Germin, a protein marker of early plant development, is an oxalate oxidase

Germin is a homopentameric glycoprotein, the synthesis of which coincides with the onset of growth in germinating wheat embryos. There have been detailed studies of germin structure, biosynthesis, homology with other proteins, and of its value as a marker of wheat development. Germin isoforms associated with the apoplast have been speculated to have a role in embryo hydration during maturation and germination. Antigenically related isoforms of germin are present during germination in all of the economically important cereals studied, and the amounts of germin-like proteins and coding elements have been found to undergo conspicuous change when salt-tolerant higher plants are subjected to salt stress. In this report, we describe how circumstantial evidence arising from unrelated studies of barley oxalate oxidase and its coding elements have led to definitive evidence that the germin isoform made during wheat germination is an oxalate oxidase. Establishment of links between oxalate degradation, cereal germination, and salt tolerance has significant implications for a broad range of studies related to development and adaptation in higher plants. Roles for germin in cell wall biochemistry and tissue remodeling are discussed, with special emphasis on the generation of hydrogen peroxide during germin-induced oxidation of oxalate.

Large-scale transcriptome analyses reveal new genetic marker candidates of head, neck, and thyroid cancer

A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.

Immunocytochemical study of Ki-67 as a prognostic marker in canine mammary neoplasia

Background: Canine mammary tumors are challenging for clinicians and pathologists because of complex histologic classification, low specificity of cytologic diagnosis, and unpredictable biological behavior. In histologic specimens, expression of tumor proliferation marker Ki-67, a nuclear nonhistone protein, has been shown to have prognostic value for canine mammary tumors and to correlate with malignancy and low survival rates. Objective: The objective of this study was to measure the proliferation index of canine mammary tumors by immunochemical detection of Ki-67 in cytologic specimens and to determine its relationship to clinical and pathologic variables and patient outcome. Methods: Spontaneous mammary tumors from 31 female dogs were surgically excised. Imprint specimens for cytologic evaluation were wet-fixed in ethanol; histologic specimens were prepared routinely. Immunostaining was performed with the PH 177 monoclonal antibody against Ki-67; proliferation index was graded from negative to +++. Dogs were followed for 18 months. Multivariate logistic regression analysis was used to determine correlations between immunocytochemical results, tumor and clinical variables, and patient outcome. Results: Ki-67 proliferation indices in cytologic specimens were significantly lower for nonmalignant tumors than for malignant tumors. High index values of Ki-67 were positively correlated with metastasis, death from neoplasia, low disease-free survival rates, and low overall survival rate. With the exception of 4 specimens for which cellularity was insufficient, positive expression of Ki-67 in cytologic specimens correlated with that of histologic specimens. Conclusions: The prognostic value of the Ki-67 index in canine mammary tumors by using wet-fixed cytology imprint specimens was similar to that observed previously for histologic specimens. Immunocytochemical detection of Ki-67 could improve the accuracy and value of cytology by providing safe and rapid information about malignancy and patient outcome. © 2004 American Society for Veterinary Clinical Pathology.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

STEAP1 protein overexpression is an independent marker for biochemical recurrence in prostate carcinoma

Aims: To investigate the prognostic value of expression levels of the genes STEAP1 and STEAP2, and of STEAP1 protein, in prostate carcinomas (PCa). Methods and results: STEAP1 and STEAP2 transcript levels were evaluated by RT-qPCR in samples from 35 PCa, 24 adjacent non-neoplastic prostate (AdjP) tissues, five cases of benign prostatic hyperplasia (BPH), and two histologically normal prostates (N). STEAP1 expression was assessed by immunohistochemistry in samples from 198 PCa, 76 AdjP, 22 BPH, and two N. The findings were compared with clinical and pathological parameters and patient outcome. STEAP1 and STEAP2 transcript analysis showed no differences between the groups tested. Although not significant, higher STEAP1 mRNA levels were detected in tumours with high Gleason scores and in patients who presented with biochemical recurrence (BCR). STEAP1 overexpression was detected in PCa, and was significantly associated with high-grade Gleason scores, seminal vesicle invasion, BCR, and worse outcome (metastasis or PCa-specific death). STEAP1 overexpression was significantly associated with shorter BCR-free survival. Multivariate analysis revealed that STEAP1 is an independent marker for BCR. Conclusions: These findings provide evidence that STEAP1 is a biomarker of worse prognosis in PCa patients. © 2013 John Wiley & Sons Ltd.

Brazilian Angiostrongylus cantonensis haplotypes, ac8 and ac9, have two different biological and morphological profiles

Angiostrongylus cantonensis is the etiologic agent of eosinophilic meningoencephalitis in humans. Cases have been recorded in many parts of the world, including Brazil. The aim of this study was to compare the differences in the biology and morphology of two different Brazilian haplotypes of A. : ac8 and ac9. A significantly larger number of L1 larvae eliminated in the faeces of rodents at the beginning of the patent period was observed for ac9 haplotype and compared to the total of L1 larvae eliminated, there was a significant difference between the two haplotypes. The ac9 haplotype showed a significant difference in the proportion of female and male specimens (0.6:1), but the same was not observed for ac8 (1.2:1). The morphometric analysis showed that male and female specimens isolated from ac8 haplotype were significantly larger with respect to body length, oesophagus length, spicule length (male) and distance from the anus to the rear end (female) compared to specimens from ac9. The morphological analysis by light microscopy showed little variation in the level of bifurcations at the lateral rays in the right lobe of the copulatory bursa between the two haplotypes. The biological, morphological and morphometric variations observed between the two haplotypes agree with the observed variation at the molecular level using the cytochrome oxidase subunit I marker and reinforce the possible influence of geographical isolation on the development of these haplotypes.

S-100 protein: is it useful as a tumour marker in diagnostic immunocytochemistry

A heterogeneous group of 159 tumours was studied for the presence of S-100 protein by the immunoperoxidase technique in order to determine whether this marker may be of value in facilitating immunocytochemical diagnosis. Among cases of melanocytic and pigmented lesions, S-100 was widely distributed and demonstrated the strongest degrees of reactivity. S-100 protein was identified in virtually all nerve sheath tumours such as schwannomas, neurofibromas, myxoid sheath nerve tumour and also in some tumours of controversial histogenesis such as granular cell tumours. The great majority of carcinomas did not express S-100, with only two cases of breast carcinoma displaying focal S-100 staining. In a miscellaneous group of tumours S-100 was demonstrated in chordomas, myoepitheliomas and Wilms' tumour with Schwann cell differentiation. Despite its presence in a wide array of cell types, S-100 protein continues to be an extremely useful marker especially for soft tissue and peripheral nervous system tumours.

Genetic Variability of Beauveria bassiana and a DNA Marker for Environmental Monitoring of a Highly Virulent Isolate Against Cosmopolites sordidus

The banana weevil Cosmopolites sordidus (Germar) is one of a number of pests that attack banana crops. The use of the entomopathogenic fungus Beauveria bassiana as a biological control agent for this pest may contribute towards reducing the application of chemical insecticides on banana crops. In this study, the genetic variability of a collection of Brazilian isolates of B. bassiana was evaluated. Samples were obtained from various geographic regions of Brazil, and from different hosts of the Curculionidae family. Based on the DNA fingerprints generated by RAPD and AFLP, we found that 92 and 88 % of the loci were polymorphic, respectively. The B. bassiana isolates were attributed to two genotypic clusters based on the RAPD data, and to three genotypic clusters, when analyzed with AFLP. The nucleotide sequences of nuclear ribosomal DNA intergenic spacers confirmed that all isolates are in fact B. bassiana. Analysis of molecular variance showed that variability among the isolates was not correlated with geographic origin or hosts. A RAPD-specific marker for isolate CG 1024, which is highly virulent to C. sordidus, was cloned and sequenced. Based on the sequences obtained, specific PCR primers BbasCG1024F (5'-TGC GGC TGA GGA GGA CT-3') and BbasCG1024R (5'-TGC GGC TGA GTG TAG AAC-3') were designed for detecting and monitoring this isolate in the field.

Genetic and morphological variation and differentiation of Raja clavata populations in the European seas: marker development and preliminary data

In this study the population structure and connectivity of the Mediterranean and Atlantic Raja clavata (L., 1758) were investigated by analyzing the genetic variation of six population samples (N = 144) at seven nuclear microsatellite loci. The genetic dataset was generated by selecting population samples available in the tissue databases of the GenoDREAM laboratory (University of Bologna) and of the Department of Life Sciences and Environment (University of Cagliari), all collected during past scientific surveys (MEDITS, GRUND) from different geographical locations in the Mediterranean basin and North-east Atlantic sea, as North Sea, Sardinian coasts, Tuscany coasts and Cyprus Island. This thesis deals with to estimate the genetic diversity and differentiation among 6 geographical samples, in particular, to assess the presence of any barrier (geographic, hydrogeological or biological) to gene flow evaluating both the genetic diversity (nucleotide diversity, observed and expected heterozygosity, Hardy- Weinberg equilibrium analysis) and population differentiation (Fst estimates, population structure analysis). In addition to molecular analysis, quantitative representation and statistical analysis of morphological individuals shape are performed using geometric morphometrics methods and statistical tests. Geometric coordinates call landmarks are fixed in 158 individuals belonging to two population samples of Raja clavata and in population samples of closely related species, Raja straeleni (cryptic sibling) and Raja asterias, to assess significant morphological differences at multiple taxonomic levels. The results obtained from the analysis of the microsatellite dataset suggested a geographic and genetic separation between populations from Central-Western and Eastern Mediterranean basins. Furthermore, the analysis also showed that there was no separation between geographic samples from North Atlantic Ocean and central-Western Mediterranean, grouping them to a panmictic population. The Landmark-based geometric morphometry method results showed significant differences of body shape able to discriminate taxa at tested levels (from species to populations).

Biological applications of plasmonic metal nanoparticles

Plasmons in metal nanoparticles respond to changes in their local environment by a spectral shift in resonance. Here, the potential of plasmonic metal nanoparticles for label-free detection and observation of biological systems is presented. Comparing the material silver and gold concerning plasmonic sensitivity, silver nanoparticles exhibit a higher sensitivity but their chemical instability under light exposure limits general usage. A new approach combining results from optical dark-field microscopy and transmission electron microscopy allows localization and quantification of gold nanoparticles internalized into living cells. Nanorods exposing a negatively charged biocompatible polymer seem to be promising candidates to sense membrane fluctuations of adherent cells. Many small nanoparticles being specific sensing elements can build up a sensor for parallel analyte detection without need of labeling, which is easy to fabricate, re-usable, and has sensitivity down to nanomolar concentrations. Besides analyte detection, binding kinetics of various partner proteins interacting with one protein of interest are accessible in parallel. Gold nanoparticles are able to sense local oscillations in the surface density of proteins on a lipid bilayer, which could not be resolved so far. Studies on the fluorescently labeled system and the unlabeled system identify an influence of the label on the kinetics.

Matrix metalloproteinase-19 is a predictive marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma

AIMS: To evaluate the expression of matrix metalloproteinase-19 (MMP-19) in oropharyngeal squamous cell carcinoma along with its association with structural features of invasiveness. To investigate whether MMP-19 expression correlates with lymphatic or systemic metastasis and prognosis in patients who have received definitive radiotherapy. METHODS AND RESULTS: The histological evaluation of the invasive front was based on Bryne's malignancy grading system. We correlated the immunohistochemical expression pattern with morphological parameters which characterize tumor invasiveness such as keratinization, nuclear polymorphism, invasion pattern, and the host inflammatory response. Local immunoreactivity for MMP-19 was positively correlated with tumor invasiveness as reflected in its structural characteristics and the degree of nuclear polymorphism, and negatively correlated with the inflammatory response of the host. No correlation existed between MMP-19 expression and clinicopathological features (TNM stage, grade of differentiation) or a patient''s outcome and prognosis. CONCLUSIONS: This latter finding probably reflects the unique change for MMPs from high immunoreactivity within healthy tissue areas and non-invasive tumor parts, through absence in the least invasive neoplastic regions, to strong re-expression at a highly invasive front of the same tumor. Our findings indicate that MMP-19 can be used as a marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

Direct metabolites of Ethanol as biological markers of alcohol use: Basic aspects and applications

In addition to self reports and questionnaires, biomarkers are of relevance in the diagnosis of and therapy for alcohol use disorders. Traditional biomarkers such as gamma-glutamyl transpeptidase or mean corpuscular volume are indirect biomarkers and are subject to the influence of age, gender and non-alcohol related diseases, among others. Direct metabolites of ethanol such as Ethyl glucuronide (EtG), ethyl sulphate (EtS) and phosphatidylethanol (PEth) are direct metabolites of ethanol, that are positive after intake of ethyl alcohol. They represent useful diagnostic tools for identifying alcohol use even more accurately than traditional biomarkers. Each of these drinking indicators remains positive in serum and urine for a characteristic time spectrum after the cessation of ethanol intake - EtG and EtS in urine up to 7 days, EtG in hair for months after ethanol has left the body. Applications include clinical routine use, emergency room settings, proof of abstinence in alcohol rehabilitation programmes, driving under influence offenders, workplace testing, assessment of alcohol intake in the context of liver transplantation and foetal alcohol syndrome. Due to their properties, they open up new perspectives for prevention, interdisciplinary cooperation, diagnosis of and therapy for alcohol-related problems.

A Cell Biological Determination of Integrator Subunit Localization

Uridine-rich small nuclear (U snRNAs), with the exception of the U6 snRNA, are RNA polymerase II (RNAPII) transcripts. The mechanism of 3’ cleavage of snRNAs has been unknown until recently. This area was greatly advanced when 12 of the Integrator complex subunits (IntS) were purified in 2005 through their interaction with the C-terminal domain (CTD) of the large subunit (RpbI) of RNAPII. Subsequently, our lab performed a genome-wide RNAi screen that identified two more members of the complex that we have termed IntS13 and IntS14. We have determined that IntS9 and 11 mediate the 3’ cleavage of snRNAs, but the exact function of the other subunits remains unknown. However, through the use of a U7 snRNA-GFP reporter and RNAi knockdown of the Integrator subunits in Drosophila S2 cells, we have shown that all subunits are required for the proper processing of snRNAs, albeit to differing degrees. Because snRNA transcription takes place in the nucleus of the cell, it is expected that all of the Integrator subunits would exhibit nuclear localization, but the knowledge of discrete subnuclear localization (i.e. to Cajal bodies) of any of the subunits could provide important clues to the function of that subunit. In this study, we used a cell biological approach to determine the localization of the 14 Integrator subunits. We hypothesized that the majority of the subunits would be nuclear, however, a few would display distinct localization to the Cajal bodies, as this is where snRNA genes are localized and transcribed. The specific aims and results are: 1. To determine the subcellular localization of the 14 Integrator subunits. To accomplish this, mCherry and GFP tagged clones were generated for each of the 14 Drosophila and human Integrator subunits. Confocal microscopy studies revealed that the majority of the subunits were diffuse in the nucleus, however, IntS3 formed discrete subnuclear foci. Surprisingly, two of the subunits, IntS2 and 7 were observed in cytoplasmic foci. 2. To further characterize Integrator subunits with unique subcellular localizations. Colocalization studies with endogenous IntS3 and Cajal body marker, coilin, showed that these two proteins overlap, and from this we concluded that IntS3 localized to Cajal bodies. Additionally, colocalization studies with mCherry-tagged IntS2 and 7 and the P body marker, Dcp1, revealed that these proteins colocalize as well. IntS7, however, is more stable in cytoplasmic foci than Dcp1. It was also shown through RNAi knockdown of Integrator subunits, that the cytoplasmic localization of IntS2 and 7 is dependent on the expression of IntS1 and 11 in S2 cells.

Biological, diagnostic and therapeutic relevance of the MET receptor signaling in head and neck cancer.

Head and neck cancer constitutes the 6th most common malignancy worldwide and affects the crucial anatomical structures and physiological functions of the upper aerodigestive tract. Classical therapeutic strategies such as surgery and radiotherapy carry substantial toxicity and functional impairment. Moreover, the loco-regional control rates as well as overall survival still need to be improved in subgroups of patients. The scatter-factor/hepatocyte growth factor receptor tyrosine kinase MET is an established effector in the promotion, maintenance and progression of malignant transformation in a wide range of human malignancies, and has been gaining considerable interest in head and neck cancer over the last 15 years. Aberrant MET activation due to overexpression, mutations, tumor-stroma paracrine loops, and cooperative/redundant signaling has been shown to play prominent roles in epithelial-to-mesenchymal transition, angiogenesis, and responses to anti-cancer therapeutic modalities. Accumulating preclinical and translational evidence highly supports the increasing interest of MET as a biomarker for lymph node and distant metastases, as well as a potential marker of stratification for responses to ionizing radiation. The relevance of MET as a therapeutic molecular target in head and neck cancer described in preclinical studies remains largely under-evaluated in clinical trials, and therefore inconclusive. Also in the context of anti-cancer targeted therapy, a large body of preclinical data suggests a central role for MET in treatment resistance towards multiple therapeutic modalities in malignancies of the head and neck region. These findings, as well as the potential use of combination therapies including MET inhibitors in these tumors, need to be further explored.

Detection of the pan neuronal marker PGP9.5 by immuno-histochemistry and quantitative PCR in eutopic endometrium from women with and without endometriosis.

PURPOSE To assess endometrial gene as well as protein expression of neuroendocrine and supposedly endometriosis-associated product PGP9.5 and pain symptoms in women with endometriosis and controls undergoing laparoscopy, using molecular biological and immuno-histochemical approaches in the same patients. METHODS Biopsy of eutopic endometrium from 29 patients by sharp curettage, and preparation of paraffin blocks. Determination of PGP9.5 gene expression and protein abundance using qPCR and immuno-histochemistry. RESULTS qPCR; The PGP9.5 mRNA expression level between women with (N = 16) and without (N = 13) endometriosis was not different, regardless of pain symptoms or menstrual cycle phase. PGP9.5 expression was higher in women who reported pain compared to those who did not; however, this association was not statistically significant. The expression of PGP9.5 mRNA was higher in women with endometriosis and pain during the proliferative than in the secretory phase (P = 0.03). Furthermore, in the first half of the cycle, the abundance of the PGP9.5 transcript was also significantly higher in endometriosis patients compared to those without (P = 0.03). Immuno-histochemistry; Thirteen of the 16 endometriosis patients showed positive PGP9.5 immuno-reactivity in the endometrium, whereas no such signal was observed in women without endometriosis. The absolute number of nerve fibres per mm(2) in women with endometriosis was similar, regardless of the pain symptoms. CONCLUSIONS PGP9.5 mRNA expression is increased in the proliferative phase of endometriotic women with pain. The presence of nerve fibres was demonstrated by a PGP9.5 protein signal in immuno-histochemistry and restricted to patients with endometriosis. Based on these results, however, there did not appear to be a direct association between the gene expression and protein abundance in women with and without endometriosis or those that experienced pain.