Comparison of Protein Expression Profiles of Novel <i>Halomonas smyrnensis</i>AAD6^Tand <i>Halomonas salina</i>DSMZ 5928^T

In this work, the protein pattern of novel Halomonas smyrnensis AAD6T was compared to that of Halomonas salina DSMZ5928T, which is the closest species on the basis of 16S rRNA sequence, to understand how AAD6T differs from type strains. Using high resolution NEPHEGE technique, the whole cell protein composition patterns of both Halomonas salina DSMZ5928T and H. smyrnensis AAD6T were mapped. The expressed proteins of the two microorganisms were mostly located at the acidic side of the gels, at molecular weight values of 60 to 17 kDa, and at isoelectric points 3.8 to 6.0, where they share a significant number of common protein spots. Identification and characterization of protein spots via whole genome sequencing data indicated that these two microorganisms used similar pathways, especially TCA cycle, for their survival;in other words, for their energy requirements. On the other hand, the protein expression differences in AAD6T and H. salina DSMZ 5928T showed that they prefer different metabolic pathways for lipid biosynthesis and in adaptation to extreme environments. Thus, we suggested that phylogenetic dissimilarities between these microorganisms could be related to the protein expression differences;in other words, metabolic flux differences in AAD6T and H. salina DSMZ 5928T. This is the first study to explain the dissimilarities of phenotypic characters and DNA-DNA hybridization between type strain and novel strain AAD6T by using protein expression differences.

Modified fibrin hydrogel for sustained delivery of RNAi lipopolyplexes in skeletal muscle

RNA interference is a promising therapeutical approach presently hindered by delivery concerns such as rapid RNA degradation and targeting of individual tissues.Injectable hydrogels are one potentially simple and direct route towards overcoming these barriers.Here we report on the utility of a combination of a mildly modified form of the clinically utilised fibrin hydrogel with Invivofectamine^(■)3.0,a lipid nonviral transfection vector,for local and sustained release.PEGylation of fibrin allowed for controlled release of small interfering RNA(siRNA)-lipopolyplexes for at least 10 days and greatly increased the stability of fibrin in vitro and in vivo.A 3D cell culture model and a release study showed transfection efficacy of siRNA-lipopolyplexes was retained for a minimum of 7 days.Injection in conjunction with PEGylated-fibrinogen significantly increased retention of siRNA-lipopolyplexes in mouse skeletal muscle and enhanced knockdown of myostatin mRNA that correlated with muscle growth.Thus,the increased efficacy observed here for the combination of a lipid nanoparticle,the only type of nonviral vector approved for the clinic,with fibrin,might allow for more rapid translation of injectable hydrogel-based RNA interference.

Requirement and Functional Redundancy of Ib Subgroup bHLH Proteins for Iron Deficiency Responses and Uptake in Arabidopsis thaliana

The Ib subgroup of the bHLH gene family in Arabidopsis contains four members （AtbHLH38, AtbHLH39, AtbHLHIO0, and AtbHLH101）. AtbHLH38 and AtbHLH39 were previously confirmed to interact with FER-like iron deft-ciency induced transcription factor （FIT）, directly functioning in activation of the expression of ferric-chelate reductase FRO2 and high-affinity ferrous iron transporter IRT1. In this work, we characterized the functions of AtbHLH100 and AtbHLH101 in the regulation of the iron-deficiency responses and uptake. Yeast two-hybrid analysis and bimolecular fluorescence complementation assay demonstrated that both AtbHLH100 and AtbHLH101 could interact with FIT. Dual expression of either AtbHLH100 or AtbHLH101 with FIT in yeast cells activated the GUS expression driven by promoters of FRO2 and IRT1. The plants overexpressing FIT together with AtbHLHI01 showed constitutive expression of FRO2 and IRT1 in roots, and accumulated more iron in shoots. Further, the single, double, and triple knockout mutants of AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101 were generated and characterized. The FRO2 and IRT1 expression in roots and the iron content in shoots were more drastically decreased in the triple knockout mutant of AtbHLH39, AtbHLH100, and AtbHLH101 than that of the other available double and triple mutants of the four genes. Comparison of the physiological responses as well as the expression of FRO2 and IRT1 in the multiple knockout mutants under iron deficiency revealed that AtbHLH100, AtbHLH38, AtbHLH101, and AtbHLH39 played the gradually increased important role in the iron-deficiency responses and uptake. Taken all together, we conclude that the four Ib subgroup bHLH proteins are required and possess redundant functions with differential significance for activation of iron-deficiency responses and uptake in Arabidopsis.

AtPRK2 Promotes ROP1 Activation via RopGEFs in the Control of Polarized Pollen Tube Growth

The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho guanine nucleotide exchange factors （RopGEFs） and in turn may be activated by an unknown factor through releasing RopGEFI＇s auto-inhibition. In this study, we found that RopGEF1 forms a complex with ROP1 and AtPRK2, a receptor-like protein kinase previously shown to interact with RopGEFs. AtPRK2 phosphorylated RopGEF1 in vitro and the atprkl,2,5 tri- ple mutant showed defective pollen tube growth, similar to the phenotype of the ropgef1,9,12,14 quadruple mutant. Overexpression of a dominant negative form of AtPRK2 （DN-PRK2） inhibited pollen germination in Arabidopsis and reduced pollen elongation in tobacco. The DN-PRK2-induced pollen germination defect was rescued by overexpressing a constitutively active form of RopGEF1, RopGEF1（90-457）, implying that RopGEF1 acts downstream of AtPRK2. Moreover, AtPRK2 increased ROP1 activity at the apical plasma membrane whereas DN-PRK2 reduced ROP1 activity. Finally, two mutations at the C-terminal putative phosphorylation sites of RopGEF1 （RopGEF1S460A and RopGEF1S480A） eliminated the function of RopGEF1 in vivo. Taken together, our results support the hypothesis that AtPRK2 acts as a positive regula- tor of the ROP1 signaling pathway most likely by activating RopGEF1 through phosphorylation.

Identification and Preliminary Characterization of a New Chemical Affecting Glucosyltransferase Activities Involved in Plant Cell Wall Biosynthesis

Chemical genetics as a part of chemical genomics is a powerful and fast developing approach to dissect biological processes that may be difficult to characterize using conventional genetics because of gene redundancy or lethality and, in the case of polysaccharide biosynthesis, plant flexibility. Polysaccharide synthetic enzymes are located in two main compartments--the Golgi apparatus and plasma membrane-and can be studied in vitro using membrane fractions. Here, we first developed a high-throughput assay that allowed the screening of a library of chemicals with a potential effect on glycosyltransferase activities. Out of the 4800 chemicals screened for their effect on Golgi glucosyltransferases, 66 compounds from the primary screen had an effect on carbohydrate biosynthesis. Ten of these compounds were confirmed to inhibit glucose incorporation after a second screen. One compound exhibiting a strong inhibition effect （ID 6240780 named chemical A） was selected and further studied. It reversibly inhibits the transfer of glucose from UDP-glucose by Golgi membranes, but activates the plasma membrane-bound callose synthase. The inhibition effect is dependent on the chemical structure of the compound, which does not affect endomembrane morphology of the plant cells, but causes changes in cell wall composition. Chemical A represents a novel drug with a great potential for the study of the mechanisms of Golgi and plasma membrane-bound glucosyltransferases.

Two-Step Processing of AtFtsH4 Precursor by Mitochondrial Processing Peptidase in Arabidopsis thaliana

Dear Editor, Most mitochondrial proteins are encoded by the nuclear genome and posttranslationally imported to mitochondria. The correct targeting is often ensured by an N-terminal exten- sion designated presequence. Upon import, the presequence is cleaved off in a reaction termed processing. Most of our knowledge regarding mitochondrial processing comes from analyses performed in Saccharomyces cerevisiae. In yeast, the mitochondrial processing peptidase （MPP） is responsible for processing of the majority of mitochondrial precursors, with a fraction of them undergoing a second step of processing, car- ried out by either metalloproteases （Octl/MIP, Icp55） or serine proteases （Imp, Pcpl） （reviewed in Teixeira and Glaser, 2012）. Unusual two-step processing in which both processing steps are carried out by MPP has been reported for yeast and human frataxin （Branda et al., 1999） and for Neurospora crassa mito- chondrial ATP synthase subunit 9 （Schmidt et al., 1984）.

Inflammasome activation under high cholesterol load triggers a protective microglial phenotype while promoting neuronal pyroptosis

Background Persistent inflammatory response in the brain can lead to tissue damage and neurodegeneration.In Alzheimer’s disease(AD),there is an aberrant activation of inflammasomes,molecular platforms that drive inflammation through caspase-1-mediated proteolytic cleavage of proinflammatory cytokines and gasdermin D(GSDMD),the executor of pyroptosis.However,the mechanisms underlying the sustained activation of inflammasomes in AD are largely unknown.We have previously shown that high brain cholesterol levels promote amyloid-β(Aβ)accumulation and oxidative stress.Here,we investigate whether these cholesterol-mediated changes may regulate the inflam-masome pathway.Methods SIM-A9 microglia and SH-SY5Y neuroblastoma cells were cholesterol-enriched using a water-soluble cholesterol complex.After exposure to lipopolysaccharide(LPS)plus muramyl dipeptide or Aβ,activation of the inflammasome pathway was analyzed by immunofluorescence,ELISA and immunoblotting analysis.Fluorescently-labeled Aβwas employed to monitor changes in microglia phagocytosis.Conditioned medium was used to study how microglia-neuron interrelationship modulates the inflammasome-mediated response.Results In activated microglia,cholesterol enrichment promoted the release of encapsulated IL-1βaccompanied by a switch to a more neuroprotective phenotype,with increased phagocytic capacity and release of neurotrophic factors.In contrast,in SH-SY5Y cells,high cholesterol levels stimulated inflammasome assembly triggered by both bacterial toxins and Aβpeptides,resulting in GSDMD-mediated pyroptosis.Glutathione(GSH)ethyl ester treatment,which recovered the cholesterol-mediated depletion of mitochondrial GSH levels,significantly reduced the Aβ-induced oxidative stress in the neuronal cells,resulting in lower inflammasome activation and cell death.Furthermore,using conditioned media,we showed that neuronal pyroptosis affects the function of the cholesterol-enriched microglia,lowering its phagocytic activity and,therefore,the ability to degrade extracellular Aβ.Conclusions Changes in intracellular cholesterol levels differentially regulate the inflammasome-mediated immune response in microglia and neuronal cells.Given the microglia-neuron cross-talk in the brain,cholesterol modulation should be considered a potential therapeutic target for AD treatment,which may help to block the aberrant and chronic inflammation observed during the disease progression.

A Plant Immune Receptor Degraded by Selective Autophagy

Plants recycle non-activated immune receptors to maintain a functional immune system.The Arabidopsis immune receptor kinase FLAGELLIN-SENSING 2 (FLS2)recognizes bacterial flagellin.However,the molecular mechanisms by which non-activated FLS2 and other non-activated plant PRRs are recycled remain not well understood.Here,we provide evidence showing thatArabidopsis orosomucoid (ORM)proteins,which have been known to be negative regulators of sphingoUpid biosynthesis,act as selective autophagy receptors to mediate the degradation of FLS2.Arabidopsis plants overexpressing ORM 1 or ORM2 have undetectable or greatly diminished FLS2 accumulation,nearly lack FLS2 signaling,and are more susceptible to the bacterial pathogen Pseudomonas syringae.On the other hand,ORM1/2 RNAi plants and orml or orrn2 mutants generated by the CRISPR/Cas9-mediated gene editing have increased FLS2 accumulation and enhanced FLS2 signaling,and are more resistant to P.syringae.ORM proteins interact with FLS2 and the autophagy-related protein ATG8.Interestingly,overexpression of ORM1 or ORM2 in autophagydefective mutants showed FLS2 abundance that is comparable to that in wild-type plants.Moreover, FLS2 levels were not decreased in Arabidopsis plants overexpressing ORM1/2 derivatives that do not interact with ATGS.Taken together,these results suggest that selective autophagy functions in maintaining the homeostasis of a plant immune receptor and that beyond sphingolipid metabolic regulation ORM proteins can also act as selective autophagy receptors.

Arabidopsis thaliana AtUTr7 Encodes a Golgi-Localized UDP-Glucose/UDP-Galactose Transporter that Affects Lateral Root Emergence

Nucleotide sugar transporters （NSTs） are antiporters comprising a gene family that plays a fundamental role in the biosynthesis of complex cell wall polysaccharides and glycoproteins in plants. However, due to the limited number of related mutants that have observable phenotypes, the biological function（s） of most NSTs in cell wall biosynthesis and assembly have remained elusive. Here, we report the characterization of AtUTr7 from Arabidopsis （Arabidopsis thaliana （L.） Heynh.）, which is homologous to multi-specific UDP-sugar transporters from Drosophila melanogaster, humans, and Caenorhabditis elegans. We show that AtUTr7 possesses the common structural characteristics conserved among NSTs. Using a green fluorescent protein （GFP） tagged version, we demonstrate that AtUTr7 is localized in the Golgi apparatus. We also show that AtUTr7 is widely expressed, especially in the roots and in specific floral organs. Additionally, the results of an in vitro nucleotide sugar transport assay carried out with a tobacco and a yeast expression system suggest that AtUTr7 is capable of transferring UDP-Gal and UDP-GIc, but not a range of other UDP- and GDP-sugars, into the Golgi lumen. Mutants lacking expression of AtUTr7 exhibited an early proliferation of lateral roots as well as distorted root hairs when cultivated at high sucrose concentrations. Furthermore, the distribution of homogalacturonan with a low degree of methyl esterification differed in lateral root tips of the mutant compared to wild-type plants, although additional analytical procedures revealed no further differences in the composition of the root cell walls. This evidence suggests that the transport of UDP-Gal and UDP-GIc into the Golgi under conditions of high root biomass production plays a role in lateral root and root hair development.