E3 ubiquitin ligase UBR5 modulates circadian rhythm by facilitating the ubiquitination and degradation of the key clock transcription factor BMAL1.

The circadian clock is the inner rhythm of life activities and is controlled by a self-sustained and endogenous molecular clock, which maintains a ~ 24 h internal oscillation. As the core element of the circadian clock, BMAL1 is susceptible to degradation through the ubiquitin-proteasome system (UPS). Nevertheless, scant information is available regarding the UPS enzymes that intricately modulate both the stability and transcriptional activity of BMAL1, affecting the cellular circadian rhythm. In this work, we identify and validate UBR5 as a new E3 ubiquitin ligase that interacts with BMAL1 by using affinity purification, mass spectrometry, and biochemical experiments. UBR5 overexpression induced BMAL1 ubiquitination, leading to diminished stability and reduced protein level of BMAL1, thereby attenuating its transcriptional activity. Consistent with this, UBR5 knockdown increases the BMAL1 protein. Domain mapping discloses that the C-terminus of BMAL1 interacts with the N-terminal domains of UBR5. Similarly, cell-line-based experiments discover that HYD, the UBR5 homolog in Drosophila, could interact with and downregulate CYCLE, the BMAL1 homolog in Drosophila. PER2-luciferase bioluminescence real-time reporting assay in a mammalian cell line and behavioral experiments in Drosophila reveal that UBR5 or hyd knockdown significantly reduces the period of the circadian clock. Therefore, our work discovers a new ubiquitin ligase UBR5 that regulates BMAL1 stability and circadian rhythm and elucidates the underlying molecular mechanism. This work provides an additional layer of complexity to the regulatory network of the circadian clock at the post-translational modification level, offering potential insights into the modulation of the dysregulated circadian rhythm.

The inhibition role of miR-22 in hepatocellular carcinoma cell migration and invasion via targeting CD147.

BACKGROUND: Recently, miR-22 is identified as a tumor-suppressing microRNA in many human cancers. CD147 is a novel cancer-associated biomarker that plays an important role in the invasion and metastasis of malignant tumor. However, the involvement of miR-22 in CD147 regulation and hepatocellular carcinoma (HCC) progression and metastasis has not been investigated. METHODS: We measured miR-22 expression level in 34 paired of HCC and matched normal tissues, HCC cell lines by real-time quantitative RT-PCR. Invasion assay, MTT proliferation assay and wound-healing assay were performed to test the invasion and proliferation of HCC cell after overexpression of miR-22. The effect of miR-22 on HCC in vivo was validated by murine xenograft model. The relationship of miR-22 and its target gene CD147 was also investigated. RESULTS: We found that the expression of miR-22 in HCC tissues and cell lines were much lower than that in normal control, respectively. The expression of miR-22 was inversely correlated with HCC metastatic ability. Moreover, overexpression of miR-22 could significantly inhibit the HCC cell proliferation, migration and invasion in vitro and decrease HCC tumor growth in vivo. Finally, we found that miR-22 interacted with CD147 and decreased its expression, via a specific target site within the CD147 3'UTR by luciferase reporter assay. The expression of CD147 was inversely correlated with miR-22 expression in HCC tissues. CONCLUSION: Our results suggested that miR-22 was downexpressed in HCC and inhibited HCC cell proliferation, migration and invasion through downregulating cancer-associated gene CD147 which may provide a new bio-target for HCC therapy.

Mechanisms of action and applications of Polygonatum sibiricum polysaccharide at the intestinal mucosa barrier: a review.

As a medicinal and edible homologous Chinese herb, Polygonatum sibiricum has been used as a primary ingredient in various functional and medicinal products. Damage to the intestinal mucosal barrier can lead to or worsen conditions such as type 2 diabetes and Alzheimer's disease. Traditional Chinese medicine and its bioactive components can help prevent and manage these conditions by restoring the integrity of the intestinal mucosal barrier. This review delves into the mode of action of P. sibiricum polysaccharide in disease prevention and management through the restoration of the intestinal barrier. Polysaccharide from P. sibiricum effectively treats conditions by repairing the intestinal mucosal barrier, offering insights for treating complex diseases and supporting the application of P. sibiricum in clinical settings.

The suppressive role of p38 MAPK in cellular vacuole formation.

Vacuolization of the cytoplasm is one of the dramatic and frequently observed phenomena in various cell types. Cellular vacuoles occur spontaneously or via a wide range of inductive stimuli, but the molecular mechanism involved in this process remains largely unknown. In this study, we investigated the role of the p38 and JNK pathways in the formation of cytoplasmic vacuoles. We found that p38 and JNK agonist anisomycin abolishes spontaneous cytoplasmic vacuolization of HepG2 cells through p38 activation, but not through JNK activation. Importantly, blocking the activity of p38 or suppression the expression of p38 elicits cytoplasmic vacuoles formation in various cancer cells. Furthermore, cytoplasmic vacuoles induced by p38 blocking are derived from the perinuclear region. These observations provide direct evidence for a role of p38 signaling in regulating the formation of cytoplasmic vacuoles.

c-Met function requires N-linked glycosylation modification of pro-Met.

c-Met, the receptor for hepatocyte growth factor (HGF), is cell surface tyrosine kinase that controls cancer cell growth, survival, invasion, and metastasis. Post-translational modification, such as glycosylation, plays an essential role in regulating the function of cell surface molecules. Whether glycosylation modification regulates the enzymatic properties of c-Met is unknown. In this study, we investigated the effect of glycosylation on the function of c-Met. We found that c-Met is an N-linked glycosylated protein. Both pro-Met and p145Met (the ß subunit of mature c-Met) have N-linked glycosylation. Glycosylation inhibitor studies revealed that the N-glycosylation modification of p145Met is from pro-Met, but not due to the further modification of pro-Met. Importantly, blocking the N-glycosylation targets pro-Met to cytoplasm and initiates its phosphorylation independent of HGF engagement. Nonglycosylated pro-Met activates c-Met downstream pathways to a certain extent to compensate for the degradation of p145Met induced by glycosylation blocking-mediated endoplasmic reticulum (ER) stress.

[Phosphorylation of eukaryotic initiation factor 2-alpha inhibits cisplatin-mediated apoptosis of hepatocellular carcinoma cells].

OBJECTIVE: To investigate whether the phosphorylation (functionally inhibitive) of eukaryotic initiation factor 2-alpha (eIF2-a) affects the molecular mechanism of cisplatin-induced cellular apoptosis in human hepatocellular carcinoma (HCC). METHODS: The human HCC cultured cell lines SMMC-7221 and HepG2 were treated with cisplatin alone (controls; 24 h) or in combination with pre-transfection of a dominant-negative eIF2-a mutant (eIF2aS51A) or pre-exposure to an eIF2-a-specific phosphatase inhibitor (salubrinal) to decrease or increase the phosphorylation level, respectively. Changes in expression of apoptosis markers were quantitatively and qualitatively assessed by flow cytometry and western blot analysis. The significance of differences among groups was assessed by analysis of variance testing and of differences between groups was assessed by t-test. RESULTS: Cisplatin treatment induced the appropriate functional-inhibitive phosphorylation of eIF2-a on serine 51. Cisplatin treatment (10 mg/ml) induced significant apoptosis in the eIF2aS51A pre-transfected SMMC-7721 (control: 21.7 +/- 1.5% vs. 50.7 +/- 2.1%, t = 19.454, P less than 0.05) and HepG2 (21.0 +/- 1.0% vs. 57.3 +/- 2.1%, t = 27.250, P less than 0.05). Salubrinal pre-treatment significantly inhibited the cisplatin (15 mg/ml)-induced apoptosis in SMMC-7721 (control: 50.3 +/- 2.5% vs. 16.3 +/- 2.1%, t = 18.031, P less than 0.05) and HepG2 (42.0 +/- 2.6% vs. 12.0 +/- 2.0%, t = 15.667, P less than 0.05). CONCLUSION: Phosphorylation of eIF2-a may act to inhibit cisplatin-induced apoptosis of HCC.

[Cross-talk between PI3K/Akt and MEK/ERK pathways regulates human hepatocellular carcinoma cell cycle progression under endoplasmic reticulum stress].

OBJECTIVE: To investigate the cross-talk between the PI3K/Akt and MEK/ERK pathways and its role in cell cycle regulation under endoplasmic reticulum stress in human hepatocellular carcinoma cells. METHODS: PI3K inhibitor LY294002 and MEK inhibitor U0126 were used to block the PI3K/Akt and MEK/ERK pathways respectively, and constitutively activated Akt mutant construct was used to activate the PI3K/Akt pathway. Western blot was used to study the potential cross-talk between the PI3K/Akt and MEK/ERK pathways under endoplasmic reticulum stress in human hepatocellular carcinoma cells. the role of the cross-talk between the PI3K/Akt and MEK/ERK pathways in cell cycle regulation was investigated by using propidium iodide staining. RESULTS: LY294002 not only blocked Akt activation efficiently but also increased ERK phosphorylation markedly under endoplasmic reticulum stress in SMMC-7721 and Hep3B cells. Furthermore, myr-Akt inhibited endoplasmic reticulum stress-mediated ERK phosphorylation. In contrast, MEK inhibitor U0126 had no effect on endoplasmic reticulum stress-induced Akt activation. It is notable that both myr-Akt overexpression and MEK inhibitor U0126 inhibited endoplasmic reticulum stress-induced G0/G1 phase arrest in SMMC-7721 cells. CONCLUSION: Endoplasmic reticulum stress-induced Akt activation is mediated through PI3K and the PI3K/Akt pathway inactivation is involved in increased ERK activity in human hepatocellular carcinoma cells. The cross-talk between the PI3K/Akt and MEK/ERK cascades plays an important role in endoplasmic reticulum stress-induced human hepatocellular carcinoma cell cycle arrest.

Novel Screw Insertion Method for Anterior Surgical Treatment of Unstable Thoracolumbar Fracture: Quadrant Positioning Method.

OBJECTIVE: To develop a novel screw positioning method to improve the treatment of unstable thoracolumbar fractures. METHODS: A total of 72 patients with unstable thoracolumbar fractures who were treated with anterior screw-rod interfixation from January 2011 to October 2015 were included in this clinical study. Those patients included 48 male and 24 female patients with an average age of 45.10 years (range, 26-63 years). Patients were randomly divided into two groups: an observation group (n = 36) and a control group (n = 36). The quadrant positioning method was used for screw insertion in the observation group during the operation, while the traditional screw positioning method was used in the control group. The quadrant positioning method targeted four quadrants, including the superior anterior (SA), superior posterior (SP), inferior anterior (IA) and inferior posterior (IP) quadrants, while for the traditional screw positioning, four screws were inserted into the vertebral bodies above and below the excision. Patients were followed up for approximately 40 months to record recovery. Clinical and radiological records, local angle and fractured vertebra body height, clinical outcomes, complications, neurological improvement, and fusion rate were recorded and compared between the two groups. RESULTS: The quadrant positioning method was successfully used for anterior screw insertion. The quadrant center in the lateral view of the vertebral body was well marked, and screws were easily located on the scheduled quadrant. Blood loss (BL), hospital stay (HS), and operation time (OP) in the observation group were 749.40 ± 379.90 mL, 17.10 ± 4.10 days, and 167.40 ± 44.70 min, respectively. While those parameters in the control group were 1198.40 ± 339.27 mL, 23.22 ± 3.77 days, and 221.47 ± 32.15 min, respectively. The average operation time and hospital stay time were significantly shorter, and blood loss was significantly less in the observation group than in the control group (P < 0.05). Local angle and vertebral body height were markedly improved and 1-2 grade improvement was achieved in patients with neurological deficits in both groups. Both groups of patients achieved bony fusion during follow-up. No incision infection or internal fixation failure was observed in the two groups, and complications including cerebrospinal fluid and chylous leakage and hemothorax were resolved. CONCLUSIONS: The quadrant positioning method can shorten operation time, reduce blood loss, and accelerate postoperative recovery. The technique provides an effective method for screw insertion for double screw-rod instrumentation fixation in the treatment of thoracolumbar fracture via the anterior approach.

[Soil Organic Carbon Distribution and Components in Different Plant Communities Along a Water Table Gradient in the Huixian Karst Wetland in Guilin].

In order to reveal the effect of vegetation type and soil physicochemical properties on the distribution of soil organic carbon and its components, a field survey was carried out on nine different plant communities along a water table gradient in the Huixian wetland with samples of soil at 0-10 cm, 10-20 cm, and 20-30 cm in depth. The soil organic carbon (SOC), light fraction organic carbon (LFOC), heavy fraction organic carbon (HFOC), easily oxidized organic carbon (EOC), dissolved organic carbon (DOC), particulate organic carbon (POC), and microbial biomass carbon (MBC) were measured. The correlations among soil organic carbon components and soil physicochemical properties were also examined. The results showed that:① The average proportion of LFOC and HFOC to SOC at 0-30 cm soil depth was 11.10% and 88.90%, respectively. The distribution ratio of the heavy component was much higher than of the light component in soils. ② The content of SOC, DOC, EOC, POC, and MBC (except in the Panicum repens community) and the values of DOC/SOC, EOC/SOC, and POC/SOC all decreased with increase of the soil depth. ③ Among the nine different plant communities, the contents of SOC, LFOC, HFOC, MBC, DOC, EOC, and POC of Cladium chinense were significantly higher than for other communities in same soil layers. ④ There were significantly positive correlations among soil organic carbon components (SOC) and soil total nitrogen (TN). LFOC, HFOC, DOC, and POC were also positively correlated with soil pH. The soil bulk density was significantly negative correlated with LFOC, HFOC, DOC, EOC, and POC, and the content of clay was also negatively correlated with LFOC, HFOC, DOC, POC, and MBC. ⑤ Path analysis showed that TN, soil pH, soil sand content, and soil water content (SWC) has indirect effects on HFOC by influencing other soil factors. Soil TN had strong positive effects on EOC, DOC, and POC, and SWC also has the largest direct negative effect on MBC. This showed that there were close interactions between soil physicochemical properties and soil organic carbon components. This study may provide a reference base for sustainable development and scientific predictions regarding the Huixian Karst wetland.

Dietary fat intake and risk of esophageal carcinoma: a meta-analysis of observational studies.

Dietary fat intake is potentially associated with the onset of esophageal carcinoma (EC), but evidence from observational studies has remained unclear. This study aimed to evaluate the role of fat intake in the development of esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). A systematic search was conducted in PubMed and Web of Science to identify all relevant studies. Study-specific relative risks (RR) for the highest versus the lowest intake categories and 95% confidence intervals (CI) were pooled using a random-effects model. Seventeen case-control studies (2058 EAC cases, 1581 ESCC cases and 11696 controls) and two prospective cohort studies (494, 978 participants and 630 EAC cases and 215 ESCC cases) were identified. In EAC, the RRs (95% CI) were 1.69 (1.14-2.50) for total fat intake, 1.88 (1.28-2.77) for saturated fat (SFA) intake, 1.04 (0.86-1.27) for polyunsaturated fat (PUFA) intake and 1.70 (1.01-2.84) for monounsaturated fat (MUFA) intake. In ESCC, the RRs (95% CI) were 1.12 (0.84-1.51) for total fat, 1.38 (0.91-2.08) for SFA, 0.95 (0.55-1.62) for PUFA and 1.04 (0.65-1.66) for MUFA. In conclusion, total fat, SFA and MUFA intake were associated with EAC risk, but fat intake showed no significant association with ESCC risk. Large-scale prospective cohort studies are needed to confirm our findings.

JNK contributes to the tumorigenic potential of human cholangiocarcinoma cells through the mTOR pathway regulated GRP78 induction.

Less is known about the roles of c-Jun N-terminal kinase (JNK) in cholangiocarcinoma (CCA). Here, we report that JNK exerts its oncogenic action in human CCA cells, partially due to the mammalian target of rapamycin (mTOR) pathway regulated glucose-regulated protein 78 (GRP78) induction. In human CCA cells, the phosphorylation of eukaryotic initiation factor alpha (eIF2α) results in the accumulation of activating transcription factor 4 (ATF4) and GRP78 independent of unfolded protein response (UPR). Suppression of GRP78 expression decreases the proliferation and invasion of human CCA cells. It's notable that mTOR is required for eIF2α phosphorylation-induced ATF4 and GRP78 expression. Importantly, JNK promotes eIF2α/ATF4-mediated GRP78 induction through regulating the activity of mTOR. Thus, our study implicates JNK/mTOR signaling plays an important role in cholangiocarcinogenesis, partially through promoting the eIF2α/ATF4/GRP78 pathway.