Alpha-Tocopherol Significantly Improved Squalene Production Yield of Aurantiochytrium sp. TWZ-97 through Lowering ROS levels and Up-Regulating Key Genes of Central Carbon Metabolism Pathways.

Media supplementation has proven to be an effective technique for improving byproduct yield during microbial fermentation. This study explored the impact of different concentrations of bioactive compounds, namely alpha-tocopherol, mannitol, melatonin, sesamol, ascorbic acid, and biotin, on the Aurantiochytrium sp. TWZ-97 culture. Our investigation revealed that alpha-tocopherol was the most effective compound in reducing the reactive oxygen species (ROS) burden, both directly and indirectly. Adding 0.7 g/L of alpha-tocopherol led to an 18% improvement in biomass, from 6.29 g/L to 7.42 g/L. Moreover, the squalene concentration increased from 129.8 mg/L to 240.2 mg/L, indicating an 85% improvement, while the squalene yield increased by 63.2%, from 19.82 mg/g to 32.4 mg/g. Additionally, our comparative transcriptomics analysis suggested that several genes involved in glycolysis, pentose phosphate pathway, TCA cycle, and MVA pathway were overexpressed following alpha-tocopherol supplementation. The alpha-tocopherol supplementation also lowered ROS levels by binding directly to ROS generated in the fermentation medium and indirectly by stimulating genes that encode antioxidative enzymes, thereby decreasing the ROS burden. Our findings suggest that alpha-tocopherol supplementation can be an effective method for improving squalene production in Aurantiochytrium sp. TWZ-97 culture.

Gancao Nourishing-Yin decoction combined with methotrexate in treatment of aging CIA mice: a study based on DIA proteomic analysis.

BACKGROUND: Elderly rheumatoid arthritis (ERA) population faces multiple treatment dilemma. Here we aim to investigate if Gancao Nourishing-Yin decoction (GCNY) added to methotrexate (MTX) exhibit better effects in an ERA mice model. METHODS: ERA mice model was established by adding D-galactose (Dgal) to collagen-induced arthritis (CIA) mice. The model was then assigned into control group (CIA + Dgal), MTX treatment group (MTX), GCNY treatment group (GCNY), and integrative treatment group (MTX + GCNY). Pathological scoring was performed to evaluate the severity between the groups. Proteomic analysis was applied to investigate the secretory phenotype of the ERA mouse model and the underlying mechanism of GCNY, MTX and their combination. Representative cytokines related to proteomic results were further validated by ELISAs. RESULTS: CIA + Dgal mice showed more aggressive joints damage than the CIA mice. Besides changes in the inflammatory pathway such as Pi3k-Akt signaling pathway in both model, differential expressed proteins (DEPs) indicated metabolism-related pathways were more obvious in CIA + Dgal mice. Low-dose MTX failed to show pathological improvement in CIA + Dgal mice, while GCNY improved joints damage significantly. Besides down-regulated inflammation-related targets, GCNY-regulated DEPs (such as Apoc1 ~ 3, Grk2 and Creb3l3) were broadly enriched in metabolism-related pathways. MTX + GCNY showed the best therapeutic effect, and the DEPs enriched in a variety of inflammatory,metabolism and osteoclast differentiation signaling pathway. Notably, MTX + GCNY treatment up-regulated Dhfr, Cbr1, Shmt1 involved in folic acid biosynthesis and anti-folate resistance pathways indicated a coincidence synergic action. ELISAs confirmed CPR and Akt that elevated in CIA + Dgal mice were significantly ameliorated by treatments, and adding on GCNY elevated folic acid levels and its regulator Dhfr. CONCLUSION: Aging aggravated joints damage in CIA, which probably due to metabolic changes rather than more severe inflammation. GCNY showed significant effects in the ERA mice model especially when integrated with MTX to obtain a synergic action.

Diet-derived antioxidants and nonalcoholic fatty liver disease: a Mendelian randomization study.

BACKGROUND: Whether supplementation with diet-derived antioxidants is beneficial for nonalcoholic fatty liver disease (NAFLD) is still controversial and we hope to answer this question using population-based genetic data. METHODS: A total of 8485 NAFLD cases and 658,849 healthy controls from four independent NAFLD genome-wide association studies were enrolled in this study. Genetic variants closely associated with the diet-derived antioxidants were selected to predict their circulating levels. A bi-directional Mendelian randomization (MR) design was employed to assess their causations. RESULTS: Genetic correlation analyses suggested inverse associations between diet-derived antioxidants and NAFLD. MR analyses indicated that the odds ratio (OR) of per standard deviation increase in genetically predicted toenail and blood selenium was 1.179 for NAFLD (95% confidence interval [1.083-1.284]). Also, the genetically elevated selenium level was causally associated with increased levels of C-reactive protein, fibrinogen, alkaline phosphatase and glycated hemoglobin. The OR of 1 µg/dL increase in genetically predicted serum lycopene was 1.082 (95%CI [1.051-1.113]). No other causal associations were found for NAFLD. However, we observed protective effects of genetically predicted ß-carotene (OR = 0.929[0.911-0.947]) and retinol (OR = 0.483[0.460-0.508]) on type 2 diabetes (T2D), and further they could reduce the serum levels of blood lipids and glucose. Reverse MR analysis suggested genetically predicated NAFLD status would not affect the levels of diet-derived antioxidants. CONCLUSION: Overall, we observed the positive associations of genetically predicted selenium and lycopene with NAFLD. However, the genetically predicted ß-carotene and retinol levels were inversely associated with the risk of T2D.

Media Supplementation with Mannitol and Biotin Enhances Squalene Production of Thraustochytrium ATCC 26185 through Increased Glucose Uptake and Antioxidative Mechanisms.

Media supplementation with exogenous chemicals is known to stimulate the accumulation of important lipids produced by microalgae and thraustochytrids. However, the roles of exogenous chemicals in promoting and preserving the terpenoids pool of thraustochytrids have been rarely investigated. Here, we realized the effects of two media supplements-mannitol and biotin-on the biomass and squalene production by a thraustochytrid strain (Thraustochytrium sp. ATCC 26185) and elucidated their mechanism of action. A significant change in the biomass was not evident with the exogenous addition of these supplements. However, with mannitol (1 g/L) supplementation, the ATCC 26185 culture achieved the best concentration (642 ± 13.6 mg/L) and yield (72.9 ± 9.6 mg/g) of squalene, which were 1.5-fold that of the control culture (non-supplemented). Similarly, with biotin supplementation (0.15 mg/L), the culture showed 459 ± 2.9 g/L and 55.7 ± 3.2 mg/g of squalene concentration and yield, respectively. The glucose uptake rate at 24 h of fermentation increased markedly with mannitol (0.31 g/Lh-1) or biotin (0.26 g/Lh-1) supplemented culture compared with non-supplemented culture (0.09 g/Lh-1). In addition, the reactive oxygen species (ROS) level of culture supplemented with mannitol remained alleviated during the entire period of fermentation while it alleviated after 24 h with biotin supplementation. The ∆ROS with mannitol was better compared with biotin supplementation. The total antioxidant capacity (T-AOC) of the supplemented culture was more than 50% during the late stage (72-96 h) of fermentation. Our study provides the potential of mannitol and biotin to enhance squalene yield and the first lines of experimental evidence for their protective role against oxidative stress during the culture of thraustochytrids.

ARTP Mutagenesis of Schizochytrium sp. PKU#Mn4 and Clethodim-Based Mutant Screening for Enhanced Docosahexaenoic Acid Accumulation.

Schizochytrium species are one of the best oleaginous thraustochytrids for high-yield production of docosahexaenoic acid (DHA, 22:6). However, the DHA yields from most wild-type (WT) strains of Schizochytrium are unsatisfactory for large-scale production. In this study, we applied the atmospheric and room-temperature plasma (ARTP) tool to obtain the mutant library of a previously isolated strain of Schizochytrium (i.e., PKU#Mn4). Two rounds of ARTP mutagenesis coupled with the acetyl-CoA carboxylase (ACCase) inhibitor (clethodim)-based screening yielded the mutant A78 that not only displayed better growth, glucose uptake and ACCase activity, but also increased (54.1%) DHA content than that of the WT strain. Subsequent optimization of medium components and supplementation improved the DHA content by 75.5 and 37.2%, respectively, compared with that of mutant A78 cultivated in the unoptimized medium. Interestingly, the ACCase activity of mutant A78 in a medium supplemented with biotin, citric acid or sodium citrate was significantly greater than that in a medium without supplementation. This study provides an effective bioengineering approach for improving the DHA accumulation in oleaginous microbes.

Potentially fatal electrolyte imbalance caused by severe hydrofluoric acid burns combined with inhalation injury: A case report.

BACKGROUND: Hydrofluoric acid (HF) is one of the most common causes of chemical burns. HF burns can cause wounds that deepen and progress aggressively. As a result, HF burns are often severe even if they involve a small area of the skin. Published cases of HF burns have mostly reported small HF burn areas. Few cases of HF inhalation injury have been reported to date. CASE SUMMARY: A 24-year-old man suffered from extensive hydrofluoric acid burns covering 60% of the total body surface area (TBSA), including deep second degree burns on 47% and third degree burns on 13% of the TBSA, after he fell into a pickling pool containing 15% HF. Comprehensive treatments were carried out after the patient was admitted. Ventricular fibrillation occurred 9 times within the first 2 h, and the lowest serum Ca2+ concentration was 0.192 mmol/L. A dose of calcium gluconate (37 g) was intravenously supplied during the first 24 h, and the total amount of calcium gluconate supplementation was 343 g. Extracorporeal membrane oxygenation (ECMO) was applied for 8 d to handle the acute respiratory distress syndrome (ARDS) induced by the HF inhalation injury. The patient was discharged after 99 d of comprehensive treatment, including skin grafting. CONCLUSION: Extensive HF burns combined with an inhalation injury led to a potentially fatal electrolyte imbalance and ARDS. Adequate and timely calcium supplementation and ECMO application were the keys to successful treatment of the patient.

Seasonal influence of scallop culture on nutrient flux, bacterial pathogens and bacterioplankton diversity across estuaries off the Bohai Sea Coast of Northern China.

In this study, we investigated the environmental impacts of scallop culture on two coastal estuaries adjacent the Bohai Sea including developing a quantitative PCR assay to assess the abundance of the bacterial pathogens Escherichia coli and Vibrio parahaemolyticus. Scallop culture resulted in a significant reduction of nitrogen, Chlorophyll a, and phosphorous levels in seawater during summer. The abundance of bacteria including V. parahaemolyticus varied significantly across estuaries and breeding seasons and was influenced by nitrate as well as nutrient ratios (Si/DIN, N/P). Bacterioplankton diversity varied across the two estuaries and seasons, and was dominated by Proteobacteria, Cyanobacteria, Actinobacteria, Bacteroidetes. Overall, this study suggests a significant influence of scallop culture on the ecology of adjacent estuaries and offers a sensitive tool for monitoring scallop contamination.

Sclerosing cholangitis secondary to bleomycin-iodinated embolization for liver hemangioma.

Sclerosing cholangitis (SC) is a rarely reported morbidity secondary to transcatheter arterial chemoembolization (TACE) with bleomycin-iodinated oil (BIO) for liver cavernous hemangioma (LCH). This report retrospectively evaluated the diagnostic and therapeutic course of a patient with LDH who presented obstructive jaundice 6 years after TACE with BIO. Preoperative imaging identified a suspected malignant biliary stricture located at the convergence of the left and right hepatic ducts. Operative exploration demonstrated a full-thickness sclerosis of the hilar bile duct with right hepatic duct stricture and right lobe atrophy. Radical hepatic hilar resection with right-side hemihepatectomy and Roux-en-Y hepaticojejunostomy was performed because hilar cancer could not be excluded on frozen biopsy. Pathological results showed chronic pyogenic inflammation of the common and right hepatic ducts with SC in the portal area. Secondary SC is a long-term complication that may occur in LCH patients after TACE with BIO and must be differentiated from hilar malignancy. Hepatic duct plasty is a definitive but technically challenging treatment modality for secondary SC.

[Effectiveness of scar split thickness skin graft combined with acellular allogeneic dermis in treatment of large deep II degree burn scar].

OBJECTIVE: To investigate the feasibility and effectiveness of using scar split thickness skin grafts combined with acellular allogeneic dermis in the treatment of large deep II degree burn scar. METHODS: Between January 2013 and December 2013, 20 cases of large deep II degree burn scar undergoing plastic operation were enrolled. There were 14 males and 6 females, aged 4 to 60 years (mean, 40 years). Burn reasons included hydrothermal burns in 10 cases, flame burns in 9 cases, and lime burns in 1 case. The burn area accounted for 70% to 96% total body surface area (TBSA) with an average of 79% TBSA. The time from wound healing to scar repair was 3 months to 2 years (mean, 7 months). Based on self-control, 0.7 mm scar split thickness skin graft was used to repair the wound at the right side of joints after scar resection (control group, n=35), 0.5 mm scar split thickness skin graft combined with acellular allogeneic dermis at the left side of joints (trial group, n=30). Difference was not statistically significant in the scar sites between 2 groups (Z=-1.152, P=0.249). After grafting, negative pressure drainage was given for 10 days; plaster was used for immobilization till wound healing; and all patients underwent regular rehabilitation exercises. RESULTS: No significant difference was found in wound healing, infection, and healing time between 2 groups (P > 0.05). All patients were followed up for 6 months. According to the Vancouver Scar Scale (VSS), the score was 5.23 ± 1.41 in trial group and was 10.17 ± 2.26 in control group, showing significant difference (t=8.925, P=0.000). Referring to Activities of Daily Living (ADL) grading standards to assess joint function, the results were excellent in 8 cases, good in 20 cases, fair in 1 case, and poor in 1 case in trial group; the results were excellent in 3 cases, good in 5 cases, fair in 22 cases, and poor in 5 cases in control group; and difference was statistically significant (Z=-4.894, P=0.000). CONCLUSION: A combination of scar split thickness skin graft and acellular allogeneic dermis in the treatment of large deep II degree burn scar is feasible and can become one of solution to the problem of skin source tension.

Expression and purification of recombinant NRL-Hsp90α and Cdc37-CRL proteins for in vitro Hsp90/Cdc37 inhibitors screening.

Hsp90 has emerged as a promising target for cancer treatment. Hsp90 interacts with co-chaperone Cdc37 to mediate the conformational maturation of its kinase client proteins. Screening small molecule inhibitors targeting Hsp90/Cdc37 might be a promising strategy for further cancer therapeutic. In order to establish a recombinant protein system, the novel cloning and purification of full-length human Hsp90α and Cdc37 from BL21 (DE3) Escherichia coli is described here. In this work, we cloned and expressed recombinant NRL-Hsp90α and Cdc37-CRL that represent the full-length human Hsp90α and Cdc37 fused with the split Renilla luciferase (RL) protein fragments. We also expressed the full-length RL protein as a control for inhibitors screening. Moreover, we confirmed that the interaction proteins were able to complement split luciferase fragments and show the RL activity when substrate was added. In comparison, two mutations NRL-Hsp90α (Q133A) and Cdc37 (R167A)-CRL retained only 20% of the complemented RL activities. Six small molecule compounds were tested using this recombinant system. Very interestingly, Sulforaphane, Withaferin A, Celastrol and EGCG all decreased the complemented NRL-Hsp90α/Cdc37-CRL activities in the concentration-dependent manner. In addition, neither Sulforaphane nor Withaferin A showed non-specific inhibition on full length RL activity. However, Celastrol and EGCG showed different RL inhibition levels. The other two compounds LBH-589 and 17-AAG showed neither NRL-Hsp90α/Cdc37-CRL nor RL inhibition activities. These results indicate that purified NRL-Hsp90α and Cdc37-CRL appeared as pure, stable and active conformation, and can be used as an in vitro bioluminescence system for Hsp90/Cdc37 inhibitors screening.

Pre-hospital emergency burn management in Shanghai: analysis of 1868 burn patients.

BACKGROUND: There are few studies reporting the level of pre-hospital emergency management of burn patients and related influencing factors in China. This study is a summary of our investigation on emergency education and people's awareness about pre-hospital emergency management of burn patients in Shanghai, China, and analyses key factors influencing pre-hospital emergency management of burn patients. METHODS: The survey was conducted by questionnaire in burn patients who sought initial clinical visits at the Burn Center of Changhai Hospital (Shanghai, China) between November 2009 and December 2010, including demographic data, burn conditions, pre-hospital emergency management and education about emergency burn management. Data were statistically treated by SPSS software. RESULTS: Altogether 1868 effective questionnaire forms were collected; 33.9% of these burn patients received cooling treatment before admission and 32.2% of them used 'folk remedies' or antibiotics to treat the wound surface. Only 12.2% of these burn patients had received education about the knowledge of emergency management, mainly through public media (38.2%), relatives and friends (24.6%), Internet (15.8%), workplace (11.4%) and schools (10.1%). The result of logistic regression analysis showed that emergency education, especially via Internet and workplace, played an important role in pre-hospital emergency management, and that different channels of emergency education affected different age groups of people: network and unit education mainly affected young adults, while relatives and friends mainly affected elderly people. In addition, educational level was an important factor favourably affecting 'cooling therapy'. CONCLUSIONS: The level of emergency burn management and related education is relatively low in China at present, and it is therefore necessary to intensify education about pre-hospital emergency management to raise the level of emergency burn management. At the same time, more attention should be paid to age- and population-specific education. Finally, universal emergency education should be included in the national basic education as a long-term strategy.

Reproducibility and discriminability of brain patterns of semantic categories enhanced by congruent audiovisual stimuli.

One of the central questions in cognitive neuroscience is the precise neural representation, or brain pattern, associated with a semantic category. In this study, we explored the influence of audiovisual stimuli on the brain patterns of concepts or semantic categories through a functional magnetic resonance imaging (fMRI) experiment. We used a pattern search method to extract brain patterns corresponding to two semantic categories: "old people" and "young people." These brain patterns were elicited by semantically congruent audiovisual, semantically incongruent audiovisual, unimodal visual, and unimodal auditory stimuli belonging to the two semantic categories. We calculated the reproducibility index, which measures the similarity of the patterns within the same category. We also decoded the semantic categories from these brain patterns. The decoding accuracy reflects the discriminability of the brain patterns between two categories. The results showed that both the reproducibility index of brain patterns and the decoding accuracy were significantly higher for semantically congruent audiovisual stimuli than for unimodal visual and unimodal auditory stimuli, while the semantically incongruent stimuli did not elicit brain patterns with significantly higher reproducibility index or decoding accuracy. Thus, the semantically congruent audiovisual stimuli enhanced the within-class reproducibility of brain patterns and the between-class discriminability of brain patterns, and facilitate neural representations of semantic categories or concepts. Furthermore, we analyzed the brain activity in superior temporal sulcus and middle temporal gyrus (STS/MTG). The strength of the fMRI signal and the reproducibility index were enhanced by the semantically congruent audiovisual stimuli. Our results support the use of the reproducibility index as a potential tool to supplement the fMRI signal amplitude for evaluating multimodal integration.

[Effect of mild hypothermia in combination of acupuncture on Bcl-2 and Bax protein expressions of local cerebral ischemia/reperfusion rats].

OBJECTIVE: To observe and compare the effects of acupuncture in combination of mild hypothermia therapy on the expressions of Bcl-2 and Bax. METHODS: Fifty rats were randomly divided into 5 groups, i.e., the control group, the model group, the acupuncture group, the mild hypothermia group, and the combination group (acupuncture in combination of mild hypothermia therapy), 10 in each group. Focal cerebral ischemia model was prepared with modified Longa's suture method. The effects of acupuncture, the mild hypothermia, and the acupuncture in combination of mild hypothermia therapy on the expressions of Bcl-2 and Bax were observed. The indices were detected using immunohistochemical assay. RESULTS: Compared with the control group, the expression of Bcl-2 in the model groups significantly decreased (P < 0.01), and the expression of Bax significantly increased (P < 0.01). Compared with the model group, the expression of Bcl-2 in the three treatment groups significantly increased (P < 0.01) and the expression of Bax significantly decreased (P < 0.01). Compared with the acupuncture group and the mild hypothermia group, the expression of Bcl-2 significantly increased and the expression of Bax significantly decreased in the combination group (both P < 0.01). Compared with the mild hypothermia group, increased expression of Bcl-2 and decreased expression of Bax in the acupuncture group were not obvious (P > 0.05). There was insignificant difference in the therapeutic efficacy between the acupuncture group and the mild hypothermia group. CONCLUSIONS: Both acupuncture and the mild hypothermia therapy could increase the expression of Bcl-2 and decrease the expression of Bax, intervene cerebral ischemia, and protect neurons. In the early cerebral ischemia/reperfusion stage, it is necessary to perform acupuncture and mild hypothermia therapy as soon as possible.

In vitro labeling of mesenchymal stem cells with superparamagnetic iron oxide by means of microbubble-enhanced US exposure: initial experience.

PURPOSE: To investigate the feasibility of magnetically labeling stem cells with superparamagnetic iron oxide (SPIO) by means of microbubble-enhanced ultrasonographic (US) exposure (MUE) and to study the effects of this approach--without secondary transfection agents--on the viability, proliferation activity, and differentiation capability of MUE-labeled stem cells. MATERIALS AND METHODS: Institutional review board approval was obtained for this study. Human mesenchymal stem cells (MSCs) ([1 to 2] x 10(6)/mL) were studied in four experiment groups: sham exposure to US with microbubbles and SPIO (group A), exposure to US with SPIO but without microbubbles (group B), exposure to US with microbubbles and SPIO (group C), and sham exposure to US without SPIO or microbubbles (group D). Intracellular iron uptake was analyzed qualitatively at light and electron microscopy. The viability and proliferation activity of MSCs were evaluated. The adipogenic and osteogenic differentiation capability of the labeled MSCs was also evaluated. Ninety-five percent confidence intervals were derived for assessment of differences in cell viability and proliferation activity between groups C and D. RESULTS: Light and electron microscopy revealed intracytoplasmic iron uptake and nearly 100% cell labeling efficiency. The MUE-labeled MSCs had unaltered viability and uncompromised proliferation activity compared with the nonlabeled MSCs. Similar to the nonlabeled MSCs, the MUE-labeled MSCs differentiated into adipogenic and osteogenic lineages. CONCLUSION: Initial study results show that stem cells can be effectively labeled with SPIO by using MUE without secondary transfection agents and thus that MUE labeling is an appealing alternative cell-labeling approach that warrants investigation for intracellular magnetic labeling of stem cells. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.2531081974/-/DC1.

[The value of spleen sub-pedicle two steps severance with LigaSure in laparoscopic splenectomy].

OBJECTIVE: To evaluate the safety and utility of the methods of spleen sub-pedicle two steps severance with LigaSure Vessel Sealing System combined with ultrasound scalpel to resect peri-splenic ligaments during laparoscopic splenectomy. METHODS: The methods and the curative effect of 32 patients with laparoscopic splenectomy performed by the way as mentioned above were summarized and analyzed. 4 males, 28 females, median 36 years (range 16 - 64 years). Mean dimensions of spleens were 17 cm (range 11 - 23 cm). Nineteen patients had idiopathic thrombocytopenia purpura (ITP), 6 hereditary spherocytosis (HS) (5 of them were the same family constellation), 3 hemolytic anemia (HA), 2 spleen injury with haematoma infection, 1 Evan syndrome, 1 leukemia. Peri-splenic ligaments were resected by LigaSure combined with ultrasound scalpel, splenic pedicle was resected by spleen sub-pedicle two steps severance with LigaSure when splenic pedicle were sufficiently free. Splenic pedicle was non-excisional occluded by LigaSure at pancreatic cauda, in the first step and was occluded and resected alongside of the spleen in the second step. RESULTS: All of the 32 cases were completely succeed, the average operating time was 70 min (55 - 130 min), the estimated intraoperative blood loss was 200 ml (50 - 600 ml), no complications such as abdominal haemorrhage, visceral injury, pancreatic leakage and infection were observed. All the patients were healed well, the average hospital stay after an operation was 6 days. The average expense of each patients could save 8050 yuan because Endo-GIA was not used. CONCLUSIONS: Spleen sub-pedicle two steps severance with LigaSure Vessel Sealing System combined with ultrasound scalpel to resect peri-splenic ligaments during laparoscopic splenectomy, which shorten the operating persistence time with less hemorrhage is a safe, effective and low-cost minimally invasive surgery in selective cases.