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"Webster, Kevin A."
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Green team!
Green team turtles are ready to have their adventure and fight with the evil robots for the first time.
Book
The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice
Clozapine-N-oxide (CNO) has long been the ligand of choice for selectively activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). However, recent studies have challenged the long-held assertion that CNO is otherwise pharmacologically inert. The present study aimed to 1) determine whether CNO is reverse-metabolized to its parent compound clozapine in mice (as has recently been reported in rats), and 2) determine whether CNO exerts clozapine-like interoceptive stimulus effects in rats and/or mice. Following administration of 10.0 mg/kg CNO, pharmacokinetic analyses replicated recent reports of back-conversion to clozapine in rats and revealed that this phenomenon also occurs in mice. In rats and mice trained to discriminate 1.25 mg/kg clozapine from vehicle, CNO (1.0–20.0 mg/kg) produced partial substitution for the clozapine stimulus on average, with full substitution being detected in some individual animals of both species at doses frequently used to activate DREADDs. The present demonstration that CNO is converted to clozapine and exerts clozapine-like behavioral effects in both mice and rats further emphasizes the need for appropriate control groups in studies employing DREADDs, and highlights the utility of the drug discrimination procedure as a tool with which to screen the off-target effects of novel DREADD agonists.
Journal Article
Robot rampage!
Donatello builds a robot to help fight evil, but when it malfunctions, the Teenage Mutant Ninja Turtles have a technological terror to battle.
Book
In Vitro and In Vivo Characterization of the Alkaloid Nuciferine
The sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays.
Nuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms.
Nuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT2A, 5-HT2C, and 5-HT2B, an inverse agonist at 5-HT7, a partial agonist at D2, D5 and 5-HT6, an agonist at 5-HT1A and D4 receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy.
The molecular profile of nuciferine was similar but not identical to that shared with several approved antipsychotic drugs suggesting that nuciferine has atypical antipsychotic-like actions.
Journal Article
Discriminative stimulus properties of the atypical antipsychotic amisulpride: comparison to its isomers and to other benzamide derivatives, antipsychotic, antidepressant, and antianxiety drugs in C57BL/6 mice
Rationale
Racemic (
RS
)-amisulpride (Solian
®
) is an atypical antipsychotic drug used to treat schizophrenia and dysthymia. Blockade of dopamine D
2
/D
3
and/or serotonin 5-HT
7
receptors is implicated in its pharmacological effects. While the (
S
)-amisulpride isomer possesses a robust discriminative cue, discriminative stimulus properties of (
RS
)-amisulpride have not been evaluated.
Objectives
The present study established (
RS
)-amisulpride as a discriminative stimulus and assessed amisulpride-like effects of amisulpride stereoisomers, other benzamide derivatives, and antipsychotic, antidepressant, and anxiolytic drugs.
Methods
Adult, male C57BL/6 mice were trained to discriminate 10 mg/kg (
RS
)-amisulpride from vehicle in a two-lever food-reinforced operant conditioning task.
Results
(
RS
)-Amisulpride’s discriminative stimulus was dose-related, time-dependent, and stereoselective. (
S
)-Amisulpride (an effective dose of 50% (ED
50
) = 0.21 mg/kg) was three times more potent than (
RS
)-amisulpride (ED
50
= 0.60 mg/kg) or (
R
)-amisulpride (ED
50
= 0.68 mg/kg). (
RS
)-Amisulpride generalized fully to the structurally related atypical antipsychotic/antidysthymia drug sulpiride (Sulpor
®
; ED
50
= 7.29 mg/kg) and its (
S
)-enantiomer (ED
50
= 9.12 mg/kg); moderate to high partial generalization [60–75% drug lever responding (%DLR)] occurred to the benzamide analogs tiapride (Tiapridal
®
) and raclopride, but less than 60% DLR to metoclopramide (Reglan
®
), nemonapride (Emilace
®
), and zacopride. Antipsychotic, antidepressant, and antianxiety drugs from other chemical classes (chlorpromazine, quetiapine, risperidone, and mianserin) produced 35–55% amisulpride lever responding. Lastly, less than 35% DLR occurred for clozapine, olanzapine, aripiprazole imipramine, chlordiazepoxide, and bupropion.
Conclusions
(
RS
)-Amisulpride generalized to some, but not all benzamide derivatives, and it failed to generalize to any other antipsychotic, antidepressant, or antianxiety drugs tested. Interestingly, the (
R
)-isomer shared very strong stimulus properties with (
RS
)-amisulpride. This finding was in contrast to findings from Donahue et al. (
Eur J Pharmacol
734:15–22,
2014
), which found that the (
R
)-isomer did not share very strong stimulus properties when the (
S
)-isomer was the training drug.
Journal Article
Direct detection of DNA methylation during single-molecule, real-time sequencing
Polymerase kinetics observed during single-molecule, real-time sequencing depend on the methylation status of the DNA template. Measurement of kinetic parameters such as interpulse duration and pulse width allows the identification of methylated adenosine in
Escherichia coli
and the distinction between 5-methylcytosine and 5-hydroxymethylcytosine in synthetic templates.
We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenine, 5-methylcytosine and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We used these kinetic signatures to identify adenine methylation in genomic samples and found that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns in even highly repetitive genomic regions.
Journal Article
Translation control of the immune checkpoint in cancer and its therapeutic targeting
Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (
MYC
Tg
) synergizes with
KRAS
G12D
to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with
KRAS
G12D
alone. Genome-wide ribosomal footprinting of
MYC
Tg
;
KRAS
G12
tumors compared with
KRAS
G12D
revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in
KRAS
G12D
tumors by functional, non-canonical upstream open reading frames in its 5′ untranslated region, which is bypassed in
MYC
Tg
;
KRAS
G12D
tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of
MYC
Tg
;
KRAS
G12D
tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for new immunotherapies.
Oncogene cooperation enhances PD-L1 translation to promote immune evasion and metastasis in a novel model of liver cancer.
Journal Article
Toward expert-level medical question answering with large language models
Large language models (LLMs) have shown promise in medical question answering, with Med-PaLM being the first to exceed a ‘passing’ score in United States Medical Licensing Examination style questions. However, challenges remain in long-form medical question answering and handling real-world workflows. Here, we present Med-PaLM 2, which bridges these gaps with a combination of base LLM improvements, medical domain fine-tuning and new strategies for improving reasoning and grounding through ensemble refinement and chain of retrieval. Med-PaLM 2 scores up to 86.5% on the MedQA dataset, improving upon Med-PaLM by over 19%, and demonstrates dramatic performance increases across MedMCQA, PubMedQA and MMLU clinical topics datasets. Our detailed human evaluations framework shows that physicians prefer Med-PaLM 2 answers to those from other physicians on eight of nine clinical axes. Med-PaLM 2 also demonstrates significant improvements over its predecessor across all evaluation metrics, particularly on new adversarial datasets designed to probe LLM limitations (
P
< 0.001). In a pilot study using real-world medical questions, specialists preferred Med-PaLM 2 answers to generalist physician answers 65% of the time. While specialist answers were still preferred overall, both specialists and generalists rated Med-PaLM 2 to be as safe as physician answers, demonstrating its growing potential in real-world medical applications.
With an improved framework for model development and evaluation, a large language model is shown to provide answers to medical questions that are comparable or preferred with respect to those provided by human physicians.
Journal Article
Mechanics and contraction dynamics of single platelets and implications for clot stiffening
Blood platelets aggregate to form clots that prevent haemorrhage. Knowledge of single-platelet mechanics is scarce, however. Atomic force microscopy experiments now show that platelets contract rapidly on contact with fibrinogen, and adhere strongly to multiple fibrin polymers, enhancing the elasticity of clots. These findings are relevant to disorders of platelet function, such as thrombosis.
Platelets interact with fibrin polymers to form blood clots at sites of vascular injury
1
,
2
,
3
. Bulk studies have shown clots to be active materials, with platelet contraction driving the retraction and stiffening of clots
4
. However, neither the dynamics of single-platelet contraction nor the strength and elasticity of individual platelets, both of which are important for understanding clot material properties, have been directly measured. Here we use atomic force microscopy to measure the mechanics and dynamics of single platelets. We find that platelets contract nearly instantaneously when activated by contact with fibrinogen and complete contraction within 15 min. Individual platelets can generate an average maximum contractile force of 29 nN and form adhesions stronger than 70 nN. Our measurements show that when exposed to stiffer microenvironments, platelets generated higher stall forces, which indicates that platelets may be able to contract heterogeneous clots more uniformly. The high elasticity of individual platelets, measured to be 10 kPa after contraction, combined with their high contractile forces, indicates that clots may be stiffened through direct reinforcement by platelets as well as by strain stiffening of fibrin under tension due to platelet contraction. These results show how the mechanosensitivity and mechanics of single cells can be used to dynamically alter the material properties of physiologic systems.
Journal Article
Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance
Mammalian biology adapts to physical activity but the molecular mechanisms sensing the activity remain enigmatic. Recent studies have revealed how Piezo1 protein senses mechanical force to enable vascular development. Here, we address Piezo1 in adult endothelium, the major control site in physical activity. Mice without endothelial Piezo1 lack obvious phenotype but close inspection reveals a specific effect on endothelium-dependent relaxation in mesenteric resistance artery. Strikingly, the Piezo1 is required for elevated blood pressure during whole body physical activity but not blood pressure during inactivity. Piezo1 is responsible for flow-sensitive non-inactivating non-selective cationic channels which depolarize the membrane potential. As fluid flow increases, depolarization increases to activate voltage-gated Ca
2+
channels in the adjacent vascular smooth muscle cells, causing vasoconstriction. Physical performance is compromised in mice which lack endothelial Piezo1 and there is weight loss after sustained activity. The data suggest that Piezo1 channels sense physical activity to advantageously reset vascular control.
The mechanisms that regulate the body’s response to exercise are poorly understood. Here, Rode et al. show that the mechanically activated cation channel Piezo1 is a molecular sensor of physical exercise in the endothelium that triggers endothelial communication to mesenteric vessel muscle cells, leading to vasoconstriction.
Journal Article