Molecular, biochemical, and proteomic analyses of transplastomic tobacco plants expressing an endoglucanase support chloroplast-based molecular farming for industrial scale production of enzymes.

The successful production of recombinant enzymes by tobacco transplastomic plants must maintain compatibility of the heterologous enzyme with chloroplast metabolism and its long-time enzyme stability. Based on previous reports, it has been taken for granted that following biolistic-transformation, homoplasticity could be obtained from the initially heteroplastic state following successive rounds of selection in the presence of the selection agent. However, several studies indicated that this procedure does not always ensure the complete elimination of unmodified wild-type plastomes. The present study demonstrates that CelK1 transplastomic plants, which were photosyntetically as active as untransformed ones, remain heteroplastomic even after repeated selection steps and that this state does not impair the relatively high-level production of the recombinant enzyme. In fact, even in the heteroplastomic state, the recombinant protein represented about 6% of the total soluble proteins (TSP). Moreover, our data also show that, while the recombinant endoglucanase undergoes phosphorylation, this post-translation modification does not have any significant impact on the enzymatic activity. Biomass storage might be required whenever the enzyme extraction process could not be performed immediately following the harvest of tobacco mature plants. In this respect, we have observed that enzyme activity in the detached leaves stored at 4 °C is maintained up to 20 weeks without significant loss of activity. These findings may have major implications in the future of chloroplast genetic engineering-based molecular farming to produce industrial enzymes in transplastomic plants.

Influence of Ag Nanostructure Location on the Absorption Enhancement in Polymer Solar Cells.

The optical absorption enhancement in Ag nanocube (NC)- and nanosphere (NS)-embedded poly[ N-9'-heptadecanyl-2,7-carbazole- alt-5,5-(4',7'-di-2-thienyl-2',1',3'-benzothiadiazole)]:[6,6]-phenyl C71-butyric acid methyl ester active layer was calculated using three-dimensional finite-difference time domain simulations. The simulations were carried out by incorporating Ag nanostructures as a two-dimensional array at various locations in the active layer matrix. High absorption enhancements of 53 and 61% were achieved with NSs and NCs, respectively, when they were incorporated at the top portion of the active layer. The influence of various passivation layers on the absorption enhancement was also investigated. The simulation results revealed that the absorption enhancement is mainly due to the near-field enhancement around the nanostructures and the backward reflection of incident light from the nanostructure array.

Organic bistable memory devices based on MoO3 nanoparticle embedded Alq3 structures.

Organic bistable memory devices were fabricated by embedding a thin layer of molybdenum trioxide (MoO3) between two tris-(8-hydroxyquinoline)aluminum (Alq3) layers. The device exhibited excellent switching characteristics with an ON/OFF current ratio of 1.15 × 103 at a read voltage of 1 V. The device showed repeatable write-erase capability and good stability in both the conductance states. These conductance states are non-volatile in nature and can be obtained by applying appropriate voltage pulses. The effect of MoO3 layer thickness and its location in the Alq3 matrix on characteristics of the memory device was investigated. The field emission scanning electron microscopy (FE-SEM) images of the MoO3 layer revealed the presence of isolated nanoparticles. Based on the experimental results, a mechanism has been proposed for explaining the conductance switching of fabricated devices.

Unexplained post-renal transplant tolerance: a case report.

Achievement of an immunosuppression-free condition defined as clinical operational tolerance is an ideal goal. We hereby report a case of clinical operational tolerance in a patient whose allograft is functioning normally, without immunosuppression, for more than 3 years. The patient withdrew from immunosuppression in 2011 following which his serum creatinine was 1.34 mg/dl and proteinuria was 178 mg/24 h. Flowcytometric studies showed an elevated number of B lymphocytes and NK cells. IL-10 cytokine levels had increased, whereas those of IFN-γ decreased, suggesting that both B lymphocytes and NK cells, with their immunoregulatory function, contribute to the maintenance of long-term graft function. Consequently, further studies in understanding the interactions of NK cells and B lymphocytes may give us a better insight into the underlying mechanisms that underpin organ tolerance.

Associations of FPG, A1C and disease duration with protein markers of oxidative damage and antioxidative defense in type 2 diabetes and diabetic retinopathy.

PURPOSE: To investigate the role of protein oxidative damage and antioxidant defense in relationship to hyperglycemia measured as fasting plasma glucose (FPG), glycated hemoglobin (A1C), and duration of disease in type 2 diabetes mellitus (DM) and diabetic retinopathy (DR). METHODS: This study recruited 23 non-diabetic subjects, 16 DM patients without any complications and 18 DR patients. The serum ischemia modified albumin (IMA) and glutathione (GSH) levels were measured. The IMA results were corrected for serum albumin. Between-group differences were studied by analysis of variance and between-variable associations were studied by Spearman's and partial correlations. RESULTS: IMA and cIMA values were elevated, whereas GSH was decreased in both patient groups vs controls (P<0.05), and the increase in IMA formation is not related to serum albumin changes. DR patients have much severe oxidative stress (OS) status with high IMA and cIMA, and low GSH than in the DM group (P<0.05). Both FPG and A1C levels were positively associated with IMA in DM group, while in the DR group, duration of disease too had a positive association with IMA. The antioxidant GSH had negative correlations with FPG (r=-0.52, P=0.02) and IMA (r=-0.49, P=0.03) in the DR group. Partial correlation analyses predicted mutual or independent associations among parameters. CONCLUSIONS: Severe OS in DR has been associated with increased FPG, A1C, and disease duration. Both hyperglycemia and elevated oxidative damage detected as IMA are collectively associated with depleted GSH status. Our study unravels the need for monitoring of OS in addition to standard glycemic management in DR.

Emerging role for αB-crystallin as a therapeutic agent: pros and cons.

HSPB5 or αB-crystallin (αBC) is a major protein of the vertebrate eye lens belonging to the small heat-shock protein family of proteins that respond to various stressful conditions. αBC also is found outside the lens in various non-ocular tissues and acts as a molecular chaperone by preventing aggregation of proteins, inhibits apoptosis and inflammation, and maintains cytoskeletal architecture. The αBC protein is phosphorylated on three serine residues S59, S45, and S19, and several functions of αBC are modulated by phosphorylation. Numerous studies have revealed the upregulation of αBC in pathological conditions such as neurodegenerative diseases, cancers, diabetes, retinal diseases, cataracts, ischemia/repurfusion, aging, and others. However, it is unknown whether the up-regulation of αBC is causative or protective for these pathological conditions. Although αBC has been shown to provide a protective effect in neurodegenerative diseases, inflammation, diabetes, and retinal diseases, other studies have described a deleterious role of αBC in cancers and pulmonary fibrosis. The therapeutic potential of αBC alone or in combination with αA-crystallin has been reported. Acetylated αBC peptides have been shown to be more potent than native αBC for chaperone as well as therapeutic activities using both in vitro and in vivo models. Further, for efficient delivery of α BC into cells, carrier molecules such as polylacticcoglycolic acid, polycaprolactone and cell penetration peptides have been used. In this review, we have summarized current data from emerging and exciting studies of the therapeutic strategies of α BC and α BC peptides and the efficient delivery strategies of these proteins in various disease models, including neurodegenerative diseases, retinal diseases, platelet aggregation, inflammation, and ischemia.

Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, ß-lactoglobulin.

Recombinant allergens and antibodies are needed for diagnostic, therapeutic, food processing and quality verification purposes. The aim of this work was to develop a barley-based production system for ß-lactoglobulin (BLG) specific immunoglobulin E antibody (D1 scFv). The expression level in the best barley cell clone was 0.8-1.2 mg/kg fresh weight, and was constant over an expression period of 21 days. In the case of barley grains, the highest stable productivity (followed up to T2 grains) was obtained when the D1 scFv cDNA was expressed under a seed-specific Glutelin promoter rather than under the constitutive Ubiquitin promoter. Translational fusion of ER retention signal significantly improved the accumulation of recombinant antibody. Furthermore, lines without ER retention signal lost D1 scFv accumulation in T2 grains. Pilot scale purification was performed for a T2 grain pool (51 g) containing 55.0 mg D1 scFv/kg grains. The crude extract was purified by a two-step purification protocol including IMAC and size exclusion chromatography. The purification resulted in a yield of 0.47 mg of D1 scFv (31 kD) with high purity. Enzyme-linked immunosorbent assay revealed that 29 % of the purified protein was fully functional. In immunoprecipitation assay the purified D1 scFv recognized the native 18 kD BLG in the milk sample. No binding was observed with the heat-treated milk sample, as expected. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by bovine milk.

Anti-thymocyte globulin versus basiliximab induction in renal transplant recipients: Long-term outcome.

Although basiliximab and rabbit anti-thymocyte globulin (ATG) are effective in delaying and reducing the incidence of acute rejection (AR) thus improving short-term graft survival, their impact on long-term graft survival has not been well established in renal transplant recipients. To evaluate the long-term efficacy after induction therapy with ATG/basiliximab in renal transplant recipients, we studied retrospectively 86 renal transplant recipients of living donor renal transplantation from 2003 to 2006; of them, 42 patients received induction with ATG three doses of 50 mg, 25 mg, 25 mg/day on 0, 1 and 2 post-operative days (POD) and 44 age-matched patients received induction with basiliximab (20 mg/day on 0 and 4 PODs). All the patients received tacrolimus, mycophenolate mofetil and corticosteroids as maintenance immunosuppressive therapy. Demographic characteristics were similar between both groups. Patient survival at 5 years was 90.5% in the ATG group and 84.1% in the basiliximab group, while graft survival was 83.4% and 77.3%, respectively. The incidence of acute rejection was 14.2% and 18.1% in the ATG and the basiliximab groups, respectively. The estimated mean glomerular filtration rates at 5 years post-transplantation was 52.1 mL/min and 49.1 mL/min and the mean serum creatinine levels were 1.55 ± 0.37 and 1.66 ± 0.51 mg/dL in the ATG and basiliximab groups, respectively. A low incidence of tuberculosis and cytomegalovirus (CMV) was observed in the ATG group. There were no significant differences between the two groups, and both induction regimens assured a safe and effective treatment and were associated with similar excellent long-term patient and graft survival.

Spectrum of malignancies in human immunodeficiency virus - positive patients at a Tertiary Care Centre in South India.

CONTEXT: India has a very large number of patients living with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome. Opportunistic infections in these patients are commonly encountered. However, malignancies in such patients also do occur. AIM: The aim was to study the spectrum of malignancies in HIV-positive patients at a tertiary health care center. SETTINGS AND DESIGN: Retrospective study. MATERIALS AND METHODS: The cases were retrieved from pathology record files at our Institute from January 2003 to December 2008. The follow-up was obtained from Medical oncology records. The morphology of each case was reviewed along with immunohistochemistry wherever done. RESULTS: There were 61 such cases (51 males, 10 females). The age range was 7-78 years with a median of 35 years. The clinical presentation varied according to the malignancy. The largest group was non-Hodgkin lymphoma (18 nodal, 23 extra-nodal). The others included carcinoma breast (4), chronic myeloid leukemia (3), Burkitt Leukemia (2), squamous cell carcinoma anal region (2), multiple myeloma (2) and one each of miscellaneous malignancies (7). CONCLUSION: Malignancies in HIV positive individual occurred in younger individuals. Non-Hodgkin lymphomas, especially extra-nodal lymphomas, were the most common malignancy. There were no cases of proven Kaposi's sarcoma or invasive cervical carcinomas. There were two cases of multiple myeloma which are infrequently reported.

Antioxidant defense in overt and subclinical hypothyroidism.

Oxidative stress as a result of disequilibrium between free radical generation and antioxidant status has been implicated in several pathologies including thyroid diseases. Studies on antioxidant status in overt (OHT) and subclinical hypothyroidism (SHT) are controversial and limited. The aim of this study was to determine the effect of OHT and SHT on antioxidant status. Thirty-six patients with OHT, 36 patients with SHT, and 39 healthy euthyroid subjects as the control group were included in the study. Plasma levels of malondialdehyde (MDA), reduced glutathione (GSH) and total antioxidant capacity (TAC) as ferric reducing ability of plasma (FRAP), and erythrocyte antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), SOD/GPx ratios, catalase (CAT), and glutathione reductase (GR) were analyzed in all groups. MDA and GPx values were elevated, while GSH, FRAP, SOD, and SOD/GPx ratio were decreased in both patient groups compared with controls. No change in activities of CAT and GR were observed in both the patient groups. Significant differences were observed between OHT and SHT groups with high MDA, GPX and low GSH, FRAP, SOD, and SOD/GPx ratio in OHT group. Thus, hypothyroid patients have a deficient antioxidant defense in the form of decreased activity of SOD, decreased levels of FRAP and GSH along with an increase in GPx activity. The severity of the disease appears to decide the degree of deficiency and our findings also point to this, in the form of decrease in SOD, FRAP, and GSH observed being more in OHT than in SHT patients. Hormonal changes and increased lipid peroxidation, which also vary with severity of disease, appear to contribute to the antioxidant deficiency.

Association of lipid peroxidation with endothelial dysfunction in patients with overt hypothyroidism.

Hypothyroidism is associated with increased oxidative stress. The mechanism underlying the endothelial dysfunction in thyroid disease is not yet clear. This study aims to investigate lipid peroxidation and its association with endothelial dysfunction in overt hypothyroidism (OHT).Plasma malondialdehyde (MDA) as a marker of oxidative stress and plasma nitrates and asymmetric dimethyl arginine levels (ADMA) as markers of endothelial dysfunction were estimated in 25 OHT patients in comparison to 25 euthyroid controls. Plasma MDA, ADMA levels were significantly increased, whereas plasma nitrates were significantly decreased in the patient group compared to control group (p<0.01). Moreover, a significant positive association between plasma MDA and ADMA was found in the patient group (ρ=0.472, p=0.036). Our results reveal the presence of endothelial dysfunction in OHT patients as evidenced by decreased plasma nitrates and increased ADMA levels. Increased levels of MDA represent an increased generation of reactive oxygen species in these patients. A finding of significant direct relation of plasma MDA with ADMA indicates that oxidative stress has a strong impact on endothelial dysfunction in overt hypothyroidism. Further studies focusing on the role of oxidative stress in endothelial dysfunction and the effects of antioxidant supplementation on endothelial function in OHT patients are required.

The quality of life of women volunteering as live-related kidney donors in India.

BACKGROUND: Women outnumber men 6:1 as live-related donors in our renal transplant programme. Women donors in developing regions are often illiterate and unemployed. This study was done to assess the change in quality of life of women who donate kidneys. METHODS: We prospectively studied 73 consecutive women volunteering as live-related kidney donors over a 6-month period using the World Health Organization Quality of Life Brief (WHO QoL Bref) Questionnaire and Hospital Anxiety and Depression Scale (HADS). Each woman was interviewed 2 weeks before and 6 months after kidney donation. RESULTS: There was a significant improvement in all the domains, namely physical (p=0.0001), psychological (p<0.0001), social relationship (p=0.037) and environment (p<0.0001) of the WHO QoL Bref questionnaire. Donors who were mothers had a greater improvement in all 4 domains than donors with other relationships. There was a significant decrease in the depression score (p<0.0001), but no change in the anxiety scores (p=0.065) following kidney donation. All donors would donate again, if possible. CONCLUSION: In live-related women kidney donors, quality of life improves and depression scores decline after kidney donation.

Risk factors for the development of new-onset diabetes mellitus in a living related renal transplant program.

New-onset diabetes mellitus is associated with considerable morbidity after transplantation. We evaluated 78 living related renal transplant recipients due to all causes except diabetic nephropathy a waiting a living related renal transplantation. We evaluated demographic characteristics, pretransplant glycemic profile, fasting C-peptide levels, plasma insulin levels, pretransplant insulin resistance, and immunosuppression protocols. Among the 16.7% of patients developing diabetes mellitus at the end of 1 year, age, family history, and impaired glucose tolerance at the time of transplantation correlated with the development of diabetes mellitus in the posttransplant period.

Crystallization and preliminary X-ray diffraction analysis of human adenovirus.

Replication-defective and conditionally replicating adenovirus (AdV) vectors are currently being utilized in approximately 25% of human gene transfer clinical trials. Unfortunately, progress in vector development has been hindered by a lack of accurate structural information. Here we describe the crystallization and preliminary X-ray diffraction analysis of a HAdV5 vector that displays a short flexible fiber derived from HAdV35. Crystals of Ad35F were grown in 100mM HEPES pH 7.0, 200mM Ca(OAc)(2), 14% PEG 550 MME, 15% glycerol in 100mM Tris-HCl 8.5. Freshly grown crystals diffracted well to 4.5A resolution and weakly to 3.5A at synchrotron sources. HAdV crystals belong to space group P1 with unit cell parameters a=854.03A, b=855.17A, c=865.24A, alpha=119.57 degrees , beta=91.71 degrees , gamma=118.08 degrees with a single particle in the unit cell. Self-rotation and locked-rotation function analysis allowed the determination of the particle orientation. Molecular replacement, density modification and phase-extension procedures are being employed for structure determination.

Memory effect in a junction-like CdS nanocomposite/conducting polymer poly[2-methoxy-5-(2-ethylhexyloxy)1,4-phenylene-vinylene] heterostructure.

The operation of a nonvolatile memory device is demonstrated using junction-like CdS nanocomposites embedded in a polymer matrix. The capacitance-voltage characteristics of Al/conducting polymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene-vinylene]/CdS nanocomposites in a polyvinyl alcohol matrix/indium tin oxide device exhibit hysteresis, which is attributed to the trapping, storage, and emission of holes in the quantized valence band energy levels of isolated CdS nanoneedles. The characteristics at different operating frequencies show that the hysteresis is due to trapping of charge carriers in CdS nanocomposites rather than in the interfacial states. The memory behavior in the inorganic/organic heterostructure is explained on the basis of a simple energy band diagram.

Molecular and Symptom Analysis Reveal the Presence of New Phytoplasmas Associated with Sugarcane Grassy Shoot Disease in India.

A total of 240 sugarcane (Saccharum officinarum) plants showing phenotypic symptoms of sugarcane grassy shoot (SCGS) disease were collected from three states of India, Maharashtra, Karnataka, and Uttar Pradesh. Phytoplasmas were detected in all symptomatic samples by the polymerase chain reaction (PCR) amplification of phytoplasma-specific 16S rRNA gene and 16S-23S rRNA spacer region (SR) sequences. No amplification was observed when DNA from asymptomatic plant samples was used as a template. Sixteen samples were selected on the basis of phenotypic symptoms and geographic location, and cloning and sequencing of the 16S rRNA and spacer regions were performed. Multiple sequence alignments of the 16S rRNA sequences revealed that they share very high sequence similarity with phytoplasmas of rice yellow dwarf, 16SrXI. However, the 16S-23S rRNA SR sequence analysis revealed that while the majority of phytoplasmas shared very high (>99%) sequence similarity with previously reported sugarcane phytoplasmas, two of them, namely BV2 (DQ380342) and VD7 (DQ380343), shared relatively low sequence similarity (79 and 84%, respectively). Therefore, these two phytoplasmas may be previously unreported ones that cause significant yield losses in sugarcane in India.

Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27.

Xylanases (EC 3.2.1.8) catalyze the hydrolysis of beta-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 A, beta = 131.2 degrees, and diffract to better than 2.2 A resolution.

Use of nesiritide (human B-type natriuretic peptide) in infants following cardiac surgery.

We report the use of nesiritide in two infants following cardiac surgery. Both infants had increased intracardiac filling pressures postoperatively, despite traditional afterload reduction and diuretics. Both infants demonstrated clinical improvement with nesiritide therapy. There is currently limited data available describing the use of nesiritide in infants.

Use of a herbicide or lysine plus threonine for non-antibiotic selection of transgenic chickpea.

A desensitized aspartate kinase (AK) gene has been developed as a non-antibiotic selection marker for use in the production of transgenic chickpea ( Cicer arietinum L.). Transgenic shoots regenerated from embryo explants bombarded with the desensitized AK gene were selected on media containing two amino acids, lysine and threonine (LT). Approximately 15% of the putative transgenic shoots of vars. P-362 and P-1042 survived after 4 weeks of growth on MSB5 medium (MS mineral salts and B5 vitamins) containing 2 microM thidiazuron (TDZ) and 2 mM lysine and 2 m M threonine. These shoots were subsequently grown on MSB5 medium supplemented with 2 micro M TDZ and 5 mM lysine and 5 mM threonine, and nearly 1% continued to grow after 16 weeks of selection. A phosphinothricin (PPT) selection system for Agrobacterium-mediated chickpea transformation was also developed. Three varieties of chickpea, P-362, P-1042 and P-1043, were successfully used for Agrobacterium transformation. Following Agrobacterium infection, 3-8% of the regenerated shoots remained green and continued to grow on MSB5 medium supplemented with 2.5 mg l(-1 )PPT. Increasing the concentrations of PPT to 15 mg l(-1) reduced transgenic shoot production in P-362, P-1042 and P-1043 to 0.7%, 1.2% and 1.1%, respectively. Selected putatively transformed shoots of all three varieties were rooted and grown to maturity. Southern hybridization analysis revealed single as well as multiple integration of genes in selected transgenic lines. The level of AK activity detected in LT-selected plants was higher than that detected in the non-transformed control.