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Control cells rely on extracellular indicators produced by the specific niche market, which state their capability to self-renew, expand and differentiate. possess not been defined previously. We performed reduction and gain of function research for LIF and present a exhaustion of NSCs, a subset of multipotential neural precursors and immature oligodendrocytes in LIF null mice. Gain of function studies showed that LIF improved the great quantity of these precursors. Our studies also show that these NPs have differential 315702-99-9 IC50 requirements for LIF and CNTF and 315702-99-9 IC50 for EGF, FGF-2 and PDGF for their propagation in vitro. Remarkably, the related cytokine, CNTF was less potent than LIF in increasing the NSCs and more potent than LIF in increasing the PDGF responsive multipotential precursors. Finally, we display that LIF raises the manifestation of the core transcription factors: and c-Myc. Completely our FACS analyses reveal that the neonatal SVZ is definitely much more heterogeneous than previously thought and our studies provide fresh information into the signals and mechanisms that regulate their self-renewal and expansion. reveal that the neonatal SVZ is definitely much more heterogeneous than previously thought provide fresh information into the signals and mechanisms that regulate their self-renewal and expansion. Materials and Methods C57BT/6 postnatal day time 7 (P7) neonatal mice generated from breeders purchased from Jackson Laboratories (Pub Harbor, Maine) hRad50 were used for neurosphere studies, FACS analysis and studies on the cellular composition of the adult SVZ. CD-1 background LIF P11 heterozygotes and nulls (bred with CD-1 mice purchased from Charles Water Laboratories (Wilmington, MA) were genotyped using primers and the polymerase chain reaction-based method, along with age-matched crazy type neonates for circulation cytometric analysis. All tests were performed in compliance with analysis suggestions established on by the IACUC panel of the New Shirt Medical College. Neurosphere Trials Spheres had been ready from G7 neonates using regular methods. After 7 days in vitro, spheres were collected, broken down with 70% power Accutase (Millipore, Billerica, MA) and plated at 5104 cells/cm2 for supplementary (2), tertiary (3) or quaternary (4) spheres. Spheres had been cultured in chemically described moderate supplemented with 20 ng/ml EGF (PeproTech, Rocky Mountain, Nj-new jersey) and 10 ng/ml FGF2 (PeproTech, Rocky Mountain, Nj-new jersey) with 2ng/ml heparin (known to as EF). Recombinant mouse LIF (rmLIF; Millipore, Billerica, MA) and recombinant rat CNTF (rrCNTF; Ur & Chemical Systems, Minneapolis, MN) had been utilized at 5 ng/ml, while soluble receptor leader for rrCNTF had been utilized at 100 ng/ml. Cells were grown for 6-7 times to evaluation past. A world was described as a free-floating, cohesive group that was at least 30 meters in size, although the bulk of spheres had been bigger in size. Plate designs were shaken to distribute the spheres before keeping track of gently. 5 arbitrary 10x areas had been measured per well and 6 wells had been examined per group. The regularity of sphere-forming cells was computed for 3 and 4 (Amount 3) by the typical amount of spheres per field, the certain area of the field and the area of the well using a Ziess Viewer.Z1. Quantity was calculated using Ziess Axiovision software program World. Amount 3 LIF Expands and Sustains Self-Renewing Multipotential Neonatal Neural Precursors Neurosphere Difference 3 spheres had been plated onto step film negatives previously covered with 1% poly-d-lysine (w/sixth is v) and 10 g/ml laminin, in ProN with 0.5% FBS without development factors. After 315702-99-9 IC50 5-7 times, step film negatives had been tarnished using O4 lifestyle supernatant (1:20) in 10% lamb serum in DMEM/Y12 with 15 315702-99-9 IC50 millimeter HEPES at area heat range (RT) for 45 minutes, cleaned in BCH (10% Baby bovine serum in DMEM/Y12 with HEPES) and after that incubated for 30 minutes in GAM IgM lissamine rhodamine sulfonyl chloride (1:200; Knutson ImmunoResearch, Western world Grove, Pennsylvania). Cells had been set in 3% paraformaldehyde, quenched with 100 millimeter glycine, rinsed with PBS and permeabilized with methanol designed for 20 minutes in -20C after that. Cells had been obstructed in TGB superblock (0.3% triton-x-100, 10% goat serum, 10% bovine serum albumin in Tris-buffer) for 45 min and immunostained using anti–Tubulin III (TUJ1, 1:300; Covance, Princeton, Nj-new jersey) and anti-GFAP (1:500; Dako, Carpenteria, California) in TGB diluent O/In at 4C. After washing thoroughly, cells were incubated for 30 min at RT in GAM dylight 488 IgG2a for TuJ1 and GAR dylight 649 IgG for GFAP (both at 1:300; Jackson ImmunoResearch, Western Grove, PA). Cells were coverslipped with ProLong Yellow metal with DAPI (4,6-diamidino-2-phenylindole; Invitrogen, Carlsbad, CA). Images of discolored cells were collected using a SenSys cooled-coupled device video camera (CRI) interfaced with IPLab medical imaging software (Scanalytics) on an Olympus AX-70 microscope. Circulation Cytometric Sample Preparation 2 or 3 spheres were dissociated.
Category: LTD4 Receptors
LTD4 Receptors
A number of enzymes become functional by presenting to zinc during
A number of enzymes become functional by presenting to zinc during their trip through the early secretory pathway. demonstrate that TNAP is normally turned on not really merely by unaggressive zinc presenting but by an complex two-step system via proteins stabilization implemented by enzyme transformation from the apo- to the holo-form with zinc packed by ZnT processes in the early secretory path. gene causes hypophosphatasia, a heritable type of rickets and osteomalacia (21, 22). Furthermore, incomplete decrease of TNAP activity and disability of osteoblast growth is normally noticed in osteoblasts differentiated from precursor cells ready from and (three-way knock-out cells (TKO cells)), and this decrease was renewed by re-expression of the individual counterparts (8, 19). Intriguingly, TKO cells exhibit various other ZnTs or transiently localised to the secretory path constitutively, such as ZnT4 and ZnT1. This led us to examine the likelihood whether they may lead to the account activation of TNAP and to investigate the molecular basis of TNAP account activation by ZnTs. In this scholarly study, we record that TNAP is normally turned on not really merely by zinc ions mobilized in the early secretory path but by an complex two-step system by particular ZnTs, through protein stabilization and service. To our knowledge, this is definitely the 1st statement to demonstrate directly that ZnTs run to regulate protein stability individually of zinc transport and how ZnTs specifically regulate the activity of a zinc-requiring enzyme. EXPERIMENTAL Methods Cell Tradition and Transfection Chicken M lymphocyte-derived DT40 cells were managed in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% heat-inactivated fetal calf serum (FCS) (JRH Biosciences, Lenexa, KS), 1% chicken serum (Invitrogen), and 50 m 2-mercaptoethanol (Sigma) at 39.5 C. TSPAN33 To generate zinc-deficient tradition medium, FCS or AP24534 (Ponatinib) supplier chicken serum was treated with Chelex-100 resin as explained previously (24). DNA transfection into DT40 cells was carried out as explained previously (8). Additional experimental strategies to generate conditional mutants are demonstrated in the supplemental numbers. Plasmid Building Info about the focusing on vectors of the chicken (c(ccDNA and genes are flanked by sequences. Therefore, the cDNA could become excised by Cre-recombinase. Plasmids for appearance of human being TNAP (hTNAP) and tamoxifen-regulated chimeric Cre recombinase (pANMerCreMre) have been explained previously (7, 19). All plasmids were linearized with appropriate restriction digestive enzymes prior to electroporation. Northern Blotting RNA (20 g) taken out from DT40 cells using Sepasol I (Nacalai Tesque) was AP24534 (Ponatinib) supplier electrophoresed on an agarose skin gels and then transferred to a nitrocellulose membrane filter in 20 SSC. The membrane was hybridized to appropriately AP24534 (Ponatinib) supplier radiolabeled cDNA probes. Radioimages were acquired as explained previously (8). Measurement of TNAP Activity Total mobile proteins ingredients had been ready from cells lysed in ALP lysis stream, and 2 or 3 g of proteins was used to measure TNAP activity as explained previously (8). Shrimp ALP (Roche Applied Technology) was used as the standard. For the zinc supplementation experiment, total cellular protein was prepared from cells incubated in the presence of 100 m ZnSO4 or 5 m zinc pyrithione for 30 min, adopted by two washes with tradition medium and incubation in medium for another 8 h at 39.5 C. Immunoblotting and Immunoprecipitation Immunoblotting and immunoprecipitation were performed as explained previously (7, 19). The blotted membrane was clogged with obstructing remedy (5% skim milk and 0.1% Tween 20 in phosphate-buffered saline) and then incubated with anti-HA HA-11 (1:1000 dilution; Covance, Emeryville, CA), anti-FLAG M2 (1:1000; Sigma), anti-Myc 9E10 (1:1000; Sigma), anti-calnexin (1:1000; StressGen, Ann Arbor, MI), anti-hTNAP (1:2000), anti-hZnT5 (1:1000), or anti-hZnT6 (1:1000; Proteintech Group Inc., Chicago) antibodies in obstructing remedy. Immobilon Western chemiluminescent HRP substrates (Millipore, Billerica, MA) were used for detection. The fluorimager was acquired using a LAS1000 Plus image analyzer (Fujifilm, Tokyo, Japan). Immunofluorescence Immunostaining for FLAG-hZnT1, hZnT2-HA, hZnT3-Myc, hZnT4-HA, or mZnT8-HA AP24534 (Ponatinib) supplier indicated in TKO cells was performed as explained previously (20). Briefly, the cells were discolored with AP24534 (Ponatinib) supplier anti-HA antibody (1:50; Sigma) for hZnT4-HA, with anti-HA tag antibody (1:100;.
LTD4 Receptors
Aberrant autophagy played an important role in the pathogenesis of autoimmune
Aberrant autophagy played an important role in the pathogenesis of autoimmune diseases, especially in systemic lupus erythematosus (SLE). exhibited MSCs have immunomodulatory effects [2]. And transplantation of umbilical cord-derived mesenchymal stem cells (UC-MSCs) has been shown effective in patients with autoimmune diseases, especially systemic lupus erythematosus (SLE) [3C5]. UC-MSCs could prevent T cell proliferation in lupus patients by secreting large Temsirolimus amounts of indoleamine 2,3-dioxygenase (IDO), which is usually enhanced by IFN-produced by CD8+T cells [6]. They also suppress W cell proliferation and differentiation [7] and regulate Treg/Th17 balance in SLE patients [8]. However, their rules on autophagy has not been assessed. Recent works indicated the important role of autophagy in SLE [9, 10], which exhibited that T cells from SLE patients have overactivated autophagy [11, 12]. Autophagy is usually the natural, destructive mechanism that disassembles, through a regulated process, unnecessary or dysfunctional cellular components. Peripheral T cells from SLE patients have been shown to have defective mitochondria, exhibited by mitochondrial hyperpolarization and ATP depletion [13]. Continuous autophagy activation usually prospects to a high turnover rate of proteins and organelles, and a high rate above the survival threshold could result in cell death [14] and thus may be related to increased apoptosis of peripheral T cells from SLE patients [15]. In this study, the link between autophagy and apoptosis of activated T cells from SLE patients was resolved and the rules of UC-MSCs on T cell autophagy was investigated. 2. Materials and Methods 2.1. Patients and Healthy Controls We included 32 SLE patients (male?:?female = 7?:?25, mean age = 36, range 16 to 49 years old) who were diagnosed according to the classification criteria of the American College of Rheumatology [16]. Current SLE disease activity was assessed using the SLE Disease Activity Index (SLEDAI) [17]. The mean standard error of mean (SEM) SLEDAI score was 6.5 0.9. Among these patients, 9 were assessed for basal autophagic activity and Temsirolimus mitochondrial mass (detailed clinical characteristics and laboratory features were shown in Table 1). 30 healthy donors (male?:?female = 8?:?22, mean age = 33, range 22 to 51 years aged) were recruited as controls. They were recruited from the Affiliated Drum Tower Hospital of Nanjing School Medical College after up to date permission was attained. The process was accepted by the Values Panel at the Associated Drum Structure Medical center of Nanjing School Medical College. Desk 1 Clinical and lab features in 9 sufferers with systemic lupus erythematosus (SLE). 2.2. UC-MSCs Solitude and Lifestyle Umbilical wires (UC) had been attained from regional mother to be clinics after regular shipping and after that broken down and cultured for 2 ages. After 2 paragraphs, UC-MSCs had been farmed. Stream cytometry studies demonstrated Compact disc29, Compact disc44, and Compact disc105 reflection >95%, in parallel with TRIB3 Compact disc45, Compact disc34, Compact disc14, and HLA-DR reflection <2%. All Temsirolimus antibodies that utilized for stream cytometry studies had been bought from eBioscience. 2.3. Traditional western Blotting Compact disc3+Testosterone levels cells had been filtered from PBMCs by microbeads and lysed with SDS test stream formulated with 20?millimeter Tris-HCl (pH 7.6), 250?mM NaCl, 0.5% NP-40, 3?mM ethylenediaminetetraacetic acidity, and 1.5?mM ethyleneglycoltetraacetic acidity with 10?mg/mL aprotinin, 10?mg/mL leupeptin, 1?mM DTT, 1?mM paranitrophenylphosphate, and 0.1?mM Na3VO4 simply because protease and phosphatase inhibitor. Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide solution electrophoresis (SDS-PAGE) and Temsirolimus transferred to a polyvinylidene difluoride (PVDF) Temsirolimus membrane (Millipore). Blots were probed by anti-LC3M antibody (Cell Signaling Technology, Inc.), anti-p62 antibodies (Cell Signaling Technology, Inc.), and anti-GAPDH antibody (Cell Signaling Technology, Inc.) before visualizing.
LTD4 Receptors
Background The chitinase-like protein, Chi3L1, is associated with increased fibrotic activity
Background The chitinase-like protein, Chi3L1, is associated with increased fibrotic activity as well as inflammatory processes. not really present in regular epidermis. Various other cells, however, added to the upregulation of Chi3T1 by OSM. Findings The emergence of cells primed to respond to OSM with improved Chi3T1 production appears to become connected with pathological processes active in SSc. General significance The presence of progenitor cells conveying the chilectin Chi3T1 in SSc pores and skin appears to play a part in the initiation of the disease process. Abbreviations: Chi3T1, chitinase 3-like protein 1; DAPI, 4,6-diamidino-2-phenylindole; ECM, extracellular matrix; IL, interleukin; mRSS, altered Rodnan pores and Rabbit polyclonal to LAMB2 skin score; OSM, oncostatin M; PDGF, platelet-derived growth element; SMA, -clean muscle mass actin; SBTI, soybean trypsin inhibitor; SSc, systemic sclerosis (scleroderma); Tie up2, tyrosine kinase with Ig and EGF homology domain names-2; TGF, changing growth element- Keywords: Scleroderma, Systemic sclerosis, Tetrahydrozoline HCl supplier Come cell, Chitinase 3-like protein 1, Cytokine, Oncostatin M Graphical subjective 1.?Intro Systemic sclerosis (scleroderma, SSc) is a compound autoimmune disease with a highly variable array of clinical features, the most characteristic being an overproduction and excessive deposition of collagen in the pores and skin and internal body organs, with a modern program and often fatal end result. It is relatively rare, influencing between 50,000 and 100,000 North People in america, and up to 250,000 Europeans [1], [2]. Although less common than additional rheumatic diseases, it offers one of the highest mortality rates [3]. Disruptions of both the vascular and defense systems are idea to contribute to the advancement of SSc. Endothelial adjustments take place early in the disease frequently, implemented by vascular harm that network marketing leads to a cascade of stimulatory adjustments culminating in tissues fibrosis [4]. This procedure consists of T lymphocytes [5], monocytes, macrophages [6] and mast cells [7] as well as fibroblasts [8]. The turned on cells secrete a range of items, including development elements, cytokines and Tetrahydrozoline HCl supplier their antagonists [9]. These chemicals trigger irritation and elevated deposit of extracellular matrix (ECM) elements, leading to modern and extensive tissues fibrosis. The heterogeneity of several forms of SSc and the problems in discerning between disease activity (factors of the disease that vary over period and are possibly reversible automatically or with medication treatment) and disease harm (permanent tissues damage that outcomes from the disease) [10] complicate research of SSc. At present there are no authenticated Tetrahydrozoline HCl supplier biomarkers which can end up being utilized to monitor disease development. There is normally an comprehensive reading, nevertheless, analyzing the romantic relationship between many of the cytokines and effector elements suggested as a factor in the several pathological procedures linked with the advancement and development of SSc [11], [12], [13], [14], [15]. The chitinase-like proteins, Chi3M1 (YKL40, HCgp39), provides been proven to end up being linked with elevated fibrotic activity as well as inflammatory procedures. Chi3M1 is normally upregulated in many pathological circumstances?[16], [17], [18], [19], [20], [21], [22], [23]. High serum amounts of Chi3M1 are linked with poor treatment, shorter recurrence-free period of time and low general success [16], [17] in sufferers with a wide range of malignancies, including breasts [16] and colorectal cancers [17]. Individuals with diseases characterized by swelling and cells fibrosis, including rheumatoid arthritis [18], osteoarthritis [19], pneumonia [20], liver cirrhosis [21] and systemic sclerosis (SSc) [22], [23], [24] are also reported to have elevated serum Chi3T1 levels. Chi3T1 goes to the family of mammalian chitinase-like healthy proteins, which share main sequence homology and three-dimensional structure with the family 18 glycohydrolases [25] but lack chitinolytic activity. The lack of catalytic activity is definitely due to two amino acid substitutions in the active site region of the protein, the most Tetrahydrozoline HCl supplier essential one becoming substitution of the catalytic glutamate residue with leucine [26]. Chi3T1 is definitely produced by macrophages, synovial cells and chondrocytes from arthritic bones [27], and neutrophils [28]. It offers been demonstrated to take action synergistically with insulin-like development aspect (IGF-1) in fibroblasts to induce cell development [29]. The protein has mitogenic effects on chondrocytes and synovial cells and promotes also.
LTD4 Receptors
Fluorescent proteins are commonly used to label cells across organisms, but
Fluorescent proteins are commonly used to label cells across organisms, but the unmodified forms cannot control biological activities. Intro Studies of multi-cellular organisms would become greatly facilitated by the ability to change the activities of genes within any cells or cell type. This is definitely demanding to accomplish in cells with varied cell types, such as the nervous system (Masland, 2004). To label and provide genetic access to varied cell types, much effort offers been dedicated to generating transgenic organisms in which transgenes are placed under the control of large genomic fragments or endogenous gene loci. Transgenic lines articulating driver genes such as transcription factors or site-specific recombinases can then SB-277011 become used to control the appearance of genes in responder cassettes. However, the energy of individual lines is definitely limited by a transgene's practical capabilities; media reporter lines articulating fluorescent proteins and histochemical digestive enzymes are useful for marking cells, but cannot currently become used to control biological activities. To change transgenes driven by the same (Kirchhofer et al., 2010). However, it was ambiguous whether any of the recognized pairs could co-occupy GFP, tolerate the addition of fusion partners and induce the development of biologically energetic processes in cells. Furthermore, many feasible GBP pair-wise combos acquired not really been examined for their capability to co-occupy GFP. To address these presssing problems, we performed an news reporter display screen for GBP pairs that could stimulate the development of an energetic transcription aspect (Amount 1 and Prolonged Experimental Techniques). The Lady4 DNA presenting domains (DBD) and VP16 account activation domains (Advertisement) (Sadowski et al., 1988) had been individually fused to GBPs in several options and positioned under control of the common CAG marketer (Niwa et al., 1991) (Amount 1B). DBD-GBP (DBDG) and AD-GBP (ADG) blend constructs had been processed through security in pair-wise combos for GFP-dependent account activation of a UAS luciferase (luc2) news reporter in 293T cells. Useful DBDG/ADG pairs shall be referred to as Transcription Gadgets Type in GFP (T-DDOG). T-DDOGs choosing GBP1+6 or GBP2+7 regularly provided the most powerful news reporter induction (Amount 1C, ?,22 and T1), and became the concentrate of this scholarly research. To state DBDG+ADG combos, the DBD-GBPX blend PAK2 is normally shown in regular font, along with the AD-GBPX blend in superscript, offering DBD-GBPXAD-GBPY. Particular T-DDOG options are tabulated in Desk Beds1. Amount 1 display screen utilized to recognize practical GBP pairs for the GFP-dependent transcription program Shape 2 SB-277011 Portrayal of the GFP-dependent transcription program Portrayal of the GFP-dependent transcription program displays. Certainly, we had been capable increase and diversify the practical repertoire of T-DDOGs. T-DDOGs using the LexA and rTetR DBDs triggered reporters bearing their particular presenting sequences, tetO (included in tetracycline response component, or TRE) and lexAop, just when GFP was present SB-277011 (Shape 3A-3E) (Butala et al., 2009; Schonig et al., 2010). The actions of rTetR-based T-DDOGs had been additional discovered to rely on doxycycline amounts (Shape 3D). This drug addiction provides temporal SB-277011 control for the operational system. Shape 3 T-DDOGs are highly adjustable T-DDOGs may end up being adjusted to alter their transcriptional strength also. The essential area for VP16AG function is situated within a 12 amino acidity peptide (VPmin) (Baron et al., 1997). We could naturally modify the transcription activity of Lady4-GBP1AD-GBP6 by either differing the quantity of VPmin repeats or the quantity of GBPs fused to the DBD (Shape 3F). We further separated powerful T-DDOGs bearing the g65AG (Schmitz and Baeuerle, 1991), an alternate to VP16AG in artificial transcription systems (Rivera, 1998) (Shape 3G). General, we regularly separated powerful T-DDOG versions using the GBP2+7 and GBP1+6 mixtures, suggesting that these pairs can effectively recruit various combinations of fusion partners onto the GFP scaffold. The GFP-dependent transcription system can be used in the mouse for cell-specific gene regulation To evaluate whether GFP can control the activity of T-DDOGs is a homeobox gene that is necessary for photoreceptor specification in the retina (Nishida et al., 2003). We used GFP to induce Cre-mediated excision of a floxed Otx2 allele (with Cre being under.
LTD4 Receptors
The cell cycle is integrated with many aspects of embryonic advancement.
The cell cycle is integrated with many aspects of embryonic advancement. AZD 2932 supplier been utilized to understand how the cell routine is certainly redesigned during vertebrate advancement and how it is certainly controlled during morphogenesis. This review represents how some of the exclusive skills of marine species have been leveraged for cell cycle research and suggests how species such as and zebrafish will continue to reveal the functions of the cell cycle in human biology and disease. a series of synchronous and rapid Mouse monoclonal antibody to PRMT6. PRMT6 is a protein arginine N-methyltransferase, and catalyzes the sequential transfer of amethyl group from S-adenosyl-L-methionine to the side chain nitrogens of arginine residueswithin proteins to form methylated arginine derivatives and S-adenosyl-L-homocysteine. Proteinarginine methylation is a prevalent post-translational modification in eukaryotic cells that hasbeen implicated in signal transduction, the metabolism of nascent pre-RNA, and thetranscriptional activation processes. IPRMT6 is functionally distinct from two previouslycharacterized type I enzymes, PRMT1 and PRMT4. In addition, PRMT6 displaysautomethylation activity; it is the first PRMT to do so. PRMT6 has been shown to act as arestriction factor for HIV replication embryonic cell cycles. These early embryonic cell cycles of frogs and other aquatic organisms are simplified versions of the cell cycles of somatic cells (Evans et al., 1983; Kane and Kimmel, 1993; Newport and Kirschner, 1982a). These cycles differ from the canonical four-phase cell cycle in four important ways. First, an autonomous biochemical oscillator pushes them, which is usually unaffected by developmental signals or checkpoints (Ikegami et al., 1997; Kane and Kimmel, 1993; Kimelman et al., 1987; Newport and Kirschner, 1982a; Newport and Kirschner, 1984). Second, they are extremely rapid and lack the gap phases (G1 and G2), which individual S-phase from mitosis (Physique 1). Third, they are characterized by a lack of growth in cytoplasmic volume, so the large egg cytoplasm is usually gradually cleaved into a large number of smaller nucleated cells. Finally, they occur without zygotic transcription, so they are entirely controlled by maternally provided mRNA and protein. For example, the frog undergoes twelve cleavage cycles to produce thousands of cells; and the zebrafish (homolog of the cell cycle kinase p34cdc2; the other protein was shown to be Cyclin W (Dunphy et al., 1988; Gautier et al., 1990; Gautier et al., 1988; Lohka et al., 1988). Together Cdc2 and Cyclin W form the prototypical Cyclin-dependent kinase (CDK) complex that pushes mitosis. Cyclin B-Cdc2 is usually just one of several CDK complexes that promote cell cycle progression; other CDKs are crucial for G1- and S-phase progression. The finding and molecular definition of MPF and Cyclin demonstrates the power of aquatic model systems for both embryology and biochemistry. Derivatives of the frog egg cytoplasmic extracts have continued to be extraordinarily productive in the cell cycle field. Strike and Laskey demonstrated that the kind of the Lohka and Masui program AZD 2932 supplier could end up being utilized to research the initiation and finalization of DNA duplication in a cell-free program (Hit and Laskey, 1986). Murray and Kirschner created the egg get program additional also, and demonstrated that their improved program could get multiple effective cell cycles (Murray and Kirschner, 1989). Variants in the egg remove protocols possess been optimized to research different factors of cell biology AZD 2932 supplier including DNA duplication licensing and initiation; sis chromatin cohesion; mitosis; the AZD 2932 supplier DNA harm and cell routine checkpoints; and DNA fix (Dasso and Newport, 1990; Hekmat-Nejad et al., 2000; Hyrien et al., 1995; Kumagai et al., 1998; Lafont et al., 2010; Raschle et al., 2008; Sheehan et al., 1988; Wally et al., 1998). Simply simply because marine model microorganisms have got been utilized to research the fundamental systems of cell routine control, they possess also been utilized to understand how the cell routine adjustments during advancement. Microorganisms such as and zebrafish possess been useful for learning how the simple embryonic cell routine is certainly redesigned into the even more complicated somatic cell routine. 3. Using Marine Model Microorganisms to Understand Cell Routine Redecorating Early advancement is certainly linked with dramatic adjustments in cell routine aspect. The cell routine is certainly redesigned throughout the cleavage, blastula, gastrula, and segmentation levels, during which period important adjustments in transcription, cell motility and cellular difference occur. The early embryonic cell cycles of cleavage and early blastula embryos, which possess been useful for elucidating the systems of the primary cell routine engine, absence extra levels of control that are discovered in somatic proliferating cells. Eventually the rapid cleavage embryo cell cycles with alternating M and S phases are transformed into.
LTD4 Receptors
Acinic cell carcinoma is an indolent form of invasive breast tumor,
Acinic cell carcinoma is an indolent form of invasive breast tumor, whereas microglandular adenosis has been shown to be a neoplastic proliferation. recurrently mutated gene (75% in microglandular adenosis 88% in acinic cell carcinomas), and mutations were consistently coupled with loss of heterozygosity of the wild-type allele. Additional somatic mutations shared by both organizations included those in and somatic mutations, and likely symbolize low-grade forms of triple-negative disease with no/minimal metastatic potential, of which a subset has the potential to progress to high-grade triple-negative breast cancer. Intro Microglandular adenosis of the breast encompasses a spectrum of lesions, ranging from genuine forms without atypia, to atypical microglandular adenosis and carcinoma-associated lesions (1). Despite becoming historically named adenosis, several studies possess reported within the progression from microglandular adenosis/atypical microglandular adenosis to invasive carcinomas, which are mostly of high histologic grade and triple-negative immunophenotype (i.e. lacking manifestation of estrogen receptor (ER), progesterone receptor (PR) and HER2) (2C5). In fact, molecular analyses have suggested that microglandular adenoses/atypical microglandular adenoses, at least those associated with carcinoma, are clonal neoplastic lesions and non-obligate precursors of high-grade triple-negative breast cancers, as synchronously diagnosed ipsilateral microglandular adenoses/atypical microglandular adenoses and invasive carcinomas display related patterns of copy number alterations and mutation profiles (6C10). Acinic cell carcinoma is definitely a rare unique histologic type of breast tumor of low-grade and indolent behavior (11), which, akin to microglandular adenoses/atypical microglandular adenoses, may progress to high-grade triple-negative breast tumor (12, 13). Indeed, metastatic potential may be limited to those instances mixed with a high-grade/non-acinic cell component (11). Despite these beneficial prognostic features, acinic cell carcinomas display complex patterns of copy number alterations and harbor highly recurrent mutations (12), paralleling the genomic profiles of common forms of triple-negative breast cancers (14). Despite conceptual variations, microglandular adenoses/atypical microglandular adenoses and acinic cell carcinomas display histologic SIB 1757 and immunohistochemical similarities (1, 11, 13, 15). Morphologically, both entities typically are characterized by an infiltrative proliferation of small glands lined by low-grade cuboidal to flattened cells lacking a myoepithelial cell coating (1, 11). Acinic cell carcinomas may however display a distinct architecture such as the hypernephroid obvious cell pattern (12), and may display intra-tumor heterogeneity with well-differentiated tubular SIB 1757 and less-differentiated solid areas (13). Immunophenotypically, microglandular adenoses/atypical microglandular adenoses and acinic cell carcinomas are characterized by strong manifestation of S100 protein (4C7, 9, 16) and most are of triple-negative immunophenotype (4, 6, 7, 9, 12, SIB 1757 13, 16). Differential analysis of microglandular adenoses/atypical microglandular adenoses and acinic cell carcinomas can be demanding (15, 16) and often relies on the recognition of diffuse serous differentiation in acinic cell carcinomas (11, 16). The second option can be defined by the presence of intracytoplasmic zymogen-type granules or manifestation of acinar differentiation markers, such as lysozyme and amylase. Microglandular adenosis/atypical microglandular adenosis cells, however, possess been shown to focally display these features (4, 5, 15). Large-scale genomic studies have been carried out in breast tumor, demonstrating that only three genes are mutated in more than 10% of unselected instances, namely (37%), (36%) and (11%) (14, 17). In the subset of triple-negative breast cancers, even greater inter-tumor heterogeneity is definitely observed. Only is highly recurrently mutated (86%), while in wild-type cancers, the p53 pathway is usually inactivated by additional mechanisms (14, 17). In addition, triple-negative breast cancers are characterized by the loss of cell cycle checkpoints and mutations (9%) or amplifications, and loss of or (14, 17, 18). Given the histologic and immunohistochemical similarities between CACNA2 microglandular adenoses/atypical microglandular adenoses and acinic cell carcinomas, and between this group of lesions and triple-negative breast cancers, and the observation that microglandular adenoses/atypical microglandular adenoses and acinic cell carcinomas may progress to high-grade triple-negative breast cancers, here we wanted to compare the genomic panorama of microglandular adenoses/atypical microglandular adenoses and.
LTD4 Receptors
Background Great needle aspiration (FNA) is normally widely used for evaluation
Background Great needle aspiration (FNA) is normally widely used for evaluation of individuals with thyroid nodules. FFPE tissues examples had been positive for the V600E mutation. From the discordant pairs, 5/6 FNAs included significantly less than 50% tumor cells. Bottom line When used in combination with indeterminate FNA examples, BRAF mutation evaluation may be a good adjunct way of confirming the medical diagnosis of malignancy within an usually equivocal case. RGFP966 Nevertheless, general tumor cell articles of some archival FNA smear slides is really a limiting aspect for mutation recognition. Background As the regularity of thyroid cancers in the overall population is fairly low, thyroid nodules certainly are a very common clinical problem, and palpable thyroid nodules can be recognized in 4C7% of all adults in the United States [1]. The prevalence of malignancy, however, in a solitary thyroid nodule is only approximately 5% in normal adults [2-4]. Consequently, the primary clinical challenge is to sort out the vast majority of nodules that are benign, which can generally be followed with surveillance, from those requiring surgical RGFP966 intervention. In 2003, approximately 75C80% of all thyroid cancers were papillary thyroid carcinoma (PTC) [5]. Among the most curable of cancers, PTC tends to remain localized in the thyroid gland, but in time it may metastasize to regional lymph nodes and, less commonly, to the lungs. At the RGFP966 time of initial assessment, most patients with PTC present with a painless, palpable, solitary thyroid nodule. As early as the1930's, studies reported on the use of fine needle aspiration (FNA) cytology for the diagnosis of thyroid carcinoma [6,7]. However, as often as 30% of the time, FNA-based evaluation of solitary thyroid nodules displays limited ability to discriminate between benign and malignant lesions and an indeterminate cytologic diagnosis is usually rendered [8]. Although surgical intervention is generally recommended following an indeterminate obtaining on FNA cytology, malignancy within indeterminate thyroid nodules varies between 3C52% [9-16]. Consequently, planning optimal surgical management in patients with an uncertain preoperative diagnosis is challenging. In view of the increasing number of thyroid nodules that require FNA evaluation, there is a clear need for the development of adjunctive diagnostic assays that would help refine indeterminate diagnoses on thyroid cytology. Recently, a single hotspot mutation at nucleotide 1799 of the BRAF gene has been identified as the most common genetic event in 29C83% of all cases of PTC [17-27]. This thymine (T) to adenine (A) transversion mutation results in the substitution of valine with glutamate (V600E) and converts BRAF into a dominant transforming protein that causes constitutive activation of the MAPK pathway, impartial of RAS activation [28]. Additionally, this mutation appears to be fairly specific for PTC. In early polymerase chain reaction (PCR) screening Mouse monoclonal to THAP11 platforms, sample DNA or RNA was amplified first and then detected in a separate step, using a technique such as gel electrophoresis to assess the size and purity of the products. Recently developed instrumentation combines PCR amplification and target nucleic acid characterization in the same closed reaction vessel [29]. Using LightCycler PCR with fluorescent melting curve analysis (LCPCR), the difference in melting profiles between mismatched probe/target and perfectly matched probe/target can be used to RGFP966 characterize amplification products and indicate the presence of a mutation [30]. The primary objective of this study was to.
LTD4 Receptors
Many genes deciding cell identity are controlled by clusters of mediator-bound
Many genes deciding cell identity are controlled by clusters of mediator-bound enhancer elements collectively known as super-enhancers. super-enhancers have already been described in lots of cell types5,6,7. Collectively these studies suggest that a relatively little group of super-enhancers become key switches to find out cell fate. Nevertheless, it really is unclear whether super-enhancers represent a fresh paradigm really, describing an operating unit that's a lot more than the amount of its parts, or if they are an set up of conventional enhancers of varying advantages8 simply. Therefore, you should determine whether you can find emergent practical properties uniquely connected with super-enhancers. Right here, using an impartial strategy1, we determined all super-enhancers in erythroid cells and discovered that both clusters of regulatory components controlling manifestation from the – and -globin genes are categorized as super-enhancers with this cell type. The analysis of mammalian enhancers can be hampered from the observation these components are described by criteria just partly or indirectly linked to their part during embryonic, fetal, and adult erythropoiesis. Both of these solid enhancers belong to a subgroup of specific erythroid enhancers that are destined by the best quantity of Mediator and the best amounts of erythroid get better at regulators. Such areas have been known as hotspots6. Significantly, no evidence is available by us of emergent functional properties through the prolonged enhancer cluster; each element seems to donate to gene manifestation as specific enhancers within an additive instead of synergistic manner. Therefore the super-enhancer Rabbit Polyclonal to STEAP4 from the globin genes could be even more simply referred to as several regular enhancers including a minumum of one solid enhancer, instead of as a fresh discrete entity with properties higher than the amount of its parts. Outcomes Five -globin regulatory components Inulin supplier type an erythroid super-enhancer Major mouse erythroid cells had been analysed to recognize and characterize super-enhancers with this cell type. All Citrine reporter assay in developing poultry embryos, the enhancer was tested by us activity of the five elements. Enhancer activity was recognized in Hamilton-Hamburger stage 9 embryos within the developing bloodstream islands, where it persisted throughout advancement (Fig. 3a), in keeping with the manifestation of globin right here. During development Later, enhancer activity was also recognized within the circulating bloodstream (Fig. 3a and Supplementary Video 1), and was most prominent within the center and mind where the biggest density of reddish colored bloodstream cells is available (Fig. 3a, sections 6-10). Manifestation was Inulin supplier most prominent for the R1 enhancer, as the activity of the R2 enhancer is detected more over the head and trunk broadly. R3 and R4 got similar patterns of manifestation to R2. Rm got the lowest degree of past due activity in areas other than bloodstream islands, with only low degrees of Citrine reporter expression detected within the relative head. No activity was noticed with the adverse control (Supplementary Fig. 2a). In keeping with our results within the PAM evaluation the consequences of such nonintegrated constructs in transient assays reveal the amounts and varieties of transcription element binding sites within each element. Shape 3 Enhancer assays of specific components Each component was examined utilizing the well-established mouse transgenic program17 also,18, in which a vector including an applicant enhancer, a minor promoter as well as the LacZ gene is built-into chromatin randomly positions within the mouse genome stably. Examination of entire embryos at E12.5 pursuing LacZ staining recommended that only R2 exhibited positive enhancer activity in hematopoietic cells at the moment point, with 5 of 7 LacZ-stained embryos exhibiting a manifestation pattern in keeping with erythroid enhancer activity (Fig. 3b and Supplementary Desk 3). We ready tissue areas from three LacZ positive mice for every enhancer construct examined; these verified solid enhancer activity for R2 and proven weaker activity for R1 also, while no activity was recognized for the rest of the three components (R3, R4 and Rm) (Fig. 3b). Therefore, despite their chromatin signatures, these three components would not become categorized as enhancers by this regular assay; just those two components with extensive transcription element binding profile as dependant on PAM evaluation scored positively within the assay. No element is crucial Inulin supplier for globin gene manifestation To look for the contribution of every enhancer to -globin transcription inside the context from the super-enhancer (Fig..
LTD4 Receptors
Background Non-invasive monitoring of liver disease remains an important health issue.
Background Non-invasive monitoring of liver disease remains an important health issue. In an independent verification group of examples, S-HPX separated the Ishak 5C6 (n?=?15) through the Ishak 3C4 (n?=?15) individuals with AuROC 0.84; at the same time, the Ishak 3C4 group was separated from disease-free settings (n?=?15) with AuROC 0.82. Summary S-HPX, a way of measuring sialylated O-glycoforms of hemopexin, gradually raises in fibrotic and cirrhotic individual of HCV etiology and may become quantified by an LCCMS/MS-MRM assay in unfractionated serum of individuals. Quantification of sialylated FG-4592 O-glycoforms of the liver organ secreted glycoprotein represents a book way of measuring the stage of liver organ disease that could possess a job in monitoring the development of liver organ pathology. Electronic supplementary materials The online edition of this content (doi:10.1186/s12014-016-9125-x) contains supplementary materials, which is open to certified users. 400C1600) was accompanied by MS/MS on 25 precursor ions in the number 100C1800, using the powerful exclusion time collection to 6?s, and 150 FG-4592 matters threshold for just two repeated precursors. Collision FG-4592 energy was collection according to charge condition and m/z of precursor ion automatically. Data depended evaluation was used to recognize main precursor and glycoforms XIC of 0.05?Da window was used to judge adjustments in glycoform amounts in liver organ FG-4592 disease (cirrhosis and HCC). Glycopeptide intensities had been normalized to an interior tryptic peptide of HPX to remove influence of adjustments in the focus of HPX proteins for the quantitative result. LC/MS3 and LCCMS/MS-MRM evaluation from the O-glycopeptides of HPX Research of S-HPX in serum of individuals were done straight without enrichment of HPX. Serum examples (2?L) were diluted in 140?L of 25?mM NH4HCO3 with 0.1?% ideals are two sided. Statistical analyses had been performed using SAS v 9.4 (SAS Institute, Cary, NC, USA). Fig.?3 Direct quantification of S-HPX at progressing stages of liver disease. Direct quantification of S-HPX in examples of the next groups of settings and HALT-C individuals in the finding sample set: a disease-free controls (n?=?23), … Table?4 ROC models comparing influence of clinical variables determined at baseline visit on separation of the FG-4592 fibrosis and cirrhosis groups in the validation set Results O-glycopeptides of HPX in liver disease Plasma samples from disease-free controls and cirrhotic patients (CIR) with or without HCC provided a baseline for our examination of O-glycoforms of HPX detectable in liver disease. HPX was isolated by hemin affinity and C18 HPLC from the pooled plasma of participant as described in Patients, materials and methods. The yield of HPX, purified to >95?% purity, was 20C25?g per 100?L of plasma. Glycopeptides of HPX were enriched by HILIC chromatography (Fig.?1) and analyzed by LCCMS/MS as described in [12, 19]. Combined analysis of all the pools led to the identification of 15 O-glycopeptides of HPX (Table?2) derived from the N-terminal tryptic peptide of HPX, TPLPPTSAHGNVAEGETKPDPVTER. We did not observe other O-glycosylated peptides of HPX in any of the samples. Estimates of the abundance of these O-glycoforms, based on intensities of precursor ions in the LCCMS/MS scans, showed that two glycoforms (HexNAc-Gal-Neu5Ac, 66?% total intensity and HexNAc-Gal-2Neu5Ac, 20?% total intensity) dominated the distribution. These two glycoforms were also clearly visible in the HILIC chromatograms by UVCVis detection (Fig.?1). The chromatogram showed that this doubly Tmem1 sialylated glycoform (HexNAc-Gal-2Neu5Ac) increases in cirrhosis and HCC samples (red and green trace) compared to disease-free controls (blue trace) while the mono-sialylated O-glycoform (HexNAc-Gal-Neu5Ac) decreases..


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Cast Members of Note- The adorable Elizabeth Rice, the usually funny Adam Goldberg, and ole' square-jaw herself, Rumer Willis.
Rumer Willis and her boyfriend laying on a hill, making out and being Goth together when out of nowhere, the guy starts to chant some creepy evil sounding prayer, pulls out a gun and tells her "I can't take your chin anymore!" and blows his head off. OMG, that's like, sooooo Emo.
|Go write a poem about it. Also, maybe grab a shower.
|"I'm after me!" Um... Say what now?
|"Me is here to kill yourself... are you scared of you?"
That being said, the movie was pretty disturbing in theme, had some good kills, and I absolutely loved the ending. I think that what the filmmakers did over the credits was a thing of beauty...
|I love creepy eyes! (I guess that's why I post pictures of just creepy eyes once a month...)
|Godless heathen!
|"Where is your God now?"
|Well, I guess that's a spoiler...
|Look at those wasted potentials.
|Christians do love their guns though!
From Within is available now on DVD and VOD.
Under the Dome's Britt Robertson, Margo Harshman, and Bruce's daughter Rumer Willis are in this.

subset: commoncrawl


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MathTree Roots utilizes real-life situations, physical objects, drawings and narratives to build a strong foundation of math in the minds and hearts of campers! Fractions and division are the focus of group instruction for this camp, while, individually, campers begin their climb up the MathTree®, our customizable skill system that adjusts to each camper's pursuit of math skills. Campers are given a short assessment on the first day of camp to properly place them in the MathTree Skill System. All campers should be fluent with single-digit multiplication prior to attendance in this camp.
MathTree Roots
for children entering Grade 4
Rates range from:
- The following rates DO NOT include Early Registration Discounts!
- Half-day, 2 week session: $465
- Full-day, 2 week session (Academic Camp + Hands-On Math): $860
- Before and After Care are available at all locations, 8 – 9 AM & 4:30-5:30 PM
- MathTree partners with several private schools during the summer. At these locations times and pricing may vary from those stated above.
Is your child ready?
To be sure your child is ready for this program, the following should be true:
- My child has fluent knowledge of multiplication facts from 2 x 2 through 9 x 9.
- My child is ready to learn about fractions or needs more help with fractions.
Too easy? Try our Roots to Leaves program.
Too hard? Try our Multiplication Matters program.
Locations, Dates, and Times
View locations and dates, then check availability by visiting our "Dates, Rates, & Times" page for specifics for your child.
Alexandria Location:
Burgundy Farm Country Day School (BFCDS)
3700 Burgundy Rd.
Dates: July 16 – 27, 2018 (Session 5 on the BFCDS summer schedule)
Arlington (North) Location:
Memorial Baptist Church
3455 N Glebe Rd.
Dates: July 9 – 20, 2018
Arlington (South) Location:
Unitarian Universalist Church of Arlington
4444 Arlington Blvd.
SESSION 1: July 30 – August 10, 2018
SESSION 2: August 13 – 24, 2018
Fairfax Location:
Trinity Christian School
11204 Braddock Rd.
Dates: July 23 – August 3, 2018
Leesburg Location:
Loudoun Country Day School (LCDS)
20600 Red Cedar Dr.
Dates: July 30 – August 10, 2018
Vienna Location:
Church of the Holy Comforter
543 Beulah Road, NE
Dates: August 6 – 17, 2018
Rockville Location:
Charles E. Smith Jewish Day School (CESJDS)
1901 E Jefferson St.
Date(s): July 16 – 27, 2018
Washington, DC – AFTER SCHOOL ENRICHMENT:
Tyler Elementary
1001 G St SE, Washington, DC 20003
Open to Tyler Elementary School Students Only.
Classes offered this semester: Bean Counting 201, Multiplication Matters, and MathTree Roots.
Times:Wednesdays, 3:30 – 5:00 PM, Cost: $285
Washington, DC – Southeast:
Hill Center (Capitol Hill)
921 Pennsylvania Ave. SE
Date(s): July 30 – August 10, 2018
*Rates show do not include discounts!
Bean Counting 201: 9 AM – 12:30 PM ($420)
Multiplication Matters: 9 AM – 12:30 PM ($465)
MathTree Roots: 1 – 4:30 PM ($465)
Roots-to-Leaves: 1 – 4:30 PM ($465) FULL at this location
A full-day experience is available by adding MathTree Hands-On Math ($395) to any of the above camps. Before and After Care are also available.


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Disruptive-behaviour problems in childhood 'predict' maladjustment (e.g. violence, criminality and school drop-out) during adolescence and adulthood. A number of intervention programmes have attempted to tackle disruptive behaviours in childhood so as to reduce these problems in later life but their long-term effects are rarely evaluated. A study of 895 boys in Montreal looked into the effect of a two-year intervention programme which the boys undertook between the ages of seven and nine. The boys were then followed up at twenty-four to see how they had got on. The intervention was made up of three different elements : social skills training aimed at promoting changes in behaviour towards peers, social acceptance and less inclination towards antisocial peers ; parenting training and the provision of information and support for teachers. Significantly more boys in the intervention group graduated from high school and fewer had a criminal record compared to the control group.
Boisjoli, Rachel ... [et al] - Impact and clinical significance of a preventive intervention for disruptive boys British Journal of Psychiatry November 2007, 164, 415-419


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Tuesday, 20 November 2012
Copying the worlds of Lynyrd Skynyrd Tuesday Gone...
Anyway today has been a good day in the sense that I had a productive day at work but also a realisation that after my op I need a night out as I got excited about a new shelf in the cabinet....I have a lot of files and they have to be done in a certain way and I was running on space! Only two more days left at work so going to try and get in earlier to make sure everything is up together.
Food wise it was another good day, I didn't take a picture of breakfast as was too busy eating it as I was hungry. I had my usual mug of coffee and then I had 40g of Dorset fruit muesli with blueberries, raspberries and strawberries and a yoghurt all mixed up.
Lunch came before I had time to breathe or go to the toilet (I was bursting haha) and I had similar to yesterday
The soup today was cauliflower cheese soup which is gorgeous. The soup was 1 syn but so worth it and I will be having the same tomorrow. My cute lunchbag is also in the background.
The afternoon was busy and unusual one of my client's has decided to call me babygirl which is kind of odd considering I am her legal representative but its also sweet as she is nice and its better than being shouted at.
So after a walk back to the car I came home to be greeted by the lovely Doug waiting for his walk, so I was happy to oblige. Some people have Christmas lights up already and its not yet December! When we got back and I got into my comfy pj's it was time to start prepping dinner as we were having spinach and cottage cheese cannelloni which is a firm favourite. The recipe makes so much though that we made three dishes of it so two have gone in the freezer. I was on a bit of a cooking frenzy so I made up two portions of fish pie. I'm not sure if I am going to like it as I'm not keen on fish but it had been in the freezer for so long I figured Id better use it. I used garlic and herb philli and chucked in some mixed veg and mushrooms. I am trying to be prepared for next week as Rich will be doing most of the cooking whilst I am recuperating.
So dinner was gorgeous I even had garlic bread using a WW petit pain for 6 syns.
I fancied something sweet after so I had a bowl of strawberries, blueberries and raspberries with a vanilla and chocolate sprinkles muller. It satisfied my craving and now I am relaxing watching Greys Anatomy.
Hope everyone reading is well tonight :D thanks for getting to this bit of the blog and still reading
2 comments:
Hi. I love reading your blog. It's fab. Can you tell me the recipe for the canneloni please. It looks amazing. Thanks.ReplyDelete
Hope your surgery goes okay and you recover quickly from it xReplies
thanks hun it did go okay now mastering one handed typing ;)Delete
the recipe is as follows
9oz/255g chopped frozen spinach, defrosted
Fry light
8oz/227 fat -free natural cottage cheese
2 egg yokes
2 crushed garlic cloves
4oz/113 g button mushrooms, finely chopped
1 onion, finely chopped
1 red pepper, deseeded and finely chopped
6oz/170g reduced-fat cheddar cheese, grated
small bunch of basil leaves, chopped (reserve some for garnish)
pinch of nutmeg (makes a big difference to the flavour)
salt & pepper
250g pack of dried cannelloni
1lb 8oz.567g passata (ideally with onion & garlic, but I used plain)
Green salad to serve
Extra Easy Free*
Green Free
*Add 5.5 Syns if you are not using the reduced fat Cheddar Cheese and a Healthy Extra
Pre-heat oven to 200c/400F/gas mark 6. Squeeze out the excess water from the spinach and spray a medium sized baking dish with Fry Light
Put the spinach, cottage cheese, egg yokes, garlic, mushrooms, onion, pepper, half the cheese and half the basil in a bowl. Add the nutmeg, season well and mix together. (use a very big bowl!)
The recipe says use a piping bag (or teaspoon) carefully fill the cannelloni tubes with the mixture. I didn't have a piping bag, the teaspoon was too fiddly, so I used my hands. Very messy, but satisfying once done. I promise you, its worth the effort! The kids might enjoy doing this bit for you!
Place the filled tubes in the baking dish in a single layer and pour over the passata making sure all the cannelloni is completely covered.
Sprinkle over the remaining cheese and chopped basil. Cover with foil and cook for 20 minutes. Remove the foil and bake for another 20 minutes, or until the pasta is tender and the top is golden.
Garnish with reserved basil leaves and finish with some black pepper. Divide between four plates and serve with a green salad.


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If you know that you have sin in your life, it is a pretty safe assumption to believe that there is sin in the life of a blogger. Read between the lines and see the reality in the pictures-not in a judgmental, sceptical way but in an honest, soul searching way. Does that make sense? Basically, we have to be discerning even when we read/write blogs.
As Ann Voskamp sometimes signs off-ALL IS GRACE! Despite the sin that saturates our everyday life, God restores joy in the mundane when we repent. We had fun catching minnows and we have a neat record of it on paper-even that is GRACE. Thank you Lord!


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Phone Control Software Computer Programming Experience
Budjetti $750-1500 USD
We are looking for someone who has computer programming and phone software experience. Similar software has already been designed but not with the following features. This software can be found on [url removed, login to view] under Phone Manager 2 and SMS Manager we are alse looking for some kind of similar program but with the following features?
SMS Features:-
Ability to send any sms to any phone number
Ability to write a script with the sms message along with phone number then
have a delay for the next message.
Prefer the sms message to be sent directly from the phone without actually
composing the letters on the phone.
Ability to compose one sms message and send to 100 mobile numbers.
Ability to design a script with 20 unique sms messages and have these mixed
in with the 100 random mobile no.s on the single message.
Ability to design a script to send an sms message and make a voice call
Ability to delete internal sms folder.
Cable type Nokia DLR-3P. Com port
Prefer to use Nokia 6310i but also have the ability to try newer
Ability to have approx 20 cables+phones connected working simultaneously.
Ability to call any number and terminate at any time.
Ability to call a number directly from phone without composing individual
numbers on the screen.
Ability to sound an audible alarm from p.c. if phone freezes or any problems
occur with phone.
Ability to have the phone power on and off.
The current phones we are using at the moment are Nokia 6310i. Please do not hestitate to contact us through GetAFreeLancer
4 freelanceria on tarjonnut keskimäärin %project_bid_stats_avg_sub_26% %project_currencyDetails_sign_sub_27% tähän työhön
i've develop similar project in java. i used siemens c55 with charging data cable. i can develop this for you too. see PM please.
I have done many projects for Blackberry , iPhone , Palm as well as Symbian enabled phones .I have also done a lot of telephony applications dealing with advanced features . I have also developed a lot of other general Lisää


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English Teacher
- Recruiter
- Location
- London (East), London (Greater)
- Salary
- ELSS scale - Equivalent to Main Pay Scale (NQT)
- Posted
- 02 Oct 2017
- Closing date
- 13 Oct 2017
- Industry
- Schools, Secondary teaching, English
- Job level
- Graduate
- Hours
- Full time
- Contract
- Permanent
- Listing type
- Job vacancy
- Education level
- Undergraduate degree (or equivalent)
The East London Science School has entered its fifth year. As our popularity grows, we are keen to recruit exceptional individuals to work with the Principal to develop this unique and exciting school as we look to our first set of GCSE results in 2018.
We are seeking to appoint an English teacher to join our team at the East London Science School in October 2017. Despite this being a science school, English is at the heart of our curriculum and our school day. Reading and writing form a key part of school life: from the morning whole class reading sessions through to drama, poetry recitals, novel projects and debating competitions.
This is an exciting and unique opportunity for an English teacher to be part of a young vibrant team who believe strongly in the power of literature to change lives and empower the pupils in our care. We teach a knowledge rich curriculum with a strong focus on chronology, rhetoric and grammar. In lessons pupils are encouraged to be both critics and creators, with teachers using challenging, high quality texts and advanced questioning to inspire a love of literature. We have also developed a strong Enrichment programme through visits to the British Library, drama workshops, literary walking tours and creative writing sessions.
Our ideal candidate should be an ambitious, confident teacher who is ready to take up the challenge of KS3 and GCSE teaching and shares our ethos of high expectations for all pupils delivered through an academic education. They should be a literary enthusiast with a drive to instil a love of English in the hearts of all our pupils.
Joining our school means joining a team of passionate subject specialists. You will have the chance not only to contribute to the development of the English curriculum but also to contribute to the building of our school as we continue to grow over the years.
Closing date: Friday 13th October 2017
Interview date : Tuesday 17th October 2017
The East London Science School is committed to safeguarding and promoting the welfare of children and applicants must be willing to undergo child protection screening appropriate to the post, including checks with past employers. All successful candidates will be subject to an Enhanced DBS disclosure.
Find us on Twitter: @ELSS_freeschool


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Making change…
Active citizens do many 'things' to improve the lives of others. They contribute to social justice in their community by taking action on issues in order to make a difference. They question the way things are done.
Active citizenship can be demonstrated globally, nationally and locally.
Global examples of active citizen groups working together include international organisations such as Oxfam, The Red Cross and Teachers without Borders.
National groups working for the common good include events such as Clean Up Australia Day, Australia's Biggest Morning Tea and the annual Red Shield Appeal.
Local active citizens may work within a school committee, church group, community organisation such as Lions or with the local council to make change in the immediate community. An example of this would be identifying and advocating to the local council to provide additional facilities such as a new school due to population growth in the area.
Schools further provide authentic opportunities for students to become active citizens. Being involved in the student representative council, taking on a school captain position or participating in peer mentoring programs allow students to develop the aforementioned skills such as uniting, connecting and making change…
Have a look at this video for some further inspiration!


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Made by the lovelies in the Bulletproof Team! @BulletproofV @MadAndrea @PrincessUnicorn @CreeTheOtaku @VKookie47 @SugaOnTop @amobigbang
Well Tae...that's so thoughtful of you! 'I brought ramen and sandwiches!! 'Tae...why ramen??' 'Oh...that's what I could do best' *Tae smiles his box smile*
'Jagi!!! Sorry I was dreaming of you!' *cant stay mad at him for long* 'Its okay Hobi....stop calling me jagi ehh....my feels' *Hobi hugs me* 'Lets have fun on our date!'
'You're pushing it Hobi...stay in your lane!' 'But....I'm your hope!!!'
*spazzes from too much feels* 'Sami, whats wrong?!' 'Sorry its just I always wanted a big bear' 'Jagi your Jimin here will always do things like this!!!' *tackles me with hugs*
0~0 '아니, we could do the Netflix but nothing else!' 'Awww jagi....please!!!!!' *is whining like a kid* 'No!!!!'
'Fetus just because you could drink now in Korea doesn't mean for you to drink the entire bottle!' *Kookie hiccups* 'I wanted to show you how much I could take!' 'Either ways both of us are not even legal age to drink in the U.S!' 'Shhh....no one has to know' *he puts a finger on my lips* 'Aish....'
'Eomma, stay in your lane!' 'You are the only other person that is as pretty as me!' *he chuckles* 'You took my first kiss....' 'Better from me than anyone else!'
'Yeobo!!!!' *Jimin starts running towards me with arms outstretched* 'Oh shit....' 'Did I surprise you??' 'Yes Jimin you did...but I'm glad you're my Valentine' 'Anyways why are you calling me yeobo?' 'Because now we are meant to get married!' 'Aish....already thinking of that!'
@Destiny1419 @RihannaTiaMay @Krin @Christianliu @Kieuseru @Herreravanessa9 @CreeTheOtaku @KellyOConnor @Amberg171997 @TiffanyBibian @Saraortiz2002 @Emealia @MadAndrea @LilySilver @ArmyofKookie @MorganElisabeth @Kpopandkimchi @Sarangseoltang @Karlythepanda66 @Nenegrint14 @mightmuffin @Ercurrent @EWillsea @jojojordy2324 @MelissaGarza @ninjamidori @musicmofo @Eliortiz13 @toitlepark @TracyLynn @MsLoyalHeart @summerblack2 @IsoldaPazo @imiebegay14 @amobigbang


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Watch Full Wild Horses in Best LookNow you can enjoy Wild Horses in HD quality with duration 100 Min and has been aired on 2015-06-05 and MPAA rating is 9.
- Original Title : Wild Horses
- Movie title in your country : Wild Horses
- Year of movie : 2015
- Genres of movie : Crime, Drama,
- Status of movie : Released
- Release date of movie : 2015-06-05
- Companies of movie : Patriot Pictures,
- Countries of movie : United States of America,
- Language of movie : English,
- Durationof movie : 100 Min
- Average vote of movie : 4.2
- Youtube ID of movie : bhaiC3_p8go
- Translation of movie : EN,ES,PT,EL,NL,RO,ZH,TR,RU,FR,
- Actors of movie :Robert Duvall (Scott Briggs), James Franco (Ben Briggs), Josh Hartnett (KC Briggs), Adriana Barraza (Mrs. Davis), Jim Parrack (Deputy Rogers), Luciana Pedraza (Samantha Payne (as Luciana Duvall)), Angie Cepeda (Maria Gonzales), Darien Willardson (Jimmy Davis), Joaquin Jackson (Ranger Jackson), Christian Jensen (Bar Guy), Devon Abner (Johnny Briggs)
Movie summary of Wild Horses :
Best Wild Horses in Best Look with movie summary "Texas Ranger Samantha Payne reopens a 15-year-old missing person case, and uncovers evidence that suggests that the boy was likely murdered on a ranch belonging to wealthy family man, Scott Briggs. When Scott's estranged son unexpectedly returns home during the investigation, Samantha becomes even more convinced that the Briggs family was involved, and will stop at nothing to discover the truth about the boy's death - even putting her own life in jeopardy." in top video format. Watch full Wild Horses in Top Quality by push of the download link.
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Director : Robert Duvall, Writer : Robert Duvall, Co-Writer : Michael Shell, Editor : Cary Gries, Director of Photography : Barry Markowitz, Original Music Composer : Tim Williams, Casting : Ed Johnston, Production Design : Adam Henderson, Art Direction : Scott Hinckley, Producer : Rob Carliner, Producer : Michael Mendelsohn, Co-Producer : Michael Shell, Co-Producer : Dama Claire, Executive Producer : Mark G. Mathis, Executive Producer : Arnold Rifkin, Line Producer : Clio Tegel, Associate Producer : Natalie Perrotta
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5-9-2008 Ohio:
COLUMBUS, Ohio – A newly proposed sex-offender law soon could include offenders living in nursing homes of other long-term care facilities.
There was a bill within the statehouse Thursday that would require neighbors to be notified of registered sex offenders living in nursing homes, NBC 4's Mike Bowersock reported.
Hundreds of people turned out for the Ohio Health Care Association's annual conference Thursday in Columbus.
On the agenda were speakers and presentations about the skilled nursing profession.
Not on the agenda was a discussion about House Bill 57 — a bill that if passed would change the way nursing facilities deal with their patients.
"It's a difficult question we're wrestling with," the association's President and CEO Peter Van Runkle said.
The difficult question? How to deal with registered sex offenders living in nursing homes?
There currently is no state law that requires residents in the homes as well as neighbors nearby to be notified about who may be living in those types of facilities.
"It kind of freaks us out a little bit, but at the same time they've paid their debt to society already," Jay Smalley said. Smalley talked with NBC 4 last year. At the time, he lived across the street from a nursing home that housed 19 sex offenders.
The bill will move from the House to the Senate and could be passed within the next month. ..more.. by Ashley Brown


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Starring Ryan Reynolds, Mark Strong, Peter Sarsgaard, Blake Lively, and the voice talents of Geoffrey Rush and Michael Clarke Duncan
Directed by Martin Campbell
Release Date:
June 17th, 2011
So far,as of June 17th 2011, www. rottentomatoes.com has accumulated an average score of 24% favorable reviews for Green Lantern, starring Ryan Reynolds, Blake Lively, Peter Sarsgaard, Mark Strong, and the voice talents of Geoffrey Rush and Michael Clarke Duncan. A lot of the reviews have claimed that the movie is a bore, that nothing is accomplished and that the "wow" factor just isn't there. We'll take a look at both the good and bad of this DC Comics adaptation.
First, the good: the movie certainly has a lot of fun. For all my own speculation and criticism, Ryan Reynolds certainly captured the cockiness and attitude that bring out the courage and heroism in Hal Jordan. Reynolds certainly brings a certain swagger and charm to Hal, and a need to be vulnerable when needed. Then there's all the CGI: 90% of it is good (the other 10....we'll talk about later). From the GL uniforms to their green constructed objects, the sights in this movie are certainly full of life and energy. One aspect I enjoyed was how the uniforms showed off a wave of energy constantly moving, a nice touch that shows that the "uniforms" are more a second skin than a latex suit.
Second, the bad: the CGI on Hal's mask, the cheesy dialogue, and a few plot holes or events that needed more explanation. Hal's mask....the movie makes a good explanation, but it still looks awful and putting the green CGI on the bridge of his nose was a bad idea. Some of the dialogue involving the characters drawing attention that they're in a comic-book movie is a little ridiculous, but I only mention it as this type of pros could have been eliminated in the revision stage of the screenplay and have made better dialogue. Then there's some plot holes; I won't mention what occurs, but you'll think about what happens and say "How did he know where _____ was?".
Still, for all its' faults, Green Lantern is an enjoyable comic-book film. Not as good as X-Men: First Class or Thor, but still entertaining.
Rating: * * * 1/2 of 5


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By Benjamin Madley
among 1846 and 1873, California's Indian inhabitants plunged from might be 150,000 to 30,000. Benjamin Madley is the 1st historian to discover the whole volume of the slaughter, the involvement of nation and federal officers, the taxpayer cash that supported the violence, indigenous resistance, who did the killing, and why the killings ended. This deeply researched publication is a accomplished and chilling historical past of an American genocide.
Madley describes pre-contact California and precursors to the genocide ahead of explaining how the Gold Rush stirred vigilante violence opposed to California Indians. He narrates the increase of a state-sanctioned killing desktop and the extensive societal, judicial, and political aid for genocide. Many participated: vigilantes, volunteer nation militiamen, U.S. military squaddies, U.S. congressmen, California governors, and others. The nation and federal governments spent not less than $1,700,000 on campaigns opposed to California Indians. along with comparing executive officers' culpability, Madley considers why the slaughter constituted genocide and the way different attainable genocides inside and past the Americas may be investigated utilizing the equipment awarded during this groundbreaking book.
Read or Download An American Genocide: The United States and the California Indian Catastrophe, 1846-1873 (The Lamar Series in Western History) PDF
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Extra resources for An American Genocide: The United States and the California Indian Catastrophe, 1846-1873 (The Lamar Series in Western History)
Example text
Yet Russians also seized and held California Indians as captive laborers. " The following year, California's Mexican governor, José Figueroa, visited another Russian ranch California Indians before 1846 37 From 1812 to 1841, Fort Ross was the southernmost outpost of Russia's North American empire. This Northern California colony was also a site to which Russians brought Pomo and Miwok people to work as unfree laborers. Duhaut- Cilly, "Vue de l'etablissement russe de la Bodega, à la Côte de la Nouvelle Albion, en 1828 [View of the Russian establishment of the Bodega, on the coast of New Albion, in 1828]," lithograph by Landais and Martenelle in A.
Such atrocities may also have been driven by genocidal impulses. 47 In sum, the period between the secularization of the missions in the 1830s and 1846 was harrowing and catastrophic for many California Indian peoples. In combination with the mass death of the mission period, devastating epidemics and violence dramatically diminished California Indian populations in the 1830s and early 1840s, destabilized many of their social structures, wreaked havoc on multiple indigenous economies, and critically weakened many California Indian peoples' ability to resist the invasion and violence that began in 1846.
In sum, according to Cook, if the "acute epidemics" of the 1830s killed roughly 60,000 California Indians "another 40,000 . . 46 Violence also took many California Indian lives under Mexican rule, albeit on a much smaller scale than did disease. In the 1820s, 1830s, and early 1840s, Mexicans killed hundreds of California Indians in battles and massacres. For example, in 1826 Lieutenant Juan Ibarra's men killed at least thirty- eight 40 California Indians before 1846 Indians—likely Quechans or Yumas—near Santa Ysabel in Southern California before sending their severed ears to Ibarra's commander.


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Cayden's Road to Rehabilitation
Meet Cayden,
Isn't he handsome, his personality just blows me away! Cayden came to us through the Great Dane Rescue of Southeast Texas, they are a great rescue organization and we couldn't be more proud to be working with them.
We do not know much about Cayden's past, but we do know he came from a family with 8 children, and from the looks of him, he put them first. Go ahead say it with me, WOW 8 children! Let me tell you, Cayden LOVES children, loves company, and loves to play. This big guy has a personality that will brighten anyones day!
Besides needing to put on a bit of weight and a few training tips he is staying with us until he finds a home, and no better place to stay than a facility designed around a Great Dane right!
To aid in putting on some weight we are currently feeding him Fromm Gold 3 cups, 4 meals a day with a few treats in between ;0). He goes on several walks a day with different staff members and gets lots of individual of play time to keep him social.
We are excited to see his changes too, so check back for weekly updates on Cayden's road to rehabilitation.


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The first video we previewed was "The Basic Math Word Problem Tutor." This video is 8 hours long with 15 sections and builds on grade 1 to 7 basic math. The word problems are more simple in the beginning and progressively get more difficult.The early segments of the video could easily be used by 3rd to 5th grade students needing help with addition, subtraction, multiplication and division word problems. The teacher uses a white board for the demonstration of the problems. The video was easy to understand and enjoyable as well.
Concepts covered include word problems with basic math and then introduces word problems with decimals, fractions, percents and ratios. The skills taught on this video help children tackle word problems more effectively which also helps to build reasoning and logic skills.
The second video we previewed was "The Algebra 2 Tutor." This DVD has 6 hours of video with 15 sections. This DVD was previewed by the men in my family that have done advanced math. They said he was interesting to listen to, the videos were very simple to follow, and understand, and kept their attention. My 17 year old said they were done very well. Examples of things covered on this video are the quadratic formula, simplifying radical expressions, and graphing inequalities.


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When one struggles continually with undocumented original film material it's a welcome change to be in a psition to positively identify a location which is indisputable as is the case with this admirable portrait of ex-GNR Ivatt D2 class 4-4-0 no. 4377 depicted alongside Potters Bar signal box. the date is more difficult to determine as the accompanying scribble stated "1930's" which is very dubious. This negative was amongst a batch of Potters Bar shots obviously taken by one photographer and probably all within a short space of time and one inclusion which will be uploaded in the near future (as at 26/2/13) features a NLR 4-4-0T which was scrapped in either 1927 or 1928 and I think that was the reason why the photographer was at that location.
The engine featured here was built at Doncaster with works no. 918 in 1901 and entered service as fleet no. 1377 but what I've found interesting is that the 100 year old table of data that I'm referring to describes this as the 'V3' class rather than 'D2'.


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We lived our villa, but our favorite thing about staying in Sunset Village was the Sunset pool, which was open to ky Sunset Villge guests. Because of this, it was more private and quiet than the other pools. Also know that each resident has something different in the backyard, be it the Sunset Pool, a tree and rope swing, a private pool, or some other view or activity.
- Free Wifi
- Free Parking


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For our public Grand Opening which will be coordinated with Robson's Marketing Department in conjunction with their opening of the 3 new models, our club will be hosting other clubs and their members. We will need volunteers to help coordinate at each set of 4 courts minimum to show what courts people have been assigned to play and to fill in if a group is short a player or two to make a foursome. I would love to have at least 32 members to help with the Grand Opening, but will welcome even more since the Grand Opening will be from 11-4.
A few teachers might be needed if someone happens to visit who doesn't know how to play. Court times will be assigned for visitors by sign in time and level of play. Please sign up at the courts or email Nancy.


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Thursday, February 18, 2010
1.21 Workday
I have been so excited to see how the future home of 1.21 church at 903 N. Cherry Street has been restored over the past months. There have been quite a few people who have put endless hours into getting the building ready to pass code, painting, construction work, cleaning up and it really looks great! This past weekend was one of the final, if not the final work day in the new building, and Craig along with other men from our church showed up to help. Speirs and I delivered lunch and one of our friends Daniel, who is a student at the School of the Arts got some really cute shots of Speirs. I am blessed to have friends with such talent!
Posted by Britt West at 1:31 PM
1 comment:
- Maria said...
Great pics, I like the second one, adorable expression and the one with his dad is priceless.
- February 28, 2010 at 8:38 PM
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It was key to this whole series. They needed this win. And they got it. Cole Hamels pitched a flawless game, except for the first inning when Atlanta scored all 4 of their runs. After that only 2 batters he faced made it on base - one on a walk and another on Wes Helms' fielding error. The Phillies have taken game 1 of the crucial 3 game set. Tomorrow Adam Eaton faces Lance Cormier. Earlier I declared that the Phils would take games 1 & 3 but lose 2. We'll find out tomorrow night. Elsewhere, the Marlins got to Billy Wagner in the 9th tonight and scored 2 runs to pull ahead of the Mets. They hung on and, as a result, 1st through 3rd in the NL East is separated by a mere 3.5 games. And if the Padres - currently tied with the Reds after 9 - lose, the Phillies are tied for the wild card lead!
Friday, August 10, 2007
Phillies take Game 1 versus Braves
Posted by J Mays at 10:41 PM Labels: Adam Eaton, Cole Hamels, J Mays, Phillies, Wes Helms
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Aftre doing the whole Home For The Holidays thing for a few days, it was time to start heading back West. If I was being totally practical, I would have gone back the way I came, taking the Southwest Chief directly back to LA. Always leery of excessive practicality, I decided to instead go back to LA via San Francisco. This was partly just to avoid taking the same route twice, but was mostly due to the fact that Amtrak's Chicago – San Francisco train is called The California Zephyr. I'll be honest, the phrase "California Zephyr" is probably what sold me on going Amtrak to begin with. I'm a sucker for cool place names, and apparently, cool train names as well.
Posts Tagged 'Chicago'
Amtrak for the Holidays, Part 2February 2, 2012
Amtrak for the Holidays, Part 1January 28, 2012
As Christmas approached, I was faced with the question of whether I could/should go home for the holidays. Having recently moved from Ohio to LA, this was substantially more troublesome all the sudden. "Going home" used to entail a short hop down 75 followed by some state routes, etc. About 180 miles in total. I was now looking at over 2,200. I took a lot of math classes in high school, all of which told me that 2,200 is more that 180.
I weighed the costs and benefits of making the trip (i.e. dithered), looking at expense, timetables, etc. Eventually, I decided that I had time and money enough to make the trip. Now that the if was answered, it was time to answer the how. By this point, it was already early December, and plane tickets would have run over $1400. Out of the question. A cross country Greyhound trip on a tight schedule, while something everyone should do once, wasn't something I was looking forward too after the last time (subject of another post perhaps). I thought about driving, but I've kind of been there and done that. It finally occurred to me that we have this thing in America called Amtrak.


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try replacing the battery for both. make sure you are not blocking the receiver - if possible make sure there is a line of sight between the mouse/keyboard and receiver.
I can't tell you how disappointed I am with this computer. On day one I noticed that the option to change screen brightness is not available. So I had to download HP My Display to be able to adjust it. But this is not the biggest problem. After a week the wireless mouse started behaving weirdly. It started to lag and move very slowly. Keyboard was working properly. Than the next day I turn on the computer and the keyboard is not working. The only 3 button that work on it are mute, + sound and - sound. I restored my computer to earlier time and keyboard started working again. After I restarted computer the same problem appeared again. Only 3 buttons on keyboard work. I don't know what the problem is. Maybe someone can help.


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Handmade Baby Photography Props Crochet Hats Cocoons and More for Moms and Photographers Too
A Special Thanks
I just received a beautiful photograph using one of my photography props and I must say the picture and the model are just beautiful and really makes my Sweet Pea Cocoon look great. With that said I'd like to give a special thanks to Kel Ward Photography for making my Cocoon look awesome. You take great Photographs Kel Ward...Keep it up
Posted by Kathy Neilsen Designs
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