EphA4 Induces the Phosphorylation of an Intracellular Adaptor Protein Dab1 via Src Family Kinases.

Dab1 is an intracellular adaptor protein essential for brain formation during development. Tyrosine phosphorylation in Dab1 plays important roles in neuronal migration, dendrite development, and synapse formation by affecting several downstream pathways. Reelin is the best-known extracellular protein that induces Dab1 phosphorylation. However, whether other upstream molecule(s) contribute to Dab1 phosphorylation remains largely unknown. Here, we found that EphA4, a member of the Eph family of receptor-type tyrosine kinases, induced Dab1 phosphorylation when co-expressed in cultured cells. Tyrosine residues phosphorylated by EphA4 were the same as those phosphorylated by Reelin in neurons. The autophosphorylation of EphA4 was necessary for Dab1 phosphorylation. We also found that EphA4-induced Dab1 phosphorylation was mediated by the activation of the Src family tyrosine kinases. Interestingly, Dab1 phosphorylation was not observed when EphA4 was activated by ephrin-A5 in cultured cortical neurons, suggesting that Dab1 is localized in a different compartment in them. EphA4-induced Dab1 phosphorylation may occur under limited and/or pathological conditions in the brain.

Naturally manufactured biochar materials based sensor electrode for the electrochemical detection of polystyrene microplastics.

In recent times, microplastics have become a disturbance to both aquatic and terrestrial ecosystems and the ingestion of these particles can have severe consequences for wildlife, aquatic organisms, and even humans. In this study, two types of biochars were manufactured through the carbonization of naturally found starfish (SF-1) and aloevera (AL-1). The produced biochars were utilized as sensing electrode materials for the electrochemical detection of ∼100 nm polystyrene microplastics (PS). SF-1 and AL-1 based biochars were thoroughly analyzed in terms of morphology, structure, and composition. The detection of microplastics over biochar based electrodes was carried out by electrochemical studies. From electrochemical results, SF-1 based electrode exhibited the detection efficiency of ∼0.2562 µA/µM∙cm2 with detection limit of ∼0.44 nM whereas, a high detection efficiency of ∼3.263 µA/µM∙cm2 was shown by AL-1 based electrode and detection limit of ∼0.52 nM for PS (100 nm) microplastics. Process contributed to enhancing the sensitivity of AL-1 based electrode might associate to the presence of metal-carbon framework over biochar's surfaces. The AL-1 biochar electrode demonstrated excellent repeatability and detection stability for PS microplastics, suggesting the promising potential of AL-1 biochar for electrochemical microplastics detection.

[Melatonin reverses oxidative damage in the submandibular gland of rats treated with Cyclophosphamide]. / Melatonina revierte el daño oxidativo en glándula submandibular de ratas tratadas con Ciclofosfamida.

Objetive: Cyclophosphamide (Cf) produces oxidative damage in rat submandibular gland (GSM). In the present work we evaluated the antioxidant protective effect of melatonin (MLT) in GSM of rats treated with Cf. Methods: 40 adult male Wistar rats were divided into 5 groups (G): G1: control; G2: Control+Ethanol: treated with 1% ethanol for 10 consecutive days. On days 11 and 12 they received a dose of saline; G3: Cf: treated with 1% ethanol for 12 days, days 11 and 12 they received an intraperitoneal (i.p.) dose of Cf 50 mg/Kg/kg of saline. ) of Cf 50 mg/kg bw; G4: Cf + MLT: MLT (5 mg/kg bw, intraperitoneal, dissolved in 1% ethanol) was administered daily, days 11 and 12 received Cf same as G3; G5: MLT: treated 12 consecutive days with MLT (same dose as G4). After 12 hours of fasting, animals were anesthetized to obtain both submandibular glands, then they were sacrificed. Uric acid (UA), lipid peroxides (LPs), aqueous peroxides (APs) and superoxide dismutase (SOD) activity were measured in submandibular gland homogenate. Statistical analysis: we used ANOVA and Bonferroni test pos hoc, considering significant p<0.05. Results: Cf treatment decreased AU concentration and SOD activity (AU, mg/mg prot., G1: 2.50±0.68; G2: 2.18±0.13; G3: 0.54±0.09* G4: 1.95±0.24#, G5: 2.64±0.47, *p<0.01 G3 vs G1, G2, G4; #p<0.01 G4 vs G3 and G5; SOD, U/mg prot, G1: 4.57±0.95, G2: 4.79±0.94, G3: 2.18±0.53*, G4: 5.13±1.10, G5: 5.09±0.39, *p< 0.01 G3 vs G1, G2, G4 and G5). MLT treatment prevented these effects. In addition, Cf increased PL and PA formation. Conclusion: MLT improved the redox status in GSM of Cf-treated rats. MLT could prevent oxidative processes in GSM produced by Cf.

Objetivo: Ciclofosfamida (Cf) produce daño oxidativo en glándula submandibular (GSM) de ratas. En el presente trabajo se evaluó el efecto protector antioxidante de melatonina (MLT) en GSM de ratas tratadas con Cf. Método: Se utilizaron 40 ratas Wistar machos adultas divididas en 5 grupos (G): G1: control; G2: Control+Etanol: tratados con etanol al 1% durante 10 días consecutivos. Los días 11 y 12 recibieron una dosis de solución salina; G3: Cf: tratados con etanol al 1% durante 12 días, días 11 y 12 recibieron una dosis intraperitoneal (i.p.) de Cf de 50 mg/Kg de pc; G4: Cf + MLT: se administró diariamente MLT (5 mg/Kg pc, intraperitoneal, disuelta en etanol al 1%), días 11 y 12 recibieron Cf igual que G3; G5: MLT: tratamiento 12 días consecutivos con MLT (igual dosis de G4). Los animales fueron anestesiados, extirpándose ambas GSM y sacrificados, previo ayuno 12 hs. Se midió la concentración de ácido úrico (AU), peróxidos lipídicos (PL) y acuosos (PA) y actividad de superóxido dismutasa (SOD) en homogenato de GSM. Análisis estadístico: ANOVA y test de bonferroni, considerando significativo p<0,05. Resultados: El tratamiento con Cf disminuyó la concentración de AU y la actividad de SOD (AU, mg/mg prot., G1: 2,50±0,68; G2: 2,18±0,13; G3: 0,54±0,09* G4: 1,95±0,24#, G5: 2,64±0,47, *p< 0,01 G3 vs G1, G2, G4; #p< 0,01 G4 vs G3 y G5; SOD, U/mg prot., G1: 4,57±0.95, G2: 4,79±0,94, G3: 2,18±0,53*, G4: 5,13±1,10, G5: 5,09±0,39, *p< 0,01 G3 vs G1, G2, G4 y G5). El tratamiento con MLT previno esos efectos. Además, Cf aumentó la formación PL y PA. Conclusión: MLT mejoró el estado redox en GSM de ratas tratadas con Cf. MLT podría prevenir los procesos oxidativos en GSM producidos por Cf.

Lipidation and PEGylation Strategies to Prolong the in Vivo Half-Life of a Nanomolar EphA4 Receptor Antagonist.

The EphA4 receptor tyrosine kinase plays a role in neurodegenerative diseases, inhibition of nerve regeneration, cancer progression and other diseases. Therefore, EphA4 inhibition has potential therapeutic value. Selective EphA4 kinase inhibitors are not available, but we identified peptide antagonists that inhibit ephrin ligand binding to EphA4 with high specificity. One of these peptides is the cyclic APY-d3 (ßAPYCVYRßASWSC-NH2), which inhibits ephrin-A5 ligand binding to EphA4 with low nanomolar binding affinity and is highly protease resistant. Here we describe modifications of APY-d3 that yield two different key derivatives with greatly increased half-lives in the mouse circulation, the lipidated APY-d3-laur8 and the PEGylated APY-d3-PEG4. These two derivatives inhibit ligand induced EphA4 activation in cells with sub-micromolar potency. Since they retain high potency and specificity for EphA4, lipidated and PEGylated APY-d3 derivatives represent new tools for discriminating EphA4 activities in vivo and for preclinical testing of EphA4 inhibition in animal disease models.

Mapping the Universe of Eph Receptor and Ephrin Ligand Transcripts in Epithelial and Fiber Cells of the Eye Lens.

The eye lens is a transparent, ellipsoid organ in the anterior chamber of the eye that is required for fine focusing of light onto the retina to transmit a clear image. Cataracts, defined as any opacity in the lens, remains the leading cause of blindness in the world. Recent studies in humans and mice indicate that Eph-ephrin bidirectional signaling is important for maintaining lens transparency. Specifically, mutations and polymorphisms in the EphA2 receptor and the ephrin-A5 ligand have been linked to congenital and age-related cataracts. It is unclear what other variants of Ephs and ephrins are expressed in the lens or whether there is preferential expression in epithelial vs. fiber cells. We performed a detailed analysis of Eph receptor and ephrin ligand mRNA transcripts in whole mouse lenses, epithelial cell fractions, and fiber cell fractions using a new RNA isolation method. We compared control samples with EphA2 knockout (KO) and ephrin-A5 KO samples. Our results revealed the presence of transcripts for 12 out of 14 Eph receptors and 8 out of 8 ephrin ligands in various fractions of lens cells. Using specific primer sets, RT-PCR, and sequencing, we verified the variant of each gene that is expressed, and we found two epithelial-cell-specific genes. Surprisingly, we also identified one Eph receptor variant that is expressed in KO lens fibers but is absent from control lens fibers. We also identified one low expression ephrin variant that is only expressed in ephrin-A5 control samples. These results indicate that the lens expresses almost all Ephs and ephrins, and there may be many receptor-ligand pairs that play a role in lens homeostasis.

RNA Modification-Related Genetic Variants in Genomic Loci Associated with Bone Mineral Density and Fracture.

Genome-wide association studies (GWASs) have identified more than 500 loci for bone mineral density (BMD), but functional variants in these loci are less known. The aim of this study was to identify RNA modification-related SNPs (RNAm-SNPs) for BMD in GWAS loci. We evaluated the association of RNAm-SNPs with quantitative heel ultrasound BMD (eBMD) in 426,824 individuals, femoral neck (FN) and lumbar spine (LS) BMD in 32,961 individuals and fracture in ~1.2 million individuals. Furthermore, we performed functional enrichment, QTL and Mendelian randomization analyses to support the functionality of the identified RNAm-SNPs. We found 300 RNAm-SNPs significantly associated with BMD, including 249 m6A-, 28 m1A-, 3 m5C-, 7 m7G- and 13 A-to-I-related SNPs. m6A-SNPs in OP susceptibility genes, such as WNT4, WLS, SPTBN1, SEM1, FUBP3, LRP5 and JAG1, were identified and functional enrichment for m6A-SNPs in the eBMD GWAS dataset was detected. eQTL signals were found for nearly half of the identified RNAm-SNPs, and the affected gene expression was associated with BMD and fracture. The RNAm-SNPs were also associated with the plasma levels of proteins in cytokine-cytokine receptor interaction, PI3K-Akt signaling, NF-kappa B signaling and MAPK signaling pathways. Moreover, the plasma levels of proteins (CCL19, COL1A1, CTSB, EFNA5, IL19, INSR, KDR, LIFR, MET and PLXNB2) in these pathways were found to be associated with eBMD in Mendelian randomization analysis. This study identified functional variants and potential causal genes for BMD and fracture in GWAS loci and suggested that RNA modification may play an important role in osteoporosis.

Expression characteristic, immune signature, and prognosis value of EFNA family identified by multi-omics integrative analysis in pan-cancer.

BACKGROUND: EphrinA (EFNA) are Eph receptor ligands that regulate various disease processes. Nonetheless, the expression characteristics of EFNAs in pan-cancer, their relationship with tumor immune microenvironment, and prognostic value landscape remain unknown. METHODS: A comprehensive landscape of EFNAs was created using various statistical data extracted from 33 cancers. Subsequently, we identified differential expression, genetic variations, potential function enrichment, tumor immune-related analysis, and drug sensitivity. Further, we investigated the clinical features and diagnostic prognostic value of EFNAs. RT-qPCR, western blot and immunohistochemistry (IHC) were used to validate the expression level and significant clinical value of EFNA5 in lung adenocarcinoma cell lines and tissues. RESULTS: EFNAs were highly mutated in various cancers. Genomic and epigenetic alterations of EFNAs were observed in various tumors, where an oncogenic mutation in specific cancer types potentially affected EFNA expression. Moreover, tumor-derived EFNAs were significantly related to the tumor immune microenvironment, suggesting that they are promising therapeutic targets. The majority of EFNA family genes were significantly linked to patient prognosis. Eventually, EFNA5 was an independent prognostic factor in lung adenocarcinoma. CONCLUSION: In summary, EFNAs are crucial in tumor immune regulation, and EFNA5 is a prognostic marker in lung adenocarcinoma. Our findings provide new insights into EFNAs from a bioinformatics standpoint and highlight the significance of EFNAs in cancer diagnosis and treatment.

Andrographolide Derivative AL-1 Ameliorates LPS-induced Acute Lung Injury by Inhibiting NLRP3 Inflammasome and Lung Permeability.

BACKGROUND: Acute lung injury (ALI) is a serious respiratory disease with a high mortality rate, and there is an urgent need for a more effective treatment strategy. Andrographolide derivative AL-1 has been identified to possess anti-inflammatory activity. However, whether it could reduce LPS-induced lung injury in mice through inhibiting NLRP3 inflammasome activation and protecting lung permeability has not yet been elucidated. In the present research, we investigated the protective effect of AL-1 on ALI mice and demonstrated the potential mechanisms. METHODS: Male Balb/c mice were anesthetized with isoflurane, and ALI mice were induced by intratracheal instillation of LPS. The mice were euthanized after LPS administration for 12 h, then bronchoalveolar lavage fluid (BALF) and lung tissues were collected. The levels of inflammatory factors were measured by ELISA assay, and HE staining and lung injury scoring were used to evaluate the pathological changes in the pulmonary tissues. Immunohistochemistry and immunofluorescence examination were conducted to detect the expression levels of related proteins. Western blot was performed to measure the levels of NLRP3 inflammasome and tight junction proteins. RESULTS: The study indicated that AL-1 effectively alleviated lung injury by reduction of proinflammatory cytokine levels, MPO activity, lung W/D ratio, and total protein levels. Furthermore, AL-1 improved pathological changes in lung tissue and significantly reduced the infiltration of inflammatory cells. Administration with AL-1 markedly inhibited the expression of NLRP3, ASC, Caspase-1, IL-1ß, gasdermin D (GSDMD), and VCAM-1 but increased the expression of ZO-1, Occludin, JAM-A, and Claudin-1. CONCLUSION: Taken together, these results demonstrated that AL-1 ameliorated pulmonary damage by inhibiting the activation of the NLRP3 inflammasome pathway and restoring TJ protein expression.

ADAM10-cleaved ephrin-A5 contributes to prostate cancer metastasis.

A disintegrin and metalloprotease-10(ADAM10) promotes the metastasis of prostate cancer (PCa), but the specific mechanism is indistinct. Herein, DU145 cell lines with stable overexpression and knockdown of ADAM10 were constructed. We found that ectopic expression of ADAM10 not only significantly facilitated cell proliferation, migration, invasion, and inhibited apoptosis, but also could specifically hydrolyze ephrin-A5 and release the ephrin-A5 soluble ectodomain into extracellular media in vitro. These effects were reversed by ADAM10 depletion or treatment of GI254023X. Meanwhile, the co-location and physical interaction among EphA3, ephrin-A5, and ADAM10 were observed in PCa cells using immunofluorescence and immunoprecipitation techniques. Interestingly, overexpression of EphA3 exerted opposite effects in DU145 (ephrin-A5 + ) cells and PC-3 (ephrin-A5 ± ) cells. In addition, the pro-tumor function of EphA3 was reversed by the treatment with the exogenous ephrin-A5-Fc, which increased the phosphorylation level of EphA3 in PC-3 (ephrin-A5 ± ) cells. In nude mice, ADAM10 accelerated growth of the primary tumor, decreased the level of ephrin-A5 in the tumor tissue, but increased the level of ephrin-A5 in the peripheral blood, accompanied with an increase in the expression of CD31 and VEGF (vascular endothelial growth factor) in the tissue. What is more, the serum ephrin-A5 content of patients with metastatic PCa was significantly higher than that of the non-metastatic group (P < 0.05). The receiver operating characteristic curve(ROC) showed that the area under the curve(AUC) of serum ephrin-A5 as a marker of PCa metastasis was 0.843, with a sensitivity of 93.5% and a specificity of 75%. It is concluded that ADAM10-mediated ephrin-A5 shedding promotes PCa metastasis via transforming the role of EphA3 from ligand-dependent tumor suppressor to ligand-independent promoter, and ephrin-A5 in the blood can be used as a new biomarker for PCa metastasis.

Manipulating the Level of Sensorimotor Stimulation during LI-rTMS Can Improve Visual Circuit Reorganisation in Adult Ephrin-A2A5-/- Mice.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique that has the potential to treat a variety of neurologic and psychiatric disorders. The extent of rTMS-induced neuroplasticity may be dependent on a subject's brain state at the time of stimulation. Chronic low intensity rTMS (LI-rTMS) has previously been shown to induce beneficial structural and functional reorganisation within the abnormal visual circuits of ephrin-A2A5-/- mice in ambient lighting. Here, we administered chronic LI-rTMS in adult ephrin-A2A5-/- mice either in a dark environment or concurrently with voluntary locomotion. One day after the last stimulation session, optokinetic responses were assessed and fluorescent tracers were injected to map corticotectal and geniculocortical projections. We found that LI-rTMS in either treatment condition refined the geniculocortical map. Corticotectal projections were improved in locomotion+LI-rTMS subjects, but not in dark + LI-rTMS and sham groups. Visuomotor behaviour was not improved in any condition. Our results suggest that the beneficial reorganisation of abnormal visual circuits by rTMS can be significantly influenced by simultaneous, ambient visual input and is enhanced by concomitant physical exercise. Furthermore, the observed pathway-specific effects suggest that regional molecular changes and/or the relative proximity of terminals to the induced electric fields influence the outcomes of LI-rTMS on abnormal circuitry.

Genomic and biological characterization of the novel phages vB_VpaP_AL-1 and vB_VpaS_AL-2 infecting Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (AHPND).

Acute hepatopancreatic necrosis disease (AHPND) is a life-threatening disease to recently stocked shrimp. This disease is mainly caused by Vibrio parahaemolyticus and, to date, it has not been effectively controlled. Bacteriophages are a promising method to control bacterial diseases in aquaculture and multiple phages that infect Asian strains of V. parahaemolyticus have been described. However, few studies have characterized the bacteriophages that infect Latin American strains. Here, two lytic Vibrio phages (vB_VpaP_AL-1 and vB_VpaS_AL-2) were isolated from estuary water in Sinaloa, Mexico. The host ranges were tested using ten AHPND-causing strains isolated from Mexico and phage AL-1 was able to infect two strains while AL-2 infected four. One-step growth curve showed that AL-1 produced 85 PFU/cell and AL-2 produced 68 PFU/cell in 30 and 40 min, respectively. Both phages were able to tolerate temperatures ranging from 20 to 50 °C and pH values ranging from 4 to 10. Phages AL-1 and AL-2 have double-stranded DNA genomes of 42,854 bp and 58,457 bp, respectively. In total, 53 putative ORFs associated with the phage structure, packing, host lysis, DNA metabolism, and additional functions were predicted in the AL-1 genome, while 92 ORFs associated with the same functions as the AL-1 and 1 tRNA were predicted in the AL-2 genome. The lifecycle was classified as virulent for both phages. Morphology, phylogeny, and comparative genomic analyses assigned phage AL-1 as a new member of the genus Maculvirus in the Autographiviridae family, and phage AL-2 as a new member of the Siphoviridae family. These findings suggest that vB_VpaP_AL-1 and vB_VpaS_AL-2 are potential biocontrol agents against AHPND-causing V. parahaemolyticus from Mexico.

EphA2 Affects Development of the Eye Lens Nucleus and the Gradient of Refractive Index.

Purpose: Our studies in mouse eye lenses demonstrate that ephrin-A5 and EphA2 are needed for normal epithelial cells and lens transparency. We sought to determine whether EphA2 and ephrin-A5 are important for lens morphometrics, nucleus formation, and refractive index. Methods: We performed tissue morphometric measurements, electron microscopy, Western blots, and interferometric measurements using an X-ray synchrotron beam source to measure the gradient of refractive index (GRIN) to compare mouse lenses with genetic disruption of EphA2 or ephrin-A5. Results: Morphometric analysis revealed that although there is no change in the overall lens volume, there is a change in lens shape in both EphA2-/- lenses and ephrin-A5-/- lenses. Surprisingly, EphA2-/- lenses had small and soft lens nuclei different from hard lens nuclei of control lenses. SEM images revealed changes in cell morphology of EphA2-/- fiber cells close to the center of the lens. Inner EphA2-/- lens fibers had more pronounced tongue-and-groove interdigitations and formed globular membrane morphology only in the deepest layers of the lens nucleus. We did not observe nuclear defects in ephrin-A5-/- lenses. There was an overall decrease in magnitude of refractive index across EphA2-/- lenses, which is most pronounced in the nucleus. Conclusions: This work reveals that Eph-ephrin signaling plays a role in fiber cell maturation, nuclear compaction, and lens shape. Loss of EphA2 disrupts the nuclear compaction resulting in a small lens nucleus. Our data suggest that Eph-ephrin signaling may be required for fiber cell membrane reorganization and compaction and for establishing a normal GRIN.

Neoadjuvant therapy alters the collagen architecture of pancreatic cancer tissue via Ephrin-A5.

BACKGROUND: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT. METHODS: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs). RESULTS: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5+ cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT. CONCLUSION: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression.

Identification and Validation of a Dysregulated miRNA-Associated mRNA Network in Temporal Lobe Epilepsy.

OBJECTIVES: This study is aimed at exploring the relationships between miRNAs and mRNAs and to characterize their biological functions in temporal lobe epilepsy (TLE). METHODS: Novel clinical significant miRNAs and target genes and their potential underlying mechanisms have been discovered and explored by mining miRNAs and mRNA expression data of TLE patients using various bioinformatics methods. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to validate the bioinformatic analysis results. RESULTS: A total of 6 dysregulated miRNAs and 442 differentially expressed genes (DEGs) related to TLE were obtained from GEO database (GSE114701 and GSE127871 datasets). A protein-protein interaction (PPI) network containing the 442 DEGs was established. mRNA response elements from the 6 dysregulated miRNAs were predicted using the miRDB and TargetScan bioinformatic tools. By merging the identified targets of the dysregulated miRNAs and the 247 downregulated DEGs, a miRNA-mRNA network was constructed revealing the interaction of miR-484 with eight mRNAs (ABLIM2, CEP170B, CTD-3193O13.9, EFNA5, GAP43, PRKCB, FXYD7, and NCAN). A weighted correlation network analysis (WGCNA) based on the eight genes was established and demonstrated that these mRNAs, except FXYD7 and NCAN, were hub genes in the network. Gene Oncology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the six hub genes were mainly involved in cellular-related biological functions and the neurotransmitter synapse pathway. The differences in expression levels of the miR-484 and the three hub genes (CTD-3193O13.9, EFNA5, and PRKCB) observed experimentally in TLE patients compared to those of healthy controls were consistent with the WGCNA prediction. CONCLUSION: Our study suggests that understanding the miRNA-mRNA interactions will provide insights into the epilepsy pathogenesis. In addition, our results indicate that miR-484 may be a promising novel biomarker for TLE.

Replicating infant-specific reactive astrocyte functions in the injured adult brain.

Infants and adults respond differently to brain injuries. Specifically, improved neuronal sparing along with reduced astrogliosis and glial scarring often observed earlier in life, likely contributes to improved long-term outcomes. Understanding the underlying mechanisms could enable the recapitulation of neuroprotective effects, observed in infants, to benefit adults after brain injuries. We reveal that in primates, Eph/ ephrin signaling contributes to age-dependent reactive astrocyte behavior. Ephrin-A5 expression on astrocytes was more protracted in adults, whereas ephrin-A1 was only expressed on infant astrocytes. Furthermore, ephrin-A5 exacerbated major hallmarks of astrocyte reactivity via EphA2 and EphA4 receptors, which was subsequently alleviated by ephrin-A1. Rather than suppressing reactivity, ephrin-A1 signaling shifted astrocytes towards GAP43+ neuroprotection, accounting for improved neuronal sparing in infants. Reintroducing ephrin-A1 after middle-aged focal ischemic injury significantly attenuated glial scarring, improved neuronal sparing and preserved circuitry. Therefore, beneficial infant mechanisms can be recapitulated in adults to improve outcomes after CNS injuries.

Mutually Repulsive EphA7-EfnA5 Organize Region-to-Region Corticopontine Projection by Inhibiting Collateral Extension.

Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered that EphA7 and EfnA5 are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets.SIGNIFICANCE STATEMENT Our findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization-both within each brain region and between connected brain regions-may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.

Aggressive and recurrent ovarian cancers upregulate ephrinA5, a non-canonical effector of EphA2 signaling duality.

Erythropoietin producing hepatocellular (Eph) receptors and their membrane-bound ligands ephrins are variably expressed in epithelial cancers, with context-dependent implications to both tumor-promoting and -suppressive processes in ways that remain incompletely understood. Using ovarian cancer tissue microarrays and longitudinally collected patient cells, we show here that ephrinA5/EFNA5 is specifically overexpressed in the most aggressive high-grade serous carcinoma (HGSC) subtype, and increased in the HGSC cells upon disease progression. Among all the eight ephrin genes, high EFNA5 expression was most strongly associated with poor overall survival in HGSC patients from multiple independent datasets. In contrast, high EFNA3 predicted improved overall and progression-free survival in The Cancer Genome Atlas HGSC dataset, as expected for a canonical inducer of tumor-suppressive Eph receptor tyrosine kinase signaling. While depletion of either EFNA5 or the more extensively studied, canonically acting EFNA1 in HGSC cells increased the oncogenic EphA2-S897 phosphorylation, EFNA5 depletion left unaltered, or even increased the ligand-dependent EphA2-Y588 phosphorylation. Moreover, treatment with recombinant ephrinA5 led to limited EphA2 tyrosine phosphorylation, internalization and degradation compared to ephrinA1. Altogether, our results suggest a unique function for ephrinA5 in Eph-ephrin signaling and highlight the clinical potential of ephrinA5 as a cell surface biomarker in the most aggressive HGSCs.

The Expression of the Cancer-Associated lncRNA Snhg15 Is Modulated by EphrinA5-Induced Signaling.

The Eph receptor tyrosine kinases and their respective ephrin-ligands are an important family of membrane receptors, being involved in developmental processes such as proliferation, migration, and in the formation of brain cancer such as glioma. Intracellular signaling pathways, which are activated by Eph receptor signaling, are well characterized. In contrast, it is unknown so far whether ephrins modulate the expression of lncRNAs, which would enable the transduction of environmental stimuli into our genome through a great gene regulatory spectrum. Applying a combination of functional in vitro assays, RNA sequencing, and qPCR analysis, we found that the proliferation and migration promoting stimulation of mouse cerebellar granule cells (CB) with ephrinA5 diminishes the expression of the cancer-related lncRNA Snhg15. In a human medulloblastoma cell line (DAOY) ephrinA5 stimulation similarly reduced SNHG15 expression. Computational analysis identified triple-helix-mediated DNA-binding sites of Snhg15 in promoters of genes found up-regulated upon ephrinA5 stimulation and known to be involved in tumorigenic processes. Our findings propose a crucial role of Snhg15 downstream of ephrinA5-induced signaling in regulating gene transcription in the nucleus. These findings could be potentially relevant for the regulation of tumorigenic processes in the context of glioma.

Zinc biosorption by Dunaliella sp. AL-1: Mechanism and effects on cell metabolism.

Phycoremediation is being considered as an eco-friendly and safe technology for toxics eradication from contaminated aquatic systems. The zinc biosorption capacity of Dunaliella sp. AL-1 was demonstrated. Zinc impacted cell growth and photosynthetic pigments accumulation showing exposure time and concentration-dependent effects. The investigation of the antioxidant protective response to zinc exposition proved a stimulation of guaiacol peroxidase (GPX) activity and an increased rate of total phenolics, flavonoids, condensed tannins and glutathione (GSH). The Box-Behnken design was used to optimize zinc removal conditions by Dunaliella sp. AL-1 strain. The maximum experimental zinc uptake was obtained when zinc concentration, algae dose, initial pH, and contact time were set at 25 mg/L, 0.5 g/L, 7.59 and 13 h 43 min, respectively. Under completely optimized conditions, the fraction of zinc removed intracellularly was much lower than the adsorbed on the cell surface. FTIR analysis Dunaliella sp. AL-1 biomass demonstrated that several functional groups as OH, CH2, CO, PO, COO and CO may participate in the biosorption process. A comparative proteomic analysis through nano-HPLC coupled to LC-MS/MS, was performed from pre- and post-zinc treatments cells. Among 199 identified proteins, 60 were differentially expressed of which 41 proteins were down-regulated against 19 up-regulated ones. Target proteins have been demonstrated to be implicated in different metabolic processes mainly photosynthesis and antioxidant defenses.

PIWIL1 interacting RNA piR-017061 inhibits pancreatic cancer growth via regulating EFNA5.

PIWI (P element induced wimpy testis) integrating RNAs (piRNAs) are small non-coding RNAs with the length of approximately 30 nucleotides that plays crucial roles in germ cells and adult stem cells. Recently, accumulating data have shown that piRNA and PIWI proteins are involved in tumorigenesis. However, the roles of PIWI proteins and piRNAs in pancreatic cancer are still elusive. Here, we showed that piR-017061 is significantly downregulated in pancreatic cancer patients' samples and pancreatic cancer cell lines. Furthermore, we studied the function of piR-017061 in pancreatic cancer and our data revealed that piR-017061 inhibits pancreatic cancer cell growth in vitro and in vivo. Moreover, we analyzed the genomic loci around piR-017061 and identified EFNA5 as a novel target of piR-017061. Importantly, our data further revealed a direct binding between piR-017061 and EFNA5 mRNA mediated by PIWIL1. Mechanically, piR-017061 cooperates with PIWIL1 to facilitate EFNA5 mRNA degradation and loss of piR-017061 results in accumulation of EFNA5 which facilitates pancreatic cancer development. Hence, our data provided novel insights into PIWI/piRNA-mediated gene regulation and their function in pancreatic cancer. Since PIWI proteins and piRNA predominately express in germline and cancer cells, our study provided novel therapeutic strategy for pancreatic cancer treatment.