TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.
21 Related JoVE Articles!
Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats
Institutions: Iowa State University.
Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 – 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6°C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5°C/min, enabling further dehydration, to a temperature of -34°C before being plunged into liquid nitrogen (-196°C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37°C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.
Developmental Biology, Issue 54, embryo, cryopreservation, cattle, sheep, goats
The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm
Institutions: Macquarie University, Macquarie University, Macquarie University.
The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e.
same finger) on some trials and incongruent (i.e.
different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.
Behavior, Issue 77, Neuroscience, Neurobiology, Medicine, Anatomy, Physiology, Psychology, Behavior and Behavior Mechanisms, Psychological Phenomena and Processes, Behavioral Sciences, rubber hand illusion, crossmodal congruency task, crossmodal congruency effect, multisensory processing, body ownership, peripersonal space, clinical techniques
Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI
Institutions: Stanford University , Duke University Medical Center, Duke University .
Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5
. In AIM MRI, Mn2+
acts a calcium analog and accumulates in depolarized neurons 6,7
. Because Mn2+
shortens the T1
tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+
clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+
does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12
. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13
. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14
. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.
Neuroscience, Issue 65, Molecular Biology, Biomedical Engineering, mouse, ultrasound, blood-brain barrier, functional MRI, fMRI, manganese-enhanced MRI, MEMRI
Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Institutions: KU Leuven.
For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα16
construct is co-transfected. Following ligand binding, activation of the Gα16
subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα16
, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.
Cellular Biology, Issue 89, G protein-coupled receptor (GPCR), calcium mobilization assay, reverse pharmacology, deorphanization, cellular expression system, HEK293T, Fluo-4, FlexStation
Introduction to Solid Supported Membrane Based Electrophysiology
Institutions: Max Planck Institute of Biophysics, Goethe University Frankfurt.
The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.
Biochemistry, Issue 75, Biophysics, Molecular Biology, Cellular Biology, Physiology, Proteins, Membrane Lipids, Membrane Transport Proteins, Kinetics, Electrophysiology, solid supported membrane, SSM, membrane transporter, lactose permease, lacY, capacitive coupling, solution exchange, model membrane, membrane protein, transporter, kinetics, transport mechanism
Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking
Institutions: Yale University, Yale University, Yale University, Yale University, Massachusetts General Hospital, University of California, Irvine.
We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute fluctuations of dopamine induced by smoking a cigarette. The smoker is imaged during a natural smoking experience while other possible confounding effects (such as head motion, expectation, novelty, or aversion to smoking repeatedly) are minimized.
We present the details of our unique analysis. Conventional methods for PET analysis estimate time-invariant kinetic model parameters which cannot capture short-term fluctuations in neurotransmitter release. Our analysis - yielding a dopamine movie - is based on our work with kinetic models and other decomposition techniques that allow for time-varying parameters 1-7
. This aspect of the analysis - temporal-variation - is key to our work. Because our model is also linear in parameters, it is practical, computationally, to apply at the voxel level. The analysis technique is comprised of five main steps: pre-processing, modeling, statistical comparison, masking and visualization. Preprocessing is applied to the PET data with a unique 'HYPR' spatial filter 8
that reduces spatial noise but preserves critical temporal information. Modeling identifies the time-varying function that best describes the dopamine effect on 11
C-raclopride uptake. The statistical step compares the fit of our (lp-ntPET) model 7
to a conventional model 9
. Masking restricts treatment to those voxels best described by the new model. Visualization maps the dopamine function at each voxel to a color scale and produces a dopamine movie. Interim results and sample dopamine movies of cigarette smoking are presented.
Behavior, Issue 78, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Medicine, Anatomy, Physiology, Image Processing, Computer-Assisted, Receptors, Dopamine, Dopamine, Functional Neuroimaging, Binding, Competitive, mathematical modeling (systems analysis), Neurotransmission, transient, dopamine release, PET, modeling, linear, time-invariant, smoking, F-test, ventral-striatum, clinical techniques
Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents
Institutions: National Institutes of Health.
The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+
and 1 H+
and the counter-transport of 1 K+
. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.
Neurobiology, Issue 78, Neuroscience, Biochemistry, Molecular Biology, Cellular Biology, Anatomy, Physiology, Biophysics, Astrocytes, Synapses, Glutamic Acid, Membrane Transport Proteins, Astrocytes, glutamate transporters, uptake, clearance, hippocampus, stratum radiatum, CA1, gene, brain, slice, animal model
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3
Institutions: The University of Texas Health Science Center at Houston.
The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+
-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+
levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+
-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.
Bioengineering, Issue 54, TRP channels, Calcium assay, FlexStation 3
Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones
Institutions: The University of Iowa, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa.
Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.
Neuroscience, Issue 59, pain, immunostaining, sensory nerve fiber, skin, bone, plantar punch, CGRP, NF200, TRPV1, Tubulin
In vivo Neuronal Calcium Imaging in C. elegans
Institutions: Boston University School of Medicine, Boston University Photonics Center.
The nematode worm C. elegans
is an ideal model organism for relatively simple, low cost neuronal imaging in vivo
. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1
and GCaMP 2
allow in vivo
imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo
using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans
and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans
presents a robust and flexible system for in vivo
neuronal imaging with advantages over other model systems in technical simplicity and cost.
Developmental Biology, Issue 74, Physiology, Biophysics, Neurobiology, Cellular Biology, Molecular Biology, Anatomy, Developmental Biology, Biomedical Engineering, Medicine, Caenorhabditis elegans, C. elegans, Microscopy, Fluorescence, Neurosciences, calcium imaging, genetically encoded calcium indicators, cameleon, GCaMP, neuronal activity, time-lapse imaging, laser ablation, optical neurophysiology, neurophysiology, neurons, animal model
The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation
Institutions: KU Leuven, Belgium, KU Leuven, Belgium, KU Leuven, Belgium.
The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1
. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2
. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3
Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4
. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.
Medicine, Issue 66, Physiology, Chemistry, cystometry, urodynamics, bladder function, bladder chemosensation, animal model, urinary tract
Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology
Institutions: University of Washington.
The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes >10 μm in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.
Physiology, Issue 76, Biophysics, Molecular Biology, Biochemistry, Genetics, Cellular Biology, Proteins, Membranes, Artificial, Lipid Bilayers, Liposomes, Phospholipids, biochemistry, Lipids, Giant Unilamellar Vesicles, liposome, electrophysiology, electroformation, reconstitution, patch clamp
Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior
Institutions: University of Florida College of Dentistry, University of Florida College of Medicine , Stoelting Co., University of Florida .
We present an operant system for the detection of pain in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) assesses pain behaviors in a more clinically relevant way by not relying on reflex-based measures of nociception. Food fasted, hairless (or shaved) rodents are placed into a Plexiglas chamber which has two Peltier-based thermodes that can be programmed to any temperature between 7 °C and 60 °C. The rodent is trained to make contact with these in order to access a reward bottle. During a session, a number of behavioral pain outcomes are automatically recorded and saved. These measures include the number of reward bottle activations (licks) and facial contact stimuli (face contacts), but custom measures like the lick/face ratio (total number of licks per session/total number of contacts) can also be created. The stimulus temperature can be set to a single temperature or multiple temperatures within a session. The OPAD is a high-throughput, easy to use operant assay which will lead to better translation of pain research in the future as it includes cortical input instead of relying on spinal reflex-based nociceptive assays.
Behavior, Issue 76, Neuroscience, Neurobiology, Anatomy, Physiology, Medicine, Biomedical Engineering, Surgery, Neurologic Manifestations, Pain, Chronic Pain, Nociceptive Pain, Acute Pain, Pain Perception, Operant, mouse, rat, analgesia, nociception, thermal, hyperalgesia, animal model
Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro
method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo
experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e.
smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo
. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro
smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro
using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro
preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers.
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo
counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure
neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic
SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications
Institutions: Purdue University, Eli Lilly and Company.
Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson's disease. Acute activation of Gαi/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro
and in vivo
following the chronic activation of several types of Gαi/o-linked receptors including D2
dopamine and μ opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gbγ signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384 well format would facilitate future studies. Using two D2
dopamine receptor cellular models (i.e
), we have converted our 48-well sensitization assay (>20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.
Bioengineering, Issue 83, adenylyl cyclase, cAMP, heterologous sensitization, superactivation, D2 dopamine, μ opioid, siRNA
Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities
Institutions: University of Iowa Carver College of Medicine, University of Bath.
Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.
Neuroscience, Issue 85, Presynaptic Terminals, Synaptic Vesicles, Microscopy, Biological Assay, Nervous System, Endocytosis, exocytosis, fluorescence imaging, FM dye, neuron, photobleaching
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
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, imaging
Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.
primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;
H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
Functional Mapping with Simultaneous MEG and EEG
Institutions: MGH - Massachusetts General Hospital.
We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.
JoVE neuroscience, Issue 40, neuroscience, brain, MEG, EEG, functional imaging


Building and Using a Groundwater Database is an introductory book that focuses on the fundamentals of groundwater database use. It is an excellent guide for people who collect and use groundwater quality data, hydrogeological data, and general geological data, as well as people who are required to prepare information about groundwater resources for others to use. The book also serves as a textbook for computer-based hydrogeology courses. Many university courses now make use of computerized groundwater data, yet no textbook exists to guide students in database use."Building and Using a Groundwater Database" provides detailed information regarding the steps and perspectives required to create a database and use it for groundwater management, land use practices, planning, cleanups, site investigations, and general hydrogeologic reporting. The book is structured to take the reader from the foundations of database development through maintenance and everyday use of the database. Actual examples from selected case studies are used to illustrate database principles.This book is unique in that it deals with the management and structuring of groundwater data, as opposed to the collection and interpretation of data. It illustrates how database software managers can be integrated with groundwater software tools. "Building and Using a Groundwater Database" provides consultants, engineers, public officials, university instructors, local and municipal water utilities, and banking and loan institutions with a clear, concise guide to using groundwater databases.
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Are you wondering what a perc test is? If you’re buying a new piece of land, it’s something you may want to know about.
A percolation test, or perc test, is a procedure performed to review water drainage in different soils. It is important for many uses, such as when designing a leach field for a septic system, planning a building design, or considering agricultural use of the land. If the water cannot drain properly, a swamp of sewer water will result. This is a foul-smelling health hazard that could cost you thousands of dollars to correct, or ruin your house or farm entirely.
Some types of soil require a percolation test to ensure sufficient drainage before a septic system can be installed. If you’re planning on building a house on a new piece of land, it is highly recommended that a perc test be performed to ensure that it passes state and local regulations. The results may determine how many bedrooms are allowed to be developed in the house. Soil that fails a perc test could require expensive septic alternatives.
Shoots Veis of Interstate Engineering has the lowdown on how an underground wastewater treatment system is designed. “(It’s) designed based on two factors: household water usage and percolation rate of the soil where the underground system is located. Using those two pieces of information, the size of drainfield area is determined.”
“If a household used a lot of water and it is discharged to a soil with a slow percolation rate, then a large drainfield area will be required. A household with small water usage and well drained soil will be able to utilize a smaller drainfield footprint.”
You can perform a perc test on any soil. Depending on your soil, the test results could be different on various parts of your property. Local building regulations may require a test be performed by a licensed professional, so check with your local zoning department to find out if this applies to you. If not, a personal perc test may do the trick.
You can perform a simple test by picking up a handful of soil and compressing it in your fist. If the soil is dry, dampen it with a little water. Soil that falls apart easily will allow water to drain quickly; soil that holds together has a large amount of clay that will not allow rapid draining.
You will need a shovel, stopwatch, yardstick, gravel, and water. Dig several holes, each 1 foot wide and up to 3 feet deep, depending on local ordinance requirements. Soak the holes overnight; remember, you must perform the test in damp soil. Place 2 inches of gravel at the bottom of the holes.
Cover the gravel with 6 inches of water and measure the depth of water every 30 minutes. Local codes will indicate passing scores for your septic system. See if your water depth makes the grade. A licensed professional may be required to perform the test and requirements vary from town to town.
Perc tests play a vital role in building design and stormwater management, just ask David Businelli, AIA. Owner of Studio 16 Architecture in Staten Island, NY.
“When designing a building, architects have to consider and deal with storm water drainage. This means that we have to calculate the amount of water from a 2” rainfall that will fall on the entire site, especially hard surfaces like the roof or paved areas.”
“Here in NYC, we have either storm sewers or combined storm and sanitary sewers into which rainwater can be discharged. But, there are many sites – especially on Staten Island that do not have storm sewers available. There are many municipalities in the country that don’t have storm sewers. Lacking them, storm water must be retained and drained on site, i.e. draining it into the ground.”
“Once an area of the site is selected, soil borings are done in order to determine the soil types and where permeable soil is. Then a percolation test is performed in order to determine how much water and at what rate the soil can absorb, in gallons per minute. This data allows us to determine the type of drainage system that is appropriate for the project, such as drywells, bioswales, retention ponds, sheet drainage over grassy areas, permeable pavement, etc. Stormwater management is an essential part of any building project, and contributes to the sustainability of the building by using the site properly.”
Scott Bergman, VP at Bergman Vineyards, has years of experience selling vineyards and wineries in Napa and Sonoma Counties.
“When someone buys a vineyard, a winery with a vineyard on it, or land that is ‘plantable’ for more vineyard, they do a detailed soil test to find the quality of the soils that the vines are growing in and the soils where future vines will be growing. This test normally takes a MINIMUM of 60 days to get the soils from the ground, categorize the soil samples, send them to the mid-west for the tests, get the results back, and then have the specialist perform a write up on the results.”
“Thankfully, a perc test can be a quick solution for someone that isn’t able to wait for the soil results 2 months later. “If there is a short (less than 60 days) timeframe to do the necessary due diligence (time for inspections of the property and improvements), a perc test can be performed much faster. The perc test will let us know how the clay is in the ground. If the water doesn’t draw down fast enough, we know that the vines could drown (not worthy to purchase).”
Any other Perc Test tips? Tell us in the comments below or on our social channels!
Search our U.S. land for sale to find the property of your dreams.
Photo Cred: wikimedia commons | Robert Young Vineyards


* Study finds second-hand smoke exposure thickens artery walls
* Raises risk of heart attacks and stroke in adulthood
* Findings lend weight to calls for car, home smoking bans
By Kate Kelland
LONDON, March 5 (Reuters) - Exposure to second-hand smoke in childhood causes irreversible damage to children's arteries - increasing their risk of heart attacks or strokes when they grow up, according to a large international study published on Wednesday.
The research, which lends weight to campaigns for smoking to be banned in private cars and homes, found passive smoking leads to a thickening of children's artery walls, adding some 3.3 years to the age of blood vessels by adulthood.
"Exposure to passive smoke in childhood causes direct and irreversible damage to the structure of the arteries," said Seana Gall, a researcher in cardiovascular epidemiology who led the study at the University of Tasmania.
She said parents, or even those thinking about becoming parents, should quit smoking - both to aid their own health and protect the future health of their children.
Smoking causes lung cancer, which is often fatal, and is the world's biggest cause of premature death from chronic conditions like heart disease, stroke and high blood pressure.
On top of the 6 million people a year killed by their own smoking, the World Health Organisation (WHO) says another 600,000 die a year as a result of exposure to other peoples' smoke - so-called second-hand or passive smoking.
Of the more than 4,000 chemicals in tobacco smoke, at least 250 are known to be harmful and more than 50 are known to cause cancer, the WHO says - and creating 100 percent smoke-free environments is the only way to protect people fully.
About 40 percent of all children are regularly exposed to second-hand smoke at home, and almost a third of the deaths attributable to second-hand smoke are in children.
This latest study, published in the European Heart Journal, was the first to follow children through to adulthood to look at links between exposure to parents' smoking and thickness of the innermost two layers of the arterial wall, known as carotid intima-media thickness (IMT).
Researchers from Finland and Australia looked at data from 2,401 people in Finland 1,375 people in Australia who were asked about their parents' smoking habits. The scientists used ultrasound to measure the thickness of the children's artery walls once they had reached adulthood.
The results showed that carotid IMT in adulthood was 0.015 millimetres thicker in those exposed to both parents smoking than in those whose parents did not smoke.
Gall said that while this was a "modest" increase, it was nonetheless an important extra and irreversible risk for suffering heart attacks or strokes later in life.
Since children of parents who smoke are also more likely to grow up to be smokers themselves, and more likely to be overweight, their heart health risks are often already raised, she said, and the second-hand smoke adds yet more risk.
The researchers said the findings showed reducing children's exposure to smoke is a public health priority.
"Legislation can reduce passive smoke exposure, with restriction of smoking in public places reducing hospitalisations for cardiovascular and respiratory disease," they wrote, adding that banning smoking in cars with children in them would also have a significant positive effect.
The United States, Australia and Canada have already banned smoking in cars carrying children, and Britain said last month that it too would be introducing a ban soon. (Editing by Keiron Henderson)
Our Standards: The Thomson Reuters Trust Principles.


The Names of the Hare
Over recent years I have spent many hours exploring the Stonehenge landscape and the ever present hares are an integral element of that region of Salisbury Plain. They are recorded as being coursed on the Downs around Amesbury in the late 16th century and in the early 19th century, and the coursing was recorded as ‘excellent.’ Amesbury Coursing Club was formed in 1822, at which time the owner of the land allowed hares to be preserved on the downland near Stonehenge. Elsewhere in the parish was also regularly used for coursing. The Altcar (Lancs) Club held a seven day meet at Amesbury in 1864 and the South of England Club used to meet at Stonehenge. Immediately southeast of Stonehenge is Coneybury Hill, where Coneybury Henge once stood. ‘Coney’ is a Middle English word deriving from Old French meaning ‘rabbits’. Hares and rabbits clearly have a long association with the Stonehenge landscape.
‘The Names of the Hare’ is a poem written in the late thirteenth century, originally in Middle English, that is attributed to a Shropshire family on the Welsh borders. The poem is said to be a ritual to be recited by a hunter on his first encounter with a hare, and the seventy seven different names given to the hare in the poem were supposed, on recital, to deliver it to the hunter’s power.
The Names of the Hare
Translation from the Middle English by Seamus Heaney
The man the hare has met
will never be the better of it
except he lay down on the land
what he carries in his hand—
be it staff or be it bow—
and bless him with his elbow
and come out with this litany
with devotion and sincerity
to speak the praises of the hare.
Then the man will better fare.
‘The hare, call him scotart,
the O’Hare, the jumper,
the rascal, the racer.
The wimount, the messer,
the skidaddler, the nibbler,
the ill-met, the slabber.
The quick-scut, the dew-flirt,
the grass-biter, the goibert,
the home-late, the do-the-dirt.
The starer, the wood-cat,
the purblind, the furze cat,
the skulker, the bleary-eyed,
the wall-eyed, the glance-aside
and also the hedge-springer.
The stubble-stag, the long lugs,
the stook-deer, the frisky legs,
the wild one, the skipper,
the hug-the-ground, the lurker,
the race-the-wind, the skiver,
the shag-the-hare, the hedge-squatter,
the dew-hammer, the dew-hoppper,
the sit-tight, the grass-bounder,
the jig-foot, the earth-sitter,
the light-foot, the fern-sitter,
the kail-stag, the herb-cropper.
The creep-along, the sitter-still,
the pintail, the ring-the-hill,
the sudden start,
The gobshite, the gum-sucker,
the scare-the-man, the faith-breaker,
the snuff-the-ground, the baldy skull,
(his chief name is scoundrel.)
The stag sprouting a suede horn,
the creature living in the corn,
the creature bearing all men’s scorn,
the creature no one dares to name.’
When you have got all this said
then the hare’s strength has been laid.
Then you might go faring forth—
east and west and south and north,
wherever you incline to go—
but only if you’re skilful too.
And now, Sir Hare, good-day to you.
God guide you to a how-d’ye-do
with me: come to me dead
in either onion broth or bread.
Source of the text – The Rattle Bag, edited by Seamus Heaney and Ted Hughes. London: Faber and Faber, 1982, pp. 305-306.
Hares are intimately woven into layer upon layer of folklore in Britain, particularly associated with the moon and witches, which I do not propose to explore here, beyond an illustrative quote of Margaret Murray’s, purportedly from seventeenth century Scottish witch trials. The method of making the ritual change into a hare (or other animal) was by the incantation:
‘I sall goe intill ane haire’
With sorrow, and sych, and meikle caire,
And I sall goe in the Divellis nam.
Ay whill I com hom againe’
To revert again to the human form the words were:
‘Hare, hare, God send thee care.
I am in hares likeness just now,
But I shall be in a woman’s likeness even now’
Below is a photograph I took recently of a 12th century carved stone depicting a hare playing a harp, originally from a cottage in Powerstock, Dorset and believed to have come from the site of Wytherston Church. It is on display at Dorset County Museum.
Below: A photograph of one of the stones in The Cove at Avebury stone circles in Wiltshire on which to the lower right hand side, can be seen the image of a hare.
Thank you to Wesley Smith of Wesverines Prehistoric Sites for use of the lead image above, a photograph of a hare in the Avebury landscape in late winter 2016, and to Sophie Kinsley for the use of her felt hare collage photograph.
Dedicated in loving memory to ‘Charlie’.


WASHINGTON: In a breakthrough, scientists have discovered a new class of antibiotics to fight deadly bacteria such as methicillin-resistant Staphylococcus aureus and other drug-resistant bacteria that threaten public health.
The new class, called oxadiazoles, was discovered by University of Notre Dame researchers led by Mayland Chang and Shahriar Mobashery in silico (by computer) screening and has shown promise in the treatment of MRSA in mouse models of infection.
Researchers who screened 1.2 million compounds found that the oxadiazole inhibits a penicillin-binding protein, PBP2a, and the biosynthesis of the cell wall that enables MRSA to resist other drugs.
The oxadiazoles are also effective when taken orally. This is an important feature as there is only one marketed antibiotic for MRSA that can be taken orally, researchers said.
MRSA has become a global public-health problem since the 1960s because of its resistance to antibiotics.
In the US alone, 278,000 people are hospitalised and 19,000 die each year from infections caused by MRSA, said researchers.
Only three drugs currently are effective treatments, and resistance to each of those drugs already exists.
The researchers have been seeking a solution to MRSA for years.
“Professor Mobashery has been working on the mechanisms of resistance in MRSA for a very long time,” Chang said.
“As we understand what the mechanisms are, we can devise strategies to develop compounds against MRSA,” said Chang.
“Mayland Chang and Shahriar Mobashery’s discovery of a class of compounds that combat drug resistant bacteria such as MRSA could save thousands of lives around the world. We are grateful for their leadership and persistence in fighting drug resistance,” said Greg Crawford, dean of the College of Science at the University of Notre Dame.
The research is published in the Journal of the American Chemical Society.


At its most basic, plain flour is flour that does not contain a raising agent.
An ingredient used in many foods, flour is a fine powder made from cereals or other starchy food sources. It is most commonly made from wheat, but also maize (a.k.a. corn), rye, barley and rice, amongst many other grasses and non-grain plants (including many Australian species of acacia). Flour is the key ingredient of bread, which is the staple food in many countries, and therefore the availability of adequate supplies of flour has often been a major economic and political issue. Flour can also be made from legumes and nuts, such as soy, peanuts, almonds, and other tree nuts.
Flour is always based on the presence of starches, which are complex carbohydrates.
Usually, the word "flour" used alone refers to wheat flour, which is one of the most important foods in European and American culture. Wheat flour is the main ingredient in most types of breads and pastries. Wheat is so widely used because of an important property: when wheat flour is mixed with water, a complex protein called gluten develops. The gluten development is what gives wheat dough an elastic structure that allows it to be worked in a variety of ways, and which allows the retention of gas bubbles in an intact structure, resulting in a sponge-like texture to the final product. This is highly desired for breads, cakes and other baked products.


Mainframe z/OS Components
Mainframe operating system z/OS is a share-everything runtime environment that provides for resource sharing through its heritage of virtualization technology. z/OS gets work done by dividing it into pieces and giving portions of the job to various system components and subsystems that function interdependently.
The workload management (WLM) component of z/OS controls system resources, while the recovery termination manager (RTM) handles system recovery.
At any point in time, one component or another gets control of the processor – makes its contribution, and then passes control along to a user program or another component. The control typically gets passed when a job has to wait for information to be read in from, or written out to, a device such as a tape drive or printer.
As with memory for a personal computer, mainframe central storage is tightly coupled with the processor itself, whereas mainframe auxiliary storage is located on (comparatively) slower, and cheaper external disk and tape drives.
Typical z/OS middleware (between the operating system and an end user or end-user applications) includes:
- Database systems
- Web servers
- Message queueing and brokering functions
- Transaction managers
- Java virtual machines
- Portal services
- XML processing functions
System Address Spaces and Master Scheduler
Many z/OS system functions run in their own address spaces. The master scheduler subsystem runs in the address space called *MASTER* and is used to establish communication between z/OS and its own address spaces. Master initialization routines initialize system services, such as the system log and communication task, and start the master scheduler address space.
Batch processing is the most fundamental function of z/OS. Many batch jobs are run in parallel and Job control language (JCL) is used to control the operation of each job.
z/OS requires the use of various subsystems, such as a primary job entry subsystem or JES. An address space is created for every batch job that runs on z/OS. Batch job address spaces are started by JES.
Multiple initiators (each in an address space) permit the parallel execution of batch jobs. Correct use of JCL parameters (especially the DISP parameter in DD statements) allows parallel, asynchronous execution of jobs that may need access to the same data sets (a technique to use batch parallelism and improve availability)
There are address spaces for middleware products such as DB2, CICS, and IMS (referred to as “secondary sub-systems). Typically an automation package starts all tasks in a controlled sequence. Then other subsystems are started. Subsystems are defined in a special file of system settings called a parameter library or PARMLIB.
Workload Management (WLM)
WLM manages the processing of workloads in the system according to the business goals, such as response time. WLM also manages the use of system resources, such as processors and storage, to accomplish these goals.
WLM has three objectives:
1. To achieve the business goals that are defined by the installation, by automatically assigning sysplex resources to workloads based on their importance and goals (goal achievement).
2. To achieve optimal use of the system resources from the system point of view (throughput).
3. To achieve optimal use of system resources from the point of view of the individual address space (response and turnaround time).
Goal achievement is the first and most important task of WLM. Optimizing throughput and minimizing turnaround times – which come after that – are essentially contradictory objectives.
Optimizing throughput means keeping resources busy. Optimizing response and turnaround time, however, requires resources to be available when they are needed. Achieving the goal of an important address space might result in worsening the turnaround time of a less important address space. Thus, WLM must make decisions that represent trade-offs between conflicting objectives.
WLM is particularly well-suited to a sysplex environment. It keeps track of system utilization and workload goal achievement across all the systems in the Parallel Sysplex and data sharing environments.
A mainframe installation can influence almost all decisions made by WLM by establishing a set of policies that allow an installation to closely link system performance to its business needs. Workloads are assigned goals (for example, a target average response time) and an importance (that is, how important it is to the business that a workload meet its goals).
I/O and data management
The input/output architecture is a major strength of the mainframe. It uses a special processor to schedule and prioritize I/O: the System Assist Processor (SAP). This processor is dedicated to drive the mainframe’s channel subsystem, up to 100,000 I/O operations per second and beyond. The channel subsystem can provide over 1000 high-speed buses, one per single server.
Data management activities can be done either manually or through the use of automated processes. When data management is automated, the system uses a policy or set of rules known as Automatic Class Selection (ACSTM) to determine object placement, manage object backup, movement, space, and security. ACS applies to all data set types including database and Unix file structures.
Storage management policies reduce the need for users to make many detailed decisions that are not related to their business objectives.
Today’s z/OS provides a disk device geometry called Extended Address Volume (EAV) that enables support for over 223 gigabytes (262,668 cylinders) per disk volume in its initial offering. This helps many larger customers having the 4-digit device number limitation to begin consolidation of disk farms.
Intelligent Resource Director (IRD)
Intelligent Resource Director can be viewed as Stage 2 of Parallel Sysplex. IRD gives the ability to move the resource to where the workload is. z/OS with WLM provides benefits from the ability to drive a processor at 100% while still providing acceptable response times for critical applications.
IRD is not a product or component, but consists of three separate but mutually supportive functions.
1. WLM LPAR CPU Management – provides a means to modify an LPAR weight to a higher value in order to move logical CPUs to that LPAR that is missing its service level goal.
2. Dynamic Channel-path Management (DCM) – designed to dynamically adjust the channel configuration in response to shifting workload patterns.
3. Channel Subsystem I/O Priority Queueing (CSS IOPQ) – z/OS uses this function to dynamically manage the channel subsystem priority of I/O operations for given workloads based on the performance goals
Predictive Failure Analysis and Health Checker for z/OS
Predictive Failure Analysis (PFA) is designed to predict whether a soft failure (abnormal yet allowable behaviors that can slowly lead to the degradation of the operating system) will occur sometime in the future and to identify the cause while keeping the base operating system components stateless. PFA is intended to detect abnormal behavior early enough to allow you to correct the problem before it affects your business.
PFA uses remote checks from IBM Health Checker for z/OS to collect data the installation. The objective of IBM Health Checker for z/OS is to identify potential problems before they impact z/OS’ availability or, in worst cases, cause outages.
Next, PFA uses machine learning to analyze this historical data to identify abnormal behavior. It issues an exception message when a system trend might cause a problem – thereby improving availability by going beyond failure detection to predict problems before they occur. To help customers correct the problem, it identifies a list of potential issues.


The New Zealand Ministry for Primary Industries (MPI) has recently released a science-based definition for identifying New Zealand Manuka honey, from the results of its science programme.
The programme found that five attributes – when identified together, at specified levels – clearly show that the honey is the real deal – authentic New Zealand Manuka honey. Unless the honey meets all five attributes, it’s not monofloral Manuka honey. But it may still pass the test for multifloral Manuka honey.
MPI proposes implementing the definition through a General Requirement for Export Notice (GREX) under the Animal Products Act 1999. The draft GREX is also designed to improve traceability, and make the food standards around honey clearer.
MPI is calling for feedback and consultation on both its definition and the new export requirements. During the consultation period (until Tuesday 23 May 2017 at 5:00pm) the Ministry is holding workshops around the New Zealand, posting locations, dates and times on the MPI website, in local papers and on social media. Three independent experts from New Zealand, Australia and Canada have already peer reviewed key aspects of the MPI science programme.
The Ministry will consider submissions and feedback before issuing the GREX – letting the apiculture industry and the public have their say. The aim is to bring in the new regulations in late July 2017.
MPI has been working with local New Zealand laboratories, so they can be ready to provide consistent, accurate Manuka honey test results for the four chemical markers and one DNA marker. Analytica Laboratories (where we test our Manuka honey) has gained MPI Limited Recognition to start testing for Manuka honey chemical markers as per the MPI definition. DNA testing will be carried out by Hill Laboratories.
The True Honey Co. is thrilled that the Ministry has made this progress and we wholeheartedly support its initiatives to make sure our industry is authentic for consumers, sustainable for the environment and ethical for all of us. As a company that proudly stands behind every jar, we can’t wait to try out the new test.
Like to know more? Visit the dedicated MPI Manuka honey page for the Science Summary Report, the draft GREX, and the discussion document.


World population is slated to top nine billion by 2050, and seeing as how arable land is being rapidly swallowed by towns and cities, oceans are increasingly overfished, and climate change is disrupting traditional farming, a new United Nations study proposes a twist on Marie Antoinette’s dietary advice: let them eat bugs.
“Common prejudice against eating insects is not justified from a nutritional point of view,” write the authors of a 191-page report (PDF) released by the United Nations Food and Agricultural Organization (FAO) entitled “Edible insects: Future prospects for food and feed security”.
Bugs are not only good eatin’, say the authors, but they’re also highly efficient sources of nutrition. Your average insect, the report claims, requires a mere two kilograms of food to produce one kilogram of what it charmingly refers to as “insect meat”, a far better feed-to-food ratio than, for example, a fatted calf, which requires eight kilograms of feed to produce one kilogram of beef.
Two billion of our current seven billion fellow travelers have already welcomed bugs into their diets, the report contends. Over 1,900 insect species find their way into our alimentary canals, with beetles being the most popular at 31 per cent, followed by caterpillars at 18 per cent; bees, wasps, and ants at 14 percent; and grasshoppers, locusts, and crickets at 13 percent………………..
full article at source.http://www.theregister.co.uk/2013/05/13/insects_as_food_and_feed/
More than 200 men, women and children appear to have been killed in this latest atrocity. Everything we have seen of the Syrian regime’s behaviour over the last seventeen months suggests that these reports are credible. They demand a united response from the international community.
We have two urgent priorities – to establish an accurate account of what happened in Tremseh so that those responsible can and will be held to account; and to agree urgent action at the United Nations Security Council.
To that end, the UN mission in Syria must be able to access Tremseh quickly and without hindrance so they can carry out an independent investigation into what has happened and who is responsible.
And we will redouble our efforts to agree a Chapter VII resolution of the UN security council. This would compel the Syrian regime to fulfil the commitments it has made under the Annan plan, to withdraw its military from residential areas, and establish a transitional government as called for by Kofi Annan and endorsed by the P5 and Arab League. It should be a legally binding security council resolution with teeth, that creates a trigger for sanctions if the Assad regime does not comply. British diplomats in New York will continue negotiations on a resolution at the security council today:
see also: http://www.washingtonpost.com/world/middle_east/2012/07/13/gJQAyfUahW_story.html
This butchery must stop and the UN should now just move in and restore order and the criminals who are responsible for these murders must be brought to justice ASAP.
The world stands condemned for standing by and allowing this genocide to continue !
The ESM has the exact same safeguards as the IMF
Question: Are they the same organisation? Immune from prosecution, from any state or government in the world? Who gave them this right? Let’s be clear the IMF and now the ESM both have immunity from all prosecution from any national government of world organization like the UN???
We cannot hand over this power to an organization that has no accountability to anybody ,That is shear madness and a total betrayal of the citizens rights enshrined in the Irish constitution.
We must not allow faceless, unaccountable financial dictators to just take our rights away from us
Vote No to this madness!
Image via Wikipedia
With the constant stream of brutal killings in the various Syrian cities and the absence of any of the world peace bodies capable of stopping this blatant Syrian Government murder I feel compelled once again to call on the forceful removal of this Butcher of his own people and pray to god that this creep will meet the same faith as Gaddaffi, the once proud master of Libya .Monsters like Assad will be brought to justice and the United Nations must now enforce the international rule of law. This is not the middle ages where the world was oblivious to the crimes of so called leaders. The Peoples of the world demand action now and the protection of the innocent.
The last slide is of a little girl holding a placard for help ,she was brutally murdered just one day after this photo.All decent people must take a good look at this little girl face and ask themselves “have I done enough to stop her murder” and many more that might come to some other child what if she was your child??? ?????
(see article below)
This innocent little girl is Martyred by the forces of Bassar al-Asad, some days ago. She was killed just one day after she was holding that board. The board says to help the Syrian children (especially in Homs). The armed forces of Syria are targeting children and killing them.
The Syrian regime says life in Homs is normal, but the pictures and reports tell a much different story. In an emotional interview, a Syrian activist gives voice to a reality of death and destruction.
Innocent men, women, and children are being slaughtered by the Assad government because they are asking for freedom.
Russia and China have this blood on their hands and should now be expelled from the UN. The UN is now a farce .Thousands of people are now dead and are now been killed! Why is this been allowed to continue? Any person who calls themselves human must condemn this genocide. The Syrian army leadership must be told that they will have to face the international court of justice for murder and crimes against humanity. These criminals must be brought to justice a face the hangman for this!
Currently, the US is following a trajectory of past unsuccessful empires that were unable to sustain themselves resulting in an eventual collapse from within. The US is currently running up a budget deficit which is not only threatening to bankrupt its entire economy, but also threatening the hegemony of its sole instrument for advantage and influence on the world stage – the US dollar. Any threat to the supremacy of the dollar is also a threat to the empire.
It is difficult to calculate the outcome of a western attack against Iran -because there are so many variables.
No moral mandate
For centuries, even Rome required a moral mandate as it conquered the known world. As was the case with the Iraqi invasion and occupation in 2003, the West and its Axis powers led by Washington will require a multi-nation coalition backed by some form of moral mandate in order to move forward with their plans.
Previously, a US-UK campaign against Iraq’s alleged weapons of mass destruction was waged through the UN, and was sufficient at the time in achieving a minimal sway in public opinion needed from both the American and British people, justifying their governments’ foreign policy goals enough to get the war off the ground. But the cost in 2012 of pushing forward under false pretences with both Afghanistan and Iraq in 2003, means that the Axis coalition powers have already played their best hand under the current social democratic system.
full article at source:http://www.marketoracle.co.uk/Article32471.html
As the Libyan conflict seems to be coming into the final stages we are now beginning to
see more and more people braving bullets in Syria .It must be made clear to the leadership
of Syria that they will be brought before the international court of justice for
the murder of their own citizens :Assad must go now!


What A Concept: Docs Trained in Genetic Testing
The current yawning gap between the availability of genetic tests for common diseases and their usefulness for patients is due in part to a lack of physician training and familiarity with these tests. Many DNA markers that convey a higher risk for diseases such as diabetes and heart disease are preliminary in terms of their true predictive power for individual patients. But so was the cholesterol test 20 years ago. It took years of using and understanding the cholesterol test–and the collection of data on thousands and later millions of patients–to establish a cholesterol threshold as an acceptable guide to a person’s heart attack risk. This process needs to happen for genetic test as well.
A helpful step in bringing genetic testing into the exam room was announced today by Boston-based Beth Isreal Deaconess Medical Center and California-based Navigenics, an online genetic company that is emphasizing alliances with medical centers and has been pushing the idea of educating doctors. Here is the announcement run on GenomeWeb Daily News:
NEW YORK (GenomeWeb News) - Navigenics and Beth Israel Deaconess Medical Center in Boston announced today that they will collaborate on training physicians in personal genomic testing.
Beth Israel has launched the Personalized Genomics and Next Generation Sequencing Training Program, which includes a series of lectures, discussions, and presentations, aimed at promoting a better understanding of the personalized genomics field and next-generation sequencing technologies. Among the specific goals of the program are fostering an understanding of issues related to the evaluation of direct-to-consumer genotyping services and familiarizing physicians with the interpretation of genomic information and its correlation with personal medical and health information.
As part of the program, residents will be given the opportunity to have their own genomes analyzed through Navigenics’ consumer genomics services.
“We believe that genetics and genomics will be critical to the future of health care,” Mark Boguski, of BIDMC’s Department of Pathology and the Center for Biomedical Informatics at Harvard Medical School, said in a statement. “Training our residents on the leading genetic services and technologies will be essential to this future.”
Geoffrey Hinton tells us why he’s now scared of the tech he helped build
“I have suddenly switched my views on whether these things are going to be more intelligent than us.”
ChatGPT is going to change education, not destroy it
The narrative around cheating students doesn’t tell the whole story. Meet the teachers who think generative AI could actually make learning better.
Meet the people who use Notion to plan their whole lives
The workplace tool’s appeal extends far beyond organizing work projects. Many users find it’s just as useful for managing their free time.
Learning to code isn’t enough
Historically, learn-to-code efforts have provided opportunities for the few, but new efforts are aiming to be inclusive.
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Approximate Acreage: 16,950 acres
Municipalities: Bridgewater, Easton, Norton, Raynham, Taunton, and West Bridgewater
The Hockomock Swamp and associated wetlands and water bodies comprise the largest vegetated freshwater wetland system in Massachusetts. The boundaries of the Hockomock Swamp ACEC include approximately 16,950 acres in the southeastern part of the state. The wetlands act as a huge water reservoir and serve as the headwaters for the Town River, which flows into the Taunton River. The wetlands and floodplains are connected hydrologically with an extensive underlying system of medium- and high-yield aquifers. There are three public water supply wells located in the ACEC, and others are proposed.
The Hockomock Swamp is a vast natural and scenic area. Because of its size, it is a unique and irreplaceable wildlife habitat. It is also the location of at least 13 rare and endangered species. According to the Massachusetts Historical Commission, the archaeological sites in the vicinity of this wetland complex are known to span a period of 9000 years; the potential quality and significance of the archaeological resources are enormous. Productive agricultural lands are located on the uplands adjacent to the wetlands, brooks, and rivers.
The Division of Fisheries and Wildlife (DFW) owns approximately 5000 acres of the Hockomock Swamp, within all six communities. The Hockomock Swamp Wildlife Management Area provides public access to the swamp and to several recreational areas. Additional public and nonprofit lands are located within the ACEC. The area is popular for hunting, fishing, boating, canoeing, swimming, and for the observation and study of flora and fauna.
Water Bodies included (partially or entirely) in the ACEC
- Rivers: Hockomock River (Bridgewater, West Bridgewater); Snake River (Norton, Taunton); Town River (Bridgewater, West Bridgewater)
- Lakes, Ponds: Gushee Pond (Raynham); Hewitts Pond (Raynham); Lake Nippenicket (Bridgewater, Raynham); Nunkets Pond (Bridgewater, Raynham)
- Wetlands: Hockomock Swamp (Bridgewater, Easton, Norton, Raynham, Taunton, West Bridgewater); Dead Swamp (Raynham); Titicut Swamp (Bridgewater, Raynham); Little Cedar Swamp (Easton)
- Brooks, Creeks: Black Brook (Easton); Coweesset Brook, West Meadow Brook, Onemile Brook (West Bridgewater)
Maps are intended to be used with the written boundary description contained in the ACEC designation document. The mapped boundary is not to be used by itself for definitive ACEC boundary delineation or regulatory interpretation. For review of site-specific projects in or bordering the ACEC, determinations must be made in the field by a certified professional in consultation with the appropriate Conservation Commission(s) and ACEC Program Staff.