R2R3-MYB transcription factor CsMYB60 controls mature fruit skin color by regulating flavonoid accumulation in cucumber.

Skin color is an important trait that determines the cosmetic appearance and quality of fruits. In cucumber, the skin color ranges from white to brown in mature fruits. However, the genetic basis for this important trait remains unclear. We conducted a genome-wide association study of natural cucumber populations, along with map-based cloning techniques, on an F2 population resulting from a cross between Pepino (with yellow-brown fruit skin) and Zaoer-N (with creamy fruit skin). We identified CsMYB60 as a candidate gene responsible for skin coloration in mature cucumber fruits. In cucumber accessions with white to pale yellow skin color, a premature stop mutation (C to T) was found in the second exon region of CsMYB60, whereas light yellow cucumber accessions exhibited splicing premature termination caused by an intronic mutator-like element insertion in CsMYB60. Transgenic CsMYB60c cucumber plants displayed a yellow-brown skin color by promoting accumulation of flavonoids, especially hyperoside, a yellow-colored flavonol. CsMYB60c encodes a nuclear protein that primarily acts as a transcriptional activator through its C-terminal activation motif. RNA sequencing and DNA affinity purification sequencing assays revealed that CsMYB60c promotes skin coloration by directly binding to the YYTACCTAMYT motif in the promoter regions of flavonoid biosynthetic genes, including CsF3'H, which encodes flavonoid 3'-hydroxylase. The findings of our study not only offer insight into the function of CsMYB60 as dominantly controlling fruit coloration, but also highlight that intronic DNA mutations can have a similar phenotypic impact as exonic mutations, which may be valuable in future cucumber breeding programs.

Insulin alleviates murine colitis through microbiome alterations and bile acid metabolism.

BACKGROUND: Insulin has been reported to exhibit anti-inflammatory activities in the context of bowel inflammation. However, the role of the interaction between insulin and the microbiota in gut health is unclear. Our goal was to investigate the mechanism of action of insulin in bowel inflammation and the relationship between insulin and the gut microbiota. METHODS: We used acute and chronic murine models of inflammatory bowel disease (IBD) to evaluate whether insulin influences the progression of colitis. Colonic tissues, the host metabolome and the gut microbiome were analyzed to investigate the relationship among insulin treatment, the microbiome, and disease. Experiments involving antibiotic (Abx) treatment and fecal microbiota transplantation (FMT) confirmed the association among the gut microbiota, insulin and IBD. In a series of experiments, we further defined the mechanisms underlying the anti-inflammatory effects of insulin. RESULTS: We found that low-dose insulin treatment alleviated intestinal inflammation but did not cause death. These effects were dependent on the gut microbiota, as confirmed by experiments involving Abx treatment and FMT. Using untargeted metabolomic profiling and 16S rRNA sequencing, we discovered that the level of the secondary bile acid lithocholic acid (LCA) was notably increased and the LCA levels were significantly associated with the abundance of Blautia, Enterorhadus and Rumi-NK4A214_group. Furthermore, LCA exerted anti-inflammatory effects by activating a G-protein-coupled bile acid receptor (TGR5), which inhibited the polarization of classically activated (M1) macrophages. CONCLUSION: Together, these data suggest that insulin alters the gut microbiota and affects LCA production, ultimately delaying the progression of IBD.

The association between Geriatric Nutritional Risk Index and the risk of Invasive Candidiasis in critically ill older adults.

BACKGROUND: Invasive candidiasis is the most common hospital-acquired fungal infection in intensive care units (ICU). The Geriatric Nutritional Risk Index (GNRI) score was developed to evaluate the nutritional status of elderly adults. We aimed to assess the association between the GNRI score and the risk of invasive candidiasis in elderly patients admitted to ICU. METHODS: Hospitalization information of elderly patients with invasive candidiasis was collected retrospectively from Medical Information Mart for Intensive Care (MIMIC) IV and MIMIC-III Clinical Database CareVue subset from 2001 to 2019. The main outcome of this study was the diagnosis of invasive candidiasis in patients. We employed a multivariable Cox regression and propensity score matching to balance the influence of confounding factors on the outcome. Furthermore, we conducted sensitivity analyses by categorizing the GNRI into classes based on thresholds of 98, 92, and 81. RESULTS: A total of 6739 patients were included in the study, among whom 134 individuals (2%) were diagnosed with invasive candidiasis. The GNRI scores of patients with invasive candidiasis upon admission to the ICU were significantly lower, measuring 88.67 [79.26-98.27], compared to the control group with a score of 99.36 [87.98-110.45] (P < 0.001). The results of the multivariable Cox regression analysis demonstrated a strong association between higher GNRI scores and a decreased risk of invasive candidiasis infection (HR: 0.98, 95% CI: 0.97-0.99, P = 0.002). Consistently, similar results were obtained when analyzing the propensity score-matched cohort (HR: 0.99, 95% CI: 0.98-1, P = 0.028). Sensitivity analyses further confirmed a significantly increased risk of invasive candidiasis infection with lower GNRI scores. Specifically, the following associations were observed: GNRI ≤ 98 (HR: 1.83, 95% CI: 1.23-2.72, P = 0.003), GNRI ≤ 92 (HR: 1.68, 95% CI: 1.17-2.4, P = 0.005), 82 ≤ GNRI ≤ 92 (HR: 1.63, 95% CI: 1.01-2.64, P = 0.046), GNRI ≤ 81 (HR: 2.31, 95% CI: 1.44-3.69, P < 0.001). CONCLUSIONS: Lower GNRI score was significantly associated with an increased risk of invasive candidiasis in elderly patients in ICU. Further research is needed to validate whether improving nutrition can prevent invasive candidiasis.

cfDNApipe: a comprehensive quality control and analysis pipeline for cell-free DNA high-throughput sequencing data.

MOTIVATION: Cell-free DNA (cfDNA) is gaining substantial attention from both biological and clinical fields as a promising marker for liquid biopsy. Many aspects of disease-related features have been discovered from cfDNA high-throughput sequencing (HTS) data. However, there is still a lack of integrative and systematic tools for cfDNA HTS data analysis and quality control (QC). RESULTS: Here, we propose cfDNApipe, an easy-to-use and systematic python package for cfDNA whole-genome sequencing (WGS) and whole-genome bisulfite sequencing (WGBS) data analysis. It covers the entire analysis pipeline for the cfDNA data, including raw sequencing data processing, QC and sophisticated statistical analysis such as detecting copy number variations (CNVs), differentially methylated regions and DNA fragment size alterations. cfDNApipe provides one-command-line-execution pipelines and flexible application programming interfaces for customized analysis. AVAILABILITY AND IMPLEMENTATION: https://xwanglabthu.github.io/cfDNApipe/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cobalt-Catalyzed Z to E Geometrical Isomerization of 1,3-Dienes.

An efficient cobalt-catalyzed geometrical isomerization of 1,3-dienes is described. In the combination of a CoCl2 precatalyst with an amido-diphosphine-oxazoline ligand, the geometrical isomerization of E/Z mixtures of 1,3-dienes proceed in a stereoconvergent manner, affording (E) isomers in high stereoselectivity. This facile transformation features a broad substrate scope with good functional group tolerance and could be scaled up to the gram scale smoothly with a catalyst loading of 1 mol %.

Parathyroid hormone-related protein prevents high-fat-diet-induced obesity, hepatic steatosis and insulin resistance in mice.

Obesity, closely related to systematic metabolic disorders, has become a major public health problem in recent decades. Here, we aimed to study the function of Parathyroid hormone-related protein (PTHrP) on high fat diet (HFD) induced murine obesity. Male C57BL/6J mice were transduced with adeno-associated virus vector encoding PTHrP (AAV-PTHrP) or adeno-associated virus control vector (AAV-Vehicle), following with HFD for 8 weeks. In addition, mice without transduction were fed on normal diet or HFD, respectively. Histological, metabolic and biochemical changes were detected. At the endpoint of experiment, body weight of mice treated with AAV-PTHrP did not increase as much as mice with AAV-Vehicle, but similar as mice with normal diet. Food efficiency ratio and weight of interscapular brown adipose tissue and epididymal white adipose tissue in mice overexpressed PTHrP were also lower than mice transducted with AAV-Vehicle. Besides, administration of AAV-PTHrP inhibited HFD-induced adipocyte hypertrophy. Protein level of PKA signaling pathway and thermogenic gene in adipose tissue exhibited a significant raise in HFD + AAV-PTHrP group, whereas transcription of inflammatory gene were decreased. Additionally, PTHrP overexpression ameliorated HFD-induced dyslipidemia, hepatic steatosis and insulin sensitivity. In HFD-induced murine obesity model, PTHrP is crucial to maintain metabolic homeostasis. PTHrP drives white adipose tissue browning and inhibits whitening of brown adipose tissue. Most importantly, PTHrP prevented HFD-induced obesity, hepatic steatosis and insulin resistance.

A novel histone deacetylase inhibitor LT-548-133-1 induces apoptosis by inhibiting HDAC and interfering with microtubule assembly in MCF-7 cells.

Many studies have indicated that histone deacetylase inhibitors (HDACis) have a significant antitumor effect in cancer. Here we report a compound named LT-548-133-1 that not only acts as an HDAC inhibitor but also interferes with microtubule assembly to inhibit MCF-7 cell proliferation and induce apoptosis. Consistent with Chidamide, LT-548-133-1 inhibited HDAC activity and increased histone H3 acetylation. But the difference is that it significantly induced cell cycle G2/M arrest while Chidamide caused G0/G1 arrest in MCF-7 cells. By Western blotting, we found the accumulation of CyclinB1 and phosphorylated histone H3 in LT-548-133-1 treated cells. Immunofluorescence based microtubule-repolymerization experiments and immunofluorescence staining of cell microtubules and nuclei showed that LT-548-133-1inhibited microtubule-repolymerization and induced mitotic abnormalities. The decreased expression of Bcl-2 and the increased expression of Bax, p53, p21, and cleaved-Caspase3 indicated the occurrence of apoptosis. Flow cytometry results also showed an increase in the proportion of apoptotic cells after administration of LT-548-133-1 or Chidamide. Therefore, we demonstrated that LT-548-133-1 could act as an HDAC inhibitor while inhibiting microtubule-repolymerization, causing mitosis to be arrested in G2/M. These two effects ultimately lead to proliferation inhibition and apoptosis of MCF-7 cells.

Chromatin remodeling enzyme Snf2h regulates embryonic lens differentiation and denucleation.

Ocular lens morphogenesis is a model for investigating mechanisms of cellular differentiation, spatial and temporal gene expression control, and chromatin regulation. Brg1 (Smarca4) and Snf2h (Smarca5) are catalytic subunits of distinct ATP-dependent chromatin remodeling complexes implicated in transcriptional regulation. Previous studies have shown that Brg1 regulates both lens fiber cell differentiation and organized degradation of their nuclei (denucleation). Here, we employed a conditional Snf2h(flox) mouse model to probe the cellular and molecular mechanisms of lens formation. Depletion of Snf2h induces premature and expanded differentiation of lens precursor cells forming the lens vesicle, implicating Snf2h as a key regulator of lens vesicle polarity through spatial control of Prox1, Jag1, p27(Kip1) (Cdkn1b) and p57(Kip2) (Cdkn1c) gene expression. The abnormal Snf2h(-/-) fiber cells also retain their nuclei. RNA profiling of Snf2h(-/) (-) and Brg1(-/-) eyes revealed differences in multiple transcripts, including prominent downregulation of those encoding Hsf4 and DNase IIß, which are implicated in the denucleation process. In summary, our data suggest that Snf2h is essential for the establishment of lens vesicle polarity, partitioning of prospective lens epithelial and fiber cell compartments, lens fiber cell differentiation, and lens fiber cell nuclear degradation.

Parathyroid Hormone-Like Hormone Induces Epithelial-to-Mesenchymal Transition of Intestinal Epithelial Cells by Activating the Runt-Related Transcription Factor 2.

Epithelial-to-mesenchymal transition (EMT) is a key contributor to fibroblast activation in fibrosis of multiple organs, including the intestine. Parathyroid hormone-like hormone (PTHLH) is an important factor in renal fibrosis and regulates several processes, including EMT. Herein, we investigated the role of PTHLH-induced EMT in intestinal fibrosis associated with Crohn disease. The expression levels of the EMT-related proteins, PTHLH, and parathyroid hormone receptor 1 (PTH1R) in intestinal tissues were determined by immunohistochemistry, and our results revealed that PTHLH and PTH1R were significantly elevated and associated with EMT marker expression. Moreover, neutralizing PTH1R and antagonizing PTHLH bioactivity prevented transforming growth factor-ß1-induced EMT. PTH1R can propagate the protein kinase A (PKA) signal and activate downstream nuclear transcription factors, including runt-related transcription factor 2 (Runx2). In addition, lentiviral vector-PTHLH-treated mice were highly sensitive to 2,4,6-trinitrobenzene sulfonic acid, and analysis of the PTHLH-PTH1R axis revealed the involvement of PKA-Runx2 in PTHLH-induced EMT. Our results indicate that PTHLH triggered EMT in intestinal epithelial cells through the PKA-Runx2 pathway, which might serve as a therapeutic target for intestinal fibrosis in Crohn disease.

The rs1024611 in the CCL2 gene and risk of gynecological cancer in Asians: a meta-analysis.

BACKGROUND: The -2518A/G (rs1024611) polymorphism of the CCL2 (C-C motif chemokine ligand 2), also known as MCP-1 (monocyte chemotactic protein-1) gene, has been reported to be associated with increased gynecological cancer risk, but the results are conflicting. METHODS: In this analysis, 1089 cases and 1553 controls from six publications were used to investigate the association between CCL2-2518A/G (rs1024611) polymorphism and the risk of gynecological cancer with a meta-analytic approach. Studies published on EBSCO, EMBASE, Web of Science, PubMed, SpringerLink, ScienceDirect, Weipu, and CNKI databases were identified (last update was on November 3, 2015). Six articles focused on the association between CCL2-2518A/G (rs1024611) polymorphism, and gynecological cancer risk was selected and data were extracted. The cancer type included endometrial cancer (n = 1), breast cancer (n = 2), ovarian cancer (n = 2), and cervical cancer (n = 1). All statistical analyses were performed using the STATA version 12.0 software. RESULTS: The meta-analysis showed that CCL2-2518A/G (rs1024611) polymorphism is associated with risk of gynecological cancer (GG vs AG + AA, OR = 1.55, 95%CI = 1.07-2.24, P < 0.05; AA vs GG, OR = 0.59 95%CI = 0.38-0.92, P < 0.05). Notably, the subgroup analysis demonstrated that the genotype AA is associated with a reduced gynecological cancer risk in Asians, but an increased risk when compared to AG in Europeans. CONCLUSIONS: Our data demonstrated the CCL2-2518A/G (rs1024611) polymorphism is significantly associated with risk of gynecological cancer, and the association differs by ethnicity.

Low-Molecular-Weight Heparins: Reduced Size Particulate Systems for Improved Therapeutic Outcomes.

A wide range of diseases have been treated using low-molecular-weight heparins (LMWHs), the drug of choice for anticoagulation. Owing to their better pharmacokinetic features compared to those of unfractionated heparin (uFH), several systems incorporating LMWHs have been investigated to deliver and improve their therapeutic outcomes, especially through development of their micro- and nano-particles. This review article describes current perspectives on the fabrication, characterization, and application of LMWHs-loaded micro- and nano-particles to achieve ameliorated bioavailability. The valuable applications of LMWH will continue to encourage researchers to identify efficient delivery systems that have specific release characteristics and ameliorated bioavailability, overcoming the challenges presented by biological obstructions and the physicochemical properties of LMWHs.

Autocrine parathyroid hormone-like hormone promotes intrahepatic cholangiocarcinoma cell proliferation via increased ERK/JNK-ATF2-cyclinD1 signaling.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is an aggressive tumor with a high fatality rate. It was recently found that parathyroid hormone-like hormone (PTHLH) was frequently overexpressed in ICC compared with non-tumor tissue. This study aimed to elucidate the underlying mechanisms of PTHLH in ICC development. METHODS: The CCK-8 assay, colony formation assays, flow cytometry and a xenograft model were used to examine the role of PTHLH in ICC cells proliferation. Immunohistochemistry (IHC) and western blot assays were used to detect target proteins. Luciferase reporter, chromatin immunoprecipitation (ChIP) and DNA pull-down assays were used to verify the transcription regulation of activating transcription factor-2 (ATF2). RESULTS: PTHLH was significantly upregulated in ICC compared with adjacent and normal tissues. Upregulation of PTHLH indicated a poor pathological differentiation and intrahepatic metastasis. Functional study demonstrated that PTHLH silencing markedly suppressed ICC cells growth, while specific overexpression of PTHLH has the opposite effect. Mechanistically, secreted PTHLH could promote ICC cell growth by activating extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, and subsequently upregulated ATF2 and cyclinD1 expression. Further study found that the promoter activity of PTHLH were negatively regulated by ATF2, indicating that a negative feedback loop exists. CONCLUSIONS: Our findings demonstrated that the ICC-secreted PTHLH plays a characteristic growth-promoting role through activating the canonical ERK/JNK-ATF2-cyclinD1 signaling pathways in ICC development. We identified a negative feedback loop formed by ATF2 and PTHLH. In this study, we explored the therapeutic implication for ICC patients.

Improved 1, 2, 4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins.

1, 2, 4-Butanetriol (BT) is a high-value non-natural chemical and has important applications in polymers, medical production and military industry. In the constructed BT biosynthesis pathway from xylose in Escherichia coli, the xylose dehydrogenase (Xdh) and the benzoylformate decarboxylase (MdlC) are heterologous enzymes and the activity of MdlC is the key limiting factor for BT production. In this study, six chaperone protein systems were introduced into the engineered E. coli harboring the recombinant BT pathway. The chaperone GroES-GroEL was beneficial to Xdh activity but had a negative effect on MdlC activity and BT titer. The plasmid pTf16 containing the tig gene (trigger factor) was beneficial to Xdh and MdlC activities and improved the BT titer from 0.42 to 0.56 g/l from 20 g/l xylose. However, co-expression of trigger factor and GroES-GroEL simultaneously reduced the activity of MdlC and had no effect on the BT production. The plasmid pKJE7 harboring dnaK-dnaJ-grpE showed significant negative effects on these enzyme activities and cell growth, leading to completely restrained the BT production. Similarly, co-expression of DnaKJ-GrpPE and GroES-GroEL simultaneously reduced Xdh and MdlC activities and decreased the BT titer by 45.2 %. The BT production of the engineered E. coli harboring pTf16 was further improved to the highest level at 1.01 g/l under pH control (pH 7). This work showed the potential application of chaperone proteins in microorganism engineering to get high production of target compounds as an effective and valuable tool.

[The mechanisms of heparin-derived oligosaccharide on the inhibition of smooth muscle cells proliferation induced by platelet-derived growth factor].

In this study, the effect of heparin-derived oligosaccharide (HDO) on platelet-derived growth factor (PDGF) induced vascular smooth muscle cells (VSMCs) proliferation and the related signal transduction mechanisms were investigated. MTT assays were used to measure VSMCs proliferation. Cell cycle distribution was analyzed by flow cytometry. The level of key regulatory proteins in PKC, MAPK and Akt/PI3K pathways were determined by RT-PCR, Western blot and immunocytochemical methods. Meanwhile, mRNA expressions of some proto-oncogenes were assayed by RT-PCR method. Our data showed that HDO (0.01, 0.1 and 1 µmol · L(-1)) inhibited 30 ng · mL(-1) PDGF-induced VSMCs proliferation in a dose-dependent manner, blocked the G1/S transition and inhibited the level of key regulatory proteins and some proto-oncogenes (P < 0.05). The results showed that HDO may decrease the key regulatory proteins expression, hence suppress the transcription of proto-oncogene and G1/S transition, finally inhibiting VSMCs proliferation.

Midpoint transverse process to pleura block for postoperative analgesia following laparoscopic renal cyst decortication: Two case reports.

BACKGROUND: The midpoint transverse process to pleura (MTP) block, a novel technique for thoracic paravertebral block (TPVB), was first employed in laparoscopic renal cyst decortication. CASE SUMMARY: Thoracic paravertebral nerve block is frequently employed for perioperative analgesia during laparoscopic cyst decortication. To address safety concerns associated with TPVBs, we administered MTP blocks in two patients prior to administering general anesthesia for laparoscopic cyst decortication. The MTP block was performed at the T9 level under ultrasound guidance, with 20 mL of 0.5% ropivacaine injected. Reduced sensation to cold and pinprick was observed from the T8 to T11 dermatome levels. Immediately postoperative Numeric Pain Rating Scale scores were 0/10 at rest and on movement, with none exceeding a mean 24 h numeric rating scale > 3. CONCLUSION: MTP block was effective technique for providing postoperative analgesia for patients undergoing laparoscopic renal cyst decortication.

Daily feeding frequency affects feed intake and body weight management of growing layers.

The objective of this study was to investigate the effect of feeding behavior on feed intake and body weight in growing layers and the underlying mechanisms, thereby providing a scientific foundation for optimal feeding practices in growing layers' management. A total of 144 Hy-line brown growing layers of 10 wk old and similar body weight, were divided into 3 treatment groups with different feeding frequency and equal cumulative daily feeding amount: the once-a-day feeding group (F1) was fed at 9:00 am every day, with feeding amount of 150 g/layer; the twice-a-day feeding group (F2) were fed at 9:00 am and 13:00 pm every day, with each feeding amount of 75 g/layer; the 4 times-a-day feeding group (F4) were fed at 9:00 am, 11:00 am, 13:00 pm, and 15:00 pm every day, with each feeding amount of 37.5 g/layer. Pre-experiment lasted for 1 wk and formal experiment lasted for 8 wk. The results indicated that the daily feed intake and body weight were decreased (P < 0.05) while feed conversion ratio was not affected (P > 0.05) as daily feeding times increased. The glandular stomach proportion was significantly increased in twice-a-day feeding group, while liver proportion and ileum length were significantly increased in 4 times-feeding group (P < 0.05). Additionally, 4 times-feeding daily resulted in a significant elevation of blood glucose levels, which may have suppressed feed intake (P < 0.05). In 4 times-feeding group, the plasma triglyceride levels increased as feeding times, accompanied by a notable up-regulation in the mRNA level of appetite-suppressing gene, hypothalamic pro-opiomelanocortin (POMC) and glandular stomach ghrelin. This modulation effectively suppressed the subsequent feed intake and body weight. Therefore, 4 times feeding daily is recommended in growing layers' management, because it reduced the feed cost without affecting the feed conversion efficiency.

Effects of high nutrient intake on the growth performance, intestinal morphology and immune function of neonatal intra-uterine growth-retarded pigs.

Intra-uterine growth-retarded (IUGR) neonates have shown an impairment of postnatal intestinal development and function. We hypothesised that the immune function of IUGR neonates might be affected by increased nutrient intake (NI) during the suckling period. Therefore, we investigated the effects of high NI (HNI) on the growth performance, intestinal morphology and immunological response of IUGR and normal-birth weight (NBW) piglets. A total of twelve pairs of IUGR and NBW piglets (7 d old) were randomly assigned to two different nutrient-level formula milk groups. After 21 d of rearing, growth performance, the composition of peripheral leucocytes, serum cytokines and intestinal innate immune-related genes involved in the Toll-like receptor (TLR)-4­myeloid differentiation factor 88­NF-κB pathway were determined. The results indicated that IUGR decreased the average daily DM intake (ADMI) and the average daily growth (ADG). However, the ADMI and ADG were increased by HNI, irrespective of body weight. Likewise, serum cytokines (TNF-α and IL-1ß) and ileal gene expressions (TLR-4, TLR-9, TRAF-6 and IL-1ß) were lower in IUGR piglets, whereas HNI significantly increased blood lymphocyte percentage and serum IL-10 concentrations, but decreased neutrophil percentage, serum IL-1ß concentrations and ileal gene expressions (NF-kB and IL-1ß). Furthermore, IUGR piglets with HNI exhibited lower serum concentrations of TNF-α and IL-1ß than NBW piglets, and these alterations in the immune traits of IUGR piglets receiving HNI were accompanied by decreasing ileal gene expressions of TLR-4, TLR-9, NF-κB and IL-1ß that are related to innate immunity. In conclusion, the present findings suggest that increased NI during the suckling period impaired the immune function of neonatal piglets with IUGR.

Modulation of miR-146b by N6-methyladenosine modification remodels tumor-associated macrophages and enhances anti-PD-1 therapy in colorectal cancer.

PURPOSE: MicroRNA-146b (miR-146b) alleviates experimental colitis in mice by mediating macrophage polarization and the release of inflammatory factors. Our goals were to evaluate the antitumor efficacy of miR-146b in colorectal cancer (CRC) and to investigate the underlying mechanisms. METHODS: We used murine models of CRC to evaluate whether miR-146b influenced the progression of tumors independent of tumor-associated macrophages (TAMs). RNA immunoprecipitation, N6-methyladenosine (m6A) RNA immunoprecipitation and in vitro pri-miRNA processing assays were conducted to examine whether m6A mediates the maturation of pri-miR-146b/miR-146b. In a series of in vitro and in vivo experiments, we further defined the molecular mechanisms of methyltransferase-like 3 (METTL3)/miR-146b-mediated antitumor immunity and its efficacy in combination with anti-PD-1 immunotherapy. RESULTS: We found that miR-146b deletion supported tumor progression by increasing the number of alternatively activated (M2) TAMs. Mechanistically, the m6A-related "writer" protein METTL3 and "reader" protein HNRNPA2B1 controlled miR-146b maturation by regulating the m6A modification region of pri-miR-146b. Furthermore, miR-146b deletion promoted the polarization of M2-TAMs by enhancing phosphoinositide 3-kinase (PI3K)/AKT signaling, and this effect was mediated by the class IA PI3K catalytic subunit p110ß, which reduced T cell infiltration, aggravated immunosuppression and ultimately promoted tumor progression. METTL3 knockdown or miR-146b deletion induced programmed death ligand 1 (PD-L1) production via the p110ß/PI3K/AKT pathway in TAMs and consequently augmented the antitumor activity of anti-PD-1 immunotherapy. CONCLUSIONS: The maturation of pri-miR-146b is m6A-dependent, and miR-146b deletion-mediated TAM differentiation promotes the development of CRC by activating the PI3K/AKT pathway, which induces upregulation of PD-L1 expression, inhibits T cell infiltration into the TME and enhances the antitumor activity of anti-PD-1 immunotherapy. The findings reveal that targeting miR-146b can serve as an adjuvant to anti-PD-1 immunotherapy.

Spatial expression patterns of autophagy genes in the eye lens and induction of autophagy in lens cells.

PURPOSE: Mutation of the autophagy gene FYVE (named after the four cysteine-rich proteins: Fab 1 [yeast orthologue of PIKfyve], YOTB, Vac 1 [vesicle transport protein], and EEA1) and coiled coil containing 1 (fyco1) causes human cataract suggesting a role for autophagy in lens function. Here, we analyzed the range and spatial expression patterns of lens autophagy genes and we evaluated whether autophagy could be induced in lens cells exposed to stress. METHODS: Autophagy gene expression levels and their spatial distribution patterns were evaluated between microdissected human lens epithelium and fibers at the mRNA and protein levels by microarray data analysis, real-time PCR and western blot analysis. Selected autophagy protein spatial expression patterns were also examined in newborn mouse lenses by immunohistochemistry. The autophagosomal content of cultured human lens epithelial cells was determined by counting the number of microtubule-associated protein 1 light chain 3B (LC3B)-positive puncta in cells cultured in the presence or absence of serum. RESULTS: A total of 42 autophagy genes were detected as being expressed by human lens epithelium and fibers. The autophagosomal markers LC3B and FYCO1 were detected throughout the newborn mouse lens. Consistently, the autophagy active form of LC3B (LC3B II) was detected in microdissected human lens fibers. An increased number of LC3B-positive puncta was detected in cultured lens cells upon serum starvation suggesting induction of autophagy in lens cells under stress conditions. CONCLUSIONS: The data provide evidence that autophagy is an important component for the function of lens epithelial and fiber cells. The data are consistent with the notion that disruption of lens autophagy through mutation or inactivation of specific autophagy proteins could lead to loss of lens resistance to stress and/or loss of lens differentiation resulting in cataract formation.

Effect of heparin-derived oligosaccharide on vascular smooth muscle cell proliferation and the signal transduction mechanisms involved.

PURPOSE: In this study, the effect of heparin-derived oligosaccharide (HDO) on vascular endothelial growth factor (VEGF) induced vascular smooth muscle cell (VSMC) proliferation and the signal transduction mechanisms involved were investigated. METHODS: MTT assays were used to measure VSMC proliferation, flow cytometry to analyze cell cycle distribution, RT-PCR for detection of gene transcript levels, and cell-based ELISA, Western blotting and immunocytochemical methods to detect the expression of PKC-α, ERK 1/2, p-ERK 1/2, Akt, p-Akt, p-PDK1 and p-GSK-3ß. RESULTS: HDO at concentrations of 0.01, 0.1 and 1 µmol·L(-1) dose-dependently inhibited VEGF-induced VSMC proliferation with inhibition indices of 6.8 %, 13.1 % and 28.9 %, respectively. Similar concentrations of HDO dose-dependently decreased the percentage of VEGF-induced cells in S phase to 3.6 %, 3.4 %, and 5.4 %, while increasing that of cells arrested in the G0/G1 phase to 80 %, 82 % and 83.6 %. HDO at 0.01, 0.1 or 1 µmol·L(-1) inhibited VEGF-induced PKC-α mRNA expression, with inhibition indices of 9.2 %, 16.1 % and 54.0 %. HDO at 0.1 or 1 µmol·L(-1) inhibited VEGF-induced proto-oncogene mRNA expression, with inhibition indices of 5.2 % and 6.6 % for c-jun, 8.8 % and 11.6 % for c-myc, and 6.5 % and 11.9 % for c-fos, respectively. Additionally, treatment with 0.01, 0.1 or 1 µmol·L(-1) HDO, inhibited VEGF-induced expression of some proliferation related proteins with inhibition indices of 33.2 %, 56.3 % and 77.0 % for PKC-α, 33.7 %, 38.7 % and 53.2 % for p-Akt, 3.5 %, 24.2 % and 49.3 % for p-ERK 1/2, 39.2 %, 71.8 % and 80.7 % for p-PDK 1 and 41.4 %, 89.4 % and 92.4 % for p-GSK-3ß, respectively. The results showed that HDO inhibited PKC-α, c-jun, c-fos and c-myc mRNA transcription, and also down-regulated phosphorylation levels of ERK 1/2 and Akt. CONCLUSION: Our study demonstrates that HDO inhibits transcription of proliferation-related proto-oncogenes and arrests G1/S transition through inhibition of the PKC, MAPK and Akt/PI3K pathways in association with inhibition of VSMC proliferation. This altered molecular signature may explain one mechanism of HDO-mediated inhibition of VSMC proliferation.