This regulation depends on Ca2+ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca2+ uniporter (MCU). Here, we have studied the mitochondrial Ca2+ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca2+ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic differences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca2+] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca2+ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca2+ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca2+ pathway.Mitochondrial [Ca The increase in mitochondrial [Ca2+] ([Ca2+]M) activates several dehydrogenases important for energy production, thus activating the respiratory chain and increasing adenosine triphosphate (ATP) production [2+]M increase is a signal of cell activation and enhances energy production in order to cover the energy requirements of the response to the stimulus. Moreover, during cell activation, mitochondrial Ca2+ uptake constitutes a mechanism of transient cytosolic Ca2+ buffering that modulates cytosolic [Ca2+]. In particular, mitochondria are very effective taking up Ca2+ from local high-Ca2+ microdomains, such as those generated by the activation of plasma membrane or ER Ca2+ channels M overload is a key factor controlling cell death during necrosis and apoptosis. During these processes, Ca2+ accumulation in mitochondria contributes to triggering of the opening of a high-conductance pore in the inner mitochondrial membrane, the so-called mitochondrial permeability transition pore, which is followed by osmotic swelling of mitochondria, rupture of the mitochondrial membranes and release of apoptotic factors to the cytosol  follows the fast oscillations of cytosolic [Ca2+] “beat-to-beat”, which is responsible for pharynx pumping [2+] in the worm pharynx of control and mcu-1 mutants, we show that the pattern of oscillatory Ca2+ dynamics in the mutants is very similar to that in the controls, although they show a smaller response to high-intensity stimulation. We conclude that MCU is essential to convey large-amplitude Ca2+ signals to the mitochondria. Instead, the low-amplitude [Ca2+]M oscillations seen in pharynx muscle during normal physiological activity are not mediated by MCU activation and use a different Ca2+ pathway to enter mitochondria.Here, we have used a knockout mutant of the MCU protein in  pumping ,17. Now,2+ sensor in the pharynx to investigate the dynamics of pharynx [Ca2+] in live worms under serotonin stimulation [2+] is able to oscillate at a high frequency in vivo, reproducing the oscillations of cytosolic [Ca2+] and behaving in a “beat-to-beat” mode at frequencies close to 1 Hz. This indicates that the mechanisms of Ca2+ uptake and release from the mitochondria are fast enough to recover the resting levels after each Ca2+ spike.We have recently used the AQ3055 strain expressing the YC3.60 Camulation . Our res2+ uniporter is the best known Ca2+ channel responsible for mitochondrial Ca2+ uptake, and therefore we wanted to study how that oscillatory Ca2+ pattern was modified in worms lacking a functional MCU protein. Surprisingly, 2+ dynamics among controls and mcu-1 mutants. Panels a and b of the figure show two typical 30 min Ca2+ records, one in the AQ3055 strain, which expresses YC3.60 targeted to the mitochondria, and the other in the mYCmcu-1 strain, which expresses YC3.60 targeted to the mitochondria in the mutant mcu-1 background. During the initial 15 min we monitored the spontaneous Ca2+ oscillations obtained in the presence of serotonin. Then, 10 mM carbachol was added to the chamber to induce a strong Ca2+ signal mediated by the activation of the nicotinic acethylcholine receptor. In this way, the first part of the experiment tests the transfer of small spontaneous cytosolic Ca2+ oscillations to the mitochondria and the second part evaluates the transfer of Ca2+ to the mitochondria during large cell stimulation.The mitochondrial Ca2+]M oscillations were not abolished by the lack of functional MCU. Panels c and d show a magnification of a small portion of each of the records in the initial part of the experiments. The figure shows the fluorescence ratio F535/F480, and also the individual F535 and F480 fluorescence data. Both wavelengths show mirror changes, as would be expected for true changes in [Ca2+]M. Our data show that the dynamics of the [Ca2+]M oscillations obtained in control worms or in worms lacking MCU was very similar. We analyzed the mean width and height of the Ca2+ oscillations, and we could only detect quite small changes in these parameters in the mutants, as seen in panel e. mYCmcu-1 mutants showed a small but significant increase in both width and height of the [Ca2+]M peaks.Our data show that the spontaneous M oscillations were secondary to changes in neuronal stimulation of the pharynx, responsible for the pumping rate, we have recorded electropharyngeograms (EPGs) in both wild-type N2 and mcu-1 mutant strains. mcu-1 mutant worms. Consistent with the changes observed in mitochondrial Ca2+ dynamics, we found a significant decrease in the mean frequency in the mcu-1 mutants, which was due to the increase in the interpumping space . In addition, mcu-1 mutants showed an increase in the pump duration, which is also consistent with the increase in the width and height of the mitochondrial [Ca2+] oscillations. There were no changes in the R/E ratio, which is the ratio of the repolarization and depolarization waves in each pump  and is a parameter that reveals possible alterations in the normal muscle contraction/relaxation. Therefore, most of the kinetic changes in the [Ca2+]M oscillations could be secondary to changes in the frequency of neuronal stimulation of the pharynx.The spontaneous Ca neurons . In orde2+ with very low affinity, the effect of the absence of MCU would be better revealed after a large stimulation of the pharynx—this was the case. Although the spontaneous Ca2+ oscillations showed few changes in the mutants, we could detect an important decrease in the response to carbachol in the mcu-1 mutants. 2+]M to carbachol addition in both AQ3055 and mYCmcu-1 mutants. In control worms, addition of carbachol increased the fluorescence ratio by 25.7 ± 3.0% (n = 8), while in the mYCmcu-1 mutants it only increased by 10.6 ± 1.0% (n = 10)  b. Therefnificant c.2+ uptake triggered by several stimuli that increased cytosolic [Ca2+] in different cell types  of around 100 nM suggests that the magnitude of the spontaneous [Ca2+]M oscillations is not higher than 200 nM. Our data show that these small changes in mitochondrial [Ca2+] occur through a different pathway and do not require the MCU. In fact, this is to be expected given MCU’s low affinity for calcium and taking into account the fact that spontaneous cytosolic [Ca2+] oscillations are also of small magnitude [2+] induced by carbachol requires the participation of MCU.In  healing , in spit 0.25 µM  and addillations . In wildagnitude . Instead2+ oscillations remained very similar in the mcu-1 mutants, they showed an increase in both width and amplitude on the mitochondrial Ca2+ peaks, and a reduction in the frequency. The mechanism of these changes is unknown, but EPG data showed also an increase in the pump duration and a decrease in the frequency of pharynx pumping, mainly due to an increase in the interpumping interval. Therefore, the changes in mitochondrial [Ca2+] can most probably be attributed to changes in the rate and intensity of neuronal stimulation arriving to the pharynx, perhaps as a consequence of the lack of MCU in the neurons.Although the spontaneous Ca2+ uptake during low-intensity physiological cell stimulation takes place in the C. elegans pharynx through a pathway independent of the MCU. If a similar pathway existed in mammals, it would explain the mild phenotype induced in mice by MCU knockout. In contrast, MCU is essential for transferring Ca2+ into the mitochondria during high-intensity stimulation, a mechanism that in some conditions may induce mitochondrial Ca2+ overload.In conclusion, our data suggest that mitochondrial Camcu-1 (ju1154), was obtained from the Caenorhabditis Genetics Center. Mutant mcu-1 strains expressing the mitochondrially targeted YC3.60 (named mYCmcu-1) were obtained by crossing AQ3055 worms with CZ19982 ones. Worms were maintained and handled as previously described  peaks in the ratio were only considered acceptable when inverted changes at both wavelengths were clearly observed. Fluorescence intensities and ratio changes were then analyzed with a specific algorithm designed to calculate off-line the width at mid-height expressed in seconds, the height obtained as the percent of ratio change and the frequency of all the Ca2+ peaks in each experiment. The frequency was measured at each peak as 9 divided by the distance among the peak 4 positions before and the peak 4 positions after. The mean frequency was calculated as the mean of all the individual frequencies higher than 5 peaks/min obtained in worms of a given age and condition.Fluorescence was recorded and analyzed using the Metafluor program , as previously described . The traTo perform electrical recordings of pharynx pumping , we placed the Nemametrix Screen Chip System on an inverted Zeiss Axiovert 200 microscope equipped with an LD A-Plan 20× objective. In order to minimize interfering electrical noise during the recordings, we used a system grounding shield. Baseline noise was typically between 10 and 40 µV. The experiments were performed at 20 °C.For each experiment, we picked 100 worms from the culture plate and washed them in 1.5 mL of 0.2 µm filtered M9 buffer +0.1% Tween. Worms were then washed 4× with 0.2 µm filtered M9 buffer, then once in M9 buffer containing 2.3 mM serotonin, and they were finally suspended in 1 mL of M9 buffer containing 2.3 mM serotonin and allowed to settle for 15 min. All the experiments were performed between 15 and 120 min of the initial serotonin exposure. We loaded a NemaMetrix screen chip system  with a fresh SC40 screen chip  and added M9 buffer containing 2.3 mM serotonin. After initiating the NemAcquire software and recording the basal power line noise, worms were vacuum-loaded onto the Nemametrix Screen Chip SC40 to start the experiment. The 1-hz high-pass and 50-hz notch filter settings were selected. A 180-s EPG recording was made for each animal, and records from 20–25 animals were obtained for each replicate. Records were analyzed with the NemAnalysis v0.2 software. All the experiments with a frequency of less than 0.1 Hz or pump duration coefficient of variation bigger than 50% were rejected.Reagents were obtained from Sigma, Madrid, Spain or Merck, Darmstadt, Germany.


These effects are dependent on transmembrane protein αKlotho, which enhances the binding affinity of FGF23 for FGF receptors (FGFR). Locally produced FGF23 in other tissues including liver or heart exerts further paracrine effects without involvement of αKlotho. Soluble Klotho (sKL) is an endocrine factor that is cleaved off of transmembrane Klotho or generated by alternative splicing and regulates membrane channels, transporters, and intracellular signaling including insulin growth factor 1 (IGF-1) and Wnt pathways, signaling cascades highly relevant for tumor progression. In mice, lack of FGF23 or αKlotho results in derangement of phosphate metabolism and a syndrome of rapid aging with abnormalities affecting most organs and a very short life span. Conversely, overexpression of anti-aging factor αKlotho results in a profound elongation of life span. Accumulating evidence suggests a major role of αKlotho as a tumor suppressor, at least in part by inhibiting IGF-1 and Wnt/β-catenin signaling. Hence, in many malignancies, higher αKlotho expression or activity is associated with a more favorable outcome. Moreover, also FGF23 and phosphate have been revealed to be factors relevant in cancer. FGF23 is particularly significant for those forms of cancer primarily affecting bone  or characterized by bone metastasis. This review summarizes the current knowledge of the significance of FGF23 and αKlotho for tumor cell signaling, biology, and clinically relevant parameters in different forms of cancer.Together with fibroblast growth factors (FGFs) 19 and 21, FGF23 is an endocrine member of the family of FGFs. Mainly secreted by bone cells, FGF23 acts as a hormone on the kidney, stimulating phosphate excretion and suppressing formation of 1,25(OH) Target (FAM20C) . In the l tubule . Moreovee kidney . In the e kidney . This wa on Ca2+ . These e on Ca2+ . Other e on Ca2+ , the ind on Ca2+ , or inhi on Ca2+ . At leas on Ca2+ . The pla on Ca2+ . In chro on Ca2+ . FGF23 p on Ca2+ . Indepen on Ca2+ , fibrosi on Ca2+  and prog on Ca2+ . In anot on Ca2+ . Dyslipi on Ca2+ . Inflamm on Ca2+ . Hence,  on Ca2+ .Klotho (referred to as KL) gene was identified in 1997. In mice, a mutation of the Kl gene causes a syndrome of rapid aging including a drastically shortened life span and further age-associated diseases and symptoms affecting most organs and tissues such as atherosclerosis, osteoporosis, skin atrophy, infertility, or emphysema 2D3 2D3 . Moreove–/– mice . Deficie in mice . These f in mice . The hum introns . It enco introns . The pro introns  . The  protein . KLB is  protein . FGF19 c  are associated with increased risk of prostate cancer  is associated with an even higher breast cancer risk of patients with BRCA1 mutation prone to developing breast cancer  are typical of the transition from normal tissue to oral dysplastic lesions to oral squamous cell carcinoma (SCC)   . KL prom results . Higher and site . Survivaand site . Moreoveand site . In lungand site . KL inhion (EMT) . Also, ion (EMT) .KL promoter may account for this  is associated with the risk of prostate cancer (odds ratio = 1.85)  . Methylaand PC-3 . The sampression . The KL thylated .KL overexpression in RCC cells down-regulates PI3K/AKT/GSK3-β/Snail signaling, thereby inhibiting cell migration, invasion, and EMT  tissue and cell lines, KL protein and mRNA expression are reduced . KL expr and EMT . Moreove and EMT .Results regarding the role of KL in ovarian cancer are controversial. According to a clinical study of 189 EOC patients, 73.5% of patients exhibit detectable KL expression. sKL is associated with high tumor grade, suboptimal tumor debulking results, disease progression [hazard ratio (HR) = 1.97], and death (HR = 2.09), possibly due to KL supporting the tumor with energy and angiogenesis . Others KL expression, depending on the age of surrounding fibroblasts  than in adipose tissue , KL overexpression attenuates IGF-1R, ERK1/2 and AKT signaling . Moreovepoptosis .As summarized in 2D3 formation  biomarker. In TIO, treatment with anti-FGF23 monoclonal antibody offers a beneficial therapeutic intervention. In other malignancies affecting bone including prostate cancer or MM, an anti-FGF23 approach may also be useful as enhanced FGF23 or FGF23 signaling is typical of these tumor entities. Clearly, this and the role of FGF23-dependent phosphate metabolism in cancer require further studies.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objectives: Due to the COVID-19 pandemic, medical curricula face major challenges. This also applies to mentoring programs, where face-to-face meetings are considered essential. Methods: The LMU Munich medical faculty mentoring program (MeCuM-Mentor) adapted to counteract the unforeseen pause of conventional course formats and associated uncertainty of many students. We here present an approach to transform the established large scale or group mentoring events of our program into online formats. Three projects are presented as examples: 1. HowTo Klink (HK), mainly informative in nature and with peer-mentoring character, 2. FacharztDuell (FAD) and 3. “Auf ein Gespräch mit... (AEGM)”, both with a focus on career counseling.Results: Initial evaluations show a similarly high participation rate and a high level of satisfaction among the participating students. Students' evaluation of whether the projects presented should take place in presence or in online format has so far shown no clear trend.Conclusion: Prospective studies are necessary to investigate the effectiveness of these online formats and analyse differences in participant behaviour. The extent to which online mentoring can replace classic mentoring functions has to be discussed anew. Over the past years, the importance of successful mentoring in health care and medical studies has been emphasized , 2]. Ac. Ac2]. Aa one-to-one mentoring program with peer-mentors and medical professionals for more than 4.000 students , a complementary second part, that comprises various projects and events with indirect mentoring character. These projects can be categorized into informative events, panel discussions on career advice and group mentoring rounds. Although most of our projects were face-to-face events to encourage personal exchange between mentors and mentees, our program also used some online elements prior to the COVID-19 pandemic. These included an online newsletter with different extracurricular or curricular voluntary offers at the medical faculty, video podcasts with brief introduction of our mentors, an online matching tool and a website for the search of open medical doctoral dissertations . In the In the following, we present three exemplary projects for different types of events:HowTo Klinik (HK) is an informative event to introduce medical students to the clinical studies of our medical school and is held twice a year at the beginning of each semester (n=150). So far, HK took place in an auditorium, was informative in nature and enabled a subsequent informal exchange between students and peer-mentors to foster the mentoring character of the event.FacharztDuell (FAD) is an event that highlights the differences between medical specialties in a moderated discussion with an additional question-and-answer session . The format is intended to advise medical students about their future academic careers . H. H12]. HFirst preliminary results of our events lead us to conclude that online formats can represent an adequate alternative to face-to-face ones. Nevertheless, long-term evaluations are still pending. These aim to answer the question of whether our online formats meet characteristics of mentoring and what effects video-based events have on the depth of conversation. Advantages and disadvantages of e-mentoring events compared to face-to-face mentoring events must therefore be analysed in order to better estimate the appropriate mix in the portfolio mentoring programs offer. The authors declare that they have no competing interests.


Euglena gracilis (Euglena) that exerts immunostimulatory activities by affecting cytokine production. This study investigated the signaling mechanisms that regulate the beneficial interactions between food compounds and the intestinal tract using cell type-specific calcium (Ca2+) imaging in vivo and in vitro. We successfully visualized Euglena- and paramylon-mediated Ca2+ signaling in vivo in intestinal epithelial cells from mice ubiquitously expressing the Yellow Cameleon 3.60 (YC3.60) Ca2+ biosensor. Moreover, in vivo Ca2+ imaging demonstrated that the intraperitoneal injection of both Euglena and paramylon stimulated dendritic cells (DCs) in Peyer’s patches, indicating that paramylon is an active component of Euglena that affects the immune system. In addition, in vitro Ca2+ imaging in dorsal root ganglia indicated that Euglena, but not paramylon, triggers Ca2+ signaling in the sensory nervous system innervating the intestine. Thus, this study is the first to successfully visualize the direct effect of β-1,3-glucan on DCs in vivo and will help elucidate the mechanisms via which Euglena and paramylon exert various effects in the intestinal tract.The intestinal tract contains over half of all immune cells and peripheral nerves and manages the beneficial interactions between food compounds and the host. Paramylon is a β-1,3-glucan storage polysaccharide from  Bifidobacterium bifidum alleviates dysbiosis and constipation in mice induced by a low-fiber diet [Aspergillus oryzae can protect against chronic constipation and gastrointestinal damage [Lactobacillus population and the number of regulatory T cells in the colon [The intestinal tract is the first line of defense against pathogenic microorganisms and manages beneficial interactions between food compounds and the host . These iber diet , while gl damage ,6. In adhe colon . Howeverhe colon ,9,10.Euglena gracilis (Euglena) is a microalga that contains a wide range of nutrients, including vitamins, minerals, amino acids, and fatty acids. Since it combines the properties of both plants and animals, it is often used as a food or dietary supplement [Euglena that has a triple-helical polymer structure composed of straight-chain β-1,3-glucans. Like other β-1,3-glucans, paramylon has various beneficial effects on health, such as modulating immune function [pplement . The stofunction ,13,14,15function ,17,18,19function ,21,22. Ffunction ,24; howe2+) signaling in specific cell populations has been visualized in conditional transgenic mice expressing Yellow Cameleon 3.60 (YC3.60) [2+ is a universal secondary messenger that performs multiple functions in most cells, including lymphocytes, epithelial cells, and neurons [2+ imaging in transgenic mice expressing YC3.60 under the control of the CD11c gene promoter has demonstrated that oral propolis administration stimulates DCs in lymphoid tissues [2+ imaging has revealed that probiotics induce Ca2+ signaling in IECs under physiological conditions [Euglena and paramylon in the intestinal tract, we performed cell type-specific Ca2+ imaging in three conditional transgenic mouse lines with specific or ubiquitous YC3.60 expression [Euglena- and paramylon-induced Ca2+ signaling in IECs, DCs in intestinal PPs, and nerve cells innervating the intestine.Calcium imaging using genetically encoded calcium biosensors has enabled nutrition-sensing mechanisms in the intestinal tract to be visualized. For example, Calcium ion (Ca(YC3.60) ,26. Ca2+ neurons ,28,29,30 tissues , while inditions . Enteroenditions ,34. To epression  to visuaflox) mouse line was crossed with cell type-specific Cre mouse lines  to produce YC3.60flox/CD11c-Cre, YC3.60flox/Nestin-Cre, and YC3.60flox/CAG-Cre mice, respectively [Conditional YC3.60 expressing transgenic mice were as described previously . The floectively ,31. Thesflox/Nestin-Cre mice as described previously [Dorsal root ganglia (DRG) cells were prepared from YC3.60eviously . Brieflyeviously . The celEuglena powder and paramylon were obtained from Euglena Co., Ltd. . The nutritional composition of the Euglena powder was as follows: carbohydrates 55.0%, protein 29.9%, and lipid 9.0%. Approximately 70–80% of the carbohydrate content was paramylon. Paramylon was prepared according to the standard method, as follows: cultured Euglena gracilis Z cells were collected by continuous centrifugation and washed with water. After being suspending in water, the cells were disintegrated using ultrasonic waves and the cell contents (containing paramylon) were collected. The crude paramylon preparation was treated with 1% sodium dodecyl sulfate (SDS) solution for 1 h at 95 °C followed by 0.1% SDS for 30 min at 50 °C to remove lipids and proteins. After centrifugation, paramylon was refined by repeated washing with water, acetone, and ether.Euglena or paramylon in phosphate buffered saline  was added to the intestinal tract, with PBS as a control. Images were acquired using a Nikon A1 laser-scanning confocal microscope with a 20× objective lens, dichronic mirrors (DM457/514), and two bandpass emission filters , as described previously [IECs and PPs from anesthetized mice were imaged as described previously . Small ieviously . The YFPEuglena or paramylon in PBS (1 mg/mL), with PBS as a control. After 2 hours, the mice were subjected to intravital imaging analysis, as described previously [To observe PPs, the peritoneal cavity of each mouse was injected with 200 μg of eviously .p < 0.05 was considered significant.Statistical analysis was performed with Pearson’s chi-square test to compare the proportions of cells. R version 3.4.1 were used to conduct the statistical analyses. Euglena and paramylon directly stimulate IECs, we carried out intravital Ca2+ imaging. Euglena induced transient Ca2+ signaling in most IECs . After 2 hours of intraperitoneal administration, intravital imaging analysis showed that Euglena increased intracellular Ca2+ levels in DCs, with 31.2% of DCs exhibiting higher intracellular Ca2+ concentrations  has been shown to control the cytokine balance between T lymphocyte Th-1 and Th-2 subsets, resulting in enhanced cellular immunity [Saccharomyces cerevisiae in 20 children with asthma increased serum levels of the anti-inflammatory cytokine IL-10 and simultaneously reduced symptom scores [Euglena and paramylon could reduce upper respiratory tract infection symptoms and protect against influenza virus infection [Euglena and paramylon injection stimulated DCs in PPs in vivo, with stronger Ca2+ signaling observed with paramylon treatment; thus, paramylon may be the active component of Euglena that affects the immunological system. This finding is consistent with previous studies indicating that β-1,3-glucans stimulate macrophages and DCs via a Dectin-1-dependent pathway [Euglena and paramylon may support a healthy immune system and protect overall health [Biologically-active polysaccharides may be a potential method of preventing dysfunctional immune homeostasis ,43. For immunity . In addim scores , while snfection ,45. Here pathway . Orally  pathway ,24. On m pathway . Alongsi pathway . Due to l health . Intravil health .Euglena, but not paramylon, directly triggers Ca2+ signaling in DRG neurons, suggesting that Euglena can excite visceral afferents. Although paramylon has various bioactive functionalities [, other bioactive components of water-soluble fraction seem to activate the neurons. Indeed, the water extract partially purified from Euglena also contains bioactive materials, as this is crucial for preventing lung carcinoma growth and intracellular lipid accumulation [2+ signaling in the intestinal tract, which is transmitted to the nervous system and results in a transient shift toward higher arousal levels in the brain [Euglena to excite neurons may underlie its beneficial effects on health-related Quality of Life observed in human subjects [Euglena responsible for these signaling responses have been identified and a method for extracting the active ingredients has been established, Euglena can be utilized to produce useful ingredients on an industrial scale [In this study, we demonstrated that nalities , other bmulation ,49. The mulation . DRG affmulation ,52. The mulation ,54, whermulation . Some prmulation , whereashe brain . Furthersubjects . In the al scale .Euglena and paramylon exert various effects from the intestinal tract.Interactions between food and the intestinal tract transmit signals via the gut–immune–brain axis by secreting multiple cytokines and hormones that modulate physiological homeostasis ,58. Thus


Hepatitis E virus (HEV) is a quasi-enveloped, positive-sense single stranded RNA virus. HEV continually expands the host ranges across animal species. In this study, the possibility of cross-species infection with swine HEV-3 was investigated using rabbits. A total of fourteen 8-week old, specific pathogen-free rabbits were divided into three experimental groups. Four rabbits were used as negative controls, four rabbits were infected with rabbit HEV as positive controls, and six rabbits were inoculated with swine HEV-3. HEV RNA were detected from serum and fecal samples after viral challenge. The levels of anti-HEV antibodies, pro-inflammatory cytokines , and liver enzymes  were determined in serum samples. Histopathological lesions were examined in liver tissues. Viral RNA and anti-HEV antibodies were identified in rabbits inoculated with swine HEV-3 demonstrating positive infectivity of the virus. However, pro-inflammatory cytokine and liver enzyme levels in serum were not significantly elevated, and only mild inflammatory lesions were detected in the liver tissues of rabbits infected with swine HEV-3. These results suggest that swine HEV-3 can engage in cross-species transmission to rabbits, but causes only mild inflammation of the liver. Hepeviridae, is a quasi-enveloped, positive-sense single stranded RNA virus with an approximately 7.2 kb-long genome [Hepatitis E virus (HEV), of the family g genome . HEV infg genome ,4,5,6. THEV, with only one serotype, has been classified into eight genotypes; HEV-1 to HEV-8 ,10. HEV-n = 4), a positive control group infected with rabbit HEV (n = 4), and a cross-species transmission group infected with swine HEV-3 (n = 6). Rabbits were intravenously infected with rabbit HEV and swine HEV-3. Rabbit HEV (GenBank No. KY496215) and swine HEV-3 (GenBank No. MF095679) used in this study were obtained from the stool sample of a rabbit and a pig, respectively, as described in our previous studies [6 genomic equivalent (GE) copy number/mL in 4% bovine albumin solution.All animal experiments were approved at 14 April, 2017 by the Institutional Animal Care and Use Committee of Konkuk University (IACUC No. KU16128). Rabbits were kept in the animal facility of Konkuk Laboratory Animal Research Center. A total of 14 8-week-old specific pathogen-free New Zealand white rabbits were used for this experiment . Rabbits were divided into three experimental groups: a negative control with mock infection , centrifuged at 3000× g for 30 min, and the supernatants were collected. The supernatants were re-centrifuged at 13,000× g for 10 min and the second supernatants were collected. All serum and fecal samples were stored at −70 °C until use. Viral RNA was extracted from 150 µL of serum and fecal samples using a Patho Gene-spin DNA/RNA kit according to manufacturer’s instructions . The extracted RNA samples were used for cDNA synthesis or stored at −70 °C.Serum and fecal samples were collected from all rabbits every week for 8 weeks post infection (wpi). Serum samples were separated from the whole blood by centrifugation at 3000× 2–103 GE copies was performed to detect partial genomic sequence of the HEV ORF2 with gene-specific primers under conditions as described previously [Nested RT-PCR with detection limits of 10eviously . The corAnti-HEV antibody titers were determined in serum samples collected from each of the rabbits in the three groups over 8 weeks using a commercial ELISA kit  according to the manufacturer’s instructions. ALT and AST levels in serum samples collected from each of the rabbits in the three experimental groups over 8 weeks were measured by a UV-assay, according to the procedure by International Federation of Clinical Chemistry and Laboratory Medicine , without pyridoxal phosphate activation.The levels of IL-1, IL-6 and TNF-α in serum samples collected at 0, 2, 3, 5, 6 and 8 wpi were measured by using ELISA kits . IFN-α levels in serum samples collected at 2, 3, 5 and 6 wpi were determined with ELISA kit (MyBioSource). All experimental procedures were conducted according to manufacturer’s instructions.Liver tissues taken at 8 wpi were fixed using 10% neutral buffered formalin and embedded with paraffin. Tissues were stained with hematoxylin and eosin (H&E) to identify inflammatory lesions, and with Masson’s trichrome for identifying fibrosis. Degree of hepatic inflammation was graded by modification of the Brunt system ,28. ScorThe HEV RNA was determined using serum and fecal samples collected over 8 weeks by nested RT-PCR. As expected, no HEV RNA was detected from negative control rabbits during the experimental period . HEV RNA was detected from the fecal or serum samples collected from all four rabbits (100%) infected with rabbit HEV from 3 to 8 wpi . In contThe presence of anti-HEV antibodies was determined from serum samples from each of the rabbits in the three groups. In negative control rabbits, anti-HEV antibody was not detected, as expected . In contThere was no elevation of ALT nor AST levels in serum of rabbits in the negative control group, as expected . While AThe levels of proinflammatory cytokines IL-1β, IL-6 and IFN-α were measured in rabbit serum samples collected at 0, 2, 3, 5, 6 and 8 wpi. IFN-α levels were determined in serum samples collected at 2, 3, 5 and 6 wpi. Serum IL-1β, IL-6, TNF, and IFN-α concentrations in rabbits infected with rabbit HEV or swine HEV were not significantly different from serum concentration in negative control rabbits . These rNo inflammatory lesions were seen in the liver tissues of rabbits in the negative control group A. ConverMultifocal and diffuse accumulations of inflammatory cells around the central veins, portal areas, and parenchyma were found in rabbits infected with rabbit HEV. Inflammatory cell infiltrates were mostly monocytes. Inflammatory lesions were also seen in liver tissue from rabbits infected with swine HEV-3 C. HoweveSeveral animal models have been used to understand the pathogenesis and immune responses of HEV. Generally, non-human primates such as chimpanzee, cynomolgus, and rhesus monkeys, are considered the best animals for HEV infection studies because these animals can be infected with various HEV genotypes and produce a similar disease to that observed in humans ,19,29. PIn this study, we demonstrated that about 67% of rabbits could be infected with swine HEV-3. Swine HEV-3 induced viremia, fecal shedding and seroconversion in virus-infected rabbits. Seroconversion patterns identified in rabbits infected with swine HEV-3 are similar to those in rabbits infected with the wild boar HEV-3 strain . HoweverElevated hepatic enzymes indicate inflammation and damage to liver tissues. It is known that the normal physiological ranges of ALT and AST are 55–260 U/L and 10–98 U/L in rabbits, respectively . In thisInduction of proinflammatory cytokines such as IL-1β, IL-6, IFN-α, and TNF-α, is indicative of acute viral infection in animals . HoweverCollectively, our results show cross-species transmission in rabbits infected with swine HEV-3. Viremia, fecal viral shedding, and seroconversion were observed, while increases in hepatic enzymes and proinflammatory cytokines did not occur in rabbits infected with swine HEV. Mild hepatic lesions and fibrosis were seen in the liver tissues of rabbits infected with swine HEV. These results indicate that swine HEV-3 can cross the species barrier to infect rabbits where it induces mild hepatitis without severe inflammation.


However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the spongiform domain of astrocytes where most tripartite synapses are located. Using 3D-STED microscopy in living organotypic brain slices, we imaged the spongiform domain of astrocytes and observed a reticular meshwork of nodes and shafts that often formed loop-like structures. These anatomical features were also observed in acute hippocampal slices and in barrel cortex in vivo. The majority of dendritic spines were contacted by nodes and their sizes were correlated. FRAP experiments and Ca2+ imaging showed that nodes were biochemical compartments and Ca2+ microdomains. Mapping astrocytic Ca2+ signals onto STED images of nodes and dendritic spines showed they were associated with individual synapses. Here, we report on the nanoscale organization of astrocytes, identifying nodes as a functional astrocytic component of tripartite synapses that may enable synapse-specific communication between neurons and astrocytes.Astrocytic Ca 2+ signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. Here, the authors report the organization of astrocytes at nanoscale level and identify nodes as a functional astrocytic component of tripartite synapses.Astrocytic Ca It assigns astrocytes an active and pivotal role in information processing in the brain, beyond their multiple homeostatic functions.The concept of the tripartite synapse posits that the flow of information at synapses is a result of dynamic signaling between pre- and post-synaptic neurons as well as astrocytes2. In addition, neurotransmitters released from presynaptic nerve terminals not only bind to postsynaptic receptors, but are also detected by astrocytes, leading to Ca2+ elevations and release of neuroactive substances, such as glutamate, D-serine, and ATP3. In turn, these gliotransmitters can influence synaptic function, including basal synaptic transmission4,5 and long-term synaptic plasticity8.The thin processes of protoplasmic astrocytes in the cortex and hippocampus, which characteristically traverse the neuropil at very high density, come into close physical contact with many synapses, appearing to wrap around them in electron microscopy images2+ elevations have long been thought of only as slow and spatially spread out, recent studies suggest that the situation in astrocytic processes may actually not be so different from neurons, which exhibit fast Ca2+ signals in microdomains16.While astrocytic Ca2+. A prime example of structural compartmentalization are mushroom-shaped dendritic spines where the spine neck reduces biochemical cross talk between neighboring spine heads, enabling synapse-specific changes in synaptic strength17. In the same vein, astrocytes also have an extremely intricate morphology, which could provide the structural basis for compartmentalized intracellular signaling18.Neurons have a very elaborate morphology, which helps them spatially compartmentalize diffusible signaling molecules or ions like Ca2+ signals have been reported in morphological enlargements on major branches of astrocytes5 and on processes of Bergmann glial cells19. However, we know very little about the structural basis of Ca2+ signals in the spongiform domain of astrocytes, where astrocytic processes come into contact with thousands of neuronal synapses they potentially regulate.Local Ca21. While EM has enough resolution, it can only provide snapshots and may give a distorted view of the morphological relationships of the tripartite synapse in the case of chemically fixed brain tissue22.The lack of information stems largely from the difficulty of visualizing the spongiform domain with conventional light microscopy, which does not have enough spatial resolution for this23 to resolve the morphological details of astrocytes and to see how they contact dendritic spines in living brain tissue.We overcame these problems by applying live-cell 3D-STED microscopyWe observed looped structures in the images of spongiform domain of astrocytes, which seemed to form continuous genuine rings, according to 3D reconstructions.We observed that the majority of contacts with dendritic spines were formed by bulbous enlargements, or nodes, which were assembled along thin astrocytic processes, or shafts. Most nodes were actually branch points that gave rise to multiple shafts. We also observed this anatomical organization in acute hippocampal slices and in barrel cortex in vivo. Large spines tended to be associated with large nodes, forming stable contacts.2+ imaging showed that nodes were diffusionally isolated compartments that hosted highly localized spontaneous Ca2+ transients. Finally, mapping the Ca2+ transients onto STED images of dendrites, these localized Ca2+ signals occurred in nodes that were in contact with dendritic spines.FRAP (fluorescence recovery after photobleaching) experiments and confocal Ca2+ activity of astrocytes in the spongiform domain, identifying astrocytic nodes as the morphological structures that host Ca2+ transients at individual tripartite synapses.Taken together, our study sheds new light on the micro-anatomical architecture and Ca23.To visualize astrocytic morphology in the spongiform domain, we used a custom-built 3D-STED microscope with a spatial resolution of around 50 nm in x–y and 200 nm in z. We labeled the astrocytes with a cytosolic fluorescent marker protein (ZsGreen) in living organotypic mouse brain slices. This sample preparation maintains an organotypic organization, yet provides nearly ideal conditions in terms of optical access and sample stability, which is critical for super-resolution imaging2)  Fig. . Based o2) Fig. .Fig. 1SpThe meshwork featured closely spaced bulbous node-like enlargements  Fig.  that freWhile the 3D-STED approach was much better at revealing morphological details than confocal microscopy  in different brain regions , more physiological preparations  and with a different fluorescent protein (Clover), indicating they are a general anatomical feature of astrocytes in the mouse brain and not just particularities or artifacts of organotypic slices or the labeling method Fig. .Fig. 2NoGiven that nodes and shafts are ubiquitous structures in the spongiform domain where most of the synaptic contacts occur, we wondered how these astrocytic structures interact with excitatory synapses formed by dendritic spines in the spongiform domain. To address this question, we performed two-color STED imaging of co-labeled astrocytes and neurons, and analyzed their spatial relationships Fig. .Fig. 3Ma23, we observed a relatively spacious distribution of the extracellular space around dendritic spines  or did not have any astrocytic structure in their vicinity (10%) Fig. . Applyinnes Fig. . This is2; median area = 0.20 µm2; Fig. r = 0.669, p < 0.0001, Fig. Given that astrocytic nodes are highly variable in size . The median value of the coverage fraction was 0.38, indicating that spine surfaces were mostly node-free Fig. . FurtherWe observed a similar structural relationship between nodes and presynaptic boutons. Most boutons (70%) were contacted by nodes, correlated in size with them and had similar coverage fractions Fig. , similar25 and hippocampal slices26.Next, we assessed the stability of the node-spine contacts by two-color time-lapse STED imaging. While nodes were more motile than spines, most contacts were maintained for at least 2.5 h Fig. . We obseGiven their bulbous shape, we wondered whether nodes could function as diffusionally isolated compartments, similar to mushroom-like spines or en passant axonal boutons, which are known to compartmentalize biochemical signals. We tested this by performing fluorescence recovery after photobleaching experiments (FRAP) in nodes and shafts Fig. .Fig. 5Asτ) quantifies the time it takes for the cytosolic fluorescent marker to equilibrate after a local drop in concentration. τ is thus a measure of biochemical compartmentalization, which should scale with the volume of the node and the diffusional resistance of the shafts27. We estimated τ by fitting the fluorescence traces with a mono-exponential decay function and calculated the width ratio (width at bleaching site divided by width of shaft), which becomes unity in the absence of a node.We applied brief two-photon laser pulses to bleach an appreciable fraction (~40%) of the ZsGreen fluorescence inside astrocytic processes and recorded the fluorescence recovery by confocal imaging in rapid line-scan mode. The time constant of recovery . This indicates that micro-anatomical features give nodes the ability to compartmentalize biochemical signals  in the spongiform domain with an automated Ca2+ detection software, we measured a median amplitude (∆F/F0) of 316%  , Ca2+ signals and astrocytic structures, we combined confocal Ca2+ imaging with STED microscopy. STED measurements of the resting GCaMP6s signal confirmed that the bright spots indeed were nodes  was confined to single nodes, while the rest also involved neighboring shafts and nodes , the temporal order could not be resolved   faster Ca2+ activity in the spongiform domain, we investigated the molecular mechanism underlying the Ca2+ transients. First, to find out if they depended on neuronal activity, we incubated the slices with TTX (1 µM), which blocks voltage-gated sodium channels and thus action potential firing in neurons, or bafilomycin A1 (2 µM), which disrupts neurotransmitter release by inhibiting vacuolar ATPases on presynaptic vesicles containing neurotransmitter.Having identified the nodes as a primary site of Ca2+ transients was unaffected by TTX , which blocks IP3 receptors (IP3R) that are the main intracellular Ca2+ release channels in astrocytes. 2APB significantly decreased the frequency of Ca2+ transients , which depletes internal Ca2+ stores by inhibiting SERCA, greatly reduced the frequency of Ca2+ transients  in the presence of TTX (1 µM). DHPG evoked Ca2+ signals suggests that nodes are the astrocytic element of the tripartite synapse. To demonstrate this more directly, we combined confocal Ca2+ imaging in astrocytes with STED microscopy of dendritic morphology. We used slices from Thy1-YFP animals where a subset of neurons express YFP and infected the astrocytes with GCaMP6s. We could map the confocal Ca2+ signals onto the morphology of nodes and spines , which is a measure of the cumulative Canes Fig. .2+ signaling at excitatory tripartite synapses, providing the structural framework and molecular machinery for synapse-specific communication between astrocytes and neurons , or reflect the fact that we focused on nodes that were in close contact (<100 nm) with spines, and not on potentially unmoored astrocytic processes. Conceivably, the contact stability we observed is provided by puncta adhaerentia that mechanically connect dendritic spines to neighboring astrocytic processes34.Previous studies reported that astrocytic processes can be relatively motile near synapses35. Cytosolic labels in hyper-thin sheets or plasmalemmal films can generate only a weak fluorescence signal, while membrane labels tend to quickly suffer from photobleaching because they do not recover by replenishment, posing a challenge for live-cell super-resolution microscopy.Our 3D-STED approach marks a major step forward in the visualization of astrocytes in living brain tissue, but it may still do injustice to some aspects of astrocytic morphology, such as sheet-like structures, which have been reported by EM22 also showed a relatively loose organization of rotund astrocytic structures close to synapses, suggesting that classic chemical fixation via cardiac perfusion introduces artifacts by compacting the tissue. In agreement with the cryogenic EM study, the nodes and shafts we observed had simple geometries, reminiscent of axonal boutons and fibers, and sizes that were well within the STED resolution. Moreover, the SUSHI images indicate that the extracellular space is relatively spacious around synapses, in line with biophysical measurements, which have reported extracellular space volume fractions of around 20% in living brain tissue36.That said, a recent EM study based on high-pressure freezing of tissue slices37.It is important to know these structural details, because the relative position, size, and shape of astrocytic processes will influence their ability to communicate with their synaptic partnersThe nodes and shafts are morphologically recognizable in several brain areas and experimental preparations. However, additional improvements in microscopic resolution and sensitivity may still be needed to provide a definitive view of astrocytic morphology in vivo. They will facilitate mechanistic analyses of the structural and functional dynamics of neuron-astrocytes interactions and along the way help settle controversies about the effects of EM protocols on tissue ultrastructure.2+ signaling in their perisynaptic processes. However, it has remained technically challenging to record Ca2+ transients and to map them onto the underlying cellular structures18. We have demonstrated here that a combination of confocal Ca2+ imaging and STED microscopy can achieve this.Astrocytes are thought to regulate synapses via Ca2+ signals in the spongiform domain were confined to single nodes and rarely spread into the intervening shafts, even when neighboring nodes were co-active. Given the dense spacing of the nodes (~1 micron), this indicates an extremely tight spatial compartmentalization, and speaks to the need for advanced imaging techniques that can resolve on a sub-micron scale the Ca2+ signals and the structures that host them18.We found that a large fraction of spontaneous Ca2+ events, given the limited sensitivity and temporal resolution of our experimental approach, which may have failed to detect very small and/or brief Ca2+ events. Yet, as it stands, our data indicate that Ca2+ events are primarily associated with nodes. For the same technical reason, we may have missed near-membrane Ca2+ events in astrocytic sheets.We cannot rule out that shafts are also capable of generating Ca2+ events in part depended on IP3Rs, which is consistent with the previous studies12, but Ca2+ influx from the extracellular space as well as mitochondria may also have contributed to the Ca2+ events38. Given their size, which is comparable to dendritic spines , and monitored daily by trained staff. All animals used were free of any disease or infection at the time of experiments. Pregnant females and females with litters were kept in cages with one male. We did not distinguish between males and females among the perinatal pups used for organotypic cultures, as potential anatomical and/or physiological differences between the two sexes were considered irrelevant in the context of our study. For in vivo experiments, only male mice were used. No animals used had been involved in previous experiments.42) and homozygous Ai6 (RRID: IMSR_JAX:007906) mice. For slices with YFP-labeled neurons, pups were produced by cross-breeding C57Bl/6J (RRID: IMSR_JAX:000664) with homozygous Thy1-YFP-H (RRID: IMSR_JAX:003782) mice. We used C57Bl/6 J wild-type for imaging in organotypic and acute slices as well as in vivo.Slices with ZsGreen-labeled astrocytes were obtained by cross-breeding tamoxifen inducible heterozygous GFAP-cre (Tg(GFAP-cre/ERT2)1Fki1D-Clover and AAV2/1.gfaABC1D-Lck-Clover were generated by replacing GFP gene of pZac2.1-gfaABC1D-GFP, and pZac2.1-gfaABC1D-Lck-GFP with the Clover sequence (Addgene plasmid #40259).To generate Sindbis-Citrine, the Citrine sequence (Addgene Plasmid #54715) was inserted to the pSinRep5 plasmid . AAV2/1.gfaABC43. In brief, the virus was injected via a pipette positioned into the CA1 area of the slice by brief pressure pulses . For morphological or Ca2+ imaging of astrocytes, AAV2/1.gfaABC1D-Lck-Clover, AAV5-gfaABC1D-Lck-GCaMP6f  or AAV9-GFAP-GCaMP6s9 was microinjected into the stratum radiatum of 2-week old slices from either WT mice or Thy1-YFP-H (JAX:003782) mice 4 to 6 weeks before the experiment. For labeling of neurons in the organotypic preparation, Sindbis-Citrine virus were microinjected into slices with ZsGreen-labeled astrocytes 1 day prior to the experiment.Microinjection of virus into the hippocampal slice was performed using a glass pipette connected to Picospritzer (Parker Hannifin)6. In brief, 4-week old mice were submitted to stereotaxic surgery and AAV2/1.gfaABC1D-Clover was injected into the hippocampus or barrel cortex with the help of a microsyringe attached to a pump. Animals were used 4 to 5 weeks after injections for acute slice and in vivo experiments.Viral delivery into the hippocampus was performed by in vivo injection23. Organotypic slices were transferred on their glass coverslip to a heated and perfused imaging chamber. The extracellular dye, 40 µM Atto-514 (Atto Tec) diluted in ACSF, was pipetted directly into the chamber while pausing the perfusion. The final fluorophore concentration was around 20 µM.Extracellular labeling was performed as follows44. Mouse pups were quickly decapitated and hippocampi were dissected out. They were subsequently cut in 350 µm coronal slices using a tissue chopper (McIlwain), and individual slices were placed on poly-L-lysine coated cover slips. To let the slices stick to the cover slips, they were embedded in chicken plasma and thrombin, which coagulated upon mixing. The coverslips with the slices were transferred to flat-bottomed culturing tubes with medium and placed in a roller drum rotating until the experiment. For experiments, a slice on a coverslip was mounted in a perfused imaging chamber, and the slice was imaged from below through the glass coverslip.Organotypic slices were isolated from 5 to 7-day old mouse pups and cultured using the roller tube method26.Acutely prepared hippocampal slices were obtained from 8 to 9-week old mice. Mice were anesthetized with isoflurane prior to decapitation. Brains were quickly removed and placed in ice-cold, oxygenated sucrose-based ACSF. 350 µm sagittal slices were cut and incubated for 1 h at 33 °C in carbogenated ACSF. Subsequently, slices were stored at room temperature and used within 4 h after preparation. Experiments were performed in a submerged recording chamber at 33 °C with continuous perfusion of carbogenated ACSF45. Mice were intraperitoneally and intramuscularly injected with buprecare and dexamethasone respectively 25 min before anesthesia induction at 4% isoflurane. Mice were maintained under anesthesia at 1.5% isoflurane, and subsequently placed on a heating pad. An anal probe to measure body temperature was inserted and eye ointment was applied. Mice were injected subcutaneously with Lidocaïne 2% and the skin was treated with Lidocaïne 2% cream. The area for incision was disinfected with betadine and the incision was made in the skin. The surface of the skull was cleaned of any remaining tissue and roughened for adhesion of the liquid glue fixing the head holder to the skull. A helmet from dental cement was created for a stable fixation of the head holder and a craniotomy of 3 mm in diameter was performed above the barrel cortex. The opened skull was rinsed with cortex buffer  and a glass coverslip with a 3 mm diameter was gently positioned on the brain and sealed with gel glue. The glue was left to dry for 10 min, after which mice were placed below the objective of the microscope for acute imaging.Cranial window was made from 8 to 9-week old mice23, the excitation light was provided by a pulsed diode laser (λ = 485 nm) and the STED light came from a Ti:Sa femtosecond laser emitting at 838 nm, which was converted to a wavelength of 597 nm by an optical parametric oscillator (OPO). A signal from the OPO was used to trigger pulsed diode laser. We used a glycerol objective with a correction collar to reduce spherical aberrations. In the case of the 2P-STED setup, the excitation light was provided by a second Ti:Sapphire laser tuned to 910 nm running at 80 MHz that was synchronized to the OPO pulses. We used water-immersion objectives with long working distances, either from Nikon 46 or Olympus 45. The STED pulses were broadened in time by passing them through glass rods and a long optical fiber (20 m) to ensure effective de-excitation and maximal resolution enhancement. Synchronization and optimal pulse delay were achieved with phase-locked loop electronics.In the case of the one-photon systemscan = 5 cm and Ftube = 25 cm, Leica Microsystems). In the case of 2P-STED microscopy47, the STED doughnut was created by passing the STED beam through a vortex phase mask, which imposed a helical 2π phase delay on the wave front. Wave plates (λ/2 and λ/4) were used to make the STED light circularly polarized before it entered into the objective.The STED beam reflected twice on two different surfaces of a single spatial light modulator (SLM)  to generate doughnut and bottle beams for 2D and 3D-STED microscopy. The SLM was conjugated to the beam scanner  via appropriate relay lenses and the scanner was conjugated to the back focal plane of the objective via the scan and tube lens combination  was injected into the optical path of the microscope using a 680 nm short-pass dichroic mirror. For two-color imaging, two fluorophores with partially overlapping excitation and emission spectra  were imaged simultaneously using a single excitation/STED laser beam pair. The emission signal was split by a 514 nm long-pass dichroic mirror, and was sent into separate photodetectors3, pixel size 19.53 nm, z plane 80 nm × 25 planes for 3D confocal STED images, (2) 20 × 20 × 2 µm3, pixel size 19.53 nm, z plane 222 nm × 9 planes for 2D-STED images, (3) 10 × 10 × 1 µm3, pixel size 19.53 nm, z plane 250 nm × 4 planes for 2 P images and (4) 10 × 10 × 1.8 µm3, pixel size pixel size 19.53 nm, z plane 300 nm × 6 planes for 2P-STED images. For time-lapse 2D-STED imaging, z stacks  were acquired every 30 min for 2.5 h.3D-STED microscopy was used for Figs. 2+ imaging  was performed for 2.5 min and corresponding STED images  were acquired subsequently. 8 Hz Ca2+ imaging  was performed for 2 min using bidirectional scanning and corresponding STED images  were acquired subsequently. STED images were always acquired after Ca2+ imaging so that Ca2+ signals were not affected by the STED laser. Image acquisition was controlled by the software Imspector.2 Hz confocal Ca2, 2 mM CaCl2, 26 mM NaHCO3, 1.25 mM NaH2PO4, 20 mM D-glucose, 1 mM Trolox; 300 mOsm; pH 7.4. Perfusion rate was 2 ml per min and the temperature 32 °C. Slices were incubated for 20 min for 1 µM TTX (Ascent scientific) and 100 µM 2APB (TOCRIS), 2 h for 2 µM Bafilomycin A1 (Sigma-Aldrich), 20 min for 2 µM Thapsigargin (TOCRIS) prior to imaging. All drugs except for Thapsigargin were present during the recording. 10 µM DHPG (Sigma-Aldrich) was bath-applied after 3 min of baseline recording for the rest of the recording time (7 min). The same concentration of DMSO was added to the control group for drugs that were dissolved in DMSO .Organotypic slice experiments except for SUSHI (described in Extracellular labelling for SUSHI) were performed in ACSF containing 125 mM NaCl, 2.5 mM KCl, 1.3 mM MgCl3, pixel size 19.53 nm, z plane 200 nm × 9 planes) was acquired prior to the FRAP experiment. Bleaching was performed by line scanning the two-photon beam for a period of 8 ms across the astrocytic structure (node or shaft) labeled with ZsGreen, concomitant with 488 nm single-photon excitation line scanning to read out fluorescence levels in confocal mode27. The bleaching period was preceded by a 0.5 s baseline scan and followed by at least 1 s of scanning to measure recovery. The line scan frequency was 277 Hz, with a pixel size of 23 nm. The time constant τ was calculated by fitting a single-exponential function (ImageJ) to fluorescence recovery curve, which was generated by plotting fluorescence intensity versus time27. To ensure the good level of fit, only the first 295 ms of the recovery phase was used for the fit. The width ratio was calculated by dividing the measured width at the bleached site by the average width of the two thickest shafts 1 µm away from the bleach site. When the shaft was shorter than 1 µm, the width of the further end of the shaft was used.A STED image of the structure  software. Loops were manually identified and their inner perimeter and area were measured using ImageJ. Nodes were manually identified based on their enlarged 3D spherical submicron structure and shafts were identified as hyperthin tubes connecting nodes. To measure the width across these astrocytic components, we used a wavelet-based custom-made ImageJ plugin2+ signals . A contact was defined by the presence of at least one pixel overlap between spines and astrocytic components (nodes or shaft). Spine heads were also manually identified and morphological parameters of spines and nodes (perimeter and area) were measured using ImageJ.Two-color images except for Fig. Coverage fraction was calculated for all planes that includes a given spine and was subsequently averaged to acquire a representative value. Spines without full 3D information were excluded from the analysis. Coverage fraction for spines was calculated by dividing node-contacting spine head perimeter by total spine head perimeter. Similar measurement was performed to calculate the coverage fraction of boutons. The analysis was performed within the domain of single astrocytes.51. After cropping out the region of interest, the images were binarized using ImageJ and the average area over time was calculated. Secondly, accumulated area and minimum area were calculated by summating all the time lapse images. Finally, The difference between accumulated area and minimum area was divided by average area to acquire motility index. Fluctuation was calculated as temporal variance of coverage over time.The motility index was calculated according to previous studies2+ events in the spongiform structure, Gaussian blur (ImageJ) was applied to the images in order to reduce noise after subtraction of background value, which was calculated by average intensity of the darkest region  in the field of view. For automatic and unbiased detection and analysis of spontaneous Ca2+ events the ImageJ plugin LC_Pro52 was used. Detected events were manually verified using Igor Pro (Wavemetrics)12. The spatial spread was defined as the maximum best-fit ellipse area above 95% confidence of signal threshold during the event interval. The frequency was calculated by the number of detected Ca2+ events in the field of view (12.5 × 25 µm2) per minute.To analyze Ca2+ signals in the spongiform domain  or non-confined (node + shaft). There were no events detected only at the shaft. Ca2+ events that occurred on undefined structures was categorized as other. Whether the event was confined or not was determined by the relative size of the Ca2+ spread  compared with the size of the node that hosted the event. When the Ca2+ spread was equal or smaller than the node, it was defined as confined. When Ca2+ spread was larger than the node, it was defined as non-confined.To determine the initiation site of the Caain Fig. , Ca2+ ev2+ spread and whose 3D structural information could be reliably captured by STED, were analyzed. We plotted the Ca2+ trace using the ImageJ plugin Time Series Analyzer V3. We used the software NeuroMatic53 to determine the onset times of the Ca2+ transients: after binominal smoothing of the trace, a sigmoid (Boltzmann) function was fitted to the data (achieving a goodness of fit (R2) of >0.95) to compute the point of maximum curvature, which was defined as the onset time. If the software detected less than 1 frame onset difference between the node and the shaft, it was categorized as Node and/or shaft initiating event. If the onset of node was equal or greater than 1 frame earlier than shaft, then it was categorized as Node initiating event. LC_Pro never detected any events that were initiated on shafts, and we also confirmed this by measuring onset times in the different compartments.The non-confined group included (i) events that initiated at the node and spread to neighboring structures, and (ii) events where the initiation site could not be determined. To distinguish between these types, we manually placed ROI (2 × 2 pixels) on nodes where LC_Pro had located the event, its neighboring shaft and node Fig. . Only st2+ signals, only node-initiating events among the non-confined group were analyzed. The amplitude ratio was calculated by dividing the amplitude at N2 (neighboring node) by the amplitude at N1 (initiating node).To correlate the shaft morphology with Ca2+ changes (ΔF) in the figures and movies, we applied a Gaussian filter and the image that preceded the Ca2+ event was subtracted from the time series. To map this Ca2+ change onto the STED image of dendritic morphology, spine signals in confocal and STED images were used to align these images by ImageJ plugin Align-image-by-line-ROI. Subsequently, the Ca2+ change was overlaid with STED spine image. Only Ca2+ signals that occurred at perisynaptic nodes, as judged by STED images of astrocytic GCaMP6s and neuronal YFP, were used for the analysis of perisynaptic Ca2+ signals. Perisynaptic Ca2+ signals were categorized as clustered when other Ca2+ events were observed in neighbor perisynaptic nodes along the same stretch of dendrite within the time frame of 2 s. If not, it was categorized as spine-specific.To illustrate the Cap < 0.05, **p < 0.01, ***p < 0.001.Data were tested for normality using D’Agostino–Pearson omnibus test and Shapiro–Wilk test, and comparisons were made by appropriate parametric or non-parametric tests, taking into account number of groups and the nature of the data (paired/non-paired). Statistical tests were performed using Graphpad Prism. The significance level was set at 5%. The size and type of individual samples, n, for given experiments is indicated and specified in the results section and in figure legends. Asterisks indicate p values as follows: *Further information on research design is available in the Supplementary InformationPeer ReviewDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Reporting Summary


Acute myeloid leukemia is a blood disease whose long-term treatment is not satisfactory due to frequent (>50%) relapse despite complete initial remission. By adopting protumoral properties, macrophages in the leukemia cell microenvironment may promote resistance to treatment leading to relapse. Our goal was to assess whether macrophages in contact with leukemia cells have an impact on blast survival and sensitivity to chemotherapy. We observed that leukemia cells can educate macrophages to support their survival and proliferation and reduce their sensitivity to therapy, as long as the CSF1 receptor remains active and they are in close contact. By inhibiting the activity of the CSF1 receptor, in the presence of GM-CSF, we could modify the macrophage phenotype and increase blast apoptosis and sensitivity to treatment. Our results indicate that leukemia therapies should not only target blasts but also their microenvironment and specifically macrophages and their receptor for CSF1.+ MΦs are abundantly present, at diagnosis, in the bone marrow of AML patients. We showed that myeloblasts, or their conditioned medium, polarize monocytes to M2-like CD163+ MΦs, induce the secretion of many protumoral factors, and promote myeloblast survival and proliferation as long as close intercellular contacts are maintained. Importantly, pharmacologic inhibition of the CSF1 receptor (CSF1R), in the presence of GM-CSF, reprogrammed MΦ polarization to an M1-like orientation, induced the secretion of soluble factors with antitumoral activities, reduced protumoral agonists, and promoted the apoptosis of myeloblasts interacting with MΦs. Furthermore, myeloblasts, which became resistant to venetoclax or midostaurin during their interplay with protumoral CD163+ MΦs, regained sensitivity to these targeted therapies following CSF1R inhibition in the presence of GM-CSF. These data reveal a crucial role of CD163+ MΦ interactions with myeloblasts that promote myeloblast survival and identify CSF1R inhibition as a novel target for AML therapy.Relapse is a major issue in acute myeloid leukemia (AML) and while the contribution of gene mutations in developing drug resistance is well established, little is known on the role of macrophages (MΦs) in an AML cell microenvironment. We examined whether myeloblasts could educate MΦs to adopt a protumoral orientation supporting myeloblast survival and resistance to therapy. Flow cytometry analyses demonstrated that M2-like CD163 AML is a very aggressive disease with poor long-term survival . SeventyChemotherapy and hematopoietic stem cell transplantation most often allow the achievement of sustained complete remission. Moreover, targeted therapies improve the AML response to chemotherapy and favorably affect survival . HoweverTAMs are often called M2-like protumoral MΦs while the classical M1-like MΦs have inflammatory, antitumor properties ,8. HowevTumor escape from immune surveillance is further promoted by the expression of the “do not eat me” signal, CD47 ,14 and t+ MΦs promotes tumor infiltration by activated T lymphocytes and tumor regression [The scavenger receptor CD163 is a marker of TAMs with protumoral characteristics ,12,13. Sgression .Targeting TAM polarization is a novel important therapeutic approach in aggressive cancers ,23. The Using human AML cell lines and primary myeloblasts, we examined the impact of AML cells on allogeneic and autologous MΦs. We showed that the co-culture of primary myeloblasts with healthy donor (HD) monocytes or with BM cells in their microenvironment induces a protumoral MΦ orientation (AML-MΦ), which supports blast cell proliferation and survival. Furthermore, using CSF1R inhibitors and granulocyte-macrophage colony-stimulating factor (GM-CSF), we succeeded in reprogramming protumoral AML-MΦs and in reversing drug-resistance induced by myeloblast cross-talk with MΦs.Heparinized blood and BM samples were obtained from 42 newly diagnosed AML patients at CHUV . Informed consent was obtained from all subjects involved in the study. After red cell lysis with ammonium chloride, the samples were processed for leukocyte immunostaining and analysis by flow cytometry (FC) . LeukemiCell proliferation, apoptosis, and immunophenotypic analyzes were performed using a Beckman Coulter Gallios flow cytometer. Patient samples were analyzed with a 10-color/24-antibody panel  accordinThe monocytic fraction was isolated by centrifuging HD PB anticoagulated with citrate phosphate dextrose-adenine 1 and diluted 1/2 with PBS for 10 min at 1000 g to prepare a buffy coat. The concentrated leukocytic fraction was then centrifuged at 400 g for 40 min on a Ficoll-Paque PLUS  gradient. Mononuclear cells were collected and plated in RPMI + 10% heat-inactivated fetal bovine serum (FBS) = plain medium (PM), see + monocytes. The medium was changed, with the added factors, every 2 days, for 7–9 days, when CD163 expression was checked by FC. Alternatively, to obtain HDAML-MΦs, HD monocytes were stimulated for 7–9 days with (1) primary myeloblast or cell line conditioned medium (CM) diluted 1:1 with PM; with (2) AML cell lines (1–5 × 104/well) or primary blasts (0.5–1 × 106/well), co-cultured in direct contact with HD monocytes; or (3) separated from MΦs by a 0.4 µm-pore membrane insert (Transwell (TW), Falcon, Dietikon, Switzerland).After overnight culture, the wells were rinsed to deplete non-adherent cells; >95% of adherent cells were CD14AML-MΦ) were washed with PBS and cultured for 7–9 more days in PM containing GM-CSF and GW2580  [For MΦ reprogramming experiments, HD MΦ monolayers initially activated with M-CSF (M-MΦ) or blast CM (HDHL-60 (AML with maturation), U937 (monoblastic leukemia), NB4 (acute promyelocytic leukemia), OCI-AML3 (NPM1 mutated) cell lines were cultured in plain medium (PM)  and MV-4Cells were labeled with 10 µM PKH26 (Sigma-Aldrich) according to the manufacturer’s protocol and immediately added to rinsed wells containing adherent MΦs for assays. A fraction of the labeled cells were fixed in 1% paraformaldehyde and used to calculate the normalized PKH mean fluorescence intensity (MFI), as PKH MFI at day 4 divided by PKH MFI at day 0. Blast cell apoptosis was assessed by FC, analyzing cells stained with annexin V .Cells were cultured in 100% PM or in 75% PM supplemented with 25% MΦ-CM. After 48 h, the cells were lysed, and the cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, immunoblotted with anti-cleaved caspase-3 antibody  followed by horseradish peroxidase-linked goat anti-rabbit immunoglobulin G (IgG) antibody , and revealed with Luminata Forte chemiluminescence  . As a loGM/GW-MΦs) was obtained after one week of culture with M-CSF, followed by another week in medium supplemented with GM-CSF and GW2580. CM from M-, GM-, and RGM/GW-MΦs were obtained from at least three different HD and pooled for cytokine assays. The CM from primary AML blasts (mononuclear cell suspension containing >95% blast cells) co-cultured with MΦ were also analyzed and processed as above.The CM of the indicated leukemia cell lines, primary myeloblasts or MΦs were collected and centrifuged twice at 1500 rpm for 10 min, twice for 10 min at 2500 rpm, and filtered with 0.22 μm low protein binding syringe filter. CM was collected at day 7, from M- or GM-MΦs cultured in medium containing M- or GM-CSF, respectively. CM from reprogrammed MΦs . X-ray films were digitalized with ImageScanner III  and dot pixel density was measured with ImageQuant software . The mean of duplicates was normalized to the mean pixel density of the six reference spots.3 NB4 or MV-4-11 were added to MΦ monolayers in PM with inhibitors . After 48 h, cells were removed from the wells, stained for AnnV and/or 7-aminoactinomycin D (7-AAD), and analyzed by FC. Experiments were repeated using MΦs from at least three different HD.HD MΦs were polarized for 7 days with M-CSF and then either reprogrammed for 7 more days in medium containing GW2580 or PLX3397 and GM-CSF or kept in M-CSF-containing medium. MΦ orientation and reprogramming was confirmed by FC. Medium was then removed, and MΦs were washed with PBS. Following this, 150 × 10t-test between parametric matched samples, or the Wilcoxon matched-pairs signed rank (WMP) test between nonparametric matched samples. p values < 0.05 were considered as significant.Statistical significance of differences between groups was examined with the Mann-Whitney (M-W) test between two nonparametric unpaired groups, the paired BM and/or PB were obtained from 42 adult patients with newly diagnosed AML . MΦs andhigh MΦs, whereas those cultured in GM-CSF differentiated into M1-like CD163low MΦs . CD163 expression on HD contact C. Simila contact . In addi contact . These rAML-MΦs  and from two AML cell lines , and other angiopoietic factors, with CXCL5 , M-CSF , and CD1AML-MΦ polarization, such as CD163, CD80, and CD206.CXCL10 could contribute to either a protumoral or an antitumoral activation, depending on the context . The relSince myeloblasts can polarize monocyte-derived MΦs C, we exaThis survival advantage was dependent on direct contact with the MΦs, as it was abrogated when blasts and MΦs were separated by TW inserts. These observations suggest that the survival of certain sub-types of AML could be dependent on direct interactions with MΦs. In contrast, the co-culture of normal HSPC, under the same conditions, with HD Mo did not affect their survival .The cross-talk between myeloblasts, MΦs, and other components of the BM microenvironment induces the secretion of multiple proinflammatory and protumoral growth factors, which promote the remodeling of the leukemia stem cell niche and may stimulate leukemia cell proliferation and/or survival . The ideThe results were compared to those of myeloblasts cultured without monocytes  and bothHGF was secreted at higher levels in co-culture with AML#23. M-CSF was upregulated in co-culture with AML#3, involved in paracrine cross-talks previously observed to drive solid tumor invasion ,49. AdheAs MΦs can contribute to creating a protumoral blast niche in BM microenvironment, we tested more specifically the effect of MΦ orientation on blast survival and proliferation. Myeloblasts were co-cultured for 4 days with HD MΦs, which had been oriented during one week in medium containing GM-CSF (GM-MΦs) or M-CSF (M-MΦs). Co-cultures were performed in PM, either in direct contact or separated with a TW.− GM-MΦs exhibited a significantly lower proliferative activity, correlated to higher PKH26 fluorescence, compared to those cultured on CD163+ M-MΦs , significantly slowed down their proliferation and promoted myeloblast apoptosis  was measured by FC.We next assessed whether MΦ orientation affects the sensitivity of myeloblasts to targeted therapy. We first examined whether MΦ polarization has an effect on myeloblast sensitivity to venetoclax, a BCL-2 inhibitor. Myeloblasts were cultured in direct contact with HD M- vs. GM-MΦs (week#1) or M- vs. R-MΦs (week#2) in presence of vehicle or escalating doses of venetoclax. After 48 h, the percentage of live cells . Direct contact contributed to the resistance of myeloblasts to venetoclax, as NB4 cells became significantly more sensitive to it, when they were separated from M-MΦs by TW inserts A. By conFLT3-mutated AML have a poor prognosis . To deteGM/GW-MΦ CM strongly decreased it . Moreover, after 48 h of incubation with MΦ CM, cleaved caspase-3 was detected by western blot ; of noteGM/GW-MΦs, we illustrated several relevant cytokines as their fold-decrease , in PM vs. PM supplemented with GM-CSF and GW2580  BM AML MΦs frequently express the protumoral orientation marker CD163 at diagnosis; (2) myeloblasts can educate HD MΦs to express CD163 in a CSF1R-dependent manner and promote their growth and survival; (3) numerous SM are secreted that support myeloblasts proliferation, survival, migration, and angiogenesis; (4) resistance to venetoclax and midostaurin is promoted by myeloblast interactions with MΦs; and (5) MΦ orientation can be reprogrammed by inhibition of CSF1R combined with GM-CSF exposure, leading to the reversal of resistance to targeted therapies and myeloblast apoptosis.MΦs are highly plastic cells that can change their phenotype and function in response to microenvironmental stimuli, which modulate their polarization . In vitrIn the majority of AML patients, we observed that the BM and PB MΦs significantly express the M2-like marker CD163 at diagnosis A. IntereAML-MΦs  and induces the biosynthesis of CCL17 in MΦs, a cytokine loop with proinflammatory properties  promotinOther mechanisms may contribute in vivo to the proapoptotic effect of R-MΦs. The proinflammatory molecule IL-6 may also contribute to immune response by promoting T-cell-mediated immune defense  and was Drug resistance is a major problem in relapsed/refractory AML. The combination of the BCL-2 inhibitor venetoclax with hypomethylating agents or low dose cytarabine demonstrated high initial efficiency in patients with de novo AML, who were ineligible for intensive chemotherapy . HoweverSeveral mechanisms of resistance to venetoclax have been reported  involvinThe ability of reprogrammed macrophages to increase the sensitivity of myeloblasts to midostaurin or venetoclax suggests a role for macrophage polarization in promoting resistance to these targeted therapies. Interestingly, close contacts between macrophages and myeloblasts were required to promote resistance to midostaurin or venetoclax, which may suggest the involvement of adhesion receptors. The presence of high soluble VCAM-1 levels in M-MΦ CM and its strong decrease in the CM of GM- and R-MΦs may suggest the involvement of VCAM-1/CD49d, which were previously reported to trigger NF-κB signaling and promote chemoresistance .Aiming to modulate the MΦ phenotype in AML is a promising therapeutic approach. Currently, clinical development of CSF1R inhibitors, including PLX3397, for AML treatment is at the early stages . The eff− MΦs exposed to CSF1R inhibitors, than on CD163+ cells. PD1/PD-L1 inhibition may improve the antitumoral activity of R-MΦs and conversely, CSF1R inhibition may improve the effect of anti-PD1/PD-L1 monoclonal antibody in relapsed/refractory AML.To reprogram human MΦs in AML, combining CSF1R inhibition with GM-CSF might be more efficient for inducing an antitumoral response. It may however not be sufficient to restore MΦ phagocytic activity and antigen-presenting capacity in vivo. Indeed, PD-L1 expression is unchanged or higher on CD163In addition to the T-cell immune checkpoints, CD47 is the dominant MΦ checkpoint, which is overexpressed in myeloid malignancies and inhibits phagocytosis through “do not eat me” signals upon its binding to SIRPα on MΦs . As obse+ MΦs predominate, at diagnosis, in the BM of AML patients and that the induction of CD163 expression on MΦs by myeloblasts was dependent on CSF1R. We observed that numerous SM were secreted by MΦs and myeloblasts, which promoted an M2-like MΦ orientation, leading to increased blast survival and drug resistance in the presence of close intercellular contacts. Finally, inhibiting CSF1R, in the presence of GM-CSF, reprogramed MΦ orientation, reversed the resistance to targeted therapies, and promoted myeloblast apoptosis. CSF1R inhibition may serve as a novel way to target the microenvironment of myeloblasts and improve the efficiency of AML therapy.Relapse and resistance to treatment remain major issues in the treatment of AML. We showed here that protumoral CD163


With-no-lysine (WNK) kinases, specifically WNK1 and WNK4, have been recognized to regulate K+ balance, in part, by orchestrating maxi K+ channel (BK)-dependent K+ secretion in the aldosterone-sensitive distal nephron (ASDN), which includes the connecting tubule and collecting duct. We recently demonstrated that the Ca2+-permeable TRPV4 channel is essential for BK activation in the ASDN. Furthermore, high K+ diet increases TRPV4 activity and expression largely in an aldosterone-dependent manner. In the current study, we aimed to test whether WNK kinases contribute to regulation of TRPV4 activity and its stimulation by aldosterone. Systemic inhibition of WNK with WNK463 (1 mg/kgBW for 3 days) markedly decreased TRPV4-dependent Ca2+ influx in freshly isolated split-opened collecting ducts. Aldosterone greatly increased TRPV4 activity and expression in cultured mpkCCDc14 cells and this effect was abolished in the presence of WNK463. Selective inhibition of WNK1 with WNK-in-11  recapitulated the effects of WNK463 on TRPV4-dependent Ca2+ influx. Interestingly, WNK-in-11 did not interfere with up-regulation of TRPV4 expression by aldosterone, but prevented translocation of the channel to the apical plasma membrane. Furthermore, co-expression of TRPV4 and WNK1 into Chinese hamster ovary (CHO) cells increased the macroscopic TRPV4-dependent cation currents. In contrast, over-expression of TRPV4 with a dominant negative WNK1 variant (K233M) decreased the whole-cell currents, suggesting both stimulatory and permissive roles of WNK1 in regulation of TRPV4 activity. Overall, we show that WNK1 is essential for setting functional TRPV4 expression in the ASDN at the baseline and in response to aldosterone. We propose that this new mechanism contributes to regulation of K+ secretion and, by extension, urinary K+ levels to maintain systemic potassium homeostasis.Kidneys play a central role in regulation of potassium homeostasis and maintenance of plasma K Indeed, genetic deletion of TRPV4 abolishes flow-dependent net K+ transport in the perfused collecting ducts i measurements closely followed the protocols previously published by our group i elevations from 110 ± 3 nM to 329 ± 7 nM  ,17. Thusye Fura2 A and the changes B, TRPV4 -/- mice  providin9 ± 7 nM B,C. This9 ± 7 nM D and the9 ± 7 nM E. TRPV4-9 ± 7 nM . Altogetc14 cells, a well-accepted model of the collecting duct principal cells [2+]i in mpkCCDc14 cell monolayers  augments GSK1016790A-induced [Ca2+]i responses nearly two-fold  similarly augments GSK1016790A [Ca2+]i responses in mpkCCDc14 cells  retention by the distal convoluted tubule thus causing little flow in the collecting duct. Interestingly, thiazide diuretics are highly effective in reducing blood pressure and also by correcting hyperkalemia in PHAII patients i responses could be attributed, at least to some extent, to the WNK actions on the non-identified targets also susceptible to stimulation by 4α-PDD and hypotonicity. In contrast, we show that deletion of TRPV4 completely abolishes [Ca2+]i elevations in response to GSK1016790A (Our results  demonstr1016790A , therefo2+ influx and decreased renal TRPV4 levels in mice treated with WNK463 (824 residue to increase channel activity in over-expression systems [We show here that intact kinase activity is necessary for regulation of TRPV4 activity by WNK1 . Thus, kh WNK463 . At this systems . It is q systems . Future + balance, WNK signaling cascades also orchestrate a highly intricate interplay between renal sodium and potassium transport to maintain systemic homeostasis. TRPV4 appears to be a vital component of the flow-induced K+ secretion and is now an identified end-effector of the aldosterone-WNK1 signaling pathway in the ASDN.This study reveals a critical role of WNK1 in promoting TRPV4 functional expression and its stimulation by aldosterone in the kidney. Moreover, we have also examined the ramifications of systemic and local WNK1 blockade on TRPV4 with the recently developed orally active antagonists with high promise to become a new class of antihypertensive drugs in clinic. Apart from its prominent role in controlling Na