Pneumonia-Induced Inflammation, Resolution and Cardiovascular Disease: Causes, Consequences and Clinical Opportunities.

Pneumonia is inflammation in the lungs, which is usually caused by an infection. The symptoms of pneumonia can vary from mild to life-threatening, where severe illness is often observed in vulnerable populations like children, older adults, and those with preexisting health conditions. Vaccines have greatly reduced the burden of some of the most common causes of pneumonia, and the use of antimicrobials has greatly improved the survival to this infection. However, pneumonia survivors do not return to their preinfection health trajectories but instead experience an accelerated health decline with an increased risk of cardiovascular disease. The mechanisms of this association are not well understood, but a persistent dysregulated inflammatory response post-pneumonia appears to play a central role. It is proposed that the inflammatory response during pneumonia is left unregulated and exacerbates atherosclerotic vascular disease, which ultimately leads to adverse cardiac events such as myocardial infarction. For this reason, there is a need to better understand the inflammatory cross talk between the lungs and the heart during and after pneumonia to develop therapeutics that focus on preventing pneumonia-associated cardiovascular events. This review will provide an overview of the known mechanisms of inflammation triggered during pneumonia and their relevance to the increased cardiovascular risk that follows this infection. We will also discuss opportunities for new clinical approaches leveraging strategies to promote inflammatory resolution pathways as a novel therapeutic target to reduce the risk of cardiac events post-pneumonia.

Inactivation of rice starch branching enzyme IIb triggers broad and unexpected changes in metabolism by transcriptional reprogramming.

Starch properties can be modified by mutating genes responsible for the synthesis of amylose and amylopectin in the endosperm. However, little is known about the effects of such targeted modifications on the overall starch biosynthesis pathway and broader metabolism. Here we investigated the effects of mutating the OsSBEIIb gene encoding starch branching enzyme IIb, which is required for amylopectin synthesis in the endosperm. As anticipated, homozygous mutant plants, in which OsSBEIIb was completely inactivated by abolishing the catalytic center and C-terminal regulatory domain, produced opaque seeds with depleted starch reserves. Amylose content in the mutant increased from 19.6 to 27.4% and resistant starch (RS) content increased from 0.2 to 17.2%. Many genes encoding isoforms of AGPase, soluble starch synthase, and other starch branching enzymes were up-regulated, either in their native tissues or in an ectopic manner, whereas genes encoding granule-bound starch synthase, debranching enzymes, pullulanase, and starch phosphorylases were largely down-regulated. There was a general increase in the accumulation of sugars, fatty acids, amino acids, and phytosterols in the mutant endosperm, suggesting that intermediates in the starch biosynthesis pathway increased flux through spillover pathways causing a profound impact on the accumulation of multiple primary and secondary metabolites. Our results provide insights into the broader implications of perturbing starch metabolism in rice endosperm and its impact on the whole plant, which will make it easier to predict the effect of metabolic engineering in cereals for nutritional improvement or the production of valuable metabolites.

Multilevel interactions between native and ectopic isoprenoid pathways affect global metabolism in rice.

Isoprenoids are natural products derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, these precursors are synthesized via the cytosolic mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways. The regulation of these pathways must therefore be understood in detail to develop effective strategies for isoprenoid metabolic engineering. We hypothesized that the strict regulation of the native MVA pathway could be circumvented by expressing an ectopic plastidial MVA pathway that increases the accumulation of IPP and DMAPP in plastids. We therefore introduced genes encoding the plastid-targeted enzymes HMGS, tHMGR, MK, PMK and MVD and the nuclear-targeted transcription factor WR1 into rice and evaluated the impact of their endosperm-specific expression on (1) endogenous metabolism at the transcriptomic and metabolomic levels, (2) the synthesis of phytohormones, carbohydrates and fatty acids, and (3) the macroscopic phenotype including seed morphology. We found that the ectopic plastidial MVA pathway enhanced the expression of endogenous cytosolic MVA pathway genes while suppressing the native plastidial MEP pathway, increasing the production of certain sterols and tocopherols. Plants carrying the ectopic MVA pathway only survived if WR1 was also expressed to replenish the plastid acetyl-CoA pool. The transgenic plants produced higher levels of fatty acids, abscisic acid, gibberellins and lutein, reflecting crosstalk between phytohormones and secondary metabolism.

Anti-inflammatory effect of rosuvastatin in patients with HIV infection: An FDG-PET pilot study.

AIMS: This study aimed to evaluate markers of systemic as well as imaging markers of inflammation in the ascending aorta, bone marrow, and spleen measured by 18F-FDG PET/CT, in HIV+ patients at baseline and following therapy with rosuvastatin. METHODS AND RESULTS: Of the 35 HIV+ patients enrolled, 17 were randomized to treatment with 10 mg/day rosuvastatin and 18 to usual care for 6 months. An HIV- control cohort was selected for baseline comparison of serum inflammatory markers and monocyte markers of inflammation. 18F-FDG-PET/CT imaging of bone marrow, spleen, and thoracic aorta was performed in the HIV+ cohort at baseline and 6 months. While CD14++CD16- and CCR2 expressions were reduced, serum levels of IL-7, IL-8, and MCP-1 were elevated in the HIV+ population compared to the controls. There was a significant drop in FDG uptake in the bone marrow (TBRmax), spleen (SUVmax) and thoracic aortic (TBRmax) in the statin-treated group compared to the control group (bone marrow: - 10.3 ± 16.9% versus 5.0 ± 18.9%, p = .0262; spleen: - 9.8 ± 20.3% versus 11.3 ± 28.8%, p = .0497; thoracic aorta: - 19.1 ± 24.2% versus 4.3 ± 15.4%, p = .003). CONCLUSIONS: HIV+ patients had significantly markers of systemic inflammation including monocyte activation. Treatment with low-dose rosuvastatin in the HIV+ cohort significantly reduced bone marrow, spleen and thoracic aortic FDG uptake.

Structure, processes, and initial outcomes of The Ottawa Hospital Multi-Specialist Limb-Preservation Clinic and Programme: A unique-in-Canada quality improvement initiative.

In 2017, The Ottawa Hospital initiated a unique-in-Canada quality improvement initiative by opening a novel, multi-specialist limb-preservation clinic. We sought to describe the structure, processes, and initial outcomes of the clinic and evaluate whether it is achieving its mandate of providing high-quality wound clinical care, education, and research. We conducted a descriptive prospective cohort study alongside a nested study of 162 clinic patients requiring serial assessments. There have been 1623 visits, mostly (72.2%) from outpatients. During 17.8% of visits, patients were evaluated by >1 specialist. Therapies provided most often included negative-pressure wound therapy (32.7%), biological wound dressings (21.6%), and total contact casting (18.5%). Furthermore, 1.2% underwent toe/ray amputations or skin grafting in clinic and 22.8% were initiated on antimicrobials. Mixed-effects models suggested that mean wound volumes for those requiring serial assessments decreased by 1.6 (95% confidence interval = -0.86 to -2.27) cm3 between visits. The clinic provided seven rotations to vascular surgery, infectious diseases, dermatology, and palliative care physicians; three nursing preceptorships; and two educational workshops. It also initiated provincial and national vascular health and wound care research initiatives. This study may be used to guide development of other limb-preservation clinics and programmes. Findings support that our programme is achieving its mandate.

Accuracy of Administrative Database Algorithms for Hospitalized Pneumonia in Adults: a Systematic Review.

BACKGROUND: Administrative data algorithms (ADAs) to identify pneumonia cases are commonly used in the analysis of pneumonia burden, trends, etiology, processes of care, outcomes, health care utilization, cost, and response to preventative and therapeutic interventions. However, without a good understanding of the validity of ADAs for pneumonia case identification, an adequate appreciation of this literature is difficult. We systematically reviewed the quality and accuracy of published ADAs to identify adult hospitalized pneumonia cases. METHODS: We reviewed the Medline, EMBase, and Cochrane Central databases through May 2020. All studies describing ADAs for adult hospitalized pneumonia and at least one accuracy statistic were included. Investigators independently extracted information about the sampling frame, reference standard, ADA composition, and ADA accuracy. RESULTS: Thirteen studies involving 24 ADAs were analyzed. Compliance with a 38-item study-quality assessment tool ranged from 17 to 29 (median, 23; interquartile range [IQR], 20 to 25). Study setting, design, and ADA composition varied extensively. Inclusion criteria of most studies selected for high-risk populations and/or increased pneumonia likelihood. Reference standards with explicit criteria (clinical, laboratorial, and/or radiographic) were used in only 4 ADAs. Only 2 ADAs were validated (one internally and one externally). ADA positive predictive values ranged from 35.0 to 96.5% (median, 84.8%; IQR, 65.3 to 89.1%). However, these values are exaggerated for an unselected patient population because pneumonia prevalences in the study cohorts were very high (median, 66%; IQR, 46 to 86%). ADA sensitivities ranged from 31.3 to 97.8% (median, 65.1%; IQR 52.5-72.4). DISCUSSION: ADAs for identification of adult pneumonia hospitalizations are highly heterogeneous, poorly validated, and at risk for misclassification bias. Greater standardization in reporting ADA accuracy is required in studies using pneumonia ADA for case identification so that results can be properly interpreted.

Evaluation of the protein gap for detection of abnormal serum gammaglobulin level: an imperfect predictor.

OBJECTIVES: The value of the serum protein gap (PG, difference between total protein and albumin) in the detection of hyper- or hypogammaglobulinemia is not well established. We assessed the performance of PG for the detection of hyper- or hypogammaglobulinemia in a large sample of patients. METHODS: We reviewed all paired measurements of serum total protein, albumin, quantitative immunoglobulins, and serum protein electrophoresis tested between March 2014 and June 2017 at the Eastern Ontario Regional Laboratory Association. Sensitivity, specificity, positive predictive value, negative predictive value and likelihood ratios of PG at thresholds between 18 and 44 g/L for the detection of hyper- and hypogammaglobulinemia were assessed. RESULTS: There were 19,575 and 5,426 simultaneous paired data points to assess hyper- and hypogammaglobulinemia identified by serum protein electrophoresis (SPE) and nephelometry, respectively. The mean PG was 36.3 g/L (SD 8.6). The prevalence of hypergammaglobulinemia (>16 g/L by SPE) and hypogammaglobulinemia (IgG <7 g/L) was 21.9 and 5.5%, respectively. High PG (≥38 g/L) had sensitivity and specificity of 76.2 and 71.5% respectively for hypergammaglobulinemia. PG ≥38 g/L had a negative predictive value (NPV) of 93.1% for monoclonal, and 96.9% for polyclonal gammopathy. A PG threshold of ≤18 g/L had of sensitivity of 0.4%, specificity of 100%, PPV of 100% and NPV of 80.1% to detect hypogammaglobulinemia (IgG <7 g/L). CONCLUSIONS: High and low PG values were not sensitive in detecting hyper- or hypogammaglobulinemia, although negative predictive values were high for both. Performance of PG should be further evaluated prospectively in specific populations at risk of for abnormal IgG levels.

Recognition motifs rather than phylogenetic origin influence the ability of targeting peptides to import nuclear-encoded recombinant proteins into rice mitochondria.

Mitochondria fulfil essential functions in respiration and metabolism as well as regulating stress responses and apoptosis. Most native mitochondrial proteins are encoded by nuclear genes and are imported into mitochondria via one of several receptors that recognize N-terminal signal peptides. The targeting of recombinant proteins to mitochondria therefore requires the presence of an appropriate N-terminal peptide, but little is known about mitochondrial import in monocotyledonous plants such as rice (Oryza sativa). To gain insight into this phenomenon, we targeted nuclear-encoded enhanced green fluorescent protein (eGFP) to rice mitochondria using six mitochondrial pre-sequences with diverse phylogenetic origins, and investigated their effectiveness by immunoblot analysis as well as confocal and electron microscopy. We found that the ATPA and COX4 (Saccharomyces cerevisiae), SU9 (Neurospora crassa), pFA (Arabidopsis thaliana) and OsSCSb (Oryza sativa) peptides successfully directed most of the eGFP to the mitochondria, whereas the MTS2 peptide (Nicotiana plumbaginifolia) showed little or no evidence of targeting ability even though it is a native plant sequence. Our data therefore indicate that the presence of particular recognition motifs may be required for mitochondrial targeting, whereas the phylogenetic origin of the pre-sequences probably does not play a key role in the success of mitochondrial targeting in dedifferentiated rice callus and plants.

The subcellular localization of two isopentenyl diphosphate isomerases in rice suggests a role for the endoplasmic reticulum in isoprenoid biosynthesis.

KEY MESSAGE: Both OsIPPI1 and OsIPPI2 enzymes are found in the endoplasmic reticulum, providing novel important insights into the role of this compartment in the synthesis of MVA pathway isoprenoids. Isoprenoids are synthesized from the precursor's isopentenyl diphosphate (IPP) and dimethylallyl diphosphosphate (DMAPP), which are interconverted by the enzyme isopentenyl diphosphate isomerase (IPPI). Many plants express multiple isoforms of IPPI, the only enzyme shared by the mevalonate (MVA) and non-mevalonate (MEP) pathways, but little is known about their specific roles. Rice (Oryza sativa) has two IPPI isoforms (OsIPPI1 and OsIPPI2). We, therefore, carried out a comprehensive comparison of IPPI gene expression, protein localization, and isoprenoid biosynthesis in this species. We found that OsIPPI1 mRNA was more abundant than OsIPPI2 mRNA in all tissues, and its expression in de-etiolated leaves mirrored the accumulation of phytosterols, suggesting a key role in the synthesis of MVA pathway isoprenoids. We investigated the subcellular localization of both isoforms by constitutively expressing them as fusions with synthetic green fluorescent protein. Both proteins localized to the endoplasmic reticulum (ER) as well as peroxisomes and mitochondria, whereas only OsIPPI2 was detected in plastids, due to an N-terminal transit peptide which is not present in OsIPPI1. Despite the plastidial location of OsIPPI2, the expression of OsIPPI2 mRNA did not mirror the accumulation of chlorophylls or carotenoids, indicating that OsIPPI2 may be a redundant component of the MEP pathway. The detection of both OsIPPI isoforms in the ER indicates that DMAPP can be synthesized de novo in this compartment. Our work shows that the ER plays an as yet unknown role in the synthesis of MVA-derived isoprenoids, with important implications for the metabolic engineering of isoprenoid biosynthesis in higher plants.

CRISPR/Cas9 mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis.

KEY MESSAGE: Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery. The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61-71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8-12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars.

Prevalence of Hypogammaglobulinemia in Adult Invasive Pneumococcal Disease.

Background: Patients with humoral immune deficiency are susceptible to invasive pneumococcal disease (IPD). This study estimates the prevalence of underlying hypogammaglobulinemia in admitted IPD cases and examines whether IPD cases had received preventative treatment. Methods: All adult IPD cases (Streptococcus pneumoniae in blood or cerebrospinal fluid) admitted to The Ottawa Hospital (TOH) from January 2013 to December 2015 were identified through the Eastern Ontario Regional Laboratory. Documented clinical demographics, S. pneumoniae serotype, serum immunoglobulins measured previously or in convalescence, and vaccination status of the cases were collected retrospectively for descriptive analyses. Results: There were 134 IPD in 133 patients (47.4% male; mean age 63, standard deviation [SD] = 15.6 years) during a 3-year observation period. All-cause mortality rate was 22.6% over a mean follow-up time of 362, SD = 345 days. Fifty-seven patients (42.9%) had serum immunoglobulin levels measured. Eighteen were either found to have hypogammaglobulinemia in convalescence (8/18) or previously known to have hypogammaglobulinemia (10/18). None of the known hypogammaglobulinemic patients had received antibiotic prophylaxis and/or immunoglobulin replacement therapy within 4 months prior to IPD. The high and low estimates of prevalence of hypogammaglobulinemia were 31.6% (of all measured) and 13.5% (of all cases). Among 18 patients with hematological malignancies in our cohort, 13 had hypogammaglobulinemia. Many isolates were vaccine serotypes; however, only 8 had documented previous pneumococcal vaccination. Conclusions: IPD has high mortality, and hypogammaglobulinemia was present in at least 13.5% of IPD cases. Secondary hypogammaglobulinemia is especially common in cases with hematological malignancy and IPD.

CRISPR/Cas9-induced monoallelic mutations in the cytosolic AGPase large subunit gene APL2 induce the ectopic expression of APL2 and the corresponding small subunit gene APS2b in rice leaves.

The first committed step in the endosperm starch biosynthetic pathway is catalyzed by the cytosolic glucose-1-phosphate adenylyl transferase (AGPase) comprising large and small subunits encoded by the OsAPL2 and OsAPS2b genes, respectively. OsAPL2 is expressed solely in the endosperm so we hypothesized that mutating this gene would block starch biosynthesis in the endosperm without affecting the leaves. We used CRISPR/Cas9 to create two heterozygous mutants, one with a severely truncated and nonfunctional AGPase and the other with a C-terminal structural modification causing a partial loss of activity. Unexpectedly, we observed starch depletion in the leaves of both mutants and a corresponding increase in the level of soluble sugars. This reflected the unanticipated expression of both OsAPL2 and OsAPS2b in the leaves, generating a complete ectopic AGPase in the leaf cytosol, and a corresponding decrease in the expression of the plastidial small subunit OsAPS2a that was only partially complemented by an increase in the expression of OsAPS1. The new cytosolic AGPase was not sufficient to compensate for the loss of plastidial AGPase, most likely because there is no wider starch biosynthesis pathway in the leaf cytosol and because pathway intermediates are not shuttled between the two compartments.

A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1.

Pepino mosaic virus (PepMV) is a mechanically-transmitted tomato pathogen of importance worldwide. Interactions between the PepMV coat protein and triple gene block protein (TGBp1) with the host heat shock cognate protein 70 and catalase 1 (CAT1), respectively, have been previously reported by our lab. In this study, a novel tomato interactor (SlTXND9) was shown to bind the PepMV TGBp1 in yeast-two-hybrid screening, in vitro pull-down and bimolecular fluorescent complementation (BiFC) assays. SlTXND9 possesses part of the conserved thioredoxin (TRX) active site sequence (W__PC vs. WCXPC), and TXND9 orthologues cluster within the TRX phylogenetic superfamily closest to phosducin-like protein-3. In PepMV-infected and healthy Nicotiana benthamiana plants, NbTXND9 mRNA levels were comparable, and expression levels remained stable in both local and systemic leaves for 10 days post inoculation (dpi), as was also the case for catalase 1 (CAT1). To localize the TXND9 in plant cells, a polyclonal antiserum was produced. Purified α-SlTXND9 immunoglobulin (IgG) consistently detected a set of three protein bands in the range of 27⁻35 kDa, in the 1000 and 30,000 g pellets, and the soluble fraction of extracts of healthy and PepMV-infected N. benthamiana leaves, but not in the cell wall. These bands likely consist of the homologous protein NbTXND9 and its post-translationally modified derivatives. On electron microscopy, immuno-gold labelling of ultrathin sections of PepMV-infected N. benthamiana leaves using α-SlTXND9 IgG revealed particle accumulation close to plasmodesmata, suggesting a role in virus movement. Taken together, this study highlights a novel tomato-PepMV protein interaction and provides data on its localization in planta. Currently, studies focusing on the biological function of this interaction during PepMV infection are in progress.

Right Heart Function During and After Community-Acquired Pneumonia in Adults.

BACKGROUND: New-onset or worsening heart failure is the most common extra-pulmonary complication of community-acquired pneumonia (CAP) during the first 30 days after diagnosis. METHODS: We evaluated the changes in the right ventricular function amongst adult CAP survivors from the time of acute infection to its resolution. We performed comprehensive transthoracic echocardiographic examinations to assess right heart function during the acute illness and the convalescent period (4 to 6 weeks after hospital discharge). RESULTS: Twenty-six patients underwent acute measurements, of which convalescent measurements were completed in 19 subjects. There was no significant change in any of the right heart function parameters from the acute to convalescent stage of CAP. CONCLUSIONS: Our results suggest that right ventricular function does not meaningfully change in the transition from the acute to convalescent stage of CAP in non-critically ill adult CAP survivors.

Cardiovascular Complications and Short-term Mortality Risk in Community-Acquired Pneumonia.

BACKGROUND.: Previous reports suggest that community-acquired pneumonia (CAP) is associated with an enhanced risk of cardiovascular complications. However, a contemporary and comprehensive characterization of this association is lacking. METHODS.: In this multicenter study, 1182 patients hospitalized for CAP were prospectively followed for up to 30 days after their hospitalization for this infection. Study endpoints included myocardial infarction, new or worsening heart failure, atrial fibrillation, stroke, deep venous thrombosis, cardiovascular death, and total mortality. RESULTS.: Three hundred eighty (32.2%) patients experienced intrahospital cardiovascular events (CVEs) including 281 (23.8%) with heart failure, 109 (9.2%) with atrial fibrillation, 89 (8%) with myocardial infarction, 11 (0.9%) with ischemic stroke, and 1 (0.1%) with deep venous thrombosis; 28 patients (2.4%) died for cardiovascular causes. Multivariable Cox regression analysis showed that intrahospital Pneumonia Severity Index (PSI) class (hazard ratio [HR], 2.45, P = .027; HR, 4.23, P < .001; HR, 5.96, P < .001, for classes III, IV, and V vs II, respectively), age (HR, 1.02, P = .001), and preexisting heart failure (HR, 1.85, P < .001) independently predicted CVEs. One hundred three (8.7%) patients died by day 30 postadmission. Thirty-day mortality was significantly higher in patients who developed CVEs compared with those who did not (17.6% vs 4.5%, P < .001). Multivariable Cox regression analysis showed that intrahospital CVEs (HR, 5.49, P < .001) independently predicted 30-day mortality (after adjustment for age, PSI score, and preexisting comorbid conditions). CONCLUSIONS.: CVEs, mainly those confined to the heart, complicate the course of almost one-third of patients hospitalized for CAP. More importantly, the occurrence of CVEs is associated with a 5-fold increase in CAP-associated 30-day mortality.

The Arabidopsis ORANGE (AtOR) gene promotes carotenoid accumulation in transgenic corn hybrids derived from parental lines with limited carotenoid pools.

KEY MESSAGE: The AtOR gene enhances carotenoid levels in corn by promoting the formation of plastoglobuli when the carotenoid pool is limited, but has no further effect when carotenoids are already abundant. The cauliflower orange (or) gene mutation influences carotenoid accumulation in plants by promoting the transition of proplastids into chromoplasts, thus creating intracellular storage compartments that act as metabolic sink. We overexpressed the Arabidopsis OR gene under the control of the endosperm-specific wheat LMW glutenin promoter in a white corn variety that normally accumulates only trace amounts of carotenoids. The total endosperm carotenoid content in the best-performing AtOR transgenic corn line was 32-fold higher than wild-type controls (~25 µg/g DW at 30 days after pollination) but the principal carotenoids remained the same, suggesting that AtOR increases the abundance of existing carotenoids without changing the metabolic composition. We analyzed the expression of endogenous genes representing the carotenoid biosynthesis and MEP pathways, as well as the plastid fusion/translocation factor required for chromoplast formation, but only the DXS1 gene was upregulated in the transgenic corn plants. The line expressing AtOR at the highest level was crossed with four transgenic corn lines expressing different carotenogenic genes and accumulating different carotenoids. The introgression of AtOR increased the carotenoid content of the hybrids when there was a limited carotenoid pool in the parental line, but had no effect when carotenoids were already abundant in the parent. The AtOR gene therefore appears to enhance carotenoid levels by promoting the formation of carotenoid-sequestering plastoglobuli when the carotenoid pool is limited, but has no further effect when carotenoids are already abundant because high levels of carotenoids can induce the formation of carotenoid-sequestering plastoglobuli even in the absence of AtOR.

Bottlenecks in carotenoid biosynthesis and accumulation in rice endosperm are influenced by the precursor-product balance.

The profile of secondary metabolites in plants reflects the balance of biosynthesis, degradation and storage, including the availability of precursors and products that affect the metabolic equilibrium. We investigated the impact of the precursor-product balance on the carotenoid pathway in the endosperm of intact rice plants because this tissue does not normally accumulate carotenoids, allowing us to control each component of the pathway. We generated transgenic plants expressing the maize phytoene synthase gene (ZmPSY1) and the bacterial phytoene desaturase gene (PaCRTI), which are sufficient to produce ß-carotene in the presence of endogenous lycopene ß-cyclase. We combined this mini-pathway with the Arabidopsis thaliana genes AtDXS (encoding 1-deoxy-D-xylulose 5-phosphate synthase, which supplies metabolic precursors) or AtOR (the ORANGE gene, which promotes the formation of a metabolic sink). Analysis of the resulting transgenic plants suggested that the supply of isoprenoid precursors from the MEP pathway is one of the key factors limiting carotenoid accumulation in the endosperm and that the overexpression of AtOR increased the accumulation of carotenoids in part by up-regulating a series of endogenous carotenogenic genes. The identification of metabolic bottlenecks in the pathway will help to refine strategies for the creation of engineered plants with specific carotenoid profiles.

Rice endosperm is cost-effective for the production of recombinant griffithsin with potent activity against HIV.

Protein microbicides containing neutralizing antibodies and antiviral lectins may help to reduce the rate of infection with human immunodeficiency virus (HIV) if it is possible to manufacture the components in large quantities at a cost affordable in HIV-endemic regions such as sub-Saharan Africa. We expressed the antiviral lectin griffithsin (GRFT), which shows potent neutralizing activity against HIV, in the endosperm of transgenic rice plants (Oryza sativa), to determine whether rice can be used to produce inexpensive GRFT as a microbicide ingredient. The yield of (OS) GRFT in the best-performing plants was 223 µg/g dry seed weight. We also established a one-step purification protocol, achieving a recovery of 74% and a purity of 80%, which potentially could be developed into a larger-scale process to facilitate inexpensive downstream processing. (OS) GRFT bound to HIV glycans with similar efficiency to GRFT produced in Escherichia coli. Whole-cell assays using purified (OS) GRFT and infectivity assays using crude extracts of transgenic rice endosperm confirmed that both crude and pure (OS) GRFT showed potent activity against HIV and the crude extracts were not toxic towards human cell lines, suggesting they could be administered as a microbicide with only minimal processing. A freedom-to-operate analysis confirmed that GRFT produced in rice is suitable for commercial development, and an economic evaluation suggested that 1.8 kg/ha of pure GRFT could be produced from rice seeds. Our data therefore indicate that rice could be developed as an inexpensive production platform for GRFT as a microbicide component.