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Plasma membrane calcium ATPase 1 (PMCA1, Atp2b1) is emerging as a key contributor to cardiac physiology, involved in calcium handling and myocardial signalling. In addition, genome wide association studies have associated PMCA1 in several areas of cardiovascular disease including hypertension and myocardial infarction. Here, we investigated the role of PMCA1 in basal cardiac function and heart rhythm stability. Cardiac structure, heart rhythm and arrhythmia susceptibility were assessed in a cardiomyocyte-specific PMCA1 deletion (PMCA1

Vitamin D is important for gonadal function in rodents, and improvement of vitamin D status in men with low sperm counts increases live birth rate. Vitamin D is a regulator of transcellular calcium transport in the intestine and kidney and may influence the dramatic changes in the luminal calcium concentration in epididymis. Here, we show spatial expression in the male reproductive tract of vitamin D receptor (VDR)-regulated factors involved in calcium transport: transient receptor potential vanilloid 5/6 , sodium/calcium exchanger 1, plasma membrane calcium ATPase 1, calbindin D9k, calcium-sensing receptor (CaSR), and parathyroid hormone-related peptide (PTHrP) in mouse and human testis and epididymis. Testicular Casr expression was lower in Vdr ablated mice compared with controls. Moreover, expression levels of Casr and Pthrp were strongly correlated in both testis and epididymis and Pthrp was suppressed by 1,25(OH)2D3 in a spermatogonial cell line. The expression of CaSR in epididymis may be of greater importance than in the gonad in mice as germ cell-specific Casr deficient mice had no major reproductive phenotype, and coincubation with a CaSR-agonist had no effect on human sperm–oocyte binding. In humans, seminal calcium concentration between 5 and 10 mM was associated with a higher fraction of motile and morphologically normal sperm cells, and the seminal calcium concentration was not associated with serum calcium levels. In conclusion, VDR regulates CaSR and PTHrP, and both factors may be involved in the regulation of calcium transport in the male reproductive tract with possible implications for sperm function and storage.

This study examined an association of ATP2B1 gene polymorphism and hypertension in the Saudi population. The 246 hypertensive cases and 300 healthy human controls were genotyped. The results showed that genotypes rs.207075 (CA + AA) [p = 0.05; OR: 95% CI, 1.5:(1.0 to 2.4) and p = 0.001, OR: 95% CI, 2.4: (1.5 to 4.0) and rs2681472 (CT + TT) [p = 0.05; OR: 95% CI, 1.5 (1.0 to 2.4) and p = 0.006 OR: 95% CI, 2.0 (1.2 to 3.1) respectively] associated with the risk of hypertension. Cases carrying the recessive models: [(CA + AA)/(CT + TT)] and [(AA)/(TT)] genotypes confer a strong susceptibility risk of hypertension [p = 0.002; OR: (95%CI) 1.8 (1.2 to 2.6) and p = 0.001; OR: (95%CI) 2.6 (1.5 to 4.7) respectively]. However, cases with body-mass-index (BMI)

The accumulation of Ca2+ and its subsequent increase in oxidative stress is proposed to be involved in selective dysfunctionality of dopaminergic neurons, the main cell type affected in Parkinson's disease. To test the in vivo impact of Ca2+ increment in dopaminergic neurons physiology, we downregulated the plasma membrane Ca2+ ATPase (PMCA), a pump that extrudes cytosolic Ca2+ , by expressing PMCARNAi in Drosophila melanogaster dopaminergic neurons. In these animals, we observed major locomotor alterations paralleled to higher cytosolic Ca2+ and increased levels of oxidative stress in mitochondria. Interestingly, although no overt degeneration of dopaminergic neurons was observed, evidences of neuronal dysfunctionality were detected such as increases in presynaptic vesicles in dopaminergic neurons and in the levels of dopamine in the brain, as well as presence of toxic effects when PMCA was downregulated in the eye. Moreover, reduced PMCA levels were found in a Drosophila model of Parkinson's disease, Parkin knock-out, expanding the functional relevance of PMCA reduction to other Parkinson's disease-related models. In all, we have generated a new model to study motor abnormalities caused by increments in Ca2+ that lead to augmented oxidative stress in a dopaminergic environment, added to a rise in synaptic vesicles and dopamine levels.

Background Developmental dysplasia of the hip (DDH) is a complex hip joint deformity with effects ranging from acetabulum malformation to irreversible hip dislocation. Previous studies suggest a significant association of four variations, teneurin transmembrane protein 3 (TENM3, OMIM * 610083) (chr4:183721398), heparan sulfate proteoglycan 2 (HSPG2, OMIM * 142461) (chr1:22201470), ATPase plasma membrane Ca2+ transporting 4 (ATP2B4, OMIM * 108732) (chr1:203682345), and prostaglandin F receptor (PTGFR, OMIM * 600563) (chr1:79002214), with DDH susceptibility in families with segregating DDH. However, the association was not validated in sporadic cases and remains controversial. To confirm the association of the reported variations in these four genes with DDH, we conducted replicative verification in 250 sporadic samples with DDH from a Chinese Han population. Methods We conducted Sanger sequencing after amplifying the variation sites. The results were compared with the reference sequence from the GRCh37 assembly in UCSC (http://genome.ucsc.edu). Results Replication analysis of 250 sporadic samples by Sanger sequencing indicated that the four variations, TENM3 (OMIM * 610083, chr4:183721398), HSPG2 (OMIM * 142461, chr1:22201470), ATP2B4 (OMIM * 108732, chr1:203682345), and PTGFR (OMIM * 600563, chr1:79002214), were not associated with the susceptibility to DDH in the Chinese Han population. Conclusions Further studies should be performed to identify other variations of these four genes that are potentially associated with DDH by whole-exome sequencing and the results should be verified in different populations.

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Background Rapid emergence of Plasmodium resistance to anti-malarial drug mainstays has driven a continual effort to discover novel drugs that target different biochemical pathway (s) during infection. Plasma membrane Calcium + 2 ATPase (PMCA4), a novel plasma membrane protein that regulates Calcium levels in various cells, namely red blood cell (RBC), endothelial cell and platelets, represents a new biochemical pathway that may interfere with susceptibility to malaria and/or severe malaria. Methods This study identified several pharmacological inhibitors of PMCA4, namely ATA and Resveratrol, and tested for their anti-malarial activities in vitro and in vivo using the Plasmodium falciparum 3D7 strain, the Plasmodium berghei ANKA strain, and Plasmodium yoelii 17XL strain as model. Results In vitro propagation of P. falciparum 3D7 strain in the presence of a wide concentration range of the inhibitors revealed that the parasite growth was inhibited in a dose-dependent manner, with IC50s at 634 and 0.231 µM, respectively. Results The results confirmed that both compounds exhibit moderate to potent anti-malarial activities with the strongest parasite growth inhibition shown by resveratrol at 0.231 µM. In vivo models using P. berghei ANKA for experimental cerebral malaria and P. yoelii 17XL for the effect on parasite growth, showed that the highest dose of ATA, 30 mg/kg BW, increased survival of the mice. Likewise, resveratrol inhibited the parasite growth following 4 days intraperitoneal injection at the dose of 100 mg/kg BW. Conclusion The findings indicate that the PMCA4 of the human host may be a potential target for novel anti-malarials, either as single drug or in combination with the currently available effective anti-malarials.

Hearing deficits impact on the communication with the external world and severely compromise perception of the surrounding. Deafness can be caused by particular mutations in the neuroplastin (Nptn) gene, which encodes a transmembrane recognition molecule of the immunoglobulin (Ig) superfamily and plasma membrane Calcium ATPase (PMCA) accessory subunit. This study investigates whether the complete absence of neuroplastin or the loss of neuroplastin in the adult after normal development lead to hearing impairment in mice analyzed by behavioral, electrophysiological, and in vivo imaging measurements. Auditory brainstem recordings from adult neuroplastin-deficient mice (Nptn−/−) show that these mice are deaf. With age, hair cells and spiral ganglion cells degenerate in Nptn−/− mice. Adult Nptn−/− mice fail to behaviorally respond to white noise and show reduced baseline blood flow in the auditory cortex (AC) as revealed by single-photon emission computed tomography (SPECT). In adult Nptn−/− mice, tone-evoked cortical activity was not detectable within the primary auditory field (A1) of the AC, although we observed non-persistent tone-like evoked activities in electrophysiological recordings of some young Nptn−/− mice. Conditional ablation of neuroplastin in Nptnlox/loxEmx1Cre mice reveals that behavioral responses to simple tones or white noise do not require neuroplastin expression by central glutamatergic neurons. Loss of neuroplastin from hair cells in adult NptnΔlox/loxPrCreERT mice after normal development is correlated with increased hearing thresholds and only high prepulse intensities result in effective prepulse inhibition (PPI) of the startle response. Furthermore, we show that neuroplastin is required for the expression of PMCA 2 in outer hair cells. This suggests that altered Ca2+ homeostasis underlies the observed hearing impairments and leads to hair cell degeneration. Our results underline the importance of neuroplastin for the development and the maintenance of the auditory system.

The study was designed to assess the effects of hypertension (HT) susceptibility genes polymorphisms in the development of preeclampsia (PE) in Caucasians from Central Russia.PE patients (n = 452) and women control group (n = 498) were genotyped for 10 polymorphisms of HT/blood pressure (BP) susceptibility genes (according to the previously published GWAS in Caucasian populations) including AC026703.1 (rs1173771), HFE (rs1799945), BAG6 (rs805303), PLCE1 (rs932764), OBFC1 (rs4387287), ARHGAP42 (rs633185), CERS5 (rs7302981), ATP2B1 (rs2681472), TBX2 (rs8068318) and RGL3 (rs167479). A logistic regression method was applied to search for associations between SNPs and PE. The relationship between SNP-SNP interactions and PE risk was analyzed by performing MB-MDR.The rs1799945 gene in HFE significantly independently increased the risk of developing PE (OR = 2.24) and rs805303 in BAG6 was associated with a reduced risk in the occurrence of PE (OR = 0.55-0.78). Among the 10 SNPs examined, nine SNPs were associated with PEs within the 10 most significant SNP-SNP interaction models. Loci rs7302981 CERS5, rs805303 BAG6 and rs932764 PLCE1 contributed to the largest number of epistatic models (50% or more).The present study is the first to report an association between polymorphisms of HT/BP susceptibility genes important for GWAS and the risk of PE in Caucasians from Central Russia. Our pathway-based functional annotation of the PE risk variants highlights the potential regulatory function (epigenetic/eQTL/sQTL/non-synonymous) that nine genetic risk markers and their 115 highly correlated variants exert on 155 genes. The study shows that these genes may function cooperatively in key signaling pathways in PE biology.

Calcium concentration must be finely tuned in all eukaryotic cells to ensure the correct performance of its signalling function. Neuronal activity is exquisitely dependent on the control of Ca

OA Förderung 2022 PMCA4 is a critical regulator of Ca2+ homeostasis in mammalian cells. While its biological and prognostic relevance in several cancer types has already been demonstrated, only preclinical investigations suggested a metastasis suppressor function in melanoma. Therefore, we studied the expression pattern of PMCA4 in human skin, nevus, as well as in primary and metastatic melanoma using immunohistochemistry. Furthermore, we analyzed the prognostic power of PMCA4 mRNA levels in cutaneous melanoma both at the non-metastatic stage as well as after PD-1 blockade in advanced disease. PMCA4 localizes to the plasma membrane in a differentiation dependent manner in human skin and mucosa, while nevus cells showed no plasma membrane staining. In contrast, primary cutaneous, choroidal and conjunctival melanoma cells showed specific plasma membrane localization of PMCA4 with a wide range of intensities. Analyzing the TCGA cohort, PMCA4 mRNA levels showed a gender specific prognostic impact in stage I–III melanoma. Female patients with high transcript levels had a significantly longer progression-free survival. Melanoma cell specific PMCA4 protein expression is associated with anaplasticity in melanoma lung metastasis but had no impact on survival after lung metastasectomy. Importantly, high PMCA4 transcript levels derived from RNA-seq of cutaneous melanoma are associated with significantly longer overall survival after PD-1 blockade. In summary, we demonstrated that human melanoma cells express PMCA4 and PMCA4 transcript levels carry prognostic information in a gender specific manner.

Genome-wide association studies for severe malaria (SM) have identified 30 genetic variants mostly located in non-coding regions. Here, we aimed to identify potential causal genetic variants located in these loci and demonstrate their functional activity. We systematically investigated the regulatory effect of the SNPs in linkage disequilibrium (LD) with the malaria-associated genetic variants. Annotating and prioritizing genetic variants led to the identification of a regulatory region containing five

We aimed to explore the associations of the ATP2B1 gene polymorphisms with eclampsia.A total of 150 patients with eclampsia (disease group) and 150 healthy pregnant women (control group) were taken as the subjects of study. The peripheral blood of the two groups of subjects was collected to extract deoxyribonucleic acids (DNAs), and the ATP2B1 gene rs71454161, rs73196661 and rs73196675 polymorphisms were detected by sequencing the Polymerase Chain Reaction (PCR) products, and then, analyzed combined with gene expression determined via Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) and clinical indicators, such as 24-h urine protein, platelets, and LDH.A difference was observed in the allele distribution of ATP2B1 gene rs71454161 (p=0.000) and rs73196661 (p=0.011) between the disease group and control group. Disease group exhibited higher frequencies of allele G of rs71454161 and allele T of rs73196661 than control group. Besides, there was a difference in the genotype distribution of ATP2B1 gene rs71454161 (p=0.000), rs73196661 (p=0.000) and rs73196675 (p=0.000) between disease group and control group. Disease group exhibited higher frequencies of genotype GG of rs71454161, genotype TT of rs73196661 and genotype CG of rs73196675 than control group. Moreover, a difference in the distributions of ATP2B1 gene rs71454161 (p=0.000) and rs73196661 (p=0.014) was found between the two groups in the dominant model. Disease group exhibited lower frequencies of AA+AG of rs71454161 and CC+CT of rs73196661 than control group in the dominant model. Differences in the distributions of haplotypes ACC (p=0.000), ATC (p=0.047) and GTC (p=0.000) of ATP2B1 gene rs71454161, rs73196661 and rs73196675 were observed between disease group and control group. Furthermore, a high degree of linkage disequilibrium was detected between rs71454161 and rs73196661 (D'=0.329). The ATP2B1 gene rs73196675 polymorphism was evidently correlated with the gene expression of ATP2B1 (p0.05), and the patients with genotype GG had a lower expression level of ATP2B1. The ATP2B1 gene rs71454161 was evidently correlated with the 24-h urinary protein in eclampsia patients (p=0.021), and the patients with genotype AG had a higher level of 24-h urinary proteins. The rs73196661 polymorphism was significantly correlated with LDH (p=0.000), and the patients with genotype CC had a higher level of LDH.The ATP2B1 gene polymorphism was significantly correlated with the occurrence and progression of eclampsia.

The genetic mechanisms regulating intracranial aneurysm (IA) formation and rupture are largely unknown. To identify germline-genetic risk factors for IA, we perform a multinational genome-wide association study (GWAS) of individuals from the United Kingdom, Finland, and Japan.To identify a shared, multinational genetic basis of IA.Using GWAS summary statistics from UK Biobank, FinnGen, and Biobank Japan, we perform a meta-analysis of IA, containing ruptured and unruptured IA cases. Logistic regression was used to identify IA-associated single-nucleotide polymorphisms. Effect size was calculated using the coefficient r , estimating the contribution of the single-nucleotide polymorphism to the genetic variance of the trait. Genome-wide significance was set at 5.0 × 10 -8 . Expression quantitative trait loci mapping and functional genomics approaches were used to infer mechanistic consequences of implicated variants.Our cohort contained 155 154 individuals (3132 IA cases and 152 022 controls). We identified 4 genetic loci reaching genome-wide: rs73392700 ( SIRT3 , effect size = 0.28, P = 4.3 × 10 -12 ), rs58721068 ( EDNRA , effect size = -0.20, P = 4.8 × 10 -12 ), rs4977574 ( AL359922.1 , effect size = 0.18, P = 7.9 × 10 -12 ), and rs11105337 ( ATP2B1 , effect size = -0.15, P = 3.4 × 10 -8 ). Expression quantitative trait loci mapping suggests that rs73392700 has a large effect size on SIRT3 gene expression in arterial and muscle, but not neurological, tissues. Functional genomics analysis suggests that rs73392700 causes decreased SIRT3 gene expression.We perform a multinational GWAS of IA and identify 4 genetic risk loci, including 2 novel IA risk loci ( SIRT3 and AL359922.1 ). Identification of high-risk genetic loci across ancestries will enable population-genetic screening approaches to identify patients with IA.

Myocardial infarction (MI) is an acute coronary syndrome that refers to tissue infarction of the myocardium. This study aimed to investigate the effect of long intergenic non‐protein‐coding RNA (lincRNA) ATPase plasma membrane Ca2+ transporting 1 antisense RNA 1 (ATP2B1‐AS1) against MI by targeting nuclear factor‐kappa‐B inhibitor alpha (NFKBIA) and mediating the nuclear factor‐kappa‐B (NF‐κB) signalling pathway. An MI mouse model was established and idenepsied by cardiac function evaluation. It was determined that ATP2B1‐AS1 was highly expressed, while NFKBIA was poorly expressed and NF‐κB signalling pathway was activated in MI mice. Cardiomyocytes were extracted from mice and introduced with a series of mouse ATP2B1‐AS1 vector, NFKBIA vector, siRNA‐mouse ATP2B1‐AS1 and siRNA‐NFKBIA. The expression of NF‐κBp50, NF‐κBp65 and IKKβ was determined to idenepsy whether ATP2B1‐AS1 and NFKBIA affect the NF‐κB signalling pathway, the results of which suggested that ATP2B1‐AS1 down‐regulated the expression of NFKBIA and activated the NF‐κB signalling pathway in MI mice. Based on the data from assessment of cell viability, cell cycle, apoptosis and levels of inflammatory cytokines, either silencing of mouse ATP2B1‐AS1 or overexpression of NFKBIA was suggested to result in reduced cardiomyocyte apoptosis and expression of inflammatory cytokines, as well as enhanced cardiomyocyte viability. Our study provided evidence that mouse ATP2B1‐AS1 silencing may have the potency to protect against MI in mice through inhibiting cardiomyocyte apoptosis and inflammation, highlighting a great promise as a novel therapeutic target for MI.

Intestinal calcium absorption from the diet plays important role in maintaining calcium homeostasis in the body. Estrogen exerts wide physiological and pathological effects in the human. Previous studies have shown that estrogen is involved in the intestinal calcium absorption. In this study, we made investigation on the mechanism of estrogen action on duodenal calcium absorption. The experiments were performed in mice, human, and human duodenal epithelial cells, SCBN cells. Murine duodenal calcium absorption was measured by using single pass perfusion of the duodenum in vivo. The calcium absorption of SCBN cells was evaluated by calcium imaging system. The expression of calcium transport proteins, transient receptor potential cation channel (TRPV6) and plasma membrane calcium pump (PMCA1b), in the duodenum or SCBN cells were analyzed by western blot. The duodenal calcium absorption in ovariectomized mice was significantly decreased, compared with control female mice, which returned to control level after 17β-estradiol replacement treatment. Estrogen regulated the expressions of TRPV6 and PMCA1b in murine and human duodenal mucosae and SCBN cells. The further results from SCBN cells showed that 17β-estradiol regulated calcium influx through the respective effects of estrogen receptor (ER) ɑ and β on TRPV6 and PMCA1b. Estrogen regulates duodenal calcium absorption through differential role of ERɑ and ERβ on duodenal epithelial cellular TRPV6 and PMCA1b. The study further elucidates the mechanism of estrogen on the regulation of intestinal calcium absorption.

Unlike other species, prion disease has never been described in dogs even though they were similarly exposed to the bovine spongiform encephalopathy (BSE) agent. This resistance prompted a thorough analysis of the canine PRNP gene and the presence of a negatively charged amino acid residue in position 163 was readily identified as potentially fundamental as it differed from all known susceptible species. In the present study, the first transgenic mouse model expressing dog prion protein (PrP) was generated and challenged intracerebrally with a panel of prion isolates, none of which could infect them. The brains of these mice were subjected to in vitro prion amplification and failed to find even minimal amounts of misfolded prions providing definitive experimental evidence that dogs are resistant to prion disease. Subsequently, a second transgenic model was generated in which aspartic acid in position 163 was substituted for asparagine (the most common in prion susceptible species) resulting in susceptibility to BSE‐derived isolates. These findings strongly support the hypothesis that the amino acid residue at position 163 of canine cellular prion protein (PrPC) is a major determinant of the exceptional resistance of the canidae family to prion infection and establish this as a promising therapeutic target for prion diseases., MINECO/FEDER. Grant Numbers: AGL2015‐65046‐C2‐1‐R, AGL2008‐05296‐C02 Interreg. Grant Number: POCTEFA EFA148/16

Severe falciparum malaria is a major cause of preventable child mortality in sub-Saharan Africa. The sequestration of parasitized erythrocytes in the microvasculature of vital organs is a central pathophysiological feature. The plasma concentration of the parasite protein P. falciparum Histidine-Rich Protein 2 (PfHRP2) has diagnostic and prognostic value in severe malaria. In the current study we investigate the potential use of plasma PfHRP2 and the sequestration index (the ratio of plasma PfHRP2 to circulating parasites) as quantitative traits in the conduct of case-only genetic association studies of severe malaria. We demonstrate the utility of this approach using data from over 2,000 Kenyan children with severe malaria, genotyped for 14 major candidate genes that were found to be associated with protection against severe malaria in previous studies. We show that PfHRP2 is a more informative quantitative trait than peripheral parasite density, and that polymorphisms in four major red cell genes (the βS sickle mutation in HBB, the blood group mutation O in ABO, the α-thalassaemia mutation in HBA, and the Dantu blood group mutation in GYP) are associated with substantially lower concentrations of plasma PfHRP2 at admission. Further, the effect sizes we observed were considerably larger than those relating to peripheral parasite density. An unexpected outlier was the rs1541255 A>G polymorphism in ATP2B4 for which we saw higher plasma PfHRP2 concentrations, lower parasite densities and a higher sequestration index. We provide testable hypotheses for how this might be explained in the context of this specific protective allele.

Mammalian hearing involves the mechanoelectrical transduction (MET) of sound-induced fluid waves in the cochlea. Essential to this process are the specialised sensory cochlear cells, the inner (IHCs) and outer hair cells (OHCs). While genetic hearing loss is highly heterogeneous, understanding the requirement of each gene will lead to a better understanding of the molecular basis of hearing and also to therapeutic opportunities for deafness. The Neuroplastin (Nptn) gene, which encodes two protein isoforms Np55 and Np65, is required for hearing, and homozygous loss-of-function mutations that affect both isoforms lead to profound deafness in mice. Here we have utilised several distinct mouse models to elaborate upon the spatial, temporal, and functional requirement of Nptn for hearing. While we demonstrate that both Np55 and Np65 are present in cochlear cells, characterisation of a Np65-specific mouse knockout shows normal hearing thresholds indicating that Np65 is functionally redundant for hearing. In contrast, we find that Nptn-knockout mice have significantly reduced maximal MET currents and MET channel open probabilities in mature OHCs, with both OHCs and IHCs also failing to develop fully mature basolateral currents. Furthermore, comparing the hearing thresholds and IHC synapse structure of Nptn-knockout mice with those of mice that lack Nptn only in IHCs and OHCs shows that the majority of the auditory deficit is explained by hair cell dysfunction, with abnormal afferent synapses contributing only a small proportion of the hearing loss. Finally, we show that continued expression of Neuroplastin in OHCs of adult mice is required for membrane localisation of Plasma Membrane Ca2+ ATPase 2 (PMCA2), which is essential for hearing function. Moreover, Nptn haploinsufficiency phenocopies Atp2b2 (encodes PMCA2) mutations, with heterozygous Nptn-knockout mice exhibiting hearing loss through genetic interaction with the Cdh23ahl allele. Together, our findings provide further insight to the functional requirement of Neuroplastin for mammalian hearing.

Background Elucidating the dynamic topological changes across different stages of breast cancer, called stage re-wiring, could lead to identifying key latent regulatory signatures involved in cancer progression. Such dynamic regulators and their functions are mostly unknown. Here, we reconstructed differential co-expression networks for four stages of breast cancer to assess the dynamic patterns of cancer progression. A new computational approach was applied to identify stage-specific subnetworks for each stage. Next, prognostic traits of genes and the efficiency of stage-related groups were evaluated and validated, using the Log-Rank test, SVM classifier, and sample clustering. Furthermore, by conducting the stepwise VIF-feature selection method, a Cox-PH model was developed to predict patients’ risk. Finally, the re-wiring network for prognostic signatures was reconstructed and assessed across stages to detect gain/loss, positive/negative interactions as well as rewired-hub nodes contributing to dynamic cancer progression. Results After having implemented our new approach, we could identify four stage-specific core biological pathways. We could also detect an essential non-coding RNA, AC025034.1, which is not the only antisense to ATP2B1 (cell proliferation regulator), but also revealed a statistically significant stage-descending pattern; Moreover, AC025034.1 revealed both a dynamic topological pattern across stages and prognostic trait. We also identified a high-performance Overall-Survival-Risk model, including 12 re-wired genes to predict patients’ risk (c-index = 0.89). Finally, breast cancer-specific prognostic biomarkers of LINC01612, AC092142.1, and AC008969.1 were identified. Conclusions In summary new scoring method highlighted stage-specific core pathways for early-to-late progressions. Moreover, detecting the significant re-wired hub nodes indicated stage-associated traits, which reflects the importance of such regulators from different perspectives.

In this review, I summarize the present knowledge of the structural and functional properties of the mammalian plasma membrane calcium pump (PMCA). It is outlined how the cellular expression of the different spliced isoforms of the four genes are regulated under normal and pathological conditions.

Calcium (Ca2+) is a universal second messenger involved in synaptogenesis and cell survival; consequently, its regulation is important for neurons. ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) belongs to the family of ATP-driven calmodulin-dependent Ca2+ pumps that participate in the regulation of intracellular free Ca2+. Here, we clinically describe a cohort of 12 unrelated individuals with variants in ATP2B1 and an overlapping phenotype of mild to moderate global development delay. Additional common symptoms include autism, seizures, and distal limb abnormalities. Nine probands harbor missense variants, seven of which were in specific functional domains, and three individuals have nonsense variants. 3D structural protein modeling suggested that the variants have a destabilizing effect on the protein. We performed Ca2+ imaging after introducing all nine missense variants in transfected HEK293 cells and showed that all variants lead to a significant decrease in Ca2+ export capacity compared with the wild-type construct, thus proving their pathogenicity. Furthermore, we observed for the same variant set an incorrect intracellular localization of ATP2B1. The genetic findings and the overlapping phenotype of the probands as well as the functional analyses imply that de novo variants in ATP2B1 lead to a monogenic form of neurodevelopmental disorder.

Mounting evidence shows that long non-coding RNAs (lncRNAs) are important to modulate the biological process of diabetic retinopathy (DR). We aimed to investigate the role of lncRNAs in DR and elucidate the exact mechanism.Real-time quantitative polymerase chain reaction was carried out to distinguish the lncRNA ATPase plasma membrane CaThe present results showed that ATP2B1-AS1 was downregulated in DR patients and high-glucose-induced HRECs. In gain- and loss-of-function assays, ATP2B1-AS1 overexpression could significantly reduce cell proliferation, migration, angiogenesis and permeability induced by high glucose in vitro. Additionally, we carried out dual-luciferase reporter experiments to determine that ATP2B1-AS1 could act as a miR-4729 sponge. ATP2B1-AS1 overexpression could rescue miR-4729 mimics and short hairpin RNA-IQGAP2 induced cell proliferation, migration and angiogenesis in HRECs.The present study showed that ATP2B1-AS1 acted as a miR-4729 sponge to regulate IQGAP2 reducing high-glucose-induced endothelial dysfunction in DR.

Placental function requires organized growth, transmission of nutrients, and an anti-inflammatory milieu between the maternal and fetal interface, but placental factors important for its function remain unclear. Renalase is a pro-survival, anti-inflammatory flavoprotein found to be critical in other tissues. We examined the potential role of renalase in placental development. PCR, bulk RNA sequencing, immunohistochemistry, and immunofluorescence for renalase and its binding partners, PMCA4b and PZP, were performed on human placental tissue from second-trimester and full-term placentas separated into decidua, placental villi and chorionic plates. Quantification of immunohistochemistry was used to localize renalase across time course from 17 weeks to term. Endogenous production of renalase was examined in placental tissue and organoids. Renalase and its receptor PMCA4b transcripts and proteins were present in all layers of the placenta. Estimated RNLS protein levels did not change with gestation in the decidual samples. However, placental villi contained more renalase immunoreactive cells in fetal than full-term placental samples. RNLS co-labeled with markers for Hofbauer cells and trophoblasts within the placental villi. Endogenous production of RNLS, PMCA4b, and PZP by trophoblasts was validated in placental organoids. Renalase is endogenously expressed throughout placental tissue and specifically within Hofbauer cells and trophoblasts, suggesting a potential role for renalase in placental development and function. Future studies should assess renalase’s role in normal and diseased human placenta.

BackgroundClear cell renal cell carcinoma (ccRCC) is one of the most lethal urologic cancer. Associations of both visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) with ccRCC have been reported, and underlying mechanisms of VAT perhaps distinguished from SAT, considering their different structures and functions. We performed this study to disclose different miRNA-mRNA networks of obesity-related ccRCC in VAT and SAT using datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA); and find out different RNAs correlated with the prognosis of ccRCC in VAT and SAT.MethodsWe screened out different expressed (DE) mRNAs and miRNAs of obesity, in both VAT and SAT from GEO datasets, and constructed miRNA-mRNA networks of obesity-related ccRCC. To evaluate the sensitivity and specificity of RNAs in networks of obesity-related ccRCC in both VAT and SAT, Receiver Operating Characteristic (ROC) analyses were conducted using TCGA datasets. Spearman correlation analyses were then performed to find out RNA pairs with inverse correlations. We also performed Cox regression analyses to estimate the association of all DE RNAs of obesity with the overall survival.Results136 and 185 DE mRNAs of obesity in VAT and SAT were found out. Combined with selected DE miRNAs, miRNA-mRNA networks of obesity-related ccRCC were constructed. By performing ROC analyses, RNAs with same trend as shown in networks and statistically significant ORs were selected to be paired. Three pairs were finally remained in Spearman correlation analyses, including hsa-miR-182&ATP2B2, hsa-miR-532&CDH2 in VAT, and hsa-miR-425&TFAP2B in SAT. Multivariable Cox regression analyses showed that several RNAs with statistically significant adjusted HRs remained consistent trends as shown in DE analyses of obesity. Risk score analyses using selected RNAs showed that the overall survival time of patients in the low‐risk group was significantly longer than that in the high‐risk group regardless of risk score models.ConclusionsWe found out different miRNA-mRNA regulatory networks of obesity-related ccRCC for both VAT and SAT; and several DE RNAs of obesity-related ccRCC were found to remain consistent performance in terms of ccRCC prognosis. Our findings could provide valuable evidence on the targeted therapy of obesity-related ccRCC.

Cardiac glycosides, such as digoxin and digitoxin, are compounds that interact with Na(+)/K(+)-ATPase to induce anti-neoplastic effects; however, these cardiac glycosides have narrow therapeutic index. Thus, semi-synthetic analogs of digitoxin with modifications in the sugar moiety has been shown to be an interesting approach to obtain more selective and more effective analogs than the parent natural product. Therefore, the aim of this study was to assess the cytotoxic potential of novel digitoxigenin derivatives, digitoxigenin-α-L-rhamno-pyranoside (1) and digitoxigenin-α-L-amiceto-pyranoside (2), in cervical carcinoma cells (HeLa) and human diploid lung fibroblasts (Wi-26-VA4). In addition, we studied the anticancer mechanisms of action of these compounds by comparing its cytotoxic effects with the potential to modulate the activity of three P-type ATPases; Na(+)/K(+)-ATPase, sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), and plasma membrane Ca(2+)-ATPase (PMCA). Briefly, the results showed that compounds 1 and 2 were more cytotoxic and selectivity for HeLa tumor cells than the non-tumor cells Wi-26-VA4. While the anticancer cytotoxicity in HeLa cells involves the modulation of Na(+)/K(+)-ATPase, PMCA and SERCA, the modulation of these P-type ATPases was completely absent in Wi-26-VA4 cells, which suggest the importance of their role in the cytotoxic effect of compounds 1 and 2 in HeLa cells. Furthermore, the compound 2 inhibited directly erythrocyte ghosts PMCA and both compounds were more cytotoxic than digitoxin in HeLa cells. These results provide a better understanding of the mode of action of the synthetic cardiac glycosides and highlights 1 and 2 as potential anticancer agents.

While many neuronal, membrane proteins undergo proteolytic cleavage, little is known about the biological significance of neuronal ectodomain shedding (ES). Here, we show that the neuronal sheddome is detectable in human cerebrospinal fluid (hCSF) and is enriched in neurodevelopmental disorder (NDD) risk factors. Among shed synaptic proteins was the ectodomain of CNTNAP2 (CNTNAP2-ecto), a prominent NDD risk factor. CNTNAP2 undergoes activity-dependent ES via MMP9 (matrix metalloprotease 9), and CNTNAP2-ecto levels are reduced in the hCSF of ASD individuals. Using mass spectrometry, we identified the PMCA Ca(2+) extrusion pumps as novel CNTNAP2-ecto binding partners. CNTNAP2-ecto enhances the activity of PMCA2 and regulates neuronal network dynamics in a PMCA2-dependent manner. Our data underscore the promise of sheddome analysis in discovering neurobiological mechanisms, provide insight into the biology of ES and its relationship with the CSF, and reveal a mechanism of regulation of Ca(2+) homeostasis and neuronal network synchrony by a shed ectodomain.

Intracellular Ca2+ signaling engendered by Ca2+ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca2+ is exported by the Na+–Ca2+ exchanger (NCX) and plasma membrane Ca2+–ATPase (PMCA) to maintain Ca2+ homeostasis. We previously demonstrated a functional coupling between Ca2+ extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca2+ concentration ([Ca2+]i). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca2+]i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1–4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca2+]i in odontoblasts in both rat and human odontoblasts. The Ca2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca2+]i increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4–8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca2+]i homeostasis in odontoblasts by Ca2+ extrusion following [Ca2+]i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca2+ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.

Background Recent genome wide analysis studies have identified a strong association between single nucleotide variations within the human ATP2B4 gene and susceptibility to severe malaria. The ATP2B4 gene encodes the plasma membrane calcium ATPase 4 (PMCA4), which is responsible for controlling the physiological level of intracellular calcium in many cell types, including red blood cells (RBCs). It is, therefore, postulated that genetic differences in the activity or expression level of PMCA4 alters intracellular Ca2+ levels and affects RBC hydration, modulating the invasion and growth of the Plasmodium parasite within its target host cell. Methods In this study the course of three different Plasmodium spp. infections were examined in mice with systemic knockout of Pmca4 expression. Results Ablation of PMCA4 reduced the size of RBCs and their haemoglobin content but did not affect RBC maturation and reticulocyte count. Surprisingly, knockout of PMCA4 did not significantly alter peripheral parasite burdens or the dynamics of blood stage Plasmodium chabaudi infection or reticulocyte-restricted Plasmodium yoelii infection. Interestingly, although ablation of PMCA4 did not affect peripheral parasite levels during Plasmodium berghei infection, it did promote slight protection against experimental cerebral malaria, associated with a minor reduction in antigen-experienced T cell accumulation in the brain. Conclusions The finding suggests that PMCA4 may play a minor role in the development of severe malarial complications, but that this appears independent of direct effects on parasite invasion, growth or survival within RBCs.

Significance Synaptic vesicle (SV) release from presynaptic terminals requires nanometer precise control of action potential (AP)–triggered calcium influx through voltage-gated calcium channels (VGCCs). SV recycling also depends on calcium signals, though in different spatiotemporal domains. Mechanisms for separate control of SV release and recycling by AP-triggered calcium influx remain elusive. Here, we demonstrate largely independent regulation of release and recycling by two different populations of VGCCs (Cav2, Cav1), identify Cav1 as one of potentially multiple calcium entry routes for endocytosis regulation, and show functional separation of simultaneous calcium signals in the nanometer space of a presynaptic terminal by the plasma membrane calcium ATPase (PMCA). The Cav2/Cav1/PMCA functional triad may provide conserved means for independent control of different vital presynaptic functions., Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca2+ signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)–triggered presynaptic Ca2+ entry remain unclear. Combining Drosophila genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Drosophila Cav2 homolog, Dmca1A or cacophony, and the Cav1 homolog, Dmca1D) but with spatial and functional separation. Cav2 within active zones is required for AP-triggered neurotransmitter release. By contrast, Cav1 localizes predominantly around active zones and contributes substantially to AP-evoked Ca2+ influx but has a small impact on release. Instead, L-type calcium currents through Cav1 fine-tune short-term plasticity and facilitate SV recycling. Separate control of SV exo- and endocytosis by AP-triggered presynaptic Ca2+ influx through different channels demands efficient measures to protect the neurotransmitter release machinery against Cav1-mediated Ca2+ influx. We show that the plasma membrane Ca2+ ATPase (PMCA) resides in between active zones and isolates Cav2-triggered release from Cav1-mediated dynamic regulation of recycling and short-term plasticity, two processes which Cav2 may also contribute to. As L-type Cav1 channels also localize next to PQ-type Cav2 channels within axon terminals of some central mammalian synapses, we propose that Cav2, Cav1, and PMCA act as a conserved functional triad that enables separate control of SV release and recycling rates in presynaptic terminals.

Clinical phenotyping of term and preterm labor is imprecise, and disagreement persists on categorization relative to underlying pathobiology, which remains poorly understood. We performed RNA sequencing (RNA-seq) of 31 specimens of human uterine myometrium from 10 term and 21 preterm cesarean deliveries with rich clinical context information. A molecular signature of 4814 transcripts stratified myometrial samples into quiescent (Q) and nonquiescent (NQ) phenotypes, independent of gestational age and incision site. Similar stratifications were achieved using expressed genes in Ca2+ signaling and TGF-β pathways. For maximal parsimony, we evaluated the expression of just 2 Ca2+ transporter genes, ATP2B4 (encoding PMCA4) and ATP2A2 (coding for SERCA2), and we found that their ratio reliably distinguished NQ and Q specimens in the current study, and also in 2 publicly available RNA-seq data sets (GSE50599 and GSE80172), with an overall AUC of 0.94. Cross-validation of the ATP2B4/ATP2A2 ratio by quantitative PCR in an expanded cohort (by 11 additional specimens) achieved complete separation (AUC of 1.00) of NQ versus Q specimens. While providing additional insight into the associations between clinical features of term and preterm labor and myometrial gene expression, our study also offers a practical algorithm for unbiased classification of myometrial biopsies by their overall contractile program.

BackgroundNeuropathic pain is often observed in individuals with multiple sclerosis (MS) and spinal cord injury (SCI) and is not adequately alleviated by current pharmacotherapies. A better understanding of underlying mechanisms could facilitate the discovery of novel targets for therapeutic interventions. We previously reported that decreased plasma membrane calcium ATPase 2 (PMCA2) expression in the dorsal horn (DH) of healthy PMCA2+/−mice is paralleled by increased sensitivity to evoked nociceptive pain. These studies suggested that PMCA2, a calcium extrusion pump expressed in spinal cord neurons, plays a role in pain mechanisms. However, the contribution of PMCA2 to neuropathic pain processing remains undefined. The present studies investigated the role of PMCA2 in neuropathic pain processing in the DH of wild-type mice affected by experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and following SCI.MethodsEAE was induced in female and male C57Bl/6N mice via inoculation with myelin oligodendrocyte glycoprotein fragment 35–55 (MOG35–55) emulsified in Complete Freund’s Adjuvant (CFA). CFA-inoculated mice were used as controls. A severe SC contusion injury was induced at thoracic (T8) level in female C57Bl/6N mice. Pain was evaluated by the Hargreaves and von Frey filament tests. PMCA2 levels in the lumbar DH were analyzed by Western blotting. The effectors that decrease PMCA2 expression were identified in SC neuronal cultures.ResultsIncreased pain in EAE and SCI was paralleled by a significant decrease in PMCA2 levels in the DH. In contrast, PMCA2 levels remained unaltered in the DH of mice with EAE that manifested motor deficits but not increased pain. Interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), and IL-6 expression were robustly increased in the DH of mice with EAE manifesting pain, whereas these cytokines showed a modest increase or no change in mice with EAE in the absence of pain. Only IL-1β decreased PMCA2 levels in pure SC neuronal cultures through direct actions.ConclusionsPMCA2 is a contributor to neuropathic pain mechanisms in the DH. A decrease in PMCA2 in DH neurons is paralleled by increased pain sensitivity, most likely through perturbations in calcium signaling. Interleukin-1β is one of the effectors that downregulates PMCA2 by acting directly on neurons.