Heterogeneous combinatorial expression of Hoxd genes in single cells during limb development.

BACKGROUND: Global analyses of gene expression during development reveal specific transcription patterns associated with the emergence of various cell types, tissues, and organs. These heterogeneous patterns are instrumental to ensure the proper formation of the different parts of our body, as shown by the phenotypic effects generated by functional genetic approaches. However, variations at the cellular level can be observed within each structure or organ. In the developing mammalian limbs, expression of Hox genes from the HoxD cluster is differentially controlled in space and time, in cells that will pattern the digits and the forearms. While the Hoxd genes broadly share a common regulatory landscape and large-scale analyses have suggested a homogenous Hox gene transcriptional program, it has not previously been clear whether Hoxd genes are expressed together at the same levels in the same cells. RESULTS: We report a high degree of heterogeneity in the expression of the Hoxd11 and Hoxd13 genes. We analyzed single-limb bud cell transcriptomes and show that Hox genes are expressed in specific combinations that appear to match particular cell types. In cells giving rise to digits, we find that the expression of the five relevant Hoxd genes (Hoxd9 to Hoxd13) is unbalanced, despite their control by known global enhancers. We also report that specific combinatorial expression follows a pseudo-time sequence, which is established based on the transcriptional diversity of limb progenitors. CONCLUSIONS: Our observations reveal the existence of distinct combinations of Hoxd genes at the single-cell level during limb development. In addition, we document that the increasing combinatorial expression of Hoxd genes in this developing structure is associated with specific transcriptional signatures and that these signatures illustrate a temporal progression in the differentiation of these cells.

Masticatory muscle architecture in a water-rat from Australasia (Murinae, Hydromys) and its implication for the evolution of carnivory in rodents.

Murines are well known for their generalist diet, but several of them display specializations towards a carnivorous diet such as the amphibious Indo-Pacific water-rats. Despite the fact that carnivory evolved repeatedly in this group, few studies have investigated associated changes in jaw muscle anatomy and biomechanics. Here, we describe the jaw muscles and cranial anatomy of a carnivorous water-rat, Hydromys chrysogaster. The architecture of the jaw musculature of six specimens captured both on Obi and Papua were studied and described using dissections. We identified the origin and insertions of the jaw muscles, and quantified muscle mass, fiber length, physiological cross-sectional area, and muscle vectors for each muscle. Using a biomechanical model, we estimated maximum incisor and molar bite force at different gape angles. Finally, we conducted a 2D geometric morphometric analyses to compare jaw shape, mechanical potential, and diversity in lever-arm ratios for a set of 238 specimens, representative of Australo-Papuan carnivorous and omnivorous murids. Our study reveals major changes in the muscle proportions among Hydromys and its omnivorous close relative, Melomys. Hydromys was found to have large superficial masseter and temporalis muscles as well as a reduced deep masseter and zygomatico-mandibularis, highlighting major functional divergence among omnivorous and carnivorous murines. Changes in these muscles are also accompanied by changes in jaw shape and the lines of action of the muscles. A more vertically oriented masseter, reduced masseteric muscles, as well as an elongated jaw with proodont lower incisors are key features indicative of a reduced propalinality in carnivorous Hydromys. Differences in the fiber length of the masseteric muscles were also detected between Hydromys and Melomys, which highlight potential adaptations to a wide gape in Hydromys, allowing it to prey on larger animals. Using a biomechanical model, we inferred a greater bite force in Hydromys than in Melomys, implying a functional shift between omnivory and carnivory. However, Melomys has an unexpected greater bite force at large gape compared with Hydromys. Compared with omnivorous Melomys, Hydromys have a very distinctive low mandible with a well-developed coronoid process, and a reduced angular process that projects posteriorly to the ascending rami. This jaw shape, along with our mechanical potential and jaw lever ratio estimates, suggests that Hydromys has a faster jaw closing at the incisor, with a higher bite force at the level of the molars. The narrowing of the Hydromys jaw explains this higher lever advantage at the molars, which constitutes a good compromise between a wide gape, a reduced anterior masseteric mass, and long fiber lengths. Lever arms of the superficial and deep masseter are less favourable to force output of the mandible in Hydromys but more favourable to speed. Compared with the small input lever arm defined between the condyle and the angular process, the relatively longer mandible of Hydromys increases the speed at the expense of the output force. This unique combination of morphological features of the masticatory apparatus possibly has permitted Hydromys to become a highly successful amphibious predator in the Indo-Pacific region.

Patterns of macroevolution among Primates inferred from a supermatrix of mitochondrial and nuclear DNA.

Here, we present a new primate phylogeny inferred from molecular supermatrix analyses of size 42 kb containing 70% of missing data, and representing 75% of primate species diversity. The supermatrix was analysed using a gene-partitioned maximum likelihood approach to obtain an exhaustive molecular phylogenetic framework. All clades recovered from recent molecular works were upheld in our analyses demonstrating that the presence of missing data did not bias our supermatrix inference. The resulting phylogenetic tree was subsequently dated with a molecular dating method to provide a timescale for speciation events. Results obtained from our relaxed molecular clock analyses concurred with previous works based on the same fossil constraints. The resulting dated tree allowed to infer of macroevolutionary processes among the primates. Shifts in diversification rate and speciation rates were determined using the SymmeTREE method and a birthdeath process. No significant asymmetry was detected for the primate clade, but significant shifts in diversification rate were identified for seven clades: Anthropoidea, Lemuriformes, Lemuridae, Galagidae, Callithrix genus, the Cercopithecinae and Asian Macaca. Comparisons with previous primate supertree results reveal that (i) there was a diversification event at the root of the Lemuriformes, (ii) a higher diversification rate is detected for Cercopithecidae and Anthropoidea and (iii) a shift in diversification is always recovered for Macaca genus. Macroevolutionary inferences and primate divergence dates show that major primate diversification events occurred after the Paleogene, suggesting the extinction of ancient primate lineages.

Short-term associations between fine and coarse particles and hospital admissions for cardiorespiratory diseases in six French cities.

OBJECTIVES: Little is known about the potential health effects of the coarse fraction of ambient particles. The aim of this study is to estimate the links between fine (PM(2.5)) and coarse particle (PM(2.5-10)) levels and cardiorespiratory hospitalisations in six French cities during 2000-2003. METHODS: Data on the daily numbers of hospitalisations for respiratory, cardiovascular, cardiac and ischaemic heart diseases were collected. Associations between exposure indicators and hospitalisations were estimated in each city using a Poisson regression model, controlling for confounding factors (seasons, days of the week, holidays, influenza epidemics, pollen counts, temperature) and temporal trends. City-specific findings were combined to obtain excess relative risks (ERRs) associated with a 10 mug/m(3) increase in PM(2.5) and PM(2.5-10 )levels. RESULTS: We found positive associations between indicators of particulate pollution and hospitalisations for respiratory infection, with an ERR of 4.4% (95% CI 0.9 to 8.0) for PM(2.5-10 )and 2.5% (95% CI 0.1 to 4.8) for PM(2.5). Concerning respiratory diseases, no association was observed with PM(2.5), whereas positive trends were found with PM(2.5-10), with a significant association for the 0-14-year-old age group (ERR 6.2%, 95% CI 0.4 to 12.3). Concerning cardiovascular diseases, positive associations were observed between PM(2.5) levels and each indicator, although some did not reach significance; trends with PM(2.5-10 )were weaker and non-significant except for ischaemic heart disease in the elderly (ERR 6.4%, 95% CI 1.6 to 11.4). CONCLUSIONS: In accordance with other studies, our results indicate that the coarse fraction may have a stronger effect than the fine fraction on some morbidity endpoints, especially respiratory diseases.

Evidence of niche partitioning among bacteria living on plastics, organic particles and surrounding seawaters.

Plastic pollution is widespread in ocean ecosystems worldwide, but it is unknown if plastic offers a unique habitat for bacteria compared to communities in the water column and attached to naturally-occurring organic particles. The large set of samples taken during the Tara-Mediterranean expedition revealed for the first time a clear niche partitioning between free-living (FL), organic particle-attached (PA) and the recently introduced plastic marine debris (PMD). Bacterial counts in PMD presented higher cell enrichment factors than generally observed for PA fraction, when compared to FL bacteria in the surrounding waters. Taxonomic diversity was also higher in the PMD communities, where higher evenness indicated a favorable environment for a very large number of species. Cyanobacteria were particularly overrepresented in PMD, together with essential functions for biofilm formation and maturation. The community distinction between the three habitats was consistent across the large-scale sampling in the Western Mediterranean basin. 'Plastic specific bacteria' recovered only on the PMD represented half of the OTUs, thus forming a distinct habitat that should be further considered for understanding microbial biodiversity in changing marine ecosystems.

Laparoscopic surgery and corticoadrenalomas.

BACKGROUND: Nowadays, laparoscopic adrenalectomy is the "gold standard" procedure for the treatment of benign lesions. However, the situation is not so clearcut when the issue is laparoscopic excision of malignant adrenal tumors. We present our results of laparoscopic adrenalectomy for treating malignant tumors over the past decade. METHODS: Between October 1995 and June 2004, 131 consecutive laparoscopic adrenalectomies were performed on 120 patients (11 synchronous bilateral procedures). All patients underwent a standardized investigation protocol during their workup for surgery. RESULTS: There were only two conversions to laparotomy (1.6%). Complications that occurred during the procedure were limited to six patients (5%). Postoperative 30-days mortality was nil. Postoperative complications occurred in five patients (4.7%) during the first 30 days of recovery. The median hospital stay for all patients was 2.5 days (range = 2-10 days). Twelve patients (9%) had a malignant tumor: nine corticoadrenalomas, one pleomorphic sarcoma, one metastatic deposit from a previously excised colonic cancer, and one malignant pheochromocytoma. At mean followup of 34 months, mean survival time was 42.3 months for corticoadrenalomas that had undergone laparoscopy versus 29.7 months for those who had had a laparotomy. Five of the nine patients are alive and well at a mean of 37 months following surgery. One patient developed pulmonary metastases one year postsurgery; they were responsive to mitotane. Five years later, the same patient had a reoperation for an intra-abdominal retrogastric recurrence of her tumor and continues to do well. Another patient developed pulmonary metastases 22 months following adrenalectomy. Two patients died of metastatic intra-abdominal disease 20 and 7 months postsurgery. CONCLUSION: When laparoscopic surgery is to used for cancer treatment, caution is the rule to maintain the primary objective of securing a survival rate at least as high as that for open surgery, without increased risk of recurrence. Considering the results presented within this study, it seems that the laparoscopic removal of a corticoadrenaloma should not worsen the prognosis, provided the surgeon respects the primary rules of oncologic resectional surgery. Any surgical conditions that would preclude the strict application of these criteria are contraindications to a laparoscopic procedure.

[Estimating mortality attribuable to PM10 particles in 9 French cities participating in the European programme Apheis]. / Estimation de la mortalité attribuable aux particules (PM10) dans les 9 villes françaises participant au programme européen Apheis.

The relationship between air pollution and mortality is now admissible with a sufficiently high level of causality proven. This link allows for health impact assessment to be carried out with a significant degree of accuracy, such as the case for the results which are presented here from the nine French cities involved in the Apheis programme. This health impact assessment is based on the methodology developed by the World Health Organization. The number of avoidable deaths is contained between categories ranging from 2.0 to 4.3, 4.0 to 8.9, and from 15.0 to 31.5 per 100,000 inhabitants according to very short term effects, short term effects and long term effects, respectively. There are two scenarios which can be envisioned for the reduction of fine particles levels which are capable of obtaining similar results for both very short term and short term effects. The first involves diminishing the daily concentrations which are above 20 microg/m3 until they reach this value, and the second entails systematically decreasing the daily levels by 5 microg/m3. The first strategy of reducing values to stabilize at 20 microg/m3 has been shown to be the one most favourable and promising for the long term effects. This strategy therefore confirms the reliability and strength of the recommendation formulated at the national level.

Submitochondrial localization and membrane topography of Ehrlich ascitic tumour cell glutaminase.

The intramitocondrial localization of the phosphate-activated glutaminase from Ehrlich cells has been examined by a combination of techniques, including: mitochondria subfractionation studies, chemical modification with sulfhydryl group reagents of different permeability, enzymatic digestion in both sides of the inner mitochondrial membrane, and immunological studies. Using alkaline extraction at high ionic strength, hypoosmotic shock and freezing-thawing cycle techniques, the enzyme was found in the particulate fraction. On the contrary, glutaminase activity was labile when subfractionation was carried out by digitonin/lubrol method; Western blot analysis localized the inactive enzyme in the matrix fraction. In addition, glutaminase was fully inactivated when mitoplasts were incubated with phospholipase A2 and phospholipase C. The enzyme also showed a non-linear Arrhenius plot with a break at 24 degrees C. The membrane-impermeant thiol reagents mersalyl and p-chloromercuriphenylsulfonic acid do not inhibit glutaminase activity in freeze-thawed mitochondria and mitoplasts, but N-ethylmaleimide, which is membrane permeant, strongly inhibited the enzyme. However, mersalyl and p-chloromercuriphenylsulfonic acid were effective inhibitors when the alkylation was performed on the matrix side of mitoplasts or using detergent-solubilized enzyme. Furthermore, trypsin digestion of mitoplasts was only effective inactivating glutaminase when the proteolysis was carried out on the matrix side of the vesicles. Enzyme-linked immunosorbent assay of the soluble and membrane fractions obtained in the preparation of submitochondrial particles, revealed that most of the enzyme was solubilized, but in the inactive form. Phase separation with Triton X-114 rendered most of the protein in the aqueous phase. These results taken together discard a transmembrane localization for the protein, whereas they are consistent with anchorage of glutaminase on the matrix side of the inner mitochondrial membrane, the matrix portion of the enzyme being relevant for its function.

Polyamine contents of human breast cancer cells treated with the cytotoxic agents chlorpheniramine and dehydrodidemnin B.

The cytotoxic agents chlorpheniramine and dehydrodidemnin B decreased the cell growth of estrogen receptor-negative human breast cancer cells MDA-MB231 and estrogen receptor-positive MCF-7, after 48 h treatment. Both agents reduced ornithine decarboxylase activity, but polyamine levels were increased in MDA-MB231 cells treated with dehydrodidemnin B. MCF-7 cells when treated with dehydrodidemnin B showed significant increases in spermidine and spermine contents. The results suggest that besides other effects, the cytotoxicity of DDB could be explained in part by the over-accumulation of spermidine and spermine.

Immunogenicity and safety of a new liquid hexavalent combined vaccine compared with separate administration of reference licensed vaccines in infants.

OBJECTIVE: The immunogenicity and safety of a new liquid hexavalent vaccine (diphtheria-tetanus-acellular pertussis-inactivated polio vaccine-hepatitis B-polyribosyl ribitol phosphate conjugated to tetanus protein; Hexavac; Aventis Pasteur MSD, Lyon, France) are compared with those of reference vaccines [diphtheria-tetanus-acellular pertussis-inactivated polio vaccine reconstituting lyophilized purified Haemophilus influenzae polysaccharide conjugated to tetanus protein vaccine (Pentavac; Aventis Pasteur MSD) and hepatitis B vaccine (H-B-Vax II; Aventis Pasteur MSD)] injected separately at the same visit in a prospective multicenter, comparative, open label trial. METHODS: Infants were randomized to receive Hexavac (n = 423) or Pentavac and H-B-Vax II (n = 425) as a primary immunization series at 2, 4 and 6 months of age. Seroprotection and seroconversion rates against all antigens at 1 month after the primary series were compared between the two vaccine groups with 95% confidence intervals (CI0.95) and were considered clinically equivalent (not inferior) when the upper limit of the 95% confidence interval on the difference (reference, hexavalent) was below predefined differences. RESULTS: Hexavac met and surpassed the pre-defined criteria for clinical equivalence to Pentavac and H-B-Vax II given concomitantly. It elicited similar seroprotection and seroconversion rates against all antigens. Seroprotection and seroconversion rates obtained 1 month after the third dose of Hexavac were >90% for all antigens. The postimmunization antibody geometric mean titers (GMT) for hepatitis B and purified Haemophilus influenzae polysaccharide were about 2-fold higher in infants who received the reference vaccines than in infants who had received Hexavac. GMTs for poliovirus antibodies tended to be enhanced in infants vaccinated with Hexavac. GMTs for all other antigens were very similar among both groups. Hexavac was generally well-tolerated. At least one local reaction was reported in 20.3% of Hexavac injections compared with 15.8% at the Pentavac injections site and 3.8% at the H-B-Vax II injections site. These reactions were generally mild and transient. At least one systemic adverse event was reported in 45.7% of Hexavac injections compared with 42.2% of Pentavac and H-B-Vax II injections (mild fever, irritability and drowsiness were most frequently reported). The frequency of adverse events was not significantly different between groups. No vaccine-related serious adverse event occurred during the study. CONCLUSION: This liquid hexavalent vaccine was generally well-tolerated and provided immune responses adequate to be protective against six infectious diseases with a single injection, given at 2, 4 and 6 months of age.

Safety and immunogenicity of three doses of a Neisseria meningitidis A + C diphtheria conjugate vaccine in infants from Niger.

BACKGROUND: High rates of endemic disease and recurrent epidemics of serogroup A and C meningococcal meningitis continue to occur in sub-Saharan Africa. A meningococcal A + C polysaccharide diphtheria-toxoid-conjugated vaccine may address this issue. METHODS: In Niger three doses of a bivalent meningococcal A + C diphtheria-toxoid-conjugated vaccine (MenD), containing 1, 4 or 16 microg of each polysaccharide per dose, administered at 6, 10 and 14 weeks of age, were compared with Haemophilus influenzae type b-tetanus toxoid-conjugated (PRP-T) vaccine given with the same schedule or with a meningococcal A + C polysaccharide vaccine (MenPS) given at 10 and 14 weeks of age. One blood sample was taken at the time of enrollment (6 weeks of age) and another was taken 4 weeks after the primary series. RESULTS: All doses of MenD were well-tolerated. After the primary series a higher proportion of infants had detectable serum bactericidal activity against serogroup A for each dose of MenD (from 94% to 100%) than for MenPS (31%) or H. influenzae type b-tetanus toxoid-conjugated vaccine (18.9%); P < or = 0.05. Significant differences were also observed for serogroup C MenD 4 microg or MenD 16 microg (100%) vs. MenPS (69.7%) or Haemophilus influenzae type b-tetanus toxoid-conjugated vaccine (24.3%); P < or = 0.05. When MenPS vaccine was given to 11-month-old children, the immune response measured by both enzyme-linked immunosorbent assay and serum bactericidal assay was greater in those previously immunized with MenD than in those immunized with MenPS vaccine. CONCLUSION: MenD was safe among infants in Niger, and immunization led to significantly greater functional antibody activity than with MenPS. The 4-microg dose of MenD for both the A and C serogroups has been selected for further studies.

Changes in mRNAs for enzymes of glutamine metabolism in the tumor-bearing mouse.

Changes in the relative mRNA levels of phosphate-activated glutaminase (PAG) and glutamine synthetase (GS) in the liver and kidney of mice bearing a highly malignant strain of Ehrlich ascites tumor cells were determined at different days after tumor transplantation. Kidney glutaminase mRNA steadily increased, reaching maximum values at day 10 of tumor growth, while those of glutamine synthetase did not change, resulting in a sustained decrease of the GS/PAG ratio in the kidneys of tumor-bearing animals compared with controls. However, the GS/PAG ratio in the liver significantly increased, mainly due to a strong decrease in PAG, whereas GS mRNA levels remained almost unaffected. These results, combined with those previously reported on enzymatic activities and glutamine concentrations in the host-tumor system, suggest a long-term regulation of the host glutaminase enzymes in order to increase the circulating glutamine levels needed for tumor growth.

Ultrasound in organic electrosynthesis.

Mechanical effects induced by ultrasonication can be very helpful for the activation of electrochemical reactions. The continuous cleaning of the electrodes by ultrasound irradiation of the electrochemical cell or the enhancement of mass transfer at the electrodes are examples of such activation. Finally, ultrasonication can play an important part for the orientation of reactions whose selectivities are very sensitive to stirring. Two very different examples have been chosen to illustrate these phenomena: the indirect electrooxidation of di-ketone-L-sorbose into the corresponding ketogulonic acid and the direct electroreduction of acetophenone into pinacol.

[Incidence of inflammatory bowel diseases in the department of Puy-de-Dôme in 1993 and 1994. EPIMICI. Epidémiologie des Maladies Inflammatoires Cryptogenetiques de l'Intestin group]. / Incidence des maladies inflammatoires du tube digestif dans le département du Puy-de-Dôme en 1993 et 1994. EPIMICI. EPidémiologie des Maladies Inflammatoires Cryptogenetiques de l'Intestin group.

UNLABELLED: The aim of this prospective epidemiological study was to investigate the incidence of Inflammatory Bowel Disease in the Puy-de-Dôme county using the same methodology as EPIMAD's registry. METHODS: From 01/01/93 to 31/12/94, each gastroenterologist (n = 22) collected patients consulting for the first time with clinical symptoms compatible with inflammatory bowel disease. Data were reported on a questionnaire by an interviewer practitioner. The final diagnosis of Crohn's disease and ulcerative colitis was made in a blind manner by two expert gastroenterologists and recorded according to the Calkin's criteria as definite, probable, or possible diagnosis, or unclassifiable chronic colitis or acute colitis. RESULTS: 167 new cases were identified: 112 (67.1%) inflammatory bowel disease for the combined group of definite and probable cases with 79 Crohn's disease (70.5%), 29 ulcerative colitis (25.9%) of which 11 ulcerative proctitis (37.9%), 4 unclassifiable chronic colitis (3.6%) and 55 acute colitis (32.9%). The crude and age-adjusted incidence (per 10(5)/year) was respectively 6.6 and 5.7 for Crohn's disease and 2.4 and 1.9 for ulcerative colitis. The highest age-specific incidence rate for Crohn's disease was between 40-49 years (14.1) and for ulcerative colitis between 80-89 years (6.8). The female/male ratio was 0.8 for Crohn's disease and 1.1 for ulcerative colitis. The median age at the time of diagnosis was 42.6 years for Crohn's disease and 35.3 years for ulcerative colitis. CONCLUSIONS: These preliminary findings revealed a high incidence of Crohn's disease and low of ulcerative colitis in this county. However, these results must be managed carefully because these data were recorded only on two years and the inflammatory bowel disease classified possible and acute colitis require a follow-up.

[Severe potassium dichromate poisoning after accidental nasal introduction]. / Intoxication grave au dichromate de potassium après introduction nasale accidentelle.

BACKGROUND: Nasal foreign body (NFB) is a common situation in pediatrics. Poisoning is a rare complication of NFB insertion. We report a case of acute potassium dichromate poisoning secondary to NFB insertion. CASE REPORT: Six days after insertion of a NFB, progressive occurrence of diarrhea, vomiting, nasal obstruction, acute renal failure, pancreatitis, hepatitis and drowsiness justified hospitalization of a 3-year-old girl in the pediatric intensive care unit. Acute potassium dichromate poisoning was confirmed by high plasma chromium level and by the spectrophotometric analysis of the crystal. Recovery was satisfactory with supportive treatment. An official survey allowed to discover that the crystal was freely sold and that its toxicity was unknown by dealers, while no information was given to the customers. CONCLUSION: Transmucosal absorption of toxics is an unusual severe potential hazard that should be evoked to allow a rapid management. After the discovery and withdrawal of a NFB, occurrence of systemic symptoms, even trivial, must make one suspect a poisoning. In this circumstance, analysis of the foreign body should be done, associated with toxicologic dosages. This case report illustrates that potassium dichromate poisoning is a severe medical condition and that its clinical presentation assume a large widespread of symptoms due to multiple organ involvement.