Decomposition stages as a clue for estimating the post-mortem interval in carcasses and providing accurate bird collision rates.

The estimation of the post-mortem interval is crucial to accurately provide bird collision rates against manmade infrastructures. Standard methodologies recommend initially clearing all carcasses to ensure that subsequent collisions can be attributed to known time intervals. In this study, we propose a more cost-efficient approach aiming to link the decomposition stages as unequivocally as possible to the most likely time elapsed since death. Factors influencing the decomposition stages of bird carcasses were evaluated by means of two experiments. Firstly, we examined carcasses of large birds in three seasons differing in temperature, sun radiation and humidity: summer, autumn and spring. Secondly, we tested the influence of body mass in the same season (spring) using small, medium-sized and large bird carcasses. Results showed that the decomposition score increased monotonically with time, attaining the highest magnitude effect. A carcass with a decomposition score ≥ 4 (skeletal reduction) was in the field for ≥ 15 days, whereas a carcass with a score < 3 (fresh or emphysematous) was exposed < 3 days. Decomposition scores were higher in summer and did not differ among carcass sizes. This study provides an alternative protocol to estimate the post-mortem interval in wild birds in studies in search of bird fatalities.

Evaluation of the biological and physicochemical properties of calcium silicate-based and epoxy resin-based root canal sealers.

Biocompatibility, dimensional stability, radiopacity, flow, and low solubility are the characteristics of an ideal endodontic sealer. This study evaluated and compared in vivo and in vitro biological and physicochemical properties of calcium silicate-based sealers: Sealer Plus BC (BC), MTA Fillapex (MF); and resin-based sealers: AH Plus (AHP) and Sealer Plus (SP). Apical papilla cells were exposed to sealer extracts and subjected to MTT, SRB, scratch, alkaline phosphatase enzyme activity (ALP) and Alizarin red staining (ALZ) assays. Sealers were histologically evaluated in connective tissue of Wistar rats in different periods. Radiopacity, film thickness, flow, setting time, pH and element analyses were investigated. BC had better results compared to AHP and MF at hour 72 for MTT assay (p < .05), and the highest cell viability under SRB (p < .05). All sealers presented ALP activity. BC presented the highest mineralized deposition under ALZ (p < .05). BC and MF promoted wound healing. All sealers induced an initial inflammation reaction that decreased over time. Eosinophils were observed at day 7 in MF (p < .05). Despite MF did not present final setting time, the sealers properties were in accordance to ISO 6876/2012 and ASTM C266-08. All sealers presented cell viability and biocompatibility. BC presented higher pH values and bioactivity. The materials tested showed physico-chemical properties in accordance with standards, except for MF setting time.

Mammalian-target of rapamycin inhibition with temsirolimus in myelodysplastic syndromes (MDS) patients is associated with considerable toxicity: results of the temsirolimus pilot trial by the German MDS Study Group (D-MDS).

The mammalian-target of rapamycin (also termed mechanistic target of rapamycin, mTOR) pathway integrates various pro-proliferative and anti-apoptotic stimuli and is involved in regulatory T-cell (TREG) development. As these processes contribute to the pathogenesis of myelodysplastic syndromes (MDS), we hypothesized that mTOR modulation with temsirolimus (TEM) might show activity in MDS. This prospective multicentre trial enrolled lower and higher risk MDS patients, provided that they were transfusion-dependent/neutropenic or relapsed/refractory to 5-azacitidine, respectively. All patients received TEM at a weekly dose of 25 mg. Of the 9 lower- and 11 higher-risk patients included, only 4 (20%) reached the response assessment after 4 months of treatment and showed stable disease without haematological improvement. The remaining patients discontinued TEM prematurely due to adverse events. Median overall survival (OS) was not reached in the lower-risk group and 296 days in the higher-risk group. We observed a significant decline of bone marrow (BM) vascularisation (P = 0·006) but were unable to demonstrate a significant impact of TEM on the balance between TREG and pro-inflammatory T-helper-cell subsets within the peripheral blood or BM. We conclude that mTOR-modulation with TEM at a dose of 25 mg per week is accompanied by considerable toxicity and has no beneficial effects in elderly MDS patients.

Selective expansion of regulatory T cells during lenalidomide treatment of myelodysplastic syndrome with isolated deletion 5q.

Lenalidomide (LEN) leads to erythroid improvement in the majority of patients with myelodysplastic syndrome and isolated deletion of the long arm of chromosome 5 (MDS-del(5q)). This effect is believed to be exerted via its immunomodulatory properties, although the precise nature is still incompletely understood. We prospectively performed immune profiling in the bone marrow and blood of MDS-del(5q) patients undergoing LEN therapy for a median of 6 cycles. Therapy with LEN led to a significant increase in the median absolute lymphocyte count (1.3-fold, p = 0.013) without changes in the distribution of the T helper cells within the entire compartment. In parallel, the frequency of Treg increased significantly during treatment both in the peripheral blood (5.0 vs. 9.6 %, p = 0.001) and bone marrow (3.4 vs. 8.1 %, p = 0.001). Surprisingly, LEN treatment led to a decrease in TGFbeta levels, both in the peripheral blood (4.9 vs. 2.3 ng/ml, p = 0.039) and bone marrow (4.5 vs. 0.8 ng/ml, p = 0.023). These changes were not associated with an increase in pro-inflammatory Th17 cells. Taken together, our results demonstrate that LEN induces a shift in lymphocytic populations towards immunosuppression in MDS-del(5q) patients.

Hsa-miR-375 is a predictor of local control in early stage breast cancer.

BACKGROUND: A long-term analysis by the Early Breast Cancer Trialist Group (EBCTG) revealed a strong correlation between local control and cancer-specific mortality. MicroRNAs (miRs), short (20-25 nucleotides) non-coding RNAs, have been described as prognosticators and predictors for breast cancer in recent years. The aim of the current study was to identify miRs that can predict local control after breast conserving therapy (BCT) in early stage breast cancer. RESULTS: Clinical data of 46 early stage breast cancer patients with local relapse after BCT were selected from the institutional database. These patients were matched to 101 control patients showing identical clinical features but without local relapse. The study was conducted in two steps. (1) In the pilot study, 32 patients (16 relapses versus 16 controls) were screened for the most de-regulated microRNAs (= candidate microRNAs) in a panel of 1250 miRs by microarray technology. Eight miRs were found to be significantly de-regulated. (2) In the validation study, the candidate microRNAs were analyzed in an independent cohort of 115 patients (30 relapses versus 85 controls) with reverse transcription quantitative polymerase chain reaction (RT-qPCR). From these eight candidates, hsa-miR-375 could be validated. Its median fold change was 2.28 (Mann-Whitney U test, corrected p value = 0.008). In the log-rank analysis, high expression levels of hsa-miR-375 correlated with a significantly higher risk of local relapse (p = 0.003). In a multivariate analysis (forward stepwise regression) including established predictors and prognosticators, hsa-miR-375 was the only variable that was able to distinguish the statistical significance between relapse and control groups (raw p value = 0.000195 HR = 0.76, 95 % CI 0.66-0.88; corrected p value = 0.005). CONCLUSIONS: Hsa-miR-375 predicts local control in patient with early stage breast cancer, especially in estrogen receptor α (ER-α)-positive patients. It can therefore serve as an additional molecular marker for treatment choice independently from known predictors and prognosticators. Validation in larger prospective studies is warranted.

Antibody validation by combining immunohistochemistry and protein extraction from formalin-fixed paraffin-embedded tissues.

AIMS: Personalized cancer treatment strategies depend on comprehensive and detailed characterization of individual human malignancies. Clinical pathology, particularly immunohistochemical evaluation of biomarkers in tissues, is considered to be the approved standard for diagnostic and therapeutic decisions, having a direct influence on patient management and therapy. Although antibody-based approaches are established and integrated successfully into both clinical and research applications, for personalized treatment regimens new demands have been placed on the quality, reproducibility and accuracy of antibody-based assays. To ensure the accuracy of specific antigen detection in immunohistochemistry, we introduce a novel approach for antibody validation. METHODS AND RESULTS: In a tandem approach we used the same archival tissue of interest for antibody validation by combining extraction of immunoreactive proteins from formalin-fixed, paraffin-embedded tissue with Western blot analysis and immunohistochemistry. This procedure allows for specification of the antigen detected and for localization of the protein in the tissue. Of the 32 antibodies tested used in research and routine diagnostics, 19 showed reliable specificity in both assays. CONCLUSION: This study emphasizes the advantage of combining suitable methods to ensure reproducibility and specific antigen detection. Based on our results, we propose a novel step-by-step strategy to validate antibody specificity and reduce variability of immunohistochemical results.

MicroRNA expression profiling of specific cells in complex archival tissue stained by immunohistochemistry.

Global implementation of molecular diagnostics in modern pathology has been limited by the use of formalin-fixed, paraffin-embedded (FFPE) tissues in current routine diagnostic procedures because of modification and degradation of nucleic acids and protein molecules. In particular, molecular analysis of a specific cell type potentially important for biomarker identification is largely prevented in highly complex, solid tissues routinely used in histopathology. Accumulating data report on the substantial contribution of microRNA molecules (miRNA) to tumor development and malignant progression of most human malignancies. Our objective was to establish a sensitive and robust procedure to quantify miRNA expression in specific cells from complex archival tumor tissues identified by immunohistochemistry. Here, we show reliable detection of miRNA expression profiles determined from limited amounts of colorectal cancer FFPE tissues after routine staining procedure. The combination of routinely used FFPE specimens stained by immunohistochemistry with the molecular analysis of laser microdissected complex tumor tissue resulted in robust miRNA expression patterns exclusively obtained from epithelial tumor cells. This approach allows for a detailed molecular analysis of cancer cells and distinct stromal cell types and their in situ interaction in solid tumors. Hence, the methodology can offer new perspectives for basic research and, by comprehensive use of present archival tissue collections linked to clinical databases, facilitate miRNA biomarker identification in defined tissue cells for future diagnostic and therapeutic strategies.

FARP2, HDLBP and PASK are downregulated in a patient with autism and 2q37.3 deletion syndrome.

We describe a patient with autism and brachymetaphalangy, meeting criteria for 2q37 deletion syndrome (also called Albright Hereditary Osteodystrophy-like syndrome or Brachydactyly-Mental Retardation syndrome, OMIM 600430). Our molecular cytogenetic studies, including array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH), define the extent of the de novo deletion to a 3.5 Mb region on 2q37.3. Although a number of reports of patients with 2q37 deletion syndrome have been published, it remains unclear if gene expression and/or translation are altered by the deletion, thus contributing to the observed phenotypes. To address this question, we selected several candidate genes for the neuropsychiatric and skeletal anomalies found in this patient (autism and brachymetaphalangy). The deleted region in 2q37.3 includes the FERM, RhoGEF and pleckstrin domain protein 2 (FARP2), glypican 1 (GPC1), vigilin (HDLBP), kinesin family member 1A (KIF1A) and proline-alanine-rich STE20-related kinase (PASK), all of which are involved in skeletal or neural differentiation processes. Expression analyses of these genes were performed using RNA from lymphoblastoid cell lines of the patient and his family members. Here we demonstrate that three of these genes, FARP2, HDLBP, and PASK, are considerably downregulated in the patient's cell line. We hypothesize that haploinsufficiency of these genes may have contributed to the patient's clinical phenotype.

The full-ORF clone resource of the German cDNA Consortium.

BACKGROUND: With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. RESULTS: Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. CONCLUSION: The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.

Cisplatin-induced apoptosis is enhanced by hypoxia and by inhibition of mitochondria in renal collecting duct cells.

Cisplatin is a widely used chemotherapeutic agent. Here we show that cisplatin induces apoptosis in renal collecting duct-derived cells (MDCK-C7 cells, resembling principal cells) in a dose-dependent manner. Additionally, we studied the role of mitochondria in this process by inhibition of the mitochondrial respiratory chain, the F1F(o)-ATP synthase or by uncoupling. The role of intra- and extracellular pH in apoptosis induction was investigated. Activation of caspase-3 and DNA ladder formation were used to monitor the apoptotic response. When cells were incubated with inhibitors of the mitochondrial respiratory chain or an inhibitor of the ATP-synthase, cisplatin-induced apoptosis was markedly enhanced. Mitochondrial blockade led to enhanced production of lactic acid. Also, anoxia potentiated the cisplatin-induced caspase-3 activation. Neither intra- nor extracellular pH had an influence on caspase-3 activation at low cisplatin concentrations. Acidic conditions (pH 6.8) potentiated the caspase-3 activation when high (100 microM) cisplatin concentrations were used. We demonstrate that intact mitochondria are important to prevent cisplatin-induced apoptosis in MDCK-C7 cells and that acidic conditions can aggravate the toxic effects of cisplatin.

Inhibition of mitochondria and extracellular acidification enhance achratoxin A-induced apoptosis in renal collecting duct-derived MDCK-C7 cells.

Ochratoxin A (OTA) is a potent nephrotoxin and suspected to be involved in the etiology of Balkan endemic nephropathy. Nanomolar concentrations of this mycotoxin induce apoptosis in renal collecting duct-derived cells (MDCK-C7 cells, resembling principal cells). We studied the role of mitochondria in this process by inhibition of the mitochondrial respiratory chain, the F1FO-ATP synthase or by uncoupling. Also, the role of intra- and extracellular pH in apoptosis induction was investigated. Activation of caspase-3 and DNA ladder formation were used to monitor the apoptotic response. When cells were incubated with inhibitors of the mitochondrial respiratory chain or an inhibitor of the ATP-synthase, OTA-induced apoptosis was enhanced dramatically. Also, mitochondrial uncoupling potentiated the effects of OTA. OTA-induced apoptosis was not dependent on a decrease of the mitochondrial potential. Mitochondrial blockade led to medium acidification due to enhanced production of lactic acid. Artificial extracellular acidification potentiated OTA-induced caspase-3 activation. Artificial extracellular alkalization had no influence on caspase-3 activity. Intracellular pH after 24 hours exposure to inhibitors of mitochondria or acidic or alkaline media did not correlate with caspase-3 activity but correlated with caspase-3 activity when OTA was present: acidic intracellular pH (pHin) was associated with higher caspase-3 activity as compared to alkaline pHin. We conclude that extra- and intracellular pH are important factors in OTA-induced apoptosis in MDCK-C7 cells. The physiologically changing pH conditions in the collecting duct can thus alter or even aggravate the toxic effects of OTA.

Aldosterone stimulates epidermal growth factor receptor expression.

The steroid hormone aldosterone plays an important role during pathological tissue modifications, similar to cardiovascular or renal fibrosis. The underlying mechanisms for the pathological actions are not understood. Interaction of aldosterone with the epidermal growth factor (EGF) receptor is an attractive hypothesis to explain pathological tissue remodeling elicited by aldosterone, because (i) mineralocorticoids can sensitize cells for EGF, (ii) mineralocorticoid receptor (MR)-antagonists reduce EGFR-mRNA expression, (iii) EGFR itself supports the development of cardiovascular or renal fibrosis, and (iv) signaling elements involved in the pathological action of aldosterone (similar to ERK1/2 or NFkB) are typical downstream modules during EGF signaling. In addition, an interaction of aldosterone and EGF with respect to ERK1/2 activation has been described. Here we show that aldosterone stimulates EGFR expression in renal tissue of adrenalectomized rats and in human renal primary cell cultures. Furthermore, Chinese hamster ovary (CHO) cells normally devoid of EGFR or MR express EGFR after transfection with human MR (CHO-MR cells) but not after transfection with human glucocorticoid receptor (CHO-GR cells). In CHO-MR cells, EGFR-expression is up-regulated by aldosterone and inhibited by spironolactone. CHO-MR cells but not CHO-GR cells respond with ERK1/2 phosphorylation to EGF exposure. The responsiveness to other peptide hormones was virtually not affected. These data suggest that EGFR is an aldosterone-induced protein and is involved in the manifold (patho)biological actions of aldosterone.

Inhibition of mitochondria prevents cell death in kidney epithelial cells by intra- and extracellular acidification.

BACKGROUND: Nephrotoxic substances like cisplatin or ochratoxin A (OTA) induce cell death in human proximal tubule-derived cells (IHKE cells). Mitochondria play a significant role in apoptosis and loss of their function may influence OTA- or cisplatin-induced apoptosis. Extracellular pH also plays an important role in tumor genesis. Therefore, we investigated the role of mitochondria and intra- and extracellular pH on cell death induction by cisplatin or OTA. METHODS: IHKE cells were incubated in the presence of OTA or cisplatin, together with inhibitors of the mitochondrial metabolism, and the activity of caspase-3 was measured and DNA laddering was monitored. Adenosine triphosphate (ATP) content of the cells, lactate release into the media, and glucose consumption was determined. In addition, media and cells were acidified or alkalized artificially to investigate the effect of intra- and extracellular pH on cell death induction. Cytochrome C was immunodetected in cellular compartments. RESULTS: Inhibition of the mitochondrial function reduced OTA- or cisplatin-induced cell death and led to considerable lactic acid production and extracellular acidification. Intra- and extracellular acidification prevented cells from cell death induced by OTA or cisplatin. No cytochrome C release from mitochondria could be detected during 24 hours of exposure to OTA or cisplatin. CONCLUSION: We conclude that OTA- or cisplatin-induced cell death is dependent on functional and intact, ATP-producing mitochondria and that intra- and extracellular pH is crucial for induction of cell death in IHKE cells.

Human epidermal growth factor receptor-1 expression renders Chinese hamster ovary cells sensitive to alternative aldosterone signaling.

The epidermal growth factor (EGF) regulates cell proliferation, differentiation, and ion transport using ERK1/2 as a downstream effector. Furthermore, the EGF receptor (EGFR) is involved in signaling by G-protein-coupled receptors, growth hormone, and cytokines via transactivation. It has been suggested that steroids interact with peptide hormones. Previously, we have shown that aldosterone modulates EGF responses in Madin-Darby canine kidney cells (Gekle, M., Freudinger, R., Mildenberger, S., and Silbernagl, S. (2002) Am. J. Physiol. 282, F669-F679). Here, we tested the hypothesis that human EGFR-1 can confer alternative aldosterone responsiveness with respect to ERK1/2 phosphorylation to Chinese hamster ovary cells, which do not express EGFR. Wild-type Chinese hamster ovary cells did not respond to EGF or aldosterone. After transfection of human EGFR-1, the cells responded to EGF, but not to aldosterone. However, when submaximal concentrations of EGF were used, nanomolar concentrations of aldosterone potentiated the action of EGF within minutes, resulting in a leftward shift of the EGF dose-response curve. This was not the case in mock-transfected cells. The EGFR kinase inhibitor tyrphostin AG1478 or the MEK1/2 inhibitor U0126 completely prevented the effect. Furthermore, aldosterone enhanced Tyr phosphorylation of c-Src and EGFR, and an inhibitor of cytosolic tyrosine kinases (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriociaine) prevented the action of aldosterone. Our data show that aldosterone uses the EGF-EGFR-MEK1/2-ERK1/2 signaling cascade to elicit its alternative effects. In the presence of EGF, aldosterone leads to EGFR transactivation via cytosolic tyrosine kinases of the Src family.