Geldanamycin ameliorates multiple organ dysfunction and microthrombosis in septic mice by inhibiting the formation of the neutrophil extracellular network by activating heat shock factor 1 HSF1.

Sepsis and septic shock are common critical illnesses in the intensive care unit with a high mortality rate. Geldanamycin (GA) has a broad spectrum of antibacterial and antiviral activity and has inhibitory effects on various viruses. However, whether GA affects sepsis due to infections remains unknown. In this study, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen and creatinine in serum; neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in the urine, cytokines (tumour necrosis factor alpha, interleukin-1ß and interleukin-6) in the bronchoalveolar lavage fluid and myeloperoxidase in the lung tissues were measured using enzyme-linked immunosorbent assay kits. Pathological injury was measured by hematoxylin and eosin staining and neutrophils were measured by flow cytometry analysis; related expressions were analysed by qPCR, western blot and immunofluorescence assay. The results showed that GA significantly ameliorated cecum ligation and puncture (CLP)-triggered liver, kidney and lung injury in septic mice. In addition, we found that GA dose-dependently inhibited microthrombosis and alleviated coagulopathy in septic mice. Further molecular mechanism analysis suggests that GA may act through upregulation of heat shock factor 1 and tissue-type plasminogen activator. In conclusion, our study elucidated the protective effects of GA in a mouse model established using CLP, and the results reveal that GA may be a promising agent for sepsis.

Screening and identification of miR-181a-5p in oral squamous cell carcinoma and functional verification in vivo and in vitro.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common malignant tumor associated with poor prognosis. MicroRNAs (miRNAs) play crucial regulatory roles in the cancer development. However, the role of miRNAs in OSCC development and progression is not well understood. METHODS: We sought to establish a dynamic Chinese hamster OSCC animal model, construct miRNA differential expression profiles of its occurrence and development, predict its targets, and perform functional analysis and validation in vitro. RESULTS: Using expression and functional analyses, the key candidate miRNA (miR-181a-5p) was selected for further functional research, and the expression of miR-181a-5p in OSCC tissues and cell lines was detected. Subsequently, transfection technology and a nude mouse tumorigenic model were used to explore potential molecular mechanisms. miR-181a-5p was significantly downregulated in human OSCC specimens and cell lines, and decreased miR-181a-5p expression was observed in multiple stages of the Chinese hamster OSCC animal model. Moreover, upregulated miR-181a-5p significantly inhibited OSCC cell proliferation, colony formation, invasion, and migration; blocked the cell cycle; and promoted apoptosis. BCL2 was identified as a target of miR-181a-5p. BCL2 may interact with apoptosis- (BAX), invasion- and migration- (TIMP1, MMP2, and MMP9), and cell cycle-related genes (KI67, E2F1, CYCLIND1, and CDK6) to further regulate biological behavior. Tumor xenograft analysis indicated that tumor growth was significantly inhibited in the high miR-181a-5p expression group. CONCLUSION: Our findings indicate that miR-181a-5p can be used as a potential biomarker and provide a novel animal model for mechanistic research on oral cancer.

CTRP6 suppresses neutrophil extracellular traps formation to ameliorate sepsis-induced lung injury through inactivation of ERK pathway.

BACKGROUND: Septic lung injury is associated with excessive neutrophil activation, while neutrophil extracellular traps formation contributes to inflammatory lung injury in sepsis. C1q/tumor necrosis factor-related protein-6 (CTRP6) is a paralog of adiponectin and exerts anti- inflammatory and antioxidant properties. The role of CTRP6 in sepsis-associated inflammatory lung injury was investigated in this study. METHODS: Mice were injected with lipopolysaccharides (LPS) intraperitoneally to establish the mouse sepsis model. They were first tail-vein injected with adenovirus-mediated overexpression CTRP6 (Ad-CTRP6) and then subjected to the LPS injection. Pathological changes in lungs were detected by hematoxylin and eosin staining. Inflammation cytokine levels in bronchoalveolar lavage fluid were assessed by qRT-PCR and ELISA. Flow cytometry was used to detect the number of neutrophils in bronchoalveolar lavage fluid, and immunofluorescence was performed to assess neutrophil extracellular traps. RESULTS: Lipopolysaccharides induced pulmonary congestion, interstitial edema, and alveolar wall thickening in the lungs, as well as upregulated lung histology score and wet/dry weight ratio. CTRP6 was reduced in lung tissues of septic mice. Injection with Ad-CTRP6 ameliorated extensive histopathological changes in LPS-induced mice and decreased lung histology score and wet/dry weight ratio. Overexpression of CTRP6 reduced the levels of TNF-α, IL-6, and IL-1ß in septic mice. Injection with Ad-CTRP6 also decreased the number of neutrophils and downregulated Cit-H3 and myeloperoxidase polymers in septic mice. Protein expression of p-ERK in septic mice was reduced by overexpression of CTRP6. CONCLUSION: CTRP6 attenuated septic lung injury, exerted anti-inflammatory effect, and suppressed neutrophil extracellular traps formation against sepsis through inactivation of extracellular signal-regulated kinase signaling.

Mitochondrial DNA Diversity of Mesocricetus auratus and Other Cricetinae Species among Cricetidae Family.

Unique anatomical and physiological features have made hamster species desirable research models. Comparative genomics and phylogenetic analysis of the hamster family members to clarify their evolution and genetic relationship, can provide a genetic basis for the comprehension of the variable research results obtained using different hamster models. The Syrian golden hamster (Mesocricetus auratus) is the most widely used species. In this study, we sequenced the complete mitochondrial genome (mitogenome) of M. auratus, compared it with the mitogenome of other Cricetinae subfamily species, and defined its phylogenetic position in the Cricetidae family. Our results show that the mitogenome organization, gene arrangement, base composition, and genetic analysis of the protein coding genes (PCGs) of M. auratus are similar to those observed in previous reports on Cricetinae species. Nonetheless, our analysis clarifies some striking differences of M. auratus relative to other subfamily members, namely distinct codon usage frequency of TAT (Tyr), AAT (Asn), and GAA (Glu) and the presence of the conserved sequence block 3 (CSB-3) in the control region of M. auratus mitogenome and other hamsters (not found in Arvicolinae). These results suggest the particularity of amino acid codon usage bias of M. auratus and special regulatory signals for the heavy strand replication in Cricetinae. Additionally, Bayesian inference/maximum likelihood (BI/ML) tree shows that Cricetinae and Arvicolinae are sister taxa sharing a common ancestor, and Neotominae split prior to the split between Cricetinae and Arvicolinae. Our results support taxonomy revisions in Cricetulus kamensis and Cricetulus migratorius, and further revision is needed within the other two subfamilies. Among the hamster research models, Cricetulus griseus is the species with highest sequence similarity and closer genetic relationship with M. auratus. Our results show mitochondrial DNA diversity of M. auratus and other Cricetinae species and provide genetic basis for judgement of different hamster models, promoting the development and usage of hamsters with regional characteristics.

Comparative responses of Sinopotamon henanense to acute and sub-chronic Cd exposure.

Studies on the freshwater crab Sinopotamon henanense have shown that acute and sub-chronic Cd2+ exposure induced differential alterations in the respiratory physiology and gill morphology. To elucidate Cd2+ toxicity under these two exposure conditions, crabs were acutely exposed to 7.14, 14.28, and 28.55 mg/L Cd2+ for 96 h and sub-chronically exposed to 0.71, 1.43, and 2.86 mg/L Cd2+ for 3 weeks. The Cd2+ accumulation, total metallothionein (MT), superoxide dismutase, and malondialdehyde (MDA) contents in the gill tissues were detected. Moreover, the glucose-6-phosphate dehydrogenase (G6PDH) activity, NADPH content, reduced glutathione (GSH), oxidized glutathione (GSSG), and GSH/GSSG ratio in the hepatopancreas were determined. The morphology of the X-organ-sinus gland complex was also observed. The results showed that sub-chronical Cd2+ exposure induced lower MT content and higher MDA level in the gills than in the acute exposure. In the hepatopancreas, acute Cd2+ exposure decreased the pentose phosphate pathway activity and NADPH content; however, an increased G6PDH activity and NADPH content were detected in sub-chronic Cd2+ exposure (2.86 mg/L). Morphological changes occurred in the sinus gland in crabs exposed to 2.86 mg/L Cd2+ for 3 weeks. The tightly packed structure composed by the axons, enlarged terminals, and glial cells, became loose and porous. Ultra-structurally, a large number of vacuoles and few neurosecretory granules were observed in the axon terminal. These effects added to our understanding of the toxic effects of Cd2+ and provide biochemical and histopathological evidence for S. henanense as a biomarker of acute or long-term waterborne Cd2+ pollution.

Animal model and bioinformatics analyses suggest the TIMP1/MMP9 axis as a potential biomarker in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck. However, the molecular mechanism underlying its development and progression is yet unclear. Genes that are differentially expressed, that is, differentially expressed genes (DEGs), between normal and diseased tissues are believed to be involved in disease development and progression. To identify the DEGs in OSCC and explore their role in occurrence and progression, we established a Chinese hamster OSCC model, determined the DEG, screened the identified DEGs, and performed Gene Ontology (GO) and KEGG enrichment analyses. A protein-protein interaction (PPI) network was generated to screen potential candidate genes. We then analyzed the expression, tumor stage and prognosis of candidate genes using the Gene Expression Profiling Interactive Analysis (GEPIA) database. Finally, we verified the candidate DEGs by quantitative real-time PCR and Gene Expression Omnibus analysis. The results showed 194 significantly DEGs, 140 enriched GO terms, and 8 KEGG pathways, which suggested that OSCC was closely related to the immune system, cell migration, and extracellular matrix. GEPIA and PPI network analysis revealed that SPP1, TNC, and ACTA1 were significantly related to tumor staging; SPP1, tissue inhibitors of matrix metallopeptidases (MMPs) 1 (TIMP1), and ACTA1 were closely related to prognosis. The scores for the top five highest degree genes were close, and the TIMP1/MMP9 axis appeared to be at the center of the PPI network, indicating that expression changes in the TIMP1/MMP9 axis and related genes may be involved in tumor invasion and metastasis. These findings provide novel insights into the mechanism of oral cancer.

Biochemical, histological and ultrastructural alterations of the alimentary system in the freshwater crab Sinopotamon henanense subchronically exposed to cadmium.

Cadmium (Cd) is one of the toxic metals in the aquatic environment. We investigated the effects of Cd on the digestive enzymes, antioxidant enzymes, malondialdehyde (MDA) content and morphology of the hepatopancreas and intestine in the freshwater crab Sinopotamon henanense. Crabs were exposed to sublethal Cd concentrations of 0, 0.725, 1.450 and 2.900 mg/L for 21 days. After Cd exposure, the activities of maltase, amylase and trypsin of two tissues were lower than the control. The activities of superoxide dismutase, catalase and glutathione peroxidase in the hepatopancreas and intestine were decreased, and the MDA concentration increased in all of the treated groups, over the experimental period. The results of light and transmission electron microscopy showed that 2.900 mg/L of Cd exposure caused profound morphological damages in the hepatopancreas and midgut. After exposure, histological abnormalities of two tissues were discovered, including cellular swelling and necrosis. Additionally, alterations in microvilli, nucleus, mitochondria, rough endoplasmic reticulum as well as Golgi complex in epithelial cells of two tissues were observed. This may be due to antioxidant enzymes activities that were reduced by Cd in the alimentary system of the crabs, and led to membrane lipid peroxidation. The membrane structure was destroyed, and caused further tissue damage, which likely made the alimentary system unable to secrete digestive enzymes, leading to further reduction of digestive enzymes.

Sublethal Cd-induced cellular damage and metabolic changes in the freshwater crab Sinopotamon henanense.

To explore whether sublethal cadmium (Cd) exposure causes branchial cellular damages and affects the metabolic activity in brachyuran crustaceans, the freshwater crab Sinopotamon henanense was exposed to 0.71, 1.43, and 2.86 mg/L Cd(2+) for 3 weeks. Gill morphology, metabolic activity (activities of isocitrate dehydrogenase (IDH), cytochrome c oxidase (CCO) and lactate dehydrogenase (LDH), mRNA expression of CCO active subunit 1 (cco-1) and ldh, as well as ATP levels) in crab muscle were investigated. The results showed that sublethal Cd exposure caused profound morphological damages in the gills. The branchial epithelial cells were disorganized and vacuolized. Ultrastructurally, a decrease in number of apical microvilli, vacuolized mitochondria, and condensed chromatin were observed in gill epithelial cells. Correspondingly, the Cd exposure also induced downregulations of cco-1 and ldh mRNA expression and reduced activities of IDH, CCO, and LDH, in accordance with the lower ATP level in crab muscle. These results led to the conclusion that gill damage caused by sublethal Cd exposure could lead to an impairment of oxygen uptake of S. henanense, and the inhibition of metabolic activity decreases the oxygen demand of the crab and assists them to survive under the condition of lower oxygen availability. These effects add to our understanding on toxic effects of Cd and survival management of S. henanense subchronically exposed to sublethal Cd.

Oxidative damages and ultrastructural changes in the sperm of freshwater crab Sinopotamon henanense exposed to cadmium.

The toxicity of several waterborne heavy metals to aquatic organisms is associated with oxidative damages due to the formation of reactive oxygen species (ROS). In the present work, the reproductive toxicity of the heavy metal cadmium was tested with the sperm of freshwater crab. The crabs were exposed to sublethal concentrations of 0, 7.25, 14.5, 29, 58 and 116 mg/L of Cd²âº for 3, 5 and 7 d. Cd²âº accumulation, ROS formation, and the total antioxidant capacity (TAC) in Sinopotamon henanense sperm were measured. Biomarkers of oxidative damage to lipid (Malondialdehyde, MDA), proteins (protein carbonyl derivates, PCO) and DNA (DNA-protein crosslinks, DPC) were investigated to address Cd²âº effects on crucial macromolecules of the S. henanense sperm. Transmission electron microscopy (TEM) was applied to assess ultrastructural changes induced by 29 and 116 mg/L Cd²âº exposure for 7 d. The results showed that sperm Cd²âº levels were significantly increased at 3, 5 and 7 d starting from the 14.5 mg L⁻¹ Cd²âº-treated groups. Meanwhile, ROS levels were significantly increased over the experimental period. In terms of TAC, statistically significant changes were observed only at day 7 with the Cd²âº concentrations of 14.5, 29, 58 and 116 mg/L. This resulted in an increase of MDA content (5 d and 7 d), PCO content (Cd²âº: 58 and 116 mg/L, 7 d), and DPC levels (Cd²âº: 116 mg/L, 3 d and 7 d), by 26.32%, 37.47%, 22.04%, respectively, in the 116 mg/L Cd²âº group at day seven. For ultrastructural observations, the sperm membrane became wrinkled and partly dissolved, the nuclear envelope turned wrinkled and the chromatin condensed, the acrosome was incomplete with a damaged acrosomal membrane in crabs treated with 29 mg/L Cd²âº for 7 d. After treatment with 116 mg/L Cd²âº for 7 d, the sperm membrane was almost dissolved, the chromatin in the nucleus was more heavily condensed, chromatin irregularities and serious acrosome damage were observed.

Oxygen consumption and metabolic responses of freshwater crab Sinopotamon henanense to acute and sub-chronic cadmium exposure.

To explore the respiratory and metabolic responses of the freshwater crab (Sinopotamon henanense) to Cd exposure, crabs were acutely exposed to 7.14, 14.28, 28.55 mg/L Cd for 96 h and subchronically exposed to 0.71, 1.43, 2.86 mg/L for three weeks. The oxygen consumption, concentrations of oxyhemocyanin, hemolymph protein, the activities of respiratory enzymes, i.e. lactate dehydrogenase (LDH), NAD-isocitrate dehydrogenase (IDH), cytochrome c oxidase (CCO), as well as cco-1(CCO active subunit 1) and ldh mRNA expression level and adenosine triphosphate (ATP) content in crab heart were assessed. Oxygen consumption, concentration of oxyhemocyanin and oxyhemocyanin/blood protein proportion were increased during acute exposure and decreased during sub-chronic exposure. Both exposure schemes induced downregulation of cco-1 gene expression and lowered CCO activity. For acute exposure, tissue ATP level was increased, in association with increased IDH activity and decreased LDH activity, whereas subchronic exposure caused decreased IDH activity accompanied with increased ldh gene expression and LDH activity, resulting in lowered ATP level. By coupling gene expression to biochemical and physiological endpoints, this work provides new insights into the mechanisms involved in metal stress and the differential respiratory and metabolic responses of S. henanense to acute and subchronic Cd exposure.

Correction: Cadmium-Induced Oxidative Stress and Apoptotic Changes in the Testis of Freshwater Crab, Sinopotamon henanense.

[This corrects the article DOI: 10.1371/journal.pone.0027853.].

Effects of cadmium exposure on digestive enzymes, antioxidant enzymes, and lipid peroxidation in the freshwater crab Sinopotamon henanense.

In this study, the effects of cadmium (Cd) stress on the activities of disaccharidases (sucrase, lactase, and maltase), amylase, trypsin, pepsase, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) content in the alimentary system of freshwater crabs Sinopotamon henanense were studied. Results showed that the enzyme activities in the stomach, intestine, and hepatopancreas changed with Cd concentration. In terms of digestive enzymes, Cd exposure had an inhibitory effect on the activities of the disaccharidases, amylase, and pepsase (only in the stomach). Significant induction of trypsin activity by Cd at a lower concentration was observed, but as Cd concentration increased, trypsin activity decreased. Maltase activity showed a slight recovery after inhibition by Cd. The activities of SOD and CAT increased initially and decreased subsequently. Cd significantly inhibited the activity of GPx. MDA content increased with increasing concentration of Cd. These results showed that acute Cd exposure led to harmful effects on the alimentary system of crabs, which are likely linked to Cd induced oxidative stress.

Cadmium-induced oxidative stress and apoptotic changes in the testis of freshwater crab, Sinopotamon henanense.

Cadmium (Cd), one of the most toxic environmental and industrial pollutants, is known to exert gonadotoxic and spermiotoxic effects. In the present study, we examined the toxic effect of Cd on the testis of freshwater crab, Sinopotamon henanense. Crabs were exposed to different Cd concentrations (from 0 to 116.00 mg·L(-1)) for 7 d. Oxidative stress and apoptotic changes in the testes were detected. The activities of SOD, GPx and CAT initially increased and subsequently decreased with increasing Cd concentrations, which was accompanied with the increase in malondialdehyde (MDA) and H(2)O(2) content in a concentration-dependent manner. Typical morphological characteristic and physiological changes of apoptosis were observed using a variety of methods (HE staining, AO/EB double fluorescent staining, Transmission Electron Microscope observation and DNA fragmentation analysis), and the activities of caspase-3 and caspase-9 were increased in a concentration-dependent manner after Cd exposure. These results led to the conclusion that Cd could induced oxidative damage as well as apoptosis in the testis, and the apoptotic processes may be mediated via mitochondria-dependent apoptosis pathway by regulating the activities of caspase-3 and caspase-9.

Effects of cadmium on carbohydrate and protein metabolisms in the freshwater crab Sinopotamon yangtsekiense.

The physiological impact of Cd(2+) on Sinopotamon yangtsekiense was evaluated through changes of selected parameters considered as key elements of carbohydrate and protein metabolisms. Crab were exposed to 0.725, 1.45, 2.9mg·L(-1) Cd(2+) for 7, 14 and 21 days. A time- and/or concentration- dependent decrease in muscle glycogen and increase in LDH activity suggested that glycolysis was accelerated during the treatments. Increased protease activity, lowering of FAA and the initially increased and subsequently decreased aminotransferase activities suggest an enhanced protein mobilization during early Cd(2+) exposure followed by a metabolic impairment during late exposure. Decreased hemolymph glucose level was observed in the crabs treated with 2.9mg·L(-1) Cd(2+) for 21d, suggesting an impaired gluconeogenesis. Ammonia level barely changed during the 14d Cd(2+) exposure most likely due to the increased urea and glutamine production; After 1.45 and 2.9mg·L(-1) Cd(2+) treatment for 21d, ammonia was observed increased followed by an exclusive increase in glutamine. Taken together, our results indicate that carbohydrate and protein are mobilized to a varying degree as a compensatory metabolism to response to the energy stress during acute Cd(2+) exposure. As the time lapsed, some symptoms on metabolism obstacle reflect the toxic effect of sublethal Cd(2+).