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(-) Remove <label class='research-domain' title='Neurosciences and Neural Disorders'>LS5 (25)</label> filter LS5 (25)
Project acronym 5HT-OPTOGENETICS
Project Optogenetic Analysis of Serotonin Function in the Mammalian Brain
Researcher (PI) Zachary Mainen
Summary Serotonin (5-HT) is implicated in a wide spectrum of brain functions and disorders. However, its functions remain controversial and enigmatic. We suggest that past work on the 5-HT system have been significantly hampered by technical limitations in the selectivity and temporal resolution of the conventional pharmacological and electrophysiological methods that have been applied. We therefore propose to apply novel optogenetic methods that will allow us to overcome these limitations and thereby gain new insight into the biological functions of this important molecule. In preliminary studies, we have demonstrated that we can deliver exogenous proteins specifically to 5-HT neurons using viral vectors. Our objectives are to (1) record, (2) stimulate and (3) silence the activity of 5-HT neurons with high molecular selectivity and temporal precision by using genetically-encoded sensors, activators and inhibitors of neural function. These tools will allow us to monitor and control the 5-HT system in real-time in freely-behaving animals and thereby to establish causal links between information processing in 5-HT neurons and specific behaviors. In combination with quantitative behavioral assays, we will use this approach to define the role of 5-HT in sensory, motor and cognitive functions. The significance of the work is three-fold. First, we will establish a new arsenal of tools for probing the physiological and behavioral functions of 5-HT neurons. Second, we will make definitive tests of major hypotheses of 5-HT function. Third, we will have possible therapeutic applications. In this way, the proposed work has the potential for a major impact in research on the role of 5-HT in brain function and dysfunction.
Serotonin (5-HT) is implicated in a wide spectrum of brain functions and disorders. However, its functions remain controversial and enigmatic. We suggest that past work on the 5-HT system have been significantly hampered by technical limitations in the selectivity and temporal resolution of the conventional pharmacological and electrophysiological methods that have been applied. We therefore propose to apply novel optogenetic methods that will allow us to overcome these limitations and thereby gain new insight into the biological functions of this important molecule. In preliminary studies, we have demonstrated that we can deliver exogenous proteins specifically to 5-HT neurons using viral vectors. Our objectives are to (1) record, (2) stimulate and (3) silence the activity of 5-HT neurons with high molecular selectivity and temporal precision by using genetically-encoded sensors, activators and inhibitors of neural function. These tools will allow us to monitor and control the 5-HT system in real-time in freely-behaving animals and thereby to establish causal links between information processing in 5-HT neurons and specific behaviors. In combination with quantitative behavioral assays, we will use this approach to define the role of 5-HT in sensory, motor and cognitive functions. The significance of the work is three-fold. First, we will establish a new arsenal of tools for probing the physiological and behavioral functions of 5-HT neurons. Second, we will make definitive tests of major hypotheses of 5-HT function. Third, we will have possible therapeutic applications. In this way, the proposed work has the potential for a major impact in research on the role of 5-HT in brain function and dysfunction.
Project acronym 5HTCircuits
Project Modulation of cortical circuits and predictive neural coding by serotonin
Summary Serotonin (5-HT) is a central neuromodulator and a major target of therapeutic psychoactive drugs, but relatively little is known about how it modulates information processing in neural circuits. The theory of predictive coding postulates that the brain combines raw bottom-up sensory information with top-down information from internal models to make perceptual inferences about the world. We hypothesize, based on preliminary data and prior literature, that a role of 5-HT in this process is to report prediction errors and promote the suppression and weakening of erroneous internal models. We propose that it does this by inhibiting top-down relative to bottom-up cortical information flow. To test this hypothesis, we propose a set of experiments in mice performing olfactory perceptual tasks. Our specific aims are: (1) We will test whether 5-HT neurons encode sensory prediction errors. (2) We will test their causal role in using predictive cues to guide perceptual decisions. (3) We will characterize how 5-HT influences the encoding of sensory information by neuronal populations in the olfactory cortex and identify the underlying circuitry. (4) Finally, we will map the effects of 5-HT across the whole brain and use this information to target further causal manipulations to specific 5-HT projections. We accomplish these aims using state-of-the-art optogenetic, electrophysiological and imaging techniques (including 9.4T small-animal functional magnetic resonance imaging) as well as psychophysical tasks amenable to quantitative analysis and computational theory. Together, these experiments will tackle multiple facets of an important general computational question, bringing to bear an array of cutting-edge technologies to address with unprecedented mechanistic detail how 5-HT impacts neural coding and perceptual decision-making.
Serotonin (5-HT) is a central neuromodulator and a major target of therapeutic psychoactive drugs, but relatively little is known about how it modulates information processing in neural circuits. The theory of predictive coding postulates that the brain combines raw bottom-up sensory information with top-down information from internal models to make perceptual inferences about the world. We hypothesize, based on preliminary data and prior literature, that a role of 5-HT in this process is to report prediction errors and promote the suppression and weakening of erroneous internal models. We propose that it does this by inhibiting top-down relative to bottom-up cortical information flow. To test this hypothesis, we propose a set of experiments in mice performing olfactory perceptual tasks. Our specific aims are: (1) We will test whether 5-HT neurons encode sensory prediction errors. (2) We will test their causal role in using predictive cues to guide perceptual decisions. (3) We will characterize how 5-HT influences the encoding of sensory information by neuronal populations in the olfactory cortex and identify the underlying circuitry. (4) Finally, we will map the effects of 5-HT across the whole brain and use this information to target further causal manipulations to specific 5-HT projections. We accomplish these aims using state-of-the-art optogenetic, electrophysiological and imaging techniques (including 9.4T small-animal functional magnetic resonance imaging) as well as psychophysical tasks amenable to quantitative analysis and computational theory. Together, these experiments will tackle multiple facets of an important general computational question, bringing to bear an array of cutting-edge technologies to address with unprecedented mechanistic detail how 5-HT impacts neural coding and perceptual decision-making.
Project acronym A-FRO
Project Actively Frozen - contextual modulation of freezing and its neuronal basis
Researcher (PI) Marta de Aragão Pacheco Moita
Summary When faced with a threat, an animal must decide whether to freeze, reducing its chances of being noticed, or to flee to the safety of a refuge. Animals from fish to primates choose between these two alternatives when confronted by an attacking predator, a choice that largely depends on the context in which the threat occurs. Recent work has made strides identifying the pre-motor circuits, and their inputs, which control freezing behavior in rodents, but how contextual information is integrated to guide this choice is still far from understood. We recently found that fruit flies in response to visual looming stimuli, simulating a large object on collision course, make rapid freeze/flee choices that depend on the social and spatial environment, and the fly's internal state. Further, identification of looming detector neurons was recently reported and we identified the descending command neurons, DNp09, responsible for freezing in the fly. Knowing the sensory input and descending output for looming-evoked freezing, two environmental factors that modulate its expression, and using a genetically tractable system affording the use of large sample sizes, places us in an unique position to understand how a information about a threat is integrated with cues from the environment to guide the choice of whether to freeze (our goal). To assess how social information impinges on the circuit for freezing, we will examine the sensory inputs and neuromodulators that mediate this process, mapping their connections to DNp09 neurons (Aim 1). We ask whether learning is required for the spatial modulation of freezing, which cues flies are using to discriminate different places and which brain circuits mediate this process (Aim 2). Finally, we will study how activity of DNp09 neurons drives freezing (Aim 3). This project will provide a comprehensive understanding of the mechanism of freezing and its modulation by the environment, from single neurons to behaviour.
When faced with a threat, an animal must decide whether to freeze, reducing its chances of being noticed, or to flee to the safety of a refuge. Animals from fish to primates choose between these two alternatives when confronted by an attacking predator, a choice that largely depends on the context in which the threat occurs. Recent work has made strides identifying the pre-motor circuits, and their inputs, which control freezing behavior in rodents, but how contextual information is integrated to guide this choice is still far from understood. We recently found that fruit flies in response to visual looming stimuli, simulating a large object on collision course, make rapid freeze/flee choices that depend on the social and spatial environment, and the fly's internal state. Further, identification of looming detector neurons was recently reported and we identified the descending command neurons, DNp09, responsible for freezing in the fly. Knowing the sensory input and descending output for looming-evoked freezing, two environmental factors that modulate its expression, and using a genetically tractable system affording the use of large sample sizes, places us in an unique position to understand how a information about a threat is integrated with cues from the environment to guide the choice of whether to freeze (our goal). To assess how social information impinges on the circuit for freezing, we will examine the sensory inputs and neuromodulators that mediate this process, mapping their connections to DNp09 neurons (Aim 1). We ask whether learning is required for the spatial modulation of freezing, which cues flies are using to discriminate different places and which brain circuits mediate this process (Aim 2). Finally, we will study how activity of DNp09 neurons drives freezing (Aim 3). This project will provide a comprehensive understanding of the mechanism of freezing and its modulation by the environment, from single neurons to behaviour.
Project acronym BRAINSIGNALS
Project Optical dissection of circuits underlying fast cholinergic signalling during cognitive behaviour
Researcher (PI) Huibert Mansvelder
Host Institution (HI) STICHTING VU
Summary Our ability to think, to memorize and focus our thoughts depends on acetylcholine signaling in the brain. The loss of cholinergic signalling in for instance Alzheimer's disease strongly compromises these cognitive abilities. The traditional view on the role of cholinergic input to the neocortex is that slowly changing levels of extracellular acetylcholine (ACh) mediate different arousal states. This view has been challenged by recent studies demonstrating that rapid phasic changes in ACh levels at the scale of seconds are correlated with focus of attention, suggesting that these signals may mediate defined cognitive operations. Despite a wealth of anatomical data on the organization of the cholinergic system, very little understanding exists on its functional organization. How the relatively sparse input of cholinergic transmission in the prefrontal cortex elicits such a profound and specific control over attention is unknown. The main objective of this proposal is to develop a causal understanding of how cellular mechanisms of fast acetylcholine signalling are orchestrated during cognitive behaviour. In a series of studies, I have identified several synaptic and cellular mechanisms by which the cholinergic system can alter neuronal circuitry function, both in cortical and subcortical areas. I have used a combination of behavioral, physiological and genetic methods in which I manipulated cholinergic receptor functionality in prefrontal cortex in a subunit specific manner and found that ACh receptors in the prefrontal cortex control attention performance. Recent advances in optogenetic and electrochemical methods now allow to rapidly manipulate and measure acetylcholine levels in freely moving, behaving animals. Using these techniques, I aim to uncover which cholinergic neurons are involved in fast cholinergic signaling during cognition and uncover the underlying neuronal mechanisms that alter prefrontal cortical network function.
Our ability to think, to memorize and focus our thoughts depends on acetylcholine signaling in the brain. The loss of cholinergic signalling in for instance Alzheimer's disease strongly compromises these cognitive abilities. The traditional view on the role of cholinergic input to the neocortex is that slowly changing levels of extracellular acetylcholine (ACh) mediate different arousal states. This view has been challenged by recent studies demonstrating that rapid phasic changes in ACh levels at the scale of seconds are correlated with focus of attention, suggesting that these signals may mediate defined cognitive operations. Despite a wealth of anatomical data on the organization of the cholinergic system, very little understanding exists on its functional organization. How the relatively sparse input of cholinergic transmission in the prefrontal cortex elicits such a profound and specific control over attention is unknown. The main objective of this proposal is to develop a causal understanding of how cellular mechanisms of fast acetylcholine signalling are orchestrated during cognitive behaviour. In a series of studies, I have identified several synaptic and cellular mechanisms by which the cholinergic system can alter neuronal circuitry function, both in cortical and subcortical areas. I have used a combination of behavioral, physiological and genetic methods in which I manipulated cholinergic receptor functionality in prefrontal cortex in a subunit specific manner and found that ACh receptors in the prefrontal cortex control attention performance. Recent advances in optogenetic and electrochemical methods now allow to rapidly manipulate and measure acetylcholine levels in freely moving, behaving animals. Using these techniques, I aim to uncover which cholinergic neurons are involved in fast cholinergic signaling during cognition and uncover the underlying neuronal mechanisms that alter prefrontal cortical network function.
Project acronym C.o.C.O.
Project Circuits of con-specific observation
Researcher (PI) Marta De Aragao Pacheco Moita
Summary A great deal is known about the neural basis of associative fear learning. However, many animal species are able to use social cues to recognize threats, a defence mechanism that may be less costly than learning from self-experience. We have previously shown that rats perceive the cessation of movement-evoked sound as a signal of danger and its resumption as a signal of safety. To study transmission of fear between rats we assessed the behavior of an observer while witnessing a demonstrator rat display fear responses. With this paradigm we will take advantage of the accumulated knowledge on learned fear to investigate the neural mechanisms by which the social environment regulates defense behaviors. We will unravel the neural circuits involved in detecting the transition from movement-evoked sound to silence. Moreover, since observer rats previously exposed to shock display observational freezing, but naive observer rats do not, we will determine the mechanism by which prior experience contribute to observational freezing. To this end, we will focus on the amygdala, crucial for fear learning and expression, and its auditory inputs, combining immunohistochemistry, pharmacology and optogenetics. Finally, as the detection of and responses to threat are often inherently social, we will study these behaviors in the context of large groups of individuals. To circumvent the serious limitations in using large populations of rats, we will resort to a different model system. The fruit fly is the ideal model system, as it is both amenable to the search for the neural mechanism of behavior, while at the same time allowing the study of the behavior of large groups of individuals. We will develop behavioral tasks, where conditioned demonstrator flies signal danger to other naïve ones. These experiments unravel how the brain uses defense behaviors as signals of danger and how it contributes to defense mechanisms at the population level.
A great deal is known about the neural basis of associative fear learning. However, many animal species are able to use social cues to recognize threats, a defence mechanism that may be less costly than learning from self-experience. We have previously shown that rats perceive the cessation of movement-evoked sound as a signal of danger and its resumption as a signal of safety. To study transmission of fear between rats we assessed the behavior of an observer while witnessing a demonstrator rat display fear responses. With this paradigm we will take advantage of the accumulated knowledge on learned fear to investigate the neural mechanisms by which the social environment regulates defense behaviors. We will unravel the neural circuits involved in detecting the transition from movement-evoked sound to silence. Moreover, since observer rats previously exposed to shock display observational freezing, but naive observer rats do not, we will determine the mechanism by which prior experience contribute to observational freezing. To this end, we will focus on the amygdala, crucial for fear learning and expression, and its auditory inputs, combining immunohistochemistry, pharmacology and optogenetics. Finally, as the detection of and responses to threat are often inherently social, we will study these behaviors in the context of large groups of individuals. To circumvent the serious limitations in using large populations of rats, we will resort to a different model system. The fruit fly is the ideal model system, as it is both amenable to the search for the neural mechanism of behavior, while at the same time allowing the study of the behavior of large groups of individuals. We will develop behavioral tasks, where conditioned demonstrator flies signal danger to other naïve ones. These experiments unravel how the brain uses defense behaviors as signals of danger and how it contributes to defense mechanisms at the population level.
Project acronym CCC
Project Cracking the Cerebellar Code
Researcher (PI) Christiaan Innocentius De Zeeuw
Host Institution (HI) ERASMUS UNIVERSITAIR MEDISCH CENTRUM ROTTERDAM
Summary Spike trains transfer information to and from neurons. Most studies so far assume that the average firing rate or "rate coding" is the predominant way of information coding. However, spikes occur at millisecond precision, and their actual timing or "temporal coding" can in principle strongly increase the information content of spike trains. The two coding mechanisms are not mutually exclusive. Neurons may switch between rate and temporal coding, or use a combination of both coding mechanisms at the same time, which would increase the information content of spike trains even further. Here, we propose to investigate the hypothesis that temporal coding plays, next to rate coding, important and specific roles in cerebellar processing during learning. The cerebellum is ideal to study this timely topic, because it has a clear anatomy with well-organized modules and matrices, a well-described physiology of different types of neurons with distinguishable spiking activity, and a central role in various forms of tractable motor learning. Moreover, uniquely in the brain, the main types of neurons in the cerebellar system can be genetically manipulated in a cell-specific fashion, which will allow us to investigate the behavioural importance of both coding mechanisms following cell-specific interference and/or during cell-specific visual imaging. Thus, for this proposal we will create conditional mouse mutants that will be subjected to learning paradigms in which we can disentangle the contributions of rate coding and temporal coding using electrophysiological and optogenetic recordings and stimulation. Together, our experiments should elucidate how neurons in the brain communicate during natural learning behaviour and how one may be able to intervene in this process to affect or improve procedural learning skills.
Spike trains transfer information to and from neurons. Most studies so far assume that the average firing rate or "rate coding" is the predominant way of information coding. However, spikes occur at millisecond precision, and their actual timing or "temporal coding" can in principle strongly increase the information content of spike trains. The two coding mechanisms are not mutually exclusive. Neurons may switch between rate and temporal coding, or use a combination of both coding mechanisms at the same time, which would increase the information content of spike trains even further. Here, we propose to investigate the hypothesis that temporal coding plays, next to rate coding, important and specific roles in cerebellar processing during learning. The cerebellum is ideal to study this timely topic, because it has a clear anatomy with well-organized modules and matrices, a well-described physiology of different types of neurons with distinguishable spiking activity, and a central role in various forms of tractable motor learning. Moreover, uniquely in the brain, the main types of neurons in the cerebellar system can be genetically manipulated in a cell-specific fashion, which will allow us to investigate the behavioural importance of both coding mechanisms following cell-specific interference and/or during cell-specific visual imaging. Thus, for this proposal we will create conditional mouse mutants that will be subjected to learning paradigms in which we can disentangle the contributions of rate coding and temporal coding using electrophysiological and optogenetic recordings and stimulation. Together, our experiments should elucidate how neurons in the brain communicate during natural learning behaviour and how one may be able to intervene in this process to affect or improve procedural learning skills.
Project acronym CMTaaRS
Project Defective protein translation as a pathogenic mechanism of peripheral neuropathy
Researcher (PI) Erik Jan Marthe STORKEBAUM
Summary Familial forms of neurodegenerative diseases are caused by mutations in a single gene. It is unknown whether distinct mutations in the same gene or in functionally related genes cause disease through similar or disparate mechanisms. Furthermore, the precise molecular mechanisms underlying virtually all neurodegenerative disorders are poorly understood, and effective treatments are typically lacking. This is also the case for Charcot-Marie-Tooth (CMT) peripheral neuropathy caused by mutations in five distinct tRNA synthetase (aaRS) genes. We previously generated Drosophila CMT-aaRS models and used a novel method for cell-type-specific labeling of newly synthesized proteins in vivo to show that impaired protein translation may represent a common pathogenic mechanism. In this proposal, I aim to determine whether translation is also inhibited in CMT-aaRS mouse models, and whether all mutations cause disease through gain-of-toxic-function, or alternatively, whether some mutations act through a dominant-negative mechanism. In addition, I will evaluate whether all CMT-aaRS mutant proteins inhibit translation, and I will test the hypothesis, raised by our unpublished preliminary data shown here, that a defect in the transfer of the (aminoacylated) tRNA from the mutant synthetase to elongation factor eEF1A is the molecular mechanism underlying CMT-aaRS. Finally, I will validate the identified molecular mechanism in CMT-aaRS mouse models, as the most disease-relevant mammalian model. I expect to elucidate whether all CMT-aaRS mutations cause disease through a common molecular mechanism that involves inhibition of translation. This is of key importance from a therapeutic perspective, as a common pathogenic mechanism allows for a unified therapeutic approach. Furthermore, this proposal has the potential to unravel the detailed molecular mechanism underlying CMT-aaRS, what would constitute a breakthrough and a requirement for rational drug design for this incurable disease.
Familial forms of neurodegenerative diseases are caused by mutations in a single gene. It is unknown whether distinct mutations in the same gene or in functionally related genes cause disease through similar or disparate mechanisms. Furthermore, the precise molecular mechanisms underlying virtually all neurodegenerative disorders are poorly understood, and effective treatments are typically lacking. This is also the case for Charcot-Marie-Tooth (CMT) peripheral neuropathy caused by mutations in five distinct tRNA synthetase (aaRS) genes. We previously generated Drosophila CMT-aaRS models and used a novel method for cell-type-specific labeling of newly synthesized proteins in vivo to show that impaired protein translation may represent a common pathogenic mechanism. In this proposal, I aim to determine whether translation is also inhibited in CMT-aaRS mouse models, and whether all mutations cause disease through gain-of-toxic-function, or alternatively, whether some mutations act through a dominant-negative mechanism. In addition, I will evaluate whether all CMT-aaRS mutant proteins inhibit translation, and I will test the hypothesis, raised by our unpublished preliminary data shown here, that a defect in the transfer of the (aminoacylated) tRNA from the mutant synthetase to elongation factor eEF1A is the molecular mechanism underlying CMT-aaRS. Finally, I will validate the identified molecular mechanism in CMT-aaRS mouse models, as the most disease-relevant mammalian model. I expect to elucidate whether all CMT-aaRS mutations cause disease through a common molecular mechanism that involves inhibition of translation. This is of key importance from a therapeutic perspective, as a common pathogenic mechanism allows for a unified therapeutic approach. Furthermore, this proposal has the potential to unravel the detailed molecular mechanism underlying CMT-aaRS, what would constitute a breakthrough and a requirement for rational drug design for this incurable disease.
Project acronym CoordinatedDopamine
Project Coordination of regional dopamine release in the striatum during habit formation and compulsive behaviour
Researcher (PI) Ingo Willuhn
Host Institution (HI) Academisch Medisch Centrum bij de Universiteit van Amsterdam
Summary The basal ganglia consist of a set of neuroanatomical structures that participate in the representation and execution of action sequences. Dopamine neurotransmission in the striatum, the main input nucleus of the basal ganglia, is a fundamental mechanism involved in learning and regulation of such actions. The striatum has multiple functional units, where the limbic striatum is thought to mediate motivational aspects of actions (e.g., goal-directedness) and the sensorimotor striatum their automation (e.g., habit formation). A long-standing question in the field is how limbic and sensorimotor domains communicate with each other, and specifically if they do so during the automation of action sequences. It has been suggested that such coordination is implemented by reciprocal loop connections between striatal projection neurons and the dopaminergic midbrain. Although very influential in theory the effectiveness of this limbic-sensorimotor "bridging" principle has yet to be verified. I hypothesize that during the automation of behaviour regional dopamine signalling is governed by a striatal hierarchy and that dysregulation of this coordination leads to compulsive execution of automatic actions characteristic of several psychiatric disorders. To test this hypothesis, we will conduct electrochemical measurements with real-time resolution simultaneously in limbic and sensorimotor striatum to assess the regional coordination of dopamine release in behaving animals. We developed novel chronically implantable electrodes to enable monitoring of dopamine dynamics throughout the development of habitual behaviour and its compulsive execution in transgenic rats - a species suitable for our complex behavioural assays. Novel rabies virus-mediated gene delivery for in vivo optogenetics in these rats will give us the unique opportunity to test whether specific loop pathways govern striatal dopamine transmission and are causally involved in habit formation and compulsive behaviour.
The basal ganglia consist of a set of neuroanatomical structures that participate in the representation and execution of action sequences. Dopamine neurotransmission in the striatum, the main input nucleus of the basal ganglia, is a fundamental mechanism involved in learning and regulation of such actions. The striatum has multiple functional units, where the limbic striatum is thought to mediate motivational aspects of actions (e.g., goal-directedness) and the sensorimotor striatum their automation (e.g., habit formation). A long-standing question in the field is how limbic and sensorimotor domains communicate with each other, and specifically if they do so during the automation of action sequences. It has been suggested that such coordination is implemented by reciprocal loop connections between striatal projection neurons and the dopaminergic midbrain. Although very influential in theory the effectiveness of this limbic-sensorimotor "bridging" principle has yet to be verified. I hypothesize that during the automation of behaviour regional dopamine signalling is governed by a striatal hierarchy and that dysregulation of this coordination leads to compulsive execution of automatic actions characteristic of several psychiatric disorders. To test this hypothesis, we will conduct electrochemical measurements with real-time resolution simultaneously in limbic and sensorimotor striatum to assess the regional coordination of dopamine release in behaving animals. We developed novel chronically implantable electrodes to enable monitoring of dopamine dynamics throughout the development of habitual behaviour and its compulsive execution in transgenic rats - a species suitable for our complex behavioural assays. Novel rabies virus-mediated gene delivery for in vivo optogenetics in these rats will give us the unique opportunity to test whether specific loop pathways govern striatal dopamine transmission and are causally involved in habit formation and compulsive behaviour.
Project Cerebellar modules and the Ontogeny of Sensorimotor Integration
Researcher (PI) Martijn Schonewille
Summary The perfect execution of a voluntary movement requires the appropriate integration of current bodily state, sensory input and desired outcome. To assure that this motor output becomes and remains appropriate, the brain needs to learn from the result of previous outputs. The cerebellum plays a central role in sensorimotor integration, yet -despite decades of studies- there is no generally excepted theory for cerebellar functioning. I recently demonstrated that cerebellar modules, identified based on anatomical connectivity and gene expression, differ distinctly in spike activity properties. It is my long-term goal to identify the ontogeny of anatomical and physiological differences between modules, and their functional consequences. My hypothesis is that these differences can explain existing controversies, and unify contradicting results into one central theory. To this end, I have designed three key objectives. First, I will identify the development of connectivity and activity patterns at the input stage of the cerebellar cortex in relation to the cerebellar modules (key objective A). Next, I will relate the differences in gene expression levels between modules to differences in basal activity and strength of plasticity mechanisms in juvenile mice (key objective B). Finally, I will determine how module specific output develops in relation to behavior and what the effect of module specific mutations is on cerebellum-dependent motor tasks and higher order functions (key objective C). Ultimately, the combined results of all key objectives will reveal how distinct difference between cerebellar modules develop, and how this ensemble ensures proper cerebellar information processing for optimal coordination of timing and force of movements. Combined with the growing body of evidence for a cerebellar role in higher order brain functions and neurodevelopmental disorders, a unifying theory would be fundamental for understanding how the juvenile brain develops.
The perfect execution of a voluntary movement requires the appropriate integration of current bodily state, sensory input and desired outcome. To assure that this motor output becomes and remains appropriate, the brain needs to learn from the result of previous outputs. The cerebellum plays a central role in sensorimotor integration, yet -despite decades of studies- there is no generally excepted theory for cerebellar functioning. I recently demonstrated that cerebellar modules, identified based on anatomical connectivity and gene expression, differ distinctly in spike activity properties. It is my long-term goal to identify the ontogeny of anatomical and physiological differences between modules, and their functional consequences. My hypothesis is that these differences can explain existing controversies, and unify contradicting results into one central theory. To this end, I have designed three key objectives. First, I will identify the development of connectivity and activity patterns at the input stage of the cerebellar cortex in relation to the cerebellar modules (key objective A). Next, I will relate the differences in gene expression levels between modules to differences in basal activity and strength of plasticity mechanisms in juvenile mice (key objective B). Finally, I will determine how module specific output develops in relation to behavior and what the effect of module specific mutations is on cerebellum-dependent motor tasks and higher order functions (key objective C). Ultimately, the combined results of all key objectives will reveal how distinct difference between cerebellar modules develop, and how this ensemble ensures proper cerebellar information processing for optimal coordination of timing and force of movements. Combined with the growing body of evidence for a cerebellar role in higher order brain functions and neurodevelopmental disorders, a unifying theory would be fundamental for understanding how the juvenile brain develops.


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In what was once America's heartland dwell the survivors of the nuclear holocaust that annihilated most of civilization, leaving a scorched land filled with roving bands of vicious outlaws and savage new breeds of deadly creatures. A team of warriors known as the Alpha Triad have vowed to defend the Home, the survivors' last refuge. Blade, Hickok and Geronimo are all that stand between a tenuous existence and certain death. The Triad's latest mission is a trek to the Twin Cities to acquire urgently needed medical supplies. But an attack by renegade soldiers will put to the test all the cunning and skills the warriors possess. If Blade and his team can't survive the ferocious battle ahead, there's no chance left for anyone back at the Home.


text:
ABQ RAW
Preferential Treatment for Attorney General's Former Chief Counsel?
Santa Fe, New Mexico - On October 23rd, 2021, Santa Fe Police officers were dispatched to a car which had crashed into another parked car on a Santa Fe street. When an officer arrived on scene he observed a male subject standing near a car which had crashed into a parked car. The officer started to investigate the scene and noticed the driver had dilated pupils. It was later identified the drivers name was Matt Baca. At the time of the incident, Baca was the Chief Counsel of the New Mexico Attorney General's office working for Hector Balderas. The officer put Baca through a variety of field sobriety tests, and the officer had enough probable cause to arrest Baca for DWI. Baca was booked into the Santa Fe County Detention Center later that evening. It took a few weeks before other media outlets reported about the arrest. Media outlets such as ourselves typically have access to criminal court records through the New Mexico Court system portal that is more detailed than nmcourts.gov The New Mexico Secure Court Case System gives credentialed media, law enforcement, and law firms access to the criminal complaints, booking sheets, motions for the case, and other paperwork pertinent to any and all cases. Throughout ABQ RAW's case reporting we have reported on criminal cases to our viewers. ABQ RAW pulls the facts of the cases from court filed criminal complaints and police reports to give readers the best look into what actually happened. DWI cases like the one involving Mr. Baca are typically uploaded the day of the arrest or a few days later due to weekends or holidays. We have been searching daily since the end of October for Matt Baca's criminal complaint and charging documents but a search through all data bases within the New Mexico Courts portals does not return Baca's case.
The Odyssey portal showing no DWI charges for Matt Baca.
We spoke with Santa Fe Police Department on policy and procedures of officers uploading court documents.
Deputy Chief of Santa Fe Police Ben Valdez in an email said his officers turn in arrest reports for incidents such as DWI are required to be completed on the day of the arrest so documents can be provided to the respective Court in a timely manner. The arresting officer did submit all the paper work to the courts the day of the arrest. We asked the New Mexico Attorney General Office when they were made aware of Baca's arrest. We asked why the criminal complaint and charging documents are omitted from the Odyssey website.
A spokesperson said they were made aware of the arrest on October 25th, 2021. They also said the Office of the Attorney General does not have jurisdiction over the Odyssey website. The spokesperson went on to say: DWI is a very serious issue and the law should always be applied equally. We cannot comment on the prosecution of this matter as it involves an individual who was an employee at the time of the incident and is being handled by the appropriate law enforcement authority. We reached out to Santa Fe Courts on the paper work missing and they have refused comment.
Video of the arrest:
Group Urges Lawmakers to Pass Stronger Laws During Session
Governor MLG's 2022 State of the State Speech
Burglary Suspect is ABQ's 7th Homicide Callout

redpajama_set_name: RedPajamaCommonCrawl


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Comparison of the E-test with the NCCLS M38-P Method for Antifungal Susceptibility Testing of Common and Emerging Pathogenic Filamentous Fungi
Ana Espinel-Ingroff
Division of Infectious Diseases, Medical College of Virginia of Virginia Commonwealth University, Richmond, Virginia 23298-0049
DOI: 10.1128/JCM.39.4.1360-1367.2001
The National Committee for Clinical Laboratory Standards (NCCLS) M38-P method describes standard parameters for testing the fungistatic antifungal activities (MICs) of established agents against filamentous fungi (molds). The present study evaluated the in vitro fungistatic activities of itraconazole and amphotericin B by the E-test and the NCCLS M38-P microdilution method against 186 common and emerging pathogenic molds (123 isolates of Aspergillus spp. [five species], 16 isolates of Fusarium spp. [two species], 4Paecilomyces lilacinus isolates, 5 Rhizopus arrhizus isolates, 15 Scedosporium spp., 18 dematiaceous fungi, and 5 Trichoderma longibrachiatumisolates). The agreement between the methods for amphotericin B MICs ranged from 70% for Fusarium solani to ≥90% for most of the other species after the first reading; agreement was dependent on both the incubation time and the species being evaluated. Major discrepancies between the amphotericin B MICs determined by the E-test and the NCCLS M38-P method were demonstrated for three of the five species of Aspergillus tested and the two species ofFusarium tested. This discrepancy was more marked after 48 h of incubation; the geometric mean MICs determined by the E-test increased between 24 and 48 h from between 1.39 and 3.3 μg/ml to between 5.2 and >8 μg/ml for Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans. The agreement between the itraconazole MICs determined by the E-test and the NCCLS M38-P method ranged from 83.3% for A. nidulansto ≥90% for all the other species tested; the overall agreement was higher (92.7%) than that for amphotericin B (87.9%). The agreement was less dependent on the incubation time. Clinical trials need to be conducted to establish the role of the results of either the E-test or the NCCLS M38-P method in vitro for molds with the two agents as predictors of clinical outcome.
A higher incidence of fungal infections has been documented since the 1980s with the parallel emergence of either new fungal pathogens or fungi that were considered nonpathogenic as etiologic agents of systemic disease, especially in the immunocompromised host (2, 4, 8, 16, 27, 28, 31). Although the volume of disseminated infections caused by the filamentous fungi (molds) is lower than that caused by yeasts, their higher incidence and the increased resistance of molds to established antifungal agents (2, 4, 9, 10, 14-16, 18, 27, 31) warrant the evaluation of the in vitro susceptibilities of these fungi to both established and investigational agents. Aspergillus fumigatus is responsible for the majority (85 to 90%) of the different clinical manifestations of severe mold infections (9). However, other Aspergillus spp.,Fusarium spp., Scedosporium apiospermum(Pseudallescheria boydii), Scedosporium prolificans, and less common molds have become important emerging pathogens (2, 4, 15, 18, 27, 28).
The National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Tests has proposed standard procedures for the antifungal susceptibility testing of molds (NCCLS M38-P document [22]). On the basis of data from several studies (12, 13), this document recommends the use of (i) standard RPMI 1640 broth; (ii) nongerminated conidial inoculum suspensions of approximately 104 CFU/m1; and (iii) incubation at 35°C for 24 h (Rhizopus spp.), 48 h (Aspergillus spp., Fusarium spp., and other opportunistic molds), and 72 h (S. apiospermum). The determination of MICs according to the instructions in the NCCLS M38-P document requires the visual examination of growth inhibition compared to the growth for the growth control (22). Some degree of correlation has been documented between the results of the M38-P method and treatment outcomes in experimental infections (24); however, the clinical value of the NCCLS methods for the testing of molds needs to be established.
The NCCLS methods are cumbersome and time-consuming, and alternative methods have been evaluated in recent years for testing of yeasts. Among these procedures, the E-test has been suggested as an alternative approach for the antifungal susceptibility testing of yeasts (11, 21, 25, 32), and more recently, this method has been evaluated for the antifungal susceptibility testing of certain molds (30). Good levels of agreement between the E-test and NCCLS methods have been demonstrated in these studies. The present study evaluated the activities of itraconazole and amphotericin B by the E-test against 186 common and emerging pathogenic molds recovered from clinical specimens during the last 5 years. The MICs obtained by the E-test were compared to those obtained by the proposed NCCLS M38-P broth microdilution method for filamentous fungi (22).
(This work was partially presented at the 96th General Meeting of the American Society for Microbiology 1996, 19 to 23 May 1996, New Orleans, La., and the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, 24 to 27 September 1998, San Diego, Calif.)
Isolates.The set of 186 isolates evaluated includedAspergillus flavus (16 isolates), A. fumigatus(69 isolates), Aspergillus nidulans (12 isolates),Aspergillus niger (10 isolates), Aspergillus terreus (16 isolates), Fusarium oxysporum (6 isolates),Fusarium solani (10 isolates), Paecilomyces lilacinus (4 isolates), Rhizopus arrhizus (5 isolates),S. apiospermum (10 isolates), S. prolificans (5 isolates), Trichoderma longibrachiatum (5 isolates), and 18 dematiaceous molds (one to three isolates each of Bipolarisspp., Cladophialophora bantiana, Cladophialophora cladosporioides, Dactylaria constricta var. gallopava, Phaeoacremonium parasiticum [Phialophora parasitica], and Wangiella dermatitidis). Each isolate originated from a different patient and was received at the Medical Mycology Research Laboratory, Medical College of Virginia, Virginia Commonwealth University, for MIC testing during the last 5 years. Isolates were maintained at −70°C until testing was performed. The reference isolate A. flavus ATCC 204304 (22) and the quality control (QC) strain Candida parapsilosis ATCC 22019 (23) were included as control isolates for both the NCCLS and the E-test methods. For the latter strain, microdilution MIC ranges of the agents evaluated in the study as well as preliminary QC ranges for the E-test are well established (3, 11). Reference MIC ranges have also been established for A. flavus ATCC 204304 on the basis of repeated testing in a prior study (13), and these values are listed in the M38-P document (22). MIC ranges for the QC and reference isolates were within established values by both methods (3, 11, 22, 23).
Antifungal agents.The E-test gradient strips of amphotericin B and itraconazole were provided by the manufacturer (AB BIODISK, Solna, Sweden). The concentration gradient for each drug ranged from 0.004 to 32 μg/ml. The strips were stored at −20°C until the day on which the test was performed. Amphotericin B (Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Conn.) and itraconazole (Janssen Pharmaceutica, Titusville, N.J.) were provided by the manufacturers as assay powders. As described in the NCCLS M38-P document (22), additive drug dilutions were prepared at 100 times the final concentrations in 100% dimethyl sulfoxide, followed by further dilutions (1:50) in the NCCLS standard RPMI 1640 medium to yield two times the final strength required for the test. The drugs were frozen at −70°C at their final concentrations (8 to 0.0078 μg/ml) for testing by the M38-P method until they were needed.
Inoculum preparations.Stock inoculum suspensions were prepared as described in the NCCLS M38-P document (22) from 7-day-old cultures grown on potato dextrose agar slants at 35°C, and the suspensions were adjusted spectrophotometrically to optical densities that ranged from 0.09 to 0.3 at 530 nm (82 to 60% transmittance); the stock suspensions contained mostly conidia. The final sizes of the stock inoculum suspensions of most of the isolates tested ranged from 0.5 × 106 to 4.5 × 106 CFU/ml, as demonstrated by quantitative colony counts on Sabouraud dextrose agar. The densities of Bipolaris sp. stock inoculum suspensions were lower (2 × 105 to 7 × 105 CFU/ml). The nongerminated conidial inoculum suspensions were diluted 1:50 in medium for testing by the M38-P method.
E-test pilot study.During an earlier investigation for testing of yeasts (11), several medium formulations were evaluated in order to identify the optimal medium for MIC determination by the E-test. In the present study, a pilot study was conducted with three media with a small sample of the 186 mold isolates evaluated. MICs were obtained by the E-test for the 30 isolates listed in Table 1(single test runs) with three medium formulations: (i) solidified (1.5%) RPMI 1640 medium with 2% dextrose (RPMI-agar), (ii) solidified antibiotic medium 3 (M-3 agar), and (iii) Casitone agar (Remel Inc., Lenexa, Kans.). Since the MICs obtained with the three media were comparable for the 30 isolates and both antifungal agents tested (see Table 1), the remaining 156 molds were tested only with the RPMI-agar. The latter medium is a more chemically defined medium than the other two and is less subject to lot-to-lot variation.
E-test procedure.The E-test was performed by following the manufacturer's instructions. Each solidified medium was inoculated by dipping a nontoxic (latex-free) sterile swab into the respective undiluted stock inoculum suspension and evenly streaking it in three directions over the entire surface of a 150-mm petri plate containing 60 ml of medium; the swab was dipped into the inoculum suspension each time that the agar surface was streaked when isolates ofBipolaris spp. were tested. The agar surface was allowed to dry for 15 min, and the strips were placed onto the inoculated agar. As described above, C. parapsilosis ATCC 22019 and A. flavus ATCC 204304 were tested each time that a set of isolates was evaluated. The plates were incubated at 35°C, and the MICs were determined following incubation for 24 h to 4 days. The MICs determined by the E-test were the lowest drug concentrations at which the border of the elliptical inhibition intercepted the scale on the antifungal strip (see Fig. 1 and 2).
NCCLS broth microdilution method (M38-P document).On the day of the test, each microdilution well that contained 100 μl of the diluted (two times) drug concentrations was inoculated with 100 μl of the diluted (two times) conidial inoculum suspensions (final volume in each well, 200 μl). Growth and sterility controls were included for each isolate tested. As described above, C. parapsilosisATCC 22019 and A. flavus ATCC 204304 were tested each time that a set of isolates was evaluated. Microdilution trays were incubated at 35°C and examined at 48 h for MIC determination; MICs for S. apiospermum were determined after 72 h of incubation, and those for C. bantiana were determined on day 4. The MICs were determined by the visual inspection of growth inhibition as described in the NCCLS M38-P document (22) and corresponded to complete growth inhibition.
Data analyses.Because the E-test strips contain a continuous gradient instead of the established twofold drug dilution schema, MICs determined by the E-test were elevated to the next twofold dilution concentration, which matched the drug dilution schema of the NCCLS M38-P method (8 to 0.0078 μg/ml). This elevation of MICs for the E-test facilitated the comparisons and presentation of results. Both on-scale and off-scale MICs were included in the analysis. As analyzed previously (11, 13), discrepancies between the MIC endpoints of no more than 3 dilutions (e.g., 0.5, 1.0, and 2 μg/ml) were used for calculation of the percent agreement. The MICs and MIC ranges determined by the E-test and the NCCLS M38-P method and the corresponding geometric mean values were obtained for each species-drug combination tested. For both the E-test and the NCCLS M38-P method, the MICs for 90% of the isolates tested (MIC90s) were determined for species for which ≥10 isolates were available; MICs for 50% of the isolates tested were obtained for species represented by less than 10 isolates.
E-test pilot study.Because optimal medium conditions had not been investigated for susceptibility testing of molds by the E-test when the present study was initiated, three medium formulations were evaluated with the first 30 mold isolates that were tested. The results for amphotericin B and itraconazole by the E-test with the three media were the same or no more than 2 dilutions different for the representative isolates of each species listed in Table1. However, the inhibition ellipses were narrower with RPMI-agar than with M-3 agar, especially for A. flavus. The trailing effect was a major problem only for testing of some R. arrhizus isolates with itraconazole. Therefore, the MIC data obtained with the RPMI-agar for 25 of the 30 molds tested in the pilot study were incorporated into the data presented in Table2, and the comparative evaluation was continued by using only RPMI-agar for the E-test. Only one other isolate of R. arrhizus was received in the Medical Mycology Research Laboratory for MIC testing during the last 3 years, and the results obtained by both procedures were similar to those presented in Table 1.
E-test pilot study resultsa
M38-P MICs for 181 opportunistic molds determined by the E-test and the NCCLS M38-P methoda
Comparison of amphotericin B MICs obtained by the E-test and the NCCLS M38-P method.Although the inhibition ellipses for amphotericin B were clear and well defined, they were usually narrower than those for itraconazole for most isolates (Fig.1 and 2), especially after the second reading. The agreement between the methods for amphotericin B MICs ranged from 70% for F. solani to ≥90% for most of the other species after the first reading; agreement was dependent on both the incubation time and the species being evaluated (Table 3). Major discrepancies between the amphotericin B MICs determined by the E-test and the NCCLS M38-P method were demonstrated for three of the five species of Aspergillus tested and the two species ofFusarium tested, for which wider and higher MICs were obtained by the E-test (Table 2). This discrepancy was more marked after 48 h of incubation; the geometric mean MICs obtained by the E-test increased between 24 and 48 h from between 1.39 and 3.3 μg/ml to between 5.2 and >8 μg/ml for A. flavus, A. fumigatus, A. nidulans, and F. oxysporum. The percent agreements were also substantially lower after 48 h of incubation than after 24 h of incubation for four of the five species ofAspergillus tested and F. solani (Table 3). The agreement between the amphotericin B MICs obtained by the two methods was good for the dematiaceous molds and other species evaluated.
Itraconazole and amphotericin B MICs at 48 h forA. fumigatus (top left), A. terreus (top right), and A. flavus (bottom), as determined by the E-test.
Amphotericin B MICs for one isolate of Aspergillus flavus: MIC at 24 h, 0.5 μg/ml (left); MIC at 48 h, 8 μg/ml (right).
Distribution of differences in MICs for 181 molds and percent agreement within 3 dilutions for the E-test and the NCCLS M38-P method
Comparison of itraconazole MICs obtained by the E-test and the NCCLS M38-P method.A major problem with antifungal susceptibility testing of yeasts with azole compounds is the trailing effect, which is due to the concentration-dependent partial inhibition of fungal growth. When one is performing the E-test procedure, microcolonies around or inside the entire inhibition ellipse are observed with trailing MICs for certain yeasts. It has been demonstrated that the trailing effect can be minimized in the testing of yeasts by the use of either RPMI-agar or Casitone agar (11). In the present study trailing was not a major problem, as it was when Candidaspp. and other yeasts were tested. In addition, there was no apparent medium dependence, as demonstrated by the results for the 30 isolates that were tested on the three agars. E-test inhibition ellipses for itraconazole were as clear and well-defined as those for amphotericin B for most of the isolates tested (Fig. 1). Small colonies were observed inside the inhibition ellipses (trailing effect) for some isolates ofF. oxysporum, P. lilacinus, and R. arrhizus; ellipses for some of these isolates were also narrow. The agreement between the itraconazole MICs obtained by the E-test and the NCCLS M38-P method ranged from 83.3% for A. nidulans and the dematiaceous fungi to ≥90% for all the other species tested; the overall agreement was higher (92.7%) than that for amphotericin B (87.9%) (Table 3). In contrast to the MICs of amphotericin B, the itraconazole MICs determined by the E-test either had no change or shifted very little between the two incubation times evaluated for most of the species (Table 2). The exception was A. fumigatus, because more discrepant MICs (>2 dilutions) were found between the two methods at 48 h (18 isolates) than at 24 h (18 isolates) (Table 3).
In the present study, the percent agreement between the results of the two methods for amphotericin B was lower (<80%) for A. flavus, A. terreus, and the two Fusarium spp. Low levels of agreement (60 to 80%) between the results of the E-test and a microdilution method have previously been reported for A. flavus and F. solani (30). However, the investigators did not evaluate isolates of either A. nidulans or F. oxysporum. Also, MICs determined by the E-test were read only after 48 h of incubation in that study. On the basis of the level of agreement between the two methods in the present study, it appears that amphotericin B MICs forAspergillus spp. should be determined by the E-test as soon as sufficient growth allows it (before 48 h). However, which of the two incubation times is providing the most clinically relevant MICs by the E-test? Although the conventional form of amphotericin B remains the drug of choice for the treatment of life-threatening fungal infections, its clinical efficacy is suboptimal in the immunocompromised host (10). In several studies (5, 17, 20, 26), death was attributed to disseminated aspergillosis in 27 of 62 patients, despite adequate treatment with amphotericin B (10). In the report of one of those studies (17), six of the seven patients whose cause of death was attributed to disseminated aspergillosis were infected with A. flavus. In the present study, the disagreement between the two methods was higher for A. flavus than for the otherAspergillus spp. tested (Table 3). The reason was that the MIC determined by the E-test shifted from ≤1.0 to ≥8 μg/ml (Fig. 2) for 4 of the 16 isolates of A. flavus tested and MICs were 2 to >8 μg/ml for the other isolates tested. Clinical resistance of this species to amphotericin B has been reported; unfortunately, the MICs for the infecting isolates were not reported (5, 17, 20, 26). It is noteworthy that the E-test appeared to be superior to the NCCLS method for yeasts (23) in its ability to detect amphotericin B-resistant Candida spp. and Cryptococcus neoformans strains (6, 21, 32). Further studies are needed to evaluate the correlation of the MICs determined by the E-test and those determined by the NCCLS M38-P method with in vivo outcome in experimental infections or clinical trials.
It has been suggested that amphotericin B MICs are not good predictors of the clinical response to treatment with this agent in patients with disseminated fusarial infections. In 56 of 73 patients who failed amphotericin B therapy for disseminated fusarial infection, the MICs for the infecting isolates were indiscriminately low (≤1.0 μg/ml) or high (≥2 μg/ml) (2, 16). However, in vitro testing was performed in those studies by using nonstandardized procedures. In addition, the status of the host and the degree of tissue involvement are also important in predicting the clinical outcome of therapy in patients with fusarial and other opportunistic infections. The amphotericin B MIC90s for F. solani determined by the E-test in the present evaluation and in the study of Szekely et al. (30) were ≥8 μg/ml. Again, the clinical relevance of the MICs for molds determined by both methods need to be established in clinical trials with standardized methods for in vitro testing.
The most important role of antifungal susceptibility testing is detection of isolates potentially resistant to the agent being evaluated. High amphotericin B MICs were obtained by both methods for most isolates of P. lilacinus and Scedosporiumspp. (MIC90s, 4 to >8 μg/ml) and some isolates ofA. terreus. The agreement between the two methods was excellent for the first three species and low for A. terreus(Table 3). In contrast, Szekely et al. (30) reported a 20% agreement between the two methods for Scedosporium spp. and an excellent agreement for A. terreus by following a procedure similar to the M38-P method (22). In their study, they prepared drug dilutions directly in RPMI 1640 broth instead of the solvent. The recent published literature regarding the results of in vitro and clinical studies indicates that the amphotericin B MICs for these four species are usually high and that infections caused by these molds are refractory to treatment with amphotericin B (1, 7, 8, 15, 18, 19, 29).
Although the NCCLS M38-P document states that azole MICs are the lowest concentrations that show a 50% inhibition of growth compared to the growth for the control, recent data developed by an NCCLS subcommittee suggest that the conventional criterion of MIC determination (100% or complete growth inhibition) could more clearly and reliably detect azole resistance (A. Espinel-Ingroff, M. S. Bartlett, V. Chaturvedi, K. Hazen, M. A. Ghannoum, M. A. Pfaller, M. G. Rinaldi, and T. J. Walsh, unpublished data). Because of that, the values depicted in Table 2 were obtained by using the 100% inhibition criterion for MIC determination. Furthermore, most itraconazole concentrations resulting in 50% inhibition (not shown in Table 2) were only 1 to 2 dilutions lower than the corresponding concentrations causing 100% inhibition.
In the present study, the overall agreement between the two methods was higher for itraconazole (92.7%) than for amphotericin B (87.9%). Similar but lower levels of agreement (71% for amphotericin B versus 88% for itraconazole) were reported in another evaluation of the E-test for susceptibility testing of Aspergillus spp.,F. solani, Scedosporium spp., and W. dermatitidis(30). The set of A. fumigatus isolates evaluated included four isolates for which high itraconazole MICs (≥8 μg/ml) have been obtained by the NCCLS and other methods by using the 100% criterion (9; Espinel-Ingroff et al., unpublished data). In the present study, the itraconazole MICs for these isolates determined by the E-test were 4 to ≥8 μg/ml at both incubation times. As reported by Denning et al. (9), two of the four isolates were recovered from patients who failed appropriate itraconazole therapy. These results suggest that both methods can identify itraconazole-resistant isolates of Aspergillus. Both methods also yielded high itraconazole MICs forFusarium spp., P. lilacinus, S.prolificans, and T. longibrachiatum (Table 2); and these results are comparable to those determined by other procedures for these species (1, 2, 7, 8, 27). E-test results have not been published for P. lilacinus, R. arrhizus, or T. longibrachiatum, and the dematiaceous species (except W. dermatitidis) evaluated in the study.
In conclusion, on the basis of data from this and another study (30), the E-test has potential value for use for the antifungal susceptibility testing of mold pathogens. Although the E-test is easier to perform, it is important to consider that, as for any test for antimicrobial susceptibility testing, the medium formulation and, in this case, the depth of the agar can influence the MIC. Therefore, the manufacturer's recommendations should be followed when attempting to obtain MICs by the E-test. Also, E-test strips are available only for investigational purposes. The wider amphotericin B MICs obtained by the E-test for some Aspergillus spp. also suggest that this method could be more useful than the NCCLS M38-P method in detecting Aspergillus isolates potentially resistant to this agent or that the latter method may not be a suitable procedure for these species. Future studies with the new triazoles and echinocandins, which are undergoing phase II and III clinical trials, will assess the value of the E-test for measurement of their in vitro activities against molds. However, in vivo-in vitro evaluations of drug efficacy also are needed to provide a better assessment of the utilities of both methods for use in the clinical laboratory as predictors of antifungal resistance in patients with mold infections.
Many thanks go to AB Biodisk and Remel Inc. for providing the E-test strips and the solified agars for the E-test.
Returned for modification 22 November 2000.
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National Committee for Clinical Laboratory Standards Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Proposed standard M38-P. 1998 National Committee for Clinical Laboratory Standards Wayne, Pa
National Committee for Clinical Laboratory Standards Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A. 1997 National Committee for Clinical Laboratory Standards Wayne, Pa
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Evaluation of possible correlations between antifungal susceptibilities of filamentous fungi in vitro and antifungal treatment outcomes in animal infection models.Antimicrob. Agents Chemother.421998282288
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Evaluation of the Etest method for determining fluconazole susceptibilities of 402 clinical yeast isolates by using three different agar media.J. Clin. Microbiol.36199825862589
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Journal of Clinical Microbiology Apr 2001, 39 (4) 1360-1367; DOI: 10.1128/JCM.39.4.1360-1367.2001
You are going to email the following Comparison of the E-test with the NCCLS M38-P Method for Antifungal Susceptibility Testing of Common and Emerging Pathogenic Filamentous Fungi
Microbial Sensitivity Tests


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The JV Baseball team has had a busy past few days leading to a very busy week. The streak of 7 games in 8 days kicked off last Friday with a game against North Montgomery. The JV Dogs had some momentum heading into the game riding a 2 game win streak. However, the offense couldn't find any holes in the Charger Defense and ended up losing 8-1 with the only run coming on a hard single by Ethan Stinson plating Cole Wills.
On Saturday the JV Dogs followed up the marathon of a Varsity game against Crawfordsville by getting the sticks going again and scoring a then season-high 14 runs. Ethan Stinson kept his good game streak alive by going 2-2 at the plate with 2 RBIs and also ended up being the winning pitcher. Hugo Alanis & Adrick Mounts each collected 2 RBIs. This coming off a solid start by Gabe Chavez who came down from the Varsity to get the start and managed to go 5 innings with 8 strikeouts before Ethan came on in relief.
Monday night the Hot Dogs traveled to West Lafayette to play in some drizzly weather and started fairly quick getting 2 quick runs but then in the bottom of the frame allowing West Lafyatte to score 7. This would only be the tip of the iceberg for the two teams as they would combine for 39 runs in the game. With the scoring onslaught, there were several guys that came up big offensively with Cole Wills collecting 4 hits, 2 walks and 7 RBIs, Hugo Alanis 4 hits and 3 RBIs, Eduardo Valdez 2 hits, 2 walks and 4 RBIs, Angel Cruz 3 hits and 3 runs scored, and Jimmy Ostler with 4 walks and 3 runs scored.
In a game that saw the JV Dogs use 4 pitchers, Cole continued the trend of a player stepping up and having a solid 1st pitching outing of the season by covering 3.1 innings in relief and kept the JV Dogs in the game. Cole ended up earning the win which was saved by Adrick Mounts who got the final 2 outs of the game. The West Lafayette win brought the JV record to 4-5 on the season.


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Every little kid knows that red + blue = purple. One of my granddaughters' favorite books is "Mix it Up! by Herve Tullet. This interactive book is all about how paint colors mix to make other colors. If you have read my other blog posts, you might think that almost every post is about mixing it up. I think that life (and construction projects) is not a straight line, yet sometimes it is the detours and mix- ups that make life worth living (and projects more successful), but I digress. Let's agree that purple is a mixed-up color.
I was driving back to my office last week, when I heard the NPR article about how Iowa is a "purple" state, but that both the legislature and the governor's office are all Republican for the first time in a long time. The democrat running for Governor could win and bring Iowa back to its "purple" status. I come from what used to be an extremely "red" state, Ohio, with the exception of Cleveland, that has always been "blue". Now Cincinnati, my home town, has become "blue" as well. Ohio is now a "purple" state.
Perhaps I've always been a little "purple". My logo was designed 25 years ago. As you probably know, it is a purple square with an orange dot. The color of the square is purple, but it really is closer to navy blue than it is to proper purple. I suppose my "blue" roots must be showing.
When I got married, I told the women in the wedding party that they could wear any style dress as long as it was in any shade of purple that pleased them. It was so much fun to see what people chose. My husband's grandmother chose a pale mauve, almost pink, and my sister chose a deep eggplant color – and we had every color in between. We bought the men purple ties. The cake was decorated in purple pansies. It all worked so well together (and so do my husband and I: we are about to celebrate 40 years of purple wedded bliss)!
In design, I often use purple as a neutral. Whether it is a pale grey with a purple cast (Benjamin Moore's Misty Memories 2118-60), a medium purply-gray (Benjamin Moore's Sea Life 2118-40), or a deep purple that is almost black (Benjamin Moore's Toucan Black 2115-20), these "purples" are so much more interesting than their counterparts of grey or black, yet they still function well as neutral "colors" that work well with any accent color in a project's palette. At one point, a long-term client said that they always searched for the "purple" on each project. I confess, I do love purple!


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Mount Olympus Lake St Clair (photograph: Dan Broun)
(Cradle Mountain - Lake St Clair National Park)
Australia's deepest lake in a wilderness carved by glaciers.
Caravan access
Overnight walk
Short walk
World Heritage Area
Lake St Clair National Park
c/o Post Office
Derwent Bridge TAS 7140
Email: lakestclair@parks.tas.gov.au
The atmosphere at Lake St Clair is moody and ethereal – there's an almost enchanted presence at this tranquil place. The Aboriginal name for Lake St Clair is leeawuleena, meaning 'sleeping water', and on still days you can truly appreciate the origins of this name.
Lake St Clair is part of the celebrated Cradle Mountain-Lake St Clair National Park – the heart of the Tasmanian Wilderness World Heritage Area. It is at Lake St Clair that Tasmania's most iconic multi-day walk, the Overland Track, comes to an end. Over six days, bushwalkers on this renowned track traverse a diverse and spectacular landscape, finishing up at the lake's edge.
In addition to the experiences to be had upon the lake, visitors can explore numerous walking trails throughout the park. A range of short and longer tracks meander off through ancient rainforests to alpine lakes or mountain summits. Regardless of which route you choose, you'll find yourself submerged in Lake St Clair's lavish wilderness.
A valid parks pass is required for entry to Tasmania's national parks.
Choose from a range of pass options to best meet your needs.
Carved out by glaciers over millions of years, Lake St Clair is Australia's deepest freshwater lake. Located at the southern end of the Cradle Mountain-Lake St Clair National Park, it forms part of Tasmania's Wilderness World Heritage Area and offers spectacular scenery, an award-winning visitor centre, and a range of beautiful walks to suit every ability.
The environment around Lake St Clair is among Tasmania's most spectacular. Deep glacial lakes are fringed by dense forests of ancient pines, and the high mountains provide a dramatic backdrop. Native wildlife is abundant in the area – Bennett's wallabies and pademelons are easily spotted by visitors to the lake, and wombats, echidnas, quolls, and platypus are also resident.
There is an excellent visitor centre at Cynthia Bay where you can learn about the history of the area, from the effects of glaciation to the ways in which Aboriginal communities interacted with the land. Information officers at the centre can provide advice about activities in the park.
The passenger ferry which operates from Cynthia Bay to Narcissus Bay at the northern end of the lake is Australia's highest altitude ferry service, and offers a leisurely way to enjoy the area's impressive scenery. The ferry timetable and booking information are available at Lake St Clair Lodge.
Several of Tasmania's Great Short Walks are within the Lake St Clair area. These include Echo Point, Mount Rufus, and Shadow Lake, which range from 3-7 hours. There are also several short walks of less than 2 hours that let you explore the striking landscapes closer to Lake St Clair: the lamairremener tabelti walk offers an insight into the Aboriginal communities who lived around the lake, the Watersmeet Walk takes you to the confluence of the Cuvier and Hugel rivers, and beyond that is a loop to Platypus Bay.
In late spring and summer, you can expect to see brilliant displays of colourful wildflowers, including waratahs, orchids and banksias.
Nature and conservation
The environment around Lake St Clair is among Tasmania's most spectacular. Deep glacial lakes are fringed by dense forests of ancient pines, and the high mountains provide a dramatic backdrop. Native wildlife is abundant in the area – Bennett's wallabies and pademelons are easily spotted by visitors to the lake, and wombats, echidnas, quolls, and platypus are also resident.
Like much of Tasmania's highlands, Lake St Clair is the result of intense glaciation in previous ice ages. The deep basin in which the lake lies was scoured out by the action of glaciers. Moraines, where debris is forced to the margins of the glacier, run along part of the length of the lake. The Watersmeet Track runs along the top of one of these moraines.
The lake is the deepest in Australia, with a maximum depth of 167 metres.
For more information on our wildlife please see Tasmania's wildlife.
Visit our caring for wildlife page for more information on interacting with and protecting our native animals.
Tasmanian Aboriginal people have a long and continuing relationship with the area around Lake St Clair. The traditional name for the lake itself is leeawuleena, meaning 'sleeping water', and today's vegetation patterns show signs of thousands of years of Aboriginal burning practices.
There are a number of Aboriginal historic sites and artefacts, including remnant stone tools, caves and rock shelters, which you can learn more about on the Aboriginal Cultural Heritage Walk which departs from Lake St Clair.
Buy a map from TasMap
Lake St Clair is at the southern end of Cradle Mountain - Lake St Clair National Park. It is 2.5 hours west of Hobart via the Lyell Highway (A10) and a similar distance from Launceston via Longford and Poatina. At Derwent Bridge turn right onto Lake St Clair Rd (C193) and follow it for 5.5 km to the visitor centre at Cynthia Bay.
Both the Lyell Highway and the access road from Derwent Bridge may occasionally be closed by snow in winter.
There is no direct road link through the Cradle Mountain - Lake St Clair National Park to join the two ends of the park. Visitors may most easily reach Cradle Mountain via the Cradle Link Road (C132) and the Murchison and Lyell Highways (A10).
Bookings for the Lake St Clair passenger ferry can be made at Lake St Clair Lodge, and fishing licences are available from Derwent Bridge Chalets.
Walker safety - alpine walks  (such as Cradle Mountain, Frenchmans Cap and Walls of Jerusalem)
Weather conditions in Tasmania can change quickly and frequently, especially in alpine areas. Snow, rain, wind and sun are all possible at any time of the year, and bushfires can occur between October and March.
When visiting alpine areas you should include at a minimum, in addition to your basic walking gear, a waterproof jacket with hood and storm front (seam sealed and breathable fabric, Gore-Tex or similar), waterproof over trousers (seam sealed and breathable fabric, Gore-Tex or similar), sturdy walking boots and warm clothing (a fleece or woollen jumper, thermal base layers, hat and gloves).
Deaths have occurred when people have been caught unprepared in cold, wet and windy weather. Children, older people and those with an illness or disability are at the greatest risk in such conditions.
Please observe all track signs – they are provided for your safety. Stay on the formed walking tracks at all times – both for your own safety and to protect the sensitive alpine region.
For more information on pack items and safety, please see Safety in parks.
The visitor centre and café at Lake St Clair are wheelchair accessible, as are the toilets within this building. There are accessible picnic tables between the visitor centre and the shore of the lake, some of which are sheltered.
The Watersmeet Trail starts from behind the visitor centre. The track is wide and mostly flat, with a fairly even surface, and is suitable for most wheelchair users, although some may require assistance. At 45 mins to an hour return, it may be too long for some visitors, but it's well worth doing even part of the walk.
Experiences in Lake St Clair (Cradle Mountain - Lake St Clair National Park)
3-4 hours one way, 11km, 20 mins by ferry (fee applies) and then walk one way to Cynthia Bay. , Grade 3
Combine a ferry cruise on Lake St Clair with a walk through the rainforest beside the lake. You leave from, and return to Cynthia Bay at the southern end of Lake St Clair.
Lake St Clair (Cradle Mountain - Lake St Clair National Park)
Echo Point
Tasmania has a wealth of excellent inland and ocean fishing locations where you can cast your line.
45 mins-1.5 hours, 4.7 km circuit. Depending on your choice of tracks: Watersmeet Track Grade 2; Platypus Bay and lamairremener tabelti, Grade 3
Lake St Clair offers three short walks for you to choose from. Pick one or do the full circuit, the choice is yours!
Lake St Clair walks
7 hour circuit, 19km circuit, Grade 4
Mount Rufus is a challenging walk that rewards well-equipped walkers with breath-taking mountain views on all sides.
Mount Rufus
1 current alerts
6 days one way, 65km one way, Grade 4
The Overland Track is Australia's premier alpine walk – a 65 km, six-day trek through the heart of the Cradle Mountain-Lake St Clair National Park, part of the magnificent Tasmanian Wilderness World Heritage Area.
Cradle Mountain (Cradle Mountain - Lake St Clair National Park)
4-5 hours circuit, 13km circuit , Grade 4
Located in the Lake St Clair National Park, the Shadow Lake walk will take you on a circuit through rainforest, sub-alpine moorland and highland eucalypt forest.
Shadow Lake
There are a number of locations around the state that offer a unique setting for your wedding ceremony.
East coast, Hobart and South, Launceston and North, North West
Lake St Clair camping
Enjoy beautiful, sheltered camp spots nestled in the trees at Lake St Clair.
Bookings and more info


The Warren Band Boosters will host their 3rd Annual Craft and Vendor Fair on Saturday April 13th 9:00 am - 3:00 pm.
Come to Warren Local High School and shop from over 140 Crafters and Vendors. This event will have EVERYTHING! Home Decor, Clothes, Jewelry, Pet Products, Easter Items and all the popular Vendor Representatives.
We currently have openings for Crafters and Vendors, but don't wait too long to contact our Coordinator because spaces fill up fast! E-mail Carol at wlmwboosters@gmail.com and she can answer questions or help you register.
Come out and see how amazing the Warren Boosters' Craft Shows are, they have been doing them for 40 years.


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Posts Tagged 'Insulin'
Latest 50p in the Innovation in Science series celebrates century of ground-breaking Insulin discovery
By Alexandra | October 14, 2021 | 4
The UK's Innovation in Science 50p Series continues as the BRAND NEW 2021 UK Discovery of Insulin 50p has just been released – commemorating 100 years since this groundbreaking finding!
Saving millions of lives around the world and triggering a century of diabetes research, the discovery of Insulin in 1921 by Sir Frederick G Banting, Charles H Best and JJR Macleod was one of the greatest medical breakthroughs in history.
2021 UK Discovery of Insulin 50p
In this special anniversary year, this life-changing hormone features on the UK's most collected coin for the very first time.
The design, by Iris De La Torre, features an artistic interpretation of the structure of Insulin along with its molecular formula.
Today, you can own the brand new 2021 UK Discovery of Insulin 50p in superior Brilliant Uncirculated quality for JUST £4.50 (+p&p).
What's more, your coin has been protectively encapsulated in Official Change Checker packaging with that all-important hologram to guarantee its superior quality.
Secure your 2021 UK Discovery of Insulin CERTIFIED BU 50p today by clicking here >>
Whilst this is the first UK coin to celebrate the discovery of Insulin, The Royal Canadian Mint issued a special collector's set earlier this year to celebrate this historic anniversary…
2021 Canada Discovery of Insulin Collector Card
Whilst 2021 marks 100 years since Sir Frederick G Banting, Charles H Best and JJR Macleod first discovered Insulin at the University of Toronto, it was later purified by James B Collip.
To celebrate this historic anniversary and one of Canada's most significant contributions to modern medicine, The Royal Canadian Mint have issued a special collector's set.
Included within this set you'll find two versions of the 2021 Discovery of Insulin $2, with a design portraying the story from laboratory to the isolation of insulin.
As well as the two Discovery of Insulin $2 coins, the set also includes five other denominations issued in 2021, perfectly displayed within the colourful collector card explaining the importance of this Canadian medical breakthrough that changed the world.
Just 100,000 sets have been issued for collectors worldwide and we've managed to secure an initial allocation of just 250 for UK collectors!
With such a significant anniversary and the inclusion of the selectively coloured $2 coin, high demand for this set is expected – so make sure you don't miss out! You can secure your 2021 Canada Discovery of Insulin Collector Card today for JUST £22.50 (+p&p) here >>
This exciting new 50p is the very latest addition to The Royal Mint's Innovation in Science series which has proven incredibly popular with collectors…
2021 UK Charles Babbage 50p
Charles Babbage was an English mathematician and inventor who originated the concept of a digital programmable computer.
In 2021, in the year marking the 150th year of his passing, The Royal Mint commemorated him on a UK 50p – an addition in their Innovation in Science series.
This 50p, designed by Nigel Tudman and Jas Bhamra, features a clever design which links Babbage's extraordinary machinery to the digital age.
You can secure this coin for your collection for JUST £4.50 (+p&p) here >>
2021 John Logie Baird 50p
It's hard to imagine life without television but back in the early 1920s, it was a complete unknown.
That was until John Logie Baird successfully produced televised objects in outline in 1924, transmitted recognisable human faces in 1925, and demonstrated the televising of moving objects in 1926.
Issued in 20201, to celebrate the life and works of the 'Father of Television', the design of this 50p coin features key milestones from Baird's life, presented between the lines of transmission radiating from the centre of the coin.
2020 Rosalind Franklin 50p
In the year that would have marked her 100th birthday, The Royal Mint released a 50p celebrating the life and crucial work of Rosalind Franklin, the first female scientist to be commemorated on a UK coin.
David Knapton's striking design of this coin, features a depiction of Rosalind Franklin's X-ray, 'Photograph 51', which revealed the helical structure of DNA, in her laboratory at King's College, London.
One of Britain's greatest scientists, Franklin made a crucial finding to the discovery of the double-helix structure of DNA.
2019 Stephen Hawking 50p
In 2019, less than a year since his death, The Royal Mint released a Stephen Hawking 50p coin, honouring his works as one of the most influential physicists of the modern age.
He became the very first person to be celebrated in The Royal Mint's Innovators in Science series and only the third person to be commemorated on a coin within a year of their death (the others being Winston Churchill and the Queen Mother!)
The reverse of the coin, designed by Edwina Ellis, features a stylised black hole and the inscription 'Stephen Hawking' . It also shows the Bekenstein-Hawking formula, which describes the thermodynamic entropy of a black hole!
The Innovation in Science series has proven incredibly popular with collectors and we're sure this brand new 2021 UK Discovery of Insulin 50p will be in high demand…
Secure the 2021 UK Discovery of Insulin 50p for your collection for JUST £4.50 (+p&p)
Secure the brand new 2021 UK Discovery of Insulin 50p in superior Brilliant Uncirculated quality for JUST £4.50 (+p&p) by clicking here >>
Posted in 50p coins, 50p coins, Blog Home, Coin Info, Coin News, General info, UK coins, Uncategorised and tagged 50p, 50p Coin, 50p coins, 50p news, 50p. fifty pence, Canadian coins, Canadian Mint, Discovery of Insulin, innovation, innovation in science, Innovative, Innovator, innovators, Insulin, Insulin 50p, new 50p, Royal Canadian Mint, Royal Mint, Science, science coin
The Insulin 50p – What we know so far…
By Alexandra | August 20, 2021 | 12
Did you know that an Insulin 50p is set to be issued this year?
The Royal Proclamation from the 23rd July 2021 confirmed that a 50p will be issued in 2021 featuring a design dedicated to the hormone, Insulin.
Whilst the design remains top secret, the Royal Proclamation reveals the coin's reverse design will feature a depiction of insulin molecules and the chemical formula for insulin.
Insulin was first discovered in 1921 by Sir Frederick G Banting, Charles H Best, and JJR Macleod at the University of Toronto.
Before this, very few people with type 1 diabetes lived more than a year or two, and it remains the only effective treatment for people with the condition to this day.
In the 100th anniversary year of its discovery, could this new 50p become the very latest issue in The Royal Mint's Innovation in Science series? It hasn't been confirmed but we certainly think it would make a great addition!
This series has brought us the 2019 Stephen Hawking 50p and the 2020 Rosalind Franklin 50p – both of which have proved incredibly popular with collectors!
We're so excited for the design of this brand new coin to be revealed – it's sure to make a huge breakthrough into the coin collecting world!
To make sure you don't miss out on adding this brand new coin to your collection as soon as it is released, you can sign up to the Change Checker Subscription Service here >>
Be one of the first to receive the brand new Insulin 50p!
So, don't miss your chance to get ahead of the crowd and be one of the very first collectors to receive the latest UK new issue coins (including the Insulin 50p) as soon as possible after their release.
The Change Checker New Issue Subscription Service allows you to own the latest coin releases, delivered to your doorstep, without any of the hassle of ordering the coin yourself!
Simply choose the Subscription that best suits you. Find out more here >>
Posted in 50p coins, 50p coins, Blog Home, Coin News, UK coins, Uncategorised and tagged 50p coins, 50p news, Insulin, Insulin 50p, new 50p, New coins, Rosalind Franklin, Rosalind Franklin 50p, Royal Proclamation, Stephen Hawking, Stephen Hawking 50p