Expression of Trichoderma reesei beta-Mannanase in Tobacco Chloroplasts and Its Utilization in Lignocellulosic Woody Biomass Hydrolysis

Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost-effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. beta-mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, the man1 gene encoding beta-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds in the selection medium showed inheritance of transgenes into the progeny without any Mendelian segregation. Expression of endo-beta-mannanase for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-beta-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-beta-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight). Chloroplast-derived mannanase had higher temperature stability (40 degrees C to 70 degrees C) and wider pH optima (pH 3.0 to 7.0) than E. coli enzyme extracts. Plant crude extracts showed 6-7 fold higher enzyme activity than E. coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different plant-derived enzymes yielded 20% more glucose equivalents from pinewood than the cocktail without mannanase. Our results demonstrate that chloroplast-derived mannanase is an important component of enzymatic cocktail for woody biomass hydrolysis and should provide a cost-effective solution for its diverse applications in the biofuel, paper, oil, pharmaceutical, coffee and detergent industries

Expression of Dengue-3 Premembrane and Envelope Polyprotein in Lettuce Chloroplasts

Dengue is an acute febrile viral disease with \u3e100 million infections occurring each year and more than half of the world population is at risk. Global resurgence of dengue in many urban centers of the tropics is a major concern. Therefore, development of a successful vaccine is urgently needed that is economical and provide long-lasting protection from dengue virus infections. In this manuscript, we report expression of dengue-3 serotype polyprotein (prM/E) consisting of part of capsid, complete premembrane (prM) and truncated envelope (E) protein in an edible crop lettuce. The dengue sequence was controlled by endogenous Lactuca sativa psbA regulatory elements. PCR and Southern blot analysis confirmed transgene integration into the lettuce chloroplast genome via homologous recombination at the trnI/trnA intergenic spacer region. Western blot analysis showed expression of polyprotein prM/E in different forms as monomers (~65 kDa) or possibly heterodimers (~130 kDa) or multimers. Multimers were solubilized into monomers using guanidine hydrochloride. Transplastomic lettuce plants expressing dengue prM/E vaccine antigens grew normally and transgenes were inherited in the T1 progeny without any segregation. Transmission electron microscopy showed the presence of virus-like particles of ~20 nm diameter in chloroplast extracts of transplastomic lettuce expressing prM/E proteins, but not in untransformed plants. The prM/E antigens expressed in lettuce chloroplasts should offer a potential source for investigating an oral Dengue vaccine

Release of Proteins from Intact Chloroplasts Induced by Reactive Oxygen Species During Biotic and Abiotic Stress

Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a novel signaling mechanism, especially for participation of chloroplast proteins (e.g. transcription factors) in retrograde signaling, thereby offering new opportunities to regulate pathways outside chloroplasts

Release of Proteins from Intact Chloroplasts Induced by Reactive Oxygen Species during Biotic and Abiotic Stress

Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a novel signaling mechanism, especially for participation of chloroplast proteins (e.g. transcription factors) in retrograde signaling, thereby offering new opportunities to regulate pathways outside chloroplasts

Anaemia in acute coronary syndrome patients: a study from rural tertiary care centre of India

Background: Acute coronary syndromes (ACS) are an imbalance between myocardial oxygen supply and demand, and the presence of anaemia further potentiates this imbalance. The burden of anaemia in patients presenting with acute coronary syndromes (ACS) is significant. Anaemia has the potential to worsen myocardial ischemic insult by decreasing the oxygen content of the blood supplied to the jeopardized myocardium.Methods: A total of 148 patients with ACS were recruited in the study from October 2016 to December 2017 in Medicine and Cardiology Department of UPUMS Saifai, India. All patients were subjected to a detailed history and thorough clinical examination and investigations after obtaining informed consent. Patient having any other diseases known to cause anaemia were excluded.Results: Mean age of patients was 58.5 years. 72.97% were vegetarian and 27.03% were non-vegetarian. Most common morphological type of anaemia was dimorphic anaemia followed by macrocytic and microcytic hypochromic respectively. Iron deficiency anaemia was most common type of anaemia followed by vitamin B12 deficiency and mixed (Iron and vitamin B12 deficiency). 45.28% anaemic patients had no symptoms of blood loss. Most common symptom of blood loss was bleeding per rectum followed by malena. Severity of acute coronary syndrome was more in subjects having anaemia which was evident by higher incidence of anaemia in subjects having ST elevation myocardial infarction (STEMI). The incidence of anaemia was low in case of Non ST elevation Myocardial Infarction (NSTEMI) and Unstable angina (UA). The results of the present study have been compared to those from India.Conclusions: Higher incidence of anaemia was reported in subjects having acute coronary syndrome. Incidence of anaemia in STEMI patients was greater than NSTEMI and unstable angina patients. Severe form of acute coronary syndrome i.e. STEMI was associated with higher incidence of anaemia.

Oral Delivery of Human Biopharmaceuticals, Autoantigens and Vaccine Antigens Bioencapsulated in Plant Cells

Among 12 billion injections administered annually, unsafe delivery leads to \u3e20 million infections and \u3e100 million reactions. In an emerging new concept, freeze-dried plant cells (lettuce) expressing vaccine antigens/biopharmaceuticals are protected in the stomach from acids/enzymes but are released to the immune or blood circulatory system when plant cell walls are digested by microbes that colonize the gut. Vaccine antigens bioencapsulated in plant cells upon oral delivery after priming, conferred both mucosal and systemic immunity and protection against bacterial, viral or protozoan pathogens or toxin challenge. Oral delivery of autoantigens was effective against complications of type 1diabetes and hemophilia, by developing tolerance. Oral delivery of proinsulin or exendin-4 expressed in plant cells regulated blood glucose levels similar to injections. Therefore, this new platform offers a low cost alternative to deliver different therapeutic proteins to combat infectious or inherited diseases by eliminating inactivated pathogens, expensive purification, cold storage/transportation and sterile injections

Role of p63 expression in non-proliferative and proliferative lesions of breast

Background: Breast lump among females, is a fairly common complaint. Breast lump have a variety of etiologies ranging from inflammatory to benign to malignant lesions. Myoepithelial markers are useful in helping to distinguish invasive carcinoma from benign proliferations with a similar morphological appearance, benign proliferative lesions and most pre-invasive lesions with an intact myoepithelium. Invasive carcinomas lack the myoepithelial cell layer that normally surrounds benign breast glands. p63 antibody is a myoepithelial cell marker that selectively stains nuclei. Also, it is negative in stromal, myofibroblastic and adipocytic cells. This makes p63 more specific and superior to other myoepithelial markers.Methods: The present study was done on a total of 151 cases of breast diseases, received in the form of core biopsy, tru cut biopsy, lumpectomy, and mastectomy specimens. Clinical history and examination findings of the patients were collected in all the cases. All specimens were routinely processed and stained with haematoxylin and Eosin (H and E) stain and only 50 cases were subjected to immunohistochemical staining for p63.Results: Out of total 151 cases, 09 were inadequate for evaluation, 96 cases benign and 46 malignant. In benign category, fibroadenoma was most common and infiltrating ductal carcinoma (NOS) was the most common in malignant category. Mean size of benign tumors was found to be less than that of malignant tumors. All malignant cases were negative for p63 expression. In the benign category, 88.6% cases showed positive expression for p63 while 11.4% were negative. Among the benign category, non-proliferative lesions were continuous positive, proliferative showed discontinuous positivity for p63.Conclusions: Myoepithelial markers are useful in helping to distinguish invasive carcinoma from benign proliferations with a similar morphological appearance, benign proliferative lesions and most pre-invasive lesions with an intact myoepithelium. Invasive carcinomas lack the myoepithelial cell layer while in the benign category, non-proliferative lesions are continuous positive, proliferative lesions show discontinuous positivity for p63

Chloroplast-Derived Enzyme Cocktails Hydrolyse Lignocellulosic Biomass and Release Fermentable Sugars

It is widely recognized that biofuel production from lignocellulosic materials is limited by inadequate technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi were expressed in E. coli or tobacco chloroplasts. A PCR based method was used to clone genes without introns from Trichoderma reesei genomic DNA. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Based on observed expression levels, up to 49, 64 and 10,751 million units of pectate lyases or endoglucanase can be produced annually, per acre of tobacco. Plant production cost of endoglucanase is 3,100-fold and pectate lyase is 1,057 or 1,480 fold lower than the same recombinant enzymes sold commercially, produced via fermentation. Chloroplast-derived enzymes had higher temperature stability and wider pH optima than enzymes expressed in E. coli. Plant crude-extracts showed higher enzyme activity than E. coli with increasing protein concentration, demonstrating their direct utility without purification. Addition of E. coliextracts to the chloroplast-derived enzymes significantly decreased their activity. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3,625%) glucose from filter paper, pine wood or citrus peel than commercial cocktails. Furthermore, pectate lyase transplastomic plants showed enhanced resistance to Erwina soft rot. This is the first report of using plant-derived enzyme cocktails for production of fermentable sugars from lignocellulosic biomass. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails

Chloroplast-Derived Enzyme Cocktails Hydrolyse Lignocellulosic Biomass and Release Fermentable Sugars

It is widely recognized that biofuel production from lignocellulosic materials is limited by inadequate technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi were expressed in E. coli or tobacco chloroplasts. A PCR based method was used to clone genes without introns from Trichoderma reesei genomic DNA. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Based on observed expression levels, up to 49, 64 and 10,751 million units of pectate lyases or endoglucanase can be produced annually, per acre of tobacco. Plant production cost of endoglucanase is 3,100-fold and pectate lyase is 1,057 or 1,480 fold lower than the same recombinant enzymes sold commercially, produced via fermentation. Chloroplast-derived enzymes had higher temperature stability and wider pH optima than enzymes expressed in E. coli. Plant crude-extracts showed higher enzyme activity than E. coli with increasing protein concentration, demonstrating their direct utility without purification. Addition of E. coliextracts to the chloroplast-derived enzymes significantly decreased their activity. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3,625%) glucose from filter paper, pine wood or citrus peel than commercial cocktails. Furthermore, pectate lyase transplastomic plants showed enhanced resistance to Erwina soft rot. This is the first report of using plant-derived enzyme cocktails for production of fermentable sugars from lignocellulosic biomass. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails

Functional validation of a novel isoform of Na<SUP>+</SUP>/H<SUP>+</SUP> antiporter from Pennisetum glaucum for enhancing salinity tolerance in rice

Salt stress is an environmental factor that severely impairs plant growth and productivity. We have cloned a novel isoform of a vacuolar Na+/H+ antiporter from Pennisetum glaucum (PgNHX1) that contains 5 transmembrane domains in contrast to AtNHX1 and OsNHX1 which have 9 transmembrane domains. Recently we have shown that PgNHX1 could confer high level of salinity tolerance when overexpressed in Brassica juncea. Here, we report the functional validation of this antiporter in crop plant rice. Overexpression of PgNHX1 conferred high level of salinity tolerance in rice. Transgenic rice plants overexpressing PgNHX1 developed more extensive root system and completed their life cycle by setting flowers and seeds in the presence of 150 mM NaCl. Our data demonstrate the potential of PgNHX1 for imparting enhanced salt tolerance capabilities to salt-sensitive crop plants for growing in high saline areas