Icariin-Curcumol promotes docetaxel sensitivity in prostate cancer through modulation of the PI3K-Akt signaling pathway and the Warburg effect.

BACKGROUND: Docetaxel (DTX) resistance reduces therapeutic efficacy in prostate cancer (PCa). Accumulating reports support the role of phytochemicals in the reversal of DTX resistance. This study aimed to determine whether Epimedium brevicornu and Curcuma zedoaria extracts (ECe), specially icariin-curcumol, attenuates DTX resistance and explore their potential mechanisms. METHODS: Regulatory pathways were predicted between ECe active ingredients and PCa using network pharmacology. DTX-resistant cell LNCaP/R were established based on DTX-sensitive LNCaP, and xenograft models were further established. Active ingredients in ECe by HLPC-MS were identified. The binding of icariin and curcumol to the target was analyzed by molecular docking. Biochemical experiments were applied to determine the possible mechanisms by which Icariin-Curcumol regulates DTX sensitivity. RESULTS: Akt1 and the PI3K-Akt signaling pathway were predicted as the primary functional target between drug and PCa. ECe and DTX inhibited xenograft tumor growth, inflammation, cell viability and promoted apoptosis. Icariin and curcumol were detected in ECe, and icariin and curcumol docked with Akt1. ECe, Icariin-Curcumol and DTX downregulated AR, PSA, PI3K, Akt1, mTOR, and HIF-1ɑ. Moreover, ECe, Icariin-Curcumol and DTX increased glucose and PDH, decreased lactic acid, ATP and LDH, and downregulated c-Myc, hnRNPs, VEGF, PFK1, and PKM2. Notably, the anti-PCa effect of DTX was attenuated compared to ECe or Icariin-Curcumol in the LNCaP/R model. The combined effect of Icariin-Curcumol and DTX was superior to that of DTX. CONCLUSION: Our data support that Icariin-Curcumol reverses DTX resistance by inhibiting the PI3K-Akt signaling and the Warburg effect, providing new ideas for improving therapeutic measures for PCa.

Phylogenomics of the Liquorilactobacillus Genus.

The genus Liquorilactobacillus is a new genus commonly found in wine and plants. Despite its significance, previous studies on Liquorilactobacillus are primarily focused on phenotypic experiments, with limited genome-level studies. This study used comparative genomics to analyze 24 genomes from the genus Liquorilactobacillus, including two novel sequenced strains (IMAU80559 and IMAU80777). A phylogenetic tree of 24 strains was constructed based on 122 core genes and divided into two clades, A and B. Significant differences in GC content were observed between the two clades (P = 10e-4). Additionally, change revealed to suggests that clade B has more exposure to prophage infection having an upgraded immune system. Further analysis of functional annotation and selective pressure suggests that clade A was subjected to greater selection pressure than B clade (P = 3.9e-6) and had higher number of functional types annotated than clade B (P = 2.7e-3), while clade B had a lower number of pseudogenes than clade A (P = 1.9e-2). The findings suggest that differently prophages and environmental stress may have influenced the common ancestor of clades A and B during evolution, leading to the development of two distinct clades.

[Effect of Polygonati Rhizoma in improving pyroptosis injury of diabetic macroangiopathy via NLRP3/caspase-1/GSDMD pathway].

This study aims to explore the influence of Polygonati Rhizoma on the pyroptosis in the rat model of diabetic macroangiopathy via the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1)/gasdermin D(GSDMD) pathway. The rat model of diabetes was established by intraperitoneal injection of streptozotocin(STZ) combined with a high-fat, high-sugar diet. The blood glucose meter, fully automated biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, and Western blot were employed to measure blood glucose levels, lipid levels, vascular thickness, inflammatory cytokine levels, and expression levels of pyroptosis-related proteins. The mechanism of pharmacological interventions against the injury in the context of diabetes was thus explored. The results demonstrated the successful establishment of the model of diabetes. Compared with the control group, the model group showed elevated levels of fasting blood glucose, total cholesterol(TC), triglycerides(TG) and low-density lipoprotein cholesterol(LDL-c), lowered level of high-density lipoprotein cholesterol(HDL-c), thickened vascular intima, and elevated serum and aorta levels of tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß) and interleukin-18(IL-18). Moreover, the model group showed increased NLRP3 inflammasomes and up-regulated levels of caspase-1 and GSDMD in aortic vascular cells. Polygonati Rhizoma intervention reduced blood glucose and lipid levels, inhibited vascular thickening, lowered the levels of TNF-α, IL-1ß, IL-18 in the serum and aorta, attenuated NLRP3 inflammasome expression, and down-regulated the expression levels of caspase-1 and GSDMD, compared with the model group. In summary, Polygonati Rhizoma can slow down the progression of diabetic macroangiopathy by inhibiting pyroptosis and alleviating local vascular inflammation.

Gut Bifidobacterium responses to probiotic Lactobacillus casei Zhang administration vary between subjects from different geographic regions.

Bifidobacteria are health-promoting human gut inhabitants, but accurate species-level composition of the gut bifidobacteria and their responses to probiotic intervention have not been fully explored. This was a follow-up work of our previous study, in which 104 volunteers from six different Asiatic regions (Singapore, Indonesia, Xinjiang, Gansu, Inner Mongolia, Mongolia) were recruited. The gut microbiota and their responses towards Lactobacillus casei Zhang (LCZ) intervention were characterized (at days 0, 7, and 14; 14 days after stopping probiotic intake), and region-based differential responses were observed after LCZ intervention. This study further investigated changes in the species-level gut bifidobacteria by PacBio small-molecule real-time sequencing (SMRT) using bifidobacteria-specific primers. Firstly, this study found that Bifidobacterium adolescentis (42.58%) and Bifidobacterium breve (26.34%) were the core species across the six Asiatic regions. Secondly, principal coordinate analysis of probiotic-induced changes in the gut bifidobacterial microbiota (represented by weighted UniFrac distances) grouped the six regions into two clusters, namely northern (Xinjiang, Gansu, Inner Mongolia, and Mongolia) and southern (Singapore, Indonesia) regions. Thirdly, LCZ intervention induced region-based differential responses of gut bifidobacterial microbiota. The relative abundance of Bifidobacterium animalis in subjects from northern but not southern region substantially increased after LCZ intervention. Moreover, LCZ intervention significantly increased the weighted UniFrac distances in the southern but not northern subjects 7 days after LCZ intervention. The gut B. adolescentis correlated significantly and negatively with the weighted UniFrac distances of the baseline gut bifidobacterial microbiota in subjects of northern but not southern region, suggesting a possible homeostatic effect of LCZ on the gut bifidobacterial population of northern but not southern subjects. Collectively, our study found that probiotic-induced responses of the gut bifidobacterial microbiota varied with subjects' geographic origins, and B. adolescentis might play a role in maintaining the overall stability of the gut bifidobacterial population. KEY POINTS: • The core species in the six Asiatic regions are Bifidobacterium adolescentis and Bifidobacterium breve. • The gut bifidobacterial microbiota in people from various geographic origins showed different responses on probiotic administration.

Untargeted metabolic footprinting reveals key differences between fermented brown milk and fermented milk metabolomes.

Fermented brown milk has gained popularity because of its unique taste and flavor. Lactobacillus bulgaricus ND02 is a starter culture that has good milk fermentation characteristics. This study aimed to profile the metabolites produced during Maillard browning and to identify metabolomic differences between fermented brown milk and fermented milk produced by the ND02 strain. This study used liquid chromatography-mass spectrometry to compare the metabolomes of milk, fermented milk, brown milk, and fermented brown milk. Significant differences were observed in the abundances of various groups of metabolites, including peptides, AA, aldehydes, ketones, organic acids, vitamins, and nucleosides. The Maillard browning reaction significantly increased the intensity of a wide spectrum of flavor compounds, including short peptides, organic acids, and compounds of aldehydes, ketones, sulfur, and furan, which might together contribute to the unique flavor of brown milk. However, Maillard browning led to an increase in Nε-(carboxymethyl)lysine, which might cause negative health effects such as diabetes, uremia, or Alzheimer's disease. On the other hand, fermenting brown milk with the ND02 strain effectively countered such an effect. Finally, 5 differentially abundant metabolites were identified between fermented brown milk and fermented milk, including l-lysine, methylglyoxal, glyoxal, 2,3-pentanedione, and 3-hydroxybutanoic acid, which might together contribute to the different nutritional qualities of fermented brown milk and fermented milk. This study has provided novel information about the Maillard reaction and compared the metabolomes of the 4 types of dairy products.

Functional analysis of the second methyltransferase in the bacteriophage exclusion system of Lactobacillus casei Zhang.

The antiphage ability is an important feature of fermentation strains in the dairy industry. Our previous work described the bacteriophage exclusion (BREX) system in the probiotic strain, Lactobacillus casei Zhang. The function of L. casei Zhang pglX gene in mediating 5'-ACRCm6AG-3' methylation was also confirmed. This study aimed to further dissect the function of the BREX system of L. casei Zhang by inactivating its second methyltransferase gene (LCAZH_2054). The methylome of the mutant, L. casei Zhang Δ2054, was profiled by single-molecule real-time sequencing. Then, the cell morphology, growth, plasmid transformation efficiency, and stability of the wildtype and mutant were compared. The mutant did not have an observable effect in microscopic and colony morphology, but it reached a higher cell density after entering the exponential phase without obvious increase in the cell viability. The mutant had fewer 5'-ACRCm6AG-3' methylation compared with the wildtype (1835 versus 1906). Interestingly, no significant difference was observed in the transformation efficiency between the 2 strains when plasmids without cognate recognition sequence (pSec:Leiss:Nuc and pG+host9) were transformed, contrasting to transforming cells with cognate recognition sequence-containing plasmids (pMSP3535 and pTRKH2). The efficiency of transforming pMSP3535 into the LCAZH_2054 mutant was significantly lower than the wildtype, whereas an opposite trend was seen in pTRKH2 transformation. Moreover, compared with the wildtype, the mutant strain had higher capacity in retaining pMSP3535 and lower capacity in retaining pTRKH2, suggesting an unequal tolerance level to different foreign DNA. In conclusion, LCAZH_2054 was not directly responsible for 5'-ACRCm6AG-3' methylation in L. casei Zhang, but it might help regulate the function and specificity of the BREX system.

Protective effects of whey protein hydrolysate on Bifidobacterium animalis ssp. lactis Probio-M8 during freeze-drying and storage.

We evaluated the potential of whey protein hydrolysate as a lyoprotectant for maintaining the cell viability of Bifidobacterium animalis ssp. lactis Probio-M8 during freeze-drying and subsequent storage. The moisture content and water activity of the lyophilized samples treated by different concentrations of whey protein hydrolysate were ≤5.23 ± 0.33 g/100 g and ≤0.102 ± 0.003, respectively. During storage at 25°C and 30°C, whey protein hydrolysate had a stronger protective effect on B. lactis Probio-M8 than the same concentration of whey protein. Using the Excel tool GinaFit, we estimated the microbial inactivation kinetics during storage. Whey protein hydrolysate reduced cell damage caused by an increase in temperature. Whey protein hydrolysate could protect cells by increasing the osmotic pressure as a compatible solute. Whey protein hydrolysate improved cell membrane integrity and reduced the amounts of reactive oxygen species and malondialdehyde produced. The findings indicated that whey protein hydrolysate was a novel antioxidant lyoprotectant that could protect probiotics during freeze-drying and storage.

Metabolic footprint analysis of volatile metabolites by gas chromatography-ion mobility spectrometry to discriminate between different fermentation temperatures during Streptococcus thermophilus milk fermentation.

Streptococcus thermophilus is widely used in the dairy industry to produce fermented milk. Gas chromatography-ion mobility spectrometry-based metabolomics was used to discriminate different fermentation temperatures (37°C and 42°C) at 3 time points (F0: pH = 6.50 ± 0.02; F1: pH = 5.20 ± 0.02; F2: pH = 4.60 ± 0.02) during S. thermophilus milk fermentation, and differences of fermentation physical properties and growth curves were also evaluated. Fermentation was completed (pH 4.60) after 6 h at 42°C and after 8 h at 37°C; there were no significant differences in viable cell counts and titratable acidity; water-holding capacity and viscosity were higher at 37°C than at 42°C. Different fermentation temperatures affected volatile metabolic profiles. After the fermentation was completed, the volatile metabolites that could be used to distinguish the fermentation temperature were hexanal, butyraldehyde, ethyl acetate, ethanol, 3-methylbutanal, 3-methylbutanoic acid, and 2-methylpropionic acid. Specifically, at 37°C of milk fermentation, branched-chain AA had higher levels, and leucine, isoleucine, and valine were involved in growth and metabolism, which promoted accumulation of some short-chain fatty acids such as 3-methylbutanoic acid and 2-methylpanprooic acid. At 42°C, at 3 different time points during fermentation, ethanol from glycolysis all presented higher levels, including acetone and 3-methylbutanal, producing a more pleasant flavor in the fermented milk. This work provides detailed insight into S. thermophilus fermented milk metabolites that differed between incubation temperatures; these data can be used for understanding and eventually predicting metabolic changes during milk fermentation.

Metabolic footprinting revealed key biochemical changes in a brown fermented milk product using Streptococcus thermophilus.

Ultra-performance liquid chromatography coupled with a quadrupole-time-of-flight mass spectrometryElevated Energy was used to investigate changes in the metabolite profile of brown milk and fermented brown milk produced using Streptococcus thermophilus S10. Samples were analyzed in both positive and negative electron ionization modes. Data were analyzed by multivariate statistical methods for biomarker metabolites that were differentially abundant in brown milk and fermented brown milk. We identified 43 differentially abundant metabolites based on mass spectrophotometry fragmentation patterns. These metabolites included peptides, AA, fatty acids and related metabolites, carbohydrate metabolites, vitamins, and nucleosides. Some of these metabolites are known to alter the sensorial quality of fermented dairy products. Thus, it is likely that some of the currently identified differentially abundant metabolites also contribute to the unique flavor, taste, and aroma of fermented brown milk. The bitterness and astringency of fermented brown milk are likely to be due to some of these peptides, whereas the sweetness and sourness could be a result of changes in carbohydrate levels. No previous study has analyzed metabolomics changes during fermentation of brown milk. Thus, our data are a valuable reference for future development and improvement of fermented brown milk products.

A Novel Bacteriophage Exclusion (BREX) System Encoded by the pglX Gene in Lactobacillus casei Zhang.

The bacteriophage exclusion (BREX) system is a novel prokaryotic defense system against bacteriophages. To our knowledge, no study has systematically characterized the function of the BREX system in lactic acid bacteria. Lactobacillus casei Zhang is a probiotic bacterium originating from koumiss. By using single-molecule real-time sequencing, we previously identified N6-methyladenine (m6A) signatures in the genome of L. casei Zhang and a putative methyltransferase (MTase), namely, pglX This work further analyzed the genomic locus near the pglX gene and identified it as a component of the BREX system. To decipher the biological role of pglX, an L. casei Zhang pglX mutant (ΔpglX) was constructed. Interestingly, m6A methylation of the 5'-ACRCAG-3' motif was eliminated in the ΔpglX mutant. The wild-type and mutant strains exhibited no significant difference in morphology or growth performance in de Man-Rogosa-Sharpe (MRS) medium. A significantly higher plasmid acquisition capacity was observed for the ΔpglX mutant than for the wild type if the transformed plasmids contained pglX recognition sites (i.e., 5'-ACRCAG-3'). In contrast, no significant difference was observed in plasmid transformation efficiency between the two strains when plasmids lacking pglX recognition sites were tested. Moreover, the ΔpglX mutant had a lower capacity to retain the plasmids than the wild type, suggesting a decrease in genetic stability. Since the Rebase database predicted that the L. casei PglX protein was bifunctional, as both an MTase and a restriction endonuclease, the PglX protein was heterologously expressed and purified but failed to show restriction endonuclease activity. Taken together, the results show that the L. casei Zhang pglX gene is a functional adenine MTase that belongs to the BREX system.IMPORTANCELactobacillus casei Zhang is a probiotic that confers beneficial effects on the host, and it is thus increasingly used in the dairy industry. The possession of an effective bacterial immune system that can defend against invasion of phages and exogenous DNA is a desirable feature for industrial bacterial strains. The bacteriophage exclusion (BREX) system is a recently described phage resistance system in prokaryotes. This work confirmed the function of the BREX system in L. casei and that the methyltransferase (pglX) is an indispensable part of the system. Overall, our study characterizes a BREX system component gene in lactic acid bacteria.

A survey of the relationship between functional genes and acetaldehyde production characteristics in Streptococcus thermophilus by multilocus sequence typing.

Streptococcus thermophilus is an important bacterium used in the production of fermented dairy products. Yogurt with good flavor is preferred by consumers; thus, variation in flavor-formation characteristics among isolates is attracting attention. Here, acetaldehyde production characteristics of 30 isolates were evaluated in parallel with genotyping and multilocus sequence typing of key functional genes involved in acetaldehyde production. The results showed that isolates could be divided into 3 phenotypically distinct groups: high-acetaldehyde-yielding isolates (>10 mg/L), medium-acetaldehyde-yielding isolates (5-10 mg/L) and low-acetaldehyde-yielding (<5 mg/L) based on evaluation of acetaldehyde production during yogurt storage. These groups, distinguishable by phenotypic characteristics, were clustered in corresponding groups based on functional gene multilocus sequence typing analysis. Combining functional gene sequence analysis of 30 Strep. thermophilus isolates with phenotypic evaluation of their flavor-related characteristics (specifically acetaldehyde production) demonstrated that groups of isolates established using genotype data analysis corresponded with groups identified based on their phenotypic traits. Interestingly, the 30 isolates of Strep. thermophilus showed significant phylogenetic clustering in acetaldehyde content by functional gene and acetaldehyde content analysis. A corresponding relationship exists between functional gene phylogenetic clustering and acetaldehyde content variation.

Analysis of the Gut Microbial Diversity of Dairy Cows During Peak Lactation by PacBio Single-Molecule Real-Time (SMRT) Sequencing.

BACKGROUND: The gut microbes of dairy cows are strongly associated with their health, but the relationship between milk production and the intestinal microbiota has seldom been studied. Thus, we explored the diversity of the intestinal microbiota during peak lactation of dairy cows. METHODS: The intestinal microbiota of nine dairy cows at peak lactation was evaluated using the Pacific Biosciences single-molecule real-time (PacBio SMRT) sequencing approach. RESULTS: A total of 32,670 high-quality 16S rRNA gene sequences were obtained, belonging to 12 phyla, 59 families, 107 genera, and 162 species. Firmicutes (83%) were the dominant phylum, while Bacteroides (6.16%) was the dominant genus. All samples showed a high microbial diversity, with numerous genera of short chain fatty acid (SCFA)-producers. The proportion of SCFA producers was relatively high in relation to the identified core intestinal microbiota. Moreover, the predicted functional metagenome was heavily involved in energy metabolism. CONCLUSIONS: This study provided novel insights into the link between the dairy cow gut microbiota and milk production.

Characteristics of Milk Fermented by Streptococcus thermophilus MGA45-4 and the Profiles of Associated Volatile Compounds during Fermentation and Storage.

The lactic acid bacterium Streptococcus thermophilus is a major starter culture for the production of dairy products. In this study, the physiochemical characteristics of milk fermented by the MGA45-4 isolate of S. thermophilus were analyzed. Our data indicate that milk fermented using S. thermophilus MGA45-4 maintained a high viable cell count (8.86 log10 colony-forming units/mL), and a relatively high pH (4.4), viscosity (834.33 mPa·s), and water holding capacity (40.85%) during 14 days of storage. By analyzing the volatile compound profile using solid-phase microextraction and gas chromatography/mass spectrometry, we identified 73 volatile compounds in the fermented milk product, including five carboxylic acids, 21 aldehydes, 13 ketones, 16 alcohols, five esters, and 13 aromatic carbohydrates. According to the odor activity values, 11 of these volatile compounds were found to play a key role in producing the characteristic flavor of fermented milk, particularly octanal, nonanal, hexanal, 2,3-butanedione, and 1-octen-3-ol, which had the highest odor activity values among all compounds analyzed. These findings thus provide more insights in the chemical/molecular characteristics of milk fermented using S. thermophilus, which may provide a basis for improving dairy product flavor/odor during the process of fermentation and storage.

Bacterial microbiota of Kazakhstan cheese revealed by single molecule real time (SMRT) sequencing and its comparison with Belgian, Kalmykian and Italian artisanal cheeses.

BACKGROUND: In Kazakhstan, traditional artisanal cheeses have a long history and are widely consumed. The unique characteristics of local artisanal cheeses are almost completely preserved. However, their microbial communities have rarely been reported. The current study firstly generated the Single Molecule, Real-Time (SMRT) sequencing bacterial diversity profiles of 6 traditional artisanal cheese samples of Kazakhstan origin, followed by comparatively analyzed the microbiota composition between the current dataset and those from cheeses originated from Belgium, Russian Republic of Kalmykia (Kalmykia) and Italy. RESULTS: Across the Kazakhstan cheese samples, a total of 238 bacterial species belonging to 14 phyla and 140 genera were identified. Lactococcus lactis (28.93%), Lactobacillus helveticus (26.43%), Streptococcus thermophilus (12.18%) and Lactobacillus delbrueckii (12.15%) were the dominant bacterial species for these samples. To further evaluate the cheese bacterial diversity of Kazakhstan cheeses in comparison with those from other geographic origins, 16S rRNA datasets of 36 artisanal cheeses from Belgium, Russian Republic of Kalmykia (Kalmykia) and Italy were retrieved from public databases. The cheese bacterial microbiota communities were largely different across sample origins. By principal coordinate analysis (PCoA) and multivariate analysis of variance (MANOVA), the structure of the Kazakhstan artisanal cheese samples was found to be different from those of the other geographic origins. Furthermore, the redundancy analysis (RDA) identified 16 bacterial OTUs as the key variables responsible for such microbiota structural difference. CONCLUSION: Our results together suggest that the diversity of bacterial communities in different groups is stratified by geographic region. This study does not only provide novel information on the bacterial microbiota of traditional artisanal cheese of Kazakhstan at species level, but also interesting insights into the bacterial diversity of artisanal cheeses of various geographical origins.

Profiles of Volatile Flavor Compounds in Milk Fermented with Different Proportional Combinations of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are key factors in the fermentation process and the final quality of dairy products worldwide. This study was performed to investigate the effects of the proportions of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus isolated from traditionally fermented dairy products in China and Mongolia on the profile of volatile compounds produced in samples. Six proportional combinations (1:1, 1:10, 1:50, 1:100, 1:1000, and 1:10,000) of L. delbrueckii subsp. bulgaricus IMAU20401 to S. thermophilus ND03 were considered, and the volatiles were identified and quantified by solid-phase microextraction and gas chromatography-mass spectrometry (SPME-GC-MS) against an internal standard. In total, 89 volatile flavor compounds, consisting of aldehydes, ketones, acids, alcohols, esters, and aromatic hydrocarbons, were identified. Among these, some key flavor volatile compounds were identified, including acetaldehyde, 3-methylbutanal, acetoin, 2-heptanone, acetic acid, butanoic acid, and 3-methyl-1-butanol. The of L. delbrueckii subsp. bulgaricus IMAU20401 to S. thermophilus ND03 influenced the type and concentration of volatiles produced. In particular, aldehydes and ketones were present at higher concentrations in the 1:1000 treatment combination than in the other combinations. Our findings emphasize the importance of selecting the appropriate proportions of L. delbrueckii subsp. bulgaricus and S. thermophilus for the starter culture in determining the final profile of volatiles and the overall flavor of dairy products.

Multilocus sequence typing of Streptococcus thermophilus from naturally fermented dairy foods in China and Mongolia.

BACKGROUND: Streptococcus thermophilus is a major dairy starter used for manufacturing of dairy products. In the present study, we developed a multilocus sequence typing (MLST) scheme for this important food bacterium. Sequences of 10 housekeeping genes (carB, clpX, dnaA, murC, murE, pepN, pepX, pyrG, recA, and rpoB) were obtained for 239 S. thermophilus strains, which were isolated from home-made fermented dairy foods in 18 different regions of Mongolia and China. METHODS: All 10 genes of S. thermophilus were sequenced, aligned, and defined sequence types (STs) using the BioNumerics Software. The nucleotide diversity was calculated by START v2.0. The population structure, phylogenetic relationships and the role of recombination were inferred using ClonalFrame v1.2, SplitsTree 4.0 and Structure v2.3. RESULTS: The 239 S. thermophilus isolates and 18 reference strains could be assigned into 119 different STs, which could be further separated into 16 clonal complexes (CCs) and 38 singletons. Among the 10 loci, a total of 132 polymorphic sites were detected. The standardized index of association (IAS=0.0916), split-decomposition and ρ/θ (relative frequency of occurrence of recombination and mutation) and r/m value (relative impact of recombination and mutation in the diversification) confirms that recombination may have occurred, but it occurred at a low frequency in these 10 loci. Phylogenetic trees indicated that there were five lineages in the S. thermophilus isolates used in our study. MSTree and ClonalFrame tree analyses suggest that the evolution of S. thermophilus isolates have little relationship with geographic locality, but revealed no association with the types of fermented dairy product. Phylogenetic analysis of 36 whole genome strains (18 S. thermophilus, 2 S. vestibularis and 16 S. salivarius strains) indicated that our MLST scheme could clearly separate three closely related species within the salivarius group and is suitable for analyzing the population structure of the other two species in the salivarius group. CONCLUSIONS: Our newly developed MLST scheme improved the understanding on the genetic diversity and population structure of the S. thermophilus, as well as provided useful information for further studies on the genotyping and evolutionary research for S. thermophilus strains with global diversity.

Microbial Pollution Tracking of Dairy Farm with a Combined PCR-DGGE and qPCR Approach.

Animal husbandry is a traditional industry with regional characteristic in the Inner Mongolia of China. Recent years, animal breeding has been one of the main pollution sources in this area, followed by domestic sewage and industrial wastewater. The pollution of livestock farm feces may accelerate the development of pathogens and antibiotic resistance genes which pose health risks to humans and animals. In present research, culture-independent molecular ecological methods based on DGGE combined with qPCR were used to investigate the pollution to surrounding environment with different degrees of livestock farm. The cluster analysis of DGGE patterns showed that the livestock farm feces from point pollution source flowed with wastewater discharge has resulted in an impacted range of at least 3000 m, but it did not cause pollution to residential water delivered from upstream of sewage drain outlet. qPCR results revealed that 5 common pathogens (Escherichia coli, Enterococcus, Staphylococcus aureus, Shigella, and Salmonella) presented decreased trend as the sampled distance from point pollution source increased. Also, qPCR assays of 10 common antibiotic resistance genes (tetO, tetL, rpp, rpoB, sul2, sulA, floR, yidY, mphA, and ermC) which cause resistance to tetracycline, rifampicin, fluoroquinolone, quinolone, and erythromycin have been found in the environmental samples. This study clearly indicates the livestock farm discharge pollutants contaminated to the surrounding environment. Our data have provided important information to pollution control in the future.

Investigation of bacterial and fungal diversity in tarag using high-throughput sequencing.

This is the first study on the bacterial and fungal community diversity in 17 tarag samples (naturally fermented dairy products) through a metagenomic approach involving high-throughput pyrosequencing. Our results revealed the presence of a total of 47 bacterial and 43 fungal genera in all tarag samples, in which Lactobacillus and Galactomyces were the predominant genera of bacteria and fungi, respectively. The number of some microbial genera, such as Lactococcus, Acetobacter, Saccharomyces, Trichosporon, and Kluyveromyces, among others, was found to vary between different samples. Altogether, our results showed that the microbial flora in different samples may be stratified by geographic region.

Icariin­curcumol promotes ferroptosis in prostate cancer cells through Nrf2/HO­1 signaling.

Ferroptosis is a form of regulatory cell death that relies on iron and reactive oxygen species (ROS) to inhibit tumors. The present study aimed to investigate whether icariin-curcumol could be a novel ferroptosis inducer in tumor inhibition. Various concentrations of icariin-curcumol were used to stimulate prostate cell lines (RWPE-2, PC-3, VCAP and DU145). Small interfering negative control (si-NC) and si-nuclear factor erythroid 2-related factor 2 (Nrf2) were used to transfect DU145 cells. Cell viability was determined by using cell counting kit-8. Ferroptosis-related factor levels were analyzed using western blotting and reverse transcription-quantitative PCR. Enzyme-linked immunosorbent assays were used to assess the ferrous (Fe2+), glutathione and malondialdehyde (MDA) content. The ROS fluorescence intensity was assessed using flow cytometry. DU145 cells were most sensitive to icariin-curcumol concentration. The Fe2+ content, ROS fluorescence intensity and MDA level gradually increased, while solute carrier family 7 member 11 (SLC7A11) level, glutathione peroxidase 4 (GPX4) level, GSH content, Nrf2 and heme oxygenase-1 (HO-1) decreased with icariin-curcumol in a dose-dependent manner. After si-Nrf2 was transfected, the cell proliferation ability, SLC7A11 and GPX4 levels declined compared with the si-NC group. In contrast to the control group, the icariin + curcumol group showed reductions in Nrf2 and HO-1 levels, cell proliferation, SLC7A11 and GPX4 levels, with an increase in Fe2+ content and ROS fluorescence intensity. Overexpression of Nrf2 reversed the regulation observed in the icariin + curcumol group. Icariin-curcumol induced ferroptosis in PCa cells, mechanistically by inhibiting the Nrf2/HO-1 signaling pathway. Icariin-curcumol could be used as a new type of ferroptosis inducer to treat PCa effectively.

Icaritin-curcumol activates CD8+ T cells through regulation of gut microbiota and the DNMT1/IGFBP2 axis to suppress the development of prostate cancer.

BACKGROUND: Prostate cancer (PCa) incidence and mortality rates are rising. Our previous research has shown that the combination of icariin (ICA) and curcumol (CUR) induced autophagy and ferroptosis in PCa cells, and altered lipid metabolism. We aimed to further explore the effects of the combination of ICA and CUR on gut microbiota, metabolism, and immunity in PCa. METHODS: A mouse subcutaneous RM-1 cell tumor model was established. 16 S rRNA sequencing was performed to detect changes in fecal gut microbiota. SCFAs in mouse feces, and the effect of ICA-CUR on T-cell immunity, IGFBP2, and DNMT1 were examined. Fecal microbiota transplantation (FMT) was conducted to explore the mechanism of ICA-CUR. Si-IGFBP2 and si/oe-DNMT1 were transfected into RM-1 and DU145 cells, and the cells were treated with ICA-CUR to investigate the mechanism of ICA-CUR on PCa development. RESULTS: After treatment with ICA-CUR, there was a decrease in tumor volume and weight, accompanied by changes in gut microbiota. ICA-CUR affected SCFAs and DNMT1/IGFBP2/EGFR/STAT3/PD-L1 pathway. ICA-CUR increased the positive rates of CD3+CD8+IFN-γ, CD3+CD8+Ki67 cells, and the levels of IFN-γ and IFN-α in the serum. After FMT (with donors from the ICA-CUR group), tumor volume and weight were decreased. SCFAs promote tumor development and the expression of IGFBP2. In vitro, DNMT1/IGFBP2 promotes cell migration and proliferation. ICA-CUR inhibits the expression of DNMT1/IGFBP2. CONCLUSIONS: ICA-CUR mediates the interaction between gut microbiota and the DNMT1/IGFBP2 axis to inhibit the progression of PCa by regulating immune response and metabolism, suggesting a potential therapeutic strategy for PCa.