Synthetic peptides corresponding to a repetitive sequence of malarial histidine rich protein bind haem and inhibit haemozoin formation in vitro

Synthetic peptides containing a repetitive hexapeptide sequence (Ala-His-His-Ala-Ala-Asp) of malarial histidine-rich protein II were evaluated for binding with haem in vitro. The pattern of haem binding suggested that each repeat unit of this sequence provides one binding site for haem. Chloroquine inhibited the haem-peptide complex formation with preferential formation of a haem chloroquine complex. In vitro studies on haem polymerisation showed that none of the peptides could initiate haemozoin formation. However, they could inhibit haemozoin formation promoted by a malarial parasite extract, possibly by competitively binding free haem. These results indicate this hexapeptide sequence represents the haem binding site of the malarial histidine-rich protein and possibly the site of nucleation for haem polymerisation.

The ins and outs of phosphatidylethanolamine synthesis in Trypanosoma brucei.

Phospholipids are not only major building blocks of biological membranes but fulfill a wide range of critical functions that are often widely unrecognized. In this review, we focus on phosphatidylethanolamine, a major glycerophospholipid class in eukaryotes and bacteria, which is involved in many unexpected biological processes. We describe (i) the ins, i.e. the substrate sources and biochemical reactions involved in phosphatidylethanolamine synthesis, and (ii) the outs, i.e. the different roles of phosphatidylethanolamine and its involvement in various cellular events. We discuss how the protozoan parasite, Trypanosoma brucei, has contributed and may contribute in the future as eukaryotic model organism to our understanding of phosphatidylethanolamine homeostasis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

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.

Molecular genetics and functional anomalies in a series of 248 Brugada cases with 11 mutations in the TRPM4 channel.

Brugada syndrome (BrS) is a condition defined by ST-segment alteration in right precordial leads and a risk of sudden death. Because BrS is often associated with right bundle branch block and the TRPM4 gene is involved in conduction blocks, we screened TRPM4 for anomalies in BrS cases. The DNA of 248 BrS cases with no SCN5A mutations were screened for TRPM4 mutations. Among this cohort, 20 patients had 11 TRPM4 mutations. Two mutations were previously associated with cardiac conduction blocks and 9 were new mutations (5 absent from ~14'000 control alleles and 4 statistically more prevalent in this BrS cohort than in control alleles). In addition to Brugada, three patients had a bifascicular block and 2 had a complete right bundle branch block. Functional and biochemical studies of 4 selected mutants revealed that these mutations resulted in either a decreased expression (p.Pro779Arg and p.Lys914X) or an increased expression (p.Thr873Ile and p.Leu1075Pro) of TRPM4 channel. TRPM4 mutations account for about 6% of BrS. Consequences of these mutations are diverse on channel electrophysiological and cellular expression. Because of its effect on the resting membrane potential, reduction or increase of TRPM4 channel function may both reduce the availability of sodium channel and thus lead to BrS.

Pulmonary Disease due to Mycobacterium tuberculosis in a Horse: Zoonotic Concerns and Limitations of Antemortem Testing.

A case of pulmonary tuberculosis caused by Mycobacterium tuberculosis was diagnosed in a horse. Clinical evaluation performed prior to euthanasia did not suggest tuberculosis, but postmortem examination provided pathological and bacteriological evidence of mycobacteriosis. In the lungs, multiple tuberculoid granulomas communicating with the bronchiolar lumen, pleural effusion, and a granulomatous lymphadenitis involving mediastinal and tracheobronchial lymph nodes were found. Serologic response to M. tuberculosis antigens was detected in the infected horse, but not in the group of 42 potentially exposed animals (18 horses, 14 alpacas, 6 donkeys, and 4 dogs) which showed no signs of disease. Diagnosis of tuberculosis in live horses remains extremely difficult. Four of 20 animal handlers at the farm were positive for tuberculous infection upon follow-up testing by interferon-gamma release assay, indicating a possibility of interspecies transmission of M. tuberculosis.

Oxygen regulates molecular mechanisms of cancer progression and metastasis

Oxygen is the basic molecule which supports life and it truly is “god's gift to life.” Despite its immense importance, research on “oxygen biology” has never received the light of the day and has been limited to physiological and biochemical studies. It seems that in modern day biology, oxygen research is summarized in one word “hypoxia.” Scientists have focused on hypoxia-induced transcriptomics and molecular–cellular alterations exclusively in disease models. Interestingly, the potential of oxygen to control the basic principles of biology like homeostatic maintenance, transcription, replication, and protein folding among many others, at the molecular level, has been completely ignored. Here, we present a perspective on the crucial role played by oxygen in regulation of basic biological phenomena. Our conclusion highlights the importance of establishing novel research areas like oxygen biology, as there is great potential in this field for basic science discoveries and clinical benefits to the society.

Reduced bone breakage and increased bone strength in free range laying hens fed omega-3 polyunsaturated fatty acid supplemented diets

INTRODUCTION The omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are the immediate precursors to a number of important mediators of immunity, inflammation and bone function, with products of omega-6 generally thought to promote inflammation and favour bone resorption. Western diets generally provide a 10 to 20-fold deficit in omega-3 PUFAs compared with omega-6, and this is thought to have contributed to the marked rise in incidence of disorders of modern human societies, such as heart disease, colitis and perhaps osteoporosis. Many of our food production animals, fed on grains rich in omega-6, are also exposed to a dietary deficit in omega-3, with perhaps similar health consequences. Bone fragility due to osteoporotic changes in laying hens is a major economic and welfare problem, with our recent estimates of breakage rates indicating up to 95% of free range hens suffer breaks during lay. METHODS Free range hens housed in full scale commercial systems were provided diets supplemented with omega-3 alpha linolenic acid, and the skeletal benefits were investigated by comparison to standard diets rich in omega-6. RESULTS There was a significant 40-60% reduction in keel bone breakage rate, and a corresponding reduction in breakage severity in the omega-3 supplemented hens. There was significantly greater bone density and bone mineral content, alongside increases in total bone and trabecular volumes. The mechanical properties of the omega-3 supplemented hens were improved, with strength, energy to break and stiffness demonstrating significant increases. Alkaline phosphatase (an osteoblast marker) and tartrate-resistant acid phosphatase (an osteoclast marker) both showed significant increases with the omega-3 diets, indicating enhanced bone turnover. This was corroborated by the significantly lower levels of the mature collagen crosslinks, hydroxylysyl pyridinoline, lysyl pyridinoline and histidinohydroxy-lysinonorleucine, with a corresponding significant shift in the mature:immature crosslink ratio. CONCLUSIONS The improved skeletal health in laying hens corresponds to as many as 68million fewer hens suffering keel fractures in the EU each year. The biomechanical and biochemical evidence suggests that increased bone turnover has enhanced the bone mechanical properties, and that this may suggest potential benefits for human osteoporosis.

Radiometrical, hormonal and biological correlates of skeletal growth in the female rat from birth to senescence

OBJECTIVE We investigated the skeletal growth profile of female rats from birth to senescence (100weeks) on the basis of sequential radiometrical, hormonal and biochemical parameters. DESIGN Weaning rats entered the study which was divided into two sections: a) sequential measurements of vertebral and tibial growths and bone mineral density (BMD), estimation of mineral content of the entire skeleton (BMC) and chemical analysis of vertebral Ca; and b) determination of basal and pulsatile growth hormone (rGH), insulin-like growth hormone (IGF-I), estradiol (E2), parathyroid hormone (PTH), osteocalcin (OC) and urinary d-pyridinoline (dp) throughout the experimental period. RESULTS Vertebral and tibial growths ceased at week 25 whereas BMD and BMC as well as total vertebral Ca exhibited a peak bone mass at week 40. rGH pulsatile profiles were significantly higher in younger animals coinciding with the period of active growth and IGF-I peaked at 7weeks, slowly declining thereafter and stabilizing after week 60. OC and dp closely paralleled IGF-I coinciding with the period of enhanced skeletal growth, remaining thereafter in the low range indicative of reduced bone turnover. E2 increased during reproductive life but the lower values subsequently recorded were still in the physiological range, strongly suggesting a protective role of this steroid on bone remodeling. PTH followed a similar profile to E2, but the significance of this after completion of growth remains unclear. CONCLUSIONS Mechanisms governing skeletal growth in the female rat appear similar to those in humans. Bone progression and attainment of peak bone mass are under simultaneous control of rGH, IGF-I and calciotropic hormones and are modulated by E2. This steroid seems to protect the skeleton from resorption before senescence whereas the role of PTH in this context remains uncertain.

Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aβ42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

A genomic perspective on a new bacterial genus and species from the Alcaligenaceae family, Basilea psittacipulmonis

BACKGROUND A novel Gram-negative, non-haemolytic, non-motile, rod-shaped bacterium was discovered in the lungs of a dead parakeet (Melopsittacus undulatus) that was kept in captivity in a petshop in Basel, Switzerland. The organism is described with a chemotaxonomic profile and the nearly complete genome sequence obtained through the assembly of short sequence reads. RESULTS Genome sequence analysis and characterization of respiratory quinones, fatty acids, polar lipids, and biochemical phenotype is presented here. Comparison of gene sequences revealed that the most similar species is Pelistega europaea, with BLAST identities of only 93% to the 16S rDNA gene, 76% identity to the rpoB gene, and a similar GC content (~43%) as the organism isolated from the parakeet, DSM 24701 (40%). The closest full genome sequences are those of Bordetella spp. and Taylorella spp. High-throughput sequencing reads from the Illumina-Solexa platform were assembled with the Edena de novo assembler to form 195 contigs comprising the ~2 Mb genome. Genome annotation with RAST, construction of phylogenetic trees with the 16S rDNA (rrs) gene sequence and the rpoB gene, and phylogenetic placement using other highly conserved marker genes with ML Tree all suggest that the bacterial species belongs to the Alcaligenaceae family. Analysis of samples from cages with healthy parakeets suggested that the newly discovered bacterial species is not widespread in parakeet living quarters. CONCLUSIONS Classification of this organism in the current taxonomy system requires the formation of a new genus and species. We designate the new genus Basilea and the new species psittacipulmonis. The type strain of Basilea psittacipulmonis is DSM 24701 (= CIP 110308 T, 16S rDNA gene sequence Genbank accession number JX412111 and GI 406042063).

Investigation of IL-23 (p19, p40) and IL-23R identifies nuclear expression of IL-23 p19 as a favorable prognostic factor in colorectal cancer: a retrospective multicenter study of 675 patients.

IL-23 is a heterodimeric cytokine involved in inflammatory diseases; its role in cancer progression is controversial. Here we analyse the expression of IL-23 subunits (p40 and p19) and IL-23R in colorectal cancer with regard to disease progression, clinical-pathological and molecular aspects. Immunohistochemistry for IL-23p19, IL-23p40, IL-23R and CD8 was performed on a multi-punch tissue microarray of 195 colorectal cancers (cohort 1), matched normal tissue, adenoma and lymph node metastases. Results were compared with clinical-pathological features and CD8+ T-cell counts, then validated on two patient cohorts (cohort 2: n=341, cohort 3: n=139). Cytoplasmic/membranous expression of IL-23 (p19 and p40 subunits) and IL-23R, respectively were over-expressed in carcinomas versus adenomas and normal tissues (p<0.0001) but were reduced in lymph node metastases (p<0.0001). Nuclear IL-23p19 expression was observed in 23.1% and was associated with early TNM stage (p=0.0186), absence of venous (p=0.0124) and lymphatic invasion (p=0.01493), favorable survival (p=0.014) and absence of distant metastasis (p=0.0146; specificity: 100%). This unexpected cellular localization was confirmed by cell fractionation. The beneficial effect of nuclear IL-23p19 was restricted to tumours with CD8+ high counts. Results were validated on Cohorts 2/3. This multicenter study underlines the possible CD8(+)--dependency and beneficial effect of nuclear IL-23p19 on overall patient survival.

Prevalence and prognostic significance of TMPRSS2-ERG gene fusion in lymph node positive prostate cancers.

BACKGROUND TMPRSS2-ERG gene fusion is the most frequent genetic alteration in prostate cancer. However, information about its distribution in lymph node positive prostate cancers and the prognostic significance in these advanced tumors is unknown. METHODS Gene fusion status was determined by fluorescence in situ hybridization on a tissue-microarray constructed from 119 hormone-naïve nodal positive, surgically treated prostate cancers containing samples from the primary tumors and corresponding lymph node metastases. Data were correlated with various tumor features (Gleason score, stage, cancer volume, nodal tumor burden) and biochemical recurrence-free, disease-specific, and overall survival. RESULTS TMPRSS2-ERG fusion was detected in 43.5% of the primary tumors. Conversely, only 29.9% of the metastasizing components showed the fusion. Concordance in TMPRSS2-ERG status between primary tumors and metastases was 70.9% (Kappa 0.39); 20.9% and 8.1% of the patients showed the mutation solely in their primary tumors and metastases, respectively. TMPRSS2-ERG fusion was not correlated with specific histopathological tumor features but predicted favorable biochemical recurrence-free, disease-specific and overall survival independently when present in the primary tumor (P < 0.05 each). CONCLUSION TMPRSS2-ERG fusion is more frequent in primary prostate cancer than in corresponding metastases suggesting no selection of fusion-positive cells in the metastatic process. The gene fusion in primary tumors independently predicts favorable outcome.

ISVI-IUA consensus document - diagnostic guidelines on vascular anomalies: vascular malformations and hemangiomas.

The diagnostic approach to vascular anomalies should include the distinction between vascular tumors (i.e. hemangiomas) and congential vascular malformations (CVMs). This step is based more on history and clinical examination rather than on instrumental evaluation. In children Duplex ultrasound and histology can be helpful to separate hypervasularized tumors from CVMs. Appropriate record of objective measures as size or flow volume is required in order to evaluate the progress of the pathology and/or to assess the results of adopted therapeutic interventions. The anatomic, pathological and hemodynamic characteristics, the secondary effects on the surrounding tissues and the systemic manifestations should be defined. Basic diagnostic tools are Duplex sonography followed by MRI or CT scanning. The definition of the vascular anomaly should be according to the Hamburg classification and should separate vascular tumors from vacular malformations followed by separation of high flow from low flow CVMs. Diagnostic investigations are best undertaken at centers where subsequent therapeutic interventions will be performed.

Characterization of small ruminant lentivirus A4 subtype isolates and assessment of their pathogenic potential in naturally infected goats.

BACKGROUND Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates. METHODS Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test. RESULTS The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed. CONCLUSIONS Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.

The teleostean liver as an immunological organ: Intrahepatic immune cells (IHICs) in healthy and benzo[a]pyrene challenged rainbow trout (Oncorhynchus mykiss).

The existence of a resident population of intrahepatic immune cells (IHICs) is well documented for mammalian vertebrates, however, it is uncertain whether IHICs are present in the liver of teleostean fish. In the present study we investigated whether trout liver contains an IHIC population, and if so, what the relative cellular composition of this population is. The results provide clear evidence for the existence of an IHIC population in trout liver, which constitutes 15-29% of the non-hepatocytes in the liver, and with a cellular composition different to that of the blood leukocyte population. We also analyzed the response of IHICs to a non-infectious liver challenge with the hepatotoxic and immunotoxic chemical, benzo[a]pyrene (BaP). Juvenile trout were treated with BaP (25 or 100mg/kgbw) at levels sufficient to induce the molecular pathway of BaP metabolism while not causing pathological and inflammatory liver changes. The IHIC population responded to the BaP treatments in a way that differed from the responses of the leukocyte populations in trout blood and spleen, suggesting that IHICs are an independently regulated immune cell population.