Autotoxicity of Endogenous Organic Acid Stress in Two Ganoderma lucidum Cultivars.

Ganoderma lucidum has been used as a rare medical mushroom for centuries in China, due to its health-promoting properties. Successive cropping obstacles are common in the cultivation of G. lucidum, although the remaining nutrients in the germ substrate are sufficient for a second fruiting. Here, we aimed to study the metabolite profile of G. lucidum via nontargeted metabonomic technology. Metabonomic data revealed that organic acids played an important role in the cropping obstacles of G. lucidum, which is accordance with the pH decrease in the germ substrate. A Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis indicated that most differential acids participated in the metabolic pathways. Five acids were all significantly upregulated by two MS with high energy (MSE) modes in two cultivars, among which 5-hydroxy-2-oxo-4-ureido-2,5-dihydro-1H-imidazole-5-carboxylic acid is also involved in purine metabolism regulation and microbial metabolism in diverse environments. Taken together, this work illustrated the organic acid stress generated by G. lucidum, which formed the autotoxicity feedback, and resulted in cropping obstacles. Determining the cause of the cropping obstacles in G. lucidum will promote the utilization rate of fungus substrate to realize the sustainable use of this resource.

Nitrogen Removal Performance of Novel Isolated Bacillus sp. Capable of Simultaneous Heterotrophic Nitrification and Aerobic Denitrification.

The control of nitrogenous pollutants is a key concern in aquaculture production. Bacillus spp. are commonly used as probiotics in aquaculture, but only a few reports have focused on the simultaneous heterotrophic nitrification and aerobic denitrification (SND) capacity of Bacillus sp. strains. In order to improve nitrogen biodegradation efficiency in the aquaculture industry, the SND capacity of Bacillus sp. strains was evaluated using both individual and mixed nitrogen sources and different sources of organic carbon. Twelve Bacillus sp. isolates were screened from aquaculture pond sediments and shrimp guts for nitrogen biodegradation. Six strains exhibited especially efficient inorganic nitrogen removal capacities in media with individual and mixed nitrogen sources. These strains comprise K8, N2, and N5 (B. subtilis), HYS (B. albus), H4 (B. amyloliquefaciens), and S1 (B. velezensis). The strains grew better when the sole nitrogen source was NH4+-N, but degraded nitrogen in the following order: nitrite nitrogen (NO2--N), ammonium nitrogen (NH4+-N), and nitrate nitrogen (NO3--N). There was no associated NO2--N accumulation, regardless of the nitrogen source. The optimal carbon source for nitrogen removal varied based on different nitrogen sources and associated metabolic pathways. The optimal carbon sources for the removal of NO3--N, NO2--N, and NH4+-N were sodium citrate, sodium acetate, and sucrose, respectively. The application of H4 in recirculating aquaculture water further demonstrated that NO2--N and NH4+-N could be effectively removed. This study thus provides valuable technical support for the bioremediation of aquaculture water.

Short-wavelength blue light contributes to the pyroptosis of human lens epithelial cells (hLECs).

PURPOSE: The purpose of this study is to examine the effect of short-wavelength blue light (SWBL) on cultured human lens epithelial cells (hLECs). The pathogenesis of cataracts after SWBL exposure is discussed. METHODS: HLE-B3 hLECs were randomly divided into 3 groups: the NC group, which was grown in a dark incubator; the acetyl (Ac)-Tyr-Val-Ala-Asp-chloromethyl ketone (AC-YVAD-CMK) treatment group; and the SWBL exposure group. After SWBL (2500 lux) irradiation (for 8, 16, 24, and 32 h), caspase-1 and gasdermin D (GSDMD) expression levels in HLE-B3 hLECs were examined using ELISA, immunofluorescence staining, and Western blotting analyses. Double-positive staining of hLECs for activated and inhibited caspase-1 was used to determine pyroptosis in HLE-B3 hLECs. RESULTS: SWBL led to hLEC death, but a caspase-1 inhibitor suppressed cell death. The flow cytometry results also confirmed the dose-dependent effect of SWBL irradiation on the pyroptotic death of hLECs. Caspase-1 and GSDMD expression levels in all hLEC groups changed with blue light exposure times (8, 16, 24, and 32 h) and were higher in the AC-YVAD-CMK and SWBL exposure groups than in the NC group. The immunofluorescence results revealed higher GSDMD-N expression in the cell membrane of both the AC-YVAD-CMK and SWBL exposure groups than in the NC group. CONCLUSIONS: Based on the data, SWBL induces pyroptotic programmed cell death by activating the GSDMD signalling axis in HLE-B3 hLECs. These results provide new insights into the exploitation of new candidates for the prevention of cataracts.

Targeting against the activity of the NLRP3 inflammasome is a potential therapy for rat testicular tissue cryopreservation and transplantation.

The objective of the present experiment was to explore the role of NLRP3 inflammasome in the testicular tissue freezing, thawing and grafting; furthermore, the potential effect of a NLRP3 inhibitor on the function of testis transplant was explored. Tissues from male Wistar rats in pre-pubertal age were cryopreserved, thawed and auto-transplanted into the scrotum treated or not treated with the MCC950 (a NLRP3 inhibitor). After grafting, cryopreserved tissue was removed and analysed. Quantitative morphometric, immunohistochemical techniques and Western blotting were used to evaluate the survival of spermatogonia and the activation of the NLRP3 inflammasome after freezing/thawing/grafting. Moreover, serum IL-1ß level was assessed with ELISA kits. The testicular transplants exhibited upregulated expression of the NLRP3 pathway meditors (NLRP3, IL-1ß). In NLRP3 inhibition group, the rate of recovered grafts, the percentage of intact tubules and spermatogonial number were significantly higher than that in cryopreserved graft group. Moreover, serum concentration of IL-1ß in NLRP3 inhibition group was significantly lower than that in cryopreserved graft group. Testicular tissue cryopreservation and transplantation exhibited upregulated expression of NLRP3 pathway and NLRP3 inflammasome blockade improves testicular graft function. These finding suggest that NLRP3 inflammasome is a therapeutic target for testicular tissue cryopreservation and transplantation.

Hydrogen gas post-conditioning attenuates early neuronal pyroptosis in a rat model of subarachnoid hemorrhage through the mitoKATP signaling pathway.

Neuronal pyroptosis serves an important role in the progress of neurologic dysfunction following subarachnoid hemorrhage (SAH), which is predominantly caused by a ruptured aneurysm. Hydrogen gas has been previously reported to be an effective anti-inflammatory agent against ischemia-associated diseases by regulating mitochondrial function. The objective of the present study was to investigate the potential neuroprotective effects of hydrogen gas post-conditioning against neuronal pyroptosis after SAH, with specific focus on the mitochondrial ATP-sensitive K+ (mitoKATP) channels. Following SAH induction by endovascular perforation, rats were treated with inhalation of 2.9% hydrogen gas for 2 h post-perforation. Neurologic deficits, brain water content, reactive oxygen species (ROS) levels, neuronal pyroptosis, phosphorylation of ERK1/2, p38 MAPK and pyroptosis-associated proteins IL-1ß and IL-18 were evaluated 24 h after perforation by a modified Garcia method, ratio of wet/dry weight, 2',7'-dichlorofluorescin diacetate, immunofluorescence and western blot assays, respectively. An inhibitor of the mitoKATP channel, 5-hydroxydecanoate sodium (5-HD), was used to assess the potential role of the mitoKATP-ERK1/2-p38 MAPK signal pathway. Hydrogen gas post-conditioning significantly alleviated brain edema and improved neurologic function, reduced ROS production and neuronal pyroptosis, suppressed the expression of IL-1ß and IL-18 whilst upregulating ERK1/2 phosphorylation, but downregulated p38 MAPK activation 24 h post-SAH. These aforementioned effects neuroprotective were partially reversed by 5-HD treatment. Therefore, these observations suggest that post-conditioning with hydrogen gas ameliorated SAH-induced neuronal pyroptosis at least in part through the mitoKATP/ERK1/2/p38 MAPK signaling pathway.

Role of Blueberry Anthocyanin Extract in the Expression of SIRT1 and NF-κB in Rat Lens Epithelial Cells in Experimentally Induced DM.

PURPOSE: To investigate the mechanism of the protective effects of blueberry anthocyanin extract (BAE) against oxidative stress and the roles of SIRT1 and NF-κB in the pathogenesis of diabetic cataracts. METHODS: Male SD rats were randomly divided into a control group (group A) and an experimental group. The rats in the experimental group were intraperitoneally injected with streptozotocin (STZ) (60 mg/kg). Rats with blood glucose levels ≥16.7 mmol/L were considered to have DM. The rats in the experimental group were subdivided into group B (distilled water by oral gavage: 10 ml/kg/day), group C (5% blueberry anthocyanin extract by oral gavage: 10 ml/kg/day), and group D (15% blueberry anthocyanin extract by oral gavage: 10 ml/kg/day), with 15 rats in each group. At the end of 8 weeks, some biochemical parameters, including the expression of SIRT1 and NF-κB by qRT-PCR and western blotting and the activity of SOD and GSH, were measured in lens epithelial cells (LECs). RESULTS: The lenses of the rats in the control group appeared transparent during the entire 8-week period. Four weeks following STZ injection, cataracts gradually progressed in the experimental rats. SIRT1 expression was upregulated in groups B, C and D compared to the control group. However, the expression of NF-κB decreased in the experimental groups with increasing doses of BAE (p < .05). Our study also showed that the activity of the SOD enzyme and GSH in the LECs of the rats in the experimental group increased with higher doses of BAE. CONCLUSIONS: The results indicated that BAE significantly delayed the progression of diabetic cataracts in rats. These effects may be due to the dose-dependent antioxidant activity of BAE, which is mediated by enhanced SOD and GSH activities, SIRT1 expression and reduced NF-κB expression. Abbreviations: SD rat: Sprague-Dawley rat; BAE: Blueberry anthocyanin extract; LECs: Lens epithelial cells; SOD: Superoxide dismutase; GSH: Glutathione; DM: Diabetes mellitus; SIRT1: Silent information regulator protein-1; STZ: Streptozotocin; PBS: Phosphate-buffered saline.

Role of short-wavelength blue light in the formation of cataracts and the expression of caspase-1, caspase-11, Gasdermin D in rat lens epithelial cells: insights into a novel pathogenic mmechanism of cataracts.

BACKGROUND: With the popularity of blue-rich light-emitting diode (LED)-backlit display devices, our eyes are now exposed to more short-wavelength blue light than they were in the past. The goal of this study was to investigate the pathogenesis of cataracts after short-wavelength light exposure. METHODS: Sprague-Dawley (SD) rats were selected and randomly divided into a control group (10 rats each for the 4-, 8-, and 12-week groups) and an experimental group (10 rats each for the 4-, 8-, and 12-week groups). The rats in the experimental group were exposed to a short-wavelength blue LED lamp for 12 h per day. After exposure to the blue LED lamp, the rats were maintained in total darkness for 12 h, after which a 12-h light/dark cycle was resumed. The intensity of the lamp was 3000 lx. At the end of the short-wavelength blue LED lamp exposure (for 4, 8, and 12 weeks), the expression levels of caspase-1, caspase-11 and gasdermin D (GSDMD) were examined in rat lens epithelial cells (LECs) using qRT-PCR and Western blot analyses. An illuminance of 2500 lx was used to study the potential effect of blue LED light on HLE-B3 hLECs in vitro. AC-YVAD-CMK, a caspase-1 inhibitor, was used to confirm the pyroptosis of LECs by flow cytometry. RESULTS: After 6 weeks, cataracts developed in the experimental rats (4/20 eyes). The clarity of the lens gradually worsened with the duration of exposure. Twelve weeks later, all of the rat eyes had developed cataracts. The expression levels of caspase-1, caspase-11 and GSDMD at 4, 8, and 12 weeks were significantly higher in the samples from rats exposed to a short-wavelength blue LED lamp than in the samples from control rats (p<0.05). The proportions of double-positive hLECs were significantly increased in the 5-h and 10-h short-wavelength blue light exposure subgroups compared with the 5-h and 10-h caspase-1 inhibitor subgroups (p < 0.05). CONCLUSION: The data indicate that pyroptosis plays a key role in cataract induction after short-wavelength blue light exposure. This study might provide new insights into a novel pathogenic mechanism of cataracts.

Targeting NRASQ61K mutant delays tumor growth and angiogenesis in non-small cell lung cancer.

Tumor cells require vascular supply for their growth, and they express proangiogenic growth factors that promote the formation of vascular networks. Many oncogenic mutations that may potentially lead to tumor angiogenesis have been identified. Somatic mutations in the small GTPase NRAS are the most common activating lesions found in human cancer and are generally associated with poor response to standard therapies. However, the mechanisms by which NRAS mutations affect tumor angiogenesis are largely unknown. Therefore, we investigated the role of NRASQ61K oncogene in tumor angiogenesis and analyzed tumors harboring NRASQ61K for potential sensitivity to a kinase inhibitor. Knock-in of the NRASQ61K allele in human normal epithelial cells triggered the angiogenic response in these cells. In cancer cells harboring oncogenic NRAS, a mitogen-activated protein kinase (MEK) inhibitor down-regulated the extracellular regulated protein kinase (ERK) pathway and inhibited the expression of proangiogenic molecules. In tumor xenografts harboring the NRASQ61K, the MEK inhibitor extensively modified tumor growth, causing abrogation of angiogenesis. Overall, our results provide a functional link between oncogenic NRAS and angiogenesis, and imply that tumor vasculature could be indirectly altered by targeting a genetic lesion on which cancer cells are dependent.

Malignant Peritoneum Mesothelioma with Hepatic Involvement: A Single Institution Experience in 5 Patients and Review of the Literature.

Malignant peritoneal mesothelioma with invasion of the liver is an invariably fatal disease. We aimed to clarify the characteristics of malignant peritoneal mesothelioma cases with liver involvement. The clinical presentation, computed tomography images, and immunohistochemical and histopathological features of 5 patients with malignant peritoneal mesothelioma and liver involvement were evaluated. The diagnosis was established by imaging and immune profiles of the tumours. A review of 8 cases with primary or invading malignant mesothelioma in liver is presented. All 5 mesothelioma cases were asbestos-related. CT images of malignant peritoneal mesothelioma with the liver involvement typically showed that the lesion grew inside the liver along the capsule and was possibly accompanied by capsule breakthrough and extrahepatic infiltration. The tumours exhibited a common epithelioid appearance in all 5 patients and most cases revealed positive Cal, CK, and MC with negative CEA and HeP. Different from our findings, the review of literature revealed that most malignant mesothelioma of liver was due to primary intrahepatic malignant mesothelioma. Finally, we concluded that the diagnosis of malignant peritoneal mesothelioma cases with liver invasion is reliably achieved by the history of asbestos exposure, the characteristic CT imaging, and immune profiles of the tumours.

Preliminary study on CT perfusion imaging in guiding biopsy of pulmonary lumps.

BACKGROUND: CT perfusion imaging (CTP) has been proved to be a powerful functional imaging technique. This study aimed to evaluate the value of CTP in guiding biopsy of pulmonary lumps. METHODS: A total of 147 patients with pulmonary lumps who had CT guided biopsies were enrolled in this study from February 2005 to June 2007. The patients were assigned to 3 groups: 33 cases guided by CTP as group I, 45 cases guided by contrast-enhanced scan of CT as group II, and 69 cases guided by plain scan of CT as group III. Each group was subdivided into central and peripheral types according to the location of the lumps. The achievement ratio of biopsy, the accuracy in grouping, and grading of lung cancer, and the incidence of complication were compared. RESULTS: The total achievement ratios of biopsy from group I to III were 100% (33/33), 91% (41/45), and 80% (55/69) respectively, and the difference was statistically significant between group I and III (P < 0.05). For the central type, they were 100% (18/18), 88% (15/17), and 79% (11/14) respectively, and the difference was also statistically significant between group I and III (P < 0.05). For the peripheral type, they were 100% (15/15), 93% (26/28), and 80% (44/55) respectivelies, and the difference was not statistically significant among the three groups. The total accuracies in grouping and grading of lung cancer from group I to III were 100% (27/27), 91% (31/34), and 72% (33/46) respectively, and the difference was statistically significant between group I and III and between group II and III (P < 0.05). For the central type, they were 100% (16/16), 94% (16/17), and 70% (8/12) respectively, and the difference was statistically significant between group I and III (P < 0.05). For the peripheral type, they were 100% (11/11), 88% (15/17), and 72% (26/36) respectively, and the difference was statistically significant between group I and III (P < 0.05). The total incidence of complication from group I to III were 15% (5/33), 27% (12/45), and 43% (30/69) respectively, and the difference was statistically significant between group I and III (P < 0.01). For the central type, they were 11% (2/18), 24% (4/17), and 57% (8/14) respectively, and the difference was statistically significant between group I and III (P < 0.01). For the peripheral type, they were 20% (3/15), 29% (8/28), and 40% (22/55) respectively, and no statistically significant difference was found among the three groups. CONCLUSIONS: CTP guided biopsy of pulmonary lumps using multi-detector row CT has the potential to improve the accuracy of histopathological diagnosis with a lower risk and higher achievement ratio. More research and technical improvements are needed before it is widely used.