DNA methylation offers an excellent example for elucidating how epigenetic information affects gene expression. β values and M values are commonly used to quantify DNA methylation. Statistical methods applicable to DNA methylation data analysis span a number of approaches such as Wilcoxon rank sum test, t-test, Kolmogorov-Smirnov test, permutation test, empirical Bayes method, and bump hunting method. Nonetheless, selection of an optimal statistical method can be challenging when different methods generate inconsistent results from the same data set.

Chronic low-grade adipose tissue and liver inflammation is a major cause of systemic insulin resistance and is a key component of the low degree of insulin sensitivity that exists in obesity and type 2 diabetes. Immune cells, such as macrophages, T cells, B cells, mast cells and eosinophils, have all been implicated as having a role in this process. Neutrophils are typically the first immune cells to respond to inflammation and can exacerbate the chronic inflammatory state by helping to recruit macrophages and by interacting with antigen-presenting cells. Neutrophils secrete several proteases, one of which is neutrophil elastase, which can promote inflammatory responses in several disease models. Here we show that treatment of hepatocytes with neutrophil elastase causes cellular insulin resistance and that deletion of neutrophil elastase in high-fat-diet–induced obese (DIO) mice leads to less tissue inflammation that is associated with lower adipose tissue neutrophil and macrophage content. These changes are accompanied by improved glucose tolerance and increased insulin sensitivity. Taken together, we show that neutrophils can be added to the extensive repertoire of immune cells that participate in inflammation-induced metabolic disease.

Programmed cell death in Caenorhabditis elegans requires activation of the caspase CED-3, which strictly depends on CED-4. CED-4 forms an octameric apoptosome, which binds the CED-3 zymogen and facilitates its autocatalytic maturation. Despite recent advances, major questions remain unanswered. Importantly, how CED-4 recognizes CED-3 and how such binding facilitates CED-3 activation remain completely unknown. Here we demonstrate that the L2' loop of CED-3 directly binds CED-4 and plays a major role in the formation of an active CED-4-CED-3 holoenzyme. The crystal structure of the CED-4 apoptosome bound to the L2' loop fragment of CED-3, determined at 3.2 Å resolution, reveals specific interactions between a stretch of five hydrophobic amino acids from CED-3 and a shallow surface pocket within the hutch of the funnel-shaped CED-4 apoptosome. Structure-guided biochemical analysis confirms the functional importance of the observed CED-4-CED-3 interface. Structural analysis together with published evidence strongly suggest a working model in which two molecules of CED-3 zymogen, through specific recognition, are forced into the hutch of the CED-4 apoptosome, consequently undergoing dimerization and autocatalytic maturation. The mechanism of CED-3 activation represents a major revision of the prevailing model for initiator caspase activation.

Bud dormancy in deciduous fruit trees is an important adaptive mechanism for their survival in cold climates. The WRKY genes participate in several developmental and physiological processes, including dormancy. However, the dormancy mechanisms of WRKY genes have not been studied in detail. We conducted a genome-wide analysis and identified 58 WRKY genes in peach. These putative genes were located on all eight chromosomes. In bioinformatics analyses, we compared the sequences of WRKY genes from peach, rice, and Arabidopsis. In a cluster analysis, the gene sequences formed three groups, of which group II was further divided into five subgroups. Gene structure was highly conserved within each group, especially in groups IId and III. Gene expression analyses by qRT-PCR showed that WRKY genes showed different expression patterns in peach buds during dormancy. The mean expression levels of six WRKY genes (Prupe.6G286000, Prupe.1G393000, Prupe.1G114800, Prupe.1G071400, Prupe.2G185100, and Prupe.2G307400) increased during endodormancy and decreased during ecodormancy, indicating that these six WRKY genes may play a role in dormancy in a perennial fruit tree. This information will be useful for selecting fruit trees with desirable dormancy characteristics or for manipulating dormancy in genetic engineering programs.

FGF21 plays a central role in energy, lipid, and glucose homeostasis. To characterize the pharmacologic effects of FGF21, we administered a long-acting FGF21 analog, PF-05231023, to obese cynomolgus monkeys. PF-05231023 caused a marked decrease in food intake that led to reduced body weight. To assess the effects of PF-05231023 in humans, we conducted a placebo-controlled, multiple ascending-dose study in overweight/obese subjects with type 2 diabetes. PF-05231023 treatment resulted in a significant decrease in body weight, improved plasma lipoprotein profile, and increased adiponectin levels. Importantly, there were no significant effects of PF-05231023 on glycemic control. PF-05231023 treatment led to dose-dependent changes in multiple markers of bone formation and resorption and elevated insulin-like growth factor 1. The favorable effects of PF-05231023 on body weight support further evaluation of this molecule for the treatment of obesity. Longer studies are needed to assess potential direct effects of FGF21 on bone in humans.

Small hive beetle (SHB), Aethina tumida can feed on honey, pollen and brood in honey bee colonies. It was endemic to Africa, but since 1996 has been detected in a number of countries worldwide, including Australia, Brazil, Canada, Italy, Mexico, South Korea, Philippines and the USA where it has had economic effects on local apiculture. To improve SHB identification, we obtained the first reference sequences from the DNA barcoding 5' COI gene region for SHB and some species of the family Nitidulidae associated with beehives. Phylogenetic analysis of SHB COI sequences (3' COI) revealed two divergent lineages, with those from Australia and USA being genetically different from the recent detection in Italy. Many countries, including New Zealand, are currently free from SHB, and require a rapid detection method for biosecurity. Here we present the development and validation of a real-time PCR assay for detection of SHB. The assay showed high specificity and sensitivity for detecting SHB, with no cross-reaction observed with closely related species, such as A. concolor. The real-time PCR is sensitive, detecting the target sequences up to 100 copies/µL. This assay should prove a useful biosecurity tool for rapid detection of SHB worldwide.

Peach is an ideal species for fruit tree research because of its small, fully sequenced genome. Chloroplast development is dependent on the tight cooperation between the nuclear and plastid genomes, and is regulated by GLK transcription factors. In this work, the pigment content was monitored and the chloroplast-to-chromoplast conversion during the fruit ripening was visualized by transmission electron microscopy. Localization and expression analyses showed that PpGLK1 was located in the nucleus and expressed mainly in the leaves and fruit skin. A transcriptome analysis showed that PpGLK1 and its target genes were significantly differentially expressed in ripening peach fruit skin. PpGLK1 silencing affected chlorophyll accumulation in peach leaves and fruits. Overexpression of PpGLK1 rescued the phenotypes of the Arabidopsis Atglk1Atglk2 double mutant and the tomato uniform ripening mutant. The results of a yeast two-hybrid analysis showed that PpGLK1 is autoactivated and that PpGLK1 (301-542 a.a.) interacted with PpARF5. Together, our results indicate that PpGLK1 regulates chloroplast development in green tissues in peach. Therefore, it may be a promising target gene for improving the production and quality of peach by genetic engineering and breeding approaches.

The Fuhrman and World Health Organization/International Society of Urological Pathology (WHO/ISUP) grading systems are widely used to predict survival for patients with conventional renal cell carcinoma. To determine the validity of nuclear grading systems (both the Fuhrman and the WHO/ISUP) and the individual components of the Fuhrman grading system in predicting the prognosis of Xp11.2 translocation renal cell carcinoma (Xp11.2 tRCC), we identified and followed up 47 patients with Xp11.2 tRCC in our center from January 2007 to June 2017. The Fuhrman and WHO/ISUP grading was reassigned by two pathologists. Nuclear size and shape were determined for each case based on the greatest degree of nuclear pleomorphism using image analysis software. Univariate and multivariate analyses were performed to evaluate the capacity of the grading systems and nuclear parameters to predict overall survival and progression-free survival. On univariate Cox regression analysis, the parameters of nuclear size were associated significantly with overall survival and progression-free survival, whereas the grading systems and the parameters of nuclear shape failed to reach a significant correlation. On multivariate analysis, however, none of the parameters was associated independently with survival. Our findings indicate that neither the Fuhrman nor the WHO/ISUP grading system is applicable to Xp11.2 tRCC. The assessment of nuclear size instead may be novel outcome predictors for patients with Xp11.2 tRCC.

Pharmacological administration of FGF21 analogues has shown robust body weight reduction and lipid profile improvement in both dysmetabolic animal models and metabolic disease patients. Here we report the design, optimization, and characterization of a long acting glyco-variant of FGF21. Using a combination of N-glycan engineering for enhanced protease resistance and improved solubility, Fc fusion for further half-life extension, and a single point mutation for improving manufacturability in Chinese Hamster Ovary cells, we created a novel FGF21 analogue, Fc-FGF21[R19V][N171] or PF-06645849, with substantially improved solubility and stability profile that is compatible with subcutaneous (SC) administration. In particular, it showed a low systemic clearance (0.243 mL/hr/kg) and long terminal half-life (~200 hours for intact protein) in cynomolgus monkeys that approaches those of monoclonal antibodies. Furthermore, the superior PK properties translated into robust improvement in glucose tolerance and the effects lasted 14 days post single SC dose in ob/ob mice. PF-06645849 also caused greater body weight loss in DIO mice at lower and less frequent SC doses, compared to previous FGF21 analogue PF-05231023. In summary, the overall PK/PD and pharmaceutical profile of PF-06645849 offers great potential for development as weekly to twice-monthly SC administered therapeutic for chronic treatment of metabolic diseases.

Phosvitin (Pv) is the principal phosphoprotein in chicken egg yolk and the most highly phosphorylated protein in nature. Pv is a good natural food antioxidant and emulsifier. However, the current extraction methods present disadvantages of complicated operation and are time-consuming. In this paper, Pv was extracted from the egg yolk by ultrasonic thermal-assisted extraction (UTAE). The effects of heating time, ultrasonic power and ultrasonic time on the extraction of Pv were investigated by a single factor. The purity of Pv, ratio of nitrogen to phosphorus (N/P), and activity were used as evaluation indexes. An efficient extraction of Pv was achieved when the sample was heated for 15 min at 80 °C and then processed for 10 min of ultrasonic treatment with an ultrasonic power of 600 W. Under optimal conditions, the purity and activity of Pv were 80% and 98%, respectively, whereas the ratio of N/P was 3.1. The obtained Pv was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Fluorescence analyses, fourier-transform infrared (FT-IR), and liquid chromatography-nanoelectrospray ionization mass spectrometry (Nano LC-ESI-MS/MS) analysis. The results showed there is no significant difference in the properties of Pv obtained by UTAE and Pv standard. The developed extraction approach is a simple, industrial compatible method without the use of any organic solvents.

Xp11.2 translocation renal cell carcinoma (tRCC) is mainly caused by translocation of the TFE3 gene located on chromosome Xp11.2 and is characterized by overexpression of the TFE3 fusion gene. Patients are diagnosed with tRCC usually before 45 years of age with poor prognosis. We investigated this disease using two tRCC cell lines, UOK109 and UOK120, in this study.

β-Hairpins in enzyme, a kind of special protein with catalytic functions, contain many binding sites which are essential for the functions of enzyme. With the increasing number of observed enzyme protein sequences, it is of especial importance to use bioinformatics techniques to quickly and accurately identify the β-hairpin in enzyme protein for further advanced annotation of structure and function of enzyme. In this work, the proposed method was trained and tested on a non-redundant enzyme β-hairpin database containing 2818 β-hairpins and 1098 non-β-hairpins. With 5-fold cross-validation on the training dataset, the overall accuracy of 90.08% and Matthew's correlation coefficient (Mcc) of 0.74 were obtained, while on the independent test dataset, the overall accuracy of 88.93% and Mcc of 0.76 were achieved. Furthermore, the method was validated on 845 β-hairpins with ligand binding sites. With 5-fold cross-validation on the training dataset and independent test on the test dataset, the overall accuracies were 85.82% (Mcc of 0.71) and 84.78% (Mcc of 0.70), respectively. With an integration of mRMR feature selection and SVM algorithm, a reasonable high accuracy was achieved, indicating the method to be an effective tool for the further studies of β-hairpins in enzymes structure. Additionally, as a novelty for function prediction of enzymes, β-hairpins with ligand binding sites were predicted. Based on this work, a web server was constructed to predict β-hairpin motifs in enzymes (http://202.207.29.251:8080/).

To detect γ-Glutamyl Transpeptidase (GGT) activity in vitro and in vivo, a bioluminescence probe with high sensitivity and specificity was well designed and synthesized. This probe can be recognized by GGT and release strong bioluminescence with its further reaction with luciferase. The performance of this probe was demonstrated in vitro and in cells. Finally, we applied the probe for detection of GGT activity in xenograft model.

Humans are inevitably exposed to ubiquitous phthalate esters (PAEs). In utero exposure to di-n-butyl phthalate (DBP) induces abnormal development of the testis and reproductive tract in male offspring, which correspond closely with the human condition of testicular dysgenesis syndrome (TDS)-like syndrome. However, the underlying mechanisms have not been elucidated in detail. In this study, pregnant rats were orally exposed to either corn oil (controls) or DBP at three different doses by gavage during Gestational Days 12.5-21.5. Pathological examinations were performed for toxicity evaluation. Proliferation and apoptosis related proteins (ras related dexamethasone induced 1 (Rasd1), mitogen-activated protein kinase kinases1/2 (MEK1/2), Bcl-2, and Bax) were measured for mechanisms exploration. The results showed that different doses of DBP caused male developmental and reproductive toxicity in rats, including the decrease of anogenital distance (AGD), the histological damage of testis, and apoptosis of seminiferous tubule cells. Our data suggested that DBP played chronic and continuous toxic roles on male reproductive system by disrupting expression of Rasd1 and MEK1/2 as well as Bcl-2/Bax ratio. Further research is warranted.

Mucor irregularis is an emerging fungal pathogen that cause cutaneous infection and could cause death. However, little is known about its mechanism of pathogenesis. There is evidence suggesting virulence vary with mating types in fungi, including the Mucorales. Here, we characterized the mating type locus of M. irregularis and the mating type ratio of 17 clinical isolates in China. Genomic data indicated M. irregularis is heterothallic having two mating types - bearing either SexP or SexM allele. Also, we employed a mice model to study the inflammation and pathological effects of different mating types. The comparison of the inflammatory response, cytokine profiles and Th-1, Th-2 and Th-17 cells numbers in each mating type treated mice showed that the severity and disease progress were enhanced in (+) mating type treated mice. One (+/0) mutant strain, with multiple mutations at the mating locus, had defects in sexual mating ability but appeared to be more virulent than the (-) mating type. Although (+) mating type appeared to be more virulent, most of our clinical isolates presented belonged to (-) mating type. Our findings support the involvement of MAT genes in sexual fertility, and the influence of mating type on the severity of cutaneous infection.

Existing literature indicated electronic cigarette users (vapers) have impaired immune response that might increase vulnerability to coronavirus disease 2019 (COVID-19) infection and death. However, whether vapers are more susceptible to COVID-19 is unknown. Using integrated data in each US state from the 2018 Behavioral Risk Factor Surveillance System (BRFSS), United States Census Bureau and the 1Point3Acres.com website, generalized estimating equation (GEE) models with negative binomial distribution assumption and log link functions were used to examine the association of statewide e-cigarette use prevalence with number of COVID-19 cases and deaths in the US on a state level from January 21, 2020 to April 25, 2020. The weighted proportion of vapers who used e-cigarettes every day or some days ranged from 2.86% to 6.42% for US states. Statistically significant associations were observed between the weighted proportion of vapers and number of COVID-19 cases as well as COVID-19 deaths in the US after adjusting for the weighted proportion of smokers and other significant covariates in the GEE models. With every one percent increase in weighted proportion of vapers in each state, the number of COVID-19 cases increase by 0.3139 (95% CI: 0.0554-0.5723) and the number of COVID-19 deaths increase by 0.3730 (95% CI: 0.0815-0.6646) in log scale in each US state. The positive associations between the proportion of vapers and the number of COVID-19 cases and deaths in each US state in this ecological study suggest an increased susceptibility of vapers to COVID-19 on a state level and warrants further investigation.

Soybean yield is a complex quantitative trait, which is greatly affected by environmental conditions. The main objective of this study is not only to identify specific traits contributing to yield in different latitudes, which can be further used in breeding, but also to identify the outperforming varieties, as this can help to select new lines with these traits. One hundred and seventy-three soybean genotypes were tested in three different ecological environments, including Harbin, Changchun, and Shenyang in China during 2015-2016 cropping seasons. The evaluation on the different agronomic and physiological traits indicated that the soybean varieties with higher plant height, more nodes of main stem, branches, pods, grains, and 100-grain weight, or longer growth periods may have higher yield. Pods, grains and 100-grain weight can be used as direct selection criteria for yield increase, and likewise the other traits such as plant height, nodes of main stem, branches, growth periods indirectly affected yield by affecting the three traits above. The effect of genotype × environment (G × E) interaction on different agronomic traits was significant. The representativeness and discriminability for grains yield per plant was the most significant in Harbin, which could be used to screen varieties with high yield and wider adaptability. Genotype "Suinong 1" was considered stable with higher value of grain yield per plant than other genotypes used in this study. As the yield of certain soybean cultivars may be significantly reduced if they are grown in a region as little as 2°N beyond its normal cultivation latitudes, therefore, the identification and analysis on the stable and widely adaptive soybean genotypes would be very important, and it would provide the significant reference accordance of soybean variety selection for the soybean breeders.

Mixed lineage kinase-4 (MLK-4) is an important member of the mixed-lineage family of kinases that regulates the extracellular signal-regulated kinases and c-Jun N-terminal kinase (JNK) signaling pathways. The functions and mechanisms of MLK-4 in cancer initiation and progression have not been well understood. The present study investigated the expression, function and regulatory mechanism of MLK-4 in gastric carcinoma cells. Biochemical data indicated that normal MLK-4 was downregulated, which exerted dominant negative effects on gastric carcinoma cell viability, migration and invasion. The experimental data demonstrated that MLK-4 supplement abrogated activity of these mutants and induced inhibitory effects on gastric carcinoma cell viabilty, migration and invasion in vitro and in vivo. In addition, to determine the regulatory mechanism of MLK-4, its signaling pathway was assessed in gastric carcinoma cancer cells by regulating MLK-4. The present observations indicated that restoring MLK-4 activity by supplemental MLK-4 reduced gastric carcinoma cell colony formation in vitro and suppressed tumor viability, migration and invasion in vivo. The results of the present study indicated that MLK-4 may be a potential protein for targeting gastric carcinoma by suppressing kinases, which may lead to reduction of JNK signaling and enhance therapeutic efficacy in gastric carcinoma.

Di-n-butyl phthalate (DBP) is considered as a potential modifier of puberty. However, different results indicate that DBP plays an accelerated, delayed, or neutral role in the initiation of puberty. Furthermore, whether the effect of DBP on puberty will disrupt the function of reproductive system in the adults is still ambiguous. Therefore, we aimed to investigate the effect of maternal exposure to DBP on the onset of puberty in male offspring mice and the subsequent changes in the development of reproductive system. Here, pregnant mice were treated with 0 (control), 50, 250, or 500 mg/kg/day DBP in 1 mL/kg corn oil administered daily by oral gavage from gestation day (GD) 12.5 to parturition. Compared with the control group, the 50 mg/kg/day DBP group accelerated puberty onset and testicular development were quite remarkable in male offspring mice during early puberty. Furthermore, in 22-day male offspring mice, 50 mg/kg/day DBP induced increased levels of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone in serum, and promoted the expression of steroidogenesis-related genes in the testes. Testicular Leydig cells (LCs) were isolated from the testes of 3-week-old mice and treated with 0 (control), 0.1, 1 mM monobutyl phthalate (MBP, the active metabolite of DBP) for 24 h. Consistent with the in vivo results, the expression of steroidogenesis-related genes and testosterone production were increased in LCs following exposure to 0.1 mM MBP. In adulthood, testes of the male offspring mice exposed to all doses of DBP exhibited adverse morphology compared with the control group. These results demonstrated that maternal exposure to 50 mg/kg/day DBP induced earlier puberty and precocious development of the testis, and eventually damaged the reproductive system in the later life.

The present systematic review and meta-analysis was undertaken to evaluate the effects of acupuncture in women with breast cancer (BC), focusing on patient-reported outcomes (PROs).