Expanding the toolbox: another auxotrophic marker for targeted gene integrations in Trichoderma reesei

Background The filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its long-term application in industry and science. A prerequisite for successful heterologous gene expression is the ability to insert a corresponding expression cassette at suitable loci in the genome of T. reesei. Results In this study, we test and demonstrate the applicability of the his1 gene [encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17), part of the histidine biosynthesis pathway] and locus for targeted gene insertion. Deletion of the his1 promoter and a part of the coding region leads to histidine auxotrophy. Reestablishment of the his1 locus restores prototrophy. We designed a matching plasmid that allows integration of an expression cassette at the his1 locus. This is demonstrated by the usage of the reporter EYFP (enhanced yellow fluorescence protein). Further, we describe a minimal effort and seamless marker recycling method. Finally, we test the influence of the integration site on the gene expression by comparing three strains bearing the same EYFP expression construct at different loci. Conclusion With the establishment of his1 as integration locus and auxotrophic marker, we could expand the toolbox for strain design in T. reesei. This facilitates future strain constructions with the aim of heterologous gene expression.

Bacillus subtilis MBI600 Promotes Growth of Tomato Plants and Induces Systemic Resistance Contributing to the Control of Soilborne Pathogens

Bacillus subtilis MBI600 (Bs MBI600) is a recently commercialized plant-growth-promoting rhizobacterium (PGPR). In this study, we investigated the effects of Bs MBI600 on the growth of tomato and its biocontrol efficacy against three main soilborne tomato pathogens (Rhizoctonia solani, Pythium ultimum, and Fusarium oxysporum f.sp. radicis-lycopersici-Forl). Furthermore, the root colonization ability of the Bs MBI600 strain on tomato roots was analyzed in vivo with a yellow fluorescence protein (yfp)-labeled strain, revealing strong colonization ability, which was affected by the root growth substrate. The application of Bs MBI600 on tomato plants resulted in significant increases in shoot and root lengths. Transcriptional activation of two auxin-related genes (SiPin6 and SiLax4) was observed. Single applications of Bs MBI600 on inoculated tomato plants with pathogens revealed satisfactory control efficacy compared to chemical treatment. Transcriptomic analysis of defense-related genes used as markers of the salicylic acid (SA) signaling pathway (PR-1A and GLUA) or jasmonic acid/ethylene (JA/ET) signaling pathway (CHI3, LOXD, and PAL) showed increased transcription patterns in tomato plants treated with Bs MBI600 or Forl. These results indicate the biochemical and molecular mechanisms that are activated after the application of Bs MBI600 on tomato plants and suggest that induction of systemic resistance (ISR) occurred.

A simple micropreparative gel electrophoresis technique for purification of proteins, nucleic acids, and bioconjugates

The biochemical and biomedical fields hinge on the ability to effectively separate and purify biological macromolecules. Though this need is largely addressed with a variety of chromatographic and electrophoretic purification techniques, such techniques are usually laborious, time-consuming, and often require complex and costly instalments that are inaccessible to most laboratories. In this work, we introduce a simple micro-preparative (MP) method based on polyacrylamide gel electrophoresis (PAGE) to purify biological samples containing proteins, nucleic acids, and complex bioconjugates. Using a conventional vertical slab system, we demonstrate the extraction of purified DNA, proteins, and DNA-protein bioconjugates from their respective mixtures using MP-PAGE. We apply this system to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using specialized commercial devices. We also demonstrate the purification of folded enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, comparable to purities achieved using a two-step size exclusion and immobilized metal-ion affinity chromatography purification procedure. Finally, we demonstrate the successful isolation of an EYFP-DNA bioconjugate sample that otherwise could not be processed using the two-step chromatography procedure. MP-PAGE thus offers a rapid and versatile means of purifying a variety of biomolecules without the need for specialized equipment.

Characterization of melanoidin formed from glucose with alanine by Fluorescence spectroscopy and redox chemistry in vitro cells: تشخيص الميلانويدين المتشكل من الكلوكوز مع الألنين بمطياف الفلوره ودراسة تأثيره التأكسدي على خلايا CHO-K1 المختبرية

Possible identify and describe a polymer formed from Glc with Ala by Fluorescence spectroscopy. Melanoidins before dialysis was higher than those of melanoidin after dialysis. Time‐resolved fluorescence has been applied in the characterization of melanoidin and was observed. Melanoidins show a decrease in the absorption after the addition of hydrogen peroxide when the concentrations of H2O2 from 100 - 200 μl are used. However, the data suggests that the melanoidin did not penetrate into the cells (CHO-K1) after 1 and 2 hours, but it can be observed after 24 hours. Abbreviation: TRES, time-resolved emission spectra ; Glc, Glucose ; Ala, Alanine ; YFP, Yellow fluorescence protein ; GFP, green fluorescence protein ; CHO, Chinese hamster ovary

A Therapeutic Role for Survivin in Mitigating the Harmful Effects of Ionizing Radiation

Background. Radiation therapy is a form of adjuvant care used in many oncological treatment protocols. However, nonmalignant neighboring tissues are harmed as a result of this treatment. Therefore, the goal of this study was to induce the production of survivin, an antiapoptotic protein, to determine if this protein could provide protection to noncancerous cells during radiation exposure.Methods. Using a murine model, a recombinant adenoassociated virus (rAAV) was used to deliver survivin to the treatment group and yellow fluorescence protein (YFP) to the control group. Both groups received targeted radiation. Visual inspection, gait analysis, and tissue histology were used to determine the extent of damage caused by the radiation.Results. The YFP group demonstrated ulceration of the irradiated area while the survivin treated mice exhibited only hair loss. Histology showed that the YFP treated mice experienced dermal thickening, as well as an increase in collagen that was not present in the survivin treated mice. Gait analysis demonstrated a difference between the two groups, with the YFP mice averaging a lower speed.Conclusions. The use of gene-modification to induce survivin expression in normal tissues allows for the protection of nontarget areas from the negative side effects normally associated with ionizing radiation.

Abstract 20201: Fibro-Adipocyte Progenitors are a Cell Source of Adipocytes in Arrhythmogenic Cardiomyopathy

Arrhythmogenic cardiomyopathy (AC) is clinically characterized by arrhythmias, heart failure and sudden death and pathologically by replacement of cardiomyocytes by fibro-adipocytes. Mutations in desmosome genes are the main causes of AC. Desmosome proteins are known to be expressed only in cardiac myocytes. Cardiac progenitor cells (CPCs) expressing mutant desmosome proteins could differentiate to adipocytes. However, genetic fate-mapping identify CPCs as the source of a fraction of adipocytes in AC. We posit that cardiac cells other than CPCs express desmosome proteins and differentiate to adipocytes in AC. We systematically determined expression of the desmosome proteins in different cardiac cells. Cardiac fibro-adipocyte progenitors (FAPs), marked by expression of platelet-derived growth factor receptor-α (PDGFRA), expressed mRNAs and proteins of major desmosome genes. FAPs isolated from the heart of Nkx2.5Cre:Dsp W/F mice, an established model of AC, showed enhanced adipogenesis associated with suppression of canonical Wnt signaling (reduced Ccnd1, Ctgf and cMyc mRNA levels by 10-, 5- and 3-fold, respectively, N=5, all p<0.001). To determine whether FAPs are a source of adipocytes in AC, we generated lineage tracer mice, utilizing the Pdgfra-Cre deleter, and conditionally deleted one copy of desmoplakin ( Dsp ) while expressing enhanced yellow fluorescence protein (EYFP) in the FAPs. Recombination efficiency (% of PDGFRA+ cells expressing EYFP) was ~75%. Six to 12 months old lineage tracer mice ( Pdgfra-Cre:Dsp W/F ) showed mild cardiac dysfunction (fractional shortening: 57±9%, N=14 vs. 66±7%, N=10 in controls, p< 0.005) and increased adipocytes in the heart (14±9, N=5 vs. 1±1, N=3 in controls, p=0.03). Co-expression of EYFP and C/EBPA adipogenic marker was detected in 54% of adipocytes in the lineage tracer mice, indicating an origin from FAPs. Dsp +/- FAPs showed enhanced differentiation to adipocytes as compared to control FAPs. The Hippo, canonical Wnt and NOTCH1 signaling pathways in mediating differentiation of Dsp +/- FAPs to adipocytes are being delineated. The findings show cardiac FAPs express desmosome proteins and when haplo-insufficient for Dsp differentiate to adipocytes. Thus, FAPs are a source of adipocytes in AC.

Transient Effects of Anesthetics on Dendritic Spines and Filopodia in the Living Mouse Cortex

Background Anesthetics are widely used to induce unconsciousness, pain relief, and immobility during surgery. It remains unclear whether the use of anesthetics has significant and long-lasting effects on synapse development and plasticity in the brain. To address this question, the authors examined the formation and elimination of dendritic spines, postsynaptic sites of excitatory synapses, in the developing mouse cortex during and after anesthetics exposure. Methods Transgenic mice expressing yellow fluorescence protein in layer 5 pyramidal neurons were used in this study. Mice at 1 month of age underwent ketamine-xylazine and isoflurane anesthesia over a period of hours. The elimination and formation rates of dendritic spines and filopodia, the precursors of spines, were followed over hours to days in the primary somatosensory cortex using transcranial two-photon microscopy. Four to five animals were examined under each experimental condition. Student t test and Mann-Whitney U test were used to analyze the data. Results Administration of either ketamine-xylazine or isoflurane rapidly altered dendritic filopodial dynamics but had no significant effects on spine dynamics. Ketamine-xylazine increased filopodial formation whereas isoflurane decreased filopodial elimination during 4 h of anesthesia. Both effects were transient and disappeared within a day after the animals woke up. Conclusion Studies suggest that exposure to anesthetics transiently affects the dynamics of dendritic filopodia but has no significant effect on dendritic spine development and plasticity in the cortex of 1-month-old mice.

AY-WB Phytoplasma Secretes a Protein That Targets Plant Cell Nuclei

The fully sequenced genome of aster yellows phytoplasma strain witches' broom (AY-WB; Candidatus Phytoplasma asteris) was mined for the presence of genes encoding secreted proteins based on the presence of N-terminal signal peptides (SP). We identified 56 secreted AY-WB proteins (SAP). These SAP are candidate effector proteins potentially involved in interaction with plant and insect cell components. One of these SAP, SAP11, contains an N-terminal SP sequence and a eukaryotic bipartite nuclear localization signal (NLS). Transcripts for SAP11 were detected in AY-WB-infected plants. Yellow fluorescence protein (YFP)-tagged SAP11 accumulated in Nicotiana benthamiana cell nuclei, whereas the nuclear targeting of YFP-tagged SAP11 mutants with disrupted NLS was inhibited. The nuclear transport of YFP-SAP11 was also inhibited in N. benthamiana plants in which the expression of importin α was knocked down using virus-induced gene silencing (VIGS). Furthermore, SAP11 was detected by immunocytology in nuclei of young sink tissues of China aster plants infected with AY-WB. In summary, this work shows that AY-WB phytoplasma produces a protein that targets the nuclei of plant host cells; this protein is a potential phytoplasma effector that may alter plant cell physiology.

In Vivo Permanent Marking of Cells with RAG1 Activation History Demonstrates That Lymphoid and Myeloid Derived Dendritic Cells Operate an Identical Developmental Program after Their Lineage Commitment.

Dendritic cells (DCs) have been shown to arise from both myeloid and lymphoid pathways, by evaluating the in vivo reconstituting potential of myeloid- and lymphoid-committed progenitor populations. However, evaluation of DC development after conventional adoptive transfer experiments may not correctly represent normal DC development including lineage contribution toward the formation of the DC pool. By crossing RAG1-Cre knockin with yellow fluorescence protein (YFP)-floxed reporter lines, we developed a mouse line in which cells with a history of RAG activation should be permanently marked with YFP as a result of lymphoid commitment. Lymphoid-derived DCs were successfully marked as YFP+ in vivo. We found that only ∼10% of conventional DCs (cDCs) and ∼20% of plasmacytoid DCs (pDCs) were YFP+, even in the thymus. This is a formal evidence that the majority of DCs in steady-state hematopoiesis originate from stages that have committed to the myeloid lineage. The lineage origin of DCs did not affect their functional abilities, including antigen-presentation and cytokine production. We then profiled the gene expression of the YFP+ and YFP- DCs at a genome wide level by DNA microarray analysis. Unexpectedly, the gene expression profiles of lymphoid and myeloid-derived DCs were virtually identical, irrespective of their organs. In contrast, the genetic programs were apparently different between splenic and thymic DCs, regardless of their lineage origin. Moreover, thymic DCs contained a number of more activated genes, such as MHC class II-related ones, as compared to splenic DCs. These results suggest that lymphoid and myeloid DCs might use a common developmental program that can be activated even after lymphoid or myeloid commitment. Thus, DCs are a unique cell population independent of other conventional lineage cells. Lineage-restricted DCs might be merged into an identical pool, distributed to various tissues, and finally fated by the microenvironment of their sites.