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MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/PHOSPHATE2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ (for isobaric tags for relative and absolute quantitation)- based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in PHOSPHATE TRANSPORTER1 (PHT1) family and PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research.

The number of genes encoding receptor-like kinases (RLKs) has expanded in the plant lineage. Their expansion has resulted in the emergence of diverse domain architectures that function in signaling cascades related to growth, development, and stress response. In this study, we focused on receptor-like cytoplasmic kinase subfamily XI (RLCK XI) in plants. We discovered an exceptionally long kinase insert domain (KID), averaging 280 amino acids, between subdomains VII and VIII of the conserved protein kinase domain. Using sequence homology search, we identified members of RLCK XI with the unique KID architecture in terrestrial plants, up to a single copy in several hornwort and liverwort species. The KID shows a high propensity for being disordered, resembling the activation segment in the model kinase domain. Several conserved sequence motifs were annotated along the length of the KID. Of note, the KID harbors repetitive nuclear localization signals capable of mediating RLCK XI translocation from the plasma membrane to the nucleus. The possible physiological implication of dual localization of RLCK XI members is discussed. The presence of a KID in RLCK XI represents a unique domain architecture among RLKs specific to land plants.

Rice (Oryza sativa) OsNLA1 has been proposed to play a crucial role in regulating phosphate (Pi) acquisition in roots, similar to that of Arabidopsis (Arabidopsis thaliana) AtNLA. However, unlike AtNLA, OsNLA1 is not a target of miR827, a Pi starvation-induced microRNA. It is, therefore, of interest to know whether the expression of OsNLA1 depends on Pi supply and how it is regulated. In this study, we provide evidence that OsNLA1 controls Pi acquisition by directing the degradation of several OsPHT1 Pi transporters (i.e. OsPT1/2/4/7/8/12). We further show that OsNLA1 has an additional function in reproduction and uncover the mechanism of its expression regulation. Analysis of mRNA levels, promoter-GUS activity, and protoplast transient expression showed that the expression of OsNLA1.1, the most abundant transcript variant, is up-regulated in response to increasing Pi supply. The OsNLA1 promoter region was found to contain an upstream open reading frame that is required for Pi-responsive expression regulation. OsNLA1 promoter activity was observed in roots, ligules, leaves, sheaths, pollen grains, and surrounding the vascular tissues of anthers, suggesting that OsNLA1 is important throughout the development of rice. Disruption of OsNLA1 resulted in increased Pi uptake from roots as well as impaired pollen development and reduced grain production. In summary, our study reveals that Pi-induced OsNLA1 expression regulated by a unique mechanism functions in Pi acquisition, Pi translocation, and reproductive success.

Scientific evidences in the literature have shown that plants treated exogenously with micromole concentration of hydrogen peroxide (H 2 O 2 ) acquire abiotic stress tolerance potential, without substantial disturbances in the endogenous H 2 O 2 pool. In this study, we enhanced the endogenous H 2 O 2 content of tobacco ( Nicotiana tabaccum L. cv. SR1) plants by the constitutive expression of a glucose oxidase ( GO ; EC 1.1.3.4) gene of Aspergillus niger and studied their cold tolerance level. Stable integration and expression of GO gene in the transgenic (T 0 –T 2 ) tobacco lines were ascertained by molecular and biochemical tests. Production of functionally competent GO in transgenic plants was confirmed by the elevated levels of H 2 O 2 in the transformed tissues. When three homozygous transgenic lines were exposed to different chilling temperatures for 12 h, the electrolyte conductivity was significantly lower in GO -expressing tobacco plants than the control plants; in particular, chilling protection was more prominent at −1°C. In addition, most transgenic lines recovered within a week when returned to normal culture conditions after −1°C–12 h cold stress. However, control plants displayed symptoms of chilling injuries such as necrosis of shoot tip, shoots and leaves, consequently plant death. The protective effect realized in the transgenic plants was comparable to cold-acclimatized wild tobacco. The chilling tolerance of transgenic lines was found associated, at least in part, with elevated levels of total antioxidant content, CAT and APX activities. Based on our findings, we predict that the transgenic expression of GO may be deployed to improve cold tolerance potential of higher plants.

Ethylene, a gaseous plant hormone, is responsible for the initiation of reproductive development in pineapple. Reproductive development can be forced in pineapple (Ananas comosus var. comosus) throughout the year with ethylene. Inhibition of natural flowering initiation with aviglycine [(S)-trans-2-amino-4-(2-aminoethoxy)-3-butenoic acid hydrochloride], an inhibitor of ethylene biosynthesis, provides evidence that reproductive development in response to cold stress and short daylength is also in response to ethylene production. We studied the effect of cold treatment of pineapple on ethylene production and flower induction by applying a short-term cold stress to stem apices. Shoot apices of pineapple treated with ice crystals also produced twice as much ethylene as did those of control plants and significantly more than was produced by “D” leaf basal tissue. Moreover, pineapple plants treated four times with ice crystals or ice water were induced to flower under field conditions and the forcing efficiency, as evaluated by the percentages of inflorescence emergence and fruit harvest, was comparable to forcing with calcium carbide (CaC₂) and ethephon. In another field experiment two applications of a 1.0% solution of CaC₂ or 0.15% ethephon applied at 48 h intervals was sufficient to force reproductive development of ‘Tainon 17'. Furthermore, 0.5 or 1.0% solutions of CaC₂ supplemented with 0.5% activated charcoal (AC) significantly improved the forcing effectiveness of CaC₂. This could/would make it possible to reduce the number or concentration, or both, of CaC₂ required to effect forcing in pineapple.

PurposeIn the KATHERINE study (NCT01772472), patients with HER2-positive early breast cancer (EBC) and residual invasive disease after neoadjuvant chemotherapy plus HER2-targeted therapy who were treated with adjuvant trastuzumab emtansine (T-DM1) had a 50% reduction in the risk of an invasive disease-free survival (IDFS) event compared to patients treated with adjuvant trastuzumab. In metastatic disease, T-DM1 has resulted in higher rates of thrombocytopenia in Asian versus non-Asian patients. Here, we report safety and efficacy in Chinese patients from KATHERINE.MethodsPatients with HER2-positive EBC and residual invasive disease after taxane- and trastuzumab-containing neoadjuvant chemotherapy followed by surgery were randomized 1:1 to 14 cycles of adjuvant T-DM1 or trastuzumab. The primary endpoint was time to an IDFS event.ResultsAmong Chinese patients (T-DM1 n = 51, trastuzumab n = 50), T-DM1 treatment resulted in a 43% reduction in risk of an IDFS event compared to trastuzumab (HR = 0.57; 95% CI 0.25–1.31), with similar results for secondary endpoints. As in the global population, Chinese patients receiving T-DM1 versus trastuzumab had more grade ≥ 3 adverse events (AEs; 39.2% versus 4.1%) and AEs leading to treatment discontinuation (27.5% versus 0%). The most common grade ≥ 3 AE with T-DM1 was thrombocytopenia (21.6%), a frequency higher than the frequency in the global population (5.7%). Grade ≥ 3 hemorrhage was reported in 1 patient (T-DM1 arm).ConclusionsIn the KATHERINE study, T-DM1 demonstrated increased efficacy compared to trastuzumab in Chinese patients. Consistent with previous data in Asian patients, T-DM1 was associated with more grade ≥ 3 AEs, and AEs leading to discontinuation, which was driven by an increase in thrombocytopenia.

To determine whether microcytic erythrocytes influence the accuracy of automated platelet (PLT) counting. We divided a total of 206 K2 ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood samples into 4 groups, as follows: In group 1 (control group), normal mean corpuscular volume (MCV > 80 fL) and PLT count equal or greater than 140,000/microL (n = 45); group 2, normal MCV, reduced PLT count (< 140 x 10(3)/microL, n = 41); group 3, microcytic samples with normal PLT count (n = 68); and group 4, microcytic samples with reduced PLT count (n = 49). We also compared the platelet counting using electroimpedance (PLT-EI), platelet count using fluorescent optical (PLT-FO), and platelet-count manual (PLT-M) methods, using the Sysmex XE 2100 automatic analyzer. Despite highly significant overall correlations between PLT-EI and PLT-FO, PLT-EI and PLT-M, and PLT-FO and PLT-M (r = 0.95 [all P < .001]), use of the PLT-EI method resulted in widely overestimated PLT counts in microcytic samples (MCV < 80 fL), compared with use of PLT-FO and PLT-M. Our results identify an MCV of 70 fL as the critical threshold below which PLT-EI became unreliable. The PLT-EI mode overestimated PLT counts compared with PLT-FO and PLT-M modes in microcytic blood. Therefore, PLT-FO is the preferred method for PLT counting in patients with microcytic anemia when using an automated analyzer.

Bruchid, Callosobruchus spp. (Coleoptera: Bruchidae), is a serious pest during storage of seeds of mungbean (Vigna radiata (L.) Wilczek) and other Vigna species. A source of resistance to this pest has been identified in Vigna sublobata (Roxb.) Bairig. accession TC1966. Two hundred recombinant inbred lines at the F₁₂ generation have been developed for molecular mapping of bruchid resistance (Br) gene in TC1966. Through bulked segregant analysis (BSA), ten randomly amplified polymorphic DNA (RAPD) markers associated with the bruchid resistance gene were successfully identified. A total of four closely linked RAPDs were cloned and transformed into sequence characterized amplified region (SCAR) and cleaved amplified polymorphism (CAP) markers. Seven CAPs developed from the identified RAPD markers showed tighter linkage with the Br gene than the original RAPD. Through transformation of RAPDs into CAPs, codominant markers for bruchid resistance were successfully obtained. Homozygous genotypes of these PCR-based markers were estimated to contribute 85% of the variance for seed damage when the insect assay was performed under favorable growth conditions for bruchid.

Hong Kong has a high burden of influenza hospitalisation. This study estimated influenza vaccine effectiveness in hospitalised Hong Kong children aged 6 months to below 6 years using data potentially obtainable from routine surveillance sources. This ‘test-negative’ case-control study was conducted over two summer and one winter influenza seasons in five public Hong Kong hospitals during 2015 and 2016. Patients admitted for febrile and/or respiratory-associated illnesses who met inclusion criteria were invited to participate. Case-patients were respiratory-associated admissions with nasopharyngeal aspirate or nasopharyngeal swab specimens obtained during the first 48 h of hospitalisation that tested positive for influenza A or B, whereas control-patients were those with specimens that tested negative for both influenza A and B. Reliability of a routinely collected influenza immunisation status form was evaluated. Vaccine effectiveness for administration of full or partial series of influenza vaccination was calculated as 1 minus the odds ratio for influenza vaccination history for case-patients versus control-patients. 2900 eligible subjects had influenza vaccination status available. A simple record form, designed to collect upon admission information on influenza vaccination status, was found to be reliable when compared to confirmed vaccination status from immunisation records and guardians’ self-reports. Influenza vaccine effectiveness for preventing influenza A or B hospitalisation in children aged from 6 months to below 6 years during the period June 2015 to November 2016 was 68% (95% confidence interval [CI]: 55%, 77%) from unconditional analyses and 64% (95% CI: 46%, 75%) from conditional analyses. Seasonal influenza vaccine was effective in preventing hospitalisation from influenza A or B in young Hong Kong children during 2015 and 2016. As influenza vaccination status is not currently routinely recorded, implementation of an influenza immunisation status form in all paediatric wards, and centralising the data in Hong Kong's central computerised database, could provide real-time monitoring of influenza vaccine effectiveness.

In order to apply a set of nine STR loci and the amelogenin locus in forensic testing, we have performed a population study on individuals from the Philippines and Thailand living in Taiwan (273 Philippine and 146 Thai individuals were typed by commercially available kits and an automated sequencer). A total of 73 alleles for all systems for both populations could be observed in these two populations. No new intermediate fragments were found. Allele frequencies showed no deviation from Hardy–Weinberg equilibrium. The mean exclusion power (MEP) ranged from 0.327 (TPOX) to 0.706 (FGA), the discriminating power (DP) ranged from 0.790 (TPOX) to 0.963 (FGA) for Philippinos, MEP ranged from 0.247 (TPOX) to 0.723 (FGA), DP ranged from 0.761 (TPOX) to 0.968 (FGA) for Thais, the combined MEP is >0.9988 and the combined DP is >0.9999999993 for both Philippinos and Thais.