Digital image analysis of AgNORs in cervical smears of women with premalignant and malignant lesions of the uterine cervix

We performed a hospital-based, unmatched case-control study to investigate the association between progressive stages of cervical neoplasia and digital analysis of cell proliferation by silver stained nucleolus organizer region associated proteins (AgNORs). We measured cell proliferation levels in the cervical epithelial cells of 10 women with low grade squamous intraepithelial lesions (LG-SIL), eight with high grade squamous intraepithelial lesions (HG-SIL), 11 with cervical cancer (CC) and eight with no cervical lesions (controls) using the AgNORs technique. Cell proliferation was measured by digital image analysis (DIA). DIA revealed increased total areas of AgNORs in HG-SIL and CC compared to LG-SIL and control patients. AgNORs with a kidney or cluster shape exhibited greater areas than those with a spherical or long shape. We propose a cut-off of 118 pixels to differentiate benign (control and LG-SIL) from malignant (HG-SIL and CC) lesions. DIA of AgNORs is a simple and inexpensive method for studying proliferation. The increased total area of AgNORs in malignant lesions provides information regarding cell behavior and may be related to cervical carcinogenesis; however, further validation studies are required to establish its usefulness in cytological analysis.     

Regional differences in WT-1 and Tcf21 expression during ventricular development: implications for myocardial compaction

Background

Morphological and functional differences of the right and left ventricle are apparent in the adult human heart. A differential contribution of cardiac fibroblasts and smooth muscle cells (populations of epicardium-derived cells) to each ventricle may account for part of the morphological-functional disparity. Here we studied the relation between epicardial derivatives and the development of compact ventricular myocardium.

Results

Wildtype and Wt1^CreERT2/+ reporter mice were used to study WT-1 expressing cells, and Tcf21^lacZ/+ reporter mice and PDGFRα^-/-;Tcf21^LacZ/+ mice to study the formation of the cardiac fibroblast population. After covering the heart, intramyocardial WT-1+ cells were first observed at the inner curvature, the right ventricular postero-lateral wall and left ventricular apical wall. Later, WT-1+ cells were present in the walls of both ventricles, but significantly more pronounced in the left ventricle. Tcf21-^LacZ + cells followed the same distribution pattern as WT-1+ cells but at later stages, indicating a timing difference between these cell populations. Within the right ventricle, WT-1+ and Tcf21-lacZ+ cell distribution was more pronounced in the posterior inlet part. A gradual increase in myocardial wall thickness was observed early in the left ventricle and at later stages in the right ventricle. PDGFRα^-/-;Tcf21^LacZ/+ mice showed deficient epicardium, diminished number of Tcf21-^LacZ + cells and reduced ventricular compaction.

Conclusions

During normal heart development, spatio-temporal differences in contribution of WT-1 and Tcf21-^LacZ + cells to right versus left ventricular myocardium occur parallel to myocardial thickening. These findings may relate to lateralized differences in ventricular (patho)morphology in humans.     

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

Biomarker responses and accumulation of hazardous substances in mussels (Mytilus trossulus) transplanted along a pollution gradient close to an oil terminal in the Gulf of Finland (Baltic Sea)

Baltic Sea blue mussels (Mytilus trossulus) were used as sentinel organisms to detect the biological effects of chemical contamination in the low salinity environment. Mussels naturally adapted to a salinity of ca. 6.0 PSU were caged for 30 days at four sites along an assumed pollution gradient (salinity ca. 4.5 PSU) in the vicinity of Finland's largest oil refinery and harbor Kilpilahti in the Gulf of Finland. Tissue concentrations and accumulation rates of especially organic contaminants (PAHs, PCBs and organotins) were clearly elevated at the innermost coastal stations near the harbor area. Biological effects of contaminant exposure on caged mussels were evaluated by measuring a suite of biomarkers including catalase, glutathione S-transferase, superoxide dismutase, glutathione reductase, lipid peroxidation, acetylcholinesterase activity and lysosomal membrane stability. Mussels transplanted near the harbor area were able to elevate their antioxidant defense in response to environmental contamination. Reduced morphometric condition index and soft tissue growth rate together with increased lipid peroxidation and low lysosomal membrane stability were also observed at the most contaminated site. The results suggest that caging of M. trossulus for four weeks at lower salinity is a feasible method for the detection of environmental pollution also in low salinity areas of the Baltic Sea.     

Production of highly 13C-labeled polyphenols in Vitis vinifera cell bioreactor cultures

The use of Vitis vinifera cells grown in a 2 l-stirred tank bioreactor for producing isotopically 13C-labeled phenolic substances is presented. Several culture parameters were optimized to achieve characteristics of growth and polyphenol metabolism similar to that recorded in shake flasks. Administration of [1-13C]L-phenylalanine (3 mM) to grape cell suspension cultures led to the production of 13C-labeled stilbenes (trans- and cis-piceids), catechins (catechin and epicatechin) and anthocyanins (delphinidin-, cyanidin-, petunidin-, peonidin- and malvidin-3-O-beta-glucosides). Incorporation of [1-13C]L-phenylalanine into polyphenols was measured by means of 13C satellites in the proton NMR spectrum and EA-IRMS. The enrichment of labeling obtained for all the compounds (between 40 and 65%) is sufficient to investigate their absorption and metabolism in humans.     

<Emphasis Type=Italic>Pseudomonas aeruginosa</Emphasis> inhibits <Emphasis Type=Italic>in-vitro Candida</Emphasis> biofilm development

Background  

Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.     

Temporal Variation in Seed Predation by Insects in a Population of Syagrus romanzoffiana (Arecaceae) in Santa Catarina Island, SC, Brazil

Insect seed predation may vary depending on seed production. The present study considers the hypothesis that the rates of seed predation tend to be smaller in years of higher fruit production. Thus, we monitored the production of fruits and predation of seeds of the palm Syagrus romanzoffiana over 2?years in the Atlantic Forest (Parque Municipal da Lagoa do Peri, Florianópolis, SC, Brazil), between July 2006 and June 2008. Plots of 0.25?m² were fitted under 20 mother plants and fruits were monthly collected for assessment of abundance and seed predation. There was variation in fruit production between the 2?years and among reproductive plants. Predation rates were high and occurred in the predispersal phase by the Curculionidae Revena rubiginosa Boheman, Anchylorhynchus aegrotus Fahraeus, and Anchylorhynchus variabilis Gyllenhal. Seed predation by these species of Anchylorhynchus is first registered in the present study. In average, about 60% of the seeds monthly produced in the population tend to escape insect predation in year of high or low production, becoming available for recruitment. The predation rate was not related to the amount of fruits produced per reproductive plant. Also, different than expected, there was a positive relation between the rates of seed predation and the total of fruits produced monthly on the plots. Thus, no evidence for the satiation of insect seed predators was found in this study with S. romanzoffiana.     

Immunological evaluation of urinary trypsin inhibitors in blood and urine: Role of N- & O-linked glycoproteins

Urinary trypsin inhibitors (uTi) suppress serine proteases during inflammation. After liberation from proinhibitors (P-alpha-I and I-alpha-I) by the white blood cell (WBC) response, uTi readily pass through the kidneys into urine. A key uTi, bikunin, is attached to O-linked and N-linked glycoconjugates. Recently, uTi inhibitors, called uristatins, were found to lack the O-linked glycoconjugates. Monoclonal antibodies were produced using purified uristatin and screened for binding differences to uristatin, bikunin, P-α-I, and I-α-I. Antibody-binding patterns were characterized using immunoaffinity binding onto protein-chip surfaces and analysis by Surface Enhanced Laser Desorption/Ionization mass spectrometry (SELDI), using specimens from patients and from purified uTi standards. Antibodies were developed and used in an enzyme-linked immunosorbent assay (ELISA) method for uTi measurement in urine and plasma specimens. ELISA was performed on specimens from normal, presumed healthy, controls and from patients who had been screened for inflammation using a high sensitivity C-reactive protein (CRP) test and a complete blood count (CBC). Polyclonal antibody against uTi showed cross-reactivity with the Tamm–Horsfall protein (THP) and with proinhibitors. Screening of anti-uTi monoclonal antibodies (Mab) revealed antibodies that did not cross-react with either of the above, thus providing a tool to measure both uristatin and bikunin in urine with Mab 3G5 and in plasma with Mab 5D11. The monoclonal antibody 5D11 cross-reacts with specific N-linked glycoconjugates of uristatin present in plasma. In ca 96% of healthy adults, uTi were present at <12 mg/l in urine and <4 mg/l in plasma. We also found that patients with an inflammation and a CRP of >2.0 mg/l had higher urinary concentrations of uTi than the control population in every subject. Free uristatin and bikunin pass readily into urine and are primarily bound to heavy chains that constitute the proinhibitor form in plasma.