Dynamic patterns of correlated activity in the prefrontal cortex encode information about social behavior.

New technologies make it possible to measure activity from many neurons simultaneously. One approach is to analyze simultaneously recorded neurons individually, then group together neurons which increase their activity during similar behaviors into an "ensemble." However, this notion of an ensemble ignores the ability of neurons to act collectively and encode and transmit information in ways that are not reflected by their individual activity levels. We used microendoscopic GCaMP imaging to measure prefrontal activity while mice were either alone or engaged in social interaction. We developed an approach that combines a neural network classifier and surrogate (shuffled) datasets to characterize how neurons synergistically transmit information about social behavior. Notably, unlike optimal linear classifiers, a neural network classifier with a single linear hidden layer can discriminate network states which differ solely in patterns of coactivity, and not in the activity levels of individual neurons. Using this approach, we found that surrogate datasets which preserve behaviorally specific patterns of coactivity (correlations) outperform those which preserve behaviorally driven changes in activity levels but not correlated activity. Thus, social behavior elicits increases in correlated activity that are not explained simply by the activity levels of the underlying neurons, and prefrontal neurons act collectively to transmit information about socialization via these correlations. Notably, this ability of correlated activity to enhance the information transmitted by neuronal ensembles is diminished in mice lacking the autism-associated gene Shank3. These results show that synergy is an important concept for the coding of social behavior which can be disrupted in disease states, reveal a specific mechanism underlying this synergy (social behavior increases correlated activity within specific ensembles), and outline methods for studying how neurons within an ensemble can work together to encode information.

Central thromboxane A2, prostaglandin F2α, prostaglandin E, and prostaglandin D contribute to the cardiovascular effects elicited by nesfatin-1.

Background/aim: Our recent study revealed that the expression of lipoxygenase (LOX) and cyclooxygenase (COX) enzymes in the hypothalamus is activated by nesfatin-1, leading to the liberation of leukotrienes and prostaglandins (PG), respectively. Moreover, our prior report explained that intracerebroventricular (ICV) nesfatin-1 treatment triggers cardiovascular responses mediated by central LOX and COX enzymes. Building upon our prior reports, the present investigation sought to clarify the role of cardiovascularly active central COX products, such as thromboxane (TX) A2, PGF2α, PGE, and PGD, in orchestrating nesfatin-1-evoked reactions in mean arterial pressure (MAP) and heart rate (HR). Materials and methods: The Sprague Dawley rats, which had guide cannula in the lateral ventricle for intracerebroventricular (ICV) injections and catheter in arteria femoralis for monitoring MAP and HR, were underwent central pretreatment with furegrelate (the TXA2 synthase inhibitor), PGF2α-dimethylamine (PGF2α-DA, the PGF2α receptor antagonist), or AH6809 (the PGE and PGD receptor antagonist), 5 min prior to ICV nesfatin-1 administration. The cardiovascular parameters were observed and recorded for 60 min posttreatment. Results: Nesfatin-1 induced cardiovascular responses in rats leading to pressor effect in MAP, and tachycardia following bradycardia in HR. Interestingly, ICV furegrelate, PGF2α-DA, or AH6809 pretreatment partially mitigated the cardiovascular effects revealed by nesfatin-1. Conclusion: The findings illuminate the role of nesfatin-1 in modulating MAP and HR through the central activation of specifically TXA2, PGF2α, PGE, and PGD from COX metabolites. Additionally, the study may also suggest the potential involvement of other central COX or LOX metabolites beyond these COX metabolites in mediating the cardiovascular effects produced by nesfatin-1.

[Mechanism of n-butanol alcohol extract of Baitouweng Decoction in treatment of vulvovaginal candidiasis based on negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis].

This study aimed to explore the mechanism of n-butanol alcohol extract of Baitouweng Decoction(BAEB) in the treatment of vulvovaginal candidiasis(VVC) in mice based on the negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis. In the experiment, female C57BL/6 mice were divided randomly into the following six groups: a blank control group, a VVC model group, high-, medium-, and low-dose BAEB groups(80, 40, and 20 mg·kg~(-1)), and a fluconazole group(20 mg·kg~(-1)). The VVC model was induced in mice except for those in the blank control group by the estrogen dependence method. After modeling, no treatment was carried out in the blank control group. The mice in the high-, medium-, and low-dose BAEB groups were treated with BAEB at 80, 40, and 20 mg·kg~(-1), respectively, and those in the fluconazole group were treated with fluconazole at 20 mg·kg~(-1). The mice in the VVC model group received the same volume of normal saline. The general state and body weight of mice in each group were observed every day, and the morphological changes of Candida albicans in the vaginal lavage of mice were examined by Gram staining. The fungal load in the vaginal lavage of mice was detected by microdilution assay. After the mice were killed, the degree of neutrophil infiltration in the vaginal lavage was detected by Papanicolaou staining. The content of inflammatory cytokines interleukin(IL)-1ß, IL-18, and lactate dehydrogenase(LDH) in the vaginal lavage was tested by enzyme-linked immunosorbent assay(ELISA), and vaginal histopathology was analyzed by hematoxylin-eosin(HE) staining. The expression and distribution of NLRP3, PKCδ, pNLRC4, and IL-1Ra in vaginal tissues were measured by immunohistochemistry(IHC), and the expression and distribution of pNLRC4 and IL-1Ra in vaginal tissues were detected by immunofluorescence(IF). The protein expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by Western blot(WB), and the mRNA expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by qRT-PCR. The results showed that compared with the blank control group, the VVC model group showed redness, edema, and white secretions in the vagina. Compared with the VVC model group, the BAEB groups showed improved general state of VVC mice. As revealed by Gram staining, Papanicolaou staining, microdilution assay, and HE staining, compared with the blank control group, the VVC model group showed a large number of hyphae, neutrophils infiltration, and increased fungal load in the vaginal lavage, destroyed vaginal mucosa, and infiltration of a large number of inflammatory cells. BAEB could reduce the transformation of C. albicans from yeast to hyphae. High-dose BAEB could significantly reduce neutrophil infiltration and fungal load. Low-and medium-dose BAEB could reduce the da-mage to the vaginal tissue, while high-dose BAEB could restore the damaged vaginal tissues to normal levels. ELISA results showed that the content of inflammatory cytokines IL-1ß, IL-18, and LDH in the VVC model group significantly increased compared with that in the blank control group, and the content of IL-1ß, IL-18 and LDH in the medium-and high-dose BAEB groups was significantly reduced compared with that in the VVC model group. WB and qRT-PCR results showed that compared with the blank control group, the VVC model group showed reduced protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues of mice and increased protein and mRNA expression of NLRP3. Compared with the VVC model group, the medium-and high-dose BAEB groups showed up-regulated protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues and inhibited protein and mRNA expression of NLRP3 in vaginal tissues. This study indicated that the therapeutic effect of BAEB on VVC mice was presumably related to the negative regulation of NLRP3 inflammasome by promoting PKCδ/NLRC4/IL-1Ra axis.

Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature.

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires the cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for the stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases the accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

Extracellular Regucalcin Suppresses the Growth, Migration, Invasion, and Adhesion of Metastatic Human Prostate Cancer Cells.

INTRODUCTION: Regucalcin plays a multifunctional role in the regulation of cellular function including metabolism, signaling process, and transcriptional activity in maintaining cell homeostasis. Downregulated expression or activity of regucalcin contributes to the development of malignancies in various types of human cancer. Survival of cancer patients, including metastatic prostate cancer, is prolonged with high expression of regucalcin in the tumor tissues. METHODS: We elucidate whether extracellular regucalcin conquers the growth, migration, invasion, and adhesion of metastatic human prostate cancer PC-3 and DU-145 cells. RESULTS: Extracellular regucalcin (0.1, 1, and 10 nM) of physiologic levels (1 nM at human serum) inhibited colony formation and growth of PC-3 and DU-145 cells, while it did not have an effect on cell death. Repressive effects of extracellular regucalcin on the proliferation were not exhibited by the presence of inhibitors of the cell cycle, intracellular signaling process, and transcriptional activity, suggesting that the signals of extracellular regucalcin are transmitted to block cell growth. Furthermore, extracellular regucalcin (0.1, 1, or 10 nM) inhibited migration, invasion, and adhesion of PC-3 and DU-145 cells. Mechanistically, extracellular regucalcin (10 nM) decreased the levels of various signaling proteins including Ras, posphatidylinositol-3 kinase, mitogen-activated protein kinase, mechanistic target of rapamycin, RSK-2, caveolin-1, and integrin ß1 in PC-3 cells. DISCUSSION AND CONCLUSION: Thus, extracellular regucalcin may play a suppressive role in growth, migration, invasion, and adhesion, which are involved in the metastatic activity of human prostate cancer cells, via affecting diverse signaling processes. This study may provide a new strategy in preventing metastatic prostate cancer with exogenous regucalcin.

Phospholamban and sarcolipin prevent thermal inactivation of sarco(endo)plasmic reticulum Ca2+-ATPases.

Na+-K+-ATPase from mice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) are small homologous proteins that regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1 h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25-35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form a protective interaction with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity.

Cartilage Acidic Protein a Novel Therapeutic Factor to Improve Skin Damage Repair?

Fish skin has been gaining attention due to its efficacy as a human-wound-treatment product and to identify factors promoting its enhanced action. Skin fibroblasts have a central role in maintaining skin integrity and secrete extra cellular matrix (ECM) proteins, growth factors and cytokines to rapidly repair lesions and prevent further damage or infection. The effects on scratch repair of the ubiquitous but poorly characterized ECM protein, cartilage acidic protein 1 (CRTAC1), from piscine and human sources were compared using a zebrafish SJD.1 primary fibroblast cell line. A classic in vitro cell scratch assay, immunofluorescence, biosensor and gene expression analysis were used. Our results demonstrated that the duplicate sea bass Crtac1a and Crtac1b proteins and human CRTAC-1A all promoted SJD.1 primary fibroblast migration in a classic scratch assay and in an electric cell impedance sensing assay. The immunofluorescence analysis revealed that CRTAC1 enhanced cell migration was most likely caused by actin-driven cytoskeletal changes and the cellular transcriptional response was most affected in the early stage (6 h) of scratch repair. In summary, our results suggest that CRTAC1 may be an important factor in fish skin promoting damage repair.

Calpastatin-Mediated Inhibition of Calpain Ameliorates Skin Scar Formation after Burn Injury.

Hypertrophic scars, the most common complication of burn injuries, are characterized by excessive deposition of fibroblast-derived extracellular matrix proteins. Calpain, a calcium-dependent protease, is involved in the fibroblast proliferation and extracellular matrix production observed in certain fibrotic diseases. However, its role in the formation of post-burn hypertrophic skin scars remains largely unknown. Here, calpain expression and activity were assessed in skin fibroblasts obtained directly from patients with third-degree burns, who consequently developed post-burn hypertrophic scars. Furthermore, the antifibrotic effect of calpastatin, an endogenous calpain inhibitor, was evaluated in human fibroblasts and a murine burn model. The activity, mRNA levels, and protein levels of calpain were markedly higher in fibroblasts from the burn wounds of patients than in normal cells. Selective calpain inhibition by calpastatin markedly reduced not only the proliferation of burn-wound fibroblasts but also the mRNA and protein expression of calpain, transforming growth factor-beta 1, α-smooth muscle actin, type I and type III collagens, fibronectin, and vimentin in burn-wound fibroblasts. The anti-scarring effects of calpastatin were validated using a murine burn model by molecular, histological, and visual analyses. This study demonstrates the pathological role of calpain and the antifibrotic effect of calpastatin via calpain inhibition in post-burn hypertrophic scar formation.

Fibulin-7 C-terminal fragment and its active synthetic peptide suppress choroidal and retinal neovascularization.

Wet age-related macular degeneration (AMD) and diabetic retinopathy are the leading causes of blindness through increased angiogenesis. Although VEGF-neutralizing proteins provide benefit, inconsistent responses indicate a need for new therapies. We previously identified the Fibulin-7 C-terminal fragment (Fbln7-C) as an angiogenesis inhibitor in vitro. Here we show that Fbln7-C inhibits neovascularization in vivo, in both a model of wet AMD involving choroidal neovascularization (CNV) and diabetic retinopathy involving oxygen-induced ischemic retinopathy. Furthermore, a short peptide sequence from Fbln7-C is responsible for the anti-angiogenic properties of Fbln7-C. Our work suggests Fbln7-C as a therapeutic candidate for wet AMD and ischemic retinopathy.

Clostridium perfringens α-toxin inhibits myogenic differentiation of C2C12 myoblasts.

Clostridium perfringens type A is the causative agent of clostridial myonecrosis, and α-toxin has been reported to be responsible for the pathogenesis. Recently, it was reported that regeneration of skeletal muscle after C. perfringens-induced muscle disorders is delayed, but the detailed mechanisms have not been elucidated. Here, we tested whether α-toxin impairs the differentiation of C2C12 myoblasts, a useful cell line to study muscle growth, maturation, and regeneration in vitro. α-Toxin dose-dependently inhibited myotube formation in C2C12 cultures after induction of their differentiation by horse serum. Also, immunoblot analysis revealed that α-toxin dose-dependently decreases the expressions of two skeletal muscle differentiation markers, myogenic differentiation 1 (MyoD) and myogenin. These results demonstrate that α-toxin impairs the myogenic differentiation of C2C12 myoblasts. To reveal the mechanism behind α-toxin-mediated impairment of myogenic differentiation, we focused on ceramide production since α-toxin is known to promote the formation of ceramide by its sphingomyelinase activity. Immunofluorescent analysis revealed that ceramide production is accelerated by treatment with α-toxin. Furthermore, a synthetic cell-permeable ceramide analog, C2-ceramide, inhibited myotube formation in C2C12 cells and decreased the expressions of MyoD and myogenin, suggesting that accelerated ceramide production is involved in the α-toxin-mediated blockage of myogenic differentiation. Together, our results illustrate that the impairment of myogenic differentiation by α-toxin might be crucial for the pathogenesis of C. perfringens to delay regeneration of severely damaged skeletal muscles.

The roles of circRFWD2 and circINO80 during NELL-1-induced osteogenesis.

Bone defects caused heavy social and economic burdens worldwide. Nel-like molecule, type 1 (NELL-1) could enhance the osteogenesis and the repairment of bone defects, while the specific mechanism remains to be elucidated. Circular RNAs (circRNAs) have been found to play critical roles in the tissue development and serve as biomarkers for various diseases. However, it remains unclear that the expression patterns of circRNAs and the roles of them played in recombinant NELL-1-induced osteogenesis of human adipose-derived stem cells (hASCs). In this study, we performed RNA-sequencing to investigate the expression profiles of circRNAs in recombinant NELL-1-induced osteogenic differentiation and identified two key circRNAs, namely circRFWD2 and circINO80. These two circRNAs were confirmed to be up-regulated during recombinant NELL-1-induced osteogenesis, and knockdown of them affected the positive effect of NELL-1 on osteogenesis. CircRFWD2 and circINO80 could interact with hsa-miR-6817-5p, which could inhibit the osteogenesis. Silencing hsa-miR-6817-5p could partially reverse the negative effect of si-circRFWD2 and si-circINO80 on the osteogenesis. Therefore, circRFWD2 and circINO80 could regulate the expression of hsa-miR-6817-5p and influence the recombinant NELL-1-induced osteogenic differentiation of hASCs. It opens a new window to better understanding the effects of NELL-1 on the osteogenic differentiation of hASCs and provides potential molecular targets and novel methods for bone regeneration efficiently and safely.

Structural basis for relief of phospholamban-mediated inhibition of the sarcoplasmic reticulum Ca2+-ATPase at saturating Ca2+ conditions.

Sarcoplasmic reticulum Ca2+-ATPase (SERCA) is critical for cardiac Ca2+ transport. Reversal of phospholamban (PLB)-mediated SERCA inhibition by saturating Ca2+ conditions operates as a physiological rheostat to reactivate SERCA function in the absence of PLB phosphorylation. Here, we performed extensive atomistic molecular dynamics simulations to probe the structural mechanism of this process. Simulation of the inhibitory complex at superphysiological Ca2+ concentrations ([Ca2+] = 10 mm) revealed that Ca2+ ions interact primarily with SERCA and the lipid headgroups, but not with PLB's cytosolic domain or the cytosolic side of the SERCA-PLB interface. At this [Ca2+], a single Ca2+ ion was translocated from the cytosol to the transmembrane transport sites. We used this Ca2+-bound complex as an initial structure to simulate the effects of saturating Ca2+ at physiological conditions ([Ca2+]total ≈ 400 µm). At these conditions, ∼30% of the Ca2+-bound complexes exhibited structural features consistent with an inhibited state. However, in ∼70% of the Ca2+-bound complexes, Ca2+ moved to transport site I, recruited Glu771 and Asp800, and disrupted key inhibitory contacts involving the conserved PLB residue Asn34 Structural analysis showed that Ca2+ induces only local changes in interresidue inhibitory interactions, but does not induce repositioning or changes in PLB structural dynamics. Upon relief of SERCA inhibition, Ca2+ binding produced a site I configuration sufficient for subsequent SERCA activation. We propose that at saturating [Ca2+] and in the absence of PLB phosphorylation, binding of a single Ca2+ ion in the transport sites rapidly shifts the equilibrium toward a noninhibited SERCA-PLB complex.

The antiapoptotic protein HAX-1 mediates half of phospholamban's inhibitory activity on calcium cycling and contractility in the heart.

The antiapoptotic protein HAX-1 (HS-associated protein X-1) localizes to sarcoplasmic reticulum (SR) in the heart and interacts with the small membrane protein phospholamban (PLN), inhibiting the cardiac sarco/endoplasmic reticulum calcium ATPase 2a (SERCA2a) in the regulation of overall calcium handling and heart muscle contractility. However, because global HAX-1 deletion causes early lethality, how much endogenous HAX-1 contributes to PLN's inhibitory activity on calcium cycling is unknown. We therefore generated a cardiac-specific and inducible knock-out mouse model. HAX-1 ablation in the adult heart significantly increased contractile parameters and calcium kinetics, associated with increased SR calcium load. These changes occurred without any changes in the protein expression of SERCA2a, PLN, and ryanodine receptor or in the PLN phosphorylation status. The enhanced calcium cycling in the HAX-1-depleted heart was mediated through increases in the calcium affinity of SERCA2a and reduced PLN-SERCA2a binding. Comparison of the HAX-1 deletion-induced stimulatory effects with those elicited by PLN ablation indicated that HAX-1 mediates ∼50% of the PLN-associated inhibitory effects in the heart. Stimulation with the inotropic and lusitropic agent isoproterenol eliminated the differences among wild-type, HAX-1-deficient, and PLN-deficient hearts, and maximally stimulated contractile and calcium kinetic parameters were similar among these three groups. Furthermore, PLN overexpression in the HAX-1-null cardiomyocytes did not elicit any inhibitory effects, indicating that HAX-1 may limit PLN activity. These findings suggest that HAX-1 is a major mediator of PLN's inhibitory activity and a critical gatekeeper of SR calcium cycling and contractility in the heart.

Activation of Notch signaling by soluble Dll4 decreases vascular permeability via a cAMP/PKA-dependent pathway.

The Notch ligand delta-like ligand 4 (Dll4), upregulated by VEGF, is a key regulator of vessel morphogenesis and function, controlling tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hypersprouting, nonfunctional, poorly perfused vessels, suggesting a role for Dll4 in the formation of mature, reactive, functional vessels, with low permeability and able to restrict fluid and solute exchange. We tested the hypothesis that Dll4 controls transvascular fluid exchange. A recombinant protein expressing only the extracellular portion of Dll4 [soluble Dll4 (sDll4)] induced Notch signaling in endothelial cells (ECs), resulting in increased expression of vascular-endothelial cadherin, but not the tight junctional protein zonula occludens 1, at intercellular junctions. sDll4 decreased the permeability of FITC-labeled albumin across EC monolayers, and this effect was abrogated by coculture with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. One of the known molecular effectors responsible for strengthening EC-EC contacts is PKA, so we tested the effect of modulation of PKA on the sDll4-mediated reduction of permeability. Inhibition of PKA reversed the sDll4-mediated reduction in permeability and reduced expression of the Notch target gene Hey1. Knockdown of PKA reduced sDLL4-mediated vascular-endothelial cadherin junctional expression. sDll4 also caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon coperfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signaling through Notch activation acts through a cAMP/PKA pathway upon intercellular adherens junctions, but not tight junctions, to regulate endothelial barrier function. NEW & NOTEWORTHY Notch signaling reduces vascular permeability through stimulation of cAMP-dependent protein kinase A.

The Effects of Systemic Therapy of PEGylated NEL-Like Protein 1 (NELL-1) on Fracture Healing in Mice.

Fractures are common, with an incidence of 13.7 per 1000 adults annually. Systemic agents have been widely used for enhancing bone regeneration; however, the efficacy of these therapeutics for the management and prevention of fracture remains unclear. NEL-like protein 1 (NELL-1) is a potent pro-osteogenic cytokine that has been modified with polyethylene glycol (PEG)ylation [PEGylated NELL-1 (NELL-PEG)] to enhance its pharmacokinetics for systemic therapy. Our aim was to investigate the effects of systemic administration of NELL-PEG on fracture healing in mice and on overall bone properties in uninjured bones. Ten-week-old CD-1 mice were subjected to an open osteotomy of bilateral radii and treated with weekly injections of NELL-PEG or PEG phosphate-buffered saline as control. Systemic injection of NELL-PEG resulted in improved bone mineral density of the fracture site and accelerated callus union. After 4 weeks of treatment, mice treated with NELL-PEG exhibited substantially enhanced callus volume, callus mineralization, and biomechanical properties. NELL-PEG injection significantly augmented bone regeneration, as confirmed by high expression of bone turnover rate, bone formation rate, and mineral apposition rate. Consistently, the immunohistochemistry results also confirmed a high bone remodeling activity in the NELL-PEG-treated group. Our findings suggest that weekly injection of NELL-PEG may have the clinical potential to accelerate fracture union and enhance overall bone properties, which may help prevent subsequent fractures.

Calmodulin-like skin protein suppresses the increase in senescence-associated ß-galactosidase induced by hydrogen peroxide or ultraviolet irradiation in keratinocytes.

Calmodulin-like skin protein (CLSP) is a secreted peptide that is produced by skin keratinocytes and some related epithelial cells. It has previously been shown that CLSP is recruited via the bloodstream into the central nervous system where it likely exerts a neuroprotective effect against toxicity related to Alzheimer's disease (AD) by binding to the heterotrimeric humanin receptor and activating intracellular survival signaling. However, it remains to be elucidated whether secreted CLSP shows a protective effect in the skin tissues. In the current study, using primary keratinocytes treated with hydrogen peroxide (H2 O2 ) or exposed to ultraviolet (UV) irradiation as senescence models of keratinocytes, we addressed whether CLSP affects senescence in skin keratinocytes. We found that CLSP expression was upregulated by H2 O2 or UV in keratinocytes. Furthermore, co-incubation with recombinant CLSP reduced the increase in senescence-associated ß-galactosidase-positivity in keratinocytes that were induced by H2 O2 or UV. These results suggest that CLSP may function as a senescence-suppressing factor in keratinocytes.

Crystal structures of the calcium pump and sarcolipin in the Mg2+-bound E1 state.

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes, and are distinct from other ATPases in that the reaction cycle includes an autophosphorylation step. The best studied is Ca(2+)-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a Ca(2+) pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates. An important outstanding structure is that of the E1 intermediate, which has empty high-affinity Ca(2+)-binding sites ready to accept new cytosolic Ca(2+). In the absence of Ca(2+) and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for Ca(2+)), and if millimolar Mg(2+) is present, one Mg(2+) is expected to occupy one of the Ca(2+)-binding sites with a millimolar dissociation constant. This Mg(2+) accelerates the reaction cycle, not permitting phosphorylation without Ca(2+) binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1·Mg(2+) state at 3.0 Å resolution in addition to crystal structures of SERCA1a in E2 free from exogenous inhibitors, and address the structural basis of the activation signal for phosphoryl transfer. Unexpectedly, sarcolipin, a small regulatory membrane protein of Ca(2+)-ATPase, is bound, stabilizing the E1·Mg(2+) state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of ß-adrenergic signal in Ca(2+) regulation in heart (for reviews, see, for example, refs 8-10), and seems to play an important role in muscle-based thermogenesis. We also determined the crystal structure of recombinant SERCA1a devoid of sarcolipin, and describe the structural basis of inhibition by sarcolipin/phospholamban. Thus, the crystal structures reported here fill a gap in the structural elucidation of the reaction cycle and provide a solid basis for understanding the physiological regulation of the calcium pump.

Direct effects of neuropeptide nesfatin-1 on testicular spermatogenesis and steroidogenesis of the adult mice.

Recent studies have revealed nesfatin-1 as a hypothalamic neuropeptide, regulating food intake, energy expenditure and reproduction primarily by acting on the hypothalamic-pituitary-gonadal axis. Nesfatin-1 is also localized in several peripheral tissues including testes. However, functional significance of nesfatin-1 in testicular activities is not yet well documented in mammals. Therefore, this study was aimed to elucidate the direct effects of nesfatin-1 on testicular markers for steroid productions, spermatogenesis, metabolic changes and oxidative stress. The results revealed the expression of both protein and mRNA of nesfatin-1 in the testes of adult mice. The testes treated in vitro with nesfatin-1 showed significant increase in testosterone production, which correlated significantly with increased expression of steroidogenic markers and insulin receptor proteins in the testes. Furthermore, the in vitro treatment with nesfatin-1 showed stimulatory effects on spermatogenesis by promoting cell proliferation (PCNA) and survival (Bcl2), while inhibiting apoptosis (caspase-3) in the testes. The nesfatin-1 treatment in vitro further increased the expression of insulin receptor and GLUT8 proteins, in parallel with increase in the intra-testicular transport of glucose and production of lactate. This nesfatin-1 induced enhanced transport of energy substrate (glucose and lactate) may be responsible for promoting spermatogenesis and steroidogenesis. Nesfatin-1 significantly reduced oxidative stress and nitric oxide, which may also be responsible for stimulatory effects on testicular activities. The present finding suggests that nesfatin-1 acts via paracrine manner to increase sperm count and fertility, thus promoting the testicular function.

Protective effects of Nesfatin-1 peptide on cerebral ischemia reperfusion injury via inhibition of neuronal cell death and enhancement of antioxidant defenses.

Nesfatin-1 is a novel peptide with anorexigenic and anti-hyperglycemic properties. According to previous studies, this multi-functional peptide protects dopaminergic cells against neurotoxicity via anti-apoptotic effects. In addition, Nesfatin-1 protects myocardial tissue after myocardial infarction via anti-inflammatory and anti-apoptotic mechanisms. In this study, we investigated the neuroprotective effects of nesfatin-1 against cerebral ischemia reperfusion injury in the CA1 area of hippocampus in rats. 56 male Wistar rats (240-270 g) were randomly selected and allocated into four groups: (1) sham, (2) nesfatin-1, (3) ischemia/reperfusion, (4) ischemia/reperfusion+nesfatin-1. Cerebral ischemia induced by the occlusion of the common carotid arteries for 20 min was followed by reperfusion. Saline as a vehicle and nesfatin-1 (20 µg/kg) were injected intraperitoneally (IP) at the start of cerebral reperfusion. Apoptotic and necrotic cell death was detected by TUNEL and Nissl staining. Malondialdehyde (MDA) and antioxidant enzymes (GSH and SOD) levels were measured by the ELISA method. The results showed that cerebral ischemia increased the apoptotic and necrotic cell death in the CA1 area of hippocampus, while, treatment with nesfatin-1significantly reduced apoptotic and necrotic cell death. Moreover, the MDA levels of the hippocampus in ischemic rats were higher, whereas in nesfatin-1-treated rats the MDA levels were decreased. Furthermore, the SOD and GSH levels in the ischemic rats were decreased, whilst in ischemic rats treated with nesfatin-1, the SOD and GSH levels were increased. This study for the first time found that nesfatin-1 treatment improves CA1 hippocampus injuries after cerebral ischemia through preventing neuronal cell death and enhancement of antioxidant defenses.

Nell-1 Enhances Osteogenic Differentiation of Pre-Osteoblasts on Titanium Surfaces via the MAPK-ERK Signaling Pathway.

BACKGROUND/AIMS: This study aimed to investigate the effect of Nell-1 on the osteogenic behaviors of pre-osteoblasts on titanium (Ti) surfaces and to identify the underlying signaling pathway. METHODS: Nell-1 at different concentrations was added to culture medium to stimulate MC3T3-E1 subclone 14 on Ti surfaces. A CCK-8 colorimetric assay was used to detect cell proliferation. Alkaline phosphatase activity (ALP) assay and enzyme-linked immunosorbent assay (ELISA) were used to evaluate ALP activity and the osteocalcin (OCN) secretion, respectively. Indicators of osteoblastic differentiation were assessed using real-time polymerase chain reaction analysis (RT-PCR). Western blot (WB) assay was used to analyze the expression changes of the osteogenic proteins and the mitogen-activated protein kinase (MAPK) pathway. RESULTS: Nell-1 significantly increased the osteogenic gene and protein expression levels of ALP, OCN, Runx2, osteoprotegerin (OPG), collagen type I (Col-I), and Osterix (Osx) in pre-osteoblasts on Ti surfaces. The optimal concentration of Nell-1 was 100 ng/ ml. In addition, Nell-1 activated ERK and JNK, but not P38, in MC3T3-E1 cells on the Ti surface. Except for ALP and Col-I, the promotive effects of Nell-1 on the expression of osteogenic markers were suppressed by ERK inhibitor U0126. CONCLUSION: Certain concentrations of Nell-1 can promote the osteogenic differentiation of pre-osteoblasts on Ti surfaces by activating the MAPK/ERK signaling pathway.