Adaptive state restricted barrier Lyapunov-based control of a Stewart platform used as ankle-controlled mobilizer.

In this research project, a closed-chain robotic active ankle orthosis with six degrees of freedom is designed, constructed, numerically valued, instrumented, and experimentally validated. The mechanical arrangement to implement the orthosis corresponds to a six-legged Stewart platform. An adaptive gain control strategy with state constraints based on a state-dependent gains control (that behaves as a diverging function as the states approach the state restrictions) operates the device's motion. The convergence to an invariant positive set centered at the origin of the tracking error space is validated using the stability analysis based on the second method of Lyapunov, with the implementation of a state barrier Lyapunov-like function. The ultimate boundedness of the tracking error is proven with an endorsed gains adjustment method leading to a reachable minimum size of the ultimate bound. Hence, the impact of the state constraints and the formal reason for applying the controller on the suggested orthosis are all established. The orthosis is also controlled using a conventional state feedback strategy to assess the tracking error for an external disturbance and contrast its performance with the proposed control approach. The technology is tested on a few carefully chosen volunteers, successfully limiting the range of motion within a pre-defined region based on the scope of movement reported by patients with ankle illnesses discovered in the literature. Based on a unique mechatronic device, the created system offers a fresh approach to treating this class of impairments.

Pulmonary Hypertension in Underrepresented Minorities: A Narrative Review.

Minoritized racial and ethnic groups suffer disproportionately from the incidence and morbidity of pulmonary hypertension (PH), as well as its associated cardiovascular, pulmonary, and systemic conditions. These disparities are largely explained by social determinants of health, including access to care, systemic biases, socioeconomic status, and environment. Despite this undue burden, minority patients remain underrepresented in PH research. Steps should be taken to mitigate these disparities, including initiatives to increase research participation, combat inequities in access to care, and improve the treatment of the conditions associated with PH.

Palmar midcarpal instability a narrative review of the literature: Have we reached a consensus on a treatment?

Palmar midcarpal instability (PMCI) is a wrist condition that requires treatment through non-surgical rehabilitation programs or surgical stabilization. This condition's natural history is poorly understood, and the optimal treatment approach remains unknown. Non-surgical treatments are initially implemented, followed by surgical stabilization if necessary. Arthrodesis and soft tissue stabilization are the two main surgical options for PMCI, with no established gold standard for treatment. A systematic review of 12 articles comparing arthrodesis and soft tissue stabilization was conducted to identify the optimal treatment approach for PMCI. Arthrodesis techniques, such as lunotriquetral arthrodesis, showed high functional outcomes but also high reintervention rates due to nonunion. Soft tissue stabilization techniques showed superior functional outcomes with less mobility loss and lower reintervention rates compared to arthrodesis. However, more studies are required to determine the optimal soft tissue technique. Based on this review we created a treatment algorithm for PMCI starting with non-surgical treatment first, followed by surgical stabilization if needed. Soft tissue stabilization techniques are preferred over arthrodesis due to better functional outcomes and lower reintervention rates. However, each patient's treatment approach should be individualized and evaluated independently to determine the best course of action. PMCI is a rare wrist condition, and further research is needed to better understand its natural history and establish a gold standard for treatment. The lack of literature comparing the two surgical options underscores the need for further research to determine the optimal treatment approach. Nonetheless, the current evidence suggests that soft tissue stabilization is a promising alternative to arthrodesis, providing superior functional outcomes and lower reintervention rates.

Molecular cloning of the gene promoter encoding the human CaVγ2/Stargazin divergent transcript (CACNG2-DT): characterization and regulation by the cAMP-PKA/CREB signaling pathway.

CaVγ2 (Stargazin or TARPγ2) is a protein expressed in various types of neurons whose function was initially associated with a decrease in the functional expression of voltage-gated presynaptic Ca2+ channels (CaV) and which is now known to promote the trafficking of the postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) towards the cell membrane. Alterations in CaVγ2 expression has been associated with several neurological disorders, such as absence epilepsy. However, its regulation at the transcriptional level has not been intensively addressed. It has been reported that the promoter of the Cacng2 gene, encoding the rat CaVγ2, is bidirectional and regulates the transcription of a long non-coding RNA (lncRNA) in the antisense direction. Here, we investigate the proximal promoter region of the human CACNG2 gene in the antisense direction and show that this region includes two functional cAMP response elements that regulate the expression of a lncRNA called CACNG2-DT. The activity of these sites is significantly enhanced by forskolin, an adenylate cyclase activator, and inhibited by H89, a protein kinase A (PKA) antagonist. Therefore, this regulatory mechanism implies the activation of G protein-coupled receptors and downstream phosphorylation. Interestingly, we also found that the expression of CACNG2-DT may increase the levels of the CaVγ2 subunit. Together, these data provide novel information on the organization of the human CACNG2-DT gene promoter, describe modulatory domains and mechanisms that can mediate various regulatory inputs, and provide initial information on the molecular mechanisms that regulate the functional expression of the CaVγ2 protein.

Sleeve gastrectomy facilitates weight loss and permits cardiac transplantation in patients with severe obesity and a left ventricular assist device (LVAD).

INTRODUCTION: Patients suffering from advanced heart failure may undergo left ventricular assist device (LVAD) placement as a bridge to cardiac transplantation. However, those with a BMI above 35 kg/m2 are generally not considered eligible for transplant due to their elevated cardiac risk. We review our experience with bariatric surgery in this high-risk population to assess its safety and efficacy in reducing BMI to permit cardiac transplantation. METHODS: We retrospectively reviewed all patients on durable LVAD support who underwent sleeve gastrectomy (SG) at Mount Sinai Hospital between August 2018 and December 2022. Electronic medical records were reviewed to analyze patient demographics, surgical details, and outcomes regarding weight loss and heart transplantation. RESULTS: We identified twelve LVAD patients who underwent SG. Three were performed laparoscopically and 9 via robotic approach. Four patients (33.3%) underwent an orthotopic heart transplant (OHTx). Half of these patients were female. For patients who underwent OHTx, mean age at LVAD placement was 41.0 (R30.6-52.2), at SG was 43.9 (R32.7-55.0) and at OHTx was 45.3 years (R33.3-56.8). Mean BMI increased from 38.8 at LVAD placement to 42.5 prior to SG. Mean time from SG to OHTx was 17.9 months (R6-7-27.5) during which BMI decreased to mean 32.8 at the time of OHTx. At most recent follow-up, mean BMI was 31.9. All patients were anticoagulated prior to surgery; one required return to the operating room on post-operative day 1 after SG for bleeding and one was re-admitted on post-operative day 7 for hematochezia treated conservatively. CONCLUSION: SG is a safe and effective operation in patients with severe obesity and heart failure requiring an LVAD. 66.7% of our cohort achieved target BMI < 35 and 33.3% underwent heart transplantation. Longer term follow-up is needed to clarify full bridge-to-transplant rate and long-term survival outcomes.

Molecular Mechanisms of Nuclear Transport of the Neuronal Voltage-gated Ca2+ Channel ß3 Auxiliary Subunit.

Previous studies have shown that in addition to its role within the voltage-gated calcium channel complex in the plasma membrane, the neuronal CaVß subunit can translocate to the cell nucleus. However, little is known regarding the role this protein could play in the nucleus, nor the molecular mechanism used by CaVß to enter this cell compartment. This report shows evidence that CaVß3 has nuclear localization signals (NLS) that are not functional, suggesting that the protein does not use a classical nuclear import pathway. Instead, its entry into the nucleus could be associated with another protein that would function as a carrier, using a mechanism known as a piggyback. Mass spectrometry assays and bioinformatic analysis allowed the identification of proteins that could be participating in the entry of CaVß3 into the nucleus. Likewise, through proximity ligation assays (PLA), it was found that members of the heterogeneous nuclear ribonucleoproteins (hnRNPs) and B56δ, a regulatory subunit of the protein phosphatase 2A (PP2A), could function as proteins that regulate this piggyback mechanism. On the other hand, bioinformatics and site-directed mutagenesis assays allowed the identification of a functional nuclear export signal (NES) that controls the exit of CaVß3 from the nucleus, which would allow the completion of the nuclear transport cycle of the protein. These results reveal a novel mechanism for the nuclear transport cycle of the neuronal CaVß3 subunit.

Identification of Dp140 and α1-syntrophin as novel molecular interactors of the neuronal CaV2.1 channel.

The primary function of dystrophin is to form a link between the cytoskeleton and the extracellular matrix. In addition to this crucial structural function, dystrophin also plays an essential role in clustering and organizing several signaling proteins, including ion channels. Proteomic analysis of the whole rodent brain has stressed the role of some components of the dystrophin-associated glycoprotein complex (DGC) as potential interacting proteins of the voltage-gated Ca2+ channels of the CaV2 subfamily. The interaction of CaV2 with signaling and scaffolding proteins, such as the DGC components, may influence their function, stability, and location in neurons. This work aims to study the interaction between dystrophin and CaV2.1. Our immunoprecipitation data showed the presence of a complex formed by CaV2.1, CaVα2δ-1, CaVß4e, Dp140, and α1-syntrophin in the brain. Furthermore, proximity ligation assays (PLA) showed that CaV2.1 and CaVα2δ-1 interact with dystrophin in the hippocampus and cerebellum. Notably, Dp140 and α1-syntrophin increase CaV2.1 protein stability, half-life, permanence in the plasma membrane, and current density through recombinant CaV2.1 channels. Therefore, we have identified the Dp140 and α1-syntrophin as novel interaction partners of CaV2.1 channels in the mammalian brain. Consistent with previous findings, our work provides evidence of the role of DGC in anchoring and clustering CaV channels in a macromolecular complex.

Antifungal Activity of Selenium Nanoparticles Obtained by Plant-Mediated Synthesis.

The continuous need to satisfy world food demand has led to the search for new alternatives to combat economic losses in agriculture caused by phytopathogenic fungi. These organisms cause plant diseases, reducing their productivity and decreasing fruit quality. Among the new tools being explored is nanotechnology. Nanoparticles with antimicrobial properties could be an excellent alternative to address this problem. In this work, selenium nanoparticles (SeNPs) were obtained using plant extracts of Amphipterygium glaucum leaves (SeNPs-AGL) and Calendula officinalis flowers (SeNPs-COF). Characterization of the SeNPs was performed and their ability as antifungal agents against two commercially relevant plant pathogenic fungi, Fusarium oxysporum and Colletotrichum gloeosporioides, was evaluated. Assays were performed with different concentrations of SeNPs (0, 0.25, 0.5, 1.0, and 1.7 mg/mL). It was observed that both SeNPs had antifungal activity against both plant pathogens at concentrations of 0.25 mg/mL and above. SeNPs-AGL demonstrated better antifungal activity and smaller size (around 8.0 nm) than SeNPs-COF (134.0 nm). FTIR analysis evidenced the existence of different functional groups that constitute both types of SeNPs. There are factors that have to be considered in the antimicrobial activity of SeNPs such as nanoparticle size and phytochemical composition of the plant extracts used, as these may affect their bioavailability.

Role of the Ca2+ channel α2δ-1 auxiliary subunit in proliferation and migration of human glioblastoma cells.

The overexpression of α2δ-1 is related to the development and degree of malignancy of diverse types of cancer. This protein is an auxiliary subunit of voltage-gated Ca2+ (CaV) channels, whose expression favors the trafficking of the main pore-forming subunit of the channel complex (α1) to the plasma membrane, thereby generating an increase in Ca2+ entry. Interestingly, TLR-4, a protein belonging to the family of toll-like receptors that participate in the inflammatory response and the transcription factor Sp1, have been linked to the progression of glioblastoma multiforme (GBM). Therefore, this report aimed to evaluate the role of the α2δ-1 subunit in the progression of GBM and investigate whether Sp1 regulates its expression after the activation of TLR-4. To this end, the expression of α2δ-1, TLR-4, and Sp1 was assessed in the U87 human glioblastoma cell line, and proliferation and migration assays were conducted using different agonists and antagonists. The actions of α2δ-1 were also investigated using overexpression and knockdown strategies. Initial luciferase assays and Western blot analyses showed that the activation of TLR-4 favors the transcription and expression of α2δ-1, which promoted the proliferation and migration of the U87 cells. Consistent with this, overexpression of α2δ-1, Sp1, and TLR-4 increased cell proliferation and migration, while their knockdown with specific siRNAs abrogated these actions. Our data also suggest that TLR-4-mediated regulation of α2δ-1 expression occurs through the NF-kB signaling pathway. Together, these findings strongly suggest that the activation of TLR-4 increases the expression of α2δ-1 in U87 cells, favoring their proliferative and migratory potential, which might eventually provide a theoretical basis to examine novel biomarkers and molecular targets for the diagnosis and treatment of GBM.

The ubiquitin E3 ligase Parkin regulates neuronal CaV1.3 channel functional expression.

Neuronal L-type Ca2+ channels of the CaV1.3 subclass are transmembrane protein complexes that contribute to the pacemaker activity in the adult substantia nigra dopaminergic neurons. The altered function of these channels may play a role in the development and progress of neurodegenerative mechanisms implicated in Parkinson's disease (PD). Although L-type channel expression is precisely regulated, an increased functional expression has been observed in PD. Previously, we showed that Parkin, an E3 enzyme of the ubiquitin-proteasome system (UPS) interacts with neuronal CaV2.2 channels promoting their ubiquitin-mediated degradation. In addition, previous studies show an increase in CaV1.3 channel activity in dopaminergic neurons of the SNc and that Parkin expression is reduced in PD. These findings suggest that the decrease in Parkin may affect the proteasomal degradation of CaV1.3, which helps explain the increase in channel activity. Therefore, the present report aims to gain insight into the degradation mechanisms of the neuronal CaV1.3 channel by the UPS. Immunoprecipitation assays showed the interaction between Parkin and the CaV1.3 channels expressed in HEK-293 cells and neural tissues. Likewise, Parkin overexpression reduced the total and membrane channel levels and decreased the current density. Consistent with this, patch-clamp recordings in the presence of an inhibitor of the UPS, MG132, prevented the effects of Parkin, suggesting enhanced channel proteasomal degradation. In addition, the half-life of the pore-forming CaV1.3α1 protein was significantly reduced by Parkin overexpression. Finally, electrophysiological recordings using a PRKN knockout HEK-293 cell line generated by CRISPR/Cas9 showed increased current density. These results suggest that Parkin promotes the proteasomal degradation of CaV1.3, which may be a relevant aspect for the pathophysiology of PD.NEW & NOTEWORTHY The increased expression of CaV1.3 calcium channels is a crucial feature of Parkinson's disease (PD) pathophysiology. However, the mechanisms that determine this increase are not yet defined. Parkin, an enzyme of the ubiquitin-proteasome system, is known to interact with neuronal channels promoting their ubiquitin-mediated degradation. Interestingly, Parkin mutations also play a role in PD. Here, the degradation mechanisms of CaV1.3 channels and their relationship with the pathophysiology of PD are studied in detail.

The role of voltage-gated calcium channels in the pathogenesis of Parkinson's disease.

Aim: Voltage-gated calcium (CaV) channels play an essential role in maintaining calcium homeostasis and regulating numerous physiological processes in neurons. Therefore, dysregulation of calcium signaling is relevant in many neurological disorders, including Parkinson's disease (PD). This review aims to introduce the role of CaV channels in PD and discuss some novel aspects of channel regulation and its impact on the molecular pathophysiology of the disease.Methods: an exhaustive search of the literature in the field was carried out using the PubMed database of The National Center for Biotechnology Information. Systematic searches were performed from the initial date of publication to May 2022.Results: Although α-synuclein aggregates are the main feature of PD, L-type calcium (CaV1) channels seem to play an essential role in the pathogenesis of PD. Changes in the functional expression of CaV1.3 channels alter Calcium homeostasis and contribute to the degeneration of dopaminergic neurons. Furthermore, recent studies suggest that CaV channel trafficking towards the cell membrane depends on the activity of the ubiquitin-proteasome system (UPS). In PD, there is an increase in the expression of L-type channels associated with a decrease in the expression of Parkin, an E3 enzyme of the UPS. Therefore, a link between Parkin and CaV channels could play a fundamental role in the pathogenesis of PD and, as such, could be a potentially attractive target for therapeutic intervention.Conclusion: The study of alterations in the functional expression of CaV channels will provide a framework to understand better the neurodegenerative processes that occur in PD and a possible path toward identifying new therapeutic targets to treat this condition.

Ion channel long non-coding RNAs in neuropathic pain.

Neuropathic pain is one of the primary forms of chronic pain and is the consequence of the somatosensory system's direct injury or disease. It is a relevant public health problem that affects about 10% of the world's general population. In neuropathic pain, alteration in neurotransmission occurs at various levels, including the dorsal root ganglia, the spinal cord, and the brain, resulting from the malfunction of diverse molecules such as receptors, ion channels, and elements of specific intracellular signaling pathways. In this context, there have been exciting advances in elucidating neuropathic pain's cellular and molecular mechanisms in the last decade, including the possible role that long non-coding RNAs (lncRNAs) may play, which open up new alternatives for the development of diagnostic and therapeutic strategies for this condition. This review focuses on recent studies associated with the possible relevance of lncRNAs in the development and maintenance of neuropathic pain through their actions on the functional expression of ion channels. Recognizing the changes in the function and spatio-temporal patterns of expression of these membrane proteins is crucial to understanding the control of neuronal excitability in chronic pain syndromes.

L5-6 Spinal Nerve Ligation-induced Neuropathy Changes the Location and Function of Ca2+ Channels and Cdk5 and Affects the Compound Action Potential in Adjacent Intact L4 Afferent Fibers.

Voltage-gated Ca2+ (CaV) channels regulate multiple cell processes, including neurotransmitter release, and have been associated with several pathological conditions, such as neuropathic pain. Cdk5, a neuron-specific kinase, may phosphorylate CaV channels, altering their functional expression. During peripheral nerve injury, upregulation of CaV channels and Cdk5 in the dorsal root ganglia (DRG) and the spinal cord, has been correlated with allodynia. We recently reported an increase in the amplitude of the C component of the compound action potential (cAP) of afferent fibers in animals with allodynia induced by L5-6 spinal nerve ligation (SNL), recorded in the corresponding dorsal roots. This was related to an increase in T-type (CaV3.2) channels generated by Cdk5-mediated phosphorylation. Here, we show that CaV channel functional expression is also altered in the L4 adjacent intact afferent fibers in rats with allodynia induced by L5-6 SNL. Western blot analysis showed that both Cdk5 and CaV3.2 total levels are not increased in the DRG L3-4, but their subcellular distribution changes by concentrating on the neuronal soma. Likewise, the Cdk5 inhibitor olomoucine affected the rapid and the slow C components of the cAP recorded in the dorsal roots. Patch-clamp recordings revealed an increase in T- and N-type currents recorded in the soma of acute isolated L3-4 sensory neurons after L5-6 SNL, which was prevented by olomoucine. These findings suggest changes in CaV channels location and function in L3-4 afferent fibers associated with Cdk5-mediated phosphorylation after L5-6 SNL, which may contribute to nerve injury-induced allodynia.

Políticas públicas sobre envejecimiento poblacional promulgadas en el contexto internacional, europeo y español entre 1982-2017 / Public policies on population ageing enacted in the international, European and Spanish contexts from 1982 to 2017

Introducción: El envejecimiento poblacional, problema de relevancia mundial, requiere acción de parte de los organismos políticos y gobiernos. Objetivo: Exponer las principales políticas dirigidas a la atención del envejecimiento poblacional promulgadas en el contexto internacional, el europeo y en las comunidades autónomas españolas entre 1982 y 2017. Métodos: Se realizó un trabajo de revisión y análisis de documentos, que incluyó la búsqueda de fuentes primarias tales como: planes y estrategias oficiales, informes, bases de datos y legislaciones en relación con el tema del envejecimiento poblacional y el envejecimiento activo. La recogida de información se ejecutó de mayo a julio de 2017. Se utilizó la técnica de análisis de contenido para identificar las políticas en el contexto internacional. La ficha diagnóstica para el análisis de las estrategias, se confeccionó a partir de una adaptación de la ficha propuesta en la Estrategia Vasca de Envejecimiento Activo. Conclusiones: El paradigma de la Organización Mundial de la Salud sobre Envejecimiento Activo promulga la perspectiva de la salud, la participación y seguridad de las personas mayores. Marca un hito en la formulación de políticas y estrategias de trabajo a nivel internacional. Las comunidades autónomas españolas han proyectado diversas estrategias, pero se requiere lograr coordinación integrada de sus actuaciones(AU)

Introduction: Population ageing, a problem of global importance, requires actions by political bodies and governments. Objective: Present the main policies aimed to the care of the population aging enacted in the international, European and Spanish autonomous communities' contexts in the period from 1982 to 2017. Methods: A review and analysis of documents was carried out, which included the search for primary sources such as: official plans and strategies, reports, databases and legislation related to the issue of population ageing and active ageing. The information collection was carried out from May to July 2017. The content analysis technique was used to identify policies in the international context; the diagnostic sheet for the analysis of strategies was prepared on the basis of an adaptation of the card proposed in the Basque Active Ageing Strategy. Conclusions: The World Health Organization paradigm on Active Ageing promulgates the perspective of the health, participation and safety of the elderly. It marks a milestone in the formulation of policies and strategies for work at the international level. The Spanish Autonomous Communities have planned various strategies, but it is necessary to achieve integrated coordination of their actions(AU)

Maltreatment and non-infant maltreatment, relevance of forensic clinical anatomy, presentation of a case / Maltrato y no maltrato infantil: relevancia de la anatomía clínica forense: presentación de un caso

In a recently published article, Andrea Porzionato et al, they expose the relevance of Forensic Clinical Anatomy as a tool in judicial strata when there are medical-legal implications for suspected child abuse and the presence of anatomical variants and traumatic injuries that at any given time are difficult to differentiate. A case is reported where the careful dissection of a minor's body reveals a congenital malformation of the genital-urinary tract that causes repeated urinary tract infections resulting in sepsis and death, based on this description and the context of death is determined that death is associated with child abuse from lack of medical attention.

En un artículo de reciente publicación, Andrea Porzionato et al, exponen la relevancia de la Anatomía Clínica Forense como herramienta en estratos judiciales cuando existen implicaciones médico-legales por sospecha de maltrato infantil y la presencia de variantes anatómicas y lesiones traumáticas que en un momento dado son difíciles de diferenciar. Se reporta un caso donde la cuidadosa disección del cuerpo de un menor revela una malformación congénita del tracto genital-urinario que ocasiona infecciones repetidas del tracto urinario resultando en sepsis y muerte, con base en esta descripción y se determina el contexto de muerte la cual está asociada con abuso infantil por falta de atención médica.

Regulation of the Ca2+ channel α2δ-1 subunit expression by epidermal growth factor via the ERK/ELK-1 signaling pathway.

Voltage-gated Ca2+ (CaV) channels are expressed in endocrine cells where they contribute to hormone secretion. Diverse chemical messengers, including epidermal growth factor (EGF), are known to affect the expression of CaV channels. Previous studies have shown that EGF increases Ca2+ currents in GH3 pituitary cells by increasing the number of high voltage-activated (HVA) CaV channels at the cell membrane, which results in enhanced prolactin (PRL) secretion. However, little is known regarding the mechanisms underlying this regulation. Here, we show that EGF actually increases the expression of the CaVα2δ-1 subunit, a key molecular component of HVA channels. The analysis of the gene promoter encoding CaVα2δ-1 (CACNA2D1) revealed binding sites for transcription factors activated by the Ras/Raf/MEK/ERK signaling cascade. Chromatin immunoprecipitation and site-directed mutagenesis showed that ELK-1 is crucial for the transcriptional regulation of CACNA2D1 in response to EGF. Furthermore, we found that EGF increases the membrane expression of CaVα2δ-1 and that ELK-1 overexpression increases HVA current density, whereas ELK-1 knockdown decreases the functional expression of the channels. Hormone release assays revealed that CaVα2δ-1 overexpression increases PRL secretion. These results suggest a mechanism for how EGF, by activating the Ras/Raf/MEK/ERK/ELK-1 pathway, may influence the expression of HVA channels and the secretory behavior of pituitary cells.

Cdk5 phosphorylates CaV1.3 channels and regulates GABAA-mediated miniature inhibitory post-synaptic currents in striato-nigral terminals.

Neurotransmission is one of the most important processes in neuronal communication and depends largely on Ca2+ entering synaptic terminals through voltage-gated Ca2+ (CaV) channels. Although the contribution of L-type CaV channels in neurotransmission has not been unambiguously established, increasing evidence suggests a role for these proteins in noradrenaline, dopamine, and GABA release. Here we report the regulation of L-type channels by Cdk5, and its possible effect on GABA release in the substantia nigra pars reticulata (SNpr). Using patch-clamp electrophysiology, we show that Cdk5 inhibition by Olomoucine significantly increases current density through CaV1.3 (L-type) channels heterologously expressed in HEK293 cells. Likewise, in vitro phosphorylation showed that Cdk5 phosphorylates residue S1947 in the C-terminal region of the pore-forming subunit of CaV1.3 channels. Consistent with this, the mutation of serine into alanine (S1947A) prevented the regulation of Cdk5 on CaV1.3 channel activity. Our data also revealed that the inhibition of Cdk5 increased the frequency of high K+-evoked miniature inhibitory postsynaptic currents in rat SNpr neurons, acting on L-type channels. These results unveil a novel regulatory mechanism of GABA release in the SNpr that involves a direct action of Cdk5 on L-type channels.

Cdk5-Dependent Phosphorylation of CaV3.2 T-Type Channels: Possible Role in Nerve Ligation-Induced Neuropathic Allodynia and the Compound Action Potential in Primary Afferent C Fibers.

Voltage-gated T-type Ca2+ (CaV3) channels regulate diverse physiological events, including neuronal excitability, and have been linked to several pathological conditions such as absence epilepsy, cardiovascular diseases, and neuropathic pain. It is also acknowledged that calcium/calmodulin-dependent protein kinase II and protein kinases A and C regulate the activity of T-type channels. Interestingly, peripheral nerve injury induces tactile allodynia and upregulates CaV3.2 channels and cyclin-dependent kinase 5 (Cdk5) in dorsal root ganglia (DRG) and spinal dorsal horn. Here, we report that recombinant CaV3.2 channels expressed in HEK293 cells are regulatory targets of Cdk5. Site-directed mutagenesis showed that the relevant sites for this regulation are residues S561 and S1987. We also found that Cdk5 may regulate CaV3.2 channel functional expression in rats with mechanical allodynia induced by spinal nerve ligation (SNL). Consequently, the Cdk5 inhibitor olomoucine affected the compound action potential recorded in the spinal nerves, as well as the paw withdrawal threshold. Likewise, Cdk5 expression was upregulated after SNL in the DRG. These findings unveil a novel mechanism for how phosphorylation may regulate CaV3.2 channels and suggest that increased channel activity by Cdk5-mediated phosphorylation after SNL contributes nerve injury-induced tactile allodynia.SIGNIFICANCE STATEMENT Neuropathic pain is a current public health challenge. It can develop as a result of injury or nerve illness. It is acknowledged that the expression of various ion channels can be altered in neuropathic pain, including T-type Ca2+ channels that are expressed in sensory neurons, where they play a role in the regulation of cellular excitability. The present work shows that the exacerbated expression of Cdk5 in a preclinical model of neuropathic pain increases the functional expression of CaV3.2 channels. This finding is relevant for the understanding of the molecular pathophysiology of the disease. Additionally, this work may have a substantial translational impact, since it describes a novel molecular pathway that could represent an interesting therapeutic alternative for neuropathic pain.

Transcription Factor Sp1 Regulates the Expression of Calcium Channel α2δ-1 Subunit in Neuropathic Pain.

High voltage-activated (HVA) Ca2+ (CaV) channels are oligomeric complexes formed by an ion-conducting main subunit (Cavα1) and at least two auxiliary subunits (Cavß and CaVα2δ). It has been reported that the expression of CaVα2δ1 increases in the dorsal root ganglia (DRGs) of animals with mechanical allodynia, and that the transcription factor Sp1 regulates the expression of the auxiliary subunit. Hence, the main aim of this work was to investigate the role of Sp1 as a molecular determinant of the exacerbated expression of CaVα2δ-1 in the nerve ligation-induced model of mechanical allodynia. Our results show that ligation of L5/L6 spinal nerves (SNL) produced allodynia and increased the expression of Sp1 and CaVα2δ-1 in the DRGs. Interestingly, intrathecal administration of the Sp1 inhibitor mithramycin A (Mth) prevented allodynia and decreased the expression of Sp1 and CaVα2δ-1. Likewise, electrophysiological recordings showed that incubation with Mth decreased Ca2+ current density in the DRG neurons, acting mostly on HVA channels. These results suggest that L5/L6 SNL produces mechanical allodynia and increases the expression of the transcription factor Sp1 and the subunit CaVα2δ-1 in the DRGs, while Mth decreases mechanical allodynia and Ca2+ currents through HVA channels in sensory neurons by reducing the functional expression of the CaVα2δ-1 subunit.

Determinación de la frecuencia de PNUs en una población heterogénea cerrada y su utilidad como marcadores para estudios de asociación genética en casos de labio y paladar hendido no sindrómico / Determination of frequency of SNP's in a population of mixed genetic origin and its use as genetic markers for association studies in cases of not syndromic cleft lip and palate

RESUMEN El labio y paladar hendido es una de las patologías congénitas con mayor prevalencia en el mundo. En el presente trabajo se hace un análisis de 12 PNU localizados en las secuencias genómicas de ABCA4, BMP4, MSX1, SUMO1, VAX1 y IRF6, bajo una perspectiva epidemiológica, de genética molecular, genómica y de genética de poblaciones; todo lo anterior aplicado a una población de Querétaro, México, de origen genético mixto. Material y métodos: Se realizó un estudio observacional, analítico y descriptivo a partir de muestras de 93 tríadas (sujetos de estudio y sus padres). Al seleccionar PNU que puedan ser diferenciados por medio de RFLP esperamos distinguir entre marcadores genéticos que: 1) cumplan con la ecuación de equilibrio de Hardy-Weinberg y 2) validarlos como potenciales marcadores genéticos para ser empleados en estudios de asociación en poblaciones cerradas de origen genético mixto con labio y paladar hendido (Amealco, Querétaro, México). De ser así, posteriormente se plantea probar las frecuencias obtenidas con una población seleccionada genéticamente cerrada de Amealco, Querétaro. Resultados: Después de realizar el análisis RFLP de 12 PNU localizados en la secuencia de genes ABCA4, BMP4, MSX1, SUMO1, VAX1 y IRF6, hallamos el mismo alelo para PNU analizado, el cual se encuentra en el 100% de la población. Conclusión: De los 12 PNU analizados, en este reporte, por primera vez se menciona la frecuencia de cinco de ellos. Los restantes siete presentaron la misma frecuencia reportada en la literatura. Aunque los PNU seleccionados no fueron de utilidad como marcadores genéticos debido a que el mismo alelo está presente en el 100% de la población general. El hecho de haberlos encontrado en el mismo genotipo de todas las muestras indica que la población de la ciudad de Querétaro es genéticamente cerrada y con base en esto extremadamente útil para futuras validaciones de otros PNU como posibles marcadores genéticos.

ABSTRACT The cleft lip and palate is one of the congenital pathologies with greater prevalence in the world. In the present work, there is an analysis of 12 SNP's located in genomic sequences of ABCA4, BMP4, MSX1, SUMO1, VAX1 andIRF6, under an epidemiological perspective, molecular genetics, genomics and population genetics. All of the above applied to a population of Queretaro, Mexico, of mixed genetic origin. Material and methods: A study was conducted of observation, analytic and descriptive study with samples from 93 triads (study subjects and their parents). When you select SNP's that can be differentiated by RFLP (Restriction Fragment Length Polymorphism)we hope to distinguish between genetic markers that: 1)comply with the equation of balance of Hardy-Weiner and 2) Validate them as potential genetic markers to be used in studies of association in closed populations of genetic origin mixed with cleft lip and palate in Amealco, Queretaro, Mexico. If so subsequently raises test the frequencies obtained with a selected population genetically closed in Amealco, Queretaro. Results: After performing the RFLP analysis of 12 SNP's located in the sequence of genes ABCA4, BMP4, MSX1, SUMO1, VAX1 and IRF6, we find the same allele for SNP analyzed which is located in the 100% of the population. Conclusion: Of the 12 SNP's analyzed in this report, for the fi rst time 5 of them are mentioned their frequency. The rest of them had the same frequency reported in the literature. Although the SNP's selected were not useful as a genetic markers due to the same allele is present in 100% of the general population. The fact of having found in the same genotype of all samples indicates that the population of the city of Queretaro is genetically closed and on the basis of this extremely useful for future validations of other SNP's as potential genetic markers.