[Variations in soil enzyme stoichiometry and microbial nutrient limitations in Camellia oleifera plantations with different ages]. / ä¸åŒæž—é¾„æ²¹èŒ¶æž—åœŸå£¤é ¶åŒ–å­¦è®¡é‡ç‰¹å¾åŠå¾®ç”Ÿç‰©å »åˆ†é™åˆ¶å› ç´ .

The alteration of stand age instigates modifications in soil properties and microbial communities. Understanding the impacts of stand age on soil enzyme stoichiometry and microbial nutrient limitations in Camellia oleifera plantation is crucial for nutrient management. Taking C. oleifera plantation across four age groups (<10 a, 15-25 a, 30-50 a, >60 a) in a subtropical red soil region as test objects, we examined the response of soil enzyme stoichiometry and microbial nutrient limitations to change in stand age and analyzed the pathways for such responses. The results showed that, compared to that of stand age <10 a, enzyme C:N in the 15-25 a was increased and enzyme N:P was significantly reduced. Microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), and microbial biomass phosphorus (MBP) exhibited a trend of initially decreasing and then increasing with stand age. MBN and MBN:MBP were significantly higher in the <10 a compared to that in the 30-50 a. MBC:MBN was significantly higher in the 30-50 a and >60 a compared to the <10 a and 15-25 a. Results of redundancy analysis revealed that soil nutrients, microbial biomass and their stoichiometry explained 92.4% of the variations in enzyme stoichiometry. Partial least squares path modeling (PLS-PM) results demonstrated that soil organic carbon (SOC) had a positive effect on microbial C limitation; MBN, MBN:MBP, MBC:MBP, SOC, and total nitrogen had a nega-tive overall effect on microbial P limitation, whereas soil C:N had a positive overall effect on microbial P limitation. There was a significant positive correlation between microbial C and P limitations. With increasing stand age, microbial nutrient limitation shifted from N and P limitation (<10 a) to C and P limitation (15-25 a, 30-50 a, >60 a).

Mitofusin 2 plays a critical role in maintaining the functional integrity of the neuromuscular-skeletal axis.

PURPOSE: Mitofusin 2 (Mfn2) is one of two mitofusins involved in regulating mitochondrial size, shape and function, including mitophagy, an important cellular mechanism to limit oxidative stress. Reduced expression of Mfn2 has been associated with impaired osteoblast differentiation and function and a reduction in the number of viable osteocytes in bone. We hypothesized that the genetic absence of Mfn2 in these cells would increase their susceptibility to aging-associated metabolic stress, leading to a progressive impairment in skeletal homeostasis over time. METHODS: Mfn2 was selectively deleted in vivo at three different stages of osteoblast lineage commitment by crossing mice in which the Mfn2 gene was floxed with transgenic mice expressing Cre under the control of the promoter for Osterix (OSX), collagen1a1, or DMP1 (Dentin Matrix Acidic Phosphoprotein 1). RESULTS: Mice in which Mfn2 was deleted using DMP1-cre demonstrated a progressive and dramatic decline in bone mineral density (BMD) beginning at 10 weeks of age (n = 5 for each sex and each genotype from age 10 to 20 weeks). By 15 weeks, there was evidence for a functional decline in muscle performance as assessed using a rotarod apparatus (n = 3; 2 males/ 1 female for each genotype), accompanied by a decline in lean body mass. A marked reduction in trabecular bone mass was evident on bone histomorphometry, and biomechanical testing at 25 weeks (k/o: 2 male/1 female, control 2 male/2 female) revealed severely impaired femur strength. Extensive regional myofiber atrophy and degeneration was observed on skeletal muscle histology. Electron microscopy showed progressive disruption of cellular architecture, with disorganized sarcomeres and a bloated mitochondrial reticulum. There was also evidence of neurodegeneration within the ventral horn and roots of the lumbar spinal cord, which was accompanied by myelin loss and myofiber atrophy. Deletion of Mfn2 using OSX-cre or Col1a1-cre did not result in a musculoskeletal phenotype. Where possible, male and female animals were analyzed separately, but small numbers of animals in each group limited statistical power. For other outcomes, where sex was not considered, small sample sizes might still limit the strength of the observation. CONCLUSION: Despite known functional overlap of Mfn1 and Mfn2 in some tissues, and their co-expression in bone, muscle and spinal cord, deletion of Mfn2 using the 8 kB DMP1 promoter uncovered an important non-redundant role for Mfn2 in maintaining the neuromuscular/bone axis.

Investigation of the oxygen coverage effect on the direct epoxidation of propylene over copper through DFT calculations.

The direct epoxidation of propylene is one of the most important selective oxidation reactions in industry. The development of high-performance copper-based catalysts is the key to the selective oxidation technology and scientific research of propylene. The mechanism of propylene's partial oxidation catalyzed by Cu(111) under different oxygen coverage conditions was studied using density functional theory calculations and microkinetic modeling. We report here in detail two parallel reaction pathways: dehydrogenation and epoxidation. The transition states and energy distributions of the intermediates and products were calculated. The present results showed that propylene oxide (PO) selectivity was high under low oxygen coverage, and increasing the oxygen coverage would decrease the PO selectivity but increase the PO activity, and there was an inverse relationship between PO selectivity and activity. Increasing oxygen coverage would reduce the energy barrier for the C-O bond formation of C3H5O due to the weaker adsorption strength of C3H5, thus decreasing the PO formation selectivity. On the other hand, increasing oxygen coverage would reduce the energy barrier for the possible reaction steps of propylene epoxidation in general, and thus increasing the catalytic activity. It might be proposed that the active site for propylene epoxidation is the metallic copper or partially oxidized copper in terms of the change of PO formation selectivity with oxygen coverage.

Transplantation Outcomes with Donor Hearts after Circulatory Death.

BACKGROUND: Data showing the efficacy and safety of the transplantation of hearts obtained from donors after circulatory death as compared with hearts obtained from donors after brain death are limited. METHODS: We conducted a randomized, noninferiority trial in which adult candidates for heart transplantation were assigned in a 3:1 ratio to receive a heart after the circulatory death of the donor or a heart from a donor after brain death if that heart was available first (circulatory-death group) or to receive only a heart that had been preserved with the use of traditional cold storage after the brain death of the donor (brain-death group). The primary end point was the risk-adjusted survival at 6 months in the as-treated circulatory-death group as compared with the brain-death group. The primary safety end point was serious adverse events associated with the heart graft at 30 days after transplantation. RESULTS: A total of 180 patients underwent transplantation; 90 (assigned to the circulatory-death group) received a heart donated after circulatory death and 90 (regardless of group assignment) received a heart donated after brain death. A total of 166 transplant recipients were included in the as-treated primary analysis (80 who received a heart from a circulatory-death donor and 86 who received a heart from a brain-death donor). The risk-adjusted 6-month survival in the as-treated population was 94% (95% confidence interval [CI], 88 to 99) among recipients of a heart from a circulatory-death donor, as compared with 90% (95% CI, 84 to 97) among recipients of a heart from a brain-death donor (least-squares mean difference, -3 percentage points; 90% CI, -10 to 3; P<0.001 for noninferiority [margin, 20 percentage points]). There were no substantial between-group differences in the mean per-patient number of serious adverse events associated with the heart graft at 30 days after transplantation. CONCLUSIONS: In this trial, risk-adjusted survival at 6 months after transplantation with a donor heart that had been reanimated and assessed with the use of extracorporeal nonischemic perfusion after circulatory death was not inferior to that after standard-care transplantation with a donor heart that had been preserved with the use of cold storage after brain death. (Funded by TransMedics; ClinicalTrials.gov number, NCT03831048.).

Hue-indexing-based absolute phase retrieval method using a discrete hue sequence.

To achieve an effective phase unwrapping for hue-based fringe projection profilometry, this paper proposes a hue-indexing-based absolute phase retrieval method using a discrete hue sequence. First, the hue component is extracted as the wrapped phase for 3D reconstruction by projecting a programmed hue fringe pattern. Afterward, a hue-indexing sequence with a random combination of six unique hues from the hue map is designed for hue unwrapping. By assigning a hue index of the fringe geometric center, the defocusing effect in the hue unwrapping is corrected, where the fringe order of the wrapped hue is then uniquely identified. The simulations show that the root mean square (RMS) of the residual error is 2.2185×10-4 r a d, and the effectiveness of the proposed method is further verified through experiments using a plaster statue and a compressor blade. The measurement comparison of the proposed method and the coordinate measuring machine is provided, where the RMS error of the measured deviation is 4.09×10-2 m m.

Rice transcription factor GAMYB modulates bHLH142 and is homeostatically regulated by TDR during anther tapetal and pollen development.

GIBBERELLIN MYB GENE (GAMYB), UNDEVELOPED TAPETUM1 (UDT1), TDR INTERACTING PROTEIN2 (TIP2/bHLH142), TAPETUM DEGENERATION RETARDATION (TDR), and ETERNAL TAPETUM 1/DELAYED TAPETUM DEGENERATION (EAT1/DTD) are important transcription factors that play a crucial role during pollen development in rice. This study demonstrates that bHLH142 acts downstream of UDT1 and GAMYB and works as a 'hub' in these two pollen pathways. We show that GAMYB modulates bHLH142 expression through specific binding to the MYB motif of the bHLH142 promoter during the early stage of pollen development, while TDR acts as a transcriptional repressor of the GAMYB modulation of bHLH142 by binding to the E-box close to the MYB motif on the promoter. Altered expression of these transcription factors highlights that a tight, precise, and coordinated regulation among them is essential for normal pollen development. Most notably, we show that the regulatory pathways of GAMYB and UDT1 rely on bHLH142 in a direct and indirect manner, respectively, and function in different tissues with distinct biological roles during pollen development. This study advances our understanding of the molecular mechanisms of rice pollen development.

Selective deletion of the receptor for CSF1, c-fms, in osteoclasts results in a high bone mass phenotype, smaller osteoclasts in vivo and an impaired response to an anabolic PTH regimen.

The receptor for Colony Stimulating Factor 1 (CSF1), c-fms, is highly expressed on mature osteoclasts suggesting a role for this cytokine in regulating the function of these cells. Consistent with this idea, in vitro studies have documented a variety of effects of CSF1 in mature osteoclasts. To better define the role of CSF1 in these cells, we conditionally deleted c-fms in osteoclasts (c-fms-OC-/-) by crossing c-fmsflox/flox mice with mice expressing Cre under the control of the cathepsin K promoter. The c-fms-OC-/- mice were of normal weight and had normal tooth eruption. However, when quantified by DXA, bone mass was significantly higher in the spine and femur of female knock out mice and in the femurs of male knock out mice. MicroCT analyses of femurs showed that female c-fms-OC-/- mice had significantly increased trabecular bone mass with a similar trend in males and both sexes demonstrated significantly increased trabecular number and reduced trabecular spacing. Histomorphometric analysis of the femoral trabecular bone compartment demonstrated a trend towards increased numbers of osteoclasts, +26% in Noc/BPm and +22% in OcS/BS in the k/o animals but this change was not significant. However, when the cellular volume of osteoclasts was quantified, the c-fms-OC-/- cells were found to be significantly smaller than controls. Mature osteoclasts show a marked spreading response when exposed to CSF1 in a non-gradient fashion. However, osteoclasts freshly isolated from c-fms-OC-/- mice had a near complete abrogation of this response. C-fms-OC-/- mice treated with (1-34)hPTH 80 ng/kg/d in single daily subcutaneous doses for 29 days showed an attenuated anabolic response in trabecular bone compared to wild-type animals. Taken together, these data indicate an important non-redundant role for c-fms in regulating mature osteoclast function in vivo.

An Unanticipated Role for Sphingosine Kinase-2 in Bone and in the Anabolic Effect of Parathyroid Hormone.

Sphingosine-1-phosphate (S1P) is an anabolic clastokine. Sphingosine kinase (SPHK) is the rate-limiting enzyme in S1P production and has 2 isoforms. To evaluate the roles of SPHK1 and SPHK2 in bone, we examined the skeletal phenotype of mice with selective deletion of SPHK1 in osteoclasts (SPHK1-Oc-/-) and mice in which the SPHK2 gene was deleted in all tissues (SPHK2-/-). SPHK1-Oc-/- had normal bone mass. By contrast, SPHK2-/- female mice had a 14% lower spinal bone mineral density (BMD; P < 0.01) and males a 22% lower BMD at the same site (P < 0.001). SPHK2-/- and control mice were subsequently treated either with daily parathyroid hormone [PTH](1-34) or vehicle for 29 days. The response to PTH was significantly attenuated in the SPHK2-/-mice. The mean femoral bone volume to total volume fraction (BV/TV) increased by 24.8% in the PTH-treated female control animals vs 10.6% in the vehicle-treated female controls (P < 0.01). In contrast, in the SPHK2-/- female mice the difference in femoral trabecular BV/TV at the end of treatment was not significant (20.5 vs13.3%, PTH vs vehicle, P = NS). The anabolic response to PTH was significantly attenuated in the spine of male SPHK2-/- mice (29.7% vs 23.1%, PTH vs vehicle, in controls, P < 0.05; 26.9% vs 19.5% PTH vs vehicle in SPHK2-/- mice, P = NS). The spine responded normally in the SPHK2-/- female mice. Interestingly, suppression of sclerostin was blunted in the SPHK2-/- mice when those animals were treated with an anabolic PTH regimen. We conclude that SPHK2 has an important role in mediating both normal bone remodeling and the anabolic response to PTH.

Introducing the crystalline phase of dicalcium phosphate monohydrate.

Calcium orthophosphates (CaPs) are important in geology, biomineralization, animal metabolism and biomedicine, and constitute a structurally and chemically diverse class of minerals. In the case of dicalcium phosphates, ever since brushite (CaHPO4·2H2O, dicalcium phosphate dihydrate, DCPD) and monetite (CaHPO4, dicalcium phosphate, DCP) were first described in 19th century, the form with intermediary chemical formula CaHPO4·H2O (dicalcium phosphate monohydrate, DCPM) has remained elusive. Here, we report the synthesis and crystal structure determination of DCPM. This form of CaP is found to crystallize from amorphous calcium hydrogen phosphate (ACHP) in water-poor environments. The crystal structure of DCPM is determined to show a layered structure with a monoclinic symmetry. DCPM is metastable in water, but can be stabilized by organics, and has a higher alkalinity than DCP and DCPD. This study serves as an inspiration for the future exploration of DCPM's potential role in biomineralization, or biomedical applications.

The contribution of cross-talk between the cell-surface proteins CD36 and CD47-TSP-1 in osteoclast formation and function.

Antibody-mediated blockade of cluster of differentiation 47 (CD47)-thrombospondin-1 (TSP-1) interactions blocks osteoclast formation in vitro and attenuates parathyroid hormone (PTH)-induced hypercalcemia in vivo in mice. Hypercalcemia in this model reflects increased bone resorption. TSP-1 has two cell-associated binding partners, CD47 and CD36. The roles of these two molecules in mediating the effects of TSP1 in osteoclasts are unclear. Osteoclast formation was attenuated but not absent when preosteoclasts isolated from CD47-/- mice were cocultured with WT osteoblasts. Suppressing CD36 in osteoclast progenitors also attenuated osteoclast formation. The hypercalcemic response to a PTH infusion was blunted in CD47-/-/CD36-/- (double knockout (DKO)) female mice but not CD47-/- mice or CD36-/- animals, supporting a role for both CD47 and CD36 in mediating this effect. Consistent with this, DKO osteoclasts had impaired resorptive activity when analyzed in vitro Inhibition of nitric oxide (NO) signaling is known to promote osteoclastogenesis, and TSP-1 suppresses NO production and signaling. An anti-TSP-1 antibody increased NO production in osteoclasts, and the inhibitory effect of anti-TSP-1 on osteoclastogenesis was completely rescued by l-nitroarginine methyl ester (l-NAME), a competitive NO synthase inhibitor. Supportive of an important role for CD36 in mediating the pro-osteoclastogenic effects of TSP-1, engaging CD36 with a synthetic agonist, p907, suppressed NO production in anti-TSP-1-treated cultures, allowing osteoclast maturation to occur. These results establish that CD36 and CD47 both participate in mediating the actions of TSP-1 in osteoclasts and establish a physiologically relevant cross-talk in bone tissue between these two molecules.

Tightly Controlled Expression of bHLH142 Is Essential for Timely Tapetal Programmed Cell Death and Pollen Development in Rice.

Male sterility is important for hybrid seed production. Pollen development is regulated by a complex network. We previously showed that knockout of bHLH142 in rice (Oryza sativa) causes pollen sterility by interrupting tapetal programmed cell death (PCD) and bHLH142 coordinates with TDR to modulate the expression of EAT1. In this study, we demonstrated that overexpression of bHLH142 (OE142) under the control of the ubiquitin promoter also leads to male sterility in rice by triggering the premature onset of PCD. Protein of bHLH142 was found to accumulate specifically in the OE142 anthers. Overexpression of bHLH142 induced early expression of several key regulatory transcription factors in pollen development. In particular, the upregulation of EAT1 at the early stage of pollen development promoted premature PCD in the OE142 anthers, while its downregulation at the late stage impaired pollen development by suppressing genes involved in pollen wall biosynthesis, ROS scavenging and PCD. Collectively, these events led to male sterility in OE142. Analyses of related mutants further revealed the hierarchy of the pollen development regulatory gene network. Thus, the findings of this study advance our understanding of the central role played by bHLH142 in the regulatory network leading to pollen development in rice and how overexpression of its expression affects pollen development. Exploitation of this novel functionality of bHLH142 may confer a big advantage to hybrid seed production.

Breast cancer-associated gene 3 interacts with Rac1 and augments NF-κB signaling in vitro, but has no effect on RANKL-induced bone resorption in vivo.

Breast cancer-associated gene 3 (BCA3) is a recently identified adaptor protein whose functions are still being defined. BCA3 has been reported to be an important regulator of nuclear factor-κB (NF-κB) signaling. It has also been reported to interact with the small GTPase, Rac1. Consistent with that observation, in the present study, BCA3 was found to interact with nuclear Rac1 in 293 cells and influence NF-κB signaling. Additional experiments revealed that depending on cell type, BCA3 augmented, attenuated or had no effect on NF-κB signaling in vitro. Since canonical NF-κB signaling is a critical downstream target from activated receptor activator of nuclear factor κB (RANK) that is required for mature osteoclast formation and function, BCA3 was selectively overexpressed in osteoclasts in vivo using the cathepsin K promoter and the response to exogenous receptor activator of nuclear factor κB ligand (RANKL) administration was examined. Despite its ability to augment NF-κB signaling in other cells, transgenic animals injected with high-dose RANKL had the same hypercalcemic response as their wild­type littermates. Furthermore, the degree of bone loss induced by a 2-week infusion of low-dose RANKL was the same in both groups. Combined with earlier studies, the data from our study data indicate that BCA3 can affect NF-κB signaling and that BCA3 plays a cell-type dependent role in this process. The significance of the BCA3/NF-κB interaction in vivo in bone remains to be determined.

Neutrophil elastase stimulates MUC5AC expression in human biliary epithelial cells: a possible pathway of PKC/Nox/ROS.

INTRODUCTION: Bacterial infection and bile flow retardation form a vicious cycle which promotes stone formation and recurrence, and it seems that mucin overexpression plays an important role in this process. However, the mechanism of increased mucus secretion in the biliary tract by bacterial infection and its treatment remain unclear. MATERIAL AND METHODS: Human biliary epithelial cells were induced by neutrophil elastase (NE), and H2O2 production in the cell supernatants was detected by a specific kit, and then cells were pretreated with a H2O2 inhibitor, and expression of MUC5AC was detected by real-time polymerase chain reaction (PCR), Western blot, and immunohistochemistry. Moreover, selective PKC and Nox inhibitors, apocynin and bisindolylmaleimide I, were used to pretreat cells and detect H2O2, MUC5AC mRNA and protein expression. Then, we pretreated cells with selective inhibitors or NE, and detected transforming growth factor α (TGF-α) using an ELISA kit. RESULTS: H2O2 production increased in an NE dose-dependent manner (p < 0.001), and NE upregulated MUC5AC expression at both mRNA and protein levels, while DMTU, could reduce this high expression (p < 0.01 at mRNA level, p < 0.001 at grey analysis for western blot and p < 0.01 at mean density for immunohistochemical staining at protein level). Moreover, apocynin and bisindolylmaleimide I could reduce the H2O2 production stimulated by NE (p < 0.05), and reduce MUC5AC high expression (p < 0.01 at mRNA level, p < 0.001 at both grey analysis for western blot and mean density for immunohistochemical staining at protein level). In addition, NE induced TGF-α production, and any of the three selective inhibitors could reduce it (p < 0.05). CONCLUSIONS: NE-induced reactive oxygen species participated in the upregulation of MUC5AC production. Moreover, protein kinase C and NADPH oxidase (Nox) regulate MUC5AC production in NE-challenged human biliary epithelial cells.

Restless legs syndrome in maintenance hemodialysis patients: an epidemiologic survey in Hefei.

OBJECTIVE: To investigate the prevalence of restless legs syndrome (RLS) in maintenance hemodialysis (MHD) patients and its possible influencing factors. METHODS: MHD patients were consecutively enrolled from five hemodialysis centers in Hefei. Clinical, demographics, and laboratory data were recorded from December 2013 to March 2014. RLS diagnosis scale, Zung Self-Rating Depression Scale (SDS), Zung Self-Rating Anxiety Scale (SAS), Pittsburgh Sleep Quality Index (PSQI), and kidney disease and quality of life (KDQOLTM-36) were used for analysis. RESULTS: A total of 269 MHD patients (81 women, 188 men) were enrolled, among which 39 patients were diagnosed as RLS. The median duration of dialysis therapy was 33 months and the prevalence of RLS was 14.5%. Compared with RLS-negative patients, RLS-positive patients had lower hemoglobin level (98.67 ± 13.50 vs 106.34 ± 17.75, P = 0.011) and higher alkaline phosphatase concentration [131.0 (98.0, 226.0) vs 94.0 (69.8, 157.5), P = 0.001]. The multivariate logistic regression showed that high hemoglobin level (OR 0.975, 95% CI 0.956-0.995, P = 0.015) was a protective factor for RLS, while high alkaline phosphatase (OR 1.003, 95% CI 1.001-1.005, P = 0.018) was an independent risk factor for RLS. RLS patients had significantly higher PSQI scores (P < 0.001), reduced subjective sleep quality (P < 0.001), increased sleep latency (P < 0.007), shorter sleep duration (P < 0.001), lower sleep efficiency (P = 0.001), higher sleep disturbances (P < 0.001), and increased daytime dysfunction (P = 0.019). CONCLUSION: Our findings demonstrated that the prevalence of RLS was 14.5% in Hefei. High hemoglobin level was a protective factor for RLS, and high alkaline phosphatase was an independent risk factor. RLS affects many aspects of quality of life and sleep quality, which may contribute to the presence of depression and anxiety.

Enhanced osteogenesis and angiogenesis by mesoporous hydroxyapatite microspheres-derived simvastatin sustained release system for superior bone regeneration.

Biomaterials with both excellent osteogenic and angiogenic activities are desirable to repair massive bone defects. In this study, simvastatin with both osteogenic and angiogenic activities was incorporated into the mesoporous hydroxyapatite microspheres (MHMs) synthesized through a microwave-assisted hydrothermal method using fructose 1,6-bisphosphate trisodium salt (FBP) as an organic phosphorous source. The effects of the simvastatin-loaded MHMs (S-MHMs) on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) and angiogenesis in EA.hy926 cells were investigated. The results showed that the S-MHMs not only enhanced the expression of osteogenic markers in rBMSCs but also promoted the migration and tube formation of EA.hy926 cells. Furthermore, the S-MHMs were incorporated into collagen matrix to construct a novel S-MHMs/collagen composite scaffold. With the aid of MHMs, the water-insoluble simvastatin was homogenously incorporated into the hydrophilic collagen matrix and presented a sustained release profile. In vivo experiments showed that the S-MHMs/collagen scaffolds enhanced the bone regeneration and neovascularization simultaneously. These results demonstrated that the water-insoluble simvastatin could be incorporated into the MHMs and maintained its biological activities, more importantly, the S-MHMs/collagen scaffolds fabricated in this study are of immense potential in bone defect repair by enhancing osteogenesis and angiogenesis simultaneously.

Deletion of Rac in Mature Osteoclasts Causes Osteopetrosis, an Age-Dependent Change in Osteoclast Number, and a Reduced Number of Osteoblasts In Vivo.

Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1(flox/flox) mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2(-/-) mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism.

AgRP Neurons Regulate Bone Mass.

The hypothalamus has been implicated in skeletal metabolism. Whether hunger-promoting neurons of the arcuate nucleus impact the bone is not known. We generated multiple lines of mice to affect AgRP neuronal circuit integrity. We found that mice with Ucp2 gene deletion, in which AgRP neuronal function was impaired, were osteopenic. This phenotype was rescued by cell-selective reactivation of Ucp2 in AgRP neurons. When the AgRP circuitry was impaired by early postnatal deletion of AgRP neurons or by cell autonomous deletion of Sirt1 (AgRP-Sirt1(-/-)), mice also developed reduced bone mass. No impact of leptin receptor deletion in AgRP neurons was found on bone homeostasis. Suppression of sympathetic tone in AgRP-Sirt1(-/-) mice reversed osteopenia in transgenic animals. Taken together, these observations establish a significant regulatory role for AgRP neurons in skeletal bone metabolism independent of leptin action.

Transcriptome dynamics of developing maize leaves and genomewide prediction of cis elements and their cognate transcription factors.

Maize is a major crop and a model plant for studying C4 photosynthesis and leaf development. However, a genomewide regulatory network of leaf development is not yet available. This knowledge is useful for developing C3 crops to perform C4 photosynthesis for enhanced yields. Here, using 22 transcriptomes of developing maize leaves from dry seeds to 192 h post imbibition, we studied gene up- and down-regulation and functional transition during leaf development and inferred sets of strongly coexpressed genes. More significantly, we developed a method to predict transcription factor binding sites (TFBSs) and their cognate transcription factors (TFs) using genomic sequence and transcriptomic data. The method requires not only evolutionary conservation of candidate TFBSs and sets of strongly coexpressed genes but also that the genes in a gene set share the same Gene Ontology term so that they are involved in the same biological function. In addition, we developed another method to predict maize TF-TFBS pairs using known TF-TFBS pairs in Arabidopsis or rice. From these efforts, we predicted 1,340 novel TFBSs and 253 new TF-TFBS pairs in the maize genome, far exceeding the 30 TF-TFBS pairs currently known in maize. In most cases studied by both methods, the two methods gave similar predictions. In vitro tests of 12 predicted TF-TFBS interactions showed that our methods perform well. Our study has significantly expanded our knowledge on the regulatory network involved in maize leaf development.

The bHLH142 Transcription Factor Coordinates with TDR1 to Modulate the Expression of EAT1 and Regulate Pollen Development in Rice.

Male sterility plays an important role in F1 hybrid seed production. We identified a male-sterile rice (Oryza sativa) mutant with impaired pollen development and a single T-DNA insertion in the transcription factor gene bHLH142. Knockout mutants of bHLH142 exhibited retarded meiosis and defects in tapetal programmed cell death. RT-PCR and in situ hybridization analyses showed that bHLH142 is specifically expressed in the anther, in the tapetum, and in meiocytes during early meiosis. Three basic helix-loop-helix transcription factors, UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141) are known to function in rice pollen development. bHLH142 acts downstream of UDT1 and GAMYB but upstream of TDR1 and EAT1 in pollen development. In vivo and in vitro assays demonstrated that bHLH142 and TDR1 proteins interact. Transient promoter assays demonstrated that regulation of the EAT1 promoter requires bHLH142 and TDR1. Consistent with these results, 3D protein structure modeling predicted that bHLH142 and TDR1 form a heterodimer to bind to the EAT1 promoter. EAT1 positively regulates the expression of AP37 and AP25, which induce tapetal programmed cell death. Thus, in this study, we identified bHLH142 as having a pivotal role in tapetal programmed cell death and pollen development.

The transcription factor T-box 3 regulates colony-stimulating factor 1-dependent Jun dimerization protein 2 expression and plays an important role in osteoclastogenesis.

Colony-stimulating factor 1 (CSF1) is known to promote osteoclast progenitor survival, but its roles in osteoclast differentiation and mature osteoclast function are less well understood. In a microarray screen, Jun dimerization protein 2 (JDP2) was identified as significantly induced by CSF1. Recent reports indicate that JDP2 is required for normal osteoclastogenesis and skeletal metabolism. Because there are no reports on the transcriptional regulation of this gene, the DNA sequence from -2612 to +682 bp (relative to the transcription start site) of the JDP2 gene was cloned, and promoter activity was analyzed. The T box-binding element (TBE) between -191 and -141 bp was identified as the cis-element responsible for CSF1-dependent JDP2 expression. Using degenerate PCR, Tbx3 was identified as the major isoform binding the TBE. Overexpression of Tbx3 induced JDP2 promoter activity, whereas suppressing Tbx3 expression substantially attenuated CSF1-induced transcription. Suppressing Tbx3 in osteoclast precursors reduced JDP2 expression and significantly impaired RANKL/CSF1-induced osteoclastogenesis. A MEK1/2-specific inhibitor was found to block CSF1-induced JDP2 expression. Consistent with these data, JDP2(-/-) mice were found to have increased bone mass. In summary, CSF1 up-regulates JDP2 expression by inducing Tbx3 binding to the JDP2 promoter. The downstream signaling cascade from activated c-Fms involves the MEK1/2-ERK1/2 pathway. Tbx3 plays an important role in osteoclastogenesis at least in part by regulating CSF1-dependent expression of JDP2.