UDP‐Api/UDP‐Xyl synthases affect plant development by controlling the content of UDP‐Api to regulate the RG‐II‐borate complex

Rhamnogalacturonan‐II (RG‐II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG‐II molecules can form an RG‐II‐borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross‐linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG‐II dimer is still unclear. In this study we investigated the two homologous UDP‐D‐apiose/UDP‐D‐xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP‐D‐apiose (UDP‐Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP‐Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG‐II‐borate complex than wild‐type Col‐0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP‐Api and RG‐II‐borate complex formation in plant growth and development.     

Auxin-mediated induction of GAL promoters by conditional degradation of Mig1p improves sesquiterpene production in Saccharomyces cerevisiae with engineered acetyl-CoA synthesis

The yeast Saccharomyces cerevisiae uses the pyruvate dehydrogenase-bypass for acetyl-CoA biosynthesis. This relatively inefficient pathway limits production potential for acetyl-CoA-derived biochemical due to carbon loss and the cost of two high-energy phosphate bonds per molecule of acetyl-CoA. Here, we attempted to improve acetyl-CoA production efficiency by introducing heterologous acetylating aldehyde dehydrogenase and phosphoketolase pathways for acetyl-CoA synthesis to enhance production of the sesquiterpene trans-nerolidol. In addition, we introduced auxin-mediated degradation of the glucose-dependent repressor Mig1p to allow induced expression of GAL promoters on glucose so that production potential on glucose could be examined. The novel genes that we used to reconstruct the heterologous acetyl-CoA pathways did not sufficiently complement the loss of endogenous acetyl-CoA pathways, indicating that superior heterologous enzymes are necessary to establish fully functional synthetic acetyl-CoA pathways and properly explore their potential for nerolidol synthesis. Notwithstanding this, nerolidol production was improved twofold to a titre of ˜ 900 mg l⁻¹ in flask cultivation using a combination of heterologous acetyl-CoA pathways and Mig1p degradation. Conditional Mig1p depletion is presented as a valuable strategy to improve the productivities in the strains engineered with GAL promoters-controlled pathways when growing on glucose.     

Clinical-scale generation of multi-specific anti-fungal T cells targeting Candida,Aspergillus and mucormycetes

Background aimsInvasive fungal infections, in particular, infections caused by Candida, Aspergillus and mucormycetes, are a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation. Adoptive transfer of donor-derived anti-fungal T cells shows promise to restore immunity and to offer a cure. Because T cells recognize only specific epitopes, the low rate of patients in which the causal fungal pathogen can be identified and the considerable number of patients with co-infection with several genera or species of fungi significantly limit the application of adoptive immunotherapy.MethodsUsing the interferon-γ secretion assay, we isolated multi-specific human anti-fungal T cells after simultaneous stimulation with cellular extracts of Aspergillus fumigatus, Candida albicans and Rhizopus oryzae. Cells were phenotypically and functionally characterized by flow cytometry.ResultsOf a total of 1.1 × 10⁹ peripheral blood mononuclear cells, a median number of 5.2 × 10⁷ CD3⁺CD4⁺ T cells was generated within 12 days. This cell population consisted of activated memory T_H1 cells and reproducibly responded to a multitude of Aspergillus spp., Candida spp. and mucormycetes with interferon-γ production. On re-stimulation, the generated T cells proliferated and enhanced anti-fungal activity of phagocytes and showed reduced alloreactivity compared with the original cell fraction.ConclusionsOur rapid and simple method of simultaneously generating functionally active multi-specific T cells that recognize a wide variety of medically relevant fungi may form the basis for future clinical trials investigating adoptive immunotherapy in allogeneic hematopoietic stem cell transplantation recipients with invasive fungal infection.     

Impact of Morphometry,Myelinization and Synaptic Current Strength on Spike Conduction in Human and Cat Spiral Ganglion Neurons

Background

Our knowledge about the neural code in the auditory nerve is based to a large extent on experiments on cats. Several anatomical differences between auditory neurons in human and cat are expected to lead to functional differences in speed and safety of spike conduction.

Methodology/Principal Findings

Confocal microscopy was used to systematically evaluate peripheral and central process diameters, commonness of myelination and morphology of spiral ganglion neurons (SGNs) along the cochlea of three human and three cats. Based on these morphometric data, model analysis reveales that spike conduction in SGNs is characterized by four phases: a postsynaptic delay, constant velocity in the peripheral process, a presomatic delay and constant velocity in the central process. The majority of SGNs are type I, connecting the inner hair cells with the brainstem. In contrast to those of humans, type I neurons of the cat are entirely myelinated. Biophysical model evaluation showed delayed and weak spikes in the human soma region as a consequence of a lack of myelin. The simulated spike conduction times are in accordance with normal interwave latencies from auditory brainstem response recordings from man and cat. Simulated 400 pA postsynaptic currents from inner hair cell ribbon synapses were 15 times above threshold. They enforced quick and synchronous spiking. Both of these properties were not present in type II cells as they receive fewer and much weaker (^∼26 pA) synaptic stimuli.

Conclusions/Significance

Wasting synaptic energy boosts spike initiation, which guarantees the rapid transmission of temporal fine structure of auditory signals. However, a lack of myelin in the soma regions of human type I neurons causes a large delay in spike conduction in comparison with cat neurons. The absent myelin, in combination with a longer peripheral process, causes quantitative differences of temporal parameters in the electrically stimulated human cochlea compared to the cat cochlea.     

Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).     

Plant secretome — From cellular process to biological activity

Recent studies suggest that plants secrete a large number of proteins and peptides into the extracellular space. Secreted proteins play a crucial role in stress response, communication and development of organisms. Here we review the current knowledge of the secretome of more than ten plant species, studied in natural conditions or during (a)biotic stress. This review not only deals with the classical secretory route via endoplasmic reticulum and Golgi followed by proteins containing a known N-terminal signal peptide, but also covers new findings about unconventional secretion of leaderless proteins. We describe alternative secretion pathways and the involved compartments like the recently discovered EXPO. The well characterized secreted peptides that function as ligands of receptor proteins exemplify the biological significance and activity of the secretome. This article is part of a Special Issue entitled: An Updated Secretome.