Liposome-lentivirus for miRNA therapy with molecular mechanism study.

BACKGROUND: Cancer stem cells (CSCs) play a vital role in the occurrence, maintenance, and recurrence of solid tumors. Although, miR-145-5p can inhibit CSCs survival, poor understanding of the underlying mechanisms hamperes further therapeutic optimization for patients. Lentivirus with remarkable transduction efficiency is the most commonly used RNA carrier in research, but has shown limited tumor-targeting capability. METHODS: We have applied liposome to decorate lentivirus surface thereby yielding liposome-lentivirus hybrid-based carriers, termed miR-145-5p-lentivirus nanoliposome (MRL145), and systematically analyzed their potential therapeutic effects on liver CSCs (LCSCs). RESULTS: MRL145 exhibited high delivery efficiency and potent anti-tumor efficacy under in vitro and in vivo. Mechanistically, the overexpressed miR-145-5p can significantly suppress the self-renewal, migration, and invasion abilities of LCSCs by targeting Collagen Type IV Alpha 3 Chain (COL4A3). Importantly, COL4A3 can promote phosphorylating GSK-3ß at ser 9 (p-GSK-3ß S9) to inactivate GSK3ß, and facilitate translocation of ß-catenin into the nucleus to activate the Wnt/ß-catenin pathway, thereby promoting self-renewal, migration, and invasion of LCSCs. Interestingly, COL4A3 could attenuate the cellular autophagy through modulating GSK3ß/Gli3/VMP1 axis to promote self-renewal, migration, and invasion of LCSCs. CONCLUSIONS: These findings provide new insights in mode of action of miR-145-5p in LCSCs therapy and indicates that liposome-virus hybrid carriers hold great promise in miRNA delivery.

Adipokine chemerin overexpression in trophoblasts leads to dyslipidemia in pregnant mice: implications for preeclampsia.

BACKGROUND: The adipokine chemerin regulates adipogenesis and the metabolic function of both adipocytes and liver. Chemerin is elevated in preeclamptic women, and overexpression of chemerin in placental trophoblasts induces preeclampsia-like symptoms in mice. Preeclampsia is known to be accompanied by dyslipidemia, albeit via unknown mechanisms. Here, we hypothesized that chemerin might be a contributor to dyslipidemia. METHODS: Serum lipid fractions as well as lipid-related genes and proteins were determined in pregnant mice with chemerin overexpression in placental trophoblasts and chemerin-overexpressing human trophoblasts. In addition, a phospholipidomics analysis was performed in chemerin-overexpressing trophoblasts. RESULTS: Overexpression of chemerin in trophoblasts increased the circulating and placental levels of cholesterol rather than triglycerides. It also increased the serum levels of lysophosphatidic acid, high-density lipoprotein cholesterol (HDL-C), and and low-density lipoprotein cholesterol (LDL-C), and induced placental lipid accumulation. Mechanistically, chemerin upregulated the levels of peroxisome proliferator-activated receptor g, fatty acid-binding protein 4, adiponectin, sterol regulatory element-binding protein 1 and 2, and the ratio of phosphorylated extracellular signal-regulated protein kinase (ERK)1/2 / total ERK1/2 in the placenta of mice and human trophoblasts. Furthermore, chemerin overexpression in human trophoblasts increased the production of lysophospholipids and phospholipids, particularly lysophosphatidylethanolamine. CONCLUSIONS: Overexpression of placental chemerin production disrupts trophoblast lipid metabolism, thereby potentially contributing to dyslipidemia in preeclampsia.

Placental trophoblast-specific overexpression of chemerin induces preeclampsia-like symptoms.

Maternal circulating levels of the adipokine chemerin are elevated in preeclampsia, but its origin and contribution to preeclampsia remain unknown. We therefore studied (1) placental chemerin expression and release in human pregnancy, and (2) the consequences of chemerin overexpression via lentivirus-mediated trophoblast-specific gene manipulation in both mice and immortalized human trophoblasts. Placental chemerin expression and release were increased in women with preeclampsia, and their circulating chemerin levels correlated positively with the soluble Fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) ratio, a well-known biomarker of preeclampsia severity. Placental trophoblast chemerin overexpression in mice induced a preeclampsia-like syndrome, involving hypertension, proteinuria, and endotheliosis, combined with diminished trophoblast invasion, a disorganized labyrinth layer, and up-regulation of sFlt-1 and the inflammation markers nuclear factor-κB (NFκB), tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß. It also led to embryo resorption, while maternal serum chemerin levels correlated negatively with fetal weight in mice. Chemerin overexpression in human trophoblasts up-regulated sFlt-1, reduced vascular endothelial factor-A, and inhibited migration and invasion, as well as tube formation during co-culture with human umbilical vein endothelial cells (HUVECs). The chemokine-like receptor 1 (CMKLR1) antagonist α-NETA prevented the latter phenomenon, although it did not reverse the chemerin-induced down-regulation of the phosphoinositide 3-kinase/Akt pathway. In conclusion, up-regulation of placental chemerin synthesis disturbs normal placental development via its CMKLR1 receptor, thereby contributing to fetal growth restriction/resorption and the development of preeclampsia. Chemerin might be a novel biomarker of preeclampsia, and inhibition of the chemerin/CMKLR1 pathway is a promising novel therapeutic strategy to treat preeclampsia.

Placenta-Specific miR-125b Overexpression Leads to Increased Rates of Pregnancy Loss in Mice.

Pregnancy loss (PL) is one of the common complications that women can experience during pregnancy, with an occurrence rate of 1 to 5%. The potential causes of pregnancy loss are unclear, with no effective treatment modalities being available. It has been previously reported that the level of miR-125b was significantly increased in placentas of PL patients. However, the role of miR-125b in the development of PL still remains unknown. In the current study, an miR-125b placenta-specific over-expression model was constructed by lentiviral transfecting zona-free mouse embryos followed by embryo transfer. On gestation day 15, it was observed that the placenta was significantly smaller in the miR-125b placenta-specific overexpression group than the control group. Additionally, the abortion rate of the miR-125b placenta-specific overexpression group was markedly higher than in the control group. The blood vessel diameter was larger in the miR-125b-overexpressing specific placenta. In addition, miR-125b-overexpressing HTR8 and JEG3 cell lines were also generated to analyze the migration and invasion ability of trophoblasts. The results showed that miR-125b overexpression significantly suppressed the migration and invasion ability of HTR8 and JEG3 cells. Overall, our results demonstrated that miR-125b can affect embryo implantation through modulating placenta angiogenesis and trophoblast cell invasion capacity that can lead to PL.

Mallotucin D, a Clerodane Diterpenoid from Croton crassifolius, Suppresses HepG2 Cell Growth via Inducing Autophagic Cell Death and Pyroptosis.

Hepatocellular carcinoma (HCC) is a major subtype of primary liver cancer with a high mortality rate. Pyroptosis and autophagy are crucial processes in the pathophysiology of HCC. Searching for efficient drugs targeting pyroptosis and autophagy with lower toxicity is useful for HCC treatment. Mallotucin D (MLD), a clerodane diterpenoid from Croton crassifolius, has not been previously reported for its anticancer effects in HCC. This study aims to evaluate the inhibitory effects of MLD in HCC and explore the underlying mechanism. We found that the cell proliferation, DNA synthesis, and colony formation of HepG2 cells and the angiogenesis of HUVECs were all greatly inhibited by MLD. MLD caused mitochondrial damage and decreased the TOM20 expression and mitochondrial membrane potential, inducing ROS overproduction. Moreover, MLD promoted the cytochrome C from mitochondria into cytoplasm, leading to cleavage of caspase-9 and caspase-3 inducing GSDMD-related pyroptosis. In addition, we revealed that MLD activated mitophagy by inhibiting the PI3K/AKT/mTOR pathway. Using the ROS-scavenging reagent NAC, the activation effects of MLD on pyroptosis- and autophagy-related pathways were all inhibited. In the HepG2 xenograft model, MLD effectively inhibited tumor growth without detectable toxicities in normal tissue. In conclusion, MLD could be developed as a candidate drug for HCC treatment by inducing mitophagy and pyroptosis via promoting mitochondrial-related ROS production.

MicroRNA-132-3p represses Smad5 in MC3T3-E1 osteoblastic cells under cyclic tensile stress.

MicroRNAs (miRNAs) regulate osteogenic differentiation of bone cells, which has applications in orthodontics. Here we evaluated the miRNA expression profile of MC3T3-E1 osteoblasts under cyclic tensile stress with chip technology and found that miR-132-3p was up-regulated by 12% cyclic tensile stress. Alkaline phosphatase activity and osteocalcin expression in MC3T3-E1 cells were decreased under these conditions. Smad2 and Smad5 were identified as potential target genes of miR-132-3p. Native and phosphorylated Smad2 and Smad5 expression was negatively correlated with miR-132-3p levels in the cells under cyclic stretch; however, only Smad5 protein level was reduced upon miR-132-3p overexpression. The luciferase reporter assay confirmed a direct interaction between miR-132-3p and Smad5. Thus, miR-132-3p maybe regulates osteoblast differentiation via Smad5 in response to cyclic tensile stress.

The excretory-secretory products of Echinococcus granulosus protoscoleces stimulated IL-10 production in B cells via TLR-2 signaling.

BACKGROUND: Excretory-secretory products released by Echinococcus granulosus protoscoleces (EgPSC-ESPs) are well-known to regulate T cell responses. However, their direct influence on the differentiation of B cell subsets remains largely elusive. This study investigated the effects of EgPSC-ESPs on the differentiation of IL-10-producing B cells (B10), and explored the possible role of Toll-like receptor 2 (TLR-2) signaling in this process. RESULTS: In comparison to phosphate buffered saline (PBS), B cells exposed to the excretory-secretory products (ESPs) generated higher percentages of B10 cells, with higher expression of IL-10 mRNA, and larger amount of IL-10 production, which were in a dose dependent way. The mRNA and protein expression of TLR-2 in the ESPs-stimulated B cells were significantly higher than those in PBS, which was consistent to the results in B cells isolated from EgPSC infected mice. Moreover, TLR-2-/- B cells in response to ESPs stimulation expressed lower levels of IL-10 mRNA and produced undetectable IL-10 in comparison to those in normal B cells. In addition, Phosphatase and tensin homolog deleted on chromosome ten/AKT/Phosphatidylinositol-3 kinase (PTEN/AKT/PI3K) pathway was activated in ESPs-treated B cells, which was also dependent on TLR-2 signaling. Pam3CSK4, the agonist of TLR-2, could mock the effects of ESPs on the expression of PTEN, AKT and PI3K. CONCLUSION: Overall, this study revealed that TLR-2 signaling was required for B10 induction mediated by EgPSC-ESPs, which might be an immunomodulatory target against the parasite infection.

Autophagy is essential for the differentiation of porcine PSCs into insulin-producing cells.

Porcine pancreatic stem cells (PSCs) are seed cells with potential use for diabetes treatment. Stem cell differentiation requires strict control of protein turnover and lysosomal digestion of organelles. Autophagy is a highly conserved process that controls the turnover of organelles and proteins within cells and contributes to the balance of cellular components. However, whether autophagy plays roles in PSC differentiation remains unknown. In this study, we successfully induced porcine PSCs into insulin-producing cells and found that autophagy was activated during the second induction stage. Inhibition of autophagy in the second stage resulted in reduced differentiational efficiency and impaired glucose-stimulated insulin secretion. Moreover, the expression of active ß-catenin increased while autophagy was activated but was suppressed when autophagy was inhibited. Therefore, autophagy is essential to the formation of insulin-producing cells, and the effects of autophagy on differentiation may be regulated by canonical Wnt signalling pathway.

Autophagy stimulated proliferation of porcine PSCs might be regulated by the canonical Wnt signaling pathway.

Porcine pancreatic stem cells (PSCs) are one kind of the potential cells for treatment of human diabetes. Autophagy is a highly conserved cellular degradation process in which it helps to maintain the balance between the synthesis, degradation and subsequent recycling of cellular components. However, how autophagy contributes to PSCs has not yet been investigated. Here, we established GFP-LC3 transfected porcine PSC lines in which the accumulation of autophagosomes can be efficiently visualized to evaluate the autophagic activity. Moreover, we observed that starved PSCs which showed increased autophagic activity exhibited an increased tendency to proliferate through the results of BrdU, flow cytometry and western blotting. Furthermore, increased expression of active ß-catenin after inducing autophagy indicated that it might be the canonical Wnt signaling that autophagy activated to exert the function on the stimulation of PSCs proliferation. Collectively, these results demonstrated that autophagy stimulated proliferation of PSCs might be regulated by the canonical Wnt signaling pathway. Our results for the first time shed light on a role of autophagy for stimulating the proliferation of porcine PSCs.

Imaging Nuclei of MDA-MB-231 Breast Cancer Cells by Chiral Ruthenium(II) Complex Coordinated by 2-(4-Phenyacetylenephenyl)-1H-imidazo[4,5f][1,10]phenanthroline.

A pair of chiral ruthenium(II) complexes, Λ- and Δ-[Ru(bpy)2(p-BEPIP)](ClO4)2 [Λ- and Δ-RM0627; bpy = 2,2-bipyridine; p-BEPIP = 2-(4-phenyacetylenephenyl)-1H-imidazo[4,5f][1,10]phenanthroline], were prepared using the Sonogashira coupling reaction under microwave irradiation. The study shows that Λ-RM0627 emitted strong phosphorescence in the range 500-700 nm with a maximum at 594 nm when excited at 365 nm (the Stokes shift is about 227 nm), which was mainly located in the cell nucleus with red phosphorescence. Further studies using real-time phosphorescence observation confirmed that Λ-RM0627 can be taken up quickly by MDA-MB-231 cells and enriched in the nucleus. The in vitro and in vivo toxicities of Λ-RM0627 were also evaluated, and it was found that Λ-RM0627 slightly inhibited the growth of MDA-MB-231 breast cancer cells and HaCaT normal human epidermal cells and had little influence on the development of Zebrafish embryos at low concentration. In conclusion, the levoisomer of chiral ruthenium complexes can act as a potential phosphorescent probe that targets nuclei of living cells with low toxicity.

mTOR signaling inhibition modulates macrophage/microglia-mediated neuroinflammation and secondary injury via regulatory T cells after focal ischemia.

Signaling by the mammalian target of rapamycin (mTOR) plays an important role in the modulation of both innate and adaptive immune responses. However, the role and underlying mechanism of mTOR signaling in poststroke neuroinflammation are largely unexplored. In this study, we injected rapamycin, a mTOR inhibitor, by the intracerebroventricular route 6 h after focal ischemic stroke in rats. We found that rapamycin significantly reduced lesion volume and improved behavioral deficits. Notably, infiltration of γδ T cells and granulocytes, which are detrimental to the ischemic brain, was profoundly reduced after rapamycin treatment, as was the production of proinflammatory cytokines and chemokines by macrophages and microglia. Rapamycin treatment prevented brain macrophage polarization toward the M1 type. In addition, we also found that rapamycin significantly enhanced anti-inflammation activity of regulatory T cells (Tregs), which decreased production of proinflammatory cytokines and chemokines by macrophages and microglia. Depletion of Tregs partially elevated macrophage/microglia-induced neuroinflammation after stroke. Our data suggest that rapamycin can attenuate secondary injury and motor deficits after focal ischemia by enhancing the anti-inflammation activity of Tregs to restrain poststroke neuroinflammation.

[Changes of Phenotype and Phagocytosis of Peritoneal Macrophages in Mice Infected with the Larval-stage of Echinococcus granulosus].

Objective: To investigate the phenotype and phagocytosis changes of the peritoneal macrophages (Mφ) in mice infected with the larval-stage Echinococcus granulosus, and explore the role of Mφ in the responses to parasite infection. Methods: Twenty-four female BALB/c mice (age of 6-8 weeks) were randomly assigned into control group and infection group (n=12 in each group). The mice in the infection group were intraperitoneally injected with 2 000 protoscoleces, while the control mice were injected with equal volume of PBS. Five months after infection, the peritoneal mononuclear cells were collected, and the percentage of Mφ and the expression of surface markers CD40, CD80, CD86, and major histocompatibility complex Ⅱ (MHCⅡ) were determined by flow cytometry. The absorbance（A490 value） of Mφ at different concentrations（1×106, 5×105, 1×105） was determined by the neutral red assay to evaluate the phagocytic ability of Mφ. Results: The Mφ constituted（30.40±3.15）% and（20.75±5.91）% in mononuclear cells in the infection and the control groups, respectively. The percentages of Mφ expressing CD40, CD80, CD86, and MHC Ⅱ were（45.33±5.51）%, （61.00±10.61）%, （56.88±10.66）% and （27.00±3.82）% in the infection group, which were all significantly higher than those in the control ［（41.43±6.19）%, （59.23±8.65）%, （10.91±1.82）% and （13.67±3.01%）］ （P<0.05）. The A490 values of Mφ at 1×106, 5×105, 1×105 were 0.41±0.03， 0.24±0.05 and 0.16±0.01 in the infection group, which were significantly lower than those in the control （0.61±0.15, 0.47±0.07 and 0.18±0.01）（P<0.01）. Conclusion: The phagocytic ability of peritoneal Mφ is dramatically weakened after infection, but the expression of activation-associated surface markers is significantly up-regulated after infection.

[Application of near infrared spectroscopy combined with particle swarm optimization based least square support vactor machine to rapid quantitative analysis of Corni Fructus].

A novel method was developed for the rapid determination of multi-indicators in corni fructus by means of near infrared (NIR) spectroscopy. Particle swarm optimization (PSO) based least squares support vector machine was investigated to increase the levels of quality control. The calibration models of moisture, extractum, morroniside and loganin were established using the PSO-LS-SVM algorithm. The performance of PSO-LS-SVM models was compared with partial least squares regression (PLSR) and back propagation artificial neural network (BP-ANN). The calibration and validation results of PSO-LS-SVM were superior to both PLS and BP-ANN. For PSO-LS-SVM models, the correlation coefficients (r) of calibrations were all above 0.942. The optimal prediction results were also achieved by PSO-LS-SVM models with the RMSEP (root mean square error of prediction) and RSEP (relative standard errors of prediction) less than 1.176 and 15.5% respectively. The results suggest that PSO-LS-SVM algorithm has a good model performance and high prediction accuracy. NIR has a potential value for rapid determination of multi-indicators in Corni Fructus.

Glial scar formation occurs in the human brain after ischemic stroke.

Reactive gliosis and glial scar formation have been evidenced in the animal model of ischemic stroke, but not in human ischemic brain. Here, we have found that GFAP, ED1 and chondroitin sulphate proteoglycans (CSPG) expression were significantly increased in the cortical peri-infarct regions after ischemic stroke, compared with adjacent normal tissues and control subjects. Double immunolabeling showed that GFAP-positive reactive astrocytes in the peri-infarct region expressed CSPG, but showed no overlap with ED1-positive activated microglia. Our findings suggest that reactive gliosis and glial scar formation as seen in animal models of stroke are reflective of what occurs in the human brain after an ischemic injury.

Age differences in the impact of dietary salt on metabolism, blood pressure and cognitive function in male rats.

The influence of salt consumption on physiological processes, especially blood pressure (BP), metabolism, and cognition, remains a topical concern. While guidelines endorse reduced salt diets, there are gaps in understanding the age-specific implications and challenges in adherence. The present study delved into the differential effects of salt intake on young adult and aged male rats over a 12-week period, using control, low-, and high-salt diets. Key metrics, such as BP, cognition, and general parameters, were monitored. Our findings revealed significant age-dependent effects of salt intake on survival rates, body weight, blood sodium, blood glucose, blood lipids, BP, heart rates, and cognition. Notably, young adult rats did not show significant sodium level changes on a high-salt diet, whereas aged rats experienced increased sodium levels even on a normal salt diet. Blood glucose levels decreased significantly in aged rats on a high-salt diet but remained stable in young adults. Aged rats had the highest survival rates on low-salt diets. Low-salt diets led to reduced BP in both age groups, more significantly in young adults. Young adult rats displayed increased BP variability on both high- and low-salt diets, while a decrease in BP variability was exclusive to aged rats on a low-salt diet. There were significant differences across age groups in short-term memory, but not in long-term memory. The study provides a nuanced understanding of the age-dependent physiological effects of salt intake, suggesting the necessity of age-specific guidelines for public health.

The impact of blood pressure variability on cognition: current limitations and new advances.

Dementia is the most common neurodegenerative disease in the aging population. Emerging evidence indicates that blood pressure (BP) variability is correlated with cognitive impairment and dementia independent of mean BP levels. The state-of-the-art review summarizes the latest evidence regarding the impact of BP variability on cognition in cognitively intact populations, patients with mild cognitive impairment, and different dementia types, focusing on the important confounding factors and new advances. This review also summarizes the potential mechanisms underlying the relationship between BP variability and cognitive impairment, and dementia, briefly discussing sex differences in the relationship. At last, current limitations and future perspectives are discussed to optimize BP management in preventing cognitive impairment and dementia.

A Self-Reinforcing Nanoplatform for Highly Effective Synergistic Targeted Combinatary Calcium-Overload and Photodynamic Therapy of Cancer.

While calcium-overload-mediated therapy (COMT) is a promising but largely untapped therapeutic strategy, combinatory therapy greatly boosts treatment outcomes with integrated merits of different therapies. Herein, a BPQD@CaO2 -PEG-GPC3Ab nanoplatform is formulated by integrating calcium peroxide (CaO2 ) and black phosphorus quantum dot (BPQD, photosensitizer) with active-targeting glypican-3 antibody (GPC3Ab), for combinatory photodynamic therapy (PDT) and COMT in response to acidic pH and near-infrared (NIR) light, wherein CaO2 serves as the reservoir of calcium ions (Ca2+ ) and hydrogen peroxide (H2 O2 ). Navigated by GPC3Ab to tumor cells at acidic pH, the nanoparticle disassembles to CaO2 and BPQD; CaO2 produces COMT Ca2+ and H2 O2 , while H2 O2 makes oxygen (O2 ) to promote PDT; under NIR irradiation BPQD facilitates not only the conversion of O2 to singlet oxygen (1 O2 ) for PDT, but also moderate hyperthermia to accelerate NP dissociation to CaO2 and BPQD, and conversions of CaO2 to Ca2+ and H2 O2 , and H2 O2 to O2 , to enhance both COMT and PDT. After supplementary ionomycin treatment to induce intracellular Ca2+ bursts, the multimodal therapeutics strikingly induce hepatocellular carcinoma apoptosis, likely through the activation of the calpains and caspases 12, 9, and 3, up-regulation of Bax and down-regulation of Bcl-2 proteins. This nanoplatform enables a mutually-amplifying and self-reinforcing synergistic therapy.

Transient filament occlusion of the middle cerebral artery in rats: does the reperfusion method matter 24 hours after perfusion?

BACKGROUND: There are two widely used transient middle cerebral artery occlusion (MCAO) methods, which differ in the use of unilateral or bilateral carotid artery reperfusion (UNICAR and BICAR). Of the two methods, UNICAR is easier to perform. This study was designed to comprehensively compare the two reperfusion methods to determine if there are any differences in outcomes. RESULTS: The UNICAR and BICAR groups each included 9 rats. At baseline, the average pO(2) was 20.54 ± 9.35 and 26.43 ± 7.39, for the UNICAR and BICAR groups, respectively (P = 0.519). Changes in pO(2), as well as other physiological parameters measured within the ischemic lesion, were similar between the UNICAR and BICAR groups during 90 min of MCAO and the first 30 min of reperfusion (all P > 0.05). Furthermore, both the Bederson score and Garcia score, which are used for neurological assessment, were also similar (both P > 0.05). There were also no significant differences in T2WI lesion volume, DWI lesion volume, PWI lesion volume, or TTC staining infarct volume between the two groups (all P > 0.05). CONCLUSION: UNICAR and BICAR have similar capability for inducing acute brain ischemic injury and can be considered interchangeable up to 24 hours after reperfusion.

Quantitative proteomics analysis of chondrogenic differentiation of C3H10T1/2 mesenchymal stem cells by iTRAQ labeling coupled with on-line two-dimensional LC/MS/MS.

The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.

Corpus callosum and experimental stroke: studies in callosotomized rats and acallosal mice.

BACKGROUND AND PURPOSE: Interhemispheric inhibition via the corpus callosum has been proposed as an exacerbating factor in outcome from stroke. METHODS: We measured infarct volume and behavioral outcome after middle cerebral artery occlusion in callosotomized rats and acallosal mice. RESULTS: Neither callosotomy in rats nor callosal agenesis in mice improved infarct volume or behavioral outcome after middle cerebral artery occlusion. CONCLUSIONS: These findings argue against a role for transcallosal projections in exacerbating focal cerebral ischemia.