Edar/Eda interactions regulate enamel knot formation in tooth morphogenesis.

tabby and downless mutant mice have apparently identical defects in teeth, hair and sweat glands. Recently, genes responsible for these spontaneous mutations have been identified. downless (Dl) encodes Edar, a novel member of the tumour necrosis factor (TNF) receptor family, containing the characteristic extracellular cysteine rich fold, a single transmembrane region and a death homology domain close to the C terminus. tabby (Ta) encodes ectodysplasin-A (Eda) a type II membrane protein of the TNF ligand family containing an internal collagen-like domain. As predicted by the similarity in adult mutant phenotype and the structure of the proteins, we demonstrate that Eda and Edar specifically interact in vitro. We have compared the expression pattern of Dl and Ta in mouse development, taking the tooth as our model system, and find that they are not expressed in adjacent cells as would have been expected. Teeth develop by a well recorded series of epithelial-mesenchymal interactions, similar to those in hair follicle and sweat gland development, the structures found to be defective in tabby and downless mice. We have analysed the downless mutant teeth in detail, and have traced the defect in cusp morphology back to initial defects in the structure of the tooth enamel knot at E13. Significantly, the defect is distinct from that of the tabby mutant. In the tabby mutant, there is a recognisable but small enamel knot, whereas in the downless mutant the knot is absent, but enamel knot cells are organised into a different shape, the enamel rope, showing altered expression of signalling factors (Shh, Fgf4, Bmp4 and Wnt10b). By adding a soluble form of Edar to tooth germs, we were able to mimic the tabby enamel knot phenotype, demonstrating the involvement of endogenous Eda in tooth development. We could not, however, reproduce the downless phenotype, suggesting the existence of yet another ligand or receptor, or of ligand-independent activation mechanisms for Edar. Changes in the structure of the enamel knot signalling centre in downless tooth germs provide functional data directly linking the enamel knot with tooth cusp morphogenesis. We also show that the Lef1 pathway, thought to be involved in these mutants, functions independently in a parallel pathway.

Early diagenesis of bone and tooth apatite in fluvial and marine settings: Constraints from combined oxygen isotope, nitrogen and REE analysis

Fossil bones and teeth of Late Pleistocene terrestrial mammals from Rhine River gravels (RS) and the North Sea (NS), that have been exposed to chemically and isotopically distinct diagenetic fluids (fresh water versus seawater), were investigated to study the effects of early diagenesis on biogenic apatite. Changes in phosphate oxygen isotopic composition (delta O-18(PO4)), nitrogen content (wt.% N) and rare earth element (REE) concentrations were measured along profiles within bones that have not been completely fossilized, and in skeletal tissues (bone, dentine, enamel) with different susceptibilities to diagenetic alteration. Early diagenetic changes of elemental and isotopic compositions of apatite in fossil bone are related to the loss of the stabilizing collagen matrix. The REE concentration is negatively correlated with the nitrogen content, and therefore the amount of collagen provides a sensitive proxy for early diagenetic alteration. REE patterns of RS and NS bones indicate initial fossilization in a fresh water fluid with similar REE compositions. Bones from both settings have nearly collagen-free, REE-, U-, F- and Sr-enriched altered outer rims, while the collagen-bearing bone compacta in the central part often display early diagenetic pyrite void-fillings. However, NS bones exposed to Holocene seawater have outer rim delta O-18(PO4) values that are 1.1 to 2.6 parts per thousand higher compared to the central part of the same bones (delta O-18(PO4) = 18.2 +/- 0.9 parts per thousand, n = 19). Surprisingly, even the collagen-rich bone compacta with low REE contents and apatite crystallinity seems altered, as NS tooth enamel (delta O-18(PO4) =15.0 +/- 0.3 parts per thousand, n=4) has about 3%. lower delta O-18(PO4) values, values that are also similar to those of enamel from RS teeth. Therefore, REE concentration, N content and apatite crystallinity are in this case only poor proxies for the alteration of delta O-18(PO4) values. Seawater exposure of a few years up to 8 kyr can change the delta O-18(PO4) values of the bone apatite by > 3 parts per thousand. Therefore, bones fossilized in marine settings must be treated with caution for palaeoclimatic reconstructions. However, enamel seems to preserve pristine delta O-18(PO4) values on this time scale. Using species-specific calibrations for modern mammals, a mean delta O-18(H2O) value can be reconstructed for Late Pleistocene mammalian drinking water of around -9.2 +/- 0.5 parts per thousand, which is similar to that of Late Pleistocene groundwater from central Europe. (c) 2008 Elsevier B.V. All rights reserved.

Evaluation of a polymerase chain reaction-restriction fragment length polymorphism assay for dermatophyte and nondermatophyte identification in onychomycosis.

BACKGROUND: Dermatophytes are the main cause of onychomycoses, but various nondermatophyte filamentous fungi are often isolated from abnormal nails. The correct identification of the aetiological agent of nail infections is necessary in order to recommend appropriate treatment. OBJECTIVE: To evaluate a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on 28S rDNA for fungal identification in nails on a large number of samples in comparison with cultures. METHODS: Infectious fungi were analysed using PCR-RFLP in 410 nail samples in which fungal elements were observed in situ by direct mycological examination (positive samples). The results were compared with those previously obtained by culture of fungi on Sabouraud agar from the same nail samples. RESULTS: PCR-RFLP identification of fungi in nails allowed validation of the results obtained in culture when Trichophyton spp. grew from infected samples. In addition, nondermatophyte filamentous fungi could be identified with certainty as the infectious agents in onychomycosis, and discriminated from dermatophytes as well as from transient contaminants. The specificity of the culture results relative to PCR-RFLP appeared to be 81%, 71%, 52% and 63% when Fusarium spp., Scopulariopsis brevicaulis, Aspergillus spp. and Candida spp., respectively, grew on Sabouraud agar. It was also possible to identify the infectious agent when direct nail mycological examination showed fungal elements, but negative results were obtained from fungal culture. CONCLUSIONS: Improved sensitivity for the detection of fungi in nails was obtained using the PCR-RFLP assay. Rapid and reliable molecular identification of the infectious fungus can be used routinely and presents several important advantages compared with culture in expediting the choice of appropriate antifungal therapy.

CAVIN-3 regulates circadian period length and PER:CRY protein abundance and interactions.

In mammals, transcriptional autorepression by Period (PER) and Cryptochrome (CRY) protein complexes is essential for the generation of circadian rhythms. We have identified CAVIN-3 as a new, cytoplasmic PER2-interacting protein influencing circadian clock properties. Thus, CAVIN-3 loss- and gain-of-function shortened and lengthened, respectively, the circadian period in fibroblasts and affected PER:CRY protein abundance and interaction. While depletion of protein kinase Cδ (PKCδ), a known partner of CAVIN-3, had little effect on circadian gene expression, CAVIN-3 required the PKCδ-binding site to exert its effect on period length. This suggests the involvement of yet uncharacterized protein kinases. Finally, CAVIN-3 activity in circadian gene expression was independent of caveolae.

Long sequence duplications, repeats, and palindromes in HIV-1 gp120: length variation in V4 as the product of misalignment mechanism.

We have shown that indels in gp120 V4 are associated to the presence of duplicated and palindromic sequences, suggesting that they may be produced by strand-slippage misalignment mechanism. Indels in V4 involved region-specific duplications 9 to 15 bp long, and repeats of various lengths, associated to trinucleotides AAT. No duplications were found in V3 and C3. The frequency of palindromic sequences in individual genes was found to be significantly higher in gp120 (p < or = 3.00E-7), and significantly lower in Tat (p < or = 9.00E-7) than the average frequency calculated over the full genome. The finding of elements of misalignment in association with indels in V4 suggests that these mutations may occur in proviral DNA after integration of HIV into the host genome. It also implies that occurrence of large indels in gp120 is not random but is directed by the presence and distribution of elements of misalignment in the HIV genome.

Cell-permeable peptides induce dose- and length-dependent cytotoxic effects.

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat(48-57) and Antp(43-58) cell-permeable peptide carriers. Both Tat(48-57) and Antp(43-58) are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 microM of Tat(48-57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic. Moreover, all peptides bound to Tat(48-57) and Antp(43-58) triggered significant and length-dependent cytotoxicity when used at concentrations above 10 microM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat(48-57) should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 microM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat(48-57)- and Antp(43-58) bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.

Length variation in HIV-1 gp120 as the product of DNA misalignment mechanism

Background: To determine whether misalignment structures such as duplications, repeats, and palindromes are associated to insertions/deletions (indels) in gp120, indicating that indels are indeed frameshift mutations generated by DNA misalignment mechanism. Methods: Cloning and sequencing of a fragment of HIV-1 gp120 spanning C2-C4 derived from plasma RNA in 12 patients with early chronic disease and naïve to antiretroviral therapy. Results: Indels in V4 involved always insertion and deletion of duplicated nucleotide segments, and AAT repeats, and were associated to the presence of palindromic sequences. No duplications were detected in V3 and C3. Palindromic sequences occurred with similar frequencies in V3, C3 and V4; the frequency of palindromes in individual genes was found to be significantly higher in structural (gp120, p ≤ 3.00E-7) and significantly lower in regulatory (Tat, p ≤ 9.00E-7) genes, as compared to the average frequency calculated over the full genome. Discussion: Indels in V4 are associated to misalignment structures (i.e. duplications repeat and palindromes) indicating DNA misalignment as the mechanism underlying length variation in V4. The finding that indels in V4 are caused by DNA misalignment has some very important implications: 1) indels in V4 are likely to occur in proviral DNA (and not in RNA), after integration of HIV into the host genome; 2) they are likely to occur as progressive modifications of the early founder virus during chronic infection, as more and more cells get infected; 3) frameshift mutations involving any number of base pairs are likely to occur evenly across gp120; however, only those mutants carrying a functional gp120 (indels as multiples of three base pairs) will be able to perpetuate the virus cycle and to keep spreading through the population.

Energy deficit and length of hospital stay can be reduced by a two-step quality improvement of nutrition therapy: the intensive care unit dietitian can make the difference.

OBJECTIVE: Critically ill patients are at high risk of malnutrition. Insufficient nutritional support still remains a widespread problem despite guidelines. The aim of this study was to measure the clinical impact of a two-step interdisciplinary quality nutrition program. DESIGN: Prospective interventional study over three periods (A, baseline; B and C, intervention periods). SETTING: Mixed intensive care unit within a university hospital. PATIENTS: Five hundred seventy-two patients (age 59 ± 17 yrs) requiring >72 hrs of intensive care unit treatment. INTERVENTION: Two-step quality program: 1) bottom-up implementation of feeding guideline; and 2) additional presence of an intensive care unit dietitian. The nutrition protocol was based on the European guidelines. MEASUREMENTS AND MAIN RESULTS: Anthropometric data, intensive care unit severity scores, energy delivery, and cumulated energy balance (daily, day 7, and discharge), feeding route (enteral, parenteral, combined, none-oral), length of intensive care unit and hospital stay, and mortality were collected. Altogether 5800 intensive care unit days were analyzed. Patients in period A were healthier with lower Simplified Acute Physiologic Scale and proportion of "rapidly fatal" McCabe scores. Energy delivery and balance increased gradually: impact was particularly marked on cumulated energy deficit on day 7 which improved from -5870 kcal to -3950 kcal (p < .001). Feeding technique changed significantly with progressive increase of days with nutrition therapy (A: 59% days, B: 69%, C: 71%, p < .001), use of enteral nutrition increased from A to B (stable in C), and days on combined and parenteral nutrition increased progressively. Oral energy intakes were low (mean: 385 kcal*day, 6 kcal*kg*day ). Hospital mortality increased with severity of condition in periods B and C. CONCLUSION: A bottom-up protocol improved nutritional support. The presence of the intensive care unit dietitian provided significant additional progression, which were related to early introduction and route of feeding, and which achieved overall better early energy balance.

Telomere length dynamics in normal individuals and in patients with hematopoietic stem cell-associated disorders.

The telomere length in nucleated peripheral blood (PB) cells indirectly reflects the mitotic history of their precursors: the hematopoietic stem cells (HSCs). The average length of telomeres in PB leukocytes can be measured using fluorescence in situ hybridization and flow cytometry (flow FISH). We previously used flow FISH to characterize the age-related turnover of HSCs in healthy individuals. In this review, we describe results of recent flow FISH studies in patients with selected hematopoietic stem cell-associated disorders: chronic myelogenous leukemia (CML) and several bone marrow failure syndromes. CML is characterized by a marked expansion of myeloid Philadelphia chromosome positive (Ph+) cells. Nevertheless, nonmalignant (Ph-) HSCs typically coexist in the bone marrow of CML patients. We analyzed the telomere length in > 150 peripheral blood leukocytes (PBLs) and bone marrow samples of patients with CML as well as samples of Ph- T-lymphocytes. Compared to normal controls, the overall telomere fluorescence in PBLs of patients with CML was significantly reduced. However, no telomere shortening was observed in Ph- T-lymphocytes. Patients in late chronic phase (CP) had significantly shorter telomeres than those assessed earlier in CP. Our data suggest that progressive telomere shortening is correlated with disease progression in CML. Within the group of patients with bone marrow failure syndromes, we only found significantly shortened telomeres (compared to age-adjusted controls) in granulocytes from patients with aplastic anemia (AA). Strikingly, the telomere length in granulocytes from AA patients who had recovered after immunosuppressive therapy (recAA) did not differ significantly from controls, whereas untreated patients and nonresponders with persistent severe pancytopenia (sAANR) showed marked and significant telomere shortening compared to healthy donors and patients with recAA. Furthermore, an inverse correlation between age-adjusted telomere length and peripheral blood counts was found in support of a model in which the degree of cytopenia and the amount of telomere shortening are correlated. These results support the concept of extensive proliferation of HSCs in subgroups of AA patients and suggest a potential use of telomere-length measurements as a prognostic tool in this group of disorders as well.

Effect of hip flexion angle on hamstring optimum length after a single set of concentric contractions.

The eccentric contraction mode was proposed to be the primary stimulus for optimum angle (angle at which peak torque occurs) shift. However, the training range of motion (or muscle excursion range) could be a stimulus as important. The aim of this study was to assess the influence of the training range of motion stimulus on the hamstring optimum length. It was hypothesised that performing a single set of concentric contractions beyond optimal length (seated at 80° of hip flexion) would lead to an immediate shift of the optimum angle to longer muscle length while performing it below (supine at 0° of hip flexion) would not provide any shift. Eleven male participants were assessed on an isokinetic dynamometer. In both positions, the test consisted of 30 consecutive knee flexions at 4.19 rad · s⁻¹. The optimum angle was significantly shifted by ∼15° in the direction of longer muscle length after the contractions at 80° of hip flexion, while a non-significant shift of 3° was found at 0°. The hamstring fatigability was not influenced by the hip position. It was concluded that the training range of motion seems to be a relevant stimulus for shifting the optimum angle to longer muscle length. Moreover, fatigue appears as a mechanism partly responsible for the observed shift.

Histological description of seminiferous epithelium and cycle length of spermatogenesis in the water shrew Neomys fodiens (Mammalia: Soricidae).

Recently, we examined the spermatogenesis cycle length in two shrews species, Sorex araneus characterized by a very high metabolic rate and a polyandric mating system (sperm competition) resulting in a short cycle and Crocidura russula characterized by a much lower metabolic rate and a monogamous mating system showing a longer cycle. In this study, we investigated the spermatogenesis cycle in Neomys fodiens showing an intermediate metabolic rate. We described the stages of seminiferous epithelium according to the spermatid morphology method and we calculated the cycle length of spermatogenesis using incorporation of 5-bromodeoxyuridine into DNA of the germ cells. Twelve males were injected intraperitoneally with 5-bromodeoxyuridine, and the testes were collected. For cycle length determination, we applied a recently developed statistical method. The calculated cycle length is 8.69 days and the total duration of spermatogenesis based on 4.5 cycles is approximately 39.1 days, intermediate between the duration of spermatogenesis of S. araneus (37.6 days) and C. russula (54.5 days) and therefore congruent with both the metabolic rate hypothesis and the sperm competition hypothesis. Relative testes size of 1.4% of body mass indicates a promiscuous mating system.

Relationships of basal metabolic rate, relative testis size and cycle length of spermatogenesis in shrews (Mammalia, Soricidae).

The aim of the present study was to determinate the cycle length of spermatogenesis in three species of shrew, Suncus murinus, Sorex coronatus and Sorex minutus, and to assess the relative influence of variation in basal metabolic rate (BMR) and mating system (level of sperm competition) on the observed rate of spermatogenesis, including data of shrew species studied before (Sorex araneus, Crocidura russula and Neomys fodiens). The dynamics of sperm production were determined by tracing 5-bromodeoxyuridine in the DNA of germ cells. As a continuous scaling of mating systems is not evident, the level of sperm competition was evaluated by the significantly correlated relative testis size (RTS). The cycle durations estimated by linear regression were 14.3 days (RTS 0.3%) in Suncus murinus, 9.0 days (RTS 0.5%) in Sorex coronatus and 8.5 days (RTS 2.8%) in Sorex minutus. In regression and multiple regression analyses including all six studied species of shrew, cycle length was significantly correlated with BMR (r2=0.73) and RTS (r2=0.77). Sperm competition as an ultimate factor obviously leads to a reduction in the time of spermatogenesis in order to increase sperm production. BMR may act in the same way, independently or as a proximate factor, revealed by the covariation, but other factors (related to testes size and thus to mating system) may also be involved.