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05). Seven metabolites associated with declines in gait speed, including 1-carboxyethylphenylalanine (P=8.8×10-5), and N-acetylaspartate, N-formylmethionine, S-adenosylhomocysteine, N-acetylneuraminate, N2,N2-dimethylguanosine, and gamma-glutamylphenylalanine (all P less then 0.05). Two metabolite modules reflecting sphingolipid and bile acid metabolism associated with physical performance (minimum P=7.6×10-4). These results add to the accumulating evidence suggesting an important role of the human metabolome in physical performance and specifically implicate lipid, nucleotide, and amino acid metabolism in early physical performance decline.Introduction The fertility preservation (FP) services offered in Hong Kong are underutilised. There have been no previous studies on Chinese medical students to investigate the underlying reasons for this underutilisation in terms of awareness, knowledge, and attitudes towards FP and age-related fertility. Methods This was a cross-sectional survey among Chinese medical students in Hong Kong. Results The majority of participants (77.8%) were not familiar with any clinics or specialists who provide FP services. The vast majority (88.1%) underestimated female infertility at age 45 years, and 89.8% overestimated the age of male fertility decline. The students' FP knowledge was mainly acquired from electronic media (58.4%) and medical school (57.6%). Medical students showed overwhelming support towards FP for medical reasons (97.9%) but had mixed responses about FP for elective reasons related to career development in women (58.8%). Of the participants, 80.2% agreed that the government should subsidise FP services for patients with medical reasons. Conclusion This study highlights the limited awareness and knowledge of FP among Chinese medical students. There is a strong worldwide need to increase education about and exposure to FP in the medical curriculum and improve medical students' knowledge.Introduction Cancer survivorship is increasingly important with advances in cancer therapeutics. Minimisation of treatment-related morbidity is an area that requires attention. This situation is most pressing in premenopausal patients with breast cancer, in whom advances in hormonal and targeted therapies have improved mortality rates. However, treatment-related infertility is still poorly addressed, and in East Asia, there is limited discussion regarding management of treatment-related infertility. Methods A search of the literature was conducted using PubMed, Google Scholar, and Science Direct using the terms "breast cancer", "fertility preservation", "oocyte and embryonic cryopreservation", "GnRH-a co-administration", "ovarian tissue cryopreservation and transplantation", "Japan", "China", "Korea", and 'Singapore". Only studies published in English from 1980-2019 were included. The focus of the review was on identifying the current fertility preservation methods available to premenopausal women with breast cancer and the barriers that impede access. Results Fertility preservation options include GnRH-a co-administration to minimise treatment-associated infertility, oocyte and embryonic cryopreservation, and emerging treatments such as ovarian tissue cryopreservation and transplantation. In East Asia, the uptake of fertility preservation options has been limited despite it being a major patient concern. A lack of awareness of fertility preservation treatments hinders discussion between patients and clinicians about fertility preservation. Conclusion Despite progress in fertility preservation technologies, their impact for patients will be minimal if there is a lack of awareness/use of the technology. This review aims to raise awareness of such technologies among clinicians, enabling discussion between patients and clinicians about fertility preservation options.In maize (Zea mays), kernel weight is an important component of yield, which has been selected during domestication. Many genes associated with kernel weight have been identified through mutant analysis. Most are involved in the biogenesis and functional maintenance of organelles or other fundamental cellular activities. However, few quantitative trait loci (QTL) underlying quantitative variation in kernel weight have been cloned. Here, we characterize a QTL, qKW9, which is associated with maize kernel weight. This QTL encodes a DYW motif pentatricopeptide repeat protein involved in C-to-U editing of ndhB, a subunit of the chloroplast NADH dehydrogenase-like complex. In a null qkw9 background, C-to-U editing of ndhB was abolished, and photosynthesis was reduced, which resulted in less maternal photosynthate available for grain filling. Characterization of qKW9 highlights the importance of optimizing photosynthesis for maize grain yield production.Pollen tube tip growth depends on balancing secretion of cell wall material with endocytic recycling of excess material incorporated into the plasma membrane (PM). The classical model of tip growth, which predicts bulk secretion occurs apically and is compensated by subapical endocytosis, has been challenged in recent years. Many signaling proteins and lipids with important functions in the regulation of membrane traffic underlying tip growth associate with distinct regions of the pollen tube PM, and understanding the mechanisms responsible for the targeting of these regulatory factors to specific PM domains requires quantitative information concerning the sites of bulk secretion and endocytosis. Here, we quantitatively characterized the spatial organization of membrane traffic during tip growth by analyzing steady-state distributions and dynamics of FM4-64-labeled lipids and YFP-tagged transmembrane (TM) proteins in tobacco (Nicotiana tabacum) pollen tubes growing normally or treated with Brefeldin A to block secretion. We established that 1) secretion delivers TM proteins and recycled membrane lipids to the same apical PM domain, and 2) FM4-64-labeled lipids, but not the analyzed TM proteins, undergo endocytic recycling within a clearly defined subapical region. We mathematically modelled the steady-state PM distributions of all analyzed markers to better understand differences between them and to support the experimental data. Finally, we mapped subapical F-actin fringe and trans-Golgi network positioning relative to sites of bulk secretion and endocytosis to further characterize functions of these structures in apical membrane traffic. Our results support and further define the classical model of apical membrane traffic at the tip of elongating pollen tubes.A study was presented in which sarcomas were microinjected simultaneously with several drugs to study the pharmacodynamic response after resection. This platform may represent a future way of probing efficacy of anticancer agents in the relevant model system human tumors.See related article by Gundle et al., p. XXX.Immunoblotting allows detection of a protein antigen immobilized on the protein-retaining membrane support such as nitrocellulose or polyvinylidene fluoride (PVDF). The detection of the protein of interest relies on the binding of an antibody that specifically recognizes the protein of interest exposed on the membrane. The protein of interest can be purified or mixed with other proteins as in cell or tissue extracts. Usually immunoblotting combines the resolution of proteins by gel electrophoresis with immunochemical detection and is referred to as "western blotting." Immunoblotting can be used to determine the presence and the steady-state level of the protein of interest in the sample, its relative molecular weight, and the distribution of the protein between cellular fractions. Immunoblotting can be performed using the antibodies raised against synthetic peptide antigens modified to mimic posttranslational modifications of proteins, such as phosphorylation and acetylation, to study these modifications in the protein of interest in vivo. Oxaliplatin RNA Synthesis inhibitor When antibodies against the protein of interest are not available, immunoblotting can be performed using antibodies that specifically recognize the recombinant epitope tags (hemagglutinin [HA]-, Flag-, cMyc-, or glutathione-S-transferase [GST]) fused to the protein of interest using recombinant DNA techniques. Immunoblotting has a variety of research, clinical, and forensic medicine applications. It is also one of the standard techniques for characterization of antibodies from different samples of polyclonal sera or hybridoma supernatants.Fusion proteins that contain a glutathione S-transferase (GST) moiety can be purified to near homogeneity by affinity chromatography on glutathione-linked resins. Glutathione immobilized on a chromatography matrix, such as agarose or Sepharose, acts as a substrate for the GST moiety of fusion proteins. Contaminating proteins are washed away, and the bound GST fusion proteins are then readily displaced from the resin by elution with buffers containing free glutathione.Immobilized metal affinity chromatography (IMAC) is based on the affinity of polyhistidine tracts for divalent metal cations (usually Ni2+) immobilized as transition metal chelate complexes on a chromatography resin. The main protocol here is optimized for use of Ni2+-NTA resin to purify soluble 6xHis-tagged proteins by a straightforward batch method during the binding step, followed by gravity flow for washes and elution. This protocol does not require any specialized equipment other than a simple glass or plastic column. IMAC resins can be used in multiple formats, including batch, gravity flow, centrifuge columns, and fast performance liquid chromatography (FPLC) systems. FPLC systems are designed specifically for the chromatographic separations of proteins and other biomolecules. These systems typically contain multiple pumps, an in-line UV absorption monitor, conductivity meter, pH meter, fraction collector, and other options that allow for the simultaneous purification, analysis, and fractionation of the sample. When linked with the appropriate instruments, an FPLC can become a high-precision, automated instrument that separates proteins at a high resolution. An alternative protocol is included here that describes 6xHis-tagged protein purification using FPLC. Procedures for the cleaning and regeneration of the IMAC resin for reuse are also described, and, finally, considerations for storing purified proteins are discussed.Preparing electrocompetent bacteria is considerably easier than preparing cells for transformation by chemical methods. Bacteria are simply grown to mid-log phase, chilled, centrifuged, washed extensively with ice-cold buffer or H2O to reduce the ionic strength of the cell suspension, and then suspended in an ice-cold buffer containing 10% glycerol. DNA may be introduced immediately into the bacteria by exposing them to a short high-voltage electrical discharge. Alternatively, the cell suspension may be snap-frozen and stored at -70°C for up to 6 mo before electroporation, without loss of transforming efficiency.This protocol differs from other transformation procedures in that the bacterial culture is grown at 18°C rather than the conventional 37°C. Otherwise, the protocol is unremarkable and follows a fairly standard course. Under standard laboratory conditions, efficiencies of 1 × 108 to 3 × 108 transformed colonies/µg of plasmid DNA are typical.