In addition, Ca2+ entry signals can eventually be amplified by ER release via calcium-induced calcium release (CICR). We have investigated the contribution of ER Ca2+ release to the action of physiological agonists in pituitary gland. Changes of [Ca2+] in the ER ([Ca2+]ER) were measured with the genetically encoded low-affinity Ca2+ sensor GAP3 targeted to the ER. We used a transgenic mouse strain that expressed erGAP3 driven by a ubiquitous promoter. Virtually all the pituitary cells were positive for the sensor. In order to mimick the physiological environment, intact pituitary glands or acute slices from the transgenic mouse were used to image [Ca2+]ER. [Ca2+]C was measured simultaneously with Rhod-2. Luteinizing hormone-releasing hormone (LHRH) or thyrotropin releasing hormone (TRH), two agonists known to elicit intracellular Ca2+ mobilization, provoked robust decreases of [Ca2+]ER and concomitant rises of [Ca2+]C. A smaller fraction of cells responded to thyrotropin releasing hormone (TRH). By contrast, depolarization with high K+ triggered a rise of [Ca2+]C without a decrease of [Ca2+]ER, indicating that the calcium-induced calcium-release (CICR) via ryanodine receptor amplification mechanism is not present in these cells. Our results show the potential of transgenic ER Ca2+ indicators as novel tools to explore intraorganellar Ca2+ dynamics in pituitary gland in situ.The adenohypophysis contains five secretory cell types , each secreting a different hormone, and controlled by different hypothalamic releasing hormones (HRHs). Exocytic secretion is regulated by cytosolic Ca The anterior pituitary (AP) is a complex organ that controls a broad array of physiological functions such as growth, lactation, metabolism, or stress response . This fu2+ and secretory responses to more than one HRH have also been identified by some authors , whose secretion is specifically regulated by a particular hypothalamic releasing hormone (HRH) , 3. Howe authors , 10, 11. authors , with ch authors , cold st authors . Multifu authors , 15 and  authors , 16–18. Recent studies using single cell transcriptomics have expanded our current knowledge on the gene expression profile associated with specific cell subtypes or AP functions in mice, rats or humans , 19–25.2+ signals ([Ca2+]C), which can be generated either by Ca2+ entry from the extracellular medium through the plasma membrane and/or by Ca2+ release from the endoplasmic reticulum (ER)  approaches 10-3 M, in contrast to the resting [Ca2+]C, which is ~10-7 M  and/or ryanodine receptors (RyRs) (2) to inositol-1, 4, 5-trisphosphate (IP3). Due to the large Ca2+ gradient between the ER and the cytosol, IP3 releases Ca2+ from the intracellular stores, and this elicits an increase in the [Ca2+]C and secretion of the corresponding hormone.AP provides an excellent model for endocrine excitation-secretion coupling. In the last decades AP studies have provided seminal insights into the mechanisms involved in endocrine stimulus-secretion coupling and regulation by ion channels activity. Exocytic secretion is regulated by cytosolic Calum (ER) . Ca2+ in ~10-7 M . This his (RyRs) . Binding2+ in an IP3-dependent manner . In frog melanotrophs, it appears that spontaneous voltage-activated Ca2+ influx is coupled to CICR, presumably through IP3Rs  that can be targeted to various organelles  and xylazine (10 mg/Kg) and transcardially perfused with 0.9% physiological saline followed by a solution of 4% paraformaldehyde (~20 ml) and then overnight post-fixed in the same solution at 4°C. The tissue was cryoprotected in 30% sucrose and then processed for immunohistochemistry. Glands were cryosectioned (10–15 μm thick) and stored at −80 °C until use. Slides were permeabilized in PBS containing 0.5% Triton X-100 for 1 h and blocked in PBS with 10% goat serum. Slides were incubated overnight with specific antibodies against each hormone  diluted  in fresh blocking buffer. Antisera was a generous gift from Dr. Parlow . As a secondary antibody, an anti-rabbit antibody coupled to Alexa Fluor 568 (Roche) diluted 1:500 in fresh blocking buffer was used, which was incubated for 30 min. Staining controls with secondary antibody alone elicited no fluorescent signal. Nuclei were stained with Hoechst 33342. Fluorescence images were collected on a Zeiss upright Axioplan 2 microscope, using a 63X W “C-Apochromat” objective (N.A. 1.2). The fluorescence filters used were: red fluorescence: Ex 546/12, Em LP590; green fluorescence: Ex 470DF35, Em 535DF35; blue fluorescence: Ex 390/22, Em 460/50 nm. For cell quantification, positive cells randomly chosen within various fields per section corresponding to various sections per gland were analyzed using the ImageJ software. For each hormone, the percentage of labeled cells was calculated by dividing by the total number of cells, evaluated from the labeled nuclei.The basic protocol was previously described elsewhere . Briefly2, 26 mM NaHCO3, 1 mM CaCl2, 10 mM glucose, 1.25 mM NaH2PO4, pH 7.4, continuously bubbled with a 95% O2/5% CO2 gas mixture at 25 °C. Slices were mounted onto the stage of a Zeiss Axioplan upright microscope equipped with a 20X objective  and a Zeiss AxioCam camera MRm (12 bit) connected through a software interface  to a Xenon fluorescent excitation source and a filter wheel. GAPs were sequentially excited at 405 and 470 nm and acquired at 518–553 nm. For simultaneous measuring of [Ca2+]ER and [Ca2+]C, slices were incubated for 1 h at room temperature with 8–12 µM Rhod-2 AM in bubbled ACSF medium. Rhod-2 was excited at 545 nm (546/12) using a dichroic mirror FT580 and light emitted was recorded above 590 nm (LP590). Pituitary slices were sequentially excited at 405, 470 (GAP) and 540 nm (Rhod). All the experiments were performed at 22–25 °C in a custom-made chamber of 42 µl volume under constant perfusion at 3 ml/min with an ‘extracellular-like solution’ containing 145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM Na-HEPES (pH 7.4). All stimuli were diluted in this extracellular-like medium and perfused for 30 s or the time indicated. Imaging of erGAP3 in the whole pituitary gland were performed similarly. Output images were captured with the AxioVision Rel 4.6.3 (Zeiss) software and pixel-to-pixel ratio analysed with ImageJ (https://imagej.nih.gov/ij/). The erGAP3 ratio R (F470/F405) was used as an index of [Ca2+]ER, and was expressed as R/R0. F540 was an index of [Ca2+]C, expressed as F/F0. R0 (or F0) was computed as the mean of the ratios (or F540) obtained during the first five to 10 frames of each experiment.Mice (P7-5 months) from transgenic line erGAP3 (L1 or L10) were sacrificed by cervical dislocation. AP gland was dissected out and sliced into 350–400 µm thick sections with a Mcllwain Tissue Chopper and quickly transferred to a fine-meshed membrane filter and maintained in artificial cerebrospinal fluid (ACSF) containing: 125 mM NaCl, 2.5 mM KCl, 1 mM MgClThe data were analysed using Origin 7 (OriginLab™) and excel. Results are expressed as mean ± SEM, as indicated2+]ER in intact AP glands we used erGAP3 expressing transgenic mice in which the biosensor was controlled by the ubiquitous promoter CAG-GS  was specifically targeted to the ER using the well-established strategy based on adding the signal peptide of calreticulin and the KDEL retention motif to the N-terminal and the C-terminal of the GAP gene, respectively , lactotrophs (20%–25%), corticotrophs (10%–20%), gonadotrophs (10%), and thyrotrophs (5%) , 42. No 2+/inositol phosphate signaling cascade . Taken together, these results show that erGAP3 expression in pituitary gland displays a performance of the Ca2+ indicator comparable to that previously obtained in other cells types such as HeLa cells, HEK293 cells, astrocytes, or hippocampal neurones  and reached a lower steady-state level. Washout of LHRH during 5 min generally failed to refill the ER, which remained half-filled after the washout. This occurred even after the stimulation of Ca2+ entry with a depolarizing pulse of high K+ (80 mM), that provoked a large cytosolic transient with no ER Ca2+ changes. The typical protocol finished with the perfusion of an ER depletion cocktail, used to determine the Rmin of erGAP3, that was composed of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor 2,5-ditert-butyl-benzo-hydro-quinone  in Ca2+-free medium. Interestingly, a quarter of the LHRH-responsive cells  also responded to thyrotropin-releasing hormone  with a rapid lumenal Ca2+ release (R/R0 (mean ± SEM) decreased from 1 down to 0.88 ± 0.05; n=18 cells; 2+ increase , this factor provoked a small and sustained increment in the cytosolic Ca2+ in a fraction of cells , neither in the cytosol nor in the ER. Spontaneous or HRH-induced cytosolic Ca2+ oscillations, either with TRH or LHRH, were observed in some cells but no ER Ca2+ changes were associated with them   responded exclusively to TRH with a rapid and large ER Ca2+ drop and this decrease was reversible by washing out the stimulus, allowing the refilling of the ER. A second group of cells  only responded to LHRH, but not to TRH. The amplitude of the ER Ca2+ release triggered by LHRH is comparable to that of TRH (~30% R/R0 change) and the ER also recovered the basal [Ca2+]ER . A third population  responded to both factors, TRH and LHRH, indicating the presence of multiresponsive cells bearing several types of HRH receptors. Addition of TRH as the first stimulus released a fraction of the stored ER Ca2+, and the second stimulus, LHRH, released an additional fraction. Interestingly, the three types of cells responded to a high K+ (80 mM) depolarization stimulus with a transient increase of 0.1 ± 0.01 , as a consequence of a transient Ca2+ uptake into the ER, and the signal quickly returned to the resting ER levels. This last result indicates that the erGAP3 sensor was not saturated at resting ER level and no sign of CICR were observed, in agreement with the results shown in slices (A higher level of tissue preservation was achieved by imaging ER Ca2 cells) Figure 62+ dynamics and elucidating the Ca2+ signals interacting between organella require specific localization of the Ca2+ sensors, especially in complex tissues and organs. Genetically encoded Ca2+ indicators (GECIs) provide a powerful tool that overcomes some of the disadvantages of the synthetic probes, such as their lack of subcellular specificity or the difficulty of loading thick tissue preparations. Transgenic mice expressing GECIs have proven to be a particularly useful technology for being minimally invasive, its ease of use and its stability and width of expression. Although the number of GECIs have dramatically increased in recent years, those optimized for high Ca2+ compartments such as the ER are more limited. Even less frequent is the application of low Ca2+ affinity indicators to transgenics, which is mostly restricted to non-mammalian organisms   , 50. We in vitro Figure 3in vitro . We did 2+ dynamics. The advantages of imaging [Ca2+]ER using these transgenic mice are several: simple tissue preparation and imaging procedures; preservation of pituitary gland organization; and sensitive, highly efficient and simultaneous ER Ca2+ imaging of multiple pituitary cells. It is well known that cell to cell contact in the intact tissue is crucial to retain many of the Ca2+ signaling patterns . In this context, the numbers of cells reported to respond to each HRH give an indication on the approximate fractions of each population but a more detailed study with a confocal or two-photon microscope would add spatial resolution required for an exact quantification. More recently, transcriptomics studies and single cell RNA-sequencing (sc-RNA) analysis of AP have proven to be excellent tools to gain insights into the expression profiles specific for each AP population –25, 59. 2+ indicator inverse pericam due to a poor SNR .JR-R and PN-N performed the experiments and analyses and participated in preparing the manuscript figures. LN participated in the initial dispersed cultures. All the authors participated in the conception and design of the study. MA and JG-S wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Spanish Ministerio de Economia y Competitividad (BFU2017-83066-P) and the Consejería de Educación, Junta de Castilla y León (GR175), and the Instituto de Salud Carlos III .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.


FLT3 mutation is present in 25–30% of all acute myeloid leukemias (AML), and it is associated with adverse outcome. FLT3 inhibitors have shown improved survival results in AML both as upfront treatment and in relapsed/refractory disease. Curiously, a variable proportion of wild-type FLT3 patients also responded to these drugs.FLT3 AMLs was performed with the Wilcoxon-rank sum test. Hierarchical clustering was used to identify FLT3-mutation like AMLs. Finally, enrichment in recurrent mutations was performed with the Fisher’s test.We analyzed 6 different transcriptomic datasets of AML cases. Differential expression between mutated and wild-type FLT3 mutation-like gene expression pattern was identified among wild-type FLT3 AMLs. This pattern was highly enriched in NPM1 and DNMT3A mutants, and particularly in combined NPM1/DNMT3A mutants.A FLT3 mutation-like gene expression pattern in AML which was highly enriched in NPM1 and DNMT3A mutations. Future analysis about the predictive role of this biomarker among wild-type FLT3 patients treated with FLT3 inhibitors is envisaged.We identified a  FLT3 internal tandem duplication (ITD) is the most common mutation, affecting 25% of de novo AML cases [FLT3-TKD) in codons D385 and I386 have been observed in 5–10% of cases [FLT3-ITD mutation confers a poor outcome in de novo and relapsed AML, and it is currently suggested to include these patients in clinical trials [FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase commonly mutated in acute myeloid leukemia (AML) . FLT3 inof cases . FLT3-ITFLT3-mutated (FLT3mut) AML significantly improved event-free survival and overall survival in all FLT3 mutation subtypes [FLT3 AML patients were reported for both gilteritinib  and quizartinib (30–36% of composite complete remissions) [FLT3 mutation in order to search for wild-type FLT3 cases that resemble FLT3mut AMLs at the transcriptomic level.A number of promising FLT3 inhibitors are under development for AML treatment. The addition of the multi-kinase inhibitor midostaurin  to standsubtypes . Severalsubtypes –11. Curiissions) , 11. It issions) . Not surissions) , but theFLT3 mutation-like gene expression pattern highly enriched in NPM1 and DNMT3A mutants. Our results indicate common deregulated pathways among these leukemias, opening the way to study the role of this biomarker in order to predict responses to FLT3 inhibitors in wild-type FLT3 AML.In this study we analyzed the transcriptomic pattern of 6 different AML cohorts, which enabled the identification of a Gene Expression Omnibus (GEO): GSE14468 , GSE10358 (188 cases of de novo AML), GSE61804 (279 de novo AML cases), GSE17855 (237 pediatric AML cases) and GSE15434 . In the case of GSE14468 and GSE10358, both FLT3-ITD and D385 mutation status were reported, whereas in the remaining datasets only the FLT3-ITD status was informed. All datasets used the same type of gene expression arrays: Affymetrix Human Genome U133 Plus 2.0 Array. Signal intensities were rank-normalized, and differential expression between FLT3mut (either ITD or TKD) and wild-type samples was performed in the largest cohort (GSE14468) using the two-sided Wilcoxon-rank sum test. P-values were adjusted for multiple testing using the Bonferroni method. Hierarchical clusterization and heatmap plots were created using the heatmaps3 package with default parameters [WebGestalt with default parameters  [We analyzed five AML gene expression datasets available in the Bamopoulos et al. (GEO identification GSE146173) [fpkm function implemented in the DESEQ2 package [FLT3-like pattern were selected, followed by standard hierarchical clusterization. Differential mutation between the FLT3-like and no-FLT3-like group was performed with Fisher’s test.RNAseq and mutation data from 246 AML patients treated with intensive chemotherapy was retrieved from E146173) . Raw couAll computations except gene ontology analysis were performed in R. All data used for this analysis is readily accessible from public repositories.FLT3mut (ITD and/or TKD) and wild-type FLT3 samples , since it contained 81.67% of all FLT3-ITD cases, 61.36% of all FLT3-TKD cases and 83.33 of composed mutants . Furthermore, 42.15% of wild-type FLT3 cases were also clustered within this group (We chose the largest database (GSE14468) as the discovery set, and we identified 911 probes mapping to 649 different genes which were differentially expressed between lue 0.03 . Hierarcis group .FLT3-ITD mutation status. In GSE61804 a cluster comprising 58.06% of all patients harbored 78% of all FLT3-ITD cases , and 53.71% of all non FLT3-ITD cases were grouped within this cluster , but it also included 39.15% of all non FLT3-ITD cases ; and additionally 49.11% of all non FLT3-ITD cases co-clustered within this group  to just 12.16% in pediatric AML (GSE17855), indicating that the existence of the FLT3-like pattern is not explained by co-occurring NPM1 mutations. Additionally, a relative enrichment in IDH1 mutants within FLT3-like leukemias was detected in the discovery set GSE14468 . Furthermore, depletion of CEBPA mutants in FLT3-like AML was a common phenomenon in all 3 cohorts that reported mutations in this gene, and a reduced frequency of KIT mutations was also discovered in the pediatric AML dataset (GSE17855).Four of the microarray databases provided data about driver mutations in a few genes . The mosBamopoulos et al. [FLT3-like leukemias were highly enriched in concurrent NPM1 and DNMT3A mutants, whereas 19.60% of FLT3-like AMLs were NPM1 and DNMT3A wild-type. Additionally, there was a tendency for an enrichment in IDH1 mutations . On the contrary, FLT3-like AMLs were depleted in ASXL1, CEBPA, RUNX1, TP53, SF3B1 and U2AF1 mutations  could smutation .FLT3 mutation-like samples were clustered near FLT3 mutants across the different cohorts, which probably reflects substantial heterogeneity between them. One of the possible explanations for this could be a differential distribution of NPM1 and DNMT3A mutants, since these are drivers of cytogenetically-normal AMLs (such as in the case of GSE15434) [FLT3-ITD annotation, and a minority of the FLT3-like cases might indeed have FLT3-TKD mutations.This study has some limitations. Firstly, a variable proportion of SE15434) . FinallyFLT3 mutation-like transcriptomic pattern with a different mutational background which clusters a proportion of wild-type FLT3 AMLs with FLT3mut samples. These leukemias were highly enriched in NPM1, and particularly in composed NPM1/DNMT3A mutants, but NPM1 status alone was insufficient to explain the existence of the FLT3-like pattern. The analysis of wild-type FLT3 AML patients treated with FLT3 inhibitors in clinical trials is envisaged in order to study its possible role as a drug response biomarker.Our results are concordant with the existence of a S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableData reported for mutations analyzed in GSE14468, GSE10358, GSE15434 and GSE17855.(XLSX)Click here for additional data file.


Here, individual and tandem fused peptides were synthesized, conjugated to keyhole limpet hemocyanin (KLH), then evaluated for immunoprotection of rabbits against swine HEV infection. Forty New Zealand White rabbits were randomly assigned to eight groups; groups 1 thru 5 received three immunizations with EPTV-KLH, VKLYTS-KLH, PSRPF-KLH, EPTVKLYTS-KLH, or EPTVKLYTSPSRPF-KLH, respectively; group 6 received truncated swine HEV ORF2 protein (sp239), and group 7 received phosphate-buffered saline. After an intravenous swine HEV challenge, all group 7 rabbits exhibited viremia and fecal virus shedding by 2–4 weeks post challenge (wpc), seroconversion by 4–9 wpc, elevated alanine aminotransferase (ALT) at 2 wpc, and severe liver lymphocytic venous periphlebitis. Only 1–2 rabbits/group in groups 1–4 exhibited delayed viremia, fecal shedding, seroconversion, increased ALT levels, and slight liver lymphocytic venous periphlebitis; groups 5–6 showed no pathogenic effects. Collectively, these results demonstrate that immunization with a polypeptide containing three genotype 4 HEV ORF2 neutralizing epitopes completely protected rabbits against swine HEV infection.Genotype 4 hepatitis E virus (HEV) is a zoonotic pathogen transmitted to humans through food and water. Previously, three genotype 4 swine HEV ORF2 peptides ( Hepatitis E virus (HEV) infects approximately 20 million people worldwide each year, causing 3.3 million cases of acute illness and 30,000–40,000 deaths annually ,2. GenerHepeviridae, which includes two genera, Orthohepevirus and Piscihepevirus [Orthohepevirus includes four HEV species (A to D), of which Orthohepevirus A, C, and D species mainly infect mammalian hosts, while the Orthohepevirus B species mainly infects chickens [Orthohepevirus A species, HEV genotypes 1 and 2 are restricted to humans [HEV belongs to the family epevirus . Orthohechickens . Within o humans , while go humans . Indeed,o humans ,12. As to humans . Recent HEV is a quasi-enveloped, positive-sense, single-stranded RNA virus ,14 that Previous reports had demonstrated rabbits to be susceptible to experimental infection by genotype 4 human and pig HEV isolates ,24,25, pMeanwhile, other reports have demonstrated that a bacterially expressed peptide fragment (designated p239) spanning the region of genotype 1 HEV capsid protein between aa 368 and aa 606 could self-assemble into virus-like particles (VLPs). This self-assembling peptide was subsequently further developed into the first vaccine against genotype 1 HEV ,28. Here407EPTV410, 410VKLYTS415, and 458PSRPF462, were characterized  host cells and purified as described previously [Swine HEV truncated ORF2 protein sp239 (aa 368-606) and ORF3 protein were expressed separately in eviously .4 genomic equivalents (GE)/mL was prepared from suspensions of fecal and bile samples collected from specific-pathogen-free (SPF) pigs infected with CHD-SD-sHEV, as previously described [Swine HEV  was isolated from a bile sample of a 32-week-old pig at a slaughterhouse in China. Viral stock containing 10escribed .A total of 40 8-weeks-old SPF New Zealand White rabbits  with initial body weights averaging 1.5 kg  were assigned to eight groups (5 rabbits per group). Rabbits were housed separately in 40 cages. Before immunization, each rabbit was screened to confirm serum negativity for anti-HEV antibodies and fecal negativity for HEV RNA using indirect ELISA and nested RT-PCR, respectively.® Alum , and two additional injections were at 2-week intervals.After one week of acclimation, 9-week-old rabbits of groups 1–6 were immunized subcutaneously with Pep VKLYTS, Pep PSRPF, Pep EPTV, Pep EPTVKLYTS, Pep EPTVKLYTSPSRPF, or sp239, respectively. Rabbits in group 7 were immunized as above, but with PBS only (negative control). Nonimmunized and unchallenged rabbits in group 8 served as normal controls. The antigen (400 μg) for each immunization was mixed with Imject4 GE/mL) prepared from a 10% fecal stock. Rabbit serum and fecal samples were collected before inoculation and weekly thereafter for 12 weeks. Viremia and fecal virus shedding were determined for each inoculated rabbit using a sensitive nested RT-PCR assay, while serum HEV RNA levels were concurrently tested using real-time RT-PCR. In addition, serum samples were tested for anti-swine HEV antibodies by indirect ELISA using swine HEV ORF3 (sHEV-ORF3) as coating antigen, and serum levels of alanine aminotransferase (ALT) were also measured. During necropsy, liver tissues were collected for routine histological examination.Rabbits in groups 1–7 were each challenged by injection via ear vein of 1 mL of swine HEV stock (102SO4 (50 μL/well), and optical density (OD) values were read at 450 nm using an automated microplate reader . Each serum sample was tested in duplicate wells. The OD values of the pre-inoculation serum samples were used to determine cutoff values.Before and after immunization and challenge, indirect ELISAs were used to measure levels of antibodies specific for peptides, sp239, or sHEV-ORF3 proteins in serum samples collected from rabbits previously immunized with BSA-conjugated antigens , as previously described by Chen et al. . BrieflySwine HEV RNA was detected in fecal and serum samples using RT-nPCR according to the method described by Huang et al. . In addiALT concentrations of serum samples collected weekly were measured  following the manufacturer’s instructions. Rabbits were considered positive for hepatitis if post-challenge ALT levels exceeded pre-challenge ALT levels by greater than two-fold .During necropsies, gross pathological lesions in the liver of each rabbit were evaluated. Liver tissues were also fixed in 10% neutral buffered formalin and processed for routine histological examination.t-tests (Microsoft Office Excel 2016) were performed to compare differences in ELISA absorbance values reflecting levels of antibodies (specific for epitopes within peptides and sp239 antigens) in pre-vaccinated versus post-vaccinated rabbit sera; antibody levels are predictive of immunoprotection conferred by immunization with peptides or sp239 protein versus that of the PBS-immunized group. ALT values among groups were also analyzed using the same method [p < 0.05 was considered significant.e method . p < 0.0All experiments in this study were designed based on the principles expressed in the Guidance for Experimental Animal Welfare and Ethical Treatment by the Ministry of Science and Technology of China. Experimental procedures and animal use and care protocols were carried out in accordance with the guidelines of the Northwest A&F University Institutional Committee for the Care and Use of Laboratory Animals and were approved by the Committee on Ethical Use of Animals of Northwest A&F University (AE124319).450mm values of immunized rabbit serum samples (groups 1–6) increased to 3.0 and remained stable after the third immunization .Serum samples of each rabbit before and at various weeks post-immunization (wpi) were collected to measure levels of antibodies specific for peptides or sp239 protein using indirect ELISA. After the second immunization (4 wpi), the ODn 6 wpi, . MeanwhiBefore the challenge, serum and fecal samples from all rabbits were negative for HEV RNA . Convers3.2–104.2 copies/mL) in all rabbits .Besides the detection of HEV RNA in serum samples by RT-nPCR, viral copy numbers were quantitatively evaluated by RT-qPCR . In grou rabbits G7. Meanwopies/mL G1,G4, whoups 1–6 . The prop < 0.05).Throughout the study, ALT levels in serum samples of all rabbits were monitored weekly. In group 7, a peak ALT level of 99-113 U/L was observed for all rabbits at 2 wpc G7. ConveThroughout the study, no gross hepatic lesions were observed in livers of any necropsied rabbits in any group. However, slight to severe lymphocytic venous periphlebitis microscopic lesions were observed in liver tissues of some rabbits, including all group 7 rabbit livers G7. By coGenotype 3 and 4 HEVs are regarded as potential zoonotic viruses, with pigs serving as a main natural reservoir for virus propagation . With thPrevious studies have pinpointed major immunodominant epitopes on the ORF2 protein that are candidate antigens for HEV vaccine development . Based oIt has been reported that immunization of SPF chickens with peptide Pep1B5-1, which includes the epitope (VKLYTS) recognized by MAb 1B5, was not protective against avian HEV challenge . In the Generally, slight to severe microscopic lesions in liver tissues were observed that demonstrated hepatitis was a complication of HEV infection, as indicated by lymphocytic venous periphlebitis, portal fibrosis, swollen hepatocytes, balloon-like lesions, and hepatocellular necrosis ,25,38. IIn general, 1–2 booster immunization were performed after the first immunization in the animal experiment. In this study, rabbits were immunized three times in total, and the antibody levels peaked at 4 wpi (after 2nd immune) and remained stable at 6 wpi (after 3rd immune) , which sImportantly, one or two rabbits in groups immunized with peptides EPTV, VKLYTS, or PSRPF showed active infection but exhibited delayed fecal virus shedding and viremia as compared with the virus-only group . These rIn summary, rabbits immunized with peptide EPTVKLYTSPSRPF or sp239 protein showed no seroconversion, fecal virus shedding, viremia, ALT level increases, or liver lesions. This study revealed that peptides synthesized by tandem neutralizing epitopes effectively protected rabbits from swine HEV infection, while also providing a foundation for future vaccine and antiviral drug development.


Membrane ion channels are structures through which ions enter or exit the cell, depending on the driving forces. The opening of transient receptor potential vanilloid 1 (TRPV1) ligand-gated ion channels facilitates transmembrane Ca2+ and Na+ entry, which modifies the delicate balance between apoptotic and proliferative signaling pathways. Proliferation is upregulated through two mechanisms: (1) ATP binding to the G-protein-coupled receptor P2Y2, commencing a kinase signaling cascade that activates the serine-threonine kinase Akt, and (2) the transactivation of the epidermal growth factor receptor (EGFR), leading to a series of protein signals that activate the extracellular signal-regulated kinases (ERK) 1/2. The TRPV1-apoptosis pathway involves Ca2+ influx and efflux between the cytosol, mitochondria, and endoplasmic reticulum (ER), the release of apoptosis-inducing factor (AIF) and cytochrome c from the mitochondria, caspase activation, and DNA fragmentation and condensation. While proliferative mechanisms are typically upregulated in cancerous tissues, shifting the balance to favor apoptosis could support anti-cancer therapies. TRPV1, through [Ca2+]i signaling, influences cancer cell fate; therefore, the modulation of the TRPV1-enforced proliferation–apoptosis balance is a promising avenue in developing anti-cancer therapies and overcoming cancer drug resistance. As such, this review characterizes and evaluates the role of TRPV1 in cell death and survival, in the interest of identifying mechanistic targets for drug discovery.Intracellular calcium (Ca Molecular mechanisms that mediate cell death and proliferation exist in balance in functional physiological systems. Proliferation is involved in structural development and renewal, while programmed cell death is necessary to eliminate defective cells and prevent uncontrolled growth. Carcinogenesis results from imbalances in the described pathways, which favor proliferation and reduce apoptosis ,2. Therec from mitochondria, which promotes caspase 9 activation i) is modulated by receptor-operated, store-operated (SOC), and voltage-sensitive ion channels, ion exchangers, pumps, Ca2+ binding proteins, mitochondrial Ca2+ ([Ca2+]m), and endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) Ca2+ ([Ca2+]ER and [Ca2+]SR) i or protein signaling, and IP3R channels, which are activated by IP3 binding, release Ca2+ from the ER and SR. Through [Ca2+]i signaling, these two channel types modulate muscle contraction and nerve impulse transmission [2+ transport from the ER or SR to the cytosol may elevate [Ca2+]m and consequently induce mitochondrial dysfunction [Calcium (CaCa2+]SR) ,8. Intrasmission ,10. Abbefunction ,12.2+]i homeostasis may also mediate cell death or proliferation. For instance, while [Ca2+]i signaling via IP3R contributes to proliferation and oncogenesis, RyR [Ca2+]i signaling supports apoptosis in lung cancer cells i-mediated cell death may be apoptotic or necrotic in nature, depending on the time of exposure and [Ca2+] i involved I signaling regulates proliferation, invasion, and metastasis in cancerous tissues [2+]i [2+]i-affecting proteins, including transmembrane ion channels, which mediate Ca2+ flow between the extracellular space and the cytosol, are potential targets for chemotherapeutic agents.Significantly, [Ca tissues . A variees [2+]i ,20. Ther2+ channels, which respond to a wide variety of environmental stimuli i, TRPV1 influences the balance between proliferation and apoptosis i through TRPV1 channel opening. Furthermore, glycolic acid-TRPV1 interactions stimulate the release of intracellular ATP molecules into the cytosol, where they bind to the membrane G protein-coupled P2Y2 receptors [2+]i and stimulated P2Y2 receptors activate phospholipase C (PLC), resulting in the upregulation of intracellular IP3 levels and subsequent store-operated Ca2+ entry [2+]i and protein signals from the P2Y2 receptor activate the phosphoinisitide-3-kinase, PI3K .,41.40,412+ entry ,57,58. Ese, PI3K ,60. PI3Kse, PI3K . PDK 1 ase, PI3K ,60,61. Cse, PI3K ,63. EGFRse, PI3K .Interestingly, in endothelial colony-forming cells (ECFC), capsaicin downregulates proliferation induced by AEA, potentially suggesting that the two TRPV1 agonists compete for binding sites and differentially influence the apoptosis–proliferation balance . Additio2+ and Na+ influx into the cell; notably, TRPV1 displays greater Ca2+ than Na+ affinity [2+ influx elevates [Ca2+]i . As TRPV1 is a ligand-gated cation channel, agonist binding results in Caaffinity . The men [Ca2+]i : ref. [3 [Ca2+]i : ref. i, resulting from TRPV1 channel opening, causes an initial increase in [Ca2+]m and subsequent downstream effects, which can be attenuated by TRPV1 antagonists  m) levels and mitochondrial ROS (ROSm) production, while Bax is upregulated via nuclear action; both GSH depletion and Bax binding to mitochondrial membrane voltage-dependent anion channels (VDAC) promote the opening of the mitochondrial permeability transition pore (PTP), through which further Ca2+ exit to the cytosol occurs m and [Na+]m cause hyperpolarization of the membrane potential, followed by the depolarization upon PTP opening m  cytosol ,86. Memb cytosol . Further cytosol ,87.i upregulates p38 and associated MAPKs, which upregulate caspase 9 and contribute to ER stress i and kinase activity (2+]i promotes the activity of the SERCA(2) pump, which facilitates Ca2+ entry into the ER/SR [2+]ER, Ca2+ is exported to the cytosol via the RyR2 (ryanodine receptor (2) channels, which are activated by increases in [Ca2+]i i . As disctransfer .Upon activation by endogenous agonists, TRPV1 protein units localized to the ER activate the eukaryotic translation initiation factor, eiF2-α, which promotes expression of activating transcription factor 4 (ATF4) ,78. ATF42+]i elevation promotes the activation of calcineurin, a protein phosphatase [Upstream proapoptotic nuclear activity results from transcription factor activation via cytosolic and ER protein signaling . [Ca2+]isphatase ,54. Calcsphatase ,52. Togesphatase ,52. ATM sphatase ,96. Actisphatase ,54,71,87i depletion chondria ,82. The chondria ,93. Togechondria ,97,98. Cchondria . With sochondria ,89,100; chondria .Caspase activity is well characterized in relation to TRPV1-mediated apoptosis : ref. 4,100,101;The collective objective of oncologic therapies is to restore and maintain a homeostatic balance between proliferation and apoptosis. In this interest, many anti-cancer compounds induce and enforce apoptosis through the targeting of p53 . The acc2+]i, a universal second messenger regulating cell death and survival, is a promising target. [Ca2+]i signaling is highly versatile, as it influences intracellular pathways for proliferation, differentiation, and apoptosis in neuroblastoma cells [2+]i signaling is modified to promote proliferation and minimize apoptosis. Modifications of this nature may downregulate Ca2+ entry into the cytosol  or eliminate downstream targets of [Ca2+]i signaling  i homeostasis (via elevation of [Ca2+]i due to transmembrane Ca2+ influx and release of Ca2+ from intracellular stores) and thereby promote apoptosis [2+]i affects mitochondrial dysfunction and ER stress along the proapoptotic pathway. [Ca2+]i is also an important regulator of the PI3K/Akt pathway, which promotes the proliferation of cancer cells i, [Ca2+]m, and [Ca2+]ER. While TRPV1 is well characterized with regards to cell death, the link between TRPV1 and proliferation has yet to be thoroughly investigated. The vast majority of studies pertaining to cell death elucidated a connection between TRPV1 activation and apoptosis in both cancerous and benign cell lines. However, some found TRPV1 activation in breast cancer cells (MCF-7) to result in necrotic cell death i. JNK and p38 (increased due ROSi) upregulate MAPK. Furthermore, TRPV1 [Ca2+]i and protein signaling activates calcineurin and ATM, which act through transcription factors to upregulate the tumor suppressor gene p53. p53 subsequently upregulates the proapoptotic proteins Bax, p16, and p21. Finally, MAPK, cytochrome c, p16, p21, and Bax activate caspases. Caspase 9 activates caspase 3; the two caspases and AIF cause DNA fragmentation and condensation in the nucleus, and ultimately apoptosis i and protein signaling; in contrast, the channel stimulates proliferation via ATP release, P2Y2 receptor activation, and EGFR transactivation, with a limited role for [Ca2+]i. As such, the activation of TRPV1 in the absence of P2Y2 and EGFR activation may result in the dominance of the apoptotic pathway. Exogenous and endogenous TRPV1 agonists, applied in conjunction with P2Y2 and EGFR antagonists, may therefore constitute a potential form of anti-cancer therapy.TRPV1 mediates apoptosis primarily through i signaling without the induction of proliferative mechanisms.The inhibition of EGFR activity may likewise support effects to curb cancer drug resistance. EGFR is a transmembrane growth factor receptor which contributes to cell proliferation via the Ras/Raf/MEK/ERK signaling pathway . This pai levels via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase upregulation i signaling and may therefore exert pleiotropic effects in cancerous tissues. In particular, the implications of TRPV1 for inflammation are multi-faceted  and downregulates the aforementioned pro-inflammatory cytokines i signaling while suppressing proliferation through inflammation reduction.TRPV1 is expressed in a wide variety of immune cells, such as macrophages, neutrophils, T cells, and dendritic cells . As suchIn the development of anti-cancer drugs to affect TRPV1, it is in all cases necessary to first characterize the apoptosis–proliferation balance that exists in the target cell line, taking into account factors such as inflammation. Depending on the balance, oncologic therapies may upregulate and stimulate TRPV1 with carefully selected agonists, downregulate or otherwise inhibit TRPV1, or target specific aspects of the TRPV1-mediated mechanisms to enhance the protein’s apoptotic effects. Recent molecular and clinical developments suggest that these objectives are achievable.Cancer management based on the targeting of TRPV1 currently encompasses a wide range of compounds with synthetic as well as natural origins. Arvanil, a synthetic TRPV1 agonist, decreases tumor weight and improves survival time in mice with implanted gliomas . MoreoveHypericum perforatum (HP) does not support the antitumor effects of 5-Fluorouracil through TRPV1 modulation. The apoptotic effects of 5-Fluorouracil, mediated by TRPV1 activation in MCF-7 cells, are downregulated by treatment with HP i signaling cascade for cancer therapy via photothermal TRPV1 activation has been developed. This cascade creates artificial Ca2+ overload stress, causing cell death via mitochondrial dysfunction  in vitro. Given TRPV1 overexpression and near-infrared (NIR) irradiation at tumor sites, a CuS@CaCO3-PEG nanoplatform can initiate the [Ca2+]i cascade in both cancerous and non-cancerous tissues. Additionally, photothermal CuS nanoparticles involved in the nanoplatform allow for three-dimensional photoacoustic imaging in vivo i, which has a dual role in that it regulates both proliferative and apoptotic mechanisms. Notably, the influence of TRPV1 on the PI3K/Akt signaling pathway may hold implications for the circumvention or minimization of cancer drug resistance. The activation of TRPV1 by agonists, in conjunction with a blockade of P13K/Akt signaling, may have therapeutic potential through the elevation of [Ca2+]i and consequent upregulation of the apoptotic pathways. The parallel involvement of TRPV1 in proliferation and apoptosis enhances the receptor’s relevance as an oncologic target. Several innovative anti-cancer strategies targeting TRPV1 are currently in development.As the objective of anti-cancer therapies is to hinder tumor growth through the enforcement of apoptosis and the minimization of proliferation, the TRPV1 ion channel constitutes a promising target. TRPV1 is constitutively expressed in a wide variety of cancerous cell lines, and the receptor’s differential expression in cancerous and healthy tissues provides insights into its role in the proliferation–apoptosis balance. Oncologic agents that target TRPV1 will exert their proapoptotic effects primarily through the modulation of [Ca


Intracellular Ca2+ dynamics in these cells are governed by the electrical activity of voltage-gated ion channels, among which ATP-sensitive K+ (KATP) channels play a crucial role. In the majority of α-cells, the global Ca2+ response to lowering external glucose occurs in the form of oscillations that are much slower than electrical activity. These Ca2+ oscillations are highly variable as far as inter-spike intervals, shapes and amplitudes are concerned. Such observations suggest that Ca2+ dynamics in α-cells are much influenced by noise. Actually, each Ca2+ increase corresponds to multiple cycles of opening/closing of voltage gated Ca2+ channels that abruptly become silent, before the occurrence of another burst of activity a few tens of seconds later. The mechanism responsible for this intermittent activity is currently unknown. In this work, we used computational modeling to investigate the mechanism of cytosolic Ca2+ oscillations in α-cells. Given the limited population of KATP channels in this cell type, we hypothesized that the stochastic activity of these channels could play a key role in the sporadic character of the action potentials. To test this assumption, we extended a previously proposed model of the α-cells electrical activity (2+ dynamics into account. Including molecular noise on the basis of a Langevin type description as well as realistic dynamics of opening and closing of KATP channels, we found that stochasticity at the level of the activity of this channel is on its own not able to produce Ca2+ oscillations with a time scale of a few tens of seconds. However, when taking into account the intimate relation between Ca2+ and ATP changes together with the intrinsic noise at the level of the KATP channels, simulations displayed Ca2+ oscillations that are compatible with experimental observations. We analyzed the detailed mechanism and used computational simulations to identify the factors that can affect Ca2+ oscillations in α-cells.In pancreatic α-cells, intracellular Caactivity  to take  Pancreatic islets respond to changes in blood glucose levels so that β-cells secrete insulin when blood glucose is elevated and α-cells secrete glucagon when it is low. Glucagon mobilizes glucose from the liver and when normoglycemia is reestablished, glucagon release from α-cells is suppressed. Extrinsic and intrinsic factors are involved in glucagon secretion . Individ2+ to enter through voltage-gated Ca2+ channels, which leads to the exocytosis of secretory granules of glucagon. In agreement with this mechanism, electrical stimulation of α-cells leads to an increase in cell membrane capacitance due to granule fusion, a well-known Ca2+ dependent process that precedes glucagon release  firing. In a low glucose medium, AP’s occur with a frequency of ∼1–3 Hz. Depolarization of the α-cell plasma membrane allows Ca release .2+ concentration ([Ca2+]i), which results from the activation of voltage-gated Ca2+ channels. Interestingly, this rise in [Ca2+]i occurs in the form of oscillations with an average frequency of ∼0.5 min–1, which is much lower than that of the AP’s. Parallel measurements of electrical activity and [Ca2+]i revealed that each oscillation corresponds to a burst of AP’s and that the amplitude of the Ca2+ increase correlates with the frequency of AP’s  play a key role in controlling α-cells electrical activity, although the details of this control are still actively debated  that couples the level of external glucose to the electrical properties of the α-cell plasma membrane, through the sensitivity of the intracellular ATP/ADP ratio to external glucose concentration. It also describes a voltage-gated Na+ current (INa), a delayed rectifying (IKDR) and a A-type K+ current (IKA), an unspecific leak current (Ileak) and a L-type (ICaL) and a T-type Ca2+ current (ICaT). We also consider an additional type of Ca2+ current, because N-type . Ca2+ concentrations are described differently just below the plasma membrane and in the cytoplasm. Ca2+ entry via the L-, T- and N-type Ca2+ channels increases Ca2+ concentration in a hypothetical sub-plasma membrane compartment. From this compartment, Ca2+ diffuses into the cytoplasm or leaves the cell via the plasma membrane Ca2+ ATPase (PMCA). Ca2+ efflux from the cell is thus accompanied by the hydrolysis of ATP into ADP. The resulting decrease in ATP concentration provokes an increase in the KATP conductance, thereby providing a feedback from Ca2+ changes on the electrical properties of the membrane. The main features of the model are described here below. Additional information about the equations and the values of the parameters can be found in the We base our model  on the me N-type  or P/Q te N-type  Ca2+ curElectrical activity is described by the following differential equation:V is the membrane voltage and Cm is the membrane capacitance set to 5 pF (IKATP (see section “ATP-Sensitive K+ Current and ATP Evolution” below), we use the model proposed by 2+ current is described as in other studies  becomes:The main goal of the present study is to assess the effect of noise on the Kξ(t) is a random function of time. The last term is a noise function with 0 mean andwhere NKATP the number of potentially openable KATP channels (with channels .f2(ATP) takes into account that KATP channels are reversibly inhibited by ATP  and cytosolic Ca2+ . It is thus defined by Eqs 1, 2, and 5.Model 2 moreover includes stochasticity in the KATP conductance through Eqs 7, 8 and 10 that are considered in addition to the equations defining Model 1. f1 and f2 are taken equal to 1.Model 3 allows to investigate the effect of the Ca2+-induced variations in ATP concentration. It is similar to Model 2, except that it includes Eq. 4 to describe the evolution of [ATP]. Changes in the concentration of this nucleotide impact on the evolutions of subplasmalemmal Ca2+ via f1 that is now given by Eq. 3 and of the KATP channel via f2 that is now given by Eq. 9.2+]i first rises just beneath the plasma membrane since Ca2+ is entering through voltage-gated channels located in this membrane. Sub-plasmalemmal Ca2+ is immediately buffered and diffuses in the cytosol where Ca2+ concentration is low at rest (∼100 nM). This attenuates Ca2+ increases below the membrane and transmits signaling to the rest of the cell. Additionally, sub-membrane Ca2+ is pumped out of the cell by Ca2+ ATPases. Model 1 takes these fluxes into account and combines a description of α-cell electrical activity with equations for the evolution of subplasmalemmal  and cytosolic Ca2+ . Simulations of Model 1 show that each AP generates one spike of Ca2+ in the subplasmalemmal space . Ca2+ concentration in the cytosol does not exceed 0.4 μM. In this model, repetitive electrical activity always leads to a sustained increase in cytosolic Ca2+, which does not correspond to the slow Ca2+ oscillations observed experimentally in most α-cells.When α-cells are electrically active,  is high (quiescent periods) because of the inhibition of the KATP current by ATP. This is visible when computing the moving average of gKATP . As a consequence of these trends, large, unlikely perturbations are necessary to switch from an inactive to an active period and vice-versa. Thus, the mutual interaction between ATP dynamics and electrical activity that occurs through the Ca2+ changes creates trends in the changes in electrical activity allowing for intermittent activity, despite the rapid fluctuations in gKATP.Although fluctuations of the Kry rapid , gKATP iof gKATP . The tre2+ oscillations shown in 2+ increases above a certain threshold , the cell membrane is most of the time hyperpolarized and infrequent, short-duration and low-amplitudes Ca2+ spikes occur. If NKATP is smaller than 10, the cell is always in a resting state, although the average value of the cell KATP conductance (0.21 nS) is in the oscillatory domain . In the deterministic analysis, this value corresponds to repetitive AP’s , which are all potentially openable. Their intrinsically low open probability and their inhibition by ATP explain why only a small fraction are open simultaneously. Moreover, we expect our value of NKATP to be underestimated since we did not take into account effective fatty acid metabolism in low glucose conditions (NKATP, as shown in 2+ oscillations in α-cells is robust.The simulations predict that the number of simultaneously active Krvations . This nunditions  nor the ATP current has been put forward to account for the irregularity of the neuronal firing pattern in hippocampal CA3 neurons (ATP current are due to rapid, random fluctuations in the concentration of ATP, which results in an irregular frequency of AP’s and not in intermittent electrical activity. In the more general context of networks dynamics, it is known that fluctuations can propagate along the different nodes of the network with a rate of decay or enhancement that depends on the network’s structure (A similar role for stochasticity at the level of the K neurons . Howevertructure .2+ channels linked to glucagon secretion. In ATP channels to simulate the changes in external glucose. However, given the reliability of the Diderichsen and Göpel’s model and the many unknowns in the field of α-cells dynamics, we considered this unchanged model as a safe starting point to explore the impact of a stochastic KATP current and of changes in ATP concentration. However, we acknowledge that values of parameters for the ionic currents obtained with better methods of α-cell identification could improve the agreement between the simulations and the experiments (Within this study, we did not investigate the behavior of the model when changing external glucose or considering paracrine signaling, which are left for further study. These limitations are due to the fact that the model used for electrical activity  does noteriments .2+ extrusion out of the cell plays a key role in the behavior predicted by the model. However, ATP synthesis is also sensitive to intracellular Ca2+ changes because mitochondrial metabolism is Ca2+-sensitive (The hydrolysis of ATP that parallels Caensitive . The ATPensitive . Furtherensitive , althougATP channel, while all molecular processes, and especially Ca2+ dynamics, are subjected to noise. This simplification was based on the experimental observation of an unusually small KATP conductance in α-cells, in particular in comparison with pancreatic β-cells that are known to be equipped with the same channels but still display very regular voltage and calcium patterns. However, the details of our conclusions may be affected by adopting a full stochastic description of the cell dynamics. In particular, it may lead to less stereotypic cytosolic Ca2+ changes, as reported in the experiments.As another limitation, we only considered noise at the level of the K2+ changes (2+ traces. It was found that glucagon secretion does not correlate with the frequency of Ca2+ oscillations. This investigation could be pursued by using Ca2+ time series obtained by simulations of the present model instead of experimental data. This could lead to identify the key elements of electrical and/or calcium activities controlling glucagon secretion.An interesting perspective would be to couple the model presented in this study to our previously proposed model that relates glucagon secretion to cytosolic Ca changes . The latThe original contributions presented in the study are included in the article/VG-V, AP, and GD contributed to the conceptualization, design of the study, development, simulation, and analysis of the models. GD and VG-V contributed to the preparation of the manuscript. All authors contributed to the article and approved the final version.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.


H2S was effective on metastatic, but not normal, cells. Therefore, we propose that exogenous administration of H2S to patients affected by metastatic colorectal carcinoma could represent a promising therapeutic alternative.Colorectal cancer (CRC) is the most common type of gastrointestinal cancer and the third most predominant cancer in the world. CRC is potentially curable with surgical resection of the primary tumor. The clinical problem of colorectal cancer, however, is the spread and outgrowth of metastases, which are difficult to eradicate and lead to a patient’s death. The failure of conventional treatment to significantly improved outcomes in mCRC has prompted the search for alternative molecular targets with the goal of ameliorating the prognosis of these patients. The present investigation revealed that exogenous delivery of hydrogen sulfide (H2S) is emerging as an alternative anticancer treatment. H2S-releasing compounds have been shown to exert a strong anticancer effect by suppressing proliferation and/or inducing apoptosis in several cancer cell types, including colorectal carcinoma (CRC). The mechanism whereby exogenous H2S affects CRC cell proliferation is yet to be clearly elucidated, but it could involve an increase in intracellular Ca2+ concentration ([Ca2+]i). Herein, we sought to assess for the first time whether (and how) sodium hydrosulfide (NaHS), one of the most widely employed H2S donors, induced intracellular Ca2+ signals in primary cultures of human metastatic CRC (mCRC) cells. We provided the evidence that NaHS induced extracellular Ca2+ entry in mCRC cells by activating the Ca2+-permeable channel Transient Receptor Potential Vanilloid 1 (TRPV1) followed by the Na+-dependent recruitment of the reverse-mode of the Na+/Ca2+ (NCX) exchanger. In agreement with these observations, TRPV1 protein was expressed and capsaicin, a selective TRPV1 agonist, induced Ca2+ influx by engaging both TRPV1 and NCX in mCRC cells. Finally, NaHS reduced mCRC cell proliferation, but did not promote apoptosis or aberrant mitochondrial depolarization. These data support the notion that exogenous administration of H2S may prevent mCRC cell proliferation through an increase in [Ca2+]i, which is triggered by TRPV1.Exogenous administration of hydrogen sulfide (H Colorectal cancer (CRC) is the most common type of gastrointestinal cancer and the third most predominant cancer in the world. In 2018, around 1.8 million cases were reported by the World Health Organization (WHO) and 862,000 deaths were registered . These numbers are expected to increase by 80% in year 2035, reaching approximately 2.4 million cases and contributing to 1.3 million deaths worldwide i) [2S is able to elevate [Ca2+]i by inducing either Ca2+ release from the endoplasmic reticulum (ER) i) . It has lum (ER)  or by prpathways , includipathways ,24,25, Tpathways , TRP Ankpathways ,28, L- a (VGCCs) . Intraceigration ,33,34,35patients . It woul2S-induced extracellular Ca2+ entry in healthy cells i in mCRC cells (p < 0.05) smaller Ca2+ response in primary CRC (pCRC) cells  larger in pCRC as compared to non-neoplastic cells  cells A,B and iC) cells A,B. Simiic cells A,B. As e2+ signals in mCRC cells. NaHS did not induce any discernible increase in [Ca2+]i at concentrations lower than 5 µM, such as 2.5 µM  difference in the percentage of responding cells in the concentration range spanning from 75 µM to 300 µM . Under such conditions, the H2S donor failed to augment [Ca2+]i in 138 out of 138 cells (100%)  A–C, whilsolution A–C. This [Ca2+]i D. The mo2S may induce extracellular Ca2+ entry by activating TRPV1 in a growing number of cell types i that exhibited different temporal patterns , while the Ca2+ response resumed upon Ca2+ restitution to the perfusate , and SB-366791  i evoked by NaHS (100 µM)  , which, y (SOCE) . Howevery (SOCE) ,36, did (100 µM) . These f2+ signals were significantly (p < 0.05) larger in mCRC cells as compared to non-neoplastic an pCRC cells . We, therefore, sought to assess whether NCX sustains TRPV1-mediated Ca2+ entry in mCRC cells i due to the reversal of Na+ gradient and reversal of NCX activity into the reverse  mode   was significantly (p < 0.05) smaller as compared to the control , a selective inhibitor of the reverse-mode NCX at this concentration [2+]i elevation in 146 out of 182 (80.2%) cells pre-treated with the blocker  mode A. At thels (73%) A,B, whilular Na+ A. The am control C. In ordntration . In agre blocker D,E. In aB-R 7943 F. + (0Na+) and KB-R 7943  interfered with the Ca2+ response to direct TRPV1 activation with capsaicin (10 μM). Under 0Na+ conditions, capsaicin induced a discernible increase in [Ca2+]i in 81 out of 144 cells  reduced  reduced  reduction in mCRC number after 24 h preincubation in the presence of 100 µM and 200 µM NaHS, while 50 µM NaHS did not affect mCRC cell viability , to inhibit TRPV1, and KB-R 7943 (10 µM), to block the reverse mode NCX. As depicted in 2S could reduce proliferation in mCRC cells by triggering Ca2+ influx through activation of TRPV1 followed by recruitment of the reverse mode NCX.Emerging evidence suggested an inhibitory effect of exogenous application of Hll lines ,17,18,19iability A. The priability A. In ordm) is regarded an widespread early marker of apoptosis i i . Furtherblastoma , renal cblastoma , and CRCblastoma . In addier cells ,49, but 2S in the form of the widely employed H2S-donor, NaHS, reliably increased the [Ca2+]i in primary cultures of mCRC cells. Conversely, the Ca2+ responses to NaHS were remarkably smaller in non-neoplastic and pCRC cells. This preliminary investigation revealed that mCRC cells displayed a higher sensitivity of exogenous delivery of H2S. An increase in [Ca2+]i was also induced by H2S release from another donor, GYY4137, in the CRC cell line, DLD1 [2+ response to H2S release [2+ signal arose at low NaHS concentration ([NaHS]) , the percentage of responding cells and the magnitude of the initial Ca2+ peak decreased by further reducing the [NaHS] to 25–50 µM. Nevertheless, the Ca2+ response was fully restored by increasing the [NaHS] to 75-300 µM. A similar U-shaped dose-response relationship has been described for NaHS-evoked intracellular Ca2+ signals in the native endothelium of rat aorta  and preliminary experiments showed that NaHS did not affect mCRC cell viability at lower doses.Exogenous administration of Hne, DLD1 . While t release , herein at aorta  and EA.hnsmitter ,73. It sell type ,22. For 26 cells , while iothelium  voltage-cilitate  or blockcilitate  CaV3.2 e2+ signatures in mCRC cells: a transient increase in [Ca2+]i, which occurred in ≈75% of the cells, and a biphasic Ca2+ signal, which arose in the remaining lower fraction of cells. Different patterns of intracellular Ca2+ signals were also elicited by NaHS in rat aortic endothelium i by both mobilizing ER stored Ca2+ through InsP3Rs and ryanodine receptors (RyRs) and activating extracellular Ca2+ entry i is triggered by extracellular Ca2+ entry. The same observation has been reported in another cancer-derived cell line, i.e., B-TECs i that was also sensitive to capsazepine, SB-366791 and ruthenium red, and to genetic deletion of TRPV1. Notably, capsaicin evoked two distinct patterns of intracellular Ca2+ signals that strongly resembled those induced by NaHS: a transient Ca2+ elevation in ≈75% cells and a biphasic Ca2+ response in the remaining ≈25% cells. One to ten µM capsaicin was shown to elicit both transient i [2+ response to capsaicin reported in the present investigation as this was observed by presenting mCRC cells with the same agonist concentration. Ten µM capsaicin was found to stimulate TRPV1 and induce long-lasting elevations in in MCF-7 breast cancer cells [2+ oscillations in some prostate cancer cell lines (DU 145 and PC-3) in one study [2+]I in another one i . Howeverer cells , while tther one , but thi2+ entry) mode NCX sustains NaHS- and capsaicin-induced extracellular Ca2+ entry. First, replacement of extracellular Na+ with an equimolar amount of choline induced a transient increase in [Ca2+]i, which reflects the directional switch of NCX from the forward  to the reverse  mode upon reversal of Na+ gradient across the plasma membrane i. These data are consistent with those reported in native endothelium of rat aorta [2S through GYY4137 induced an increase in resting [Ca2+]i that was associated to the switch of NCX activity into the reverse mode. Furthermore, earlier work demonstrated that H2S release was able to increase the expression levels of NCX1 i, which persist as long as the stimulus is presented to the cells i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (Ratio (F340/F380)). An increase in [Ca2+]i causes an increase in the ratio [Calsewhere ,36. BrieCells were lysed in ice-cold RIPA buffer  added of protease and phosphatase inhibitors. Upon protein quantification, the samples were dissociated by addition of half volume of SDS-sample buffer 3× , separated by SDS-PAGE on a 10% or 6% polyacrylamide gel, and blotted on a PVDF membrane. Membrane probing was performed using the different antibodies diluted 1:1000 in TBS  containing 5% BSA and 0.1% Tween-20 in combination with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies  plus 0.1% Tween-20). The following antibodies were used: anti-TRPV1 (ab3487) from ABCAM , HRP-conjugated anti-GAPDH (sc365062), anti-Akt 1/2/3 (sc8312), anti-Erk 2 (sc-154) and anti-mTOR (sc1549) from Santa Cruz Biotechnology ; the specific phospho-antibodies: phospho-Akt (Ser473) (#9271), phospho-Erk 1/2 (Thr 202/Tyr 204) (#9101) and phosphor-mTOR (Ser 2481) (#2974) from Cell Signaling Technology . The chemiluminescence reaction was performed using Immobilon Western  and images were acquired by ChemiDoc XRS . Membrane was then re-probed with an HRP-conjugated anti-GAPDH antibody as equal loading control. Cells were grown for 24 h on 15 mm glass coverslips in a 12-well plate. Samples were then fixed with 3% PFA in PBS, permeabilized with 0.25% ice-cold TRITON X100 in PBS and blocked with 1% BSA in PBS. The coverslips were stained with a rabbit anti-human TRPV1 antibody  for 1 h at room temperature and then incubated with TRITC-anti rabbit secondary antibody  in the dark for 1 h at room temperature. Negative controls were prepared by omitting the primary antibody. Nuclei were stained with 1 µg/mL Hoechst 33342  and cell membrane with 2 µM PKH67 . Finally, the coverslips were mounted on glass slides using ProLong Gold antifade reagent  as mounting medium. Immunofluorescence images were acquired using an Olympus BX51 microscope, whereas the confocal microscopy images were captured using a Leica TCS SP8 Microscope and analyzed with LAS X software .The siRNA targeting TRPV1 was purchased from Sigma-Aldrich Inc. MISSION esiRNA (human TRPV1) (EHU073721). Scrambled siRNA was used as negative control. In brief, once the seeded mCRC cells had reached 50% confluency, the medium was replaced with Opti-MEM, the serum in the medium reduced without antibiotics . siRNAs  were diluted with Opti-MEM I reduced serum medium and mixed with Lipofectamine™ RNAiMAX transfection reagent  pre-diluted in Opti-MEM), according to the manufacturer’s instructions. After 20 min incubation at room temperature, the mixtures were added to the cells and incubated at 37 °C for 5 h. Transfection mixes were then completely removed and fresh culture media was added again and silenced cells were used 48 h after transfection. The effectiveness of silencing was determined by immunoblotting see . 5 cells)  and with 7-ADD (5 µL/5 × 105 cells) . Metastatic CRC cells maintained in the absence and presence of NaHS  were incubated for 15 min at resting temperature and then analyzed by Beckman Coulter Navios according to manufactures’ instructions.Metastatic CRC cells at confluence were detached by trypsinization and incubated with FITC-conjugated Annexin V  was evaluated by the Student’s t-test for unpaired observations or one-way ANOVA analysis followed by the post-hoc Dunnett’s or Bonferroni tests, as required. The number of cells measured for each experimental condition is indicated in, or above, the corresponding bar histogram.All the data have been obtained from mCRC cells plated on at least three coverslips and deriving from three distinct donors. Each trace shown is representative of multiple cells displaying a similar Ca2S-releasing compounds NaHS elicit intracellular Ca2+ signals in primary cultures of mCRC, but not of non-neoplastic and pCRC cells, by stimulating TRPV1 to mediate extracellular Ca2+ entry and induce NCX to switch into the reverse (Ca2+ entry) mode. NaHS-induced TRPV1 activation, in turn, inhibited mCRC cell proliferation. The anti-proliferative effect of NaHS was not due to mCRC cell apoptosis. These data lend further support to the exogenous delivery of H2S as a novel therapeutic strategy to treat mCRC.This study demonstrates for the first time that the H


Targeted treatment with all-trans-retinoic acid (ATRA) and ATRA combined with arsenic trioxide significantly improved the survival of AML M3 patients. Unknown prognostic factors could contribute to the early death of these patients.Co-occurrence of cytogenetic and molecular abnormalities is frequently seen in patients with acute myeloid leukemia (AML). The clinical outcome and genetic abnormalities of AML may vary; therefore, genetic investigation must be complex, using several techniques, to have an appropriate characterization of the AML genome and its clinical impact. The available molecular markers can predict prognosis only partially. Acute promyelocytic leukemia subtype M3 We present the case of a young female (20 years old) patient, who presented at the emergency department 5 months after giving birth to her first child, complaining of asthenia, fatigue, general musculoskeletal pain, and fever (38°C), symptoms having been present for the previous 6 days. The patient denied any chronic diseases in her medical and family history.PML-RARA gene fusion, and fms-like tyrosine kinase 3 (FLT3) gene mutation. The immunophenotypic analysis was also suggestive for AML M3 according to the FAB classification.Laboratory analysis revealed severe pancytopenia. Cytogenetic and molecular analyzes revealed chromosomal abnormalities (trisomy 8), Specific treatment was initiated for AML M3 and for secondary conditions. Molecular and cytogenetic analyzes were performed to have a more detailed characterization of the patient's genome.Seventy-two hours after admission, she developed psychomotor agitation, confusion, coma, and convulsion. Subsequent deterioration and early death were caused by intracerebral hemorrhage with multiple localization and diffuse cerebral edema.FLT3 internal tandem duplication (ITD) mutation may explain the rapid and progressive degradation of this AML M3 case and it may be used as a prognostic marker even when co-occuring with other markers such as PML-RARA gene fusion and trisomy 8. We consider that FLT3 ITD mutation analysis in young patients with AML should be performed as soon as possible. New strategies for patients’ education, AML  prevention, and treatment are needed.The presence of  Molecular markers, such as fms-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1), CCAAT/enhancer-binding protein alpha (CEBPA), runt-related transcription factor 1 (RUNX1), additional sex combs like 1, and tumor protein p53 (TP53) genes, were introduced in the latest European LeukemiaNet (ELN) recommendations. Co-occurrence of cytogenetic and molecular abnormalities are frequently present in AML patients. Moreover, the clinical outcome and genetic abnormalities are different according to the age of the patients. Therefore, the genetic investigations of patients with AML should be complex, using various techniques, to have an appropriate characterization of the AML genome and its clinical impact, because the available molecular markers can only partially predict prognosis for AML. Acute promyelocytic leukemia subtype M3 (AML M3) is a subtype of AML characterized by the presence of promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) genes fusion.,8 Targeted treatment with all-trans retinoic acid (ATRA) and ATRA combined with arsenic trioxide significantly improved the survival of AML M3 patients. However, unknown prognostic factors could contribute to the early death of these patients.Acute myeloid leukemia (AML) is a heterogeneous disease characterized by various cytogenetic and molecular abnormalities.PML-RARA gene fusion, and FLT3 internal tandem duplication (ITD) mutation, who was diagnosed with AML 5 months after she gave birth to her first child. Genetic investigations included conventional cytogenetic analysis and molecular techniques, such as ligation-dependent reverse transcription polymerase chain reaction (LD-RT PCR), multiplex ligation–dependent probe amplification (MLPA), and NGS.Here, we describe the clinical outcome of a young AML M3 female patient, with chromosome 8 trisomy, 2The Ethics Committee of the Clinical and Emergency County Hospital from TârguMureş, Romania, approved this study (No. 10665/2019). The patient has provided informed consent for publication of the case and signed the informed written consent for genetic testing.2.1Immunophenotyping analysis was made from bone marrow aspirate.2.2,10 In parallel, we initiated molecular analysis for FLT3 (ITD and D835), NPM1 c.863_864ins, and DNMT3A R882 mutations by using different polymerase chain reaction (PCR) methods , as previously reported. In the second day, we performed MLPA analysis using 3 different kits  and LD-RT PCR to detect 57 specific acute leukemia gene fusions according to the protocol previously described (capillary sequencing was performed to sequence the amplicon).–13 NGS was carried out using a community panel, Ion AmpliSeq AML Research Panel, and Ion Proton system (Thermo Fisher Scientific USA).Conventional cytogenetic analysis was performed from bone marrow obtained by biopsy and peripheral leucocytes according to protocols previously described.2.3Methicillin-sensitive Staphylococcus aureus (MSSA) and targeted treatment was initiated. In the following hours, the patient presented disseminated intravascular coagulation and received treatment consisted of pooled platelets and red blood cells, fresh blood plasma, and hemostatic agents.The patient, a young female (20 years old), presented to the Emergency Department complaining of asthenia, fatigue, general musculoskeletal pain, and fever (38°C), symptoms having been present for the previous 6 days. The patient denied any chronic diseases in her medical and family history. Laboratory analysis revealed severe pancytopenia. The patient was immediately admitted to the hematology department for further investigations and treatment. Before initiating any treatment, bone marrow aspiration was performed and sent for immunophenotypic, conventional cytogenetic, and molecular analyzes. The immunophenotypic analysis revealed a percentage of 86% myeloid elements positive for CD45, CD33, CD13, CD11b+ (4.1%), CD11c (10%), CD64 (81%), CD34 (5%), CD117 (15%), CD123, CD38 (30%), CD4 (8%), CD22 (9.5%), ic MPO and negative for CD15, CD14, CD16, CD36, HLA-DR, CD3, CD5, CD7, CD19, CD10, ic CD3, and ic CD79a. The results were suggestive for AML M3 according to the FAB classification. Therefore, the patient received treatment with ATRA and idarubicin. Laboratory analysis  revealed high values of white blood cells count, MR-pro-atrial natriuretic peptide, and ultra sensitive C-reactive protein and low values of red blood cells and platelets. Blood culture analysis showed positive results for FLT3 ITD mutation. For confirmation and quantification of the mutant clone, fragment analysis was performed. Figure Molecular analysis by PCR technique detected the presence of the CEBPA, RUNX1, ASXL, TP53, and other 15 genes, which might be difficult to analyze with other molecular techniques. Five missense variants were identified by NGS analysis, namely rs2276599 , rs17253672 , rs34402524 , rs2454206 , and rs1042522 . According to VarSome and ClinVar, the TP53 c.215C>G variant has an uncertain significance, and the other identified variants are considered benign.After short-term culture of bone marrow cells, the conventional cytogenetic analysis confirmed chromosome 8 trisomy. NGS analysis was performed to investigate genes such as After 72 hours, the molecular results  were communicated to the hematology department. At the same time, the patient developed psychomotor agitation, confusion, coma, and convulsion. Specific treatment was administrated, and brain computed tomography was performed. Intracerebral hemorrhage with multiple localization and diffuse cerebral edema were the cause of her deterioration. The patient was transferred to the neurology department due to her poor medical status, but unfortunately, she died within 24 hours.3PML-RARA gene fusion and FLT3 ITD mutations (VAF = 50%) simultaneously.Our investigated young patient with AML presented chromosome 8 trisomy,  its prognostic significance is not completely cleared. Moreover, one study concludes that trisomy 8 cannot be considered as a prognostic marker, since this chromosomal aberration co-occurs with other genetic alterations such as monosomy 5, monosomy 7, several gene fusions, or gene mutations. One previous study suggests that prognosis of patients with AML with trisomy 8 is determined by the accompanying genetic alterations (if any exist). Another study suggests that trisomy 8 is an intermediate prognostic factor in patients with AML and a poor prognosis factor for chronic myeloid leukemia (being associated with blastic phase).Although chromosome 8 trisomy is the most frequent genetic aberration found in AML patients, Chromosome 8 trisomy influences the expression of genes localized on chromosome 8, alters global gene expression, and seems to have various consequences in the different AML subtypes. Therefore, the genetic investigation of patients with AML with trisomy 8 (and not only) should be complex using various techniques to overcome the limitation of each of them.The fact that chromosome 8 trisomy usually coexists with other genetic alterations, is currently accepted. Trisomy 8 is considered to be a disease-modulating secondary event, that may associate cryptic translocations, deletions (del), or gene mutations as the most important primary events.PML-RARA fusion has a favorable prognosis even in the presence of a secondary abnormality such as trisomy 8.,20FLT3 ITD mutation is considered an unfavorable marker associated with short overall survival and disease-free survival, as well as with high risk for AML relapse.,21–23 According to ELN 2017, patients with AML with allelic ratio greater than 0.5 for FLT3 ITD mutation and negative for NPM1 mutation are considered to have an adverse risk status.,24 The presence of FLT3 ITD mutation in our patient could explain the rapid and progressive degradation, and it could be used as an adverse prognostic marker even if it co-occurs with favorable prognostic markers such as PML-RARA fusion and chromosome 8 trisomy. Frequent co-occurrence of trisomy 8 and FLT3 ITD mutation in young AML patients has been previously described. Therefore we consider that FLT3 ITD mutation analysis in young patients with AML should be performed as soon as possible. In our laboratory molecular genetic tests  were completed in 72 hours, but for patients with AML, we should be able to detect mutations more quickly and more efficiently, thus improving the outcome and health education of these patients. Also, there may be insufficient awareness of leukemia and of cancer in general among young individuals. Previous studies on female breast cancer conclude that the occupation and education level of the patients are independent factors for tumor staging; therefore, new strategies, publicity, and education programs are needed focusing on cancer prevention and treatment.,27In most cases, the  Therefore, it is recommended for patients who present nonspecific symptoms for prolonged periods, to consult a physician as soon as possible for investigations. One possible solution may be represented by personalized treatment, like FLT3 tyrosine kinase inhibitors in adult patients with AML with FLT3 ITD mutation.Moreover, one study investigating the causes of early death in AML M3 patients, concludes that “the current therapeutic strategies to reduce the incidence of early death in these cases are not adequate and will benefit from focused research attention.”,30 which could be performed for rapid identification of aneuploidy in patients with leukemia by using subtelomeric (P036 and P070) or centromeric (P181 or P182) MLPA probe mixes as previously described by Vázquez-Reyes et al. As we previously reported, MLPA is an efficient technique for AML patient's investigation, the presence of CNCs in patients with Eastern Cooperative Oncology Group performance status ≥3 being correlated with higher risk for death. LD-RT PCR is a promising low-cost (approximately 15 euro/patient) and rapid  multiplex technique, which may be used for the investigation of gene fusions in patients with AML and not only.,13 We consider that fragment analysis is the criterion standard for FLT3 (ITD and D835) and NPM1 mutation investigation, being able to predict a more appropriate outcome.Regarding molecular techniques used for patient investigations, we admit that MLPA is a fast multiplex technique, useful, and cost-effectiveFLT3 ITD mutation with a high VAF, associated with PML-RARA gene fusion and chromosome 8 trisomy represents a poor prognostic for AML and in young patients may lead to early death. The genetic investigation of patients with AML with trisomy 8 (and not only) must be complex, to overcome the limits of each technique, to have an appropriate characterization of the genome, a realistic prognosis, and personalized treatment. New strategies for patients’ education, AML prevention, and treatment are needed.In conclusion, we consider that Conceptualization: Florin Tripon, Johanna Sándor-Kéri, Claudia Banescu.Data curation: Florin Tripon, Alina Boglis, George Andrei Crauciuc, István Jr Benedek.Formal analysis: Florin Tripon, Alina Boglis, George Andrei Crauciuc, István Jr Benedek.Funding acquisition: Florin Tripon.Investigation: Valeriu Moldovan, Johanna Sándor-Kéri, István Jr Benedek, Claudia Banescu.Methodology: István Jr Benedek, Claudia Banescu.Project administration: Florin Tripon.Resources: Florin Tripon.Software: Alina Boglis, George Andrei Crauciuc.Supervision: Claudia Banescu.Validation: Adrian Pavel Trifa, Claudia Banescu.Visualization: Adrian Pavel Trifa.Writing – original draft: Florin Tripon.Writing – review & editing: Valeriu Moldovan, Adrian Pavel Trifa, Claudia Banescu.Florin Tripon orcid: 0000-0002-4297-9988.


Urticaria is a refractory dermatosis with long duration and a high recurrence rate. More to the point, medication of acute urticaria always demands high doses, which may cause some adverse effects. Acupuncture, with a history for over 2000 years, has been utilized in clinical practice as an alternative treatment strategy for dermatologic diseases.A 26-year-old male nurse on duty suddenly fell sick during the night-shift in the ward. Wheals began spreading all over his body, but he had no urticaria medicine on hand. The unbearable itching made it extremely difficult for him to resume working.The patient was diagnosed with acute urticaria according to the symptoms.A 30-minute acupuncture treatment was performed.About 5 minutes after needles were inserted into the acupoints, the patient felt significantly relieved of itching sensation; moreover, he could even subjectively control himself from scratching and could calmly wait for gradual disappearance of the wheals. Then 30 minutes later, the wheals almost faded away.The results suggest that acupuncture may be a promising alternative therapy to treat acute urticaria, especially for patients who cannot receive drug treatment. Eliminating inducements or suspicious causes is conducive to the natural disappearance of urticaria. As for the treatment of acute urticaria, the 2nd-generation nonsedating antihistamine is the 1st choice, and glucocorticoids could be adopted if symptoms cannot be effectively controlled. As a nonpharmaceutical therapy, acupuncture has very few adverse effects and is extensively utilized in urticaria treatment in Asia.–9 According to the collected data, no relevant clinical report over urticarial treatment through acupuncture on Yuji point (LU1O) has been reported. In this report, we describe a 26-year-old male nurse on duty who got acute urticaria and treated with acupuncture alone in the absence of medication, achieving good curative effects.Acute urticaria, a skin disease, is a localized edema reaction resulting from dilation and increasing permeability of skin and mucosal small blood vessels. Nearly 20% of people get acute urticaria at least once in their lifetime. This disease is mainly manifested with wheals in various sizes and unendurable itching.2am. Scratching could not alleviate itching, and wheals gradually swelled up and spread to the knee joint, side waist and especially the elbow part. The itching was unbearable and agonizing  utilized to evaluate wheals and itching, he was diagnosed with acute urticaria.UAS: 4 points.2.2The nurse received acupuncture immediately for once and was informed of the detailed treatment procedures.Due to quite a few body parts showing symptoms, it was difficult to realize local acupoints selection. As a result, we adopted the “Five Shu points”  selection method in treatment. According to traditional meridian theory, Xing-spring point Yuji point (LU10) on Lung meridian was chosen.Upon disinfection of the operator's hands and the acupoints, 2 disposable stainless needles  were stabbed into the bilateral Yuji (LU10) on the patient's hands with the depth of 10 mm . Acute urticaria is commonly featured by wind-induced diseases At present, mainstream scholars are convinced that the activated mast cells induced by the immunologic and nonimmunologic mechanisms are the key effector cells in urticaria pathogenesis. Abnormal activation of the coagulation system is also believed to be involved in urticaria onset.–17 It has been abundantly reported in recent literatures that acupuncture and moxibustion are effective in treating urticaria.,19 Acupuncture can activate endomorphine-like substances, inhibit and block the transmission of pain and itching signals, thus realizing the analgesia and antipruritic effects, regulating the immune system and inhibiting allergic reactions. Scholars have found that acupuncture treatment of urticaria shows bidirectional regulation on the immune system, enabling it to work in the normal or optimal status.It is believed in modern medicine that the causes of urticaria are complicated, which are normally exogenous and endogenous according to different sources.Restricted by a single case without a long-term follow-up, large-scale randomized clinical trials shall be conducted in the future. As the 1st report on acute urticaria treatment via acupuncture at the Yuji (LU10), this research could provide guidance for our future research, which will be centered on the molecular biologic mechanism of acupuncture treatment of acute urticaria.Conceptualization: Mingxin Xue.Writing – original draft: Shuai Zhou, Ruirui Zhao.Writing – review & editing: Shuai Zhou, Ruirui Zhao.