The stabilizing effect of resting egg banks of the <Emphasis Type="Italic">Daphnia longispina</Emphasis> species complex for longitudinal taxon heterogeneity in long and narrow reservoirs

The aim of this study was to determine the effect of monsoonal spates and land uses on abundance, richness, diversity, and evenness of benthic macroinvertebrates in streams, and whether these factors interacted with each other to influence community structure. Two groups of streams with different underlying disturbance profiles were studied. One group was in a native forest (non-degraded area) and experienced only spate, and the other group drained an area degraded by agriculture and cattle grazing, and so was subject to two types of disturbances. Spates produced a strong decline in invertebrate abundance and an increase in evenness, whereas changes in land uses caused an increase in abundance of tolerant taxa and a significant decline in richness, diversity, and evenness. Differences in richness and Oligochaeta abundance between land uses were mostly evident in the pre-spate period due to low water levels magnifying some physicochemical changes caused by ecosystem degradation. Canonical correspondence analysis indicated that degraded sites had the highest conductivity, nitrate, pH, and water temperature. Upstream non-degraded streams generally exhibited higher dissolved oxygen values. Taxa such as Psychodidae and Tipulidae, Camelobaetidius penai, Austrelmis spp. (adult), Macrelmis spp. (adult), and Marilia spp. dominated in non-degraded sites, while Tricorythodes popayanicus, Caenis ludicra, Dodecabates dodecaporus, Atractides sp., and Torrenticola columbiana were abundant in downstream degraded streams. These two groups of taxa may be useful biological indicators of water quality in streams of northwestern Argentina.     

Dietary Exposure to PCDD/Fs Through Animal Products in a Highly Polluted Area in Turkey

We present an estimation of dietary exposure to PCDD/Fs by animal products in Kocaeli, a highly polluted area in Turkey, based on current food data consumption. The calculation of the PCDD/F intakes by an exposure methodology concerning consumption habits of different receptor groups in Kocaeli was included. The data relate to the PCDD/F levels in food groups of animal origin (milk, egg, meat, chicken, and fish), food consumption rates, and the fractions of locally grown foods in total consumption were statistically assessed. Monte Carlo simulation was used to estimate the ranges of PCDD/F doses through the consumption of animal products. The PCDD/F intakes through the consumption of animal products were calculated to average between 0.4–1.8 pg WHO-TEQ.kg^?1 bodyweight (bw).day^?1. The results are within the range of 1–4 pg WHO-TEQ.kg^?1 bw.day^?1, proposed as the tolerable daily intake by the World Health Organization. On the other hand, contributions of the consumption of different foods to the total PCDD/F intake and the results of sensitivity analysis showed that the PCDD/F intake in semi-urban and rural settings was determined primarily by the consumption of milk products, while consumption of meat and fish had a greater importance in urban settings.     

The prevalence of TNFα-induced necrosis over apoptosis is determined by TAK1-RIP1 interplay

Death receptor-induced programmed necrosis is regarded as a secondary death mechanism dominating only in cells that cannot properly induce caspase-dependent apoptosis. Here, we show that in cells lacking TGFβ-activated Kinase-1 (TAK1) expression, catalytically active Receptor Interacting Protein 1 (RIP1)-dependent programmed necrosis overrides apoptotic processes following Tumor Necrosis Factor-α (TNFα) stimulation and results in rapid cell death. Importantly, the activation of the caspase cascade and caspase-8-mediated RIP1 cleavage in TNFα-stimulated TAK1 deficient cells is not sufficient to prevent RIP1-dependent necrosome formation and subsequent programmed necrosis. Our results demonstrate that TAK1 acts independently of its kinase activity to prevent the premature dissociation of ubiquitinated-RIP1 from TNFα-stimulated TNF-receptor I and also to inhibit the formation of TNFα-induced necrosome complex consisting of RIP1, RIP3, FADD, caspase-8 and cFLIP(L). The surprising prevalence of catalytically active RIP1-dependent programmed necrosis over apoptosis despite ongoing caspase activity implicates a complex regulatory mechanism governing the decision between both cell death pathways following death receptor stimulation.     

Potential of Raloxifene in reversing osteoarthritis-like alterations in rat chondrocytes: An in vitro model study

The aim of this study was to investigate the effects of Raloxifene (Ral) on degeneration-related changes in osteoarthritis (OA)-like chondrocytes using two- and three-dimensional models. Five-azacytidine (Aza-C) was used to induce OA-like alterations in rat articular chondrocytes and the model was verified at molecular and macrolevels. Chondrocytes were treated with Ral (1, 5 and 10 μM) for 10 days. Caspase-3 activity, gene expressions of aggrecan, collagen II, alkaline phosphatase (ALP), collagen X, matrix metalloproteinases (MMP-13, MMP-3 and MMP-2), and MMP-13, MMP-3 and MMP-2 protein expressions were studied in two-dimensional model. Matrix deposition and mechanical properties of agarose-chondrocyte discs were evaluated in three-dimensional model. One μM Ral reduced expression of OA-related genes, decreased apoptosis, and MMP-13 and MMP-3 protein expressions. It also increased aggrecan and collagen II gene expressions relative to untreated OA-like chondrocytes. In three-dimensional model, 1 μM Ral treatment resulted in increased collagen deposition and improved mechanical properties, although a significant increase for sGAG was not observed. In summation, 1 μM Ral improved matrix-related activities, whereas dose increment reversed these effects except ALP gene expression and sGAG deposition. These results provide evidence that low-dose Ral has the potential to cease or reduce the matrix degeneration in OA.     

Health Risk Assessment of Workers' Exposure to Organic Compounds in a Tire Factory

This study focuses on a health risk assessment related to chemical exposure via inhalation for workers in a tire factory. Specifically, several volatile organic compounds (VOCs) and semi-volatile organic compounds (SVOCs) were measured in the four different points of the vulcanization unit. A chemical transport model was developed in order to better represent the workers' exposure to the chemicals. Then, a risk assessment methodology was employed to evaluate the potential adverse health effects of the chemicals according to their carcinogenicities. Concentrations measured near the milling machine and press in the vulcanization unit were generally higher than the respective occupational exposure limit values. The corresponding estimated cumulative cancer risks for the carcinogens at the each sampling point were higher than the designated acceptable risk level of 1 × 10^{? 4}. With respect to non-carcinogenic risks, the hazard indexes, both individually and cumulatively, were lower than the specified level of one. The high cancer risk estimated in this study suggests that the VOCs and SVOCs exposure for workers in the vulcanization unit should not be neglected. The results obtained in this study are valuable to plant managers, government officials, and regulators in the risk evaluation process.     

Profiles of serum microRNAs; miR-125b-5p and miR223-3p serve as novel biomarkers for HBV-positive hepatocellular carcinoma

Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions including cancer. Certain microRNAs (miRNAs) have been shown early diagnostic potential for many types of cancer. The objective of this study was to investigate the potential of certain serum/plasma miRNAs as novel non-invasive biomarkers for early diagnosis of hepatitis B virus (HBV) related hepatocellular carcinoma (HCC). For this reason, the expression levels of 24 miRNA (let-7c, miR-92a-3p, 423-5p, 150-5p, 223-3p, 125b-5p, 342-3p, miR-206, 122-5p, 375, 223-5p, 10a-5p, 23b-5p, 99a-5p, 23a-5p, 10a-3p, 122-3p, 125b-1-3p, 23b-3p, 125b-2-3p, 23a-3p, 92a-1-5p, 92a-2-5p, 99a-3p) were analyzed in plasma of patients with chronic hepatitis B, HBV-positive cirrhosis and HBV-positive HCC and compared with control group samples. Totally 94 plasma samples; 28 control and 66 patient plasma (24 CHB, 22 HBV-positive cirrhosis, 20 HBV-positive HCC) and were included in this study. The expression levels of 24 miRNAs were detected for all control and patient group plasma samples by qRT-PCR using BioMark? 96.96 Dynamic Array (Fluidigm Corporation) system. The expression levels of miR-125b-5p were detected 2.85 fold, 2.46 fold and 1.89 fold (p = 0.01513, p = 0.0009440, p = 0.0001446) up regulated in CHB, HBV-positive cirrhosis and HBV-positive HCC, respectively when compared versus control group individually by Mann–Whitney U test. The expression levels of miR-223-3p were detected 5.55 fold, 13.88 fold and 12.65 fold (p = 0.01513, p = 0.0009440, p = 0.0001446) down regulated in same comparisons. When all groups were compared versus control group by one-way ANOVA test, the expression levels of miR-223-3p were also found statistically significant (p < 0.05). Although not statistically significant, miR-125b-5p tended to be upregulated. (p = 0.07192). These results significantly imply that miR-125b-5p and miR223-3p could be used as novel non-invasive biomarkers of HBV-positive HCC in very early, even at CHB stage of liver disease.     

De Novo Proteome Analysis of Genetically Modified Tumor Cells By a Metabolic Labeling/Azide-alkyne Cycloaddition Approach

Activin receptor type II (ACVR2) is a member of the transforming growth factor type II receptor family and controls cell growth and differentiation, thereby acting as a tumor suppressor. ACVR2 inactivation is known to drive colorectal tumorigenesis. We used an ACVR2-deficient microsatellite unstable colon cancer cell line (HCT116) to set up a novel experimental design for comprehensive analysis of proteomic changes associated with such functional loss of a tumor suppressor. To this end we combined two existing technologies. First, the ACVR2 gene was reconstituted in an ACVR2-deficient colorectal cancer (CRC) cell line by means of recombinase-mediated cassette exchange, resulting in the generation of an inducible expression system that allowed the regulation of ACVR2 gene expression in a doxycycline-dependent manner. Functional expression in the induced cells was explicitly proven. Second, we used the methionine analog azidohomoalanine for metabolic labeling of newly synthesized proteins in our cell line model. Labeled proteins were tagged with biotin via a Click-iT chemistry approach enabling specific extraction of labeled proteins by streptavidin-coated beads. Tryptic on-bead digestion of captured proteins and subsequent ultra-high-performance LC coupled to LTQ Orbitrap XL mass spectrometry identified 513 proteins, with 25 of them differentially expressed between ACVR2-deficient and -proficient cells. Among these, several candidates that had already been linked to colorectal cancer or were known to play a key role in cell growth or apoptosis control were identified, proving the utility of the presented experimental approach. In principle, this strategy can be adapted to analyze any gene of interest and its effect on the cellular de novo proteome.Human tumors acquire a large number of genetic and epigenetic alterations that arise during progression from preneoplastic lesions to metastatic disease. However, the diversity of these alterations reflects the intratumoral heterogeneity and represents the genomic landscape of tumors. Among a high background number of irrelevant passenger alterations, only a limited number of genetic alterations are considered to be driving events that confer a selective advantage to tumor cells. Major signaling pathways affected by such driver mutations include the TGFβ, BMP, Activin, Wnt, and Notch pathways, abrogating normal regulation of key cellular processes such as cell fate, cell survival, and genome maintenance.Both tumor-relevant driver mutations in a major signaling receptor and tumor-irrelevant passenger mutations can cause changes at the proteomic level. Passenger-mutation-associated proteomic patterns are propagated randomly and do not represent generic tumor-associated changes (1). Therefore, a focus on proteome alterations associated with single driver mutations is necessary in order for specific changes that underlie tumor development to be identified. However, such analyses encounter two major limitations at different levels.At the molecular level, the genetic heterogeneity of tumors—especially those of the microsatellite unstable and mutator phenotype—poses a significant problem in determining mutation-specific effects. Two principal strategies for detecting cellular consequences of a single mutation have been applied. First, targeted gene knock-out in target-gene-proficient cell lines by means of homologous recombination, adeno-associated viral delivery, or zinc finger nucleases has been used successfully (2–4). However, these approaches are often limited by their low efficiency, are laborious and time-consuming, and bear the potential for confounding off-target effects. Second, transfer of the target gene into deficient cell lines via gene insertion or gene targeting methods has been extensively applied. Unfortunately, insertion methods are often affected by random insertion, a variable number of integrated gene copies per cell, and inconsistent integration sites, eventually resulting in unpredictable expression patterns (5). However, many non-integrating vectors, such as adenoviral DNA, are not often replicated during cell division, which limits their use in basic research.At the protein level, sample complexity is a major limiting factor. In addition to prefractionation methods, metabolic labeling is a versatile tool in work focusing on proteomic changes induced by gene activation. Because the activation of tumor suppressor pathways directly regulates target gene expression, analysis of tumor-suppressor-dependent alterations of newly synthesized proteins via metabolic labeling is a reasonable approach for restricting proteomic complexity. Conventional methods for metabolic labeling usually rely on amino acids containing either radioactive or stable isotopes. Although radioactive labeling enables extremely sensitive detection methods, its use for proteomic analysis is limited because of the need for special handling and precautions against contamination of the analytical instrumentation. Stable isotopic labeling, in particular the SILAC methodology, is currently the preferred method for most metabolic labeling approaches in proteomic analyses, and especially for cell lines (6). However, when applying the SILAC technology, mass spectrometric detection of labeled peptides has to be conducted in the presence of numerous irrelevant, unlabeled peptides, which hampers the detection of labeled low-abundance peptides. A relatively new method, termed Click-iT labeling, that enables labeling of nascent proteins comparable to that by a radioactive compound can overcome this problem, because upon incorporation of the labeled compound a handle for specific extraction of the labeled protein is worked in. The click reaction makes use of a methionine derivative that is functionalized with an azide (l-azidohomoalanine (AHA)).¹ During protein synthesis AHA is incorporated into proteins, which can then be tagged with a biotin alkyne (PEG4 carboxamide-propargyl biotin), resulting in the specific biotinylation of the metabolically labeled proteins (7). The final step is extraction of the labeled proteins by streptavidin beads.In the present study we pursued a completely new strategy to determine proteomic changes caused by a tumor-specific mutation in a major signaling pathway exemplified by the activin receptor type II (ACVR2) tumor suppressor. Disruption of activin signaling is a frequent event during colorectal tumorigenesis and can be caused by different molecular mechanisms. In colon tumors with chromosomal instability but microsatellite stability, ACVR2 inactivation mainly occurs via epigenetic mechanisms, whereas in colon cancers with a high level of microsatellite instability (MSI) loss of ACVR2 emerges from frameshift mutation (8). MSI has been defined as a change in length (insertions/deletions) of small repetitive DNA sequences (microsatellites) that arises specifically in tumor cells with impaired DNA mismatch repair functions (mutator phenotype) but not in corresponding normal tissue (9, 10). The A8 coding microsatellite in exon 10 of the ACVR2 gene has been identified as one of the most frequent mutation targets in high-MSI colorectal tumors that abrogates normal ACVR2 protein expression (11–13). As a consequence, phosphorylation of downstream signaling mediators such as Smad2/3, as well as regulation of several target genes such as SERPINE, SMAD7, and MYC, is significantly impaired, and this contributes to MSI colorectal tumorigenesis.Our strategy relied on a combination of two established technologies. First, we employed a recombination-mediated genomic targeting approach (14–16) to generate a genetically modified MSI colorectal tumor cell line that enabled doxycycline-inducible and reversible expression of an ACVR2 transgene and activation of ligand-dependent signaling in an isogenic background. Second, we performed metabolic labeling via a click-chemistry approach (7) in this MSI cell line to uncover alterations in the pattern of newly synthesized proteins (de novo proteome). In particular, we determined the constituents of the total cellular de novo proteome and subsets derived thereof that were regulated in an ACVR2-dependent manner. Our combinatorial strategy represents a versatile approach allowing one to overcome the limitations inherent in the genetic heterogeneity and proteomic complexity of MSI tumor cells. Moreover, this model system allows one to address any tumor target gene and thereby possesses broad applicability for studies of cancer proteome complexity, which is the logical starting point for identifying diagnostic biomarkers and therapeutic targets for cancer.     

Structure based discovery of small molecules to regulate the activity of human insulin degrading enzyme

Background

Insulin-degrading enzyme (IDE) is an allosteric Zn⁺² metalloprotease involved in the degradation of many peptides including amyloid-β, and insulin that play key roles in Alzheimer''s disease (AD) and type 2 diabetes mellitus (T2DM), respectively. Therefore, the use of therapeutic agents that regulate the activity of IDE would be a viable approach towards generating pharmaceutical treatments for these diseases. Crystal structure of IDE revealed that N-terminal has an exosite which is ∼30 Å away from the catalytic region and serves as a regulation site by orientation of the substrates of IDE to the catalytic site. It is possible to find small molecules that bind to the exosite of IDE and enhance its proteolytic activity towards different substrates.

Methodology/Principal Findings

In this study, we applied structure based drug design method combined with experimental methods to discover four novel molecules that enhance the activity of human IDE. The novel compounds, designated as D3, D4, D6, and D10 enhanced IDE mediated proteolysis of substrate V, insulin and amyloid-β, while enhanced degradation profiles were obtained towards substrate V and insulin in the presence of D10 only.

Conclusion/Significance

This paper describes the first examples of a computer-aided discovery of IDE regulators, showing that in vitro and in vivo activation of this important enzyme with small molecules is possible.