Knockdown of GFAT disrupts chitin synthesis in Hyphantria cunea larvae.

Glutamine-fructose-6-phosphate transaminase (GFAT) has been reported to regulate the hexosamine biosynthetic pathway as the first rate-limiting enzyme. As a key enzyme that catalyzes the substrate of glycosylation modification, which has a wide-ranging effect on cellular functions. However, there are few studies on the relationship between GFAT and chitin metabolism in insects. In the present study, the GFAT gene from Hyphantria cunea was identified based on transcriptome and bioinformatic analysis. The role of HcGFAT in regulating development and chitin synthesis was analyzed by RNA interference (RNAi) in H. cunea larvae. The full-length HcGFAT gene (2028 bp) encodes a 676 amino acid (aa) polypeptide had typical structural features of the SIS and Gn_AT_II superfamily. Phylogenetic analyses showed that GFAT of H. cunea shares the highest homology and identity with GFAT of Ostrinia furnacalis. Expression profiles indicated that HcGFAT was expressed throughout larval, pupal and three tissues (midgut, fat body, epidermis), and highly expressed in the last instar of larvae and strongly expressed in epidermis among three tissues. Bioassay results showed that knockdown of HcGFAT repressed larval growth and development, resulting in a significant loss of larval body weight. Meanwhile, HcGFAT knockdown also significantly caused larval developmental deformity. Knockdown of HcGFAT regulated the expression of four other critical genes in the chitin synthesis pathway (HcGNA, HcPAGM, HcUAP, HcCHSA), and ultimately resulted in decreased chitin content in the epidermis. In summary, these findings indicated that GFAT plays a critical role in larval growth and development, as well as chitin synthesis in H. cunea.

Avermectin stress varied structure and function of gut microbial community in Lymantria dispar asiatica (Lepidoptera: Lymantriidae) larvae.

Lymantria dispar asiatica is a globally distributed herbivorous pest. Avermectin is a highly effective, broad-spectrum insecticide. In this study, fourth instar L. dispar asiatica larvae were exposed to a LC30 dose of avermectin. The structure and function of larval gut microbial community was analyzed to examine how gut microbiota in L. dispar asiatica larvae responded to avermectin stress. Results showed that the structure and function of gut microbial community in L. dispar asiatica larvae were varied by avermectin stress. To be precise, more than half quantity of the observed Optical Taxonomic Units (OTUs) showed significantly different abundances under avermectin stress. Linear discriminant analysis effect size (LEfSe) suggested nine bacterial genera and 12 fungal genera contributed to the different gut microbial community structure in L. dispar asiatica larvae. Gut microbial function classification (PICRUSt and FUNGuild) suggested that three bacterial function categories and a fungal function guild were significantly increased, and two fungal function guilds were significantly decreased by avermectin stress. This study furthers our understanding of the physiology of L. dispar asiatica larvae under avermectin stress, and is an essential step towards future development of potential pesticide targets.

Phase I study of high-dose ascorbic acid with mFOLFOX6 or FOLFIRI in patients with metastatic colorectal cancer or gastric cancer.

BACKGROUND: Preclinical studies suggest synergistic effectiveness of ascorbic acid (AA, vitamin C) and cytotoxic agents in gastrointestinal malignancies. This phase 1 study aimed to establish the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of AA combined with mFOLFOX6 or FOLFIRI regimens in patients with metastatic colorectal cancer (mCRC) or gastric cancer (mGC). METHODS: In the dose-escalation phase, patients received AA (0.2-1.5 g/kg, 3-h infusion, once daily, days 1-3) with mFOLFOX6 or FOLFIRI in a 14-day cycle until the MTD was reached. In the speed-expansion phase, AA was administered at the MTD or at 1.5 g/kg if the MTD was not reached at a fixed rate of 0.6, 0.8 or 1 g/min. Pharmacokinetics and preliminary efficacy were also assessed. RESULTS: Thirty-six patients were enrolled. The MTD was not reached. The RP2D was established as AA at 1.5 g/kg/day, days 1-3, with mFOLFOX6 or FOLFIRI. No dose-limiting toxicity (DLT) was detected during dose escalation. The most common treatment-emergent adverse events (TRAEs) were sensory neuropathy (50%), nausea (38.9%), vomiting (36.1%) and neutropenia (27.8%). Grade 3-4 TRAEs were neutropenia (13.9%), sensory neuropathy (2.8%), vomiting (2.8%), diarrhea (2.8%) and leukopenia (2.8%). AA exposure was dose-proportional. The objective response rate was 58.3%, and the disease control rate was 95.8%. No difference in efficacy was found between mCRC patients with wild-type RAS/BRAF and mutant RAS or BRAF. CONCLUSIONS: The favorable safety profile and preliminary efficacy of AA plus mFOLFOX6/FOLFIRI support further evaluation of this combination in mCRC or mGC. TRIAL REGISTRATION: ClinicalTrial.gov Identifier: NCT02969681 .

Cu/ZnSOD always responded stronger and rapider than MnSOD in Lymantria dispar larvae under the avermectin stress.

Metalloenzyme SODs play important roles in insects dealing with environmental stress. Here, we cloned the Cu/ZnSOD (LdCZS) and MnSOD (LdMS) mRNA of Lymantria dispar by rapid amplification of cDNA ends (RACE). Afterwards their expression patterns were detected by quantitative real-time polymerase chain reaction (qPCR) after bioinformatic analysis. We found that both LdCZS and LdMS were widely detected in all gypsy moth larvae and all five tissues that we analyzed, and both of them were up-regulated after larvae were fed with avermectin of sublethal concentration and LC10. The LdCZS expression value are always higher than LdMS after treating with avermectin of sublethal concentrations. In addition, temporal expression profile in avermectin treated larvae showed that LdCZS expressed highest at 2nd hour, and LdMS expressed highest at 6th hour. The cuticulas transcribed LdCZS and LdMS significantly higher than heads, fat bodies, Malpighian tubes, and midguts after spraying avermectin of sublethal concentration. These results suggested that both Cu/ZnSOD and MnSOD are important antioxidant enzymes in L. dispar defensing against pesticide stress, and LdCZS always responded rapider and stronger than LdMS.

[Construction of multi-dimensional teaching reform system of Biochemistry based on outcome-based education].

Biochemistry is an important fundamental course of biology related majors, and has the characteristics of speedy development, massive information, sound theoretical basis and feasible applicability. It is difficult for students to learn well in the process of teaching. In addition, the experimental course lacks integrity, comprehensiveness and design experiments. Under the guidance of outcome-based education (OBE) concept, we established a multi-dimensional teaching reform system through theoretically and practically introducing of various teaching methods, online courses, bilingual teaching, stronger practical teaching and optimized assessment mode. The teaching reform system could effectively allow students to change from "passive learning" to "active learning" through activating learning enthusiasm, resulting in cultivated innovation ability. This system could play very important role in promoting quality of talent training of colleges and universities.

Utility of dermoscopy for evaluating the therapeutic efficacy of tacrolimus ointment plus 308-nm excimer laser combination therapy in localized vitiligo patients.

The aim of the present study was to assess the function of dermoscopy in evaluating the therapeutic efficacy of tacrolimus ointment plus 308-nm excimer laser combination therapy in patients with localized vitiligo. A total of 147 patients with localized vitiligo (progressive disease, n=92; stable period, n=55) were enrolled and received combination therapy for 12 weeks. The condition of the skin lesions was monitored by dermoscopy and visual observation. At the initial visit, skin lesions were observed in 61 progressive and 19 stable patients. Residual perifollicular pigmentation was more abundant in progressive-stage patients than in stable-stage patients, whereas the presence of perilesional hyperpigmentation was obviously lower in patients with progressive vitiligo. After 12 weeks of combination therapy, marked differences in residual perifollicular pigmentation were identified between the progressive- and stable-stage patients. Dermoscopy and visual observation indicated that the 12-week treatment efficacy in patients with progressive disease was significantly higher than in those with stable disease and that assessment by dermoscopy was superior to visual observation at 8 or 12 weeks of treatment. Binary logistic regression analysis revealed that the disease stage, vitiliginous areas and disease course were risk factors associated with the treatment efficacy of the combination therapy. In conclusion, dermoscopy may be used as an effective means of vitiligo therapy assessment to provide an accurate and scientific evaluation of treatment efficacy for localized vitiligo patients.

Fentanyl Enhances Hepatotoxicity of Paclitaxel via Inhibition of CYP3A4 and ABCB1 Transport Activity in Mice.

Fentanyl, a potent opioid analgesic that is used to treat cancer pain, is commonly administered with paclitaxel in advanced tumors. However, the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanism of action is not well studied. The purpose of this study was to investigate the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanisms of action. Pharmacokinetic parameters of paclitaxel were tested using reversed phase high-performance liquid chromatography (RP-HPLC). Aspartate transaminase (AST), alanine aminotransferase (ALT), and mouse liver histopathology were examined. Moreover, the cytotoxicity of anti-carcinogens was examined using 1-(4, 5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), and the intracellular accumulation of doxorubicin and rhodamine 123 was detected by flow cytometry. Furthermore, the expression of ABCB1 and the activity of ABCB1 ATPase and CYP3A4 were also examined. In this study, the co-administration of fentanyl and paclitaxel prolonged the half-life (t1/2) of paclitaxel from 1.455 hours to 2.344 hours and decreased the clearance (CL) from 10.997 ml/h to 7.014 ml/h in mice. Fentanyl significantly increased the levels of ALT in mice to 88.2 U/L, which is more than 2-fold higher than the level detected in the control group, and it increased the histological damage in mouse livers. Furthermore, fentanyl enhanced the cytotoxicity of anti-carcinogens that are ABCB1 substrates and increased the accumulation of doxorubicin and rhodamine 123. Additionally, fentanyl stimulated ABCB1 ATPase activity and inhibited CYP3A4 activity in the liver microsomes of mice. Our study indicates that the obvious hepatotoxicity during this co-administration was due to the inhibition of CYP3A4 activity and ABCB1 transport activity. These findings suggested that the accumulation-induced hepatotoxicity of paclitaxel when it is combined with fentanyl should be avoided.

Quantification of photocyanine in human serum by high-performance liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.

Photocyanine is a novel anticancer drug. Its pharmacokinetic study in cancer patients is therefore very important for choosing doses, and dosing intervals in clinical application. A rapid, selective and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the determination of photocyanine in patient serum. Sample preparation involved one-step protein precipitation by adding methanol and N,N-dimethyl formamide to 0.1 mL serum. The detection was performed on a triple quadrupole tandem mass spectrometer operating in multiple reaction-monitoring (MRM) mode. Each sample was chromatographed within 7 min. Linear calibration curves were obtained for photocyanine at a concentration range of 20-2000 ng/mL (r > 0.995), with the lower limit of quantification (LLOQ) being 20 ng/mL. The intrabatch accuracy ranged from 101.98% to 107.54%, and the interbatch accuracy varied from 100.52% to 105.62%. Stability tests showed that photocyanine was stable throughout the analytical procedure. This study is the first to utilize the HPLC-MS/MS method for the pharmacokinetic study of photocyanine in six cancer patients who had received a single dose of photocyanine (0.1 mg/kg) administered intravenously.

Chlorbipram: a novel PDE4 inhibitor with improved safety as a potential antidepressant and cognitive enhancer.

Major depressive disorder is a common, but serious, psychiatric dysfunction that affects 21% of the population worldwide. Rolipram, a first-generation phosphodiesterase-4 (PDE4) inhibitor, has been shown to have significant antidepressant and cognitive enhancement effects; however, it was unsuccessful in clinic trials because of PDE4-dependent side effects such as nausea and emesis. In this study, we investigated the neuropharmacology of the novel PDE4 inhibitor chlorbipram and the classical PDE4 inhibitor rolipram. Using antidepressant-sensitive behavioral tests, we demonstrated that the acute single administration of chlorbipram (0.075-0.6 mg/kg) produced antidepressant-like effects, as evidenced by decreases in the duration of immobility in Kunming mice in the forced swim and tail suspension tests, and no significant changes in locomotor activity. Scopolamine-induced cognitive dysfunction was also significantly attenuated in the Morris water maze test after the treatment of Sprague Dawley rats with different doses of chlorbipram (0.5-1.5mg/kg). Furthermore, we evaluated the emetic potential of chlorbipram in beagle dogs. After oral administration, 0.5mg/kg rolipram showed emetic profiles in all dogs within 20 minutes, whereas chlorbipram did not induce any emesis during the 120-min observation period, even at the 1.0mg/kg dose. Together, our data suggest that chlorbipram is a novel antidepressant and cognitive enhancer with little or no emetic potency.

Promotion of ß-amyloid production by C-reactive protein and its implications in the early pathogenesis of Alzheimer's disease.

C-reactive protein (CRP) and ß-amyloid protein (Aß) are involved in the development of Alzheimer's disease (AD). However, the relationship between CRP and Aß production is unclear. In vitro and in vivo experiments were performed to investigate the association of CRP with Aß production. Using the rat adrenal pheochromocytoma cell line (PC12 cells) to mimic neurons, cytotoxicity was evaluated by cell viability and supernatant lactate dehydrogenase (LDH) activity. The levels of amyloid precursor protein (APP), beta-site APP cleaving enzyme (BACE-1), and presenilins (PS-1 and PS-2) were investigated using real-time polymerase chain reaction and Western blotting analysis. Aß1-42 was measured by enzyme-linked immunosorbent assay. The relevance of CRP and Aß as well as potential mechanisms were studied using APP/PS1 transgenic (Tg) mice. Treatment with 0.5-4.0 µM CRP for 48 h decreased cell viability and increased LDH leakage in PC12 cells. Incubation with CRP at a sub-toxic concentration of 0.2 µM increased the mRNA levels of APP, BACE-1, PS-1, and PS-2, as well as Aß1-42 production. CRP inhibitor reversed the CRP-induced upregulations of the mRNA levels of APP, BACE-1, PS-1, and PS-2, and the protein levels of APP, BACE-1, PS-1, and Aß1-42, but did not reversed Aß1-42 cytotoxicity. The cerebral levels of CRP and Aß1-42 in APP/PS1 Tg mice were positively correlated, accompanied with the elevated mRNA expressions of serum amyloid P component (SAP), complement component 1q (C1q), and tumor necrosis factor-α (TNF-α). These results suggest that CRP cytotoxicity is associated with Aß formation and Aß-related markers expressions; CRP and Aß were relevant in early-stage AD; CRP may be an important trigger in AD pathogenesis.