Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor.

The clinical application of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) is restricted by its short serum half-life. Herein, site-selective modification of the N-terminus of rhG-CSF with PAL-PEG3-Ph-CHO was used to develop a long-acting rhG-CSF. The optimized conditions for rhG-CSF modification with PAL-PEG3-Ph-CHO were: reaction solvent system of 3% (w/v) Tween 20 and 30 mM NaCNBH3 in acetate buffer (20 mmol/L, pH 5.0), molar ratio of PAL-PEG3-Ph-CHO to rhG-CSF of 6:1, temperature of 20°C, and reaction time of 12 h, consequently, achieving a PAL-PEG3-Ph-rhG-CSF product yield of 70.8%. The reaction mixture was purified via preparative liquid chromatography, yielding the single-modified product PAL-PEG3-Ph-rhG-CSF with a HPLC purity exceeding 95%. The molecular weight of PAL-PEG3-Ph-rhG-CSF was 19297 Da by MALDI-TOF-MS, which was consistent with the theoretical value. The circular dichroism analysis revealed no significant change in its secondary structure compared to unmodified rhG-CSF. The PAL-PEG3-Ph-rhG-CSF retained 82.0% of the in vitro biological activity of unmodified rhG-CSF. The pharmacokinetic analyses showed that the serum half-life of PAL-PEG3-Ph-rhG-CSF was 7.404 ± 0.777 h in mice, 4.08 times longer than unmodified rhG-CSF. Additionally, a single subcutaneous dose of PAL-PEG3-Ph-rhG-CSF presented comparable in vivo efficacy to multiple doses of rhG-CSF. This study demonstrated an efficacious strategy for developing long-acting rhG-CSF drug candidates.

Mac-1 deficiency ameliorates pressure overloaded heart failure through inhibiting macrophage polarization and activation.

Persistent pressure overload commonly leads to pathological cardiac hypertrophy and remodeling, ultimately leading to heart failure (HF). Cardiac remodeling is associated with the involvement of immune cells and the inflammatory response in pathogenesis. The macrophage-1 antigen (Mac-1) is specifically expressed on leukocytes and regulates their migration and polarization. Nonetheless, the involvement of Mac-1 in cardiac remodeling and HF caused by pressure overload has not been determined. The Mac-1-knockout (KO) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) for 6 weeks. Echocardiography and pressure-volume loop assessments were used to evaluate cardiac function, and cardiac remodeling and macrophage infiltration and polarization were estimated by histopathology and molecular techniques. The findings of our study demonstrated that Mac-1 expression was markedly increased in hearts subjected to TAC treatment. Moreover, compared with WT mice, Mac-1-KO mice exhibited dramatically ameliorated TAC-induced cardiac dysfunction, hypertrophy, fibrosis, oxidative stress and apoptosis. The potential positive impacts may be linked to the inhibition of macrophage infiltration and M1 polarization via reductions in NF-kB and STAT1 expression and upregulation of STAT6. In conclusion, this research reveals a new function of Mac-1 deficiency in reducing pathological cardiac remodeling and HF caused by pressure overload. Additionally, inhibiting Mac-1 could be a potential treatment option for patients with HF in a clinical setting.

CD11b mediates hypertensive cardiac remodeling by regulating macrophage infiltration and polarization.

INTRODUCTION: Leukocyte infiltration is an early event during cardiac remodeling frequently leading to heart failure (HF). Integrins mediate leukocyte infiltration during inflammation. However, the importance of specific integrins in hypertensive cardiac remodeling is still unclear. OBJECTIVES: To elucidate the significance of CD11b in hypertensive cardiac remodeling. METHODS: Angiotensin (Ang II) or deoxycorticosterone acetate (DOCA)-salt was used to induce cardiac remodeling in mice of gene knockout (KO), bone marrow (BM) chimera, and the CD11b neutralizing antibody or agonist leukadherin-1 (LA1) treatment. RESULTS: Our microarray data showed that integrin subunits Itgam (CD11b) and Itgb2 (CD18) were the most highly upregulated in Ang II-infused hearts. CD11b expression and CD11b/CD18+ myelomonocytes were also time-dependently increased. KO or pharmacological blockade of CD11b greatly attenuated cardiac remodeling and macrophage infiltration and M1 polarization induced by Ang II or DOCA-salt. This protection was verified in wild-type mice transplanted with CD11b-deficient BM cells. Conversely, administration of CD11b agonist LA1 showed the opposite effects. Further, CD11b KO reduced Ang II-induced macrophage adhesion and M1 polarization, leading to reduction of cardiomyocyte enlargement and fibroblast differentiation in vitro. The numbers of CD14+CD11b+CD18+ monocytes and CD15+CD11b+CD18+ granulocytes were obviously higher in HF patients than in normal controls. CONCLUSION: Our data demonstrate an important role of CD11b+ myeloid cells in hypertensive cardiac remodeling, and suggest that HF may benefit from targeting CD11b.

Cytokine-responsive T- and NK-cells portray SARS-CoV-2 vaccine-responders and infection in multiple myeloma patients.

Patients with multiple myeloma (MM) routinely receive mRNA-based vaccines to reduce COVID-19-related mortality. However, whether disease- and therapy-related alterations in immune cells and cytokine-responsiveness contribute to the observed heterogeneous vaccination responses is unclear. Thus, we analyzed peripheral blood mononuclear cells from patients with MM during and after SARS-CoV-2 vaccination and breakthrough infection (BTI) using combined whole-transcriptome and surface proteome single-cell profiling with functional serological and T-cell validation in 58 MM patients. Our results demonstrate that vaccine-responders showed a significant overrepresentation of cytotoxic CD4+ T- and mature CD38+ NK-cells expressing FAS+/TIM3+ with a robust cytokine-responsiveness, such as type-I-interferon-, IL-12- and TNF-α-mediated signaling. Patients with MM experiencing BTI developed strong serological and cellular responses and exhibited similar cytokine-responsive immune cell patterns as vaccine-responders. This study can expand our understanding of molecular and cellular patterns associated with immunization responses and may benefit the design of improved vaccination strategies in immunocompromised patients.

Eldecalcitol prevents muscle loss and osteoporosis in disuse muscle atrophy via NF-κB signaling in mice.

We investigated the effect of eldecalcitol on disuse muscle atrophy. C57BL/6J male mice aged 6 weeks were randomly assigned to control, tail suspension (TS), and TS-eldecalcitol-treated groups and were injected intraperitoneally twice a week with either vehicle (control and TS) or eldecalcitol at 3.5 or 5 ng for 3 weeks. Grip strength and muscle weights of the gastrocnemius (GAS), tibialis anterior (TA), and soleus (SOL) were determined. Oxidative stress was evaluated by malondialdehyde, superoxide dismutase, glutathione peroxidase, and catalase. Bone microarchitecture was analyzed using microcomputed tomography. The effect of eldecalcitol on C2C12 myoblasts was analyzed by measuring myofibrillar protein MHC and the atrophy markers Atrogin-1 and MuRF-1 using immunofluorescence. The influence of eldecalcitol on NF-κB signaling pathway and vitamin D receptor (VDR) was assessed through immunofluorescence, (co)-immunoprecipitation, and VDR knockdown studies. Eldecalcitol increased grip strength (P < 0.01) and restored muscle loss in GAS, TA, and SOL (P < 0.05 to P < 0.001) induced by TS. An improvement was noted in bone mineral density and bone architecture in the eldecalcitol group. The impaired oxidative defense system was restored by eldecalcitol (P < 0.05 to P < 0.01 vs. TS). Eldecalcitol (10 nM) significantly inhibited the expression of MuRF-1 (P < 0.001) and Atrogin-1 (P < 0.01), increased the diameter of myotubes (P < 0.05), inhibited the expression of P65 and P52 components of NF-κB and P65 nuclear location, thereby inhibiting NF-κB signaling. Eldecalcitol promoted VDR binding to P65 and P52. VDR signaling is required for eldecalcitol-mediated anti-atrophy effects. In conclusion, eldecalcitol exerted its beneficial effects on disuse-induced muscle atrophy via NF-κB inhibition.

Mechanism of Al toxicity alleviation in acidic red soil by rice-straw hydrochar application and comparison with pyrochar.

In the past decade, biochar has been widely regarded as a new type of soil conditioner that can effectively control soil acidification and alleviate Al toxicity. Hydrochar is identified as a more economical carbon material than pyrochar, but its effect on Al toxicity and the associated mechanism have not been studied. Thus, a two-stage indoor incubation experiment was conducted to investigate the effect of rice-straw hydrochar (HC, application rate: 1/2/3 %) on maize seedling root growth, soil solution Al activity, soil exchangeable Al and pH buffering ability in acidic red soils from two sites. We also used pyrochar (PC, application rate: 3 %) produced from the same rice straw for comparison. Except for HC-1 %, both hydrochar and pyrochar addition significantly stimulated relative root elongation (136.36 % ~ 284.09 %), diminished the cell death ratio (27.96 % ~ 85.56 %) and Al content in root tips (18.80 % ~ 80.11 %) by decreasing the total Al content (44.78 % ~ 76.10 %) and the proportion of Al3+ species (27 % ~ 32 %) in soil solution. Hydrochar did not significantly promote the soil pH buffer capacity (pH-BC) or effective cation exchange capacity (ECEC), while PC-3 % did. The DOC (dissolved organic carbon) content of soil solution was dramatically elevated by 203.9 % ~ 783.2 % after hydrochar addition. Hydrochar mitigates Al activity in soil solution mainly through Al-DOC complexation and adsorption, thus suppressing the Al toxicity of maize roots. Hydrochar may be an economical soil amendment for ameliorating Al toxicity despite its overall alleviation effect on Al toxicity being lower than pyrochar.

Blocking VCAM-1 ameliorates hypertensive cardiac remodeling by impeding macrophage infiltration.

Cardiac remodeling is an important mechanism of heart failure, which frequently results from leukocyte infiltration. Vascular cellular adhesion molecule-1 (VCAM-1) plays a critical role in leukocyte adhesion and transmigration. However, the importance of VCAM-1 in the development of angiotensin II (Ang II)-induced cardiac remodeling remains unclear. Wild-type (WT) mice were infused with Ang II (1,000 ng/kg/min) for 14 days and simultaneously treated with VCAM-1 neutralizing antibody (0.1 or 0.2 mg) or IgG control. Systolic blood pressure (SBP) and cardiac function were detected by a tail-cuff and echocardiography. Cardiac remodeling was evaluated by histological staining. Adhesion and migration of bone marrow macrophages (BMMs) were evaluated in vitro. Our results indicated that VCAM-1 levels were increased in the serum of patients with heart failure (HF) and the hearts of Ang II-infused mice. Furthermore, Ang II-caused hypertension, cardiac dysfunction, hypertrophy, fibrosis, infiltration of VLA-4+ BMMs and oxidative stress were dose-dependently attenuated in mice administered VCAM-1 neutralizing antibody. In addition, blocking VCAM-1 markedly alleviated Ang II-induced BMMs adhesion and migration, therefore inhibited cardiomyocyte hypertrophy and fibroblast activation. In conclusion, the data reveal that blocking VCAM-1 ameliorates hypertensive cardiac remodeling by impeding VLA-4+ macrophage infiltration. Selective blockage of VCAM-1 may be a novel therapeutic strategy for hypertensive cardiac diseases.

Eldecalcitol prevents muscle loss by suppressing PI3K/AKT/FOXOs pathway in orchiectomized mice.

Elderly male patients are susceptible to develop osteoporosis and sarcopenia, especially those with fragility fractures, hypogonadism, and prostate cancer with androgen deprivation therapy. However, at present, very few treatments are available for men with sarcopenia. Previous preclinical studies in ovariectomized rats have shown the promising effects of eldecalcitol in ameliorating the bone strength and muscle atrophy. We thus investigated the effects of eldecalcitol on androgen-deficient male mice. Six-week-old male mice underwent orchiectomy (ORX) or sham surgery. Mice were randomly divided into 4 groups (n = 12/per group), including 1) sham mice, 2) ORX group, 3) ORX eldecalcitol 30 ng/kg, and 4) ORX eldecalcitol 50 ng/kg. Eldecalcitol increased bone mass and strength of femur in ORX mice. Eldecalcitol 30 ng/kg dose completely rescued ORX-induced muscle weakness. The RT-qPCR showed that eldecalcitol enhanced the mRNA levels of type I and IIa fibers. The expression levels of MuRF1 and Atrogin-1 of gastrocnemius in the eldecalcitol groups were much lower than that of the ORX group. It is assumed that eldecalcitol potentially acts via PI3K/AKT/FOXOs signaling pathway. These findings provide evidence for evaluating eldecalcitol as an investigational treatment for male patients with sarcopenia and osteoporosis.

Association between Serum Oxytocin, Bone Mineral Density and Body Composition in Chinese Adult Females.

Background and Objectives: Oxytocin (OT) is a neuropeptide hormone which is known for its classical effects in pregnancy and lactation. Recently, growing evidence demonstrated a close relation between OT and bone. The present study aimed to explore the relationship between OT, bone and osteoporosis risk in Chinese adult females. Materials and Methods: in total, 149 adult females were enrolled. The serum OT levels were measured using ELISA kits. Bone mineral density (BMD) and body composition were measured by dual-energy X-ray absorptiometry (DXA). The study subjects were divided into two groups according to their menopause status and then divided into tertiles based on their serum OT level. Results: Serum OT, serum estradiol and BMD at three skeletal sites were significantly higher in the premenopausal group than in the postmenopausal group (p < 0.001, p = 0.008 and p < 0.001, respectively). In the tertile analysis, relative to tertile 1, significant associations were found for tertile 3 for OT levels and higher BMD in the femoral neck and total hip, in both pre- and postmenopausal groups. Using logistic regression analysis, tertile 3 appeared less likely to have low-BMD osteoporosis than tertile 1 (OR = 0.257, 95% CI = 0.073, 0.910). In multivariate stepwise regression analysis, OT and total lean mass were two positive determinants of BMD in the femoral neck and total hip in the premenopausal group (adjusted R2 for the model = 0.232 and 0.199, respectively; both p < 0.001). Conclusion: Our study demonstrated positive associations between serum OT levels and BMD in a Chinese (non-Caucasian) population. OT appeared to be more strongly associated with hip BMD in premenopausal females. These results may suggest a protective role and potential therapeutic use of OT in osteoporosis, especially for premenopausal women.

Risk factors of mortality and second fracture after elderly hip fracture surgery in Shanghai, China.

INTRODUCTION: Hip fracture is one of the leading causes of death and disability in the elderly. We analyzed the risk factors of mortality and second fracture within 2 years after hip fracture surgery in elderly Chinese patients. MATERIALS AND METHODS: A total of 613 elderly patients after hip fracture surgery were selected, including 181 males and 432 females, and the patients were followed for at least 24 months. Information about patients and surgery was collected from medical records. Information on death, secondary fracture, and postoperative activities of daily living (ADL) was obtained by telephone follow-up. Cox regression was performed to identify risk factors associated with mortality and second fracture, measured by hazard ratio (HR). RESULTS: The 1-year and 2-year mortality rates after hip fracture were 13.4% and 20.7%, respectively. The second fracture rate within 2 years was 9.5%. Male gender (HR 1.51, P = 0.035), increased age (HR 1.07, P < 0.001), preoperative hypoalbuminemia (HR 1.79, P = 0.004), preoperative pneumonia (HR 2.60, P = 0.005) and poor ADL (P = 0.048) were independent risk factors for 2-year mortality, while high preoperative hemoglobin (HR 0.98, P = 0.002), high preoperative eGFR (HR 0.99, P = 0.031), high preoperative LVEF (HR 0.92, P = 0.048) were protective factors for 2-year mortality. Poor ADL (P = 0.002) was the independent risk factor for second fracture within 2 years. CONCLUSIONS: The 2-year mortality rate and second fracture rate after hip fracture in elderly remained high, which was related to old age and complications exists. Postoperative rehabilitation and improving ADL were very important to reduce mortality and second fracture.

Isolation of murine bone marrow hematopoietic stem and progenitor cell populations via flow cytometry.

Flow cytometry is a powerful technology that allows not only multiparameter quantitative data analysis at single cell resolution but also simultaneous cell separation of different populations of interest at high speed. It has been rapidly employed in biological research and clinical diagnostics. This technology has enabled the thorough understanding of murine hematopoiesis, especially the physiology of surface marker-defined hematopoietic stem and progenitor cell populations. The isolation of these populations has been well established over the last three decades with a large consensus among leading laboratories. Here, we describe a detailed methodology protocol of two different state-of-the-art approaches to isolate bone marrow cells and purify hematopoietic stem and progenitor cells via flow cytometry. Different gating schemes are introduced to identify well-defined populations of murine hematopoietic stem and multipotent progenitor cells.

Preparation and Characterization of Site-Specific Fatty Chain-Modified Recombinant Human Granulocyte Colony Stimulating Factor.

The clinical use of recombinant human granulocyte colony-stimulating factor (rhG-CSF) is limited by its short serum half-life. In this study, a long-acting strategy for site-specific modification of rhG-CSF with 1-pentadecyl-1H-pyrrole-2,5-dione (C15 fatty chain-maleimide, C15-MAL) was studied in mixed DMSO-aqueous solutions. The factors influencing the conjugation reaction were investigated and optimized, and a high yield of the desired product (C15-rhG-CSF) was achieved. Subsequently, C15-rhG-CSF product was efficiently purified using preparative liquid chromatography, and further characterized. Circular dichroism spectroscopy analysis showed that the secondary structure of C15-rhG-CSF had no significant difference from unmodified rhG-CSF. C15-rhG-CSF retained 87.2% of in vitro bioactivity of unmodified rhG-CSF. The pharmacokinetic study showed that the serum half-life of C15-rhG-CSF in mice was 2.08-fold longer than that of unmodified rhG-CSF. Furthermore, C15-rhG-CSF by single-dose subcutaneous administration showed better in vivo efficacy than those of both PEG10k-rhG-CSF by single-dose administration and rhG-CSF by multiple doses administration. This study demonstrated the potential of C15-rhG-CSF being developed into a novel drug candidate as well as an efficient process for the development of long-acting protein and peptide drugs.

Blocking VCAM-1 Prevents Angiotensin II-Induced Hypertension and Vascular Remodeling in Mice.

Adhesion of monocytes to the vascular endothelium frequently leads to an inflammatory response, which contributes to hypertension and vascular remodeling. Vascular cellular adhesion molecule-1 (VCAM-1) plays an important role in leukocyte adhesion and migration during inflammatory diseases. However, its role in angiotensin (Ang) II -induced hypertension and vascular dysfunction remains largely unknown. Wild-type (WT) mice were administered a VCAM-1 neutralizing antibody (0.1 or 0.2 mg/mouse/day) or IgG control and then infused with Ang II (490 ng kg-1 min-1) or saline continuously for 14 days. Systolic blood pressure (SBP) was measured with a tail-cuff system, pathological changes in the aorta were assessed by histological staining, and vascular relaxation was analyzed an aortic ring assay. Our results indicated that compared with saline infusion, Ang II infusion significantly upregulated VCAM-1 expression in the mouse aorta and serum. Moreover, Ang II infusion markedly increased arterial hypertension, wall thickness, fibrosis, infiltration of Mac-2+ macrophages, reactive oxygen species (ROS) production and vascular relaxation dysfunction. Conversely, blockade of VCAM-1 with a neutralizing antibody substantially alleviated these effects. In vitro experiments further confirmed that the VCAM-1 neutralizing antibody inhibited Ang II-induced macrophage adhesion and migration and DNA damage and oxidative stress in endothelial cells (ECs). In conclusion, these results indicate that blockade of VCAM-1 exerts a protective effect against Ang II-induced arterial hypertension and dysfunction by regulating monocytes adhesion and infiltration into the endothelium and represents a novel therapeutic approach for hypertension.

Angiotensin II Induces Cardiac Edema and Hypertrophic Remodeling through Lymphatic-Dependent Mechanisms.

Cardiac lymphatic vessel growth (lymphangiogenesis) and integrity play an essential role in maintaining tissue fluid balance. Inhibition of lymphatic lymphangiogenesis is involved in cardiac edema and cardiac remodeling after ischemic injury or pressure overload. However, whether lymphatic vessel integrity is disrupted during angiotensin II- (Ang II-) induced cardiac remodeling remains to be investigated. In this study, cardiac remodeling models were established by Ang II (1000 ng/kg/min) in VEGFR-3 knockdown (Lyve-1Cre VEGFR-3f/-) and wild-type (VEGFR-3f/f) littermates. Our results indicated that Ang II infusion not only induced cardiac lymphangiogenesis and upregulation of VEGF-C and VEGFR-3 expression in the time-dependent manner but also enhanced proteasome activity, MKP5 and VE-cadherin degradation, p38 MAPK activation, and lymphatic vessel hyperpermeability. Moreover, VEGFR-3 knockdown significantly inhibited cardiac lymphangiogenesis in mice, resulting in exacerbation of tissue edema, hypertrophy, fibrosis superoxide production, inflammation, and heart failure (HF). Conversely, administration of epoxomicin (a selective proteasome inhibitor) markedly mitigated Ang II-induced cardiac edema, remodeling, and dysfunction; upregulated MKP5 and VE-cadherin expression; inactivated p38 MAPK; and reduced lymphatic vessel hyperpermeability in WT mice, indicating that inhibition of proteasome activity is required to maintain lymphatic endothelial cell (LEC) integrity. Our results show that both cardiac lymphangiogenesis and lymphatic barrier hyperpermeability are implicated in Ang II-induced adaptive hypertrophic remodeling and dysfunction. Proteasome-mediated hyperpermeability of LEC junctions plays a predominant role in the development of cardiac remodeling. Selective stimulation of lymphangiogenesis or inhibition of proteasome activity may be a potential therapeutic option for treating hypertension-induced cardiac remodeling.

Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53.

Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein-protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.

Fe3O4@Polydopamine-Labeled MSCs Targeting the Spinal Cord to Treat Neuropathic Pain Under the Guidance of a Magnetic Field.

PURPOSE: Neuropathic pain causes great distress among patients; however, its response to traditional analgesia techniques remains sub-optimal. There has been progress in stem cell research for neuropathic pain treatment; however, effective homing remains problematic. This study aimed to establish Fe3O4@polydopamine(PDA)-labeled mesenchymal stem cells (MSCs); moreover, we aimed to guide MSCs using a magnetic field to the spinal cord segments showing pain-related responses to allow MSC homing and gathering, in advance, in order to fully employ their repair function. MATERIALS AND METHODS: Fe3O4@PDA-labeled MSCs were characterized using transmission electron microscopy. We analyzed the characteristics of MSCs, as well as the nanoparticle effects on MSC activity, differentiation, and proliferation, using the CCK-8 method, flow cytometry, and staining. Using rats, we performed behavioral tests of mechanical and thermal pain hypersensitivity. Serum inflammatory markers were detected using ELISA. Finally, changes in proteins associated with spinal cord pain were detected through quantitative reverse transcription PCR, histology, and immunohistochemistry. RESULTS: Fe3O4@PDA did not affect the characteristics and viability of MSCs. The magnetic field guidance improved the therapeutic effect of Fe3O4@PDA-labeled MSCs as indicated by the paw withdrawal threshold. Fe3O4@PDA-labeled MSCs decreased spinal nerve demyelination and c-Fos expression (a pain molecule); moreover, they inhibited microglia and astrocyte activation. CONCLUSION: Fe3O4@PDA-labeled MSCs showed better homing to the spinal cord under magnetic field guidance. Moreover, they inhibited microglial and astrocyte activation, as well as played an early and continuous role in neuropathic pain treatment.

GPR182 is an endothelium-specific atypical chemokine receptor that maintains hematopoietic stem cell homeostasis.

G protein-coupled receptor 182 (GPR182) has been shown to be expressed in endothelial cells; however, its ligand and physiological role has remained elusive. We found GPR182 to be expressed in microvascular and lymphatic endothelial cells of most organs and to bind with nanomolar affinity the chemokines CXCL10, CXCL12, and CXCL13. In contrast to conventional chemokine receptors, binding of chemokines to GPR182 did not induce typical downstream signaling processes, including Gq- and Gi-mediated signaling or ß-arrestin recruitment. GPR182 showed relatively high constitutive activity in regard to ß-arrestin recruitment and rapidly internalized in a ligand-independent manner. In constitutive GPR182-deficient mice, as well as after induced endothelium-specific loss of GPR182, we found significant increases in the plasma levels of CXCL10, CXCL12, and CXCL13. Global and induced endothelium-specific GPR182-deficient mice showed a significant decrease in hematopoietic stem cells in the bone marrow as well as increased colony-forming units of hematopoietic progenitors in the blood and the spleen. Our data show that GPR182 is a new atypical chemokine receptor for CXCL10, CXCL12, and CXCL13, which is involved in the regulation of hematopoietic stem cell homeostasis.

The JmjC-domain protein NO66/RIOX-1 affects the balance between proliferation and maturation in acute myeloid leukemia.

As epigenetic regulators are frequently dysregulated in acute myeloid leukemia (AML) we determined expression levels of the JmjC-protein NO66 in AML cell lines and sub fractions of healthy human hematopoietic cells. NO66 is absent in the AML cell lines KG1/KG1a which consist of cells with the immature CD34+/CD38- phenotype and is regarded as a "stem cell-like" model system. Similarly, NO66 is not detectable in CD34+/CD38- cells purified from healthy donors but is clearly expressed in the more committed CD34+/CD38+ cell population. Loss of NO66 expression in KG1/KG1a cells is due to hyper-methylation of its promoter and is released by DNA-methyltransferase inhibitors. In KG1a cells stably expressing exogenous wild type (KG1a66wt) or enzymatically inactive mutant (KG1a66mut) NO66, respectively, the wild type protein inhibited proliferation and rDNA transcription. Gene expression profiling revealed that the expression of NO66 induces a transcriptional program enriched for genes with roles in proliferation and maturation (e.g.EPDR1, FCER1A, CD247, MYCN, SNORD13). Genes important for the maintenance of stem cell properties are downregulated (e.g. SIRPA, Lin28B, JAML). Our results indicate that NO66 induces lineage commitment towards myeloid progenitor cell fate and suggest that NO66 contributes to loss of stem cell properties.

Association between LGR4 polymorphisms and peak bone mineral density and body composition.

INTRODUCTION: Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) could affect differentiation of osteoblasts and bone mass through potentiating Wnt/ß-catenin signaling. LGR4 is also relevant to glycolipid metabolism. The present study aims to explore the relationship between genetic variations in LGR4 gene and peak bone mineral density (peak BMD) and body composition phenotypes in Chinese nuclear families. MATERIALS AND METHODS: 22 single-nucleotide polymorphisms (SNPs) were selected and five blocks were constructed in LGR4. Body composition (lean mass and fat mass) and peak BMD were measured by dual-energy X-ray absorptiometry (DXA). Quantitative transmission disequilibrium test (QTDT) analysis was used to explore the relationship between LGR4 genotypes and the mentioned phenotypes. RESULTS: For QTDT analysis after 1000 permutations, significant within-family associations were observed between rs11029986 and total fat mass (TFM) and percentage of TFM (PFM) (P = 0.014 and 0.011, respectively), rs12787344, rs4128868, rs4923445, and rs7936621 and body mass index (BMI) (P = 0.008, 0.003, 0.046, and 0.003, respectively), rs11029986 and total hip BMD (P = 0.026), and rs12796247, rs2219783, and lumbar spine BMD (P = 0.013 and 0.027, respectively). Haplotypes GCGT and AAGC (both in block 3) were observed in significant within-family association with BMI (P = 0.003 and 0.002, respectively). CONCLUSION: It is the first family-based association analysis to explore and demonstrate significant associations between LGR4 genotypes and variations of peak BMD and body composition in young Chinese men. The results are consistent with the findings that recent studies revealed, and confirm the critical relationship between LGR4 gene and both BMD and body composition.

Reports of 17 Chinese patients with tumor-induced osteomalacia.

Tumor-induced osteomalacia (TIO) is a rare acquired form of hypophosphatemic osteomalacia, which is usually attributed to the overproduction of fibroblast growth factor 23 (FGF-23) by benign mesenchymal neoplasms. Localization and thereafter surgical resection of tumors lead to a cure. The present study aimed to investigate the clinical data, diagnostic methods, and follow-up after tumor resection at one medical center in Shanghai to characterize the profile of this rare disorder and to share our successful experience in diagnosis and treatment. Twenty-three patients with adult-onset hypophosphatemia osteomalacia seen in Shanghai Sixth People's Hospital from 2009 to 2014 and 95 normal individuals were enrolled. After taking a medical history and performing a physical examination, we analyzed the laboratory results (including the serum FGF-23 levels) and localized the tumors by 18F-fluorodeoxyglucose positron emission tomography and computed tomography (18F-FDG PET/CT), 99mTc-octreotide (99mTc-OCT) scintigraphy, and magnetic resonance imaging (MRI). On the basis of the results of laboratory tests and imaging findings, tumor resection was conducted in 17 patients with a certain diagnosis of TIO. The results demonstrated that the 17 patients (nine men and eight women, average age 46.6 ± 12.9 years) had TIO. FGF-23 level was elevated in 94.1 % of patients (16 of 17 patients) . Serum phosphorus level decreased in 100 % of patients. 18F-FDG PET/CT revealed five tumors, 99mTc-OCT scintigraphy revealed two tumors, physical examination revealed nine tumors, and MRI revealed one tumor, among which 58.8 % of the causative tumors (10 of 17 tumors) were located in the lower extremities. After tumor resection, serum phosphorus levels normalized in 100 % of patients (all 17 patients) in 4-21 days and FGF-23 levels decreased in 90 % of patients (nine of ten patients). We found 64.7 % of the tumors (11 of 17 tumors) were phosphaturic mesenchymal tumors or a phosphaturic mesenchymal tumor mixed connective tissue variant. Measurement of serum phosphorus and FGF-23 levels in patients with suspected TIO is of paramount importance for diagnosing of TIO. 18F-FDG PET/CT, 99mTc-OCT scintigraphy, and physical examination play a considerable role in revealing TIO-associated tumors. TIO-associated tumors were more frequently located in the lower extremities than in other places; thus, the lower extremities need to be carefully checked. Complete surgical resection results in normalization of parameters in laboratory tests and relief of symptoms of TIO patients.