USP18 enhances the resistance of BRAF-mutated melanoma cells to vemurafenib by stabilizing cGAS expression to induce cell autophagy.

This study aims to discern the possible molecular mechanism of the effect of ubiquitin-specific peptidase 18 (USP18) on the resistance to BRAF inhibitor vemurafenib in BRAF V600E mutant melanoma by regulating cyclic GMP-AMP synthase (cGAS). The cancer tissues of BRAF V600E mutant melanoma patients before and after vemurafenib treatment were collected, in which the protein expression of USP18 and cGAS was determined. A BRAF V600E mutant human melanoma cell line (A2058R) resistant to vemurafenib was constructed with its viability, apoptosis, and autophagy detected following overexpression and depletion assays of USP18 and cGAS. Xenografted tumors were transplanted into nude mice for in vivo validation. Bioinformatics analysis showed that the expression of cGAS was positively correlated with USP18 in melanoma, and USP18 was highly expressed in melanoma. The expression of cGAS and USP18 was up-regulated in cancer tissues of vemurafenib-resistant patients with BRAF V600E mutant melanoma. Knockdown of cGAS inhibited the resistance to vemurafenib in A2058R cells and the protective autophagy induced by vemurafenib in vitro. USP18 could deubiquitinate cGAS to promote its protein stability. In vivo experimentations confirmed that USP18 promoted vemurafenib-induced protective autophagy by stabilizing cGAS protein, which promoted resistance to vemurafenib in BRAF V600E mutant melanoma cells. Collectively, USP18 stabilizes cGAS protein expression through deubiquitination and induces autophagy of melanoma cells, thereby promoting the resistance to vemurafenib in BRAF V600E mutant melanoma.

Adaptive evolutionary strategy coupled with an optimized biosynthesis process for the efficient production of pyrroloquinoline quinone from methanol.

BACKGROUND: Pyrroloquinoline quinone (PQQ), a cofactor for bacterial dehydrogenases, is associated with biological processes such as mitochondriogenesis, reproduction, growth, and aging. Due to the extremely high cost of chemical synthesis and low yield of microbial synthesis, the election of effective strains and the development of dynamic fermentation strategies for enhancing PQQ production are meaningful movements to meet the large-scale industrial requirements. RESULTS: A high-titer PQQ-producing mutant strain, Hyphomicrobium denitrificans FJNU-A26, was obtained by integrating ARTP (atmospheric and room­temperature plasma) mutagenesis, adaptive laboratory evolution and high-throughput screening strategies. Afterward, the systematic optimization of the fermentation medium was conducted using a one-factor-at-a-time strategy and response surface methodology to increase the PQQ concentration from 1.02 to 1.37 g/L. The transcriptional analysis using qRT-PCR revealed that the expression of genes involved in PQQ biosynthesis were significantly upregulated when the ARTP-ALE-derived mutant was applied. Furthermore, a novel two-stage pH control strategy was introduced to address the inconsistent effects of the pH value on cell growth and PQQ production. These combined strategies led to a 148% increase in the PQQ concentration compared with that of the initial strain FJNU-6, reaching 1.52 g/L with a yield of 40.3 mg/g DCW after 144 h of fed-batch fermentation in a 5-L fermenter. CONCLUSION: The characteristics above suggest that FJNU-A26 represents an effective candidate as an industrial PQQ producer, and the integrated strategies can be readily extended to other microorganisms for the large-scale production of PQQ.

Unilateral Sciatic Nerve Crush Induces White Blood Cell Infiltration of the Contralateral Nerve.

Nerve injury leads to the accumulation of white blood cells derived from the bone marrow in the lesioned nerve, but it is still unknown whether there are similar responses in unlesioned nerves. To address this question, sciatic nerves of mice expressing enhanced green fluorescent protein (EGFP) in their bone marrow were crushed unilaterally to observe the invasion of bone marrow-derived cells into the contralateral unlesioned nerve. Two days after surgery, EGFP+ cells began to infiltrate both the damaged and undamaged nerves. These cells gradually amplified to the highest point within 14 days and slowly lowered. In ipsilateral (lesioned) and contralateral (unlesioned) nerves, the time course of infiltration of EGFP+ cells was similar, but the magnitude was much less for the unlesioned one. Through CD68 staining, some cells were identified as macrophages. Transmission electron microscopy revealed slight demyelination and phagocytosing macrophages in the contralateral nerve. The data showed that infiltration by white blood cells is a response to nerve injury, even in uninjured nerves.

Efficacy and Safety of High Vs Standard Daptomycin Doses Examined in Chinese Patients With Severe Burn Injuries by Pharmacokinetic Evaluation.

Previous studies and the concentration-dependent antibacterial actions of daptomycin suggested that a high dose would be needed for difficult-to-treat infections in burn patients. Here, we evaluated the effects of administration of low and high doses of daptomycin in patients with severe burn injuries. The study retrospectively analyzed 10 patients with severe burn injuries, using pharmacokinetic (PK) and pharmacodynamic (PD) evaluations of daptomycin doses given to combat serious infections. Daptomycin was administered as a single dose or by multiple doses intravenously at a standard dose of 6 mg/kg/d or a high dose of 12 mg/kg/d for 7 to 14 days. The serum concentrations of daptomycin from patients were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Burn injury patients treated with high-dose daptomycin had a linear PK profile and a negative correlation between the AUC0-24 and Baux score (R2 = .953 and R2 = .801). The Cmax, AUC0-24, and t(h)½ increased significantly compared with patients given a standard dose. The efficacy of daptomycin against Staphylococcus aureus showed significantly higher rates of (AUC0-24)/MIC and Cmax/MIC after high-dose daptomycin compared with the standard dose, reflected in a significant correlation between a high dose and the Baux score (r = .976, P < .001). Positive S. aureus cultures from two of three high-dose and none of two daptomycin low-dose patients converted from positive to negative after therapy. No serious adverse events or discontinuation of the drug occurred during the treatment period. Daptomycin doses up to 12 mg/kg/d were well tolerated in Chinese patients with severe burn injuries, which were complicated by infections with S. aureus.

GDC-0084 inhibits cutaneous squamous cell carcinoma cell growth.

GDC-0084 is a novel and potent small-molecule PI3K-mTOR dual inhibitor. The present study examined its potential activity in cutaneous squamous cell carcinoma (cSCC) cells. Our results show that GDC-0084 treatment at nanomole concentrations potently inhibited survival and proliferation of established (A431, SCC-13 and SCL-1 lines) and primary human cSCC cells. GDC-0084 induced apoptosis activation and cell cycle arrest in the cSCC cells. It was more efficient than other known PI3K-Akt-mTOR inhibitors in killing cSCC cells, but was non-cytotoxic to the normal human skin fibroblasts/keratinocytes. In A431 cells and primary cSCC cells, GDC-0084 blocked phosphorylation of key PI3K-Akt-mTOR components, including p85, Akt, S6K1 and S6. GDC-0084 also inhibited DNA-PKcs activation in cSCC cells. Significantly, restoring DNA-PKcs activation by a constitutively active-DNA-PKcs (S2056D) partially inhibited GDC-0084-induced cell death and apoptosis in A431 cells. In vivo, GDC-0084 daily gavage potently inhibited A431 xenograft tumor growth in mice. In GDC-0084-treated tumor tissues PI3K-Akt-mTOR and DNA-PKcs activation were significantly inhibited. In summary, GDC-0084 inhibits human cSCC cell growth in vitro and in vivo through blocking PI3K-Akt-mTOR and DNA-PKcs signalings.

Roles of p300 and cyclic adenosine monophosphate response element binding protein in high glucose-induced hypoxia-inducible factor 1α inactivation under hypoxic conditions.

AIMS/INTRODUCTION: Given the high prevalence of diabetes and burn injuries worldwide, it is essential to dissect the underlying mechanism of delayed burn wound healing in diabetes patients, especially the high glucose-induced hypoxia-inducible factor 1 (HIF-1)-mediated transcription defects. MATERIALS AND METHODS: Human umbilical vein endothelial cells were cultured with low or high concentrations of glucose. HIF-1α-induced vascular endothelial growth factor (VEGF) transcription was measured by luciferase assay. Immunofluorescence staining was carried out to visualize cyclic adenosine monophosphate response element binding protein (CREB) localization. Immunoprecipitation was carried out to characterize the association between HIF-1α/p300/CREB. To test whether p300, CREB or p300+CREB co-overexpression was sufficient to rescue the HIF-1-mediated transcription defect after high glucose exposure, p300, CREB or p300+CREB co-overexpression were engineered, and VEGF expression was quantified. Finally, in vitro angiogenesis assay was carried out to test whether the high glucose-induced angiogenesis defect is rescuable by p300 and CREB co-overexpression. RESULTS: Chronic high glucose treatment resulted in impaired HIF-1-induced VEGF transcription and CREB exclusion from the nucleus. P300 or CREB overexpression alone cannot rescue high glucose-induced HIF-1α transcription defects. In contrast, co-overexpression of p300 and CREB dramatically ameliorated high glucose-induced impairment of HIF-1-mediated VEGF transcription, as well as in vitro angiogenesis. Finally, we showed that co-overexpression of p300 and CREB rectifies the dissociation of HIF-1α-p300-CREB protein complex in chronic high glucose-treated cells. CONCLUSION: Both p300 and CREB are required for the function integrity of HIF-1α transcription machinery and subsequent angiogenesis, suggesting future studies to improve burn wound healing might be directed to optimization of the interaction between p300, CREB and HIF-1α.

[Effect of salvia miltiorrhiza and Ligustrazine injection on the early myocardial damage of patients with severe burn].

OBJECTIVE: To observe the effect of salvia miltiorrhiza and ligustrazine injection on the early myocardial damage of severely burned patients. METHODS: Twenty severely burned patients hospitalized from January 2010 to August 2011, with burn area equal to or more than 50% TBSA, were divided into two groups following hospitalization sequence, with odd number patients entering treatment group (T, n = 10) and even number patients entering control group (C, n = 10). Patients in C group were treated with routine methods, including fluid resuscitation based on the Third Military Medical University formula, anti-infection treatment, support treatment, and organ-protection treatment, etc. In addition to routine treatment methods, patients in T group received intravenous infusion of 250 mL glucose injection (50 g/L) containing 10 mL salvia miltiorrhiza and ligustrazine concoction, once a day, and continued for three days. Venous blood of patients was drawn at post burn hour (PBH) 12, 24, 48, and 72 to determine the plasma levels of cardiac troponin I (cTnI), creatine kinase isozyme MB (CK-MB), and atrial natriuretic peptide (ANP). Data were processed with t test. RESULTS: At each time point, levels of cTnI, CK-MB, and ANP were lower in T group than in C group. Differences in contents of these parameters between two groups were statistically significant at most time points, with t values from 2.136 to 2.918, P < 0.05 or P < 0.01. Plasma levels of cTnI, CK-MB, and ANP in both groups peaked at PBH 12, which were respectively (28 ± 10) ng/mL, (76 ± 13) U/L, (430 ± 87) pg/mL in T group, and (38 ± 11) ng/mL, (87 ± 10) U/L, (453 ± 91) pg/mL in C group. From PBH 24 to 72, contents of above-mentioned parameters decreased gradually in both groups. CONCLUSIONS: Early use of salvia miltiorrhiza and ligustrazine injection in severely burned patients can effectively reduce myocardial damage, thus protect the myocardium from injury.