Estimates of heavy metal tolerance and chromium(VI) reducing ability of Pseudomonas aeruginosa CCTCC AB93066: chromium(VI) toxicity and environmental parameters optimization

The potential role of parameters in the reduction of hexavalent chromium [Cr(VI)] by Pseudomonas aeruginosa is not well documented. In this study, laboratory batch studies were conducted to assess the effect of a variety of factors, e.g., carbon sources, salinity, initial Cr(VI) concentrations, co-existing ions and a metabolic inhibitor, on microbial Cr(VI) reduction to Cr(III) by P. aeruginosa AB93066. Strain AB93066 tolerated up to 400 mg/L of Cr(VI) in nutrient broth medium compared to only 150 mg/L of Cr(VI) in nutrient agar. This bacteria exhibited different levels of resistance against Pb(II) (200 mg/L), Cd(II) (100 mg/L), Ni(II) (100 mg/L), Cu(II) (100 mg/L), Co(II) (50 mg/L) and Hg(II) (5 mg/L). Cr(VI) reduction was significantly promoted by the addition of glucose and glycerine but was strongly inhibited by the presence of methanol and phenol. The rate of Cr(VI) reduction increased with increasing concentrations of Cr(VI) and then decreased at higher concentrations. The presence of Ni(II) stimulated Cr(VI) reduction, while Pb(II), Co(II) and Cd(II) had adverse impact on reduction ability of this strain. Cr(VI) reduction was also inhibited by high levels of NaCl, various concentrations of sodium azide and 20 mM of SO₄ ^2?, MoO₄ ^2?, NO₃ ^?, PO₄ ^3?. No significant relationship was observed between Cr(VI) reduction and redox potential of the culture medium. Scanning electron microscopy showed visible morphological changes in the cells due to chromate stress. Fourier transform infrared spectroscopy analysis revealed chromium species was likely to form complexes with certain functional groups such as carboxyl and amino groups on the surface of P. aeruginosa AB93066. Overall, above results are beneficial to the bioremediation of chromate-polluted industrial wastewaters.     

Speciation of chromium in chromium yeast

High-performance liquid chromatography was used to separate Cr(III) and Cr(VI) in samples with detection by inductively coupled plasma mass spectrometry(ICP-MS). The separation was achieved on a weak anion exchange column. The mobile phase was pH 7.0 ammonium nitrate solution. The redox reaction between Cr(III) and Cr(VI) was avoided during separation and determination. This separation method could be used to separate the samples with large concentration differences between Cr(III) and Cr(VI). The alkaline digestion was used to extract chromium in solid sample, which had no effect on the retention time and the peak area of the Cr(VI). However, the conversion of Cr(VI) from Cr(III) was observed during alkaline digestion, which displayed positive relation with the ratio of Cr(III) and Cr(VI) in samples. Both Cr(III) and Cr(VI) contents of chromium yeasts cultured in media with different chromium additions were determined. The spike recoveries of Cr(VI) for chromium yeasts were in the range of 95–108 %.     

Metabolic reduction of chromium,as related to its carcinogenic properties

At variance with Cr(III), Cr(VI) compounds easily cross cell membranes and exert genotoxic effects. No metabolic oxidation of Cr(III) could be detected, whereas Cr(VI) reduction was observed in the presence of body fluids and subcellular fractions of various tissues from several animal species. The differential efficiency of this process may account for the selection of target tissues in Cr(VI) carcinogenesis. For instance, reduction by saliva and gastric juice may explain a lack of carcinogenicity by the oral route; reduction inside erythrocytes may explain a lack of carcinogenicity at a distance from administration sites; reduction by the epithelial-lining fluid of terminal airways and by alveolar macrophages may be consistent with the occurrence of thresholds in lung carcinogenesis. Liver preparations displayed the top efficiency in reducing Cr(VI), whereas skeletal muscle, i.e., a typical target in experimental Cr(VI) carcinogenesis, had no detectable activity. Bronchial tree and peripheral lung parenchyma preparations from almost 100 individuals reduced Cr(VI) to a variable extent. The efficiency of lung parenchyma and of isolated alveolar macrophages was enhanced in cigarette smokers. In rats, Cr(VI) reduction by lung preparations was significantly stimulated by the repeated i.t. instillation of Cr(VI) itself. Among the electron donors (chiefly GSH) and enzymatic mechanisms responsible for the intracellular Cr(VI) reduction, such as cytochrome P-450 reductase, glutathione redactase, and aldehyde oxidase, an important role can be ascribed to cytosolic DT diaphorase activity, usually catalyzing a 2-electron reduction.     

Removal of Cr(VI) from aqueous solutions by fruiting bodies of the jelly fungus (Auricularia polytricha)

The aim of this study was to investigate the potential to remove chromium (Cr) from aqueous solutions using the fruiting body of Auricularia polytricha. Batch experiments were conducted under various conditions, and different models were used to characterize the biosorption process. Results showed that, for both fresh and dried fruiting bodies of A. polytricha, removal efficiencies of Cr(VI) and total Cr reached maximum values at pH values of 1 and 2, respectively. The process of Cr(VI) removal by A. polytricha included the sorption process as well as the reduction of Cr(VI) to Cr(III). Spectra of X-ray photoelectron spectroscopy of the biosorbent revealed that most of the Cr loaded on the biomass surface was in the trivalent form. The Freundlich model fitted the isotherm process better than the Langmuir model in the concentration range examined. The pseudo-second-order model well described the adsorption process of Cr onto the biomass. The biosorption capacity of Cr(VI) by fruiting bodies was much higher than that by most of other biosorbents reported. The results suggest that the fruiting bodies of A. polytricha should be a promising biomaterial for Cr removal from water contaminated by the heavy metal.     

Inhibitory and stimulating effect of single and multi-metal ions on hexavalent chromium reduction by Acinetobacter sp. Cr-B2

Potential application of chromium reducing bacteria for industrial scale wastewater treatment demands that effect of presence of other metal ions on rate of Cr(VI) reduction be investigated, as industrial wastewaters contain many toxic metal ions. In the current study, the effect of different heavy metal ions (nickel, zinc, cadmium, copper, lead, iron) on chromium reduction by a novel strain of Acinetobacter sp. Cr-B2 that shows high tolerance up to 1,100 mg/L and high Cr(VI) reducing capacity was investigated. The alteration in Cr(VI) reduction capacity of Cr-B2 was studied both in presence of individual metal ions and in the presence of multi-metal ions at different concentrations. The study showed that the Cr(VI) reduction rates decreased in presence of Ni²⁺, Zn²⁺ and Cd²⁺ when present individually. Pb²⁺ at lower concentration did not show significant effect while Cu²⁺ and Fe³⁺ stimulated the rate of Cr(VI) reduction. In the studies on multi-metal ions, it was observed that in presence of Cu²⁺ and Fe³⁺, the inhibiting effect of Ni²⁺, Zn²⁺, Cd²⁺ and Pb²⁺ on Cr(VI) reduction was reduced. Each of these metals affect the overall rate of Cr(VI) reduction by Cr-B2. This work highlights the need to consider the presence of other heavy metal ions in wastewater when assessing the bioreduction of Cr(VI) and while designing the bioreactors for the purpose, as rate of reduction is altered by their presence.     

Increased Expression of Angiogenic Factors in Cultured Human Brain Arteriovenous Malformation Endothelial Cells

To compare the mRNA level of angiogenic factor vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-2, and MMP-9 in cultured human brain arteriovenous malformation (AVM) endothelial cells (ECs) and normal brain endothelial cells (BECs). Tissue explants both from deformed vessels of AVM and normal microvessel were put into culture for endothelial cells. After the monolayer adherent ECs reached confluence, they were tested with endothelial specific marker CD34 and von Willebrand factor (vWF) by immunochemical assay. mRNA levels of VEGF-A, MMP-2, and MMP-9 in AVM endothelial cells (AVMECs) and BECs were measured by PCR. Immunostaining confirmed that more than 95 % of the cultured cells were CD34 (Fig. 1b) and/or vWF positive. Expression levels of VEGF-A and MMP-2 mRNAs were significantly higher in AVMECs than in BECs. The MMP-9 level was also increased in AVMECs, but the difference was not statistically significant. Vascular tissue explants adherent method is a better approach for isolation and culture of AVMECs. Cultured AVMECs expressed higher angiogenic factors (VEGF, MMP-2) than the controlled BECs, implicating angiogenesis plays an important role in the pathogenesis of AVM.     

Dietary l-glutamine supplementation modulates microbial community and activates innate immunity in the mouse intestine

This study was conducted to determine effects of dietary supplementation with 1 % l-glutamine for 14 days on the abundance of intestinal bacteria and the activation of intestinal innate immunity in mice. The measured variables included (1) the abundance of Bacteroidetes, Firmicutes, Lactobacillus, Streptococcus and Bifidobacterium in the lumen of the small intestine; (2) the expression of toll-like receptors (TLRs), pro-inflammatory cytokines, and antibacterial substances secreted by Paneth cells and goblet cells in the jejunum, ileum and colon; and (3) the activation of TLR4-nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), and phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt) signaling pathways in the jejunum and ileum. In the jejunum, glutamine supplementation decreased the abundance of Firmicutes, while increased mRNA levels for antibacterial substances in association with the activation of NF-κB and PI3K-Akt pathways. In the ileum, glutamine supplementation induced a shift in the Firmicutes:Bacteroidetes ratio in favor of Bacteroidetes, and enhanced mRNA levels for Tlr4, pro-inflammatory cytokines, and antibacterial substances participating in NF-κB and JNK signaling pathways. These results indicate that the effects of glutamine on the intestine vary with its segments and compartments. Collectively, dietary glutamine supplementation of mice beneficially alters intestinal bacterial community and activates the innate immunity in the small intestine through NF-κB, MAPK and PI3K-Akt signaling pathways.     

Inhibition of MMP-2 but not MMP-9 Influences Inner Ear Spiral Ganglion Neurons In Vitro

Matrix metalloproteinases (MMPs) play an important role in modeling of the extracellular matrix. There is increasing evidence that these proteases are important in neurite elongation and axonal guidance during development in the central nervous system and retina. Moreover, they are also expressed after acute injury and can be the key mediators of pathogenesis. However, the role of MMPs in the inner ear is largely unknown. Our group recently demonstrated that general inhibition of MMPs resulted in auditory hair cell loss in vitro. In the present study, we investigated the role of MMPs in inner ear spiral ganglion neuron (SGN) survival, neuritogenesis and neurite extension by blocking MMPs known to be involved in axonal guidance, neurite elongation, and apoptosis in other neuronal systems. Spiral ganglion (SG) explants from 5-day-old Wistar rats were treated with different concentrations of the general MMP inhibitor GM6001, a specific MMP-2 inhibitor, and a specific MMP-9 inhibitor, in vitro. The general inhibitor of MMPs and the specific inhibition of MMP-2 significantly reduced both the number of neurites that extended from SG explants, as well as the length of individual neurites. However, neither the general inhibitor of MMPs nor the specific inhibition of MMP-2 influenced SGN survival. Inhibition of MMP-9 had no influence on SGNs. The data suggest that MMPs, and more specifically MMP-2, influence the growth of developing afferent neurites in the mammalian inner ear in vivo.     

Platelet-derived growth factor mediates interleukin-13-induced collagen I production in mouse airway fibroblasts

Interleukin-13 (IL-13) is associated with the production of collagen in airway remodelling of asthma. Yet, the molecular mechanisms underlying IL-13 induction of collagen remain unclear; the aim of this study is to address this issue. IL-13 dose- and time-dependently-induced collagen I production in primary cultured airway fibroblasts; this was accompanied with the STAT6 phosphorylation, and pre-treatment of cells with JAK inhibitor suppressed IL-13-induced collagen I production. Further study indicated that IL-13 stimulated JAK/STAT6-dependent PDGF production and subsequent ERK1/2 MAPK activation in airway fibroblasts, and the presence of either PDGF receptor blocker or MEK inhibitor partially suppressed IL-13-induced collagen I production. Taken together, our study suggests that activation of JAK/STAT6 signal pathway and subsequent PDGF generation and resultant ERK1/2 MAPK activation mediated IL-13-induced collagen I production in airway fibroblasts.     

Deep-sea water inhibits metastatic potential in HT-29 human colorectal adenocarcinomas via MAPK/NF-κB signaling pathway

A previous investigation showed that deep-sea water (DSW) can affect the expression of genes that regulate metastasis, including cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP-2), urokinase plasminogen activator (uPA) and uPA receptor (uPAR), in HT-29 human colorectal adenocarcinomas. In the present study, we investigated the effects of DSW on inducible nitric oxide synthase (iNOS) expression and cell migration and also explored the mechanism of DSW-induced anti-metastatic potential in HT-29 human colorectal adenocarcinomas. Cytokine-induced expression of iNOS, which is highly expressed in colon cancer and enhances cancer growth and metastasis, was decreased in a hardness-dependent manner by DSW. Also, the wound healing assay revealed that DSW inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration in a hardness-dependent manner. DSW also decreased the phosphorylation of various MAPKs, including p38, ERK and JNK, and suppressed the nuclear translocation of NF-κB but not c-Jun. The results suggest that DSW may inhibit cancer cell growth related to iNOS overexpression and PKC-mediated cell migration in HT-29 human colorectal adenocarcinomas and the antimetastatic potential of DSW may be regulated by prevention of NF-κB nuclear translocation via inhibition of p38, ERK and JNK phosphorylation. In conclusion, the present investigation demonstrates that DSW inhibits cancer growth and metastasis via down-regulation of iNOS expression and the MAPK/NF-κB signaling pathway.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells

Cystic fibrosis transmembrane conductance regulator (CFTR) acts as a cAMP-dependent chloride channel, has been studied in various types of cells. CFTR is abundantly expressed in vascular smooth muscle cells and closely linked to vascular tone regulation. However, the functional significance of CFTR in basilar vascular smooth muscle cells (BASMCs) remains elusive. Accumulating evidence has shown the direct role of CFTR in cell apoptosis that contributes to several main pathological events in CF, such as inflammation, lung injury and pancreatic insufficiency. We therefore investigated the role of CFTR in BASMC apoptotic process induced by hydrogen peroxide (H₂O₂). We found that H₂O₂-induced cell apoptosis was parallel to a significant decrease in endogenous CFTR protein expression. Silencing CFTR with adenovirus-mediated CFTR specific siRNA further enhanced H₂O₂-induced BASMC injury, mitochondrial cytochrome c release into cytoplasm, cleaved caspase-3 and -9 protein expression and oxidized glutathione levels; while decreased cell viability, the Bcl-2/Bax ratio, mitochondrial membrane potential, total glutathione levels, activities of superoxide dismutase and catalase. The pharmacological activation of CFTR with forskolin produced the opposite effects. These results strongly suggest that CFTR may modulate oxidative stress-related BASMC apoptosis through the cAMP- and mitochondria-dependent pathway and regulating endogenous antioxidant defense system.     

Prioritizing non-marine invertebrate taxa for Red Listing

The IUCN Red List of threatened species is biased towards vertebrate animals, a major limitation on its utility for overall biodiversity assessment. There is a need to increase the representation of invertebrates (currently 21 % of species assessed on the List; <1 % of all invertebrates). A prioritisation system of terrestrial and freshwater groups is presented here, categorising taxa by species richness, assessment practicality, value for human land use and bioindication, and potential to act as conservation flagships. 25 major taxonomic groupings were identified as priorities, including the Annelida, Arthropoda, Mollusca, and Onycophora. Of these, the high-level taxa that emerge as highest priorities are Odonata (dragonflies and damselflies), Araneae (spiders), Mantophasmatodea (heelwalkers), Plecoptera (stoneflies), non-marine Mollusca (Bivalvia and Gastropoda), Trichoptera (caddisflies), Coleoptera (beetles), Lepidoptera (moths and butterflies), Oligochaetes (earthworms), Orthoptera (grasshoppers and crickets), Decapoda (crayfish, crabs, shrimps) and Diptera (flies). Of these Red Listing is well advanced for Decapoda, freshwater Mollusca and Odonata. This leaves eight higher taxa with currently a minimum or patchy Red List assessment coverage. We recommend that Red List assessments in future focus on these groups, as well as completion of assessments for terrestrial Molluscs and Odonata. However, we also recommend realism, and as some of groups are very large, it will be necessary to focus on subsets such as certain functionally important or charismatic taxa or on a sampled subset which is representative of a larger taxon.     

Basic amino acids and dimethylarginines targeted metabolomics discriminates primary hepatocarcinoma from hepatic colorectal metastases

Hepatocellular carcinoma (HCC) is a very aggressive neoplasia requiring early and accurate diagnosis to improve patient outcomes with timely treatment. The liver is also very frequently colonized by metastases, and the most frequent differential diagnosis is HCC against intrahepatic cholangiocarcinoma or metastatic adenocarcinoma. Metabolomics is a powerful tool for identification of altered biomarkers in cancer, and to evaluate the efficacy of drug treatments. Here we analyzed by HILIC-MS/MS methylated arginines, basic amino acids (Arg, Cit, Orn), and their ratios in the extracts of primary HCC tissues, liver metastases from colorectal carcinoma (MET), cirrhotic related hepatitis-C-virus (CIR), and non-cirrhotic normal liver (NT) adjacent tissues. We found high levels of Arg (p < 0.0001) and Arg/Orn (p < 0.01) in MET compared to other tissues. In MET, compared to NT and CIR, Arg concentration was fivefold higher, while in HCC it was twofold higher. ADMA increased twofold compared to NT and CIR, while in HCC it was 50 % higher. Arg/Cit and ADMA/SDMA ratios were significantly higher in MET compared to NT and CIR (p < 0.005). Arg/Orn, Arg/Cit, and ADMA/SDMA ratios increased progressively from NT, CIR, HCC, to MET tissues. Arg/Cit correlated significantly with Arg/Orn ratios (r = 0.77; p < 0.0001), and discriminates tumor from non-tumor samples. In addition, the discriminant lactate/glucose ratio we previously found by NMR, also correlated significantly with the Arg levels (r = 0.64; p < 0.0001), and discriminated MET from all other tissues. The results indicated that Arg in MET is higher than other tissue classes, suggesting that, together with the lactate/glucose ratio, it can be considered a further biomarker for HCC-metastases differentiation.     

The stimulatory effects of alpha1-adrenergic receptors on TGF-beta1, IGF-1 and hyaluronan production in human skin fibroblasts

Skin fibroblasts modulate tissue repair, wound healing and immunological responses. Adrenergic receptors (ARs) mediate important physiological functions, such as endocrine, metabolic and neuronal activity. In this study, the expression α1A-ARs in human skin fibroblasts is examined and verified. Regulatory effects of α1-agonist cirazoline on cell migration and the production of transforming growth factor β1 (TGF-β1), insulin-like growth factor 1 (IGF-1), hyaluronan (HA), fibronectin and procollagen type I carboxy-terminal peptide (PIP) by human skin fibroblasts are assessed and validated. α1A-AR mRNA and protein were found in human skin fibroblasts WS1. Exposure of cirazoline doubled skin fibroblast migration and the increase in cell migration was attenuated by α1-antagonist prazosin. TGF-β1 mRNA and production were enhanced after exposure to cirazoline and IGF-1 production was also increased after treatment with cirazoline. Exposure to cirazoline also enhanced HA and PIP production. The increases in TGF-β1, IGF-1, HA and PIP production were partially abolished in fibroblasts transfected with α1A-AR short interfering RNAs, indicating that α1A-ARs are involved in the cirazoline-induced increases in TGF-β1, IGF-1, HA and PIP production. Thus, α1A-ARs are stably expressed and stimulate cell migration and TGF-β1, IGF-1, HA and PIP production in human skin fibroblasts. Moreover, TGF-β1, IGF-1, HA and PIP production and the cell migration of human skin fibroblasts are possibly modulated by natural catecholamines produced by the endocrine system or sympathetic innervation, which could directly or indirectly participate in cytokine secretion, fibroblast migration and matrix production of wound healing in the skin.     

Expression status of let-7a and miR-335 among breast tumors in patients with and without germ-line BRCA mutations

The genetic factors of cancer predisposition remain elusive in the majority of familial and/or early-onset cases of breast cancer (BC). This type of BC is promoted by germ-line mutations that inactivate BRCA1 or BRCA2. On the other hand, recent studies have indicated that alterations in the levels of miRNA expression are linked to this disease. Although BRCA1 and BRCA2 gene mutations have been reported to commonly lead to alterations in genes that encode cancer-related proteins, little is known regarding the putative impact of these mutations on noncoding miRNAs. In the present study, we aimed to determine whether miRNA dysregulation is involved in the pathogenesis of BRCA-mutated BC. An expression analysis of 14 human miRNAs previously shown to be related to BC diagnosis, prognosis, and drug resistance was conducted using tissues from 60 familial and/or early-onset patients whose peripheral blood samples had been screened for BRCA1 and BRCA2 mutations through sequence analysis. Let-7a and miR-335 expression levels were significantly downregulated in the tumors of patients with a BRCA mutation compared with those of patients without a BRCA mutation (P = 0.04 and P = 0.02, respectively). Our results defined the associations between the expression status of let-7a and miR-335 and BRCA mutations. The expression analysis of these miRNAs might be used as biomarkers of the BRCA mutation status of early-onset and/or familial BC.     

Age-related changes in the distribution of transient receptor potential vanilloid 4 channel (TRPV4) in the central nervous system of rats

Transient receptor potential vanilloid type 4 (TRPV4) channels are expressed in the central nervous system, but their role in regulating the aging process under physiological and pathological conditions is still largely unknown. To identify age-related changes in the TRPV4 channel that contribute to the central nervous system, we investigated the distribution of TRPV4 in the brain and spinal cord regions of adult and aged rats. The expression of TRPV4 in the brain and spinal cord of adult and aged Sprague–Dawley rats was compared using immunohistochemistry performed with antibodies recognizing TRPV4 on free floating sections and western blotting analysis. TRPV4 immunoreactivity was significantly increased in the cerebral cortex, hippocampal formation, thalamus, basal nuclei, cerebellum and spinal cord of aged rats compared with adult control rats. In the cerebral cortex, TRPV4 immunoreactivity was significantly increased in pyramidal cells of aged rats. In addition, TRPV4 immunoreactivity was increased in the spinal cord, hippocampal formation, thalamus, basal nuclei and cerebellum of aged rats. This first demonstration of age-related increases in TRPV4 expression in the brain and spinal cord may provide useful data for investigating the pathogenesis of age-related neurodegenerative diseases. The exact regulatory mechanism and its functional significance require further elucidation.