Generation of mice with functional inactivation of talpid3, a gene first identified in chicken

Specification of digit number and identity is central to digit pattern in vertebrate limbs. The classical talpid(3) chicken mutant has many unpatterned digits together with defects in other regions, depending on hedgehog (Hh) signalling, and exhibits embryonic lethality. The talpid(3) chicken has a mutation in KIAA0586, which encodes a centrosomal protein required for the formation of primary cilia, which are sites of vertebrate Hh signalling. The highly conserved exons 11 and 12 of KIAA0586 are essential to rescue cilia in talpid(3) chicken mutants. We constitutively deleted these two exons to make a talpid3(-/-) mouse. Mutant mouse embryos lack primary cilia and, like talpid(3) chicken embryos, have face and neural tube defects but also defects in left/right asymmetry. Conditional deletion in mouse limb mesenchyme results in polydactyly and in brachydactyly and a failure of subperisoteal bone formation, defects that are attributable to abnormal sonic hedgehog and Indian hedgehog signalling, respectively. Like talpid(3) chicken limbs, the mutant mouse limbs are syndactylous with uneven digit spacing as reflected in altered Raldh2 expression, which is normally associated with interdigital mesenchyme. Both mouse and chicken mutant limb buds are broad and short. talpid3(-/-) mouse cells migrate more slowly than wild-type mouse cells, a change in cell behaviour that possibly contributes to altered limb bud morphogenesis. This genetic mouse model will facilitate further conditional approaches, epistatic experiments and open up investigation into the function of the novel talpid3 gene using the many resources available for mice.     

The relevance of non-human primate and rodent malaria models for humans

At the 2010 Keystone Symposium on "Malaria: new approaches to understanding Host-Parasite interactions", an extra scientific session to discuss animal models in malaria research was convened at the request of participants. This was prompted by the concern of investigators that skepticism in the malaria community about the use and relevance of animal models, particularly rodent models of severe malaria, has impacted on funding decisions and publication of research using animal models. Several speakers took the opportunity to demonstrate the similarities between findings in rodent models and human severe disease, as well as points of difference. The variety of malaria presentations in the different experimental models parallels the wide diversity of human malaria disease and, therefore, might be viewed as a strength. Many of the key features of human malaria can be replicated in a variety of nonhuman primate models, which are very under-utilized. The importance of animal models in the discovery of new anti-malarial drugs was emphasized. The major conclusions of the session were that experimental and human studies should be more closely linked so that they inform each other, and that there should be wider access to relevant clinical material.     

DNA damage during meiosis induces chromatin remodeling and synaptonemal complex disassembly

DNA damage to the germline genome must be accurately repaired to ensure transmission of intact genetic information to following generations. Meiosis presents challenges to the DNA damage response (DDR) because it universally requires changes to chromosome structure that can affect DNA repair outcomes. We report the existence of a meiotic DDR at chromosome axes that results in chromatin remodeling, synaptonemal complex disassembly, and axis separation in response to irradiation at late pachytene stages in C. elegans. The axis component HTP-3 is required for germline acquisition of H2AacK5, an axis-specific chromatin mark that is DNA damage responsive. Irradiated wild-types show reduction of H2AacK5 and axis separation that are dependent on the acetyltransferase MYS-1/TIP60. Restoration of H2AacK5 levels requires ATM-1 kinase and correlates with resynapsis. We propose that the meiotic DDR involves early chromatin remodeling at chromosome axes to dismantle structures promoting interhomolog recombination and facilitate efficient nonhomolog-based repair before pachytene exit.     

Comparative analysis of 3D expression patterns of transcription factor genes and digit fate maps in the developing chick wing

Hoxd13, Tbx2, Tbx3, Sall1 and Sall3 genes are candidates for encoding antero-posterior positional values in the developing chick wing and specifying digit identity. In order to build up a detailed profile of gene expression patterns in cell lineages that give rise to each of the digits over time, we compared 3 dimensional (3D) expression patterns of these genes during wing development and related them to digit fate maps. 3D gene expression data at stages 21, 24 and 27 spanning early bud to digital plate formation, captured from in situ hybridisation whole mounts using Optical Projection Tomography (OPT) were mapped to reference wing bud models. Grafts of wing bud tissue from GFP chicken embryos were used to fate map regions of the wing bud giving rise to each digit; 3D images of the grafts were captured using OPT and mapped on to the same models. Computational analysis of the combined computerised data revealed that Tbx2 and Tbx3 are expressed in digit 3 and 4 progenitors at all stages, consistent with encoding stable antero-posterior positional values established in the early bud; Hoxd13 and Sall1 expression is more dynamic, being associated with posterior digit 3 and 4 progenitors in the early bud but later becoming associated with anterior digit 2 progenitors in the digital plate. Sox9 expression in digit condensations lies within domains of digit progenitors defined by fate mapping; digit 3 condensations express Hoxd13 and Sall1, digit 4 condensations Hoxd13, Tbx3 and to a lesser extent Tbx2. Sall3 is only transiently expressed in digit 3 progenitors at stage 24 together with Sall1 and Hoxd13; then becomes excluded from the digital plate. These dynamic patterns of expression suggest that these genes may play different roles in digit identity either together or in combination at different stages including the digit condensation stage.     

A new approach to dengue fatal cases diagnosis: NS1 antigen capture in tissues

Abstract/Background

Dengue is the most important arthropod borne viral disease worldwide in terms of morbidity and mortality and is caused by any of the four serotypes of dengue virus (DENV-1 to 4). Brazil is responsible for approximately 80% of dengue cases in the Americas, and since the introduction of dengue in 1986, a total of 5,944,270 cases have been reported including 21,596 dengue hemorrhagic fever and 874 fatal cases. DENV can infect many cell types and cause diverse clinical and pathological effects. The goal of the study was to investigate the usefulness of NS1 capture tests as an alternative tool to detect DENV in tissue specimens from previously confirmed dengue fatal cases (n = 23) that occurred in 2002 in Brazil.

Methodology/Principal Findings

A total of 74 tissue specimens were available: liver (n = 23), lung (n = 14), kidney (n = 04), brain (n = 10), heart (n = 02), skin (n = 01), spleen (n = 15), thymus (n = 03) and lymph nodes (n = 02). We evaluated three tests for NS1 antigen capture: first generation Dengue Early ELISA (PanBio Diagnostics), Platelia NS1 (BioRad Laboratories) and the rapid test NS1 Ag Strip (BioRad Laboratories). The overall dengue fatal case diagnosis based on the tissues analyzed by Dengue Early ELISA, Platelia NS1 and the NS1 Ag Strip was 34.7% (08/23), 60.8% (14/23) and 91.3% (21/23), respectively. The Dengue Early ELISA detected NS1 in 22.9% (17/74) of the specimens analyzed and the Platelia NS1 in 45.9% (34/74). The highest sensitivity (78.3%; 58/74) was achieved by the NS1 Ag Strip, and the differences in the sensitivities were statistically significant (p<0.05). The NS1 Ag Strip was the most sensitive in liver (91.3%; 21/23), lung (71.4%; 10/14), kidney (100%; 4/4), brain (80%; 8/10), spleen (66.6%, 10/15) and thymus (100%, 3/3) when compared to the other two ELISA assays.

Conclusions/Significance

This study shows the DENV NS1 capture assay as a rapid and valuable approach to postmortem dengue confirmation. With an increasing number of DHF and fatal cases, the availability of new approaches useful for cases confirmation plays an important tool for the disease surveillance.     

Mitosis-specific phosphorylation of amyloid precursor protein at Threonine 668 leads to its altered processing and association with centrosomes

Background

Atypical expression of cell cycle regulatory proteins has been implicated in Alzheimer's disease (AD), but the molecular mechanisms by which they induce neurodegeneration are not well understood. We examined transgenic mice expressing human amyloid precursor protein (APP) and presenilin 1 (PS1) for changes in cell cycle regulatory proteins to determine whether there is a correlation between cell cycle activation and pathology development in AD.

Results

Our studies in the AD transgenic mice show significantly higher levels of cyclin E, cyclin D1, E2F1, and P-cdc2 in the cells in the vicinity of the plaques where maximum levels of Threonine 668 (Thr668)-phosphorylated APP accumulation was observed. This suggests that the cell cycle regulatory proteins might be influencing plaque pathology by affecting APP phosphorylation. Using neuroglioma cells overexpressing APP we demonstrate that phosphorylation of APP at Thr668 is mitosis-specific. Cells undergoing mitosis show altered cellular distribution and localization of P-APP at the centrosomes. Also, Thr668 phosphorylation in mitosis correlates with increased processing of APP to generate Aβ and the C-terminal fragment of APP, which is prevented by pharmacological inhibitors of the G1/S transition.

Conclusions

The data presented here suggests that cell cycle-dependent phosphorylation of APP may affect its normal cellular function. For example, association of P-APP with the centrosome may affect spindle assembly and cell cycle progression, further contributing to the development of pathology in AD. The experiments with G1/S inhibitors suggest that cell cycle inhibition may impede the development of Alzheimer's pathology by suppressing modification of βAPP, and thus may represent a novel approach to AD treatment. Finally, the cell cycle regulated phosphorylation and processing of APP into Aβ and the C-terminal fragment suggest that these proteins may have a normal function during mitosis.