The chaperone micronemal protein Hsp70-1 from Plasmodium berghei ookinetes is shed during gliding on solid surface sustrata.

Plasmodium the causative agent of malaria is a member of the phylum Apicomplexa, where all invasive forms have a substrate-dependent motility called gliding, key to malaria transmission. Gliding allows parasite host-cell recognition, binding, cell entry and trespassing the cytoplasm. In this process Plasmodium releases molecules from micronemes and the cell surface that are deposited on trails left behind on the substratum as the parasite progresses. Previously we identified the heat shock protein 70-1 (HSP 70-1) on the surface and micronemes of P. berghei ookinetes, the parasite form that invades the mosquito midgut. To investigate if this protein is shed of from the parasite during invasion, we searched HSP 70-1 in gliding trails deposited on a solid surface by P. berghei ookinetes.

An extended core nanocoax pillar architecture for enhanced molecular detection.

Biosensors that incorporate nanomaterials and nanofabrication techniques enable molecular detection of chemical and biological macromolecules with a high degree of specificity and ultrasensitivity. Here, we present a novel fabrication process that yields a nanostructure capable of detecting biological macromolecules. The extended core nanocoax (ECC) structure builds on a previously reported nanocoaxial-based sensor. The fabrication of the device incorporates an extended inner pillar, with controllable extension above the annulus and into the surrounding solution. This new design eliminates structural constraints inherent in the original nanocoax architecture. We also provide results demonstrating improvement in biosensing capability. Specifically, we show the capability of the new architecture to detect the B subunit of the Vibrio cholerae toxin at improved sensitivity (100 pg/ml) in comparison to optical enzyme-linked immunosorbant assay (1 ng/ml) and previously reported coaxial nanostructures (2 ng/ml).

Unilateral absence of mandibular condyle in a Bronze Age male skeleton from Portugal.

In 2009, a pit burial dated to the Bronze Age was excavated in Monte do Gato de Cima 3 (Portugal). The purpose of this paper is to describe the pathological absence of the left mandibular condyle noted in an adult male skeleton and to discuss possible diagnoses, including subcondylar fracture, cystic defect, congenital absence, condylar aplasia and mandibular condylysis. The most likely explanation for the pathological alteration is subcondylar fracture with non-union. Although the occurrence of non-union and slight osteoarthritic alterations in the left glenoid fossa were evident, this mandible was likely functional, as can be inferred from dental wear and muscle attachment sites. This trauma probably occurred before adult age when remodelling capacity is still high. Thus, bones and muscles adequately compensated for the trauma and only minor asymmetry developed. Consequently, this injury seems not to have greatly influenced masticatory functions. This is in accordance with clinical data, which demonstrate that, in growing patients, conservative treatment (non-surgical) results in good remodelling and patient recovery. In addition, in the few paleopathological cases published, the healing capacity of these types of mandibular fractures seems to be good, as can be inferred by evidence from the bone.

Predicting soil water content at - 33 kPa by pedotransfer functions in stoniness 1 soils in northeast Venezuela.

Soil water content is a key property in the study of water available for plants, infiltration, drainage, hydraulic conductivity, irrigation, plant water stress and solute movement. However, its measurement consumes time and, in the case of stony soils, the presence of stones difficult to determinate the water content. An alternative is the use of pedotransfer functions (PTFs), as models to predict these properties from readily available data. The present work shows a comparison of different widely used PTFs to estimate water content at-33 kPa (WR-33kPa) in high stoniness soils. The work was carried out in the Caramacate River, an area of high interest because the frequent landslides worsen the quality of drinking water. The performance of all evaluated PTFs was compared with a PTF generated for the study area. Results showed that the Urach's PTF presented the best performance in relation to the others and could be used to estimate WR-33kPa in soils of Caramacate River basin. The calculated PTFs had a R2 of 0.65. This was slightly higher than the R2 of the Urach's PTF. The inclusion of the rock fragment volume could have the better results. The weak performance of the other PTFs could be related to the fact that the mountain soils of the basin are rich in 2:1 clay and high stoniness, which were not used as independent variables for PTFs to estimate the WR-33kPa.

Integrating genomic alterations in diffuse large B-cell lymphoma identifies new relevant pathways and potential therapeutic targets.

Genome studies of diffuse large B-cell lymphoma (DLBCL) have revealed a large number of somatic mutations and structural alterations. However, the clinical significance of these alterations is still not well defined. In this study, we have integrated the analysis of targeted next-generation sequencing of 106 genes and genomic copy number alterations (CNA) in 150 DLBCL. The clinically significant findings were validated in an independent cohort of 111 patients. Germinal center B-cell and activated B-cell DLBCL had a differential profile of mutations, altered pathogenic pathways and CNA. Mutations in genes of the NOTCH pathway and tumor suppressor genes (TP53/CDKN2A), but not individual genes, conferred an unfavorable prognosis, confirmed in the independent validation cohort. A gene expression profiling analysis showed that tumors with NOTCH pathway mutations had a significant modulation of downstream target genes, emphasizing the relevance of this pathway in DLBCL. An in silico drug discovery analysis recognized 69 (46%) cases carrying at least one genomic alteration considered a potential target of drug response according to early clinical trials or preclinical assays in DLBCL or other lymphomas. In conclusion, this study identifies relevant pathways and mutated genes in DLBCL and recognizes potential targets for new intervention strategies.

Collective secondary cremation in a pit grave: a unique funerary context in Portuguese Chalcolithic burial practices.

Perdigões is a large site with a set of ditched enclosures located at Reguengos de Monsaraz, Alentejo, South Portugal. Recently at the central area of this site burnt human remains were found in a pit (#16). This structure had inside human remains, animal bones (namely pig, sheep or goat, cattle, dog, deer and rabbit), shards, ivory idols and arrowheads. All have been subjected to fire and later deposited in that pit, resulting in a secondary disposal of human bones. The recovered fragmented human bones (4845.18 g) correspond to a minimal number of 9 individuals: 6 adults and 3 sub-adults. The aim of this work is to document and interpret this funerary context based on the study of the recovered human remains. For that purpose, observations of all alterations due to fire, such as colour change and type of bone distortion, as well as anthropological data were collected. The data obtained suggest that these human remains were probably intentionally cremated, carefully collected and finally deposited in this pit. The cremation was conducted on probably complete corpses, some of them still fairly fresh and fleshed, as some bones presented thumbnail fractures. The collective cremation of the pit 16 represents an unprecedented funerary context for Portuguese, and Iberian Peninsula, Chalcolithic burial practices. Moreover, it is an example of the increasing diversity of mortuary practices of Chalcolithic human populations described in present Portuguese territory, as well as, in the Iberian Peninsula.

Systemic infection induced by intranasal inoculation of Bovine herpesvirus 1.1 in pregnant and non-pregnant rabbits.

Bovine herpesvirus (BoHV) type 1.1 (BoHV-1.1) causes repeated outbreaks of upper respiratory disease and abortion in cattle. The systemic effects of BoHV-1.1 in rabbits, using intranasal inoculation are reported. Female rabbits were divided into four groups and inoculated with the virus 10 days before mating, and at 15 or 22 days of pregnancy. Studies of the clinical signs, antibody production, virus isolation, and DNA detection as well as histological and immunohistochemical studies were carried out on lungs, kidneys, spleen, placentas, uteri and foetal tissues. All virus-inoculated animals developed respiratory clinical signs and a humoral response. BoHV-1.1 was isolated from nasal swabs and plasma rich in leukocytes, and viral DNA was detected in blood, dead foetuses and placentas. Histopathological lesions were found in the respiratory tract and some placentas and foetuses were immunohistochemically positive. Intranasal inoculation might be useful to study the systemic effects of BoHV-1.1 infection in the rabbit model.

Conserved peptide sequences bind to actin and enolase on the surface of Plasmodium berghei ookinetes.

The description of Plasmodium ookinete surface proteins and their participation in the complex process of mosquito midgut invasion is still incomplete. In this study, using phage display, a consensus peptide sequence (PWWP) was identified in phages that bound to the Plasmodium berghei ookinete surface and, in selected phages, bound to actin and enolase in overlay assays with ookinete protein extracts. Actin was localized on the surface of fresh live ookinetes by immunofluorescence and electron microscopy using specific antibodies. The overall results indicated that enolase and actin can be located on the surface of ookinetes, and suggest that they could participate in Plasmodium invasion of the mosquito midgut.

Activation of the EGFR/ERK pathway in high-grade mucoepidermoid carcinomas of the salivary glands.

BACKGROUND: Mucoepidermoid carcinoma (MEC) shows differences in biological behaviour depending mainly on its histological grade. High-grade tumours usually have an aggressive biological course and they require additional oncological treatment after surgery. METHODS: In a series of 43 MECs of the salivary glands, we studied the epidermal growth factor receptor (EGFR) gene by using dual-colour chromogenic in situ hybridisation (CISH). Moreover, we assessed the protein expressions of the EGFR and the activated extracellular signal-regulated kinases (pERK1/2) by using immunohistochemistry. These results were correlated with the histological grade of the tumours and the outcome of the patients. RESULTS: The CISH study demonstrated a high-EGFR gene copy number, with balanced chromosome 7 polysomy, in 8 out of 11 high-grade MECs (72.7%), whereas 27 low-grade and 15 intermediate-grade tumours had a normal EGFR gene copy number (P<0.001). The EGFR gene gains correlated with disease-free interval (P=0.003) and overall survival of the patients (P=0.019). The EGFR protein expression had a significant correlation with the histological grade of the tumours but not with the outcome of the patients. The pERK1/2 expression correlated with histological grade of tumours (P<0.001), disease-free interval (P=0.004) and overall survival (P=0.001). CONCLUSIONS: The EGFR/ERK pathway is activated in high-grade MECs with aggressive behaviour. Patients with these tumours who require oncological treatment in addition to surgery could benefit from EGFR and mitogen-activated protein kinase pathway inhibitors.

A simple method of infecting rabbits with Bovine herpesvirus 1 and 5.

This report describes an alternative technique to inoculate rabbits and to reproduce infection by Bovine herpesvirus 1 and 5. First, the nostrils are anaesthetized by aspersion with local anaesthetic. A few seconds later, and after proving the insensitivity of the zone, the rabbits are put on their back legs with their nostrils upwards and the inoculum is introduced slowly into each nostril by using disposable droppers. Clinical signs, viral isolation from nasal swabs, histological lesions found, positive polymerase chain reaction and antibodies production confirm the infection. This very simple and bloodless technique, where the animals are exposed to minor distress, may be useful for evaluating the virulence of BoHV-1 and BoHV-5 strains, to study the establishment of latent virus infection and to test the potential of experimental vaccines or properties of antiviral drugs. It may be also suitable for experimental infection with other respiratory viruses in this animal model.

[Intravenous cyclophosphamide in lupus nephritis: twenty years reducing dose]. / Ciclofosfamida intravenosa en nefritis lúpica: veinte años reduciendo dosis.

The prognosis for patients with proliferative glomerulonephritis associated with systemic lupus erythematosus has dramatically improved over recent decades. We review our experience with intermittent pulse therapy with intravenous cyclophosphamide (IC) in 97 patients (75 female) aged over 20 years. The series was divided into three groups. Group A (n=39) received monthly IC pulses (begin 1 g) for up to 24 months between 1985-1991. Group B (n=47) received monthly IC pulses (1 g) for six months with additional quarterly doses for a maximum of 18 months, depending on the therapeutic response (from 1991). From 1999, Group C (n=11) patients were treated with low-dose IC (3 g in three months) followed by azathioprine (2 mg/kg) or mycophenolate mofetil (1.5-2.0 g/day) for 12-18 months. The total IC doses (g) administered were: Group A, 15.1+/-9.0; Group B, 8.5+/-3.5; and Group C, 3.0+/-0.0. These figures show the trend progressive reduction in exposure to IC. Overall, treatment with the different IC regimens achieved satisfactory control of lupus nephritis in 76% of the patients. Comparison of the values at baseline and after 24 months showed that the serum creatinine (mg/dl) fell in Group A from 1.77+/-1.06 to 1.09+/-0.63, in Group B from 1.22+/- 0.85 to 0.95+/- 0.45, and in Group C from 0.90+/-0.23 to 1.17+/-0.54 (p<0.05). In the same period, proteinuria (g/day) fell in Group A from 6.19+/-4.31 to 0.79+/-1.76, in Group B from 4.43+/- 3.17 to 2.08+/-3.65, and in Group C from 5.43+/- 3.37 to 3.22+/-4.00 (p=0.05). There was not differences between the three groups in both variables. The adverse effects were mainly viral and bacterial infections, with no intergroup differences. Avascular osteonecrosis requiring hip replacement and early menopause were more frequent in Group A. Nine patients died, seven due to cardiovascular causes and two with infection. No differences were detected between the three groups when analyzing the overall patient survival at 5, 10 and 15 years (95%, 92%, and 84%, respectively). The likelihood of maintaining serum creatinine within normal ranges or less than twice the baseline range was similar in the three groups at 5, 10 and 15 years (92%, 72% and 66%, respectively). There were 47 episodes of relapse, with no differences between the three groups. In Summary, treatment with different regimens of intermittent IC is relatively safe and efficient to control the disease and lupus nephritis in SLE patients even with progressively smaller doses. The price paid concerned infectious complications, and bone and ovarian toxicity. New alternatives should at least maintain the same efficacy, but with fewer adverse effects and relapses.

Cocaine- and amphetamine-regulated transcript is overexpressed in the anteroventral periventricular nucleus of pregnant rats.

The anteroventral periventricular nucleus (AVPV) is sexually dimorphic, presenting a higher neuronal density in females. The AVPV contains a dense collection of oestrogen and progesterone receptors and has been related to the modulation of gonadotrophin-releasing hormone (GnRH) secretion and gene expression in response to circulating hormonal levels. It has been suggested that cocaine- and amphetamine-regulated transcript (CART) is also related to reproductive control because CART immunoreactive fibres are in close apposition with GnRH neurones. A portion of these fibres originate in the AVPV but its role in mediating hormonal action needs to be better explored. We hypothesised that CART expression in the AVPV would be influenced by the reproductive state and, consequently, by hormonal levels. To test this hypothesis, we analysed CART expression in the AVPV of female rats in different reproductive states (pro-oestrous, pregnancy and lactation). We found that, on the 19th day of pregnancy, female rats presented increased CART expression. Our findings indicate that AVPV CART expression is influenced by the reproductive state and that CART neurones in the AVPV may play a role in the hormonal mechanisms involved in the induction of maternal behaviour.

The ubiquitin-proteasome system in Huntington's disease.

The main histopathological feature of Huntington's disease (HD) is the presence of protein aggregates that are gathered into inclusion bodies. So far the mechanisms that lead to inclusion formation as well as their role in the pathogenesis of HD are not totally understood. However, it is well established that inclusion bodies contain components of the ubiquitin-proteasome system. Accordingly, it has been postulated that impairment of this machinery can be one of the causes of this disorder. In this review, the authors summarize the state of current knowledge about this hypothesis.

[Survival of myeloma patients treated with dialysis]. / Supervivencia de pacientes con mieloma tratados con diálisis.

BACKGROUND: Renal failure is a common complication of myeloma. Renal replacement therapy in these patients is controversial due to poor survival outcomes and low tolerance to treatment. We reviewed our experience on patients with myeloma undergoing dialysis therapy at one centre. PATIENTS AND METHODS: Between 1980 and 2000, 28 patients (21 men and 7 women) with myeloma were admitted to chronic dialysis programme and the following variables were analysed: sex, age when starting dialysis, lapse of time between diagnosis of myeloma and admission to dialysis (TD), disease stage, comorbity, mode of presentation, calcium, creatinine at diagnostic, albumin and Hb at the beginning of dialysis, and cause of death. We studied survival among these patients (Kaplan-Meier), identified predictors of survival outcome (Cox's regression) and compared survival between the two decades studied. RESULTS: Mean age was 65 years, median TD was 0.4 months, and modes of presentation were: end-stage renal failure (18 patients), acute renal failure (8), amyloldosis (2). Eleven patients (39%) had myeloma IgG, four (14%) IgA and thirteen (46%) had light chains. Kappa light chain was the most frequent one. In 75% of patients myeloma was at IIIb stage. Cause of death were: Cardiovascular disease (5 patients), infections (4), suspension of treatment (4), tumours (4), and others causes (2). Median survival for all patients was 16.8 months (range 0.4-78) and 25% survived over 39 months. Hb level was the only significant predictor in the multivariant analysis (p = 0.02). In the 80's median survival was 6.17 months versus 17 months in the 90's but this difference was not significant with long-rank test. CONCLUSION: Although survival of patients with myeloma treated with dialysis is still short, 25 percent survive over 3 years, being Hb level the only predictive factor. Moreover, we observed an improvement of survival in recent years.

Involvement of an F-actin skeleton on the acrosome reaction in guinea pig spermatozoa.

The acrosome reaction (AR) is a regulated exocytotic process. In several cell types, an actin network situated under the plasma membrane (PM) acts as a physical barrier to prevent this exocytosis. In seeking a function for a cortical skeleton in guinea pig spermatozoa, the PM and the outer acrosomal membrane (OAM) were investigated for the presence of F-actin and spectrin, proteins generally found in cell cortical skeletons. Both membrane types were visualized in whole-mount preparations by electron microscopy. PM proteins gave positive reaction to the Na(+),K(+)-ATPase antibody and the OAM proteins did not react to the antibody. Furthermore, a Triton X-100-resistant skeleton was obtained from both membrane types. Using gold immunoelectron microscopy, F-actin was visualized in the PM and in the OAM skeletons, while spectrin was only detected in the PM skeleton. The presence of an F-actin cortical skeleton in the sperm PM suggests that F-actin may be involved in the AR. The significantly higher number of AR elicited by cytochalasin D (Cyt-D) treatment(P<0.005) and data showing a significant (P>0.03) decrease in F-actin relative concentration in capacitating spermatozoa, agree with this suggestion. Furthermore, the proposal is strengthened by the fact that stabilization of F-actin by phalloidin (Ph) significantly (P>0.01) diminished AR induced by Ca(2+) in a streptolysin O (SLO)-permeabilized sperm model.

Genetic structure of natural populations of Escherichia coli in wild hosts on different continents.

Current knowledge of genotypic and phenotypic diversity in the species Escherichia coli is based almost entirely on strains recovered from humans or zoo animals. In this study, we analyzed a collection of 202 strains obtained from 81 mammalian species representing 39 families and 14 orders in Australia and the Americas, as well as several reference strains; we also included a strain from a reptile and 10 from different families of birds collected in Mexico. The strains were characterized genotypically by multilocus enzyme electrophoresis (MLEE) and phenotypically by patterns of sugar utilization, antibiotic resistance, and plasmid profile. MLEE analysis yielded an estimated genetic diversity (H) of 0.682 for 11 loci. The observed genetic diversity in this sample is the greatest yet reported for E. coli. However, this genetic diversity is not randomly distributed; geographic effects and host taxonomic group accounted for most of the genetic differentiation. The genetic relationship among the strains showed that they are more associated by origin and host order than is expected by chance. In a dendrogram, the ancestral cluster includes primarily strains from Australia and ECOR strains from groups B and C. The most differentiated E. coli in our analysis are strains from Mexican carnivores and strains from humans, including those in the ECOR group A. The kinds and numbers of sugars utilized by the strains varied by host taxonomic group and country of origin. Strains isolated from bats were found to exploit the greatest range of sugars, while those from primates utilized the fewest. Toxins are more frequent in strains from rodents from both continents than in any other taxonomic group. Strains from Mexican wild mammals were, on average, as resistant to antibiotics as strains from humans in cities. On average, the Australian strains presented a lower antibiotic resistance than the Mexican strains. However, strains recovered from hosts in cities carried significantly more plasmids than did strains isolated from wild mammals. Previous studies have shown that natural populations of E. coli harbor an extensive genetic diversity that is organized in a limited number of clones. However, knowledge of this worldwide bacterium has been limited. Here, we suggest that the strains from a wide range of wild hosts from different regions of the world are organized in an ecotypic structure where adaptation to the host plays an important role in the population structure.

Transgenic mice in the analysis of metabolic regulation.

In normal animals, the extracellular concentration of glucose is maintained within a very narrow range by the matching of glucose flux into and out of the extracellular space through the tightly coordinated secretion of insulin and glucagon. Functional alterations in beta-cells, liver, or skeletal muscle and adipose tissue may disrupt glucose homeostasis and lead to the development of non-insulin-dependent diabetes mellitus (type 2 diabetes). This review outlines the contribution of these organs and tissues to the control of glucose homeostasis. We discuss new insights obtained through studies of transgenic mice that overexpress or show decreased expression of putative key genes in the regulation of pancreatic beta-cell function, in the control of hepatic glucose uptake and output, and in the regulation of glucose uptake and utilization by skeletal muscle and adipose tissue.

Evidence from transgenic mice that interferon-beta may be involved in the onset of diabetes mellitus.

A number of cytokines have been shown to alter the function of pancreatic beta-cells and thus might be involved in the development of type 1 diabetes. Interferon-beta (IFN-beta) expression is induced in epithelial cells by several viruses, and it has been detected in islets of type 1 diabetic patients. Here we show that treatment of isolated mouse islets with this cytokine was able to alter insulin secretion in vitro. To study whether IFN-beta alters beta-cell function in vivo and leads to diabetes, we have developed transgenic mice (C57BL6/SJL) expressing IFN-beta in beta-cells. These mice showed functional alterations in islets and impaired glucose-stimulated insulin secretion. Transgenic animals presented mild hyperglycemia, hypoinsulinemia, hypertriglyceridemia, and altered glucose tolerance test, all features of a prediabetic state. However, they developed overt diabetes, with lymphocytic infiltration of the islets, when treated with low doses of streptozotocin, which did not induce diabetes in control mice. In addition, about 9% of the transgenic mice obtained from the N3 back-cross to outbred albino CD-1 mice spontaneously developed severe hyperglycemia and hypoinsulinemia and showed mononuclear infiltration of the islets. These results suggest that IFN-beta may be involved in the onset of type 1 diabetes when combined with either an additional factor or a susceptible genetic background.