A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.

We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.

Differential sodium and potassium transport selectivities of the rice OsHKT2;1 and OsHKT2;2 transporters in plant cells.

Na(+) and K(+) homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na(+) transport or Na(+) and K(+) transport when expressed in Xenopus laevis oocytes and yeast. However, the Na(+)/K(+) selectivities of the K(+)-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5' untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K(+) permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na(+) uptake, but little Rb(+) uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na(+)-K(+) cotransport in plant cells such that extracellular K(+) stimulated OsHKT2;2-mediated Na(+) influx and vice versa. Furthermore, at millimolar Na(+) concentrations, OsHKT2;2 mediated Na(+) influx into plant cells without adding extracellular K(+). This study shows that the Na(+)/K(+) selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K(+) and Ca(2+) down-regulated OsHKT2;1-mediated Na(+) influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.

Macrophage PPAR gamma is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones.

PPAR gamma is required for fat cell development and is the molecular target of antidiabetic thiazolidinediones (TZDs), which exert insulin-sensitizing effects in adipose tissue, skeletal muscle, and liver. Unexpectedly, we found that inactivation of PPAR gamma in macrophages results in the development of significant glucose intolerance plus skeletal muscle and hepatic insulin resistance in lean mice fed a normal diet. This phenotype was associated with increased expression of inflammatory markers and impaired insulin signaling in adipose tissue, muscle, and liver. PPAR gamma-deficient macrophages secreted elevated levels of factors that impair insulin responsiveness in muscle cells in a manner that was enhanced by exposure to FFAs. Consistent with this, the relative degree of insulin resistance became more severe in mice lacking macrophage PPAR gamma following high-fat feeding, and these mice were only partially responsive to TZD treatment. These findings reveal an essential role of PPAR gamma in macrophages for the maintenance of whole-body insulin action and in mediating the antidiabetic actions of TZDs.

Normal hematopoiesis after conditional targeting of RXRalpha in murine hematopoietic stem/progenitor cells.

Because of the retinoic acid receptor-alpha (RARalpha) gene's involvement in acute promyelocytic leukemia, the important role of RARs in hematopoiesis is now well established. However, relatively few studies of hematopoiesis have focused on the role of the retinoid X receptors (RXRs), the obligate heterodimeric partners of the RARs. We sought to establish whether conditional targeting of RXRalpha in early hematopoietic progenitors, ideally to the level of the hematopoietic stem cell (HSC), would compromise hematopoiesis. For hematopoietic targeting of RXRalpha, we characterized IFN-inducible MxCre mice for use in studying the role of RXRalpha in hematopoiesis. We established that MxCre executes recombination of loxP-flanked RXRalpha in hematopoietic progenitors immunophenotypically enriched for HSC, leading to widespread and sustained targeting of RXRalpha in hematopoietic cells. However, we found no evidence of hematologic compromise in mice lacking RXRalpha, suggesting that RXRalpha is dispensable for normal murine hematopoiesis. Nonetheless, RXRalpha null bone marrow cells cultured in methylcellulose form colonies more efficiently than bone marrow cells obtained from control mice. This result suggests that although RXRalpha is not required for murine hematopoiesis, there may be hematopoietic signaling pathways that respond selectively to RXRalpha or settings in which combined expression of RXR (alpha, beta, and gamma) is limiting.

A Murine Model of Lipopolysaccharide-Induced Peri-Implant Mucositis and Peri-Implantitis.

Dental implants are a widely used treatment option for tooth replacement. However, they are susceptible to inflammatory diseases such as peri-implant mucositis and peri-implantitis, which are highly prevalent and may lead to implant loss. Unfortunately, the understanding of the pathogenesis of peri-implant mucositis and peri-implantitis is fragmented and incomplete. Therefore, the availability of a reproducible animal model to study these inflammatory diseases would facilitate the dissection of their pathogenic mechanisms. The objective of this study is to propose a murine model of experimental peri-implant mucositis and peri-implantitis. Screw-shaped titanium implants were placed in the upper healed edentulous alveolar ridges of C57BL/6J mice 8 weeks after tooth extraction. Following 4 weeks of osseointegration, Porphyromonas gingivalis -lipolysaccharide (LPS) injections were delivered to the peri-implant soft tissues for 6 weeks. No-injections and vehicle injections were utilized as controls. Peri-implant mucositis and peri-implantitis were assessed clinically, radiographically (microcomputerized tomograph [CT]), and histologically following LPS-treatment. LPS-injections resulted in a significant increase in soft tissue edema around the head of the implants as compared to the control groups. Micro-CT analysis revealed significantly greater bone loss in the LPS-treated implants. Histological analysis of the specimens demonstrated that the LPS-group had increased soft tissue vascularity, which harbored a dense mixed inflammatory cell infiltrate, and the bone exhibited noticeable osteoclast activity. The induction of peri-implant mucositis and peri-implantitis in mice via localized delivery of bacterial LPS has been demonstrated. We anticipate that this model will contribute to the development of more effective preventive and therapeutic approaches for these 2 conditions.

Effect of supplementation of leukemia inhibitory factor and epidermal growth factor on murine embryonic development in vitro, implantation, and outcome of offspring.

OBJECTIVES: To evaluate the supplementation of leukemia inhibitory factor (LIF) or epidermal growth factor (EGF) in a single culture system (IVF50) and a sequential culture system (IVF50-S2) to determine whether they had any additional benefit on embryo development and implantation. DESIGN: Prospective controlled animal study. SETTING: University research laboratory. SUBJECT(S): Two-cell embryos from F1(CBA x C57BL) mice. INTERVENTION(S): Embryos were randomly cultured in different growth factor-treated or untreated in vitro systems. MAIN OUTCOME MEASURE(S): Blastocyst formation and morphology, total cell numbers of day 5 blastocysts, birth rates, and characteristics of the offspring. RESULT(S): The beneficial effects of LIF or EGF on blastocyst development and morphology were observed only in IVF50 medium but not in sequential IVF50-S2 media. In addition, blastocysts generated from the LIF-supplemented cultures had higher total cell numbers and higher total birth rates after transfer compared to those from IVF50 medium alone. No significant effect on fetal development was observed but pups born after treatment in LIF-supplemented sequential cultures had prolonged gestation and increased birth weights. CONCLUSION(S): The beneficial effects of LIF and EGF on in vitro blastocyst development are mainly seen under suboptimal culture conditions (simple medium only) and their effects are masked in an improved in vitro system (sequential culture media). Supplementation of culture media with such factors, however, is still an attractive approach in enhancing embryo viability through increased total cell numbers. The beneficial effect of LIF in improving implantation and subsequent increased birth rates should be further explored.

Mechanical stability and clinical applicability assessment of novel orthodontic mini-implant design.

OBJECTIVE: To compare the stability and clinical applicability of a novel orthodontic mini-implant design (N2) with the most widely used commercially available (CA) design. MATERIALS AND METHODS: Two groups of mini-implants were tested: a CA design (1.5-mm diameter, 6-mm length) and N2 (3-mm diameter, 2-mm length, tapered shape). Implants were inserted in bone blocks of cortical bone simulation with varying densities (20 pounds per cubic foot [pcf], 30 pcf, and 40 pcf). A torque test was used to measure maximum insertion torque (MIT) and maximum removal torque (MRT). Compression and tension force vectors were applied at angles of 10°, 20°, 30°, and 40° using customized load pins to determine primary stability. RESULTS: Mean MIT and MRT were higher in the N2 than the CA design at all three cortical bone densities except MRT in 20 pcf bone (not statistically significant). The mean compression force required to displace the N2 at all distances and angulations was greater for the N2 than the CA design. At all displacement distances, the highest mean tension force required for N2 displacement was at 10° angulation, whereas at 30° and 40°, the mean tension force required to displace the CA design was greater. CONCLUSIONS: The primary stability of the N2 is superior to that of the CA design and is promising for both orthodontic and orthopedic clinical applicability, especially under compression force. The short length of the N2 reduces risk of damage to anatomic structures and root proximity during placement and orthodontic treatment. The stability of the N2 may be compromised in areas of high bone density and highly angulated tension force.

A new multiple regression approach for the construction of genetic regulatory networks.

OBJECTIVE: Reconstruction of a genetic regulatory network from a given time-series gene expression data is an important research topic in systems biology. One of the main difficulties in building a genetic regulatory network lies in the fact that practical data set has a huge number of genes vs. a small number of sampling time points. In this paper, we propose a new linear regression model that may overcome this difficulty for uncovering the regulatory relationship in a genetic network. METHODS: The proposed multiple regression model makes use of the scale-free property of a real biological network. In particular, a filter is constructed by using this scale-free property and some appropriate statistical tests to remove redundant interactions among the genes. A model is then constructed by minimizing the gap between the observed and the predicted data. RESULTS: Numerical examples based on yeast gene expression data are given to demonstrate that the proposed model fits the practical data very well. Some interesting properties of the genes and the underlying network are also observed. CONCLUSIONS: In conclusion, we propose a new multiple regression model based on the scale-free property of real biological network for genetic regulatory network inference. Numerical results using yeast cell cycle gene expression dataset show the effectiveness of our method. We expect that the proposed method can be widely used for genetic network inference using high-throughput gene expression data from various species for systems biology discovery.

Rice OsHKT2;1 transporter mediates large Na+ influx component into K+-starved roots for growth.

Excessive accumulation of sodium in plants causes toxicity. No mutation that greatly diminishes sodium (Na+) influx into plant roots has been isolated. The OsHKT2;1 (previously named OsHKT1) transporter from rice functions as a relatively Na+-selective transporter in heterologous expression systems, but the in vivo function of OsHKT2;1 remains unknown. Here, we analyzed transposon-insertion rice lines disrupted in OsHKT2;1. Interestingly, three independent oshkt2;1-null alleles exhibited significantly reduced growth compared with wild-type plants under low Na+ and K+ starvation conditions. The mutant alleles accumulated less Na+, but not less K+, in roots and shoots. OsHKT2;1 was mainly expressed in the cortex and endodermis of roots. (22)Na+ tracer influx experiments revealed that Na+ influx into oshkt2;1-null roots was dramatically reduced compared with wild-type plants. A rapid repression of OsHKT2;1-mediated Na+ influx and mRNA reduction were found when wild-type plants were exposed to 30 mM NaCl. These analyses demonstrate that Na+ can enhance growth of rice under K+ starvation conditions, and that OsHKT2;1 is the central transporter for nutritional Na+ uptake into K+-starved rice roots.

Calcium regulation of sodium hypersensitivities of sos3 and athkt1 mutants.

T-DNA disruption mutations in the AtHKT1 gene have previously been shown to suppress the salt sensitivity of the sos3 mutant. However, both sos3 and athkt1 single mutants show sodium (Na+) hypersensitivity. In the present study we further analyzed the underlying mechanisms for these non-additive and counteracting Na+ sensitivities by characterizing athkt1-1 sos3 and athkt1-2 sos3 double mutant plants. Unexpectedly, mature double mutant plants grown in soil clearly showed an increased Na+ hypersensitivity compared with wild-type plants when plants were subjected to salinity stress. The salt sensitive phenotype of athkt1 sos3 double mutant plants was similar to that of athkt1 plants, which showed chlorosis in leaves and stems. The Na+ content in xylem sap samples of soil-grown athkt1 sos3 double and athkt1 single mutant plants showed dramatic Na+ overaccumulation in response to salinity stress. Salinity stress analyses using basic minimal nutrient medium and Murashige-Skoog (MS) medium revealed that athkt1 sos3 double mutant plants show a more athkt1 single mutant-like phenotype in the presence of 3 mM external Ca2+, but show a more sos3 single mutant-like phenotype in the presence of 1 mM external Ca2+. Taken together multiple analyses demonstrate that the external Ca2+ concentration strongly impacts the Na+ stress response of athkt1 sos3 double mutants. Furthermore, the presented findings show that SOS3 and AtHKT1 are physiologically distinct major determinants of salinity resistance such that sos3 more strongly causes Na+ overaccumulation in roots, whereas athkt1 causes an increase in Na+ levels in the xylem sap and shoots and a concomitant Na+ reduction in roots.

Enhanced salt tolerance mediated by AtHKT1 transporter-induced Na unloading from xylem vessels to xylem parenchyma cells.

AtHKT1 is a sodium (Na+) transporter that functions in mediating tolerance to salt stress. To investigate the membrane targeting of AtHKT1 and its expression at the translational level, antibodies were generated against peptides corresponding to the first pore of AtHKT1. Immunoelectron microscopy studies using anti-AtHKT1 antibodies demonstrate that AtHKT1 is targeted to the plasma membrane in xylem parenchyma cells in leaves. AtHKT1 expression in xylem parenchyma cells was also confirmed by AtHKT1 promoter-GUS reporter gene analyses. Interestingly, AtHKT1 disruption alleles caused large increases in the Na+ content of the xylem sap and conversely reduced the Na+ content of the phloem sap. The athkt1 mutant alleles had a smaller and inverse influence on the potassium (K+) content compared with the Na+ content of the xylem, suggesting that K+ transport may be indirectly affected. The expression of AtHKT1 was modulated not only by the concentrations of Na+ and K+ but also by the osmolality of non-ionic compounds. These findings show that AtHKT1 selectively unloads sodium directly from xylem vessels to xylem parenchyma cells. AtHKT1 mediates osmolality balance between xylem vessels and xylem parenchyma cells under saline conditions. Thus AtHKT1 reduces the sodium content in xylem vessels and leaves, thereby playing a central role in protecting plant leaves from salinity stress.