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Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na⁺/H⁺ exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J–resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J–susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.

The mammalian Na+/H+ exchanger isoform one (NHE1) is a plasma membrane protein that is ubiquitously present in human cells. It functions to regulate intracellular pH removing an intracellular proton in exchange for one extracellular sodium and is involved in heart disease and in promoting metastasis in cancer. It is made of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. The membrane domain is thought to have 12 transmembrane segments and a large membrane-associated extracellular loop. Early studies demonstrated that in mice, disruption of the NHE1 gene results in locomotor ataxia and a phenotype of slow-wave epilepsy. Defects included a progressive neuronal degeneration. Growth and reproductive ability were also reduced. Recent studies have identified human autosomal homozygous recessive mutations in the NHE1 gene (SLC9A1) that result in impaired development, ataxia and other severe defects, and explain the cause of the human disease Lichtenstein-Knorr syndrome. Other human mutations have been identified that are stop codon polymorphisms. These cause short non-functional NHE1 proteins, while other genetic polymorphisms in the NHE1 gene cause impaired expression of the NHE1 protein, reduced activity, enhanced protein degradation or altered kinetic activation of the protein. Since NHE1 plays a key role in many human physiological functions and in human disease, genetic polymorphisms of the protein that significantly alter its function and are likely play significant roles in varying human phenotypes and be involved in disease.

The NHE1 isoform of the mammalian Na+/H+ exchanger is a ubiquitous plasma membrane protein that regulates intracellular pH in mammalian cells by removing one intracellular proton in exchange for one extracellular sodium. Deletion of the NHE1 gene (SLC9A1) affects the growth and motor ability of mice and humans but mutations and polymorphisms of the gene are only beginning to be characterized. NHE1 has a cytosolic C-terminal regulatory tail of approximately 315 amino acids and a 500 amino acid membrane domain. We examined the functional effects of three human stop codon mutations at amino acids 321, 449 and 735 in comparison with a mutant that had a shortened tail region (543 stop codon). The short mutants, 321, 449 and 543 stop codon mutant proteins, lost NHE1 activity and expression, and did not target to the plasma membrane. Protein for these short mutants was more rapidly degraded than the wild type and 735 ending proteins. The 735 terminating mutant, with the membrane domain and much of the cytosolic tail, had reduced protein expression and activity. The results demonstrate that early stop codon polymorphisms have significant and deleterious effects on the activity of the SLC9A1 protein product. The 735-NHE1 mutant, without the last 80 amino acids, had more minor defects. Surprisingly, retention of a proximal 43 amino acids adjacent to the membrane domain did little to maintain NHE1 expression, targeting and activity.

We previously found that deficiency of the sodium–hydrogen exchanger 1 (NHE1) gene prevented hypoxia-induced pulmonary hypertension and vascular remodeling in mice, which were accompanied by a significantly reduced proliferation of pulmonary artery smooth muscle cells (PASMCs), and which decreased the medial-wall thickness of pulmonary arteries. That finding indicated the involvement of NHE1 in the proliferation and hypertrophy of PASMCs, but the underlying mechanism was not fully understood. To define the mechanism by which the inhibition of NHE1 decreases hypoxic pulmonary hypertension and vascular remodeling, we investigated the role of E2F1, a nuclear transcription factor, in silencing the NHE1 gene–induced inhibition of the proliferation, hypertrophy, and migration of human PASMCs. We found that: (1) silencing of NHE1 by short, interfering RNA (siRNA) significantly inhibited PASMC proliferation and cell cycle progression, decreased hypoxia-induced hypertrophy (in terms of cell size and protein/DNA ratio) and migration (in terms of the wound-healing and migration chamber assays); (2) hypoxia induced the expression of E2F1, which was reversed by NHE1 siRNA; and (3) the overexpression of E2F1 blocked the inhibitory effect of NHE1 siRNA on the proliferation, hypertrophy, and migration of PASMCs. The present study determined that silencing the NHE1 gene significantly inhibited the hypoxia-induced proliferation, hypertrophy, and migration of human PASMCs via repression of the nuclear transcription factor E2F1. This study revealed a novel mechanism underlying the regulation of hypoxic pulmonary hypertension and vascular remodeling via NHE1.

As a kind of binding protein, the type 1 Na + /H + exchanger (NHE1) is a receptor for the highly pathogenic Avian leukosis viruses-J subgroup (ALV-J) in chicken. In order to investigate the potential effect of chicken NHE1 gene on leukosis, we compared its expression between ALV-J-affected and -unaffected chicken, screened variations across the whole gene, and then performed association analysis with ALV-J affected/unaffected trait in three un-related chicken populations. We found that the NHE1 gene expressed in four immune tissues including spleen, bursa fabricius, liver, and thymus, and its expression was significantly up-regulated in liver and thymus of ALV-J-affected chickens (with leukosis phenotype) compared to -unaffected ones (ALV-J-negative controls). Thirty-six single nucleotide polymorphisms (SNP) were identified in a 6,105 bp region of the chicken NHE1 gene, giving rise to every 170 bp per SNP. Two SNP of g.4405A>G and g.5886C>G were genotyped with PCR-RFLP method. Results showed that g.4405A>G was significantly associated ( P  < 0.05) with ALV-J infection in all of the three chicken populations, including White Recessive Rock (WRR), Dwarf Yellow (DY) and Shiki Yellow (SY), while g.5886C>G was significantly associated ( P  < 0.05) with ALV-J infection in SY. These results indicated that the NHE1 gene was related to ALV-J infection in chicken.

It has long been speculated that intracellular pH is a critical regulator of both invertebrate and vertebrate sperm motility, and sodium-hydrogen exchange has been suggested as a mediator of such pH sub(i) regulation in various instances. Two sodium-hydrogen exchangers (NHE1 and NHE5) are expressed in spermatozoa. However, elimination of the NHE1 gene fails to cause infertility, suggesting that normal sperm function is maintained in NHE1-null animals. Here, we used a functionally unbiased signal peptide trap screen to identify a novel sperm-specific NHE. The NHE contains 14 predicted transmembrane segments, including a potential voltage sensor and a consensus cyclic nucleotide-binding motif. Testis histology, sperm numbers and morphology were normal, but NHE-null males were completely infertile with severely diminished sperm motility. The addition of ammonium chloride, which elevates intracellular pH, partially rescued the motility and fertility defects. Surprisingly, cyclic AMP analogues almost completely rescued the motility and infertility phenotypes. The existence of this new sperm NHE provides an attractive contraceptive target, given its cell-specific expression and absolute requirement for fertility.

Microglial Na/H exchanger-1 (NHE1) protein, encoded by Slc9a1 , plays a role in white matter demyelination of ischemic stroke brains. To explore underlying mechanisms, we conducted single cell RNA-seq transcriptome analysis in conditional Slc9a1 knockout (cKO) and wild-type (WT) mouse white matter tissues at 3 days post-stroke. Compared to WT, Nhe1 cKO brains expanded a microglial subgroup with elevated transcription of white matter myelination genes including Spp1 , Lgals3 , Gpnmb , and Fabp5 . This subgroup also exhibited more acidic pH i and significantly upregulated CREB signaling detected by ingenuity pathway analysis and flow cytometry. Moreover, the Nhe1 cKO white matter tissues showed enrichment of a corresponding oligodendrocyte subgroup, with pro-phagocytosis and lactate shuffling gene expression, where activated CREB signaling is a likely upstream regulator. These findings demonstrate that attenuation of NHE1-mediated H + extrusion acidifies microglia/macrophage and may underlie the stimulation of CREB1 signaling, giving rise to restorative microglia-oligodendrocyte interactions for remyelination.

Sodium glucose cotransporter 2 inhibitors (SGLT2i) constitute the only medication class that consistently prevents or attenuates human heart failure (HF) independent of ejection fraction. We have suggested earlier that the protective mechanisms of the SGLT2i Empagliflozin (EMPA) are mediated through reductions in the sodium hydrogen exchanger 1 (NHE1)-nitric oxide (NO) pathway, independent of SGLT2. Here, we examined the role of SGLT2, NHE1 and NO in a murine TAC/DOCA model of HF. SGLT2 knockout mice only showed attenuated systolic dysfunction without having an effect on other signs of HF. EMPA protected against systolic and diastolic dysfunction, hypertrophy, fibrosis, increased Nppa/Nppb mRNA expression and lung/liver edema. In addition, EMPA prevented increases in oxidative stress, sodium calcium exchanger expression and calcium/calmodulin-dependent protein kinase II activation to an equal degree in WT and SGLT2 KO animals. In particular, while NHE1 activity was increased in isolated cardiomyocytes from untreated HF, EMPA treatment prevented this. Since SGLT2 is not required for the protective effects of EMPA, the pathway between NHE1 and NO was further explored in SGLT2 KO animals. In vivo treatment with the specific NHE1-inhibitor Cariporide mimicked the protection by EMPA, without additional protection by EMPA. On the other hand, in vivo inhibition of NOS with L-NAME deteriorated HF and prevented protection by EMPA. In conclusion, the data support that the beneficial effects of EMPA are mediated through the NHE1-NO pathway in TAC/DOCA-induced heart failure and not through SGLT2 inhibition.

The peroxisome proliferators-activated receptor ( PPAR) γ pathway is involved in cancer, but it appears to have both tumor suppressor and oncogenic functions. In neuroblastoma cells, miR-27b targets the 3′ untranslated region of PPAR γ and inhibits its mRNA and protein expression. miR-27b overexpression or PPAR γ inhibition blocks cell growth in vitro and tumor growth in mouse xenografts. PPAR γ activates expression of the pH regulator NHE1 , which is associated with tumor progression. Lastly, miR-27b through PPAR γ regulates nuclear factor-κB activity and transcription of inflammatory target genes. Thus, in neuroblastoma, miR-27b functions as a tumor suppressor by inhibiting the tumor-promoting function of PPAR γ, which triggers an increased inflammatory response. In contrast, in breast cancer cells, PPAR γ inhibits NHE1 expression and the inflammatory response, and it functions as a tumor suppressor. We suggest that the ability of PPAR γ to promote or suppress tumor formation is linked to cell type-specific differences in regulation of NHE1 and other target genes.

Nutraceuticals exert a series of health benefits, including protection against cardiovascular diseases. In this study, naringin, naringenin, and quercetin were tested for their safety and efficacy in ameliorating angiotensin (Ang) II-induced cardiac hypertrophy through carbonic anhydrase II (CA-II) inhibition. In silico molecular docking and MD simulations exhibited that naringin strongly binds CA-II with a docking score of -9.55 kcal/mol and hydrogen bonding energy of -6.07 kcal/mol. Naringin formed stable hydrogen bond interactions with Asn62, Trp5, and N-acetyl His4 via catalytic water molecule, and a continuous interaction via major water bridge with N-acetyl His4, His4, and Trp5. Moreover, naringin effectively inhibited CA-II activity with an IC 50 value of 82.99 ± 4.92 nM, followed by naringenin and quercetin. Of note, all the tested nutraceuticals were found to be safe as evident from the cell viability assays. Further, naringin effectively attenuated cardiac hypertrophy, as indicated by the reductions in the Ang II-induced increases in cell surface area of H9c2 cardio myoblasts (165.6 ± 1.26% Ang II vs. 109.8 ± 1.88% Ang II + naringin), followed by naringenin and quercetin. Furthermore, naringin significantly inhibited CA-II activity (191.77 ± 7.69% Ang II vs. 120.16 ± 5.52% Ang II + naringin) and suppressed Ang II-induced CA-II and Na + /H + exchanger 1 (NHE1) protein expression. Besides, naringin suppressed Ang II-induced CA-II, NHE1, Na + /Ca 2+ exchanger 1 (NCX1), and angiotensin-converting enzyme (ACE1) mRNA expression. Collectively, naringin when compared to naringenin and quercetin effectively attenuated Ang II-induced cardio myoblast hypertrophy, CA-II activity, CA-II, and NHE1 expression. The naringin-mediated attenuation of cardiac hypertrophy might be through the inhibition of CA-II enzyme activity, and the suppression of NHE1, and NCX1.