Daptomycin dosing based on ideal body weight versus actual body weight: comparison of clinical outcomes.

Daptomycin use at our institution changed to ideal body weight dosing based on a published analysis of pharmacokinetic-pharmacodynamic efficacy target attainment, bacterial ecology, and a desire to reduce drug toxicity. The current study compared outcomes between actual body weight and ideal body weight dosing of daptomycin before and after this intervention. In the evaluable group, 69 patients received doses based on actual body weight and 48 patients received doses based on ideal body weight. Patients were treated for documented Enterococcus species, Staphylococcus aureus, or coagulase-negative Staphylococcus infections, including bloodstream, intraabdominal, skin and soft tissue, urinary, and bone. There was no statistically significant difference in clinical success between the groups (88.9% for actual body weight compared to 89.1% for ideal body weight, P = 0.97). After we adjusted for gender, age, body mass index, concomitant 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, infection type, and organism type, clinical success rates remained similar between groups (adjusted odds ratio of 0.68 in favor of actual body weight, 95% confidence interval [CI] of 0.13 to 3.55). Microbiological outcomes, length of stay, mortality, and adverse effects were also similar between groups. Further studies are warranted to confirm that ideal body weight dosing provides similar outcomes to actual body weight dosing for all patients and types of infections and organisms.

Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice.

INTRODUCTION: The controlled expression of many genes, including G-protein coupled receptors (GPCRs), is important for delineating gene functions in complex model systems. Binary systems for inducible regulation of transgene expression are widely used in mice. One system is the tTA/TRE expression system, composed of a tetracycline-dependent DNA binding factor and a separate tetracycline operon. However, the requirement for two separate transgenes (one for each tTA or TRE component) makes this system less amenable to models requiring directed cell targeting, increases the risk of multiple transgene integration sites, and requires extensive screening for appropriately-functioning clones. METHODS: We developed a single, polycistronic tetracycline-inducible expression platform to control the expression of multiple cistrons in mammalian cells. This platform has three basic constructs: regulator, responder, and destination vectors. The modular platform is compatible with both the TetOff (tTA) and TetOn (rtTA) systems. The modular Gateway recombineering-compatible components facilitate rapidly generating vectors to genetically modify mammalian cells. We apply this system to use the elongation factor 1α (EF1α) promoter to drive doxycycline-regulated expression of both the fluorescent marker mCherry and an engineered Gs-coupled GPCR "Rs1" separated by a 2A ribosomal skip site. RESULTS: We show that our combined expression construct drives expression of both the mCherry and Rs1 transgenes in a doxycycline-dependent manner. We successfully target the expression construct into the Rosa26 locus of mouse embryonic stem (ES) cells. Rs1 expression in mouse ES cells increases cAMP accumulation via both basal and ligand-induced Gs mechanisms and is associated with increased embryoid body size. Heterozygous mice carrying the Rs1 expression construct showed normal growth and weight, and developed small increases in bone formation that could be observed in the calvaria. CONCLUSIONS: Our results demonstrate the feasibility of a single-vector strategy that combines both the tTA and TRE tetracycline-regulated components for use in cells and mouse models. Although the EF1α promoter is useful for driving expression in pluripotent cells, a single copy of the EF1α promoter did not drive high levels of mCherry and Rs1 expression in the differentiated tissues of adult mice. These findings indicate that promoter selection is an important factor when developing transgene expression models.

Modifying ligand-induced and constitutive signaling of the human 5-HT4 receptor.

G protein-coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their in vivo functions have been hampered by the molecular and functional diversity of GPCRs and the paucity of ligands with specific signaling effects. To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT(4b) receptor, a GPCR with high constitutive G(s) signaling and strong ligand-induced G-protein activation of the G(s) and G(s/q) pathways. The first receptor in this series, 5-HT(4)-D(100)A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced G(s) signaling, but only a few (e.g., zacopride) also induced signaling via the G(q) pathway. Zacopride-induced G(q) signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT(2C) receptor. Additional point mutations (D(66)A and D(66)N) blocked constitutive G(s) signaling and lowered ligand-induced G(q) signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT(1A) conferred ligand-mediated G(i) signaling. This G(i)-coupled RASSL, Rs1.3, exhibited no measurable signaling to the G(s) or G(q) pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection.

Man1, an inner nuclear membrane protein, regulates vascular remodeling by modulating transforming growth factor beta signaling.

A growing number of integral inner nuclear membrane (INM) proteins have been implicated in diverse cellular functions. Man1, an INM protein, has recently been shown to regulate transforming growth factor (Tgf) beta superfamily signaling by interacting with receptor-associated Smads. However, the in vivo roles of Man1 have not been fully characterized. Here, we show that Man1 regulates vascular remodeling by analyzing Man1-deficient embryos lacking the Smad interacting domain. Man1-deficient embryos die at midgestation because of defects in embryonic vasculature; the primary capillary plexus forms, but subsequent remodeling is perturbed. It has been proposed that the angiogenesis process is divided into two balanced phases, the activation and resolution/maturation phases, both of which are regulated by Tgfbeta1. We have demonstrated, in Man1-deficient embryos, the expression of Tgfb1 is upregulated and Smad2/3 signaling is abnormally activated, resulting in increased extracellular matrix deposition, a hallmark of the resolution phase of angiogenesis. We have also showed that the recruitment of mural cells to the vascular wall is severely disturbed in mutants, which may lead to disruption of intercellular communication between endothelial and mural cells required for proper vascular remodeling. These results have revealed a novel role for Man1 in angiogenesis and provide the first evidence that vascular remodeling can be regulated at the INM through the interaction between Man1 and Smads.

Prelamin A and lamin A appear to be dispensable in the nuclear lamina.

Lamin A and lamin C, both products of Lmna, are key components of the nuclear lamina. In the mouse, a deficiency in both lamin A and lamin C leads to slow growth, muscle weakness, and death by 6 weeks of age. Fibroblasts deficient in lamins A and C contain misshapen and structurally weakened nuclei, and emerin is mislocalized away from the nuclear envelope. The physiologic rationale for the existence of the 2 different Lmna products lamin A and lamin C is unclear, although several reports have suggested that lamin A may have particularly important functions, for example in the targeting of emerin and lamin C to the nuclear envelope. Here we report the development of lamin C-only mice (Lmna(LCO/LCO)), which produce lamin C but no lamin A or prelamin A (the precursor to lamin A). Lmna(LCO/LCO) mice were entirely healthy, and Lmna(LCO/LCO) cells displayed normal emerin targeting and exhibited only very minimal alterations in nuclear shape and nuclear deformability. Thus, at least in the mouse, prelamin A and lamin A appear to be dispensable. Nevertheless, an accumulation of farnesyl-prelamin A (as occurs with a deficiency in the prelamin A processing enzyme Zmpste24) caused dramatically misshapen nuclei and progeria-like disease phenotypes. The apparent dispensability of prelamin A suggested that lamin A-related progeroid syndromes might be treated with impunity by reducing prelamin A synthesis. Remarkably, the presence of a single Lmna(LCO) allele eliminated the nuclear shape abnormalities and progeria-like disease phenotypes in Zmpste24-/- mice. Moreover, treating Zmpste24-/- cells with a prelamin A-specific antisense oligonucleotide reduced prelamin A levels and significantly reduced the frequency of misshapen nuclei. These studies suggest a new therapeutic strategy for treating progeria and other lamin A diseases.

Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice.

Zmpste24 is a metalloproteinase required for the processing of prelamin A to lamin A, a structural component of the nuclear lamina. Zmpste24 deficiency results in the accumulation of prelamin A within cells, a complete loss of mature lamin A, and misshapen nuclear envelopes. Zmpste24-deficient (Zmpste24(-/-)) mice exhibit retarded growth, alopecia, micrognathia, dental abnormalities, osteolytic lesions in bones, and osteoporosis, which are phenotypes shared with Hutchinson-Gilford progeria syndrome, a human disease caused by the synthesis of a mutant prelamin A that cannot undergo processing to lamin A. Zmpste24(-/-) mice also develop muscle weakness. We hypothesized that prelamin A might be toxic and that its accumulation in Zmpste24(-/-) mice is responsible for all of the disease phenotypes. We further hypothesized that Zmpste24(-/-) mice with half-normal levels of prelamin A (Zmpste24(-/-) mice with one Lmna knockout allele) would be subjected to less toxicity and be protected from disease. Thus, we bred and analyzed Zmpste24(-/-)Lmna(+/-) mice. As expected, prelamin A levels in Zmpste24(-/-)Lmna(+/-) cells were significantly reduced. Zmpste24(-/-)Lmna(+/-) mice were entirely normal, lacking all disease phenotypes, and misshapen nuclei were less frequent in Zmpste24(-/-)Lmna(+/-) cells than in Zmpste24(-/-) cells. These data suggest that prelamin A is toxic and that reducing its levels by as little as 50% provides striking protection from disease.

Characterization of NF-kappa B/I kappa B proteins in zebra fish and their involvement in notochord development.

Although largely involved in innate and adaptive immunity, NF-kappa B plays an important role in vertebrate development. In chicks, the inactivation of the NF-kappa B pathway induces functional alterations of the apical ectodermal ridge, which mediates limb outgrowth. In mice, the complete absence of NF-kappa B activity leads to prenatal death and neural tube defects. Here, we report the cloning and characterization of NF-kappa B/I kappa B proteins in zebra fish. Despite being ubiquitously expressed among the embryonic tissues, NF-kappa B/I kappa B members present distinct patterns of gene expression during the early zebra fish development. Biochemical assays indicate that zebra fish NF-kappa B proteins are able to bind consensus DNA-binding (kappa B) sites and inhibitory I kappa B alpha proteins from mammals. We show that zebra fish I kappa B alphas are degraded in a time-dependent manner after induction of transduced murine embryo fibroblasts (MEFs) and that these proteins are able to rescue NF-kappa B activity in I kappa B alpha(-/-) MEFs. Expression of a dominant-negative form of the murine I kappa B alpha (mI kappa B alpha M), which is able to block NF-kappa B in zebra fish cells, interferes with the notochord differentiation, generating no tail (ntl)-like embryos. This phenotype can be rescued by coinjection of the T-box gene ntl (Brachyury homologue), which is typically required for the formation of posterior mesoderm and axial development, suggesting that ntl lies downstream of NF-kappa B . We further show that ntl and Brachyury promoter regions contain functional kappa B sites and NF-kappa B can directly modulate ntl expression. Our study illustrates the conservation and compatibility of NF-kappa B/I kappa B proteins among vertebrates and the importance of NF-kappa B pathway in mesoderm formation during early embryogenesis.

MKP3 mediates the cellular response to FGF8 signalling in the vertebrate limb.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol-3-OH kinase (PI3K)/Akt pathways are involved in the regulatory mechanisms of several cellular processes including proliferation, differentiation and apoptosis. Here we show that during chick, mouse and zebrafish limb/fin development, a known MAPK/ERK regulator, Mkp3, is induced in the mesenchyme by fibroblast growth factor 8 (FGF8) signalling, through the PI3K/Akt pathway. This correlates with a high level of phosphorylated ERK in the apical ectodermal ridge (AER), where Mkp3 expression is excluded. Conversely, phosphorylated Akt is detected only in the mesenchyme. Constitutively active Mek1, as well as the downregulation of Mkp3 by small interfering RNA (siRNA), induced apoptosis in the mesenchyme. This suggests that MKP3 has a key role in mediating the proliferative, anti-apoptotic signalling of AER-derived FGF8.

The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10.

A major gap in our knowledge of development is how the growth and identity of tissues and organs are linked during embryogenesis. The vertebrate limb is one of the best models to study these processes. Combining mutant analyses with gain- and loss-of-function approaches in zebrafish and chick embryos, we show that Tbx5, in addition to its role governing forelimb identity, is both necessary and sufficient for limb outgrowth. We find that Tbx5 functions downstream of WNT signaling to regulate Fgf10, which, in turn, maintains Tbx5 expression during limb outgrowth. Furthermore, our results indicate that Tbx5 and Wnt2b function together to initiate and specify forelimb outgrowth and identity. The molecular interactions governed by members of the T-box, Wnt and Fgf gene families uncovered in this study provide a framework for understanding not only limb development, but how outgrowth and identity of other tissues and organs of the embryo may be regulated.