The Electronic Medical Record Search Engine (EMERSE) is a software tool built to aid research spanning cohort discovery, population health, and data abstraction for clinical trials. EMERSE is now live at three academic medical centers, with additional sites currently working on implementation. In this report, we describe how EMERSE has been used to support cancer research based on a variety of metrics.We identified peer-reviewed publications that used EMERSE through online searches as well as through direct e-mails to users based on audit logs. These logs were also used to summarize use at each of the three sites. Search terms for two of the sites were characterized using the natural language processing tool MetaMap to determine to which semantic types the terms could be mapped.We identified a total of 326 peer-reviewed publications that used EMERSE through August 2019, although this is likely an underestimation of the true total based on the use log analysis. Oncology-related research comprised nearly one third  of all research output. The use logs showed that EMERSE had been used by multiple people at each site  who had collectively logged into the system > 100,000 times. Many user-entered search queries could not be mapped to a semantic type, but the most common semantic type for terms that did match was “disease or syndrome,” followed by “pharmacologic substance.”EMERSE has been shown to be a valuable tool for supporting cancer research. It has been successfully deployed at other sites, despite some implementation challenges unique to each deployment environment. However, for all the benefits of EHRs, persistent challenges remain in leveraging EHR data for cancer research. This is because a substantial number (up to 80% by some estimates)Key ObjectiveTo demonstrate the utility of an information retrieval system, the Electronic Medical Record Search Engine (EMERSE), in the context of supporting cancer research.Knowledge GeneratedAn analysis of audit logs and peer-reviewed publications demonstrated that EMERSE is being used to support cancer research for a broad array of research projects and tasks, ranging from cohort identification to data abstraction for elements that may not be found in a structured form. Users are searching for a wide variety of concepts, including “pharmacologic substance,” “neoplastic process,” and “sign or symptom.”RelevanceInformation retrieval systems such as EMERSE have the potential to be powerful and easy-to-use software tools for supporting cancer research. EMERSE is available at no cost and has been successfully implemented at multiple medical centers, so it is a viable option for sites seeking to provide additional software tools for supporting cancer research.3 For example, symptomatic data are often recorded exclusively in the free text.4 One study found that free text from EHRs was required for resolving nearly 60% of eligibility criteria for a chronic lymphocytic leukemia clinical trial and almost 80% of eligibility criteria for a prostate cancer trial.5 Another such study listed 10 data elements derived from the free text related to bone marrow biopsy findings, including biopsy blast counts, biopsy cellularity, fibrosis grade, and aspirate cellularity.6 A study about engraftment syndrome after allogeneic hematopoietic cell transplantation used concepts found in the free text, such as engraftment failure, stool output, lymphocyte recovery, cytokine storm, disorientation, capillary leak, effusions, fevers, and rashes.7 Furthermore, the accuracy of the readily accessible structured data from EHRs may be low in some cases.8 For example, one study found that up to 20% of patients at one medical center had a medication listed in their unstructured data that was not in the structured medication list.9 Another study of cancer staging found that nearly 84% of patients had conflicting statements about staging in their records, necessitating an algorithm to infer the most likely staging for each patient.10Ignoring the free text in EHRs can be problematic.11 but widespread use remains limited. The quality of NLP results have been mixed, with some acknowledging the complexity and “inherent difficulty of natural language processing in this domain.” This complexity results from a variety of factors, including understanding temporal relationships, ambiguous abbreviations, and anaphoric references. Other challenges include issues of replicability across algorithms and institutions12 and the need for large manually annotated data sets for new use cases,11 especially because these systems perform best when tailored to a specific task or domain.13 The lack of available experts to architect and deploy NLP systems is also a limiting factor.To help the research community use the free text in EHRs, substantial resources have been devoted to develop natural language processing (NLP) tools. NLP remains promising for oncology research,To address the immediate needs of the cancer research community, members of which often lack the resources, time, and access to NLP experts, we developed a simpler approach using information retrieval for concept identification in free text. The Electronic Medical Record Search Engine (EMERSE) is a general-purpose term-searching system tailored to the needs of the medical research community to help researchers quickly find information buried in EHR free text. In general, information retrieval is like search engines such as Google that help people find information quickly, but it does not attempt to code the data, the latter of which falls within the domain of NLP. General familiarity with tools such as Google is thus an advantage. EMERSE uses an index of terms coupled with the capacity for query expansion using locally customized or standardized terminologies.14 EMERSE has been in use at the University of Michigan for 15 years and has supported a wide variety of clinical research, including oncology research. EMERSE is being implemented at other academic medical centers. Our report covers details about the system, including metrics based on use logs and publications, an analysis of search terms entered, and ongoing development work supported by the National Cancer Institute Informatics Technology for Cancer Research program.Rather than an example of an artificial intelligence system, EMERSE is more like an augmented intelligence system, wherein the software helps a person perform his or her work more efficiently but does not completely remove that person from the workflow. With EMERSE, the person is needed for the complex task of making sense of nuanced prose, a task that remains formidable for machines.EMERSE is a Web-based application that provides an easy-to-use interface for either (1) identifying a cohort among all patients in the EHR or identifying concepts within the clinical unstructured notes of an existing defined patient cohort. EMERSE indexes free-text data from EHR notes, with additional metadata related to the notes . The software is based on Apache Solr (an open-source search engine), but a substantial user interface has been built to provide study management features, visualization of results, and a query expansion feature.15 EMERSE maintains detailed audit logs for all user sessions. 16Technical details about EMERSE can be found in a prior publication.17 and plans to use EMERSE in a pilot program for training medical students about research software and as part of its health informatics training program.EMERSE is currently in use at three academic medical centers: University of Michigan, University of North Carolina at Chapel Hill, and University of Cincinnati . Other sPeer-reviewed publications using EMERSE were identified via manual searches for “EMERSE” or “electronic medical record search engine” in both PubMed and Google Scholar. Searches were conducted between August and September 2019. Each article identified was reviewed to confirm EMERSE use. To identify additional peer-reviewed publications without mention or citation of EMERSE, all principal investigators at the University of Michigan who had used EMERSE for research within the prior 5-year period (n = 600) were sent an e-mail in July/August 2019 to inquire about the use of EMERSE for their work and what publications arose from that use. The e-mail contained personalized audit logs to remind them about the use. A follow-up e-mail to nonresponders was sent in early September 2019. For all articles identified, the titles and abstracts were read to determine if they were cancer related.To characterize how EMERSE was used to support various research initiatives, 47 recent cancer-related peer-reviewed publications published within the last 2 years were reviewed. Among these, 11 were summarized with respect to their descriptions of how EMERSE was used. These 11 articles were selected to showcase a diversity of use cases, were from a variety of research teams from different disciplines, and had enough details described in their methods sections to understand the contribution of EMERSE.18 was used to process the search terms from two of the sites . For this analysis, the “-a -N” flags were used. The “-a” flag enables the use of variants of acronyms and abbreviations, and the “-N” flag modifies how the output is displayed. Prior studies have shown that MetaMap can perform comparably to other NLP tools, such as cTAKES.19Use logs were extracted to characterize the total number of users and the number of EMERSE logins over the past 5 years . The search terms  entered within this timeframe were also extracted. The NLP tool MetaMap20 and, if the concept could be identified, to what semantic type it belonged. Because MetaMap outputs a list of potential CUI candidates, only the top-scoring candidate was selected. For ties among top-scoring candidates, only the first was selected. The results across the two sites were merged, and the relative frequencies of the top 20 most common UMLS semantic types were visualized using RAWGraphs.21MetaMap processed each search term to determine if MetaMap could map the query to a concept unique identifier (CUI) within the Unified Medical Language System (UMLS)16 Summaries of how EMERSE was used for 11 selected oncology-related articles are provided in A total of 222 peer-reviewed publications were identified through manual searches using PubMed and Google Scholar through September 19, 2019. For the e-mail survey that was conducted to gain additional data about publications, 337 (56.2%) of the 600 principal investigators responded, revealing an additional 105 peer-reviewed publications that did not cite or mention EMERSE, bringing the total number of publications to 326. Of the 326 publications, 105 (32.2%) were oncology related. An additional 285 studies were still in progress, with potential publications coming at a later date. The current list of known peer-reviewed publications can be found on the EMERSE project Web site.NCT01865747),22 another that used EMERSE for 58 sessions and 26 hours ,23 and a third that used EMERSE for 398 user sessions and 166 hours .24The audit logs revealed substantial use of EMERSE for cancer-related work that did not acknowledge EMERSE use within publications. This included multisite clinical trials where EMERSE was used at a single site (University of Michigan). These publications could be identified via unique data, such as National Clinical Trial numbers, which were sometimes mentioned in the publications. Examples include one study that used EMERSE for 31 sessions, with a total session time of 13 hours , EMERSE has proven to be a useful tool for supporting cancer research. Furthermore, EMERSE has been successfully deployed at three academic medical centers to date, including the University of North Carolina, with additional centers in process, leading to multiple peer-reviewed publications.Through several rounds of implementation work with other sites , we have learned a great deal about the complexities of enterprise-wide software implementation. We describe a few of the most important insights, provided as guidance for others who might be interested in implementing EMERSE or other centrally managed research tools.Environments at each site are highly variable, including servers, storage, access to EHR documents, formats of these documents, and regulatory requirements. Although there is no cost per se for the software, the resources needed for implementation are not free. Competing priorities, institutional review board requirements, small teams, security reviews, and the need to obtain buy-in from leadership can delay implementation for months. There is no single solution to overcoming these challenges, but we have made efforts to reduce the burden on implementing sites, including providing installation and setup documentation, training materials for end users, and a messaging forum for technical teams.Because EMERSE is meant to be user facing, preserving the original document formatting helps users understand the data in the notes. Modern EHRs, such as Epic, allow for documentation using rich text formatting, in which notes can be made with tables, line breaks, and other formatting . However, the Epic analytics database, Clarity, almost universally stores a version of the notes stripped of all formatting. The University of North Carolina at Chapel Hill has avoided using Clarity and is using the live production database, Chronicles, instead.26 standard that should solve this challenge by extracting formatted notes in bulk. This approach is aligned with priorities of the US National Institutes of Health to “explore the use of the Fast Healthcare Interoperability Resources (FHIR) standard to capture, integrate, and exchange clinical data for research purposes and to enhance capabilities to share research data.” Other sites, such as University of Cincinnati, have used simple logic and regular expressions to rebuild functional formatting in the notes.The University of Utah, one of our partners, is working on a solution based on application program interfaces compliant with the Health Level Seven Fast Healthcare Interoperability ResourcesContrary to when EMERSE was first developed and deployed, security considerations are becoming a top priority, as they are for any software that contains protected health information within a medical center. This focus on security requires substantial, ongoing resources for conducting repeated scans for vulnerabilities that exist in the underlying open-source components, as well as in the system configuration, code reviews, penetration testing, and other measures. This work adds to the development costs but is a necessary component that other sites are requiring before considering a deployment. The importance of software security, as well as local institutional policies, should not be underestimated.Finally, demonstrating the value, effectiveness, and return on investment of software such as EMERSE remains challenging, especially if one considers peer-reviewed publications to be the gold standard of evidence. As demonstrated by the number of times the tool was used but never cited or mentioned, referencing software tools are not a top priority for many in the research community. However, this type of attribution is important to ensure future funding for software development teams, which can be expensive.For the analysis of semantic types, it is worth noting that only a few of the semantic types identified are for data typically found in the structured section of EHRs . Many of the other concepts are likely to be found only in the free-text notes. Furthermore, many of the terms entered by users were not mappable by the popular NLP tool MetaMap. This could be because of limitations of current NLP tools or because users of EMERSE are searching for concepts that do not have a matching CUI or semantic type within UMLS.The performance of MetaMap in our case likely could have been improved by adding an additional preprocessing step wherein incorrectly spelled terms would be mapped back to their correct spellings. Even though signs and symptoms are almost exclusively noted in the narrative portion of the medical record, these did not represent the most frequent semantic type. However, this may be because our analysis was performed on a unique list of terms in the search logs, and there may be far fewer signs and symptoms than there are disease or drug names.28 and PCORnet,29 but the novelty with the EMERSE-based network is the focus on free-text notes. This should be useful for finding rare cancer cases where structured data are not specific enough. For example, there is no specific code in the ICD (version 10) for endometrial stromal sarcoma, because the parent code C54.1 represents multiple types of endometrial neoplasms.Additional work under way involves securely networking sites for obfuscated counts. This feature will be similar to other cohort discovery networks currently based on structured data, such as i2b2 ACTIt is important to point out that EMERSE is not meant to be a replacement for NLP systems, and NLP will be a preferable option in certain use cases. For relatively small numbers of patients  and where accuracy is important enough to warrant human review, EMERSE may be the tool of choice. In other situations, such as automatically coding data across hundreds of thousands or millions of patients, NLP may be a preferable option. There is no one-size-fits-all solution, and multiple tools can benefit the research enterprise.In conclusion, EMERSE can be a valuable tool to support cancer research as well as other clinical domains. This is a simple-to-operate, self-service tool that is powerful, scalable, and generalizable across use cases, allowing for teams from various fields to increase their productivity and gain access to accurate patient data that normally would have required a manual approach for identification. In addition, it has many data security features. Successful implementation at other locations has demonstrated that EMERSE can be deployed and used outside its original site. Groups interested in adopting EMERSE can contact the EMERSE team at the University of Michigan for a working virtual machine for testing, demonstrations, advice, and other details.


The initiation of the first spike of Ca2+ oscillation was substantially delayed in egg fused with eCs-KO sperm, despite normal expression of sperm factor phospholipase C zeta 1. The eCs-KO male mice were initially fertile, but the fertility dropped with age. Metabolomic analysis of aged sperm revealed that the loss of eCS enhances TCA cycle in the mitochondria with age, presumably leading to depletion of extra-mitochondrial citrate. The data suggest that eCS suppresses age-dependent male infertility, providing insights into the decline of male fertility with age.Men and women become infertile with age, but the mechanism of declining male fertility, more specifically, the decrease in in sperm quality, is not well known. Citrate synthase (CS) is a core enzyme of the mitochondrial tricarboxylic acid (TCA) cycle, which directly controls cellular function. Extra-mitochondrial CS (eCS) is produced and abundant in the sperm head; however, its role in male fertility is unknown. We investigated the role of eCS in male fertility by producing  2+ oscillation in a phospholipase C zeta 1 -independent manner. Moreover, the fertility of eCs-knockout male mice dropped with age, indicating that eCS suppresses age-dependent sperm dysfunction by accelerating Ca2+ oscillation.Citrate synthase is a core enzyme of tricarboxylic acid cycle, but the role of extra-mitochondrial CS (eCS) is less clear. eCS, abundant in the sperm head, triggers a first spike of egg Ca Age affects male fertility indirectly and directly. Female fertility declines after the age of 35, thus indirectly affecting and often diminishing the male fertility . More di2+) are released from the intracellular stores, such as the endoplasmic reticulum i) rise. CS is localized in the mitochondrial matrix, where it catalyzes the formation of citric acid from acetyl-coenzyme A (CoA) and oxaloacetate  and boiling for 10 min at 95 °C. The protein samples were resolved by SDS polyacrylamide gel electrophoresis on 12% acrylamide gel, and immunoblotted. Detection of the proteins and the relevant primary Abs (0.1 μg/ml) was based on enzyme-linked color development with horseradish peroxidase-conjugated secondary Abs .The sperm were collected from the epididymis of 8–12-week-old ICR male mice, and fixed with 4% paraformaldehyde in HEPES-buffered saline for 1 h at 4 °C. The sperm were then incubated in TYH medium containing primary Abs (0.5 μg/ml) for 1 h at 4 °C. The sperm were next treated with secondary Abs  for 1 h at 4 °C, and then washed three times with TYH medium, prior to the acquisition of fluorescent images using a laser scanning confocal microscope . In some experiments, the sperm were visualized by immunostaining with Alexa Fluor 488-conjugated peanut lectin PNA  on ice for 1 h, and washing with PBS. Fluorescent images were then acquired using a laser scanning confocal microscope .Csl (eCs) (Csltm1(KOMP)Vlcg; ID14519) obtained from the Knockout Mouse Project (KOMP) repository . Homozygous mice (C57BL/6 genetic background) were generated by subsequent intercrosses of heterozygous animals.Mutant mice were generated from C57BL/6-derived embryonic stem cell clones by injection into blastocysts from C57BL/6 mouse with a genetically deleted The sperm were collected from the epididymis of 8–12-week-old male mice and fixed in 50 mM sodium cacodylate, pH 7.2, containing 2.5% glutaraldehyde and 2% sucrose, for 2 h at 4 °C, as described previously . The fixTo measure sperm motility, computer assisted sperm analysis operated by IVOS software (Hamilton-Thorne Biosciences) was used as previously described . An aliqeCs-KO females (8–12-week old) 14–16 h after hCG injection, and placed in a 100-μl drop of TYH medium under paraffin oil , equilibrated with 5% CO2 in air at 37 °C. The sperm collected from the epididymides of C57BL/6N and eCs-KO males  were induced to capacitate by incubating in TYH medium for 90 min under an atmosphere of 5% CO2 in air at 37 °C. The eggs were then inseminated with sperm (150 sperm/μl) and two-cell embryos were observed under an Olympus IX71 microscope (Olympus) after a 24-h incubation in TYH medium at 37 °C under 5% CO2 in air. The 60, 122, 78, and 68 eggs were inseminated with WT, eCs-KO, WT , and eCs-KO sperm , respectively. These experiments were performed in quintuplicate experiments.The eggs were collected from the oviductal ampulla region of superovulated C57BL/6N and g for 10 min. CS activity was determined using the CS activity colorimetric assay kit  according to the protocol provided by the manufacturer. The sperm (2 × 106) were homogenized in CS buffer and incubated on ice for 10 min. After centrifugation at 10,000 × g for 5 min, enzyme activity was measured in the collected supernatant. The rate of color development was measured at 405 nm using a spectrophotofluorometer .The cauda epididymal sperm  were collected in phosphate-buffered saline and washed with phosphate-buffered saline by centrifugation at 600 × 2+ concentration, eggs with intact ZP  were incubated in TYH medium containing the Ca2+-sensitive fluorescent dye Oregon green 488 BAPTA-1 AM  for 15 min at 37 °C in a CO2 incubator. They were then washed three times with TYH medium (5 min each). After washing, capacitated sperm (150 sperm/μl) were added to TYH medium. Fluorescent images of the eggs were captured using a highly sensitive CCD camera , using software to operate the camera , every 10 s. Fluorescence intensity was measured by using the imaging software Andor IQ (Andor Technology). Each fluorescent image (F) was subtracted from the image before injection or the image with the lowest fluorescence intensity (F0). The fluorescence intensity for individual eggs was measured within a user-selected region that covered the majority of the area of each egg. The mean intensity over the same area for each of the images in a time series was analyzed automatically. Changes in fluorescence intensity are reported as the F/F0 ratio after insemination with the sperm, as previously described .The GC–MS approach allowed us to identify and quantify 46 metabolites  is a precursor of nicotinamide adenine dinucleotide as a precursor of adenosine triphosphate (ATP) . Niacinaerm Fig. . By conterm Fig. , implyineCs-KO and WT sperm  cycle, because succinate semialdehyde is a metabolite of GABA, which leads to succinate production . GABA leerm Fig. . Converserm Fig. .eCs-KO sperm from 3-month-old mice  in Fig. The data presented herein suggest the possibility that at least two sperm factors, eCS and PLCz1, are involved in CaeCs and Plcz1-KO sperm elicit delayed triggering of Ca2+ oscillation, they exhibit completely different patterns of Ca2+ oscillation. Even though the first spike was delayed in an egg inseminated with eCs- and Plcz1-KO sperm, a relatively regular oscillation occurred after the initiation of Ca2+ oscillation in the egg inseminated with eCs-KO sperm , whereas PLCz1 could continue to regulate the latter step of Ca2+ oscillation .Although both erm Fig. . By contort time . Hence, eCs-KO sperm induced insufficient Ca2+ oscillation, the eCs-KO males were fully fertile, but the period of fertility was relatively shortened. The age-dependent reduction of litter size may be related to the ATP production in the egg. In human granulosa–luteal cells, ATP evokes Ca2+ oscillation in the absence of extracellular Ca2+, while sustained Ca2+ oscillation requires extracellular Ca2+ [2+ rise, and the second ATP increase takes place 1 h later [2+ rise depends on ATP production. By contrast, the second and additional increases of the Ca2+ levels are independent of ATP production but depend on the extracellular Ca2+ concentration. Based on previous reports and the findings of current study, we propose that eCS-driven ATP production triggers the first Ca2+ rise that corresponds to Ca2+ release from intracellular storage; the ATP demand then becomes lower, presumably shifting the mechanism of increasing Ca2+ levels from dependence on intracellular Ca2+ to dependence on extracellular Ca2+ [(II) in Fig. eCs-KO sperm  is restricted in human tissues [The expression of an alternatively spliced form of  tissues , and the tissues  can funceCs-KO sperm with age, resulting from CS deficiency in the TCA cycle. The changes in citrate levels in eCs-KO sperm may reflect the inactivity of the TCA cycle and could be used as markers to discriminate the aging infertile male from aging fertile male (Fig. In this study, we showed the role of eCS in age-related changes in male fertility. Importantly, we demonstrated an abnormal metabolism in the ale Fig. .2+ oscillation was significantly delayed in eggs fused with eCs-KO sperm despite the normal expression of Plcz1. This result offers a novel insight into the role of sperm factor, because eCs-KO males were fertile initially. Aberrant function of Plcz is able to explain egg activation failure in infertile patients [eCs-KO male mice are useful for analyzing unexplained male infertility with age, presumably contributing to development of diagnostic methods in patients with age-dependent male infertility.In the present study, we showed that initiation of Capatients . OtherwiRevised supplementary Materials


In vivo, AZA prolonged survival and attenuated AML by decreasing CD33+ immunophenotyping in the bone marrow of a patient-derived xenograft AML model. Furthermore, mice in the AZA-treated group had an increased antioxidant capacity and Prdx2/Prdx3 upregulation. The findings indicate that AZA may be a potential agent against AML by regulating the Prdxs/ROS signaling pathway.Acute myeloid leukemia (AML) is a hematological malignancy with a poor prognosis attributed to elevated reactive oxygen species (ROS) levels. Thus, agents that inhibit ROS generation in AML should be exploited. Azelaic acid (AZA), a small molecular compound, can scavenge ROS and other free radicals, exerting antitumor effects on various tumor cells. Herein, this study evaluated the antileukemic activity of AZA against AML via regulation of the ROS signaling pathway. We found that AZA reduced intracellular ROS levels and increased total antioxidant capacity in AML cell lines and AML patient cells. AZA suppressed the proliferation of AML cell lines and AML patient cells, expending minimal cytotoxicity on healthy cells. Laser confocal microscopy showed that AZA-treated AML cells surged and ruptured gradually on microfluidic chips. Additionally, AZA promoted AML cell apoptosis and arrested the cell cycle at the G1 phase. Further analysis demonstrated that peroxiredoxin (Prdx) 2 and Prdx3 were upregulated in AZA-treated AML cells.  Acute myeloid leukemia (AML) is one of the most common hematological malignancies with a rapidly progressive and poor prognosis. Larger numbers of blasts accumulate in the bone marrow and infiltrate other tissues, thus inhibiting hematopoietic functions and inducing subsequent hemorrhage and severe infection. The annual mortality rate is 2.2 per 100,000 [Jab1), in AML patient relapse, and the upregulated Jab1 can regulate Trx by binding to Trx1, which contributes to the poor survival [Reactive oxygen species (ROS) are small short-lived oxygen-containing molecules that regulated many processes, such as cell growth and death, inflammation, stem cell renewal, tumorigenesis, oxygen sensing, angiogenesis, and immune responses , 3. Elevsurvival . Additiosurvival .The roles of ROS depend on their intracellular levels. Low ROS levels are required to initiate and promote tumor growth; moderate ROS levels are involved in the inflammatory response, while high ROS levels contribute to apoptosis and autophagy . Both ROMalassezia [Azelaic acid (AZA) is a natural, nontoxic, saturated, nine-carbon dicarboxylic acid that was first recognized as a secondary metabolite in fungal infections with lassezia . AZA, aslassezia  and othelassezia . Prior slassezia , 28. AZAlassezia . Furtherlassezia , malignalassezia , lymphomlassezia , and humlassezia , by inhilassezia , 33. A plassezia . Howeverβ-actin (Cat# 14395-1) and Prdx2 (Cat# 10545-2) were from ProteinTech (USA).DMSO (Cat# D2650) and AZA (Cat# 95054) were purchased from Sigma (USA). PrimeScript™ RT reagent kit with gDNA Eraser was from Takara (Cat# RR047A). Annexin V-FITC Apoptosis Detection Kit was from KeyGEN Biotech . Cell Cycle Staining Kit was from MultiSciences Biotech . Antibodies to the following proteins were used: Prdx3 was from CUSABIO ; AML cell lines U937, HL60, THP-1, and Molm-13 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37°C. Different types of AML patient primary cells (AML-PC) according to the FAB classification and healthy peripheral mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation followed by the manufacturer's recommendation ; the detailed AML patient information can be found in Table μM for 20 minutes at 37°C to assess ROS-mediated oxidation of the fluorescence compound DCF-DA. ROS levels were determined by detecting the fluorescence intensity of the oxidized DCF cytometer, and the flow cytometry analysis results were quantified by using CytExpert2.0 software.Intracellular ROS levels were measured using the Reactive Oxygen Species Assay Kit . Briefly, HL60, U937, THP-1, and AML-PC cells were treated with AZA for 24 h, and the collected cells were then washed with serum-free RPMI-1640 and incubated with 2,7-dichlorodihydrofluorescein diacetate (DCF-DA) at the concentration of 5 HL60, U937, and Molm-13 cells were pretreated with 5 mM AZA for 24 h; cells were collected and washed twice by PBS. Cells were lysed by using the ultrasonic wave breaking, and cell homogenate was centrifuged for 15 minutes at 12,000 rpm at 4°C. Thereafter, supernatant was harvested and stored at -20°C. In vivo experiment, peripheral blood of mice was centrifuged for 15 minutes at 12,000 rpm at 4°C in an anticoagulant tube and the serum was collected from liquid supernatant. The harvested cell supernatant and serum were prepared for the following study. The total antioxidant capacity , superoxide dismutase  activity, glutathione peroxidase  content, and malondialdehyde  levels were assayed according to the manufacturer's protocol as described previously  .μL CCK8 was added to each plate and incubated for additional 2 h. The cell viability was measured by reading the absorbance at 450 nm.Cell viability was measured by using a Cell Counting Kit-8 . HL60, THP-1, Molm-13, and different types of AML-PC cells were seeded in a 96-well plate and treated with different concentrations of AZA for 24 h. Thereafter, 10 +-THP-1 cell was dead, the green fluorescence disappeared. The cell viability can be assayed by comparing the green fluorescence intensity between two groups.To observe the AML cell viability after AZA treatment, THP-1 cells transfected with GFP were used in our study and a DAPI stain was used as a control for indicating all cell numbers in the field. When the GFPTo detect the cytotoxic effect of AZA on AML cells, a microfluidic chip was designed to capture the cells and allowing the injection of AZA. The clip size was designed according to the cell size as described previously , 36. BriEarly apoptosis analysis was measured by using the Mitochondrial Membrane Potential Assay Kit with JC-1 . JC-1 aggregates in the matrix of mitochondria to form a polymer (J-aggregates) and can emit red fluorescence at normal MMP but gets converted to monomer when MMP decreased and then can emit green fluorescence. HL60 cells were treated with 5 mM AZA for 24 h; cells were collected and stained with JC-1 dye. The change in mitochondrial membrane potential was analyzed by flow cytometry.μL Annexin V-FITC and 5 μL propidium iodide and then incubated for 5 minutes at 25°C. The apoptotic rate was measured by calculating the percentage of FITC and PI-positive cells with flow cytometry.The apoptotic rate of U937 cells was detected with the Annexin V/FITC Apoptosis Detection Kit. U937 cells were seeded in 6-well plates and treated with 5 mM AZA for 24 h. The collected cells were washed twice by PBS and stained with 5 μL propidium iodide for 30 minutes. The number of cells in different phases of the cell cycle was analyzed by flow cytometry. Data were analyzed by using CytExpert2.0 software.Cell cycle was measured by using a Cell Cycle Staining Kit. U937 cells were pretreated and collected as above described. Cells were then fixed by 1 mL staining buffer and incubated with 10 http://www.peptideatlas.org/PASS/PASS01499.THP-1 cells were treated with 5 mM AZA for 24 h, and total proteins were extracted by RIPA buffer. Then, the peptides from the AZA-treated group and the control group were labeled with isotopomeric dimethyl label. The labeled samples were analyzed by using a hybrid Quadrupole-TOF Mass Spectrometer as described previously   and peroxiredoxin 3 (Prdx3) expression using the SYBR GREEN MIXTURE kit according to the manufacturer's recommendations. The expression level was analyzed using the 2μg) were resolved by SDS gel electrophoresis and transferred to PVDF membranes. The membranes were blocked by 5% nonfat milk followed by probing with primary and secondary antibodies.Molm-13, THP-1, and AML-PC cells were seeded in 6-well plates and treated with 5 mM AZA for 24 h. Total proteins were extracted by RIPA and 10 mM PMSF. Thereafter, protein concentration was determined by the BCA protein assay kit . The proteins (20 7 human AML cells/per mouse intravenously within 24 h for the development of leukemic disease. The patient-derived xenograft (PDX) AML model was successfully established when AML-PC cells could be observed on the peripheral blood (PB) and bone marrow (BM) smears. Then, mice were randomly divided into two groups with 6 mice in each group. The mice in the AZA group were treated with 10 mg/kg AZA by intraperitoneal injection , while the control group (n = 8) received saline with the same volume and frequency. At the end of experiments, mice were sacrificed and the tissues were harvested for further study. Importantly, all animal studies were approved by the Institutional Animal Care and Use Committee of Wuhan University (2017048).The B-NSG mice  were obtained from the Animal Research Center of Wuhan University. AML-PC cells were used to construct the model which were isolated from a hyperleukocytic AML patient sample after undergoing leukapheresis by using a Fresenius COM.TEC machine. Mice were given 1.5 Gy X-ray and injected with 1.0 × 10After injecting mice with AML-PC cells for one week, we randomly selected one mouse which was humanly killed, and its PB and BM were harvested. PB and BM smears were stained with Wright's stain and observed microscopically to measure the proportion of leukemia cells and determine whether the PDX model was constructed successfully .Tissues collected from the mice were fixed, trimmed, processed, dewaxed, and rehydrated, then under pretreated for antigen retrieval in citrate buffer at pH 6.0 at 100°C for 30 minutes. Thereafter, tissues were blocked with primary antibodies  overnight, then probed with secondary antibodies. Images were photographed using a Nikon microscope at the Hematology Department, Wuhan University, Zhongnan Hospital, Wuhan, China.http://wego.genomics.org.cn/), and analyzed with R code by creating a heat map. The protein-protein interaction networks were analyzed using STRING v11.0 (https://string-db.org/cgi/network). Data are presented as the means ± standard deviations and were analyzed via Student's t-test and one-way analysis of variance using GraphPad Prism 7 and IBM SPSS. P < 0.05 was considered statistically significant.Differentially expressed proteins were identified via LC-MS, annotated by WEGO analysis  were identified after AZA treatment. The DEPs were then annotated by WEGO analysis as described previously . Five huWe performed quantitative real-time PCR and western blotting to confirm this finding. Consistent with our MS results, AZA induced higher Prdx2 and Prdx3 RNA and protein expressions Figures . Finallyvivo, we constructed a PDX AML model as per a previous report [7 CD33+ AML patient primary cells after 1.5 Gy irradiation. When PB and BM smears indicated illness, mice were randomly divided into two groups (n = 8). AZA was administered every three days for 2 weeks; saline was administered as a negative control. The PDX AML models progressed rapidly, and most mice died within 18-27 days; thus, all mice were sacrificed on day 21, and their tissues were harvested for further study  can be used only in patients with FLT-3 mutations and patients who develop resistance . Immune 2O2-eliminating enzyme involved in detoxifying 90% of H2O2 [2O2 accumulation and elevated ROS levels in the mitochondria [2O2 [ROS are generated from multiple sources. The main source of intracellular ROS is the mitochondria. Healthy cells control the intracellular ROS balance via the scavenging system . One maj of H2O2 . Deletinchondria . Prdx2 iria [2O2 . Early sria [2O2 . In addiria [2O2 . Moreoveria [2O2 , 49. In Identifying agents that can decrease intracellular ROS levels is a potential method of treating cancer. Lycopene can suppress the progression of prostate carcinoma by decreasing oxidative DNA damage and scavenging ROS , 51. SomAZA exerts antitumor effects at micromole concentration, it was usually topical application in the form of water-miscible or gel for the treatment of skin disorder with only mild additional side effects; however, when it was administrated orally or intraperitoneal injection in the form of solution, it could cause noticeable weight loss and acid-base imbalance , 56. TheOur MS detection results showed that AZA upregulated other antioxidant enzymes such as CAT and SOD2. These antioxidant enzymes interact with Prdx3 and Prdx2, but how they act in synergy with Prdxs to maintain intracellular redox equilibrium is being studied. Several immune-related signaling pathways were active after AZA treatment as shown on the heat map; the most obvious was Notch. Notch can maintain low ROS levels to promote cell development and survival . In addiIn summary, we provided a potential novel agent and therapeutic approach to treating AML by regulating the Prdxs/ROS signaling pathway, which may provide insight into ROS-eliminating strategies.in vitro and in vivo. AZA is a potential agent for treating AML; ROS-eliminating strategies may be promising strategies for treating AML.AZA decreased intracellular ROS levels and increased antioxidant capacity by upregulating Prdx2 and Prdx3, which maintained the intracellular redox balance and further suppressed AML


How these transients are generated and regulate brain function in vivo remains unclear. Here we show that Drosophila astrocytes exhibit spontaneous, activity-independent microdomain Ca2+ transients in their fine processes. Astrocyte microdomain Ca2+ transients are mediated by the TRP channel TrpML, stimulated by reactive oxygen species (ROS), and can be enhanced in frequency by the neurotransmitter tyramine via the TyrRII receptor. Interestingly, many astrocyte microdomain Ca2+ transients are closely associated with tracheal elements, which dynamically extend filopodia throughout the central nervous system (CNS) to deliver O2 and regulate gas exchange. Many astrocyte microdomain Ca2+ transients are spatio-temporally correlated with the initiation of tracheal filopodial retraction. Loss of TrpML leads to increased tracheal filopodial numbers, growth, and increased CNS ROS. We propose that local ROS production can activate astrocyte microdomain Ca2+ transients through TrpML, and that a subset of these microdomain transients promotes tracheal filopodial retraction and in turn modulate CNS gas exchange.Astrocytes exhibit spatially-restricted near-membrane microdomain Ca Octopamine and tyramine stimulate cell-wide astrocyte Ca2+ increase through the dual-specificity Octopamine-Tyramine Receptor (Oct-TyrR) and the TRP channel Water witch (Wtrw). This astrocyte-mediated signaling event downstream of octopamine and tyramine is critical for in vivo neuromodulation: astrocyte-specific elimination of Oct-TyrR or Wtrw in larvae blocks the ability of octopamine and tyramine to silence downstream dopaminergic neurons, and alters both simple chemosensory behavior and a touch-induced startle response  in astrocytes using the astrocyte-specific alrm-Gal4 driver. We acutely dissected 3rd instar larval CNS and live-imaged myr-GCaMP5a signals in the ventral nerve cord (VNC)  seconds  in the astrocytes of intact L1 (1st instar) larvae   . We collrd (VNC) . We founquencies . The ave seconds . Microdog window , and Ca2) larvae , suggest (LaCl3) , suggest2+ transients spanned astrocyte-astrocyte cell boundaries, or if they appeared only within the domain of single cells. We used a flippable construct expressing either QF or Gal4 under the control of the alrm promoter, along with two genetically encoded Ca2+ indicators: QUAS::myr-GCaMP5a and UAS::myr-R-GECO1  or octopamine (nM18Tβh) did not significantly alter the frequency of astrocyte microdomain Ca2+ transients, nor did mutations in Oct-TyrR, which we previously showed was essential for activation of whole-cell Ca2+ transients in astrocytes  in astrocytes rescued decreased microdomains in trpml mutants  , and astby TrpA1 . The molr larvae  and acutr larvae . Astrocy by ~60% , and exp mutants , arguing reduced , suggest2+ events in astrocyte cell bodies, and found that tyramine induced comparable Ca2+ rise in astrocytes in control and 1trpml mutants  led to a TrpML-dependent increase in astrocyte microdomain Ca2+ events, while, reciprocally, addition of the antioxidant N-acetyl cysteine completely abolished them  may serve as factors to coordinate their activity.Previous work has shown that reactive oxygen species (ROS) can activate TrpML , and aste to ROS . We therhed them  and 3. TDrosophila infiltrate the CNS neuropil where they associate with neural processes, synapses, and tracheal elements , retraction (20.7%), extension and retraction (75.3%), or stationary (2.3%) . The vas2+ transients. Interestingly, 52% of tracheal filopodial tips overlapped, at some point, with an astrocyte microdomain Ca2+ transient  and more broadly distributed  within an circular area beginning 7.5 μm away from filapodial tips and extending outward , we examined tracheal structure in larval CNS. We first counted the total number of protrusions from a pair of most posterior ganglion trachea (mpgTr) that innervate a few segments from A5 to A8/9 in the ventral nerve cord. We found that mpgTr in the ventral nerve cord in 1trpml mutants exhibited increased total length compared to those in control animals . In live preparations of 1trpml mutants we found a ~ 3 fold increase in oxidized DHE+ puncta, likely in mitochondria  demonstrated individual astrocyte microdomain Ca2+ transients can span astrocyte-astrocyte boundaries, which might argue for an extrinsic mechanism regulating their production. Alternatively, it might indicate that astrocyte-astrocyte coupling of Ca2+ signaling events is strong during their production. Astrocyte microdomain Ca2+ transients are not suppressed by blockade of action potentials with tetrodotoxin, suggesting they are not activity dependent, although we also cannot formally rule out a role for spontaneous release of neurotransmitters at synapses.Astrocyte microdomain Ca mammals . They arTdc2 mutants, which lack tyramine and octopamine in CNS, we conclude that spontaneous astrocyte microdomain Ca2+ transients do not require the production of tyramine or octopamine in vivo. Nevertheless, and similar to application of norepinephrine to mouse preparations  to indirectly activate Ca2+ entry through other cation channels at the cell surface. Alternatively, TrpML might function at the cell surface to directly drive Ca2+ entry, or stimulate entry from the extracellular space by other channels, or release from intracellular stores. It is also important to note that while we have assayed Ca2+ entry, TrpML is also capable of passing several other cations . We previously showed either of these can activate the Oct-TyrR receptor and the TRP channel Waterwitch on astrocytes, and in turn through the adenosine receptor AdoR, mediate many of the physiological and behavioral changes exerted by tyramine and octopamine  and they are sensitive to ROS.Astrocyte microdomain Caof event . In mamme to ROS . Similartopamine . In cont2+ transients are associated with mitochondria, which have been proposed to serve as a source of ROS, potentially through transient opening of the mPTP  could explain why although only half of all observed astrocyte microdomain Ca2+ transients are associated with tracheal filopodia, all appear to be potently regulated by ROS. Perhaps the remaining half of astrocyte microdomain Ca2+ transients not associated with trachea are responding to mitochondria-based metabolic needs through ROS signaling. Finally, approximately half of tracheal filopodia we observed were not visibly associated with astrocyte microdomain Ca2+ transients, yet they were able to extend and retract. This argues that other mechanisms exist to modulate tracheal dynamics in the CNS and astrocyte microdomain Ca2+ transients only represent one regulatory mechanism.In mouse, a subset of microdomain Cathe mPTP . These echondria . Multipl2-consuming metabolism is thought to fluctuate vigorously in response to neural activity. The close association of astrocyte microdomain Ca2+ transients with trachea, the larval breathing apparatus, and with tracheal retraction in particular, suggests a role in modulating CNS gas exchange. Increase in O2 delivery to tissues can lead to hyperoxia and elevated production of ROS. TrpML has recently been found to be a ROS sensor , which argues that astrocyte-tracheal signaling through TrpML can regulate long-term structural changes in the tracheal morphology. Based on these findings, we propose that an important role for a subset of astrocyte microdomain Ca2+ transients in the larval CNS is to coordinate gas exchange through regulation of tracheal dynamics, thereby balancing O2 delivery/CO2 removal according to local metabolic needs.Maintaining a healthy, spatiotemporally regulated normoxic environment to prevent either hypoxia or hyperoxia in CNS is an enormous and ongoing challenge, as the OS sensor , which w a model  where a Drosophila strains used include: Bloomington stock center 1trpml (28992), 2trpml (42230), JF01239trpml (31294), JF01878tyrR (25857), JF02749tyrRII (27670), 1trpA1 (36342), 2trpm (35527), 3nompC (42258), 1trp (5692), 302trpl (31433), 1pkd2 (24495), JF01244trpγ (31299), JF01242pyx (31297), btl-Gal4 (8807), UAS-Lifeact-GFP (57326), UASp-αTub-GFP (7373), 10XUAS-IVS-myr::tdTomato (32222), UAS-trpml-myc (57372), UAS-GFP-Lamp1 (42714). honoOct-TyrR  to generate constructs pUAST-myr::GCaMP5a, pQUAST-myr::GCaMP5a, pUAST-myr::R-GECO1, pJFRC19-13XLexAop2-IVS-myr::GCaMP6s for injection. The transgenic flies were injected and recovered by Rainbow Transgenic Flies, Inc .The full-length ORFs of GCaMP5a, R-GECO1, GCaMP6s with an in-frame DNA fragment encoding the myristoylation signal peptide at 5’-end were cloned into vectors pUAST, pQUAST, pJFRC19  was sandwiched in 30 µl halocarbon oil 27  between a slide and a 22 × 22 mm coverslip , then a 3 min time-lapse video was taken immediately on a spinning disk confocal microscope equipped with a 40X oil immersion objective.Card instar larvae expressing myr-GCaMP5a in astrocytes  was performed in the imaging buffer (pH7.2) containing 110 mM NaCl, 5.4 mM KCl, 0.3 mM CaCl2, 0.8 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, the CNS was immediately transferred to a silicone coated petri dish, immersed in 100 µl imaging buffer (1.2 mM CaCl2), and immobilized gently by sticking the attached nerves onto the silicone surface with forceps. The petri dish then was placed on the stage of a spinning disk confocal microscope equipped with a 40X water dipping objective. The focal plane was fixed around where most of the dorsal lateral astrocytes start to appear in the field of view. After 4 min acclimation and stabilization, a 6 min movie  was taken for analysis. The 488 nm and 516 nm channels were alternated for imaging both the microdomain Ca2+ transients in astrocytes and the dynamics of tracheal filopodia. To keep the tracheal filopodia in focus during the course of extension and retraction, images spanning 5 µM in z depth were taken.CNS dissection from early 32+ transients per minute) of microdomain Ca2+ transients in each preparation was initially quantified as follows: a 100 µm X 100 µm window was cropped from each movie and resulted in nine smaller, side-to-side 100pixel X 100pixel windows (line drawing over movies) in which the number of microdomain Ca2+ transients was counted manually. The total number of microdomain Ca2+ transients in each preparation (100 µm X 100 µm) was acquired by adding up all the numbers counted in these 9 100pixel X 100pixel windows. The cutoff for defining a Ca2+ transient is 5% change in delta F/F0, which is evaluated by post hoc calculations after manually selecting active events. After the automatic Ca2+ signal analysis software Astrocyte Quantitative Analysis (AQuA), and we didn’t find a significant difference . The intensity of microdomain Ca2+ transients was measured with software Volocity , and the amplitude of microdomain Ca2+ transients was defined by (Ft-F0)/F0  as percentage. The full width at half maximum (FWHM) of astrocyte microdomains was acquired using software Igor Pro .The frequency (the number of microdomain Cais (AQuA) became p2+) containing drugs  was directly applied onto the preparations, then imaging continued for another 3 min. The chemicals used for bath application experiments include: Tocris, tetrodotoxin . Sigma-Aldrich, lanthanum chloride , acetylcholine , γ-aminobutyric acid , glutamate , tyramine , octopamine , N-acetyl cysteine , hydrogen peroxide .For bath application of compounds, halfway through the 6 min imaging window (~3 min), 100 µl imaging buffer  was immediately transferred in 4% formaldehyde for fixation for 20 min . Washing in PBS for 3 × 10 min. Permeabilization in PBS + 0.3% Triton X-100 for 2 hr. Primary antibody  incubation at 4 ℃ for ~72 hr. Secondary antibody incubation at room temperature for ~2 hr. Tracheal branches in ventral nerve cord were illuminated with 408 nm laser light and emitting autofluorescence was imaged. Each individual branch was then traced manually with Simple Neurite Tracer (Fiji).The CNS dissected in PBS from 3rd instar larvae  were made exactly in the same way for Ca2+ imaging. Incubation in 100 µl imaging buffer (1.2 mM Ca2+) containing 30 µM DHE for 8 min before imaging . The puncta were automatically counted by the segmentation tool in Slidebook.The CNS preparations from 3post hoc tests, or unpaired t-test. Comparison within groups was tested by paired t-test. p<0.05 was considered statistically significant. *p<0.05, **p<0.01.All statistics were performed in Graphpad. No data were excluded for analyses. 2–3 replications were successfully performed for each experiment. Comparison between groups was tested by one-way ANOVA with Tukey’s Source data for  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:trpml in regulating astrocyte microdomain calcium oscillations in Drosophila. They link this biology to regulation of local tracheal filopodial dynamics and ROS activity, suggesting one role for astrocyte calcium dynamics is to control local gas exchange in Drosophila larvae. This study represents a significant advance in understanding how microdomain calcium transients are generated and regulate CNS physiology and function.The manuscript by Ma and Freeman describes a role for the TRP channel Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Ronald Calabrese as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Brian A MacVicar (Reviewer #2).Thank you for submitting your article "TrpML-mediated astrocyte microdomain Ca 2+ transients regulate astrocyte-tracheal interactions" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Summary:2+ transients in the Drosophila nervous system and their relationship to the physiology and behavior of CNS trachea. Using genetic Ca2+ reporters, they first show that astrocyte processes exhibit transient changes in Ca2+ concentration within microdomains. They present results suggesting that extracellular Ca2+ is required for Ca2+ changes within microdomains, and they identify the TrpML ion channel, in a targeted genetic screen, as a critical component of the mechanism regulating Ca2+ within microdomains. Importantly, Ca2+ transients are almost eliminated with astrocyte-specific knockdown of 1trpml. The authors further show that tyramine, acting through a fly tyramine receptor (TyrRII), can enhance Ca2+ transients but that it is not required for such transients. They demonstrate that the Ca2+ microdomains are in close proximity to filopodia of tracheal tubules, and go on to show that transient changes in Ca2+ within such microdomains can influence the behavior of tracheal filopodia.Ma and Freeman present results from studies of astrocyte Ca2+ within such microdomains is strongly correlated with filapodial retraction events. Additional findings show that extension of tracheal filopodia is enhanced in the 1trpml mutant, whereas retraction events are normal, suggesting that the TrpML channel is important for inhibition of extension events. Finally, the authors show that there is overgrowth of trachea and higher levels of reactive oxygen species in the 1trpml mutant, suggesting a role of the TrpML channel and Ca2+ in tracheal growth and gas exchange.Their results suggest that increased Ca2+ regulation in astrocytes. The results are important as similar astrocyte Ca2+ microdomains regulate CNS physiology in mammals, and Drosophila provides an important genetic model for the analysis of astrocyte Ca2+ signaling. However, there are a few issues and suggestions for improvement, as well as points that need clarification prior to publication.The manuscript is clearly written and represents a significant advance in understanding CaEssential revisions:trpml directly mediates calcium microdomain influx or tone down conclusion and add more to the Discussion that discusses alternative models.1) Based on the data presented, it is not clear whether the TRP channel is the primary source of calcium influx for microdomain oscillations, or secondarily coupled to the process to control the frequency of the events only. Please clarify this or provide more convincing data that trpml localized in Drosophila astrocytes? All reviewers agree that it is important to know whether TrpML is acting in astrocyte lysosomes or at the cell membrane to regulate CNS and tracheal functions. Localizing it would help, as would measuring the amount of calcium influx signal in the absence of the channel --- the frequency goes down, but they still appear to be present. See reviewer comments for specific comments and suggestions.2) Where is 3) Please address whether astrocytes are regulating trachea in the mature larval CNS, as detailed in comments by reviewer 3. Alternatively, if the alteration of tracheal function is a consequence of abnormal astrocyte development.Drosophila and mouse astrocytes need to be clarified in some cases, and the authors should be more careful in discussing the different types of mouse and rat astrocyte calcium signals (see specific comments by reviewer 2)4) The comparisons made between the results in Reviewer #1:2+ regulation in astrocytes. The results are important as similar astrocyte Ca2+ microdomains regulate CNS physiology in mammals, and Drosophila provides an important genetic model for the analysis of astrocyte Ca2+ signaling.The manuscript of Ma and Freeman is clearly written and represents a significant advance in understanding CaThe major issues detailed below should be addressed prior to publication:2+ transients within astrocyte microdomains. The presentation of these results leads the reader to think that TrpML acts at the astrocyte cellular membrane to regulate Ca2+ entry. While this might be true, The TrpML channel has been described as a lysosomal membrane rather than a cellular membrane channel, and it is clearly required for cell migration and phagocytosis via regulation of actomyosin contractility . Is there any evidence that TrpML is even present at the cellular membrane? Have the authors determined localization of the channel in astrocytes? If not, the presentation will lead most readers to think its site of action is the cellular membrane when this has not been demonstrated and is likely not to be the case. A more detailed model for TrpML, taking its localization into account, is needed.1) The authors show that elimination of extracellular and the TrpML channel inhibit Ca2+ transients is to modulate CNS gas exchange through TrpML and ROS signaling." This suggests that TrpML might have a modulatory role in the mature nervous system. However, it seems that all of the authors' results about TrpML could be interpreted as a requirement for the channel in astrocytes during development with a consequent effect on physiology of the tracheal system. Indeed, the authors show that tracheal overgrowth occurs in the channel mutant. I wonder if there is any evidence for a role of TrpML in the mature nervous system. Did the authors attempt a conditional knockdown of TrpML in the mature larval CNS? What about inhibition of TrpML in mature CNS astrocytes using an antagonist that blocks Ca2+ release from lysosomes? Or, activation of TrpML with an agonist in the mature CNS? Or, transient blockade of Ca2+ entry in the mature larval CNS? Is it known whether such manipulations alter tracheal function? It would seem important to determine whether astrocytes actually modulate trachea and/or gas exchange in the mature CNS since this is the implication of the authors' model.2) The authors propose "that one important physiological role for astrocyte microdomain Ca2+ permeable, it is also permeable to other cations. It is described in FlyBase as a cation channel involved in regulation of endosomal trafficking, autophagy, lateral inhibition and TOR signaling in hemocytes and perhaps other cell types. Is regulation of astrocytes and tracheal function as simple as modulation of Ca2+ entry or is it more complicated than that? I wonder if Ca2+ microdomains are affected indirectly because of an effect on endosomal trafficking. Some discussion of this is needed.3) Although TrpML is known to be Ca1trpml mutant or 1trpml knockdown flies. Does the CNS appear to be completely normal with absence of TrpML?4) There is no mention of CNS abnormalities in the Reviewer #2:Drosophila astrocytes occur, are triggered by reactive oxygen species (ROS), are enhanced in frequency by the neurotransmitter tyramine via the TyrRII receptor and that they decrease in frequency by ~70% to 80% in trpml loss of function mutants. The authors also show that tracheal growth is modified by these calcium signals in astrocytes. These data provide intriguing counterparts to observations in mammalian astrocytes with respect to calcium signaling. However, there are comparisons that the authors make between the results in Drosophila and mouse astrocytes that need to be clarified in some cases and the authors should be more careful in discussing the different types of mouse and rat astrocyte calcium signals. Finally, the TRPML1 channel is expressed on lysosomes according to the report they cite from Zhang et al., 2016. The results presented here indicate expression on the plasma membrane based on the block by lanthanum and effects of removing external calcium. This puzzle and apparent discrepancy should be addressed by the authors.This is very interesting paper that provides convincing data that calcium microdomains in 2+ events decrease by ~70% to 80% in trpml loss of function mutants, in both intact 1st instar larvae  and acute CNS preparations from 3rd instar larvae  "While knockout of 11 of these TRP channels had no effect, we found that microdomain CaDrosophila as a model system.This is a key finding of this study and this careful analysis shows the strength and power of working with 2+ transients in Drosophila share many features with those observed in mammals."2) "Astrocyte microdomain CaThe fly astrocyte microdomains have overlap with the observations more recently in mammalian astrocytes but the authors should be careful to differentiate between the older observations of IP3-mediated calcium release from ER  and the observations of calcium entry dependent microdomains. I don't think the microdomains were observed using the older dye loading techniques. For example, the Nett et al. paper showed almost complete block of the astrocyte calcium signals by the IP3-R antagonist heparin. In my opinion these fly microdomains that are described here are most similar to the microdomains first reported by Srinivasan et al., 2015. Rungta et al., 2016, supports Srinivasan et al. and their data also point to a calcium entry pathway independent of IP3R mediated release and not altered in TRPA1-/- mice. Of course, there appear to be multiple sources of calcium that generate microdomains and Agarwal et al., 2017, point to a mechanism of calcium release from mitochondria. The differences between these various mechanisms generating calcium transients should be clearly stated because different types of calcium signals generated in different regions of astrocytes may potentially have different functions (as would be expected in neurons).3) This observation of ROS sensitivity in this study is consistent with the report by Zhang et al., 2016, describing the ROS sensitivity of TRPMl1 channels. However, there is a significant difference in that Zhang et al. describe the calcium signals as arising from the ROS triggered release of calcium from lysosomes into the cytoplasm not as a plasma membrane channel leading to entry from the extracellular space. The authors should reconcile this important difference and describe which signals are lysosomal release dependent.4) "We note that those that exhibited only retraction did so very early in the imaging window, which could indicate that we began our imaging after extension had been initiated."This paragraph is not critical but is confusing. It's clear that 6 min is sufficient to observe a cycle of extension -retraction but what is surprising is that retractions alone occur at a higher rate than extensions alone. Wouldn't these be the same if the probability was similar? Alternatively could imaging the tissue may make retractions much more likely perhaps by generating ROS itself from the tissue illumination in the spinning disk confocal?5) "Together, these observations indicate that a large fraction of astrocyte2+ transients are spatiotemporally correlated with the retraction ofmicrodomain Caadjacent tracheal filopodia."This conclusion seems reasonable based on the imaging data.Reviewer #3:trpml in regulating astrocyte microdomain calcium oscillations in Drosophila. They link this biology to regulation of local tracheal filopodial dynamics and ROS activity, suggesting one role for astrocyte calcium dynamics is to control local gas exchange in Drosophila larvae. The authors' data set is quite interesting and relevant to the field. I have a few suggestions for improvement and some general clarification points as well.The manuscript by Ma and Freeman describes a role for the TRP channel trpml channel directly mediates microdomain calcium influx in astrocytes, but I don't think their data separates a role for the channel being the source of calcium influx or secondarily coupled to the process to control the frequency of the events only. As such, the authors need to state this or provide more convincing data that trpml directly mediates calcium microdomain influx.1) The authors imply that the trpml knockdowns and mutants as they do for wildtype in Figure 1B. Is trpml required only for the frequency of these events, or also the amplitude. The authors must have this data already in hand. If the amplitude of the residual events in the same, it seems more likely that trpml is controlling when these events occur, rather than being the direct channel for gating the calcium events themselves.2) Related to the point above, the authors should document and show the amplitude of calcium transients in trpml localized in Drosophila astrocytes – is it on the plasma membrane at sites of calcium influx? If it is located on lysosomes or internal compartments, I have a hard time understanding how it could be directly mediating calcium influx from the extracellular space.3) Similarly, where is trpml RNAi line from Figure 2D and Figure 4B for the retraction/extension assays in Figure 4A. In 4A, only the trpml mutant is used to show defects in tracheal filopodia. If this biology requires trpml in astrocytes, knocking it down only in these cells should trigger this phenotype. Alternatively, this biology could be coming from an independent role of trpml in tracheal cells. This experiment is important, especially given how small some of the reported changes appear to be. Since the authors used trpml RNAi knockdown in astrocytes for all there other manipulations, it raises a question as to why not here in one of the most important experiments.4) It would be helpful to use the 5) The most difficult part of the paper for me is Figure 3D. I have a hard time finding compelling images here to support the model. From the bottom astrocyte GCaMP traces (red) and tracheal filopodial length (red) traces, only pair 2 looks compelling – red goes up, green goes down. The rest are bouncing around all over the place and it is hard to see a compelling correlation. Would it be possible to add a supplementary figure showing more of just these traces for all (or a larger representative sample) of the data included in Figure 3E. That figure shows a perfect correlation, but the raw data provided do not look convincing. Seeing more of those raw data would be very helpful for convincing the readers of this correlation. Essential revisions:1) Based on the data presented, it is not clear whether the TRP channel is the primary source of calcium influx for microdomain oscillations, or secondarily coupled to the process to control the frequency of the events only. Please clarify this or provide more convincing data that trpml directly mediates calcium microdomain influx or tone down conclusion and add more to the Discussion that discusses alternative models.We agree that TrpML may be the channel, or coupled to the channel that drive the majority of calcium influx. Given that we cannot test this directly, we have toned down these conclusions and added a discussion of these alternative models to the Discussion.2) Where is trpml localized in Drosophila astrocytes? All reviewers agree that it is important to know whether TrpML is acting in astrocyte lysosomes or at the cell membrane to regulate CNS and tracheal functions. Localizing it would help, as would measuring the amount of calcium influx signal in the absence of the channel --- the frequency goes down, but they still appear to be present. See reviewer comments for specific comments and suggestions.This is an interesting question that we have addressed by assaying localization of TrpML in astrocytes. In new data  we show that TrpML-MYC is localized (1) near the membrane of astrocyte cell bodies, and appears to be sub-cortical based on co-staining with plasma membrane-tethered GFP; and (2) in a punctate pattern throughout astrocytes, which largely overlaps with the endo-lysosomal marker GFP-Lamp1.3) Please address whether astrocytes are regulating trachea in the mature larval CNS, as detailed in comments by reviewer 3. Alternatively, if the alteration of tracheal function is a consequence of abnormal astrocyte development.rd larval instar. For instance, tracheal morphologies were quantified in Figure 4—figure supplement 1B in 3rd instar larvae.Astrocyte development based on the analysis of a number of markers and cellular morphology appears normal in TrpML mutants . We have no evidence that loss of TrpML (in mutants or RNAi lines) results in developmental defects in astrocytes. Astrocyte modulation of trachea occurs at all larval stages tested, including the 34) The comparisons made between the results in Drosophila and mouse astrocytes need to be clarified in some cases, and the authors should be more careful in discussing the different types of mouse and rat astrocyte calcium signals (see specific comments by reviewer 2)We have clarified this point throughout the manuscript according to reviewer #2’s request.Reviewer #1:2+ regulation in astrocytes. The results are important as similar astrocyte Ca2+ microdomains regulate CNS physiology in mammals, and Drosophila provides an important genetic model for the analysis of astrocyte Ca2+ signaling.The manuscript of Ma and Freeman is clearly written and represents a significant advance in understanding CaThe major issues detailed below should be addressed prior to publication:2+ transients within astrocyte microdomains. The presentation of these results leads the reader to think that TrpML acts at the astrocyte cellular membrane to regulate Ca2+ entry. While this might be true, The TrpML channel has been described as a lysosomal membrane rather than a cellular membrane channel, and it is clearly required for cell migration and phagocytosis via regulation of actomyosin contractility . Is there any evidence that TrpML is even present at the cellular membrane? Have the authors determined localization of the channel in astrocytes? If not, the presentation will lead most readers to think its site of action is the cellular membrane when this has not been demonstrated and is likely not to be the case. A more detailed model for TrpML, taking its localization into account, is needed.1) The authors show that elimination of extracellular and the TrpML channel inhibit CaIn the revised manuscript we have added data to address this directly by assaying localization of TrpML in astrocytes. In new data  we show that TrpML-MYC is localized (1) near the membrane of astrocyte cell bodies, and appears to be sub-cortical based on co-staining with membrane-tethered GFP; and (2) in a punctate pattern throughout astrocytes, which largely overlaps with the endo-lysosomal marker GFP-Lamp1. As such, both possibilities exist—that TrpML is functioning near/at the membrane or at endo-lysosomes. We have included this possibility in our model for proposed TrpML function.2+ transients is to modulate CNS gas exchange through TrpML and ROS signaling." This suggests that TrpML might have a modulatory role in the mature nervous system. However, it seems that all of the authors' results about TrpML could be interpreted as a requirement for the channel in astrocytes during development with a consequent effect on physiology of the tracheal system. Indeed, the authors show that tracheal overgrowth occurs in the channel mutant. I wonder if there is any evidence for a role of TrpML in the mature nervous system. Did the authors attempt a conditional knockdown of TrpML in the mature larval CNS? What about inhibition of TrpML in mature CNS astrocytes using an antagonist that blocks Ca2+ release from lysosomes? Or, activation of TrpML with an agonist in the mature CNS? Or, transient blockade of Ca2+ entry in the mature larval CNS? Is it known whether such manipulations alter tracheal function? It would seem important to determine whether astrocytes actually modulate trachea and/or gas exchange in the mature CNS since this is the implication of the authors' model.2) The authors propose "that one important physiological role for astrocyte microdomain Catrpml mutants or when trpml is depleted from astrocytes . Our best evidence for active regulation of tracheal elements by astrocytes in the mature (3rd instar) larval nervous system is that bath application of tyramine, which activates microdomain transients, drives filopodial retraction in live preparations . We included our new data in the revised manuscript that showed trpml knockdown specifically at the 3rd instar larval stage did result in faster tracheal filopodial growth. Interestingly, such knockdown in trachea also make trachea filopodia grow more rapidly. This suggests that trpml function both in astrocytes and trachea controlling tracheal filopodial dynamics through increasing growth rate. Finally, All of the approaches suggested above to manipulate calcium would likely work in live preparations  for blockade of different aspects of calcium signaling in astrocytes, but it would not spare trachea from influence, and more importantly not be technically feasible for us to do this chronically, allow animals to grow, and then observe how this alters tracheal morphology. Nevertheless, in response to this concern we will temper our conclusions regarding active regulation of gas exchange and leave open alternative possibilities for the phenotypes we observe.Our assumption is that by modulating tracheal growth, astrocytes can thereby regulate gas exchange by increasing or decreasing tracheal filopodial coverage of the CNS, since trachea is the primary route of gas exchange in the brain. Our argument is based on the increased ROS in 2+ permeable, it is also permeable to other cations. It is described in FlyBase as a cation channel involved in regulation of endosomal trafficking, autophagy, lateral inhibition and TOR signaling in hemocytes and perhaps other cell types. Is regulation of astrocytes and tracheal function as simple as modulation of Ca2+ entry or is it more complicated than that? I wonder if Ca2+ microdomains are affected indirectly because of an effect on endosomal trafficking. Some discussion of this is needed.3) Although TrpML is known to be Catrpml mutants, it is the frequency that changes ; (2) tyramine stimulates normal somatic calcium signal in trpml mutant astrocytes. ; (3) astrocyte development even in trpml mutants appears normal, and one might expect developmental defects if endosomal trafficking was significantly perturbed.The reviewer is correct, TrpML may exert its effects through other ions, or by moving multiple ions. It may also be more complicated than the model we propose, and we have discussed these points in the revised manuscript. We do not think there is a general disruption of calcium signaling based on the following evidence: (1) in new data we show that the amplitude of calcium transients in astrocytes in not altered in 1 mutant or trpml1 knockdown flies. Does the CNS appear to be completely normal with absence of TrpML?4) There is no mention of CNS abnormalities in the trpmltrpml do mimic mucolipidosis type IV, in that we can visualize autofluorescent material in the CNS. We also note  that the lethality of trpml mutants is a result of defective astrocyte function—we are able to rescue ~100% of trpml mutants to adulthood by astrocyte-specific expression of TrpML .Overall the CNS appears grossly normal based on size and the morphology of glia, and major tracheal branches (minor overgrowth phenotypes). However, we note that some phenotypes in Reviewer #2:This is very interesting paper that provides convincing data that calcium microdomains in Drosophila astrocytes occur, are triggered by reactive oxygen species (ROS), are enhanced in frequency by the neurotransmitter tyramine via the TyrRII receptor and that they decrease in frequency by ~70% to 80% in trpml loss of function mutants. The authors also show that tracheal growth is modified by these calcium signals in astrocytes. These data provide intriguing counterparts to observations in mammalian astrocytes with respect to calcium signaling. However, there are comparisons that the authors make between the results in Drosophila and mouse astrocytes that need to be clarified in some cases and the authors should be more careful in discussing the different types of mouse and rat astrocyte calcium signals.Thank you for making this point. We have tried to clarify this comparison in the revised manuscript to be accurate and fair. We welcome any additional feedback from the reviewer on clarification if appropriate..Finally, the TRPML1 channel is expressed on lysosomes according to the report they cite from Zhang et al., 2016. The results presented here indicate expression on the plasma membrane based on the block by lanthanum and effects of removing external calcium. This puzzle and apparent discrepancy should be addressed by the authors.In the revised manuscript we have added data to address this directly by assaying localization of TrpML in astrocytes. In new data  we show that TrpML-MYC is localized (1) near the membrane of astrocyte cell bodies, and appears to be sub-cortical based on co-staining with membrane-tethered GFP; and (2) in a punctate pattern throughout astrocytes, which largely overlaps with the endo-lysosomal marker GFP-Lamp1. As such, both possibilities exist—that TrpML is functioning near/at the membrane or at endo-lysosomes. We have included this possibility in our model for proposed TrpML function.2+ events decrease by ~70% to 80% in trpml loss of function mutants, in both intact 1st instar larvae  and acute CNS preparations from 3rd instar larvae  "While knockout of 11 of these TRP channels had no effect, we found that microdomain CaThis is a key finding of this study and this careful analysis shows the strength and power of working with Drosophila as a model system.We thank the reviewer for the enthusiasm and support for the model.2+ transients in Drosophila share many features with those observed in mammals "Astrocyte microdomain Ca2015)."The fly astrocyte microdomains have overlap with the observations more recently in mammalian astrocytes but the authors should be careful to differentiate between the older observations of IP3-mediated calcium release from ER  and the observations of calcium entry dependent microdomains. I don't think the microdomains were observed using the older dye loading techniques. For example, the Nett et al. paper showed almost complete block of the astrocyte calcium signals by the IP3-R antagonist heparin. In my opinion these fly microdomains that are described here are most similar to the microdomains first reported by Srinivasan et al., 2015. Rungta et al., 2016, supports Srinivasan et al. and their data also point to a calcium entry pathway independent of IP3R mediated release and not altered in TRPA1-/- mice. Of course, there appear to be multiple sources of calcium that generate microdomains and Agarwal et al., 2017, point to a mechanism of calcium release from mitochondria. The differences between these various mechanisms generating calcium transients should be clearly stated because different types of calcium signals generated in different regions of astrocytes may potentially have different functions (as would be expected in neurons).This is an excellent point raised by the reviewer. In the revised manuscript we have more carefully discussed the diversity of types of calcium signals, and focused primarily on comparison to Agarwal .3) This observation of ROS sensitivity in this study is consistent with the report by Zhang et al., 2016, describing the ROS sensitivity of TRPMl1 channels. However, there is a significant difference in that Zhang et al. describe the calcium signals as arising from the ROS triggered release of calcium from lysosomes into the cytoplasm not as a plasma membrane channel leading to entry from the extracellular space. The authors should reconcile this important difference and describe which signals are lysosomal release dependent.Based on the comments of several reviewers we have now included localization studies for TrpML (see above). Based on its localization we believe it could be working by lysosomal release, or by release from the sub-cortical region of the plasma membrane. At the moment we cannot resolve which compartment is the crucial one for astrocyte signaling, so we have left open both possibilities in our model.2+ transients using trpml-GCaMP5G  and myr-R-GECO1 (which should be limited to the plasma membrane)  . In many4) "We note that those that exhibited only retraction did so very early in the imaging window, which could indicate that we began our imaging after extension had been initiated."This paragraph is not critical but is confusing. It's clear that 6 min is sufficient to observe a cycle of extension -retraction but what is surprising is that retractions alone occur at a higher rate than extensions alone. Wouldn't these be the same if the probability was similar? Alternatively could imaging the tissue may make retractions much more likely perhaps by generating ROS itself from the tissue illumination in the spinning disk confocal?The reviewer is correct in their assumption. We did indeed observe a statistically equal number of extension and retraction events  throughout our 6-min imaging window. We apologize for our unclear description and have tried to clarify this. The point we were trying to make is that there are very few filopodia that only retract, and usually they appear at the very beginning of imaging.5) "Together, these observations indicate that a large fraction of astrocyte2+ transients are spatiotemporally correlated with the retraction ofmicrodomain Caadjacent tracheal filopodia."This conclusion seems reasonable based on the imaging data.Reviewer #3:The manuscript by Ma and Freeman describes a role for the TRP channel trpml in regulating astrocyte microdomain calcium oscillations in Drosophila. They link this biology to regulation of local tracheal filopodial dynamics and ROS activity, suggesting one role for astrocyte calcium dynamics is to control local gas exchange in Drosophila larvae. The authors' data set is quite interesting and relevant to the field. I have a few suggestions for improvement and some general clarification points as well.1) The authors imply that the trpml channel directly mediates microdomain calcium influx in astrocytes, but I don't think their data separates a role for the channel being the source of calcium influx or secondarily coupled to the process to control the frequency of the events only. As such, the authors need to state this or provide more convincing data that trpml directly mediates calcium microdomain influx.We agree with this point, which was also raised by another reviewer, and have clarified this in our revised manuscript.2) Related to the point above, the authors should document and show the amplitude of calcium transients in trpml knockdowns and mutants as they do for wildtype in Figure 1B. Is trpml required only for the frequency of these events, or also the amplitude. The authors must have this data already in hand. If the amplitude of the residual events in the same, it seems more likely that trpml is controlling when these events occur, rather than being the direct channel for gating the calcium events themselves.trpml mutants are not significantly different .We thank the reviewer for pointing this out. In the revised manuscript we show that the amplitude of calcium transients in control and 3) Similarly, where is trpml localized in Drosophila astrocytes – is it on the plasma membrane at sites of calcium influx? If it is located on lysosomes or internal compartments, I have a hard time understanding how it could be directly mediating calcium influx from the extracellular space.In the revised manuscript we have added data to address this directly by assaying localization of TrpML in astrocytes. In new data  we show that TrpML-MYC is localized (1) near the membrane of astrocyte cell bodies, and appears to be sub-cortical based on co-staining with membrane-tethered GFP; and (2) in a punctate pattern throughout astrocytes, which largely overlaps with the endo-lysosomal marker GFP-Lamp1. As such, both possibilities exist—that TrpML is functioning near/at the membrane or at endo-lysosomes. We have included this possibility in our model for proposed TrpML function.4) It would be helpful to use the trpml RNAi line from Figure 2D and Figure 4B for the retraction/extension assays in Figure 4A. In 4A, only the trpml mutant is used to show defects in tracheal filopodia. If this biology requires trpml in astrocytes, knocking it down only in these cells should trigger this phenotype. Alternatively, this biology could be coming from an independent role of trpml in tracheal cells. This experiment is important, especially given how small some of the reported changes appear to be. Since the authors used trpml RNAi knockdown in astrocytes for all there other manipulations, it raises a question as to why not here in one of the most important experiments.trpml mutants. These data indicate that astrocyte TrpML can indeed regulate tracheal growth. It seems that tracheal TrpML can also influence growth to some extent. The full effect in the trpml mutant is therefore likely due to additivity between astrocytic and tracheal TrpML.This is an excellent point. We have done these experiments and the data are now included in Figure 4—figure supplement 1A. We found that astrocyte knockdown increased extension rate similar to the 5) The most difficult part of the paper for me is Figure 3D. I have a hard time finding compelling images here to support the model. From the bottom astrocyte GCaMP traces (red) and tracheal filopodial length (red) traces, only pair 2 looks compelling – red goes up, green goes down. The rest are bouncing around all over the place and it is hard to see a compelling correlation. Would it be possible to add a supplementary figure showing more of just these traces for all (or a larger representative sample) of the data included in Figure 3E. That figure shows a perfect correlation, but the raw data provided do not look convincing. Seeing more of those raw data would be very helpful for convincing the readers of this correlation.We have included an additional set of traces and video frames.


The objective of the current study was to evaluate the effect of a topical, commercial solution made of essential oils, plant-extracted polyunsaturated fatty acids, and Vitamin E on rabbits suffering from skin problems.Rabbits often experience skin diseases. The beneficial effects of plant extracts and essential oils are well known in other species, but the properties of these natural ingredients have not been evaluated in rabbits Thirty New Zealand rabbits (no sex distinction) were included in this study, with an average weight of 2–3 kg. The rabbits were divided into two groups: The first group was treated with a topical solution made from natural ingredients, and the second was a control group. The rabbits’ hair and skin conditions were evaluated on days 1, 14, 28, and 35 after treatment. Data were analyzed using a Kruskal–Wallis range test.Significant differences were determined in terms of glossy hair variability on days 28 and 35 (p≤0.0001). On days 14-35, hair loss was determined to have reduced (p=0.001), and flaking and odor improved in the treatment group, showing increased scores and significant differences (p=0.0001). By contrast, the control group showed stable overall skin and hair score and an increase in the dryness score.The topical application of essential oils and polyunsaturated fatty acids with Vitamin E was able to improve hair shine and skin hydration and reduce flaking, bad odor, and hair loss, improving the general, and cutaneous aspect of rabbits. Oryctolagus cuniculus) have been part of the daily lives of humans .ad libitum. The rabbits did not receive any pharmacological or systemic treatment previous to the study. The inclusion criteria were as follows: Rabbits with dry skin, loss of gloss, opaque or brittle coat, excessive hair loss, and bad odor. All rabbits underwent individual physical evaluation. The animals were registered in files with their corresponding data.This study was conducted in the UAEM Amecameca University Center, Mexico, from November to December 2018. All animals and procedures were performed in compliance with the practices for the care of experimental animals and recommendations according to the Mexican norm (NOM-062-ZOO-1999). We included 30 rabbits of an indistinct genus, New Zealand breed, with an average weight of 2-3 kg and with cutaneous disorders. The rabbits were provided a diet based on commercial rabbit feed and water Thirty rabbits were randomly divided into two groups. Fifteen were treated with the topical solution containing essential oils , essential fatty acids (hemp seed and neem seed oils), and Vitamin E. The spot-on solution was applied directly to the skin by separating the hair from the interscapular region, at a dosage according to the animal’s weight. Rabbits weighing up to 5 kg were treated with 0.06 ml of solution/kg. The solution was applied once per week for 4 weeks. Meanwhile, the remaining 15 rabbits formed the control group. All 30 rabbits were evaluated on days 1, 14, 28, and 35 using a clinical score for areas such as the face, head, neck, sternum, chest, groin, abdomen, back, sides, hind limbs, posterior end, perianal, perigenital, and tail. A description of the fur and skin conditions of each animal was made using the following criteria and scores: Very dull (1) to very shiny (7) for coat shine, very severe (1) to absent (7) for hair loss and flaking, and very bad (1) to very pleasant (7) for body odor. Skin and hair balance were also scored as very greasy (1), greasy (2), slightly greasy (3), and normal (4) and very dry (1), dry (2), slightly dry (3), and normal (4), respectively. The evaluations of the rabbit’s skin were performed by the same researcher for each rabbit. This protocol was applied according to the manufacturer’s recommendations.The Shapiro–Wilk normality test was applied to determine the distribution of the data. The data did not present a normal distribution. The data were analyzed using the Kruskal–Wallis test to compare the variables between the two groups, with an alpha of 0.05 set for statistical significance.All rabbits concluded the study. Skin evaluations were performed on all rabbits every 2 weeks and again on day 35 (1 week after the end of treatment). All 30 rabbits presented dermatological disorders with no difference in any of the evaluation criteria on day 1. For the coat shine criteria, no significant differences were determined between groups on day 14. However, there were significant differences between days 28 and 35. The scores of the treated group increased at each time point, whereas little variation was observed in the control group scores. The scores for hair loss, flaking, and odor all improved, with significant differences in the treated group at all-time points on days 14, 28, and 35.Conversely, the control group scores have decreased for all criteria, showing significant differences in hair loss, flaking, and odor between groups at all-time points on days 14, 28, and 35. For skin balance, between greasiness and dryness, scores improved in the treated group and stabilized, indicating a normalized skin and hair condition (4). The scores for the control group, however, deteriorated, indicating drier or greasy skin and hair, with significant differences on days 28 and 35. The scores for the treatment group increased for each evaluated criterion, whereas the control group scores decreased .In rabbits, skin diseases are identified as one of the most common reasons for veterinary consultations. Oils and plant extracts have been administered orally for a wide variety of purposes since they were reported to possess antimicrobial activities and even anticoccidial effects in rabbits . To the In vitro, they have been shown to have a high antimicrobial effect against a wide range of dermatological pathogens [et al. [Essential oils have been reported to offer various benefits because of its analgesic, antiseptic, antimicrobial, antifungal, carminative, diuretic, spasmolytic, hyperemic, and stimulant properties . In vitrathogens . Transepathogens . A study [et al.  evaluate [et al. .et al. [Psychrobacter and Pseudomonas) in the skin of bloodhounds was associated with cutaneous odor and that the use of a topical product based on essential oils and essential fatty acids derived from plants, similar to that used in the present study, significantly improved cutaneous odor and modified the structure of the bacterial community in the treatment group after four topical applications once per week.Bensignor and Bordeau , in two et al.  showed tnd week of treatment.Because of its role as a barrier, the skin is often exposed to an oxidative environment, which includes air pollutants, ultraviolet radiation, released oxidants , parasites, and aerobic microorganisms . VitaminOne factor that should be considered when practicing medicine in rabbits is handling, as rabbits can be easily stressed, including bathing . The appOur findings showed that the topical cutaneous administration of essential oils, fatty acids, and Vitamin E applied weekly as a spot-on treatment exhibited clinical improvement of the cutaneous aspect in rabbits. As no adverse reactions were observed, this was identified to be a safe and effective treatment.GSW and AMC: Participated in writing, interpreting, and editing the manuscript. CRN: Substantial contribution to the concept and design of the study; contribution to data collection, interpretation, and contribution to manuscript preparation and critical revision. LMC: Participated in writing, interpreting, and editing the manuscript and critical revision. RHC: Substantial contribution to the statistical data analysis and interpretation and critical revision. LGBG: Contribution to data collection and critical revision. All authors read and approved the final manuscript.