The extracellular vesicle proteomes of Sorghum bicolor and Arabidopsis thaliana are partially conserved.

Plant extracellular vesicles (EVs) are membrane-bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. Here, we describe the isolation and characterization of EVs from sorghum (Sorghum bicolor). Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, calcein acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also found in the EV proteome of Arabidopsis (Arabidopsis thaliana). These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

Ophthalmology as a career choice among medical students in Eastern India - A cross-sectional study.

Purpose: Several factors influence medical students to choose a specialty branch for post-graduate training, and it is important to understand them so that strategies can be made to make it more attractive and hence the shortage in the workforce can be addressed. This study aimed to identify the factors that influenced under-graduate medical students to choose ophthalmology as their specialty for post-graduate training. Methods: It was a descriptive, cross-sectional, questionnaire-based study, carried out among medical students at a tertiary academic institute in eastern India. In this survey tool, 25 questions were divided into different sections: demographic data, factors influencing the choice and perceptions about ophthalmology as a career option, and the impact of rotatory internship posting on the choice of subject. The various factors were then scored and indexed appropriately. Results: There were 515 participants. The median age was 23 years. The major influencing factor for choosing ophthalmology was "adequate time" (52.04%), and the discouraging factor was "steep learning curve" (58.64%). Age had an odds ratio of 0.781, showing that the lower the age, the higher the chances of being positively inclined toward ophthalmology as a career choice. Similarly, major deciding factors, perceptions, and experiences in ophthalmology had odds ratios of 1.841, 1.725, and 2.057, respectively, showing a positive inclination. Conclusion: The study states that a steep learning curve, personal attitudes, and misconceptions may dissuade the students from taking this subject, but adequate clinical exposure and experience, as well as role models during the internship, can create a positive impact on career choice.

Pathway Engineering, Re-targeting, and Synthetic Scaffolding Improve the Production of Squalene in Plants.

Plants are increasingly becoming an option for sustainable bioproduction of chemicals and complex molecules like terpenoids. The triterpene squalene has a variety of biotechnological uses and is the precursor to a diverse array of triterpenoids, but we currently lack a sustainable strategy to produce large quantities for industrial applications. Here, we further establish engineered plants as a platform for production of squalene through pathway re-targeting and membrane scaffolding. The squalene biosynthetic pathway, which natively resides in the cytosol and endoplasmic reticulum, was re-targeted to plastids, where screening of diverse variants of enzymes at key steps improved squalene yields. The highest yielding enzymes were used to create biosynthetic scaffolds on co-engineered, cytosolic lipid droplets, resulting in squalene yields up to 0.58 mg/gFW or 318% higher than a cytosolic pathway without scaffolding during transient expression. These scaffolds were also re-targeted to plastids where they associated with membranes throughout, including the formation of plastoglobules or plastidial lipid droplets. Plastid scaffolding ameliorated the negative effects of squalene biosynthesis and showed up to 345% higher rates of photosynthesis than without scaffolding. This study establishes a platform for engineering the production of squalene in plants, providing the opportunity to expand future work into production of higher-value triterpenoids.

Invasive fungal disease misdiagnosed as tumour in association with orbital apex syndrome.

Invasive sino-orbital aspergillosis is a rare cause of orbital apex syndrome (OAS) in immunocompetent patients and often misdiagnosed as tumour because of its aggressive nature and invasive patterns. We report a 23-year-old immunocompetent man presenting with painful progressive loss of vision, ophthalmoplegia and proptosis of the right eye suggestive of OAS. MRI with gadolinium contrast showed an enhancing heterogeneous mass filling the paranasal sinuses, extraconal space and extending up to the right orbital apex. A functional endoscopic biopsy reported as invasive sino-orbital aspergillosis. He was started on intravenous voriconazole and maximal surgical debridement was done. He gradually regained his vision to 20/30 in the right eye. A review of literature reported several such cases which were managed medically or surgically but with poor visual recovery. This case highlights the need for awareness among clinicians for early diagnosis and treatment to prevent vision loss and better survival.

Phosphoglucoisomerase Is an Important Regulatory Enzyme in Partitioning Carbon out of the Calvin-Benson Cycle.

Phosphoglucoisomerase (PGI) isomerizes fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) in starch and sucrose biosynthesis. Both plastidic and cytosolic isoforms are found in plant leaves. Using recombinant enzymes and isolated chloroplasts, we have characterized the plastidic and cytosolic isoforms of PGI. We have found that the Arabidopsis plastidic PGI K m for G6P is three-fold greater compared to that for F6P and that erythrose 4-phosphate is a key regulator of PGI activity. Additionally, the K m of spinach plastidic PGI can be dynamically regulated in the dark compared to the light and increases by 200% in the dark. We also found that targeting Arabidopsis cytosolic PGI into plastids of Nicotiana tabacum disrupts starch accumulation and degradation. Our results, in combination with the observation that plastidic PGI is not in equilibrium, indicates that PGI is an important regulatory enzyme that restricts flow and acts as a one-way valve preventing backflow of G6P into the Calvin-Benson cycle. We propose the PGI may be manipulated to improve flow of carbon to desired targets of biotechnology.

Resolution of grade IV hypertensive retinopathy in an adult with pheochromocytoma: post-tumor resection.

A 64-year-old hypertensive woman presented with complaints of a painless, progressive decrease in vision, headache, dizziness for the last month. She was a known case of diabetes mellitus on insulin therapy. On examination, best-corrected visual acuity was 6/12 in the right eye and 6/18 in the left eye. The fundus examination in both eyes revealed features of grade IV hypertensive retinopathy. Systemic examination revealed the raised blood pressure of 200/110 mm Hg. Diagnosis of pheochromocytoma was made on the basis of increased urinary norepinephrine (892.8 mg/dL) and mass in the left adrenal gland (measuring 31×28 mm) at contrast-enhanced CT. Medical management to control hypertension was done and ultimately, she underwent left laparoscopic adrenalectomy. After 10 months of surgery, the patient was asymptomatic, blood pressure was within normal limit and her vision improved to 6/6 in both the eyes. The retinal features of hypertensive retinopathy had completely disappeared.

Plastidic glucose-6-phosphate dehydrogenases are regulated to maintain activity in the light.

Glucose-6-phosphate dehydrogenase (G6PDH) can initiate the glucose-6-phosphate (G6P) shunt around the Calvin-Benson cycle. To understand the regulation of flux through this pathway, we have characterized the biochemical parameters and redox regulation of the three functional plastidic isoforms of Arabidopsis G6PDH. When purified, recombinant proteins were measured, all three exhibited significant substrate inhibition by G6P but not NADP+, making the determination of enzyme kinetic parameters complex. We found that the half-saturation concentration of G6PDH isoform 1 is increased under reducing conditions. The other two isoforms exhibit less redox regulation, however, isoform 2 is strongly inhibited by NADPH. Redox regulation of G6PDH1 can be partially reversed by hydrogen peroxide or protected against by the presence of its substrate, G6P. Overall, our results support the conclusion that G6PDH can have significant activity throughout the day and can be dynamically regulated to allow or prevent flux through the glucose-6-phosphate shunt.

Cytosolic lipid droplets as engineered organelles for production and accumulation of terpenoid biomaterials in leaves.

Cytosolic lipid droplets are endoplasmic reticulum-derived organelles typically found in seeds as reservoirs for physiological energy and carbon to fuel germination. Here, we report synthetic biology approaches to co-produce high-value sesqui- or diterpenoids together with lipid droplets in plant leaves. The formation of cytosolic lipid droplets is enhanced in the transient Nicotiana benthamiana system through ectopic production of WRINKLED1, a key regulator of plastid fatty acid biosynthesis, and a microalgal lipid droplet surface protein. Engineering of the pathways providing the universal C5-building blocks for terpenoids and installation of terpenoid biosynthetic pathways through direction of the enzymes to native and non-native compartments boost the production of target terpenoids. We show that anchoring of distinct biosynthetic steps onto the surface of lipid droplets leads to efficient production of terpenoid scaffolds and functionalized terpenoids. The co-produced lipid droplets "trap" the terpenoids in the cells.

Engineering modular diterpene biosynthetic pathways in Physcomitrella patens.

MAIN CONCLUSION: Modular assembly and heterologous expression in the moss Physcomitrella patens of pairs of diterpene synthases results in accumulation of modern land plant diterpenoids. Physcomitrella patens is a representative of the ancient bryophyte plant lineage with a genome size of 511 Mb, dominant haploid life cycle and limited chemical and metabolic complexity. For these plants, exceptional capacity for genome editing through homologous recombination is met with recently demonstrated in vivo assembly of multiple heterologous DNA fragments. These traits earlier made P. patens an attractive choice as a biotechnological chassis for photosynthesis-driven production of recombinant peptides. The lack of diterpene gibberellic acid phytohormones in P. patens combined with the recent targeted disruption of the single bifunctional diterpene synthase yielded lines devoid of endogenous diterpenoid metabolites and well-suited for engineering of terpenoid production. Here, we mimicked the modular nature of diterpene biosynthetic pathways found in modern land plants by developing a flexible pipeline to install three combinations of class II and class I diterpene synthases in P. patens to access industrially relevant diterpene biomaterials. In addition to a well-established neutral locus for targeted integration, we also explored loci created by a class of Long Terminal Repeat Retrotransposon present at moderate number in the genome of P. patens. Assembly of the pathways and production of the enzymes from the neutral locus led to accumulation of diterpenes matching the reported activities in the angiosperm sources. In contrast, insights gained with the retrotransposon loci indicate their suitability for targeting, but reveal potentially inherent complications which may require adaptation of the experimental design.

P450s controlling metabolic bifurcations in plant terpene specialized metabolism.

ABSTRACT: Catalyzing stereo- and regio-specific oxidation of inert hydrocarbon backbones, and a range of more exotic reactions inherently difficult in formal chemical synthesis, cytochromes P450 (P450s) offer outstanding potential for biotechnological engineering. Plants and their dazzling diversity of specialized metabolites have emerged as rich repository for functional P450s with the advances of deep transcriptomics and genome wide discovery. P450s are of outstanding interest for understanding chemical diversification throughout evolution, for gaining mechanistic insights through the study of their structure-function relationship, and for exploitation in Synthetic Biology. In this review, we highlight recent developments and examples in the discovery of plant P450s involved in the biosynthesis of industrially relevant monoterpenoids, sesquiterpenoids, diterpenoids and triterpenoids, throughout 2016 and early 2017. Examples were selected to illustrate the spectrum of value from commodity chemicals, flavor and fragrance compounds to pharmacologically active terpenoids. We focus on a recently emerging theme, where P450s control metabolic bifurcations and chemical diversity of the final product profile, either within a pathway, or through neo-functionalization in related species. The implications may inform approaches for rational assembly of recombinant pathways, biotechnological production of high value terpenoids and generation of novel chemical entities.

Sustainable Development for Whom and How? Exploring the Gaps between Popular Discourses and Ground Reality Using the Mexican Jatropha Biodiesel Case.

In the last decade, jatropha-based bioenergy projects have gotten significant attention as a solution to various social, economic, and environmental problems. Jatropha's popularity stemmed out from different discourses, some real and some perceived, in scientific and non-scientific literature. These discourses positioned jatropha as a crop helpful in producing biodiesel and protecting sustainability by reducing greenhouse gas emissions compared to fossil fuels and increasing local, rural development by creating jobs. Consequently, many countries established national policies that incentivized the establishment of jatropha as a bioenergy feedstock crop. In this paper, we explore the case of jatropha bioenergy development in Yucatan, Mexico and argue that the popular discourse around jatropha as a sustainability and rural development tool is flawed. Analyzing our results from 70 semi-structured interviews with community members belonging to a region where plantation-scale jatropha projects were introduced, we found that these projects did not have many significant social sustainability benefits. We conclude from our case that by just adding bioenergy projects cannot help achieve social sustainability in rural areas alone. In ensuring social sustainability of bioenergy projects, future policymaking processes should have a more comprehensive understanding of the rural socioeconomic problems where such projects are promoted and use bioenergy projects as one of the many solutions to local problems rather than creating such policies based just on popular discourses.

Correction: Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity.

[This corrects the article DOI: 10.1371/journal.pone.0161534.].

Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity.

Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity.

Aliphatic glucosinolate synthesis and gene expression changes in gamma-irradiated cabbage.

Glucosinolates, found principally in the plant order Brassicales, are modulated by different post-harvest processing operations. Among these, ionizing radiation, a non-thermal process, has gained considerable interest for ensuring food security and safety. In gamma-irradiated cabbage, enhanced sinigrin, a major glucosinolate, has been reported. However, the molecular basis of such a radiation induced effect is not known. Herein, the effect of radiation processing on the expression of glucosinolate biosynthetic genes was investigated. RT-PCR based expression analysis of seven glucosinolate biosynthetic pathway genes (MYB28, CYP79F1, CYP83A1, SUR1, UGT74B1, SOT18 and TGG1) showed that CYP83A1, MYB28, UGT74B1, CYP79F1 and SUR1 were up-regulated in irradiated cabbage. The content of jasmonates, signalling molecules involved in glucosinolate induction was, however, unaffected in irradiated cabbage suggesting their non-involvement in glucosinolate induction during radiation processing. This is the first report on the effect of gamma irradiation on the expression of glucosinolate biosynthetic genes in vegetables.

Shelf life extension of minimally processed ready-to-cook (RTC) cabbage by gamma irradiation.

Gamma irradiation (0.5-2.5 kGy) in combination with low temperature (4-15 °C) storage was attempted to increase shelf life of ready-to-cook shredded cabbage wrapped in cling films. A maximum extension in shelf life of 8 days, while retaining the microbial and sensory quality, was obtained with an irradiation dose of 2 kGy and storage at 10 °C. Gamma irradiation also inhibited browning of shredded cabbage at their cut edges resulting in enhanced visual appeal. An increase in total antioxidant activity was observed with respect to DPPH and hydroxyl radical scavenging ability while the nitric oxide radical scavenging activity and ferric reducing property remained unaffected with irradiation. Total phenolic, flavonoid and vitamin C content remained unchanged due to irradiation. No significant migration of additives from cling films into stimulant water was observed up to a radiation dose of 2 kGy thus demonstrating the feasibility of such films for above applications.

Allyl isothiocyanate enhances shelf life of minimally processed shredded cabbage.

The effect of allyl isothiocyanate (AITC), in combination with low temperature (10°C) storage on post harvest quality of minimally processed shredded cabbage was investigated. An optimum concentration of 0.05µL/mL AITC was found to be effective in maintaining the microbial and sensory quality of the product for a period of 12days. Inhibition of browning was shown to result from a down-regulation (1.4-fold) of phenylalanine ammonia lyase (PAL) gene expression and a consequent decrease in PAL enzyme activity and o-quinone content. In the untreated control samples, PAL activity increased following up-regulation in PAL gene expression that could be linearly correlated with enhanced o-quinone formation and browning. The efficacy of AITC in extending the shelf life of minimally processed shredded cabbage and its role in down-regulation of PAL gene expression resulting in browning inhibition in the product is reported here for the first time.

Gamma irradiation inhibits wound induced browning in shredded cabbage.

Gamma-radiation induced browning inhibition in minimally processed shredded cabbage stored (10 °C) for up to 8 days was investigated. γ-irradiation (2 kGy) resulted in inhibition of browning as a result of down-regulation (1.4-fold) in phenylalanine ammonia lyase (PAL) gene expression and a consequent decrease in phenylalanine ammonia lyase (PAL) activity. Activity of polyphenol oxidase and peroxidase, total and individual phenolic content as well as o-quinone concentration were, however, unaffected. In the non-irradiated samples, PAL activity increased as a consequence of up-regulation of PAL gene expression after 24 and 48 h by 1.2 and 7.7-fold, respectively, during storage that could be linearly correlated with enhanced quinone formation and browning. Browning inhibition in radiation processed shredded cabbage as a result of inhibition of PAL activity was thus clearly demonstrated. The present work provides an insight for the first time on the mechanism of browning inhibition at both biochemical and genetic level.

Policies for the Sustainable Development of Biofuels in the Pan American Region: A Review and Synthesis of Five Countries.

Rapid growth of biofuel production in the United States and Brazil over the past decade has increased interest in replicating this success in other nations of the Pan American region. However, the continued use of food-based feedstock such as maize is widely seen as unsustainable and is in some cases linked to deforestation and increased greenhouse gas emissions, raising further doubts about long-term sustainability. As a result, many nations are exploring the production and use of cellulosic feedstock, though progress has been extremely slow. In this paper, we will review the North-South axis of biofuel production in the Pan American region and its linkage with the agricultural sectors in five countries. Focus will be given to biofuel policy goals, their results to date, and consideration of sustainability criteria and certification of producers. Policy goals, results, and sustainability will be highlighted for the main biofuel policies that have been enacted at the national level. Geographic focus will be given to the two largest producers-the United States and Brazil; two smaller emerging producers-Argentina and Canada; and one stalled program-Mexico. However, several additional countries in the region are either producing or planning to produce biofuels. We will also review alternative international governance schemes for biofuel sustainability that have been recently developed, and whether the biofuel programs are being managed to achieve improved environmental quality and sustainable development.

Effect of post harvest radiation processing and storage on the volatile oil composition and glucosinolate profile of cabbage.

Effect of radiation processing (0.5-2 kGy) and storage on the volatile oil constituents and glucosinolate profile of cabbage was investigated. Among the volatile oil constituents, an enhancement in trans-hex-2-enal was noted on irradiation that was attributed to the increased liberation of precursor linolenic acid mainly from monogalactosyl diacyl glycerol (MGDG). Irradiation also enhanced sinigrin, the major glucosinolate of cabbage that accounted for the enhanced allyl isothiocyanate (AITC) in the volatile oils of the irradiated vegetable. During storage the content of trans-hex-2-enal increased immediately after irradiation and then returned to the basal value within 24h while the content of sinigrin and AITC increased post irradiation and thereafter remained constant during storage. Our findings on the enhancement in potentially important health promoting compounds such as sinigrin and AITC demonstrates that besides extending shelf life and safety, radiation processing can have an additional advantage in improving the nutritional quality of cabbage.