The Protein Kinase C Inhibitor Aeb071 (Sotrastaurin) Modulates Migration and Superoxide Anion Production by Human Neutrophils In Vitro

We examined the effect of the protein kinase C-selective inhibitor AEB071 (sotrastaurin) on neutrophil functions in vitro. Pre-incubation with AEB071 at concentrations similar to those reached during in vivo therapy significantly reduced cell capacity to migrate toward three different chemo-attractants and to produce superoxide anions (O2) in response to phorbol myristate acetate (PMA) or to iV-formyl-methionyl-leucyl-phenylalanine (fMLP). AEB071 also significantly inhibited the O−2 "overproduction induced by fMLP in neutrophils primed with tumor necrosis factor alpha (TNF-α) or granulocyte/macrophage-colony stimulating factor (GM-CSF). This inhibition was not linked to fMLP-receptor down-regulation since the drug had no effect on either fMLP-receptors or fMLP-induced CD11b membrane expression. When the activity of AEB071 was compared to that of the conventional protein kinase C (PKC) inhibitor Gö6850 (which, like sotrastaurin, inhibits classical and novel PKC isoforms), Gö6976 (an inhibitor of α and β PKC isoforms) and rottlerin (a prevailing δ PKC isoform inhibitor), AEB071 at an equimolar concentration of 3 μM (close to the maximum drug concentration reached in patients treated with AEB071) caused significantly more inhibition on both chemotactic response and superoxide production. These in vitro findings suggest that neutrophils may offer a cellular target for AEB071 activity in vivo

Bioprinting of three-dimensional dentin-pulp complex with local differentiation of human dental pulp stem cells

Numerous approaches have been introduced to regenerate artificial dental tissues. However, conventional approaches are limited when producing a construct with three-dimensional patient-specific shapes and compositions of heterogeneous dental tissue. In this research, bioprinting technology was applied to produce a three-dimensional dentin-pulp complex with patient-specific shapes by inducing localized differentiation of human dental pulp stem cells within a single structure. A fibrin-based bio-ink was designed for bioprinting with the human dental pulp stem cells. The effects of fibrinogen concentration within the bio-ink were investigated in terms of printability, human dental pulp stem cell compatibility, and differentiation. The results show that micro-patterns with human dental pulp stem cells could be achieved with more than 88% viability. Its odontogenic differentiation was also regulated according to the fibrinogen concentration. Based on these results, a dentin-pulp complex having patient-specific shape was produced by co-printing the human dental pulp stem cell-laden bio-inks with polycaprolactone, which is a bio-thermoplastic used for producing the overall shape. After culturing with differentiation medium for 15 days, localized differentiation of human dental pulp stem cells in the outer region of the three-dimensional cellular construct was successfully achieved with localized mineralization. This result demonstrates the possibility to produce patient-specific composite tissues for tooth tissue engineering using three-dimensional bioprinting technology

The effect of the DLTIDDSYWYRI motif of the human laminin α2 chain on implant osseointegration

Considerable effort has been directed towards replacing lost teeth using tissue-engineering methods such as titanium implants. A number of studies have tried to modify bioinert titanium surfaces by coating them with functionally bioactive molecules for faster and stronger osseointegration than pure titanium surfaces. Recently, peptides have been recognized as valuable scientific tools in the field of tissue-engineering. The DLTIDDSYWYRI motif of the human laminin-2 α2 chain has been previously reported to promote the attachment of various cell types; however, the in vivo effects of the DLTIDDSYWYRI motif on new bone formation have not yet been studied. To examine whether a laminin-2-derived peptide can promote osseointegration by accelerating new bone formation in vivo, we applied titanium implants coated with the DLTIDDSYWYRI motif in a rabbit tibia model. The application of the DLTIDDSYWYRI motif-treated implant to tibia wounds enhanced collagen deposition and alkaline phosphatase expression. It significantly promoted implant osseointegration compared with treatment with scrambled peptide-treated implants by increasing the bone-to-implant contact ratio and bone area. These findings support the hypothesis that the DLTIDDSYWYRI motif acts as an effective osseointegration accelerator by enhancing new bone formation.Considerable effort has been directed towards replacing lost teeth using tissue-engineering methods such as titanium implants. A number of studies have tried to modify bioinert titanium surfaces by coating them with functionally bioactive molecules for faster and stronger osseointegration than pure titanium surfaces. Recently, peptides have been recognized as valuable scientific tools in the field of tissue-engineering. The DLTIDDSYWYRI motif of the human laminin-2 α2 chain has been previously reported to promote the attachment of various cell types; however, the in vivo effects of the DLTIDDSYWYRI motif on new bone formation have not yet been studied. To examine whether a laminin-2-derived peptide can promote osseointegration by accelerating new bone formation in vivo, we applied titanium implants coated with the DLTIDDSYWYRI motif in a rabbit tibia model. The application of the DLTIDDSYWYRI motif-treated implant to tibia wounds enhanced collagen deposition and alkaline phosphatase expression. It significantly promoted implant osseointegration compared with treatment with scrambled peptide-treated implants by increasing the bone-to-implant contact ratio and bone area. These findings support the hypothesis that the DLTIDDSYWYRI motif acts as an effective osseointegration accelerator by enhancing new bone formation.Tissue-engineeringThis work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by MEST (Grant No. 2011-0007662) and the Mid-career Researcher Program through NRF, funded by MEST (Grant No. 2010-0014662).OAIID:oai:osos.snu.ac.kr:snu2013-01/102/2008003883/1SEQ:1PERF_CD:SNU2013-01EVAL_ITEM_CD:102USER_ID:2008003883ADJUST_YN:NEMP_ID:A078517DEPT_CD:861CITE_RATE:7.404FILENAME:Biomaterials 201305 34(16) 4027-37.pdfDEPT_NM:치의학과EMAIL:[email protected]_YN:YCONFIRM:

Differences in Circulating Dendritic Cell Subtypes in Pregnant Women, Cord Blood and Healthy Adult Women

Different subtypes of dendritic cells (DC) influence the differentiation of naíve T lymphocytes into T helper type 1 (Th1) and Th2 effector cells. We evaluated the percentages of DC subtypes in peripheral blood from pregnant women (maternal blood) and their cord blood compared to the peripheral blood of healthy non pregnant women (control). Circulating DC were identified by flow cytometry as lineage (CD3, CD14, CD16, CD19, CD20, and CD56)-negative and HLA-DR-positive cells. Subtypes of DC were further characterized as myeloid DC (CD11c+/CD123±), lymphoid DC (CD11c-/CD123+++) and less differentiated DC (CD11c-/CD123±). The frequency of DC out of all nucleated cells was significantly lower in maternal blood than in control (P<0.001). The ratio of myeloid DC/lymphoid DC was significantly higher in maternal blood than in control (P<0.01). HLA-DR expressions of myeloid DC as mean fluorescence intensity (MFI) were significantly less in maternal blood and in cord blood than in control (P<0.001, respectively). The DC differentiation factors, TNF-α and GM-CSF, released from mononuclear cells after lipopolysaccharide stimulation were significantly lower in maternal blood than in control (P<0.01). The distribution of DC subtypes was different in maternal and cord blood from those of non-pregnant women. Their role during pregnancy remains to be determined

Is Myocardial Infarction in Patients without Significant Stenosis on a Coronary Angiogram as Benign as Believed?

The present study aimed to investigate the clinical characteristics and 1-year outcomes of acute myocardial infarction (AMI) patients without significant stenosis on a coronary angiogram comparison with the clinical characteristics and outcomes of patients with significant coronary artery stenosis. A total of 1,220 patients with AMI were retrospectively classified into Group I (≥50% diameter stenosis, n=1,120) and Group II (<50%, n=100). Group II was further divided into two subgroups according to the underlying etiology: cryptogenic (Group II-a, n=54) and those with possible causative factors (Group II-b, n=46). Patients in Group II were younger, were more likely to be women, and were less likely to smoke and to have diabetes mellitus than were patients in Group I. The levels of cardiac enzymes, LDL-cholesterol levels, and the apo-B/A1 ratio were lower in Group II. However, 1-month and 12-month rates of major adverse cardiac events (MACE) were not significantly different between the two groups. The Group II-b subgroup comprised 29 patients with vasospasm, 11 with myocardial bridge, and 6 with spontaneous thrombolysis. Left ventricular ejection fraction and creatinine clearance were lower and levels of N-terminal pro-brain natriuretic peptide (NT-proBNP) and high-sensitivity C-reactive protein (hs-CRP) were higher in Group II-a than in Group II-b. However, outcomes including MACE and mortality at 12 months were not significantly different between the two subgroups. The 1-year outcomes of patients in Group II were similar to those of patients in Group I. The clinical outcomes in Group II-a were also similar to those of Group II-b, although the former group showed higher levels of NT-proBNP and hs-CRP

Functional genomics reveals serine synthesis is essential in PHGDH-amplified breast cancer

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation[superscript 1, 2]. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes[superscript 3]. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of α-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.Susan G. Komen Breast Cancer Foundation (Fellowship)Life Sciences Research Foundation (Fellowship)W. M. Keck FoundationDavid H. Koch Cancer Research FundAlexander and Margaret Stewart TrustNational Institutes of Health (U.S.) (Grant CA103866

Systemic AAV vectors for widespread and targeted gene delivery in rodents

We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery