated in both root and shoot after heat treatment with the notable exception of CAM5 in root and shoot, and CAM1 in shoot where the mRNA levels were reduced. At 25 degrees C all cam mutants showed varying levels of mRNA for corresponding CAM genes with the highest levels of CAM5 gene mRNA being found in cam5-1 and cam5-3. CAM5 gene mRNA was not observed in the cam5-4 allele which harbors a T-DNA insertion in exon II. The level of respective CAM gene mRNAs were reduced in all cam alleles compared to levels in wild type except for increased expression of CAM5 in roots and shoots of cam5-1 and cam5-3. Compared to wild type, the level of mRNA for all CAM genes varied in each cam mutant, but not in a systematic way. In general, any non-exonic T-DNA insertion produced a decrease in the mRNA levels of the CAM2 and CAM3 genes, and the levels of CAM gene mRNAs were the same as wild type or lower in the cam1, cam4, cam5-2, and cam6-1 non-exonic mutant alleles. However, the level of mRNA for all genes except CAM2 and CAM3 genes was up-regulated in all cam2 and cam3 alleles and in the cam5-1 and cam5-3 alleles. During heat stress at 42 degrees C the level of CAM gene mRNAs were also variable between insertional mutants, but the level of CAM1 and CAM5 gene mRNAs were consistently greater in response to heat stress in both root and shoot. These results suggest differential tissue-specific expression of CAM genes in root and shoot tissues, and specific regulation of CAM gene mRNA levels by heat. Each of the CAM genes appears to contain noncoding regions that play regulatory roles resulting in interaction between CAM genes leading to changes in specific CAM gene mRNA levels in Arabidopsis. Only exonic insertion in CAM5 gene resulted in a loss-of-function of CAM5 gene among the mutants we surveyed in this study.

Nanoparticle-induced controlled biodegradation and its mechanism in poly(epsilon-caprolactone).

Poly(epsilon-caprolactone) (PCL)/layered silicate nanocomposites have been prepared via solution route. Two different organically modified nanoclays were used to compare the variation in properties based on organic modifications. The nanostructures, as observed from wide-angle X-ray diffraction and transmission electron microscopy, indicate intercalated and partially exfoliated hybrids depending on the nature of organic modification in nanoclay. The nanohybrids exhibit significant improvement in thermal and mechanical properties of the matrix as compared to neat polymer. The nanoclays act as nucleating agent for the crystallization of PCL. The biodegradability of pure PCL and its nanocomposites have been studied under controlled conditions in enzyme, pure microorganism (fungi), compost, Ganges water, and alkaline buffer solution. The rate of biodegradation of PCL has enhanced dramatically in nanohybrids and depends strongly on the media used. Scanning confocal, electron, and atomic force microscopes have used to demarcate the nature of biodegradation of pristine PCL and its nanocomposites. The change in biodegradation is rationalized in terms of the crystallization behavior and organic modification in nanoclays of the nanohybrids vis-a-vis the neat polymer. The extent of compatibility was measured quantitatively through the interaction parameter for two different nanoclays to compare and establish the reason for variation in their properties in nanohybrids. A biodegradation mechanism has been revealed for PCL and its nanocomposites through enzyme activity in varying pH environment.

Nitrate uptake and utilization is modulated by exogenous gamma-aminobutyric acid in Arabidopsis thaliana seedlings.

Exogenously applied GABA modulates root growth by inhibition of root elongation when seedlings were grown in vitro on full-strength Murashige and Skoog (MS) salts, but root elongation was stimulated when seedlings were grown on 1/8 strength MS salts. When the concentration of single ions in MS salts was individually varied, the control of growth between inhibition and stimulation was found to be related to the level of nitrate (NO(3)(-)) in the growth medium. At NO(3)(-) concentrations below 40 mM (full-strength MS salts level), root growth was stimulated by the addition of GABA to the growth medium; whereas at concentrations above 40 mM NO(3)(-), the addition of GABA to the growth medium inhibited root elongation. GABA promoted NO(3)(-) uptake at low NO(3)(-), while GABA inhibited NO(3)(-) uptake at high NO(3)(-). Activities of several enzymes involved in nitrogen and carbon metabolism including nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and phosphoenol pyruvate carboxylase (PEPCase) were regulated by GABA in the growth medium. Supplementing 1/8 strength MS medium with 50 mM GABA enhanced the activities of all of the above enzymes except ICDH activities in root tissues. However, at full-strength MS, GABA showed no inhibitory effect on the activities of these enzymes, except on GS in both root and shoot tissues, and PEPCase activity in shoot tissues. Exogenous GABA increased the amount of NR protein rather than its activation status in the tissues. This study shows that GABA affects the growth of Arabidopsis, possibly by acting as a signaling molecule, modulating the activity of enzymes involved in primary nitrogen metabolism and nitrate uptake.