Deubiquitinase USP20 promotes breast cancer metastasis by stabilizing SNAI2.

SNAI2/SLUG, a metastasis-promoting transcription factor, is a labile protein that is degraded through the ubiquitin proteasome degradation system. Here, we conducted comprehensive gain- and loss-of-function screens using a human DUB cDNA library of 65 genes and an siRNA library of 98 genes, and identified USP20 as a deubiquitinase (DUB) that regulates SNAI2 ubiquitination and stability. Further investigation of USP20 demonstrated its function in promoting migration, invasion, and metastasis of breast cancer. USP20 positively correlates with SNAI2 protein level in breast tumor samples, and higher USP20 expression is associated with poor prognosis in ER- breast cancer patients.

Rates of pulmonary vein reconnection at repeat ablation for recurrent atrial fibrillation and its impact on outcomes among females and males.

BACKGROUND: Several studies have demonstrated that females have a higher risk of arrhythmia recurrence after pulmonary vein (PV) isolation for atrial fibrillation (AF). There are limited data on sex-based differences in PV reconnection rates at repeat ablation. We aimed to investigate sex-based differences in electrophysiological findings and atrial arrhythmia recurrence after repeat AF ablation METHODS: We conducted a retrospective study of 161 consecutive patients (32% female, age 65 ± 10 years) who underwent repeat AF ablation after index PV isolation between 2010 and 2022. Demographics, procedural characteristics and follow-up data were collected. Recurrent atrial tachycardia (AT)/AF was defined as any atrial arrhythmia ≥30 s in duration. RESULTS: Compared to males, females tended to be older and had a significantly higher prevalence of prior valve surgery (10 vs. 2%; P = .03). At repeat ablation, PV reconnection was found in 119 (74%) patients. Males were more likely to have PV reconnection at repeat ablation compared to females (81 vs. 59%; P = .004). Excluding repeat PV isolation, there were no significant differences in adjunctive ablation strategies performed at repeat ablation between females and males. During follow-up, there were no significant differences in freedom from AT/AF recurrence between females and males after repeat ablation (63 vs. 59% at 2 years, respectively; P = .48). CONCLUSIONS: After initial PV isolation, significantly fewer females have evidence of PV reconnection at the time of repeat ablation for recurrent AF. Despite this difference, long-term freedom from AT/AF was similar between females and males after repeat ablation.

Melatonin protects oogenesis from hypobaric hypoxia-induced fertility damage in mice.

Environmental hypoxia adversely affects reproductive health in humans and animals at high altitudes. Therefore, how to alleviate the follicle development disorder caused by hypoxia exposure and to improve the competence of fertility in plateau non-habituated female animals are important problems to be solved urgently. In this study, a hypobaric hypoxic chamber was used for 4 weeks to simulate hypoxic conditions in female mice, and the effects of hypoxia on follicle development, proliferation and apoptosis of granulosa cells, reactive oxygen species (ROS) levels in MII oocyte and 2-cell rate were evaluated. At the same time, the alleviating effect of melatonin on hypoxic exposure-induced oogenesis damage was evaluated by feeding appropriate amounts of melatonin daily under hypoxia for 4 weeks. The results showed that hypoxia exposure significantly increased the proportion of antral follicles in the ovary, the number of proliferation and apoptosis granulosa cells in the follicle, and the level of ROS in MII oocytes, eventually led to the decline of oocyte quality. However, these defects were alleviated when melatonin was fed under hypoxia conditions. Together, these findings suggest that hypoxia exposure impaired follicular development and reduced oocyte quality, and that melatonin supplementation alleviated the fertility reduction induced by hypoxia exposure.

Mechanical strength changes of combustible municipal solid waste components during their early pyrolysis stage and mechanism analysis.

Implementation of municipal solid waste (MSW) source segregation leads to a more convenient recycle of combustible MSW components. Textiles, plastics and papers are commonly available combustible components in MSW. Their shredding is conducive to resources recovery. But these components usually have high tensile strengths and are difficult to shred. To understand their mechanical strength changes in their early pyrolysis stage will help to address this problem. In this study, a universal electronic testing machine was used to determine the breaking strengths of the materials including cotton towel, polyethylene glycol terephthalate (PET), ivory board (IB), kraft paper (KP) and wool scarf in the temperature range of 30-250°C under N2 atmosphere, and the mechanisms of their strength changes were explored. The reaction force field molecular dynamics (ReaxFF-MD) simulation was used to explain the decomposition behaviours of different sugar groups of hemicellulose in cotton and paper and the change of van der Waals energy of wool during their early pyrolysis stages. The results showed that breaking strengths of all the combustible MSW components reduced as the temperature increased. The breaking strength of PET was found to have the highest descent rate with increasing temperature, then the descent rates of wool and cotton came as the second and third, respectively. Compared with cotton, the breaking strengths of KP and IB decreased more slowly. As the temperature increased, the breaking strength of cotton reduced mainly due to the decomposition of the glucuronic acid in hemicellulose, and the reduction was characterized by CO2 release. The breaking strength reduction of PET was caused by its molecular chain being relaxed. The breaking strength reduction of wool was firstly caused by the decrease in the van der Waals energy between its molecules, and then caused by molecular chain breaking. In addition, in order to understand the influence of material size on the breaking strength change during thermal treatment, the breaking strengths of cotton yarn bundles were correlated with their yarn number and temperature. This study lays the foundation for understanding changes in mechanical strengths of combustible MSW components during their early pyrolysis stage.

Metabolic phenotype analysis of Trichophyton rubrum after laser irradiation.

BACKGROUND: Biological phenotypes are important characteristics of microorganisms, and often reflect their genotype and genotype changes. Traditionally, Trichophyton rubrum (T. rubrum) phenotypes were detected using carbon source assimilation tests, during which the types of tested substances are limited. In addition, the operation is complicated, and only one substance can be tested at once. To observe the changes of the metabolic phenotype of T. rubrum after laser irradiation, a high-throughput phenotype microarray system was used to analyze the metabolism of different carbon, nitrogen, phosphorus and sulfur source substrates in a Biolog metabolic phenotyping system. RESULTS: The strain of T. rubrum used in this study can effectively utilize 33 carbon, 20 nitrogen, 16 phosphorus, and 13 sulfur source substrates prior to laser irradiation. After laser irradiation, the strain was able to utilize 10 carbon, 12 nitrogen, 12 phosphorus, and 8 sulfur source substrates. The degree of utilization was significantly decreased compared with the control. Both groups efficiently utilized saccharides and organic acids as carbon sources as well as some amino acids as nitrogen sources for growth. The number of substrates utilized by T. rubrum after laser irradiation were significantly reduced, especially carbon substrates. Some substrates utilization degree in the laser treated group was higher than control, such as D-glucosamine, L-glutamine, D-2-Phospho-Glyceric Acid, D-glucosamine-6-phosphate, and D-methionine. CONCLUSION: Laser irradiation of T. rubrum may lead to changes in the metabolic substrate and metabolic pathway, thus weakening the activity of the strain.

Effects of fermentation chamber temperature on microbes and quality of cigar wrapper tobacco leaves.

The natural fermentation of cigar tobacco leaves usually utilizes natural temperature and humidity for fermentation. Cigars produced in China are often fermented in winter, and the low environmental temperatures can lead to slow heating of the tobacco stack, affecting the cigar tobacco leaves quality. This study aimed to determine the minimum chamber temperature required to initiate the process of fermentation for cigar tobacco leaves and to explore the impact of temperature on the microbial community of tobacco leaves. Here, the cigar variety "Dexue 1" were subjected to stacking fermentation under three temperature parameters (20 ℃, 27 ℃, 34 ℃). With an increase in environmental temperature, the temperature inside the stack of cigar leaves increased significantly, the protein, total sugar, starch, and total alkaloid content in fermented tobacco leaves decreased, and the aroma components and amino acid content increased. Microbial richness and community diversity associated with fermented tobacco were highest at chamber temperatures of above 27 ℃. The relative abundance of Chryseobacterium and Rhodococcus was significantly negatively correlated with protein, alkaloids, total sugar, and starch, and positively correlated with amino acids and aroma components. Chryseobacterium and Rhodococcus may be responsible for the degradation of macromolecular substances and the conversion of favorable aromatic substances, thus improving the tobacco leaves quality. This study demonstrated that increasing the fermentation chamber temperature above 27 ℃ was conductive to raising the inner-stack temperature, increased microbial diversity and aromatic quality, reduced the strength and irritation, and extremely enhanced the overall quality of fermented cigar tobacco leaves. KEY POINTS: • The environmental temperature of the fermentation chamber has a significant impact on the quality of tobacco • Temperature > 27 ℃ can initiate the process of cigar tobacco leaves fermentation and increase inner-stack temperature and microbial diversity and abundance • Chryseobacterium and Rhodococcus may be related to the degradation of macromolecular substances and the transformation of aromatic substances, thereby improving the quality of tobacco leaves.

Tuning Ionic Liquid-Based Catalysts for CO2 Conversion into Quinazoline-2,4(1H,3H)-diones.

Carbon capture and storage (CCS) and carbon capture and utilization (CCU) are two kinds of strategies to reduce the CO2 concentration in the atmosphere, which is emitted from the burning of fossil fuels and leads to the greenhouse effect. With the unique properties of ionic liquids (ILs), such as low vapor pressures, tunable structures, high solubilities, and high thermal and chemical stabilities, they could be used as solvents and catalysts for CO2 capture and conversion into value-added chemicals. In this critical review, we mainly focus our attention on the tuning IL-based catalysts for CO2 conversion into quinazoline-2,4(1H,3H)-diones from o-aminobenzonitriles during this decade (2012~2022). Due to the importance of basicity and nucleophilicity of catalysts, kinds of ILs with basic anions such as [OH], carboxylates, aprotic heterocyclic anions, etc., for conversion CO2 and o-aminobenzonitriles into quinazoline-2,4(1H,3H)-diones via different catalytic mechanisms, including amino preferential activation, CO2 preferential activation, and simultaneous amino and CO2 activation, are investigated systematically. Finally, future directions and prospects for CO2 conversion by IL-based catalysts are outlined. This review is benefit for academic researchers to obtain an overall understanding of the synthesis of quinazoline-2,4(1H,3H)-diones from CO2 and o-aminobenzonitriles by IL-based catalysts. This work will also open a door to develop novel IL-based catalysts for the conversion of other acid gases such as SO2 and H2S.

High-Throughput Screening Using a Synchronized Pulsed Self-aspiration Vacuum Electrospray Ionization Miniature Mass Spectrometer.

With the advantages of rapid analysis, high sensitivity, and multicomponent identification, mass spectrometry (MS) is recognized as an appealing choice for high-throughput screening (HTS) analysis. Aiming at the small size, simple operation, and adequate performance, the development of miniature mass spectrometers has made great progress over the last 2 decades. Besides the essential analytical performance, simple operation and HTS capability are two other crucial features desired in miniature MS instruments. In this paper, an induced self-aspiration vacuum electrospray ionization source (ISA-VESI) was developed and coupled to a miniature ion trap mass spectrometer. A special timing sequence was designed to synchronize all the operation steps in each measurement, including dual-pulse sample injection, multipulse gas injection, MS analysis, and the movement of the homemade HTS platform used as the sampler. Then, the automatic high-throughput analysis of multiple samples can be accomplished with close coordination among the sample delivery, the sample introduction and ionization, and the ion trap operation. The measurement time of each ISA-VESI-MS analysis was about 7 s, with a sample consumption of less than 100 nL.

Growth inhibition of Trichophyton rubrum by laser irradiation: exploring further experimental aspects in an in vitro evaluation study.

BACKGROUND: According to the results of the clinical trials, laser therapy is effective for the treatment of onychomycosis, but the in vitro findings are inconsistent among studies. This study aimed to explore the experimental conditions of laser for the inhibition of Trichophyton rubrum growth in vitro. A 1064-nm neodymium-doped yttrium aluminum garnet (Nd:YAG) laser was used to irradiate colonies using a small (6-mm diameter) or large (13-mm diameter) area, and using 300, 408, or 600 J/cm2. The surface temperature of the colony was measured after irradiation and every 2 min. The growth area was calculated until the 7th or 10th day of incubation daily. RESULTS: For the small area group, at 300 J/cm2, the immediate surface temperature was 25.2 ± 0.2°C, but without effect on growth (P = 0.516). At 408 J/cm2, the immediate surface temperature was 32.0 ± 0.4°C; growth was inhibited for 7 days (P < 0.001). At 600 J/cm2, the immediate surface temperature was 38.1 ± 0.4°C; the growth was completely stopped for at least 10 days (P < 0.001). For the large area group, the temperature patterns were similar to those of the small area group, but the highest temperature was lower than in the small area groups, and no growth inhibition effect was observed (all P > 0.05). CONCLUSIONS: When the irradiation area is small, a 1064-nm Nd:YAG laser at 408 or 600 J/cm2 can be effective in suppressing T. rubrum growth in vitro.

Next-generation sequencing identification and multiplex RT-PCR detection for viruses infecting cigar and flue-cured tobacco.

BACKGROUND: Early, precise and simultaneous identification of plant viruses is of great significance for preventing virus spread and reducing losses in agricultural yields. METHODS AND RESULTS: In this study, the identification of plant viruses from symptomatic samples collected from a cigar tobacco planting area in Deyang and a flue-cured tobacco planting area in Luzhou city, Sichuan Province, China, was conducted by deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform, and plant virus-specific contigs were generated based on virus-derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis were performed to determine the species or strains of these viruses. A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples, with a total of 105 contigs mapped to the closest plant viruses with lengths ranging from 34 ~ 1720 nt. The results indicated that the major viruses were potato virus Y, Chilli veinal mottle virus, tobacco vein banding mosaic virus, tobacco mosaic virus and cucumber mosaic virus. Subsequently, a fast and sensitive multiplex reverse transcription polymerase chain reaction assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan. CONCLUSIONS: These results provide a theoretical basis and convenient methods for the rapid detection and control of viruses in cigar- and flue-cured tobacco.

Tuning Functionalized Ionic Liquids for CO2 Capture.

The increasing concentration of CO2 in the atmosphere is related to global climate change. Carbon capture, utilization, and storage (CCUS) is an important technology to reduce CO2 emissions and to deal with global climate change. The development of new materials and technologies for efficient CO2 capture has received increasing attention among global researchers. Ionic liquids (ILs), especially functionalized ILs, with such unique properties as almost no vapor pressure, thermal- and chemical-stability, non-flammability, and tunable properties, have been used in CCUS with great interest. This paper focuses on the development of functionalized ILs for CO2 capture in the past decade (2012~2022). Functionalized ILs, or task-specific ILs, are ILs with active sites on cations or/and anions. The main contents include three parts: cation-functionalized ILs, anion-functionalized ILs, and cation-anion dual-functionalized ILs for CO2 capture. In addition, classification, structures, and synthesis of functionalized ILs are also summarized. Finally, future directions, concerns, and prospects for functionalized ILs in CCUS are discussed. This review is beneficial for researchers to obtain an overall understanding of CO2-philic ILs. This work will open a door to develop novel IL-based solvents and materials for the capture and separation of other gases, such as SO2, H2S, NOx, NH3, and so on.

Environmental impacts and nutrient distribution routes for food waste separated disposal on large-scale anaerobic digestion/ composting plants.

Centralized biological treatments, i.e., anaerobic digestion (AD) and in-vessel composting (IVC), were supposed to be the promising processes for the disposal of food waste (FW) after source separation, while the systematic benefits were unclear for FW with high water content, salt and oil and thus influenced the selection by the local decision-makers. In this study, two large-scale working AD and IVC plants were compared for environmental impacts, nutrient recovery and economic benefits. For unit amount of FW, 89.26 kg CO2-eq was released in IVC mainly due to 47.89 kWh electricity consumption, and 57.02 kg CO2-eq was produced in AD. With the application of compost and energy recovery, 26.88 and 93.55 kg CO2-eq savings were obtained in IVC and AD, respectively. NH3 emissions were the main contributor to acidification (0.35 kg SO2-eq) in IVC, while AD exerted less impact on acidification (0.09 kg SO2-eq) and nutrient enrichment (0.25 kg NO3-eq) attributed to the counteract of energy recovery. 2029 would be the inflection point for global warming potential in AD with more clean energy applied in electricity mix in China. For nutrient recovery, more C (8.3%), N (37.9%) and P (66.7%) could be recovered in compost, while those were discharged via leachate and biogas residue in AD. The cost of IVC was 16 CNY/t (2.40 USD/t) lower than AD. Combing the three key indexes and the sale routes of products, IVC was recommended to be used in areas dominated by agriculture and forestry industries, and AD was more suitable for large cities.

Molecular transformation and composition flow of dissolved organic matter in four typical concentrated leachates from the multi-stage membrane system.

Concentrated leachate (CL), characterized with high content salts and compositional complexity of dissolved organic matter (DOM), is difficult to degrade. Understanding the CL from molecular insight level is the requirement for further disposal based on their components. Here, typical CL samples were collected from the multi-stage membrane separation process in a large-scale leachate plant, including nanofiltration (NF), primary ultrafiltration (PUF), secondary nanofiltration (SNF), and reverse osmosis (RO). More than 95% of DOM was removed from raw CL, of which about 3/4 flowed into PUFCL and 1/5 flowed into SNFCL. DOM with macro-molecular weight (>500 Da, 30.46%) and highly unsaturated compounds (double-bond equivalents >15) were detected in PUFCL. Nearly half of DOM was CHO-only compounds (42.04%) in SNFCL. PUFCL was abundant in heteroatom species with higher-order oxygen (O ≥ 10), which was coincident with the trend of humic substance distribution (humic substance >1/2). Based on these properties results, advanced oxidation processes, such as ozonation, might be the right process for SNFCL rich in heteroatom species with low-order oxygen (O < 10). Abundant disulfides (S2O2-6 classes, 20.19%) and monovalent salts existed in ROCL, which should be removed from the system. These findings might provide basic information for the treatment of CLs from different membranes.

The effect of sludge retention time (SRT) on the Nitrifier typical kinetics at ambient temperature under the low ammonia density.

Sludge retention time (SRT) regulation is one of the essential management techniques for refined control of the main-sidestream treatment process under the low ammonia density. It is indispensable to understand the effect of SRTs changes on the Nitrifier kinetics to obtain the functional separation of the Nitrifier and the refined control of the nitrification process. In this study, Nitrifier was cultured with conditions of 35 ± 0.5 °C, pH 7.5 ± 0.2, DO 5.0 ± 0.5 mg-O/L, and SRTs were controlled for 40 d, 20 d, 10 d, and 5 d. The net growth rate (µm), decay rate (b), specific growth rate (µ), the yield of the Nitrifier (YA), temperature parameter (TA), and inhibition coefficient (KI) have been measured and extended with the SRT decreases. Instead, the half-saturation coefficient (KS) decreased. In addition, the limited value of pH inhibition occurs (pHUL), and the pH of keeping 5% maximum reaction rate (pHLL) was in a relatively stable state. The trade of kinetics may be induced by the change of species structure of Nitrifier. The Nitrosomonas proportion was increased, and the Nitrospira was contrary with the SRT decreasing. It is a match for the functional separation of Nitrifier when SRTs was 20 d at ambient temperature under the low ammonia density. The kinetics of ammonia-oxidizing organisms (AOO) and nitrite-oxidizing organisms (NOO) in Nitrifier under different SRT conditions should be measured respectively to the refined control of the partial nitrification process in future study.

A novel approach to estimate and control denitrification performance in activated sludge systems with respirogram technology.

Respirogram technology has been widely applied for aerobic process, however, the response of respirogram to anoxic denitrification is still unclear. To reveal such response may help to design a new method for the evaluation of the performance of denitrification. The size distribution of flocs measured at different denitrification moments demonstrated a clear expansion of flocs triggered by denitrification, during which higher specific endogenous and quasi-endogenous respiration rates (SOURe and SOURq) were also observed. Furthermore, SOURq increases exponentially with the specific denitrification rate (SDNR), suggesting that there should be a maximum SDNR in conventional activated sludge systems. Based on these findings, an index Rq/t, defined as the ratio of quasi-endogenous (OURq) to maximum respiration rate (OURt), is proposed to estimate the denitrification capacity that higher Rq/t indicates higher denitrification potential, which can be readily obtained without complex measurement or analysis, and it offers a novel and promising respirogram-based approach for denitrification estimation and control by taking measures to extend anoxic time to maintain its value at a high level within a certain range.

More frequent olive oil intake is associated with reduced platelet activation in obesity.

BACKGROUND AND AIMS: Obesity is an independent risk factor for atherosclerotic cardiovascular disease (CVD), and platelet hyperactivation in obesity may contribute to this association. Olive oil consumption is associated with lower cardiovascular disease (CVD) risk in the general population. However, little is known for individuals with obesity. We investigated whether olive oil intake is associated with platelet activation in obesity. METHODS AND RESULTS: We assessed platelet activation (surface P-selectin expression) with and without thrombin exposure and diet composition in 63 patients with severe obesity. Among 63 subjects with obesity, the mean age was 32.2 ± 8.0 years and BMI 44.1 ± 8.5 kg/m2. Olive oil intake was stratified into <1 time/week (n = 21), 1-3 times/week (n = 18), ≥4 times/week (n = 24). Strata did not differ by age, BMI or platelet count. Unstimulated P-selectin expression did not differ by olive oil consumption. Subjects with more frequent olive oil intake exhibited lower P-selectin expression on submaximal thrombin exposure. CONCLUSIONS: More frequent olive oil intake is associated with reduced thrombin-induced platelet activation in obesity.

Risk factors for intracerebral hemorrhage in patients with COVID-19.

Intracerebral hemorrhage (ICH) can be a devastating complication of coronavirus disease (COVID-19). We aimed to assess risk factors associated with ICH in this population. We performed a retrospective cohort study of adult patients admitted to NYU Langone Health system between March 1 and April 27 2020 with a positive nasopharyngeal swab polymerase chain reaction test result and presence of primary nontraumatic intracranial hemorrhage or hemorrhagic conversion of ischemic stroke on neuroimaging. Patients with intracranial procedures, malignancy, or vascular malformation were excluded. We used regression models to estimate odds ratios and 95% confidence intervals (OR, 95% CI) of the association between ICH and covariates. We also used regression models to determine association between ICH and mortality. Among 3824 patients admitted with COVID-19, 755 patients had neuroimaging and 416 patients were identified after exclusion criteria were applied. The mean (standard deviation) age was 69.3 (16.2), 35.8% were women, and 34.9% were on therapeutic anticoagulation. ICH occurred in 33 (7.9%) patients. Older age, non-Caucasian race, respiratory failure requiring mechanical ventilation, and therapeutic anticoagulation were associated with ICH on univariate analysis (p < 0.01 for each variable). In adjusted regression models, anticoagulation use was associated with a five-fold increased risk of ICH (OR 5.26, 95% CI 2.33-12.24, p < 0.001). ICH was associated with increased mortality (adjusted OR 2.6, 95 % CI 1.2-5.9). Anticoagulation use is associated with increased risk of ICH in patients with COVID-19. Further investigation is required to elucidate underlying mechanisms and prevention strategies in this population.

Molecular mechanism of activation of human musk receptors OR5AN1 and OR1A1 by (R)-muscone and diverse other musk-smelling compounds.

Understanding olfaction at the molecular level is challenging due to the lack of crystallographic models of odorant receptors (ORs). To better understand the molecular mechanism of OR activation, we focused on chiral (R)-muscone and other musk-smelling odorants due to their great importance and widespread use in perfumery and traditional medicine, as well as environmental concerns associated with bioaccumulation of musks with estrogenic/antiestrogenic properties. We experimentally and computationally examined the activation of human receptors OR5AN1 and OR1A1, recently identified as specifically responding to musk compounds. OR5AN1 responds at nanomolar concentrations to musk ketone and robustly to macrocyclic sulfoxides and fluorine-substituted macrocyclic ketones; OR1A1 responds only to nitromusks. Structural models of OR5AN1 and OR1A1 based on quantum mechanics/molecular mechanics (QM/MM) hybrid methods were validated through direct comparisons with activation profiles from site-directed mutagenesis experiments and analysis of binding energies for 35 musk-related odorants. The experimentally found chiral selectivity of OR5AN1 to (R)- over (S)-muscone was also computationally confirmed for muscone and fluorinated (R)-muscone analogs. Structural models show that OR5AN1, highly responsive to nitromusks over macrocyclic musks, stabilizes odorants by hydrogen bonding to Tyr260 of transmembrane α-helix 6 and hydrophobic interactions with surrounding aromatic residues Phe105, Phe194, and Phe207. The binding of OR1A1 to nitromusks is stabilized by hydrogen bonding to Tyr258 along with hydrophobic interactions with surrounding aromatic residues Tyr251 and Phe206. Hydrophobic/nonpolar and hydrogen bonding interactions contribute, respectively, 77% and 13% to the odorant binding affinities, as shown by an atom-based quantitative structure-activity relationship model.

Establishment of a feeder and serum-free culture system for human embryonic stem cells.

Stem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cells in vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and α-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and α-fetoprotein were expressed in H9 cells, suggesting that the cells maintained in vitro differentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs.

Genetic Transformation and Green Fluorescent Protein Labeling in Ceratocystis paradoxa from Coconut.

Ceratocystis paradoxa, the causal agent of stem-bleeding disease of the coconut palm, causes great losses to the global coconut industry. As the mechanism of pathogenicity of C. paradoxa has not been determined, an exogenous gene marker was introduced into the fungus. In this study, pCT74-sGFP, which contains the green fluorescent protein (GFP) gene, and the hygromycin B resistance gene as a selective marker, was used as an expression vector. Several protoplast release buffers were compared to optimize protoplast preparation. The plasmid pCT74-sGFP was successfully transformed into the genome of C. paradoxa, which was verified using polymerase chain reaction and green fluorescence detection. The transformants did not exhibit any obvious differences from the wild-type isolates in terms of growth and morphological characteristics. Pathogenicity tests showed that the transformation process did not alter the virulence of the X-3314 C. paradoxa strain. This is the first report on the polyethylene glycol-mediated transformation of C. paradoxa carrying a 'reporter' gene GFP that was stably and efficiently expressed in the transformants. These findings provide a basis for future functional genomics studies of C. paradoxa and offer a novel opportunity to track the infection process of C. paradoxa.