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Local apoptotic-like mechanisms underlie complement-mediated synaptic pruning
Edited by Charles F. Stevens, Salk Institute for Biological Studies, La Jolla, CA, and approved May 9, 2018 (received for review December 29, 2017)
Synaptic pruning is dominant in early ontogenesis when a large number of unnecessary synapses are eliminated, and it maintains synaptic plasticity in the mature healthy brain, e.g., in memory processes. Its malfunction is involved in degenerative diseases such as Alzheimer's disease. C1q, a member of the immune complement system, plays a central role in the selective pruning of synapses by microglial phagocytosis. Understanding the molecular aspects of complement-mediated synapse elimination is of high importance for developing effective therapeutic interventions in the future. Our analysis on C1q-tagged synaptosomes revealed that C1q label-based synaptic pruning is linked to local apoptotic-like processes in synapses.
C1q, a member of the immune complement cascade, is implicated in the selective pruning of synapses by microglial phagocytosis. C1q-mediated synapse elimination has been shown to occur during brain development, while increased activation and complement-dependent synapse loss is observed in neurodegenerative diseases. However, the molecular mechanisms underlying C1q-controlled synaptic pruning are mostly unknown. This study addresses distortions in the synaptic proteome leading to C1q-tagged synapses. Our data demonstrated the preferential localization of C1q to the presynapse. Proteomic investigation and pathway analysis of C1q-tagged synaptosomes revealed the presence of apoptotic-like processes in C1q-tagged synapses, which was confirmed experimentally with apoptosis markers. Moreover, the induction of synaptic apoptotic-like mechanisms in a model of sensory deprivation-induced synaptic depression led to elevated C1q levels. Our results unveiled that C1q label-based synaptic pruning is triggered by and directly linked to apoptotic-like processes in the synaptic compartment.
The complement cascade, as part of the innate immune system, plays a role in the recognition and removal of invading pathogens and stimulating further components of the immune system. It has also been shown that certain complement system molecules are constitutively expressed in diseased and healthy brain (1). Specifically, C1q, the first component of the classical complement pathway, C3, and complement receptor 3 participate in postnatal synaptic development and pruning (2). Schafer et al. (3) reported the role of microglia cells in synapse elimination in the developing murine visual system in a synaptic activity-dependent and complement-mediated manner. Stevens et al. (4) showed the synaptic localization of C1q and its expression in a period of postnatal development of the retina and brain, and they demonstrated that C1q is the pruning-initiator label on certain synapses. Erroneous synapse remodeling was observed in C1q knock-out mice (4). Stephan et al. (5) showed that correlating with cognitive decline, the C1q level dramatically increases during normal aging of mouse and human brain and that C1q is localized in close proximity to synapses. Most recently, participation of C1q-mediated synaptic pruning was described in early-stage synapse loss in animal models of Alzheimer's disease (6) and in frontotemporal dementia (7).
It is well-known that synaptic turnover can occur at high rates even in adulthood (8) and that synaptic C1q, as a molecular "tag," mediates the selective pruning of unnecessary synapses by the adjacent microglia (6, 7). However, the molecular changes attracting the C1q tag to a synapse are still unknown. In the present work, we addressed the question of what kind of specific synaptic proteome alterations could induce the C1q tagging of a synapse. We identified the subsynaptic localization of C1q and elaborated a technique of separating C1q-labeled murine synaptosomes by fluorescence-activated cell sorting (FACS). We applied 2D differential gel electrophoresis (2D-DIGE) and mass spectrometry (MS) for quantitative synaptic proteome change discovery. Our results revealed the significance of apoptotic-like mechanisms in the background of synaptic C1q tagging, which was verified experimentally. To our knowledge, this is a unique report on the direct link between synaptic local apoptotic-like processes and C1q-mediated synaptic pruning mechanisms.
Presence of C1q in Synaptic and Subsynaptic Fractions—Preferential Recognition of the Presynapse by C1q.
This study was critically dependent on the appropriate purification of synaptosomes from the cerebral cortices of mice. Visual analysis of electron micrographs of the samples demonstrated that ∼87% of the particles were synaptosomes (SI Appendix, Fig. S1 A–C). We also investigated the enrichment of well-known synaptic protein markers in the cortical synaptosome fraction in comparison with whole cortical tissue homogenate. Our results demonstrated prominent enrichment of the postsynaptic marker postsynaptic density protein 95 (Psd95) and a significantly elevated level of the presynaptic synaptophysin (Syp) in the fraction of synaptosomes (SI Appendix, Fig. S1D). Synaptosomes were visible via differential interference contrast (DIC) light microscopy, as they formed large aggregates in PBS medium (SI Appendix, Fig. S1E). Confocal microscopy experiments also confirmed sample purity by labeling the synapse-specific Syp protein (SI Appendix, Fig. S1E).
Another crucial element was the demonstration of the presence of C1q protein in the synaptosome fraction. Our immunoblotting experiments indicated the presence of C1q in the whole cerebral cortex tissue samples. Moreover, C1q was localized to the cerebral cortical synapses of healthy adult mice (SI Appendix, Fig. S1F). In addition, the presence of brain-derived, and particularly synapse-attached C1q in transcardially perfused mice without complement contamination from the blood confirmed our observations on synaptosomes. Individual C1q-tagged synaptosomes have also been observed in sucrose/EDTA/Tris (SET) buffer using confocal microscopy (Fig. 1A). C1q and Syp were clearly colocalized (Fig. 1A), which is in accordance with previous data (5). These results confirmed the presence of a pool of secreted C1q molecules that directly bind to synapses in the adult brain.
C1q has been observed previously in colocalization with and in close vicinity of pre- and postsynaptic molecular markers using different techniques (4, 5). To estimate precisely the distribution of C1q between the two sides of synapses, we fractionated pre- and postsynaptic membranes (PRE and PSD, respectively) from the synaptosomes. Western blot analysis (Fig. 1B) verified the enrichment of the mostly presynapse-derived Syp in the PRE fraction and the postsynapse-marker Psd95 in the PSD fraction (Fig. 1C). Our results revealed that C1q is predominantly presynaptically bound (PSD/PRE C1q levels = 0.26 ± 0.08, mean ± SEM; P = 0.0013; Fig. 1C). These results suggest that C1q-dependent elimination of synapses originates from the presynaptic compartment in the cerebral cortex.
Sorting of C1q-Tagged Synaptosomes.
Analysis (and sorting) of synaptosomes using flow cytometry has been conducted by few groups before (9⇓–11). However, experimental designs varied considerably between the studies (e.g., detection of fluorescence by antibody-based method or by the presence of transgenically engineered fluorescent protein); thus, we had to optimize the method for our purposes. In contrast to the previous practice, we used SET buffer instead of PBS throughout the immunolabeling of synaptosomes to prevent their aggregation (12) (Fig. 1A). Synaptosome samples were filtered through a 5-µm pore size membrane to remove large contaminants and aggregated synaptosomes. This step has been successfully used for synaptosome purification previously (13). This protocol enabled the analysis of individual synaptosomes in FACS experiments (SI Appendix, Fig. S2A). Immunolabeled synaptosomes formed a homogeneous population by size and inner complexity in flow cytometry analysis (SI Appendix, Fig. S2B). The diameter of the synaptosomes was in the range of 0.5–1 µm according to our tests with differently sized flow cytometry calibration beads, which is in accordance with previously published results (9). We excluded the possibility that detection of cellular debris could compromise our observations in flow cytometry experiments because the detected particles could almost be completely labeled with the viability dye calcein-acetoxymethyl (AM) ester (SI Appendix, Fig. S2C). In addition, electron microscopy examinations revealed intact synaptosomes after the sorting procedure (SI Appendix, Fig. S2D). Gating of fluorescently labeled synaptosomes was performed strictly eliminating the false positives based on the histogram of the negative control samples (Fig. 2A). Summarizing our flow cytometry data, 15.60 ± 3.03% (mean ± SEM) of the synaptosomes were labeled with anti-C1qA antibody (SI Appendix, Fig. S3). The specificity of the antibody in this experiment was verified in a parallel investigation, as we detected a similar percentage of C1q-positive synaptosomes using an alternative, highly specific, monoclonal anti-C1q antibody (5) (SI Appendix, Fig. S3). In addition, we also assessed the percentage of C1q-positive synaptosomes isolated from the cerebral cortices of newborn mice [at postnatal day 5 (P5)] and observed a lower amount of C1q-positive synaptosomes in comparison with the results obtained using adult mice (SI Appendix, Fig. S3), which is in accordance with the already described notable increase in C1q expression during aging (5). Western blot analysis also verified the reliability of sorting purity via the successful detection of C1q-signal from C1q-tagged synaptosome homogenates and the lack of a detectable signal from the untagged ones (Fig. 2B). In conclusion, immunolabeling of the complement tag of synapses and application of the FACS methodology provided sorted synaptosome samples suitable for characterizing the molecular composition of C1q-tagged synapses.
An Altered Proteome Characterizes Synapses Tagged with C1q.
Proteomics experiments were performed on 2 million C1q-tagged and 2 million untagged sorted synaptosomes. The 2D-DIGE proteomics investigation utilized gel-based protein separation and protein abundance quantification coupled with protein identification via MS. These experiments were carried out on six independent parallels (six mice) resulting in 410 quantifiable spots of different proteins (the criterion was to be comparable on at least five samples out of six). Out of these spots, 25 exhibited significant intensity differences between tagged and untagged samples. Reliable protein identification was possible for 19 protein spots (SI Appendix, Table S1). Ten spots exhibited significantly lower levels and 9 spots indicated significantly higher levels in C1q-tagged synaptosomes in comparison with untagged ones. Fold changes were between −1.23 to −1.6 and 1.32–2.24 (Fig. 3A and SI Appendix, Fig. S4). Altogether, we have identified 18 proteins with different abundance between the two sorted synaptosome subpopulations using MS (Fig. 3A and SI Appendix, Fig. S4).
The proteins with altered levels were assigned into functional groups according to their roles in physiological cellular mechanisms (Fig. 3B). Our results revealed that many aspects of synaptic and general cellular functions are different between the two synaptic subpopulations. Affected proteins are directly linked to synaptic transmission (Cplx1, Nptx1, Sncb, and Syn2), carbohydrate and energy metabolism (Aco2, Aldoa, Atp5a1, and Eno1), signal transduction (Gnb1, Pitpna, and Ppp3r1), the cytoskeleton (Actb and Tmod2), protein degradation (Psma5 and Uchl1), oxidative stress response (Prdx6), and ion transport (Vdac1), while the cellular function of Phpt1 is not well-characterized. Taken together, our data suggest that disturbances in several cellular mechanisms are present in synapses assigned for complement-mediated phagocytosis.
We have revealed decreased neuronal pentraxin 1 (Nptx1) in C1q-tagged synapses. Proteins of the pentraxin family are regulators of complement in the periphery, e.g., pentraxin-3 is an interacting partner of C1q (14). Nptx1, Nptx2, and neuronal pentraxin receptor are implicated in synaptic transmission and plasticity, e.g., participating in the trafficking of AMPA receptors (15). They are involved in developmental synaptic maturation (16) and in the refinement of the visual system (17). Nptx1 and Nptx2 were hypothesized to be binding partners of C1q (4, 18), but these data need to be confirmed. Interestingly, we have found a decreased level of Nptx1 in C1q-tagged synapses in one spot in the 2D-DIGE experiment. However, using 2D immunoblotting, we have identified several posttranslationally modified forms (SI Appendix, Fig. S5). The difference in their isoelectric points possibly indicates phosphorylation as modification. These data suggest that posttranslational modification of Nptx1 could be an important factor in C1q tagging or untagging of a synapse. Considering the predicted significance of neuronal pentraxins in complement regulation and their importance in synaptic transmission and synaptic pruning, these data have to be deciphered in the future.
Local Apoptotic-Like Mechanisms Are Linked to Synaptic C1q Tagging.
To reveal the regulatory mechanisms that are responsible for the molecular disturbances in C1q-tagged synapses, bioinformatics analyses were conducted using the Pathway Studio knowledge tool (Elsevier Life Science Solutions). The investigation involved the identification of possible regulatory components (i.e., small molecules, proteins, molecular complexes, and cellular processes) downstream of the altered proteins. Surprisingly, the second highest-ranked term after "synaptic plasticity" was "apoptosis" among the possible downstream elements regulated by the identified proteins (SI Appendix, Fig. S6 and Dataset S1A). Investigating the literature, it was revealed that 15 of the 18 proteins were reported to be involved in apoptotic processes, particularly Vdac1, Prdx6, Uchl1, Eno1, Ppp3r1, and Nptx1 (for the list of references, see Dataset S1B). These results suggested that synaptic C1q tagging plays a role in the recognition of synapses in which local apoptotic-like processes have been initiated. Regarding the possible upstream elements, the highest-ranked term was "Ca2+" (SI Appendix, Fig. S6 and Dataset S1 A and B). This finding also strengthens the possibility that a limited synaptic apoptotic-like mechanism characterizes C1q-tagged synapses, in light of the fact that local calcium transients in the dendritic arborization can activate apoptotic molecular mechanisms ultimately leading to dendrite pruning (19).
The coexistence of apoptotic-like mechanisms and C1q tagging in the synapse required experimental verification. Flow cytometry experiments were conducted to define the colocalization of C1q with apoptotic markers, cleaved (or active) caspase-3 and annexin V. Caspase-3, once it becomes activated by cleavage, is a central player in the execution of apoptosis (20). Moreover, in the brain, caspase-3 is present at its second highest level in the subcellular compartment of nerve endings after the cytosol (21). Annexin V selectively recognizes externalized phosphatidylserine, a phospholipid translocated from the intracellular to the extracellular side of the plasma membrane during apoptosis; thus, it is frequently used in apoptosis detection assays (22). The majority of C1q-labeled synaptosomes exhibit the molecular signs of apoptotic-like mechanisms in comparison with nonlabeled ones, as revealed by double labeling for C1q and cleaved caspase-3 (Fig. 4 A and B) and for C1q and annexin V (Fig. 4 C and D). In contrast, C1q-untagged synaptosomes were less likely to show the molecular signs of apoptotic processes (Fig. 4 A–D). Interestingly, we found a moderate-to-strong positive correlation between synaptic cleaved caspase-3 and C1q levels (R = 0.61 ± 0.01; mean ± SEM; Pearson correlation coefficient; n = 4 independent experiments). We also have to note that it is highly probable that the amount of C1q- and annexin V-double-positive synaptosomes was even underestimated. This assumption is based on the fact that besides annexin V, C1q is also reported to bind phosphatidylserine, but with lower affinity (23). Therefore, annexin V could dissociate C1q from its common binding partner at the surface of synaptosomes. This hypothesis is also supported by our observation that anti-C1qA labeling without the addition of annexin V resulted in a remarkably increased number of C1q-positive synaptosomes (15.60 ± 3.03%, mean ± SEM) relative to after double labeling (3.71 ± 0.71%). These results raise the possibility that phosphatidylserine, a nonprotein apoptotic marker, could be an important binding partner of C1q.
To confirm our observations with an independent method, we performed immunostaining of brain sections for C1q, cleaved caspase-3, and Syp. In agreement with our flow cytometry data, we demonstrated the colocalization of cleaved caspase-3 in C1q-tagged synapses (Fig. 4E and SI Appendix, Fig. S7). Importantly, our translational experiment confirmed the abundant colocalization of C1q, cleaved caspase-3, and Syp in human cerebral cortex (Fig. 4E and SI Appendix, Fig. S8). The activation of the molecular machinery, well known in apoptosis, has already been described under synaptic weakening and long-term depression (as excellently reviewed in ref. 24). To test the hypothesis that synaptic downscaling could result in activation of an apoptotic-like molecular process ending up with C1q tagging of synapses involved, we studied a well-established model of synaptic depression in the barrel cortex (25, 26). In this model, whisker removal induces synaptic weakening and degeneration of synaptic boutons and axonal branches in the sensory deprived primary somatosensory barrel cortex (25, 26). Our investigation revealed the elevation of C1q and cleaved caspase-3 levels in homogenates of synaptosomes isolated from affected barrel cortices in comparison with unaffected ones (Fig. 5). Moreover, the level of synaptic vesicle-localized Syp decreased (Fig. 5), which is in accordance with the previously reported presynaptic dysfunction in this model (25). In conclusion, our data strongly suggest that the selective C1q tagging of synapses depends on the occurrence of local apoptotic-like processes.
Synaptic pruning is a physiological process necessary in early ontogenesis to shape functional neuronal circuits (27) and to promote synaptic plasticity in the mature central nervous system as it maintains a kinetic balance in synapse turnover (28). However, unbalanced synaptic turnover shifted toward pruning is a hallmark of neurodegenerative diseases, e.g., Alzheimer's disease (29), and schizophrenia as well (30). The classical pathway of the complement system has been repeatedly implicated in the process of synaptic pruning since the pioneering study of Stevens et al. (4). A detrimental role of the excessive activation of C1q and C3 (6), and other downstream complement components (31, 32) leading to synapse and/or neuronal loss has been identified in neurodegenerative pathology. Despite the significance of complement-mediated synapse elimination in health and disease, knowledge about the molecular changes leading to C1q-tagged synapses is limited.
Our investigations were performed on synaptosomes containing both the pre- and postsynaptic neuronal elements, which can be compartmentalized, and the localization of C1q can be examined (Fig. 1A and SI Appendix, Fig. S1). The presence of C1q attached to individual synaptosomes is in accordance with the results of previous studies that revealed detectable C1q levels in the brains of healthy adult rodents and of human subjects as well (5, 6). Separation of the two sides of the synapse unveiled that the C1q tag is dominantly localized to the presynaptic part of the labeled synapse (Fig. 1 B and C). Therefore, complement-mediated synaptic pruning might be directly initiated by presynaptic processes. This finding is supported by the fact that microglia engulf specific presynaptic elements (3). Although several interacting partners of C1q are known (33), they were identified in the periphery, not in the central nervous system. Our data suggest that it is worth searching for presynapse-specific interacting partners of C1q in the future.
In this study, we have combined FACS with proteomics techniques to reveal synapse-specific protein alterations in the subset of synapses that are assigned for elimination via the local complement system. The experimental procedure enabled the multiparametric analysis of individual synaptosomes (Fig. 2A and SI Appendix, Fig. S2) and the pure separation of C1q-tagged ones (Fig. 2B). Synaptic proteins of altered abundance in the C1q-tagged synapses (Fig. 3A and SI Appendix, Fig. S4) were assigned to functional groups (Fig. 3B), which indicated impaired cellular functions. One of the interesting findings was that our data suggest a general down-regulation of synaptic transmission. The low level of synaptic transmission in the C1q-tagged group of synapses is in accordance with the hypothesis that lower synaptic strength predisposes to complement-dependent microglial synaptic phagocytosis (34). Specifically, disturbances in synaptic vesicle recycling could be noted because major participants in this cellular process, such as Sncb (35), Syn2 (36), and Cplx1 (37), were affected in C1q-tagged synapses. Moreover, we have revealed a different level of Nptx1, a potential C1q-binding protein based on its homology to the well-known C1q-interacting peripheral pentraxins. Nptx1 could be present intracellularly, exhibiting marked proapoptotic function (38), and in a secreted form, is attached to the synaptic surface (15); therefore, the mechanistic rules underlying its trafficking have to be uncovered in future studies.
The functions of altered proteins suggested several interesting possibilities for cellular processes taking place in a C1q-tagged synapse. Applying knowledge-based tools to proteomic changes provided a better understanding of the general mechanisms underlying selective C1q tagging and synaptic pruning. This analysis noted apoptosis with a high rank among all of the possible downstream entities regulated by the proteins identified in our proteomics study (SI Appendix, Fig. S6). To verify the presence of apoptotic-like mechanisms in the C1q-tagged synapses, we investigated the colocalization of apoptosis markers, either cleaved caspase-3 or annexin V, with C1q. It turned out that double-labeled synaptosomes were greatly enriched in the C1q-tagged fraction of synapses (Fig. 4 A–D). Furthermore, the positive correlation between synaptosomal C1q and cleaved caspase-3 amounts suggests that the extent of caspase-3 activation influences the level of synaptic C1q deposition, thereby affecting synaptic pruning. The abundant presence of both cleaved caspase-3 and C1q double-positive synapses was also verified with immunohistochemistry and confirmed on human brain sections as well (Fig. 4E). Based on our proteomic data, we also propose that the initiation of local apoptotic-like mechanisms might be linked to synaptic calcium ion influx because calcium ion is a common regulator of an outstanding proportion of altered proteins and of apoptosis itself (SI Appendix, Fig. S6). The elevation of the local calcium ion level is already known to be involved in dendrite pruning in Drosophila sensory neurons (19).
The phenomenon of local or limited apoptosis at the level of individual synapses was reported by previous studies (39, 40) (and also termed "synaptosis") (41), but it has never been linked to C1q tagging. The significance of synaptically localized apoptotic mechanisms has been revealed in brain disorders such as Alzheimer's disease (40) and schizophrenia (42). Notably, a causal relationship was uncovered previously between local caspase-3 activation and synaptic failure in a mouse model of Alzheimer's disease (10). C1q is known as a potent recognition molecule of apoptotic cells in the periphery (43). It has also been described that C1q is able to recognize apoptotic cultured neurons and assign them for microglial clearance (44). Focusing on synapse physiology, it has been demonstrated that synapse weakening relies on local synaptic activation of apoptotic-like processes, with a key role of caspase-3 in it (reviewed in refs. 24 and 45). The activation of this molecular machinery does not necessarily lead to cell death, but has central roles in normal synaptic functions. Hence, local activation of caspase-3 is indispensable in the induction of long-term depression (46) and its spatially restricted activation is tightly controlled to prevent neuronal cell death (39). In a model of lower synaptic activation, we provided additional evidence for the induction of apoptotic molecular machinery, and, importantly, we described a concomitant elevation in C1q level (Fig. 5). These results are in agreement with previously demonstrated phenomena in which increased synaptic activity can suppress intrinsic apoptotic pathways (47), whereas C1q tagging likely affects weak synaptic connections (34). Our current study is unique to our knowledge in demonstrating that local synaptic apoptotic-like mechanisms are correlated with the complement-mediated recognition of synapses for their removal. Therefore, our unique results provide the link between synapse removal and C1q tagging via the identification of synaptic apoptotic-like mechanisms.
Taking together the experimental and bioinformatics results, we propose that synaptic transmission downscaling or any other damaging processes affecting normal synaptic functions could induce apoptotic-like mechanisms in the synaptic region. The initiated cascade triggers the binding of C1q to the presynapse, leading to the selective elimination of the synapse by the adjacent microglia.
For fully detailed methods, please refer to SI Appendix.
Human Samples and Ethics Statement.
Control human cortical tissue was obtained from a 72-y-old male subject, who died from causes not related to any brain disease and had no history of neurological disorders, by autopsy in Saint Borbála Hospital, Department of Pathology, Tatabánya, Hungary. All procedures were approved by the Regional and Institutional Committee of Science and Research Ethics of the Scientific Council of Health [ETT TUKEB 31443/2011/EKU (518/PI/11)] and performed in accordance with the ethical standards of the institutional and/or national research committee and with the Declaration of Helsinki and its amendments or comparable ethical standards. According to Hungarian law (1997 CLIV and 18/1998/XII.27. EÜM Decree), no informed consent is required to study postmortem human tissues.
Preparation of the Whole Tissue Samples, Synaptosomes, and Pre- and Postsynaptic Membrane Fractions.
Adult and newborn (at postnatal day 5) male Crl:NMRI BR mice were used in the experiments. Mouse cortical whole tissue samples were prepared via mechanical homogenization in lysis buffer. Proteins of the samples were acetone precipitated and stored at −80 °C. The fraction of synaptosomes was prepared using a sucrose density gradient centrifugation method, while the separation of pre- and postsynaptic membranes was carried out based on the treatment of synaptic junctions with detergents at different pH.
Immunolabeling of Synaptosomes and Assessment of Apoptotic Processes and Viability.
Synaptosomes were gently fixed and immunolabeled in SET buffer with anti-C1qA primary and appropriate secondary antibodies. When additional labeling of the intracellular Syp and cleaved caspase-3 proteins was conducted, a permeabilization step was applied using 0.2% Tween-20. Annexin V labeling was performed using annexin V-FITC protein, and for viability tests, calcein-AM labeling was applied.
Fluorescence-Activated Cell Sorting.
Flow cytometry was performed on a BD FACSAria III sorter coupled with BD FACSDiva data acquisition and analysis software. The population of synaptosomes was designated based on their FSC and SSC characteristics compared with that of the buffer alone, and further gating of fluorescent and nonfluorescent particles was performed among the members of this population. Fluorescently labeled, C1q-tagged and unlabeled untagged synaptosomes were collected separately and used for the proteomics and validation experiments. To determine the extent of synaptic colocalization of C1q with apoptotic markers, additional flow cytometry experiments were carried out.
Proteomics Experiments.
Two-dimensional-DIGE was conducted to compare the proteomes of sorted C1q-tagged and untagged synaptosomes from six mice. Proteins from 2 to 2 million C1q-tagged and untagged synaptosomes were labeled with Cy5, and the pooled sample (2 to 2 million from each sample) was labeled with Cy3 fluorescent dye. C1q-tagged and untagged synaptosome samples were prepared and run on separate gels together with the same amount of a pooled sample. The fluorescence intensity of the protein spots was detected, and individual spots were identified, matched, and manually verified using the DeCyder 2D Differential Analysis software. For mass spectrometric protein identification, HPLC-MS/MS analysis of the tryptic peptide mixtures was performed using a nanoflow ultra (U)HPLC system coupled to a high-resolution quadrupole time-of-flight mass spectrometer.
Bioinformatics Analysis.
Functional classification of the altered proteins was conducted using the GeneOntology (www.geneontology.org) and UniProt (www.uniprot.org) databases as well as with published literature. Common regulator and common target analyses of the differentially regulated proteins were conducted using the Pathway Studio software.
We thank Péter Kovács (CRU Hungary, Ltd.) for generous financial support for the group at ELTE. This work was supported by the National Research, Development, and Innovation Office of Hungary Grants KTIA_NAP_13-2-2015-0003 (to L.D., P.G., and G.J.), KTIA_NAP_13-2-2014-0017 and 2017-1.2.1-NKP-2017-00002 (to B.A.G., J. Kun, É.B., A.M., and J. Kardos, and funding for STED microscopy), K_120391 (to J. Kun, É.B., J. Kardos, and A.M.), FIEK_16-1-2016-0005 (to Z.B., J. Kun, G.J., K.A.K., and J. Kardos), and VEKOP-2.3.3-15-2016-00007 for confocal microscopy. A.M. is supported by the Bolyai János fellowship of the Hungarian Academy of Sciences. Human tissue was provided by the Department of Pathology of the Saint Borbála Hospital, Tatabánya, Hungary.
↵1K.A.K. and J. Kardos contributed equally to this work.
- ↵2To whom correspondence should be addressed. Email: kardos{at}elte.hu.
Author contributions: B.A.G., Z.B., J.M., L.D., G.J., K.A.K., and J. Kardos designed research; B.A.G., J. Kun, G.T., P.G., V.K., P.S., L.D., and K.A.K. performed research; B.A.G., J. Kun, G.T., É.B., Z.B., P.G., V.K., A.M., J.M., G.J., K.A.K., and J. Kardos analyzed data; and B.A.G., G.J., K.A.K., and J. Kardos wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1722613115/-/DCSupplemental.
- Copyright © 2018 the Author(s). Published by PNAS.
This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
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1966 through the eyes of an Englishman
|Warning Copyright Contact Prev Next Index Home
The BEV Retrospective - 1966.
Topics this year are:
Vietnam War.
In his State of the Union address in January, LBJ left no doubt about his Vietnam policy:
"As the assault [of the NLF] mounted, our choice gradually became clear. We could leave, abandoning South Vietnam to its attackers and to certain conquest, or we could stay and fight beside the people of South Vietnam. We stayed. And we will stay until aggression has stopped."
A few days later, another 8,000 US soldiers arrived in Vietnam. They were joined by more Australian troops in March. BY the end of April the US has 250,000 troops in Vietnam.
The war was not getting any more popular at home. There were many rallies against it, nation wide, in March, and in May, tens of thousands rallied against it at the Washington Monument. The next Day Martin Luther King made his first speech on the subject.
Later that month, a division of the North Vietnamese Army crossed into the south and clashed with a US Marine battalion. The marines were reinforced by most of the 3rd Marine Division, and the largest battle of the war to that date ensued. The NVA were driven back into the north.
In June, the US bombing of the north intensified, with attacks on Hanoi and Haiphong. Starting June 8th, on National Route 13, a major north-south highway in Binh Long Province, two battles were fought by the US 1st Infantry Division both initiated by NLF ambushes. Only superior American artillery and air support prevented a complete disaster.
On Independence Day, North Vietnam declared general mobilization, and 1,300 NVA troops were killed near Con Thien that month.
The following month, D Company, 6th Battalion of the Royal Australian Regiment, met and defeated a Viet Cong force estimated to be 4 times larger, at the Battle of Long Tan in Phuoc Tuy Province. The Australians always were formidable opponents when hard pressed.
By the end of the year, American forces in Vietnam had reached 385,000 men, plus 60,000 sailors offshore. More than 6,000 Americans have been killed during the year, and 30,000 wounded. It was believed that 61,000 Vietcong had been killed, though such casualties were very difficult to pin down. However, the number of NLF troops continued to increase, and was now over 280,000.
The US was also concerned about the communist insurgency in Malaysia, and extracted a promise from the British government that British troops would stay there until the situation was stabilized.
China's Cultural Revolution.
In 1958 the Chinese leader Mao Zedong had initiated a program he called the "Great Leap Forward". This proposed to use collectivisation, and a system of communes, to kick start the Chinese economy into a modern, agriculturalized and industrialized society. It had been an abject failure, and has been credited with the death by starvation of perhaps 40 million people.
As a consequence, Mao had lost a significant proportion of his power within the Communist Party to his rivals Liu Shaoqi and Deng Xiaoping. His 'Great Proletarian Cultural Revolution' was presumably intended to deal with this problem. His plan was to achieve this by the influencing the thoughts and mobilizing the actions of China's youth, to form Red Guards groups around the country. They would set about destroying the 'Four Olds' - Old Customs, Old Culture, Old Habits, and Old Ideas.
On August 8th, Mao, who still had the army behind him, pushed a motion through CCP called its "Decision Concerning the Great Proletarian Cultural Revolution". On the 16th, about 11 million Red Guards came to Beijing to see their leader, then went back to their homes to instigate a reign of terror against all who practiced the old ways.
To add power to their elbow, Mao issued a public notice, which stopped "all police intervention in Red Guard tactics and actions." Any in the police force who defied this notice, were to be labeled "counter-revolutionaries" - an automatic target for the Red Guard.
White Separatists in Rhodesia.
In January, the United Kingdom announced that it would cease all trade with Rhodesia, and in April it asked the UN Security Council for authority to use force to stop oil tankers that violated the embargo. This authority was given.
Not to be put off, Rhodesian security forces killed 7 men in combat; 'Chimurenga', the rebellion, had begun.
In May, African members of the UN Security Council said that the British army should blockade Rhodesia, and in July, Zambia threatened to leave the Commonwealth of Nations because of alleged British peace overtures to Rhodesia. Such demands continued, and in November, 38 African states demanded that the United Kingdom use force against the Rhodesian government.
None of this did much good, Prime Minister Harold Wilson had too much adverse public opinion at home to allow him to use force. In December he met the Rhodesian Prime minister Ian Smith on HMS Tiger in the Mediterranean for negotiations. The results were inconclusive.
Later that month, the UN Security Council approved an oil embargo against Rhodesia, but South Africa refused to join the trade embargo against Rhodesia, which effectively negated its effects since the two countries had a common border.
Toward the end of the month, Wilson withdrew all his previous offers to the Rhodesian government, and announced that he will agree to independence only after the founding of a Black majority government. It was hot air. Ian Smith simply declared that Rhodesia was already a republic.
Politics and Happenings in the US.
In January, the first 'Acid Test' was held at the Fillmore Hotel, San Francisco. These events were LSD parties, and were a marker of the beginning of a transition from a rock-and-roll/beat culture to the time of the 'hippies'. LSD was still legal in the US at this time.
In St. Louis, Missouri, during May, Vice-President Hubert Humphrey attended the dedication of the iconic 'Gateway Arch' - a part of the Jefferson National Expansion Memorial
The rule know as 'Miranda Rights' was effectively imposed in June when the US Supreme Court ruled police must inform suspects of their rights before questioning them.
In October, LBJ signed a bill creating the United States Department of Transportation. its objective was to "Serve the United States by ensuring a fast, safe, efficient, accessible and convenient transportation system that meets our vital national interests and enhances the quality of life of the American people, today and into the future." The extent to which it has succeeded in achieving this is not entirely clear to me.
In November, an amiable looking actor called Ronald Reagan, was elected as republican Governor of California.
Politics and Happenings in the UK.
In March of this year, in a well publicized interview published in The London Evening Standard, John Lennon was quoted as saying, "We're more popular than Jesus now." The remark was quoted out of context by an American teen magazine, and caused an outcry in the US bible belt. Their records were banned on some radio stations, and live performances were cancelled. Partly as a result of this, the Beatles gave up touring.
The same month, a bomb placed by ex-members of the destroyed Nelson's Pillar in Dublin. An organization called the Ulster Volunteer Force was set up in Northern Ireland this same year, and it's possible that the two events may not have been just coincidental. The UVF killed two catholic men in West Belfast in the June of this year, signaling the start of 'The Troubles' in Northern Ireland.
Harold Wilson called for an election this year because he had a very tight majority in parliament - 4 seats, and because he had some grounds for optimism. The election was held on March 31st, and the Labour party won handsomely, giving Wilson a majority of 96.
On October 21st, the spoil heap from a coal mine in Aberfan, in South Wales, was subject to a land slip which demolished a school, some cottages, and a farm. The slip killed 144 people, 116 of who were children, buried by the debris.
Other Significant World Events.
On January 10th, peace negotiations between India and Pakistan reached a successful conclusion.
Later in the month, Indira Gandhi was elected as Prime Minister of India when the then Prime Minister Shastri died. She was sworn in on the 24th. She became the second woman prime minister after Sirimavo Bandaranaike of Sri Lanka, who became prime minister of that country in 1960.
She proceeded to visit Washington in March, and then Moscow in July.
There were a number of skirmishes between Israeli and Syrian forces during this year, some about water rights. Then in November, the discomfort of the Israelis was increased when the Syrians entered into a joint defence pact with Egypt.
The space effort continued apace.
In February, the unmanned Soviet Luna 9 spacecraft made the first controlled landing on the Moon. Later in the month, the Soviet probe Venera 3, launched the tear before, crashed on Venus as intended, becoming the first spacecraft to reach the surface of another planet. In March, Luna 10 was launched, and would become the first space probe to enter orbit around the Moon.
The NASA Gemini program was very active again in this year:
Surveyor 1 was launched in June - the first launch in a program to explore the moon. It would soft-land in Oceanus Procellarum. As contributions to the same general objective, Lunar Orbiter 1 followed in August becoming the first US spacecraft to orbit the moon. Lunar Orbiter 2 followed in November.
In the UK, the worlds first military service VTOL aircraft, the Harrier GR1, was ordered from Hawker Aircraft by the RAF. A regular hovercraft passenger service began across the English Channel - it would run for more than three decades.
In Germany, the first practicable EFI system was developed by Bosch. The D-Jetronic was first used in the VW 1600TL in 1967, so was presumably developed at this time.
Nerdland was quiet, though Martin Richards designed a programming language called BCPL at the University of Cambridge. This was significant because it would be the grandfather of one of the worlds most widely used languages - 'C'. The Richards BCPL compiler was also the first one to generate intermediate code for a 'virtual machine'. To port the language to another computer architecture it was then only necessary to write code that would convert this intermediate code to the native code of the new system. In doing this it also established a precedent for another of the worlds most widely used languages - 'Java'.
Escape from South Yorkshire was now a priority for both of us. We didn't like where we lived, the work was boring, and we had no friends. I started job hunting again in earnest, and after a while, I got an interview with the North Eastern Region Scientific Services Department - SSD- of the CEGB in Leeds. It went OK, and I was offered a job in the Chemistry Division, starting in March.
Although we didn't like the area, there was plenty of local interest in the house now that the estate it was part of was more populated and established than it had been when we moved in. We soon found a buyer. Once we had, Elaine, now in the later stages of pregnancy again, high tailed it up to Yorkshire, and left me to wind things up.
Inflation was low in 1966, but house prices were doing better than inflation, so when the house was sold, we would not have any difficulty with a deposit on another one back in West Yorkshire. We looked around in an area that was suitably positioned for access to both Leeds and Bingley and Shipley. We found one in a small town called Guiseley, which is to the north west of Leeds, and to the north east of shipley. It was on an estate that was established, but also quite new, and there were decent bus services in both directions.
I made my apologies for my short stay at the United Steel Companies, worked out my notice time, then rode with the removers van up to Guiseley. We did not move into the house immediately. The general thinking was that it would be better if we waited until Elaine had the baby. So we all stayed at the Stead's house for a while. My son Richard Charles William (I think Elaine had an attack of Christian name escalation syndrome) was born on the 13th of April - all went well, and in mid May, we moved in to number 16 Silverdale Road. My first memory of the place was that Elaine almost immediately succeeded in blocking the drains by flushing disposable baby nappies down the toilet. I had to open it up and do some shit bailing. Otherwise the house - another semi-detached, with three bedrooms - was fine. We had a policeman living on one side, and a schoolteacher on the other.
It was about five minutes walk from the main road between Leeds and Ilkley, just off a road that cuts across from the Leeds road to the Bradford Road. So you had a five minute walk to get to the bus for either Leeds or Bradford. To go to work, I'd take the walk to the Leeds Road, and catch a bus that would drop me right outside the gates of Kirkstall Power Station. The SSD buildings had been built in the grounds of the station a couple of years before.
The CEGB was a nationalized industry, being considered by the post-war governments as too strategic to be out of government control. As such,it had a research arm copied without too much modification from the civil service research establishments like the AERE at Harwell, and similar ethics. There was a central research laboratory site - CERL - at Leatherhead in Surrey, and other laboratories elsewhere, and then more recently, the Scientific Services Departments had been created in each of the CEGB regions to provide more immediate technical support to the power stations of the region, or to do research on problems or opportunities peculiar to the area.
The grading system was broadly similar, and I was employed as a 'band 3' scientific officer - the entry level grade for an SO. I worked in the Chemistry Division under a man called Jim Dunbar. His boss was the 'Scientific Services Controller', Leslie Young. His post made him a member of the Regional Executive, though to be fair, not a very influential one. The real raw power in the CEGB was wielded by the Power Station Superintendents. They were like ships captains at sea and had within their vested authority the power to do whatever they thought necessary to keep their plant running and the electricity on.
The focus of Chemistry Division's work was on corrosion of the steam generator tubes of the region's coal fired boilers. These boilers operated at a pressure of about 2000psi, so if one of the tubes corroded enough so it burst, the ensuing steam leak would soon cut holes in surrounding tubes and create a real mess, so you had to take the boiler out of service.
When this happened at one of the large power stations like Drax that were being built in the Aire Valley to the east, it meant that older, less efficient, plant had to be brought on line instead. This cost much more to run, and thus lost the industry money hand over fist.
I spent much of my time climbing up and down the gantries of the boilers at Ferrybridge 'C' power station collecting water samples from sampler tubes inserted into boiler tubes. Then we'd take the samples back to the labs and analyse the substances dissolved in the water to attempt to understand the corrosion process. It was not high-tech work, except possibly for the analysis, but it was interesting even if only because the plant itself was quite spectacularly large and powerful. A 500MW steam boiler/turbine combination is an impressive beast.
At this time of my life I first encountered haemorrhoids - piles. Climbing up and down steel staircases close to a hot boiler with sweat running down the crack of your arse is pretty good way to get them. Fortunately, when I stopped the boiler climbing, they went away.
What I missed in Guiseley was a good pub. The town was full of them but I was not impressed by any. I went to one on the Leeds Road called the "Yorkshire Rose" for a while, but it didn't have a good regular crowd. However, wherever I've lived I've always been an explorer, and while walking at the weekend I discovered a pub called "The Commercial Inn" at a little village called Esholt off the Bradford road half way down Hollings Hill. This was too far to walk for a drink in the evening, but was OK for the weekend.
Though I worked close to Leeds, we favoured Bradford as a sopping centre, since it was the city we'd been brought up with, and was actually a little closer. Now, in the new job we were a little better off, and things weren't as much of a stretch as they'd been in North Anston.
My classical music collection had expanded, and now I also had some of the music of Brahms, Dvorak, and Mozart. I would also listen to performances on Radio 3, and to the Henry Wood Promenade concerts when I got the chance
So life was quite agreeable, with challenges in the new job, my daughter, another baby, the new house, and with the ability to see friends at the weekend. 1966 slipped away quite quickly.
This years number ones in the UK, and the odd number two were as follows:
As you can see, the British groups had lost their grip to some extent, with new and established American performers pushing back in. The Beatles and the Stones were there less, and not there for as long.
It seemed in fact that the British invasion had triggered a sudden flowering of talent in the US, with so much good music being produced that it really is impossible to give an overall impression of it here. If you want to get a more detailed view, look at the Wiki 1966 in Music Page.
Apart from the UK chart list, the recordings I'd pick out as interesting were:
Interestingly, Herb Alpert & the Tijuana Brass set a world record this year by having five albums simultaneously on Billboard's Pop Album Chart. Their music outsold The Beatles by two-to-one.
In May of this year, two further pirate radio stations - Swinging Radio England and Britain Radio - appeared, both broadcast from the same ship anchored off the south coast of England in international waters.
So, films? Well in that respect, in the US, it was a lacklustre year. The award winner was "A Man for All Seasons", the story of Sir Thomas More. The only other title I recognize from the top 10 was Who's Afraid of Virginia Woolf?, but I don't think I ever saw it.
The British list looked better. "A Man for All Seasons" was, of course, a British movie, then the others I remember were "Alfie" - Michael Caine does Don Juan, Antonioni's "Blowup", and "The Blue Max".
In these years, I did not get out a lot in the evenings, so Television was important. Among other things, 1966 was a world cup year, and that provided its fair share of entertainment, since England beat West Germany 4 - 2 in the final, some 33 million people watched it.
The important thing though was regular content. BBC broadcasting was generally pretty good in the 60s. The Wednesday play was a staple, and the outstanding example in this year was "Cathy Come Home".
Regular weekly drama series included "The Avengers", "Maigret" - a TV adaptation of the works of French novelist George Simenon, "Dr Finlay's Casebook", and "Z Cars".
The comedy sitcom series of the time was "Till Death Us Do Part". For a simple good laugh you could watch the "Morecambe and Wise Show", and for topical satire, you could watch "Not Only - But Also...".
News and current affairs has always been strong on British TV, and a good example at this time was Granada TVs "World in Action". For nature, science, and technology you could watch "Horizon" or "Tomorrow's World".
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Tuesday, March 29, 2011
does that make me a bad mama?
it's hard work being responsible for two little beings all day long. feed them, clean them up, dress them, clean up their messes, entertain them, entertain them some more, feed them some more, clean them and their messes up some more...it's hard hard work.
i have once again misplaced my camera memory card so i can't upload any new pictures onto the computer. which sucks, cuz there are some cute ones i want to share.
anyway...the Kitty House is coming along. rob's almost done the plumbing, and i finally see the light at the end of the tunnel. i won't jinx it by writing a move-in date yet, but i'm trusting it will be sooner than later.
p.s. as i type this i hear devin waking up. that would be THREE times in the last 1.5 hours.
7 comments:
- Shannon said...
They are SO CUTE when they sleep! Especially when that little mouth is open. My fave time as well.
YAY to the Kitty House being done soon!! Can't wait to see the photos when the memory card is found.
- Audrey said...
It is true....such hard work. And it's non-stop! That's why you too can run away for a weekend when Devin is almost 3 years old!
That is such good news that the plumbing is almost done. Take pictures....it will be good to chronicle all the progress.
- Sonia said...
I completely agree. I do a little dance when the kids are in bed. It's so nice to have some "me" time.
We all know how much mommy's love their kids, but after a full day with them, they look much cuter when they are sleeping. :)
- ChantaleP said...
Hooray for the kitty house! haha, I love that you call it the kitty house. Mm, I also love it when my kiddo's sleeping.. Esp when she snores. That's the best! Tiny snore from a tiny nose and throat.
Man, just having one kid is hard enough work, I can't imagine another one. We are both so over winter boots and crap, I can't tell you how much we're over it! lol.
Can't wait to see kitty house pics too!
- joyce said...
Even Hudson has said we should stop calling it the Kitty House because no kitties live there anymore...the name has just STUCK!!!
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Develop your artistic eye and your sketching techniques with local artist Patricia Lightfoot while you learn to draw, sketch, and watercolor from the world around us.
We'll take short walking trips to the beach, boat harbor, and El Estero lake for some landscape and wildlife drawing, and head indoors for portraits and still-life drawing. Be it animal, mineral, or vegetable, real or fantasy, students will learn to develop their sketches into full color watercolor paintings using nature as their inspiration.
Ages: 10 - 14
Dates: Jun 11 - 15, 2018
Hours: 9:00am - 3:00pm
Location: Lyceum
Instructor: Pat Lightfoot
Class Size: Limited to 8 Students
Fee: $275


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Chris Staples from the Gosport and Fareham Breathe Easy group is encouraging people to get active
Support for the Gosport and Fareham Breathe Easy group continues to grow among sufferers and clinicians.
Mal North, lead nurse for the Trooper study, spoke at a previous meeting about this and other ways of being more self supportive at home.
Being self supportive does not mean NOT seeing your GP/respiratory nurse,it means working with them.
It is the time of year to be booking flu and pneumonia jabs, so why not ask about pulmonary rehab courses as well? These are a series of controlled exercises over a six-week period run by physiotherapists which your GP can refer you to.
The exercises are designed so that anyone can do them and the physiotherapists tailor them to individual needs and constantly monitor during the activity. Some of you will automatically say you can't do exercises, but can you go up and down on tiptoe while the kettle is boiling?
You can use the worktop as a support if you want. Just a minute of this will help.
Try standing,looking up to the ceiling then gently down to the ground for a few times. This is part of the warm up activity the groups use.
Any sufferer can ask to go on the programme, smoker or non-smoker, no-one is barred, but of course it would not be expected for smokers to go out for a cigarette during the hour-long session.
The exercises will not cure but could help a person cope a little better during the more adverse weather conditions and in a group situation with others with similar problems, there are many other benefits besides just the medical side.
The physiotherapists are very dedicated and conscientious and provide a weekly contact for any worries/concerns, advising and directing a person to relevant sources if necessary.
The Breathe Easy meetings provide a meeting point for sufferers, carers and those who are worried about someone with breathing problems along with various clinicians who may be able to help and advise or put your mind at rest.
At our last meeting, Jo the nutritionist, gave an excellent talk to both patients and carers.
Jess Williams, Senior Respiratory Research nurse also gave a presentation about how researchers at QA are now seeking patient input to direct their work with research fellows from the hospital and clinicians.We anticipate our group will be fully involved in this innovative approach.
Our next meeting will be on Thursday, November 5. Everyone is welcome.


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KJ Apa & Camila Mendes Bring 'Riverdale' To the AMAs 2017
KJ Apa suits up sharp for the 2017 American Music Awards held at the Microsoft Theater on Sunday (November 19) in Los Angeles.
The Riverdale actor was joined by his gorgeous co-stars Lili Reinhart, Madelaine Petsch and Camila Mendes at the annual event, where the group will be presenting together.
PHOTOS: Check out the latest pics of Lili Reinhart
Hosted by black-ish star Tracee Ellis Ross, make sure to tune in for the AMAs, tonight at 8/7c on ABC!
FYI: Lili wore a Cushnie et Ochs black dress, Stuart Weitzman shoes, a Swarovski ring, and an Edie Parker bag for the event. Madelaine wore a Fabiana dress with Loriblu heels, jewelry from Gilan, Djula, Joelle and Established, and an Emm Kuo bag.


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Business Secretary Welcomed to JCB for Skills Insight
Business Secretary Dr Vince Cable visited Staffordshire for an insight into JCB's development of skills for the future and its global manufacturing operations.
The Secretary of State for Business, Innovation & Skills was first of all welcomed by JCB Chairman Lord Bamford at the JCB Academy in Rocester, the UK's first academy dedicated to developing the engineers and business leaders of the future by delivering a technical education to 14 to 19-year-olds.
He was shown around classrooms equipped with more than £1 million worth of modern engineering equipment by Chair of Governors David Bell and Academy Principal Jim Wade. He also met a number of students during afternoon lessons.
After his whistlestop tour, Dr Cable was welcomed to the nearby JCB World Headquarters by JCB CEO Graeme Macdonald where he reviewed a line-up of innovative JCB machines for construction and agricultural markets.
The Secretary of State was also given a full tour of the Story of JCB, a permanent exhibition which charts the history of the Bamford family from the 1820s when they were blacksmiths in Uttoxeter, through to the present day and JCB's rise to the world's third largest manufacturer of construction equipment.
He was also introduced to four apprentices: Leila Worsey, Josh Machin, Dan Shaw and Dan Hodgkinson. Leilia and Josh are both former students at the JCB Academy.
The visit came 48 hours after Lord Bamford delivered his maiden speech in the House of Lords in which he highlighted the importance of technical education to the national economy and called for a coherent long-term industrial strategy to support the future growth of manufacturing as a vital component of the UK economy in the future.


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We comfort ourselves with pretty little lies. Make them our truth. But it can't be. It leads to an inner conflict with the real truth. A struggle ensues. Denial and anger are fighting alongside those lies.
Our world view is under fire. A threat to our belief. Instead of welcoming new knowledge from which to gain better understanding and new perspectives, we reject it. Strangers are not welcome in a state of ignorance. Pitch forks and torches will drive out anyone who is different. Who thinks differently. Who acts differently. The different ones. The crazy ones. The daredevils. The thinkers. Welcome them, beware of them. They might just change the way we see the world. They might change everything. Be ready. Don't send them away. Welcome them and listen to what they have to say. Decide whether or not to act upon their wisdom and knowledge. Their words can be poison or it can be the antidote. It can kill us or it can kill our ignorance.
Ignorance will cause us to die without knowing why we lived in the first place.
Directing energy into the wrong channels will fire a beam of hatred, ignorance and bigotry. Oblivious of our own doing. And the flames create a scorching sky, blocking the sun, blocking hope. Not letting love through. It is easier that way. Destroy everything before it has the chance to change a life. To make a difference. To change the course of someone else's life.
At the very end a moment of clarity. A ray of light will break through a lifetime of clouded ignorance only to reveal the sad remains of an unlived life. A temple destroyed and burned to the ground. Too late to rebuild. It can't be rebuild. You can't build upon destruction. Only more rubble and debris will be added onto the never-ending pile. Every destroyed building resembles a dream, an idea never coming to fruition. It's a ghost town. A personal hell created out of obliviousness. It is the end and a life ends with regrets.
Don't scorch your sky. Look at it. Marvel at it. Don't let your life be reduced to one fully lived day. There is more to life than just one perfect day. It only seems perfect, because the rest of them are not.
Create and help others create. Don't destroy and don't let others destroy. If you keep creating, you can't destroy. If you keep destroying, you can't create.
Let your own light shine upon a marvelous city, where people want to live in. Where they feel welcome, regardless of their origin. Different viewpoints are welcomed in the halls and opinions are discussed with open minds. A change for the better. The common good in mind and only together will this be achieved. The sum is greater than its parts. Encourage outspokenness. Let people be themselves and let them show their true selves. There can only be benefit.
Smiles on the street. Children playing and being raised in understanding and acceptance. They will carry the future into better times. You will find support in every corner of your city. It only depends on how you build it.


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CATEGORIES: Asian / Sushi / Indian | Patio | Happy Hours
Welcome to Sherpa's! All food is true to authentic recipes and, in most cases, made to order including fresh breads, sauces, and desserts. Sherpa's features an authentic, heart-healthy, low -fat, charcoal fired, Tandoor clay oven. In addition to our great food, feel free to take advantage of our Traveler's Library & Lounge, peruse the displayed Sherpa history and pictures, explore our expedition, trekking & volunteer trip booking opportunities, and look for our list of guest speakers. Thanks for coming in and we hope to see you often. Pemba, Pete & the staff at Sherpa's.
Sherpa's upstairs Camp1 private room is the perfect place to hold your business meeting, birthday party, wedding reception, family get-together, and graduation parties! The area is one of Boulder's largest private party rooms, has fantastic views of the Flatirons, has outstanding pictures of Nepal, and a private patio to enjoy fresh breezes. The private room allows you and your loved ones to enjoy yourselves, the best Nepalese Indian and Tibetan food in Boulder, all on your own private floor!
HAPPY HOUR SPECIALS: Daily 5 - 7pm | Drinks: $2 Select Draft Beers


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Match winner Jon Colman has done consistently well all series to finish in a well deserved third place overall
22nd April 2018
Jon Colman finished the championship series in style with a superb win, recording the heaviest match weight of the series at Wood Lane on Sunday. Feeding and fishing hard 6ml pellet on two lines, he switched between them to take a steady run of carp for a level 85-0-0. In second place, only eight ounces behind Jon with 84-8-0 was Glen Briggs who caught carp fishing long to the island then and had a great last hour catching some decent fish down the edge to boost his weight. Third on the day was Dave Cook who fished shallow to the rushes in permanent peg twelve to take carp to double figures and record a final weight of 69-2-0. Richard day did well to finish fourth with 67-14-0 from a peg with no real form. Richard fed 4ml pellets on the waggler line with some success, but when he switched to fishing a 4ml hookbait shallow, his catch rate improved dramatically. Glen Briggs is this year's club champion with Graham Fox finishing as runner up, a superb performance in his debut season. Jon Colman has had an excellent series and is steadily improving his game each year. Match captain Fred Fairman finished fourth and the up-and-coming Declan Kelly finished in fifth place. This has been another excellent series that has been very well supported by the match group with some great attendances and some great banter. Roll on the next one….
1 – Jon Colman – 85-0-0 21
2 – Glen Briggs – 84-8-0 6
3 – Dave Cook – 69-2-0 11
Ron Chenery – Carp – 13-8-0
For the full match results click here.


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Behold: results of the fall's mad landscaping adventure!
Still need to sweep stone dust and sand between the pavers and put in the concrete toe. I am ever hopeful that I might conceivably get this done before baby #2 arrives in July.
Eremurus! Ahahahahahahahahaaaaaaa! And two of the three have TWO leaf spikes coming up - if that ends up meaning two flower spikes I will faint dead away from delight. Then again I will probably do that if they bloom at all.
In the backyard, meanwhile:
Not much to look at yet at this time of year, but I can already tell it has increased in gorgeousness. In my spring cleanup I have tried to sweep the pine needles onto the paths so as to make a nice thick (and somewhat contrasting) mulch. We'll see how that works out, but if it actually deters weeds I'd be glad to have a use for the stuff, because the tree certainly sheds enough of it.
Details of the beds, mostly just for posterity at this point, I might come back and add ruminating at some point when it's not almost 11 p.m. on a work night:
One note I will add here, regarding the lily patch: I raked as many pine needles out of the area as I could and doused everything with neem oil. Result: ~10 lily beetles were disturbed out of their hiding places and hunted to their doom. IT STARTS. Siiiiiiigh.
Behold the trench!
Aaaaaand one detail: bloodroot, very briefly (and rather gorgeously) in flower.
Sunday, May 1, 2011
2 comments:
- AnonymousMay 2, 2011 at 6:11 PM
none of the photos show up :(ReplyDelete
auntie naomi
- AnonymousMay 3, 2011 at 5:25 PM
lovely! i really like the front paving blocks you've put in. i look forward to more developments. :)ReplyDelete
auntie naomi
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Tous La Frite! - NYE at Point Ephémère
Point Ephémère rounds off its Belgium season (lasting through December) with a juicy lineup of DJs from that country, running the gamut from electro rock to deep house. Named 'Tous La Frite', with tongue firmly in cheek, in reference to the Belgian fondness for chips, it's shaping up to be a lively affair.
|Event website:


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When you think of parasites, you might think of things like fleas and ticks – parasites that tend to bother cats and dogs – but there are also some that can bother humans. Intestinal parasites are some of the most common parasite infections in humans, though they can attack other systems in the body as well.
Parasites tend to thrive in the more tropical regions of the world, though they can also occur in the United States. Keep reading to learn more about the different types of parasites as well as some natural remedies to clear your infection.
A parasite is an organism that lives on or in another organism, often feeding on it. In humans, parasite infections are rarely fatal, though they can cause some very unpleasant symptoms. There are three distinct types of disease-causing parasites:
Protozoa – These are single-celled organism that are only capable of dividing and multiplying inside the host (Plasmodium, the parasite that causes malaria is an example).
Helminths – These are multicellular worm parasites such as tapeworms, pinworms, and roundworms.
Ectoparasites – These are parasites that live on rather than in their hosts, such as fleas or lice.
Though there are only three main categories of parasites, there are many individual parasites that can cause infection. Here Is a list of the most common parasite infections in humans:
- Coccidia – An intestinal parasite found around the world; this parasite enters the body through food contaminated with human or animal feces.
- Giardia – A parasite that affects the small intestine, giardia are transmitted through contaminated food or water.
- Malaria – Spread through the Anopheles mosquito, malaria is an infection that affects the red blood cells.
- Toxoplasmosis – Also known as parasitic pneumonia, this parasite infection affects the liver, heart, eyes, and brain – it is spread through ingesting raw or undercooked pork or lamb.
- Pinworm – This parasite affects the intestines and anus and is very common in temperate regions.
- Fasciolopsiasis – Common in East Asia, this intestinal parasite is transmitted through contaminated water plants or water.
Now that you know a little more about what parasites are and what kind of infections are common, you may be wondering how to spot a parasite infection. Keep reading to learn about the symptoms of parasite infections.
Parasite infections can be spread in a number of different ways. Protozoa and helminths, for example, can be spread through contaminated food, water, soil, or blood – some can also be transmitted through sexual contact. Other parasites are spread through insects such as mosquitos.
Each parasite infection is a little bit different, but many of them have overlapping symptoms. Here are the most common symptoms associated with parasite infections:
- Itching of the anus or vagina
- Abdominal pain
- Weight loss or changes in appetite
- Diarrhea or vomiting
- Sleep problems
- Anemia
- General malaise
- Allergies
- Aches and pains
- Anxiety
There are more than 1,000 parasites known to affect humans, so symptoms may vary according to the specific parasite and its location in or on the body. There are numerous tests your doctor may run to diagnose your infection.
10 Natural Home Remedies for Parasites
Now that you understand the signs and symptoms of parasite infections, you may be wondering how to handle them. Treatment options may vary according to the type of parasite, though traditional medical treatments typically include some kind of medication.
Unfortunately, these medications often come with side effects and they may interact poorly with other drugs or supplements you are taking. The ten natural remedies listed below will help to clear up your parasite infection without the risk for dangerous interactions or unpleasant side effects.
- Shredded Coconut – A natural anti-parasitic agent, coconut can be very powerful against parasite infections (especially intestinal infections). To use this natural remedy, take a tablespoon of shredded unsweetened coconut in the morning then drink a glass of water with two tablespoons of castor oil three hours later.
- Papaya – This tropical fruit has been used in Ayurvedic medicine for centuries and has been shown to help expel intestinal worms. Take one tablespoon of fresh papaya juice (from an unripe papaya) and one tablespoon of honey and mix with ¼ cup of hot water. Drink this mixture in the morning on an empty stomach and follow it with a glass of warm milk two hours later.
- Garlic – Another natural anti-parasitic agent, raw garlic is great for intestinal worms. Simply chew three cloves of raw garlic on an empty stomach once a day for seven days.
- Carrots – These orange vegetables are rich in beta-carotene, a nutrient that is a precursor for vitamin A which helps to kill parasite eggs. Simply grate two carrots and eat them in the morning on an empty stomach. Do not eat anything for several hours after and repeat once daily for a week.
- Pumpkin Seeds – These seeds contain a compound called cucurbitacin which has natural anti-parasitic properties. Grind 2 tablespoons of pumpkin seeds and mix it with 3 cups of boiling water. Steep for 30 minutes then cool and drink slowly.
- Cloves – This aromatic spice has natural antibacterial, antiseptic, and anti-parasitic properties. Crush some dried cloves and stir one teaspoon of the powder into a cup of hot water then steep for 20 minutes and drink three times daily.
- Pomegranate – All parts of the pomegranate plant are effective in killing parasites, but the bark is the most potent. Take a daily decoction of 90 to 180 ml of pomegranate bark three times daily, with the treatments spaced an hour apart.
- Turmeric – This yellow spice is naturally antiseptic and antimicrobial, plus it helps to kill intestinal worms. Simply mix a half teaspoon of ground turmeric with a pinch of salt in a half cup of warm water and drink once daily for five days.
- Wormwood – The flowers of the wormwood plant are very powerful in fighting parasites. Dry and crush the flowers into a powder and take 8 to 16 grams per day for roundworms and tapeworms.
- Hyssop – To get rid of roundworms and other intestinal parasites, take an infusion of hyssop leaves three times daily.
Kate Barrington
Latest posts by Kate Barrington (see all)
- Pink Eye - June 18, 2017
- Parasites - June 18, 2017
- Sore Throat - May 25, 2017


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* Main index ends slightly lower after big early drop
* TSX down 2.8 percent for the week
* Financial sector leads Friday's fall, down 2.5 percent
* Materials group up 4.9 percent on rising gold price (Adds quote, details)
By Jennifer Kwan
TORONTO, Oct 24 (Reuters) - The Toronto Stock Exchange's main index closed only slightly lower on Friday, erasing most of the 7.5 percent loss it started the day with, rattled by the recession fears that sparked big selloffs on Asian and European markets and on Wall Street.
The key S&P/TSX composite index .GSPTSE closed down 37.26 points, or 0.4 percent, at 9,294.09. Just after the open it plunged more than 700 points to touch its lowest level in four years.
Gold mining shares aided the comeback, with bullion prices rising as safe-haven buying emerged on fears that global stock markets were heading into freefall. The gold-mining subindex was up 7.9 percent. [ID:nL1449620]
Barrick Gold (ABX.TO) rose 8.9 percent to C$25.75, and Agnico-Eagle (AEM.TO) was up 12.8 percent at C$33.86.
"There always is a disconnect between commodity prices and the underlying companies, and maybe today (the market) felt the disconnect between gold and the gold companies was far enough that there's an opportunity there," said Brian Pow, vice-president, research and equity analyst at Acumen Capital Partners, in Calgary, Alberta.
The broader mining-heavy materials sector was up 4.9 percent.
Analysts had said the TSX had arrived at the "capitulation" phase as global markets have swooned on fears of a worldwide downturn. Others said hedge funds were unwinding and margin calls were some contributing factors.
The market had taken its earlier cue from the United States, which saw overnight selling so steep that U.S. stock index futures were halted until after the opening bell.
"By any form of measurement, this market is deeply, deeply oversold," said Peter Chandler, senior vice-president at Canaccord Capital in Waterloo, Ontario.
"We will get to a point, that I will suggest will be within days, where the market is exhausted on the selling side and at a minimum you'll get a significant trading bounce."
For the week, the TSX was down 2.8 percent. For the year it is down 32.8 percent.
The financial services group fell 2.5 percent, with Manulife Financial (MFC.TO) off 3.9 percent at C$25.00 as several analysts downgraded their share-price targets for the company. [ID:nN24523458]
The oil and gas sector slid 0.2 percent as U.S. crude dropped $3.69 to $64.15 a barrel despite an OPEC decision to cut production in a bid to stem the oil price slide [ID:nN24513540].
EnCana Corp (ECA.TO) rose 1.3 percent to C$55.78.
Market volume was 539.4 million shares worth C$7.92 billion. Decliners outpaced advancers 1,088 to 472.
The blue chip S&P/TSX 60 index .TSE60 closed 1.13 points lower, or 0.2 percent, at 561.39.
The market retreat was far more pronounced south of the border because of fears of recession and a darker outlook for corporate profits.
The Dow Jones industrial average .DJI fell 312.30 points, or 3.59 percent, at 8,378.95, while the Nasdaq composite index .IXIC ended down 51.88 points, or 3.23 percent, at 1,552.03. ($1=$1.27 Canadian) (Reporting by Jennifer Kwan; editing by Rob Wilson)


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Karen S. Murley
Accounting Manager
Phone: (614) 799-9800 ext. 19
Email: firstname.lastname@example.com
22 Year Member of The Case Bowen Company Team
Karen has been with The Case Bowen Company since 1995. She attended Columbus State College with a major in Accounting and came to us with vast background in accounting; this was her first exposure to real estate and community management. Karen is currently in charge of the financial reports for 25 condominium associations. Karen's accurate and timely reports keep board members informed of operating costs, reserve funds and delinquent accounts. She takes the time to customize reports to satisfy special needs or requests of the board. In addition to financial reporting, Karen manages the complete computer set-up when new properties are taken on and is the lead trainer for the accounting department. She also works with title agencies regarding condo sales on a daily basis. Karen is a valuable and experienced part of our Accounting Department and The Case Bowen Company.
Quality Control Standards
The Case Bowen Company's Accounting Department is committed to providing the highest quality accounting, budgetary, financial management, and advisory services. Our team brings a wide range of experience and skills to our clients. We believe in the importance of relationships, and view every client as a partnership. For your assurance, we comply with exacting quality control standards in the conduct of our accounting practice and submit to periodic on-site reviews as well as internal checks and balance procedures as a fiduciary responsibility to our clients.
"Live as if you were to die tomorrow. Learn as if you were to live forever." ~ Mahatma Gandhi
Giving Back to the Community
Karen strongly believes in giving back to the community and has been a volunteer for Meals on Wheels for 20 years. She enjoys working in the flower garden and reading. Has a great passion for golf and being outdoors walking, biking, and camping with her grandkids.
Business Acumen
We take pride in our monthly statements and processes, as we report to federal/state/local auditors and tax professionals on your behalf continuously. The excellence of our accounting team comes from years of advanced training, technical experience and financial acumen. Our continual investment of time and resources in professional continuing education, state-of-the-art computer technology and extensive business relationships is indicative of our commitment to excellence.


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Kanye West and Spike Jonze's Team Up
After seeing Where The Wild Things Are, I've decided to follow anything Spike Jonze related. Then I discovered this short,We Were Once a Fairytale that Jonze did featuring Kanye West. The short tries and humanize (successfully to a degree) Kanye's ego and life. Fortunately, Kanye isn't asked to say many lines beyond "Where's the bathroom," or "I'm Kanye," or "This is my SONG!," but nonetheless, with Jonze's direction, and cinematography, the short proves to be worthy of a look. Let me know what you think:


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Brooke Hogan Writes Poem In defense Of Hulk Hogan Following Racist comment Scandal
July 28, 2015
Hulk Hogan has been on the receiving end of a controversy on the grounds that offensive feedback had been released to the public. Hogan got here below hearth ultimate week when a transcript of him pronouncing racially insensitive issues about his daughter's ex-boyfriend was made public.
RadarOnline has launched extra comments from the tape that contained the racist comments made in 2006. Hogan talks about an come across with a gay man
Hogan was once discussing his fact show, Hogan knows highest and had the following to say:
"VH1 wanted me to do a tremendous thing and go back to the house I grew up in. So we knock on the door and a huge f*g lives there now! This half of-gay used to be enamored with Linda."
the next statements occur after Hogan's original rant on a mysterious "billionaire black man" who had it appears been funding Brooke Hogan's attempted musical occupation.
"Fucking n*gger… he had Jamie Foxx coming in on the twenty second observe…"
"I didn't even tell Brooke about it. F*ck her. Brooke and (identify withheld by way of Radar and The ENQUIRER) met in Miami."
"Brooke f*cks up a ten million dollar deal I had with the Saudis. Brooke says, 'F*ck you dad.' She's never stated that. She flipped a hen at me."
"i've this enormous f*cking home in Miami. My household never comes residence. They went to LA. F*ck 'em."
Brooke (Hogan) Bollea came to her father's defense on fb in a poetic standing replace. She wrote a poem titled "in case you Knew My Father" and revealed it on social media, including fb:
~when you Knew My Father~by means of Brooke BolleaIf you knew the dad I knew,you'd know his soft heart.He'd never wish to…
Posted through Brooke Hogan on Friday, July 24, 2015
~if you Knew My Father~
through Brooke Bollea
in the event you knew the dad I knew,
you'd comprehend his mushy coronary heart.
He'd by no means need to harm his lovers,
or family from the start.
if you knew my father,
you could know the way arduous he fought…
and the way in which it brought a smile to individuals light, medium and darkish.
We all the time combat a fight
that people by no means see,
and every so often when you're hurting, you don't suppose logically.
Human isn't perfect,
and excellent will not be he,
but i will be able to tell you one thing, it's simply now not what it seems.
lead to if you knew the dad I knew,
you'd be aware of he raised me well.
He taught me other folks are a lot more
than colorations might ever inform.
And when you knew my father,
you could possibly comprehend he's all the way down to earth.
He can have slammed the large,
but remembers life's proper value.
when you knew my father,
if you just walked in his shoes…
then you can understand that microscope that comes with yellow boots.
should you knew my father,
you could possibly see your individual in mine.
And if he was once your father, you have to by no means find divide.
result in me- I wager your father
or anyone that you love
possibly regrets something
they need they'd never performed.
So if you understand my father,
take into account that he's a person,
and you could crave reduction if individuals judged the whole lot you did.
The lord says to forgive them,
don't be the one to stone…
so please remember his strong arms whilst you were all by myself.
The lives he's changed, the buddies he's made,
the folks he's inspired…
It makes me sad to look my dad
defeated and so tired.
for those who knew my father,
you possibly can be aware of just how he's hurting…
For he loves every person of you, despite your story.
And when you met my father,
i do know he'd offer you love!
He'd raise you up, ask for forgiveness and offer you all a hug.
for those who think you already know my father,
needless to say who you are.
we all can make mistakes and elevate unsightly scars.
as a result of i do know my father,
i will promise this-
similar to you and i, issues we don't mean can every so often slip.
all of us keep learning.
This lifestyles will also be so rough…
So if you know my father, please are attempting to not be difficult.
My father has a daughter,
and i have feelings too.
And if I knew your father, i'd do the same for you.
Kevin Nash posted the following remarks on Hulk Hogan:
I've spent the final 23 years of my existence with Hulk. i have been in the most various of scenarios and never heard Hulk use a racial slur.
— Kevin Nash (@RealKevinNash) July 27, 2015
under no circumstances dismissing the remarks.simply taking a look at the scenario. intercourse tape thats set up.Tapes which can be simply edited. content that was once pushed
— Kevin Nash (@RealKevinNash) July 27, 2015
I've watched Hulk do 10,000 make awish greetings. religion creed or color. never changed the love he shared.choose a man put him with a baby
— Kevin Nash (@RealKevinNash) July 27, 2015
unless u went through this,proper loyality and love has a special which means,it makes u want to are living and no longer die,4those that never left me i am
— Hulk Hogan (@HulkHogan) July 27, 2015
unless u went through this,proper loyality and love has a unique meaning,it makes u want to reside and now not die,4those that by no means left me i am
— Hulk Hogan (@HulkHogan) July 27, 2015
for people who left me and failed to stand by way of my facet,u will see and i'll show,who i actually am and u can even take into account my love for u 0LHH
— Hulk Hogan (@HulkHogan) July 27, 2015
[photograph credit score: supply]
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