Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution.

The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A's protease domain (LC/A) could expand its therapeutic applications; however, LC/A's extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A retains its ability to form full-length holotoxin, infiltrate neurons, and cleave SNAP23. The identification of substrate control loops outside BoNT/A's active site could guide the design of improved BoNT proteases and inhibitors.

Aquaporin-4 autoantibodies in neuromyelitis optica spectrum disorders: comparison between tissue-based and cell-based indirect immunofluorescence assays.

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSD) are severe central nervous system inflammatory demyelinating disorders (CNS IDD) characterized by monophasic or relapsing, longitudinally extensive transverse myelitis (LETM) and/or optic neuritis (ON). A significant proportion of NMOSD patients are seropositive for aquaporin-4 (AQP4) autoantibodies. We compared the AQP4 autoantibody detection rates of tissue-based indirect immunofluorescence assay (IIFA) and cell-based IIFA. METHODS: Serum of Chinese CNS IDD patients were assayed for AQP4 autoantibodies by tissue-based IIFA using monkey cerebellum and cell-based IIFA using transfected HEK293 cells which express human AQP4 on their cell membranes. RESULTS: In total, 128 CNS IDD patients were studied. We found that 78% of NMO patients were seropositive for AQP4 autoantibodies by cell-based IIFA versus 61% by tissue-based IFA (p = 0.250), 75% of patients having relapsing myelitis (RM) with LETM were seropositive by cell-based IIFA versus 50% by tissue-based IIFA (p = 0.250), and 33% of relapsing ON patients were seropositive by cell-based IIFA versus 22% by tissue-based IIFA (p = 1.000); however the differences were not statistically significant. All patients seropositive by tissue-based IIFA were also seropositive for AQP4 autoantibodies by cell-based IIFA. Among 29 NMOSD patients seropositive for AQP4 autoantibodies by cell-based IIFA, 20 (69%) were seropositive by tissue-based IIFA. The 9 patients seropositive by cell-based IIFA while seronegative by tissue-based IIFA had NMO (3), RM with LETM (3), a single attack of LETM (1), relapsing ON (1) and a single ON attack (1). Among 23 NMO or RM patients seropositive for AQP4 autoantibodies by cell-based IIFA, comparison between those seropositive (n = 17) and seronegative (n = 6) by tissue-based IIFA revealed no differences in clinical and neuroradiological characteristics between the two groups. CONCLUSION: Cell-based IIFA is slightly more sensitive than tissue-based IIFA in detection of AQP4 autoantibodies, which are highly specific for NMOSD.

Epigenetic regulation of pluripotent genes mediates stem cell features in human hepatocellular carcinoma and cancer cell lines.

Activation of the stem cell transcriptional circuitry is an important event in cancer development. Although cancer cells demonstrate a stem cell-like gene expression signature, the epigenetic regulation of pluripotency-associated genes in cancers remains poorly understood. In this study, we characterized the epigenetic regulation of the pluripotency-associated genes NANOG, OCT4, c-MYC, KLF4, and SOX2 in a variety of cancer cell lines and in primary tumor samples, and investigated the re-activation of pluripotency regulatory circuits in cancer progression. Differential patterns of DNA methylation, histone modifications, and gene expression of pluripotent genes were demonstrated in different types of cancers, which may reflect their tissue origins. NANOG promoter hypomethylation and gene upregulation were found in metastatic human liver cancer cells and human hepatocellular carcinoma (HCC) primary tumor tissues. The upregulation of NANOG, together with p53 depletion, was significantly associated with clinical late stage of HCC. A pro-metastatic role of NANOG in colon cancer cells was also demonstrated, using a NANOG-overexpressing orthotopic tumor implantation mouse model. Demethylation of NANOG promoter was observed in CD133+(high) cancer cells. In accordance, overexpression of NANOG resulted in an increase in the population of CD133+(high) cells. In addition, we demonstrated a cross-regulation between OCT4 and NANOG in cancer cells via reprogramming of promoter methylation. Taken together, epigenetic reprogramming of NANOG can lead to the acquisition of stem cell-like properties. These results underscore the restoration of pluripotency circuits in cancer cells as a potential mechanism for cancer progression.

A subpopulation of CD26+ cancer stem cells with metastatic capacity in human colorectal cancer.

Recent evidence suggests that a subpopulation of cancer cells, cancer stem cells (CSCs), is responsible for tumor growth in colorectal cancer. However, the role of CSCs in colorectal cancer metastasis is unclear. Here, we identified a subpopulation of CD26(+) cells uniformly present in both the primary and metastatic tumors in colorectal cancer patients with liver metastasis. Furthermore, in patients without distant metastasis at the time of presentation, the presence of CD26(+) cells in their primary tumors predicted distant metastasis on follow-up. Isolated CD26(+) cells, but not CD26(-) cells, led to development of distant metastasis when injected into the mouse cecal wall. CD26(+) cells were also associated with enhanced invasiveness and chemoresistance. Our findings have uncovered a critical role of CSCs in metastatic progression of cancer. Furthermore, the ability to predict metastasis based on analysis of CSC subsets in the primary tumor may have important clinical implication as a selection criterion for adjuvant therapy.

AH receptor agonist activity in human blood measured with a cell-based bioassay: evidence for naturally occurring AH receptor ligands in vivo.

In the present study, an aryl hydrocarbon receptor (AHR)-driven reporter gene bioassay was used to measure the activity, measured as an induction equivalent (IEQ) as compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or IEQ concentration in human blood samples from 10 volunteers under different dietary regimens. Blood concentrations of polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) and polychlorinated biphenyls (PCBs), as determined by analytical chemistry (HR-GC/MS), and expressed as toxic equivalents (TEQs) with the use of TCDD equivalency factors (TEFs), were within a range that has been reported in the general US population, ranging from 0.022 to 0.119 ppt (whole blood basis). However, the human blood IEQ measured directly via bioassay ranged from 13.4 to 218 ppt (whole blood basis). These order of magnitude greater IEQs compared to the TEQs for dioxins, furans, and certain PCBs suggests that human blood contains a relatively high level of AHR agonists able to activate the CYP1A1 dioxin response element (DRE)-linked reporter gene bioassay and that this AHR activity is not accounted for by PCDDs/Fs and dioxin-like PCBs based on standard HR-GC/MS and TEF analysis. When study participants switched from a "baseline" to a high-vegetable diet, increases in bioassay IEQ were observed that were statistically significant (P<0.05). In addition, IEQ activity was elevated above levels observed following dietary intervention in two subjects given indole-3-carbinol (I3C) supplements. We conclude that a substantial portion of the IEQ activity occurred as a result of the increased intake of natural AHR agonists (NAHRAs) present in many fruits, vegetables. and herbs. Our findings also suggest that dietary NAHRAs constitute a substantial daily dietary intake of AHR-active compounds, and these NAHRAs could influence AHR status in humans and play a role in a basal level of AHR activation.

Mathematical model developed for environmental samples: prediction of GC/MS dioxin TEQ from XDS-CALUX bioassay data.

Remediation of hazardous waste sites requires efficient and cost-effective methods to assess the extent of contamination by toxic substances including dioxin-like chemicals. Traditionally, dioxin-like contamination has been assessed by gas chromatography/high-resolution mass spectrometry (GC/MS) analysis for specific polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyl congeners. Toxic equivalency factors for these congeners are then used to estimate the overall dioxin toxic equivalency (TEQ) of complex mixtures found in samples. The XDS-CALUX bioassay estimates contamination by dioxin-like chemicals in a sample extract by measuring expression of a sensitive reporter gene in genetically engineered cells. The output of the XDS-CALUX assay is a CALUX-TEQ value, calibrated based on TCDD standards. Soil samples taken from a variety of hazardous waste sites were measured using the XDS-CALUX bioassay and GC/MS. TEQ and CALUX-TEQ from these methods were compared, and a mathematical model was developed describing the relationship between these two data sets: log(TEQ) = 0.654 x log(CALUX-TEQ) + 0.058-(log(CALUX-TEQ))2. Applying this equation to these samples showed that predicted and GC/MS measured TEQ values strongly correlate (R2 = 0.876) and that TEQ values predicted from CALUX-TEQ were on average nearly identical to the GC/MS-TEQ. The ability of XDS-CALUX bioassay data to predict GC/MS-derived TEQ data should make this procedure useful in risk assessment and management decisions.

TNP-470 blockage of VEGF synthesis is dependent on MAPK/COX-2 signaling pathway in PDGF-BB-activated hepatic stellate cells.

Angiogenesis is a key pathogenic event in hepatic fibrogenesis, which is mediated by activated hepatic stellate cells (HSCs). TNP-470 is a known anti-angiogenic agent in cancer, and in this study, we investigated the regulatory mechanisms of TNP-470 blockage of vascular endothelial growth factor (VEGF) synthesis in activated HSCs. Primary HSCs were isolated from rat liver, cultured in vitro, and activated with platelet-derived growth factor-BB (PDGF-BB). After treatment with TNP-470, Nimesulide, PD98059, SB203580 or SP600125, activated HSCs were analyzed by immunoblotting, quantitative RT-PCR, and ELISA for mitogen-activated protein kinase (MAPK) family [ERKs, JNK, and p38], cyclooxygenase-2 (COX-2), and VEGF levels. Phosphorylation of p44/42 MAPK, which was followed by increased expressions of COX-2 and VEGF, was observed in PDGF-BB-activated HSCs; these events could be ameliorated by addition with TNP-470 in time- and dose-dependent manners. TNP-470 also inhibited the secretion of VEGF from activated HSCs into culture supernatant. Furthermore, TNP-470 blockage of VEGF production in activated HSCs could be nullified by exogenous inoculation with prostaglandin E(2). In summary, our findings suggest that TNP-470 exhibits the observed anti-angiogenic properties in activated HSCs by targeting the COX-2/phospho-p44/42 MAPK pathway to inhibit VEGF production.

Clinical phenotypes of a large Chinese multigenerational kindred with autosomal dominant familial ALS due to Ile149Thr SOD1 gene mutation.

About 10% of amyotrophic lateral sclerosis (ALS) cases are familial. We identified a five-generation Chinese family with autosomal dominant familial ALS (FALS). We performed a detailed family study, clinical and electromyographic validation, and SOD1, VEGF and CNTF mutation analyses. Forty-five living members (16 affected) were studied and DNA samples collected. Genealogical data were collected for deceased members. Based on the duration between symptom onset to ventilator dependence, they were divided into rapidly progressive (range 1-18 months, mean (SD) duration = 12.08 (+/-6.10) months, mean (SD) age of symptom onset = 39.75 (+/-9.84) years) and slowly progressive groups (>18 months; mean (SD) age of onset = 37.25 (+/-5.32) years old). We identified a heterozygous mutation of ATT to ACT of SOD1 gene at codon 149 in exon 5 resulting in substitution of isoleucine to threonine. It co-segregated with all affected members and 11 non-symptomatic members. We report a large multigenerational Chinese FALS kindred with I149T mutation in SOD1. No polymorphisms or mutations were found to date in two known modifier genes, namely, VEGF and CNTF, which were associated with heterogeneity in the phenotype within this kindred. Follow-up of the family will be helpful to explore any potential disease markers.

Regulatory role of vHL/HIF-1alpha in hypoxia-induced VEGF production in hepatic stellate cells.

Activated hepatic stellate cells (HSCs) produce cyclooxygenase-2 (COX-2) protein to induce vascular endothelial growth factor (VEGF) production that participates in angiogenesis in injured liver. To reveal the unknown regulatory mechanism, we used hypoxic atmosphere mimicking injured-tissue microenvironment to induce VEGF expression in a rat hepatic stellate cell line (T6-HSCs). The present study showed that hypoxia up-regulated the protein levels of COX-2 and hypoxia-inducible factor-1-alpha (HIF-1alpha), but rapidly effected degradation of von Hippel-Lindau (vHL) protein. As a result, expression of VEGF in HSCs was markedly elevated; and pretreatment with COX-2 inhibitors (nimesulide or indomethacin) could significantly ameliorate the angiogenic event. Collectively, hypoxic HSCs increased accumulation of HIF-1alpha protein and induced VEGF expression in a time-dependent manner. Inhibition of COX-2 activities would prevent vHL protein from degradation and suppress HIF-1alpha up-regulation. Thus, vHL/HIF-1alpha has a regulatory role in COX-2-mediated VEGF production in hypoxic stellate cells in injured liver.