DNA sequencing has unveiled extensive tumor heterogeneity in several different cancer types, with many exhibiting diverse subclonal populations. Identifying and tracing mutations throughout the expansion and progression of a tumor represents a significant challenge. Furthermore, prioritizing the subset of such mutations most likely to contribute to tumor evolution or that could serve as potential therapeutic targets represents an ongoing problem.Here, we describe OncoGEMINI, a new tool designed for exploring the complex patterns and trajectory of somatic and inherited variation observed in heterogeneous tumors biopsied over the course of treatment. This is accomplished by creating a searchable database of variants that includes tumor sampling time points and allows for filtering methods that reflect specific changes in variant allele frequencies over time. Additionally, by incorporating existing annotations and resources that facilitate the interpretation of cancer mutations , OncoGEMINI enables rapid searches for, and potential identification of, mutations that may be driving subclonal evolution.github.com/fakedrtom/oncogemini.By combining relevant genomic annotations alongside specific filtering tools, OncoGEMINI provides powerful and customizable approaches that enable the quick identification of individual tumor variants that meet specified criteria. It can be applied to a wide range of tumor-derived sequence data, but is especially designed for studies with multiple samples, including longitudinal datasets. It is available under an MIT license at The online version contains supplementary material available at 10.1186/s13073-021-00854-6. Cancers arise from a variety of genetic alterations and, over time, often accumulate a substantial mutational load leading to subclonal diversity , 2. As aRecognizing this need, we introduce OncoGEMINI  as a newOncoGEMINI addresses these limitations and enables rapid variant exploration in tumor sequencing studies, especially those featuring longitudinal data across multiple time points from a single patient. To better select or prioritize tumor variants, OncoGEMINI integrates several cancer-relevant genomic annotations that serve as searchable terms to differentiate variants from one another. Additionally, OncoGEMINI provides multiple filtering tools that search for various signatures of tumor heterogeneity, including observable allele frequency changes across multiple samples. These include the bottleneck, loh, truncal, and unique tools. By using these filtering tools in combination with specific cancer annotations, OncoGEMINI can effectively prioritize tumor variants that may drive tumor progression or be potential treatment targets.OncoGEMINI employs the same general functionality as GEMINI and imports variant information, including sample genotypes, from a VCF file into a searchable SQLite database. OncoGEMINI is intended to be used alongside the VCF annotation tool, vcfanno , and theAn important change to the existing GEMINI framework that is necessary for some of OncoGEMINI’s functionality is the inclusion of a tumor-specific sample manifest file used during database loading. OncoGEMINI utilizes a user-generated sample manifest file to describe the distinctive relationships that may exist between tumor samples. This manifest retains the same basic structure of common pedigree files with additional columns describing patient_id, time, and lastly, purity. Given that multiple samples may be derived from the same patient, the patient_id column ensures that sample relatedness is properly cataloged. Since these samples may be obtained through a series of biopsies at different time points, the time column describes the temporal relationship between samples for a given patient. While OncoGEMINI does not compute tumor purity estimates, if such estimates are known, they may be included in the purity column and invoked as a command line parameter to alter allele frequencies accordingly. Altogether, these extra columns give OncoGEMINI greater flexibility and specificity when performing queries or employing different filtering schemes.Diverse databases and annotations have been developed to help interpret identified tumor variants with their specific relevance to various aspects of tumor origin, growth, and treatment –17. Suchhttps://github.com/fakedrtom/crab). While the relevance of each annotation varies, especially with regard to particular cancer types, they provide valuable insights into the pathogenicity and frequency of genetic variants, the propensity for certain genes to harbor mutations in specific cancer types, or known drug susceptibilities and interactions with genes or individual variants. From each of these, we have converted selected information into a series of summarized files that can then be used to annotate a VCF file in preparation for loading into an OncoGEMINI database. Given the inherent flexibility of vcfanno, any combination of these annotations or any additional ones not included here may be selected and added to a VCF. We expect that this will be a valuable resource even outside of use within OncoGEMINI and are eager to add more annotations, references, and data to improve its utility.We have integrated a number of cancer-relevant annotation resources including the Cancer Genome Interpreter (CGI) , ClinicaCombining tumor variants with genome annotations into an OncoGEMINI database enables the identification and prioritization of tumor-relevant genetic variants. Once loaded, the database is populated with the information contained within the selected VCF. Variants from the VCF become rows in the database, and annotations are stored as columns within the OncoGEMINI database that can be useful for isolating variants of interest.Via OncoGEMINI’s query tool, users are able to impose specific search parameters to identify variants meeting custom search criteria. For example, if one wanted to filter all variants in an OncoGEMINI database to only those with the highest validated clinical association as documented in CIViC , the following query would restrict the output to the genomic coordinates, reference and alternate alleles, and gene (if applicable), for all variants that match those in the CIViC database with an evidence level of “A”:oncogemini query -q “select chrom, start, end, ref, alt, gene from variants where civic_evi_level = ‘A’” oncogemini.dbThe query tool can take advantage of the flexibility of OncoGEMINI’s SQL language to build sophisticated search commands. Anticipating common searches pertaining to tumor growth and propagation, we have also developed a suite of tools that identify variants that follow patterns of particular interest in tumor evolution and alleviate the need to repeatedly craft lengthy search queries. Each of the tools described in the following sections is capable of further customization with additional tool-specific parameters and via the annotations included in the database. Together, these options provide the ability to design explicit search queries that can effectively reduce the list of variants, thereby narrowing in on potential variants of interest.As individual tumor cells replicate, they may acquire private mutations that give rise to subclonal structures and heterogeneity within tumors. Over time, owing to drift or the selective pressure of drug and other treatments, the prevalence of certain tumor variants can be reduced or increased. Thus, the ability to determine the frequency at which any given tumor variant exists is both a function of sampling time and efficiency. Variants which increase in frequency over time, especially across different interventions, may represent potential contributors to the proliferation and survivability of a given tumor. We have developed the bottleneck tool to identify variants exhibiting this increased allele frequency over time. This tool relies upon having information regarding the sequential order that tumor samples arose; therefore, this tool is most applicable to longitudinal data, but may be useful with other studies, including metastatic studies, provided temporal information is otherwise appropriately known or ascertained. The bottleneck tool scans each variant in the database for samples that exhibit increasing allele frequencies over the sampling time indicated in the sample manifest file Fig. . To accoLoss of heterozygosity (LOH) is a common genomic alteration in cancer genomes that leads to the loss of one allele. LOH events can result in functional consequences including reduced gene expression, haploinsufficiency, or acting as the second “hit” in a tumor suppressor gene. LOH mutations can indicate causative variants or otherwise serve as biomarkers for cancer identification and potential patient care. One method for identifying LOH relies upon observing genetic loci that appear heterozygous in germline DNA but are not heterozygous in tumor DNA. The loh tool identifies potential LOH variants by observing allele frequency changes that are consistent with heterozygosity present in the normal tissue samples, but absent in all specified tumor samples Fig. .Somatic mutations that arose early in tumor development are likely to be present in all sequenced samples. Such mutations may serve as important therapeutic targets since they are present throughout all samples, rather than localized and private to any specific subclones. For example, this subset of variants may harbor potential neoantigens which represent ideal targets for patient-specific T cell-based cancer immunotherapy . The truWhile one of the key features of analyses with OncoGEMINI is the inclusion of multiple samples from different spatial and temporal biopsies, the unique tool also allows OncoGEMINI to highlight variants that are specific to a single sample or group of samples. This tool requires desired samples to be listed and returns all variants that are present in that samples and absent from all others. Similar to the truncal tool, this is done by specifying a minimum allele frequency (default is 0) that must be exceeded in the listed samples, but not be met in all other samples.OncoGEMINI will evaluate all variants within the database and select those that meet specified tool and annotation filter requirements. Thus, if the VCF used to create the database contained both germline and somatic mutations, both mutation types would be considered by OncoGEMINI commands. To focus solely on somatic mutations, it is recommended that the VCF used for the creation of a OncoGEMINI database be pre-filtered to only include somatic mutations. Somatic mutations may also be labeled with a specific identifier in the “INFO” field of the VCF, thereby enabling facile filtering for such mutations within the database. Alternatively, the set_somatic tool may be employed to annotate variants as somatic within an existing OncoGEMINI database, based on genotypes or user-defined criteria regarding normal and tumor sample sequencing depths and allele frequencies. The set_somatic tool creates an “is_somatic” annotation within the database. OncoGEMINI tools may then take advantage of the --somatic-only parameter to restrict variant evaluations to only those variants that have been marked as somatic in the database by the set_somatic tool.OncoGEMINI’s integration of genetic variants identified across one or more biopsies with annotations relevant to cancer enables a wide range of analyses and variant prioritization strategies. Furthermore, the OncoGEMINI framework can be used to study variation observed in diverse study designs, including studies of a single tumor and normal biopsy and multiple tumor and/or ascite biopsies from the same patient over the course of treatment. Existing tools –26 are wWe first highlight OncoGEMINI’s utility with an example analysis that identifies and filters variants of interest from longitudinal biopsies obtained from three breast cancer patients . We specFor each VCF, variants were annotated in two distinct steps. First, consistent with GEMINI, OncoGEMINI also incorporates standard variant effect predictions made by SnpEff  or VEP , and in To further reduce this number and reveal potential variants of interest , we required certain filters via the included annotations to be met and utilized many of the previously described OncoGEMINI tools. As expected, the count of variants that OncoGEMINI returns depends upon the number of annotation filters and tools that are specified where, generally speaking, the more filters alongside tools that are required, the more the list of returned variants is reduced.Specifying that variants be filtered to only those with a SnpEff impact prediction of “medium” or “high” (impact_severity != ‘LOW’) drastically reduced the number of returned variants to 20,580 and 19,516, respectively. Also, restricting variants to those found in genes with previous implications towards tumorigenesis in certain cancer types via CGI’s Catalog of Cancer Genes (cgi_gene != ‘’) and genes with high CIViC evidence levels or ratings (civic_gene_max_level == ‘A’ or civic_gene_max_level == ‘B’ or civic_gene_max_rating >= 4) refined the number of variants to 140 and 149. Combining these annotation filters with each of the OncoGEMINI bottleneck, loh, and truncal tools resulted in a total of three variants from patient #1 and 2 variants from patient #3 being returned which accounted for three of the nine previously reported variants. To try and recover the remaining 6 previously reported variants, we relaxed the filter requirements by removing the CIViC evidence level and rating annotation filters. This increased the number of resulting variants to 1245 and 1253, respectively, but by once again passing them through the bottleneck, loh, and truncal tools only 17 and 27 variants remained, including eight of the nine previously reported variants. Altogether with just a few commands that returned results in seconds of time, we were able to filter millions of tumor variants to a much more manageable number in each patient, which included nearly all of the previously reported variants ” patient1.dbThis command will return the chromosome, start and end positions, reference and alternate alleles, and the gene for the two truncal variants in patient #1 that meet these criteria. This allows for maximum customization into a single command when devising search specifications.OncoGEMINI also provides a means to evaluate multiple tumor samples as is common for many metastatic and other regional tumor studies. Here, we demonstrate an example spatial analysis that focuses on four individual breast cancer patients, identified as ER1, ER2, ER3, and TN1, that each exhibited extensive metastatic expansions from a primary tumor to other organs while receiving various treatments and interventions over the course of several months or years . MultiplIn total, 48 tumor sample biopsies were obtained from these four patients. Whole-exome sequencing was performed for all samples, including a blood sample from each patient, and patient-specific, joint-called VCFs were generated for all patient biopsies using FreeBayes . FreeBaySPEN (K1838X) mutation in the ER3 patient does appear as truncal, but only if the first metastatic sample, ER3_M1, is omitted from the analysis. As mentioned previously, ER3_M1 was the first biopsy taken from this patient, and it would appear that this SPEN mutation was either not sampled in ER3_M1 or it developed later and persisted in all subsequent biopsies. Similarly, an ITPR1 mutation in TN1 fails to be recovered by the truncal tool, because it appears in only two of the four primary tumor samples that were included, and is also absent from one metastatic sample. It is possible that the lack of evidence for these mutations in these samples can be attributed to variant calling differences employed here versus the original publication. By removing the samples in question using the --samples parameter, these mutations are then recovered by the truncal tool.The truncal tool is ideally suited to recover variants that are present in all tumor samples, but absent from any other samples included. For each patient, the truncal tool (with the --maxNorm parameter set to 0.05) recovered nearly all previously reported mutations that fit this expected mutation profile with a few exceptions Table . The fouIdentifying mutations specific to groups of samples is precisely what the unique tool is designed to do and is therefore appropriate for the identification of subclonal mutations that are specific to certain metastatic samples, but absent from all others (including the primary tumor samples). For example, ER1 has five metastatic samples labeled as ER1_A1, ER1_A2, ER3_A3, ER1_A4, and ER1_A5 within the ER1 OncoGEMINI database, and the following command can use the unique tool to identify variants with a minimum sequencing depth of 10 across all samples and that are present in all indicated metastatic biopsies, but absent from all other included samples: oncogemini unique --minDP 10 --specific ER1_A1,ER1_A2,ER3_A3,ER1_A4,ER1_A5 --columns “chrom,start,end,ref,alt,gene” ER1.dbSPOP (Y87F) variant.This command returns the chromosome, start and end positions, reference and alternate alleles, and the gene name (if the variant is within a gene region) for all variants that are found in the indicated metastatic samples from ER1. By using similarly structured commands (including the --maxOthers parameter set at 0.05) that specify only the metastatic samples for each patient, we expect to find certain alterations in the metastatic samples of ER1 and ER2 and we are able to recover all of these using the unique tool Table . The uniConsistent with the previous longitudinal data analysis, the tools provided by OncoGEMINI are capable of greatly reducing the number of variants, but when used alongside annotation filters, the number of returned variants is further refined Tables  and 3. WBuilding upon the GEMINI framework, OncoGEMINI is ideally suited to the exploration and prioritization of tumor mutations. Cancer research increasingly requires the integration of data from not only multiple biological samples, but also the accumulated genomic information that is found in a variety of distinct databases. We have designed OncoGEMINI to combine information from numerous sources alongside longitudinal tumor sequence data to enable the rapid identification of variants that match specific patterns and criteria. Therefore, OncoGEMINI provides a unique tool that assists in complex cancer analyses.While much of OncoGEMINI’s functionality is applicable to a variety of data from different tumor studies, it is optimized to incorporate and analyze data from multiple tumor samples belonging to the same patient and is thus most powerful when used with longitudinal data that has been collected over various time points and treatments. OncoGEMINI offers a number of specific filtering tools that focus on variant allele frequency changes between samples and each tool can be further adjusted with included options and parameters. Additionally, using the vcfanno tool, user-defined OncoGEMINI databases can be built that enable different genomic annotations to be incorporated and used in identifying relevant tumor mutations, thus empowering powerful cancer-specific queries. We have suggested and prepared specific cancer annotations, but given the inherent flexibility afforded by vcfanno, custom annotations provided or created by users can be included. This allows OncoGEMINI greater versatility and suitability to a wide range of cancer analysis projects. By combining annotation information with OncoGEMINI tools, we demonstrated that individual tumor variants can be identified with simple commands that run quickly in a matter of seconds.APC V1822D) because it was a germline variant that increased in frequency over the time course of the tumor samples (Fig. Even though OncoGEMINI is not clinically deterministic, it can help rapidly sort through tumor variants from sequencing data and identify individual variants fitting specific requirements that may be indicative of variants with potential clinical significance. The default settings of the OncoGEMINI tools aim to pinpoint such likely relevant variants, but being primarily an exploratory tool, OncoGEMINI’s individual tool settings can be adjusted, as is appropriate, to expand query criteria. For example, the last remaining variant from the previously discussed longitudinal example was not recovered in the described analysis (patient #3, https://github.com/fakedrtom/oncogemini.OncoGEMINI is an open-source software package, and it is freely available. Source code and further documentation can be found at Additional file 1. : Supplementary Methods for the running of FreeBayes.


Apophysomyces or Saksenaea complex. In addition, new risk factors are reported from Asia, including post-pulmonary tuberculosis and chronic kidney disease. New emerging species include Rhizopus homothallicus, Thamnostylum lucknowense, Mucor irregularis and Saksenaea erythrospora. Diagnosis of mucormycosis remains challenging. Clinical approach to diagnosis has a low sensitivity and specificity, it helps however in raising suspicion and prompting the initiation of laboratory testing. Histopathology, direct examination and culture remain essential tools, although the molecular methods are improving. The internal transcribed spacer (ITS) region is the most widely sequenced DNA region for fungi and it is recommended as a first-line method for species identification of Mucorales. New molecular platforms are being investigated and new fungal genetic targets are being explored. Molecular-based methods have gained acceptance for confirmation of the infection when applied on tissues. Methods on the detection of Mucorales DNA in blood have shown promising results for earlier and rapid diagnosis and could be used as screening tests in high-risk patients, but have to be validated in clinical studies. More, much needed, rapid methods that do not require invasive procedures, such as serology-based point-of-care, or metabolomics-based breath tests, are being developed and hopefully will be evaluated in the near future.Mucormycosis is an angioinvasive fungal infection, due to fungi of the order Mucorales. Its incidence cannot be measured exactly, since there are few population-based studies, but multiple studies have shown that it is increasing. The prevalence of mucormycosis in India is about 80 times the prevalence in developed countries, being approximately 0.14 cases per 1000 population. Diabetes mellitus is the main underlying disease globally, especially in low and middle-income countries. In developed countries the most common underlying diseases are hematological malignancies and transplantation. Τhe epidemiology of mucormycosis is evolving as new immunomodulating agents are used in the treatment of cancer and autoimmune diseases, and as the modern diagnostic tools lead to the identification of previously uncommon genera/species such as  Lichtheimia corymbifera. Over time, more cases were diagnosed, and the incidence of the disease has increased [Aspergillus, leading to invasive fungal disease in patients with malignancies or transplantation [Apophysomyces [Saksenaea complex [Mucormycosis is an angioinvasive fungal infection due to fungi of the order Mucorales. Depending on the clinical presentation it is classified as rhinocerebral, pulmonary, cutaneous, gastrointestinal, disseminated or other, which includes uncommon rare forms, such as endocarditis, osteomyelitis, peritonitis, renal, etc. The disease was first described in 1876 when Fürbinger described in Germany a patient who died of cancer and in whom the right lung showed a hemorrhagic infarct with fungal hyphae and a few sporangia . His drancreased . Currentantation . The incantation , which iwww.zygomyco.net) and published 230 cases from Europe in 2011 [Most of the data regarding the epidemiology of mucormycosis originate from case reports and case series. The first extensive review of the literature was made by Roden et al. in 2005 . The ana in 2011 .The incidence of mucormycosis is increasing ,15,16,17The order Mucorales comprises 261 species in 55 genera, 38 of which have been associated with human infections . Their tRhizopus arrhizus (formerly Rhizopus oryzae). Other isolated fungi belong to the genera Lichtheimia, Mucor, Rhizomucor, Cunninghamella, Saksenaea, Apophysomyces, Cokeromyces, Actinomucor and Syncephalastrum. In the global review by Jeong et al. Rhizopus spp., Lichtheimia spp. and Mucor spp. accounted for 75% of all cases [Rhizopus spp. were isolated in 34% of cases, Lichtheimia spp. in 19% and Mucor spp. in 19% [Rhizopus spp. were the causative agents in 52%, while the second most common genus was Lichtheimia (29%) [Apophysomyces spp. [Apophysomyces and Saksenaea spp. are almost always responsible for cutaneous mucormycosis in immunocompetent patients, and are mainly found in Asia [Rhizopus homothallicus [Thamnostylum lucknowense [Mucor irregularis [Saksenaea erythrospora [The fungal spores enter the human organism by inhalation, ingestion or direct inoculation. The most common species all over the world is ll cases . The age. in 19% . In the ia (29%) . In the ces spp. . Apophys in Asia ,22. New hallicus , Thamnosknowense , Mucor iegularis  and SaksThe most important conditions that predispose to mucormycosis, according to various studies , includeRisk factors for mucormycosis vary considerably by geographical area. In studies from Europe ,14,27 thSeveral studies have shown that the underlying disease is correlated to the site of infection ,9,14. HeDiabetes mellitus is the leading underlying disease in patients with mucormycosis globally ,9. AccorHematological malignancies (HMs) and hematopoietic stem cell transplantation (HSCT) are the most common underlying diseases in mucormycosis in Europe, USA and Australia . In IndiSolid organ malignancies (SOMs) and solid organ transplantation (SOT), although not as common as HM and HSCT, are also important risk factors for mucormycosis. The reported prevalence varies, ranging from 0.6% to 23% , but theChronic administration of corticosteroids and other immunosuppressive agents is an important risk factor for mucormycosis. They are used in the treatment of malignancies, transplantation and autoimmune diseases. Corticosteroids impair migration, ingestion and phagolysosome fusion in macrophages. In addition they may lead to drug-induced diabetes. Prolonged (>3 weeks) high-dose systemic corticosteroids are risk factors for mucormycosis . HoweverIncreased serum iron is a risk factor for mucormycosis, as iron plays a crucial role in the pathogenesis of this infection . Iron isAspergillus but not Mucorales (voriconazole and echinocandins) [Another factor that may predispose to mucormycosis is the use of antifungal prophylaxis or treatment, which is effective against candins) ,58,59,60candins) . Breakthcandins) ,63,64. Icandins) . A recencandins) .Other diseases associated with mucormycosis are intravenous drug use, AIDS, renal failure, liver diseases, chronic alcoholism, malnutrition and low birth weight infants . MucormyPatients with a history of IVDU who develop mucormycosis, most often present with isolated cerebral infection . ConversApophysomyces elegans were reported after the tsunami in Sri-Lanka [Syncephalastrum racemosum was isolated from respiratory samples following hurricane Katrina [In a significant proportion of cases mucormycosis develops in immunocompetent patients. In the two large reviews by Roden et al. and Jeong et al., 19% and 18% of patients respectively, had no underlying disease. In such cases trauma or burns is the usual predisposing factor, resulting in cutaneous disease. The trauma can be minor  or major, including motor vehicle accident, natural disasters and surgery . In caseri-Lanka  and Sync Katrina .Mucor irregularis (formerly Rhizomucor variabilis), leading to primary cutaneous mucormycosis, which progresses very slowly, in contrast to the classical presentation of mucormycosis [Mucormycosis has also been reported following combat-related injury . Anotherrmycosis ,74.(a)The use of non-sterile products is the most commonly suspected cause of infection . Bandage(b)Various procedures and medical devices, such as catheters, insulin pumps and finger sticks , and ins(c)Environmental factors may also be a source of infection. Molds may be found in the air, dust, water or any surfaces in the hospital. Construction works increase the risk of invasive fungal infections. Outbreaks have been linked to defective ventilation systems and water leakage.There have been multiple reports of healthcare-associated mucormycosis, either as isolated cases or as outbreaks. In a publication from India, 75 cases of mucormycosis were reported during an eighteen-month period, of which 9% were nosocomial . HealthcIn the analysis of 169 cases by Rammaert et al. 22% of the patients had diabetes mellitus, 24% had a solid organ transplant, 12% had a malignancy, 37% had a history of prolonged steroid therapy and 5% had other immunodepression factors .The clinical presentation varies, depending on the source of infection. Infections due to bandages, adhesives or contaminated wound dressings are mostly cutaneous. Percutaneous exposure in immunocompromised patients has led to disseminated disease. Inhalation leads to pulmonary and rhino-cerebral infection, while ingestion of tablets or food, and the use of tongue depressors, are responsible for gastrointestinal mucormycosis. Dialysis catheters have been linked to peritonitis .Early diagnosis of mucormycosis is of utmost importance, since it may improve outcome. Studies have shown that it increases survival , and it Aspergillus, Fusarium, Pseudallescheria and Scedosporium species [Pseudomonas aeruginosa causing ecthyma gangrenosum may also produce a similar picture. Although the clinical signs and symptoms are non-specific, some of them may have a potentially high predictive value. In a susceptible host, a history of prophylaxis with voriconazole or the emergence of breakthrough fungal infection while receiving agents active against Aspergillus but not Mucorales, suggests the presence of mucormycosis [The clinical approach to diagnosis has low sensitivity and specificity. The hallmark of mucormycosis is tissue necrosis resulting from angioinvasion and thrombosis, the absence, however, of a necrotic eschar does not preclude the diagnosis. Necrotic cutaneous lesions in immunocompromised patients may be due to mucormycosis, but the differential diagnosis includes other pathogens, such as  species . Pseudomrmycosis ,59,60,85A patient with diabetes and sinusitis should be thoroughly examined for possible mucormycosis. Corzo-Leon et al.  proposedIn clinical practice, laboratory diagnosis of mucormycosis includes histopathology, direct examination of wet mounts and culture.Mucorales genera produce typically non-pigmented, wide (5–20 μm), thin-walled, ribbon-like hyphae with no or few septations (pauciseptate) and right-angle branching [Aspergillus species or other hyaline molds, which are typically 3–5 μm wide, septate and form acute-angle branching. Routine hematoxylin and eosin (H&E) stains may show only the cell wall with no structures inside, or occasionally, very degenerate hyphae. Stains that can help highlight the fungal wall include Grocott methenamine-silver (GMS) and periodic acid-Schiff PAS stains, although PAS gives a better visualization of the surrounding tissue compared to GMS [A definitive diagnosis is based on the demonstration of fungal hyphae typical for mucormycetes in biopsies of affected tissues, or bronchoalveolar lavage (BAL) in patients with pulmonary mucormycosis. Histopathology is a very important diagnostic tool since it distinguishes the presence of the fungus as a pathogen in the specimen from a culture contaminant and is indispensable to define whether there is blood vessel invasion . It can ranching , in contd to GMS .R. arrhizus  can aid in the diagnosis when cultures are negative and has been proven useful for differentiating aspergillosis from mucormycosis  [For a rapid presumptive diagnosis of mucormycosis direct microscopy of KOH wet mounts can be used. It can be applied to all materials sent to the clinical laboratory, preferably using fluorescent brighteners such as Blankophor and Calcofluor White together with KOH, which enhance the visualization of the characteristic fungal hyphae, in this case requiring a fluorescent microscope . Direct mycosis) ,96,97 anmycosis) .Mucorales are thermotolerant and are able to grow rapidly at temperatures of 37 °C. They grow on virtually any carbohydrate substrate, colonies appearing usually within 24–48 h and identification is based on colonial and microscopic morphology and growth temperature [Mucorales is a promising method for those laboratories that are accordingly equipped [Culture of specimens is essential for the diagnosis of mucormycosis since it allows identification to the genus and species level, and eventually antifungal susceptibility testing. Most medically important perature . Matrix equipped , but theequipped .A positive culture from a sterile site confirms the diagnosis, while a positive culture from a non-sterile site could be due to a contaminant and must be combined with clinical and radiological data to establish a probable diagnosis . Hence, Mucorales, [The major concern about culture, however, is its low sensitivity, as it can be falsely negative in up to 50% of mucormycosis cases ,99. Thiscorales, ,99 or evMolecular methods have evolved as a useful tool to confirm the infection and identify the strains involved. There are methods therefore, developed on the one hand to accurately identify to the species level strains that already have grown in cultures, and on the other hand methods to detect mucormycetes in tissues.Mucorales [The ITS region is the most widely sequenced DNA region for fungi in general. ITS sequencing is a reliable method and has typically been the most useful for molecular systematics at the species level, and even within species and it is recommended as a first-line method for species identification of ucorales .rnl [For the detection in tissues, several methods have been developed, including PCR based techniques such as nested PCR, real-time PCR (qPCR), nested PCR combined with RFLP , PCR cournl , the cytrnl  or the Mrnl .R. oryzae, R. microsporus and Mucor spp. [Mucorales specific real-time qPCR assays, targeting two different regions of the multicopy ribosomal operon-18S and 28S, that are able to detect DNA from a broad range of clinically relevant Mucorales species.Kasai et al. developed a specific qPCR targeting the 28S rDNA , Bernal-cor spp.  and Spricor spp.  develope®, PathoNostics, Maastricht, The Netherlands) appeared to be a fast diagnostic test with an overall sensitivity of 75% tested on serial blood samples from patients with culture-positive invasive mucormycosis, often preceding the final diagnosis by several days to weeks [Mucor-Rhizopus spp., Lichtheimia spp., Rhizomucor spp. and Cunninghamella spp. [The in-house tests that have been developed so far use different probes and primers, therefore the lack of standardization makes their implementation in the clinical laboratory difficult. Furthermore, they are cumbersome, costly and need expertise. A new pan-Mucorales real-time (qRCR) commercial kit . Despite the angioinvasive nature of the infection, blood cultures remain negative. In fact, only a handful of cases with a positive blood culture have been described so far. ,116,117.Mucorales PCR has been shown to be a highly reliable tool for the diagnosis of invasive mucormycosis in immunocompromised patients. Millon et al. in a study using three qPCR (for Absidia corymbifera (Lichtheimia), Mucor/Rhizopus and Rhizomucor 18S ribosomal RNA genes) on sera of mucormycosis patients showed that this method was highly sensitive, had a low detection level and could detect infection 3–68 days earlier than the conventional methods [Mucorales DNA (cmDNA) allowed earlier diagnosis of invasive wound mucormycosis (IWM) in severely ill burn patients and earlier treatment initiation. [Mucorales in blood and tissue samples from hematological patients with invasive mucormycosis, using a fast (4 h turnaround time) probe-based Mucorales-specific real-time PCR assay (Muc18S) [Serum  methods . These f methods . This me methods . Legrandtiation. . Springe(Muc18S) .Methods of qPCR in blood are rapid (about 3 h turnaround time). Although their sensitivity is lower than in tissues, they are highly specific. Furthermore, they are able to diagnose mucormycosis in immunocompromised patients earlier than the conventional mycological methods (8 days) or imaging  for Aspergillus. However, GM testing in blood and BAL in hematology patients or patients with compatible chest CT imaging results may be used to decrease the likelihood of mucormycosis [d-glucan test does not detect antigen components of the Mucorales.There are no commercially available antigen markers to detect rmycosis . FurtherMucor spp. [In search of new targets for serologic tests, Burnha-Maurish et al. evaluated a monoclonal antibody (2DA6) in a sandwich ELISA and found it to be highly reactive with purified fucomannan of the cor spp. .Mucorales cell wall fucomannan in clinical samples and demonstrated that LFIA was more convenient to use than ELISA and had the potential to be used as a point-of-care test on BAL, serum, urine and tissue. The test was able to rapidly and accurately identify R. delemar, L. corymbifera, M. circinelloides and C. bertholletiae, early after infection (within 3–4 days of infection) in murine models [They subsequently constructed a lateral flow immunoassay (LFIA) for detection of e models .Mucorales species that most commonly cause human IM—Rhizopus arrhizus var. arrhizus, R. arrhizus var. delemar and R. microsporus, by thermal desorption gas chromatography/tandem mass spectrometry (GC–MS) [Aspergillus fumigatus were used as controls. They also analyzed breath volatile metabolites from five patients eventually diagnosed with proven IM caused by R. microsporus, sampled prospectively. The findings showed that the three Mucorales species had distinct breath profiles of the volatile metabolite sesquiterpene, which could be used to identify these infections in vivo. These profiles distinguished the infections from each other and from aspergillosis, therefore this method has the potential to diagnose fungal infection non-invasively, and perhaps monitor response to therapy. Additionally, it could be used in a high risk population such as patients with neutropenia due to treatment for leukemia or those undergoing hematopoietic cell transplantation, to screen for mold infections additionally to Aspergillus galactomannan. This method appears to be very appealing and promising, but needs further evaluation.In an experimental murine model of invasive mucormycosis (IM), Koshy et al. examined breath volatile metabolite profiles, using the three  (GC–MS) . Mice inRhizopus homothallicus, Thamnostylum lucknowense, Mucor irregularis and Saksenaea erythrospora.Epidemiology of mucormycosis is evolving. In light of new evidence, diabetes mellitus remains the main underlying disease globally. As diabetes is rising, especially in low and middle-income countries, a rise in mucormycosis cases is expected and this should be alarming. In developed countries, the most common underlying diseases are hematological malignancies, with acute myeloid leukemia the most frequent. IVDU is the main risk factor for isolated cerebral mucormycosis, and post-pulmonary tuberculosis and chronic kidney disease are emerging risk factors in developing countries. New emerging species include Diagnosis of mucormycosis remains challenging. Histopathology, direct examination and culture remain essential tools, although the molecular methods are improving. New molecular platforms are being investigated and new fungal genetic targets are being explored. Molecular-based methods have gained acceptance for confirmation of the infection when applied to tissues. Methods on detection of Mucorales DNA in blood have shown promising results for earlier and rapid diagnosis and could be used as screening tests in high-risk patients, but have to be validated in clinical studies. More, much needed, rapid methods that do not require invasive procedures, such as serology-based point-of-care, or metabolomics-based breath tests, are being developed and hopefully will be evaluated in the near future.


The current study investigated telocytes (TCs) in the intestinal bulb of Grass carp using light microscopy (LM), Transmission electron microscopy (TEM), scanning electron microscopy, and immunohistochemistry (IHC). By LM, TCs were distinguished by the typical morphological features that had a cell body and telopodes using HE, toluidine blue, methylene blue, Marsland silver stain, Grimelius’s silver nitrate, Giemsa, PAS, combined AB pH2,5/PAS, Crossmon’s and Mallory triple trichrome, Van Gieson stains, Verhoeff’s stain, Sudan black, osmic acid, performic acid with methylene blue and bromophenol blue. TCs were identified under the epithelium as an individual cell or formed a TCs sheath. They detected in the lamina propria, between muscle fibers, around the myenteric plexus and fibrous tissue. TCs acquired immunological features of endocrine cells that exhibited high affinity for silver stain, performic acid with methylene blue, Marsland stain, and immunohistochemical staining using chromogranin A. Sub epithelial TCs were closely related to the endocrine cells. TCs and their secretory activities were recognized using acridine orange. TCs were identified by IHC using CD34, CD117, S100-protein, desmin. TCs formed a3D network that established contact with macrophage, mast cells, dendritic cells, lymphocytes, smooth muscle fibers, fibroblast, Schwann cells and nerve fibers. In conclusion, the localization of TCs in relation to different types of immune cells indicated their potential role in the maintenance of intestinal immunity. Telocytes (TCs) are the cell population that forms a network through a labyrinthine system formed by telopodes. They are long and slender prolongations reached up to hundreds of microns. Telopodes are identified by their segments; the thin segment, podomer and the thick segment, the podoms. TCs established contact to other resident cells or to wandering cells via their telopodes or via the cell body. Telopodes network provide—long-distance cell–cell signaling (intercellular communication)2 and proteomics analysis3 are investigated for TCs. The functional contributions of TCS are supposed to be related to angiogenesis4, and development, maintenance of homeostatic balance4, immunosurveillance5, tissue regeneration and repair through providing adequate microenvironment for stem cell niche and promoting their differentiation7.Paracrine signaling is critical for TCS function. TCs produce several secretory molecules and factors contributing to the functional significance.Gene analysis11, avian13, reptiles14, Amphibians15 and aquatic species17 and parasitic worm18. They are located in the trachea and lungs19, heart20 and the blood vessels21, kidney, ureter, urinary bladder22, tongue23, oesophagus, stomach and small and large intestines24, liver , pancreas14, testis25, prostate26, efferent ductules27 ovaries, oviducts, uterus, vagina, mammary glands and placenta26, spleen28.TCs are identified in a wide variety of organs from diverse species including mammals29. TCs express other markers such as desmin, vimentin, SMA, tubulin, VEGF, and others27. The current study investigates the distribution, morphology, immunohistochemical characterization of TCs in the intestinal bulb of Grass carp.TCs immunoprofile is recognized in several species. CD34/PDGFRα is specific markers for TCsCtenopharyngodon idella)  with the average mean standard length was 36.40 ± 3.01 cm, and the mean standard body mass was 506.40 ± 9.60 g.The present work was carried out on eight specimens of adult grass carp or white amur (All fish were anaesthetized using benzocaine (4 mg/L) and decapitated. The gastrointestinal tract was carefully excised after abdominal incision. The intestinal bulb, an anterior intestinal dilatation, was obtained and incised to wash and remove the mucous coat of the mucosal surface.Four fish were taken for light microscopic examination, two fish for Scanning electron micrograph, and two fish transmission electron microscopic examination.30 as the following: The fixed samples thoroughly washed in 70% ethanol (3 × 24 h) to get rid of the fixative before subsequent processing. The samples were dehydrated in ascending grades of alcohol , cleared in methyl benzoate, and then embedded in paraffin wax for three hrs. Serial transverse and longitudinal sections of 5–7 µm were prepared by using a Richert Leica RM 2125 Microtome, Germany. For general histological examination, paraffin representative sections were stained by Hematoxylin and Eosin satin31.Samples from the intestinal bulb were fixed in Bouin’s solution Table . Samples32: trichrome according to Crossmon’s33 and Mallory triple trichrome stain34, PAS35, Combined Alcian Blue pH 2.5 Periodic acid Schiff (AB pH 2.5 /PAS) technique36, Wiegert37, Verhoeff’s counter stain by Van Gieson38, Long ziehl neelsen, Giemsa stain, Grimelius’s silver nitrate39 and Marsland. Gless and Erikson method40.The following histochemical stain used was cited in32All staining methods were cited byLeitz Dialux 20 Microscope provided with a Canon digital camera (Canon Power shot A95) used to examine the stained sections and capture the images.43] .After fixation of a small piece about 0.0.5 × 0.5 × 0.5 cm sample in 10% Neutral buffer formalin, they placed in osmium tetroxide for demonstration of fat by the method that allows paraffin embedded in the tissue ). Interstitial cells of Cajal of connective tissue septa (ICC-SEP) occur in the connective tissue septa. Intramuscular interstitial cells of Cajal (ICC-IM) are interstitial cells of Cajal of the circular muscle (ICC-CM) and interstitial cells of Cajal of the longitudinal muscle (ICC-LM). Interstitial cells of Cajal of the deep muscular plexus (ICC-DMP) locate in the deep muscular plexus. Interstitial cells of Cajal of the submucosa (ICC-SM) and interstitial cells of Cajal of the submucosal plexus (ICC-SMP) occur in the submucosa and submucosal plexus, respectively. ICC of the subserosa locates in the subserosa77.In the current study, TCs were located under the epithelium as an individual cell or formed TCs in the lamina propria, between muscle fibers, between muscle bundles, around the myenteric plexus and in the fibrous tissue. The distribution of TCs in the intestinal bulb of the Grass carp was similar to other tubular organs in mammals. In the bovine uterine tubes, TCs are located under the epithelium forming a subepithelial sheath as well as three other sheath; an outer perimuscular, inner perimuscular and intramuscular sheath. TCs are also distributed in the lamina propria, between the SMF and in the serosa27. CD34 is a transmembrane phosphoglycoprotein that commonly identified in hematopoietic stem cells and is detected in other progenitor cells such as interstitial cell progenitors, muscle satellite cells, epithelial progenitors, corneal keratocytes, and vascular endothelial progenitors78.CD-34 is frequently used as a marker for TCs in mammalian and fish species79. Chromogranin A involved in the initiation and regulation of biogenesis of secretory granules and sequestration of hormones in neuroendocrine cells80.TCs expressed chromogranin A that is a highly acidic secretory glycoprotein and is expressed by most neuroendocrine cells. Chromogranin A is closely associated and packed with neurotransmitter peptides and monoamines in secretory granules or synaptic vesicles81. Moreover, in vitro studies support TCs role in the regulation of immune response via the paracrine pathway. Uterine TCs have a major role in the activation of peritoneal macrophages. Mouse peritoneal macrophages acquired abundant pseudopodia and cytoplasmic secretory granules when co-cultured with TCs. Macrophages increase the section of several cytokines including TNF-α, IL1-R1, and IL-10, but not TGF-β1, IL-1β, IL-23α, and IL-18, suggesting potential role in immunoregulatory and immunosurveillance mechanism82. TCs implicated to triggers tissue changes in dendritic-linked immune disease83.TCs established direct contact with different types of immune cells in the intestinal blub of the Grass carp. They were connected to mast cells, dendritic cells, and lymphocytes indicating a contribution in the immune response of the intestinal bulb. TCs–immune cells contact occurs as uniform or multicontact synapses that resemble juxtacrine cell-to-cell signaling sites or chemical synapses. Different types of immune cells are mentioned in contact with TCs, such as lymphocytes, plasma cells, eosinophils, basophils, macrophages, and mast cells85. CD117 interacts with the ligand stem cell factor to stimulate cellular differentiation, proliferation, chemotaxis, cell adhesion, and apoptosis86. Telocytes are commonly express c-kit. c-kit is a member of the transmembrane kinase receptor. c-kit plays a critical role in TCs functions87. C-kit tyrosine kinase is involved in the transduction of intracellular signaling88. Telocytes are categorized depending on c-kit immunoaffinity. Some TCs were CD34-positive/c-kit-negative in the human bladder89.C-kit or CD117 is strong markers of stem cells90. S-100 proteins are a member of Ca+2-binding proteins. They function as Ca+2sensor proteins and promote Ca+2signal transduction. S-100 proteins have intracellular and extracellular functions. They combine into specific proteins in cells, e.g., desmin, tubulin, and others91. S-100 proteins bind to different types of cell receptors93. S-100 proteins are implicated in the regulation of cell dynamics and shape through the interaction of cytoskeletal proteins including microfilaments, microtubules, and intermediate filaments95. They interact with proteins that act as protein Players that regulate the secretory pathway93. Thus, S-100 proteins may participate in the regulation of the TC morphology and the secretory functions of TCS. TCS express desmin in the intestinal bulb of the Grass carp. Desmin is a member of the intermediate filament. As the other cytoskeletal elements, desmin has a supportive function to the cells96. TCs express desmin in the uterine tube of bovine8.TCs had high affinity for immunostaining of cytoskeletal components, particularly the intermediate filaments desmin, and S-100 protein. The term S-10 protein is used based on the physical properties of the protein that is soluble in a 100% saturated solution of ammonium sulfate at neutral pHThe current study presented the relation of TCs with different types of immune cells in the intestinal bulb of the Grass carp. We suggest that TCs may have an essential role to maintain intestinal immunity. TCs share histochemical and immunological features of endocrine cells. This may reveal that TCS had endocrine properties.Further studies should investigate TCs proteins profile linked to endocrine function.Supplementary information.


There are multiple etiologies and phenotypes of age-related hearing loss or presbyacusis. In this review we summarize findings from animal and human studies of presbyacusis, including those that provide the theoretical framework for distinct metabolic, sensory, and neural presbyacusis phenotypes. A key finding in quiet-aged animals is a decline in the endocochlear potential (EP) that results in elevated pure-tone thresholds across frequencies with greater losses at higher frequencies. In contrast, sensory presbyacusis appears to derive, in part, from acute and cumulative effects on hair cells of a lifetime of environmental exposures , which often result in pronounced high frequency hearing loss. These patterns of hearing loss in animals are recognizable in the human audiogram and can be classified into metabolic and sensory presbyacusis phenotypes, as well as a mixed metabolic+sensory phenotype. However, the audiogram does not fully characterize age-related changes in auditory function. Along with the effects of peripheral auditory system declines on the auditory nerve, primary degeneration in the spiral ganglion also appears to contribute to central auditory system aging. These inner ear alterations often correlate with structural and functional changes throughout the central nervous system and may explain suprathreshold speech communication difficulties in older adults with hearing loss. Throughout this review we highlight potential methods and research directions, with the goal of advancing our understanding, prevention, diagnosis, and treatment of presbyacusis. The Nat+) recycling and ion homeostasis , supporting a single-gene mechanism for some cases of progressive hearing loss. It is likely that both common variants with small effect size and rare variants with moderate effects are involved in presbyacusis in the population. Furthermore, it is likely that many of the same genes can be involved in both presbyacusis and childhood deafness, making the latter group good candidates for involvement in presbyacusis  as well as in a milder phenotype - postlingual onset progressive hearing loss  for a disease if it lies within the sequenced regions. Exome sequence focusses upon the 2% of the genome that covers the coding regions (exons) so it will often miss regulatory variants that affect mRNA expression levels of genes or the distribution of the resulting proteins in specific cell types, because regulatory regions will very often be in the 98% of the genome outside of the sequence available in exomes. The difficulty with using sequence data is not the detection of variants but assessing the variants for their potential to contribute to presbyacusis. For example, a recent exome sequence analysis of a small set of people with presbyacusis revealed very large numbers of predicted pathogenic variants in genes known to underlie Mendelian deafness ; this fiAs genomic analysis becomes less expensive and cohorts of people with well-characterized auditory phenotypes are assembled, both GWAS and sequence analysis will be useful to build our understanding of the genetic architecture of presbyacusis, providing the information and targets that are needed for development of treatments. We anticipate that these genetic findings will be complemented by human temporal bone studies and validated in mice carrying candidate human variants where the mechanisms and consequences of these genetic variants can be observed.2.3The availability and study of large numbers of different hearing-impaired mouse mutants has given us insight into the wide range of pathological processes underlying progressive hearing loss with age e.g., . These c33.1Otos gene show elevated thresholds across frequencies and degeneration of lateral wall fibrocytes with no apparent pathology in the stria vascularis or neurosensory epithelium, suggesting a metabolic disorder  channels in the stereocilia and a fixed resistance across the basolateral surface of the OHC  representing the cation  on the EP during sound stimulation effectively results in the CM present within scala media . Thus, tincrease under these conditions. In metabolic presbyacusis, the strial battery is weakened by fewer active and passive ion channels in the stria vascularis, effectively yielding an increased source resistance and a decreased EP; however, because the voltage fluctuations under a varying load are greater with a higher source resistance, the CM magnitude can 4.3Another example of how the battery model can lend clarity relates to the audiograms of older adults with a history of noise exposure, whose audiometric thresholds at frequencies below ∼2.0 kHz are often near normal in the presence of large losses of basal OHCs , which is needed to depolarize IHCs, to the point where the low-SR fibers are no longer activated. Auditory nerve fibers in mammals can be classified by their SR . High-SRIt has been shown in some animal species that low-level noise trauma can reduce the numbers of low-SR fibers with minimal threshold shift . Underst6.2About 95% of auditory nerve fibers in a variety of mammals, including human, are myelinated by Schwann cells , althoug6.3Auditory nerve function can be characterized in humans using the N1 of the CAP or the equivalent Wave I of the auditory brainstem response ABR; . These eStudying neural presbyacusis in humans can be difficult because peripheral auditory system declines may affect auditory nerve function. These effects can be addressed by comparing response amplitudes from younger and older adults with relatively normal pure-tone thresholds. Acoustic information is encoded by differences in the spike timing and rates across auditory nerve fibers. The redundancy of afferent information coded in auditory nerve fibers means that a loss of as much as 80% of auditory nerve synapses can occur without affecting detection of pure-tone signals . However6.4Age-related inner ear declines are thought to initiate changes throughout the central auditory system and perhaps across other neural systems. Evidence for these widespread effects in humans includes associations between hearing thresholds and brain morphology. A close examination of these findings and their putative mechanisms is important because some suggest that these findings can explain age-related changes in speech recognition, as well as associations between elevated pure-tone thresholds and lower general cognitive function (Associations between pure-tone thresholds and auditory cortex gray matter volume have been reported in multiple cross-sectional studies [; but notIt is tempting to interpret the hearing loss and brain morphology findings as reflecting the consequences of sensory presbyacusis. However, it is possible that inner ear and auditory cortex morphology are independently affected by age-correlated factors. This perspective is supported by evidence that the strength of associations between hearing thresholds and primary auditory cortical thickness was similar to the strength of associations between hearing thresholds and primary motor and somatosensory cortical thickness . MoreoveAn additional interesting finding from the The significance of the gross brain structure and hearing associations to age-related differences in speech communication and cognition also remains unclear. Expanded lateral ventricles have been related to lower general cognitive function in people whose post-mortem brains exhibited lacunar infarcts, neuritic plaques, and/or cortical alpha-synuclein pathology . Relativ7The importance of identifying the different mechanisms and consequences of presbyacusis is underscored by diminished quality of life for older adults with hearing loss and their family members. Here, we have described different presbyacusis phenotypes that have distinct patterns of audiometric expression with specific pathophysiology identified in animals. Differentiating these subtypes of age-related hearing loss may lead to development of tailored forms of intervention to optimize healthy aging, perhaps through targeting genetic mechanisms contributing to declines and protection from declines.There are multiple mechanisms for hearing loss due to genetic mutations that are expressed with aging and environmental exposures, including noise trauma, that can affect different functions of the inner ear. In humans, these factors are likely additive and perhaps interacting to create a complex audiologic phenotype. This premise is supported by parallel findings in animals and human data demonstrating age-related hearing loss that occurs with a modestly sloping audiogram with loss across frequencies can be explained by age-related pathology of non-sensory cells in the cochlear lateral wall with attendant changes in K+ recycling mechanisms and reductions in the EP. These changes deprive the cochlear amplifier of its essential power supply and promote a loss or inactivity of low-SR auditory nerve fibers. In addition, hearing loss is often related to noise exposure and OHC loss, particularly in males with elevated high frequency thresholds. A similar audiometric pattern can be seen in animals with hair cell loss after exposure to noise or ototoxic drugs. Variation in the sensory phenotype may be dependent on genetic interactions that either increase susceptibility or provide protection to/from noise and may be associated with effects throughout the auditory system. It is also possible that genetic mutations contribute to the loss of hair cells and produce the sensory presbyacusis phenotype in humans.Audiometric measures of pure-tone thresholds do not fully reflect the functional consequences of age-related pathology of the inner ear. Primary neural degeneration, as well as secondary neural declines due to EP loss and/or synaptopathy and hair cell loss, contributes to declines in auditory nerve function. These declines, which appear to include glial cell pathology and demyelination, likely contribute to the communication difficulties that older adults experience. New approaches for measuring and analyzing the CAP show promise in characterizing the function of the auditory nerve, including perhaps the ability to differentiate the health of low-SR fibers that may be particularly vulnerable to effects of aging.The primary remedial options currently available for presbyacusis are devices such as hearing aids and cochlear implants, which may provide some benefits but do not restore normal function. There is a large unmet need for biologic approaches to slow or reverse progressive hearing loss, or delay its onset. Developing effective treatments will require knowledge of the molecular and cellular basis of presbyacusis, as well as functional metrics that are sensitive to presbyacusis phenotypes to diagnose and track treatment outcomes.Mark A. Eckert: Conceptualization; Judy R. Dubno, Mark A. Eckert, Kelly C. Harris, Hainan Lang, Morag A. Lewis, Richard A. Schmiedt, Bradley A. Schulte, Karen P. Steel, Kenneth I. Vaden, Jr.: Writing - Original Draft; Judy R. Dubno, Mark A. Eckert, Kelly C. Harris, Hainan Lang, Morag A. Lewis, Richard A. Schmiedt, Bradley A. Schulte, Karen P. Steel, Kenneth I. Vaden, Jr.: Writing - Review & EditingNone.


Significance: The retina is critical for vision, and several diseases may alter its biomechanical properties. However, assessing the biomechanical properties of the retina nondestructively is a challenge due to its fragile nature and location within the eye globe. Advancements in Brillouin spectroscopy have provided the means for nondestructive investigations of retina biomechanical properties.Aim: We assessed the biomechanical properties of mouse retinas using Brillouin microscopy noninvasively and showed the potential of Brillouin microscopy to differentiate the type and layers of retinas based on stiffness.Approach: We used Brillouin microscopy to quantify stiffness of fresh and paraformaldehyde (PFA)-fixed retinas. As further proof-of-concept, we demonstrated a change in the stiffness of a retina with Results: We found that the retina layers with higher cell body density had higher Brillouin modulus compared to less cell-dense layers. We have also demonstrated that PFA-fixed retina samples were stiffer compared with fresh samples. Further, NMDA-induced neurotoxicity leads to retinal ganglion cell (RGC) death and reactive gliosis, increasing the stiffness of the RGC layer.Conclusion: Brillouin microscopy can be used to characterize the stiffness distribution of the layers of the retina and can be used to differentiate tissue at different conditions based on biomechanical properties. The retina is a part of the central nervous system and is composed of a single glial population as well as six neuronal cell types. Among these cells, retinal ganglion cells (RGCs) convey visual information, which is transmitted through the optic nerve to the visual cortex. Retinal diseases, such as glaucoma and retinal ischemia, are significant causes of blindness worldwide. In these conditions, RGCs die and vision is permanently lost since they cannot be replaced.The retina constantly experiences static and dynamic forces,Cell replacement therapies for retinal diseases have been developed and are underway in various stages of clinical trials.Compared with the choroid and sclera, the retina is softer and much more fragile, so assessing its biomechanical properties is a challenge. Early studies on retinal elasticity utilized conventional mechanical testing,Brillouin microscopy is a noninvasive, high-resolution, optical elastography technique that has shown promising applications in determining tissue elasticity.This study demonstrates the layer-by-layer biomechanical characterization of retinal tissue stiffness. Brillouin microscopy was used to measure the change in the Brillouin modulus between fresh and paraformaldehyde (PFA)-fixed retinal explants. We also performed biomechanical characterization of a mouse retina with 21.1All procedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. Adult C57/BL6J mice were euthanized by μL of 100 mM NMDA. The mouse was euthanized 48 h postinjection, and the eye-globes were enucleated. Then, the retinas were dissected and mounted fresh on a glass slide as described earlier.Four-week-old C57/BL6J mice were injected intravitreally with 2 2.2Imaging was performed with a home-built Brillouin microscopy system based on a two-stage virtually imaged phased array (VIPA) spectrometer.3To demonstrate a proof-of-concept for detecting retinal damage with Brillouin microscopy, we characterized the biomechanical properties of NMDA-induced damage to the mouse retina. The dissected retinas were kept on a glass slide where the top layer was the NFL/RGC, and the photoreceptor side was at the bottom. 4,In this work, we demonstrated all-optical noninvasive elastography of fresh murine retinal tissue using Brillouin microscopy. The retina is a layered heterogeneous tissue, and each layer has different characteristic biomechanical properties, which are demonstrated by our results. The stiffness of the layers is highly dependent on cell body density.,In the second part of this work, we compared the biomechanical properties of PFA-fixed and fresh mouse retinas. PFA is a cross-linking fixative that covalently bonds proteins within tissue, thereby conferring rigidity.,Finally, we performed a pilot study on the effects of NMDA-induced retinal damage on the stiffness of the retina in a mouse model. The tissue was assessed 48-h post-NMDA injection in a young mouse. The results showed a very drastic increase in the Brillouin modulus of the RGC layer and/or NFL in the damaged retina as compared with its contralateral control. NMDA can induce damage to retinal blood vessels,OCT is used routinely for live imaging of retinal damage, but biomechanical assessments of retinal tissue are generally qualitative only, rely heavily on other factors such as geometry,One major limitation of this work is that Brillouin microscopy can only provide measurements of the tissue bulk modulus instead of the quantitative measurement of elasticity. We have previously demonstrated quantitative measurements of crystalline lens elasticity by combining Brillouin microscopy with OCE.5in vivo studies of the long-term effects of NMDA-induced retinal damage in a mouse model and combining Brillouin microscopy with OCT for noninvasive and all-optical co-registered structural and biomechanical maps of the retina.We noninvasively characterized retinal tissue stiffness using Brillouin microscopy on freshly harvested mouse retinas. The results show that Brillouin microscopy can map the mechanical properties of the different retinal layers. The stiffness of the layers correlated with the cellular density, and fixing the retinas in PFA also increased the stiffness. Furthermore, we demonstrated an increase in the stiffness of the RGC and/or NFL layer due to NMDA-induced damage in a mouse model. This work was a preliminary study to demonstrate noninvasive biomechanical characterization of retinal tissue using Brillouin microscopy. Our future work is focused on


Retinitis pigmentosa (RP) is an inherited retinal dystrophy (IRD) with a prevalence of 1:4000, characterized by initial rod photoreceptor loss and subsequent cone photoreceptor loss with accompanying nyctalopia, visual field deficits, and visual acuity loss. A diversity of causative mutations have been described with autosomal dominant, autosomal recessive, and X-linked inheritance and sporadic mutations. The diversity of mutations makes gene therapy challenging, highlighting the need for mutation-agnostic treatments. Neural leucine zipper (NRL) and NR2E3 are factors important for rod photoreceptor cell differentiation and homeostasis. Germline mutations in NRL or NR2E3 leads to a loss of rods and an increased number of cones with short wavelength opsin in both rodents and humans. Multiple groups have demonstrated that inhibition of NRL or NR2E3 activity in the mature retina could endow rods with certain properties of cones, which prevents cell death in multiple rodent RP models with diverse mutations. In this review, we summarize the literature on NRL and NR2E3, therapeutic strategies of NRL/NR2E3 modulation in preclinical RP models, as well as future directions of research. In summary, inhibition of the NRL/NR2E3 pathway represents an intriguing mutation agnostic and disease-modifying target for the treatment of RP.  Retinitis pigmentosa (RP) encompasses a genetically heterogeneous collection of inherited progressive retinal degenerative disorders affecting approximately 1:4000 individuals . DespiteTo date, nearly 100 unique genes have been implicated in RP pathogenesis, with significant overlap among genes shared with RP and the related IRDs including Leber’s Congenital Amaurosis (LCA), cone-rod dystrophy, and macular dystrophy . InheritConceptually, therapeutic strategies that would be effective across all forms of RP can be divided into three broad categories: (1) neuroprotective strategies to prevent photoreceptor death; (2) regenerative medicine approaches to replace lost photoreceptors or prevent photoreceptor death; and (3) optogenetic approaches to endow cells that are normally not light sensitive  and are not affected by RP to be sensitive to light by gene therapy or chemical means. Unfortunately, there have been no successful Phase 3 trials using these strategies thus far . CiliaryPde6β-/- mice there was preliminary evidence of functional photoreceptor integration into the mouse retina [Surgical cell/tissue transplantation generally aims to replace degenerated photoreceptors or RPE from various donor sources. This approach is thought to either replace dying photoreceptors or RPE or secrete neurotrophic factors which may prevent photoreceptor death. Preclinical models of stem cell-derived retinal cells have shown early promise that has not yet been demonstrated in human clinical trials. When human embryonic stem cells (hESC) or induced pluripotent stem cell (iPSC)-derived photoreceptors were transplanted in retinal degeneration e retina . Intravie retina . Previoue retina . Fetal te retina . SeveralA strategy termed optogenetics has emerged as a promising strategy for conferring light sensitivity into the surviving non-photoreceptor cells in retinal degenerative disease. This can occur either through the genetic introduction of light-sensitive ion channels such as channelrhodopsin, or through small molecule approaches using light-sensitive small molecules which can open ion channels to depolarize neurons ,40. ChanMutation-independent treatments are attractive for the prospect of treating greater numbers of RP patients with a single generic treatment. At present, however, none specifically target the pathologic biology of photoreceptor degeneration. A greater understanding of photoreceptor molecular biology is likely to yield additional clinically relevant insights. To this end, the transcription factor NRL and orphan nuclear receptor NR2E3 transcription factors are the subject of extensive investigation in photoreceptor transcriptional regulation ,44,45 anThe NRL/NR2E3 pathway has been implicated in differentiation and subsequent maintenance of rod photoreceptors throughout the lifetime of the cell. Mice which lack NRL or NR2E3 activity do not have rods and instead have an increased number of cone-like cells expressing S-opsin. Physiologically, human patients with mutations in NRL/NR2E3 have phenotypic similarities in reduced or absent rod function with increased S-cone function. This suggests that blocking the NRL/NR2E3 pathway may endow rods with certain cone-like features that may prevent cell death by reducing or abolishing the manifestation of rod degenerative phenotypes. Here we review the scientific premise and evidence behind this therapeutic strategy. Human mutations in the NRL/NR2E3 pathway leading to RP phenotypes provide proof of principle for the role of this pathway in photoreceptor development and homeostasis. Mutations in NRL have been implicated in both autosomal recessive (A76V)  and auto-/-Nrl mouse [There are few reports of autosomal recessive RP due to NRL, with one report describing patients with homozygous mutations in both NRL as well as PABPN1, a cause of oculopharyngeal muscular dystrophy ,51,52. T/- mouse . Recessive and dominant mutations in human NR2E3 have also been associated with a wider spectrum of retinal disease. Pathogenic NR2E3 mutations have been associated with enhanced S-cone syndrome (ESCS)/Goldman-Favre syndrome ,54, clum-/- retina, with gene ontology analysis revealing that affected genes were involved in signal transduction and transcriptional regulation, including Rho [-/- mouse revealed absent rod activity and abnormally high S-cone activity ; the rod pigment rhodopsin could not be detected immunohistochemically, leading the authors to conclude that NRL was critical for differentiation of rod photoreceptors. NRL absence favored development of rods with cone-predominant physiology, including cone-like nuclear morphology and increased blue light-adapted ERG cone activity, suggesting unexpectedly elevated cone-like light response in the retina [Mammalian photoreceptors are a type of specialized postmitotic neuroepithelial cell originating from a common photoreceptor precursor, generating the first cones by 8 weeks’ gestation and first rods by 10 weeks’ gestation in the developing human retina . Recent ding Rho ,63. In 2ding Rho . Interese retina . Indeed,e retina ,20. Evide retina ; importae retina  and appee retina . Thus, N-/- mice [-/- mouse retina, as well as in wildtype Xenopus retina [-/- mice prevented retinal degeneration, supporting the hypothesis of NR2E3 favoring rod homeostasis [NR2E3 encodes the orphan nuclear receptor NR2E3 specific to rod photoreceptor cell nuclei that is conserved among vertebrates, where it suppresses transcription of cone-specific genes in developing rod photoreceptors  Figure . The rd7-/- mice , suggest-/- mice . Ectopics retina ,71. NR2Es retina . Indeed,s retina . Under teostasis . Thus, eeostasis  as a dowGermline mutations in NRL or NR2E3 cause RP by perturbing the normal balance of photoreceptor cell fate and interfering with rod homeostasis in the mature retina. One potential therapeutic strategy is that inhibition of NRL or NR2E3 expression may reduce rod gene expression and/or disinhibit cone transcription, thus reducing the potential for disease manifestation .-/- mice. When the adult NRL knockout experiment was repeated on the Rho-/- retinal degeneration mouse model at P25, the knockout prevented retinal degeneration, preserved retinal laminar architecture, and restored photopic cone ERG physiology when assayed at P90 [-/- germline knockout mice, CRISPR/Cas9 NRL knockout at P14 or P28 significantly slowed the loss of ERG light-adapted response and increased the ONL surviving photoreceptor density. In the Rd10 rod degeneration mouse model caused by mutation in Pde6β, CRISPR-Cas9 NRL knockout at P14 prevented both rod and cone degeneration and improved light-adapted ERG response. Finally, CRISPR/Cas9 NRL knockout at P14 in the RhoP347S rod degeneration model significantly slowed light-adapted b-wave ERG degeneration over time and protected photoreceptors from apoptosis [-/- retinal degeneration mouse models [-/- mice. Moreover, epigenetic modifications to photoreceptor genes made in development may render adult cells less flexible to cone reprogramming versus the developing retina.Multiple efforts from independent investigators have validated this proof-of-concept approach in preclinical rodent RP models . Montanad at P90 . This sud at P90 . A smalld at P90 . Importapoptosis . A double models . ERG phoe models . Thus, ie models ,76. ThisP23H rod degeneration mice and in the Pde6brd1 rod degeneration mouse [+ cells in culture and in vivo following systemic PR3 treatment of wt mice [P23H rod degeneration mouse and also improved photopic and scotopic function on ERG [Unlike NRL, NR2E3 inhibition strategies include both small-molecule and genetic modalities . Nakamuron mouse . Thus, t wt mice . Systemin on ERG . Althoug+ and rhodopsin+ cells, and improved ERG response, which the authors attributed to increased recruitment of phototransduction-relevant transcription factors in a number of rodent preclinical models of RP [The benefits of genetic manipulation of the NE2E3 are less clear. Naessens et al. provided proof-of-concept of an antisense oligonucleotide capable of NE2E3 knockdown in vitro but did not investigate functional consequences . Interesls of RP . This suls of RP , whereasls of RP .RP presents a significant multifaceted challenge for developing therapeutics in ophthalmic genetics. Genetic heterogeneity among diagnosed cases complicates gene therapies and favors the most prevalent autosomal recessive mutations that are amenable to gene replacement drug development. However, patients harboring less prevalent mutations or mutations with dominant inheritance are unlikely to benefit from currently available gene therapies. Thus, recent research has focused on mutation-agnostic treatments. Channelrhodopsin gene therapies under investigation look to confer photoreceptor properties on retinal cells such as interneurons or retinal ganglion cells that do not degenerate in RP although with likely poor visual sensitivity and resolution. Clinical trials of neuroprotective small molecules have yielded disappointing results, and to date none have gained FDA approval. In the natural history of RP progression, initial rod dysfunction and degeneration precedes later cone degeneration and worsening loss of central vision . Prevent-/- rods compared to controls [What might be the molecular mechanisms behind photoreceptor survival when inhibiting the NRL pathway? This remains an unanswered question in the field. One possibility is that decreased expression of rod genes such as rhodopsin, whose protein levels are exquisitely regulated to prevent photoreceptor degeneration , may deccontrols , but low-/- germline knockout mice have abnormal retinal lamination, photoreceptor death, as well as Müller glia dysfunction and changes in vascular permeability [-/- photoreceptors [One other unanswered question is whether long term inhibition of the NRL pathway will lead to deleterious effects on the retina such as retinal degeneration. NRLeability . Howevereability , suggesteceptors . TherefoIn conclusion, inhibition of the NRL/NR2E3 pathway represents an intriguing approach for the treatment of retinitis pigmentosa. It represents an attractive target of future investigation for its potential to modify the natural history of RP retinal degeneration from multiple genetic etiologies. We hope that further research will refine this approach and allow for testing of this hypothesis in clinical trials, which brings the possibility of preserving sight for the many patients with untreated retinal degenerative diseases.


We live in a world continuously immersed in noise, an environmental, recreational, and occupational factor present in almost every daily human activity. Exposure to high-level noise could affect the auditory function of individuals at any age, resulting in a condition called noise-induced hearing loss (NIHL). Given that by 2018, more than 400 million people worldwide were suffering from disabling hearing loss and that about one-third involved noise over-exposure, which represents more than 100 million people, this hearing impairment represents a serious health problem. As of today, there are no therapeutic measures available to treat NIHL. Conventional preventive measures, including public awareness and education and physical barriers to noise, do not seem to suffice, as the population is still being affected by damaging noise levels. Therefore, it is necessary to develop or test pharmacological agents that may prevent and/or diminish the impact of noise on hearing. Data availability about the pathophysiological processes involved in triggering NIHL has allowed researchers to use compounds, that could act as effective therapies, by targeting specific mechanisms such as the excess generation of free radicals and blood flow restriction to the cochlea. In this review, we summarize the advantages/disadvantages of these therapeutic agents, providing a critical view of whether they could be effective in the human clinic. Noise over-exposure is the major avoidable cause of permanent hearing loss , and its removal from cells in the auditory receptor. ROS build up in stressed mitochondria has been proposed as a major player at the origin of NIHL  and the most abundant endogenous free radical scavenger in humans, was the first antioxidant evaluated for the treatment of NIHL , a cysteine precursor, is an antioxidant molecule that enhances the production of glutathione  is an excellent scavenger of singlet oxygen, blocking and/or reverting lipid peroxidation in the plasma membrane  which seem to have protective effects in the noise-exposed cochlea , have a good safety profile, reduced drug acquisition costs, and few or no side effects. Antioxidants such as N-acetylcysteine, HK-2, vitamins A, C, and E and the vasodilator MgJA and VF-S: drafting of the manuscript and design of figures. JA, VF-S, and JJ: critical revision of the manuscript for important intellectual content.JA, VF-S, and JJ are co-inventors of the US Patents 9, 889, 156, “Method for treating NIHL” and 9, 919, 008, “Methods for treating ARHL.” Both patents are based on the use of oral ACEMg, but currently, they are not involved in any trials testing this compound or any other commercial exploitation.


There is an increased interest in the impact that hearing loss has on general well-being, including overall quality of life (QOL). The Quality of Life Inventory (QOLI), the Charlson Comorbidity Index and an Accessibility to Health Care questionnaire were administered to 108 participants. For adults with hearing loss who did not have access to hearing health care, lower QOL scores were reported compared to those with access to hearing health care, but this finding was not significant. Effect size calculations indicated that adults with hearing loss who lived in the most rural regions of Alabama, had lower reported QOL scores than their counterparts who had hearing within normal limits. Finally, those with higher incomes, who were older, and who had fewer physical disorders reported higher QOL compared to those with lower incomes, were younger, and who had more physical ailments. Part of a symposium sponsored by the Mental Health Practice and Aging Interest Group.