prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What do dendritic spines contain?

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Context 1: JoVE | Peer Reviewed Scientific Video Journal - Methods and Protocols
A role for thrombospondin-1 deficits in astrocyte-mediated spine and synaptic pathology in Downs syndrome  Octavio Garcia, Maria Torres, Pablo Helguera, Pinar Coskun, Jorge Busciglio 
PUBLISHED: 07-02-2010	Downs syndrome (DS) is the most common genetic cause of mental retardation  Reduced number and aberrant architecture of dendritic spines are common features of DS neuropathology  However, the mechanisms involved in DS spine alterations are not known  In addition to a relevant role in synapse formation and maintenance, astrocytes can regulate spine dynamics by releasing soluble factors or by physical contact with neurons  We have previously shown impaired mitochondrial function in DS astrocytes leading to metabolic alterations in protein processing and secretion  In this study, we investigated whether deficits in astrocyte function contribute to DS spine pathology 
Analysis of Dendritic Spine Morphology in Cultured CNS Neurons Authors: Deepak P  Srivastava, Kevin M  Woolfrey, Peter Penzes  Published: 07-13-2011 JoVE Neuroscience
Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic compartment of synapses  These structures are rich in actin and have been shown to be highly dynamic  In response to classical Hebbian plasticity as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural circuits and the processing and storage of information within the brain  Within dendritic spines, a complex network of proteins link extracellular signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number  Neuropathological studies have demonstrated that a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers  Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders  In order to study the potential role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages  Firstly, this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells  Secondly, this in vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological treatments  These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these proteins may function in vivo 
Play ButtonIsolation and Culture of Mouse Cortical AstrocytesAuthors: Sebastian Schildge, Christian Bohrer, Kristina Beck, Christian Schachtrup  Institutions: University of Freiburg , University of Freiburg  Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics  In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail  This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups  The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture  Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes  Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers  This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology Neuroscience, Issue 71, Neurobiology, Cellular Biology, Medicine, Molecular Biology, Anatomy, Physiology, brain, mouse, astrocyte culture, astrocyte, fibroblast, fibrinogen, chondroitin sulfate proteoglycan, neuronal regeneration, cell culture, animal model50079Play ButtonImaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination MicroscopyAuthors: Marijn Schouten, Giulia M  R  De Luca, Diana K  Alatriste González, Babette E  de Jong, Wendy Timmermans, Hui Xiong, Harm Krugers, Erik M  M  Manders, Carlos P  Fitzsimons  Institutions: University of Amsterdam, University of Amsterdam Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain  Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity  The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit  Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy  However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm 
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines  These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen  Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures  Possible applications of SIM overlap with those of confocal microscopy  However, the two techniques present different applicability  SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light  In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM  The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal  After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software Neuroscience, Issue 87, Dendritic Spine, Microscopy, Confocal, Fluorescence, Neurosciences, hippocampus, primary neuron, super resolution microscopy, structured illumination microscopy (SIM), neuroscience, dendrite51276Play ButtonSetting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated TransportAuthors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky  Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259 The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue  Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer  Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture  RBEC were isolated from 5-week-old rat cortices  The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion  Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue  The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix  The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium  They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells  RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders  The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average  The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0 26 ± 0 11 x 10-3 cm/min  Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer  In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),51278Play ButtonInducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing RatesAuthors: Alison X  Xie, Kelli Lauderdale, Thomas Murphy, Timothy L  Myers, Todd A  Fiacco  Institutions: University of California Riverside, University of California Riverside, University of California Riverside Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter  However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention  Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices  Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy  In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs  Applications of the technique for study of astrocytes are discussed  Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse51458Play ButtonInhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA ReceptorsAuthors: Laura E  Brown, Celine Fuchs, Martin W  Nicholson, F  Anne Stephenson, Alex M  Thomson, Jasmina N  Jovanovic  Institutions: University College London Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks  GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain  It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons  Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials  During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes  This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other  To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes  Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification  Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses  Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts  Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line52115Play ButtonTwo-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull PreparationAuthors: Xinzhu Yu, Yi Zuo  Institutions: University of California, Santa Cruz In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses  Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity  As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses  Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain  Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo  This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex  Therefore, images can be acquired immediately after surgery is performed  The experimental procedure can be performed repetitively over various time intervals ranging from hours to years  The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions Neuroscience, Issue 87, dendritic spine, mouse cortex, in vivo, two-photon microscopy, thinned-skull, imaging51520Play ButtonModeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered MiceAuthors: Robert S  McNeill, Ralf S  Schmid, Ryan E  Bash, Mark Vitucci, Kristen K  White, Andrea M  Werneke, Brian H  Constance, Byron Huff, C  Ryan Miller  Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development  In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture  Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes  The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro  Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo  Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma  Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time  Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft51763Play ButtonPaired Whole Cell Recordings in Organotypic Hippocampal SlicesAuthors: Chantelle Fourie, Marianna Kiraly, Daniel V  Madison, Johanna M  Montgomery  Institutions: University of Auckland, Stanford University Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron  When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased  This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue  A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation  Paired whole cell recordings are often perceived as too challenging to perform  While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed  The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings  While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation  In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology Neuroscience, Issue 91, hippocampus, paired recording, whole cell recording, organotypic slice, synapse, synaptic transmission, synaptic plasticity51958Play ButtonImaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium IndicatorsAuthors: Ruotian Jiang, Martin D  Haustein, Michael V  Sofroniew, Baljit S  Khakh  Institutions: University of California Los Angeles, University of California Los Angeles Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals  It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules  However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series  The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia  In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described  We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity  We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ  The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets  The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry Neuroscience, Issue 93, astrocyte, calcium, striatum, GECI, GCaMP3, AAV2/5, stereotaxic injection, brain slice, imaging51972Play ButtonMethods to Assess Subcellular Compartments of Muscle in C  elegansAuthors: Christopher J  Gaffney, Joseph J  Bass, Thomas F  Barratt, Nathaniel J  Szewczyk  Institutions: University of Nottingham Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state  The loss of muscle mass and function with disease and age are significant public health burdens  We currently understand little about the genetic regulation of muscle health with disease or age  The nematode C  elegans is an established model for understanding the genomic regulation of biological processes of interest  This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species  Since C  elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo  Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo  These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance  Thus, C  elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function  Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism Developmental Biology, Issue 93, Physiology, C  elegans, muscle, mitochondria, sarcomeres, ageing52043Play ButtonImproved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term PotentiationAuthors: Agnès Villers, Laurence Ris  Institutions: University of Mons Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding  LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied  However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood  This could be partly due to the various experimental conditions used by different laboratories  Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity  It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection 
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation  This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices  It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena Neuroscience, Issue 76, Neurobiology, Anatomy, Physiology, Biomedical Engineering, Surgery, Memory Disorders, Learning, Memory, Neurosciences, Neurophysiology, hippocampus, long-term potentiation, mice, acute slices, synaptic plasticity, in vitro, electrophysiology, animal model50483Play ButtonIn Vivo Modeling of the Morbid Human Genome using Danio rerioAuthors: Adrienne R  Niederriter, Erica E  Davis, Christelle Golzio, Edwin C  Oh, I-Chun Tsai, Nicholas Katsanis  Institutions: Duke University Medical Center, Duke University, Duke University Medical Center Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish  Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated 1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling  Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders  Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish  This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants  Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype  Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf)  This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol  We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others  Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model50338Play ButtonDirect Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)Authors: Samira Samtleben, Juliane Jaepel, Caroline Fecher, Thomas Andreska, Markus Rehberg, Robert Blum  Institutions: University of Wuerzburg, Max Planck Institute of Neurobiology, Martinsried, Ludwig-Maximilians University of Munich Visualization of calcium dynamics is important to understand the role of calcium in cell physiology  To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular  Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types  To date, TED was used in cell lines, glial cells, and neurons in vitro  TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES)  The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging  The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red  At the beginning of the procedure, cells are transduced with a lentivirus  Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging  Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM  The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen  After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope  Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging  Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity  Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0 Cellular Biology, Issue 75, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging50317Play ButtonPreparation of Dissociated Mouse Cortical Neuron CulturesAuthors: Lutz G  W  Hilgenberg, Martin A  Smith  Institutions: University of California, Irvine (UCI) This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain  These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation  These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation  The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared  Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells  To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step  This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death  Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons Neuroscience, Issue 10, cellular, molecular, neurobiology, neuron, calcium/sodium imaging, primary cultures, mouse562Play ButtonAnalysis of Schwann-astrocyte Interactions Using In Vitro AssaysAuthors: Fardad T  Afshari, Jessica C  Kwok, James W  Fawcett  Institutions: University of Cambridge Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries  Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4  In addition they myelinate the demyelinated axons at the site of injury 5 
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7  This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally  Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13 
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour 
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts  As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide  This allows the behaviour of the two cellular populations to be analyzed at the boundary  Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries 
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15  This assay is commonly known as inverted coverslip assay  Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip 
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation  Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19 
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur Cellular Biology, Issue 47, Schwann cell, astrocyte, boundary, migration, repulsion2214Play ButtonQuantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse NumberAuthors: Dominic M  Ippolito, Cagla Eroglu  Institutions: Duke University, Duke University One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation  As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity  Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry  Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization  We define synapses as points of colocalization between the signals generated by these markers  The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c eroglu@cellbio duke edu) under the ImageJ analysis software platform  The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers  This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development Neuroscience, Issue 45, synapse, immunocytochemistry, brain, neuron, astrocyte2270Play ButtonPreparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid PathologyAuthors: Diana M  Mathis, Jennifer L  Furman, Christopher M  Norris  Institutions: University of Kentucky College of Public Health, University of Kentucky College of Medicine, University of Kentucky College of Medicine The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity  The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid  Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments  The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory  Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds  This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3  Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research  Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity  Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment  Here, we discuss the steps taken during the dissection procedure to minimize these problems  Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments  The possible impact of other methodological factors on synaptic function in these animal models (e g  recording solution components, stimulation parameters) are also discussed  While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models Neuroscience, Issue 49, aging, amyloid, hippocampal slice, synaptic plasticity, Ca2+, CA1, electrophysiology2330Play ButtonMesenteric Artery Contraction and Relaxation Studies Using Automated Wire MyographyAuthors: Lakeesha E  Bridges, Cicely L  Williams, Mildred A  Pointer, Emmanuel M  Awumey  Institutions: North Carolina Central University, Durham, North Carolina Central University, Durham, Wake Forest University School of Medicine Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance  These small vessels of between 100-400 μm in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body  The rat mesenteric artery has a diameter greater than 100 μm  The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2  The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented  Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires  Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3 The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors 
Medicine, Issue 55, cardiovascular, resistant arteries, contraction, relaxation, myography3119Play ButtonVisualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero ElectroporationAuthors: Emilie Pacary, Matilda A  Haas, Hendrik Wildner, Roberta Azzarelli, Donald M  Bell, Djoher Nora Abrous, François Guillemot  Institutions: MRC National Institute for Medical Research, National Institute for Medical Research, Université de Bordeaux In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5  To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8  Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions 
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development  Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells  In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines Neuroscience, Issue 65, Developmental Biology, Molecular Biology, Neuronal development, In utero electroporation, dendrite, spines, hippocampus, cerebral cortex, gain and loss of function4163Play ButtonImaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture SystemAuthors: Haruki Higashimori, Yongjie Yang  Institutions: Tufts University, Tufts Sackler School of Graduate Biomedical Sciences Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS)  This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2   Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology  Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia  What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i e , axon, dendrite, or soma)3  This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites  In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia  Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell 
In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP)  In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels  In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels  In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth  The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces Neuroscience, Issue 68, Molecular Biology, Cellular Biology, Biophysics, Microfluidics, Microfluidic culture platform, Compartmented culture, Neuron to glia signaling, neurons, glia, cell culture4448Play ButtonFluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal NeuronsAuthors: Chan-Ying Zheng, Ronald S  Petralia, Ya-Xian Wang, Bechara Kachar  Institutions: National Institutes of Health, Bethesda FRAP has been used to quantify the mobility of GFP-tagged proteins  Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest  The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest  The mobility of the protein is then analyzed by measuring the fluorescence recovery rate  This technique could be used to characterize protein mobility and turnover rate 
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data Neuroscience, Issue 50, Spine, FRAP, hippocampal neurons, live cell imaging, protein mobility2568Play ButtonPrimary Neuronal Cultures from the Brains of Late Stage Drosophila PupaeAuthors: Beatriz Sicaeros, Jorge M  Campusano, Diane K  O'Dowd  Institutions: University of California, Irvine (UCI) In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae  The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation  The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium  The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated  The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating  Images show live cultures at 2 days  Neurons continue to elaborate processes during the first week in culture  Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as GFP or RFP  Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses  A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons  These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses 
Context 2: Vitamin K - Wikipedia
(Redirected from Vitamin k)
This article needs more medical references for verification or relies too heavily on primary sources  Please review the contents of the article and add the appropriate references if you can  Unsourced or poorly sourced material may be challenged and removed  (November 2015)
This article is about the family of vitamers  For vitamin K1 the form usually used as a supplement, see Phytomenadione 
Vitamin K structures  MK-4 and MK-7 are both subtypes of K2 
Vitamin K deficiency, Warfarin overdose
Vitamin K is a group of structurally similar, fat-soluble vitamins the human body requires for complete synthesis of certain proteins that are prerequisites for blood coagulation and which the body also needs for controlling binding of calcium in bones and other tissues  The vitamin K-related modification of the proteins allows them to bind calcium ions, which they cannot do otherwise  Without vitamin K, blood coagulation is seriously impaired, and uncontrolled bleeding occurs  Low levels of vitamin K also weaken bones and promote calcification of arteries and other soft tissues[citation needed] 
Chemically, the vitamin K family comprises 2-methyl-1,4-naphthoquinone (3-) derivatives  Vitamin K includes two natural vitamers: vitamin K1 and vitamin K2 [1] Vitamin K2, in turn, consists of a number of related chemical subtypes, with differing lengths of carbon side chains made of isoprenoid groups of atoms 
Vitamin K1, also known as phylloquinone, is made by plants, and is found in highest amounts in green leafy vegetables because it is directly involved in photosynthesis  It may be thought of as the plant form of vitamin K  It is active as a vitamin in animals and performs the classic functions of vitamin K, including its activity in the production of blood-clotting proteins  Animals may also convert it to vitamin K2 
Bacteria in the gut flora can also convert K1 into vitamin K2  In addition, bacteria typically lengthen the isoprenoid side chain of vitamin K2 to produce a range of vitamin K2 forms, most notably the MK-7 to MK-11 homologues of vitamin K2  All forms of K2 other than MK-4 can only be produced by bacteria, which use these forms in anaerobic respiration  The MK-7 and other bacterially derived forms of vitamin K2 exhibit vitamin K activity in animals, but MK-7's extra utility over MK-4, if any, is unclear and is a matter of investigation 
Three synthetic types of vitamin K are known: vitamins K3, K4, and K5  Although the natural K1 and all K2 homologues and synthetic K4 and K5 have proven nontoxic, the synthetic form K3 (menadione) has shown toxicity [2]
1 2 Cardiovascular health
1 4 Coumarin poisoning
4 1 Conversion of vitamin K1 to vitamin K2
4 2 Vitamin K2
6 Absorption and dietary need
7 Dietary reference intake
10 Biochemistry
10 1 Function in animals
10 2 Gamma-carboxyglutamate proteins
10 3 Methods of assessment
10 4 Function in bacteria
11 Injection in newborns
11 3 Controversy
A review of 2014 concluded that there is positive evidence that monotherapy using MK-4, one of the forms of Vitamin K2, reduces fracture incidence in post-menopausal women with osteoporosis, and suggested further research on the combined use of MK-4 with bisphosphonates  In contrast, an earlier review article of 2013 concluded that there is no good evidence that vitamin K supplementation helps prevent osteoporosis or fractures in postmenopausal women [3]
A Cochrane systematic review of 2006 suggested that supplementation with Vitamin K1 and with MK4 reduces bone loss; in particular, a strong effect of MK-4 on incident fractures among Japanese patients was emphasized [4]
A review article of 2016 suggested to consider, as one of several measures for bone health, increasing the intake of foods rich in vitamins K1 and K2 [5]
Cardiovascular health[edit]
Adequate intake of vitamin K is associated with the inhibition of arterial calcification and stiffening,[6] but there have been few interventional studies and no good evidence that vitamin K supplementation is of any benefit in the primary prevention of cardiovascular disease [7]
One 10-year population study, the Rotterdam Study, did show a clear and significant inverse relationship between the highest intake levels of menaquinone (mainly MK-4 from eggs and meat, and MK-8 and MK-9 from cheese) and cardiovascular disease and all-cause mortality in older men and women [8]
Vitamin K has been promoted in supplement form with claims it can slow tumor growth; there is however no good medical evidence that supports such claims [9]
Coumarin poisoning[edit]
Vitamin K is part of the suggested treatment regime for poisoning by rodenticide (coumarin poisoning) [10]
Although allergic reaction from supplementation is possible, no known toxicity is associated with high doses of the phylloquinone (vitamin K1) or menaquinone (vitamin K2) forms of vitamin K, so no tolerable upper intake level (UL) has been set [11]
Blood clotting (coagulation) studies in humans using 45 mg per day of vitamin K2 (as MK-4)[12] and even up to 135 mg per day (45 mg three times daily) of K2 (as MK-4),[13] showed no increase in blood clot risk  Even doses in rats as high as 250 mg/kg, body weight did not alter the tendency for blood-clot formation to occur [14]
Unlike the safe natural forms of vitamin K1 and vitamin K2 and their various isomers, a synthetic form of vitamin K, vitamin K3 (menadione), is demonstrably toxic at high levels  The U S  FDA has banned this form from over-the-counter sale in the United States because large doses have been shown to cause allergic reactions, hemolytic anemia, and cytotoxicity in liver cells [2]
Phylloquinone (K1)[15][16] or menaquinone (K2) are capable of reversing the anticoagulant activity of the anticoagulant warfarin (tradename Coumadin)  Warfarin works by blocking recycling of vitamin K, so that the body and tissues have lower levels of active vitamin K, and thus a deficiency of vitamin K 
Supplemental vitamin K (for which oral dosing is often more active than injectable dosing in human adults) reverses the vitamin K deficiency caused by warfarin, and therefore reduces the intended anticoagulant action of warfarin and related drugs [17] Sometimes small amounts of vitamin K are given orally to patients taking warfarin so that the action of the drug is more predictable [17] The proper anticoagulant action of the drug is a function of vitamin K intake and drug dose, and due to differing absorption must be individualized for each patient [citation needed] The action of warfarin and vitamin K both require two to five days after dosing to have maximum effect, and neither warfarin or vitamin K shows much effect in the first 24 hours after they are given [18]
The newer anticoagulants dabigatran and rivaroxaban have different mechanisms of action that do not interact with vitamin K, and may be taken with supplemental vitamin K [19][20]
Vitamin K2 (menaquinone)  In menaquinone, the side chain is composed of a varying number of isoprenoid residues  The most common number of these residues is four, since animal enzymes normally produce menaquinone-4 from plant phylloquinone 
A sample of phytomenadione for injection, also called phylloquinone
The three synthetic forms of vitamin K are vitamins K3 (menadione), K4, and K5, which are used in many areas, including the pet food industry (vitamin K3) and to inhibit fungal growth (vitamin K5) [21]
Conversion of vitamin K1 to vitamin K2[edit]
Vitamin K1 (phylloquinone) – both forms of the vitamin contain a functional naphthoquinone ring and an aliphatic side chain  Phylloquinone has a phytyl side chain 
The MK-4 form of vitamin K2 is produced by conversion of vitamin K1 in the testes, pancreas, and arterial walls [22] While major questions still surround the biochemical pathway for this transformation, the conversion is not dependent on gut bacteria, as it occurs in germ-free rats[23][24] and in parenterally-administered K1 in rats [25][26] In fact, tissues that accumulate high amounts of MK-4 have a remarkable capacity to convert up to 90% of the available K1 into MK-4 [27][28] There is evidence that the conversion proceeds by removal of the phytyl tail of K1 to produce menadione as an intermediate, which is then condensed with an activated geranylgeranyl moiety (see also prenylation) to produce vitamin K2 in the MK-4 (menatetrione) form [29]
Vitamin K2[edit]
Main article: Vitamin K2
Vitamin K2 (menaquinone) includes several subtypes  The two subtypes most studied are menaquinone-4 (menatetrenone, MK-4) and menaquinone-7 (MK-7) 
Vitamin K1, the precursor of most vitamin K in nature, is a stereoisomer of phylloquinone, an important chemical in green plants, where it functions as an electron acceptor in photosystem I during photosynthesis  For this reason, vitamin K1 is found in large quantities in the photosynthetic tissues of plants (green leaves, and dark green leafy vegetables such as romaine lettuce, kale and spinach), but it occurs in far smaller quantities in other plant tissues (roots, fruits, etc )  Iceberg lettuce contains relatively little  The function of phylloquinone in plants appears to have no resemblance to its later metabolic and biochemical function (as "vitamin K") in animals, where it performs a completely different biochemical reaction 
Vitamin K (in animals) is involved in the carboxylation of certain glutamate residues in proteins to form gamma-carboxyglutamate (Gla) residues  The modified residues are often (but not always) situated within specific protein domains called Gla domains  Gla residues are usually involved in binding calcium, and are essential for the biological activity of all known Gla proteins [30]
At this time[update], 17 human proteins with Gla domains have been discovered, and they play key roles in the regulation of three physiological processes:
Blood coagulation: prothrombin (factor II), factors VII, IX, and X, and proteins C, S, and Z[31]
Bone metabolism: osteocalcin, also called bone Gla protein (BGP), matrix Gla protein (MGP),[32] periostin,[33] and the recently discovered Gla-rich protein (GRP) [34][35]
Vascular biology: growth arrest-specific protein 6 (Gas6)[36]
Unknown function: proline-rich γ-carboxyglutamyl proteins (PRGPs) 1 and 2, and transmembrane γ-carboxy glutamyl proteins (TMGs) 3 and 4 [37]
Like other lipid-soluble vitamins (A, D and E), vitamin K is stored in the fatty tissue of the human body 
Absorption and dietary need[edit]
Previous theory held that dietary deficiency is extremely rare unless the small intestine was heavily damaged, resulting in malabsorption of the molecule  Another at-risk group for deficiency were those subject to decreased production of K2 by normal intestinal microbiota, as seen in broad spectrum antibiotic use [38] Taking broad-spectrum antibiotics can reduce vitamin K production in the gut by nearly 74% in people compared with those not taking these antibiotics [39] Diets low in vitamin K also decrease the body's vitamin K concentration [40] Those with chronic kidney disease are at risk for vitamin K deficiency, as well as vitamin D deficiency, and particularly those with the apoE4 genotype [41] Additionally, in the elderly there is a reduction in vitamin K2 production [42]
The National Academy of Medicine (NAM) updated an estimate of what constitutes an adequate intake (AI) for vitamin K in 2001  The NAM does not distinguish between K1 and K2 – both are counted as vitamin K  At that time there was not sufficient evidence to set the more rigorous estimated average requirement (EAR) or recommended dietary allowance (RDA) given for most of the essential vitamins and minerals  The current daily AIs for vitamin K for adult women and men are 90 μg and 120 μg respectively  The AI for pregnancy and lactation is 90 μg  For infants up to 12 months the AI is 2–2 5 μg, and for children aged 1 to 18 years the AI increases with age from 30 to 75 μg  As for safety, the FNB also sets tolerable upper intake levels (known as ULs) for vitamins and minerals when evidence is sufficient  In the case of vitamin K no UL is set, as evidence for adverse effects is not sufficient  Collectively EARs, RDAs, AIs and ULs are referred to as dietary reference intakes [43] The European Food Safety Authority reviewed the same safety question and did not set an UL [44]
For U S  food and dietary supplement labeling purposes, the amount in a serving is expressed as a percentage of daily value (%DV)  For vitamin K labeling purposes the daily value was 80 μg, but as of May 2016 it has been revised upwards to 120 μg  A table of the pre-change adult daily values is provided at reference daily intake  Food and supplement companies have until 28 July 2018 to comply with the change 
See also: Vitamin K2 § Dietary sources
K1 (μg)[45]
Kale, cooked
Collards, cooked
Collards, raw
Swiss chard, cooked
Swiss chard, raw
Turnip greens, raw
Romaine lettuce, raw
Table from "Important information to know when you are taking: Warfarin (Coumadin) and Vitamin K", Clinical Center, National Institutes of Health Drug Nutrient Interaction Task Force [46]
Vitamin K1 is found chiefly in leafy green vegetables such as dandelion greens (which contain 778 4 μg per 100 g, or 741% of the recommended daily amount), spinach, swiss chard, lettuce and Brassica vegetables (such as cabbage, kale, cauliflower, broccoli, and brussels sprouts) and often the absorption is greater when accompanied by fats such as butter or oils; some fruits, such as avocados, kiwifruit and grapes, are also high in vitamin K  By way of reference, two tablespoons of parsley contains 153% of the recommended daily amount of vitamin K [47] Some vegetable oils, notably soybean oil, contain vitamin K, but at levels that would require relatively large calorie consumption to meet the USDA-recommended levels [48] colonic bacteria synthesize a significant portion of humans' vitamin K needs; newborns often receive a vitamin K shot at birth to tide them over until their colons become colonized at five to seven days of age from the consumption of breast milk 
The tight binding of vitamin K1 to thylakoid membranes in chloroplasts makes it less bioavailable  For example, cooked spinach has a 5% bioavailability of phylloquinone, however, fat added to it increases bioavailability to 13% due to the increased solubility of vitamin K in fat [49]
Main article: Vitamin K deficiency
Average diets are usually not lacking in vitamin K, and primary deficiency is rare in healthy adults  Newborn infants are at an increased risk of deficiency  Other populations with an increased prevalence of vitamin K deficiency include those who suffer from liver damage or disease (e g  alcoholics), cystic fibrosis, or inflammatory bowel diseases, or have recently had abdominal surgeries  Secondary vitamin K deficiency can occur in people with bulimia, those on stringent diets, and those taking anticoagulants  Other drugs associated with vitamin K deficiency include salicylates, barbiturates, and cefamandole, although the mechanisms are still unknown  Vitamin K1 deficiency can result in coagulopathy, a bleeding disorder [50]Symptoms of K1 deficiency include anemia, bruising, nosebleeds and bleeding of the gums in both sexes, and heavy menstrual bleeding in women 
Osteoporosis[51][52] and coronary heart disease[53][54] are strongly associated with lower levels of K2 (menaquinone)  Vitamin K2 (as menaquinones MK-4 through MK-10) intake level is inversely related to severe aortic calcification and all-cause mortality [8]
Function in animals[edit]
Mechanism of action of vitamin K1 
The function of vitamin K2 in the animal cell is to add a carboxylic acid functional group to a glutamate (Glu) amino acid residue in a protein, to form a gamma-carboxyglutamate (Gla) residue  This is a somewhat uncommon posttranslational modification of the protein, which is then known as a "Gla protein"  The presence of two −COOH (carboxylic acid) groups on the same carbon in the gamma-carboxyglutamate residue allows it to chelate calcium ions  The binding of calcium ions in this way very often triggers the function or binding of Gla-protein enzymes, such as the so-called vitamin K-dependent clotting factors discussed below 
Within the cell, vitamin K undergoes electron reduction to a reduced form called vitamin K hydroquinone, catalyzed by the enzyme vitamin K epoxide reductase (VKOR) [55] Another enzyme then oxidizes vitamin K hydroquinone to allow carboxylation of Glu to Gla; this enzyme is called gamma-glutamyl carboxylase[56][57] or the vitamin K-dependent carboxylase  The carboxylation reaction only proceeds if the carboxylase enzyme is able to oxidize vitamin K hydroquinone to vitamin K epoxide at the same time  The carboxylation and epoxidation reactions are said to be coupled  Vitamin K epoxide is then reconverted to vitamin K by VKOR  The reduction and subsequent reoxidation of vitamin K coupled with carboxylation of Glu is called the vitamin K cycle [58] Humans are rarely deficient in vitamin K1 because, in part, vitamin K1 is continuously recycled in cells [59]
Warfarin and other 4-hydroxycoumarins block the action of VKOR [60] This results in decreased concentrations of vitamin K and vitamin K hydroquinone in tissues, such that the carboxylation reaction catalyzed by the glutamyl carboxylase is inefficient  This results in the production of clotting factors with inadequate Gla  Without Gla on the amino termini of these factors, they no longer bind stably to the blood vessel endothelium and cannot activate clotting to allow formation of a clot during tissue injury  As it is impossible to predict what dose of warfarin will give the desired degree of clotting suppression, warfarin treatment must be carefully monitored to avoid overdose 
Gamma-carboxyglutamate proteins[edit]
Main article: Gla domain
The following human Gla-containing proteins ("Gla proteins") have been characterized to the level of primary structure: blood coagulation factors II (prothrombin), VII, IX, and X, anticoagulant proteins C and S, and the factor X-targeting protein Z  The bone Gla protein osteocalcin, the calcification-inhibiting matrix Gla protein (MGP), the cell growth regulating growth arrest specific gene 6 protein (Gas6), and the four transmembrane Gla proteins (TMGPs), the function of which is at present unknown  Gas6 can function as a growth factor to activate the Axl receptor tyrosine kinase and stimulate cell proliferation or prevent apoptosis in some cells  In all cases in which their function was known, the presence of the Gla residues in these proteins turned out to be essential for functional activity 
Gla proteins are known to occur in a wide variety of vertebrates: mammals, birds, reptiles, and fish  The venom of a number of Australian snakes acts by activating the human blood-clotting system  In some cases, activation is accomplished by snake Gla-containing enzymes that bind to the endothelium of human blood vessels and catalyze the conversion of procoagulant clotting factors into activated ones, leading to unwanted and potentially deadly clotting 
Another interesting class of invertebrate Gla-containing proteins is synthesized by the fish-hunting snail Conus geographus [61] These snails produce a venom containing hundreds of neuroactive peptides, or conotoxins, which is sufficiently toxic to kill an adult human  Several of the conotoxins contain two to five Gla residues [62]
Methods of assessment[edit]
Vitamin K status can be assessed by:
The prothrombin time (PT) test measures the time required for blood to clot  A blood sample is mixed with citric acid and put in a fibrometer; delayed clot formation indicates a deficiency  This test is insensitive to mild deficiency, as the values do not change until the concentration of prothrombin in the blood has declined by at least 50% [63]
Undercarboxylated prothrombin (PIVKA-II); in a study of 53 newborns, found "PT (prothrombin time) is a less sensitive marker than PIVKA II",[64] and as indicated above, PT is unable to detect subclinical deficiencies that can be detected with PIVKA-II testing 
Plasma phylloquinone was found to be positively correlated with phylloquinone intake in elderly British women, but not men,[65] but an article by Schurgers et al  reported no correlation between FFQ[further explanation needed] and plasma phylloquinone [66]
Urinary γ-carboxyglutamic acid responds to changes in dietary vitamin K intake  Several days are required before any change can be observed  In a study by Booth et al , increases of phylloquinone intakes from 100 μg to between 377 and 417 μg for five days did not induce a significant change  Response may be age-specific [67]
Undercarboxylated osteocalcin (UcOc) levels have been inversely correlated with stores of vitamin K[68] and bone strength in developing rat tibiae  Another study following 78 post-menopausal Korean women found a supplement regimen of vitamins K and D, and calcium, but not a regimen of vitamin D and calcium, was inversely correlated with reduced UcOc levels [69]
Function in bacteria[edit]
Many bacteria, such as Escherichia coli found in the large intestine, can synthesize vitamin K2 (menaquinone-7 or MK-7, up to MK-11),[70] but not vitamin K1 (phylloquinone)  In these bacteria, menaquinone transfers two electrons between two different small molecules, during oxygen-independent metabolic energy production processes (anaerobic respiration) [71] For example, a small molecule with an excess of electrons (also called an electron donor) such as lactate, formate, or NADH, with the help of an enzyme, passes two electrons to menaquinone  The menaquinone, with the help of another enzyme, then transfers these two electrons to a suitable oxidant, such fumarate or nitrate (also called an electron acceptor)  Adding two electrons to fumarate or nitrate converts the molecule to succinate or nitrite plus water, respectively 
Some of these reactions generate a cellular energy source, ATP, in a manner similar to eukaryotic cell aerobic respiration, except the final electron acceptor is not molecular oxygen, but fumarate or nitrate  In aerobic respiration, the final oxidant is molecular oxygen (O2), which accepts four electrons from an electron donor such as NADH to be converted to water  E  coli, as facultative anaerobes, can carry out both aerobic respiration and menaquinone-mediated anaerobic respiration 
Injection in newborns[edit]
The blood clotting factors of newborn babies are roughly 30–60% that of adult values; this may be due to the reduced synthesis of precursor proteins and the sterility of their guts  Human milk contains 1–4 μg/L of vitamin K1, while formula-derived milk can contain up to 100 μg/L in supplemented formulas  Vitamin K2 concentrations in human milk appear to be much lower than those of vitamin K1  Occurrence of vitamin K deficiency bleeding in the first week of the infant's life is estimated at 0 25–1 7%, with a prevalence of 2–10 cases per 100,000 births [72] Premature babies have even lower levels of the vitamin, so they are at a higher risk from this deficiency 
Bleeding in infants due to vitamin K deficiency can be severe, leading to hospitalization, blood transfusions, brain damage, and death  Supplementation can prevent most cases of vitamin K deficiency bleeding in the newborn  Intramuscular administration is more effective in preventing late vitamin K deficiency bleeding than oral administration [73][74]
As a result of the occurrences of vitamin K deficiency bleeding, the Committee on Nutrition of the American Academy of Pediatrics has recommended 0 5–1 mg of vitamin K1 be administered to all newborns shortly after birth [74]
In the UK vitamin K supplementation is recommended for all newborns within the first 24 hours [75] This is usually given as a single intramuscular injection of 1 mg shortly after birth but as a second-line option can be given by three oral doses over the first month [76]
Controversy arose in the early 1990s regarding this practice, when two studies suggested a relationship between parenteral administration of vitamin K and childhood cancer,[77] however, poor methods and small sample sizes led to the discrediting of these studies, and a review of the evidence published in 2000 by Ross and Davies found no link between the two [78] Doctors reported emerging concerns in 2013,[79] after treating children for serious bleeding problems  They cited lack-of newborn vitamin K administration, as the reason that the problems occurred, and recommended that breastfed babies could have an increased risk unless they receive a preventative dose 
In the early 1930s, Danish scientist Henrik Dam investigated the role of cholesterol by feeding chickens a cholesterol-depleted diet [80] He initially replicated experiments reported by scientists at the Ontario Agricultural College (OAC) [81] McFarlane, Graham and Richardson, working on the chick feed program at OAC, had used chloroform to remove all fat from chick chow  They noticed that chicks fed only fat-depleted chow developed hemorrhages and started bleeding from tag sites [82] Dam found that these defects could not be restored by adding purified cholesterol to the diet  It appeared that – together with the cholesterol – a second compound had been extracted from the food, and this compound was called the coagulation vitamin  The new vitamin received the letter K because the initial discoveries were reported in a German journal, in which it was designated as Koagulationsvitamin  Edward Adelbert Doisy of Saint Louis University did much of the research that led to the discovery of the structure and chemical nature of vitamin K [83] Dam and Doisy shared the 1943 Nobel Prize for medicine for their work on vitamin K (K1 and K2) published in 1939  Several laboratories synthesized the compound(s) in 1939 [84]
For several decades, the vitamin K-deficient chick model was the only method of quantifying vitamin K in various foods: the chicks were made vitamin K-deficient and subsequently fed with known amounts of vitamin K-containing food  The extent to which blood coagulation was restored by the diet was taken as a measure for its vitamin K content  Three groups of physicians independently found this: Biochemical Institute, University of Copenhagen (Dam and Johannes Glavind), University of Iowa Department of Pathology (Emory Warner, Kenneth Brinkhous, and Harry Pratt Smith), and the Mayo Clinic (Hugh Butt, Albert Snell, and Arnold Osterberg) [85]
The first published report of successful treatment with vitamin K of life-threatening hemorrhage in a jaundiced patient with prothrombin deficiency was made in 1938 by Smith, Warner, and Brinkhous [86]
The precise function of vitamin K was not discovered until 1974, when three laboratories (Stenflo et al ,[87] Nelsestuen et al ,[88] and Magnusson et al [89]) isolated the vitamin K-dependent coagulation factor prothrombin (factor II) from cows that received a high dose of a vitamin K antagonist, warfarin  It was shown that, while warfarin-treated cows had a form of prothrombin that contained 10 glutamate (Glu) amino acid residues near the amino terminus of this protein, the normal (untreated) cows contained 10 unusual residues that were chemically identified as γ-carboxyglutamate (Gla)  The extra carboxyl group in Gla made clear that vitamin K plays a role in a carboxylation reaction during which Glu is converted into Gla 
The biochemistry of how vitamin K is used to convert Glu to Gla has been elucidated over the past thirty years in academic laboratories throughout the world 
^ "Vitamin K Overview"  University of Maryland Medical Center  ^ a b Higdon, Jane (Feb 2008)  "Vitamin K"  Linus Pauling Institute, Oregon State University  Retrieved 12 Apr 2008  ^ Hamidi, M  S ; Gajic-Veljanoski, O ; Cheung, A  M  (2013)  "Vitamin K and bone health"  Journal of Clinical Densitometry (Review)  16 (4): 409–413  doi:10 1016/j jocd 2013 08 017  PMID 24090644  ^ Cockayne, S ; Adamson, J ; Lanham-New, S ; Shearer, M  J ; Gilbody, S; Torgerson, D  J  (Jun 2006)  "Vitamin K and the prevention of fractures: systematic review and meta-analysis of randomized controlled trials"  Archives of Internal Medicine (Review)  166 (12): 1256–1261  doi:10 1001/archinte 166 12 1256  PMID 16801507  ^ O'Keefe, J  H ; Bergman, N ; Carrera Bastos, P ; Fontes Villalba, M ; Di Nicolantonio, J  J ; Cordain, L  (2016)  "Nutritional strategies for skeletal and cardiovascular health: hard bones, soft arteries, rather than vice versa"  Open Heart (Review)  3 (1): e000325  doi:10 1136/openhrt-2015-000325  PMC 4809188  PMID 27042317  ^ Maresz, K  (Feb 2015)  "Proper Calcium Use: Vitamin K2 as a Promoter of Bone and Cardiovascular Health"  Integrative Medicine (Review)  14 (1): 34–39  PMC 4566462  PMID 26770129  ^ Hartley, L ; Clar, C ; Ghannam, O ; Flowers, N ; Stranges, S ; Rees, K  (Sep 2015)  "Vitamin K for the primary prevention of cardiovascular disease"  The Cochrane Database of Systematic Reviews (Systematic review)  9 (9): CD011148  doi:10 1002/14651858 CD011148 pub2  PMID 26389791  ^ a b Geleijnse, J  M ; Vermeer, C ; Grobbee, D  E ; Schurgers, L  J ; Knapen, M  H ; van der Meer, I  M ; Hofman, A ; Witteman, J  C  (Nov 2004)  "Dietary intake of menaquinone is associated with a reduced risk of coronary heart disease: the Rotterdam Study"  Journal of Nutrition  134 (11): 3100–3105  PMID 15514282  ^ Ades, T  B , ed  (2009)  "Vitamin K"  American Cancer Society Complete Guide to Complementary and Alternative Cancer Therapies (2nd ed )  American Cancer Society  pp  558–563  ISBN 978-0-944235-71-3  ^ Lung, D  (Dec 2015)  Tarabar, A , ed  "Rodenticide Toxicity Treatment & Management"  Medscape  WebMD  ^ Rasmussen, S  E ; Andersen, N  L ; Dragsted, L  O ; Larsen, J  C  (Mar 2006)  "A safe strategy for addition of vitamins and minerals to foods"  European Journal of Nutrition  45 (3): 123–135  doi:10 1007/s00394-005-0580-9  PMID 16200467  ^ Ushiroyama, T ; Ikeda, A ; Ueki, M (Mar 2002)  "Effect of continuous combined therapy with vitamin K2 and vitamin D3 on bone mineral density and coagulofibrinolysis function in postmenopausal women"  Maturitas  41 (3): 211–221  doi:10 1016/S0378-5122(01)00275-4  PMID 11886767  ^ Asakura, H ; Myou, S ; Ontachi, Y ; Mizutani, T ; Kato, M ; Saito, M ; Morishita, E ; Yamazaki, M ; Nakao, S  (Dec 2001)  "Vitamin K administration to elderly patients with osteoporosis induces no hemostatic activation, even in those with suspected vitamin K deficiency"  Osteoporosis International  12 (12): 996–1000  doi:10 1007/s001980170007  PMID 11846334  ^ Ronden, J  E ; Groenen-van Dooren, M  M ; Hornstra, G ; Vermeer, C  (Jul 1997)  "Modulation of arterial thrombosis tendency in rats by vitamin K and its side chains"  Atherosclerosis  132 (1): 61–67  doi:10 1016/S0021-9150(97)00087-7  PMID 9247360  ^ Ansell, J ; Hirsh, J ; Poller, L ; Bussey, H ; Jacobson, A ; Hylek, E (Sep 2004)  "The pharmacology and management of the vitamin K antagonists: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy"  Chest  126 (3 Suppl ): 204S–233S  doi:10 1378/chest 126 3_suppl 204S  PMID 15383473  ^ Crowther, M  A ; Douketis, J  D ; Schnurr, T ; Steidl, L ; Mera, V ; Ultori, C ; Venco, A ; Ageno, W  (Aug 2002)  "Oral vitamin K lowers the international normalized ratio more rapidly than subcutaneous vitamin K in the treatment of warfarin-associated coagulopathy  A randomized, controlled trial"  Annals of Internal Medicine  137 (4): 251–254  doi:10 7326/0003-4819-137-4-200208200-00009  PMID 12186515  ^ a b "Important Information to Know When You Are Taking: Warfarin (Coumadin) and Vitamin K" (PDF)  National Institute of Health Clinical Center Drug-Nutrient Interaction Task Force  Retrieved 17 Apr 2015  ^ "Guidelines For Warfarin Reversal With Vitamin K" (PDF)  American Society of Health-System Pharmacists  Retrieved 17 Apr 2015  ^ "Pradaxa Drug Interactions"  Pradaxapro com  19 Mar 2012  Retrieved 21 Apr 2013  ^ Bauersachs, R ; Berkowitz, S  D ; Brenner, B ; Buller, H  R ; Decousus, H ; Gallus, A  S ; Lensing, A  W ; Misselwitz, F ; Prins, M  H ; Raskob, G  E ; Segers, A ; Verhamme, P ; Wells, P ; Agnelli, G ; Bounameaux, H ; Cohen, A ; Davidson, B  L ; Piovella, F ; Schellong, S  (Dec 2010)  "Oral rivaroxaban for symptomatic venous thromboembolism"  New England Journal of Medicine  363 (26): 2499–2510  doi:10 1056/NEJMoa1007903  PMID 21128814  ^ McGee, W  (1 Feb 2007)  "Vitamin K"  MedlinePlus  Retrieved 2 Apr 2009  ^ Shearer, M  J ; Newman, P  (Oct 2008)  "Metabolism and cell biology of vitamin K"  Thrombosis and Haemostasis  100 (4): 530–547  doi:10 1160/TH08-03-0147  PMID 18841274  ^ Davidson, R  T ; Foley, A  L ; Engelke, J  A ; Suttie, J  W  (Feb 1998)  "Conversion of dietary phylloquinone to tissue menaquinone-4 in rats is not dependent on gut bacteria"  Journal of Nutrition  128 (2): 220–223  PMID 9446847  ^ Ronden, J  E ; Drittij-Reijnders, M  J ; Vermeer, C ; Thijssen, H  H  (Jan 1998)  "Intestinal flora is not an intermediate in the phylloquinone–menaquinone-4 conversion in the rat"  Biochimica et Biophysica Acta  1379 (1): 69–75  doi:10 1016/S0304-4165(97)00089-5  PMID 9468334  ^ Thijssen, H   H ; Drittij-Reijnders, M  J  (Sep 1994)  "Vitamin K distribution in rat tissues: dietary phylloquinone is a source of tissue menaquinone-4"  The British Journal of Nutrition  72 (3): 415–425  doi:10 1079/BJN19940043  PMID 7947656  ^ Will, B  H ; Usui, Y ; Suttie, J  W  (Dec 1992)  "Comparative metabolism and requirement of vitamin K in chicks and rats"  Journal of Nutrition  122 (12): 2354–2360  PMID 1453219  ^ Davidson, R  T ; Foley, A  L ; Engelke, J  A ; Suttie, J  W  (Feb 1998)  "Conversion of dietary phylloquinone to tissue menaquinone-4 in rats is not dependent on gut bacteria"  Journal of Nutrition  128 (2): 220–223  PMID 9446847  ^ Ronden, J  E ; Drittij-Reijnders, M  J ; Vermeer, C ; Thijssen, H  H  (Jan 1998)  "Intestinal flora is not an intermediate in the phylloquinone-menaquinone-4 conversion in the rat"  Biochimica et Biophysica Acta  1379 (1): 69–75  doi:10 1016/S0304-4165(97)00089-5  PMID 9468334  ^ Al Rajabi, Ala (2011)  The Enzymatic Conversion of Phylloquinone to Menaquinone-4 (PhD thesis)  Tufts University, Friedman School of Nutrition Science and Policy  ^ Furie, B ; Bouchard, B  A ; Furie, B  C  (Mar 1999)  "Vitamin K-dependent biosynthesis of gamma-carboxyglutamic acid"  Blood  93 (6): 1798–1808  PMID 10068650  ^ Mann, K  G  (Aug 1999)  "Biochemistry and physiology of blood coagulation"  Thrombosis and Haemostasis  82 (2): 165–174  PMID 10605701  ^ Price, P  A  (1988)  "Role of vitamin-K-dependent proteins in bone metabolism"  Annual Review of Nutrition  8: 565–583  doi:10 1146/annurev nu 08 070188 003025  PMID 3060178  ^ Coutu, D  L ; Wu, J  H ; Monette, A ; Rivard, G  E ; Blostein, M  D ; Galipeau, J (Jun 2008)  "Periostin, a member of a novel family of vitamin K-dependent proteins, is expressed by mesenchymal stromal cells"  Journal of Biological Chemistry  283 (26): 17991–18001  doi:10 1074/jbc M708029200  PMID 18450759  ^ Viegas, C  S ; Simes, D  C ; Laizé, V ; Williamson, M  K ; Price, P  A ; Cancela, M  L  (Dec 2008)  "Gla-rich protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates"  Journal of Biological Chemistry  283 (52): 36655–36664  doi:10 1074/jbc M802761200  PMC 2605998  PMID 18836183  ^ Viegas, C  S ; Cavaco, S ; Neves, P  L ; Ferreira, A ; João, A ; Williamson, M  K ; Price, P  A ; Cancela, M  L ; Simes, D  C  (Dec 2009)  "Gla-rich protein is a novel vitamin K-dependent protein present in serum that accumulates at sites of pathological calcifications"  American Journal of Pathology  175 (6): 2288–2298  doi:10 2353/ajpath 2009 090474  PMC 2789615  PMID 19893032  ^ Hafizi, S ; Dahlbäck, B  (Dec 2006)  "Gas6 and protein S  Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily"  The FEBS Journal  273 (23): 5231–5244  doi:10 1111/j 1742-4658 2006 05529 x  PMID 17064312  ^ Kulman, J  D ; Harris, J  E ; Xie, L ; Davie, E  W  (May 2007)  "Proline-rich Gla protein 2 is a cell-surface vitamin K-dependent protein that binds to the transcriptional coactivator Yes-associated protein"  Proceedings of the National Academy of Sciences of the United States of America  104 (21): 8767–8772  doi:10 1073/pnas 0703195104  PMC 1885577  PMID 17502622  ^ "Vitamin K"  MedlinePlus  US National Library of Medicine, National Institutes of Health  Sep 2016  Retrieved 26 May 2009  ^ Conly, J; Stein, K  (Dec 1994)  "Reduction of vitamin K2 concentrations in human liver associated with the use of broad spectrum antimicrobials"  Clinical and Investigative Medicine  17 (6): 531–539  PMID 7895417  ^ Ferland, G ; Sadowski, J  A ; O'Brien, M  E  (Apr 1993)  "Dietary induced subclinical vitamin K deficiency in normal human subjects"  Journal of Clinical Investigation  91 (4): 1761–1768  doi:10 1172/JCI116386  PMC 288156  PMID 8473516  ^ Holden, R  M ; Morton, A  R ; Garland, J  S ; Pavlov, A ; Day, A  G ; Booth, S  L  (Apr 2010)  "Vitamins K and D status in stages 3-5 chronic kidney disease"  Clinical Journal of the American Society of Nephrology  5 (4): 590–597  doi:10 2215/CJN 06420909  PMC 2849681  PMID 20167683  ^ Hodges, S  J ; Pilkington, M  J ; Shearer, M  J ; Bitensky, L ; Chayen, J (Jan 1990)  "Age-related changes in the circulating levels of congeners of vitamin K2, menaquinone-7 and menaquinone-8"  Clinical Science  78 (1): 63–66  PMID 2153497  ^ "Vitamin K"  Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc (PDF)  National Academy Press  2001  p  162–196  ^ Tolerable Upper Intake Levels For Vitamins And Minerals (PDF), European Food Safety Authority, 2006 ^ a b Rhéaume-Bleue, p  42
^ "Important information to know when you are taking: Warfarin (Coumadin) and Vitamin K" (PDF)  National Institutes of Health Clinical Center  ^ "Nutrition Facts and Information for Parsley, raw"  Nutritiondata com  Retrieved 21 Apr 2013  ^ "Nutrition facts, calories in food, labels, nutritional information and analysis"  Nutritiondata com  13 Feb 2008  Retrieved 21 Apr 2013  ^ "Vitamin K"  Vivo colostate edu  2 Jul 1999  Retrieved 21 Apr 2013  ^ "Vitamin K"  Micronutrient Data Centre  ^ Ikeda, Y ; Iki, M ; Morita, A ; Kajita, E ; Kagamimori, S ; Kagawa, Y ; Yoneshima, H  (May 2006)  "Intake of fermented soybeans, natto, is associated with reduced bone loss in postmenopausal women: Japanese Population-Based Osteoporosis (JPOS) Study"  Journal of Nutrition  136 (5): 1323–1328  PMID 16614424  ^ Katsuyama, H ; Ideguchi, S ; Fukunaga, M ; Saijoh, K ; Sunami, S  (Jun 2002)  "Usual dietary intake of fermented soybeans (Natto) is associated with bone mineral density in premenopausal women"  Journal of Nutritional Science and Vitaminology  48 (3): 207–215  doi:10 3177/jnsv 48 207  PMID 12350079  ^ Sano, M ; Fujita, H ; Morita, I ; Uematsu, H ; Murota, S  (Dec 1999)  "Vitamin K2 (menatetrenone) induces iNOS in bovine vascular smooth muscle cells: no relationship between nitric oxide production and gamma-carboxylation"  Journal of Nutritional Science and Vitaminology  45 (6): 711–723  doi:10 3177/jnsv 45 711  PMID 10737225  ^ Gast, G  C ; de Roos, N  M ; Sluijs, I ; Bots, M  L ; Beulens, J  W ; Geleijnse, J  M ; Witteman, J  C ; Grobbee, D  E ; Peeters, P  H ; van der Schouw, Y  T  (Sep 2009)  "A high menaquinone intake reduces the incidence of coronary heart disease"  Nutrition, Metabolism, and Cardiovascular Diseases  19 (7): 504–510  doi:10 1016/j numecd 2008 10 004  PMID 19179058  ^ Oldenburg, J ; Bevans, C  G ; Müller, C  R ; Watzka, M  (2006)  "Vitamin K epoxide reductase complex subunit 1 (VKORC1): the key protein of the vitamin K cycle"  Antioxidants & Redox Signaling  8 (3–4): 347–353  doi:10 1089/ars 2006 8 347  PMID 16677080  ^ Suttie, J  W  (1985)  "Vitamin K-dependent carboxylase"  Annual Review of Biochemistry  54: 459–477  doi:10 1146/annurev bi 54 070185 002331  PMID 3896125  ^ Presnell, S  R ; Stafford, D  W  (Jun 2002)  "The vitamin K-dependent carboxylase"  Thrombosis and Haemostasis  87 (6): 937–946  PMID 12083499  ^ Stafford, D  W  (Aug 2005)  "The vitamin K cycle"  Journal of Thrombosis and Haemostasis  3 (8): 1873–1878  doi:10 1111/j 1538-7836 2005 01419 x  PMID 16102054  ^ Rhéaume-Bleue, p  79 
^ Whitlon, D  S ; Sadowski, J  A ; Suttie, J  W  (Apr 1978)  "Mechanism of coumarin action: significance of vitamin K epoxide reductase inhibition"  Biochemistry  17 (8): 1371–1377  doi:10 1021/bi00601a003  PMID 646989  ^ Terlau, H ; Olivera, B  M  (Jan 2004)  "Conus venoms: a rich source of novel ion channel-targeted peptides"  Physiological Reviews  84 (1): 41–68  doi:10 1152/physrev 00020 2003  PMID 14715910  ^ Buczek, O ; Bulaj, G ; Olivera, BM (Dec 2005)  "Conotoxins and the posttranslational modification of secreted gene products"  Cellular and Molecular Life Sciences  62 (24): 3067–3079  doi:10 1007/s00018-005-5283-0  PMID 16314929  ^ "Prothrombin Time"  WebMD  ^ Dituri, F ; Buonocore, G ; Pietravalle, A ; Naddeo, F ; Cortesi, M; Pasqualetti, P; Tataranno M  L ; R , Agostino (Sep 2012)  "PIVKA-II plasma levels as markers of subclinical vitamin K deficiency in term infants"  Journal of Maternal, Fetal & Neonatal Medicine  25 (9): 1660–1663  doi:10 3109/14767058 2012 657273  PMID 22280352  ^ Thane, C  W ; Bates, C  J ; Shearer, M  J ; Unadkat, N; Harrington, D  J ; Paul, A  A ; Prentice, A ; Bolton-Smith, C  (Jun 2002)  "Plasma phylloquinone (vitamin K1) concentration and its relationship to intake in a national sample of British elderly people"  British Journal of Nutrition  87 (6): 615–622  doi:10 1079/BJNBJN2002582  PMID 12067432  ^ McKeown, N  M ; Jacques, P  F ; Gundberg, C  M ; Peterson, J  W ; Tucker, K  L ; Kiel, D  P ; Wilson, P  W ; Booth, SL (Jun 2002)  "Dietary and nondietary determinants of vitamin K biochemical measures in men and women" (PDF)  Journal of Nutrition  132 (6): 1329–1334  PMID 12042454  ^ Yamano, M ; Yamanaka, Y ; Yasunaga, K ; Uchida, K  (Sep 1989)  "Effect of vitamin K deficiency on urinary gamma-carboxyglutamic acid excretion in rats"  Nihon Ketsueki Gakkai Zasshi  52 (6): 1078–1086  PMID 2588957  ^ Matsumoto, T ; Miyakawa, T ; Yamamoto, D  (Mar 2012)  "Effects of vitamin K on the morphometric and material properties of bone in the tibiae of growing rats"  Metabolism  61 (3): 407–414  doi:10 1016/j metabol 2011 07 018  PMID 21944271  ^ Je, S -H ; Joo, N -S ; Choi, B -H ; Kim, K -M ; Kim, B -T ; Park, S -B ; Cho, D -Y ; Kim, K -N ; Lee, D -J  (Aug 2011)  "Vitamin K supplement along with vitamin D and calcium reduced serum concentration of undercarboxylated osteocalcin while increasing bone mineral density in Korean postmenopausal women over sixty-years-old"  Journal of Korean Medical Science  26 (8): 1093–1098  doi:10 3346/jkms 2011 26 8 1093  PMC 3154347  PMID 21860562  ^ Bentley, R ; Meganathan, R  (Sep 1982)  "Biosynthesis of vitamin K (menaquinone) in bacteria" (PDF)  Microbiological Reviews  46 (3): 241–280  PMC 281544  PMID 6127606  ^ Haddock, B  A ; Jones, C  W  (Mar 1977)  "Bacterial respiration" (PDF)  Bacteriological Reviews  41 (1): 47–99  PMC 413996  PMID 140652  ^ Shearer, M  J  (Jan 1995)  "Vitamin K"  Lancet  345 (8944): 229–234  doi:10 1016/S0140-6736(95)90227-9  PMID 7823718  ^ Greer, J  P ; Foerster, J ; Lukens, J  N ; Rodgers, G  M ; Paraskevas, F ; Glader, B  (eds )  Wintrobe's Clinical Hematology (11th ed )  Philadelphia, Pennsylvania: Lippincott, Williams and Wilkens  ^ a b American Academy of Pediatrics Committee on Fetus Newborn  (Jul 2003)  "Controversies concerning vitamin K and the newborn  American Academy of Pediatrics Committee on Fetus and Newborn" (PDF)  Pediatrics  112 (1 1): 191–192  doi:10 1542/peds 112 1 191  PMID 12837888  ^ Logan, S ; 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Binkley, S  B ; Thayer, S  A ; Doisy, E  A  (1939)  "On the constitution of Vitamin K1"  Journal of the American Chemical Society  61 (7): 1928–1929  doi:10 1021/ja01876a510  ^ Fieser, L  F  (1939)  "Synthesis of Vitamin K1"  Journal of the American Chemical Society  61 (12): 3467–3475  doi:10 1021/ja01267a072  ^ Dam, C  P  H  (12 Dec 1946)  "The discovery of vitamin K, its biological functions and therapeutical application" (PDF)  Nobel Prize lecture  ^ Warner, E  D ; Brinkhous, K  M ; Smith, H  P  (1938)  "Bleeding Tendency of Obstructive Jaundice"  Proceedings of the Society of Experimental Biology and Medicine  37 (4): 628–630  doi:10 3181/00379727-37-9668P  ^ Stenflo, J; Fernlund, P ; Egan, W ; Roepstorff, P  (Jul 1974)  "Vitamin K dependent modifications of glutamic acid residues in prothrombin"  Proceedings of the National Academy of Sciences of the United States of America  71 (7): 2730–2733  doi:10 1073/pnas 71 7 2730  PMC 388542  PMID 4528109  ^ Nelsestuen, G  L ; Zytkovicz, T  H ; Howard, J  B  (Oct 1974)  "The mode of action of vitamin K  Identification of gamma-carboxyglutamic acid as a component of prothrombin" (PDF)  Journal of Biological Chemistry  249 (19): 6347–6350  PMID 4214105  ^ Magnusson, S ; Sottrup-Jensen, L ; Petersen, T  E ; Morris, H  R ; Dell, A  (Aug 1974)  "Primary structure of the vitamin K-dependent part of prothrombin"  FEBS Letters  44 (2): 189–193  doi:10 1016/0014-5793(74)80723-4  PMID 4472513  Bibliography[edit]
Rhéaume-Bleue, Kate (2012)  Vitamin K2 and the Calcium Paradox  John Wiley & Sons, Canada  ISBN 1-118-06572-7  External links[edit]
"Vitamin K: Another Reason to Eat Your Greens"  v
TPP / ThDP (B1)
FMN, FAD (B2)
NAD+, NADH, NADP+, NADPH (B3)
Coenzyme A (B5)
PLP / P5P (B6)
THFA / H4FA, DHFA / H2FA, MTHF (B9)
AdoCbl, MeCbl (B12)
Phylloquinone (K1), Menaquinone (K2)
non-vitamins
Coenzyme B
Heme / Haem (A, B, C, O)
Molybdopterin/Molybdenum cofactor
THMPT / H4MPT
Fe2+, Fe3+
vitamins: see vitamins
Antihemorrhagics (B02)
(coagulation)
Phytomenadione (K1)
Menadione (K3)
intrinsic: IX/Nonacog alfa
VIII/Moroctocog alfa/Turoctocog alfa
extrinsic: VII/Eptacog alfa
common: X
II/Thrombin
I/Fibrinogen
XIII/Catridecacog
combinations: Prothrombin complex concentrate (II, VII, IX, X, protein C and S)
Carbazochrome
thrombopoietin receptor agonist (Romiplostim
Eltrombopag)
Tetragalacturonic acid hydroxymethylester
Epinephrine/Adrenalone
amino acids (Aminocaproic acid
Aminomethylbenzoic acid)
serpins (Aprotinin
Alfa1 antitrypsin
Camostat) 
Context 3: My Aspergers Child: COMMENTS & QUESTIONS [for Feb , 2017]
I emailed you a while back and you mentioned that I could email when I needed to  Thank you  I last wrote you in December that my son became involved in a dispute involving the local police  We have had 3 court dates  It keeps delaying due to not being able to come to an agreement  But the attorney, even though he was just vaguely familiar with Aspergers, has been very good with Craig  He has the compassion and excellence that is needed here  What started out very bad is turning into a good thing  It will probably take another 90 days or more 
But Craig is working hard  Too hard sometimes  He goes to therapy 3 times a week  Doing excellent  He's more focused and can calm down easier  He's got a lot on his plate but has support from his family  From his attorney  From therapy  And from his work 
He has been renting a room from a lady who has a son with ADHD  It is good for him  I'm a little worried though because since she smokes he wants to find his own place  With all the costs he has to balance it out financially  That is good  I can't help him more than I am which is good  He is stepping up and taking responsibility  He is listening much better 
He is going to have an evaluation today to get an accurate diagnosis  I understand that is a little difficult since he is an adult  Also the PTSD may cover it over  The attorney stated it would help to have the diagnosis 
Aware this is a long update, but thanks for reading  I am fighting much guilt still but I have a lot of peace now  My daughter and her 4 year old son also have Aspergers symptoms  So my life chapters may not close for a while  :-)
My name is Mac  I'm sure you're quite busy, so I'll get right to it I just wanted to pass on compliments on My Aspergers Child and your post, How to Implement the GFCF Diet: Tips for Parents of Autistic Children 
Me and my wife absolutely loved it!
I got a facebook message from him today begging to be able to come home saying he misses home and he will change  He says he will follow rules now  I stated to him the simple rules he has to follow which were - No weed in my house, or smoked in my house, coming home at curfew, going to school, no skipping, no drugs at school, and to drop the attitude of I am 17 I can do whatever I want 
I have made it very clear that if I see any drugs in my home I will be calling the police, as well as if I see signs of it being sold by him I will report him  (He has never had selling amounts in my house,     I believe it's being kept at his "friends" which of course I have no proof of    I just know it is not here 
I know my battle is not over by a long shot, I am sure we will have more consequences and possibly another being kicked out, but I am going to think positive and hope that he learned some form of a valuable lesson here 
Thank you so much for the guidance, never in a million years did I ever think I'd be on this side, (the one needing the help, as I am the one who helps )
I am going to go back to the start of the program like I said earlier and keep notes close by for reference 
Thanks for all you do, helping us all with ODD children/teens
I have a small company providing educational support services to a few families who have children with various disabilities in Ohio  One of the families has multiple adopted children of whom several have significant attachment disorders including RAD  As an experienced teacher and foster parent I have some experience in working with children who have extensive trauma backgrounds  However, I could use additional training  Also working with these children are two staff members with minimal background in attachment disorders who would also benefit from training primarily in behavior management  The primary caregiver to the children does a wonderful job managing their needs  In order to further develop team cohesion, I'm hoping to include her in any training as well 
Is it possible to schedule such a training session with you? If so, please let us know what will work for you including time, place, and cost  Thank you for your assistance 
I just listed to your tapes on dealing with an out of control, defiant teen  I'd like to ask your advice on a particular situation we have  Our 15 year old daughter is smoking pot almost every day at school  Because we had no way to control the situation, we told her, fine, go ahead and smoke weed  However, you will no longer receive the same support from us  You will not have your phone, lunch money to go off campus (she has an account at the school for the cafeteria she can use), and you will be grounded until you can pass a drug test  We will not be testing you except for when you tell us you are ready to be tested  She is now saying she's suicidal because she feels so isolated, yet she continues to smoke weed  In fact, she tried to sneak out last night but was foiled by our alarm system  For the particular drug test we have, I read it takes about 10 days of not smoking to pass the test  What would you do? Please advise 
I am having a problem with my 18 year old son, Danny, with high functioning autism  We finally had him diagnosed when he was 16 years old  I always knew something was going on with him but the doctors misdiagnosed him as bipolar  It's been 2 years now and he will not accept his diagnosis  He won't talk about it and when I try to bring it up he gets very angry  I've tried telling him that it's not a bad thing, that there's been many, many very successful people with Aspergers  He won't tell anyone and refuses to learn about managing life with it  He once shared with me that the other kids at school use it as an insult, like saying someone is so autistic when they do something they don't approve of  So he doesn't want anyone to know  He's turned down services that could help him  He has a girlfriend, going on 8 months  He won't tell her and they're having problems arguing a lot and I wonder if it would help for her to know 
I'm sad that he thinks it's a life sentence to something horrible instead of accepting, embracing it and learning about it more so he maybe can understand why he's struggling  I told him that he doesn't need to shout it out to the whole world but he won't even accept it himself 
I don't know how to help him with it and because he's almost 19 I have limited control now  It's made my life easier knowing what we're dealing with and I think his life would be easier is he accepted it 
Please help me help him 
I am a clinical psychologist in NYC who now has several (!!) children I see who have RAD  In 20 years of practice, I’d seen only one case  Now, I have at least three children with this  I have no training, per se, in working with this children though I know about setting structure, consistency, etc  I do a lot of work with parents about parenting  I work primarily within the school setting in a charter school whose mission is to educate children on the autism spectrum in a mainstream setting  We use Michelle Garcia Winner’s social thinking program with our ASD kids  I also work with gen ed kids in the school who are at-risk; the school is in the inner city from where the majority of our non-ASD kids live 
It would have been so much easier to mention to my adult son that I think (I know he does, but want to ease into the subject)
he has Asperger's when we were living together two years ago  He has since moved to Tennessee working in his field of interest
which is 3-D printing and software development  I am so happy for him that he has found his way into a job that he truly enjoys
even though he's socially isolated 
He's not diagnosed and does not know he has it  How I know is his classic symptoms being sensory issues (fabric feeling like sandpaper)
communication difficulties, meltdowns and much more  Throughout his childhood I just felt he was a bit different  Nothing major stood out and time
just passes, misdiagnosis of ADHD, low frustration, etc  We've talked about his ADHD numerous times (which I now know he doesn't have) 
It's so much easier to communicate with him now that I know he has Asperger's  I keep it "slow and low" in talking, with long moments
of silence and then we connect  It's really too bad that Asperger's got a diagnostic code back in the 90's, yet all the so called doctors,
physiologist's, etc, didn't know how to diagnose it  Too bad 
There seems to be no one answer to "should I tell my adult son he has Asperger's" from a few specialists I asked  He is typical Asperger,
complicated, highly intelligent (high IQ), anxiety at times, socially isolated, hard to make friends  Not knowing how he will react is the hard part 
How will he be better off knowing he has it? Do I wait to tell him in person, or ease into it with him over Skype? He likes direct, honest, concrete communication 
Why is this so hard for me? Maybe because no one know's if he is going to be better off knowing or not  Do you know if people are better off
knowing? I try to get up the courage to just let him know, then I back down 
I have been searching the web looking for advice and came upon your site  I am trying to read blogs, websites, books, and articles to help guide me  I was so happy when you said that I could ask you a question  My husband and I are struggling with my 27 year old son who lives with us 
Kyle is the youngest of 4 sons  He is a college graduate but never could find the "right" job  He has always been quiet and never had a lot of friends  Two years ago, his girlfriend broke up with him  Kyle had an online gambling addiction and was using pot all the time  After the breakup, Kyle was very depressed and started using heroin and finally told my husband he was using  He is now seeing a psychiatrist who has him on suboxone and antidepressants  He is also seeing a psychologist weekly for counseling but it does not seem to be helping 
Last October,, Kyle lost his job, got drunk, and was agitated and came home , fighting with us, damaging our home and being verbally abusive  My other son , age 32, who also lives with us called the police and Kyle got arrested  He is currently in the family court system  He went through an anger management course and now is in substance abuse classes  Kyle continues to verbally abusive to me and blame me for everything  He says he "hates me "and calls me terrible names  At times, he pushes my husband and intimidates me  My husband and I are so upset  We just hired an attorney for him because since he has been going to these classes, he is getting more depressed and not getting better  Kyle continues to drink while taking his meds prescribed by the psychiatrist and then he has his "moods " My husband and I have met once with the psychiatrist just to give him background information when Kyle started with him 
At this point, we do not know what to do  We never thought at this stage of our life, we would be supporting and spending our retirement money on adult children  I do not know why Kyle hates me, I could not have been a better mom  My husband and I have no life and just do not know what it the right path we should take  Kyle does not want anything to do with us  He spends all his time in his room playing football online We have tried tough love versus caring and love and understanding  Do you have any advice for me?
This whole ODD and ADHD is killing me as a parent  I work in the field of adult psych and addictions so I am well educated  I have been dealing with my teen being like this for almost 3 years and I totally lost my cool today with my 17-year-old son to the point I told him he is out of the house  He can never simple rules, comes and goes as he pleases sometimes doesn't come home, just recently back in school from several suspension for drug related    I am just so exhausted  He has made me hate life, hate being a parent and sometimes I just feel like not even being here  I bought your program in hopes to it would help, I am at week three and I feel things are getting worse    what am I doing wrong??
My partner hasn't been diagnosed yet but I know he has aspergers   day to day is a struggle   I feel I'm going crazy with how he makes me feel Feel let down constantly  He lies alot but I've been told they can't but I know he does I just feel trapped and unloved We have a 4yr old daughter together and my main worry with how he is that it will effect our daughter ; (his skills as a parent are so weak He can't disapline at all Feel so alone  he hides it well too I just wondered if things will get worse? He's angry so quick in arguments Scares me etc I can't leave as he's the main bread winner and our daughter loves him to bits Don't know why I'm writing this  Sorry if I'm going on and not making sense :(
I wanted to let you know about a research opportunity for children, teens, and young adults with autism  I am studying the effects of Brazilian Jiu Jitsu, and psychotherapy on helping people with autism develop subjective awareness of others 
I am writing you to see if this might help someone in your practice, or to see if you might know of someone with autism who may benefit from participating in this study  The requirements of the study will be:
1  A participant should be between 7-21 years of age and have a diagnosis of Autism Spectrum Disorder 
2  The participant should enroll in an approved Jiu Jitsu Academy and attend at least two sessions a week for a period of six months 
3  The participant should enroll in social skills groups, provided by my office or be in a steady psychotherapeutic relationship in your office, at least once a week, or minimally two to three times a month 
4  The participant will be given a SRS (Social Responsiveness Scale) test at the beginning of the study, at three months, and again at six months 
If you know of anyone who might benefit from this novel approach to helping to develop social awareness in autism, please do not hesitate to contact me for further information 
I have a 10 year old daughter who has outbursts with prolonged crying almost like tantrums that 2 year olds have when they cannot express themselves 
I had her in therapy from age 6-8 years old for the same thing but I feel that the sessions didn't really help much 
She has severe sensitivities to light, sound, vibration, frequencies which trigger irritability and crying 
We changed her diet and tried getting her involved with activities but she is anti-social and prefers reading than being social  She is terrified of change even in daily routine (even that will trigger prolonged crying) 
It frustrates me because I don't know what else to do with her behavior 
I've tried acupuncture (she refused at the first session); she refuses massage too 
She is an honor-roll student at school and has very minimal issues at school but if she has had a bad day it does result in a tantrum or crying and defiance 
How can I get her tested for Asperger's Syndrome?
Last night our 24 year old son with Aspergers told his dad and I that he is pulling out of the 4 college classes that he recetnly enrolled in because he has not been attending class or turning in his assignments  He paid $2800 (his own money) for tuition and I reminded him of this when he told us but it did not seem to bother him 
This is the 3rd time he has started college courses and has not completed them  (He also took some concurrent college classes while he was in high school that he failed)  This is a son who basically had a 4 0 grade point average through 10th grade and got a 34 on the ACT the first time he took it 
With the news that he was once again not sticking with college courses I did not sleep well  When I got up this mornning I began looking online for help in how to deal with his situation  I found your "Launching Adult Children With Aspergers" and purchased it  Most of what is included are things we have done or did with our son throughout his life  I was hoping for more help so I am emailing you now in hopes of more specific ideas 
We noticed some things with our son, Taylor, as a yound child but as we had not heard of Aspergers at that time we just did what we thought would help him  As a toddler and a child at pre-school he generally went off on his own to play  When I talked to his pre-school teacher about my concerns (that I was worried he would end up a hermit) she said she did not see him being a loner and that he seemed to interact fine with others in many situations  We worked with him on making eye contact when talking with others  We explained different emotions in people's faces and mannerisms to help him know how to interact with others  We discussed the fact that people would say things that did not mean what they souneded like - such as "I'm so hungry I could eat a horse"  As we did these things he worked hard to better understand communication with others 
During his 4th grade year I had a teacher from the gifted program ask me if I had ever heard of Aspergers  I told her that I had not heard of it  She proceeded to read me some of the charateristics and so many of them described my son  So we had him tested by the school district during the summer between 4th and 5th grade and they did find that he had Aspergers but that he was high functioning  We then set him up with and EIP which stayed with him until his sophomore year  We pulled him from it at that time because we had moved and the new district was requiring him to take one class a day that was a study class  This reduced the number of required classes he could take and he was doing fine with his studies at the time 
It was during the 2nd half of his Junior year that we noticed some of his grades going down  Then during his Senior year is when he started skipping classes and not doing assignments  We had not realized it before then but we soon became aware that he was addicted to gaming  He would go to the library or somewhere else on campus and play games on the computer rather than go to class  It was also at this time that he began lying about his actions (so as not to get in trouble) 
Based on his grades and his ACT score he received offers from colleges for full tuition scholarships  He chose the college where he had taken concurrent classes during his high school years  But he proceeded to skip class and not turn in assignments so he lost his scholarship and quit attending college  During this time he was only able to find employment through an employment agency where he was mostly sent to manuel labor type jobs (which is not something he enjoys but he did it anyway)  It was during this time that at one place had gone to on numerous occasions he was told if he came late one more time they would tell the emplyment agency they did not want him to come there anymore  (This seemed to make an impression on him because he has continued to be reliable and responsbile at his places of employment) 
At 19 1/2 he left to serve a 2 year full-time mission for our church  He completed his mission successfully  (I don't think it was without some struggle, stress and depression, but he was able to pick himself up and move on from those times) 
When he came home he started working for the employment agency again but began looking for employment elsewhere  He got a job at a local Chick Fil-A where he has worked for 3 years  He started college again shortly after he came home but as before it was short lived  He did finish out the semester but failed most of the classes due to his skipping class and not turning in assignments



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Question: What are the three synthetic types of vitamin K?

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Context 3:  When he skipped class he would usually sleep in his car 
Taylor's life consists of working (where to the best of our knowledge) he does well, he is reliable and his employer likes him  When he comes home from work he either sleeps or plays video games or other games - such as kakuro  He spendes most of his time in the basement where his bedroom is and this is where he games  Taylor owns his own car, bought his own laptop and very rarely spends money  He pays us $200 /month to still live at home, unloads the dishwasher on a regular basis and does the weekly garbage  However, his room is a mess and he only cleans his bathroom when I tell him he needs to clean it 
Taylor used to read quite a bit and loved to learn  It has just been in his adult years that he has not read as much - I think because of his gaming addiction  Taylor goes to church on a regular basis but sleeps through the main meeting  In Sunday class room settings he stays awake - I think because he is able to particpate in discussions 
Taylor has only had 2 real friends since entering Junior High school  And as of now he only keeps in contact with one of them who still lives in Georgia  We have lived in Utah since the summer of 2007 and he has never had a friend to do things with since we have lived here  He has two younger siblings, a brother 22 and a sister 20  They love Taylor and spend time with him when they are home  They are both at college and doing well 
Throughout Taylor's school years he has seen a counsleor on a fairly regular basis  One summer during junior high he attended a weekly class where he interacted with other kids with Aspergers  We did see a lot of change in him from this group  After he returned from his mission he went to see a counselor for a short period - this counselor tried to help him with some social skills  His dad and I went with him the first 3 or 4 times but we found out that after we quit going with him he only went a few more times and then scheduled appointments but did not show a couple of the times  We only found this out when a bill came for a "no show" appointment 
I don't know if this is too much information but were are in dire need of help for him  In the information that we purchased from you you mentioned that you do coaching for Aspergers adults  I don't know if you can help us but I thought I would check with you just in case 
Alas I think I have found your information too late to save my marriage but I am hoping to save myself 
I am currently going through a very very painful separation after a 27 year relationship with my husband whom I am convinced has aspergers syndrome  It is a long and painful story and I am desperately trying to process it all alongside dealing with a very conflictual separation  My partner is angry non communicative and totally dismissive of me and our long shared history 
He walked out last year after I discovered he had been visiting massage parlours and developed a relationship with an illegal Chinese escourt whom he subsequently moved in with  He had been seeing this woman behind my back for over 18 months  The pain of all this indescribable and his dismissal of my pain and very existence beyond belief 
Leading up to this I had been battling anxiety and depression which my husband found very hard to cope with 
Over the years of our relationship I knew something was off but I just could not put my finger on it  I often felt a complete lack of validation and empathy  Communication was also difficult as my husband was defensive and unwilling to look at issues in our marriage 
Please Mark could you help me validate some of this pain and try and make dense of 27 years of my life without drowning in fear guilt and despair about my future 
Thank you for listening and your site 
I have had problems with drunkenness, being late for school, not handing in school work, buying pot from a dealer etc  I chose to focus on the drinking and did the grounding then (grounding happened 3 times)  I also stopped sleep overs at friends 100%  I have stopped handing out money for no reason or even buying treats like chocolate 
I did lose it one evening (and didn't do the poker face) when I was trying to unplug the internet at midnight on a school night (she’s always late for school so I am trying to get her to sleep at a reasonable hour)  I was physically stopped and pushed around so I slapped my daughter (it was not hard)  This ended up with her saying she didn’t want to come home (the next day after school)  By this stage, I also had enough and didn’t go get her  I thought I am not begging  You will run out of money soon  It was quite a relief to have some peace  Daughter’s Dad was in town (from another country) and called a family meeting with the counsellor  To cut a long story short, daughter and her counsellor put it on the table that daughter wants to go live somewhere else (with her friends family) because of the stress at home with me (we live on our own) (i e  stricter rules and her bucking up against it) 
I didn’t really want this but made a compromise that daughter would go there Tues morning – Friday afternoon as the friend is an A student whereas my daughter is failing  They do the same subjects  I made the decision at the end of the day based on what is good for me – some time away from the daughter  I also thought of your book when the child went to live with the grandparents – daughter will dig her own hole over at the friend’s house  They have a week day no going out policy which made me think it is OK  I went and discussed with them the problems experienced (drinking, pot, late nights, not handing in work)
I am also trying to follow the let go of school thing per your book  I find it really difficult to remain calm when I can see daughter on her phone and watching series (when I have her on the weekends) when I know there are projects due  I hired her a private tutor once a week for help with a subject  The tutor has just fired my daughter for not handing in work and being not committed  It’s not the first time private tutoring has not been appreciated  The school give me a report back on a Friday as to whether everything is handed in  The deal is – if the work is not handed in – no pocket money and no Friday night out)  Her school is a "progressive" school and there are no repercussions for her being late or not handing in work  I would change schools if I could but there are only 8 months left of school (she turns 18 in August) 
We have just completed the first week and beginning week two of your material  We are agreeing with your take and see our son and ourselves in most of what you are saying  Prior to finding your material and starting your program we had been having extreme out of control behaviors and had to call the police because he was breaking things in our house and pushed my husband  This happened three weeks ago  After that incident we took away privileges ie  PS4, phone (which had already been taken for a few days), and friends  So, last week while doing your program he already didn’t have privileges and has continued with poor behavior – name calling, throwing things, slamming doors  We are not sure when to give privileges back  He has been given the privilege of playing with friends on occasion  His 13th birthday is tomorrow  This past weekend, for his birthday my husband and he went boar hunting  Of course we debated about it but decided to go ahead since it was his bday  We are cooking some of the meet on the grill tomorrow night for his bday and inviting a couple of his friends over for a cookout  No more gifts other than cards and balloons  We are wondering if we should go ahead and give him his privileges back and not sure how to do it  Last Friday morning we attempted to talk giving him a date to return privileges and that conversation ended with him getting angry but he gathered from our conversation that he is getting his stuff back on his bday  We are starting week 2 assignments today but not sure how to handle what was already in place  Of course, we aren’t seeing the respect and responsibility we are looking for but realize it has been a long time  We were wanting him to pay for his phone and thought it might be a good time to introduce that idea  Allowing him to earn his phone  We expect that he will be angry with this idea and not sure how to implement 
My son and myself are interested in a inpatient Aspergers program  We line in Calif which is preferable  My son is very high functioning and was diagnosed dry late  He was eight years old  He has never been in or attended a full day of class  Partially due to depression,anxiety, and trouble with his ADHD also his aversion and being bullied and of course his Aspergers  He will not attend his freshmen year due to surgery on both Achilles' tendons from walking on his toes  With physical therapy he should be ready by his sophomore year! We all feel he needs in patient therapy to give him the tools on how to work with his issues in a structured setting and a place that will give him tools for the rest of his life 
In my utter desperation to find a way to get some help for my daughter's increasingly challenging behaviour I trawled the internet to see if I could find some strategies that would provide specific methods on dealing with teenagers with Asperger's syndrome  When I came across your website, I couldn't believe that every statement you made was exactly what I have been going through with my daughter  She has just turned 14 last week, and was diagnosed with Asperger's/ Autism Spectrum Disorder 15 months ago  I have already been seeing a child psychologist for the past five months, however the methods she has been advising have not been very effective 
Our main difficulty with our daughter is her overwhelming obsession to use her cell phone (and to a lesser extent her laptop) constantly  Without any restriction, she will be on it every minute of the day, and will be awake until the early hours every day  We have tried to incorporate her input around rules as to when she has to give in her phone, but she is unwilling to compromise on a time that she should give it to us, believing that she should have unlimited use  I believe she is unable to do any adequate study or homework, as she is constantly having to look at the phone  We have tried to put rules in place that she has to give in her phone and laptop on school nights at 22:15  If she is able to do this then she is given rewards, and if she doesn't then she knows that there will be consequences  The consequence has been restricted use the following day  However, this is usually where we fail, because taking her phone away from her results in tantrums, screaming, and even threatening to harm herself  This behaviour is relentless to the point where the whole family becomes deeply distressed, and inevitably results in her getting the phone back 
This obsession is affecting her schoolwork, and more severely her eyesight  She has become very shortsighted, and her eyesight continues to deteriorate as a result of holding the phone or laptop very close, and mostly in the dark without any lights on  My husband and I have a constant battle on our hands daily, in all areas of discipline with our daughter, but our main concern is that we have been unable to find a way to minimise this obsessive behaviour centred around her phone and laptop  Please can you provide some strategies that can help us specifically with this problem 
First of all, I thank you for developing this program and I am only at the first stage of assignment 1  I have loads of books I have bought, attended psychiatrists for my son and myself, family therapy, occupational therapy, begged and prayed for change but have been dealing with behavioural issues for so long I am definitely exhausted and resentful 
I am a mum to a 15 yr old boy with ASD, dyslexia, OCD and ODD  Sorry to focus on the labels but just to give you an idea of what I am dealing with  I also have a 13 yr old son whom finds his brother’s behaviours difficult, embarassing and challenging  My husband whom is not in great health ( he had a cerebral aneurysm clamped two years ago and has two further aneurysms that are inoperable so endures fatigue, headaches and stress)  We have however a pet cat that is very social and a calming influence in the home! I was fortunate enough to have loving parents but I lost both my mum and dad in 2008 and 2015  My inlaws are elderly and quite directly say they are too old to help us so it feels we are alone in dealing with the issues we have 
I am desperate for change as the household is one of stress and anger and I feel all the control lies in my son Patrick’s hands  I am hopeful your programme can make life better for all of us but I wonder if it is too early to ask you two questions?
The first lies with what to do when Patrick goes into my other son Brendan’s room and will either turn on a light when he is sleeping, yell when he is on his phone or create some disturbance  He will not leave the room when asked to do so and the situation always escalates into yelling and Brendan attempting to physically remove him  This happens regularly and always ends badly with doors slamming, my husband being woken and myself in tears feeling the lack of control and also I admit I seem to think “Why me?” which rationally I know is of no help 
The second problem is leaving the house for school  Patrick refuses personal hygiene (either morning or night) and any request to even brush his teeth is fraught with swearing and abuse  If I can get him to shower, he will watch the water roll down the drain and turn up the water really high temp (mu husband has had to turn down the thermostat on the hot water service) without so much as getting wet  My husband leaves for work at 6am but I leave at 745 to work as a nurse in a busy outpatients department in the Alfred Hospital (Melbourne)  My work is my sanity as it is a paid break from home but most days I am late which is causing considerable stress and anxiety not to mention my responsibility to do my job  Patrick simply refuses to leave the house and as much as I am tempted to just walk out and leave I know the house would be left unlocked and wonder if Patrick would even attend school  The time I need to leave is not negotiable but Patrick uses this to his advantage and seems to delight in stressing me out and subsequently speeding to work in a frazzled mess 
The interesting and frustrating element in all of this is that although he is socially isolated at school (he has no friends) and academically challenged his behaviour at school is not a problem  He is quiet and his teachers report he does his best and is compliant and well mannered  It is like a Jekyll and Hyde situation where another side of him at home is so angry and abusive yet at school this behaviour does not happen 
I’m Jackie, I now work primarily as a freelance tech writer, after starting my career in software development and moving on to teach IT to young adults at a variety of colleges and schools 
My freelance work is pretty varied and looks at many aspects of the computer industry as a whole, and I’ve just recently completed a piece which gives help and advice to anyone wanting to become a game designer, which you can read here: http://www gamedesigning org/become-a-game-designer/  It highlights the hard work and effort it takes to get into such a role, and also how you can further your career and continue to learn and improve as you go  I hope you’ll agree it shows that starting work in the industry takes dedication and skill and that becoming a game designer isn’t just a fly-by-night job!
If you’d be interested in sharing a quick mention of my work on your blog that would be really wonderful and I’d appreciate the chance to get my work out there to a wider audience  Alternatively, I’d be happy to write a short blurb or paragraph or two (or a longer piece - just let me know) highlighting the key points because I think some of your readers might get a lot of value from it 
My son just turned 15 and is a freshman in high school  Although this is his first year in a general ed environment, he is struggling with behaviors in school  He has meltdowns and does not express why he would have them until much later  Once we all know what caused it, the school will accommodate him and try to "change up" things so as not to cause his meltdown  Once that is resolved, another issue comes up and causes him to melt down  He is a high functioning and academically does well, when he wants to do the work  We battle at home over homework  He does not care how it is done, as long as he hands it in  He thinks failing a test is ok, at least he took the test  Homework is never on his mind when he gets home from school  If I never prompt him, he would never open is backpack  He can be aggressive but is never intentionally trying to hurt anyone  He may push over a chair in school, but it is not directed at anyone  We know how that in itself could hurt someone who gets hit by it though  He is defiant in that he only wants to do what interests him  He does not go out by himself (still immature), or abuse alcohol or drugs and never curses  He is a very funny kid and very talented  His main problems are task avoidance and seeking attention  He can be disrespectful to adults in that he is "cheeky" with them, trying to be funny or cute  And he has no "filters" 
I’ve just finished reading your Living with an Aspergers Partner ebook  I found it so informative, thank you 
You offered some personal advise, and i wanted to run a situation past you and seek your input as to a strategy for what to do next 
I’ve been seeing a guy for about 7 months now who I believe has Aspergers  I came to this conclusion months ago and I don’t think he realizes, (or acknowledges) although he is aware he has some traits 
He’s highly intelligent and successful, a pattern seeker, has a tendency to focus on the project to hand to the total exclusion of all else for as long sit takes (work or home) socially awkward (has learned coping strategies), sensitive to loud noise, high anxiety with control strategies, black and white thinking etc  He’s currently not working and I’ve seen a slow withdrawal over the last 6 weeks, including the need to ‘escape’ and leave a situation at least once 
He also has a bipolar ex overseas who has primary custody one daughter where there has been ongoing patterns of drama which has recently increased 
Over the past couple of months (since stopping work and drama increase) I’ve gone from being ‘wonderful’ in his eyes to him now being sorry and not having the ‘urge’ to spend close/intimate time with me and offering friendship  Since he shared that with me in a message he’s stonewalled and has retreated to the safety of minimal messages and talks about not knowing what best to say and not being able to find the right words somehow 
He’s a good kind man who I feel is struggling  I’m concerned about his anxiety and possibly the risk of depression  I’m fairly resilient and whilst i’m disappointed he doesn’t want to pursue a relationship with me, i’m concerned for him and his well being  One of his very few close friends is also just leaving the country to live overseas 
The strategy I’ve used so far is simply to back off and give him space  I’ve asked to take him up on an original offer he made to talk but haven’t pushed it  I also haven’t been aggressive or accusatory in the few messages i’ve sent 
Any advise you could give would be greatly appreciated,
Carli who is 10 years old and has had behavioral issues her whole life  The other night she came home very upset after having a conflict with a friend  She was at her friend's house and her and her friend wanted to get on the computer and the older sister was using it  Carli made up a story that someone was at the door to get the older sister off the computer  Her friend didn't understand that she was making up a story to get the sister off the computer  She got excited that someone was at the door and ran downstairs to answer the door  In the process of getting the door, she fell and yelled at Carli  Carli became extremely upset  She was able to control her feelings at her friend's house, but when she came home, she proceeded to cry extremely loudly for over an hour  Her dad spent most of that time with her, talking to her and trying to calm her down  After an hour, I asked him if he could please tell her to be more quiet because the other members of the household were trying to go to sleep 
My question is    how do I as the girlfriend, handle this? He did not like that I asked her to be quiet  We have a rule that if she is having bad behavior, and can't calm down in 5 minutes, he takes her out of the house because her yelling doesn't stop for a long time and is very upsetting to everyone in the household  I would like to ask him to do this with this kind of situation as well  Is this a reasonable request? His thought was that she shouldn't be made to calm down, because everyone handles being upset in a different way  But, she was literally sobbing and wailing very loudly 
My other question is should she have been told that if she wouldn't have lied, this wouldn't have happened? She has a history of lying and of not accepting responsibility for her actions  My boyfriend became very upset with me when I brought this up  He was being very sympathetic and understanding to her  I feel like he was giving her negative attention, and being an over indulgent parent by not putting his foot gown and saying, "you can't carry on like this, even though you are upset"  Please let me know how we can handle these situations better 
I am contacting you for help with adult AS  I am taking initiative to pre screen potential therapists to help my current boyfriend get therapy and help with Adult AS 
He has seen many therapists, but it seems like they aren’t really helping him with his problems  They don’t seem to understand how his (undiagnosed) AS would affect therapy approaches  For example, he may not share enough in therapy session and I’m assuming an AS therapist would recognize that is part of the AS and employ strategies to get information from him that helps with treatment  Sometime he tunes out when he is processing something heavy or that he doesn’t want to hear necessarily, or he gets distracted and I’m hoping an As therapist would recognize that and get that he may need repeated something for example, if this is happening 
He is currently suffering from depression that appears clinical in nature as well as reoccurring negative thoughts about something specific that has been worrying him about our relationship  Today he told me these reoccurring thoughts happen during all waking hours unless he watches TV, he never gets a break from them and they make him feel like he is going crazy  As his girlfriend, I am extremely concerned that he cannot get relief from these thoughts and that the therapists he is seeing are unable to help him with his problems  Therefore, I am taking initiative to try and help him find better therapy options, because I want to see him someone who can better help him get to the bottom of things and help him with the challenges he is facing  He really needs an advocate that will help him go deep to figure things out and not just assume therapies are working well, without seeing changes or getting supporting feedback from him in that regard 
Here are some questions I am trying to ask in advance to find the right people to help us with this  As you may know, insurance for these therapies are not often available  We don’t have a lot of money to go from therapist to therapist to find the right person and are hoping prescreening will help 
I recently downloaded your e-book and listened to your talks and your information is by far the most helpful I have been able to find to date  It's very accurately describes my situation as an NT wife married to a very probable AS husband  I think you for taking the time to write this and sharing your insights as well as the experiences of many of your clients  It has really helped me understand the last 32 years of our marriage and get a grasp on how to move forward 
One area that is of primary concern to me, that I did not see addressed, is stimming  I believe that is the behavior my husband is showing through constant vocal singing, repetition of words, shouting out, as well as slapping himself in the chest and general nervous activity  It is very loud and disruptive to our household and it is often a relief when he is not at home  I think there may be a level of Tourette's syndrome as well 
I did some searches on the Internet and could not find anything that really describes his behavior  Most of what I found was flapping or children's behavior  I understand that it is a release of nervous tension but I am really trying to find some strategies to help him stop this behavior as it is extremely frustrating and builds my resentment in dealing with it daily  A lot of it is embarrassing as well and sounds childish to me 
He usually does this when close family members are around and will reign himself in if he is around other people besides us  When we are home it is constant  He also has a lot of anger, mostly at himself, and blows up at unimportant things, it is as if he has a ton of negative energy inside him that need to get out and stimming is one outlet 
I will try to build my acceptance of it, but I also would just like him to stop especially the loudest and most annoying portions  Would you have any resources you could point me to?



ground_truth: Vitamins K3, K4, and K5.

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What is the research opportunity that is mentioned?

Read the following text and answer briefly.

Context 4: 
Senate Floor Amendments of 8/20/10 revise the statute of
limitations and remedies for specified housing-related
ANALYSIS : The Planning and Zoning Law requires cities
and counties to prepare and adopt a general plan, including
a housing element, to guide the future growth of a
community  Following a staggered statutory schedule,
cities and counties located within the territory of a
metropolitan planning organization (MPO) must revise their
housing elements every eight years, and cities and counties
in rural non-MPO regions must revise their housing elements
every five years  These five- and eight-year periods are
known as the housing element planning period 
Before each revision, each community is assigned its fair
share of housing for each income category through the
regional housing needs assessment (RHNA) process  A
housing element must identify and analyze existing and
projected housing needs, identify adequate sites with
appropriate zoning to meet its share of the RHNA, and
ensure that regulatory systems provide opportunities for,
and do not unduly constrain, housing development  The
reviews both draft and adopted housing elements to
determine whether or not they are in substantial compliance
with the law  The Planning and Zoning Law and the Subdivision Map Act
also includes a number of sections governing zoning and
entitlements specifically related to housing, including:
? The Housing Accountability Act, which requires a city or
county to make one or more specified findings in order to
disapprove a particular housing development 
? A provision requiring cities and counties, when adopting
an ordinance which limits the number of housing units
which may be constructed on an annual basis, to make
findings as to the public health, safety, and welfare
benefits that justify reducing the housing opportunities
of the region  ? Density bonus law, which requires cities and counties to
grant a developer a density bonus, incentives, and
concessions when the developer proposes to include
specified percentages of affordable housing within a
development  ? The Least Cost Zoning Law, which requires cities and AB 602
counties to designate and zone sufficient vacant land for
residential use with appropriate standards to meet
housing needs for all income categories and to contribute
to producing housing at the lowest possible cost 
? A requirement that, when determining whether to approve a
tentative subdivision map, a city or county shall apply
only those ordinances, policies, and standards in effect
as of the date the developer’s application is deemed
Prior to a recent court decision, it was understood that
current law allowed a party to challenge the adequacy of a
city’s or county’s housing element at any time during a
planning period, provided that the challenger brought the
action “in support of or to encourage or facilitate the
development of housing that would increase the community’s
supply of [affordable] housing ” The challenging party was
required first to serve the city or county with a notice
identifying the deficiencies in the housing element  After
60 days or the date on which the city or county took final
action in response to the notice, whichever occurred first,
the challenging party had one year to file the action in
court  This process and statute of limitations also
applied to actions brought pursuant to the housing-related
statutes listed above  In 2006 Urban Habitat Program brought suit to challenge the
City of Pleasanton’s housing policies, including the city’s
annual cap on housing permits and the city’s cap on the
aggregate number of permissible housing units, both of
which Urban Habitat claimed were insufficient to allow the
city to meet its RHNA obligation  In 2008, the First
District California Court of Appeals issued an unpublished
decision in the case of Urban Habitat Program v  City of
Pleasanton allowing the case to proceed with respect to
some causes of action, but ruling that the challenge to the
housing element itself was time-barred  The court stated:
Although the statute does not specify the time within
which [a deficiency] notice must be given, it is our
conclusion that the statute must be interpreted as
containing a time limit within which this requirement
must be met? In sum, a party bringing a challenge AB 602
governed by section 65009, subdivision (d), has 90
days from the date a legislative action is taken or
approval is given to notify the local land use
authority of any claimed deficiencies in such an
action or approval  Its claim then accrues 60 days
after it gives this notice 
In other words, instead of being able to initiate a
challenge to a deficient housing element at any time during
the planning period, housing advocates and other interested
parties may now only initiate such a challenge by
submitting a deficiency notice within 90 days of the
housing element’s adoption 
1 Removes from the current list of city or county actions
which may be challenged pursuant to Government Code 65009
notice and accrual provisions those actions related to
the Housing Accountability Act, the Subdivision Map Act,
and the application of a Density Bonus ordinance to a
particular project, all of which are project-specific
actions  The bill maintains the ability to use these
notice and accrual provisions to challenge the adequacy
of a city’s or county’s density bonus ordinance
2 Extends lengthening the time in which a deficiency notice
may be served to cover all remaining city or county
actions described in this section of law, as opposed to
just housing element challenges  In other words, the
amendments apply the longer timeframe to serve the
deficiency notice to actions relating to the Least Cost
Zoning Law, annual limits on housing permits, and the
adequacy of a density bonus ordinance, in addition to
housing element law  3 Provides that an entity challenging such an action in
support of affordable housing may serve the deficiency
notice up to five years after the city’s or county’s
action  After 60 days or the date on which the city or
county takes final action in response to the notice,
whichever occurs first, the challenging party has one
year to file an action in court, except that the lawsuit AB 602
may not be filed more than five years after the city’s or
county’s action  In other words, the entity must file
the lawsuit within one year of the expiration of the
deficiency notice or within five years of the city’s or
county’s action, whichever occurs first 
4 Provides that a housing element from a prior planning
period may not be challenged if the city or county has
adopted a revised housing element for the new planning
Government Code 65755   Current law requires a court, if it
finds any portion of a general plan, including a housing
element, out of compliance with the law, to include within
its order or judgment one or more of the following remedies
for any or all types of developments or any or all
geographic segments of the city or county until the city or
county has complied with the law:
? Suspend the authority of the city or county to
issue building permits 
grant zoning changes and/or variances 
grant subdivision map approvals 
? Mandate the approval of building permits for
residential housing that meet specified criteria 
? Mandate the approval of final subdivision maps for
housing projects that meet specified criteria 
? Mandate the approval of tentative subdivision maps
for residential housing projects that meet specified
This bill clarifies that in any action or proceeding
brought pursuant to the notice and accrual provisions of
Government Code Section 65009 described above, neither the
court remedies described above nor any injunction against
the development of a housing project shall abrogate,
impair, or otherwise interfere with the full exercise of
the rights and protections granted to an applicant for a
tentative map or a vesting tentative map under specified
provisions of the Subdivision Map Act or to a developer
under a specified provision relating to development AB 602
Under current law, HCD operates a number of grant programs
to which cities and counties may apply  In many cases, the
law requires a city or county to have an HCD-approved
housing element in order to be eligible for funding  This bill provides that if a third-party challenges the
adequacy of a housing element in court and the court finds
that the housing element substantially complies with all of
the requirements of housing element law, the element shall
be deemed to be in compliance for purposes of state housing
The statutory language interpreted by the court and at
issue in this bill was added to statute by AB 998 (Waters),
Chapter 1138, Statutes of 1983, a bill sponsored by the
League of California Cities and the California Building
Industry Association  AB 998 created a short statute of
limitations period for land use decisions generally but
provided a specific exception to protect the ability to
challenge deficient housing elements  The Senate Housing
and Land Use Committee and the Senate Third Reading
analysis of the bill stated that the bill:
Specifies that for challenges in support of low- and
moderate-income housing requirements, the petitioner
shall notice local government 60 days prior to filing
action  The [one-year] statute of limitations then
begins on the first day the legislative body fails to
In the intervening 25 years prior to the Urban Habitat
ruling, housing advocates filed and successfully settled at
least ten cases in which the 60-day deficiency notice was
sent more than 90 days after adoption of the city’s or
county’s housing element  In none of these cases was the
timeliness on the advocates’ suit contested  Likewise, six
bills amended other portions of this statute during those
intervening years, and there was never any controversy
surrounding the lack of a deadline for housing advocates to
serve a deficiency notice nor any attempt to change the AB 602
statute in this regard  Current level of housing element compliance   According to
HCD’s website as of June 7, 2010, only 46 percent of cities
and counties have adopted an HCD-approved housing element
for the current planning period that began in 2005 for the
San Diego region, 2008 for the Southern California, Fresno,
Kern, and Sacramento regions, and the summer of 2009 for
the remaining areas of the state  Unlocking the private market   The purpose of housing
element law is to create opportunities for the private
housing market to function  Builders cannot build without
access to appropriately zoned land, and current land use
plans in many cities and counties in California fail to
provide sufficient opportunities to accommodate projected
population growth  The San Diego Association of
Governments’ Regional Comprehensive Plan describes this
typical California paradox in the following way:
Under current plans and policies, more than 90 percent
of [the San Diego region’s] remaining vacant land
designated for housing is planned for densities of
less than one home per acre, and most is in the rural
back country areas dependent upon scarce groundwater
supplies  And of the remaining vacant land planned for
housing in the 18 incorporated cities, only about
seven percent is planned for multifamily housing  When
taken together, the current land use plans of the 19
local jurisdictions do not accommodate the amount of
growth anticipated in our region  SANDAG’s population
forecast, which reflects the current adopted local
land use plans in the region, projects that while
population will increase by 37 percent by 2030,
housing will grow by just 30 percent  The forecast
shows that if local plans are not changed, demand for
housing will continue to outpace the supply, just as
Housing element law addresses this problem directly by
requiring cities and counties to zone land at appropriate
densities to accommodate the projected housing needs of all
income groups and to remove constraints that prevent such
sites from being developed at the allowed densities  AB 602
Cities and counties, however, are not required to build
housing because that is the role of private developers 
The law holds cities and counties accountable only for that
which they control: zoning and land use entitlements 
Without the ability to enforce housing element law, the
market’s ability to meet housing demand may well remain
locked up 
FISCAL EFFECT : Appropriation: No Fiscal Com : No
SUPPORT : (Verified 8/23/10)
California Rural Legal Assistance Foundation (co-source)
Housing California (co-source)
Advocates for Affordable Homes in Fremont
California Coalition for Rural Housing
Community Housing Improvement Program
Community Housing Works
Eden Housing
Fair Housing of Marin
Grassroots Leadership Network of Marin
Kennedy Commission
Public Advocates, Inc
San Diego Housing Federation
Self-Help Enterprises
Sierra Club of California
American Planning Association, California Chapter
JA:nl 8/23/10 Senate Floor Analyses SUPPORT/OPPOSITION: SEE ABOVE
pasoobserver says:	09/11/2010 at 11:17 pm
To whatisup — Thank you for your response to my comments  However, you failed to answer some of my questions that I mentioned to you  It’s almost like dealing with some City officials  They just let the public vent at their bimonthly council meetings  In my opinion, it’s difficult to deal with narcissism and arrogance  Over the years, there has been some very good input to our elected officials on how to proceed on the Nacimiento water pipeline,but it fell on deaf ears  You wanted me to answer some of your questions,but you did not answer some of my questions  Again, are you willing to subsidize new development?,Yes?or No?, are you willing to pay for a commodity that you are not receiving? Yes?or No? and another question for you  Are you willing to pay over 300% on your water bills within the five (5) year plan that the City has proposed? Also, the water rates will be subject to later increases too  By the way, I do concur with the city’s plan of “you pay for the amount of water units you use”  (748 gal=one unit)  However, the higher water rates are not good for our senior citizens on fixed incomes and other struggling families in our community  My first suggestion years ago was desalination  The response was it was too expensive  Of course, now it is more expensive  I would suggest that our elected officials recall the existing bonds (The bonds can be recalled early)  The City council can explain to the citizens in detail with financing of new bonds at a lower interest rate as of now for the sewer plant and Nacimiento water pipeline and present their new proposal in compliance with Proposition 218  Let the citizens of Paso VOTE on the financing bonds for their approval  Most of the citizens,that I had spoken to were not happy with the way our City Council handled the Nacimiento water pipeline project  The citizens of Paso didn’t give our City Council a “BLANK CHECK” for $176 million to spend without voter approval  I would suggest that it be a “special tax” or “an assessment” be levied on our property taxes  A percentage of those bonds can be deducted on Federal Income taxes  As it is now, a” fee” on a capital funding project is not deductible  Of course, there are homeowners would not go for this suggestion due to our poor economy  My analogy mentioned above would be, you would get something back on a “special tax” or an “assessment” verses nothing on a “fee”  What say you?
whatisup says:	09/12/2010 at 9:02 am
Unfortunately the law says we have to subsidize new development in California  I don’t like it, but it is the law  I know paying using the property taxes was bandied about  The argument against it was it would mean some would be paying for water they aren’t using and others could be big water users, but pay a small special assessment on their property taxes  I think the decision that was made to base it on usage was out of fairness  It seems to me if people are using water and not paying their share of the costs it is not fair  The Senior issue is very difficult  If someone is retired for twenty years is it realistic to think prices don’t go up during the 20 years of retirement  Think what prices were in 1990 compared to today  Should Seniors never have to pay for capital improvements? Paso Robles also had very low water rates  Rates that are no longer possible given the circumstances  Desalination will happen eventually  California is out of water  If you want to pay $1,000,000 a gallon there is no more allotable water of any consequence in California  The expense will be tremendous — still have to build a desalination plant, still have to build a pipeline  I don’t know if the plant has to be built along the ocean or if the salt water could be piped over to Paso Robles  If it has to be built along the ocean, Paso Robles doesn’t own land on the ocean and, in any case, the environmentalists will keep it in courts for years as they have done so for other proposed desalination plants in Southern California  Eventually necessity will force desalination past the environmentalists, but not yet 
pasojim says:	09/13/2010 at 7:46 am
Whatisup – On one of your previous post you made the comment you haven’t heard any of the legal suggestions for the water issue, But you obviously have  That is a good thing  So we can move the discussion ahead 
Once, again this was handled incorrectly by our city custodians from the beginning  And now here we are  The public is not supporting this very expensive, very limited benefit project  As you said, until a plan is developed that the public can support, things don’t look good 
All this discussion about the water issue has only reinforced my opinion the issue hasn’t been about water, only how the plan should be paid for  Or more specifically, to what extent do we allow our elected custodians and our un-elected GOD tzar decide which laws they will follow and which laws they will ignore  When the City GOD tzar tell citizens at a council meeting if we don’t agree with the City’s plan, then we should just sue him, and when the City Attorney explains to a citizen at a City Council meeting that she does have to respond to their questions because she does NOT work for them  When the project is voted down by the citizens and the council brings it right back up, it is clear that our elected representatives are not doing their job providing direction to their employees and listening to and representing the CITIZENS 
The subject of the original post was the need to elect different representation  I think with all the conversation made on this post, as well as the post on Cal Coast about the hiring of the new legal firm you were involved in, Supports my original opinion 



ground_truth: A study on the effects of Brazilian Jiu Jitsu and psychotherapy on people with autism.