Temperature multiscale entropy analysis: a promising marker for early prediction of mortality in septic patients.

A few studies estimating temperature complexity have found decreased Shannon entropy, during severe stress. In this study, we measured both Shannon and Tsallis entropy of temperature signals in a cohort of critically ill patients and compared these measures with the sequential organ failure assessment (SOFA) score, in terms of intensive care unit (ICU) mortality. Skin temperature was recorded in 21 mechanically ventilated patients, who developed sepsis and septic shock during the first 24 h of an ICU-acquired infection. Shannon and Tsallis entropies were calculated in wavelet-based decompositions of the temperature signal. Statistically significant differences of entropy features were tested between survivors and non-survivors and classification models were built, for predicting final outcome. Significantly reduced Tsallis and Shannon entropies were found in non-survivors (seven patients, 33%) as compared to survivors. Wavelet measurements of both entropy metrics were found to predict ICU mortality better than SOFA, according to a combination of area under the curve, sensitivity and specificity values. Both entropies exhibited similar prognostic accuracy. Combination of SOFA and entropy presented improved the outcome of univariate models. We suggest that reduced wavelet Shannon and Tsallis entropies of temperature signals may complement SOFA in mortality prediction, during the first 24 h of an ICU-acquired infection.

Early embryonic lethality in genetically engineered mice: diagnosis and phenotypic analysis.

Embryonic lethality is a common phenotype that occurs in mice that are homozygous for genetically engineered mutations. These phenotypes highlight the time and place that a gene is first required during embryogenesis. Early embryonic lethality (ie, before and up to mid-gestation) can be straightforward to analyze because the stage at which death occurs suggests why an embryo has failed. Here we summarize general strategies for analyzing early embryonic lethal phenotypes in genetically engineered mouse mutants.

The oocyte population is not renewed in transplanted or irradiated adult ovaries.

BACKGROUND: According to conventional theory, the oocyte population is not renewed in mammalian ovaries after birth. A new hypothesis proposes that oocytes are generated continuously from haematopoietic progenitor cells. There is, however, no evidence that they can ovulate, although they may partially restore fertility by organizing 'helper follicles'. The hypothesis that follicles can form de novo in adult ovaries has been tested in a transplant model. METHODS: Ovaries from adult mice were transplanted under the kidney capsule or into the ovarian bursa of histocompatible, transgenic CAG::H2B-EGFP host animals. Some donors were sterilized before transplantation by X-irradiation to ensure 'empty niches' were available for repopulation. The phenotype of follicular oocytes at 2, 4 and 8 weeks post-transplantation was scored by epifluorescence. RESULTS: A total of 819 oocytes were examined in 30 ovarian grafts. None expressed green fluorescence, as would be predicted if they had formed de novo from germ cell progenitors in the systemic circulation of the host. Furthermore, small follicles eliminated by irradiation were not replaced in transplanted ovaries, and the few growing follicles present were apparently survivors of the original population. CONCLUSIONS: No evidence was found to support the hypothesis that progenitor cells from extra-ovarian sources can repopulate the adult ovary. The findings are consistent with the conventional view that a limited number of oocytes are formed before birth and declines with age. The study did not, however, rule out the possibility that germline stem cells may reside in the adult ovary.

Effect of a policy for restriction of selected classes of antibiotics on antimicrobial drug cost and resistance.

Based on the instructions of the National Organization of Pharmaceutical Agents (Greece) from July 1, 2003, quinolones, 3( rd )and 4(th )generation cephalosporins, carbapenems, monobactams, glycopeptides, oxazolidinones, and streptogramins were considered as "restricted" antibiotics that could be used only with the approval of an Infectious Disease specialist. We analyzed the effect of the policy on the consumption and cost of antibiotics as a group and of specific classes, adjusted for the patient load, as well as on the antimicrobial resistance of isolated bacteria. We analyzed 5 trimesters (2 prior and 3 after the implementation of the new policy). A 20% and 16% reduction in adjusted consumption [in daily defined doses (DDDs)] and cost, respectively, of the restricted antibiotics was accomplished during the first trimester after implementation of the new policy. However, this was accompanied by a 36% and 56% increase in adjusted consumption and cost, respectively, of unrestricted antibiotics. A logistic regression model that we performed showed that the new policy had an independent positive effect on the in vitro antimicrobial susceptibility of Pseudomonas aeruginosa (p=0.051) but not of Acinetobacter baumannii and Escherichia coli isolates. Our data suggest that there are considerable limitations to the programs aiming to reduce the consumption of restricted antibiotics through the approval of their use by specialists, at least in some settings.

T-box genes in development: from hydra to humans.

The T-box gene family was uncovered less than a decade ago but has been recognized as important in controlling many and varied aspects of development in metazoans from hydra to humans. Extensive screening and database searching has revealed several subfamilies of genes with orthologs in species as diverse as Caenorhabditis elegans and humans. The defining feature of the family is a conserved sequence coding for a DNA-binding motif known as the T-box, named after the first-discovered T-box gene, T or Brachyury. Although several T-box proteins have been shown to function as transcriptional regulators, to date only a handful of downstream target genes have been discovered. Similarly, little is known about regulation of the T-box genes themselves. Although not limited to the embryo, expression of T-box genes is characteristically seen in dynamic and highly specific patterns in many tissues and organs during embryogenesis and organogenesis. The essential role of several T-box genes has been demonstrated by the developmental phenotypes of mutant animals.

DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1.

The DiGeorge/velocardiofacial syndrome (DGS/VCFS) is a relatively common human disorder, usually associated with deletions of chromosome 22q11. The genetic basis for the wide range of developmental anomalies in the heart, glands and facial structures has been elusive. We have investigated the potential role of one candidate gene, Tbx1, which encodes a transcription factor of the T-box family, by producing a null mutation in mice. We found that mice heterozygous for the mutation had a high incidence of cardiac outflow tract anomalies, thus modeling one of the major abnormalities of the human syndrome. Moreover, Tbx1-/- mice displayed a wide range of developmental anomalies encompassing almost all of the common DGS/VCFS features, including hypoplasia of the thymus and parathyroid glands, cardiac outflow tract abnormalities, abnormal facial structures, abnormal vertebrae and cleft palate. On the basis of this phenotype in mice, we propose that TBX1 in humans is a key gene in the etiology of DGS/VCFS.

Mapping and expression analysis of the mouse ortholog of Xenopus Eomesodermin.

The T-box gene family has been conserved throughout metazoan evolution. The genes code for putative transcription factors which share a uniquely defining DNA binding domain, known as the T-box ([Bollag et al., 1994]). They are implicated in the control of diverse developmental processes by their highly specific expression patterns throughout gastrulation and organogenesis in mouse and other species ([Chapman et al., 1996]) ([Gibson-Brown et al., 1998]), and by mutations in T-box genes that have profound developmental effects ([Papaioannou, 1997]; [Chapman and Papaioannou, 1998]; [Papaioannou and Silver, 1998]). In this report, we describe the mapping and expression pattern of the mouse ortholog of a gene, Eomesodermin, first identified in Xenopus ([Ryan et al., 1996]). The mouse gene was previously reported ([Wattler et al., 1998]) under the name MmEomes. The gene maps to mouse chromosome 9 in a region syntenic with human chromosome 3p. Mouse eomesodermin is expressed in the trophoblast of the blastocyst and in its derivative, the chorionic ectoderm. At gastrulation, eomesodermin is expressed in the primitive streak and embryonic mesoderm as well, but this expression disappears prior to the end of gastrulation. Later, eomesodermin is expressed in the developing forebrain, in a pattern largely overlapping a closely related T-box gene, Tbr1 ([Bulfone et al., 1995]), and is also seen in a localized area of each limb.

Cloning, mapping, and expression analysis of TBX15, a new member of the T-Box gene family.

The T-box gene family has been conserved throughout metazoan evolution and codes for putative transcription factors that share a uniquely defining DNA-binding domain. We have previously uncovered six mouse T-box genes with discrete spatial and temporal patterns of expression during embryogenesis. Here, we report a novel mouse T-box gene, Tbx15. The Tbx15 gene produces a 3.7-kb transcript with an open reading frame coding for a polypeptide with 602 amino acid residues. Phylogenetic analysis places the Tbx15 gene into a T-box subfamily that also includes mouse Tbx1, Drosophila H15, and nematode Ce-tbx-12 genes. We have mapped mouse Tbx15 to chromosome 3, at a position 49 cM from the centromere. During development, Tbx15 transcripts are first detected at embryonic day 9.5. The gene is expressed primarily in the cranio-facial region and in the developing limbs. An isolated human homolog, TBX15, has been mapped by in situ hybridization to chromosomal band 1p13. TBX15 appears to be an excellent candidate for the dominantly expressed acromegaloid facial appearance syndrome, which also maps to the short arm of human chromosome 1 and, like TBX15, is expressed prominently in the eyebrow regions.

Expression of T-box genes Tbx2-Tbx5 during chick organogenesis.

T-box genes encode putative transcription factors implicated in diverse developmental processes (Papaioannou, V.E. and Silver, L.M., 1998. BioEssays 20, 9-19). We have previously reported the embryonic expression patterns of T-box genes in mice (Chapman, D.L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S.I., Gibson-Brown, J.J., Cebra-Thomas, J., Bollag, R.J., Silver, L.M., Papaioannou, V.E., 1996. Dev. Dyn. 206, 379-390; Chapman, D.L., Agulnik, I., Hancock, S., Silver, L.M. and Papaioannou, V.E., 1996. Dev. Biol. 180, 534-542; Gibson-Brown, J.J., Agulnik, S.I., Chapman, D.L., Alexiou, M., Garvey, N., Silver, L.M., Papaioannou, V.E., 1996. Mech. Dev. 56, 93-101). Four of these genes (Tbx2-Tbx5) are represented in the mouse genome as two cognate, linked gene pairs (Agulnik, S.I., Garvey, N., Hancock, S., Ruvinsky, I., Chapman, D.L., Agulnik, I., Bollag, R., Papaioannou, V.E., Silver, L.M., 1996. Genetics 144, 249-254), and have all been implicated in playing important roles in limb development (Gibson-Brown, J.J., Agulnik, S.I., Chapman, D.L., Alexiou, M., Garvey, N., Silver, L.M., Papaioannou, V.E., 1996. Mech. Dev. 56, 93-101). To investigate the role of these genes in limb development, we cloned the chicken orthologs and report functional studies, as well as patterns of expression in the developing limbs, elsewhere (Gibson-Brown, J.J., Agulnik, S.I., Silver, L.M., Niswander, L., Papaioannou, V.E., Development, in press). This report details the patterns of expression of Tbx2-Tbx5 in chick embryonic tissues other than the limbs.

Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb specification and development.

We have recently shown in mice that four members of the T-box family of transcription factors (Tbx2-Tbx5) are expressed in developing limb buds, and that expression of two of these genes, Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. In this report, we investigate the role of these genes in limb specification and development, using the chick as a model system. We induced the formation of ectopic limbs in the flank of chick embryos to examine the relationship between the identity of the limb-specific T-box genes being expressed and the identity of limb structures that subsequently develop. We found that, whereas bud regions expressing Tbx4 developed characteristic leg structures, regions expressing Tbx5 developed characteristic wing features. In addition, heterotopic grafts of limb mesenchyme (wing bud into leg bud, and vice versa), which are known to retain the identity of the donor tissue after transplantation, retained autonomous expression of the appropriate, limb-specific T-box gene, with no evidence of regulation by the host bud. Thus there is a direct relationship between the identity of the structures that develop in normal, ectopic and recombinant limbs, and the identity of the T-box gene(s) being expressed. To investigate the regulation of T-box gene expression during limb development, we employed several other embryological manipulations. By surgically removing the apical ectodermal ridge (AER) from either wing or leg buds, we found that, in contrast to all other genes implicated in the patterning of developing appendages, maintenance of T-box gene expression is not dependent on the continued provision of signals from the AER or the zone of polarizing activity (ZPA). By generating an ectopic ZPA, by grafting a sonic hedgehog (SHH)-expressing cell pellet under the anterior AER, we found that Tbx2 expression can lie downstream of SHH. Finally, by grafting a SHH-expressing cell pellet to the anterior margin of a bud from which the AER had been removed, we found that Tbx2 may be a direct, short-range target of SHH. Our findings suggest that these genes are intimately involved in limb development and the specification of limb identity, and a new model for the evolution of vertebrate appendages is proposed.

The T-box gene family.

A novel family of transcription factors that appears to play a critical role in the development of all animal species was recently uncovered on the basis of homology of the DNA binding domain of the Brachyury, or T locus, gene product. Phylogenetic studies have shown the ancient origin of this gene family, which has been named the T-box family, prior to the divergence of metazoa from a common ancestral type. T-box genes have now been identified in the genomes of C. elegans, Drosophila, sea urchin, ascidian, amphioxus, Xenopus, chick, zebrafish, mouse, and human and will probably be found in the genomes of all animals. Although functional analyses of T-box family members have just begun, the results show a wide range of roles in developmental processes that extend over time from the unfertilized egg through organogenesis. Only a few mutations in T-box genes are known, but all have drastic effects on development, including a targeted mutation in mice causing an embryonic lethal phenotype, and two human T-box gene mutations that results in developmental syndromes. This review presents a current overview of progress made in the analysis of T-box genes and their products in a variety of model systems.

Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6.

Somites, segmented mesodermal units of the vertebrate embryo, are the precursors of adult skeletal muscle, bone and cartilage. During embryogenesis, somite progenitor cells ingress through the primitive streak, move laterally to a paraxial position (alongside the body axis) and segment into epithelial somites. Little is known about how this paraxial mesoderm tissue is specified. We have previously described a mouse T-box gene, Tbx6, which codes for a putative DNA-binding protein. The embryonic pattern of expression of Tbx6 in somite precursor cells suggests that this gene may be involved in the specification of paraxial mesoderm. We now report the creation of a mutation in Tbx6 that profoundly affects the differentiation of paraxial mesoderm. Irregular somites form in the neck region of mutant embryos, whereas more posterior paraxial tissue does not form somites but instead differentiates along a neural pathway, forming neural-tube-like structures that flank the axial neural tube. These paraxial tubes show dorsal/ventral patterning that is characteristic of the neural tube, and have differentiated motor neurons. These results indicate that Tbx6 is needed for cells to choose between a mesodermal and a neuronal differentiation pathway during gastrulation; Tbx6 is essential for the specification of posterior paraxial mesoderm, and in its absence cells destined to form posterior somites differentiate along a neuronal pathway.

Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizygous embryos.

Mutations of the human BRCA1 and BRCA2 genes encoding tumor suppressors have been implicated in inherited predisposition to breast and other cancers. Disruption of the homologous mouse genes Brca1 and Brca2 by targeting showed that they both have indispensable roles during embryogenesis, because nullizygous embryos become developmentally retarded and disorganized, and die early in development. In Brca1 mutants, the onset of abnormalities is earlier by one day and their phenotypic features and time of death are highly variable, whereas the phenotype of Brca2 null embryos is more uniform, and they all survive for at least 8.5 embryonic days. Observations with Brca1/Brca2 double nullizygotes raise the possibility that the two developmental pathways could be linked. Interestingly, the impact of the Brca1 or Brca2 null mutation is less severe in a p53 null background.

Tbx6, a mouse T-Box gene implicated in paraxial mesoderm formation at gastrulation.

The T-box genes constitute an evolutionarily conserved family of putative transcription factors which are expressed in discrete domains during embryogenesis, suggesting that they may play roles in inductive interactions. Members have been identified by virtue of their homology to the prototypical T-box gene, T or Brachyury, which is required for mesoderm formation and axial elongation during embryogenesis. We have previously reported the discovery of six new mouse T-box genes, Tbx1-Tbx6, and described the expression patterns of Tbx1-Tbx5 (Bollag et al., 1994; Agulnik et al., 1996; Chapman et al., 1996; Gibson-Brown et al., 1996). We have obtained cDNA clones encoding the full-length Tbx6 protein from screens of gastrulation-stage mouse cDNA libraries and determined the spatial and temporal distribution of Tbx6 transcripts during embryogenesis. The gene codes for a 1.9-kb transcript with an open reading frame coding for a 540-amino acid protein, with a predicted molecular weight of 59 kDa. Tbx6 maps to chromosome 7 and does not appear to be linked to any known mutation. Unlike other members of the mouse T-box gene family which are expressed in a wide variety of tissues derived from all germ layers, Tbx6 expression is quite restricted. Tbx6 transcripts are first detected in the gastrulation stage embryo in the primitive streak and newly recruited paraxial mesoderm. Later in development, Tbx6 expression is restricted to presomitic, paraxial mesoderm and to the tail bud, which replaces the streak as the source of mesoderm. Expression in the tail bud persists until 12. 5 days postcoitus. Tbx6 expression thus overlaps that of Brachyury in the primitive streak and tail bud, although Brachyury is expressed earlier in the primitive streak. Brachyury is also expressed in a second domain, the node and notochord, that is not shared with Tbx6. The onset of Tbx6 expression is not affected in homozygous null Brachyury mutant embryos at 7.5 days postcoitus. However, Tbx6 expression is extinguished in mutant embryos as soon as the Brachyury phenotype becomes evident at 8.5 days postcoitus, indicating that the continued expression of Tbx6 is directly or indirectly dependent upon Brachyury expression.