CACHD1 is an α2δ-Like Protein That Modulates CaV3 Voltage-Gated Calcium Channel Activity.

The putative cache (Ca2+ channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca2+ channel auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with CaV3.1, CaV3.2, and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in CaV3.1, CaV3.2, or CaV3.3. A comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased CaV3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates CaV3 voltage-gated calcium channel activity.SIGNIFICANCE STATEMENT This is the first study to characterize the Ca2+ channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (CaV3, T-type) calcium channels, in particular to CaV3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases CaV3 calcium current. CACHD1 increases the presence of CaV3.1 at the cell surface, forms complexes with CaV3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates CaV3 activity.

Functional confirmation that the R1488* variant in SCN9A results in complete loss-of-function of Nav1.7.

BACKGROUND: Individuals with an extremely rare inherited condition, termed Congenital Insensitivity to Pain (CIP), do not feel pain in response to noxious stimuli. Variants in SCN9A, encoding the transmembrane voltage-gated sodium channel Nav1.7, have previously been reported in subjects with CIP accompanied by anosmia, which are typically transmitted in a recessive pattern. Functional characterisations of some of these SCN9A mutations show that they result in complete loss-of-function of Nav1.7. METHODS: In a consanguineous family we performed whole exome sequencing of three members who have a diagnosis of CIP and one unaffected family member. The functional effects of the segregating variant in SCN9A were determined using patch clamp electrophysiology in human embryonic kidney (HEK) 293 cells transfected with the variant. RESULTS: We found that each CIP subject was homozygous for a putatively nonsense variant, R1488*, in SCN9A. This variant was reported elsewhere in a subject with CIP, though the functional effect was not determined. Using electrophysiology, we confirm that this variant results in a complete loss-of-function of Nav1.7. CONCLUSIONS: We confirm through electrophysiological analysis that this R1488* variant in SCN9A results in complete loss-of-function of Nav1.7, which is consistent with reports on other variants in this gene in subjects with CIP.

Big Data in Drug Discovery.

Interpretation of Big Data in the drug discovery community should enhance project timelines and reduce clinical attrition through improved early decision making. The issues we encounter start with the sheer volume of data and how we first ingest it before building an infrastructure to house it to make use of the data in an efficient and productive way. There are many problems associated with the data itself including general reproducibility, but often, it is the context surrounding an experiment that is critical to success. Help, in the form of artificial intelligence (AI), is required to understand and translate the context. On the back of natural language processing pipelines, AI is also used to prospectively generate new hypotheses by linking data together. We explain Big Data from the context of biology, chemistry and clinical trials, showcasing some of the impressive public domain sources and initiatives now available for interrogation.

The "ischial limb": a landmark on anterior ultrasound scanning used to assess reduction in developmental dysplasia of the hip.

BACKGROUND: Early recognition of failure of reduction during abduction splinting in developmental dysplasia of the hip (DDH) is essential to avoid inappropriate prolonged damaging harness use. Graf ultrasound scanning (USS) is an accepted gold standard for assessing neonatal hip dysplasia but cannot verify reduction in the abducted position of a neonatal hip in a Pavlik harness. We describe a new anatomic landmark of the hip using anterior USS-the "ischial limb," correlate this to the position of the femoral head in reduced and dislocated positions, and assess the application of this in the assessment of DDH. METHODS: A porcine model was used to identify anatomic, radiologic, and ultrasonic features comparable to the human neonatal hip. A pilot clinical study was conducted of 50 neonates (11M:39F) treated for DDH in Pavlik harness, utilizing weekly anterior USS to determine the concentricity of femoral head reduction relative to the ischial limb and whether this could identify any failures to achieve reduction. RESULTS: The animal model identified an ultrasound landmark, termed the ischial limb that represents the ossification zone of the ischial contribution to the tri-radiate acetabular cartilage, and approximates to the center of the acetabulum. Anterior USS clearly demonstrated this ischial limb in clinical practice and its relation to the femoral head in all dysplastic hips (n=79). Immediate concentric reduction was observed in 11 patients, more gradual reduction over 3 weeks in 37 patients, and failure to achieve stable reduction was verified in 2 patients. The technique was reproducible in 100% of patients (n=50). Mean additional clinic time was 5 minutes per visit. CONCLUSIONS: The ischial limb, identified on anterior hip USS, delineates the position of the tri-radiate cartilage. This technique efficiently assesses hip reduction during Pavlik harness treatment and can differentiate between failure to achieve reduction or maintain a stable reduction at an earlier stage than conventional US methods. LEVEL OF EVIDENCE: Development of diagnostic criteria on basis of consecutive patients: level 2 evidence.

A rare case of mobile diplopodia mistaken for polydactyly.

We present a rare case of a mobile diplopodia in an infant with disorganization syndrome. This was initially mistaken for polydactyly due to the more typical association between these conditions. The resulting corrective surgery was more extensive and complicated than anticipated, with the removal of a partial foot duplication and reconstruction of residual hindfoot structures, rather than the planned digit amputation. We highlight the association of diplopodia with disorganization syndrome, discuss differentiating diplopodia from polydactyly and describe the surgical management of an unusual case.

The chimeric approach reveals that differences in the TRPV1 pore domain determine species-specific sensitivity to block of heat activation.

The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.

Combined syringe cement pressurisation and intra-osseous suction: an effective technique in total knee arthroplasty.

Our study looked at the short and medium-term results of a cost-effective technique of bone surface preparation and cement introduction in total knee arthroplasty (TKA). Numerous factors determine the outcome following TKA. Early failure and component loosening have been attributed to inadequate bone-cement and prosthesis-cement interfaces, established at the time of surgery. Various operative techniques have been reported to achieve effective cement penetration and inter-digitation into cancellous bone, with techniques of cement pressurization and intra-osseous suction being employed. We have devised a technique of cement pressurization using a modified standard 20 ml syringe, combined with intra-osseous suction. Retrospective evaluation of a series of 50 post-operative radiographs has shown that effective and even penetration of cement to a depth of 8.0 to 10.6 mm can be achieved consistently with this technique, during TKA, without the use of tourniquet. In addition evaluation of post-operative plain radiographs with minimum follow-up of 5 years showed 16 knees with minor, non-progressive lucent lines around the tibial component with a maximum Knee Society TKA Roentgenographic score of 2. To conclude we propose that this simple technique of surface preparation and cement introduction produces good results in the short and medium-term.

The effect of deletion of the orphan G - protein coupled receptor (GPCR) gene MrgE on pain-like behaviours in mice.

BACKGROUND: The orphan GPCR MrgE is one of an extended family of GPCRs that are expressed in dorsal root ganglia (DRG). Based on these expression patterns it has been suggested that GPCRs like MrgE may play a role in nociception however, to date, no direct supporting evidence has emerged. We generated mutant mice lacking MrgE and examined the effects of deletion of this gene in three pain behavioural models. The effect of MrgE gene deletion on expression of Mrgs and genes involved in sensory neurone function was also investigated. RESULTS: The absence of MrgE had no effect on the development of pain responses to a noxious chemical stimulus or an acute thermal stimulus. However, in contrast, the development but not the maintenance of neuropathic pain was affected by deletion of MrgE. The expression of Mrg genes was not significantly affected in the MrgE knockout (KO) mice with the sole exception of MrgF. In addition, the expression of 77 of 84 genes involved in sensory neuron development and function was also unaffected by deletion of MrgE. Of the 7 genes affected by MrgE deletion, 4 have previously been implicated in nociception. CONCLUSION: The data suggests that MrgE may play a role in selective pain behavioural responses in mice.

[3H] pregabalin binding is increased in ipsilateral dorsal horn following chronic constriction injury.

Pregabalin, a 3-substituted analogue of gamma-amino butyric acid has recently been approved for treatment of neuropathic pain. We have investigated the anatomical binding profile of [(3)H] pregabalin following chronic constriction injury (CCI) and compared this with alpha 2 delta 1 subunit expression using in situ hybridisation. We report here that the intensity and distribution pattern of [(3)H] pregabalin binding is altered in the ipsilateral dorsal horn following CCI and this is associated with a corresponding increase in alpha 2 delta 1 mRNA in the ipsilateral dorsal root ganglion (DRG). It is likely that increased DRG mRNA production leads to increased alpha 2 delta 1 protein production and subsequent transport by primary afferents to the dorsal horn. The increased expression of calcium channel subunits and protein in central terminals is interesting, given that abnormal activity within sensory nerves is likely to significantly contribute to the symptomatology of neuropathic pain. The upregulation of pregabalin binding sites in sensory nerve terminals may occur as part of the response to nerve damage in neuropathic pain patients, and therefore, preferential actions of pregabalin at these sites may contribute to its mechanism of action in man.

A Ketone Ester Drink Increases Postexercise Muscle Glycogen Synthesis in Humans.

INTRODUCTION: Physical endurance can be limited by muscle glycogen stores, in that glycogen depletion markedly reduces external work. During carbohydrate restriction, the liver synthesizes the ketone bodies, D-ß-hydroxybutyrate, and acetoacetate from fatty acids. In animals and in the presence of glucose, D-ß-hydroxybutyrate promotes insulin secretion and increases glycogen synthesis. Here we determined whether a dietary ketone ester, combined with plentiful glucose, can increase postexercise glycogen synthesis in human skeletal muscle. METHODS: After an interval-based glycogen depletion exercise protocol, 12 well-trained male athletes completed a randomized, three-arm, blinded crossover recovery study that consisted of consumption of either a taste-matched, zero-calorie control or a ketone monoester drink, followed by a 10-mM glucose clamp or saline infusion for 2 h. The three postexercise conditions were control drink then saline infusion, control drink then hyperglycemic clamp, or ketone ester drink then hyperglycemic clamp. Skeletal muscle glycogen content was determined in muscle biopsies of vastus lateralis taken before and after the 2-h clamps. RESULTS: The ketone ester drink increased blood D-ß-hydroxybutyrate concentrations to a maximum of 5.3 versus 0.7 mM for the control drink (P < 0.0001). During the 2-h glucose clamps, insulin levels were twofold higher (31 vs 16 mU·L, P < 0.01) and glucose uptake 32% faster (1.66 vs 1.26 g·kg, P < 0.001). The ketone drink increased by 61 g, the total glucose infused for 2 h, from 197 to 258 g, and muscle glycogen was 50% higher (246 vs 164 mmol glycosyl units per kilogram dry weight, P < 0.05) than after the control drink. CONCLUSION: In the presence of constant high glucose concentrations, a ketone ester drink increased endogenous insulin levels, glucose uptake, and muscle glycogen synthesis.

Discovery of a Series of Indazole TRPA1 Antagonists.

A series of TRPA1 antagonists is described which has as its core structure an indazole moiety. The physical properties and in vitro DMPK profiles are discussed. Good in vivo exposure was obtained with several analogs, allowing efficacy to be assessed in rodent models of inflammatory pain. Two compounds showed significant activity in these models when administered either systemically or topically. Protein chimeras were constructed to indicate compounds from the series bound in the S5 region of the channel, and a computational docking model was used to propose a binding mode for example compounds.

Genome-wide association analysis of pain severity in dysmenorrhea identifies association at chromosome 1p13.2, near the nerve growth factor locus.

Dysmenorrhea is a common chronic pelvic pain syndrome affecting women of childbearing potential. Family studies suggest that genetic background influences the severity of dysmenorrhea, but genetic predisposition and molecular mechanisms underlying dysmenorrhea are not understood. In this study, we conduct the first genome-wide association study to identify genetic factors associated with dysmenorrhea pain severity. A cohort of females of European descent (n = 11,891) aged 18 to 45 years rated their average dysmenorrhea pain severity. We used a linear regression model adjusting for age and body mass index, identifying one genome-wide significant (P < 5 × 10) association (rs7523086, P = 4.1 × 10, effect size 0.1 [95% confidence interval, 0.074-0.126]). This single nucleotide polymorphism is colocalising with NGF, encoding nerve growth factor. The presence of one risk allele corresponds to a predicted 0.1-point increase in pain intensity on a 4-point ordinal pain scale. The putative effects on NGF function and/or expression remain unknown. However, genetic variation colocalises with active epigenetic marks in fat and ovary tissues, and expression levels in aorta tissue of a noncoding RNA flanking NGF correlate. Participants reporting extreme dysmenorrhea pain were more likely to report being positive for endometriosis, polycystic ovarian syndrome, depression, and other psychiatric disorders. Our results indicate that dysmenorrhea pain severity is partly genetically determined. NGF already has an established role in chronic pain disorders, and our findings suggest that NGF may be an important mediator for gynaecological/pelvic pain in the viscera.

Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis, Axonal Conduction and Presynaptic Release.

Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7's role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.

Verification of hip reduction using anterior ultrasound scanning during Pavlik harness treatment of developmental dysplasia of the hip.

AIM: Ultrasound scanning (USS) is used for diagnosis and surveillance in developmental dysplasia of the hip (DDH). Lateral coronal scanning is performed with the hip flexed, in neutral adduction. In this position an unstable hip may dislocate, failing to demonstrate a reducible hip, leading to abandonment of harness treatment. Anterior ultrasound permits imaging of the flexed abducted hip in harness. This study evaluates the role of anterior & lateral USS in determining duration of treatment and reduction in DDH. METHOD: Between 1997 & 2010, 233 patients requiring harness treatment received lateral USS, with dislocated & dysplastic hips re-imaged fortnightly. From 2005, anterior USS was used additionally to assess reduction in harness. RESULTS: One-hundred and eighteen patients (167 hips) received lateral USS, 115 (160 hips) received both. In the lateral cohort, 103 (140 hips) were treated successfully, mean duration 66.2 days (95% CI 60.2-72.1), with 15 (26 hips) failures (15.5%), mean 30 (CI 95% 19.3-40.6). In the anterior cohort, 107 (150 hips) were treated successfully, mean 53.3 (95% CI 49.8-56.7), with 8 (10 hips) failures (6.25%), mean 35.3 (CI 95% 25.5-44.9). Children receiving an anterior USS had a shorter duration of treatment (p = 0.011) and no difference in failures (p = 0.21). CONCLUSIONS: A reduced duration of treatment for Graf 3 hips was observed. Anterior ultrasound allows earlier recognition of hips that fail to stabilize, via two observed modes of failure; failure of hip reduction and failure to stabilize after reduction.

Ion channels as therapeutic targets: a drug discovery perspective.

Ion channels are membrane proteins expressed in almost all living cells. The sequencing of the human genome has identified more than 400 putative ion channels, but only a fraction of these have been cloned and functionally tested. The widespread tissue distribution of ion channels, coupled with the plethora of physiological consequences of their opening and closing, makes ion-channel-targeted drug discovery highly compelling. However, despite some important drugs in clinical use today, as a class, ion channels remain underexploited in drug discovery and many existing drugs are poorly selective with significant toxicities or suboptimal efficacy. This Perspective seeks to review the ion channel family, its structural and functional features, and the diseases that are known to be modulated by members of the family. In particular, we will explore the structure and properties of known ligands and consider the future prospects for drug discovery in this challenging but high potential area.

Histologic examination of the eye of acid-sensing ion channel 1a knockout mice.

Various subtypes of the acid sensing ion channel have been detected in the retina of rodents and other mammalian species, but the functional importance of this finding is not clearly understood. The purpose of the study was to determine if retinal degeneration was present in ASIC1a-/- mice. The eyes of ASIC1a-/- mice, heterozygote ASIC1a+/- mice, and wild type ASIC1a+/+ mice that were 5 or 22-27 weeks old were processed by routine histotech-nological methods and examined for histologic changes in the retina and other portions of the eye. Additional sections of eyes from ASIC1a-/- and ASIC1a+/+ mice were labeled with peanut agglutinin (PNA) to evaluate cone pho-toreceptors. The retinas of ASIC1a-/-, ASIC1a+/-, and ASIC1a+/+ mice at 5 or 22-27 weeks of age were unremarkable and no morphologic changes in cone photo receptors were detected. Additional findings detected in the eye of ASIC1a+/+ mice included swelling of lens fibers or cataract that were also detected in some of the ASIC1a-/- or ASIC1a+/- mice. Lenticular findings were not considered to be associated with an absence of ASIC1a.

Identification of the alpha2-delta-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin.

Neuropathic pain is a debilitating condition affecting millions of people around the world and is defined as pain that follows a lesion or dysfunction of the nervous system. This type of pain is difficult to treat, but the novel compounds pregabalin (Lyrica) and gabapentin (Neurontin) have proven clinical efficacy. Unlike traditional analgesics such as nonsteroidal antiinflammatory drugs or narcotics, these agents have no frank antiinflammatory actions and no effect on physiological pain. Although extensive preclinical studies have led to a number of suggestions, until recently their mechanism of action has not been clearly defined. Here, we describe studies on the analgesic effects of pregabalin in a mutant mouse containing a single-point mutation within the gene encoding a specific auxiliary subunit protein (alpha2-delta-1) of voltage-dependent calcium channels. The mice demonstrate normal pain phenotypes and typical responses to other analgesic drugs. We show that the mutation leads to a significant reduction in the binding affinity of pregabalin in the brain and spinal cord and the loss of its analgesic efficacy. These studies show conclusively that the analgesic actions of pregabalin are mediated through the alpha2-delta-1 subunit of voltage-gated calcium channels and establish this subunit as a therapeutic target for pain control.

New targets for neuropathic pain therapeutics.

Neuropathic pain (NeP) is initiated by a lesion or dysfunction in the nervous system. Unlike physiological pain it serves no useful purpose and is usually sustained and chronic. NeP encompasses a wide range of pain syndromes of diverse aetiologies which together account for > 12 million sufferers in the US. Currently, there are a number of therapies available for NeP, including gabapentin, pregabalin, anticonvulsants (tiagabine HCl), tricyclic antidepressants (amitriptyline, nortriptyline) and acetaminophen/opioid combination products (Vicodin, Tylenol #3). However, these products do not provide sufficient pain relief and a significant proportion of sufferers are refractory (60%). Therefore, there is a need for new therapies that provide more predictable efficacy in all patients with improved tolerability. Over the last decade, understanding of the basic mechanisms contributing to the generation of NeP in preclinical animal models has greatly improved. Together with the completion of the various genome sequencing projects and significant advances in microarray and target validation strategies, new therapeutic approaches are being rigourously pursued. This article reviews the rationale behind a number of these mechanism-based approaches, briefly discusses specific challenges that they face, and finally, speculates on the potential of emerging technologies as alternative therapeutic strategies to the traditional 'small-molecule' approach.