Dynamics of Antibacterial Drone Establishment in Staphylococcus aureus: Unexpected Effects of Antibiotic Resistance Genes

For a particulate therapeutic agent, such as the ABD, accurate enumeration of particles is critical to enable evaluation of preparative procedures and calculation of therapeutic dosages. It is equally important that a selective marker used for these two purposes be biologically inert.

A novel dominant selection system for plant transgenics based on phosphite metabolism catalyzed by bacterial alkaline phosphatase

Selective markers are generally indispensable in plant genetic transformation, of which the frequently used are of antibiotic or herbicide resistance. However, the increasing concerns on transgenic biosafety have encouraged many new and safe selective markers emerging, with an eminent representative as phosphite (Phi) in combination to its dehydrogenase (PTDH, e.g. PtxD). As bacterial alkaline phosphatase (BAP) can resemble PtxD to oxidatively convert toxic Phi into metabolizable phosphate (Pi), herein we harnessed it as the substitute of PtxD to develop an alternative Phi-based selection system. We first validated the Escherichia coli BAP (EcBAP) did own an extra enzymatic activity of oxidizing Phi to Pi. We further revealed EcBAP could be used as a dominant selective marker for Agrobacterium-mediated tobacco transformation. Although the involved Phi selection for transformed tobacco cells surprisingly required the presence of Pi, it showed a considerable transformation efficiency and dramatically accelerated transformation procedure, as compared to the routine kanamycin selection and the well-known PtxD/Phi system. Moreover, the EcBAP transgenic tobaccos could metabolize toxic Phi as a phosphorus (P) fertilizer thus underlying Phi-resistance, and competitively possess a dominant growth over wild-type tobacco and weeds under Phi stress. Therefore, this novel BAP/Phi-coupled system, integrating multiple advantages covering biosafe dominant selective marker, plant P utilization and weed management, can provide a PTDH-bypass technological choice to engineer transgenic plant species, especially those of great importance for sustainable agriculture.

The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR/Cas9 and Cre/loxP systems

Phytopathogenic fungi belonging to the Colletotrichum genus cause devastating damage for many plant species. Among them, Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to efficiently generate multiple knockout mutants of C. orbiculare. We first found that CRISPR/Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR/Cas9 vector, more than 90 % of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre/loxP marker recycling system for C. orbiculare because limited availability of desired selective markers hampers sequential gene disruption. In this system, integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling reuse of the same selective marker for the next transformation. Using our CRISPR/Cas9 and Cre/loxP systems, we attempted to identify functional sugar transporters in C. orbiculare. Multiple disruptions of putative quinate transporter genes restrict fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. Our analyses revealed that quinate acquisition is dispensable during fungal infection because this mutant displayed normal virulence to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants established in this study, we inferred that repetitive rounds of gene disruption using CRISPR/Cas9 and Cre/loxP systems do not cause negative effects for fungal virulence and growth. Therefore, these systems will be powerful tools to perform systematic gene targeting approach for C. orbiculare.

Genome Editing in Filamentous Fungus Penicillium Verruculosum Using the CRISPR/Cas9 System

PurposeTo adapt CRISPR/Cas9 genome editing method for use in filamentous fungus Penicillium verruculosum, which is industrial producer of carbohydrases. ResultsFor the first time the CRISPR/Cas9 method was adapted for genome editing in the filamentous fungi Penicillium verrucullosum. Using the nitrate reductase gene (niaD) as a selective marker with the CRISPR/Cas9 system we performed double knockout of niaD and cellobiohydrolase 1 (cbh1) genes. The efficiency of double editing was 50%. At the same time, it was unexpected that the specific cellobiohydrolase activity rised after knockout of cbh1 gene due to the increase CBH2 expression. ConclusionWe developed effective method for genome editing in P. verruculosum, which can be used to improve qualities of industrial strains.

Secretagogin marks amygdaloid PKCδ interneurons and modulates NMDA receptor availability

The perception of and response to danger is critical for an individual’s survival and is encoded by subcortical neurocircuits. The amygdaloid complex is the primary neuronal site that initiates bodily reactions upon external threat with local-circuit interneurons scaling output to effector pathways. Here, we categorize central amygdala neurons that express secretagogin (Scgn), a Ca2+-sensor protein, as a subset of protein kinase Cδ (PKCδ)+ interneurons, likely “off cells.” Chemogenetic inactivation of Scgn+/PKCδ+ cells augmented conditioned response to perceived danger in vivo. While Ca2+-sensor proteins are typically implicated in shaping neurotransmitter release presynaptically, Scgn instead localized to postsynaptic compartments. Characterizing its role in the postsynapse, we found that Scgn regulates the cell-surface availability of NMDA receptor 2B subunits (GluN2B) with its genetic deletion leading to reduced cell membrane delivery of GluN2B, at least in vitro. Conclusively, we describe a select cell population, which gates danger avoidance behavior with secretagogin being both a selective marker and regulatory protein in their excitatory postsynaptic machinery.

Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3′-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.

Midfrontal neural dynamics distinguish between general control and inhibition-specific processes in the stopping of motor actions

Action inhibition, the suppression of action impulses, is crucial for goal-directed behaviour. In order to dissociate neural mechanisms specific to motor stopping from general control processes which are also relevant for other types of conflict adjustments, we compared midfrontal oscillatory activity in human volunteers via EEG between action inhibition and two other types of motor conflicts, unexpected action activation and unexpected action change. Error rates indicated that action activation was significantly easier than the other two equally demanding tasks. Midfrontal brain oscillations were significantly stronger for inhibition than for both other conflict types. This was driven by increases in the delta range (2–3 Hz), which were higher for inhibition than activation and action change. Increases in the theta range (4–7 Hz) were equally high for inhibition and change, but lower for action activation. These findings suggest that inhibition is facilitated by neural mechanisms specific to motor-stopping, with midfrontal delta being a potentially selective marker of motor inhibition.