The landscape of scientific research and funding is in flux as a result of tight budgets, evolving models of both publishing and evaluation, and questions about training and workforce stability. As future leaders, junior scientists are uniquely poised to shape the culture and practice of science in response to these challenges. A group of postdocs in the Boston area who are invested in improving the scientific endeavor, planned a symposium held on October 2 and 3 , 2014, as a way to join the discussion about the future of US biomedical research. Here we present a report of the proceedings of participant-driven workshops and the organizers' synthesis of the outcomes.

We describe the development and utilization of a new imaging technology for plant biology, optical coherence microscopy (OCM), which allows true in vivo visualization of plants and plant cells. This novel technology allows the direct, in situ (e.g. plants in soil), three-dimensional visualization of cells and events in shoot tissues without causing damage. With OCM we can image cells or groups of cells that are up to 1 mm deep in living tissues, resolving structures less than 5 m in size, with a typical collection time of 5 to 6 min. OCM measures the inherent light-scattering properties of biological tissues and cells. These optical properties vary and provide endogenous developmental markers. Singly scattered photons from small (e.g. 5 ϫ 5 ϫ 10 m) volume elements (voxels) are collected, assembled, and quantitatively false-colored to form a threedimensional image. These images can be cropped or sliced in any plane. Adjusting the colors and opacities assigned to voxels allows us to enhance different features within the tissues and cells. We show that light-scattering properties are the greatest in regions of the Arabidopsis shoot undergoing developmental processes. In large cells, high light scattering is produced from nuclei, intermediate light scatter is produced from cytoplasm, and little if any light scattering originates from the vacuole and cell wall. OCM allows the rapid, repetitive, non-destructive collection of quantitative data about inherent properties of cells, so it provides a means of continuously monitoring plants and plant cells during development and in response to exogenous stimuli.Studies in plant physiology and development characteristically follow changes in space and time that occur as part of normal plant activity or in response to exogenous stimuli. Typical studies require the destruction and analysis of a plant or a tissue sample, followed by the collection and analysis of a second distinct plant or sample. Thus, biological responses or changes are inferred by comparing different plants or samples. Such approaches have been used for centuries and have produced a great deal of knowledge. However, when scientists are able to nondestructively follow biological changes, important concepts and insights have emerged. For example, critical genes involved in programmed cell death were found in Caenorhabditis elegans partially because the developing nematode is nearly transparent, allowing the fate of each cell to be followed in vivo by light microscopy (Gilbert, 1998). Similarly, an elegant fate map for Arabidopsis roots was constructed because the relatively transparent roots allow changes in individual plants to be followed continuously (Dolan et al., 1993). This study led to new discoveries such as the presence of downward communication between mature root cells and the root apical meristem and short-range control of differentiation signals (van den Berg et al., 1997a(van den Berg et al., , 1997b.Except for the relatively transparent Arabidopsis root, plants provide a challenge for in vivo an...

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BackgroundAnthocyanins such as cyanidin 3-O-glucoside (C3G) have wide applications in industry as food colorants. Their current production heavily relies on extraction from plant tissues. Development of a sustainable method to produce anthocyanins is of considerable interest for industrial use. Previously, E. coli-based microbial production of anthocyanins has been investigated extensively. However, safety concerns on E. coli call for the adoption of a safe production host. In the present study, a GRAS bacterium, Corynebacterium glutamicum, was introduced as the host strain to synthesize C3G. We adopted stepwise metabolic engineering strategies to improve the production titer of C3G.ResultsAnthocyanidin synthase (ANS) from Petunia hybrida and 3-O-glucosyltransferase (3GT) from Arabidopsis thaliana were coexpressed in C. glutamicum ATCC 13032 to drive the conversion from catechin to C3G. Optimized expression of ANS and 3GT improved the C3G titer by 1- to 15-fold. Further process optimization and improvement of UDP-glucose availability led to ~ 40 mg/L C3G production, representing a > 100-fold titer increase compared to production in the un-engineered, un-optimized starting strain.ConclusionsFor the first time, we successfully achieved the production of the specialty anthocyanin C3G from the comparatively inexpensive flavonoid precursor catechin in C. glutamicum. This study opens up more possibility of C. glutamicum as a host microbe for the biosynthesis of useful and value-added natural compounds.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0990-z) contains supplementary material, which is available to authorized users.

The landscape of scientific research and funding is in flux and affected by tight budgets, evolving models of both publishing and evaluation, and questions about training and workforce stability. As future leaders, junior scientists are uniquely poised to shape the culture and practice of science in response to these challenges. A group of postdocs in the Boston area who are invested in improving the scientific endeavor, planned a symposium held on October 2 nd and 3 rd, 2014, as a way to join the discussion about the future of US biomedical research. Here we present a report of the proceedings of participant-driven workshops and the organizers’ synthesis of the outcomes.

Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into ␤-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.Organophosphates have been widely used as nerve agents and pesticides. Examples of these compounds include the pesticides coumaphos and parathion and the nerve gas agents soman, sarin, and VX. These compounds irreversibly inhibit acetylcholine degradation in the human body and can kill quickly by causing persistent and uncontrolled muscle stimulation. Parathion, methyl parathion, and paraoxon, three insecticides, are common model organophosphates because they are much less toxic (in rats, the 50% lethal dose of paraoxon is 1.8 mg/kg, compared to 0.001 mg/kg for VX) and less volatile than many nerve agents. Synthetic in origin, many organophosphates are persistent in the environment and resist degradation by naturally extant microorganisms.Although incineration and chemical hydrolysis have been widely used to destroy these toxic compounds (20), many microorganisms can detoxify organophosphates by hydrolyzing them using organophosphate acid anhydrases. In general, these enzymes have a broad substrate range and can hydrolyze a number of organophosphate contaminants.

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In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5' mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work 11 , and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy. Video LinkThe video component of this article can be found at