Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1alpha

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1alpha) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1alpha was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.

Proteome and radioimmunoassay analyses of pituitary hormones and proteins in response to feed restriction of dairy cows

The hypothalamic-pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2-DE followed by MALDI-TOF-MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non-esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine-rich element-binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co-transcribed homolog, peroxiredoxin III, beta-tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood.

Virtual histology assessment of cardiac allograft vasculopathy following introduction of everolimus--results of a multicenter trial

In this 12-month multicenter Scandinavian study, 78 maintenance heart transplant (HTx) recipients randomized to everolimus with reduced calcineurin inhibitor (CNI) exposure or continued standard CNI-therapy underwent matched virtual histology (VH) examination to evaluate morphological progression of cardiac allograft vasculopathy (CAV). Parallel measurement of a range of inflammatory markers was also performed. A similar rate of quantitative CAV progression was observed in the everolimus (n = 30) and standard CNI group (n = 48) (plaque index 1.9 ± 3.8% and 1.6 ± 3.9%, respectively; p = 0.65). However, VH analysis revealed a significant increase in calcified (2.4 ± 4.0 vs. 0.3 ± 3.1%; p = 0.02) and necrotic component (6.5 ± 8.5 vs. 1.1 ± 8.6%; p = 0.01) among everolimus patients compared to controls. The increase in necrotic and calcified components was most prominent in everolimus patients with time since HTx >5.1 years and was accompanied by a significant increase in levels of von Willebrand (vWF) factor (p = 0.04) and vascular cell adhesion molecule (VCAM) (p = 0.03). Conversion to everolimus and reduced CNI is associated with a significant increase in calcified and necrotic intimal components and is more prominent in patients with a longer time since HTx. A significant increase in vWF and VCAM accompanied these qualitative changes and the prognostic implication of these findings requires further investigation.

Body contouring surgery following bariatric surgery and dietetically induced massive weight reduction: a risk analysis

BACKGROUND: This study analyzed the impact of weight reduction method, preoperative, and intraoperative variables on the outcome of reconstructive body contouring surgery following massive weight reduction. METHODS: All patients presenting with a maximal BMI >/=35 kg/m(2) before weight reduction who underwent body contouring surgery of the trunk following massive weight loss (excess body mass index loss (EBMIL) >/= 30%) between January 2002 and June 2007 were retrospectively analyzed. Incomplete records or follow-up led to exclusion. Statistical analysis focused on weight reduction method and pre-, intra-, and postoperative risk factors. The outcome was compared to current literature results. RESULTS: A total of 104 patients were included (87 female and 17 male; mean age 47.9 years). Massive weight reduction was achieved through bariatric surgery in 62 patients (59.6%) and dietetically in 42 patients (40.4%). Dietetically achieved excess body mass index loss (EBMIL) was 94.20% and in this cohort higher than surgically induced reduction EBMIL 80.80% (p < 0.01). Bariatric surgery did not present increased risks for complications for the secondary body contouring procedures. The observed complications (26.9%) were analyzed for risk factors. Total tissue resection weight was a significant risk factor (p < 0.05). Preoperative BMI had an impact on infections (p < 0.05). No impact on the postoperative outcome was detected in EBMIL, maximal BMI, smoking, hemoglobin, blood loss, body contouring technique or operation time. Corrective procedures were performed in 11 patients (10.6%). The results were compared to recent data. CONCLUSION: Bariatric surgery does not increase risks for complications in subsequent body contouring procedures when compared to massive dietetic weight reduction.

Unique posttranslational modifications in eukaryotic translation factors and their roles in protozoan parasite viability and pathogenesis.

Protozoan parasites are one of the major causes of diseases worldwide. The vector transmitted parasites exhibit complex life cycles involving interactions between humans, protozoa, and arthropods. In order to adapt themselves to the changing microenvironments, they have to undergo complex morphological and metabolic changes. These changes can be brought about by expressing a new pool of proteins in the cell or by modifying the existing repertoire of proteins via posttranslational modifications (PTMs). PTMs involve covalent modification and processing of proteins thereby modulating their functions. Some of these changes may involve PTMs of parasite proteins to help the parasite survive within the host and the vector. Out of many PTMs known, three are unique since they occur only on single proteins: ethanolamine phosphoglycerol (EPG) glutamate, hypusine and diphthamide. These modifications occur on eukaryotic elongation factor 1A (eEF1A), eukaryotic initiation factor 5A (eIF5A) and eukaryotic elongation factor 2 (eEF2), respectively. Interestingly, the proteins carrying these unique modifications are all involved in the elongation steps of translation. Here we review these unique PTMs, which are well conserved in protozoan parasites, and discuss their roles in viability and pathogenesis of parasites. Characterization of these modifications and studying their roles in physiology as well as pathogenesis will provide new insights in parasite biology, which may also help in developing new therapeutic interventions.

Scp160p is required for translational efficiency of codon-optimized mRNAs in yeast

The budding yeast multi-K homology domain RNA-binding protein Scp160p binds to > 1000 messenger RNAs (mRNAs) and polyribosomes, and its mammalian homolog vigilin binds transfer RNAs (tRNAs) and translation elongation factor EF1alpha. Despite its implication in translation, studies on Scp160p's molecular function are lacking to date. We applied translational profiling approaches and demonstrate that the association of a specific subset of mRNAs with ribosomes or heavy polysomes depends on Scp160p. Interaction of Scp160p with these mRNAs requires the conserved K homology domains 13 and 14. Transfer RNA pairing index analysis of Scp160p target mRNAs indicates a high degree of consecutive use of iso-decoding codons. As shown for one target mRNA encoding the glycoprotein Pry3p, Scp160p depletion results in translational downregulation but increased association with polysomes, suggesting that it is required for efficient translation elongation. Depletion of Scp160p also decreased the relative abundance of ribosome-associated tRNAs whose codons show low potential for autocorrelation on mRNAs. Conversely, tRNAs with highly autocorrelated codons in mRNAs are less impaired. Our data indicate that Scp160p might increase the efficiency of tRNA recharge, or prevent diffusion of discharged tRNAs, both of which were also proposed to be the likely basis for the translational fitness effect of tRNA pairing.

Tyrosine kinase receptor B (TrkB) expression in colorectal cancers highlights anoikis resistance as a survival mechanism of tumour budding cells.

AIMS Tumour buds in colorectal cancer represent an aggressive subgroup of non-proliferating and non-apoptotic tumour cells. We hypothesize that the survival of tumour buds is dependent upon anoikis resistance. The role of tyrosine kinase receptor B (TrkB), a promoter of epithelial-mesenchymal transition and anoikis resistance, in facilitating budding was investigated. METHODS AND RESULTS Tyrosine kinase receptor B immunohistochemistry was performed on a multiple-punch tissue microarray of 211 colorectal cancer resections. Membranous/cytoplasmic and nuclear expression was evaluated in tumour and buds. Tumour budding was assessed on corresponding whole tissue slides. Relationship to Ki-67 and caspase-3 was investigated. Analysis of Kirsten Ras (KRAS), proto-oncogene B-RAF (BRAF) and cytosine-phosphate-guanosine island methylator phenotype (CIMP) was performed. Membranous/cytoplasmic and nuclear TrkB were strongly, inversely correlated (P < 0.0001; r = -0.41). Membranous/cytoplasmic TrkB was overexpressed in buds compared to the main tumour body (P < 0.0001), associated with larger tumours (P = 0.0236), high-grade budding (P = 0.0011) and KRAS mutation (P = 0.0008). Nuclear TrkB was absent in buds (P <0.0001) and in high-grade budding cancers (P =0.0073). Among patients with membranous/cytoplasmic TrkB-positive buds, high tumour membranous/cytoplasmic TrkB expression was a significant, independent adverse prognostic factor [P = 0.033; 1.79, 95% confidence interval (CI) 1.05-3.05]. Inverse correlations between membranous/cytoplasmic TrkB and Ki-67 (r = -0.41; P < 0.0001) and caspase-3 (r =-0.19; P < 0.05) were observed. CONCLUSIONS Membranous/cytoplasmic TrkB may promote an epithelial-mesenchymal transition (EMT)-like phenotype with high-grade budding and maintain viability of buds themselves.

Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions

BACKGROUND Dysregulation of the coagulation system due to inflammatory responses and cross-species molecular incompatibilities represents a major obstacle to successful xenotransplantation. We hypothesized that complement inhibition mediated by transgenic expression of human CD46 in pigs might also regulate the coagulation and fibrinolysis cascades and tested this in ex vivo human-to-pig xenoperfusions. METHODS Forelimbs of wild-type and hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused for 12 hours with whole heparinized human blood. Muscle biopsies were stained for galactose-α1,3-galactose, immunoglobulin M, immunoglobulin G, complement, fibrin, tissue factor, fibrinogen-like protein 2, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI)-1. The PAI-1/tPA complexes, D-dimers, and prothrombin fragment F1 + 2 were measured in plasma samples after ex vivo xenoperfusion. RESULTS No differences of galactose expression or deposition of immunoglobulin M and immunoglobulin G were found in xenoperfused tissues of wild type and transgenic limbs. In contrast, significantly lower deposition of C5b-9 (P < 0.0001), fibrin (P = 0.009), and diminished expression of tissue factor (P = 0.005) and fibrinogen-like protein 2 (P = 0.028) were found in xenoperfused tissues of transgenic limbs. Levels of prothrombin fragment F1 + 2 (P = 0.031) and D-dimers (P = 0.044) were significantly lower in plasma samples obtained from transgenic as compared to wild-type pig limb perfusions. The expression of the fibrinolytic marker tPA was significantly higher (P = 0.009), whereas PAI-1 expression (P = 0.022) and PAI-1/tPA complexes in plasma (P = 0.015) were lower after transgenic xenoperfusion as compared to wild-type xenoperfusions. CONCLUSIONS In this human-to-pig xenoperfusion model, complement inhibition by transgenic hCD46 expression led to a significant inhibition of procoagulant and antifibrinolytic pathways.

Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. (1) In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. (1) In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Evidence for chronic low-grade systemic inflammation in individuals with agoraphobia from a population-based prospective study.

BACKGROUND Anxiety disorders have been linked to an increased risk of incident coronary heart disease in which inflammation plays a key pathogenic role. To date, no studies have looked at the association between proinflammatory markers and agoraphobia. METHODS In a random Swiss population sample of 2890 persons (35-67 years, 53% women), we diagnosed a total of 124 individuals (4.3%) with agoraphobia using a validated semi-structured psychiatric interview. We also assessed socioeconomic status, traditional cardiovascular risk factors (i.e., body mass index, hypertension, blood glucose levels, total cholesterol/high-density lipoprotein-cholesterol ratio), and health behaviors (i.e., smoking, alcohol consumption, and physical activity), and other major psychiatric diseases (other anxiety disorders, major depressive disorder, drug dependence) which were treated as covariates in linear regression models. Circulating levels of inflammatory markers, statistically controlled for the baseline demographic and health-related measures, were determined at a mean follow-up of 5.5 ± 0.4 years (range 4.7 - 8.5). RESULTS Individuals with agoraphobia had significantly higher follow-up levels of C-reactive protein (p = 0.007) and tumor-necrosis-factor-α (p = 0.042) as well as lower levels of the cardioprotective marker adiponectin (p = 0.032) than their non-agoraphobic counterparts. Follow-up levels of interleukin (IL)-1β and IL-6 did not significantly differ between the two groups. CONCLUSIONS Our results suggest an increase in chronic low-grade inflammation in agoraphobia over time. Such a mechanism might link agoraphobia with an increased risk of atherosclerosis and coronary heart disease, and needs to be tested in longitudinal studies.

Pathologic Markers of Prognosis in Ampullary Carcinoma

Ampullary cancer is a rare gastrointestinal malignancy that can be curable with surgical resection of localized disease. The benefit of adjuvant therapy, however, remains unknown in these patients partly because of difficulty in stratifying which patients are at high risk for recurrence. To better identify those patients who may benefit from adjuvant therapy, I conducted a retrospective analysis the pathology reports from 176 patients with surgically resected ampullary cancer who had not received any neoadjuvant therapy, the systemic therapy given, and the patient outcomes. A tissue microarray (TMA) of 95 surgically resected ampullary specimens was also constructed to examine whether there is a correlation between classical immunohistochemical profiles for intestinal and pancreaticobiliary tumors and their histologic classification. In this study, I confirmed the prognostic value of advanced T-stage, nodal metastases, and lymphovascular invasion. Patients whose tumors had “high risk” features had a significantly worse overall survival (p=.002). Furthermore, my research highlighted the importance of histology and its impact on survival, with pancreaticobiliary-like features being a negative prognostic factor (p=0.001). Importantly, patients whose tumors have pancreaticobiliary histology appear to benefit from adjuvant therapy, further implicating histology as an important pathologic marker (p=0.053). In addition, the TMA confirmed a correlation between classical immunohistochemical profiles for intestinal and pancreaticobiliary tumors and histologic classification. My research findings suggest that histology subtypes, T-stage, nodal metastases, and lymphovascular invasion should all be taken into consideration when determining which patients with ampullary cancer may benefit from further adjuvant therapy.

Risk factors for inflammatory breast cancer

Background: Inflammatory breast cancer (IBC) is rare and accounts for 2.5% of all invasive breast cancers. The 5-year survival rates are significantly lower than for other types of breast cancer, highlighting the significance of cancer prevention in IBC. The comprehensive multi-disciplinary team Morgan Welch Inflammatory Breast Cancer Research Program and Clinic at University of Texas MD Anderson Cancer Center treats the largest number of Inflammatory Breast patients in a single center. Because of this unique center, large patient resources, and good medical and epidemiological records, we were able to conduct the largest single center case-control and case-case study on IBC. Methods: We identified 246 patients diagnosed with IBC and 397 cancer free patients seen at the Dan L Duncan Cancer Prevention Clinic. Breast cancer reproductive risk factors and lifestyle risk factors were compared between tumor subtypes of IBC patients (Estrogen Receptor positive (ER+) and/or Progesterone Receptor positive (PR+), Human Epidermal Growth Factor 2 positive (HER2+)), and (ER -/PR-/HER2-)) and cancer free controls. Results: Breastfeeding was the only significant risk factor (p<0.01) between tumor subtypes in IBC patients. In the case-control study that included all IBC patients and cancer free patients the descriptive statistics indicate significant difference in BMI, history of smoking, number of children, age of first pregnancy, any breastfeeding and total time breastfeeding (p<0.05). No differences were found in the frequency of other breast cancer risk factors. Conclusion: The associations determined between cancer free controls and IBC patients have identified previously unknown risk factors for IBC. The risk factors identified by the case control study suggest BMI, history of smoking, and the protective effect of breastfeeding as potential modifiable risk factors that can be used to decrease the incidence of IBC. Impact: These results highlight the importance of evaluating epidemiologic risk factors of IBC, which could lead to the identification of distinct etiologic pathways that could be targeted for prevention.^