Cellular Alterations Research Articles

Pathophysiology of drug-resistant canine epilepsy

Drug resistance continues to be a major clinical problem in the therapeutic management of canine epilepsies with substantial implications for quality of life and survival times. Experimental and clinical data from human medicine provided evidence for relevant contributions of intrinsic severity of the disease as well as alterations in pharmacokinetics and -dynamics to failure to respond to antiseizure medications. In addition, several modulatory factors have been identified that can be associated with the level of therapeutic responses. Among others, the list of potential modulatory factors comprises genetic and epigenetic factors, inflammatory mediators, and metabolites. Regarding data from dogs, there are obvious gaps in knowledge when it comes to our understanding of the clinical patterns and the mechanisms of drug-resistant canine epilepsy. So far, seizure density and the occurrence of cluster seizures have been linked with a poor response to antiseizure medications. Moreover, evidence exists that the genetic background and alterations in epigenetic mechanisms might influence the efficacy of antiseizure medications in dogs with epilepsy. Further molecular, cellular, and network alterations that may affect intrinsic severity, pharmacokinetics, and -dynamics have been reported. However, the association with drug responsiveness has not yet been studied in detail. In summary, there is an urgent need to strengthen clinical and experimental research efforts exploring the mechanisms of resistance as well as their association with different etiologies, epilepsy types, and clinical courses.

Memantine and its role in parkinsonism, seizure, depression, migraine headache, and Alzheimer’s disease

Alzheimer’s disease (AD) is a neurological disorder characterized by mental and behavioral changes that develop progressively with a decline in brain function. Dysfunctions in glutamatergic and cholinergic pathways, along with an increased concentration of beta-amyloid protein (Aβ), lead to synapses that are full of phosphorylated protein. These changes result in several pathological, biochemical, cellular, and molecular alterations that increase neural excitation directly or indirectly at the neural level, affecting the synapse, axons, signal transmission, and all parts of neurons. All these alterations, with continuous excitatory effects, eventually lead to neural loss and degradation due to stimulation of the immune response. However, memantine is a non-competitive antagonist of N-methyl-D-aspartic acid (NMDA) glutamatergic receptors of moderate affinity and voltage-dependent that blocks the effects of pathologically elevated glutamate tonic levels, which can lead to neuronal dysfunction. Memantine has shown improvement in cognition, global clinical status, activities of daily living, and behavioral disturbances in moderate and severe AD. In this review, we will discuss the effects of memantine use and side effects, as well as its application in treating other diseases or pathological conditions with the prospective use of memantine or an alternative. Memantine is generally well-tolerated, and the most common adverse reactions are vertigo, headache, and hallucinations, which are usually mild.

Serum/glucose starvation strikingly reduce heterogeneous nuclear ribonucleoprotein A1 protein and its target, cyclin D1.

Our investigation to explore cellular alterations related to undernutrition in cancer cells revealed that the protein level of heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) is drastically decreased by serum/glucose starvation. Its loss was reversible, serum/glucose starvation-specific, and universal throughout cell types and species. The hnRNP A1 mRNA level and hnRNP A1 mRNA/protein stability were not altered under this condition. CCND1 mRNA, which we newly identified as the binding target of hnRNP A1, was decreased by serum/glucose starvation. Under similar conditions, CCND1 protein was reduced in vitro and in vivo, whereas hnRNP A1 mRNA level and CCND1 mRNA level revealed no correlation in most clinical samples. Functional analyzes revealed that CCND1 mRNA stability is certainly dependent on hnRNP A1 protein level and that RNA recognition motif-1 (RRM1) in hnRNP A1 plays a central role in maintaining CCND1 mRNA stability and subsequent protein expression. The injection of RRM1-deleted hnRNP A1-expressing cancer cells in the mouse xenograft model did not form any tumors, and that of hnRNP A1-expressing cancer cells retained CCND1 expression at the lesion adjacent to necrosis with a slight increase of tumor volume. Furthermore, RRM1 deletion caused growth suppression with induction of apoptosis and autophagy, whereas CCND1 restoration completely recovered it. Our results indicate that serum/glucose starvation triggers entire hnRNP A1 protein loss, and its loss may play a role in CCND1 mRNA destabilization and CCND1-mediated cellular event inhibition, i.e., growth promotion, apoptosis induction, and autophagosome formation.

Tri-modal single cell (TEA-seq) profiling reveals a novel, epigenetically distinct T cell subset in children and global molecular reprogramming across age

Abstract Most single cell analyses of human T cell heterogeneity use a single modality (i.e., RNA or ATAC or protein), with limited ability to deconvolute complex cellular and molecular alterations that can occur during human aging and disease. Here, we use a new trimodal single cell technology (TEA-seq) to elucidate the interplay between the surface proteome, transcriptome, and epigenome in more than 300,000 T cells from healthy children (11–13 yrs) and older adults (55–65 yrs). We uncover that age universally drives molecular reprogramming across all T cell subsets towards a more activated basal state. Naive CD4 T cells, considered relatively resistant to aging, exhibited far more pronounced transcriptional reprogramming than naive CD8 T cells, in which alterations are preferentially driven by compositional shifts in naive-like memory subsets. We further discover a novel naïve-like CD8aa T cell subset lost with age that displays an innate-like transcriptome, a KLF/SP-rich epigenome and high IL-21 receptor expression. These data reveal the complex molecular landscape of the human T cell compartment that may contribute to differential immune responses across age. Supported by grants from NIH (K01 AG068373 to CEG) and the Allen Institute

Role of Flavonoids in Management of Various Biological Targets in Alzheimer's Disease: Evidence from Preclinical to Clinical Studies.

More than 10 million people worldwide have Alzheimer's disease (AD), a degenerative neurological illness and the most prevalent form of dementia. AD's progression in memory loss, cognitive deterioration, and behavioral changes are all symptoms. Amyloid-beta 42 (Aβ42), the hyperphosphorylated forms of microtubule-associated tau protein, and other cellular and systemic alterations are all factors that contribute to cognitive decline in AD. Rather than delivering a possible cure, present therapy strategies focus on reducing disease symptoms. It has long been suggested that various naturally occurring small molecules (plant extract products and microbiological isolates, for example) could be beneficial in preventing or treating disease. Small compounds, such as flavonoids, have attracted much interest recently due to their potential to alleviate cellular stress. Flavonoids have been proven helpful in various ways, including antioxidants, anti-inflammatory agents, and anti-apoptotic agents, but their mechanism remains unknown. The flavonoid therapy of Alzheimer's disease focuses on this review, which includes a comprehensive literature analysis.

Sett priming with salicylic acid improves salinity tolerance of sugarcane (Saccharum officinarum L.) during early stages of crop development

Sugarcane (Saccharum officinarum L.), a globally cultivated carbohydrate producing crop of industrial importance is being challenged by soil salinity due to its glycophytic nature. Water stress coupled with cellular and metabolic alterations resulting from excess sodium (Na+) ion accumulation is irreversibly damaging during early crop developmental stages that often results in complete crop failure. This study therefore aimed to explore the potential of salicylic acid as a sett priming material to mitigate the negative effects of salt stress on sugarcane during germination and early growth stages. Five doses of salicylic acid (0 [hydropriming] [control], 0.5 mM, 1 mM, 1.5 mM and 2 mM) were tested against three levels of salinity (0.5 dS m−1 [control], 4 dS m−1, and 8 dS m−1) within a polyhouse environment. Results revealed 11.2%, 18.5%, 25.4%, and 38.6%, average increase in final germination, germination energy, seedling length and seedling vigor index respectively with a subsequent reduction of 21% mean germination time. Investigations during early seedling growth revealed 21.6%, 17.5%, 27.0%, 39.9%, 10.7%, 11.5%, 17.5%, 47.9%, 35.3% and 20.5% overall increase in plant height, total leaf area, shoot dry matter, root dry matter, leaf greenness, relative water content, membrane stability index, proline content, total antioxidant activity and potassium (K+) ion accumulation respectively with a subsequent reduction of 24.9% Na+ ion accumulation and 35.8% Na+/K+ ratio due to salicylic acid priming. Germination, seedling growth and recovery of physiochemical traits were highly satisfactory in primed setts than non-primed ones even under 8 dS m−1 salinity level. This study should provide useful information for strategizing salinity management approaches for better productivity of sugarcane.

Alteration of metabolite landscape in hypoxia replicates immune cell programs indicative of dysfunctional immunity in severe COVID-19

Abstract Our prior work has demonstrated that altered metabolic programs in T cells define dysregulated immunity in acute SARS-CoV-2 infection. Apoptotic T cells were defined by high expression of the mitochondrial membrane protein voltage-dependent anion channel 1 (VDAC1). These cells were markedly increased in elderly patients and correlated with lymphopenia. We devised in vitro systems in healthy T cells to investigate the mechanism driving this unique phenotype. While SARS-CoV-2 did not directly induce this T cell phenotype, factors that enhanced the development of VDAC1+ T cells, including hypoxia, advanced age of healthy T cell donor, and COVID-19 patient-derived plasma. Analysis of patient-derived plasma revealed robust differences in cytokine and metabolomic profiles between people who did or did not exhibit apoptotic T cells. Increased flux through the tryptophan:kynurenin pathway has been suggested as a biomarker of severe COVID-19 and was further enhanced in individuals with apoptotic T cells. Kynurenine directly induced VDAC1+ T cells in a dose dependent manner in vitro. Together, our work indicates metabolic alterations combined with cellular age and hypoxia may induce apoptosis of T cells following SARS-CoV-2 infection. This work highlights mechanisms leading to cellular alterations following infection and highlights key aspects of the host environment that could be targeted therapeutically. U54CA260492

Cytoskeletal Remodeling and Gap Junction Translocation Mediates Blood-Brain Barrier Disruption by Non-invasive Low-Voltage Pulsed Electric Fields.

High-voltage pulsed electric fields (HV-PEF) delivered with invasive needle electrodes for electroporation applications is known to induce off-target blood-brain barrier (BBB) disruption. In this study, we sought to determine the feasibility of minimally invasive PEF application to produce BBB disruption in rat brain and identify the putative mechanisms mediating the effect. We observed dose-dependent presence of Evans Blue (EB) dye in rat brain when PEF were delivered with a skull mounted electrode used for neurostimulation application. Maximum region of dye uptake was observed while using 1500V, 100 pulses, 100µs and 10Hz. Results of computational models suggested that the region of BBB disruption was occurring at thresholds of 63V/cm or higher; well below intensity levels for electroporation. In vitro experiments recapitulating this effect with human umbilical vein endothelial cells (HUVEC) demonstrated cellular alterations that underlie BBB manifests at low-voltage high-pulse conditions without affecting cell viability or proliferation. Morphological changes in HUVECs due to PEF were accompanied by disruption of actin cytoskeleton, loss of tight junction protein-ZO-1 and VE-Cadherin at cell junctions and partial translocation into the cytoplasm. Uptake of propidium iodide (PI) in PEF treated conditions is less than 1% and 2.5% of total number of cells in high voltage (HV) and low-voltage (LV) groups, respectively, implying that BBB disruption to be independent of electroporation under these conditions. 3-D microfabricated blood vessel permeability was found to increase significantly following PEF treatment and confirmed with correlative cytoskeletal changes and loss of tight junction proteins. Finally, we show that the rat brain model can be scaled to human brains with a similar effect on BBB disruption characterized by electric field strength (EFS) threshold and using a combination of two bilateral HD electrode configurations.

Biological injection therapy with leukocyte-poor platelet-rich plasma induces cellular alterations, enhancement of lubricin, and inflammatory downregulation in vivo in human knees: A controlled, prospective human clinical trial based on mass spectrometry imaging analysis.

To investigate the in vivo biological effects of leukocyte-poor platelet-rich plasma (LpPRP) treatment in human synovial layer to establish the cellular basis for a prolonged clinical improvement. Synovial tissues (n = 367) were prospectively collected from patients undergoing arthroscopic surgery. Autologous-conditioned plasma, LpPRP, was injected into the knees of 163 patients 1-7 days before surgery to reduce operative trauma and inflammation, and to induce the onset of regeneration. A total of 204 patients did not receive any injection. All samples were analyzed by mass spectrometry imaging. Data analysis was evaluated by clustering, classification, and investigation of predictive peptides. Peptide identification was done by tandem mass spectrometry and database matching. Data analysis revealed two major clusters belonging to LpPRP-treated (LpPRP-1) and untreated (LpPRP-0) patients. Classification analysis showed a discrimination accuracy of 82%-90%. We identified discriminating peptides for CD45 and CD29 receptors (receptor-type tyrosine-protein phosphatase C and integrin beta 1), indicating an enhancement of musculoskeletal stem cells, as well as an enhancement of lubricin, collagen alpha-1-(I) chain, and interleukin-receptor-17-E, dampening the inflammatory reaction in the LpPRP-1 group following LpPRP injection. We could demonstrate for the first time that injection therapy using "autologic-conditioned biologics" may lead to cellular changes in the synovial membrane that might explain the reported prolonged beneficial clinical effects. Here, we show in vivo cellular changes, possibly based on muscular skeletal stem cell alterations, in the synovial layer. The gliding capacities of joints might be improved by enhancing of lubricin, anti-inflammation by activation of interleukin-17 receptor E, and reduction of the inflammatory process by blocking interleukin-17.

Morphological and functional alterations in hemocytes of Mytilus galloprovincialis exposed in high-impact anthropogenic sites

The work aimed to study the induction of morphological alterations in M. galloprovincialis in the field and its suitability to be integrated into a sensitive, simple, and cost-effective cell-based multimarker approach for the detection of the stress status induced by pollution in coastal marine environments in view of ecotoxicological biomonitoring and assessment application. Cellular morphometric alterations was paralleled by the analysis of standardized biomarkers such as lysosomal membrane destabilization, and genotoxocity biomarkers such as micronuclei and binuclated cells frequencies were investigated. The study was carried out by means of a transplanting experiment in the field, using caged organisms from an initial population exposed in the field in two multi-impacted coastal sites of the central Mediterranean area, Bagnoli in the eastern Tyrrhenian Sea and Augusta-Melilli-Priolo in the western Ionian Sea. Capo Miseno (NA) for the Tyrrhenian area and Brucoli (ME) for the Ionian area were chosen as control sites.Hemocyte enlargement and filopodial elongation increased frequencies were observed in organisms exposed to the impacted sites. These morphometric alterations showed strong agreement with the lysosomal membrane destabilization and biomarkers of genotoxicity, suggesting their usefulness in detecting the pollutant-induced stress syndrome related to genotoxic damage.

Attention-Deficit/Hyperactivity Disorder Animal Model Presents Retinal Alterations and Methylphenidate Has a Differential Effect in ADHD versus Control Conditions.

Attention-Deficit/Hyperactivity Disorder (ADHD) is one of the most prevalent neurodevelopmental disorders. Interestingly, children with ADHD seem to experience more ophthalmologic abnormalities, and the impact of methylphenidate (MPH) use on retinal physiology remains unclear. Thus, we aimed to unravel the retina's structural, functional, and cellular alterations and the impact of MPH in ADHD versus the control conditions. For that, spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were used as animal models of ADHD and the controls, respectively. Animals were divided into four experimental groups as follows: WKY vehicle (Veh; tap water), WKY MPH (1.5 mg/kg/day), SHR Veh, SHR MPH. Individual administration was performed by gavage between P28-P55. Retinal physiology and structure were evaluated at P56 followed by tissue collection and analysis. The ADHD animal model presents the retinal structural, functional, and neuronal deficits, as well as the microglial reactivity, astrogliosis, blood-retinal barrier (BRB) hyperpermeability and a pro-inflammatory status. In this model, MPH had a beneficial effect on reducing microgliosis, BRB dysfunction, and inflammatory response, but did not correct the neuronal and functional alterations in the retina. Curiously, in the control animals, MPH showed an opposite effect since it impaired the retinal function, neuronal cells, and BRB integrity, and also promoted both microglia reactivity and upregulation of pro-inflammatory mediators. This study unveils the retinal alterations in ADHD and the opposite effects induced by MPH in the retina of ADHD and the control animal models.

Abnormal glucose metabolism in virus associated sepsis.

Sepsis is identified as a potentially lethal organ impairment triggered by an inadequate host reaction to infection (Sepsis-3). Viral sepsis is a potentially deadly organ impairment state caused by the host's inappropriate reaction to a viral infection. However, when a viral infection occurs, the metabolism of the infected cell undergoes a variety of changes that cause the host to respond to the infection. But, until now, little has been known about the challenges faced by cellular metabolic alterations that occur during viral infection and how these changes modulate infection. This study concentrates on the alterations in glucose metabolism during viral sepsis and their impact on viral infection, with a view to exploring new potential therapeutic targets for viral sepsis.

In vitro to in vivo extrapolation and high-content imaging for simultaneous characterization of chemically induced liver steatosis and markers of hepatotoxicity

Chemically induced steatosis is characterized by lipid accumulation associated with mitochondrial dysfunction, oxidative stress and nucleus distortion. New approach methods integrating in vitro and in silico models are needed to identify chemicals that may induce these cellular events as potential risk factors for steatosis and associated hepatotoxicity. In this study we used high-content imaging for the simultaneous quantification of four cellular markers as sentinels for hepatotoxicity and steatosis in chemically exposed human liver cells in vitro. Furthermore, we evaluated the results with a computational model for the extrapolation of human oral equivalent doses (OED). First, we tested 16 reference chemicals with known capacities to induce cellular alterations in nuclear morphology, lipid accumulation, mitochondrial membrane potential and oxidative stress. Then, using physiologically based pharmacokinetic modeling and reverse dosimetry, OEDs were extrapolated from data of any stimulated individual sentinel response. The extrapolated OEDs were confirmed to be within biologically relevant exposure ranges for the reference chemicals. Next, we tested 14 chemicals found in food, selected from thousands of putative chemicals on the basis of structure-based prediction for nuclear receptor activation. Amongst these, orotic acid had an extrapolated OED overlapping with realistic exposure ranges. Thus, we were able to characterize known steatosis-inducing chemicals as well as data-scarce food-related chemicals, amongst which we confirmed orotic acid to induce hepatotoxicity. This strategy addresses needs of next generation risk assessment and can be used as a first chemical prioritization hazard screening step in a tiered approach to identify chemical risk factors for steatosis and hepatotoxicity-associated events.

Biomarkers of aging.

Aging biomarkers are a combination of biological parameters to (i) assess age-related changes, (ii) track the physiological aging process, and (iii) predict the transition into a pathological status. Although a broad spectrum of aging biomarkers has been developed, their potential uses and limitations remain poorly characterized. An immediate goal of biomarkers is to help us answer the following three fundamental questions in aging research: How old are we? Why do we get old? And how can we age slower? This review aims to address this need. Here, we summarize our current knowledge of biomarkers developed for cellular, organ, and organismal levels of aging, comprising six pillars: physiological characteristics, medical imaging, histological features, cellular alterations, molecular changes, and secretory factors. To fulfill all these requisites, we propose that aging biomarkers should qualify for being specific, systemic, and clinically relevant.

SARS-CoV-2 infection aggravates cigarette smoke-exposed cell damage in primary human airway epithelia

BackgroundThe coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a worldwide pandemic with over 627 million cases and over 6.5 million deaths. It was reported that smoking-related chronic obstructive pulmonary disease (COPD) might be a crucial risk for COVID-19 patients to develop severe condition. As cigarette smoke (CS) is the major risk factor for COPD, we hypothesize that barrier dysfunction and an altered cytokine response in CS-exposed airway epithelial cells may contribute to increased SARS-CoV-2-induced immune response that may result in increased susceptibility to severe disease. The aim of this study was to evaluate the role of CS on SARS-CoV-2-induced immune and inflammatory responses, and epithelial barrier integrity leading to airway epithelial damage.MethodsPrimary human airway epithelial cells were differentiated under air-liquid interface culture. Cells were then exposed to cigarette smoke medium (CSM) before infection with SARS-CoV-2 isolated from a local patient. The infection susceptibility, morphology, and the expression of genes related to host immune response, airway inflammation and damages were evaluated.ResultsCells pre-treated with CSM significantly caused higher replication of SARS-CoV-2 and more severe SARS-CoV-2-induced cellular morphological alteration. CSM exposure caused significant upregulation of long form angiotensin converting enzyme (ACE)2, a functional receptor for SARS-CoV-2 viral entry, transmembrane serine protease (TMPRSS)2 and TMPRSS4, which cleave the spike protein of SARS-CoV-2 to allow viral entry, leading to an aggravated immune response via inhibition of type I interferon pathway. In addition, CSM worsened SARS-CoV-2-induced airway epithelial cell damage, resulting in severe motile ciliary disorder, junctional disruption and mucus hypersecretion.ConclusionSmoking led to dysregulation of host immune response and cell damage as seen in SARS-CoV-2-infected primary human airway epithelia. These findings may contribute to increased disease susceptibility with severe condition and provide a better understanding of the pathogenesis of SARS-CoV-2 infection in smokers.

Spatially multicellular variability of intervertebral disc degeneration by comparative single-cell analysis.

Previous studies have revealed cellular heterogeneity in intervertebral discs (IVDs). However, the cellular and molecular alteration patterns of cell populations during degenerative progression remain to be fully elucidated. To illustrate the cellular and molecular alteration of cell populations in intervertebral disc degeneration (IDD), we perform single cell RNA sequencing on cells from four anatomic sites of healthy and degenerative goat IVDs. EGLN3+ StressCs, TGFBR3+ HomCs and GPRC5A+ RegCs exhibit the characteristics associated with resistance to stress, maintaining homeostasis and repairing, respectively. The frequencies and signatures of these cell clusters fluctuate with IDD. Notably, the chondrogenic differentiation programme of PROCR+ progenitor cells is altered by IDD, while notochord cells turn to stemness exhaustion. In addition, we characterise CAV1+ endothelial cells that communicate with chondrocytes through multiple signalling pathways in degenerative IVDs. Our comprehensive analysis identifies the variability of key cell clusters and critical regulatory networks responding to IDD, which will facilitate in-depth investigation of therapeutic strategies for IDD.

Abstract 68: Dissecting cellular and molecular alterations in pre-invasive to invasive lung cancer progression at single-cell resolution

Abstract Non-small cell lung cancer (NSCLC) accounts for most cancer-related deaths worldwide, largely due to diagnosis at advanced metastatic stages. Increased implementation of low-dose computed tomography (CT)-guided screening has led to the identification of non-solid lesions or ground-glass opacities (GGOs) that are not visible on plain chest radiography and can harbor very early-stage tumors. Growth of the lesions and/or development of a solid component can indicate more aggressive behavior. Using the low-dose CT reduced lung cancer mortality by >20%, underscoring the need for early detection and intervention. However, the cellular and molecular determinants that contribute to the malignant progression of those early preinvasive lesions remain poorly understood. To assess intratumoral heterogeneity and associated dynamic changes in the immune contexture during early lung carcinogenesis, we performed single-cell RNA-seq and Hyperion imaging on surgically resected freshly harvested tissues from 21 patients (matched solid and non-solid components of lesions and adjacent non-involved lungs), representing a spectrum from preneoplasia to invasive disease. Analysis of >300K cells identified cellular, transcriptomic, and metabolic alterations as well as tumor-stroma cross-talk pathways. Specifically, preinvasive to invasive disease progression was associated with significant changes in the immune landscape determined by an increase in exhausted CD4/CD8 T-cells, suppressive T-regs, polymorphonuclear myeloid-derived suppressor cells, and M2-like macrophages; and a decrease in cytotoxic CD8 T-cells, NK cells, and inflammatory cDC2 cells, suggesting that activation of immunosuppressive/anti-inflammatory microenvironment and suppression of immune activation microenvironment may enable tumor progression. Citation Format: Liron Yoffe, Alain Borczuk, Timothy E. McGraw, Olivier Elemento, Nasser K. Altorki, Vivek Mittal. Dissecting cellular and molecular alterations in pre-invasive to invasive lung cancer progression at single-cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 68.

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner.

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1-10 μM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca2+ influx. Yoda1 induced Ca2+ -dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo-/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.

Translational Neuroscience Approaches to Understanding Autism

While autism spectrum disorder affects nearly 2% of children in the United States, little is known with certainty concerning the etiologies and brain systems involved. This is due, in part, to the substantial heterogeneity in the presentation of the core symptoms of autism as well as the great number of co-occurring conditions that are common in autistic individuals. Understanding the neurobiology of autism is further hampered by the limited availability of postmortem brain tissue to determine the cellular and molecular alterations that take place in the autistic brain. Animal models therefore provide great translational value in helping to define the neural systems that constitute the social brain and mediate repetitive behaviors or interests. If they are based on genetic or environmental factors that contribute to autism, organisms from flies to nonhuman primates may serve as models of the neural structure or function of the autistic brain. Ultimately, successful models can also be employed to test the safety and effectiveness of potential therapeutics. This is an overview of the major animal species that are currently used as models of autism, including an appraisal of the advantages and limitations of each.

Transmembrane Mucin Response in Conjunctival Epithelial Cells Exposed to Wall Shear Stresses

Human conjunctival epithelium cells (HCEC) line the inner surface of the eyelid and cover the sclera and are continuously subjected to wall shear stresses (WSS). The effects of external forces on the conjunctival epithelium are not fully known. The conjunctival epithelium contains stratified squamous cells that synthesize the membrane-spanning mucins MUC1 and MUC16, which play important roles in protecting the ocular surface. Alterations in both gel-forming and membrane-tethered mucins occur in drying ocular surface diseases. The aim of this study was to explore the mechanobiological characteristics of transmembrane mucin secretion and cellular alterations of primary HCEC exposed to airflow-induced WSS perturbations. We exposed the HCEC to a steady WSS of 0.5 dyne/cm2 for durations of 15 and 30 min. Cytoskeletal alterations and MUC1 secretions were studied using immunohistochemically fluorescent staining with specific antibodies. We investigated for the first time an in vitro model of membrane-tethered mucin secretion by HCEC in response to WSS. The exposure of HCEC to WSS increased the polymerization of F-actin, altered the cytoskeletal shape and reduced the secretion of membrane-tethered MUC1.