A comparative study of bacterial diversity based on culturable and culture-independent techniques in the rhizosphere of maize (Zea mays L.)

ObjectiveMaize is an important crop for fodder, food and feed industry. The present study explores the plant-microbe interactions as alternative eco-friendly sustainable strategies to enhance the crop yield.MethodologyBacterial diversity was studied in the rhizosphere of maize by culture-dependent and culture-independent techniques by soil sampling, extraction of DNA, amplification of gene of interest, cloning of desired fragment and library construction.ResultsCulturable bacteria were identified as Achromobacter, Agrobacterium, Azospirillum, Bacillus, Brevibacillus, Bosea, Enterobacter, Microbacterium, Pseudomonas, Rhodococcus, Stenotrophomonas and Xanthomonas genera. For culture-independent approach, clone library of 16S ribosomal RNA gene was assembled and 100 randomly selected clones were sequenced. Majority of the sequences were related to Firmicutes (17%), Acidobacteria (16%), Actinobacteria (17%), Alpha-Proteobacteria (7%), Delta-proteobacteria (4.2%) and Gemmatimonadetes (4.2%) However, some of the sequences (30%) were novel that showed no homologies to phyla of cultured bacteria in the database. Diversity of diazotrophic bacteria in the rhizosphere investigated by analysis of PCR-amplified nifH gene sequence that revealed abundance of sequences belonging to genera Azoarcus (25%), Aeromonas (10%), Pseudomonas (10%). The diazotrophic genera Azotobacter, Agrobacterium and Zoogloea related nifH sequences were also detected but no sequence related to Azospirillum was found showing biasness of the growth medium rather than relative abundance of diazotrophs in the rhizosphere.ConclusionThe study provides a foundation for future research on focussed isolation of the Azoarcus and other diazotrophs found in higher abundance in the rhizosphere.     

Bioethanol production with carboxymethylcellulase of Pseudomonas poae using castor bean (Ricinus communis L.) cake

Increased consumption of fossil fuels is an emerging problem. Scientists look for the existence of other alternatives to fossil fuels, including so-called renewable energy. Accordingly, we report the production of bio-ethanol from the remnants of castor oil bean seed cake (CBC) by the carboxymethylcellulase enzyme (CMCase). A bacterial strain isolated from rice straw showing higher CMCase activity was identified. The 16S rRNA result showed a 93% homology with the 16SrRNA gene sequences of Pseudomonas poae RE11-1-14, the strain was identified as Pseudomonas poae AB3. In addition, our results showed that the highest enzyme activity was achieved after 48 h and inoculum size of 3.7 × 10⁵ CFU. The optimum temperature, pH and Carboxymethylcellulose (CMC) concentration for the highest enzyme activity was 25 °C, pH 7 and 10 g/l respectively. Furthermore, The CMCase was purified by ammonium sulphate at a concentration of 60%. The SDS-PAGE of the purified enzyme showed a molecular weight of 88 kDa. Additionally, the (CBC) was hydrolyzed by the purified CMCase at the enzyme optimum conditions. The results showed the liberation of 5.2 g/L of reducing sugar by using dinitrosalicylic acid (DNS) assay. Finally, the total sugar produces 35 g/L after 48 h when Saccharomyces cerevisiae was used as a fermentation agent. Hence for the first time, we have been successfully able to produce bioethanol from CBC with CMCase of Pseudomonas poae.     

Pseudomonas putida modulates the expression of miRNAs and their target genes in response to drought and salt stresses in chickpea (Cicer arietinum L.)

MicroRNAs are small non-coding regulatory RNA molecules that play an important role in the modulation of gene expression during various environmental stresses. Pseudomonas putida RA, a plant growth promoting rhizobacteria (PGPR) colonizes the root surface of plants improving their growth and development during abiotic stresses modulating the expression of stress-responsive genes; however, the impact of RA on stress responsive-miRNA remains elusive. The present study was an attempt to delineate the role of PGPR in modulating stress responsive-miRNAs in a tolerant desi chickpea genotype exposed to drought and salt stresses. The existence of variable expression patterns of individual miRNAs and their target genes under these stresses at different time points indicate a distinct miRNA-mediated perception and response mechanisms operating under these stresses in the presence or absence of RA in chickpea.     

RNA sequencing analysis of salt tolerance in soybean (Glycine max)

Salt stress causes foliar chlorosis and scorch, plant stunting, and eventually yield reduction in soybean. There are differential responses, namely tolerance (excluder) and intolerance (includer), among soybean germplasm. However, the genetic and physiological mechanisms for salt tolerance is complex and not clear yet. Based on the results from the screening of the RA-452 x Osage mapping population, two F_4:6 lines with extreme responses, most tolerant and most sensitive, were selected for a time-course gene expression study in which the 250 mM NaCl treatment was initially imposed at the V1 stage and continued for 24 h (hrs). Total RNA was isolated from the leaves harvested at 0, 6, 12, 24 h after the initiation of salt treatment, respectively. The RNA-Seq analysis was conducted to compare the salt tolerant genotype with salt sensitive genotype at each time point using RNA-Seq pipeline method. A total of 2374, 998, 1746, and 630 differentially expressed genes (DEGs) between salt-tolerant line and salt-sensitive line, were found at 0, 6, 12, and 24 h, respectively. The expression patterns of 154 common DEGs among all the time points were investigated, of which, six common DEGs were upregulated and seven common DEGs were downregulated in salt-tolerant line. Moreover, 13 common DEGs were dramatically expressed at all the time points. Based on Log2 (fold change) of expression level of salt-tolerant line to salt-sensitive line and gene annotation, Glyma.02G228100, Glyma.03G226000, Glyma.03G031000, Glyma.03G031400, Glyma.04G180300, Glyma.04G180400, Glyma.05 g204600, Glyma.08G189600, Glyma.13G042200, and Glyma.17G173200, were considered to be the key potential genes involving in the salt-tolerance mechanism in the soybean salt-tolerant line.     

Effect of synthetic surfactants and soapwort (Saponaria officinalis L.) extract on skin-mimetic model lipid monolayers

The effect of a saponin-rich extract from rhizomes of Soapwort (Saponaria officinalis L) and four synthetic surfactants: sodium lauryl sulphate (SLS), sodium laureth sulphate (SLES), ammonium lauryl sulphate (ALS) and cocamidopropyl betaine (CAPB) on two model lipid monolayers is analyzed using surface pressure, surface dilatational rheology and fluorescence microscopy. The following monolayers were employed: dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3 (DPPC/CHOL) and Ceramide [AP]/stearic acid/cholesterol in a molar ratio of 14:14:10 (CER/SA/CHOL). They mimicked a general bilayer structure and an intercellular lipid mixture, respectively. Both lipid mixtures on Milli-Q water were first compressed to the initial surface pressure, Π₀ = 30 mN/m and then the subphase was exchanged with the respective (bio)surfactant solution at 1% (w/w). All four synthetic surfactants behaved in a similar way: they increased surface pressure to about 40 mN/m and reduced the storage modulus of surface dilational surface rheology, E′, to the values close to zero. The corresponding fluorescence microscopy pictures confirmed that the lipids mimicking the stratum corneum components were almost completely removed by the synthetic surfactants under the present experimental conditions. The components of the Soapwort extract (SAP) increased surface pressure to significantly higher values than the synthetic surfactants, but even more spectacular increase was observed for the storage modulus of the SAP-penetrated lipid monolayers (up to E′= 715 mN/m).     

Reproductive biology and morphology of Apis mellifera jemenitica (Apidae) queens and drones

The current study aimed to investigate the important reproductive biology and morphology of A.m. jemenitica queens and drones through measuring the weight of virgin and mated queens, size and weight of spermathecae, weight of ovaries, number of ovarioles, quantity and viability of semen in queen and drones. Accordingly, the average weights of 0.139 ± 0.01 g and 0.143 ± 0.013 g recorded for virgin and mated queens respectively. The sizes of spermathecae were 1.248 ± 0.103 mm and 1.25 ± 0.022 mm for virgin and mated queens respectively. The mean weight of ovaries was 0.013 ± 0.003 g and the numbers of ovarioles varied from 124 to 163 with the mean of 142.9 ± 9.47 and with no significant difference between virgin and mated queens. The average number of stored sperm per spermathecae of mated queen was estimated to be 4.202 ± 0.613 million with the viability of 80.39%. The average number of sperm per drone recorded was 8,763,950 ± 1,633,203.15 with viability of 79.54 ± 6.70%. In general, the current study revealed that the values recorded for reproductive biology and morphological characters of A. m. jemenitica queens and drones were relatively lower than values recorded for other Apis mellifera races. This mainly could be associated with the body size of the race which is known to be the smallest race among A. mellifera races. Moreover, the harsh environmental conditions of the regions, high temperature, low humidity and limited resources may have contributed for the smaller biological and morphological values. The information will serve as a base in future selection and breeding of program of the race.     

Occurrence model of first instars of Ricania shantungensis (Hemiptera: Ricaniidae)

An invasive planthopper, Ricania shantungensis, is an important pest in agriculture and forestry in Korea. Best target stage for insecticide application is known to be newly hatched first instar. Thus, the objective of the present study was to predict the occurrence of first instars of R. shantungensis. Effects of temperature on development and survival of R. shantungensis eggs were examined at seven constant temperatures (12.4, 16.4, 20.4, 24.8, 28.3, 32.4, and 36.9 °C). Development and survival of R. shantungensis eggs were quantitatively described by applying empirical models as a function of temperature over a wide thermal range. Lower developmental threshold, thermal constant, optimal developmental temperature, and upper developmental threshold were estimated to be 12.1 °C, 202 DD, 31 °C, and 36.9 °C, respectively. Survivorship was the highest at 23.3 °C. The models well predicted timing of field occurrences at three sites (Buyeo, Gwangyang, and Habcheon) in Korea. Therefore, results of this study would increase the prediction accuracy of R. shantungensis occurrence and management efficiency of R. shantungensis.     

Microbial production of cyanophycin: From enzymes to biopolymers

Cyanophycin is an attractive biopolymer with chemical and material properties that are suitable for industrial applications in the fields of food, medicine, cosmetics, nutrition, and agriculture. For efficient production of cyanophycin, considerable efforts have been exerted to characterize cyanophycin synthetases (CphAs) and optimize fermentations and downstream processes. In this paper, we review the characteristics of diverse CphAs from cyanobacteria and non-cyanobacteria. Furthermore, strategies for cyanophycin production in microbial strains, including Escherichia coli, Pseudomonas putida, Ralstonia eutropha, Rhizopus oryzae, and Saccharomyces cerevisiae, heterologously expressing different cphA genes are reviewed. Additionally, chemical and material properties of cyanophycin and its derivatives produced through biological or chemical modifications are reviewed in the context of their industrial applications. Finally, future perspectives on microbial production of cyanophycin are provided to improve its cost-effectiveness.     

Ras-related proteins (Rab) are key proteins related to male fertility following a unique activation mechanism

Ras-related protein Rab (Rab) proteins, member of Ras superfamily of monomeric G proteins, are well known key regulators of intracellular vesicular transport. Recently, it has been reported that Rab 2A and 3A are related to acrosomal exocytosis in spermatozoa and Rab 2A can be used to fertility-related biomarker in male. However, the role and mechanism of Rab proteins in spermatozoa has not been fully understood yet. Therefore, the study to analyze the expression and location of various Rab proteins in spermatozoa is required to understand the role and mechanism of Rab proteins in spermatozoa. In present study, to analyze the expression level and location of various Rab proteins (Rab 2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, and Rab34) and Rab protein regulators (RabGAP, RabGDI, RabGEF) in spermatozoa following capacitation, immunofluorescence and western blot analysis were performed. All of 11 Rab proteins were expressed in acrosomal region and tail of spermatozoa. Furthermore, all Rab proteins and Rab protein regulators, except RabGAP, have decreased expression patterns after capacitation. Taken together, Rab proteins were located in sperm head and tail. In addition, expression patterns of Rab proteins in spermatozoa were altered following capacitation. Therefore, our results suggested that Rab proteins may be key proteins related with capacitation as well as playing important role with uniquely activation pathway for male fertility.     

Olfactory responses of adult Potato Tuber Moth,Phthorimaea operculella (Zeller) measured by attraction relative to the tomato leaf volatiles

The potato tuber moth (PTM) is an oligophagous herbivore and a severe pest of solanaceous crops in many countries of the world. Previously, we reported host expansion and damage potential of PTM on tomato, a congeneric crop of potato. Here we tested adult olfactory behaviour of PTM to leaves of five different cultivated tomato varieties including Moneymaker, Campari, Ailsa craig, LA3475 and E6203, and one wild species, Solanum pimpinellifolium. Tomato leaf hydro-distilled oils of Moneymaker, Campari, Ailsa craig, S. pimpinellifolium and E6203 showed strong attractiveness and LA3475 exhibited repulsiveness for adult PTM of both sexes in two-armed bioassays. Volatiles of Moneymaker, Campari, Ailsa Craig, S. pimpinellifolium and E6203 showed attractiveness for mated adult PTM of both sexes (> 70%). The extracted oil of all tomato leaves contains monoterpenes, sesquiterpenes, diterpenes and C₆ alcohols as well as other components. Phytol is one of the common and major compounds in all tomato varieties. Phytol showed weak attractiveness: 60.8–63.6% and 57.6–60.6% for male and female PTM, respectively when tested at concentrations of 0.1–10 mg/mL. Therefore, the attractiveness of tomato leaf volatiles might be due to synergistic effects of plant volatile mixtures. Identification PTM attractant volatile in tomato leaf is important in its control in environmentally friendly manner.     

Bifidobacterium primatium sp. nov., Bifidobacterium scaligerum sp. nov., Bifidobacterium felsineum sp. nov. and Bifidobacterium simiarum sp. nov.: Four novel taxa isolated from the faeces of the cotton top tamarin (Saguinus oedipus) and the emperor tamarin (Saguinus imperator)

Four novel Gram-stain-positive, non spore forming and fructose-6-phosphate phosphoketolase-positive strains were isolated from the faeces of a cotton top tamarin (Saguinus oedipus) and an emperor tamarin (Saguinus imperator). Phylogenetic analyses based on 16S rRNA revealed that bifidobacterial strains TRE 1^T exhibit close phylogenetic relatedness to Bifidobacterium catulorum DSM 103154 (96.0%) and Bifidobacterium tissieri DSM 100201 (96.0%); TRE D^T and TRE H^T were closely related to Bifidobacterium longum subsp. longum ATCC 15708^T with similarity values of 97.4% and 97.5%, respectively; TRI 7^T was closely related to Bifidobacterium tissieri DSM 100201 (96.0%). The Average Nucleotide Identity (ANI) and in silico DDH (isDDH) analysis with closest neighbour supported an independent phylogenetic position of all strains with values ranged from 74 to 85% for ANI and from 24 to 28% for isDDH. DNA base composition of the four strains was in the range of 58.3–63.5 mol% G + C. Based on the phylogenetic, genotypic and phenotypic data, the strains TRE 1^T, TRE D^T, TRE H^T and TRI 7^T clearly represent four novel taxa within the genus Bifidobacterium for which the names Bifidobacterium primatium sp. nov. (type strain TRE 1^T = DSM 100687^T = JCM 30945^T), Bifidobacterium scaligerum sp. nov. (type strain TRE D^T = DSM 103140^T = JCM 31792^T), Bifidobacterium felsineum sp. nov. (type strain TRE H^T = DSM 103139^T = JCM 31789^T) and Bifidobacterium simiarum sp. nov. (type strain TRI 7^T = DSM 103153^T = JCM 31793) are proposed.     

Development of single nucleotide polymorphism markers specific to Apis mellifera (Hymenoptera: Apidae) line displaying high hygienic behavior against Varroa destructor,an ectoparasitic mite

To control Varroa destructor, an ectoparasitic mite, a honey bee line possessing high hygienic behavior (HHB) against this mite has been bred in South Korea. However, a method that can diagnose and assess the HHB line from control (the low hygienic behavior, LHB) line has not been reported yet. Thus, the objective of this study was to develop single nucleotide polymorphism (SNP) markers through whole-genome sequencing of worker bees from HHB line of A. mellifera caucasica and LHB line of A. m. carnica (Hymenoptera: Apidae). A total of 319,445,977 sequence reads were mapped to the known A. mellifera reference genome (average coverage of 87.46%). In 2,316,128 and 3,266,756 SNPs from HHB and LHB line, respectively, 20 SNPs that showed homozygosity in each line were selected and eight SNPs were used to diagnose the HHB line either by typical PCR-restriction fragment length polymorphism or allele-specific PCR. Six of remaining SNPs were of different sizes, enabling relatively easy differentiation of these two honey bee lines on typical agarose gel. Another remaining six SNPs had different sequences, including SNP sites. These SNP markers can be used to diagnose and assess V. destructor-specific HHB line of honey bees.     

Phylogenetic analyses of Bradyrhizobium symbionts associated with invasive Crotalaria zanzibarica and its coexisting legumes in Taiwan

In this study, the genetic diversity and identification of Bradyrhizobium symbionts of Crotalaria zanzibarica, the most widely-distributed invasive legume in Taiwan, and other sympatric legume species growing along riverbanks of Taiwan were evaluated for the first time. In total, 59 and 54 Bradyrhizobium isolates were obtained from C. zanzibarica and its coexisting legume species, respectively. Based on the multilocus sequence analysis (MLSA) of concatenated four housekeeping genes (dnaK-glnII-recA-rpoB gene sequences, 1901 bp), the 113 isolates displayed 53 unique haplotypes and grouped into 21 clades. Of these clades, 11 were found to be congruent to already defined Bradyrhizobium species, while the other 10 clades were found to not be congruent to any defined species. In particular, the C. zanzibarica isolates belong to 14 MLSA clades, six of which overlapped with the isolates of coexisting legumes. According to the nodA gene sequences (555 bp) obtained from the 105 isolates, these isolates were classified into three known nodA clades, III.2, III.3 and VII and were further clustered into 10 groups. Furthermore, the C. zanzibarica isolates were clustered into 8 nodA groups, five of which overlapped with the isolates from coexisting legumes. Additionally, the nodA genes of the isolates from native species were dominated by Asian origin, while those from C. zanzibarica were dominated by American origin. In conclusion, C. zanzibarica is a promiscuous host capable of recruiting diverse Bradyrhizobium symbionts, some of which are phylogenetically similar to the symbionts of coexisting legumes in Taiwan.     

2-(Bipiperidin-1-yl)-5-(nitroaryl)-1,3,4-thiadiazoles: Synthesis,evaluation of in vitro leishmanicidal activity,and mechanism of action

The development of novel leishmanicidal agents that are capable of being replaced by the available therapeutic options has become a priority. In the present study, the synthesis and leishmanicidal activity of a series of 5-(nitroheteroaryl-2-yl)-1,3,4-thiadiazole derivatives are described. All compounds appeared to be potent anti-leishmanial agents against both promastigote and amastigote forms of Leishmania major (L. major). Amongst the synthesized compounds, 2-([1,4′-bipiperidin]-1′-yl)-5-(5-nitrofuran-2-yl)-1,3,4-thiadiazole (IIa) and 1-(5-(1-methyl-5-nitro-1H-imidazole-2-yl)-1,3,4-thiadiazol-2-yl)-4-(piperidine-1-yl) piperidine (IIc) are the most effective. Infection index was statistically declined in the presence of all compounds. The analysis of redox-related factors revealed that exposure of L. major cells to IIa and IIc led to an increase in reactive oxygen species (ROS). Furthermore, two compounds were able to increase ROS and NO levels in infected macrophages in a dose-independent manner. In addition, we showed that these compounds induced cell death in promastigotes. Altogether, our results indicated the anti-leishmanial potential of IIa and IIc is mediated by apoptosis through an imbalance in the redox system resulting in the elevation of ROS. This new class of compound seems to hold great promise for the development of new and useful anti-leishmanial agents.     

Self-assembly of trimethyl chitosan and poly(anionic amino acid)-peptide antigen conjugate to produce a potent self-adjuvanting nanovaccine delivery system

Short peptides derived from virulent pathogen proteins are promising antigens for the development of vaccines against infectious diseases. However, in order to mimic the danger signals associated with natural infection and stimulate an adaptive immune response, peptide antigens must be co-delivered with immune adjuvants. In this study, a group A streptococcus (GAS) M-protein derived B-cell epitope: J8, and universal T-helper epitope P25 containing peptides, were chemically coupled with different anionic amino acid-based polymers. The poly(anionic amino acid)-peptide antigen conjugates were mixed with trimethyl chitosan (TMC) to produce self-adjuvanting nanoparticulate vaccine candidates. TMC from two different sources were used to analyse their effect on immunogenicity. The nanoparticles produced from a peptide modified with 10 residues of polyglutamic acid and fungal TMC (NP5) stimulated production of the highest levels of serum antibodies in outbred mice. These antibodies were opsonic against all clinical GAS isolates tested.     

Identification of the amino acids in the Major Histocompatibility Complex class II region of Scottish Blackface sheep that are associated with resistance to nematode infection

Lambs with the Major Histocompatibility Complex DRB1*1101 allele have been shown to produce fewer nematode eggs following natural and deliberate infection. These sheep also possess fewer adult Teladorsagia circumcincta than sheep with alternative alleles at the DRB1 locus. However, it is unclear if this allele is responsible for the reduced egg counts or merely acts as a marker for a linked gene. This study defined the MHC haplotypes in a population of naturally infected Scottish Blackface sheep by PCR amplification and sequencing, and examined the associations between MHC haplotypes and faecal egg counts by generalised linear mixed modelling. The DRB1*1101 allele occurred predominately on one haplotype and a comparison of haplotypes indicated that the causal mutation or mutations occurred in or around this locus. Additional comparisons with another resistant haplotype indicated that mutations in or around the DQB2*GU191460 allele were also responsible for resistance to nematode infections. Further analyses identified six amino acid substitutions in the antigen binding site of DRB1*1101 that were significantly associated with reductions in the numbers of adult T. circumcincta.     

Enantioselective total synthesis,divergent optimization and preliminary biological evaluation of (indole-N-alkyl)-diketopiperazines

The first enantioselective total synthesis of the antifungal natural product (indole-N-isoprenyl)-tryptophan-valine diketopiperazine 5 was accomplished. Four stereoisomers of 5 were intentionally prepared, and the (R, R)-isomer is more favorable in enhancing the antifungal bioactivity. Divergent structural optimization of this attractive model was conducted from the chiral pool amino acids. Fine-tuning of the structure protruded the broad-spectrum antifungal 6b, which also showed good preventative efficacy against Sclerotinia scleotiorum. Compound 5d could accelerate both hypocotyl elongation and root growth of Eclipta prostrata even at the concentration of <2.5 ppm. This unique and easily accessible scaffold will be of prime importance in achieving agrochemical candidates with the novel scaffold.     

Role of iron metabolism in heart failure: From iron deficiency to iron overload

Iron metabolism is a balancing act, and biological systems have evolved exquisite regulatory mechanisms to maintain iron homeostasis. Iron metabolism disorders are widespread health problems on a global scale and range from iron deficiency to iron-overload. Both types of iron disorders are linked to heart failure. Iron play a fundamental role in mitochondrial function and various enzyme functions and iron deficiency has a particular negative impact on mitochondria function. Given the high-energy demand of the heart, iron deficiency has a particularly negative impact on heart function and exacerbates heart failure. Iron-overload can result from excessive gut absorption of iron or frequent use of blood transfusions and is typically seen in patients with congenital anemias, sickle cell anemia and beta-thalassemia major, or in patients with primary hemochromatosis. This review provides an overview of normal iron metabolism, mechanisms underlying development of iron disorders in relation to heart failure, including iron-overload cardiomyopathy, and clinical perspective on the treatment options for iron metabolism disorders.     

Acetylation as a major determinant to microtubule-dependent autophagy: Relevance to Alzheimer's and Parkinson disease pathology

Protein post-translational modifications (PTMs) that potentiate protein aggregation have been implicated in several neurological disorders, including Alzheimer's (AD) and Parkinson's disease (PD). In fact, Tau and alpha-synuclein (ASYN) undergo several PTMs potentiating their aggregation and neurotoxicity.Recent data posits a role for acetylation in Tau and ASYN aggregation. Herein we aimed to clarify the role of Sirtuin-2 (SIRT2) and HDAC6 tubulin deacetylases as well as p300 acetyltransferase in AD and PD neurodegeneration. We used transmitochondrial cybrids that recapitulate pathogenic alterations observed in sporadic PD and AD patient brains and ASYN and Tau cellular models.We confirmed that Tau protein and ASYN are microtubules (MTs)-associated proteins (MAPs). Moreover, our results suggest that α-tubulin acetylation induced by SIRT2 inhibition is functionally associated with the improvement of MT dynamic determined by decreased Tau phosphorylation and by increased Tau/tubulin and ASYN/tubulin binding. Our data provide a strong evidence for a functional role of tubulin and MAPs acetylation on autophagic vesicular traffic and cargo clearance. Additionally, we showed that an accumulation of ASYN oligomers imbalance mitochondrial dynamics, which further compromise autophagy. We also demonstrated that an increase in Tau acetylation is associated with Tau phosphorylation. We found that p300, HDAC6 and SIRT2 influences Tau phosphorylation and autophagic flux in AD. In addition, we demonstrated that p300 and HDAC6 modulate Tau and Tubulin acetylation.Overall, our data disclose the role of Tau and ASYN modifications through acetylation in AD and PD pathology, respectively. Moreover, this study indicates that MTs can be a promising therapeutic target in the field of neurodegenerative disorders in which intracellular transport is altered.     

Identification of recombinant AtPYL2, an abscisic acid receptor,in E. coli using a substrate-derived bioactive small molecule,a biotin linker with alkyne and amino groups,and a protein cross-linker

Target protein identification of bioactive small molecules is one of the most important research in forward chemical genetics. The affinity chromatography technique to use a resin bound with a small molecule is often used for identification of a target protein of a bioactive small molecule. Here we report a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, protein cross-linker containing disulfide bond, and a bioactive small molecule with an azido group (azido probe). After an azido probe is associated with a target protein, the complex of a target protein and azido probe is covalently bound through the biotin linker by azide-alkyne Huisgen cycloaddition and protein cross-linker containing disulfide bond. This ternary complex is immobilized on an affinity matrix with streptavidin, and then the target protein is selectively eluted with a buffer containing a reducing agent for cleavage of disulfide bonds. This method uses a probe having an azido group, which a small functional group, and has the possibility to be a solution strategy to overcome the hindrance of a functional group introduced into the probe that reduces association a target protein. The effectiveness of the method in this study was shown using linker 1, 3′-azidoabscisic acid 3, and protein cross-linker containing a disulfide bond (DTSSP 5).