A receptor signaling polarity in several neurological disorders, but these have not yet been explored in AD. NKCC1 and KCC2 regulate intracellular chloride [Cl−]i by accumulating and extruding Cl−, respectively. Increased NKCC1 expression in mature neurons has been reported in these disease conditions, and bumetanide, an NKCC1 inhibitor, is suggested to show potential therapeutic benefits. This study used primary mouse hippocampal neurons to explore if KCC2 and NKCC1 expression levels are altered following beta-amyloid (Aβ1-42) treatment and the potential neuroprotective effects of bumetanide. KCC2 and NKCC1 expression levels were also examined in 18-months-old male C57BL/6 mice following bilateral hippocampal Aβ1-42 stereotaxic injection. No change in KCC2 and NKCC1 expression levels were observed in mouse hippocampal neurons treated with 1 nM Aβ1-42, but NKCC1 expression increased 30-days post-Aβ1-42-injection in the CA1 region of the mouse hippocampus. Primary mouse hippocampal cultures were treated with 1 nM Aβ1-42 alone or with various concentrations of bumetanide  to investigate the effect of the drug on cell viability. Aβ1-42 produced 53.1 ± 1.4% cell death after 5 days, and the addition of bumetanide did not reduce this. However, the drug at all concentrations significantly reduced cell viability, suggesting bumetanide is highly neurotoxic. In summary, these results suggest that chronic exposure to Aβ1-42 alters the balance of KCC2 and NKCC1 expression in a region-and layer-specific manner in mouse hippocampal tissue; therefore, this process most likely contributes to altered hippocampal E/I balance in this model. Furthermore, bumetanide induces hippocampal neurotoxicity, thus questioning its suitability for AD therapy. Further investigations are required to examine the effects of Aβ1-42 on KCC2 and NKCC1 expression and whether targeting CCCs might offer a therapeutic approach for AD.Alzheimer’s disease (AD) is a neurodegenerative disorder with an increasing need for developing disease-modifying treatments as current therapies only provide marginal symptomatic relief. Recent evidence suggests the γ-aminobutyric acid (GABA) neurotransmitter system undergoes remodeling in AD, disrupting the excitatory/inhibitory (E/I) balance in the brain. Altered expression levels of K-Cl-2 (KCC2) and N-K-Cl-1 (NKCC1), which are cation–chloride cotransporters (CCCs), have been implicated in disrupting GABAergic activity by regulating GABA While studies have historically pointed to dysfunction in the cholinergic and glutamatergic neurotransmitter systems, it has become increasingly apparent that the GABAergic system is also affected in AD.Alzheimer’s disease (AD) is a neurodegenerative disorder affecting the aging population. It is associated with the progressive loss of neurons within the hippocampus, resulting in the clinical manifestation of memory loss and cognitive impairment. The hallmarks of AD pathology are well-known as the progressive accumulation of extracellular beta-amyloid (Aβ) plaques and intracellular neurofibrillary tangles (NFTs), which severely affect the hippocampus in the disease ,2. The hAR subunits; indicating elaborate GABAergic remodeling in AD [Recent evidence indicates altered GABAergic signaling may be a key player in exacerbating aberrant network activity in AD ,10. GABAng in AD ,10,15,16− across cellular membranes to regulate cell volume and modulate neuronal excitability. In the developing neuron NKCC1 is the primary Cl− importer which cotransports 1 potassium (K+), 1 sodium (Na+), and 2 Cl− into the neuron. KCC2, while lowly expressed in the immature neuron, becomes the predominant Cl− exporter in mature neurons where it cotransports K+ and Cl− in a 1:1 ratio [AR signaling in a Huntington’s disease (HD) mouse model [GABA) in these mice, which skews GABA to become excitatory [Extensive GABAergic remodeling is apparent in AD, but the cation–chloride cotransporters (CCCs) K-Cl-2 (KCC2) and N-K-Cl-1 (NKCC1) have also been implicated in disease progression by altering GABA signaling polarity in several neurological disorders . CCCs, c:1 ratio ,18. Wher:1 ratio ,20. Evid:1 ratio . In such:1 ratio . A gain-:1 ratio ,23. Recese model . Electrocitatory ,25,26,27citatory . While tcitatory ,35,36,37citatory .1-42 following 5-day exposure in vitro, but after 30 days in vivo significantly increased NKCC1 expression. Furthermore, bumetanide does not improve the survival of primary mouse hippocampal neurons treated with neurotoxic Aβ1-42, but further reduces cell viability and has neurotoxic properties on healthy neurons.This study investigated the hippocampal expression of KCC2 and NKCC1 and the potential neuroprotective properties of bumetanide in an in vitro and in vivo AD mouse model. Here we demonstrate KCC2 and NKCC1 expression is unchanged in the presence of AβHippocampal cultures were fixed at 7 and 19 DIV and fluorescently labeled for NeuN and KCC2 to evaluate neuronal expression of the cotransporter. KCC2 staining was localized to the cell body with some neurons expressing KCC2 proximally and distally along neuronal processes A,B.Hippocampal cultures were also double labeled with either GAD65/67 or VGluT1, and KCC2 to investigate which neuronal subtypes express KCC2. GABAergic, or GAD65/67-positive, hippocampal neurons show KCC2 expression on the cell body and sparsely along the proximal processes C. Based Expression of NKCC1 was also explored in hippocampal cultures by double-labeling with NeuN and NKCC1. NKCC1 staining was observed on the cell bodies and along the neuronal processes of NeuN-positive cells in these cultures A,B. Unli1-42 and bumetanide on KCC2 and NKCC1 expression, hippocampal cultures treated with Aβ1-42 and bumetanide were triple-labeled with KCC2, NKCC1 and NeuN. Signal density measurements of KCC2 showed that 5-day treatment with 1 nM Aβ1-42 did not change KCC2 expression in hippocampal cultures compared to the vehicle control group . Adding bumetanide to Aβ1-42-treated cells at 1 µM , 10 µM , 100 µM , or 1 mM bumetanide  did not alter KCC2 expression compared to vehicle control either. Similarly, administering bumetanide alone did not alter KCC2 expression at 1 µM , 10 µM , 100 µM , or 1 mM , compared to vehicle controls. The high variability in staining density for each treatment group might indicate that subpopulations of hippocampal neurons express different levels of KCC2. Overall, KCC2 expression levels were unchanged across all treatment groups.To determine the effect of Aβ1-42 did not significantly change NKCC1 expression on hippocampal neurons compared to controls . In addition, there was no change in NKCC1 expression following administration of Aβ1-42 and bumetanide at 1 µM , 10 µM , 100 µM , or 1 mM bumetanide  compared to controls. Finally, NKCC1 expression levels were unaffected by bumetanide alone at 1 µM , 10 µM , 100 µM , and 1 mM . Overall, there was no change in NKCC1 expression across all treatment groups.Treatment with Aβ1-42-induced cell death using the ReadyProbes Live/Dead cell viability assay. After 5 days, hippocampal cultures treated with Aβ1-42 and bumetanide showed no improvement in neuronal survival compared to Aβ1-42 alone. Furthermore, all concentrations of bumetanide showed a trend towards increasing cell death, with concentrations 1 µM and 1 mM leading to a significant increase in cell death of 11%  and 11.1%  compared to Aβ1-42 alone, respectively. Interestingly, treatment with bumetanide alone significantly increased cell death to 61.5% at 1 µM , 65.4% at 10 µM , 64.6% at 100 µM , and 59.8% at 1 mM  compared to the vehicle controls. Moreover, bumetanide appeared to be more neurotoxic than Aβ1-42 alone, with cell death increasing by 8.4%, 12.3% (** p = 0.0017), 11.4% (** p = 0.0023), and 6.7% with 1 µM, 10 µM, 100 µM, and 1 mM bumetanide, respectively, compared to Aβ1-42 alone. Overall, these results suggest bumetanide is not neuroprotective against Aβ1-42 neurotoxicity and on its own is neurotoxic to hippocampal neurons.Bumetanide was added to hippocampal cultures at 1 µM, 10 µM, 100 µM, and 1 mM to determine whether the drug could prevent Aβ1-42-injected mice for KCC2 and NKCC1. We determined whether the bilateral stereotaxic injection of Aβ1-42 affected KCC2 and NKCC1 expression in the stratum (str.) oriens, str. pyramidale, and str. radiatum of the CA1 and CA3 regions; and the str. moleculare, str. granulosum, and hilus of the DG in the mouse hippocampus compared to naïve and ACSF controls 30-days post-Aβ1-42-injection.Expression of KCC2 and NKCC1 was further investigated in an in vivo AD model by labeling mouse hippocampal sections from naïve, ACSF-injected, and AβKCC2 immunoreactivity in the CA1 and CA3 regions appeared punctate in the str. oriens and str. radiatum and localized to the neuronal soma in the str. pyramidale and str. radiatum A,B. Neur1-42-injected mice compared to naïve and ACSF controls , and Aβ1-42-injected mice , ACSF , and scrAβ-injected mice . In the str. pyramidale and str. radiatum NKCC1 expression levels were greater in Aβ1-42-injected mice compared to naïve , ACSF , and scrAβ  groups 30-days post- Aβ1-42-injection, respectively  or insulin-like growth factor 1 are involved in KCC2 developmental upregulation [1-42 injection. Most interestingly, it appeared as though KCC2 was downregulated specifically at the CA1 injection site. Full length APP is capable of modulating KCC2 expression by stabilizing KCC2 on the cell membrane and thus reducing its degradation, and by post-transcriptional mechanisms [1-42 this may switch into more localized modulation of KCC2 expression. KCC2 might become less stable on the cell membrane and more prone to degradation following Aβ1-42-injection [1-42 administration in the in vivo model, to observe whether bumetanide can affect the apparent localized modulation of KCC2. However, the possible neurotoxic effects of the drug have to be explored as well.In the in vitro AD mouse model, we conclude that Aβegulated . The durgulation ,40,46. Igulation . Investichanisms ,48. Perhnjection . KCC2 manjection . Lack ofnjection ,40,46. F− influx, a less negative EGABA, and depolarizing GABA activity. Throughout development, NKCC1 expression diminishes as GABA becomes hyperpolarizing, but is still present [We also report NKCC1 is expressed on neuronal cell bodies and processes in immature and mature neurons, with mature neurons predominantly exhibiting expression on the cell body. NKCC1 also appeared to be expressed on non-neuronal cells, which we speculate are endothelial cells in our cultures. As NKCC1 is shown to be expressed by a diverse population of cells such as vascular endothelial cells, this finding was not unexpected ,35,49. I present . Thus, w present . PlotkinIn the mouse hippocampus NKCC1 was expressed along neuronal processes of pyramidal neurons; and punctate in the str. oriens and str. radiatum of the CA1 region. Previous studies in the adult rat brain have also shown specific expression of NKCC1 on the dendrites of CA1 and CA3 pyramidal neurons ,49. The 1-42, suggesting Aβ1-42 does not affect NKCC1 expression. The addition of bumetanide at four concentrations had no effect on NKCC1 expression either alone or in conjunction with Aβ1-42 treatment. It is plausible that NKCC1 is regulated by transcriptional mechanisms like KCC2. NKCC1 activation relies on phosphorylation and thus kinase activity [1-42, particularly in an in vivo model. In the in vivo AD mouse model NKCC1 expression appeared to undergo layer and region-specific increases in the CA1 region, particularly in the CA1 injection site of Aβ1-42-injected mice 30-days post-injection. Specifically, NKCC1 density was enhanced in the str. pyramidale and str. radiatum of the CA1 region of Aβ1-42-injected mice compared to naïve and ACSF controls. The inhibitory-to-excitatory switch in GABAergic transmission may be a local effect of Aβ1-42 and the limited duration of cell culture experiments may be insufficient for Aβ1-42 to have an effect, which supports the speculation that NKCC1 is transcriptionally regulated like KCC2. Elucidating which kinases are involved in NKCC1 upregulation will be challenging as several kinases have been implicated in regulating NKCC1 expression [GABA in mature CA1 hippocampal neurons, which consequently promotes GABAA-mediated neuronal excitability [In the in vitro AD mouse model, there was no change in NKCC1 expression following a 5-day exposure to Aβactivity . A studyactivity . And likactivity . As for pression . Regardipression . Westerntability .1-42-induced cell death in this in vitro AD model and may even exacerbate cell death. We hypothesized that the E/I imbalance and depolarizing GABA activity in AD may be influenced by altered expression of KCC2 and NKCC1. Increased NKCC1 expression would increase the intracellular Cl− concentration, disrupt inhibition, and increase cellular excitation [− rises during reoxygenation and/or reducing of Na+ influx via NKCC1 and preventing excitotoxicity. Therefore, in AD bumetanide might also protect against Aβ1-42-induced excitotoxicity by restoring the E/I imbalance. DS mouse models have shown bumetanide could rescue hippocampal-dependent memory and synaptic plasticity [GABA, loss of inhibitory drive, and increased neuronal excitability. As treatment with bumetanide abolished these deficits, it became apparent that NKCC1 triggered GABAA-mediated excitation [1-42 alone. This suggests bumetanide is capable of inducing cell death, and the presence of NKCC1 in mature neurons, although lowly expressed, are necessary for normal physiological function. Therefore, blocking its activity via bumetanide may be detrimental and promote cell death. Indeed, impairing NKCC1 function has been shown to disrupt K+ influx and secretion into the endolymph involved in hearing in NKCC1 knock-out mice, leading to deafness and balance impairments [− influx, promoting KCC2-mediated Cl− efflux and thus GABAAR-mediated hyperpolarization. Increased inhibition in a DS mouse model has been associated with hippocampal learning and memory deficits [+ uptake leading to cell shrinkage, which was postulated to promote generation of reactive oxygen intermediates and oxidative stress [Finally, we showed that bumetanide is not neuroprotective against Aβcitation . The neucitation ,56. The asticity . In addiasticity  has showcitation . Howeverairments . Presentdeficits . As celldeficits . Anotherdeficits  used 5 µe stress . Althouge stress  reportede stress ,60. Suche stress . In our 1-42 and bumetanide does not protect primary hippocampal neurons from Aβ1-42 induced neurotoxicity. Importantly, bumetanide reduces cell viability in the presence of Aβ1-42 and is highly neurotoxic on its own to hippocampal primary neurons. NKCC1 is upregulated in the CA1 region of the hippocampus following Aβ1-42 administration in an in vivo AD mouse model. These findings indicate more research is required to elucidate the effects of Aβ1-42 on KCC2 and NKCC1 expression in the human AD brain to explore if targeting CCCs might offer a therapeutic approach for AD, as we demonstrate altered CCC expression may be a contributing factor to the E/I imbalance in AD. If bumetanide has a therapeutic potential for AD, the neurotoxicity on hippocampal neurons suggest caution as to its suitability, therefore more work is required to understand its mechanism of action on neurons.In summary, we demonstrate that KCC2 and NKCC1 expression is not altered by Aβad libitum. Postnatal (P0) male C57/BL/6 mice were used to culture mouse hippocampal neurons while male C57BL/6 mice at 18-months of age were used in vivo experiments . A total of 75 mice were used form the in vitro experiments and 24 aged mice used for the in vivo study. The aged mice were categorized into four groups, NC (n = 6), ACSF- injected (n = 6), scrAβ1–42-injected (n = 6) and Aβ1–42-injected (n = 6).All experiments were approved by the University of Auckland Animal Ethics Committee . Mice were housed at the University of Auckland Vernon Jansen Unit under standard laboratory conditions and maintained in a 12 h light-dark cycle with food and water 2/95% O2 cell culture incubator for 4 h to allow for cell adhesion. After this, the plating media was removed and replaced with fresh Neurobasal medium , 2 µg/mL gentamicin , and 2 mM glutamine . Every 3-4 days half the media was exchanged. To inhibit glial cell proliferation and reduce the presence of astrocytes in culture, 5 µM of cytosine arabinofuranoside  was used at 3 days in vitro (DIV). Neurons could be maintained for up to 4 weeks, sufficient to differentiate, and develop axons, dendrites, and synaptic connections.Cell culture procedures were adapted from Beaudoin et al.  with adj1-42 was produced as a recombinant protein with maltose binding protein (MBP) to establish a highly soluble recombinant fusion protein, which was then expressed in Escherichia coli. The product was then purified on an amylose column which binds to the MBP portion. The pure fusion protein was extracted from the amylose resin using maltose and concentrated using ammonium sulphate precipitation. Factor X protease was used to cleave the carrier MBP off the fusion protein, then the Aβ1-42 protein was further isolated and purified using hydrophobic chromatography with 0–50% v/v acetonitrile/0.1% v/v TFA using FPLC. Fractions with pure Aβ1-42 were detected immunologically using antibodies against residues 17–24 of Aβ1-42, which were lyophilized to remove the solvent. The expected molecular ion for the product was determined using mass spectrometry. Prior to in vitro application, Aβ1-42 was dissolved in artificial cerebrospinal fluid (aCSF), diluted culture medium, and aged by incubating in a 37 °C water bath for 48 h to increase the neurotoxicity of Aβ1-42 by facilitating the formation of toxic soluble aggregates [1-42 was dissolved in aCSF and ‘aged’ as above. The optimal incubation time for Aβ1-42 preparations to produce toxic oligomers varies between preparations but ranges from 48–120 h. Yeung et al. [1-42. Western blots of an aging profile of Aβ1-42 showed the Aβ1-42 monomer decreases and the oligomer appears by 48 h in non-dissociating gels. Whereas in SDS gels, the dimer and trimer of Aβ1-42, and a higher molecular weight oligomer, can be observed at 5 days. This preparation of Aβ1-42 has been shown to be highly neurotoxic both in vitro and in vivo [Aβgregates . Whereasg et al.  previous in vivo ,64,65,661-42 at 14 DIV, when neurons represent an ‘adult’ state, for 5 days to establish an in vitro AD mouse model [1-42 and 1 µM, 10 µM, 100 µM, and 1 mM bumetanide in 500 µL of culture medium for 5 days.Primary mouse hippocampal neurons were treated with 1 nM of aged Aβse model . Culturese model . The NKC1-42 and/or bumetanide using the ReadyProbes Cell Viability Kit , as per the manufacturer’s instructions. Wells were randomly allocated for treatment with different bumetanide concentrations  alone or with 1 nM Aβ1-42, 1 nM Aβ1-42 alone, or as controls. Control wells were treated with an equal volume of culture medium, which was used to dissolve Aβ1-42 and bumetanide, to mimic treatment administration. At least 8-13 wells were allocated per treatment group. At 19 DIV, 15 µL of the NucBlue® Live reagent and 15 µL of the NucGreen® Dead reagent was added to each well containing 500 µL of culture medium. Cells were returned to the incubator for 15 min to facilitate reagent staining prior to imaging. The NucBlue® reagent labels the nuclei of all cells (live and dead) to fluoresce blue, while the NucGreen® reagent labels the nuclei of dead cells only to fluoresce green. Cells were imaged using the ImageXpress Micro XLS  using the DAPI filter to acquire the NucBlue® staining and the FITC filter from the Quad5 filter cube to acquire the NucGreen® staining. Each well was imaged across 16 regions of interest using the 20×/0.45 NA CFI Plan Fluor ELWD ADM (phase contrast) objective lens. Cells were counted manually and based on neuronal morphology to determine the total number of cells based on NucBlue® staining and the total number of dead cells based on NucGreen® staining.Cell viability was determined 5 days after treatment with Aβ1-42 and/or bumetanide. Cells were also stained for glutamatergic neurons using vesicular glutamate transporter 1 (VGluT1), GABAergic neurons using glutamic acid decarboxylase (GAD)65/67, and cholinergic neurons using choline acetyltransferase (ChAT). Cells were washed thrice for 5 min in PBS containing 0.05% Tween 20 (PBT) and incubated in PBS containing 0.2% Triton-X 100 (PBST) for 5 min on a slow rocker at room temperature (RT). Cells were then washed thrice for 5 min with PBT and blocked with 1% BSA-PBT (immunobuffer) for 1 h at RT before adding primary antibodies for KCC2 , NKCC1 , NeuN , VGluT1 , GAD65/67 , and ChAT . Specificity of the primary antibodies has been tested and reported previously  and stored in PBS-azide at 4 °C. KCC2 and NKCC1 expression levels were characterized in control cultures, and cultures treated with Aβy . The moThe cells were stored in PBS and imaged using the ImageXpress Micro XLS  across nine regions of interest per well with the 20×/0.45 NA CFI Plan Fluor ELWD ADM (phase contrast) objective lens. The DAPI, TxRed, and CY5 excitation filters were used to acquire Hoechst, NeuN, and KCC2 or NKCC1 staining, respectively. Image acquisition settings were optimized for each antibody and identical across all experimental and treatment replicates.Signal intensity was measured over the whole image across nine regions of interest per well specifically on neurons. For each image, a mask was created based on NeuN staining to identify neurons only. The NeuN mask was then overlaid onto the corresponding KCC2 or NKCC1 fluorescent images and density measurements captured based on the NeuN mask to determine neuronal transporter expression only. The raw intensity data was normalized by dividing it by the area as the area for density measurements differed between sites. Densitometry measurements of KCC2 and NKCC1 staining, based on a NeuN staining mask was conducted using ImageJ Fiji software . It was ensured the researchers were blinded to the treatment groups during analysis and the threshold was kept identical across all the wells analysed. Three experimental replicates, each with 8–13 wells per treatment group, were used for this experiment. Two hippocampi dissected from one mouse was sufficient to culture 12 wells of primary neurons in a 24-well plate.1-42, scrambled Aβ1-42 , or ACSF in to the CA1 region of the hippocampus, located relative to the bregma , at a speed of 0.5 µL/min [1-42 administration animals were anaesthetized via ketamine and domitor (12.5 mL/kg) and transcardially perfused with 4% PFA. Brains were then extracted, postfixed in 4% PFA solution for 2 h at RT and then incubated in 30% sucrose in TBS  overnight at 4 °C, cut into 30 μm sections using a freezing microtome, and stored in antifreeze solution at −20 °C. Mice were treated with aCSF, Aβ1-42 alone, or scrAβ. aCSF and no treatment (naïve) mice were used as controls [The surgery and treatments, collection, and preparation of mouse tissue used in this study was done as described in Calvo-Flores Guzmán et al. . Mice we5 µL/min . Anaesthcontrols .1-42-injected, and scrAβ-injected mice. Two coronal mouse brain sections containing the hippocampus were washed once for 5 min in PBS and twice for 5 min in PBS containing 0.1% Triton X-100 (PBST) at RT. Sections were blocked for 1 h at RT using PBS containing 10% goat serum and 0.1% Triton X-100 prior to adding primary antibodies for KCC2 , NKCC1 , and NeuN . Primary antibodies were diluted in 1% BSA in PBST and added for 24 h at 4 °C. The following day sections were washed thrice for 10 min with PBS and incubated in Alexa Fluor secondary antibodies diluted 1:500 in PBST for 2 h at RT on a slow rocker. The secondary antibodies used were goat anti-mouse 647 , goat anti-rabbit 488 , and goat anti-guinea pig 594 . Sections were then incubated in Hoechst33342  for 30 min at RT and washed thrice for 10 min in PBS. Sections were mounted on gelatin-coated slides, allowed to air-dry overnight, rehydrated using Milli-Q water, coverslipped using Mowiol488 mounting medium  and finally sealed with nail varnish. Control sections were incubated in 1% BSA in PBST in place of primary antibody to determine the degree of non-specific secondary antibody binding. The omission of the primary antibodies resulted in complete absence of immunoreactivity  in the following areas in each layer: 21,352 μm2 for the CA1 region, 4761 μm2 for the CA3 region, and 12,295 μm2 for the DG to quantify KCC2 and NKCC1 expression. Intensity measurements were performed in the following CA1 and CA3 layers: str. oriens, str. pyramidale, and str. radiatum; and in the following DG layers: molecular layer, granular layer, and hilus. Intensity was measured adjacent to the injection site, 50–500 µm from the needle track [Both hippocampi on each section was imaged using the Zeiss 710 confocal laser-scanning microscope  at the CA1, CA3, and DG regions at 20× magnification using the dry 20×/0.8NA objective lens , and using the 405 nm laser for Hoechst, 488 nm laser for AlexaFluorp ≤ 0.05 considered significant. All figures and images were prepared using Adobe Photoshop CC (2021) . All experimental data is presented as the mean ± the Standard Error of Mean (SEM).For cell viability, immunocyto- and histochemistry experiments, a one-way ANOVA with a Tukey’s multiple comparisons test was used to determine differences between treatment groups. All statistical analyses were conducted using GraphPad Prism version 8.2.1  with a


Bacteria with the ability to remove nitrogen (N) have been isolated to reduce water pollution caused by the excessive use of N fertilizer. To obtain plant growth-promoting rhizobacteria (PGPR) with salt tolerance and NO3–-N removal abilities, bacterial strains were isolated from plant rhizosphere soils, their plant growth-promoting effects were evaluated using tomato in plate assays, and their NO3–-N removal abilities were tested under different salinity, initial pH, carbon source, and agriculture wastewater conditions. The results obtained showed that among the seven strains examined, five significantly increased the dry weight of tomato plants. Two strains, Pseudomonas stutzeri NRCB010 and Bacillus velezensis NRCB026, showed good plant growth-promoting effects, salinity resistance, and NO3–-N removal abilities. The maximum NO3–-N removal rates from denitrifying medium were recorded by NRCB010 (90.6%) and NRCB026 (92.0%) at pH 7.0. Higher NO3–-N removal rates were achieved using glucose or glycerin as the sole carbon source. The total N (TN) removal rates of NRCB010 and NRCB026 were 90.6 and 66.7% in farmland effluents, respectively, and 79.9 and 81.6% in aquaculture water, respectively. These results demonstrate the potential of NRCB010 and NRCB026 in the development of novel biofertilizers and their use in reducing N pollution in water.Excess nitrate (NO However, nitrogen loss via agricultural nonpoint sources may lead to eutrophication in aquatic ecosystems  at 7% salinity and pH 7.0 and 72.2% of TN at 3% salinity and pH 9.0 , denitrifiers applied to the field may accelerate the mineralization of biologically available N. Denitrifying bacteria reduce NO3–-N removal rate previously reached 90.0% for Fusarium solani RADF-77 when glucose was used as the C source, but was only 19.7% when lactose was the C source  source play important roles in the N removal process. pH is one of the primary external factors that affects the growth and process of denitrification  rhizosphere soil collected in Nanjing  of Jiangsu Province, China. To isolate NRCB026 and NRCB030, 1 g of plant rhizosphere soil was added to 100 mL of 0.86% NaCl and incubated at 30°C for 7 days. The soil-NaCl suspension was shaken at 200 rpm for 20 min, and approximately 80 mL of the suspension was then transferred to a culture flask, to which 40 mL of 2.1 μmol L–1 sodium nitrate and 40 mL of 1.4 μmol L–1 sodium succinate were added. The suspension was incubated statically at 30°C for 24 h. One hundred microliters of five serial supernatant dilutions were plated on nitrogen free broth (NBF) and 1/10 modified nutrient broth containing sodium nitrate and sodium succinate (NBNS) . After an incubation at 30°C for 2 days, colonies that differed in appearance were isolated and maintained in 30% (w/v) glycerol at –80°C for further characterization.Seven bacterial strains were used in the present study. Five bacterial strains, NRCB001, NRCB010, NRCB023, NRCB024, and NRCB025, were previously isolated from rice rhizosphere soil collected in Yixing  of Jiangsu Province, China  tests were performed according to Bergey’s Manual of Systematic Bacteriology , followed by shaking at 30°C and 200 rpm for 48 h. The cultured suspension was centrifuged at 6,000 rpm for 10 min, and 2 mL of the supernatant was mixed with 2 mL of Salkowski reagent (1 mL of 0.5 mol L–1 FeCl3+50 mL 35% HClO4) and incubated at 37°C for 1 h in the dark. The concentration of IAA was measured at 530 nm using a spectrophotometer . IAA concentrations were assessed using an IAA standard curve ranging between 10 and 500 μg mL–1. Each treatment was performed in triplicate.Indole acetic acid (IAA) production was assessed using the method described by 3(PO4) was used as the sole phosphorus source. The production of siderophore and 1-aminocyclopropane-1-carboxyacetate (ACC) was analyzed according to the method described by Phosphate-solubilizing capacity was measured according to 600 of 1.0 and diluted 100 times. Seeds were washed in 2.5% sodium hypochlorite solution for 10 min and further washed with sterile distilled water. Surface-sterilized seeds were soaked in a 100-fold diluted strain suspension for 30 min, after which the inoculated seeds were placed on 1/10 MS medium . Seeds were cultured at 24°C for 10 days under a 16-h photoperiod, after which their height, root length, and dry weight were measured. Each treatment was performed in triplicate.To evaluate plant growth-promoting effects, seven bacteria were inoculated into tomato seeds  on agar plates. In brief, strains were cultured in NBNS liquid medium, centrifuged, and resuspended in sterile 1/10 Murashige and Skoog (MS) salt  (Hope Bio-Technology) at pH 5.8. Strain suspensions were adjusted to an OD–1, yeast extract 5 g L–1, and NaCl 10 g L–1) for 12 h. One milliliter of the pre-cultured strain (an OD600 of approximately 1.0) was added to 100 mL of LB-based liquid medium (tryptone 10 g L–1 and yeast extract 5 g L–1) supplemented with six different salinities , followed by shaking at 30°C and 200 rpm for 48 h. OD600 was measured using a spectrophotometer. Each treatment was performed in quadruplicate.To evaluate salinity effects, strains were precultured in LB liquid medium  in a 250-mL shaker flask as described by –1 NO3–-N) was used as the sole N source and the initial pH of DM medium was adjusted to 6.8. The initial salinities of DM were 0, 0.2, 0.4, 0.6, and 0.8 mol L–1 NaCl. Bacteria were cultured at 30°C and 200 rpm for 48 h, after which OD600 and pH were measured. The suspensions were centrifuged, NO3–-N in the supernatant was measured by UV spectrophotometry at wavelengths of 220 and 275 nm  according to the manufacturer’s protocol. The 16S rRNA gene was amplified using an S1000 Thermal Cycler (Bio-Rad) in 10-μL reaction volumes containing 5 μL of 2×Taq Master Mix (Dye Plus), 200 ng of template DNA, 1 μL of each primer (10 μmol L–1), and sterile distilled water. Primers and thermal profiles are listed in http://www.ncbi.nlm.nih.gov/BLAST) in order to compare the strains. The phylogenetic tree was constructed in MEGA 5.0 using the neighbor-joining method with 1,000 bootstrap replicates.Genomic DNA was extracted from NRCB010 and NRCB026 using the FastPureDNA gyrase subunit A gene (gyrA) and DNA gyrase subunit B gene (gyrB) were amplified and sequenced as described above. The gyrA and gyrB sequences obtained were used for a multilocus sequence analysis (MLSA). The nucleotide sequence alignment of concatenated genes consisted of 2,033 bp . The phylogenetic tree was constructed as described above.To identify more intimate inter- and intra-specific relationships for NRCB026, the P. stutzeri NRCB010 and Bacillus velezensis NRCB026 were evaluated under 0.6 mol L–1 NaCl conditions. To test the initial pH effects, the pH values of DM were adjusted to 6.0, 7.0, 8.0, 9.0, and 10.0. To test the C source effects, glucose, glycerin, and sodium acetate were selected as C sources to replace sodium succinate in DM. One milliliter of the strain suspension  was inoculated into 100 mL sterile medium and cultured at 30°C and 200 rpm for 72 h. OD600, NO3–-N, and NO2–-N were measured spectrophotometrically after an incubation for 0, 6, 12, 24, 30, 36, 48, and 72 h. Each treatment was performed in triplicate.The effects of initial pH and C sources in culture medium on N removal by 600=1.0) were inoculated into 250 mL of each water sample, and non-inoculated water was used as the control. Water samples were incubated at 30°C and 200 rpm for 72 h. Water samples were sampled at 6, 12, 24, 36, 48, 60, and 72 h to measure TN using the potassium persulfate oxidation-ultraviolet spectrophotometric method .IBM SPSS statistics for Windows  of IAA were produced by the seven isolates, among which NRCB010, NRCB023, and NRCB026 showed higher concentrations than the other strains (–1), siderophore activities (29.26–52.3%), and ACC deaminase activities.Varying concentrations (1.19–33.33 μg mL strains . The sevP<0.05) (P<0.05) (P<0.05), respectively. NRCB010 and NRCB026 exerted stronger plant growth-promoting effects than the other strains.In comparisons with the non-inoculated control, the height and root length of tomato seedlings significantly increased after inoculations with the seven isolates (P<0.05) a and b. (P<0.05) c. Dry we–1 NaCl, six isolates, except for NRCB001, showed good growth (OD600 higher than 1.0)  . Under 0isolates .600 values of these isolates generally decreased when the concentration of NaCl was ≥0.4 mol L–1  and NRCB026 (92.0%) at an initial pH of 7.0 after a 72-h incubation. NO2–-N concentrations exponentially increased within ~12 h of the incubation, reaching a maximum after a 24-h incubation, and decreasing thereafter  at pH 7.0–10.0 and 3.5% salinity. The salinity levels of coastal aquaculture wastewater range between 2 and 35 g LnapA, norB, and nosZ genes were successfully amplified from NRCB010  generally involves a series of reducing enzymatic reactions of microbes under anaerobic conditions; however, NO3– removal tests were performed under aerobic conditions in the present study. Additional experiments need to be conducted to confirm that the strains are aerobic denitrifiers, such as an oxygen sensitivity test on the transcription of denitrification functional genes. Since only nirS was detected in NRCB026 using the current primers  Simultaneous Nitrogen Removal and Plant Growth Promotion Using Salt-tolerant Denitrifying Bacteria in Agricultural Wastewater. https://doi.org/10.1264/jsme2.ME22025Supplementary Material


Arachis hypogaea L.) is a widespread oilseed crop of high agricultural importance in tropical and subtropical areas. It plays a major role in the food supply in the Democratic Republic of Congo (DRC). However, one major constraint in the production of this plant is the stem rot (white mold or southern blight) disease caused by Athelia rolfsii which is so far controlled mainly using chemicals. Considering the harmful effect of chemical pesticides, the implementation of eco-friendly alternatives such as biological control is required for disease management in a more sustainable agriculture in the DRC as in the other developing countries concerned. Bacillus velezensis is among the rhizobacteria best described for its plant protective effect notably due to the production of a wide range of bioactive secondary metabolites. In this work, we wanted to evaluate the potential of B. velezensis strain GA1 at reducing A. rolfsii infection and to unravel the molecular basis of the protective effect.Peanut (B. velezensis to reduce peanut disease caused by A. rolfsii both via direct antagonism against the fungus and by stimulating systemic resistance in the host plant. As treatment with pure surfactin yielded a similar level of protection, we postulate that this lipopeptide acts as main elicitor of peanut resistance against A. rolfsii infection.Upon growth under the nutritional conditions dictated by peanut root exudation, the bacterium efficiently produces the three types of lipopeptides surfactin, iturin and fengycin known for their antagonistic activities against a wide range of fungal phytopathogens. By testing a range of GA1 mutants specifically repressed in the production of those metabolites, we point out an important role for iturin and another unidentified compound in the antagonistic activity against the pathogen. Biocontrol experiments performed in greenhouse further revealed the efficacy of  The stem rot disease is characterized by yellowing and wilting of the main stem, lateral branches or the whole plant. It can also attack pods in the soil leading to losses estimated at 10-40% according to the production area   responsible for the biosynthesis of bioactive secondary metabolites (BSMs). This secondary metabolome largely contributes to the biocontrol activities of the strains and is chemically diverse, including volatiles, terpenes, non-ribosomal peptides (NRP), cyclic lipopeptides (CLPs) and polyketides (PKs), but also ribosomally-synthesized and post translationally-modified bacteriocins and lantibiotics (RiPPs)  . B. vele (RiPPs) . Cyclic  (RiPPs) . B. vele iturins . They co iturins . These C iturins B. velezensis for the biocontrol of peanut disease caused by A. rolfsii. To that end, we used the genetically amenable, natural rhizosphere isolate GA1 as representative of the species. Indeed, based on genome mining, this strain produces the whole panoply of bioactive secondary metabolites typically formed by members of this species  the test of pure CLP in planta for assessing the efficiency to reduce plant infection via the stimulation of systemic resistance.With this work, our objective was to evaluate the understudied potential of  species . Therefo22.1Bacillus velezensis strains used in this study was B. velezensis GA1 and its mutants (600nm) and adjusted to the desired concentration for all subsequent experiments.Bradyrhizobium arachidis (provided by the Belgian Co-Ordinated Collections of Microorganisms “BCCM”/Bacteria Collection LMG26795) was used in this study. Before each experiments, the bacteria were streaked on Yeast Extract Mannitol Agar (YEMA) ; 1 ml of Solution B (Per 100 ml: 5.28 g of CaCl2.2H2O); 1 ml of Solution C (Per 100 ml: 666 mg of FeCl3.6H2O); 20 g of agar). One colony was used to inoculate Yeast Extract Mannitol broth (YEM) for 3 to 7 days at 30°C and the pre-culture was washed with YEM three times. Cell concentration was evaluated (OD600nm) and adjusted to the desired concentration for all subsequent experiments2.2Athelia rolfsii (Curzi) Tu & Kimbrough  was reactivated on V8 juice agar . After this step, fungus was cultivated on Oatmeal agar OMA  to produce mycelia and sclerotia at 28°C. Inoculated petri dishes were stored at 4°C or collected sclerotia were retained in peptone water  at the same temperature for the next experiments.Fusarium oxysporum, Aspergillus niger and Rhizoctonia solani (lab collection) were cultivated on Potato Dextrose Agar PDA  to produce mycelia. Cultivated fungi were stored at 4°C for subsequent experiments.2.3Arachis hypogaea L.) cultivar JL24  - M’vuazi in Kongo Central (DRC)) were used. Peanut seeds were surface disinfected during 2 minutes in 70% ethanol prior to another 2 minutes in 5% Sodium Hypochlorite and finally three times washed in sterile distilled water. The effect of surface sterilization was checked by spotting 50 µl of the final rinse water onto LB plates. After sterilization, seeds were deposited in a petri dish containing sterile filter paper soaked with sterile water and placed in the dark at 22°C during 5 to 7 days until the emergence of an approx. 2 cm-long radicle. Sterile seedlings were then transferred in vials container covered with aluminum foil paper to avoid light filled with 15 ml Murashige and Skoog medium (MS) for hydroponic cultivation (growth chamber conditions: 16h-day and 8h-night cycle at 22°C). After 15 days of cultivation, natural peanut root exudates (NPRE) were filtrated through a membrane (0.22 µm) and stored at 4°C for further experiments.Seeds from healthy and highly quality pods of peanut  was extracted using 1.2 ml of methanol by vortexing 1 minute and then sonicated at room temperature for 20 min. After centrifugation, 500 µL of the supernatant was transferred into two centrifuge tubes for measurement of sugars and acids. For sugars, 20 µL of 3 mg/ml phenyl-β-D-glucopyranoside and 20 µL of 0.1 mg/ml 3-(4-hydroxyphenyl)-propionic acid were added as internal standards. The derivatization of sugars was performed by adding 120 μL methoxyamine-hydrochloride (20 mg/ml in pyridine) for 60 min at 30°C and with 120 μL N,O-bis (trimethylsilyl) trifluoro-acetamide (BSTFA) for 120 min at 45°C, 700 rpm. For amino acids and organic acids, 20 µL of 0.1 mg/ml 3-(4-hydroxyphenyl)-propionic acid was added to the sample as the internal standard and the derivatization was performed by adding 120 µL of methoxylamin-hydrochloride (20 mg/ml in pyridine) for 90 min at 37°C, 700 rpm, followed by addition of 120µl of dimethyl-tert-butylsilyl (TBDMS) and incubated for 30 min at 60°C, 700 rpm. After centrifuging, 100 µL of the supernatant was transferred to vials for detection using TSQ Duo GC-MS/MS (Thermo Fisher Scientific).4·7H2O; 0.34 g/l; K2HPO4; 3-morpholino-1-propanesulfonic acid MOPS 10.5 g/l; 0.25 g/l KCl, 1 g/l (NH4)2SO4; 1.2 mg/l Fe2(SO4)3; 0.4 mg/l MnSO4; 1.6 mg/l CuSO4, 4 mg/l Na2 MoO4, pH adjusted to 7. In case of assay on solid MMPRE, 20 g/l of agar was used for this preparation.Based on NPRE composition, medium mimicking peanut root exudates (MMPRE) was recomposed as follows: Maltose 1.09 g/l; Glucose 0.88 g/l; Arabinose 0.67 g/l; Fructose 0.28 g/l; Trehalose 0.14 g/l; Myo-inositol 0.07 g/l; Lactic acid 2.73 g/l; Succinic acid 0.13 g/l; Malonic acid 0.11 g/l; Fumaric acid 0.07 g/l; Pyruvic acid 0.03 g/l; Pyroglutamic acid 0.02 g/l; Malic acid 0.02 g/l; Citric acid 0.02 g/l; Valine 1.05 g/l; Leucine 0.57 g/l; Threonine 0.57 g/l; Isoleucine 0.42 g/l; Alanine 0.32 g/l; Phenylalanine 0.32 g/l; Tyrosine 0.15 g/l; Serine 0.14 g/l; Glycine 0.11 g/l; Asparagine 0.08 g/l; Glutamic acid 0.03 g/l. Salt and micronutrients were added with these proportions: 0.25 g/l MgSO2.5600nm) after 48 hours culture at 30°C. Relative quantification of CLP production was evaluated by UPLC-MS  in positive mode. MS parameters were set up as follows: capillary voltage: 3.5 kV; nebulizer pressure: 35 psi; drying gas: 8 L/min; drying gas temperature: 300°C; flow rate of sheath gas: 11 L/min; sheath gas temperature: 350°C; fragmentor voltage: 175 V; skimmer voltage: 65 V; Accurate mass spectra were recorded in the range of m/z = 100-1700. Five µL of samples were injected and separated using a C18 Acquity UPLC BEH column, 2.1 × 50 mm × 1.7 μm (Waters) at a flow rate of 0.6 mL/min and a temperature of 40°C. A gradient of 0.1% formic acid (solvent A) and acetonitrile acidified with 0.1% formic acid (solvent B) was used. First, 10% B was applied during 1 min before increasing to 100% in 20 min and maintained during 3.5 min before going back to initial ratio in 0.5 min. Identification of compounds was based on retention time (in-house database) and accurate mass. Relative quantification was made according to the peak area of each metabolite.Wild type strain GA1 and GA1-mutants were cultivated in liquid to evaluate bacterial growth and secondary metabolite production on NPRE and liquid MMPRE. Bacterial growth was evaluated by turbidity measurement (OD2.6600nm = 0.05) prepared as detailed in section 2.1. After this step, inoculated plantlets were transferred to vials containing 15 ml of sterile liquid Murashige and Skoog for 15 days in a growth chamber (day 16h/night 8h at 22°C). To evaluate the population, plants roots were immersed in 10 ml of peptone water solution supplemented with tween 80 (1 g/L) in which 10-15 sterile glassy beads were added to properly detach the bacterial cells from the root surface. Serial dilutions were prepared and 100 µl of each dilution were plated on solid medium LB. Plated petri dishes were then incubated at 28°C and cell emitting green fluorescence were counted using imaging system (Fusion Fx (Vilber Lourmat), AlexaFluor 488-Blue_AppStd). Bacillus population data were expressed to CFU/g root Dry Matter and calculated from two samples having a similar weight of root dry matter.Sterile peanut plantlets (see section 2.3) were immersed during 1 hour in 1 ml GFP tagged GA1 suspension (OD2.7600nm = 1) prepared as described in section 2.1 were spotted at 4.5 cm of the pathogen (A. rolfsii) on gelosed MMPRE. Fusarium oxysporum, Aspergillus niger and Rhizoctonia solani antagonism were tested in the same conditions on PDA plates. The plates were sealed with parafilm and incubated at 28°C for 6 days. The inhibition of mycelial growth was evaluated by measuring the distance between bacteria and the advancing front of the fungus. This experiment was performed using 6 independent repetitions. Impact of Bacillus volatiles in antagonism was performed using bipartite petri dishes. In this case, one compartment containing 10 ml of gelosed MMPRE was inoculated with 10 µl of GA1 culture (OD600nm = 1) prepared as detailed in section 2.1 and sclerotia were deposited at 4.5 cm from bacteria in the other part of the petri dish. The petri dishes were doubled sealed with parafilm and incubated at 28°C for 6 days. Inhibition rate (IR) was calculated with the formula: IR (%) = (S1-S2/S1) x 100 where S1: represents the fungal area growth without bacteria, and S2 represents the fungal area growth in inoculated plates. This experiment was performed using 6 independent repetitions.Ten µL of GA1 WT or mutants’ strains (OD2.8Bacillus suspension (50 ml OD600nm = 1) for 10 minutes and then sown directly into plastic pots  filled with 1 kg of sterilized soil . Before sowing one seed per pot, 10 ml of a bacterial suspension of B. arachidis (OD600nm = 1) were deposited on the seedbed of each seed of peanut. 14 days after sowing, 3 sclerotia of A. rolfsii were placed on the plant collar and 10 ml of bacterial suspension of B. arachidis were again inoculated at the stem basis. Three days after this step, B. arachidis suspension (10 ml OD600nm = 1/plant) were inoculated again by watering the stem basis and after 5 days, B. velezensis GA1 (50 ml OD600nm = 1/plant) was applied. These two microorganisms were applied once a week for three weeks. Assessment of disease incidence (DI) and disease severity (DS) . The characteristics of the soil used in this experiment were: pH: 5.35; Organic Carbon (%): 4.60; P available (ppm): 7.38; Nitrogen (%): 0.11; Potassium (ppm): 0.52; conductivity (µS/cm): 23.8. Twenty surface sterilized seeds (see section 2.3) were coated with a A. rolfsii, a system with two concentric plastic cups was used (adapted from B. arachidis (10 ml OD600nm = 1/plant) was poured in each large pot. Five days after seedlings transfer, GA1 suspension (50 ml OD600nm = 1/plant) or purified surfactin  was applied once again. The application of B. arachidis, GA1 and surfactin was performed once a week for three weeks. Assessment of DI and DS . One-way ANOVA  followed by Dunn’s multiple comparisons in GraphPad software were used specially to compare DS between treatments. The proportions of diseased plants and healthy plants (DI) after treatment by B. velezensis GA1 were assessed by Pearson’s chi-squared test followed by the pairwise tests of independence for nominal data. These tests were applied with the “pairwiseNominalIndependence” and the “cldList” functions of the R companion package   within 4 days post inoculation. We next determined the pattern of BSMs formed by the bacterium in these conditions and UPLC-qTOFMS analysis of the corresponding cell-free crude extract revealed significant amounts of the lipopeptides iturin and surfactin but not fengycin or other non-ribosomal products potentially produced by the strain . The amounts of plant chemicals utilizable by bacteria to form biomass are usually quite low in these naturally produced exudates but we observed a substantial development of GA1 with final cell density that reached an OD600nm = 0.73) upon growth of GA1 in this mimicking NPRE (MMPRE) together with increased amounts of CLPs compared with the production in NPRE. The relative proportions of iturins and surfactins as well as the homologue distribution within each family were similar to NPRE but detectable amounts of fengycins were observed in these conditions  and severity (p<0.0001)  was observed by comparing A rolfsii-infected plants (disease control) and infected plants previously treated with GA1   in the genome  also belonging to the Fabaceae family (Importantly, a successful biocontrol agent must meet a number of criteria, including being ecologically compatible with other plant-beneficial bacteria such as rhizobia in leguminous plants . Upon inn peanut  and soybe family .B. velezensis to protect peanut against an important fungal disease. This further support the potential of the species as powerful biocontrol agent keeping in mind that rational management of its mode of application is required to avoid negative impact on natural symbiosis, which is crucial for the ecology and health of such kind of crop.A better understanding of the molecular processes driving such multitrophic interactions between the host plant and mutualistic, symbiotic and pathogenic microbes is necessary, but our work provide new insights into the mechanisms used by The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.VKA performed most of the microbiology and greenhouse experiments with the technical support of CH and advisory help of PD. AAA performed metabolomics using UPLC-MS and data analysis. BR and BC performed the analysis for root exudate composition. VKA, AAA, and MO mainly wrote the manuscript. MO supervised the study. All authors contributed to the article and approved the submitted version.


Ferlin is a single transmembrane protein with multiple Ca2+-binding C2 domains, and there are six ferlins, dysferlin (DYSF), otoferlin (OTOF), myoferlin (MYOF), fer-1–like 4 (FER1L4), FER1L5, and FER1L6, in mammals. Dysf, Otof, and Myof knockout mice have been generated, and each knockout mouse line exhibited membrane fusion disorders such as muscular dystrophy in Dysf, deafness in Otof, and abnormal myogenesis in Myof. Here, by generating mutant mice of Fer1l4, Fer1l5, and Fer1l6, we found that only Fer1l5 is required for male fertility. Fer1l5 mutant spermatozoa could migrate in the female reproductive tract and reach eggs, but no acrosome reaction took place. Even a Ca2+ ionophore cannot induce the acrosome reaction in Fer1l5 mutant spermatozoa. These results suggest that FER1L5 is the missing link between Ca2+ and the acrosome reaction.Spermatozoa need to undergo an exocytotic event called the acrosome reaction before fusing with eggs. Although calcium ion (Ca 2+-activated exocytosis in spermatozoa.FER1L5, a single transmembrane protein with multiple C2 domain, regulates Ca Fertilization is a union of two gametes, spermatozoa, and oocytes. To fertilize eggs, spermatozoa need to travel a long distance in the female reproductive tract using flagella. During the migration, the acrosome located anterior to the nucleus in sperm heads needs to undergo an exocytotic event called the acrosome reaction to fertilize eggs  (v2.3Ca or Plcd4 result in impaired acrosome reaction in response to solubilized zona pellucida (ZP), the extracellular matrix surrounding the eggs  proteins that are involved in fusing vesicles with the target membrane . Each mutant mice were viable and had no gross abnormality in appearance or behavior. We then analyzed male fertility by mating each mutant male with wild-type females. While Fer1l4 and Fer1l6 mutant males were fertile (fig. S2A), the fertility of Fer1l5 mutant males was severely impaired . We then focused on the analysis of Fer1l5. In contrast to male fertility, Fer1l5 mutant females were fertile (fig. S2B). To ensure that Fer1l5 is responsible for impaired male fertility, we generated transgenic (Tg) mice that expressed FLAG-tagged or PA-tagged Fer1l5 under testis-specific calmegin (Clgn) promoter (fig. S2C). We detected FER1L5 in the testis of Tg mice with immunoblotting following immunoprecipitation  and confirmed that both Fer1l5-FLAG and Fer1l5-PA transgenes rescued impaired male fertility of Fer1l5 mutant mice (fig. S2F).To analyze the function of impaired . BecauseF domain  and starF domain , the funFer1l5 mutant mice, we analyzed testis and cauda epididymis sections, but no apparent abnormalities were found in Fer1l5 mutant mice , but a few ZP-free eggs were fertilized . These results indicate that FER1L5 is required for the acrosome reaction and functions downstream of Ca2+ influx.Because immunostaining of IZUMO1 of fixed spermatozoa cannot tell the differences between live and dead spermatozoa, we also analyzed the acrosome reaction rates of live spermatozoa using Red Body Green Sperm (RBGS) Tg mice, whose spermatozoa lose the enhanced green fluorescent protein (EGFP) fluorescence signal after the acrosome reaction , Fer1l5 Fer1l5 mutant spermatozoa  cells and performed an immunoprecipitation analysis using an anti-FLAG antibody  and regulates Caantibody . After iantibody . In contantibody  (34). ThFer1l5 mutant spermatozoa could pass through the utero-tubal junction (UTJ) that connect the uterus and oviduct (fig. S5B) and reach the ZP surrounding the eggs in the ampulla  injection].  ampulla , indicat-reacted . These rC. elegans and MISFIRE regulating the sperm plasma membrane breakdown in D. melanogaster. These results indicate that ferlin function in spermatozoa is evolutionarily conserved, although vesicle fusion during fertilization takes place at different times. In C. elegans, vesicle fusion occurs during spermatid activation . acrosome .C. elegans, it has been reported that FER-1 is localized in the membranous organelles . In contrast, a few pups were obtained from Fer1l5 mutant males in vivo  or Japan SLC . Mice were maintained under specific pathogen–free conditions in a temperature-controlled environment, 12-hour light/12-hour dark cycle, and ad libitum feeding. All animal experiments were approved by the Animal Care and Use Committee of the Research Institute for Microbial Diseases, Osaka University (#Biken-AP-H30-01) and were conducted in accordance with the guidelines established by the Research Institute for Microbial Diseases, Osaka University.Fer1l6) (www.mousephenotype.org/) (Fer1l4 and Fer1l5) (www.ncbi.nlm.nih.gov/nuccore/378747675) (Fer1l4 mutant mice and EGR-G01 for generating Fer1l5 and Fer1l6 mutant mice) ] signal under the mouse Clgn promoter (fig. S2C) was prepared. The linearized DNA was injected into one of the pronuclei of fertilized eggs that were obtained by the mating of superovulated B6D2F1 females and B6D2F1 males, and the zygotes were cultivated in KSOM to two-cell stage and then transferred into pseudo-pregnant females.Mouse 2.HEK293T cells (Drosophila MISFIRE (NP_001137919.1), human MYOF (NP_038479.1), mouse MYOF (NP_001093104), zebrafish MYOF (NP_957169.2), human DYSF (NP_001124459.1), mouse DYSF (NP_067444.2), zebrafish DYSF (XP_005172350.1), human FER1L5 (NP_001280012.1), mouse FER1L5 (NP_001264005.1), nematode FER-1 (NP_492337.1), human FER1L6 (NP_001034201.2), mouse FER1L6 (NP_001357841.1), zebrafish FER1L6 (XP_021334459.1), human OTOF (NP_919224.1), mouse OTOF (NP_114081.2), zebrafish OTOF (NP_001025283.1), zebrafish OTOFB (XP_017207363.1), mouse FER1L4 (NP_001130028.1), and zebrafish FER1L4 (XP_021335523.1).The phylogenetic trees of candidate genes were created using the neighbor-joining method with the GENETYX software . Amino acid sequences of each protein were obtained from the National Center for Biotechnology Information Entrez Protein database. The accession numbers of each amino acid sequence used were as follows: Mouse RNAs were prepared from multiple adult tissues of C57BL/6N or testes from 1- to 5-week-old males using TRIzol . The obtained RNA was immediately reverse-transcribed to cDNA with SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific) using oligo(dT) as primers. PCR primers for each gene are shown in table S1.Fer1l5 mutant mice, plugs were checked every morning.Sexually matured male mice were individually caged with two 8-week-old B6D2F1 female mice for 2 months, and the number of pups was counted. For Male mice (12 to 18 weeks old) were euthanized, and testes and cauda epididymides were dissected. Testes and cauda epididymides were fixed overnight at 4°C in Bouin’s fluid , embedded in paraffin, and sectioned at a thickness of 5 μm. Paraffin sections were then rehydrated, treated with 1% periodic acid for 20 min at room temperature, and incubated with Schiff’s reagent  for 20 min at room temperature. The sections were stained with Mayer’s hematoxylin solution  and observed using a BX-53 microscope .Spermatozoa were collected from the cauda epididymis and dispersed in phosphate-buffered saline (PBS) (Thermo Fisher Scientific). The spermatozoa were observed using a BX-53 microscope (Olympus).2. Ten minutes, 2 hours, or 4 hours after incubation, spermatozoa were collected from the top of the drop. Sperm motility was analyzed using the CEROS sperm analysis system . Analysis settings were used as described previously (Spermatozoa obtained from the cauda epididymis were incubated in a drop of Toyoda Yokoyama Hosi (TYH) medium  HyperOva  was injected into the abdominal cavity of B6D2F1 female mice, followed by hCG  with 48-hour interval. Eggs were then collected from the superovulated females, treated with hyaluronidase  for 10 min to remove the cumulus cells (cumulus-free eggs) or with collagenase  for 10 min to remove the ZP (ZP-free eggs). The spermatozoa were added to the TYH drops that contain intact, cumulus-free, or ZP-free eggs at a final density of 2 × 105 or 2 × 106 spermatozoa/ml and incubated at 37°C under 5% CO2. The formation of pronuclei was observed 6 hours after insemination for ZP-free eggs. Two-cell embryos were counted the next day for intact or cumulus-free eggs.Spermatozoa collected from the cauda epididymis were incubated in a drop of TYH medium for 2 hours at 37°C under 5% CO2. To induce the acrosome reaction with Ca2+ ionophore, 20 μM A23187  was added after 4 hours of incubation. The spermatozoa were spotted onto slides, air-dried, fixed with 4% paraformaldehyde for 10 min, and washed in PBS for 5 min. The slides were blocked with PBS containing 5% bovine serum albumin and 10% goat serum for 1 hour at room temperature. The slides were incubated with anti-IZUMO1 antibody (1:500)   was added after 4 hours of incubation. Propidium iodide (PI) (10 μg/ml) was added to the drop, and an aliquot of the sperm suspension was placed on a glass slide. Acrosome reaction rates were determined by observing EGFP signals while distinguishing viable cells with PI staining with a BX-53 microscope (Olympus). Spermatozoa with EGFP signals in the acrosome were counted as acrosome intact. More than 200 spermatozoa were counted for each trial.B6D2F1 female mice were superovulated as mentioned above and mated with male mice that contained the RBGS transgene. Two hours after observation of vaginal plugs or 20 hours after hCG injection, female reproductive tracts were dissected and spermatozoa inside the oviduct or ampulla were observed. To confirm that the spermatozoa were alive, motile spermatozoa were observed using a Nikon Eclipse Ti microscope connected to a Nikon C2 confocal module . Spermatozoa with EGFP signals in the acrosome were counted as acrosome intact.2. To induce the acrosome reaction with a Ca2+ ionophore, 20 μM A23187 (Merck) was added after 4 hours of incubation. The spermatozoa were washed once with cold PBS and then with electron microscope (EM) buffer . The samples were fixed with 1% glutaraldehyde in EM buffer for 1 hour at 4°C and washed once with EM buffer. Spermatozoa with a final concentration of 2 × 106 spermatozoa/ml in EM buffer were spotted onto a cover slip coated with 0.001% poly-l-lysine using Cytospin 4 (1000 rpm for 3 min) (Thermo Fisher Scientific). The samples were washed three times for 5 min each in 0.1 M phosphate buffer (pH 7.4) containing 4% sucrose and postfixed in 1% OsO4 and 0.5% potassium ferrocyanide of 0.1 M phosphate buffer (pH 7.4) for 60 min at room temperature. After washing in distilled water, samples were dehydrated in a graded series of ethanol solutions: 50, 70, and 90% ethanol on ice and three times in 100% ethanol for 10 min at room temperature. Dehydrated samples were incubated in 50% epoxy resin mixture dissolved in 100% ethanol (ethanol:resin = 1:1) for 60 min at room temperature. Samples were incubated twice in pure epoxy resin mixture for 60 min each at room temperature and embedded in epoxy resin for 2 days at 60°C. Ultrathin sections (80 nm in thickness) were obtained and stained with 8% uranyl acetate solution for 30 min, briefly washed three times in distilled water, stained with lead staining solution for 2 min, and washed three times in distilled water. The ultrathin sections were observed using a JEM-1400 plus EM  at 80 kV with a charge-coupled device Veleta 2K × 2K camera (Olympus).Cauda epididymal spermatozoa were dispersed in a drop of TYH medium for 10 min or 4 hours of incubation at 37°C under 5% COg for 15 min at 4°C to collect supernatants. Samples were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) under reduced conditions and transferred to polyvinylidene difluoride membrane using the Trans-Blot Turbo system . The blots were blocked with 10% skimmed milk or Blocking One , incubated with primary antibodies  overnight at 4°C, and incubated with secondary antibodies conjugated to horseradish peroxidase (1:5000) for 2 hours at room temperature. The antibodies used can be found in table S2. The signals were detected with Chemi-Lumi One Super  or Chemi-Lumi One Ultra .For analyzing tyrosine phosphorylation, spermatozoa were collected from the cauda epididymis and incubated in TYH medium for 10 min, 1 hour, or 2 hours. The spermatozoa were then lysed with a sample buffer  and centrifuged at 15,300g for 15 min at 4°C to collect the supernatants.Testes were lysed with a solution containing 1% Triton X-100, 50 mM NaCl, 20 mM tris-HCl (pH 7.4), and protease inhibitor mixture ; incubated for 1 hour at 4°C; and centrifuged at 15,300Fer1l5, Stx2, and Adam1b were amplified from mouse testis (C57BL/6N) and cloned into FLAG-tagged (C terminus), PA-tagged (C terminus), and untagged pCAG vectors, respectively, that contain the cytomegalovirus immediate enhancer/β-actin (CAG) promoter and a rabbit globin poly(A) signal  or anti-PA antibody –conjugated Dynabeads (Thermo Fisher Scientific). The complexes were washed three times with a solution containing 40 mM tris-HCl, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol and eluted with a sample buffer. Immunoblotting with collected samples were performed as mentioned above.g for 15 min at 4°C to collect supernatants. The samples were subjected to MS analyses directly or after SDS-PAGE under reduced condition. To visualize proteins in gels, silver staining was performed following the manufacturer’s protocol . Mass spectrometry analyses were performed as described previously with some modifications , and protease inhibitor mixture ; incubated for 2 hours at 4°C; and centrifuged at 15,300t test was performed using Excel . Significant differences were considered at *P < 0.05, **P < 0.01, and ***P < 0.001. Data represent the means ± SD.A two-tailed unpaired Student’s


Agrobacterium tumefaciens has frequently been found in Agrobacterium-mediated plant transformation. This overgrowth can reduce transformation efficiency and even lead to explant death. Therefore, this research investigates an alternative way to mitigate or eliminate Agrobacterium after transformation using a bacteriophage. To develop this alternative method, we conducted effectiveness studies of two lytic bacteriophages (ΦK2 and ΦK4) and performed an application test to control Agrobacterium growth after transformation. According to plaque morphological characterization and molecular analysis, the two bacteriophages used in this experiment were distinct. Moreover, some stability physicochemical and growth kinetics, such as adsorption time and susceptibility test, also showed that both bacteriophages differed. On the other hand, the optimum temperature and pH of both phages were the same at 28–30 °C and pH 7. Further investigation showed that both ΦK2 and ΦK4 were able to reduce the overgrowth of A. tumefaciens post transformation. Moreover, applying the cocktail (mixture of ΦK2 and ΦK4) with antibiotic application eradicated A. tumefaciens (0% overgrowth percentage). This result indicates that the application of bacteriophage could be used as an alternative way to eradicate the overgrowth of A. tumefaciens subsequent to transformation.Overgrowth of  Agrobacterium tumefaciens-mediated transformation is commonly used in plant genetic engineering ). Restrictions followed the manufacturer’s protocol; DNA (1 µg/µL) was digested individually with 10 U of enzyme in a total reaction of 20 µL and incubated at 37 °C for 3 h. After incubation, the restriction results were electrophoresed using 1% (w/v) agarose at a voltage of 80 V for 60 min and stained with ethidium bromide. The agarose gel was then visualized with UV Transilluminator gel .The bacteriophage genome was isolated using the WizardRAPD markers were performed by amplifying DNA using GoTaq Green Master Mix (Promega) in a 10 µL reaction with the following composition: 1 µL of DNA template (5 ng/μL), 5 µL of master mix, 1 µL of primer, and 3 µL of nuclease-free water (Promega). The PCR conditions for RAPD35 (CCGCAGCCAA) and RAPD37 (AACGCGCAAC) were as follows: pre-denaturation at 95 °C, 3 min; denaturation at 95 °C, 3 s; annealing at 48 °C, 30 s; extension at 72 °C, 1 min; post extension at 72 °C, 8 min. The PCR results were visualized using electrophoresis following the same method as above.Agrobacterium and Escherichia  and E. coli . Furthermore, observations were made and classified as a function of the degree of spot brightness into two different classes: (+) bacteriophage-susceptible; (−) bacteriophage-resistant [Host range testing using the spot test method refers to Narulita et al. . The bacherichia . Specifiesistant .2/MgCl2 buffer, it was incubated at 28 °C, 30 °C, 40 °C, 50 °C, and 60 °C for 60 min. Furthermore, plaque calculations were performed using a standard double layer [A total of 100 µL of bacteriophage was added to 900 µL of LB, adjusting the pH from 3 to 12. The mixture was incubated at 28 °C for 60 min and continued with a double layer. To test the temperature stability of bacteriophage stock in CaClle layer .A. tumefaciens AGLI (108 CFU/mL) and bacteriophage (103 PFU/mL). The mixture was incubated at 28 °C. Subsequently, sampling was performed at 0, 5, 10, 15, 20, 25, and 30 min and centrifuged at 13,000× g for 10 min to separate the adsorbed bacteriophages. Then, 100 µL of supernatant was taken, double-layered, and counted for non-adsorbed bacteriophage particles. The number of non-adsorbed bacteriophages and bacterial hosts at time 0 was 100% [The adsorption kinetics test used was 100% .8 CFU/mL were mixed with bacteriophages (103 PFU/mL), and then left for 10 min for adsorption onto the bacterial cell. Then, 1 mL of the mixture was taken and put into 9 mL of medium in a new flask and left for another 10 min. The same procedure was repeated until the dilution was 10−4. The mixed cultures were incubated at 28 °C for 6 h. Samples of up to 100 µL were taken from each flask at a predetermined time to avoid the PFU/mL value being too numerous to count [Bacteria that entered the mid-log phase of 10to count . The burto count .5, 106, or 107 PFU/mL and A. tumefaciens (106 CFU/mL) were mixed, whereas the cocktail treatment was performed using a bacteriophage concentration of 107 PFU/mL. As a control, LB was only inoculated with bacteria. All treatments were incubated at 28 °C and observed for 12 h. The OD600 was observed every 1 h interval using a 96-well plate. All treatments were repeated three times.Several concentrations of bacteriophage were used to calculate the susceptibility of each isolate according Muturi et al.  with minA. tumefaciens bacteria were cultured overnight and then diluted with sterile distilled water to obtain an OD600 = 0.01 (106 CFU/mL). Next, a mixed culture was generated with bacteriophages at several concentration values, namely 107, 108, and 109 PFU/mL. The mixed culture was then incubated for 180 min. The mixed culture measurements were taken at 40 and 180 min. A total of 1 mL of mixed culture was taken at each predetermined sampling time, and then a plaque assay was performed by pouring 4 mL of top agar over the basal LB. Overgrowth was indicated by counting the number of colonies that grew on the medium after incubation for 72 h at 28 °C. Tests were also carried out using a cocktail of ΦK2 and ΦK4 at 108 PFU/mL with a 1:1 (v/v) ratio. The test was carried out using three individual replications.This method was developed by PT Wilmar Benih Indonesia, Cikarang-Indonesia. A. tumefaciens OD600 = 0.1 carrying the plasmid pCAMBIA1300-eGFP-hpt in 50 mL of AAM + 100 mM acetosyringone medium (USA) [Transformation explants used calluses derived from the scutellum of the Nipponbare rice plant . A totalum (USA) . For thev/v) with a phage cell density of 1011 PFU/mL for 40 min. For the control treatment, washing was carried out using only phage buffer, i.e., CaCl2/MgCl2  buffer; the treatment details are shown in According to Tamzil et al. , the calThe number of contaminated calluses was observed for 4 weeks. Furthermore, the percentage overgrowth was calculated using the following formula: percentage contamination = / × 100%. The data were analyzed using one-way ANOVA followed by a Tukey HSD test (α = 0.05). All data were visualized using Origin Pro 2019b software .In this paper, we concluded that the two bacteriophage isolates were different on the basis of several characterization results. As a function of the morphological plaque, both ΦK2 and ΦK4 were categorized as lytic bacteriophages with different plaque sizes . Furthermore, the molecular analysis supported the morphological observation result, with both phages showing a different pattern of RFLP and RAPD.The effectiveness of phage applications depends on several factors, such as the environmental conditions (pH and temperature), phage adsorption rate, burst size, and latent period. The optimal temperature and pH of isolates were at 28–30 °C and pH 7. The optimal adsorption times for ΦK2 and ΦK4 were 27 min and 20 min, respectively. In addition, a susceptibility test showed that ΦK4 had greater inhibitory properties than ΦK2. The information gained in the effectiveness studies was used for the application test.A. tumefaciens growth post transformation. During the single application test, ΦK2 and ΦK4 could significantly reduce the overgrowth compared to the control, and there was no significant difference between ΦK2 and ΦK4. Interestingly, the application of a cocktail without antibiotics significantly reduced the Agrobacterium growth compared to the control. This phage cocktail application could banish the side-effects of antibiotic application. Nevertheless, the highest suppression was shown in the cocktail treatment combined with antibiotics (percentage overgrowth of 0%). This result indicates that bacteriophage application could be used as an alternative to eradicate the Agrobacterium tumefaciens overgrowth post transformation.In the application test, the percentage overgrowth showed that ΦK2 and ΦK4 or a cocktail could reduce or even eliminate the


A major challenge in the treatment of liver cancer is that a large proportion of patients fail to achieve long-term disease control, with death from liver cancer cell migration and invasion. Acid-sensitive ion channel 1α (ASIC1α) is involved in the migration, invasion, and proliferation of liver cancer cells. Therefore, we explored the mechanism of ASIC1α-mediated liver cancer cell migration and invasion. We determined the levels of ASIC1α by western blotting and immunofluorescence in HepG2 and SK-Hep1 cells cultured in various acidic conditions. In addition, wound healing assay, transwell invasion assay, and MTT assay were conducted to assess the migration, invasion, and proliferation abilities of liver cancer cells. Western blotting was conducted to determine the levels of MMP2, MMP9, ASIC1α, p-PI3Kp85, t-PI3Kp85, p-AKT(Ser473), t-AKT, p-mTOR (Ser2448), t-mTOR. We first found that the levels of ASIC1α in the HepG2 and SK-Hep1 cells in acidic conditions (pH 6.5) were significantly increased. Inhibition and knockdown of ASIC1α down-regulated MMP-2/9 expression and inhibited the migration, invasion, and proliferation of HepG2 and SK-Hep1 cells; overexpression of ASIC1α had the opposite effect. We further demonstrated that ASIC1α up-regulates MMP-2/9 via activation of the PI3K/AKT/mTOR pathway, thereby promoting migration, invasion, and proliferation of liver cancer cells. Overexpression of MMP-2/9 and activation of AKT reversed these effects on liver cancer cells caused by inhibition of ASIC1α. We conclude that ASIC1α can regulate migration, invasion, and proliferation of liver cancer cells through the MMP-2/9/PI3K/AKT/mTOR pathway. These observations may provide a new reference for liver cancer chemotherapy.The online version contains supplementary material available at 10.1186/s12885-022-09874-w. Liver cancer is the most common lethal malignancy in the world . MalignaAcid-sensing ion channels (ASICs) are cation-permeable protein complexes that belong to the epithelial-channel mutagenic protein ion channel superfamily. ASICs play an important role in sensing the pH of body fluids and regulating physiological functions such as pain and sour taste. ASIC1α is involved in pathological behaviours and regulation of biological functions that are identified with ASIC subunits 1α, 1b, 2α, 2b, 3, and 4 . The higMatrix metalloproteinases (MMPs) maintain the homeostasis of extracellular matrices and play a key role in tissue remodelling, organogenesis, inflammation, and tumour progression –16. MMP2Abnormal activation of the PI3K/AKT/mTOR pathway has a key role in biological behaviours, such as proliferation, migration, invasion, and apoptosis of tumour cells –26. DownIn this study, we identified the function of ASIC1α, explored the role of ASIC1α in the migration, invasion, and proliferation of HepG2 and SK-Hep1 cells, and elaborated on a molecular mechanism involved. This information may lead to a new way to control the progression of malignant liver cancer and to treat patients with malignant liver cancer to improve their prognosis.Concentrated hydrochloric acid was slowly added to 20 mL of normal culture (pH 7.4) RPMI 1640 medium, and the pH was determined with a pH meter. When the pH reached 6.5, the added amount of concentrated hydrochloric acid was recorded. Then, concentrated hydrochloric acid was added to the RPMI 1640 medium according to the volume ratio of the medium to the concentrated hydrochloric acid to achieve a medium with pH 6.5.2. The HepG2 and SK-Hep1 cells were cultured in acidic (pH 6.5) medium for 4 h to activate ASIC1a.The human liver cell line L-02 was received from the Chinese Academy of Sciences ; the human liver cancer cell line SK-Hep1 was obtained from Cellcook Biotech Co., Ltd. ; and the HepG2 cell line was acquired from Mingjin Biotech Co., Ltd. . The cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS at 37 ℃ with 5% COA potent and selective ASIC1α blocker, Psalmotoxin 1 (PcTx1) (#B5796), and a type IV collagen-specific MMP-2 and MMP-9 inhibitor, cis-ACCP (#C4501), were purchased from APExBIO Technology LLC . LY294002 (a broad-spectrum PI3K inhibitor) (#S1105), MK-2206 (a AKT inhibitor) (#S1078), Rapamycin (a specific inhibitor of mTOR) (#S1039) and NVP-BEZ235  (#S1009) were obtained from Selleck Chemicals . SC79 (a brain-permeable AKT phosphorylation activator) (#HY-18749) was obtained from MedChem Express . Primary antibodies for p-PI3Kp85 (#4228), t-PI3Kp85 (#4292), p-AKT(Ser473) (#4060), t-AKT (#4691), p-mTOR(Ser2448) (#2971) and t-mTOR (#2972) were purchased from Cell Signaling Technology . Antibodies to MMP-2 (#ab92536) and MMP-9 (#ab283575) were obtained from Abcam Biological Technology . Antibody against ASIC1a (#ASC-014) was purchased from Alomone Labs Company . Antibody to β-actin (#AF5003) was received from Biosharp Life Science . The BCA-200 protein assay kit  was obtained from Thermo Scientific . Phosphatase inhibitor tablets (#P1045) were obtained from Beyotime Biotechnology . ASIC1α-specific shRNA lentiviral particles, ASIC1α gene-lentiviral particles, MMP-2 gene-lentiviral particles, and MMP-9 gene-lentiviral particles were obtained from Genechem Co., Ltd .HepG2, SK-Hep1, and L-02 cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, permeated with 0.5% Triton-100 for 10 min, and blocked with 5% BSA for 1 h at room temperature. Anti-ASIC1α (1:200) was incubated at 4 °C overnight, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000) was incubated in the dark for 1 h at room temperature. Finally, the cells were stained with DAPI  for 15 min and imaged with an inverted fluorescence microscope.The ASIC1a-specific shRNA lentiviral particles were transfected into cells according to the manufacturer’s protocol. The shRNA for gene silencing areASIC1a shRNA: CCGGCTATGGAAAGTGCTACACGTTCTCGAGAACGTGTAGCACTTTCCATAGTTTTT.The MMP-2, MMP-9, and ASIC1a lentiviral particles was transfected into cells as according to the manufacturer’s protocol. The primers for MMP-2, MMP-9 and ASIC1a overexpression are5′-GATACCCCTTTGACGGTAAGGA-3′ (forward)and 5′-CCTTCTCCCAAGGTCCATAGC-3′ (reverse);5′-GTGCTGGGCTGCTTTGCTG-3′ (forward) and5'-GTCGCCCTCAAAGGTTTGGAAT-3′ (reverse);5′-GAGGATCCCCGGGTACCGGTCGCCACCATGGAACTGAAGGCCGAGGAGGAG-3′ (forward)5′-TCCTTGTAGTCCATACCGCAGGTAAAGTCCTCGAACGTG-3′ (reverse), respectively.HepG2, SK-Hep1, and L-02 cells were inoculated into six-well plates with 90% confluence the next day. The cells were scraped with a 20 µL plastic tip. After the cells were washed with PBS, photographs were taken at 0 h and 48 h. Scratch-healing ability was expressed as migration area relative to 0 h.BD Matrigel  was held at 4 °C to become liquid and added to the serum-free medium at a ratio of 1: 5 and mixed . One hundred microliters of the mixed solution were added to the upper chamber and placed in a 37 °C incubator for 5 h. The cells were digested, counted, and prepared for cell suspension. The experiment thereafter proceeded in accordance with our described method .5 cells/well overnight, and 10 µL MTT solution  were added and cultured for 4 h. Dimethylsulfoxide  was added and measured at OD 490 nm. Blank wells  and control wells  were set. Cell viability was calculated as (OD in test well-OD in blank well) / (OD in control well-OD in blank well) × 100%.Cells were inoculated into 96-well plates at a density of 1 × 10http://imagej.nih.gov/ij/). Due to insufficient experimental funds, in the western blot assay, we cut the PVDF membrane into a membrane small enough to incubate the antibody according to the molecular weight of the incubated antibody and the protein molecular weight marker. In addition, protein samples of related research topics that were conducted simultaneously were added to the same gel for protein electrophoresis.Cultured cells were lysed in microcentrifuge tubes containing a mixture of protease and phosphatase inhibitors. The protein concentration was determined with a BCA-200 Protein Assay Kit. Various concentrations of SDS-PAGE were used to separate the protein samples, which were electrotransferred onto PVDF membranes. After sealing for 1 h at room temperature with 5% skim milk dissolved in TBST, the membranes were reacted overnight at 4 °C, followed by the incubation of secondary antibodies at room temperature for one hour. The density of the target band was quantified with an ImageJ digital imaging system  or pH 6.5 medium. As shown in Figs. .4 or pH P < 0.05), whereas 10 nM PcTx1inhibited the migration of SK-Hep1 cells (P < 0.01). Transwell invasion assays were then used to analyze invasion of the cells. As displayed in Figs. P < 0.01).  Lastly, MTT assay was used to analyze proliferation of various groups of cells. As illustrated in Figs. The ability of PcTx1 (a potent and selective ASIC1α blocker) to inhibit ASIC1α in the HepG2 and SK-Hep1 liver cancer cells was measured with western blotting. When the concentration of PcTx1 reached 20 nM, ASIC1α was inhibited in pH 6.5 medium  in the HepG2 (pH 6.5)-shASIC1α cells compared with migration in the HepG2 (pH 6.5) cells  in the HepG2 (pH 6.5)-shASIC1α cells compared with invasion in the HepG2 (pH 6.5) cells. Similar results were obtained with SK-Hep1 cells. Lastly, we used the MTT assay to test the proliferation of HepG2 and SK-Hep1 cells after treating them at 5 time points . As shown in Fig. Since the expression of ASIC1α was significantly upregulated in liver cancer cells cultured in pH 6.5 medium, we silenced the ASIC1α gene by shRNA to study the effect of the ASIC1α protein on migration, invasion, and proliferation of the HepG2 and SK-Hep1 cells. The silencing effect was detected by western blotting. As shown in Figs. ls Figs. B1 and B2ls Figs. C1 and C2P < 0.01)   . Thus, ASIC1α overexpression resulted in increased migration and invasion of both liver cancer cell lines, HepG2 and SK-Hep1. Finally, we tested the proliferation ability of the liver cancer cells with MTT assay at 5 time points  and found that overexpression of ASIC1α enhanced proliferation of the cells  Figs. B1 and B21) Figs. C1 and C25) Figs. D1 and D2P < 0.001) and MMP-2 (P < 0.001) in HepG2 (pH 6.5) + cis-ACCP (a type IV collagen-specific MMP-2 and MMP-9 inhibitor), HepG2 (pH 6.5) + PcTx1 (20 nM) group, and HepG2 (pH6.5)-ASIC1α shRNA group were significantly decreased, while the expression levels of MMP-9 and MMP-2 in the HepG2 (pH 7.4)-ASIC1α cells were not significantly different. Similar results were obtained with SK-Hep1 cells. These data indicated that ASIC1α could regulate the expression of MMP-2/9. Next, we examined the migration ability of liver cancer cells. As shown in Figs. P < 0.05). Similar results were obtained with SK-Hep1 (pH 6.5) cells. We also examined the invasion abilities of HCC cells, as shown in Figs. P < 0.05) in HepG2 (pH 6.5) + cis-ACCP cells, HepG2 (pH 6.5) + PcTx1(20 nM) cells, and HepG2 (pH6.5)-ASIC1α shRNA cells. Similar results were obtained with SK-Hep1 (pH 6.5) cells.  We then measured the proliferation ability of the various cells with MTT assay. ASIC1α treatment resulted in enhanced proliferation ability of liver cancer cells , whereas the migration area relative to 0 h of overexpression of MMP-2/9 and activation of AKT cells were neither significantly increased nor decreased  after overexpression of MMP-2/9 and activation of AKT in HepG2 (pH 7.4) cells and SK-Hep1 (pH 7.4) cells. As illustrated in Figs. ed Figs. E1 and E2ed Figs. F abilityThe migration and invasion properties of liver cancer cells are key attributes for promoting liver cancer metastasis and malignant progression. The extracellular acidic microenvironment (about pH 6.5) is a common feature of most solid tumours and plays an important role in tumour growth, migration, and invasion –30. ASICCancer is caused by genetic mutations and disruption of cellular homeostasis. These alterations create an extracellular environment called the tumour microenvironment, which includes surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, various signaling molecules, and the extracellular matrix, and affects tumour development and metastasis. The acidic pH of the tumour microenvironment caused by altered tumour cell metabolism is characteristic of abnormal cell–cell interactions and disruption of homeostasis. In this state, tumour cells preferentially utilize glycolysis over oxidative phosphorylation as the primary means of energy release, an effect known as anaerobic glycolysis. The anaerobic glycolysis results in about a tenfold increase in the lactate load in the extracellular environment and the diffusion and transport of H + ions into the intercellular substance to avoid autoacidosis. These cellular responses also cause the pH of the tumour microenvironment to decrease and the overall environment to be acidic. In this study, we found that the expression levels of ASIC1α were significantly higher in liver cancer cells cultured in pH 6.5 than in pH 7.4, and the levels were especially higher than in the human normal liver cell line L-02. These results were consistent with those of previous studies . On the However, it is not clear whether ASIC1α can significantly affect the migration, invasion, and proliferation of HepG2 and SK-Hep1 cells. To address this deficit, we first tested the effect of inhibiting the activity of ASIC1α (with PcTx1) on the liver cancer cells with the wound healing assay, transwell invasion assay, and MTT assay. The results revealed that in an acidic medium pH 6.5), reducing the activity of ASIC1α enhanced the inhibition of migration, invasion, and proliferation of the HepG2 and SK-Hep1 cells. Also, silencing the ASIC1α gene enhanced these activities in the HepG2 and SK-Hep1 cells. Conversely, overexpression of the ASIC1α gene attenuated the inhibition of migration, invasion, and proliferation in the HepG2 and SK-Hep1 cells. These results suggest that activation of ASIC1α is a mechanism by which the metastasis and malignant progression of liver cancer is promoted. As illustrated in Fig. , reducinMMP-2/9 is a major member of the matrix metalloproteinase family. The MMPs are upregulated in liver cancer cells, and inhibition of their expression inhibits the migration and invasion of liver cancer cells –35. InhiThe PI3K/AKT/mTOR pathway is abnormally activated in most tumour cells and mediates tumour cell growth, migration, invasion, and anti-apoptosis –40. AlsoSince the two compounds have different dose relationships that may lead to different results, we analyzed the dose relationship of the two compounds, the Akt activator, SC79, and the ASIC1α inhibitor, PcTx1, based on dose–response results at the beginning the study and finally determined a combination with the best dose–response results. Activators affect protein activity, such as acetylation, phosphorylation, and ubiquitination activation, whereas gene amplification technology directly increases protein expression. In this study, we needed to examine the reversal role of Akt protein activity (phosphorylation) on ASIC1α inhibition. Therefore, we used the Akt activator SC79 without the gene amplification technique used in the MMP group.Due to limitations of laboratory conditions, we selected target cell lines based on the existing cells in the laboratory, not by detecting the expression of ASIC1α on all liver cancer cells. We admit that this is a limitation of our study, which we will correct in future research. In addition, drug resistance of liver cancer cells is an extremely important factor in promoting tumor progression. We will explore the relationship between ASIC1α and the drug resistance of liver cancer cells and its molecular mechanism in the next study.Overexpression of the acid-sensitive ion channel 1α (ASIC1α) affects the molecular mechanisms responsible for the migration, invasion, and proliferation of liver cancer cells. This action is achieved through modulation of the MMP-2/9/PI3K/AKT signaling pathway in the extracellular acidic microenvironment. Appreciation of this mechanism may be useful in the diagnosis and treatment of liver cancer.Additional file 1.