TMD1 domain and CRAC motif determine the association and disassociation of MxIRT1 with detergent-resistant membranes.

Iron is essential for most living organisms. The iron-regulated transporter1 (IRT1) plays a major role in iron uptake in roots, and its trafficking from endoplasmic reticulum (ER) to plasma membrane (PM) is tightly coordinated with changes in iron environment. However, studies on the IRT1 response are limited. Here, we report that Malus xiaojinesis IRT1 (MxIRT1) associates with detergent-resistant membranes (DRMs, a biochemical counterpart of PM microdomains), whereas the PM microdomains are known platforms for signal transduction in the PM. Depending on the shift of MxIRT1 from microdomains to homogeneous regions in PM, MxIRT1-mediated iron absorption is activated by the cholesterol recognition/interaction amino acid consensus (CRAC) motif of MxIRT1. MxIRT1 initially associates with DRMs in ER via its transmembrane domain 1 (TMD1), and thus begins DRMs-dependent intracellular trafficking. Subsequently, MxIRT1 is sequestered in COPII vesicles via the ER export signal sequence in MxIRT1. These studies suggest that iron homeostasis is influenced by the CRAC motif and TMD1 domain due to their determination of MxIRT1-DRMs association.

Neuroprotective Effects of Baicalein on Acrolein-induced Neurotoxicity in the Nigrostriatal Dopaminergic System of Rat Brain.

Elevated levels of acrolein, an α,ß-unsaturated aldehyde are detected in the brain of patients with Parkinson's disease (PD). In the present study, the neuroprotective effect of baicalein (a phenolic flavonoid in the dried root of Scutellaria baicalensis Georgi) on acrolein-induced neurodegeneration of nigrostriatal dopaminergic system was investigated using local infusion of acrolein in the substantia nigra (SN) of rat brain. Systemic administration of baicalein (30 mg/kg, i.p.) significantly attenuated acrolein-induced elevations in 4-hydroxy-2-noneal (a product of lipid peroxidation), N-(3-formyl-3,4-dehydropiperidino)lysine (a biomarker of acrolein-conjugated proteins), and heme-oxygenase-1 levels (a redox-regulated protein) in the infused SN, indicating that baicalein inhibited acrolein-induced oxidative stress and protein conjugation. Furthermore, baicalein reduced acrolein-induced elevations in glial fibrillary acidic protein (a biomarker of activated astrocytes), ED-1 (a biomarker of activated microglia), and mature cathepsin B levels (a cysteine lysosomal protease), suggesting that baicalein attenuated acrolein-induced neuroinflammation. Moreover, baicalein attenuated acrolein-induced caspase 1 activation (a pro-inflammatory caspase) and interleukin-1ß levels, indicating that baicalein prevented acrolein-induced inflammasome activation. In addition, baicalein significantly attenuated acrolein-induced caspase 3 activation (a biomarker of apoptosis) as well as acrolein-induced elevation in receptor interacting protein kinase (RIPK) 3 levels (an initiator of necroptosis), indicating that baicalein attenuated apoptosis and necroptosis. At the same time, baicalein mitigated acrolein-induced reduction in dopamine levels in the striatum ipsilateral to acrolein-infused SN. In conclusion, our data suggest that baicalein is neuroprotective via inhibiting oxidative stress, protein conjugation, and inflammation. Furthermore, baicalein prevents acrolein-induced program cell deaths, suggesting that baicalein is therapeutically useful for slowing PD progression.

Acrolein acts as a neurotoxin in the nigrostriatal dopaminergic system of rat: involvement of α-synuclein aggregation and programmed cell death.

Clinical studies report significant increases in acrolein (an α,ß-unsaturated aldehyde) in the substantia nigra (SN) of patients with Parkinson's disease (PD). In the present study, acrolein-induced neurotoxicity in the nigrostriatal dopaminergic system was investigated by local infusion of acrolein (15, 50, 150 nmoles/0.5 µl) in the SN of Sprague-Dawley rats. Acrolein-induced neurodegeneration of nigrostriatal dopaminergic system was delineated by reductions in tyrosine hydroxylase (TH) levels, dopamine transporter levels and TH-positive neurons in the infused SN as well as in striatal dopamine content. At the same time, apomorphine-induced turning behavior was evident in rats subjected to a unilateral infusion of acrolein in SN. Acrolein was pro-oxidative by increasing 4-hydroxy-2-nonenal and heme oxygenase-1 levels. Furthermore, acrolein conjugated with proteins at lysine residue and induced α-synuclein aggregation in the infused SN. Acrolein was pro-inflammatory by activating astrocytes and microglia. In addition, acrolein activated caspase 1 in the infused SN, suggesting acrolein-induced inflammasome formation. The neurotoxic mechanisms underlying acrolein-induced neurotoxicity involved programmed cell death, including apoptosis and necroptosis. Compared with well-known Parkinsonian neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and rotenone which do not exist in the SN of PD patients, our in vivo study shows that acrolein acts as a Parkinsonian neurotoxin in the nigrostriatal dopaminergic system of rat brain.

Zea mays Fe deficiency-related 4 (ZmFDR4) functions as an iron transporter in the plastids of monocots.

Iron (Fe)-homeostasis in the plastids is closely associated with Fe transport proteins that prevent Fe from occurring in its toxic free ionic forms. However, the number of known protein families related to Fe transport in the plastids (about five) and the function of iron in non-green plastids is limited. In the present study, we report the functional characterization of Zea mays Fe deficiency-related 4 (ZmFDR4), which was isolated from a differentially expressed clone of a cDNA library of Fe deficiency-induced maize roots. ZmFDR4 is homologous to the bacterial FliP superfamily, coexisted in both algae and terrestrial plants, and capable of restoring the normal growth of the yeast mutant fet3fet4, which possesses defective Fe uptake systems. ZmFDR4 mRNA is ubiquitous in maize and is inducible by iron deficiency in wheat. Transient expression of the 35S:ZmFDR4-eGFP fusion protein in rice protoplasts indicated that ZmFDR4 maybe localizes to the plastids envelope and thylakoid. In 35S:c-Myc-ZmFDR4 transgenic tobacco, immunohistochemistry and immunoblotting confirmed that ZmFDR4 is targeted to both the chloroplast envelope and thylakoid. Meanwhile, ultrastructure analysis indicates that ZmFDR4 promotes the density of plastids and accumulation of starch grains. Moreover, Bathophenanthroline disulfonate (BPDS) colorimetry and inductively coupled plasma mass spectrometry (ICP-MS) indicate that ZmFDR4 is related to Fe uptake by plastids and increases seed Fe content. Finally, 35S:c-Myc-ZmFDR4 transgenic tobacco show enhanced photosynthetic efficiency. Therefore, the results of the present study demonstrate that ZmFDR4 functions as an iron transporter in monocot plastids and provide insight into the process of Fe uptake by plastids.

Expression of Malus xiaojinensis IRT1 (MxIRT1) protein in transgenic yeast cells leads to degradation through autophagy in the presence of excessive iron.

Iron is essential for plants, but highly toxic when present in excess. Consequently, iron uptake by root transporters must be finely tuned to avoid excess uptake from soil under iron excess. The iron-regulated transporter of Malus xiaojinensis (MxIRT1), induced in roots under iron deficiency, is a highly effective iron(II) transporter. Here, we investigated how the presence of excessive iron leads to MxIRT1 degradation in yeast expressing this plant iron transporter protein. To determine the relationship between iron abundance and MxIRT1 degradation, relative levels of autophagy-related gene-8 (ATG8) mRNA and the active ATG8-phosphatidylethanolamine-conjugated (PE) protein were measured in wild-type yeast and the autophagic mutant strains atg1∆, atg5∆, atg7∆, ypt7∆ and tor1∆ under normal and excessive iron conditions. The data showed that the exposure of MxIRT1-eGFP-transformed wild-type and tor1∆ strains to excessive iron led to significantly increased levels of ATG8 transcript and ATG8-PE protein, which resulted in enhanced MxIRT1 degradation. Co-localization of mCherry-ATG8 and MxIRT1-eGFP provided evidence that these proteins interact during autophagy in yeast. While inhibition of autophagic initiation, autophagosome formation and vacuole fusion all decreased MxIRT1 degradation. PMSF inhibition of autophagy prevented degradation, leading to the accumulation of MxIRT1-containing vesicles in the vacuoles. MxIRT1-vesicles were sorted into autophagosomes for iron-induced degradation in yeast, whereas the endogenous iron(II) transporter Fet4 was degraded in an autophagy-independent manner. Moreover, immunoprecipitation showed that multimono-ubiquitins provided MxIRT1 with the ubiquitination signal. Together, three factors, iron excess, autophagy and mono-ubiquitination, affect the functional activity and stability of exogenous MxIRT1 in yeast, thereby preventing iron uptake via this root transporter.

OsSEC24, a functional SEC24-like protein in rice, improves tolerance to iron deficiency and high pH by enhancing H(+) secretion mediated by PM-H(+)-ATPase.

Iron is abundant in the soil, but its low solubility in neutral or alkaline soils limits its uptake. Plants can rely on rhizosphere acidification to increase iron solubility. OsSEC27p was previously found to be a highly up-regulated gene in iron-deficient rice roots. Here, pH-dependent complementation assays using yeast mutants sec24Δ/SEC24 and sec27Δ/SEC27 showed that OsSEC27 could functionally complement SEC24 but not SEC27 in yeast; thus, it was renamed as OsSEC24. We found that OsSEC24-transgenic tobacco plants increased the length and number of roots under iron deficiency at pH 8.0. To explore how OsSEC24 confers tolerance to iron deficiency, we utilized transgenic tobacco, rice and rice protoplasts. H(+) flux measurements using Non-invasive Micro-test Technology (NMT) indicated that the transgenic OsSEC24 tobacco and rice enhanced H(+) efflux under iron deficiency. Conversely, the application of plasma membrane PM-H(+)-ATPase inhibitor vanadate elucidated that H(+) secretion increased by OsSEC24 was mediated by PM-H(+)-ATPase. OsPMA2 was used as a representative of iron deficiency-responsive PM-H(+)-ATPases in rice root via RT-PCR analysis. In transgenic rice protoplasts OsPMA2 was packaged into OsSEC24 vesicles after export from the ER through confocal-microscopy observation. Together, OsSEC24 vesicles, along with PM-H(+)-ATPases stimulate roots formation under iron deficiency by enhancing rhizosphere acidification.

Over-expression of the MxIRT1 gene increases iron and zinc content in rice seeds.

Iron and zinc are essential in plant and human nutrition. Iron deficiency has been one of the causes of human mortality, especially in developing countries with high rice consumption. MxIRT1 is a ferrous transporter that has been screened from an iron-efficient genotype of the apple tree, Malus xiaojinensis Cheng et Jiang. In order to produce Fe-biofortified rice with MxIRT1 to solve the Fe-deficiency problem, plant expression vectors of pCAMBIA1302-MxIRT1:GFP and pCAMBIA1302-anti MxIRT1:GFP were constructed that led to successful production of transgenic rice. The transgenic plant phenotypes showed that the expression of endogenous OsIRT1 was suppressed by anti-MxIRT1 in antisense lines that acted as an opposing control, while sense lines had a higher tolerance under Zn- and Fe-deficient conditions. The iron and zinc concentration in T3 seeds increased by three times in sense lines when compared to the wild type. To understand the MxIRT1 cadmium uptake, the MxIRT1 cadmium absorption trait was compared with AtIRT1 and OsIRT1 in transgenic rice protoplasts, and it was found that MxIRT1 had the lowest Cd uptake capacity. MxIRT1 transgenic tobacco-cultured bright yellow-2 (BY-2) cells and rice lines were subjected to different Fe conditions and the results from the non-invasive micro-test technique showed that iron was actively transported compared to cadmium as long as iron was readily available in the environment. This suggests that MxIRT1 is a good candidate gene for plant Fe and Zn biofortification.

[Complete nucleotide sequence of a human coxsackievirus B3 strain A103/KM/09 isolated in Yunnan province, 2009].

OBJECTIVE: To analyze the genetic characterization of the complete genome from a human coxsackievirus B3 strain A103/KM/09 isolated in Yunnan province, 2009. METHODS: By using RT-PCR, all the eight fragments which containing about 1000 nucleotides and covering full viral genome, were sequenced. By using Mega 5.05,Geneious, RDP 3 and SimPlot 3.5.1 software, sequences were aligned with other enterovirus reference sequences. Phylogenetic and recombination analysis were also carried out. RESULTS: The A103/KM/09 isolate genome showed 7389 nucleotides in length , encoding for 2185 amino acids. In the complete genome, the homology of nucleotide and amino acid among the seven coxsackievirus B3 isolates were 81.0%-88.0% and 95.7%-98.0%, respectively. There appeared 81.0% and 95.7% homology when compared with that of Nancy prototype strain. Results from the Phylogenetic analysis showed that the coxsackievirus B3 formed five distinct clades, I-V. Nucleotide divergence rates between clades were 16.2%-24.3% . The A103/KM/09 strain belonged to clade V. Clade V was further divided into four sub-clades,A-D. The nucleotide divergence between sub-clades was 4.3%-11.4%. Putative recombinant event for A103/ KM/09 was detected. CONCLUSION: All coxsackievirus B3 isolates could be divided into five clades, with A103/KM/09 strain belonged to Clade V-D. Evolution of coxsackievirus B3 had occurred in China.

A new method for species identification via protein-coding and non-coding DNA barcodes by combining machine learning with bioinformatic methods.

Species identification via DNA barcodes is contributing greatly to current bioinventory efforts. The initial, and widely accepted, proposal was to use the protein-coding cytochrome c oxidase subunit I (COI) region as the standard barcode for animals, but recently non-coding internal transcribed spacer (ITS) genes have been proposed as candidate barcodes for both animals and plants. However, achieving a robust alignment for non-coding regions can be problematic. Here we propose two new methods (DV-RBF and FJ-RBF) to address this issue for species assignment by both coding and non-coding sequences that take advantage of the power of machine learning and bioinformatics. We demonstrate the value of the new methods with four empirical datasets, two representing typical protein-coding COI barcode datasets (neotropical bats and marine fish) and two representing non-coding ITS barcodes (rust fungi and brown algae). Using two random sub-sampling approaches, we demonstrate that the new methods significantly outperformed existing Neighbor-joining (NJ) and Maximum likelihood (ML) methods for both coding and non-coding barcodes when there was complete species coverage in the reference dataset. The new methods also out-performed NJ and ML methods for non-coding sequences in circumstances of potentially incomplete species coverage, although then the NJ and ML methods performed slightly better than the new methods for protein-coding barcodes. A 100% success rate of species identification was achieved with the two new methods for 4,122 bat queries and 5,134 fish queries using COI barcodes, with 95% confidence intervals (CI) of 99.75-100%. The new methods also obtained a 96.29% success rate (95%CI: 91.62-98.40%) for 484 rust fungi queries and a 98.50% success rate (95%CI: 96.60-99.37%) for 1094 brown algae queries, both using ITS barcodes.