Despite an increase in the number of molecular epidemiological studies conducted in recent years to evaluate the association between human papillomavirus (HPV) and the risk of breast carcinoma, these studies remain inconclusive. Here we aim to detect HPV DNA in various tissues from patients with breast carcinoma using the method of HPV capture combined with massive paralleled sequencing (MPS). To validate the confidence of our methods, 15 cervical cancer samples were tested by PCR and the new method. Results showed that there was 100% consistence between the two methods.DNA from peripheral blood, tumor tissue, adjacent lymph nodes and adjacent normal tissue were collected from seven malignant breast cancer patients, and HPV type 16 (HPV16) was detected in 1/7, 1/7, 1/7 and 1/7 of patients respectively. Peripheral blood, tumor tissue and adjacent normal tissue were also collected from two patients with benign breast tumor, and 1/2, 2/2 and 2/2 was detected to have HPV16 DNA respectively. MPS metrics including mapping ratio, coverage, depth and SNVs were provided to characterize HPV in samples. The average coverage was 69% and 61.2% for malignant and benign samples respectively. 126 SNVs were identified in all 9 samples. The maximum number of SNVs was located in the gene of E2 and E4 among all samples. Our study not only provided an efficient method to capture HPV DNA, but detected the SNVS, coverage, SNV type and depth. The finding has provided further clue of association between HPV16 and breast cancer.

LSH, a SNF2 family DNA helicase, is a key regulator of DNA methylation in mammals. How LSH facilitates DNA methylation is not well defined. While previous studies with mouse embryonic stem cells (mESc) and fibroblasts (MEFs) derived from Lsh knockout mice have revealed a role of Lsh in de novo DNA methylation by Dnmt3a/3b, here we report that LSH contributes to DNA methylation in various cell lines primarily by promoting DNA methylation by DNMT1. We show that loss of LSH has a much bigger effect in DNA methylation than loss of DNMT3A and DNMT3B. Mechanistically, we demonstrate that LSH interacts with UHRF1 but not DNMT1 and facilitates UHRF1 chromatin association and UHRF1-catalyzed histone H3 ubiquitination in an ATPase activity-dependent manner, which in turn promotes DNMT1 recruitment to replication fork and DNA methylation. Notably, UHRF1 also enhances LSH association with the replication fork. Thus, our study identifies LSH as an essential factor for DNA methylation by DNMT1 and provides novel insight into how a feed-forward loop between LSH and UHRF1 facilitates DNMT1-mediated maintenance of DNA methylation in chromatin.

Reconstituted high density lipoprotein (rHDL) has been regarded as a promising brain-targeting vehicle for anti-glioma drugs under the mediation of apolipoprotein A-I (apoA-I). However, some stability issues relating to drug leakage and consequent reduced targeting efficiency in the course of discoidal rHDL (d-rHDL) circulating in blood hinder its broad application. The objective of the study was to develop a novel stabilized d-rHDL by replacing cholesterol and apoA-I with mono-cholesterol glutarate (MCG) modified apoA-I (termed as mA) and to evaluate its allosteric behavior and glioma targeting. MCG was synthesized through esterifying the hydroxyl of cholesterol with glutaric anhydride and characterized by FI-IR and 1H NMR. d-rHDL assembled with mA (termed as m-d-rHDL) presented similar properties such as minute particle size and disk-like appearance resembling nascent HDL. Morphological transformation observation and in vitro release plots convinced that the modification of cholesterol could effectively inhibit the remolding of d-rHDL. The uptake of m-d-rHDL by LCAT-pretreated bEND.3 cells was significantly higher than that of d-rHDL, thereby serving as another proof for the capability of m-d-rHDL in enhancing targeting property. Besides, apoA-I anchoring into m-d-rHDL played a critical role in the endocytosis process into bEND.3 cells and C6 cells, which implied the possibility of traversing blood brain barrier and accumulating in the brain and glioma. These results suggested that the modification toward cholesterol to improve the stability of d-rHDL is advantageous, and that this obtained m-d-rHDL revealed great potential for realization of suppressing the remolding of d-rHDL in the brain-targeted treatment of glioma for drug delivery.

Global DNA hypomethylation is a most common epigenetic alteration in cancer, but the mechanism remains elusive. Previous studies demonstrate that UHRF1 but not UHRF2 is required for mediating DNA maintenance methylation by DNMT1. Here we report unexpectedly a conserved function for UHRF1 and UHRF2: inhibiting de novo DNA methylation by functioning as E3 ligases promoting DNMT3A degradation. UHRF1/2 are frequently overexpressed in cancers and we present evidence that UHRF1/2 overexpression downregulates DNMT3A proteins and consequently leads to DNA hypomethylation. Abrogating this negative regulation on DNMT3A or overexpression of DNMT3A leads to increased DNA methylation and impaired tumor growth. We propose a working model that UHRF1/2 safeguards the fidelity of DNA methylation and suggests that UHRF1/2 overexpression is likely a causal factor for widespread DNA hypomethylation in cancer via suppressing DNMT3A.

Reconstituted lipoproteins (rLips) based on endogenous lipid nanostructures has been increasingly regarded as an excellent and promising antitumor drug delivery. However, some problems relating to the main component, apolipoprotein, for instance, rare source, unaffordable price, and low specificity of relevant receptor expression, become chief obstacles to its broad development and application.

In mammals it is unclear if UHRF1-mediated DNA maintenance methylation by DNMT1 is strictly dependent on histone H3K9 methylation. Here we have generated an Uhrf1 knockin (KI) mouse model that specifically abolishes the H3K9me2/3-binding activity of Uhrf1. The homozygous Uhrf1 KI mice are viable and fertile, and exhibit ∼10% reduction of DNA methylation in various tissues. The reduced DNA methylation occurs globally in the genome and does not restrict only to the H3K9me2/3 enriched repetitive sequences. In vitro UHRF1 binds with higher affinity to reconstituted nucleosome with hemi-methylated CpGs than that with H3K9me2/3, although it binds cooperatively to nucleosome with both modifications. We also show that the nucleosome positioning affects the binding of methylated DNA by UHRF1. Thus, while our study supports a role for H3K9 methylation in promoting DNA methylation, it demonstrates for the first time that DNA maintenance methylation in mammals is largely independent of H3K9 methylation.

Previous studies have implicated an essential role for UHRF1-mediated histone H3 ubiquitination in recruiting DNMT1 to replication sites for DNA maintenance methylation during S phase of the cell cycle. However, the regulatory mechanism on UHRF1-mediated histone ubiquitination is not clear. Here we present evidence that UHRF1 and USP7 oppositely control ubiquitination of histones H3 and H2B in S phase of the cell cycle and that DNMT1 binds both ubiquitinated H3 and H2B. USP7 knockout markedly increased the levels of ubiquitinated H3 and H2B in S phase, the association of DNMT1 with replication sites and importantly, led to a progressive increase of global DNA methylation shown with increased cell passages. Using DNMT3A/DNMT3B/USP7 triple knockout cells and various DNA methylation analyses, we demonstrated that USP7 knockout led to an overall elevation of DNA methylation levels. Mechanistic study demonstrated that USP7 suppresses DNMT1 recruitment and DNA methylation through its deubiquitinase activity and the interaction with DNMT1. Altogether our study provides evidence that USP7 is a negative regulator of global DNA methylation and that USP7 protects the genome from excessive DNA methylation by attenuating histone ubiquitination-dependent DNMT1 recruitment.

Chlorogenic acid (CGA), as one of the richest polyphenol compounds in nature, has broad applications in many fields due to its various biological properties. However, initial data on the effects of dietary CGA on protein synthesis and related basal metabolic activity has rarely been reported. The current study is aimed at (1) determining whether dietary CGA supplementation improves the growth performance and carcass traits, (2) assessing whether dietary CGA alters the free amino acid profile, and (3) verifying whether dietary CGA promotes muscle protein synthesis in finishing pigs. Thirty-two (Large × White × Landrace) finishing barrows with an average initial body weight of 71.89 ± 0.92 kg were randomly allotted to 4 groups and fed diets supplemented with 0, 0.02%, 0.04%, and 0.08% CGA, respectively. The results indicated that, compared with the control group, dietary supplementation with 0.04% CGA slightly stimulated the growth performance of pigs, whereas no significant correlation was noted between the dietary CGA levels and animal growth (P > 0.05). Furthermore, the carcass traits of pigs were improved by 0.04% dietary CGA (P < 0.01). In addition, dietary CGA significantly improved the serum free amino acid profiles of pigs (P < 0.01), while 0.04% dietary CGA promoted more amino acids to translocate to skeletal muscles (P < 0.05). The relative mRNA expression levels of SNAT2 in both longissimus dorsi (LD) and biceps femoris (BF) muscles were augmented in the 0.02% and 0.04% groups (P < 0.05), and the LAT1 mRNA expression in the BF muscle was elevated in the 0.02% group (P < 0.05). We also found that dietary CGA supplementation at the levels of 0.04% or 0.08% promoted the expression of p-Akt and activated the mTOR-S6K1-4EBP1 axis in the LD muscle (P < 0.05). Besides, the MAFbx mRNA abundance in the 0.02% and 0.04% groups was significantly lower (P < 0.05). Our results revealed that dietary supplementation with CGA of 0.04% improved the free amino acid profile and enhanced muscle protein biosynthesis in the LD muscle in finishing pigs.

Alpha-ketoglutarate (AKG), a critical molecule in the tricarboxylic acid cycle, is beneficial to intestinal functions. However, its influence on intestinal microbiota and metabolism is not fully understood. We investigated the effects of a low-protein (LP) diet supplemented with AKG on cecal microbial communities and the parameters of microbial metabolism in growing pigs. Twenty-seven young pigs (Large White × Landrace) with an average initial body weight of 11.96 ± 0.18 kg were randomly allotted into three groups (n = 9): a normal protein (NP) diet containing 20% crude protein (CP); LP diet formulated with 17% CP (LP diet); or LP diet supplemented with 10 g kg-1 of AKG (ALP diet). After a 35-day trial period, the digesta of the cecum were collected to analyze the concentrations of ammonia and short-chain fatty acids (SCFAs). We also performed a microbial analysis. Although no significant differences were found in performance among the diet groups, pigs fed the ALP diet had greater average daily gain (ADG) when compared with those in the LP group. Experimental diet did not affect cecal bacterial richness or diversity, as determined by Chao1 and ACE species richness measures and Shannon and Simpson indices, respectively. The predominant phyla Firmicutes, Bacteroidetes, and Proteobacteria increased in relative abundances in the cecum of pigs fed ALP diet. At the genus level, compared to the LP diet, the ALP diet significantly increased the abundances of Lachnospiraceae UCG-005, Lachnospiraceae NK4A136 group, Phascolarctobacterium and Parabacteroides, while decreased Vibrio and Maritalea. Pigs fed the ALP diet increased Oribacterium and Lachnoclostridium when compared with the NP diet. Non-metric multidimensional scaling analysis revealed that the distribution of microbiota at each group was distinctly clustered separately along principal coordinate. In addition, quantitative PCR revealed that the ALP diet was also associated with increases in the amounts of Bacteroides, Bifidobacterium, and Lactobacillus, but a decrease in the level of Escherichia coli. Compared with the NP diet, the ALP diet enhanced the concentrations of valerate and propionate. This ALP diet also increased the concentrations of valerate and isobutyrate when compared with the LP diet. Moreover, the ALP diet was linked with a significant decline in the concentration of ammonia in the cecum. These results indicate that a LP diet supplemented with AKG can alter the balance in microbial communities, increasing the population of SCFA-producing bacteria and the amounts of Bacteroides and Bifidobacterium, while reducing the counts of Escherichia coli and the amount of ammonia in the cecum.

Staphylococcus aureus is a zoonotic pathogen that causes various life-threatening diseases. The mechanisms of action of amoxicillin against S. aureus are unclear. Here, we established a rabbit tissue cage infection model to evaluate the relationship between the pharmacokinetic/pharmacodynamic (PK/PD) parameters of amoxicillin and selective enrichment of resistant strains of S. aureus and to elucidate the evolution of its resistance to amoxicillin. S. aureus was injected into the tissue cages at 1010 colony forming units (CFU)/mL. We injected different intramuscular concentrations of amoxicillin at doses of 5, 10, 20, and 30 mg/kg body weight once a day for 5 days and 5, 10, 20, and 30 mg/kg body weight twice a day for 2.5 days. Differences in gene expression between two differentially resistant strains and a sensitive strain were evaluated using Illumina sequencing followed by COG and KEGG analysis. RT-qPCR was carried out to validate the difference in protein translation levels. Our results demonstrated that the emergence of resistant bacteria was dose dependent within a given time interval. In the same dosage group, the appearance of resistant bacteria increased with time. The resistant bacteria showed cumulative growth, and the level of resistance increased over time. The resistant bacteria were completely inhibited when the cumulative percentage of time over a 24-h period that the drug concentration exceeded the mutant prevention concentration (MPC) (%T > MPC) was ≥52%. We also found that mecA and femX in S. aureus played a leading role in the development of resistance to amoxicillin. In conclusion, it provide references for optimizing amoxicillin regimens to treat infections caused by S. aureus.

This study was conducted to explore the regulatory mechanism of leucine (Leu) on lipid metabolism of finishing pigs. Twenty-four Duroc × Landrace × Large cross pigs with an average body weight of 68.33 ± 0.97 kg were randomly allocated into 3 treatment groups with 8 replicates per group (1 pig per replicate). The dietary treatments were as follows: control group (CON), 0.25% Leu group and 0.50% Leu group. The experimental period was 42 d. The results showed as follows. (1) Compared with the CON, 0.25% and 0.50% Leu increased (P < 0.01) the average daily gain (ADG), while the average backfat thickness (ABT) and the ratio of feed intake to body weight gain (F:G ratio) were decreased (P < 0.05). (2) In the 0.25% Leu group, the relative mRNA expression levels of sterol regulatory element binding protein-1c (SREBP1c), recombinant fatty acid transport protein 1 (FATP1), chemerin and peroxisome proliferator-activated receptor γ (PPARγ) were decreased but the level of fatty acid binding protein 4 (FABP4) and fatty acid translocase (FAT/CD36) were increased in backfat tissue. In the 0.25% Leu group, the protein levels of p-Rictor, p-Raptor, p-eIF4E-binding protein 1 (p-4EBP1), p-silent mating type information regulator 2 homolog 1 (p-SIRT1) and acetylation ribosome s6 protein kinase 1 (Ac-S6K1) were increased (P < 0.05). (3) Compared to the CON, the diversity of gut microbiota in the 0.25% Leu group was increased. Principal component analysis showed that the relative abundance of Bacteroidetes, Lactobacillus and Desulfovibrio was higher in the 0.25% Leu group than the CON, but the relative abundance of Firmicutes, Treponema and Shigella was lower than in the CON (P < 0.05). (4) Four different metabolites were screened out from the serum of finishing pigs including allolithocholic acid (alloLCA), isolithocholic acid (isoLCA), ursodeoxycholic acid (UDCA) and hyodeoxycholic acid (HDCA), which correlate to various degrees with the above microorganisms. In conclusion, Leu could promote adipose tissue lipolysis of finishing pigs through the mTOR-SIRT1 signaling pathway, and S6K1 is acetylated at the same time, and the interaction between gut microbiota and bile acid metabolism is also involved.

Faithful inheritance of DNA methylation across cell division requires DNMT1 and its accessory factor UHRF1. However, how this axis is regulated to ensure DNA methylation homeostasis remains poorly understood. Here we show that SET8, a cell-cycle-regulated protein methyltransferase, controls protein stability of both UHRF1 and DNMT1 through methylation-mediated, ubiquitin-dependent degradation and consequently prevents excessive DNA methylation. SET8 methylates UHRF1 at lysine 385 and this modification leads to ubiquitination and degradation of UHRF1. In contrast, LSD1 stabilizes both UHRF1 and DNMT1 by demethylation. Importantly, SET8 and LSD1 oppositely regulate global DNA methylation and do so most likely through regulating the level of UHRF1 than DNMT1. Finally, we show that UHRF1 downregulation in G2/M by SET8 has a role in suppressing DNMT1-mediated methylation on post-replicated DNA. Altogether, our study reveals a novel role of SET8 in promoting DNA methylation homeostasis and identifies UHRF1 as the hub for tuning DNA methylation through dynamic protein methylation.

We evaluated the association between plasma levels of mac-2 binding protein (M2BP) with the risk of in-stent restenosis (ISR) after percutaneous coronary intervention (PCI).

Sepsis is a condition that is associated with high rates of mortality. It is characterized by serious systemic inflammatory responses induced by pathogenic invasion. Although microRNA-150 (miR-150) has been previously reported to be involved in the modulation of sepsis, the underlying molecular mechanism in sepsis remains poorly understood. In the present study, the human monocytic cell line THP-1 was treated with LPS to mimic sepsis in vitro, following which miR-150 and STAT1 expression were measured using reverse transcription-quantitative PCR or western blotting. Secretion of inflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) into the medium were measured by ELISA. The potential relationship between STAT1 and miR-150 was determined using dual-luciferase reporter and RNA immunoprecipitation assays. miR-150 expression was found to be was downregulated by LPS treatment in THP-1 cells in both dose- and time-dependent manners. LPS treatment also induced IL-1β, IL-6 and TNF-α secretion in a manner that could be inhibited by miR-150 overexpression and enhanced by transfection with the miR-150 inhibitor. miR-150 was revealed to directly target STAT1 by negatively regulating its expression. In addition, STAT1 expression was demonstrated to be upregulated by LPS treatment. STAT1 overexpression reversed the inhibitory effects of miR-150 overexpression on IL-1β, IL-6 and TNF-α secretion whilst STAT1 knockdown attenuated IL-1β, IL-6 and TNF-α secretion induced by miR-150 inhibitor transfection. In conclusion, the present study suggested that miR-150 regulates the inflammatory response in macrophages following LPS challenge by regulating the expression of STAT1.

Some research has suggested that miRNA-10a (miR-10a-5p) had an inhibitory function in proliferation and invasion of cancers. Whereas the role of miR-10a-5p in melanoma has not been fully explored. This study aims to confirm LIN28B as the targeted gene of miR-10a-5p which was explored in melanoma cells. In addition, upstream regulatory molecule of miR-10a-5p was also investigated in melanoma cells.

Circular RNAs (circRNAs) have been reported to exert critical functions in tumorigenesis and development. However, the underlying mechanism by which circRNAs regulate melanoma progression remain to be elucidated.