Cafestol inhibits colon cancer cell proliferation and tumor growth in xenograft mice by activating LKB1/AMPK/ULK1-dependent autophagy.

Chemotherapy failure in colorectal cancer patients is the major cause of recurrence and poor prognosis. As a result, there is an urgent need to develop drugs that have a good chemotherapy effect while also being extremely safe. In this study, we found cafestol inhibited colon cancer growth and HCT116 proliferation in vivo and in vitro, and improved the composition of intestinal flora. Further metabolomic data showed that autophagy and AMPK pathways were involved in the process of cafestol's anti-colon cancer effects. The functional validation studies revealed that cafestol increased autophagy vesicles and LC3B-II levels. The autophagic flux induced by cafestol was prevented by using BafA1. The autophagy inhibitor 3-MA blocked the cafestol-induced increase in LC3B-II and cell proliferation inhibition. Then we found that cafestol induced the increased expressions of LKB1, AMPK, ULK1, p-LKB1, p-AMPK, and p-ULK1 proteins in vivo and in vitro. Using the siRNA targeted to the Lkb1 gene, the levels of AMPK, ULK1, and LC3B-II were suppressed under cafestol treatment. These results indicated that the effect of cafestol is through regulating LKB1/AMPK/ULK1 pathway-mediated autophagic death. Finally, a correlation matrix of the microbiome and autophagy-related proteins was conducted. We found that cafestol-induced autophagic protein expression was positively correlated with the beneficial intestinal bacteria (Muribaculaceae, Bacteroides, Prevotellacece, and Alloprevotella) and negatively correlated with the hazardous bacteria. Conclusions: This study found that cafestol inhibited colon cancer in vitro and in vivo by the mechanism that may be related to LKB1/AMPK/ULK1 pathway-mediated autophagic cell death and improved intestinal microenvironment.

Life's Essential 8, Life's Simple 7 and the odds of hyperuricaemia: results from the China Multi-Ethnic Cohort Study.

Objective: Life's Essential 8 (LE8) is a new comprehensive metric based on Life's Simple 7 (LS7). Few studies have investigated the association between LE8 and the odds of hyperuricaemia (HUA). This study examined the association between LE8, LS7 with odds of HUA. Methods: We cross-sectionally analysed data from the China Multi-Ethnic Cohort (CMEC) study. LE8 and LS7 were categorized as low, moderate and high. The CMEC provided an ideal and unique opportunity to characterize the association between LE8, LS7 and the odds of HUA. Results: Of the 89 823 participants, 14 562 (16.2%) had HUA. A high level of LE8 was associated with lower odds of HUA after full adjustment. The adjusted odds ratios (ORs) were 1 (reference), 0.70 (95% CI 0.67, 0.73) and 0.45 (0.42, 0.48) across low, moderate and high LE8 groups, respectively (Ptrend < 0.001). Similar results were observed in LS7 and HUA. The adjusted ORs were 1 (reference), 0.68 (95% CI 0.65, 0.71) and 0.46 (95% CI 0.43, 0.49) across low, moderate and high LS7 groups, respectively (Ptrend < 0.001). There were significant interactions between LE8 and age, gender, ethnicity and drinking habits on HUA. Receiver operating characteristics analysis showed that the area under the curve for LE8 and LS7 were similar (0.638 and 0.635, respectively). Conclusion: This study indicated a clearly inverse gradient association between the cardiovascular health metrics LE8 and LS7 and the odds of HUA.

Associations of long-term exposure to particulate matter with gallstone risks in Chinese adults: A large cross-sectional study.

BACKGROUND: Epidemiological evidence regarding the relation of exposure to ambient particulate matter (PM) with gallstone disease (GSD) risk remains lacking. We tested the hypothesis that long-term exposure to PM is related to the development of GSD and that dyslipidemia can mediate the effect of PM-associated GSD formation. METHODS: We extracted related information on the basis of the baseline survey of the China Multi-Ethnic Cohort Study. The exposure levels of PM (PM1, PM2.5, and PM10) were assessed by validated spatiotemporal models. The relation of exposure to ambient PM with GSD risks was analyzed by non-conditional logistic regression models. Additionally, mediation analysis was conducted to assess whether dyslipidemia was related to the relation of PM exposure with GSD risks. RESULTS: A total of 72,893 participants were included. Increased ambient PM exposure was positively associated with a higher GSD risk, with ORs (and 95% CI) of 1.17 (1.06, 1.28), 1.10 (1.05, 1.15), and 1.07 (1.04, 1.10) for every 10 µg/m3 increment in PM1, PM2.5, and PM10, separately. The association was more remarkable in males, drinkers, and central obesity participants. Dyslipidemia significantly mediated the association between PM and GSD, with mediating proportions of 5.37%, 9.13%, and 7.66% in PM1, PM2.5, and PM10, respectively. CONCLUSION: Exposure to PM may relate to the increased risk of GSD in Chinese adults, especially among males, drinkers, and central obesity participants. Dyslipidemia may partially mediate the effect of PM-associated GSD development. Our results might provide epidemiological evidence for the progression of GSD related to PM and give new insights into GSD prevention and screening priorities.

Mediation Effect of Obesity on the Association of Age at Menarche With Blood Pressure Among Women in Southwest China.

Background Previous studies have been inconsistent about the association between age at menarche and high blood pressure. Little is known about such association across a wide range of menarcheal ages in less developed ethnic minority regions in China. We aimed to explore the association between age at menarche and high blood pressure (BP; ≥140/90 mm Hg) and to examine the mediating effect of obesity and the moderating effect of menopausal status on this association. Methods and Results A total of 45 868 women from the baseline data of the CMEC (China Multi-Ethnic Cohort) were included in this study. Binary logistic regression was used to analyze the relationship between age at menarche and high BP, and the mediation model was used to evaluate the mediating effects of body mass index and waist circumference on the association of age at menarche with high BP. The mean age at enrollment and age at menarche of participants in our study were 49.3 (SD=10.7) and 14.7 (SD=2.1) years, respectively. Late menarche was associated with a lower risk of high BP (odds ratio, 0.831 [95% CI, 0.728-0.950]). The risk of high BP decreased by 3.1% with each year's delay in the onset of menarche (P for trend <0.001). Body mass index and waist circumference could partially mediate the association of age at menarche and high BP with the indirect effect of body mass index (odds ratio, 0.998 [95% CI, 0.997-0.998]) and waist circumference (odds ratio, 0.999 [95% CI, 0.998-0.999]). In addition, the mediation effects were modified by the status of menopause. Conclusions Women with late menarche have a lower risk of high BP, and obesity could be one of the important mediators. Obesity prevention is an efficient strategy to reduce the association between age at menarche and high BP, especially in premenopausal women.

Correlation between adipocyte fatty acid binding protein and glucose dysregulation is closely associated with obesity and metabolic syndrome: A cohort of Han Chinese population from Yunnan plateau.

The present study investigated the correlation of plasma A-FABP with glucose dysregulation under different body mass index (BMI) and metabolic states in a Han Chinese population from Yunnan plateau. We cross-sectionally analyzed data from the China Multi Ethnic Cohort, Yunnan province. Participants were divided into two groups. Group A contained 297 obese individuals with metabolic syndrome (MetS). Group B contained 326 age-, sex-, and region-matched normal BMI subjects without MetS. Glucose dysregulation was defined as elevated fasting plasma glucose (FPG) (FPG ≥ 5.6 mmol/L or current use of oral hypoglycemic agents or insulin). Circulating A-FABP were assayed by ELISA method. Binary and multiple regression analyses were preformed to evaluate the correlation between A-FABP and glucose dysregulation. Plasma A-FABP level was significantly higher in group A compared with group B (p < 0.001). Plasma A-FABP level correlated positively with elevated FPG in group A (r = 0.120, p = 0.039), but negatively with elevated FPG in group B (r = -0.115, p = 0.039). Multiple logistic regression analysis revealed that A-FABP was an independent predictor for elevated FPG in group A (ß, 0.028; 95% CI, 1.001-1.056; p < 0.05), but not in group B (ß, -0.008; 95% CI, 0.882-1.117; p > 0.05). In this study, A-FABP was an independent risk factor for glucose dysregulation in obese individuals with MetS living in the Yunnan plateau, but not for those without obesity and MetS.

Cross-sectional association between gamma-glutamyl transferase and hyperuricaemia: the China Multi-Ethinic Cohort (CMEC) study.

OBJECTIVES: Several studies have demonstrated the association between gamma-glutamyl transferase (GGT) and hyperuricaemia, but little is known about such relation in less-developed ethnic minority regions. DESIGN: We cross-sectionally analysed data from the China Multi-Ethnic Cohort (Yunnan region). SETTING: Cross-sectional study. PARTICIPANTS: 22 020 participants aged 30-79 years from Han ethnicity, Yi ethnicity and Bai ethnicity. OUTCOMES: The serum level of uric acid, GGT and other metabolic parameters were tested. Weight, height and blood pressure were measured. Smoking, drinking, ethnicity, education and medical history were obtained from questionnaires. RESULTS: In the crude model, compared with the lowest quintile, the second, third, fourth and fifth quintiles of serum GGT exhibited a positive association with hyperuricaemia risk (OR=1.69, 2.90, 4.34 and 7.70, 95% CI=1.42 to 2.01, 2.47 to 3.42, 3.71 to 5.09 and 6.60 to 8.98, respectively, p-trend<0.0001). In fully adjusted model, compared with the lowest quintile, the second, third, fourth and fifth quintiles of serum GGT also exhibited a positive association with hyperuricaemia risk (OR=1.26, 1.68, 2.02 and 3.02, 95% CI=1.04 to 1.51, 1.40 to 2.00, 1.69 to 2.42 and 2.51 to 3.64, respectively, p-trend<0.0001). Logistic regression model was conducted separately in ethnic groups. Compared with first quintile, the highest GGT level were related to higher risk of hyperuricaemia in three ethnic groups (OR (95% CI): 2.89 (2.26 to 3.68), 2.81 (1.93 to 4.11) and 3.04 (1.91 to 4.84) for Han, Yi and Bai ethnicity, respectively, p-trend <0.0001). The relationship between GGT and hyperuricaemia was also observed in different age groups or gender groups. CONCLUSIONS: High serum GGT level was related to a higher risk of hyperuricaemia in less-developed ethnic minority regions in China.

Association of Sedentary Behavior and Physical Activity With Hyperuricemia and Sex Differences: Results From the China Multi-Ethnic Cohort Study.

OBJECTIVE: To determine the association of physical activity (PA) and sedentary time (ST; leisure and total ST), commuting mode with hyperuricemia in a multiethnic Chinese population, and to analyze the difference between sexes. METHODS: Baseline data were analyzed from 22,094 participants from the China Multi-Ethnic Cohort study in the Yunnan region, China. PA and sedentary behavior were assessed through questionnaires. Hyperuricemia was defined as serum urate > 7.0 mg/dL among men and > 6.0 mg/dL among women. A restricted cubic spline (RCS) was created to model the possible nonlinear relationship of PA and ST with hyperuricemia. Logistic regression was used to estimate the odds ratio (OR) and 95% CI. RESULTS: Hyperuricemia prevalence in the observed population was 15.5% (men 25.5%, women 10.7%). Compared to those with light PA, participants with moderate-to-vigorous PA had lower odds of hyperuricemia (adjusted ORs were 0.85 [95% CI 0.77-0.94] and 0.88 [95% CI 0.79-0.97]). However, RCS showed a U-shaped nonlinear relationship between PA and hyperuricemia, and a linear relationship between hyperuricemia prevalence and increasing ST. Total ST ≥ 4 hours/day increased the risk of hyperuricemia in women but not in men. Mode of transportation revealed that sedentary behavior increased the risk of hyperuricemia, but there were inconsistent results based on sex. CONCLUSION: Moderate PA may be more beneficial in reducing the risk of hyperuricemia. Reducing ST may have a greater effect on preventing hyperuricemia in females than in males.

Evidence for Persistent Heteroplasmy and Ancient Recombination in the Mitochondrial Genomes of the Edible Yellow Chanterelles From Southwestern China and Europe.

Mitochondrial genes and genomes have patterns of inheritance that are distinctly different from those of nuclear genes and genomes. In nature, the mitochondrial genomes in eukaryotes are generally considered non-recombining and homoplasmic. If heteroplasmy and recombination exist, they are typically very limited in both space and time. Here we show that mitochondrial heteroplasmy and recombination may not be limited to a specific population nor exit only transiently in the basidiomycete Cantharellus cibarius and related species. These edible yellow chanterelles are an ecologically very important group of fungi and among the most prominent wild edible mushrooms in the Northern Hemisphere. At present, very little is known about the genetics and population biology of these fungia cross large geographical distances. Our study here analyzed a total of 363 specimens of edible yellow chanterelles from 24 geographic locations in Yunnan in southwestern China and six geographic locations in five countries in Europe. For each mushroom sample, we obtained the DNA sequences at two genes, one in the nuclear genome and one in the mitochondrial genome. Our analyses of the nuclear gene, translation elongation factor 1-alpha (tef-1) and the DNA barcode of C. cibarius and related species, suggested these samples belong to four known species and five potential new species. Interestingly, analyses of the mitochondrial ATP synthase subunit 6 (atp6) gene fragment revealed evidence of heteroplasmy in two geographic samples in Yunnan and recombination within the two new putative species in Yunnan. Specifically, all four possible haplotypes at two polymorphic nucleotide sites within the mitochondrial atp6 gene were found distributed across several geographic locations in Yunnan. Furthermore, these four haplotypes were broadly distributed across multiple phylogenetic clades constructed based on nuclear tef-1 sequences. Our results suggest that heteroplasmy and mitochondrial recombination might have happened repeatedly during the evolution of the yellow chanterelles. Together, our results suggest that the edible yellow chanterelles represent an excellent system from which to study the evolution of mitochondrial-nuclear genome relationships.

The Association Between Long-term Exposure to Ambient Air Pollution and Bone Strength in China.

CONTEXT: Evidence regarding the association of long-term exposure to air pollution on bone strength or osteoporosis is rare, especially in highly polluted low- and middle-income countries. Little is known about whether the association between air pollution and bone strength changes at different bone strength distributions. OBJECTIVE: Using the baseline data from the China Multi-Ethnic Cohort, we investigated the association between long-term air pollution exposure and bone strength. METHODS: We used multiple linear models to estimate the association between air pollution and bone strength, and we conducted quantile regression models to investigate the variation of this association in the distribution of bone strength. The 3-year concentrations of PM1, PM2.5, PM10, and NO2 for each participant were assessed using spatial statistical models. Bone strength was expressed by the calcaneus quantitative ultrasound index (QUI) measured by quantitative ultrasound, with higher QUI values indicating greater bone strength. RESULTS: A total of 66 598 participants were included. Our analysis shows that every 10 µg/m3 increase in 3-year average PM1, PM2.5, PM10, and NO2 was associated with -5.38 units (95% CI: -6.17, -4.60), -1.89 units (95% CI: -2.33, -1.44), -0.77 units (95% CI: -1.08, -0.47), and -2.02 units (95% CI: -2.32, -1.71) changes in the QUI, respectively. In addition, populations with higher bone strength may be more susceptible to air pollution. CONCLUSION: Long-term exposure to PM1, PM2.5, PM10, and NO2 was significantly associated with decreased bone strength in southwestern China adults. Air pollution exposure has a more substantial adverse effect on bones among populations with higher bone strength.

Exploring the Species Diversity of Edible Mushrooms in Yunnan, Southwestern China, by DNA Barcoding.

Yunnan Province, China, is famous for its abundant wild edible mushroom diversity and a rich source of the world's wild mushroom trade markets. However, much remains unknown about the diversity of edible mushrooms, including the number of wild edible mushroom species and their distributions. In this study, we collected and analyzed 3585 mushroom samples from wild mushroom markets in 35 counties across Yunnan Province from 2010 to 2019. Among these samples, we successfully obtained the DNA barcode sequences from 2198 samples. Sequence comparisons revealed that these 2198 samples likely belonged to 159 known species in 56 different genera, 31 families, 11 orders, 2 classes, and 2 phyla. Significantly, 51.13% of these samples had sequence similarities to known species at lower than 97%, likely representing new taxa. Further phylogenetic analyses on several common mushroom groups including 1536 internal transcribed spacer (ITS) sequences suggested the existence of 20 new (cryptic) species in these groups. The extensive new and cryptic species diversity in wild mushroom markets in Yunnan calls for greater attention for the conservation and utilization of these resources. Our results on both the distinct barcode sequences and the distributions of these sequences should facilitate new mushroom species discovery and forensic authentication of high-valued mushrooms and contribute to the scientific inventory for the management of wild mushroom markets.

Daytime Napping Duration Is Positively Associated With Risk of Hyperuricemia in a Chinese Population.

CONTEXT: Loss of sleep or disturbance of sleep-wake cycles has been related to metabolic impairments. However, few studies have investigated the association between daily sleep duration and hyperuricemia. OBJECTIVE: We investigated daily sleep duration (daytime napping and nocturnal sleep) with hyperuricemia risk. METHODS: We cross-sectionally analyzed data from the China Multi-Ethnic Cohort (CMEC), Yunnan region. A total of 22 038 participants aged 30 to 79 years were recruited in 2018. Hyperuricemia was defined as serum uric acid (SUA) above 7.0 mg/dL in men and above 6.0 mg/dL in women. Outcomes were associations between daily sleep duration and hyperuricemia. RESULTS: We found that the longest daytime napping duration was associated with a higher risk of hyperuricemia in the crude model (odds ratio [OR] [95% CI], 2.22 [1.88-2.61], P < .001) and in a multivariable adjustment model (OR, 1.69; 95% CI, 1.41-2.01, P < .001) after adjusting for demographic, sleep habits, and metabolic risk factors. The association was moderately attenuated with additionally adjusted for serum creatinine (OR, 1.54; 95% CI, 1.28-1.86, P < .001). Longer daytime napping duration was also related to higher risk of hyperuricemia combined with metabolic syndrome (MetS). Respondents in the group with daytime napping duration greater than or equal to 90 minutes presented with a higher risk of hyperuricemia combined with MetS (OR, 1.39; 95% CI, 1.06-1.79; P < .001) in the fully adjusted model. We did not observe any relation between nocturnal sleep duration and risk of hyperuricemia in the study. CONCLUSION: Longer daytime napping duration (but not nocturnal sleep duration) was independently associated with risk of hyperuricemia in a Chinese population.

Aeromicrobium chenweiae sp. nov. and Aeromicrobium yanjiei sp. nov., isolated from Tibetan antelope (Pantholops hodgsonii) and plateau pika (Ochotona curzoniae), respectively.

Four novel strains (592T, S592, MF47T and SMF47) were isolated from Tibetan antelopes (Pantholops hodgsonii) and plateau pikas (Ochotona curzoniae), respectively. The cells were aerobic, non-motile, Gram-stain- and catalase-positive, rod-shaped bacteria. The 16S rRNA gene sequences of the four strains showed highest similarities to Aeromicrobium fastidiosum DSM 10552T (98.1, 98.6, 98.7 and 98.7 %, respectively), and the phylogenetic analyses based on 16S rRNA gene and genomic sequences indicated that strains 592T and MF47T represent two novel species. The four isolates produced acid from l-rhamnose, d-xylose and cellobiose, but were unable to reduce nitrate. The DNA G+C contents of strains 592T and MF47T were 70.3 and 69.8 mol%, respectively. The digital DNA-DNA hybridization value between strains 592T and MF47T was 32.6 %, lower than the threshold of 70 %, indicating they belong to different species. The four strains' genomes displayed less than 24.6 % DNA-DNA relatedness with all available genomes of the genus Aeromicrobium in the NCBI database, including Aeromicrobium fastidiosum NBRC 14897T and Aeromicrobium ginsengisoli JCM 14732T. The major fatty acids of the four strains were C18 : 1 ω9c and C18 : 0 10-methyl, and the main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. The predominant respiratory quinones were MK-9(H4) and MK-8(H4). The cell-wall peptidoglycan contained ll-diaminopimelic acid. Based on these genotypic, phenotypic and biochemical analyses, it is proposed that the four unidentified bacteria be classified as two novel species, Aeromicrobium chenweiae sp. nov. and Aeromicrobium yanjiei sp. nov. The type strains are 592T (=CGMCC1.16526T=DSM 106289T) and MF47T (=CGMCC 1.17444T=JCM 33790T), respectively.

Agromyces badenianii sp. nov., isolated from plateau pika (Ochotona curzoniae).

Two aerobic, Gram-stain-positive, catalase-positive, non-motile and rod-shaped bacterial strains, designated MF30-AT and MF845, were isolated from the intestinal contents of plateau pika collected from the Qinghai-Tibet Plateau. Optimal growth of these two strains was observed under aerobic conditions at pH 7.0 and 28 °C. The 16S rRNA gene sequences of the isolates had highest similarities of 98.5 and 98.4 % to Agromyces fucosus, respectively. In the 16S rRNA gene and polygenetic trees, strains MF30-AT and MF845 were clearly distinct from other species. The two strains could not produce acid from arbutin, d-fructose, D-sucrose, glycogen, salicin or starch. Production of ß-glucosidase by these strains was negative. The major fatty acids of these strains were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. Strain MF30-AT contained galactose, rhamnose and ribose as cell wall sugars and MK-12 and MK-11 as predominant menaquinones. The major polar lipids in strain MF30-AT were diphosphatidylglycerol, phosphatidylglycerol and a glycolipid, while the peptidoglycan contained alanine, glutamic acid, glycine and 2,4-diaminobutyric acid. The G+C contents of the DNA of strains MF30-AT and MF845 were 69.8 mol% and 69.7 mol%, respectively. The average nucleotide identity and digital DNA-DNA relatedness values of the two strains with all available genomes of the genus Agromyces were far below the respective thresholds of 95 and 70 %, respectively. All genotypic and phenotypic data indicated that strains MF30-AT and MF845 should be classified as novel members of the genus Agromyces, for which the name Agromyces badenianii sp. nov. is proposed. The type strain is MF30-AT (=CGMCC 1.16469T=DSM 106183T).

Analyses of the Global Multilocus Genotypes of the Human Pathogenic Yeast Candida tropicalis.

Candida tropicalis is a globally distributed human pathogenic yeast, especially prevalent in tropical and sub-tropical regions. Over the last several decades, a large number of studies have been published on the genetic diversity and molecular epidemiology of C. tropicalis from different parts of the world. However, the global pattern of genetic variation remains largely unknown. Here we analyzed the published multilocus sequence data at six loci for 876 isolates from 16 countries representing five continents. Our results showed that 280 of the 2677 (10.5%) analyzed nucleotides were polymorphic, resulting in a mean of 82 (a range of 38-150) genotypes per locus and a total of 633 combined diploid sequence types (DSTs). Among these, 93 combined DSTs were shared by 336 strains, including 10 by strains from different continents. Analysis of Molecular Variance (AMOVA) showed that 89% of the observed genetic variations were found within regional and national populations while < 10% was due to among-country separations. Pairwise geographic population analyses showed overall low but statistically significant genetic differentiation between most geographic populations, with the Singaporean and Indian populations being the most distinct from other populations. However, the Mantel test showed no significant correlation between genetic distance and geographic distance among the geographic populations. Consistent with high genetic variation within and limited variations among geographic populations, results from STRUCTURE analyses showed that the 876 isolates could be grouped into 15 genetic clusters, with each cluster having a broad geographic distribution. Together, our results suggest frequent gene flows among certain regional, national, and continental populations of C. tropicalis, resulting in abundant regional and national genetic diversities of this important human fungal pathogen.

Secretion of interleukin-6 by bone marrow mesenchymal stem cells promotes metastasis in hepatocellular carcinoma.

Mesenchymal stem cells (MSCs) interact with tumor cells and regulate tumorigenesis and metastasis. As one of the important components of the tumor microenvironment, MSC-secreted cytokines play a critical role in cancer development. However, whether and how bone marrow MSCs (BMSCs) and their secreted cytokines participate in hepatocellular carcinoma (HCC) progression, still remains largely unknown. In the present study, we first measured the concentration of interleukin-6 (IL-6) in BMSC conditioned medium (BMSC-CM). Next, we assessed the changes of invasion ability in response to treatment of BMSC-CM or recombinant IL-6 in two human HCC cell lines Bel-7404 and HepG2. Then we analyzed the level of key components of the IL-6 signal pathway, including IL-6 receptor and signal transducer (i.e. IL-6R and gp130), a transcription factor STAT3 (signal transducer and activator of transcription 3), as well as its target genes BCL2, CCND1, MCL1 and MMP2, in BMSC-CM or recombinant IL-6 treated Bel-7404 and HepG2 cells. Results showed that a considerable amount of IL-6 was secreted by BMSCs, and BMSC-CM markedly elevated Bel-7404 cell invasion rate and stimulated the signal transduction of IL-6/STAT3 pathway. Neutralizing the secreted IL-6 bioactivity by the anti-IL-6 antibody diminished the invasion-promoting effect and down-regulated IL-6/STAT3 pathway of BMSC-CM treated Bel-7404 cells. In conclusion, we found that BMSCs may activate the IL-6/STAT3 signaling pathway and promote cell invasion in Bel-7404 cells, suggesting that this protumor effect should be seriously considered before clinical application of MSC-mediated cancer therapy.

Frequent heteroplasmy and recombination in the mitochondrial genomes of the basidiomycete mushroom Thelephora ganbajun.

In the majority of sexual eukaryotes, the mitochondrial genomes are inherited uniparentally. As a result, individual organisms are homoplasmic, containing mitochondrial DNA (mtDNA) from a single parent. Here we analyzed the mitochondrial genotypes in Clade I of the gourmet mushroom Thelephora ganbajun from its broad geographic distribution range. A total of 299 isolates from 28 geographic locations were sequenced at three mitochondrial loci: the mitochondrial small ribosomal RNA gene, and the cytochrome c oxidase subunits I (COX1) and III (COX3) genes. Quantitative PCR analyses showed that the strains had about 60-160 copies of mitochondrial genomes per cell. Interestingly, while no evidence of heteroplasmy was found at the 12S rRNA gene, 262 of the 299 isolates had clear evidence of heterogeneity at either the COX1 (261 isolates) or COX3 (12 isolates) gene fragments. The COX1 heteroplasmy was characterized by two types of introns residing at different sites of the same region and at different frequencies among the isolates. Allelic association analyses of the observed mitochondrial polymorphic nucleotide sites suggest that mtDNA recombination is common in natural populations of this fungus. Our results contrast the prevailing view that heteroplasmy, if exists, is only transient in basidiomycete fungi.

Human lactoferrin efficiently targeted into caprine beta-lactoglobulin locus with transcription activator-like effector nucleases.

OBJECTIVE: To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF) with transcription activator-like effector nucleases. METHODS: TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG) locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT). RESULTS: The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23%) were confirmed pregnant at 30 d. In second round SCNT, 7 (53%), 4 (31%), and 3 (23%) recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d. CONCLUSION: This finding signifies the combined use of TALENs and SCNT can generate bi-allelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.

Efficient TALEN-mediated myostatin gene editing in goats.

BACKGROUND: Myostatin (MSTN) encodes a negative regulator of skeletal muscle mass that might have applications for promoting muscle growth in livestock. In this study, we aimed to test whether targeted MSTN editing, mediated by transcription activator-like effector nucleases (TALENs), is a viable approach to create myostatin-modified goats (Capra hircus). RESULTS: We obtained a pair of TALENs (MTAL-2) that could recognize and cut the targeted MSTN site in the goat genome. Fibroblasts from pedigreed goats were co-transfected with MTAL-2, and 272 monoclonal cell strains were confirmed to have mono- or bi-allelic mutations in MSTN. Ten cell strains with different genotypes were used as donor cells for somatic cell nuclear transfer, which produced three cloned kids (K179/MSTN(-/-), K52-2/MSTN (+/-), and K52-1/MSTN (+/+)). CONCLUSIONS: The results suggested that the MTAL-2 could disrupt MSTN efficiently in the goat genome. The mutated somatic cells could be used to produce MSTN-site mutated goats without developmental disruption. Thus, TALENs is an effective method for accurate genome editing to produce site-modified goats.

Evidence for Inbreeding and Genetic Differentiation among Geographic Populations of the Saprophytic Mushroom Trogia venenata from Southwestern China.

During the past 40 years, more than 400 Sudden Unexplained Deaths (SUDs) have occurred in Yunnan, southwestern China. Epidemiological and toxicological analyses suggested that a newly discovered mushroom called Trogia venenata was the leading culprit for SUDs. At present, relatively little is known about the genetics and natural history of this mushroom. In this study, we analyzed the sequence variation at four DNA fragments among 232 fruiting bodies of T. venenata collected from seven locations. Our ITS sequence analyses confirmed that all the isolates belonged to the same species. The widespread presence of sequence heterozygosity within many strains at each of three protein-coding genes suggested that the fruiting bodies were diploid, dikaryotic or heterokaryotic. Within individual geographic populations, we found significant deviations of genotype frequencies from Hardy-Weinberg expectations, with the overall observed heterozygosity lower than that expected under random mating, consistent with prevalent inbreeding within local populations. The geographic populations were overall genetically differentiated. Interestingly, while a positive correlation was found between population genetic distance and geographic distance, there was little correlation between genetic distance and barium concentration difference for the geographic populations. Our results suggest frequent inbreeding, geographic structuring, and limited gene flow among geographic populations of T. venenata from southwestern China.