Potential factors affecting the success rate of indirect pulp therapy in primary molars with deep caries: a retrospective study.

Indirect pulp therapy (IPT) is a common conservative treatment for deep dental caries. However, the potential risk factors for the prognosis of IPT have not been well studied. This study retrospectively investigated the success rate of IPT in treating primary molars with deep caries and the factors potentially affecting the two-year success rate. A total of 303 primary molars in 202 children (106 boys and 96 girls) were included in this study. These primary molars were identified as having deep caries by clinical and radiographic examinations and were treated with IPT. The factors potentially affecting the IPT success rate were analyzed after two years of follow-up. The results indicated that the two-year IPT success rate was 86% (262/303). The success rate of primary molars with and without stainless steel crowns was 96% (120/125) and 80% (142/178), respectively. Primary molars treated with stainless steel crowns showed a significantly lower risk of failure (hazard ratio (HR) = 0.18, 95% confidence interval (CI): (0.10, 0.34), p = 0.01). There were no significant differences in other factors, including gender (male vs. female), age (preschool vs. school age), cooperation level (Frankl 2 vs. 3 or 4 scales), arch type (maxillary vs. mandibular), tooth type (first vs. second primary molar), or pulp capping material (calcium hydroxide vs. glass ionomer cement). IPT is an effective, conservative treatment modality for primary molars with deep caries. Stainless steel crowns could significantly improve the IPT success rate.

Comparative Evaluation of STEAP1 Targeting Chimeric Antigen Receptors with Different Costimulatory Domains and Spacers.

We have developed a chimeric antigen receptor (CAR) against the six-transmembrane epithelial antigen of prostate-1 (STEAP1), which is expressed in prostate cancer, Ewing sarcoma, and other malignancies. In the present study, we investigated the effect of substituting costimulatory domains and spacers in this STEAP1 CAR. We cloned four CAR constructs with either CD28 or 4-1BB costimulatory domains, combined with a CD8a-spacer (sp) or a mutated IgG-spacer. The CAR T-cells were evaluated in short- and long-term in vitro T-cell assays, measuring cytokine production, tumor cell killing, and CAR T-cell expansion and phenotype. A xenograft mouse model of prostate cancer was used for in vivo comparison. All four CAR constructs conferred CD4+ and CD8+ T cells with STEAP1-specific functionality. A CD8sp_41BBz construct and an IgGsp_CD28z construct were selected for a more extensive comparison. The IgGsp_CD28z CAR gave stronger cytokine responses and killing in overnight caspase assays. However, the 41BB-containing CAR mediated more killing (IncuCyte) over one week. Upon six repeated stimulations, the CD8sp_41BBz CAR T cells showed superior expansion and lower expression of exhaustion markers (PD1, LAG3, TIGIT, TIM3, and CD25). In vivo, both the CAR T variants had comparable anti-tumor activity, but persisting CAR T-cells in tumors were only detected for the 41BBz variant. In conclusion, the CD8sp_41BBz STEAP1 CAR T cells had superior expansion and survival in vitro and in vivo, compared to the IgGsp_CD28z counterpart, and a less exhausted phenotype upon repeated antigen exposure. Such persistence may be important for clinical efficacy.

Facile synthesis of δ-MnO2 biotemplated by waste tobacco stem-silks for enhanced removal of Sb(III).

The elimination of antimony pollution has attracted increasing concerns because of its high toxicity to human health and the natural environment. In this work, biomimetic δ-MnO2 was synthesized by using waste tobacco stem-silks as biotemplate (Bio-δ-MnO2) and used in the capture of Sb(III)from aqueous solution. The tobacco stem-silks not only provided unique wrinkled morphologies but also contained carbon element self-doped into the resulting samples. The maximum Sb(III) adsorption capacity reached 763.4 mg∙g -1, which is 2.06 times higher than δ-MnO2 without template (370.0 mg∙g -1), 4.53 times than tobacco stem-silks carbon (168.5 mg∙g -1), and 10.39 times than commercial MnO2 (73.5 mg∙g -1), respectively. The isotherm and kinetic studies indicated that the adsorption behavior was consistent with the Langmuir isotherm model and the pseudo-second-order kinetic equation. As far as we are aware, the adsorption capacity of Bio-δ-MnO2 is much higher than that of most Sb(III) adsorbents. FT-IR, XPS, SEM, XRD, and Zeta potential analyses showed that the main mechanism for the adsorption of Sb(III) by Bio-δ-MnO2 includes electrostatic attraction, surface complexation, and redox. Overall, this study provides a new sustainable way to convert agricultural wastes to more valuable products such as biomimetic adsorbent for Sb(III) removal in addition to conventional activated carbon and biochar.

Identification of potential molecular mechanisms and candidate drugs for radiotherapy- and chemotherapy-induced mucositis.

BACKGROUND: Radiotherapy-induced oral mucositis (RIOM) and chemotherapy-induced oral mucositis (CIOM) are common complications in cancer patients, leading to negative clinical manifestations, reduced quality of life, and unsatisfactory treatment outcomes. OBJECTIVE: The present study aimed to identify potential molecular mechanisms and candidate drugs by data mining. METHODS: We obtained a preliminary list of genes associated with RIOM and CIOM. In-depth information on these genes was explored by functional and enrichment analyses. Then, the drug-gene interaction database was used to determine the interaction of the final enriched gene list with known drugs and analyze the drug candidates. RESULTS AND CONCLUSION: This study identified 21 hub genes that may play an important role in RIOM and CIOM, respectively. Through our data mining, bioinformatics survey, and candidate drug selection, TNF, IL-6, and TLR9 could play an important role in disease progression and treatment. In addition, eight candidate drugs (olokizumab, chloroquine, hydroxychloroquine, adalimumab, etanercept, golimumab, infliximab, and thalidomide) were selected by the drug-gene interaction literature search additionally, as candidates for treating RIOM and CIOM.

Development of a TGFß-IL-2/15 Switch Receptor for Use in Adoptive Cell Therapy.

Therapy employing T cells modified with chimeric antigen receptors (CARs) is effective in hematological malignancies but not yet in solid cancers. CAR T cell activity in solid tumors is limited by immunosuppressive factors, including transforming growth factor ß (TGFß). Here, we describe the development of a switch receptor (SwR), in which the extracellular domains of the TGFß receptor are fused to the intracellular domains from the IL-2/15 receptor. We evaluated the SwR in tandem with two variants of a CAR that we have developed against STEAP1, a protein highly expressed in prostate cancer. The SwR-CAR T cell activity was assessed against a panel of STEAP1+/- prostate cancer cell lines with or without over-expression of TGFß, or with added TGFß, by use of flow cytometry cytokine and killing assays, Luminex cytokine profiling, cell counts, and flow cytometry phenotyping. The results showed that the SwR-CAR constructs improved the functionality of CAR T cells in TGFß-rich environments, as measured by T cell proliferation and survival, cytokine response, and cytotoxicity. In assays with four repeated target-cell stimulations, the SwR-CAR T cells developed an activated effector memory phenotype with retained STEAP1-specific activity. In conclusion, the SwR confers CAR T cells with potent and durable in vitro functionality in TGFß-rich environments. The SwR may be used as an add-on construct for CAR T cells or other forms of adoptive cell therapy.

Two Dual-Function Zr/Hf-MOFs as High-Performance Proton Conductors and Amines Impedance Sensors.

In the field of sensing, finding high-performance amine molecular sensors has always been a challenging topic. Here, two highly stable 3D MOFs DUT-67(Hf) and DUT-67(Zr) with large specific surface areas and hierarchical pore structures were conveniently synthesized by solvothermal reaction of ZrCl4/HfCl4 with a simple organic ligand, 2,5-thiophene dicarboxylic acid (H2TDC) according to literature approach. By analyzing TGA data, it was found that the two MOFs have defects (unsaturated metal sites) that can interact with substrates (H2O and volatile amine gas), which is conducive to proton transfer and amine compound identification. Further experiments showed that at 100 °C and 98% relative humidity (RH), the optimized proton conductivities of DUT-67(Zr) and DUT-67(Hf) can reach the high values of 2.98 × 10-3 and 3.86 × 10-3 S cm-1, respectively. Moreover, the room temperature sensing characteristics of MOFs' to amine gases were evaluated at 68, 85 and 98% RHs, respectively. Impressively, the prepared MOFs-based sensors have the desired stability and higher sensitivity to amines. Under 68% RH, the detection limits of DUT-67(Zr) or DUT-67(Hf) for volatile amine gases were 0.5 (methylamine), 0.5 (dimethylamine) and 1 ppm (trimethylamine), and 0.5 (methylamine), 0.5 (dimethylamine) and 0.5 ppm (trimethylamine), respectively. As far as we know, this is the best performance of ammonia room temperature sensors in the past proton-conductive MOF sensors.

Phenol-soluble modulin contributes to the dispersal of Staphylococcus epidermidis isolates from catheters.

Staphylococcus epidermidis (S. epidermidis), a human commensal, has been implicated in invasive infection in humans due to their ability to form biofilm. It is assumed that when a biofilm is dispersed it will subsequently cause a more severe infection. The clinical significance of S. epidermidis isolated from sterile body fluid (BF) remains unclear, and might be related to dispersal from catheter-associated biofilm infection. To evaluate this relationship, we evaluated S. epidermidis isolates from catheters (CA) or BF in hospitalized patients. Sequence type 2 (ST2) is the most prevalent type isolated from infection sites. Although the specific STs were also observed in isolates from different sites, we observed that the main sequence type was ST2, followed by ST59, among all the 114 isolates from different infection sites. Interestingly, ST2 strains isolated from BF exhibited significantly thicker biofilm than those from CA. The thicker biofilm was due to the higher expression of accumulation-associated protein (aap) but not intercellular adhesion (ica) operon. Moreover, the transcription of PSMδ and PSMε were significantly increased in ST2 strains isolated from BF. Although the bacterial loads on catheters were similar infected by CA- or BF-originated strains in mouse biofilm-associated infection model, we observed a higher CFU in peri-catheter tissues infected by ST2 clones isolated from BF, suggesting that S. epidermidis with thicker biofilm formation might be able to disperse. Taken together, our data suggested that S. epidermidis originated from diverse infection sites exhibited different biofilm forming capacity. The major ST2 clone isolated from BF exhibited thicker biofilm by increasing the expression of Aap. The higher expression of PSM of these strains may contribute to bacteria dispersal from biofilm and the following bacterial spread.

Development of STEAP1 targeting chimeric antigen receptor for adoptive cell therapy against cancer.

Chimeric antigen receptors (CARs) that retarget T cells against CD19 show clinical efficacy against B cell malignancies. Here, we describe the development of a CAR against the six-transmembrane epithelial antigen of prostate-1 (STEAP1), which is expressed in ∼90% of prostate cancers, and subgroups of other malignancies. STEAP1 is an attractive target, as it is associated with tumor invasiveness and progression and only expressed at low levels in normal tissues, apart from the non-vital prostate gland. We identified the antibody coding sequences from a hybridoma and designed a CAR that is efficiently expressed in primary T cells. The T cells acquired the desired anti-STEAP1 specificity, with a polyfunctional response including production of multiple cytokines, proliferation, and the killing of cancer cells. The response was observed for both CD4+ and CD8+ T cells, and against all STEAP1+ target cell lines tested. We evaluated the in vivo CAR T activity in both subcutaneous and metastatic xenograft mouse models of prostate cancer. Here, the CAR T cells infiltrated tumors and significantly inhibited tumor growth and extended survival in a STEAP1-dependent manner. We conclude that the STEAP1 CAR exhibits potent in vitro and in vivo functionality and can be further developed toward potential clinical use.

Stress-Mediated Reprogramming of Prostate Cancer One-Carbon Cycle Drives Disease Progression.

One-carbon (1C) metabolism has a key role in metabolic programming with both mitochondrial (m1C) and cytoplasmic (c1C) components. Here we show that activating transcription factor 4 (ATF4) exclusively activates gene expression involved in m1C, but not the c1C cycle in prostate cancer cells. This includes activation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) expression, the central player in the m1C cycle. Consistent with the key role of m1C cycle in prostate cancer, MTHFD2 knockdown inhibited prostate cancer cell growth, prostatosphere formation, and growth of patient-derived xenograft organoids. In addition, therapeutic silencing of MTHFD2 by systemically administered nanoliposomal siRNA profoundly inhibited tumor growth in preclinical prostate cancer mouse models. Consistently, MTHFD2 expression is significantly increased in human prostate cancer, and a gene expression signature based on the m1C cycle has significant prognostic value. Furthermore, MTHFD2 expression is coordinately regulated by ATF4 and the oncoprotein c-MYC, which has been implicated in prostate cancer. These data suggest that the m1C cycle is essential for prostate cancer progression and may serve as a novel biomarker and therapeutic target. SIGNIFICANCE: These findings demonstrate that the mitochondrial, but not cytoplasmic, one-carbon cycle has a key role in prostate cancer cell growth and survival and may serve as a biomarker and/or therapeutic target.

Regulation of the unfolded protein response through ATF4 and FAM129A in prostate cancer.

Cancer cells exploit many of the cellular adaptive responses to support their survival needs. One such critical pathway in eukaryotic cells is the unfolded protein response (UPR) that is important in normal physiology as well as disease states, including cancer. Since UPR can serve as a lever between survival and death, regulated control of its activity is critical for tumor formation and growth although the underlying mechanisms are poorly understood. Here we show that one of the main transcriptional effectors of UPR, activating transcription factor 4 (ATF4), is essential for prostate cancer (PCa) growth and survival. Using systemic unbiased gene expression and proteomic analyses, we identified a novel direct ATF4 target gene, family with sequence similarity 129 member A (FAM129A), which is critical in mediating ATF4 effects on prostate tumorigenesis. Interestingly, FAM129A regulated both PERK and eIF2α in a feedback loop that differentially channeled the UPR output. ATF4 and FAM129A protein expression is increased in patient PCa samples compared with benign prostate. Importantly, in vivo therapeutic silencing of ATF4-FAM129A axis profoundly inhibited tumor growth in a preclinical PCa model. These data support that one of the canonical UPR branches, through ATF4 and its target gene FAM129A, is required for PCa growth and thus may serve as a novel therapeutic target.

Essential role of Cdc42 in cardiomyocyte proliferation and cell-cell adhesion during heart development.

Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cell migration, proliferation, differentiation and survival. However, the role of Cdc42 in heart development remains largely unknown. To determine the function of Cdc42 in heart formation, we have generated a Cdc42 cardiomyocyte knockout (CCKO) mouse line by crossing Cdc42 flox mice with myosin light chain (MLC) 2a-Cre mice. The inactivation of Cdc42 in embryonic cardiomyocytes induced lethality after embryonic day 12.5. Histological analysis of CCKO embryos showed cardiac developmental defects that included thin ventricular walls and ventricular septum defects. Microarray and real-time PCR data also revealed that the expression level of p21 was significantly increased and cyclin B1 was dramatically decreased, suggesting that Cdc42 is required for cardiomyocyte proliferation. Phosphorylated Histone H3 staining confirmed that the inactivation of Cdc42 inhibited cardiomyocytes proliferation. In addition, transmission electron microscope studies showed disorganized sarcomere structure and disruption of cell-cell contact among cardiomyocytes in CCKO hearts. Accordingly, we found that the distribution of N-cadherin/ß-Catenin in CCKO cardiomyocytes was impaired. Taken together, our data indicate that Cdc42 is essential for cardiomyocyte proliferation, sarcomere organization and cell-cell adhesion during heart development.

Transformation of Iodide by Carbon Nanotube Activated Peroxydisulfate and Formation of Iodoorganic Compounds in the Presence of Natural Organic Matter.

In this study, we interestingly found that peroxydisulfate (PDS) could be activated by a commercial multiwalled carbon nanotube (CNT) material via a nonradical pathway. Iodide (I-) was quickly and almost completely oxidized to hypoiodous acid (HOI) in the PDS/CNT system over the pH range of 5-9, but the further transformation to iodate (IO3-) was negligible. A kinetic model was proposed, which involved the formation of reactive PDS-CNT complexes, and then their decomposition into sulfate anion (SO42-) via inner electron transfer within the complexes or by competitively reacting with I-. Several influencing factors (e.g., PDS and CNT dosages, and solution pH) on I- oxidation kinetics by this system were evaluated. Humic acid (HA) decreased the oxidation kinetics of I-, probably resulting from its inhibitory effect on the interaction between PDS and CNT to form the reactive complexes. Moreover, adsordable organic iodine compounds (AOI) as well as specific iodoform and iodoacetic acid were appreciably produced in the PDS/CNT/I- system with HA. These results demonstrate the potential risk of producing toxic iodinated organic compounds in the novel PDS/CNT oxidation process developed very recently, which should be taken into consideration before its practical application in water treatment.

Oxidation of the odorous compound 2,4,6-trichloroanisole by UV activated persulfate: Kinetics, products, and pathways.

The transformation efficiency and products of an odorous compound 2,4,6-trichloroanisole (TCA) at the wavelength of 254 nm in the presence of persulfate were investigated for the first time. The effects of water matrix (i.e., natural organic matter (NOM), pH, carbonate/bicarbonate (HCO3(-)/CO3(2-)), and chloride ions (Cl(-))) were evaluated. The second order rate constant of TCA reacting with sulfate radical (SO4(-)) was determined to be (3.72 ± 0.10) × 10(9) M(-1) s(-1). Increasing dosage of persulfate increased the observed pseudo-first-order rate constant for TCA degradation (kobs), and the contribution of SO4(-) to TCA degradation was much higher than that of HO at each experimental condition. Degradation rate of TCA decreased with pH increasing from 4.0 to 9.0, which could be explained by the lower radical scavenging effect of dihydrogen phosphate than hydrogen phosphate in acidic condition (pH < 6). NOM significantly decreased kobs due to the effects of radical scavenging and UV absorption with the former one being dominant. kobs decreased from 2.32 × 10(-3) s(-1) to 0.92 × 10(-3) s(-1) with the CO3(2-)/HCO3(-) concentration increased from 0.5 mM to 10 mM in the UV/persulfate process, while kobs slightly decreased from 2.54 × 10(-3) s(-1) in the absence of Cl(-) to 2.10 × 10(-3) s(-1) in the presence of 10 mM Cl(-). Most of these kinetic results could be described by a steady-state kinetic model. Furthermore, liquid chromatography/electrospray ionization-triple quadrupole mass spectrometry at powerful precursor ion scan approach was used to selectively detect oxidation products of TCA. It was found that 2,4,6-trichorophenol (TCP) was the major oxidation product (i.e., the initial yield of TCP was above 90%). The second order rate constant between TCP and SO4(-) was estimated to be (4.16 ± 0.20) × 10(9) M(-1) s(-1). In addition, three products (i.e., 2,6-dichloro-1,4-benzoquinone and two aromatic ring-opening products) were detected in the reaction of TCP with SO4(-), which also appeared in the oxidation of TCA in the UV/persulfate process. A tentative pathway was proposed, where the initial one-electron oxidation of TCA by SO4(-) and further reactions (e.g., ipso-hydroxylation and aromatic ring-cleavage) of the formed cation intermediate TCA were involved.

Stromal interaction molecule 1 (STIM1) and Orai1 mediate histamine-evoked calcium entry and nuclear factor of activated T-cells (NFAT) signaling in human umbilical vein endothelial cells.

Histamine is an important immunomodulator involved in allergic reactions and inflammatory responses. In endothelial cells, histamine induces Ca(2+) mobilization by releasing Ca(2+) from the endoplasmic reticulum and eliciting Ca(2+) entry across the plasma membrane. Herein, we show that histamine-evoked Ca(2+) entry in human umbilical vein endothelial cells (HUVECs) is sensitive to blockers of Ca(2+) release-activated Ca(2+) (CRAC) channels. RNA interference against STIM1 or Orai1, the activating subunit and the pore-forming subunit of CRAC channels, respectively, abolishes this histamine-evoked Ca(2+) entry. Furthermore, overexpression of dominant-negative CRAC channel subunits inhibits while co-expression of both STIM1 and Orai1 enhances histamine-induced Ca(2+) influx. Interestingly, gene silencing of STIM1 or Orai1 also interrupts the activation of calcineurin/nuclear factor of activated T-cells (NFAT) pathway and the production of interleukin 8 triggered by histamine in HUVECs. Collectively, these results suggest a central role of STIM1 and Orai1 in mediating Ca(2+) mobilization linked to inflammatory signaling of endothelial cells upon histamine stimulation.

Inactivation of Cdc42 in neural crest cells causes craniofacial and cardiovascular morphogenesis defects.

Neural crest cells (NCCs) are physically responsible for craniofacial skeleton formation, pharyngeal arch artery remodeling and cardiac outflow tract septation during vertebrate development. Cdc42 (cell division cycle 42) is a Rho family small GTP-binding protein that works as a molecular switch to regulate cytoskeleton remodeling and the establishment of cell polarity. To investigate the role of Cdc42 in NCCs during embryonic development, we deleted Cdc42 in NCCs by crossing Cdc42 flox mice with Wnt1-cre mice. We found that the inactivation of Cdc42 in NCCs caused embryonic lethality with craniofacial deformities and cardiovascular developmental defects. Specifically, Cdc42 NCC knockout embryos showed fully penetrant cleft lips and short snouts. Alcian Blue and Alizarin Red staining of the cranium exhibited an unfused nasal capsule and palatine in the mutant embryos. India ink intracardiac injection analysis displayed a spectrum of cardiovascular developmental defects, including persistent truncus arteriosus, hypomorphic pulmonary arteries, interrupted aortic arches, and right-sided aortic arches. To explore the underlying mechanisms of Cdc42 in the formation of the great blood vessels, we generated Wnt1Cre-Cdc42-Rosa26 reporter mice. By beta-galactosidase staining, a subpopulation of Cdc42-null NCCs was observed halting in their migration midway from the pharyngeal arches to the conotruncal cushions. Phalloidin staining revealed dispersed, shorter and disoriented stress fibers in Cdc42-null NCCs. Finally, we demonstrated that the inactivation of Cdc42 in NCCs impaired bone morphogenetic protein 2 (BMP2)-induced NCC cytoskeleton remodeling and migration. In summary, our results demonstrate that Cdc42 plays an essential role in NCC migration, and inactivation of Cdc42 in NCCs impairs craniofacial and cardiovascular development in mice.

Deletion of Cdc42 enhances ADAM17-mediated vascular endothelial growth factor receptor 2 shedding and impairs vascular endothelial cell survival and vasculogenesis.

Cdc42 is a Ras-related GTPase that plays an important role in the regulation of a range of cellular functions, including cell migration, proliferation, and survival. Consistent with its critical functions in vitro, the inactivation of Cdc42 in mice has been shown to result in embryonic lethality at embryonic day 6.5 (E6.5) before blood vessel formation. To determine the role of Cdc42 in new blood vessel formation, we have generated vascular endothelial cell (EC)-specific Cdc42 knockout mice by crossing Cdc42(flox/flox) mice with Tie2-Cre mice. The deletion of Cdc42 in ECs caused embryonic lethality with vasculogenesis and angiogenesis defects. We observed that Cdc42 is critical for EC migration and survival but not for cell cycle progression. Moreover, we found that the inactivation of Cdc42 in ECs decreased the level of vascular endothelial growth factor receptor 2 (VEGFR2) protein on the EC surface and promoted the production of a 75-kDa membrane-associated C-terminal VEGFR2 fragment. Using cultured primary mouse ECs and human umbilical vein ECs, we have demonstrated that the deletion of Cdc42 increased ADAM17-mediated VEGFR2 shedding. Notably, inhibition of ADAM17 or overexpression of VEGFR2 can partially reverse Cdc42 deletion-induced EC apoptosis. These data indicate that Cdc42 is essential for VEGFR2-mediated signal transduction in blood vessel formation.

ERK-mediated phosphorylation of fibroblast growth factor receptor 1 on Ser777 inhibits signaling.

Fibroblast growth factor 1 (FGF1) controls cellular activities through the activation of specific cell-surface FGF receptors (FGFRs). Transphosphorylation of tyrosine residues in the kinase domain of FGFRs leads to activation of intracellular signaling cascades, including those mediated by mitogen-activated protein kinases (MAPKs). FGFRs also contain a serine-rich C-terminal tail. We identified a regulatory mechanism of FGFR signaling involving phosphorylation of Ser(777) in the C-terminal region of FGFR1 by the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2. Prevention of the phosphorylation of Ser(777) in FGFR1 or mutation of Ser(777) to alanine enhanced FGF-stimulated receptor tyrosine phosphorylation and increased cell proliferation, cell migration, and axonal growth. A form of FGFR1 with a phosphomimetic mutation at Ser(777) exhibited reduced signaling. Activation of MAPKs by other receptor tyrosine kinases also resulted in phosphorylation of Ser(777) in FGFR1, thereby enabling crosstalk regulation of FGFR activity by other signaling pathways. Our data reveal a negative feedback mechanism that controls FGF signaling and thereby protects the cell from excessive activation of FGFR.

Inactivation of Cdc42 in embryonic brain results in hydrocephalus with ependymal cell defects in mice.

The establishment of a polarized cellular morphology is essential for a variety of processes including neural tube morphogenesis and the development of the brain. Cdc42 is a Ras-related GTPase that plays an essential role in controlling cell polarity through the regulation of the actin and microtubule cytoskeleton architecture. Previous studies have shown that Cdc42 plays an indispensable role in telencephalon development in earlier embryo developmental stage (before E12.5). However, the functions of Cdc42 in other parts of brain in later embryo developmental stage or in adult brain remain unclear. Thus, in order to address the role of Cdc42 in the whole brain in later embryo developmental stage or in adulthood, we used Cre/loxP technology to generate two lines of tissue-specific Cdc42-knock-out mice. Inactivation of Cdc42 was achieved in neuroepithelial cells by crossing Cdc42/ flox mice with Nestin-Cre mice and resulted in hydrocephalus, causing death to occur within the postnatal stage. Histological analyses of the brains from these mice showed that ependymal cell differentiation was disrupted, resulting in aqueductal stenosis. Deletion of Cdc42 in the cerebral cortex also induced obvious defects in interkinetic nuclear migration and hypoplasia. To further explore the role of Cdc42 in adult mice brain, we examined the effects of knocking-out Cdc42 in radial glial cells by crossing Cdc42/flox mice with human glial fibrillary acidic protein (GFAP)-Cre mice. Inactivation of Cdc42 in radial glial cells resulted in hydrocephalus and ependymal cell denudation. Taken together, these results highlight the importance of Cdc42 for ependymal cell differentiation and maintaining, and suggest that these functions likely contribute to the essential roles played by Cdc42 in the development of the brain.

Nuclear import of exogenous FGF1 requires the ER-protein LRRC59 and the importins Kpnα1 and Kpnß1.

Fibroblast growth factor 1 (FGF1) taken up by cells into endocytic vesicles can be translocated across vesicular membranes into the cytosol and the nucleus where it has a growth regulatory activity. Previously, leucine-rich repeat containing 59 (LRRC59) was identified as an intracellular binding partner of FGF1, but its biological role remained unknown. Here, we show that LRRC59 is strictly required for nuclear import of exogenous FGF1. siRNA-mediated depletion of LRRC59 did not inhibit the translocation of FGF1 into cytosol, but blocked the nuclear import of FGF1. We also found that an nuclear localization sequence (NLS) in FGF1, Ran GTPase, karyopherin-α1 (Kpnα1), and Kpnß1 were required for nuclear import of FGF1. Nuclear import of exogenous FGF2, which depends on CEP57/Translokin, was independent of LRRC59, but was dependent on Kpnα1 and Kpnß1, while the nuclear import of FGF1 was independent of CEP57. LRRC59 is a membrane-anchored protein that localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE). We found that LRRC59 possesses NLS-like sequences in its cytosolic part that can mediate nuclear import of soluble LRRC59 variants, and that the localization of LRRC59 to the NE depends on Kpnß1. We propose that LRRC59 facilitates transport of cytosolic FGF1 through nuclear pores by interaction with Kpns and movement of LRRC59 along the ER and NE membranes.

Isolation and characterization of vascular endothelial cells from murine heart and lung.

The formation of blood vessel networks is a fundamental event in vertebrate embryo development. Angiogenesis and vasculogenesis are the essential processes in vascular formation. Endothelial cells play a key role during angiogenesis and vasculogenesis, and cultured vascular endothelial cells provide an indispensable model for exploring the molecular mechanisms of angiogenesis and vasculogenesis. In this chapter, we described a protocol using PECAM-1-coated Dynabeads for the isolation of vascular endothelial cells from mouse heart and lung. This method will provide up to 10(7) endothelial cells with high purity (>85%). The isolated endothelial cells retain their in vivo characteristics, such as the expression of the cell surface markers PECAM-1 and ICAM-2.