A truncation in the Aryl Hydrocarbon Receptor of the CRL:WI(Han) rat does not affect the developmental toxicity of TCDD

The Aryl Hydrocarbon Receptor (AhR) is required for the toxicity of TCDD, and so the AhR of CRL:WI and CRL:WI(Han) rats was characterised. Western blot showed AhR proteins of ~110 and ~97 kDa in individual rats from both strains. The AhR cDNA from a CRL:WI(Han) rat with the ~110kDa protein revealed a sequence that was identical to that of the CRL:WI and SD rat. However, cloning of the AhR from a rat with the ~97kDa protein revealed a point mutation, and five variants encoding two C-terminally truncated variants of the AhR protein, arising from a point mutation in the intron/exon junction and consequent differential splicing. These C-terminally truncated variants were expressed and shown to give rise to a protein of ~97kDa; the recombinant AhR bound TCDD with an affinity that was not statistically different from the full-length protein. A single-nucleotide polymorphism (SNP) assay was developed, and showed that both alleles were represented in a Hardy-Weinberg equilibrium in samples of CRL:WI and CRL:WI(Han) populations; both alleles are abundant. Rats from two studies of TCDD developmental toxicity were genotyped, and the association with toxicity investigated using statistical analysis. There was no plausible evidence that the AhR allele had a significant effect on the toxic endpoints examined. These data show that the two AhR alleles are common in two strains of Wistar rat, and that the AhR alleles had no effect on TCDD-induced developmental toxicity in two independent studies.

Neustonic distribution of decapod planktonic stages and competence of brachyuran megalopae in coastal waters

Settlement rate may not reflect larval supply to coastal waters in different marine invertebrates and demersal fishes. The importance of near-shore oceanography and behaviour of late larval stages may be underestimated. The present study conducted neustonic sampling over station grids and along full-length transects at two embayments in south-eastern Brazil to (1) compare diurnal and nocturnal occurrence of most frequent decapod stages to assess their vertical movements, (2) describe the formation of larval patches and (3) measure competence of crab megalopae according to their distance to recruitment grounds. Several shrimp species apparently undergo a diel vertical migration, swimming crab megalopae showed no vertical movements and megalopae of the intertidal crab Pachygrapsus transversus revealed a reversed vertical migration. During the day, crab megalopae aggregated in convergence zones just below surface slicks. These larvae consisted of advanced, pre-moult stages, at both mid-bay and near-shore patches. Competence, measured as the time to metamorphosis in captivity, was similar between larval patches within each taxon. Yet, subtidal portunids moulted faster to juveniles than intertidal grapsids, possibly because they were closer to settlement grounds. Megalopae of Pachygrapsus from benthic collectors moulted faster than those from bay areas. These results suggest that alternative vertical migration patterns of late megalopae favour onshore transport, and actual competence takes place very close to suitable substrates, where larvae may remain for days before settlement. Lack of correlation between larval supply and settlement for Pachygrapsus suggests that biological processes, besides onshore transport, may play an important role in determining settlement success of coastal crabs.

Sloshing and slamming oscillations in collapsible channel flow

We consider laminar high-Reynolds-number flow through a finite-length planar channel, where a portion of one wall is replaced by a thin massless elastic membrane that is held under longitudinal tension T and subject to an external pressure distribution. The flow is driven by a fixed pressure drop along the full length of the channel. We investigate the global stability of two-dimensional Poiseuille flow using a method of matched local eigenfunction expansions, which is compared to direct numerical simulations. We trace the neutral stability curve of the primary oscillatory instability of the system, illustrating a transition from high-frequency ‘sloshing’ oscillations at high T to vigorous ‘slamming’ motion at low T . Small-amplitude sloshing at high T can be captured using a low-order eigenmode truncation involving four surface-based modes in the compliant segment of the channel coupled to Womersley flow in the rigid segments. At lower tensions, we show that hydrodynamic modes contribute increasingly to the global instability and we demonstrate a change in the mechanism of energy transfer from the mean flow, with viscous effects being destabilising. Simulations of finite-amplitude oscillations at low T reveal a generic slamming motion, in which the the flexible membrane is drawn close to the opposite rigid wall before rapidly recovering. A simple model is used to demonstrate how fluid inertia in the downstream rigid channel segment, coupled to membrane curvature downstream of the moving constriction, together control slamming dynamics.

Insights on epidemiology and control of Marek’s disease through traditional and novel molecular tools

Marek's disease (MD) is a contagious, lymphoproliferative and neuropathic disease of poultry caused by a ubiquitous lymphotropic and oncogenic virus, Gallid alphaherpesvirus 2 (GaHV-2). MD has been reported in all poultry-rearing countries and is among the viral diseases with the highest economic impact in the poultry industry worldwide, including Italy. MD has been also recognized as one of the leading causes of mortality in backyard poultry. The present doctoral thesis aimed at exploring Marek's disease virus molecular epidemiology in Italian commercial and backyard chicken flocks and, for the first time, in commercial turkeys affected by clinical MD. Molecular biology techniques targeting the full-length meq gene, the major GaHV-2 oncogene, were used to detect and characterize the circulating GaHV-2 strains searching for genetic markers of virulence. A final study focused on the development of rapid, sensitive, and species-specific loop-mediated isothermal amplification assays coupled with a lateral flow device readout for the detection of conventional and recombinant HVT-based vaccines is included in the thesis. HVT vaccines, currently used to protect chickens from MD, are referred to as "leaky", as they do not impede the infection, replication, and shedding of field GaHV-2: vaccinal and field viruses can coexist in the vaccinated host and molecular tests able to discriminate between GaHV-2 and HVT are required. These new simple, fast, and accurate tests for the monitoring of MD vaccination success in the field could be greatly beneficial for field veterinarians, small laboratories, and more broadly for resource-limited settings.

Identification of Human T-lymphotropic Virus Type I (HTLV-I) Subtypes Using Restrited Fragment Length Polymorphism in a Cohort of Asymptomatic Carriers and Patients with HTLV-I-associated Myelopathy/tropical Spastic Paraparesis from São Paulo, Brazil

Although human T-lymphotropic virus type I (HTLV-I) exhibits high genetic stability, as compared to other RNA viruses and particularly to human immunodeficiency virus (HIV), genotypic subtypes of this human retrovirus have been characterized in isolates from diverse geographical areas. These are currently believed not to be associated with different pathogenetic outcomes of infection. The present study aimed at characterizing genotypic subtypes of viral isolates from 70 HTLV-I-infected individuals from São Paulo, Brazil, including 42 asymptomatic carriers and 28 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), using restricted fragment length polymorphism (RFLP) analysis of long terminal repeat (LTR) HTLV-I proviral DNA sequences. Peripheral blood mononuclear cell lysates were amplified by nested polymerase chain reaction (PCR) and amplicons submitted to enzymatic digestion using a panel of endonucleases. Among HTLV-I asymptomatic carriers, viral cosmopolitan subtypes A, B, C and E were identified in 73.8%, 7.1%, 7.1% and 12% of tested samples, respectively, whereas among HAM/TSP patients, cosmopolitan A (89.3%), cosmopolitan C (7.1%) and cosmopolitan E (3.6%) subtypes were detected. HTLV-I subtypes were not statistically significant associated with patients' clinical status. We also conclude that RFLP analysis is a suitable tool for descriptive studies on the molecular epidemiology of HTLV-I infections in our environment.

Ectopic human telomerase catalytic subunit expression maintains telomere length but is not sufficient for CD8+ T lymphocyte immortalization.

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8(+) T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8(+) lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.

Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry.

To measure the average length of telomere repeats at chromosome ends in individual cells we developed a flow cytometry method using fluorescence in situ hybridization (flow FISH) with labeled peptide nucleic acid (PNA) probes. Results of flow FISH measurements correlated with results of conventional telomere length measurements by Southern blot analysis (R = 0.9). Consistent differences in telomere length in CD8+ T-cell subsets were identified. Naive and memory CD4+ T lymphocytes in normal adults differed by around 2.5 kb in telomere length, in agreement with known replicative shortening of telomeres in lymphocytes in vivo. T-cell clones grown in vitro showed stabilization of telomere length after an initial decline and rare clones capable of growing beyond 100 population doublings showed variable telomere length. These results show that flow FISH can be used to measure specific nucleotide repeat sequences in single cells and indicate that the very large replicative potential of lymphocytes is only indirectly related to telomere length.

The gene for the ligand binding chain of the human interferon gamma receptor.

In order to characterize the gene encoding the ligand binding (1(st); alpha) chain of the human IFN-gamma receptor, two overlapping cosmid clones were analyzed. The gene spans over 25 kilobases (kb) of the genomic DNA and has seven exons. The extracellular domain is encoded by exons 1 to 5 and by part of exon 6. The transmembrane region is also encoded by exon 6. Exon 7 encodes the intracellular domain and the 3' untranslated portion. The gene was located on chromosome 6q23.1, as determined by in situ hybridization. The 4 kb region upstream (5') of the gene was sequenced and analyzed for promoter activity. No consensus-matching TATA or CAAT boxes in the 5' region were found. Potential binding sites for Sp1, AP-1, AP-2, and CREB nuclear factors were identified. Compatible with the presence of the Sp1/AP-2 sites and the lack of TATA box, S1-nuclease mapping experiments showed multiple transcription initiation sites. Promoter activity of the 5' flanking region was analyzed with two different reporter genes: the Escherichia coli chloramphenicol acetyltransferase and human growth hormone. The smallest 5' region of the gene that still had full promoter activity was 692 base pairs in length. In addition, we found sequences belonging to the oldest family of Alu repeats, 2 - 3 kb upstream of the gene, which could be useful for genetic studies.

Mental imagery for full and upper human bodies: common right hemisphere activations and distinct extrastriate activations.

The processing of human bodies is important in social life and for the recognition of another person's actions, moods, and intentions. Recent neuroimaging studies on mental imagery of human body parts suggest that the left hemisphere is dominant in body processing. However, studies on mental imagery of full human bodies reported stronger right hemisphere or bilateral activations. Here, we measured functional magnetic resonance imaging during mental imagery of bilateral partial (upper) and full bodies. Results show that, independently of whether a full or upper body is processed, the right hemisphere (temporo-parietal cortex, anterior parietal cortex, premotor cortex, bilateral superior parietal cortex) is mainly involved in mental imagery of full or partial human bodies. However, distinct activations were found in extrastriate cortex for partial bodies (right fusiform face area) and full bodies (left extrastriate body area). We propose that a common brain network, mainly on the right side, is involved in the mental imagery of human bodies, while two distinct brain areas in extrastriate cortex code for mental imagery of full and upper bodies.

Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding

Trypanosoma cruzi and Trypanosoma rangeli are human-infective blood parasites, largely restricted to Central and South America. They also infect a wide range of wild and domestic mammals and are transmitted by a numerous species of triatomine bugs. There are significant overlaps in the host and geographical ranges of both species. The two species consist of a number of distinct phylogenetic lineages. A range of PCR-based techniques have been developed to differentiate between these species and to assign their isolates into lineages. However, the existence of at least six and five lineages within T. cruzi and T. rangeli, respectively, makes identification of the full range of isolates difficult and time consuming. Here we have applied fluorescent fragment length barcoding (FFLB) to the problem of identifying and genotyping T. cruzi, T. rangeli and other South American trypanosomes. This technique discriminates species on the basis of length polymorphism of regions of the rDNA locus. FFLB was able to differentiate many trypanosome species known from South American mammals: T. cruzi cruzi. T. cruzi marinkellei, T. dionisii-like, T. evansi, T. lewisi, T. rangeli, T. theileri and T. vivax. Furthermore, all five T. rangeli lineages and many T. cruzi lineages could be identified, except the hybrid lineages TcV and TcVI that could not be distinguished from lineages III and II respectively. This method also allowed identification of mixed infections of T. cruzi and T. rangeli lineages in naturally infected triatomine bugs. The ability of FFLB to genotype multiple lineages of T. cruzi and T. rangeli together with other trypanosome species, using the same primer sets is an advantage over other currently available techniques. Overall, these results demonstrate that FFLB is a useful method for species diagnosis, genotyping and understanding the epidemiology of American trypanosomes. (C) 2010 Elsevier B.V. All rights reserved.

Fluorescent amplified fragment length polymorphism genotyping of human and animal Staphylococcus aureus isolates from dairy farms with manual milking

Fluorescence amplified fragment length polymorphism (fAFLP) was used to assess the genetic relatedness of 40 Staphylococcus aureus strains isolated from human and animal skin samples in seven dairy farms with manual milking. S. aureus was isolated from 11 out of 30 (36%) human skin samples and from 29 out of 100 (29%) teat skin samples from apparently healthy cows. Genomic DNA from each isolate was double-digested with EcoRI and MseI and complementary oligonucleotide adaptors were ligated to the restriction fragments. Pre-selective and selective, amplification reactions were performed, the amplified fragments were separated by electrophoresis in an ABI377 sequencer and analysed using GeneScan 3.1 and Genotyper 2.5. Three single isolates (a-c), a predominant cluster with 35 isolates (d) and another cluster with two isolates (e) were identified. Both clusters d and e included human and animal isolates genetically related, because the profiles had 90-100% homology. Since no cluster was comprised uniquely of human or animal isolates and given the close genetic relatedness among human and animal samples in the farms, the present findings support the. hypothesis that dairy workers can spread S. aureus through manual milking. (C) 2005 Elsevier B.V. All rights reserved.

Taenia saginata: Differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay

Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected patients. A different Dral-RFLP pattern: a two-band pattern (421 and 100 bp) for T saginata and a three-band pattern (234, 188, and 99 bp) for T solium was observed allowing the two species to be separated.. The lower detection limit of the PCR-RFLP using a non-infected fecal sample prepared with a given number of T saginata eggs was 34 eggs in 2 g stool sediment. The 521 bp mtDNA fragment was detected in 8 out of 12 Taenia sp. carriers (66.6%). of these, three showed a T solium pattern and five a T saginata pattern. (c) 2005 Elsevier B.V. All rights reserved.

Influence of growth rate and length on fluoride detection in human nails

This study aimed to determine the lag time between increased fluoride (F) intake and F detection in human nails, as well as the influence of nails growth rate and length on this. Ten 20- to 35-year-old volunteers received 1.8 mg F daily, for 30 days. Nail growth rate and length were determined for all fingernails and toenails. Nail samples were collected at the beginning of the study and every 2 weeks (15 collections in all) and F concentrations were determined. The growth rate was statistically higher in fingernails than in toenails. No statistically significant differences were observed between right and left sides. Growth rate was significantly greater for big toenails than for the other toenails, but this pattern was not found for fingernails. The estimated mean lag times for F detection in fingernails and toenails were 101 and 123 days, respectively. An apparent increase in fingernail F concentrations was observed 84 days after the beginning of the study, although this was not statistically different from baseline. For toenails, statistically significant increases in F concentration in relation to baseline were observed 112 and 140 days after increased F ingestion. These increases occurred within the 95% confidence intervals for the calculated mean lag time for fluoride detection in nails. Considering the large amount of sample provided by the big toenails, together with their faster growth rate, as well as the fact that toenails are less prone to environmental contamination, our data suggest that big toenails are more suitable biomarkers of fluoride intake.

Genotyping of human and porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis

In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.

Telomere length in human natural killer cell subsets

Natural killer (NK) cells are cytotoxic cells that play a critical role in the innate immune response against infections and tumors. In the elderly, the cytotoxic function of NK cells is often compromised. Telomeres progressively shorten with each cell division and with age in most somatic cells eventually leading to chromosomal instability and cellular senescence. We studied the telomere length in NK cell subsets isolated from peripheral blood using "flow FISH," a method in which the hybridization of telomere probe in cells of interest is measured relative to internal controls in the same tube. We found that the average telomere length in human NK cells decreased with age as was previously found for human T lymphocytes. Separation of adult NK cells based on CD56 and CD16 expression revealed that the telomere length was significantly shorter in CD56(dim)CD16(+) (mature) NK cells compared to CD56(bright)CD16(-) (immature) NK cells from the same donor. Furthermore, sorting of NK cells based on expression of activation markers, such as NKG2D and LFA-1, revealed that NK cells expressing these markers have significantly shorter telomeres. Telomere fluorescence was very heterogeneous in NK cells expressing CD94, killer inhibitory receptor (KIR), NKG2A, or CD161. Our observations indicate that telomeric DNA in NK cells is lost with cell division and with age similar to what has been observed for most other hematopoietic cells. Telomere attrition in NK cells is a plausible cause for diminished NK cell function in the elderly.