A Type I-F Anti-CRISPR Protein Inhibits the CRISPR-Cas Surveillance Complex by ADP-Ribosylation.

CRISPR-Cas systems are bacterial anti-viral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here, we report a novel mechanism by which AcrIF11 inhibits the type I-F CRISPR system. Our structural and biochemical studies demonstrate that AcrIF11 functions as a novel mono-ADP-ribosyltransferase (mART) to modify N250 of the Cas8f subunit, a residue required for recognition of the protospacer-adjacent motif, within the crRNA-guided surveillance (Csy) complex from Pseudomonas aeruginosa. The AcrIF11-mediated ADP-ribosylation of the Csy complex results in complete loss of its double-stranded DNA (dsDNA) binding activity. Biochemical studies show that AcrIF11 requires, besides Cas8f, the Cas7.6f subunit for binding to and modifying the Csy complex. Our study not only reveals an unprecedented mechanism of type I CRISPR-Cas inhibition and the evolutionary arms race between phages and bacteria but also suggests an approach for designing highly potent regulatory tools in the future applications of type I CRISPR-Cas systems.

AcrIF5 specifically targets DNA-bound CRISPR-Cas surveillance complex for inhibition.

CRISPR-Cas systems are prokaryotic antiviral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here we present structural and functional analyses of AcrIF5, exploring its unique anti-CRISPR mechanism. AcrIF5 shows binding specificity only for the target DNA-bound form of the crRNA-guided surveillance (Csy) complex, but not the apo Csy complex from the type I-F CRISPR-Cas system. We solved the structure of the Csy-dsDNA-AcrIF5 complex, revealing that the conformational changes of the Csy complex caused by dsDNA binding dictate the binding specificity for the Csy-dsDNA complex by AcrIF5. Mechanistically, five AcrIF5 molecules bind one Csy-dsDNA complex, which destabilizes the helical bundle domain of Cas8f, thus preventing subsequent Cas2/3 recruitment. AcrIF5 exists in symbiosis with AcrIF3, which blocks Cas2/3 recruitment. This attack on the recruitment event stands in contrast to the conventional mechanisms of blocking binding of target DNA. Overall, our study reveals an unprecedented mechanism of CRISPR-Cas inhibition by AcrIF5.

Direct White-Light Emitting From a Single Metal-Organic Framework with Dual Phosphorescence Peaks.

Single component white-light-emitting (SCWLE) materials are extremely desired in the field of solid-state lighting. However, pure-phosphorescent SCWLE has rarely been reported. Herein, one halogen-bonding-containing MOF [Cd(5-BIPA)(phen)] (1) has been synthesized, which shows efficient white-light emission originating from dual phosphorescence bands with different wavelengths and lifetimes. The fabrication of a phosphor-converted white-light-emitting diode device driven by pulsing current enables this MOF to be a promising phosphor.

Fast photocatalytic degradation of rhodamine B using indium-porphyrin based cationic MOF under visible light irradiation.

A broad light-harvesting range and efficient charge separation are two main ways to enhance the visible photocatalytic performance of semiconductors. Herein, an ionic porphyrin MOF [In(TPyP)]·(NO3) (1) (TPyP = 5,10,15,20-tetrakis(4-pyridyl)-21H,23H-porphyrin) was synthesized via in situ metalation. The orderly arranged porphyrin photosensitizer and the internal electric field between the MOF host and NO3- guests enable effective visible light response and electron-hole separation. Consequently, the as-synthesized MOF shows efficient photocatalytic degradation of rhodamine B (RhB), methyl orange (MO) and methylene blue (MB) organic pollutants. It can degrade 99.07% of RhB within only 20 minutes under visible light irradiation (λ > 420 nm) with a high chemical reaction rate constant of 0.2400 min-1. The photocatalytic activity of the title MOF is more efficient than those of other reported MOFs, COFs and even inorganic semiconductors. The reusability, energy level, band gap, charge distribution and main degradation mechanisms of the photocatalyst were well studied.

Structural insights into the inactivation of the type I-F CRISPR-Cas system by anti-CRISPR proteins.

Phage infection is one of the major threats to prokaryotic survival, and prokaryotes in turn have evolved multiple protection approaches to fight against this challenge. Various delicate mechanisms have been discovered from this eternal arms race, among which the CRISPR-Cas systems are the prokaryotic adaptive immune systems and phages evolve diverse anti-CRISPR (Acr) proteins to evade this immunity. Until now, about 90 families of Acr proteins have been identified, out of which 24 families were verified to fight against subtype I-F CRISPR-Cas systems. Here, we review the structural and biochemical mechanisms of the characterized type I-F Acr proteins, classify their inhibition mechanisms into two major groups and provide insights for future studies of other Acr proteins. Understanding Acr proteins in this context will lead to a variety of practical applications in genome editing and also provide exciting insights into the molecular arms race between prokaryotes and phages.

Screen for Potential Candidate Alternatives of Sargentodoxa cuneata from Its Six Adulterants Based on Their Phenolic Compositions and Antioxidant Activities.

Daxueteng, the liana stem of Sargentodoxa cuneata, is a widely used Traditional Chinese Medicine facing the overflow of its commercial adulterants. A method for discriminating adulterants and screening potential candidate alternatives of S. cuneata was thus established. Total phenols and flavonoids of S. cuneata and its six adulterants and their abilities to scavenge DPPH• and ABTS•+, to absorb peroxyl radicals (ORAC), and to inhibit AAPH-induced supercoiled plasmid DNA strand scission were comprehensively assessed. Polygonum cuspidatum and Bauhinia championii, two of the six adulterants of S. cuneate, shared considerably higher antioxidant activities as well as phenolic contents and, therefore, were considered as potential candidate alternatives. Phenolic compositions of the two potential candidate alternatives and S. cuneata itself were further determined by UPLC-QTOF-MS/MS. Totally 38 phenolics, including four hydroxybenzoic acids, two tyrosols, two caffeoylquinic acids, seven flavanol or its oligomers, two lignans, three hydroxycinnamic acids, six stilbenes, seven anthraquinones, and five flavanones were determined from three species. Furthermore, contents of different phenolic categories were semi-quantified and the major antioxidant contributors of S. cuneata and the two potential candidate alternatives were subsequently determined. It is concluded that tyrosols and caffeoylquinic acids were unique categories making great antioxidant contributions in S. cuneata and thus were considered as effective biomarkers in distinguishing its potential candidate alternatives.

Comparison of Free, Esterified, and Insoluble-Bound Phenolics and Their Bioactivities in Three Organs of Lonicera japonica and L. macranthoides.

Dried flower buds of Lonicera japonica and L. macranthoides have long been used as herbs in numerous Chinese traditional medicines. Comparisons of three phenolic fractions (i.e., free, esterified, and insoluble-bound phenolics) in three different organs (i.e., flower, leaf, and stem) of the two species revealed that the free phenolics were the highest in terms of total phenol and total flavonoid content, composed of the most numerous phenolics and flavonoids; thus, they exhibited the most excellent antioxidant activities (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), and oxygen radical absorbance capacity (ORAC)), as well as protective effects on DNA damage induced by free radicals. In identical free and esterified phenolics of a same organ, higher contents and bioactivities were observed in L. macranthoides than in L. japonica. Phenolics identified by ultra-performance liquid chromatography with a diode array detector, alongside tandem mass spectrometry coupled with a quadrupole time-of-flight mass spectrometer (UPLC-DAD⁻QTOF-MS/MS) mainly included chlorogenic acid and its five derivatives, three flavonoids that were only found in the free phenolic fraction and closely correlated with its bioactivity, and caffeic acid that was the major contributor to antioxidant activity of the esterified and insoluble-bound phenolic fractions. It was, thus, concluded that, like L. japonica, L. macranthoides, which was underestimated since being separately listed by the 2010 edition of the Chinese Pharmacopoeia, is also a good (and better) herbal medicine.

Identification of bioactive phenolics from Porana sinensis Hemsl. stem by UPLC-QTOF-MS/MS and the confirmation of anti-inflammatory indicators using LPS-induced RAW264.7 cells.

To characterise bioactive phenolics and confirm anti-inflammatory indicators in Porana sinensis stem, 23 phenolics were identified by UPLC-QTOF-MS/MS from crude extract (CE) prepared optimally with 80% methanol. Further fractionalisation using D101 macroporous resin resulted in predominant enrichment of total phenols and flavonoids into Fr.II. Correspondingly, the bioactive components-enriched Fr.II exhibited the lowest IC50 for scavenging DPPH and ABTS and the highest oxygen radical absorbance capacity or ORAC followed by Fractions Fr.I + Fr.II, CE and Fr.I, implying that certain phenolics possessing lower antioxidant activity completely remained in CE. Anti-inflammatory tests with LPS-stimulated RAW264.7 cells showed that CE possessed the highest inhibition of NO-production followed by Fr.II and Fr.I, meaning that CE might contain compounds that expressed higher anti-inflammatory but lower antioxidant activities or possessed synergistic interactions but were not fractionated together. Quantitative determination of nine major phenolics revealed that caffeic acid and 3-, 4- and 5-caffeoylquinic acids were concentrated into Fr.I, whereas scopolin, scopoletin and 3,5-, 3,4- and 4,5-dicaffeoylquinic acids were enriched into Fr.II. Further experiments with three selected major phenolics reduced the proposed synergistic interactions. Anti-inflammatory tests of the nine major phenolics evidenced that caffeic acid and the six caffeoylquinic acids produced higher, and the three dicaffeoylquinic acids at 140 µΜ showed even more significant activities in suppressing NO-production and mRNA expression of iNOS, TNF-α, COX-2, and IL-6, suggesting that these three dicaffeoylquinic acids could be indicators of the anti-inflammatory potential of P. sinensis stem. These findings provided novel insights for potential use of P. sinensis or liana, as an important source of natural antioxidants, against inflammation.

Hypertrophied myocardium is vulnerable to ischemia/reperfusion injury and refractory to rapamycin-induced protection due to increased oxidative/nitrative stress.

Left ventricular hypertrophy (LVH) is causally related to increased morbidity and mortality following acute myocardial infarction (AMI) via still unknown mechanisms. Although rapamycin exerts cardioprotective effects against myocardial ischemia/reperfusion (MI/R) injury in normal animals, whether rapamycin-elicited cardioprotection is altered in the presence of LVH has yet to be determined. Pressure overload induced cardiac hypertrophied mice and sham-operated controls were exposed to AMI by coronary artery ligation, and treated with vehicle or rapamycin 10 min before reperfusion. Rapamycin produced marked cardioprotection in normal control mice, whereas pressure overload induced cardiac hypertrophied mice manifested enhanced myocardial injury, and was refractory to rapamycin-elicited cardioprotection evidenced by augmented infarct size, aggravated cardiomyocyte apoptosis, and worsening cardiac function. Rapamycin alleviated MI/R injury via ERK-dependent antioxidative pathways in normal mice, whereas cardiac hypertrophied mice manifested markedly exacerbated oxidative/nitrative stress after MI/R evidenced by the increased iNOS/gp91phox expression, superoxide production, total NO metabolites, and nitrotyrosine content. Moreover, scavenging superoxide or peroxynitrite by selective gp91phox assembly inhibitor gp91ds-tat or ONOO- scavenger EUK134 markedly ameliorated MI/R injury, as shown by reduced myocardial oxidative/nitrative stress, alleviated myocardial infarction, hindered cardiomyocyte apoptosis, and improved cardiac function in aortic-banded mice. However, no additional cardioprotective effects were achieved when we combined rapamycin and gp91ds-tat or EUK134 in ischemic/reperfused hearts with or without LVH. These results suggest that cardiac hypertrophy attenuated rapamycin-induced cardioprotection by increasing oxidative/nitrative stress and scavenging superoxide/peroxynitrite protects the hypertrophied heart from MI/R.

Hypoglycemic Effects in Alloxan-Induced Diabetic Rats of the Phenolic Extract from Mongolian Oak Cups Enriched in Ellagic Acid, Kaempferol and Their Derivatives.

Our previous reports showed that crude extract prepared with 50% ethanol (ethanol crude extract, ECE) from Mongolian oak cups possessed excellent in vitro antioxidant capacities as well as inhibitory activities against α-glucosidase, α-amylase and protein glycation caused by its enrichment in phenolics, including mainly ellagic acid, kaempferol and their derivatives. Nevertheless, few in vivo studies on antidiabetic activities of these phenolics were conducted. The present study investigated hypoglycemic effects with normal and diabetic rats being administrated orally without or with ECE at 200 and 800 mg/kg for 15 days. In normal rats, no significant differences were exhibited after ECE administration in body weight, fasting blood glucose level, levels of cholesterol, triglyceride, LDL and AST in serum, organ indexes, and levels of GSH and MDA in organs. In diabetic rats, the fasting blood glucose level, indexes of heart and liver, and levels of cholesterol and triglyceride in serum and MDA in heart tissue were significantly decreased. Moreover, HDL levels in serum and SOD activities in the four organs of diabetic rats were significantly improved after ECE administration at 800 mg/kg. Thus, in addition to inhibiting α-glucosidase, α-amylase and protein glycation reported previously, oak cups might contain novel dietary phytonutrients in preventing abnormal changes in blood glucose and lipid profile and attenuating oxidant stress in vivo. The results also implied that it is ellagic acid, kaempferol and their derivatives enriched in ECE that might play vital roles in managing type 1 as well as type 2 diabetes.

IL-1ß-induces NF-κB and upregulates microRNA-372 to inhibit spinal cord injury recovery.

Excessive inflammation including IL-1ß-initiated signaling is among the earlies reactions that can cause neuronal damage following spinal cord injury (SCI). It has been suggested that microRNAs may participate in stem cell repair to facilitate functional recovery following SCI. In this study we have shown that in cultured human neural stem cells (hNSC), IL-1ß reduced the expression of both KIF3B (kinesin family member 3B) and NOSIP (nitric oxide synthase-interacting protein), two key modulators for restricting inflammation and promoting neuronal regeneration. The induction of microRNA-372 (miR-372) by IL-1ß is specifically responsible for the inhibition of KIF3B and NOSIP. The 3'-untranslated regions (UTRs) of both KIF3B and NOSIP contain targeting sequences to miR-372 that directly inhibit their expression. Moreover, we found that the expression of miR-372 was stimulated in hNSC by IL-1ß through an NF-κB binding site at its promoter region. Finally, stable overexpression of miR-372 inhibitor in hNSC rescued the IL-1ß-induced impairment as shown by significant improvements in tissue water content, myeloperoxidase activity, and behavioral assessments in SCI rats. These findings suggest a critical role of miR-372 in inflammatory signaling and pinpoint a novel target for the treatment of acute SCI.NEW & NOTEWORTHY Our data demonstrate that IL-1ß can impair the functional recovery of neural stem cell transplant therapy for spinal cord injury (SCI) treatment in rats. This effect is dependent on microRNA-372 (miR-372)-dependent gene repression of KIF3B and NOSIP. Therefore, specific knockdown of miR-372 may provide benefits for SCI treatments.

Flaxseed oil ameliorates alcoholic liver disease via anti-inflammation and modulating gut microbiota in mice.

BACKGROUND: Alcoholic liver disease (ALD) represents a chronic wide-spectrum of liver injury caused by consistently excessive alcohol intake. Few satisfactory advances have been made in management of ALD. Thus, novel and more practical treatment options are urgently needed. Flaxseed oil (FO) is rich in α-linolenic acid (ALA), a plant-derived n-3 polyunsaturated fatty acids (PUFAs). However, the impact of dietary FO on chronic alcohol consumption remains unknown. METHODS: In this study, we assessed possible effects of dietary FO on attenuation of ALD and associated mechanisms in mice. Firstly, mice were randomly allocated into four groups: pair-fed (PF) with corn oil (CO) group (PF/CO); alcohol-fed (AF) with CO group (AF/CO); PF with FO group (PF/FO); AF with FO group (AF/FO). Each group was fed modified Lieber-DeCarli liquid diets containing isocaloric maltose dextrin a control or alcohol with corn oil and flaxseed oil, respectively. After 6 weeks feeding, mice were euthanized and associated indications were investigated. RESULTS: Body weight (BW) was significantly elevated in AF/FO group compared with AF/CO group. Dietary FO reduced the abnormal elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in chronic ethanol consumption. Amelioration of these parameters as well as liver injury via HE staining in dietary FO supplementation in ALD demonstrated that dietary FO can effectively benefit for the protection against ALD. To further understand the underlying mechanisms, we investigated the inflammatory cytokine levels and gut microbiota. A series of inflammatory cytokines, including TNF-α, IL-1ß, IL-6 and IL-10, were determined. As a result, TNF-α, IL-1ß and IL-6 were decreased in AF/FO group compared with control group; IL-10 showed no significant alteration between AF/CO and AF/FO groups (p > 0.05). Sequencing and analysis of gut microbiota gene indicated that a reduction of Porphyromonadaceae and Parasutterella, as well as an increase in Firmicutes and Parabacteroides, were seen in AF group compared with PF control. Furthermore, dietary FO in ethanol consumption group induced a significant reduction in Proteobacteria and Porphyromonadaceae compared with AF/CO group. CONCLUSION: Dietary FO ameliorates alcoholic liver disease via anti-inflammation and modulating gut microbiota, thus can potentially serve as an inexpensive interventions for the prevention and treatment of ALD.

Ryanodine Receptor Type 2 Plays a Role in the Development of Cardiac Fibrosis under Mechanical Stretch Through TGFß-1.

Ryanodine receptor type 2 (RyR-2), the main Ca2+ release channel from sarcoplasmic reticulum in cardiomyocytes, plays a vital role in the regulation ofmyocardial contractile function and cardiac hypertrophy. However, the role of RyR-2 in cardiac fibrosis during the development of cardiac hypertrophy remains unclear.In this study, we examined whether RyR-2 regulates TGFß1, which is secreted from cardiomyocytes and exerts on cardiac fibrosis using cultured cardiomyocytes and cardiac fibroblasts of neonatal rats. The expression of RyR-2 was found only in cardiomyocytesbut not in cardiac fibroblasts. Mechanical stretch induced upregulation of TGFß1 in cardiomyocytes and RyR-2 knockdown significantly suppressed the upregulation of TGFß1 expression. The transcript levels of collagen genes were also decreased in fibroblasts compare with wild type, although the expression of both two kinds was higher than those in stationary cardiomyocytes (non-stretch). With the inhibition of the TGFß1-neutralizing antibody, the expression of collagen genes has no significant difference between the mechanically stretched cardiomyocytes and non-stretchedones. These results indicate that RyR-2 regulated TGFß1 expression in mechanically stretched cardiomyocytes and TGFß1 promoted collagen formation of cardiac fibroblasts by a paracrine mechanism.RyR-2 in mechanical stretch could promote the development of cardiac fibrosis involving TGFß1-dependent paracrine mechanism. Our findings provided more insight into comprehensively understanding the molecular role of RyR-2 in regulating cardiac fibrosis.

Response Surface Methodology Optimization of Ultrasonic-Assisted Extraction of Acer Truncatum Leaves for Maximal Phenolic Yield and Antioxidant Activity.

This study is the first to report the use of response surface methodology to improve phenolic yield and antioxidant activity of Acer truncatum leaves extracts (ATLs) obtained by ultrasonic-assisted extraction. The phenolic composition in ATLs extracted under the optimized conditions were characterized by UPLC-QTOF-MS/MS. Solvent and extraction time were selected based on preliminary experiments, and a four-factors-three-levels central composite design was conducted to optimize solvent concentration (X1), material-to-liquid ratio (X2), ultrasonic temperature (X3) and power (X4) for an optimal total phenol yield (Y1) and DPPH• antioxidant activity (Y2). The results showed that the optimal combination was ethanol:water (v:v) 66.21%, material-to-liquid ratio 1:15.31 g/mL, ultrasonic bath temperature 60 °C, power 267.30 W, and time 30 min with three extractions, giving a maximal total phenol yield of 7593.62 mg gallic acid equivalent/100 g d.w. and a maximal DPPH• antioxidant activity of 74,241.61 µmol Trolox equivalent/100 g d.w. Furthermore, 22 phenolics were first identified in ATL extract obtained under the optimized conditions, indicating that gallates, gallotannins, quercetin, myricetin and chlorogenic acid derivatives were the main phenolic components in ATL. What's more, a gallotannins pathway existing in ATL from gallic acid to penta-O-galloylglucoside was proposed. All these results provide practical information aiming at full utilization of phenolics in ATL, together with fundamental knowledge for further research.

The effects of different angiotensin II type 1 receptor blockers on the regulation of the ACE-AngII-AT1 and ACE2-Ang(1-7)-Mas axes in pressure overload-induced cardiac remodeling in male mice.

Angiotensin II (AngII) type 1 receptor blockers (ARBs) have been effectively used in hypertension and cardiac remodeling. However, the differences among them are still unclear. We designed this study to examine and compare the effects of several ARBs widely used in clinics, including Olmesartan, Candesartan, Telmisartan, Losartan, Valsartan and Irbesartan, on the ACE-AngII-AT1 axis and the ACE2-Ang(1-7)-Mas axis during the development of cardiac remodeling after pressure overload. Although all of the six ARBs, attenuated the development of cardiac hypertrophy and heart failure induced by transverse aortic constriction (TAC) for 2 or 4weeks in the wild-type mice evaluated by echocardiography and hemodynamic measurements, the degree of attenuation by Olmesartan, Candesartan and Losartan tended to be larger than that of the other three drugs tested. Additionally, the degree of downregulation of the ACE-AngII-AT1 axis and upregulation of the ACE2-Ang(1-7)-Mas axis was higher in response to Olmesartan, Candesartan and Losartan administration in vivo and in vitro. Moreover, in angiotensinogen-knockdown mice, TAC-induced cardiac hypertrophy and heart failure were inhibited by Olmesartan, Candesartan and Losartan but not by Telmisartan, Valsartan and Irbesartan administration. Furthermore, only Olmesartan and Candesartan could downregulate the ACE-AngII-AT1 axis and upregulate the ACE2-Ang(1-7)-Mas axis in vitro. Our data suggest that Olmesartan, Candesartan and Losartan could effectively inhibit pressure overload-induced cardiac remodeling even when with knockdown of Ang II, possibly through upregulation of the expression of the ACE2-Ang(1-7)-Mas axis and downregulation of the expression of the ACE-AngII-AT1 axis. In contrast, Telmisartan, Valsartan and Irbesartan only played a role in the presence of AngII, and Losartan had no effect in the presence of AngII in vitro.

Nucleosome Assembly Protein 1-Like 1 (Nap1l1) Regulates the Proliferation of Murine Induced Pluripotent Stem Cells.

BACKGROUND/AIMS: To investigate whether nucleosome assembly protein 1-like 1 (Nap1l1) regulates the proliferation of induced pluripotent stem cells (iPSC) and the potential mechanisms. METHODS: Nap1l1-knockdown-iPSC and Nap1l1-overexpression-iPSC were constructed by transfection of lentiviral particles. The proliferation of iPSC was detected by MTT analysis, and cell cycle was analyzed by flow cytometry. RESULTS: Nap1l1 overexpression promoted iPSC proliferation and induced G2/M transition compared to their control iPSC while Nap1l1-knockdown-iPSC dramatically displayed the reduced proliferation and accumulated G2/M phase cells. Further analysis showed that Nap1l1 overexpression in iPSC increased the expression of cyclin B1, downregulated the expression of p21 and p27, while knockdown of Nap1l1 showed the opposite effects. In addition, overexpression of Nap1l1 promoted the phosphorylation of AKT and ERK in iPSC, while knockdown of Nap1l1 inhibited the effects. However, these effects displayed in Nap1l1-overexpression-iPSC were greatly suppressed by the inhibition of AKT or ERK signaling. CONCLUSIONS: The results indicate that Nap1l1 promotes the proliferation of iPSC attributable to G2/M transition caused by downregulation of p27 and p21, and upregulation of cyclin B1, the activation of AKT or ERK is involved in the process. The present study has revealed a novel molecular mechanism involved in the proliferation of iPSC.

Curcumin protects human adipose-derived mesenchymal stem cells against oxidative stress-induced inhibition of osteogenesis.

The detrimental effects of oxidative stress on the skeletal system have been documented, and understanding the mechanisms is important to design a therapeutic strategy. As an antioxidant and anti-inflammatory agent, the active ingredient of turmeric curcumin has been used as medication for numerous complications including bone loss. However, it is unclear if curcumin could influence the osteogenic potential of mesenchymal stem cells (MSCs), particularly in oxidative injuries. Here we demonstrate that curcumin treatment protects cell death caused by hydrogen peroxide (H2O2) exposure in human adipose-derived MSCs in vitro. Importantly, curcumin is able to enhance the osteoblast differentiation of human adipose-derived MSCs that is inhibited by H2O2. Notably, both oxidative stress and the inhibition of Wnt/ß-catenin signaling are attenuated by curcumin treatment. These results suggest that curcumin can promote osteoblast differentiation of MSCs and protect the inhibitory effect elicited by oxidative injury. The findings support potential use of curcumin or related antioxidants in MSC-based bone regeneration for disease related with oxidative stress-induced bone loss.

Knockdown of nucleosome assembly protein 1-like 1 induces mesoderm formation and cardiomyogenesis via notch signaling in murine-induced pluripotent stem cells.

Low efficiency of cardiomyocyte differentiation from induced pluripotent stem cells (iPSCs) hinders the clinical application of iPSC technology for cardiac repair strategy. Recently, we screened out nucleosome assembly protein 1-like 1 (Nap1l1), which was downregulated during the differentiation of P19CL6 cells into cardiomyocytes. Here, we attempted to study the role of Nap1l1 in cardiomyogenesis of iPSC. Nap1l1 was downregulated during the differentiation of iPSC. Knockdown of Nap1l1 dramatically enhanced the differentiation of iPSC into functional cardiomyocytes while overexpression of Nap1l1 sharply lowered the differentiation. Moreover, although Nap1l1-knockdown had little effect on endoderm differentiation, the Nap1l1 modulation significantly accelerated mesoderm development. Re-expressing Nap1l1 in Nap1l1-knockdown-iPSC rescued the effects of Nap1l1. Inducibly overexpressing Nap1l1 at early stage of differentiation greatly inhibited mesoderm induction and cardiogenesis of iPSC. However, mesoderm stem cells (Flk-1-positive cells) originated from Nap1l1-knockdown- or -overexpression-iPSC showed no difference in further cardiomyocyte differentiation compared with that of control-iPSC. Further study revealed that Nap1l1-overexpression increased γ-secretase activity and the expression of Notch intracellular domain (NICD) and downstream genes during the differentiation of iPSC. γ-Secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester) greatly suppressed the production of NICD and abolished the inhibitory effects of Nap1l1-overexpression on mesoderm induction and cardiogenesis. These findings demonstrate that downregulation of Nap1l1 significantly enhances mesodermal induction and subsequent cardiogenesis of murine iPSC via inhibition of γ-secretase-regulated Notch signaling, which would facilitate the application of iPSC in heart diseases.

Combination Treatment With Antihypertensive Agents Enhances the Effect of Qiliqiangxin on Chronic Pressure Overload-induced Cardiac Hypertrophy and Remodeling in Male Mice.

We previously showed that Qiliqiangxin (QL) capsules could ameliorate cardiac hypertrophy and remodeling in a mouse model of pressure overload. Here, we compared the effects of QL alone with those of QL combined with the following 3 types of antihypertensive drugs on cardiac remodeling and dysfunction induced by pressure overload for 4 weeks in mice: an angiotensin II type 1 receptor (AT1-R) blocker (ARB), an angiotensin-converting enzyme inhibitor (ACEI), and a ß-adrenergic receptor (ß-AR) blocker (BB). Adult male mice (C57B/L6) were subjected to either transverse aortic constriction or sham operation for 4 weeks, and the drugs (or saline) were orally administered through gastric tubes. Cardiac function and remodeling were evaluated through echocardiography, catheterization, histology, and analysis of hypertrophic gene expression. Cardiomyocyte apoptosis and autophagy, AT1-R and ß1-AR expression, and cell proliferation-related molecules were also examined. Although pressure overload-induced cardiac remodeling and dysfunction, hypertrophic gene reprogramming, AT1-R and ß1-AR expression, and ERK phosphorylation were significantly attenuated by QL alone, QL + ARB, QL + ACEI, and QL + BB, the attenuation was stronger in the combination treatment groups. Moreover, apoptosis was reduced to a larger extent by each combination treatment than by QL alone, whereas autophagy was more strongly attenuated by either QL + ARB or QL + ACEI. None of the treatments significantly upregulated ErbB2 or ErbB4 phosphorylation, and none significantly downregulated C/EBPß expression. Therefore, the effects of QL on chronic pressure overload-induced cardiac remodeling may be significantly increased when QL is combined with an ARB, an ACEI, or a BB.

Insulin-like growth factor binding protein 4 enhances cardiomyocytes induction in murine-induced pluripotent stem cells.

Insulin-like growth factor binding protein 4 (IGFBP4) has been reported to play critical role in cardiomyocytes differentiation of embryonic stem cells (ESCs). But whether it promotes cardiomyocytes induction of iPSCs is unclear. In the present study, we aim to explore the role of IGFBP4 in the cardiogenesis of mouse iPSCs. We observed that IGFBP4 treatment at late stage during differentiation process of mouse iPSCs greatly enhanced the beating frequency of embryoid bodies (EBs). The expressions of Nkx2.5 (cardiac-specific transcription factor), α-MHC, α-actinin, and Troponin I (cardiac-specific protein) were significantly enhanced by IGFBP4 treatment. Immunostaining analysis showed that α-MHC, TNNT2 and connexin 43, typical cardiac markers, were obviously expressed in isolated cardiomyocytes from iPSCs with or without IGFBP4 treatment. Further study revealed that IGFBP4 had little effect on the apoptosis of EBs, but it significantly promoted the proliferation of cardiomyocytes from iPSCs characterized by higher ratio EdU positive cells in differentiated cardiomyocytes. We next observed that IGFBP4 inhibited ß-catenin expression in cytosol of EBs at late stage during differentiation of iPSCs. Knockdown of ß-catenin using a siRNA technique promoted the proliferation of differentiated cardiomyocytes and enhanced cardiomyocytes induction of iPSCs, suggesting that the effect of IGFBP4 on cardiomyocytes differentiation of iPSCs has relationship with ß-catenin signaling pathway. In conclusion, IGFBP4 promotes cardiogenesis of iPSCs by enhancing the proliferation of differentiated cardiomyocytes through inhibiting ß-catenin signaling.