The potential of plant systems to break the HIV‐TB link

Tuberculosis (TB) and human immunodeficiency virus (HIV) can place a major burden on healthcare systems and constitute the main challenges of diagnostic and therapeutic programmes. Infection with HIV is the most common cause of Mycobacterium tuberculosis (Mtb), which can accelerate the risk of latent TB reactivation by 20‐fold. Similarly, TB is considered the most relevant factor predisposing individuals to HIV infection. Thus, both pathogens can augment one another in a synergetic manner, accelerating the failure of immunological functions and resulting in subsequent death in the absence of treatment. Synergistic approaches involving the treatment of HIV as a tool to combat TB and vice versa are thus required in regions with a high burden of HIV and TB infection. In this context, plant systems are considered a promising approach for combatting HIV and TB in a resource‐limited setting because plant‐made drugs can be produced efficiently and inexpensively in developing countries and could be shared by the available agricultural infrastructure without the expensive requirement needed for cold chain storage and transportation. Moreover, the use of natural products from medicinal plants can eliminate the concerns associated with antiretroviral therapy (ART) and anti‐TB therapy (ATT), including drug interactions, drug‐related toxicity and multidrug resistance. In this review, we highlight the potential of plant system as a promising approach for the production of relevant pharmaceuticals for HIV and TB treatment. However, in the cases of HIV and TB, none of the plant‐made pharmaceuticals have been approved for clinical use. Limitations in reaching these goals are discussed.     

Phylogenetic analyses of Vitis (Vitaceae) based on complete chloroplast genome sequences: effects of taxon sampling and phylogenetic methods on resolving relationships among rosids

Background  

The Vitaceae (grape) is an economically important family of angiosperms whose phylogenetic placement is currently unresolved. Recent phylogenetic analyses based on one to several genes have suggested several alternative placements of this family, including sister to Caryophyllales, asterids, Saxifragales, Dilleniaceae or to rest of rosids, though support for these different results has been weak. There has been a recent interest in using complete chloroplast genome sequences for resolving phylogenetic relationships among angiosperms. These studies have clarified relationships among several major lineages but they have also emphasized the importance of taxon sampling and the effects of different phylogenetic methods for obtaining accurate phylogenies. We sequenced the complete chloroplast genome of Vitis vinifera and used these data to assess relationships among 27 angiosperms, including nine taxa of rosids.     

Oral delivery of bioencapsulated exendin‐4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta‐TC6 cells

Glucagon‐like peptide (GLP‐1) increases insulin secretion but is rapidly degraded (half‐life: 2 min in circulation). GLP‐1 analogue, exenatide (Byetta) has a longer half‐life (3.3–4 h) with potent insulinotropic effects but requires cold storage, daily abdominal injections with short shelf life. Because patients with diabetes take >60 000 injections in their life time, alternative delivery methods are highly desired. Exenatide is ideal for oral delivery because insulinotropism is glucose dependent, with reduced risk of hypoglycaemia even at higher doses. Therefore, exendin‐4 (EX4) was expressed as a cholera toxin B subunit (CTB)–fusion protein in tobacco chloroplasts to facilitate bioencapsulation within plant cells and transmucosal delivery in the gut via GM1 receptors present in the intestinal epithelium. The transgene integration was confirmed by PCR and Southern blot analysis. Expression level of CTB‐EX4 reached up to 14.3% of total leaf protein (TLP). Lyophilization of leaf material increased therapeutic protein concentration by 12‐ to 24‐fold, extended their shelf life up to 15 months when stored at room temperature and eliminated microbes present in fresh leaves. The pentameric structure, disulphide bonds and functionality of CTB‐EX4 were well preserved in lyophilized materials. Chloroplast‐derived CTB‐EX4 showed increased insulin secretion similar to the commercial EX4 in beta‐TC6, a mouse pancreatic cell line. Even when 5000‐fold excess dose of CTB‐EX4 was orally delivered, it stimulated insulin secretion similar to the intraperitoneal injection of commercial EX4 but did not cause hypoglycaemia in mice. Oral delivery of the bioencapsulated EX4 should eliminate injections, increase patient compliance/convenience and significantly lower their cost.     

Mycobacteria induce IFN-gamma production in human dendritic cells via triggering of TLR2

IFN-gamma is of central importance for the induction of robust cell-mediated immunity and for the activation of APC. Recent studies using experimental murine systems have now suggested a fundamental role for APC-derived IFN-gamma during infection with intracellular pathogens. It is currently unknown whether human dendritic cells (DC) can respond to bacterial stimulation with production of IFN-gamma. To test this question, we used human monocyte-derived DC stimulated by Mycobacterium bovis bacillus Calmette-Guérin as a model system. We demonstrate production of IFN-gamma mRNA and protein on the single cell level. IFN-gamma in DC cultures was not simply produced by contaminating lymphocytes because production of DC-IFN-gamma could also be demonstrated in highly purified DC cultures containing virtually no T, B, and NK cells. TLR2 was identified as a key receptor involved in triggering production of DC-IFN-gamma. Interestingly, DC-IFN-gamma seems to participate in an autocrine DC activation loop, and production of DC-IFN-gamma could be enhanced by costimulation of DC with IL-12/IL-15/IL-18. In conclusion, we have demonstrated production of IFN-gamma by human DC on the single cell level, identified TLR2 as a pattern recognition receptor involved in this process, and elucidated some of the functional consequences of autocrine IFN-gamma production by human DC.     

Metabolite secretion in microorganisms: the theory of metabolic overflow put to the test

Introduction

Microbial cells secrete many metabolites during growth, including important intermediates of the central carbon metabolism. This has not been taken into account by researchers when modeling microbial metabolism for metabolic engineering and systems biology studies.

Materials and Methods

The uptake of metabolites by microorganisms is well studied, but our knowledge of how and why they secrete different intracellular compounds is poor. The secretion of metabolites by microbial cells has traditionally been regarded as a consequence of intracellular metabolic overflow.

Conclusions

Here, we provide evidence based on time-series metabolomics data that microbial cells eliminate some metabolites in response to environmental cues, independent of metabolic overflow. Moreover, we review the different mechanisms of metabolite secretion and explore how this knowledge can benefit metabolic modeling and engineering.

     

Pinellia ternata agglutinin expression in chloroplasts confers broad spectrum resistance against aphid, whitefly, Lepidopteran insects, bacterial and viral pathogens

Broad spectrum protection against different insects and pathogens requires multigene engineering. However, such broad spectrum protection against biotic stress is provided by a single protein in some medicinal plants. Therefore, tobacco chloroplasts were transformed with the agglutinin gene from Pinellia ternata (pta), a widely cultivated Chinese medicinal herb. Pinellia ternata agglutinin (PTA) was expressed up to 9.2% of total soluble protein in mature leaves. Purified PTA showed similar hemagglutination activity as snowdrop lectin. Artificial diet with purified PTA from transplastomic plants showed marked and broad insecticidal activity. In planta bioassays conducted with T0 or T1 generation PTA lines showed that the growth of aphid Myzus persicae (Sulzer) was reduced by 89%-92% when compared with untransformed (UT) plants. Similarly, the larval survival and total population of whitefly (Bemisia tabaci) on transplastomic lines were reduced by 91%-93% when compared with UT plants. This is indeed the first report of lectin controlling whitefly infestation. When transplastomic PTA leaves were fed to corn earworm (Helicoverpa zea), tobacco budworm (Heliothis virescens) or the beet armyworm (spodoptera exigua), 100% mortality was observed against all these three insects. In planta bioassays revealed Erwinia population to be 10,000-fold higher in control than in PTA lines. Similar results were observed with tobacco mosaic virus (TMV) challenge. Therefore, broad spectrum resistance to homopteran (sap-sucking), Lepidopteran insects as well as anti-bacterial or anti-viral activity observed in PTA lines provides a new option to engineer protection against biotic stress by hyper-expression of an unique protein that is naturally present in a medicinal plant.     

Molecular strategies for gene containment in transgenic crops

The potential of genetically modified (GM) crops to transfer foreign genes through pollen to related plant species has been cited as an environmental concern. Until more is known concerning the environmental impact of novel genes on indigenous crops and weeds, practical and regulatory considerations will likely require the adoption of gene-containment approaches for future generations of GM crops. Most molecular approaches with potential for controlling gene flow among crops and weeds have thus far focused on maternal inheritance, male sterility, and seed sterility. Several other containment strategies may also prove useful in restricting gene flow, including apomixis (vegetative propagation and asexual seed formation), cleistogamy (self-fertilization without opening of the flower), genome incompatibility, chemical induction/deletion of transgenes, fruit-specific excision of transgenes, and transgenic mitigation (transgenes that compromise fitness in the hybrid). As yet, however, no strategy has proved broadly applicable to all crop species, and a combination of approaches may prove most effective for engineering the next generation of GM crops.     

Three-dimensional Microorganization of the Soil–Root–Microbe System

Soils contain the greatest reservoir of biodiversity on Earth, and the functionality of the soil ecosystem sustains the rest of the terrestrial biosphere. This functionality results from complex interactions between biological and physical processes that are strongly modulated by the soil physical structure. Using a novel combination of biochemical and biophysical indicators and synchrotron microtomography, we have discovered that soil microbes and plant roots microengineer their habitats by changing the porosity and clustering properties (i.e., spatial correlation) of the soil pores. Our results indicate that biota act to significantly alter their habitat toward a more porous, ordered, and aggregated structure that has important consequences for functional properties, including transport processes. These observations support the hypothesis that the soil–plant–microbe complex is self-organized.     

Whole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9‐edited cotton plants

The CRISPR/Cas9 system has been extensively applied for crop improvement. However, our understanding of Cas9 specificity is very limited in Cas9‐edited plants. To identify on‐ and off‐target mutation in an edited crop, we described whole genome sequencing (WGS) of 14 Cas9‐edited cotton plants targeted to three genes, and three negative (Ne) control and three wild‐type (WT) plants. In total, 4188–6404 unique single‐nucleotide polymorphisms (SNPs) and 312–745 insertions/deletions (indels) were detected in 14 Cas9‐edited plants compared to WT, negative and cotton reference genome sequences. Since the majority of these variations lack a protospacer‐adjacent motif (PAM), we demonstrated that the most variations following Cas9‐edited are due either to somaclonal variation or/and pre‐existing/inherent variation from maternal plants, but not off‐target effects. Of a total of 4413 potential off‐target sites (allowing ≤5 mismatches within the 20‐bp sgRNA and 3‐bp PAM sequences), the WGS data revealed that only four are bona fide off‐target indel mutations, validated by Sanger sequencing. Moreover, inherent genetic variation of WT can generate novel off‐target sites and destroy PAMs, which suggested great care should be taken to design sgRNA for the minimizing of off‐target effect. These findings suggested that CRISPR/Cas9 system is highly specific for cotton plants.