A new polymodal gating model of the proton-activated chloride channel.

The proton-activated chloride (PAC) channel plays critical roles in ischemic neuron death, but its activation mechanisms remain elusive. Here, we investigated the gating of PAC channels using its novel bifunctional modulator C77304. C77304 acted as a weak activator of the PAC channel, causing moderate activation by acting on its proton gating. However, at higher concentrations, C77304 acted as a weak inhibitor, suppressing channel activity. This dual function was achieved by interacting with 2 modulatory sites of the channel, each with different affinities and dependencies on the channel's state. Moreover, we discovered a protonation-independent voltage activation of the PAC channel that appears to operate through an ion-flux gating mechanism. Through scanning-mutagenesis and molecular dynamics simulation, we confirmed that E181, E257, and E261 in the human PAC channel serve as primary proton sensors, as their alanine mutations eliminated the channel's proton gating while sparing the voltage-dependent gating. This proton-sensing mechanism was conserved among orthologous PAC channels from different species. Collectively, our data unveils the polymodal gating and proton-sensing mechanisms in the PAC channel that may inspire potential drug development.

Identification and targeting of a unique NaV1.7 domain driving chronic pain.

Small molecules directly targeting the voltage-gated sodium channel (VGSC) NaV1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to NaV1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the NaV1.7 CRS over other NaV isoforms. Substitution of the NaV1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased NaV1.7 currents. A cell-penetrant decoy peptide corresponding to the NaV1.7-CRS reduced NaV1.7 currents and trafficking, decreased presynaptic NaV1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the NaV1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a NaV1.7 -targeted gene therapy, we packaged a plasmid encoding the NaV1.7-CRS in an AAV virus. Treatment with this virus reduced NaV1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

Human Hsp70 Substrate-Binding Domains Recognize Distinct Client Proteins.

The 13 Hsp70 proteins in humans act on unique sets of substrates with diversity often being attributed to J-domain-containing protein (Hsp40 or JDP) cofactors. We were therefore surprised to find drastically different binding affinities for Hsp70-peptide substrates, leading us to probe substrate specificity among the 8 canonical Hsp70s from humans. We used peptide arrays to characterize Hsp70 binding and then mined these data using machine learning to develop an algorithm for isoform-specific prediction of Hsp70 binding sequences. The results of this algorithm revealed recognition patterns not predicted based on local sequence alignments. We then showed that none of the human isoforms can complement heat-shocked DnaK knockout Escherichia coli cells. However, chimeric Hsp70s consisting of the human nucleotide-binding domain and the substrate-binding domain of DnaK complement during heat shock, providing further evidence in vivo of the divergent function of the Hsp70 substrate-binding domains. We also demonstrated that the differences in heat shock complementation among the chimeras are not due to loss of DnaJ binding. Although we do not exclude JDPs as additional specificity factors, our data demonstrate substrate specificity among the Hsp70s, which has important implications for inhibitor development in cancer and neurodegeneration.

C-H Functionalization-Enabled 11-Step Semisynthesis of (-)-Veragranine A and Characterization of Synthetic Analogs in Osteoarthritis-related Pain Treatment.

We report an efficient semisynthesis of the cholestane steroidal alkaloid (-)-veragranine A with a 6/6/6/5/6/6 hexacyclic ring system, eight stereocenters, and a unique C12-C23 linkage. Our synthesis features a Schönecker-Baran C-H oxidation at C12, a Suzuki-Miyaura cross-coupling to form the C12-C23 bond, and a hydrogen atom transfer (HAT)-initiated Minisci C-H cyclization to forge the C20-C22 bond with desired stereochemistry at C20. These enabling transformations significantly enhanced the overall synthetic efficiency and delivered (-)-veragranine A in 11 steps and over 200 mg from cheap and readily available dehydroepiandrosterone. In addition, this approach allowed flexible syntheses of novel synthetic analogs for biological evaluations in sensory neurons in vitro and in an in vivo model of arthritic pain, from which two novel lead compounds were identified for further development.

Missense variants in DPYSL5 cause a neurodevelopmental disorder with corpus callosum agenesis and cerebellar abnormalities.

The collapsin response mediator protein (CRMP) family proteins are intracellular mediators of neurotrophic factors regulating neurite structure/spine formation and are essential for dendrite patterning and directional axonal pathfinding during brain developmental processes. Among this family, CRMP5/DPYSL5 plays a significant role in neuronal migration, axonal guidance, dendrite outgrowth, and synapse formation by interacting with microtubules. Here, we report the identification of missense mutations in DPYSL5 in nine individuals with brain malformations, including corpus callosum agenesis and/or posterior fossa abnormalities, associated with variable degrees of intellectual disability. A recurrent de novo p.Glu41Lys variant was found in eight unrelated patients, and a p.Gly47Arg variant was identified in one individual from the first family reported with Ritscher-Schinzel syndrome. Functional analyses of the two missense mutations revealed impaired dendritic outgrowth processes in young developing hippocampal primary neuronal cultures. We further demonstrated that these mutations, both located in the same loop on the surface of DPYSL5 monomers and oligomers, reduced the interaction of DPYSL5 with neuronal cytoskeleton-associated proteins MAP2 and ßIII-tubulin. Our findings collectively indicate that the p.Glu41Lys and p.Gly47Arg variants impair DPYSL5 function on dendritic outgrowth regulation by preventing the formation of the ternary complex with MAP2 and ßIII-tubulin, ultimately leading to abnormal brain development. This study adds DPYSL5 to the list of genes implicated in brain malformation and in neurodevelopmental disorders.

Chronic pain recruits hypothalamic dynorphin/kappa opioid receptor signalling to promote wakefulness and vigilance.

Increased vigilance in settings of potential threats or in states of vulnerability related to pain is important for survival. Pain disrupts sleep and conversely, sleep disruption enhances pain, but the underlying mechanisms remain unknown. Chronic pain engages brain stress circuits and increases secretion of dynorphin, an endogenous ligand of the kappa opioid receptor (KOR). We therefore hypothesized that hypothalamic dynorphin/KOR signalling may be a previously unknown mechanism that is recruited in pathological conditions requiring increased vigilance. We investigated the role of KOR in wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep in freely moving naïve mice and in mice with neuropathic pain induced by partial sciatic nerve ligation using EEG/EMG recordings. Systemic continuous administration of U69,593, a KOR agonist, over 5 days through an osmotic minipump decreased the amount of NREM and REM sleep and increased sleep fragmentation in naïve mice throughout the light-dark sleep cycle. We used KORcre mice to selectively express a Gi-coupled designer receptor activated by designer drugs (Gi-DREADD) in KORcre neurons of the hypothalamic paraventricular nucleus, a key node of the hypothalamic-pituitary-adrenal stress response. Sustained activation of Gi-DREADD with clozapine-N-oxide delivered in drinking water over 4 days, disrupted sleep in these mice in a similar way as systemic U69,593. Mice with chronic neuropathic pain also showed disrupted NREM and total sleep that was normalized by systemic administration of two structurally different KOR antagonists, norbinaltorphimine and NMRA-140, currently in phase II clinical development, or by CRISPR/Cas9 editing of paraventricular nucleus KOR, consistent with endogenous KOR activation disrupting sleep in chronic pain. Unexpectedly, REM sleep was diminished by either systemic KOR antagonist or by CRISPR/Cas9 editing of paraventricular nucleus KOR in sham-operated mice. Our findings reveal previously unknown physiological and pathophysiological roles of dynorphin/KOR in eliciting arousal. Physiologically, dynorphin/KOR signalling affects transitions between sleep stages that promote REM sleep. Furthermore, while KOR antagonists do not promote somnolence in the absence of pain, they normalized disrupted sleep in chronic pain, revealing a pathophysiological role of KOR signalling that is selectively recruited to promote vigilance, increasing chances of survival. Notably, while this mechanism is likely beneficial in the short-term, disruption of the homeostatic need for sleep over longer periods may become maladaptive resulting in sustained pain chronicity. A novel approach for treatment of chronic pain may thus result from normalization of chronic pain-related sleep disruption by KOR antagonism.

Graphene based T-shaped monopole antenna sensor for food quality evaluation.

BACKGROUND: Food adulteration has long been considered a major problem. It compromises the quality, safety, and nutritional value of food, posing significant risks to public health. Novel techniques are required to control it. RESULTS: A graphene-based T-shaped monopole antenna sensor was tested for its ability to detect adulteration in liquid foods. Mustard oil was the pure reference sample used for product quality analysis. Olive oil and rice bran oil were adulterants added to the pure sample. It was found that the sensor could be immersed easily in the liquid sample and provided precise results. CONCLUSION: The graphene-based T-shaped monopole antenna sensor can be used for the quality assessment of liquid food products and is suitable for real-time monitoring. © 2024 Society of Chemical Industry.

Glutamatergic and GABAergic neurons in the vLGN mediate the nociceptive effects of green and red light on neuropathic pain.

Phototherapy is an emerging non-pharmacological treatment for depression, circadian rhythm disruptions, and neurodegeneration, as well as pain conditions including migraine and fibromyalgia. However, the mechanism of phototherapy-induced antinociception is not well understood. Here, using fiber photometry recordings of population-level neural activity combined with chemogenetics, we found that phototherapy elicits antinociception via regulation of the ventral lateral geniculate body (vLGN) located in the visual system. Specifically, both green and red lights caused an increase of c-fos in vLGN, with red light increased more. In vLGN, green light causes a large increase in glutamatergic neurons, whereas red light causes a large increase in GABAergic neurons. Green light preconditioning increases the sensitivity of glutamatergic neurons to noxious stimuli in vLGN of PSL mice. Green light produces antinociception by activating glutamatergic neurons in vLGN, and red light promotes nociception by activating GABAergic neurons in vLGN. Together, these results demonstrate that different colors of light exert different pain modulation effects by regulating glutamatergic and GABAergic subpopulations in the vLGN. This may provide potential new therapeutic strategies and new therapeutic targets for the precise clinical treatment of neuropathic pain.

Dysregulation of serum prolactin links the hypothalamus with female nociceptors to promote migraine.

Migraine headache results from activation of meningeal nociceptors, however, the hypothalamus is activated many hours before the emergence of pain. How hypothalamic neural mechanisms may influence trigeminal nociceptor function remains unknown. Stress is a common migraine trigger that engages hypothalamic dynorphin/kappa opioid receptor (KOR) signalling and increases circulating prolactin. Prolactin acts at both long and short prolactin receptor isoforms that are expressed in trigeminal afferents. Following downregulation of the prolactin receptor long isoform, prolactin signalling at the prolactin receptor short isoform sensitizes nociceptors selectively in females. We hypothesized that stress may activate the kappa opioid receptor on tuberoinfundibular dopaminergic neurons to increase circulating prolactin leading to female-selective sensitization of trigeminal nociceptors through dysregulation of prolactin receptor isoforms. A mouse two-hit hyperalgesic priming model of migraine was used. Repeated restraint stress promoted vulnerability (i.e. first-hit priming) to a subsequent subthreshold (i.e. second-hit) stimulus from inhalational umbellulone, a TRPA1 agonist. Periorbital cutaneous allodynia served as a surrogate of migraine-like pain. Female and male KORCre; R26lsl-Sun1-GFP mice showed a high percentage of KORCre labelled neurons co-localized in tyrosine hydroxylase-positive cells in the hypothalamic arcuate nucleus. Restraint stress increased circulating prolactin to a greater degree in females. Stress-primed, but not control, mice of both sexes developed periorbital allodynia following inhalational umbellulone. Gi-DREADD activation (i.e. inhibition through Gi-coupled signalling) in KORCre neurons in the arcuate nucleus also increased circulating prolactin and repeated chemogenetic manipulation of these neurons primed mice of both sexes to umbellulone. Clustered regularly interspaced short palindromic repeats-Cas9 deletion of the arcuate nucleus KOR prevented restraint stress-induced prolactin release in female mice and priming from repeated stress episodes in both sexes. Inhibition of circulating prolactin occurred with systemic cabergoline, a dopamine D2 receptor agonist, blocked priming selectively in females. Repeated restraint stress downregulated the prolactin receptor long isoform in the trigeminal ganglia of female mice. Deletion of prolactin receptor in trigeminal ganglia by nasal clustered regularly interspaced short palindromic repeats-Cas9 targeting both prolactin receptor isoforms prevented stress-induced priming in female mice. Stress-induced activation of hypothalamic KOR increases circulating prolactin resulting in trigeminal downregulation of prolactin receptor long and pain responses to a normally innocuous TRPA1 stimulus. These are the first data that provide a mechanistic link between stress-induced hypothalamic activation and the trigeminal nociceptor effectors that produce trigeminal sensitization and migraine-like pain. This sexually dimorphic mechanism may help to explain female prevalence of migraine. KOR antagonists, currently in phase II clinical trials, may be useful as migraine preventives in both sexes, while dopamine agonists and prolactin/ prolactin receptor antibodies may improve therapy for migraine, and other stress-related neurological disorders, in females.

Neuronal allodynic mechanisms of Slc7a5 (LAT1) in the spared nerve injury rodent model of neuropathic pain.

High-impact chronic pain is suffered by 1 in 5 patients in the USA and globally. Effective, non-addictive, non-opioid therapeutics are urgently needed for the treatment of chronic pain. Slc7a5 (Lat1), also known as system L-neutral amino acid transporter, is involved in a number of physiological processes related to inflammation. Transcriptomics studies have shown that Slc7a5 and its binding partner Slc3a2 are expressed in neurons of the dorsal root ganglia (DRG) and spinal dorsal horn, which are critical to the initiation and maintenance of nociception and pathophysiology of chronic pain. In addition, Slc7a5 is a transporter for the first-line anti-allodynic gabapentinoid drugs and binds to ion channels implicated in nociception and chronic pain including the voltage-gated sodium channel Nav1.7 and the voltage-gated potassium channels Kv1.1 and Kv1.2. We found that blocking Slc7a5 with intrathecal administration of the drug JPH203 alleviated allodynia in the spared nerve injury (SNI) rodent model of neuropathic pain. Western blot and immunohistochemistry studies revealed an increase in Slc7a5 protein levels in the spinal cord and DRGs of SNI mice compared to control mice. Using whole-cell current-clamp electrophysiology, we observed that JPH203 treatment reduced excitability of small-diameter (< 30 µm) DRG neurons from SNI mice, in agreement with its behavioral effects. Voltage-clamp recordings from JPH203-treated naïve rat DRGs identified an effect on tetrodotoxin-resistant (TTX-R) sodium currents. Altogether, these results demonstrate that Slc7a5 is dysregulated in chronic neuropathic pain and can be targeted to provide relief of hypersensitivity.

Mutant huntingtin does not cross the mitochondrial outer membrane.

Mutant huntingtin (mHTT) is associated with mitochondria, but the exact mitochondrial location of mHTT has not been definitively established. Recently, it was reported that mHTT is present in the intermembrane space and inhibits mitochondrial protein import by interacting with TIM23, a major component of mitochondrial protein import machinery, but evidence for functional ramifications were not provided. We assessed mHTT location using synaptic and nonsynaptic mitochondria isolated from brains of YAC128 mice and subjected to alkali treatment or limited trypsin digestion. Mitochondria were purified either with discontinuous Percoll gradient or with anti-TOM22-conjugated iron microbeads. We also used mitochondria isolated from postmortem brain tissues of unaffected individuals and HD patients. Our results demonstrate that mHTT is located on the cytosolic side of the mitochondrial outer membrane (MOM) but does not cross it. This refutes the hypothesis that mHTT may interact with TIM23 and inhibit mitochondrial protein import. The levels of expression of nuclear-encoded, TIM23-transported mitochondrial proteins ACO2, TUFM, IDH3A, CLPP and mitochondrially encoded and synthesized protein mtCO1 were similar in mitochondria from YAC128 mice and their wild-type littermates as well as in mitochondria from postmortem brain tissues of unaffected individuals and HD patients, supporting the lack of deficit in mitochondrial protein import. Regardless of purification technique, mitochondria from YAC128 and WT mice had similar respiratory activities and mitochondrial membrane potentials. Thus, our data argue against mHTT crossing the MOM and entering into the mitochondrial intermembrane space, making it highly unlikely that mHTT interacts with TIM23 and inhibits protein import in intact mitochondria.

Alternaria alternata-induced airway epithelial signaling and inflammatory responses via protease-activated receptor-2 expression.

Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A. alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A. alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A. alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation and maintenance remains undefined. Airway epithelial cells provide the first encounter with A. alternata and are thought to play an important role in initiating the physiologic response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR-/-) from a model bronchial parental line (16HBE14o-). Comparison of in vitro physiologic responses in these cell lines demonstrated a complete loss of PAR2 agonist (2at-LIGRL-NH2) response and significantly attenuated protease (trypsin and elastase) and A. alternata responses in the 16HBEPAR-/- line. Apical application of A. alternata to 16HBE14o- and 16HBEPAR2-/- grown at air-liquid interface demonstrated rapid, PAR2-dependent and independent, inflammatory cytokine, chemokine and growth factor basolateral release. In conclusion, the novel human PAR2-deficient cell line allows for direct in vitro examination of the role(s) for PAR2 in allergen challenge with polarized human airway epithelial cells.

A prolactin-dependent sexually dimorphic mechanism of migraine chronification.

OBJECTIVE: Determination of possible sex differences in mechanisms promoting migraine progression and the contribution of prolactin and the prolactin long (PRLR-L) and short (PRLR-S) receptor isoforms. BACKGROUND: The majority of patients with chronic migraine and medication overuse headache are female. Prolactin is present at higher levels in women and increases migraine. Prolactin signaling at the PRLR-S selectively sensitizes nociceptors in female rodents, while expression of the PRLR-L is protective. METHODS: Medication overuse headache was modeled by repeated sumatriptan administration in male and female mice. Periorbital and hindpaw cutaneous allodynia served as a surrogate of migraine-like pain. PRLR-L and PRLR-S isoforms were measured in the trigeminal ganglion with western blotting. Possible co-localization of PRLR with serotonin 5HT1B and 5HT1D receptors was determined with RNAscope. Cabergoline, a dopamine receptor agonist that inhibits circulating prolactin, was co-administered with sumatriptan. Nasal administration of CRISPR/Cas9 plasmid was used to edit expression of both PRLR isoforms. RESULTS: PRLR was co-localized with 5HT1B or 5HT1D receptors in the ophthalmic region of female trigeminal ganglion. A single injection of sumatriptan increased serum PRL levels in female mice. Repeated sumatriptan promoted cutaneous allodynia in both sexes but down-regulated trigeminal ganglion PRLR-L, without altering PRLR-S, only in females. Co-administration of sumatriptan with cabergoline prevented allodynia and down-regulation of PRLR-L only in females. CRISPR/Cas9 editing of both PRLR isoforms in the trigeminal ganglion prevented sumatriptan-induced periorbital allodynia in females. INTERPRETATION: We identified a sexually dimorphic mechanism of migraine chronification that involves down-regulation of PRLR-L and increased signaling of circulating prolactin at PRLR-S. These studies reveal a previously unrecognized neuroendocrine mechanism linking the hypothalamus to nociceptor sensitization that increases the risk of migraine pain in females and suggest opportunities for novel sex-specific therapies including gene editing through nasal delivery of CRISPR/Cas9 constructs.

Defining the Kv2.1-syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant.

The neuronal cell death-promoting loss of cytoplasmic K+ following injury is mediated by an increase in Kv2.1 potassium channels in the plasma membrane. This phenomenon relies on Kv2.1 binding to syntaxin 1A via 9 amino acids within the channel intrinsically disordered C terminus. Preventing this interaction with a cell and blood-brain barrier-permeant peptide is neuroprotective in an in vivo stroke model. Here a rational approach was applied to define the key molecular interactions between syntaxin and Kv2.1, some of which are shared with mammalian uncoordinated-18 (munc18). Armed with this information, we found a small molecule Kv2.1-syntaxin-binding inhibitor (cpd5) that improves cortical neuron survival by suppressing SNARE-dependent enhancement of Kv2.1-mediated currents following excitotoxic injury. We validated that cpd5 selectively displaces Kv2.1-syntaxin-binding peptides from syntaxin and, at higher concentrations, munc18, but without affecting either synaptic or neuronal intrinsic properties in brain tissue slices at neuroprotective concentrations. Collectively, our findings provide insight into the role of syntaxin in neuronal cell death and validate an important target for neuroprotection.

Neurodegenerative Diseases: From Dysproteostasis, Altered Calcium Signalosome to Selective Neuronal Vulnerability to AAV-Mediated Gene Therapy.

Despite intense research into the multifaceted etiology of neurodegenerative diseases (ND), they remain incurable. Here we provide a brief overview of several major ND and explore novel therapeutic approaches. Although the cause (s) of ND are not fully understood, the accumulation of misfolded/aggregated proteins in the brain is a common pathological feature. This aggregation may initiate disruption of Ca++ signaling, which is an early pathological event leading to altered dendritic structure, neuronal dysfunction, and cell death. Presently, ND gene therapies remain unidimensional, elusive, and limited to modifying one pathological feature while ignoring others. Considering the complexity of signaling cascades in ND, we discuss emerging therapeutic concepts and suggest that deciphering the molecular mechanisms involved in dendritic pathology may broaden the phenotypic spectrum of ND treatment. An innovative multiplexed gene transfer strategy that employs silencing and/or over-expressing multiple effectors could preserve vulnerable neurons before they are lost. Such therapeutic approaches may extend brain health span and ameliorate burdensome chronic disease states.

Effect of morphine and ibuprofen on nociceptive behavior, preening and motor activity following tonic chemical pain in the Japanese quail (Coturnix japonica).

OBJECTIVE: To establish a tonic chemical model of pain in quail and evaluate the efficacy of opioid and non-steroidal anti-inflammatory drugs. STUDY DESIGN: A randomized, blinded, experimental study design. ANIMALS: A total of 120 male Japanese quail, aged 7 weeks. METHODS: A formalin solution (0.3%, 0.6% and 0.9%; total volume of 40 µL) was injected subcutaneously (SC) into the medial aspect of the right metatarsus (shank). Foot lift and preening activities were recorded for 45 minutes following injection of formalin and scored by an investigator blinded to the treatment. An open field test was used to evaluate motor activity. Treatments included SC saline, SC morphine (1.25, 2.5 and 5 mg kg-1) and oral ibuprofen (5 and 10 mg kg-1). The treatment effect was analyzed by one-way anova and the time course effect analyzed using repeated measures anova, both followed by Dunnett's post hoc test (p < 0.05). RESULTS: All formalin concentrations induced significant foot lifting activity in the first phase (0-5 minutes), whereas only 0.6% and 0.9% formalin elicited responses in both the first and second (25-45 minutes) phases. Neither morphine nor ibuprofen affected phase 1 of the formalin test. Morphine (2.5 and 5 mg kg-1) and ibuprofen (5 and 10 mg kg-1) significantly reduced foot lift responses. Preening activity was significantly decreased following injection of 0.6% and 0.9% formalin. Preening was normalized with ibuprofen, but not with morphine. Morphine, but not ibuprofen, reduced quail activity. CONCLUSION AND CLINICAL RELEVANCE: These results suggest that the formalin test was a reliable method for assessing tonic pain behavior in quail. The acute phase of the formalin test was not affected by morphine or ibuprofen. Although ibuprofen reduced the pain response in phase 2, the analgesic effects of morphine were not conclusive because morphine appeared to induce sedation.

Novel monkey-wrench-shaped microstrip patch sensor for food evaluation and analysis.

BACKGROUND: Microwave sensor technology is considered to be a non-destructive and hygienic means for food evaluation and analysis. The rapid progress in microwave sensor technologies motivated us to present a novel monkey-wrench-shaped microstrip patch sensor for evaluating food quality. The proposed antenna is considered as a liquid sensor to detect adulteration in liquids by examining the relationship between concentration, shift in resonant frequency, and variation in reflection coefficient. RESULTS: The sensor has a compact size of 17 × 14 mm2 imprinted on an FR4 substrate of thickness 1.57 mm. This microwave system is proposed for monitoring overall milk quality using a microstrip sensor and has considerably good numerical sensitivity and accuracy (13.11% and 88.5% respectively), which makes the system attractive for detecting adulteration. Further, the Q-factor for the proposed sensor is 209 and has a standard deviation less than the difference between non-adulterated and adulterated values, giving resolution high enough to distinguish adulteration with an acceptable statistical accuracy. In this study, different milk categories (buffalo, goat, and cow milk) are considered as liquid samples to detect adulteration by demonstrating the variation in reflection coefficient due to change in dielectric properties for the following different cases: (i) adulteration of milk with water; (ii) adulteration of milk with synthetic milk powder; (iii) adulteration of milk with caustic soda; and (iv) adulteration of milk with vegetable oil. The referred variation in reflection coefficient and resonant frequency is due to a change in the dielectric properties of a liquid when a varying concentration of an adulterant/solute is added to the liquid sample. CONCLUSION: The simulation and measured results show good agreement that validates the proposed sensor for food adulteration detection with high sensitivity. In terms of performance, the proposed sensor shows accuracy with high spatial resolution and reduced penetration depth to detect the adulteration in various milk samples (i.e. buffalo, goat, and cow milk). © 2021 Society of Chemical Industry.

Post-nephrectomy pulmonary thromboembolism.

The association of pulmonary thromboembolism with nephrectomy is well documented in malignant conditions. However, the data for incidence of pulmonary thromboembolism following nephrectomy in nonmalignant conditions remains scarce. We report the first case of an incidence of pulmonary thromboembolism following nephrectomy in a patient with a nonfunctional kidney due to multiple renal calculi.

Sculpting Dendritic Spines during Initiation and Maintenance of Neuropathic Pain.

Accumulating evidence has established a firm role for synaptic plasticity in the pathogenesis of neuropathic pain. Recent advances have highlighted the importance of dendritic spine remodeling in driving synaptic plasticity within the CNS. Identifying the molecular players underlying neuropathic pain induced structural and functional maladaptation is therefore critical to understanding its pathophysiology. This process of dynamic reorganization happens in unique phases that have diverse pathologic underpinnings in the initiation and maintenance of neuropathic pain. Recent evidence suggests that pharmacological targeting of specific proteins during distinct phases of neuropathic pain development produces enhanced antinociception. These findings outline a potential new paradigm for targeted treatment and the development of novel therapies for neuropathic pain. We present a concise review of the role of dendritic spines in neuropathic pain and outline the potential for modulation of spine dynamics by targeting two proteins, srGAP3 and Rac1, critically involved in the regulation of the actin cytoskeleton.