The Active Ingredient Catalpol in Rehmannia glutinosa Reduces Blood Glucose in Diabetic Rats via the AMPK Pathway.

Background: Type 2 diabetes mellitus (T2DM) poses a huge threat to population health globally, and more drugs need to be explored for treatment. In this study, we investigated the mechanism of active ingredient catalpol in Rehmannia glutinosa on reduces blood glucose in diabetic. Methods: The T2DM model was constructed by intraperitoneal injection of streptozotocin into Sprague-Dawley (SD) rats, which were randomly grouped into diabetes model group, pioglitazone group, Rehmannia glutinosa group, catalpol high-dose group, catalpol low-dose group and normal control group.The intervention was continued for 28 d, and changes in body weight, fasting blood glucose, insulin and lipid levels were observed. Results: Of all the drugs, pioglitazone had the most pronounced hypoglycemic effect, which began to decline after 2 weeks of treatment in the low-dose catalpol group and had no hypoglycemic effect in the high-dose catalpol group. Among them, Rehmannia glutinosa was able to increase serum triglyceride level, and pioglitazone effectively reduced total cholesterol level in rats. The low dose of catalpol decreased the concentration of low-density lipoprotein cholesterol (LDL), while the high dose of catalpol increased the concentration of LDL. Conclusion: As an active ingredient in Rehmannia glutinosa, catalpol has the potential to lower blood glucose and improve blood lipids in diabetes treatment, and its action may be achieved by regulating the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway, which provides a new idea for the development of new diabetes therapeutic approaches.

Water conservation pattern of Fangcheng River Basin in Beibu Gulf and its response to precipitation.

Assessing the spatiotemporal patterns of watershed water conservation under the influence of the South Asian monsoon climate and its response to precipitation is essential for revealing the evolving patterns of water conservation under different temporal scales. Following the principles of water balance and using the Soil and Water Assessment Tool (SWAT) model, we investigated the spatiotemporal patterns of water conservation and its response to precipitation in the Fangcheng River Basin of Beibu Gulf. The results showed that water conservation in Fangcheng River Basin calculated by SWAT model were 1637.4 mm·a-1, accounting for 50.7% of the mean annual precipitation. The variation of water conservation in different sub-basins was obviously different. Sub-basins with high forest coverage and steep slopes exhibited higher water conservation, while sub-basins with other land use types (such as cropland and grassland), gentle slopes, and intense human activities showed lower water conservation. At the monthly scale, both water conservation and its variation showed similar response characteristics to precipitation in the basin. The response of water conservation variation to sub-precipitation events could be classified into two types. For the short-term rainfall events (duration≤2 days), water conservation variation showed a linear relationship. For the medium to long-term rainfall events (2 days

LncRNA SLC7A11-AS1 promotes the progression of hepatocellular carcinoma by mediating KLF9 ubiquitination.

Hepatocellular carcinoma (HCC) is a malignant tumor, which seriously threatens the life of patients. LncRNA SLC7A11-AS1 was reported to be abnormally expressed in HCC. Here, the functions and relative molecular regulatory mechanism of SLC7A11-AS1 in HCC were investigated. Nude mice and HCC cells were used as the experimental subjects. Knockdown or overexpression of exogenous genes was conducted in HCC cells. RT-qPCR, IHC, and western blot were employed to evaluate the abundance of genes and proteins. The malignant behaviors were evaluated using CCK-8, clone formation, wound-healing, and Transwell. The locations of SLC7A11-AS1 and KLF9 in cells were determined by FISH and IF assays. The total m6A level was evaluated by dot-blot assay. m6A modification of SLC7A11-AS1 was detected using RNA MeRIP. The interactions among molecules were validated by RIP, ChIP, dual luciferase reporter assay, and co-IP. SLC7A11-AS1 was elevated apparently in HCC cells and HCC tissues from mice. SLC7A11-AS1 silencing could suppress HCC progression, which was validated in in vivo and in vitro experiments. Furthermore, METTL3 mediated m6A modification of SLC7A11-AS1 to elevate its expression. In addition, SLC7A11-AS1 downregulated KLF9 expression by affecting STUB1-mediated ubiquitination degradation and KLF9 enhanced PHLPP2 expression to inactivate the AKT pathway. Eventually, rescue experiments revealed that KLF9 knockdown abolished SLC7A11-AS1 silencing-mediated suppression of HCC progression in vivo and in vitro. Our results unveiled that m6A-modified SLC7A11-AS1 promoted HCC progression by regulating the STUB1/KLF9/PHLPP2/AKT axis, indicating that targeting SLC7A11-AS1 might alleviate HCC progression.

Dipeptidyl peptidase-8 induces sorafenib resistance via binding with c-Rel to mediate NF-κB signaling in hepatocellular carcinoma.

Sorafenib is the important first-standard drug for patients with advanced hepatocellular carcinoma (HCC). A major obstacle to successful treatment is sorafenib resistance. However, the mechanism of sorafenib resistance is unclear. The present study aimed to determine the involvement of dipeptidyl peptidase-8 (DPP8) in sorafenib resistance. DPP8 expression was detected using quantitative real-time PCR (qPCR) and western blot analysis. The effect of DPP8 on sorafenib resistance was examined using terminal deoxynulceotidyl transferase nick-end-labeling (TUNEL), colony formation, flow cytometry, luciferase reporter, immunofluorescence, and immunoprecipitation (IP) assays. We found that DPP8 mRNA and protein levels were dramatically upregulated in HCC. Gene set enrichment analysis (GSEA) illustrated that DPP8 might be involved in apoptosis regulation. Downregulation of DPP8 substantially promoted the sensitivity of HCC cells to sorafenib. Further analysis showed that DPP8 might regulate nuclear factor kappa B (NF-κB) signaling, which was confirmed using a luciferase reporter assay. Downregulation of DPP8 decreased the expression levels of downstream genes of the NF-κB pathway. IP showed that DPP8 can interact with NF-κB subunit c-Rel, an important protein of NF-κB signaling. Finally, a drug combination of sorafenib and Val-boroPro induced higher mortality of HCC cells than sorafenib alone in DPP8-upregulated cells. Our findings indicated that using the inhibitor Val-boroPro might be a promising method to enhance sorafenib sensitivity in advanced HCC.

Clinical significance of serum S100A10 in lung cancer.

OBJECTIVE: To investigate the clinical significance of serum S100 calcium-binding protein A10 (S100A10) levels in lung cancer. METHODS: This prospective study enrolled patients with lung cancer, patients with benign lung nodules and healthy control subjects. Serum S100A10 levels and three biomarkers were measured and compared between the groups. Associations between serum S100A10 and clinical characteristics in patients with lung cancer were investigated. The diagnostic efficacy of serum S100A10 and carcinoembryonic antigen for lung cancer was calculated. RESULTS: The study enrolled 82 patients with lung cancer, 21 with benign lung nodules and 50 healthy controls. Serum S100A10 levels were significantly higher in patients with lung cancer compared with patients with benign lung nodules and healthy control subjects. Serum S100A10 levels of patients with advanced lung cancer were significantly higher than those with early stage disease. Patients with lymph node metastases had significantly higher serum S100A10 levels than patients without lymph node metastases. The cut-off serum S100A10 value for lung cancer detection was 1.34 ng/ml, which had a sensitivity of 48.2%, a specificity of 76.2% and an area under the curve of 0.63. CONCLUSION: Serum S100A10 was significantly correlated with disease stage and lymph node metastasis. It has the potential to be a tumour biomarker for lung cancer.

Amplification of DDR2 mediates sorafenib resistance through NF-κB/c-Rel signaling in hepatocellular carcinoma.

Sorafenib was the first systemic therapy approved by the Food and Drug Administration to treat advanced hepatocellular carcinoma (HCC). However, sorafenib therapy is frequently accompanied by drug resistance. We aimed to explore the mechanisms of sorafenib resistance and provide feasible solutions to increase the response to sorafenib in patients with advanced HCC. The expression profile of discoidin domain receptor 2 (DDR2) in HCC tissues and cells was detected using quantitative real-time PCR (qPCR) and western blotting assays. The effects of DDR2 on sorafenib resistance were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, TdT-mediated dUTP nick end labeling, and flow cytometry assays. The effect of DDR2 on the nuclear factor kappa B (NF-κB) signaling pathway was evaluated by luciferase reporter, immunofluorescence, qPCR and flow cytometry assays. We demonstrated that DDR2 expression was dramatically upregulated in sorafenib-resistant HCC tissues relative to sensitive tissues. Downregulation of DDR2 sensitized HCC cell lines to sorafenib cytotoxicity. Further analysis showed that DDR2 could increase the nuclear location of REL proto-oncogene, a NF-κB subunit, to mediate NF-κB signaling. Blocking NF-κB signaling using the NF-κB signaling inhibitor, bardoxolone methyl, increased the response of HCC cells to sorafenib. Further analysis showed that DNA amplification of DDR2 is an important mechanism leading to DDR2 overexpression in HCC. Our results demonstrated that DDR2 is a potential therapeutic target in patients with HCC, and targeting DDR2 represents a promising approach to increase sorafenib sensitivity in patients with HCC.

Demodex-induced Lupus miliaris disseminatus faciei: A case report.

RATIONALE: Lupus miliaris disseminatus faciei (LMDF) is an inflammatory granulomatous skin disease without a clear etiology that frequently involves the middle area of the face and the upper eyelids. Pathological features of the disease include caseation necrosis and epithelioid granuloma. Consensus treatment for LMDF is currently unavailable. PATIENT CONCERNS: A 47-year-old Chinese female patient who presented with facial pruritic, erythematous papules 8 months before this study. She was diagnosed with skin tuberculosis at another hospital and given antituberculosis medication. However, the treatment was not efficacious. DIAGNOSES: In this study, the diagnosis of Demodex-induced LMDF was made by a dermatologist according to physical examination, skin biopsy pathology, and microscopic examination. INTERVENTIONS: The patient was given ornidazole tablets (500 mg twice a day) and recombinant bovine basic fibroblast growth factor gel (0.2 g/cm twice a day) for an 8-week period. OUTCOMES: Eight weeks after the treatment, the facial erythematous papules were improved, and no new skin lesions were observed. The patient showed no signs of recurrence during the 6-month follow-up. LESSONS SUBSECTIONS: This case showed that ornidazole combined with recombinant bovine basic fibroblast growth factor gel might be useful in treatment of Demodex-induced LMDF. In addition, the results suggested that pathological caseation necrosis was caused by a series of inflammatory and immune responses to Demodex infection.

Improved telangiectasia and reduced recurrence rate of rosacea after treatment with 540 nm-wavelength intense pulsed light: A prospective randomized controlled trial with a 2-year follow-up.

The aim of the present study was to evaluate the clinical efficacy and safety of 540 nm-wavelength intense pulsed light (IPL) for the treatment of telangiectasia in late-stage rosacea. Between July 2013 and January 2016, patients with rosacea who tested positive for Demodex folliculorum were recruited. Patients received anti-mite therapy and were then randomly apportioned to receive either three 540 nm-IPL treatments at 4-week intervals (IPL group), or no treatment (control group). Telangiectasia was assessed by the same clinician at baseline and at follow-up intervals over 2 years, where ≥90% clearance of telangiectasia was considered to indicate effective treatment. The rates of effective treatment, improvement (≥30% clearance) and recurrence (original or neo-location) were compared in both groups. After 33 patients were lost during follow-up, the IPL and control groups were comprised of 107 and 120 patients for the final analysis, respectively. The rates of effective treatment and total efficacy in the IPL group (66.36 and 95.33%, respectively) were found to be significantly higher compared with those of the control group (0 and 30.83%, respectively). By contrast, the rates of recurrence were found to be lower in the IPL group (8.41%) compared with the control group (48.33%). Redness-to-blisters associated with IPL treatment (9.7% of analyzed patients) subsided within one week and hyperpigmentation (1.9%) within 3 months. To conclude, treatment with 540 nm-IPL improved facial telangiectasia in late-stage rosacea that remained after sequential anti-mite therapy and effectively reduced the recurrence of rosacea. The present study was registered into the Chinese Clinical Trial Registry under the title 'Sequential therapy for mites folliculitis' (Trial registration number: ChiCTR-IPR-15006451; approved May 27, 2015).

bHLH transcription factor SmbHLH92 negatively regulates biosynthesis of phenolic acids and tanshinones in Salvia miltiorrhiza.

Objective: Salvia miltiorrhiza is a valuable herbal medicine with tanshinone and phenolic acid as the main biological active ingredients. The biosynthetic regulation of these bioactive compounds is controlled by a set of transcription factors (TFs). The basic helix-loop-helix (bHLH) transcription factor plays an important role in various physiological and biochemical processes in plants. However, research on bHLH TFs regulating phenolic acid or tanshinone biosynthesis in S. miltiorrhiza is limited. Methods: qRT-PCR was used for gene expression analysis. The subcellular localization of SmbHLH92 was detected by SmbHLH92-GFP transient transformation into tobacco leaves, and its fluorescence was observed using a confocal laser scanning microscope. The transcriptional activity of SmbHLH92 was confirmed in the AH109 yeast strain. RNA interference hairy roots of SmbHLH92-RNAi transgenic lines were obtained through Agrobacterium-mediated genetic transformation. Ultra performance liquid chromatography (UPLC) was used to detect the changes of phenolic acids and tanshinones. Results: SmbHLH92 is a bHLH transcription factor that is highly expressed in the root and phloem of S. miltiorrhiza. The subcellular localization and transcriptional activity of SmbHLH92 indicated that SmbHLH92 was located in the nucleus and may be a transcription factor. RNA interference (RNAi) of SmbHLH92 in hairy roots of S. miltiorrhiza significantly increased the accumulation of phenolic acid and tanshinone. Quantitative RT-PCR (RT-qPCR) analysis showed the transcription level of genes encoding the key enzymes involved in the phenolic acid and tanshinone biosynthetic pathways was increased in the hairy roots of the SmbHLH92-RNAi transgenic line, comparing with the control line. Conclusion: These data indicate that SmbHLH92 is a negative regulator involved in the regulation of phenolic acid and tanshinone biosynthesis in S. miltiorrhiza.

Comprehensive analysis of the differential expression profile of microRNAs in missed abortion.

To screen the key circulating microRNAs (miRNAs) involved in missed abortion (MA) and explore their role in MA process. We examined the miRNA profile from the serum of three MA patients and three early pregnancy induced abortion patients (controls) by next-generation sequencing. We analyzed the target genes of the differentially expressed (DE) miRNAs to analyze the function and pathway enrichment using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, respectively. We validated five candidate miRNAs by real time-qPCR. Integrated miRNA-mRNA-pathway network analysis was performed to show the interaction network of the candidate miRNAs and their target genes of interest with the involved pathways. It was observed that 227 miRNAs were differently expressed between the MA group and the early pregnancy control group, with 58 miRNAs downregulated and 169 miRNAs upregulated in the MA group. Real-time qPCR results revealed that expression of the five candidate miRNAs, namely hsa-miR-22-3p, hsa-miR-145-3p, hsa-miR-107, hsa-miR-361-3p, and hsa-miR-378c, was consistent with the miRNA data obtained by sequencing. Integrated miRNA-mRNA-pathway network analysis illustrated that target genes of the candidate miRNAs were mainly involved in the PI3K-Akt signaling pathway, HIF-1 signaling pathway, and VEGF signaling pathway, which would have potential significance in pregnancy and MA. We are the first to reveal the DE miRNAs involved in MA and illustrate their functional interaction network. These results might provide potential circulating biomarkers and new therapeutic targets for MA.

Effect of recombinant bovine basic fibroblast growth factor gel on repair of rosacea skin lesions: A randomized, single-blind and vehicle-controlled study.

The aim of the present study was to assess the effect of topical use of recombinant bovine basic fibroblast growth factor (rbFGF) gel on the repair of facial skin lesions in patients with rosacea. In the present single-blind study, a total of 1,287 patients with Demodex mite-induced rosacea who received treatment with ornidazole tablets were randomized to rbFGF gel treatment group (n=651) or control group (n=636) without revealing the group identity. Patients in the treatment group were treated with topical application of rbFGF gel over the skin lesions (0.2 g/cm2) for up to 8 weeks, whereas patients in the control group received gel vehicle treatment unless ulceration occurred. Skin lesions of all patients were scored prior to and following treatment with rbFGF gel and subjected to histological analysis. All patients were followed up for 6 months. Significant improvement in the total effective rates for erythema, papules, desquamation and dryness were observed in the rbFGF treatment group. At the end of the 2, 4 and 6 months of follow-up, the total effective rates for patients in the treatment group were significantly higher than those in the control group (81.67 vs. 28.84%; 85.11 vs. 40.81%, and 96.56 vs. 55.82%, respectively). Following treatment for 6 months, none of the patients in the rbFGF group exhibited ulceration or scar formation. In the control group, 61% of patients experienced exacerbation of skin lesions, of which, 12% exhibited ulceration and were treated with rbFGF gel to prevent scar formation. Histological analysis revealed gradual reduction in epidermal hyperplasia and resolution of dermal edema in skin lesions treated with rbFGF gel. In conclusion, rbFGF gel may improve the repair of facial rosacea skin lesions in patients treated with anti-Demodex.

Using the entrapment sequence method as a standard to evaluate key steps of proteomics data analysis process.

BACKGROUND: The mass spectrometry based technical pipeline has provided a high-throughput, high-sensitivity and high-resolution platform for post-genomic biology. Varied models and algorithms are implemented by different tools to improve proteomics data analysis. The target-decoy searching strategy has become the most popular strategy to control false identification in peptide and protein identifications. While this strategy can estimate the false discovery rate (FDR) within a dataset, it cannot directly evaluate the false positive matches in target identifications. RESULTS: As a supplement to target-decoy strategy, the entrapment sequence method was introduced to assess the key steps of mass spectrometry data analysis process, database search engines and quality control methods. Using the entrapment sequences as the standard, we evaluated five database search engines for both the origanal scores and reprocessed scores, as well as four quality control methods in term of quantity and quality aspects. Our results showed that the latest developed search engine MS-GF+ and percolator-embeded quality control method PepDistiller performed best in all tools respectively. Combined with efficient quality control methods, the search engines can improve the low sensitivity of their original scores. Moreover, based on the entrapment sequence method, we proved that filtering the identifications separately could increase the number of identified peptides while improving the confidence level. CONCLUSION: In this study, we have proved that the entrapment sequence method could be an useful strategy to assess the key steps of the mass spectrometry data analysis process. Its applications can be extended to all steps of the common workflow, such as the protein assembling methods and data integration methods.

Effectiveness of cognitive-coping therapy and alteration of resting-state brain function in obsessive-compulsive disorder.

BACKGROUND: Cognitive-coping therapy (CCT), integrating cognitive theory with stress-coping theory, is an efficacious therapy for obsessive-compulsive disorder (OCD). However, the potential brain mediation for the effectiveness remains unclear. We sought to investigate differences of resting-state brain function between OCD and healthy controls and if such differences would be changed by a four-week CCT. PATIENTS AND METHODS: Thirty-one OCD patients were recruited and randomized into CCT (n=15) and pharmacotherapy plus CCT (pCCT, n=16) groups, together with 25 age-, gender- and education-matched healthy controls. The Yale-Brown Obsessive Compulsive Scale (Y-BOCS) was scored to evaluate the severity in symptoms. Resting-state functional magnetic resonance imaging was scanned pre- and post-treatment. RESULTS: For patients, Y-BOCS scores were reduced during four-week treatment for CCT and pCCT (P<0.001), but no group difference was observed. No differences in amplitude of low-frequency fluctuation (ALFF) values were found between CCT and pCCT either pre- or post-treatment. Compared to controls, ALFF in OCD patients was higher in the left hippocampus, parahippocampus, and temporal lobes, but lower in the right orbitofrontal cortex, rectus, bilateral calcarine, cuneus, lingual, occipital, left parietal, postcentral, precentral, and parietal (corrected P<0.05). The ALFF in those regions was not significantly correlated to the severity of OCD symptoms. After a 4-week treatment, the ALFF differences between OCD patients and controls disappeared. LIMITATIONS: The pharmacotherapy group was not included since OCD patients generally do not respond to pharmacotherapy in four weeks. CONCLUSIONS: Our data indicated that resting-state brain function was different between OCD and controls; such differences disappeared after OCD symptoms were relieved.

Highly efficacious cognitive-coping therapy for overt or covert compulsions.

Pharmacotherapy and cognitive-behavioral therapy (CBT) present limitations when they are used to treat obsessive-compulsive disorder (OCD), a severe and debilitating psychiatric disorder. To search for more efficacious treatment, we investigated the effects of pharmacotherapy plus cognitive-coping therapy (pCCT) on adult OCD patients with overt or covert compulsions. Two hundred and fifteen OCD patients were randomized into pharmacotherapy plus psychological support (PPS, n=107) and pCCT (n=108). The Yale-Brown Obsessive Compulsive Scale (Y-BOCS) was used to measure severity of symptoms in the OCD patients. The Y-BOCS scores were significantly lower in pCCT than in PPS in both acute term (<3 months) and long-term follow-up. In pCCT, severity of symptoms was not different between those with covert compulsions and those with overt compulsions, but was significantly reduced at any post-treatment time-point. Y-BOCS scores in the two subtype compulsions were significantly lower in pCCT than in PPS at any post-treatpost-treatment time-point. Compared with PPS, effect size, response rate and remission rate were significantly higher in pCCT. Our findings corroborated with the hypothesis that pCCT could efficaciously treat OCD with overt compulsions or covert compulsion, suggesting that pCCT might be a potential option for adult OCD.

Partial protective effect of intranasal immunization with recombinant Toxoplasma gondii rhoptry protein 17 against toxoplasmosis in mice.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects a variety of mammals, including humans. An effective vaccine for this parasite is therefore needed. In this study, RH strain T. gondii rhoptry protein 17 was expressed in bacteria as a fusion with glutathione S-transferase (GST) and the recombinant proteins (rTgROP17) were purified via GST-affinity chromatography. BALB/c mice were nasally immunised with rTgROP17, and induction of immune responses and protection against chronic and lethal T. gondii infections were investigated. The results revealed that mice immunised with rTgROP17 produced high levels of specific anti-rTgROP17 IgGs and a mixed IgG1/IgG2a response of IgG2a predominance. The systemic immune response was associated with increased production of Th1 (IFN-γand IL-2) and Th2 (IL-4) cytokines, and enhanced lymphoproliferation (stimulation index, SI) in the mice immunised with rTgROP17. Strong mucosal immune responses with increased secretion of TgROP17-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes were also observed in these mice. The vaccinated mice displayed apparent protection against chronic RH strain infection as evidenced by their lower liver and brain parasite burdens (59.17% and 49.08%, respectively) than those of the controls. The vaccinated mice also exhibited significant protection against lethal infection of the virulent RH strain (survival increased by 50%) compared to the controls. Our data demonstrate that rTgROP17 can trigger strong systemic and mucosal immune responses against T. gondii and that ROP17 is a promising candidate vaccine for toxoplasmosis.

Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection.

Nasal vaccination is an effective therapeutic regimen for preventing certain infectious diseases. The mucosal immune response is important for resistance to Toxoplasma gondii infection. In this study, we evaluated the immune responses elicited in BALB/c mice by nasal immunisation with recombinant T. gondii receptor for activated C kinase 1 (rTgRACK1) and their protective efficacy against T. gondii RH strain during both chronic and lethal infections. Nasal vaccination with rTgRACK1 increased the level of secretory IgA in nasal, intestinal and vesical washes, and the level of IFN-γ and IL-2 in intestinal washes, indicating that rTgRACK1 vaccination promotes mucosal immune responses. The mice immunised with rTgRACK1 also displayed increased levels of rTgRACK1-specific IgA, total IgG, IgG1 and in particular IgG2a in their blood sera, increased production of IFN-γ, IL-2 and IL-4 but not IL-10 from their isolated spleen cells, and enhanced splenocyte proliferation in vitro. rTgRACK1-vaccinated mice were effectively protected against infection with T. gondii RH strain, showing over 50% reduction of tachyzoite burdens in their liver and brain tissues during a chronic infection, and also a 45% increase in their survivals during a lethal challenge. These results indicate that rTgRACK1 might represent an intriguing immunogen for developing a mucosal vaccine against toxoplasmosis.

Megakaryocytic differentiation of K562 cells induced by PMA reduced the activity of respiratory chain complex IV.

Mitochondria are involved in the regulation of cell differentiation processes, but its function changes and molecular mechanisms are not yet clear. In this study, we found that mitochondrial functions changed obviously when K562 cells were induced to megakaryocytic differentiation by phorbol 12-myristate 13-acetate (PMA). During the cell differentiation, the reactive oxygen species (ROS) level was increased, mitochondrial membrane potential declined and respiratory chain complex IV activity was decreased. Treatment with specific inhibitor of mitochondrial respiratory chain complex IV led to a significant inhibition in mitochondrial membrane potential and reduction of PMA-induced cell differentiation. However, treatment with cyclosporine A, a stabilization reagent of mitochondrial membrane potential, did not improve the down-regulation of mitochondrial respiratory chain complex IV induced by PMA. Furthermore, we found that the level of the complex IV core subunit COX3 and mitochondrial transport-related proteins Tim9 and Tim10 were decreased during the differentiation of K562 cells induced by PMA, suggesting an important role of these factors in mitochondrial functional changes. Our results suggest that changes in mitochondrial functions are involved in the PMA-induced K562 cell differentiation process, and the maintenance of the steady-state of mitochondrial functions plays a critical role in the regulation of cell differentiation.

Serine 249 phosphorylation by ATM protein kinase regulates hepatocyte nuclear factor-1α transactivation.

Hepatocyte nuclear factor-1 alpha (HNF1α) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1α. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1α at Ser249. We also found that the ATM protein kinase phosphorylated HNF1α at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1α at Ser249 in vivo. Coimmunoprecipitation assays confirmed the association between HNF1α and ATM. Moreover, ATM enhanced HNF1α transcriptional activity in a dose-dependent manner, whereas the ATM kinase-inactive mutant did not. The use of KU55933 confirmed our observation. Compared with wild-type HNF1α, a mutation in Ser249 resulted in a pronounced decrease in HNF1α transactivation, whereas no dominant-negative effect was observed. The HNF1αSer249 mutant also exhibited normal nuclear localization but decreased DNA-binding activity. Accordingly, the functional studies of HNF1αSer249 mutant revealed a defect in glucose metabolism. Our results suggested that ATM regulates the activity of HNF1α by phosphorylation of serine 249, particularly in glucose metabolism, which provides valuable insights into the undiscovered mechanisms of ATM in the regulation of glucose homeostasis.

Hepassocin activates the EGFR/ERK cascade and induces proliferation of L02 cells through the Src-dependent pathway.

Hepassocin (HPS) is a secreted protein with mitogenic activity on primary hepatocytes and protects hepatocytes from chemically-induced injury. Our previous studies showed that HPS stimulates proliferation of hepatocytes in an ERK pathway-dependent manner. However, the molecular mechanism of HPS-induced activation of the ERK pathway remains unclear. In this study, we found that HPS induced the phosphorylation of the epidermal growth factor receptor (EGFR) in the human L02 hepatocyte cell line, and this event was concomitant with the activation of the non-receptor tyrosine kinase Src. Specific inhibition of EGFR kinase activity by gefitinib or down-regulation of EGFR by specific EGFR siRNAs prevented HPS-induced activation of the ERK pathway and proliferation of L02 cells. Furthermore, inhibition of Src activity significantly blocked HPS-induced activation of the EGFR, which was suggestive of a ligand-independent transactivation mechanism of EGFR itself as well as ERK phosphorylation and proliferation of L02 cells. These results indicate that EGFR plays an important role in the mitogenic signaling induced by HPS in L02 cell lines and may further stimulate research on the role of HPS in hepatocytes within biological processes in human health and disease.

Effects of THAP11 on erythroid differentiation and megakaryocytic differentiation of K562 cells.

Hematopoiesis is a complex process regulated by sets of transcription factors in a stage-specific and context-dependent manner. THAP11 is a transcription factor involved in cell growth, ES cell pluripotency, and embryogenesis. Here we showed that THAP11 was down-regulated during erythroid differentiation but up-regulated during megakaryocytic differentiation of cord blood CD34+ cells. Overexpression of THAP11 in K562 cells inhibited the erythroid differentiation induced by hemin with decreased numbers of benzidine-positive cells and decreased mRNA levels of α-globin (HBA) and glycophorin A (GPA), and knockdown of THAP11 enhanced the erythroid differentiation. Conversely, THAP11 overexpression accelerated the megakaryocytic differentiation induced by phorbol myristate acetate (PMA) with increased percentage of CD41+ cells, increased numbers of 4N cells, and elevated CD61 mRNA levels, and THAP11 knockdown attenuated the megakaryocytic differentiation. The expression levels of transcription factors such as c-Myc, c-Myb, GATA-2, and Fli1 were changed by THAP11 overexpression. In this way, our results suggested that THAP11 reversibly regulated erythroid and megakaryocytic differentiation.