186 research outputs found
Establishment of a human cell-based in vitro battery to assess developmental neurotoxicity hazard of chemicals
Developmental neurotoxicity (DNT) is a major safety concern for all chemicals of the human exposome. However, DNT data from animal studies are available for only a small percentage of manufactured compounds. Test methods with a higher throughput than current regulatory guideline methods, and with improved human relevance are urgently needed. We therefore explored the feasibility of DNT hazard assessment based on new approach methods (NAMs). An in vitro battery (IVB) was assembled from ten individual NAMs that had been developed during the past years to probe effects of chemicals on various fundamental neurodevelopmental processes. All assays used human neural cells at different developmental stages. This allowed us to assess disturbances of: (i) proliferation of neural progenitor cells (NPC); (ii) migration of neural crest cells, radial glia cells, neurons and oligodendrocytes; (iii) differentiation of NPC into neurons and oligodendrocytes; and (iv) neurite outgrowth of peripheral and central neurons. In parallel, cytotoxicity measures were obtained. The feasibility of concentration-dependent screening and of a reliable biostatistical processing of the complex multi-dimensional data was explored with a set of 120 test compounds, containing subsets of pre-defined positive and negative DNT compounds. The battery provided alerts (hit or borderline) for 24 of 28 known toxicants (82% sensitivity), and for none of the 17 negative controls. Based on the results from this screen project, strategies were developed on how IVB data may be used in the context of risk assessment scenarios employing integrated approaches for testing and assessment (IATA).European Food Safety Authority (EFSA-Q-2018-00308), the Danish Environmental Protection Agency (EPA), Denmark, under the grant number MST-667-00205, the State Ministry of Baden-Wuerttemberg, Germany, for Economic Affairs, Labour and Tourism (NAM-Accept), the project CERST (Center for Alternatives to Animal Testing) of the Ministry for culture and science of the State of North-Rhine Westphalia, Germany (file number 233–1.08.03.03- 121972/131–1.08.03.03–121972), the European Chemical Industry Council Long-Range Research Initiative (Cefic LRI) under the project name AIMT11 and the BMBF (NeuroTool). It has also received funding from the European Union's Horizon 2020 research and innovation program under grant agreements No. 964537 (RISK-HUNT3R), No. 964518 (ToxFree), No. 101057014 (PARC) and No. 825759 (ENDpoiNTs)
Expanding the Repertoire of Modified Vaccinia Ankara-Based Vaccine Vectors via Genetic Complementation Strategies
nkara (MVA) is a safe, highly attenuated orthopoxvirus that is being developed as a recombinant vaccine vector for immunization against a number of infectious diseases and cancers. However, the expression by MVA vectors of large numbers of poxvirus antigens, which display immunodominance over vectored antigens-of-interest for the priming of T cell responses, and the induction of vector-neutralizing antibodies, which curtail the efficacy of subsequent booster immunizations, remain as significant impediments to the overall utility of such vaccines. Thus, genetic approaches that enable the derivation of MVA vectors that are antigenically less complex may allow for rational improvement of MVA-based vaccines. during infection, and that the processes governing the generation of antiviral antibody responses are more readily saturated by viral antigen than are those that elicit CD8+ T cell responses. deletion, enables the generation of novel replication-defective MVA mutants and expands the repertoire of genetic viral variants that can now be explored as improved vaccine vectors
Mechanisms of Intragastric pH Sensing
Luminal amino acids and lack of luminal acidity as a result of acid neutralization by intragastric foodstuffs are powerful signals for acid secretion. Although the hormonal and neural pathways underlying this regulatory mechanism are well understood, the nature of the gastric luminal pH sensor has been enigmatic. In clinical studies, high pH, tryptic peptides, and luminal divalent metals (Ca2+ and Mg2+) increase gastrin release and acid production. The calcium-sensing receptor (CaSR), first described in the parathyroid gland but expressed on gastric G cells, is a logical candidate for the gastric acid sensor. Because CaSR ligands include amino acids and divalent metals, and because extracellular pH affects ligand binding in the pH range of the gastric content, its pH, metal, and nutrient-sensing functions are consistent with physiologic observations. The CaSR is thus an attractive candidate for the gastric luminal sensor that is part of the neuroendocrine negative regulatory loop for acid secretion
Tryptophan Scanning Analysis of the Membrane Domain of CTR-Copper Transporters
Membrane proteins of the CTR family mediate cellular copper uptake in all eukaryotic cells and have been shown to participate in uptake of platinum-based anticancer drugs. Despite their importance for life and the clinical treatment of malignancies, directed biochemical studies of CTR proteins have been difficult because high-resolution structural information is missing. Building on our recent 7Å structure of the human copper transporter hCTR1, we present the results of an extensive tryptophan-scanning analysis of hCTR1 and its distant relative, yeast CTR3. The comparative analysis supports our previous assignment of the transmembrane helices and shows that most functionally and structurally important residues are clustered around the threefold axis of CTR trimers or engage in helix packing interactions. The scan also identified residues that may play roles in interactions between CTR trimers and suggested that the first transmembrane helix serves as an adaptor that allows evolutionarily diverse CTRs to adopt the same overall structure. Together with previous biochemical and biophysical data, the results of the tryptophan scan are consistent with a mechanistic model in which copper transport occurs along the center of the trimer
Self-renewal and chemotherapy resistance of p75NTR positive cells in esophageal squamous cell carcinomas
<p>Abstract</p> <p>Background</p> <p>p75<sup>NTR </sup>has been used to isolate esophageal and corneal epithelial stem cells. In the present study, we investigated the expression of p75<sup>NTR </sup>in esophageal squamous cell carcinoma (ESCC) and explored the biological properties of p75<sup>NTR+ </sup>cells.</p> <p>Methods</p> <p>p75<sup>NTR </sup>expression in ESCC was assessed by immunohistochemistry. p75<sup>NTR+ </sup>and p75<sup>NTR- </sup>cells of 4 ESCC cell lines were separated by fluorescence-activated cell sorting. Differentially expressed genes between p75<sup>NTR+ </sup>and p75<sup>NTR- </sup>cells were determined by real-time quantitative reverse transcription-PCR. Sphere formation assay, DDP sensitivity assay, <sup>64</sup>copper accumulation assay and tumorigenicity analysis were performed to determine the capacity of self-renewal, chemotherapy resistance and tumorigenicity of p75<sup>NTR+ </sup>cells.</p> <p>Results</p> <p>In ESCC specimens, p75<sup>NTR </sup>was found mainly confined to immature cells and absent in cells undergoing terminal differentiation. The percentage of p75<sup>NTR+ </sup>cells was 1.6%–3.7% in Eca109 and 3 newly established ESCC cell lines. The expression of Bmi-1, which is associated with self-renewal of stem cells, was significantly higher in p75<sup>NTR+ </sup>cells. p63, a marker identified in keratinocyte stem cells, was confined mainly to p75<sup>NTR+ </sup>cells. The expression of CTR1, which is associated with cisplatin (DDP)-resistance, was significantly decreased in p75<sup>NTR+ </sup>cells. Expression levels of differentiation markers, such as involucrin, cytokeratin 13, β1-integrin and β4-integrin, were lower in p75<sup>NTR+ </sup>cells. In addition, p75<sup>NTR+ </sup>cells generated both p75<sup>NTR+ </sup>and p75<sup>NTR- </sup>cells, and formed nonadherent spherical clusters in serum-free medium supplemented with growth factors. Furthermore, p75<sup>NTR+ </sup>cells were found to be more resistant to DDP and exhibited lower <sup>64</sup>copper accumulation than p75<sup>NTR- </sup>cells.</p> <p>Conclusion</p> <p>Our results demonstrated that p75<sup>NTR+ </sup>cells possess some characteristics of CSCs, namely, self-renewal and chemotherapy resistance. Chemotherapy resistance of p75<sup>NTR+ </sup>cells may probably be attributable to decreased expression of CTR1.</p
Prolonged Depression-Like Behavior Caused by Immune Challenge: Influence of Mouse Strain and Social Environment
Immune challenge by bacterial lipopolysaccharide (LPS) causes short-term
behavioral changes indicative of depression. The present study sought to explore
whether LPS is able to induce long-term changes in depression-related behavior
and whether such an effect depends on mouse strain and social context. LPS (0.83
mg/kg) or vehicle was administered intraperitoneally to female CD1 and C57BL/6
mice that were housed singly or in groups of 4. Depression-like behavior was
assessed with the forced swim test (FST) 1 and 28 days post-treatment.
Group-housed CD1 mice exhibited depression-like behavior 1 day post-LPS, an
effect that leveled off during the subsequent 28 days, while the behavior of
singly housed CD1 mice was little affected. In contrast, singly housed C57BL/6
mice responded to LPS with an increase in depression-like behavior that was
maintained for 4 weeks post-treatment and confirmed by the sucrose preference
test. Group-housed C57BL/6 mice likewise displayed an increased depression-like
behavior 4 weeks post-treatment. The behavioral changes induced by LPS in
C57BL/6 mice were associated with a particularly pronounced rise of
interleukin-6 in blood plasma within 1 day post-treatment and with changes in
the dynamics of the corticosterone response to the FST. The current data
demonstrate that immune challenge with LPS is able to induce prolonged
depression-like behavior, an effect that depends on genetic background (strain).
The discovery of an experimental model of long-term depression-like behavior
after acute immune challenge is of relevance to the analysis of the epigenetic
and pathophysiologic mechanisms of immune system-related affective
disorders
Structure and functional characterization of pyruvate decarboxylase from Gluconacetobacter diazotrophicus
BACKGROUND: Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially
mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas
mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC
has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in
metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC),
G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance.
RESULTS: This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter
diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (KM 0.06 mM at pH 5), high
catalytic efficiencies, pHopt of 5.5 and Topt at 45 degrees C. The enzyme is not thermostable (T of
18 minutes at 60 degrees C) and the calculated number of bonds between monomers and dimers do not give clear indications
for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z.
mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 A for C? when comparing GdPDC to that
of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci,
involving the regions Thr341 to Thr352 and Asn499 to Asp503.
CONCLUSIONS: This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future
research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily
closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the
enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the
host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity
on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in
G. diazotrophicus.IS
Evidence-Based Assessment of Child Obsessive Compulsive Disorder: Recommendations for Clinical Practice and Treatment Research
Obsessive-compulsive disorder (OCD) presents heterogeneously and can be difficult to assess in youth. This review focuses on research-supported assessment approaches for OCD in childhood. Content areas include pre-visit screening, diagnostic establishment, differential diagnosis, assessment of comorbid psychiatric conditions, tracking symptom severity, determining psychosocial functioning, and evaluating clinical improvement. Throughout this review, similarities and differences between assessment approaches geared towards clinical and research settings are discussed
Combined modalities of resistance in an oxaliplatin-resistant human gastric cancer cell line with enhanced sensitivity to 5-fluorouracil
To identify mechanisms underlying oxaliplatin resistance, a subline of the human gastric adenocarcinoma TSGH cell line, S3, was made resistant to oxaliplatin by continuous selection against increasing drug concentrations. Compared with the parental TSGH cells, the S3 subline showed 58-fold resistance to oxaliplatin; it also displayed 11-, 2-, and 4.7-fold resistance to cis-diammine-dichloroplatinum (II) (CDDP), copper sulphate, and arsenic trioxide, respectively. Interestingly, S3 cells were fourfold more susceptible to 5-fluorouracil-induced cytotoxicity due to downregulation of thymidylate synthase. Despite elevated glutathione levels in S3 cells, there was no alteration of resistant phenotype to oxaliplatin or CDDP when cells were co-treated with glutathione-depleting agent, l-buthionine-(S,R)-sulphoximine. Cellular CDDP and oxaliplatin accumulation was decreased in S3 cells. In addition, amounts of oxaliplatin- and CDDP–DNA adducts in S3 cells were about 15 and 40% of those seen with TSGH cells, respectively. Western blot analysis showed increased the expression level of copper transporter ATP7A in S3 cells compared with TSGH cells. Partial reversal of the resistance of S3 cells to oxaliplatin and CDDP was observed by treating cell with ATP7A-targeted siRNA oligonucleotides or P-type ATPase-inhibitor sodium orthovanadate. Besides, host reactivation assay revealed enhanced repair of oxaliplatin- or CDDP-damaged DNA in S3 cells compared with TSGH cells. Together, our results show that the mechanism responsible for oxaliplatin and CDDP resistance in S3 cells is the combination of increased DNA repair and overexpression of ATP7A. Downregulation of thymidylate synthase in S3 cells renders them more susceptible to 5-fluorouracil-induced cytotoxicity. These findings could pave ways for future efforts to overcome oxaliplatin resistance
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