Spearman’s correlation indicated a good correlation between the two assays (rs = 0.88 for ADL and rs = 0.93 for IFX), and McNemar’s test revealed significant differences (p = 0.016) when classifying IFX samples between therapeutic and supra-therapeutic ranges but no significant differences were found among the other ranges for either IFX or ADL. These results show that we should be cautious when using these rapid measurement methods, and new targets should probably be defined for IFX when using this new analytical method.The introduction of point-of-care (POC) assays into clinical practice in patients with inflammatory disease enables on-demand therapeutic decision making. The aim of this study was to compare the POC test Quantum blue (Bühlmann Laboratories) for infliximab (IFX), adalimumab (ADL), and its anti-drug antibodies with the traditional ELISA assay (Promonitor). A total of 200 serum samples were analyzed. Samples were classified into the following three different groups; sub-therapeutic range (IFX < 3 μg/mL and ADL < 5 μg/mL); therapeutic range (IFX: 3–7 μg/mL and ADL: 5–12 μg/mL) and supra-therapeutic range (IFX levels > 7 μg/mL and ADL levels > 12 μg/mL). Significant higher values were measured using the POC test ( Inflammatory bowel disease (IBD) is a chronic immune-mediated inflammatory disease affecting the gastrointestinal tract that englobes two entities, Crohn’s disease (CD) and ulcerative colitis (UC), which are both characterized by recurrent and destructive pathological inflammation that causes significant morbidity and impacts the quality of life. While it is currently an incurable disease, there are several treatments that target clinical symptoms. The introduction of biologic drugs has revolutionized the approach to the treatment of IBD. This has led to improved and timely treatment responses, lower hospitalization rates, reduced surgical requirements, and remarkable outcomes, such as complete mucosal histologic healing and an improvement in quality of life . Anti-tuThe discovery of new mechanisms of action and the optimization of conventional treatments has greatly improved the situation; however, approximately 20–30% of patients with IBD show a primary non-response to biologic therapies, and up to 50% of patients discontinue treatment because of a secondary loss of response or a serious adverse event after an initial clinical response . Many stTherapeutic drug monitoring (TDM), defined as the assessment of drug concentrations and the detection of ADAs, is an important tool for optimizing biologic therapy . In a re® (QB) , and Rida®Quick (RQ) . They vary in their range of detection in the samples required for detection (serum/plasma/whole blood).The analysis of blood concentrations of monoclonal antibodies is performed routinely using the enzyme-linked immunosorbent assay (ELISA). This requires the logistic accumulation of samples that are necessary to make it a cost-efficient technique, which is why these methods are commonly centralized. However, it does not lend itself to prompt decision making, often delaying clinical decisions that demand precision, agility, and practicality. Point-of-care (POC) tests allow the measurement of IFX and ADL through concentrations and the rapid detection of ADAs on the same day of consultation, providing instant results in less than half an hour. POCs are rapid immunochromatographic assays based on lateral flow technology for the quantitative detection of drugs and qualitative detection in the case of ADAs. Currently, there are three rapid detection tests that are commercialized: the Promonitor Quick assay (PQ) , Quantum BlueComparative studies between both methods are needed to provide solid evidence to incorporate POCs into daily clinical practice and, thus, reduce response times. In addition, it is important to elucidate whether the therapeutic ranges used, based primarily on ELISA results, are also applicable to these new rapid methods. There are some published studies on a short series of samples ,18,19,20The aim of this study is to compare the POC test Quantum Blue for quantitative IFX and ADL and qualitative anti-IFX and anti-ADL analysis with the traditional ELISA assay (Promonitor) to evaluate their concordance in the measurement of trough levels, the detection of antibodies, and whether using one method or the other implies changes in therapeutic decisions.This was a retrospective study in which patients with IBD were selected, and their ADL and IFX trough levels were monitored following the hospital protocol using the ELISA technique. The study and the data collection strictly adhered to the declaration of Helsinki principles. Ethical approval was given by the local Institutional Review Board and the autonomous region of Galicia (2018/077).A total of 200 serum samples  from 200 different patients were analyzed from the IBD unit. One thousand five hundred samples for concentration analysis were classified into three different groups according to their sub-therapeutic (IFX levels < 3 μg/mL and ADL levels < 5 μg/mL), therapeutic (IFX levels 3–7 μg/mL and ADL levels 5–12 μg/mL) and supra-therapeutic (IFX levels > 7 μg/mL and ADL levels > 12 μg/mL) ranges, and samples from the total amount of each group were randomly selected. Also, 40 samples were randomly selected for anti-IFX and 40 samples for anti-ADL analysis .g for 10 min within 4 h after sample collection and stored at −80 °C before analysis. ELISA analyses were performed following routine clinical practice with the Promonitor ELISA kit according to the manufacturer’s instructions , and all were run on a semi-automated ELISA processor . The dilution of the serum samples was modified to 1:400 and 1:10 for IFX and ADL trough levels and to 1:1 and 1:10 for IFX and ADL ADA. For quantitative analysis, according to the package insert  the lower limit of quantification (LLoQ) for ADL was 0.017 μg/mL, and the LLoQ for Promonitor-IFX was determined to be 0.3 μg/mL. In the case of the ADA assay, the LLoQ of Promonitor anti-IFX was determined to be 2 AU/mL, and the LLoQ of Promonitor anti-ADL was determined to be 3.7 AU/mL.Promonitor is a capture (IFX kit) or sandwich (ADL kit) ELISA in which microwell strips are provided and pre-coated with an anti-TNF human F(ab’)2 fragment bound to a recombinant human TNF. Both Promonitor anti-IFX and anti-ADL bridge ELISA in which microwell strips are provided and pre-coated with IFX and ADL, respectively. Samples were centrifuged at 2280× ® (QB) is an in vitro diagnostic lateral flow immunoassay for the quantitative determination of trough levels of ADL and IFX and qualitative determination of ADAs detection in serum samples , POC analysis was made following the manufacturer’s instructions. Briefly, serum samples for the determination of IFX and ADL were diluted with an assay buffer (1:20). For high-concentration samples, an additional dilution of 1:200 was made. In the case of ADA analysis, the dilution was 1:10. Then, diluted serum samples were incubated for 15 min at room temperature before the results were interpreted by the Quantum Blue Reader . According to the package insert, the lower limit of quantitation for QB-ADL was 1.3 μg/mL, and the LLoQ for QB-IFX was 0.32 μg/mL. As QB only shows qualitative results for ADA analysis, the cut-off point was set at 0.2 μgeq/mL for the QB anti-ADL assay and 1.3 μgeq/mL for the QB anti-IFX assay to distinguish between negative and positive results.Quantum BlueNormality was tested using the Shapiro–Wilk test and correlation was evaluated with Spearman’s rank correlation coefficient (rs). The Wilcoxon rank sum test was used to detect differences between the drug trough levels. A passing-Bablock regression was performed to estimate the line of best fit by comparing the ranks of observations between the two variables. Also, Bland–Altman analysis was conducted to assess the agreement between the two quantitative measurements. A Bland–Altman plot showed the difference between these two measurements (y-axis), and its mean (x-axis) represented the mean difference between the two measurements, and the limits of agreement were expressed as the mean difference plus or minus the two standard deviations of differences. The concordance between the ELISA and QB classification of patients was placed into three different therapeutic groups for both drugs assessed through weighted Cohen’s kappa (κ), and the differences in classification for each group were assessed using McNemar’s test. Also, the concordance between the detection of ADAs with ELISA and QB for both drugs was run using Cohen’s kappa (κ).According to the classification of the different ranges, the 120 samples for quantitative analysis were distributed as follows: 20 samples for the IFX sub-therapeutic range, 19 for the ADL sub-therapeutic range, 18 samples for the IFX therapeutic range, 21 for the ADL therapeutic range, 22 samples for the IFX supra-therapeutic range and 20 for the ADL supra-therapeutic range.p = 0.3101). Also, 60 samples of patients treated with IFX were measured via the Promonitor and Quantum Blue assays. The median values were 4.86 µg/mL (IQR: 2.22–9.31) for the Promonitor assay and 6.15 µg/mL (IQR: 2.9–12.92) for the Quantum Blue assay, with significantly higher values measured using the QB test (p < 0.001). Spearman’s rank correlation coefficient indicated a good correlation for ADL trough levels between the two assays (rs = 0.88), which was even higher between IFX trough levels (rs = 0.93).ADL trough concentrations were measured using the two-method analysis in 60 samples. The median values were 10 µg/mL (IQR: 3.87–13.25) for the Promonitor assay and 8.85 µg/mL (IQR: 3.67–13.62) for the Quantum Blue assay. No differences in ADL trough levels were observed between the Promonitor and QB  when classifying IFX samples between therapeutic and supra-therapeutic ranges, but no significant differences were found among the other ranges for either IFX or ADL (The therapeutic strategy decision could be different in 13 samples (21.6%) for each treatment. To complete the analysis, McNemar’s test was performed to assess whether each method resulted in discordant classifications considering the sub-therapeutic and therapeutic range and therapeutic and supra-therapeutic range. McNemar’s test revealed that there were significant differences (X or ADL .p = 0.1336) or anti-IFX (p = 0.0771) tests. Descriptive analysis and kappa statistics results are shown in A total of 40 samples from IBD patients who received ADL and IFX and had sub-therapeutic levels were tested using the Promonitor kit and Quantum Blue assays. As Quantum Blue is a qualitative test for ADAs’ determination, a qualitative comparison was conducted between these two methods. A moderate agreement (κ = 0.536 ± 0.136) between the two assays was revealed for infliximab ADAs. In the case of adalimumab ADAs, the agreement was substantial (κ = 0.793 ± 0.095). Also, McNemar’s test was performed to complete the comparative classification, and no statistically significant differences were found for either anti-ADL (The management of inflammatory bowel disease (IBD) has advanced significantly in recent years, with the increasing availability of biologic agents, but compared to other immune-mediated pathologies, they are still insufficient . TherefoIn the case of ADL, our results show a good correlation and no significant differences in the measurement of trough levels between the two methods, as well as in the stratification of patients in different therapeutic ranges. This has been also observed in another study , where tIn our study, QB systematically provided higher IFX trough values than those determined by ELISA, which led us to conclude that the target value of IFX values in TDM, according to different measurements, are different depending on the method. In our study, 5/20 (25%) patients with sub-therapeutic ranges measured by ELISA were classified as therapeutic by QB, and 7/18 (38.8%) in a therapeutic range by ELISA were classified as supra-therapeutic by QB. This led us to the fact that, in cases of doubt, it is always necessary to confirm with the reference method before taking any clinical decision and to be cautious to not rely solely on rapid measurement methods. Also, it may be necessary to redefine the target ranges of POC analytical methods, at least with the IFX test.Finally, antibody detection using Promonitor ELISA kits is a robust method with high sensitivity and specificity, but, as with its drug testing counterpart, multiple samples need to be accumulated to make it cost-effective. The introduction of POCs into daily clinical practice would allow this to be resolved at the patient’s bedside in a few minutes; this is especially important in patients under ADL treatment because, due to their schedule of 40 mg every other week, physicians can make decisions before the next drug injection. In the case of antibody detection by both methods, no statistically significant differences were found for either anti-IFX or anti-ADL. One of the limitations of this assay is that we have not analyzed the effect of the presence of drugs in the sample, despite the fact that all of them came from sub-therapeutic levels or from other interfering substances in the detection of ADAs. In this sense, one prior study has already shown a correlation between these two analytical methods, but only regarding the detection of anti-IFX antibodies . Lastly,The routine TDM of ADL can be performed with Promonitor and QB assays since trough levels and patient classification into therapeutic ranges are comparable. In the case of IFX trough levels, we must be cautious when using these new methods due to the differences found when classifying patients between the therapeutic and supra-therapeutic ranges. New ranges should probably be defined for using different methods and not be considered interchangeable, especially when monitoring patients’ maintenance therapies. On the other hand, QB appears to be a suitable method for the qualitative determination of ADAs and could be used individually or as an add-on to the measurement of levels via traditional methods. Nevertheless, further studies with larger sample sizes and comparing all currently available analytical methods in a simultaneous manner are needed to establish the interchangeability between them and, if necessary, new therapeutic ranges according to the analytical method used.


Mucorales order, is an insidious infection that manifests in different clinical forms. Even the most benign form, the cutaneous mucormycosis, can present with severe complications and a fatal outcome in patients with a suppressed immune system and underlining comorbidities. We present a rare case of a proven primary multifocal cutaneous mucormycosis in a child with newly diagnosed acute leukemia without multiorgan dissemination. Various laboratory techniques  were used to detect and confirm the diagnosis. Etiological therapy  combined with surgical intervention were used to manage the infection. The case shows that a rapid and complex diagnostic approach is of crucial importance for the timely initiation of adequate therapy, as well as for the successful management of this life-threatening fungal infection.Mucormycosis, caused by the widespread molds of the  Candida and Aspergillus associated infections [Mucoraceae family of order Mucorales, which inhabit the soil and organic matter [Rhizopus arrhizus (Rhizopus oryzae), it can also occur due to some representatives of the genera Mucor, Lichthemia (Absidia) and Cunninghamella [Mucormycosis  is the third most common invasive fungal infection after fections . Its cauc matter . Except c matter . The firc matter . Years lc matter . While mghamella . The infghamella . Regardlghamella .We present a rare case of multifocal cutaneous mucormycosis without dissemination in multiple organs or the central nervous system in a 2-year-old child with newly diagnosed acute lymphoblastic leukemia.A 2-year-old boy was admitted to the Pediatric Hematology and Oncology ward with fever, drowsiness, decreased appetite and gingivorragia. The symptoms first appeared a week before hospitalization. Ambulatory blood tests revealed severe anemia, thrombocytopenia and lymphocytosis. The patient was in an impaired general condition. A single indurated lesion was found in the left cubital fossa. The child’s family, perinatal, growth, and developmental histories were unremarkable. He had a complete vaccination record for his age.A bone marrow examination revealed total bone marrow blast cell infiltration. The flowcytometry confirmed the diagnosis of acute B-cell lymphoblastic leukemia. The patient started induction chemotherapy  according to the ALL-IC BFM 2009 IA protocol along with standard supportive therapy. Laboratory blood test results at admission and during the hospital stay are presented in Mucor sp., was observed. Microscopic examination of the samples with lactophenol cotton blue revealed that nonseptate fungal hyphae were forming oval sporangia  [During the induction chemotherapy, an increase in the diameter of the infiltrate located in the left cubital fossa and change in its color from red to black was observed . Aspiratporangia . Rhizoidla spp.) . The folMucoraceae was observed on SDA. The histological examination revealed fatty necrosis, leukocyte infiltrates, an abundance of Periodic acid–Schiff stain (+), coarse, nonseptate, branching at right angles of fungal hyphae, and angioinvasion  was initiated and surgical intervention was performed on all seven skin lesions. Biopsy samples were sent for microbiological and histological examination. The cultural method confirmed the result from the initial microbiological testing performed before antifungal and surgical treatment. After 48 h of incubation at 30 °C, the fungal growth characteristic of invasion .Along with the classical microbiological and histological examinations, as well as PCR, several additional tests were carried out, including the serum galactomannan antigen test, multiple blood cultures, cerebrospinal fluid, urine, throat and nasal cultures, fecal screening for colonization by multidrug-resistant microorganisms, and latent viral infections tests . All results were negative. Computed tomography scans of the chest, abdomen, and pelvis did not reveal abnormalities. Apart from the skin lesions, no other primary or secondary source of fungal infection were found during the patient’s hospital stay.Mucor sp. with no involvement of internal organs or central nervous system was established [Based on the data obtained from the laboratory tests and using the revised and updated guidelines of the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium, a diagnosis of proven primary multifocal cutaneous mucormycosis caused by ablished .After surgical excision and antifungal therapy, healthy granulation of the surgical wounds were observed with no new lesions. Antifungal therapy with liposomal amphotericin B continued for 4 months. Following a re-evaluation, the patient was assessed as successfully cured.Mucormycosis is an exogenous, life-threatening and opportunistic infection associated with high morbidity and mortality in children and adults . It is cPredisposing factors for the development of mucormycosis are hematological malignancies, transplantation, diabetes, iron overload and deferoxamine use . The rhiThe cutaneous form of mucormycosis manifests with a red and swollen plaque, which can later ulcerate and necrotize. Blood vessels, subcutaneous layers and muscles may also be affected . Upon hoAuthors from different parts of the world  have described cases of cutaneous mucormycosis in pediatric patients with various risk factors. In concordance with our case, most of them were with hematological malignancies  or had underwent hematopoietic stem cell transplantation, but the cutaneous infection was presented only by a single focus without spreading to other skin areas and mainly affected the arm or the face ,16,17,18Aspergillus spp. and Fusarium spp. The coarse and nonseptate hyphae of the representatives of Mucorales spp. are easily differentiated from the narrow and septate hyphae of Aspergillus spp. and Fusarium spp. [Mucorales spp. branch at a right angle (90°), while the hyphae of the other molds tend to branch at 45° [Diagnosis of mucormycosis requires a combined approach with the inclusion of imaging, histological and microbiological methods. The histopathological examination presents wide, coarse, and ribbon-like nonseptate hyphae. Angioinvasion and necrosis are often observed. When clinical samples are inoculated on agar media, visible fungal growth is detected in only 1/3 of the cases. Isolating the pathogen on artificial media is challenging because of the fragile nature of the hyphae, which are quickly destroyed when processing the material . If succium spp. . Additioh at 45° . In addih at 45° . Matrix-h at 45° .Aspergillus galactomannan being the most commonly used. However, these fungal cell components are not present in the Mucorales molds. Although negative in mucormycosis, when the positive result is obtained, this can rule out Mucorales infection. Currently, no serum markers are in use to diagnose mucormycosis [The serologic tests are another aspect of the complex diagnostic approach of the invasive fungal diseases. Various serum markers can be screened and monitored when invasive fungal infections occur with 1, 3-beta-D-glucan (BDG) and rmycosis .Typical for the histological presentation of mucormycosis, we observed fat necrosis, leukocyte infiltrates, fungal angioinvasion, an abundance of nonseptate, and coarse branching at 90° mycotic hyphae. Mold growth was documented and detected on the SDA and DNA of clinically significant molds associated with mucormycosis.Mucorales are naturally resistant to several antifungal agents, such as flucytosine, echinocandins  and representatives of the azole group (fluconazole and voriconazole) [Candida spp. and Aspergillus spp. in severely immunocompromised individuals, and the lack of activity of this agent against Mucorales molds.Therapy of cutaneous mucormycosis is complex and usually includes surgical and antifungal treatment . Accordionazole) . Moreoveonazole) . AnalyziHowever, if a good therapeutic response to amphotericin B monotherapy is not achieved, some authors recommend a combination of amphotericin B with azole anti-fungal agents (posaconazole or itraconazole). When the combination treatment is not effective, a salvage triple therapy including agents from three classes of antifungals  can be initiated. There are no specific recommendations for the duration of mucormycosis treatment. The process can take months or even a year .One of the most severe complications of cutaneous mucormycosis is the hematogenous dissemination of the pathogen, observed mainly in individuals with underlying diseases and various risk factors . When diTo the best of our knowledge, this is the first report in Europe of primary multifocal cutaneous mucormycosis without systemic dissemination in a child with hematological malignancy. A rapid and complex diagnostic approach is of crucial importance for the timely initiation of an adequate therapy and successful management of this invasive fungal disease.


In silico WB-subtraction identified 90 high-priority proteins with retina-enriched expression at stringency criteria of ≥2.5 average spectral counts, ≥2.0 fold-enrichment, False Discovery Rate <0.01. These top candidates represent a pool of retina-enriched proteins, several of which are associated with retinal biology and/or defects , indicating the effectiveness of this approach. Importantly, in silico WB-subtraction also identified several new high-priority candidates with potential regulatory function in retina development. Finally, proteins exhibiting expression or enriched-expression in the retina are made accessible in a user-friendly manner at iSyTE (https://research.bioinformatics.udel.edu/iSyTE/), to allow effective visualization of this information and facilitate eye gene discovery.To expedite gene discovery in eye development and its associated defects, we previously developed a bioinformatics resource-tool iSyTE (integrated Systems Tool for Eye gene discovery). However, iSyTE is presently limited to lens tissue and is predominantly based on transcriptomics datasets. Therefore, to extend iSyTE to other eye tissues on the proteome level, we performed high-throughput tandem mass spectrometry (MS/MS) on mouse embryonic day (E)14.5 retina and retinal pigment epithelium combined tissue and identified an average of 3,300 proteins per sample (n=5). High-throughput expression profiling-based gene discovery approaches-involving either transcriptomics or proteomics–pose a key challenge of prioritizing candidates from thousands of RNA/proteins expressed. To address this, we used MS/MS proteome data from mouse whole embryonic body (WB) as a reference dataset and performed comparative analysis-termed “ We developed a user-friendly web-based resource-tool called iSyTE (integrated Systems Tool for Eye gene discovery) to address these challenges in the eye; however, the earlier versions were focused on the lens  as well as led to the characterization of several regulatory pathways associated with eye/lens development and defects  can identify several genes linked to retina biology and defects and is in itself helpful, in silico WB-subtraction provides another effective approach in prioritizing key candidates that are not necessarily among the highest expressed proteins in the retina. We generated new expression tracks at the University of California at Santa Cruz (UCSC) Genome Browser and make this new data accessible through iSyTE.The present version of iSyTE is restricted to data on the lens, and furthermore, it is predominantly based on transcriptomic data in the form of microarrays or RNA-seq , with liMice of the background C57BL/6J, obtained from The Jackson Laboratory, were used as wild-type animals in this study, and were bred and maintained at the University of Delaware Center for Animal research facility. The Institutional Animal Care and Use Committee (IACUC) approved the animal protocol (AUP#1226). All the animal experiments described in this study were performed in adherence to the guidelines in the Association of Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research.g, 2 min., room temperature) followed by heating (90°C for 15 min.). Sample protein quantification was estimated by BCA protein assay kit (Thermo Fisher Cat. No. 23225). For each biological replicate , 55 μg of protein/sample was subjected to trypsinization as previously described  0.5, and tissues was collected at E14.5. Whole retina tissue , henceforth referred to as “retina”) were micro-dissected from E14.5 mouse embryos and stored at −80°C until further processing. Whole embryonic tissue minus the eye at E14.5 was considered as “whole embryonic body” (WB). Five biological replicates with each replicate consisting of two retinas isolated from the same embryo were collected. Tissues were processed as previously described . Brieflyescribed . Brieflyescribed .https://www.ebi.ac.uk/pride/) (Mass spectrometry (MS) was performed as previously described . Briefly/pride/)  partner https://github.com/pwilmart/fasta_utilities.git) was used to download a canonical mouse reference proteome  from UniProt. To this, a concatenated sequence-reversed decoy database was added along with common contaminants (179 sequences) to obtain 44,932 entries. Peptide sequences were assigned to the MS2 spectra (PSMs) using the search engine Comet (MSConvert (Proteowizard toolkit) was used to convert RAW files to MS2 format for the samples as described . The retne Comet , which wne Comet  and werehttps://github.com/pwilmart/PAW_pipeline.git) and the target/decoy method described previously  were measured. Prior to protein inference, the SpC for individual samples were tallied and they independently validated the peptide assay results. Next, the retina and the WB samples were matched by subjecting the individual samples to be scaled to the average total spectral count per sample. Both the retina and the WB samples had about 3,300 protein identifications per sample. Next, the proteins with enriched expression in the retina compared to WB were determined as follows: For individual proteins, the average SpC for all samples was computed from the scaled data, and only values greater than 2.5  were considered in the differential expressed enrichment analysis between the retina and WB. The bioconductor package, edgeR, was used for differential gene expression analysis . The defin silico WB-subtraction . Benjamini corrected significant p-values were considered for prioritization of the pathways and GO categories identified from this analysis as previously described  categories, a cluster-based analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID v6 .8)  was perfescribed .in silico WB-subtraction , 55 μg of protein were subjected to eFASP (enhanced filter-aided sample preparation) and digestion by trypsin. After digestion, equal amounts of peptides were used for high-throughput tandem mass spectrometry (MS/MS) and spectral count (SpC) data were generated. Application of stringent criteria  to the resulting data led to enrichment analysis of 2,675 proteins in the E14.5 retina (Supplementary Table S1). Across the samples, on average ~35K SpC were detected. Total average SpC was subjected to TMM  normalization using edgeR package  between the retina and WB . To examine whether specific pathways relevant to retina biology were enriched in this dataset, a cluster-based analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID v6 .8) for functional annotation by gene ontology (GO) categories.e.g., “GO:0003723 RNA-binding”, “GO:0030529 intracellular ribonucleoprotein complex”, “GO:0006397 mRNA processing” and “GO:0051028 mRNA transport”  data for each protein. Those peptides that passed the filtration criteria of ≥2.5 SpC were considered in the analysis. This approach identified 2675 proteins that could be tested for differential expression between the retina and WB samples. At ≥2.0 fold-enrichment and FDR <0.01 cut-off, 90 proteins had enriched expression in the retina compared to WB  did not contain a single protein that has been associated with retina development or defects/disease  candidates that have been associated with retinal biology and/or defects/disease  retina-enriched proteins prioritized by the  defects . The top defects . The enr defects , 1997. APE cells . FurtherPE cells .in silico-WB subtraction is the epidermal growth factor receptor (Egfr), which is associated with retinal cell fate determination , are associated with retinal dystrophy in two patients with Costello syndrome . Anothermination . Additiomination . The recmination . Furthermination . Morphol(Xenopus . Recessi(Xenopus .Xenopus retina (Some RNA-binding proteins (RBPs) involved in post-transcriptional gene expression control that are linked to retina development and differentiation were also among the prioritized proteins. Musashi homolog 1 (Msi1) is an RBP whose deficiency causes photoreceptor morphogenesis defect in mice . Anothers retina . Among os retina .Several proteins associated with the cell membrane and/or cytoskeleton were identified. Patients with mutation in the gap junction alpha-1 protein (Gja1) exhibit optic nerve and retinal dysplasia . The ankSimilarly, extracellular proteins as well as other cellular proteins with relevance to retinal biology were identified. Genetic variants in fibrillin-2 C-terminal peptide (FBN2) are reported to contribute to AMD . CytoplaSome genes that function in early retina development were also identified. For example, the transcriptional repressor CTCF (Ctcf) was among these candidates, and its deletion in mouse forebrain at E8.5 causes apoptosis and reduced retinal tissue by E13.5 . FurtherSeveral other proteins were identified that are independently reported to be expressed in retina, but their function has not been examined in detail. We identified several crystallins  that are known to be expressed in the retina and have been associated with retinal ganglion cell survival and regeneration . InteresA few other proteins identified here have been reported to be associated with eye defects or disease, but their specific function in the retina has not been explored in detail. For example, FRAS1-related extracellular matrix protein 2 (Frem2) is associated with Cryptophthalmos . Interesrd of the top 90 proteins identified by in silico-WB subtraction, renders confidence that other candidates may also have key roles in the retina and may be linked to its defects.Together, this documented association to retina biology of nearly 1/3in silico WB-subtraction to retina biology, a cluster-based analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID v6 .8) for functional annotation by gene ontology (GO) categories . As examples, the enrichment and expression in the retina, of proteins previously linked to retina defects, e.g., Hras and Tyr, are shown as visualized in iSyTE  such as Otx2, Sox2 and Vsx2 with key roles in the retina were not detected. This may be due to the following reasons. While they may be enriched in tissues, TFs are often in lower abundance compared to other expressed proteins . Although 2675 quantifiable proteins  were identified in the present study, since the above mentioned TFs were not among these proteins, this suggests that more sensitive methods would be needed to detect these proteins in future studies. Together, these datasets and their ready accessibility through the web-based ocular gene discovery tool iSyTE represent a rich resource for prioritizing candidates for future hypothesis-driven studies in retina development. Finally, this study serves as a proof of the principle that in silico subtraction can also be applied to the retina and RPE to identify promising new candidates in these tissues. In the future, this approach will be expanded to prioritize candidates in other developmental stages of the retina.Recent studies have highlighted that post-transcriptional regulation of gene expression plays a key role in determining the cellular proteome in eye development. Therefore, it is important to include ocular proteome data to the existing RNA-based profiling datasets to gain new insights into eye development. As a proof-of-principal we previously generated proteomic profiles for the mouse lens and the embryonic whole body (WB) and effectively applied proteins . Further


Pediatric sudden sensorineural hearing loss (SSNHL) is a rare otological emergency. With the emergence of the Coronavirus 19 pandemic, alcohol-based hand sanitizers are among the essential household items. Many hand sanitizers are frequently coupled with scents that young children may find pleasant.A 5-year-old girl presented to our clinic with hearing loss after the consumption of alcohol-based hand sanitizer. A pure tone audiogram showed bilateral SSNHL. The child was prescribed systemic corticosteroids resulted in a slight improvement in hearing thresholds. The child was followed up at 6 and 18 months showing no further improvement in hearing thresholds.Although various infective, vascular, and immune responses have been proposed, alcohol-based hand sanitizer consumption has not been reported to present with SSNHL to the best of our knowledge. In the current scenario of the Coronavirus pandemic, otorhinolaryngologists must keep in mind that SSNHL may occur as a result of hazardous alcohol-based hand disinfectant consumption. Sudden sensorineural hearing loss (SSNHL) is most commonly defined as a sensorineural hearing loss of 30dB or greater over at least three contiguous audiometric frequencies occurring within a 72-hr period . SometimThe COVID-19 (Coronavirus Disease-2019) pandemic has become a major worldwide public health concern, prompting widespread usage of hand disinfectants due to its contagious nature. Hand sanitizers are typically divided into two types: alcohol-based (ABHS) and alcohol-free. An ABHS may contain up to 60%-95% ethanol or isopropyl alcohol by volume, to be applied to the hands to eliminate microorganisms and temporarily limit their multiplication. ABHS can efficiently and swiftly eliminate germs over a broad antimicrobial spectrum without the need for water or towel drying. Alcohol-free sanitizers consist of specialized chemicals having antimicrobial activity . Many hand sanitizers are frequently coupled with scents that young children may find pleasant. Alcohol hand sanitizer consumption is linked to worse consequences than nonalcohol hand sanitizer exposure .Alcohol is quickly absorbed into the circulation from the stomach and small intestine . It has The cytotoxic effect of ethanol on vestibulocochlear ganglion causing hearing and balancing problems have been established . There hA 5-year-old female patient presented to pediatric emergency in June 2021 with chief complaints of nausea and vomiting for the last 6 hours with a history of consumption of sanitizer orally. According to the history given by her parents, she was drinking liquid from a bottle that was being used for sanitization during the ongoing Coronavirus pandemic. It contained 150 ml of liquid sanitizer consisting of Ethyl alcohol I.P 70% v/v with emollients and moisturizer. She had consumed 50-60 ml according to her parents. The general condition of the patient was fair. The patient was admitted to the pediatrics department and started intravenous fluids. All routine investigations  were sent to our laboratory and were at normal levels. The patient underwent gastric lavage. The ENT department was consulted for hearing loss developed while discharging the patient after 24 hours.A detailed history was taken, there was no history of hard of hearing before. Neonatal records of the patient were reviewed showing normal neonatal screening tests. Her speech and language milestones were normal. There was no history of tinnitus or vertigo. Also, there was no history of any medications other than intravenous fluids during admission to the pediatrics department before hearing loss. There was no past history pertaining to any neurological disorder or developmental abnormality. Otoscopy and other ENT examinations were normal. The patient underwent pure tone audiometry showing a bilateral sudden sensorineural hearing loss. Pure tone averages of 0.5, 1, 2, 4 kHz  for the Speech Discrimination Score (SDS) was 75% and 78% in the right ear and left ear respectively. Speech Reception Threshold (SRT) was 55 dB and 50 dB in the right ear and left ear respectively. Tympanometry showed Type A curve. st and 11th day audiograms. On the 11th day, Pure tone averages of 0.5, 1, 2, 4 kHz for the right and left ears were 40 dB and 36 dB (The patient was prescribed oral prednisolone of 1mg/kg/day for 10 days and then tapered. The patient was followed up with alternate-day audiogram, hearing improvement was around 15 dB comparing 1nd 36 dB . th day. The patient was prescribed bilateral conventional hearing aids. The patient was followed up after 6 months and 18 months, audiometric results were approximately the same as on 11th day.SDS was 85% & 90% and SRT was 40 dB & 35dB in the right and left ear respectively on the 11However, the major limitation of this case report is that we couldn’t use high frequency for a hearing evaluation. Also, we didn’t evaluate the disability by using any questionnaire.Studies on SSNHL in the pediatric population are sparse and have poor treatment outcomes . Common Pitaro J et al in a study established viruses as the most common etiology for SSNHL in the pediatric group . On reviDemir E et al. reported a case of a 6-year-old girl developing bilateral SNHL after drinking electronic cigarette liquid. The patient was treated with intravenous methylprednisolone 1mg/kg in tapering dose for 10 days with daily audiogram follow-up, with approximately 20 dB of hearing improvement . The nicAccidental ingestion of hand disinfectants causes an increase in blood alcohol levels leading to apnea, acidosis, delirium, vomiting, abdominal pain, nausea, throat irritation, drowsiness, loss of consciousness, and even death -22. In 2013, the Victorian Poisons Information Centre received 15729 calls pertaining to children under the age of five, with topical antiseptics/hand sanitizers being the seventh most common source of poison to which this age group was exposed . However, as in our case, unanticipated side effects can occur. Using alcohol hand sanitizers carefully, under adult supervision, and with suitable child safety precautions, as well as keeping them out of the reach of small children, may help to avoid unforeseen negative repercussions.Sudden sensorineural hearing loss is a medical emergency. Prompt diagnosis and early intervention may prevent permanent hearing loss. In the current scenario of coronavirus pandemic, otolaryngologists must keep in mind that SSNHL may occur as a result of hazardous alcohol-based hand disinfectant consumption.Patient’s parent’s perspectiveOur child consumed some amount of sanitizer after which she had nausea and vomiting for which we visited the hospital. After receiving treatment, she was well, but the next day she was not able to hear our words properly for which we were referred to ENT specialist. We were asked various questions regarding the birth and growth of the child and our child underwent various investigations. We got worried after knowing that investigations are in favor of sensorineural hearing loss. After consulting the ENT specialist, our child was given oral drug therapy and had hearing tests conducted every alternate day. After the completion of treatment, our child had a slight improvement in hearing. The doctor advised us to use hearing aids for both ears for proper speech and language development of the child and we were advised to follow up regularly for a hearing evaluation. We are thankful to doctors for guiding us.


Northwestern Pacific (NWP) tropical cyclones (TCs) impose a severe threat to the life and economy of the people living in East Asian countries. The microphysical features, mainly the raindrop size distributions (RSD) of TCs that improve the modeling simulation and rainfall estimation algorithms, are limited to case studies, and an extensive understanding of TCs’ RSD is still scarce over the northwest Pacific. Here, we examine a comprehensive outlook on disparities in microphysical attributes of NWP TCs with radial distance and storm type, using sixteen years of disdrometer, ground-based radar, and reanalysis datasets in north Taiwan. We find that dominant stratiform precipitation in the inner rainbands leads to the occurrence of more bigger drops in the inner rainbands than the inner core and outer rainbands. Moreover, a decrease in mass-weighted mean diameter and rainfall rate with radial distance is associated with a reduction in moisture availability for various circumstances, and this association is deceptive in intense storms. Our findings give an insight into crucial processes governing microphysical inequalities in different regions of NWP TCs, with implications for the ground-based and remote-sensing rainfall estimation algorithms. The precipitation microphysics of TCs, especially the raindrop size distribution (RSD), has been a paramount consideration in advancing the radar rainfall estimation algorithms and microphysical parameterization12.The Northwestern Pacific (NWP) Tropical cyclones (TCs)  associated with torrential rainfall attribute a severe threat to the life and economy of people living in East Asian countries17. Conversely, most previous observations were conducted with RSD samples using a limited number of TCs or a portion of TC rainbands. Studies performed with numerical simulations and remote sensing data demonstrated the disparities in convection and precipitation distribution with radial distance from the TC center24. In addition, despite the limited number of TCs measurements, recent studies have hinted at the inequalities in TC’s RSD with radial distance17. Nonetheless, up to now, an extensive framework that emphasizes the RSD features of TCs with their radial distance and intensity is yet to be known.Owing to the significant contribution of TC RSDs in hydrometeorology and earth surfaces process, there has been increasing interest in exploring the RSD information of TCs from different oceanic regionsTherefore, it is imperative to study the RSD of TCs in a long-term perspective to deduce their robust characteristics at different radial distances and category types. In this work, using long-term ground-based (disdrometer and radar) and re-analysis data sets, we elucidate the microphysical processes liable for the RSD changes with TCs radial distance and intensity. The results depict a decrease in rainfall rate and mass-weighted mean diameter with radial distance from the TC center, with a substantial decreasing pattern for intense TCs (CAT15) than the tropical storm category TCs. The microphysical attributions and rainfall retrieval relations disclosed for different intensities and radial distances can improve the TCs modeling simulations and radar precipitation estimation algorithms. The results present in this study provide conceivable microphysical attributes responsible for the RSDs variations at different radial distances and offer possible implications for the rain retrieval algorithms of ground-based and remote sensing radars.10R, R is in mm h–1), mass-weighted mean diameter , normalized intercept parameter , and mean radar reflectivity  profiles at 10 km radial distances from TC center. For the total precipitation of all TCs  gradually increases while moving radially outward from TC center. The contour plot of mean Z profiles for every 10 km shows higher Z values within 50 km, 100–130 km, and around 200 km, and gradually decrease beyond 200 km from the TC center. In the cold rain regions of TCs , the mean reflectivity values greater than 25 dBZ are visible around 50 km from the TC center. However, in the warm rain region of TCs, mean Z values greater than 25 dBZ are apparent around 50 km, 130 km, and 200 km. A further classification of all TCs precipitation into stratiform and convective types shows that the RSD parameters , and mean Z profiles in stratiform rain follow a similar distribution pattern to that of the total rainfall of all TCs. In contrast to stratiform precipitation, the RSD parameters in convective precipitation show a sharp decrease from TC center to 50 km, and these parameters increase above 200 km from the TC center. In the warm rain region of all TCs convective precipitation, the mean Z profiles show higher values (> 35 dBZ) up to 100 km, and above 200 km from the TC center. The extent of deep convective cores with Z values greater than 35 dBZ into the cold rain region is more predominant, especially from 340 to 450 km from the TC center.Figure 10R, Dm, and log10Nw) for total, stratiform and convective precipitations of different category TCs. For all TCs  than inner regions (RD1 and RD2)  depicts that the RSD parameters  of TDs have an inhomogeneous distribution with radial distance. However, for TSs, median values of Dm and log10R decrease and log10Nw increase, except at RD4. In the case of TCs of CAT15, Dm and log10R median values increase (RD1–RD2) and then decrease, and log10Nw values increase. Segregation of TCs of different categories into two precipitation (stratiform and convective) types shows that the RSD parameters  in stratiform precipitation  decrease (RD1–RD3) and then increase (RD3–RD5). Whereas, for TCs of CAT15, the median values of the three RSD parameters decrease from the TC center. Despite that, for the first two radial distances (RD1–RD2), TCs of CAT15 show higher (lower) median values of Dm (log10Nw) than TDs and TSs, and an opposite feature appears for the last two radial distances (RD4 and RD5)  in TSs than CAT15.Observed NWP TCs are segregated to tropical depressions (TDs), tropical storms (TSs), and category 1–5 (CAT15) with radial distances of 100 km each from the TC center  in TSs (TDs). In the case of stratiform precipitation value in the OB (IB). On the other hand, the RSD parameters show decreasing (increasing and then decreasing) tendency in TSs (TDs) from IC to OB. In the case of convective precipitation  values in the IB (IC) region. However, for TDs, three RSD parameters increase and then decrease from IC to OB. Figure To further investigate the disparities in the RSDs of TCs in different rain regions , the distribution of the RSD parameters at three rain regions of WP TCs are illustrated in Fig. Dm values, radial decrease (increase) in Dm (log10Nw) values signifies the increase in small drops leading to reduction in the rainfall amount from TC center to outer regions, i.e., from RD1 to RD5  characteristics of NWP TCs at different radial distances RD1–RD5) and rain regions  can be apprehended with the changes in thermodynamics and microphysics of precipitation. These changes can be related to crucial parameters like radar reflectivity profiles, moisture availability, melting layer height, and total column liquid/ice water content. Distributions of these cloud and precipitation parameters  at different radial distances (RD1–RD5) and rain regions  are portrayed in Fig. –RD5 and 20, the RD1 (0–100 km), RD2 (100–200 km), and RD3–RD5 (200–500 km) can be approximated to the inner core, inner rainbands, and outer rainbands. In the cold regions (above the melting layer) of CAT15 TCs, greater reflectivity values in the inner core suggest that the inner core is characterized by strong convective activity than inner rainbands, and this intense convection can radially transfer the small ice particle in the higher altitudes of the inner core to inner rainbands22. The small ice particles radially transferred from inner core to inner rainbands by deep and intense convection can suppress the vertical extent of convection and enhance robust stratiform precipitation in the inner rainbands. Further, relatively higher zero-degree isotherms  measured over Eastern China. Moreover, the RSD features documented for different rain regions of southern Ocean TCs are likewise to that of the present study16. Variability in RSD parameters with radial distance gives a clue to estimate the region-specific rainfall estimation relations such as radar reflectivity–rainfall rate (Z–R), shape–slope (μ–Λ), and mass-weighted mean diameter–rainfall rate (Dm–R) relations. The Z–R, μ–Λ, and Dm–R relations estimated for NWP TCs of different intensity and radial distances are provided in Table Z–R relations (Z = 300R1.4 for convective and Z = 32.5R1.65 for stratiform precipitation) or elsewhere relations (Z = 32.5R1.65/Z = 300R1.25)29. It is important to note that the Z–R relations adopted in Taiwan QPESUMS are quite different from the Z–R relations estimated in this study. Moreover, even though there were reports on the Z–R relations of NWP TCs for the Taiwan region, such estimates were constrained to the RSD samples of limited TCs11. Hence, the Z–R relations estimated in this paper could offer better QPE results for Taiwan typhoon rainfall events. The slope-shaper relations established for different rain regions of TCs of different intensity categories could advance the TCs’ RSD estimates for the ground-based polarimetric radars30. Furthermore, shape-slope and R–Dm relations derived in this study can progress the precipitation estimation algorithm of remote-sensing instrumentation 31.Because of the adaptation of present TCs’ rain region segregation into IC, IB, and OB using Yang et al.http://www.usno.navy.mil/NOOC/nmfc-ph/RSS/jtwc/best_tracks/), which provides TC’s center, longitude, latitude, maximum sustained surface wind speed, and minimum central pressures  track information (for the years 2005–2020) is obtained from the best track archive of the U.S. Navy’s Joint Typhoon Warning Center , are used for the TCs rainy periods during 2005–2020 , mm–1 m–3), rainfall rate , radar reflectivity factor , liquid water content , mass-weighted mean diameter , normalized intercept parameter , shape parameter , slope parameter 35. The 1-min RSD samples with rainfall rates greater than 0.1 mm h–1 are considered in the present study. A good agreement between the disdrometer measured daily accumulated rainfall amounts and the collocated rain gauge for the considered TCs gives the trustworthiness of the disdrometer measurements for further analysis  measurements from the Joss‐Waldvogel Disdrometer 20.The RSD measurements of the disdrometer are considered as TC’s RSDs if the distance between the disdrometer site and the TC center is ≤ 500 km. Furthermore, based on the distance between the disdrometer site and TC center, the rain regions of the NWP TCs are classified into five radial distances, i.e., radial distance 1 (RD1), radial distance 2 (RD2), radial distance 3 (RD3), radial distance 4 (RD4) and radial distance 5 (RD5), if the distances between the TC center and disdrometer site are 0–100 km, 100–200 km, 200–300 km, 300–400 km, and 400–500 km, respectively  were used to generate the contour frequency by altitude diagrams (CFADs) over the disdrometer site  are obtained from six ground-based radars’ (red color triangles in supplementary Fig. Supplementary Information.


This article highlights the importance of early identification and surgical treatment for extremely rare traumatic perilymphatic fistula (TPF) caused by an earpick, which can pose the risk of irreversible hearing loss. Herein, we have described two cases of TPF and reviewed the literature primarily based on surgical treatment for penetrating ear trauma-induced TPF. We highlight the case of two females who sustained an accidental penetrating injury in the ear caused by the introduction of an earpick, leading to hearing loss and dizziness. Pure tone audiometry detected elevation of the bone-conduction thresholds. Computed tomography of Labyrinth revealed pneumolabyrinth in one case. Both patients underwent exploratory surgery, we completely repositioned the stapes that had invaginated into the vestibule in one case, in the other case, we reconnected the disarticulated incudostapedial joint and sealed perilymph fistula caused by rupture of the oval window. Both patients achieved hearing improvement and complete relief from the vestibular symptoms.The literature review indicated that a scar on the posterior aspect of the tympanic membrane was found in 44.4% of cases. Hearing improvement was observed in 45.5% and 25.0% of cases with invagination of stapes and fractured footplates by fistula repair, respectively. In terms of handling stapes dislocation, the hearing improvement rate was better in cases of complete stapes repositioning (66.7%) than those of complete or partial stapes removal (16.7%). Preoperative mild bone-conduction hearing loss or localized pneumolabyrinth are favorable factors for satisfactory hearing. When surgery is performed within 11 days of the injury, satisfactory hearing improvement can be expected. Penetrating trauma through the ear can injure the middle ear structure, potentially leading to perilymphatic fistula. However, it can occasionally be missed or misdiagnosed because of the complicated signs associated with mixed impairment in the ossicles or inner ear. For cochlear dysfunction, once traumatic perilymphatic fistula (TPF) develops, sudden or progressive sensorineural hearing loss can manifest in the days after trauma ,2.Earpicks are sticks used for cleaning ear wax and are more popular in Asian countries. They are usually made of harder materials, including wood, metal, or plastic, and have a slightly sharper tip compared to a cotton swab. While inserting the earpick, unfortunately, forces the sharp tip into the ear canal, such as falling or someone hitting the arm, the tip can easily wound the deeper external ear meatus. In such cases, the middle ear structures are likely to be injured through the perforation of the tympanic membrane (TM). This trauma can simultaneously interrupt the ossicular chain, which poses the risk of stapes dislocation and resultant TPF; however, such cases are extremely rare, and reviews related to this topic are scarce.We encountered two cases of TPF that required urgent exploratory tympanotomy. Further, the present article discusses the surgical strategy for penetrating trauma-induced TPF.Case 1A 31-year-old woman sustained a forceful penetrating injury in the external ear meatus caused by an earpick while cleaning her ear. She visited a hospital the same day because of dizziness and tinnitus. Otoscopy revealed swelling of the posterosuperior aspect of the right TM without perforation. She had horizontal-rotatory positional nystagmus to the right. Pure-tone audiometry (PTA) detected moderate bone-conduction hearing loss Figure . The airCase 2A 39-year-old woman accidentally thrust an earpick deep into the left ear canal while scratching there. She experienced dizziness immediately after the accident, prompting her to visit the hospital on the same day. Otoscopy revealed a perforation in the posterosuperior aspect of the TM. She had horizontal-rotatory gaze nystagmus to the left. The PTA revealed left mixed hearing loss Figure . DespiteWe present the cases of two patients with rare TPF caused by earpick injury who achieved a satisfactory outcome in hearing and vestibular symptoms following surgical restoration. Literature related to penetrating injury-induced TPF confirmed by surgery was reviewed Table  1-16].-16.1-16]Symptoms and clinical findingsAll patients with penetrating trauma-induced TPF suffered from vestibular and cochlear symptoms, including conductive, sensorineural, or mixed hearing loss. In terms of otoscopic views, scars on the posterior aspect of the TM were present in 44.4% cases Table . AccuratRole of computed tomographyHRCT can confirm the configurational abnormality of stapes, such as the stapediovestibular luxation or fractures. In typical external ear trauma, stapes dislocation is rarely encountered because the stapes is protected from direct injury by the overhanging scutum ; howeverIndication and optimal time to surgeryCurrently, there is no specified protocol towards surgical intervention for TPF. Given the lack of definitive diagnostic criteria in practice, conservative treatment is initially preferred. Although there may be an impact of publishing bias, most published cases ultimately progressed to surgical treatment. Contrary to idiopathic fistulae, disrupted annular ligaments or fractured footplates in TPF may cause more copious perilymph leakage, eventually leading to complete inner ear dysfunction . TherefoRepair for injured stapesThe repair method for dislocated stapes in TPF remains unstandardized and variable, without achieving consistent hearing outcomes. Table There has been controversy in treating invaginated stapes. One surgeon advocated that removal of the invaginated stapes should be avoided due to risk of further sensorineural hearing loss . MeanwhiComorbid sensorineural hearing lossThe hearing outcome may be related to the severity of preoperative sensorineural hearing deterioration. Our cases and the review support the notion that satisfactory hearing outcome is anticipated for TPF with a mildly elevated bone-conduction threshold . MeanwhiPneumolabyrinthA critical sign of perilymphatic fistula is pneumolabyrinth, which was observed in 83.3% of cases Table . AeratioMeanwhile, the presence of pneumolabyrinth is not indicative of active TPF. Some researchers ,12 have Limitations First, publication bias was evident in our review. When a favorable prognosis was not expected, clinicians generally did not recommend surgery. This might limit the generalizability of our review to patients who do not undergo surgery because of profound hearing loss. Second, exploratory tympanotomy is mandatory for diagnosis of TPF; however, surgical indications may be specific to each center. Therefore, cases managed with conservative treatment may have been unintentionally excluded from our review, resulting in the exclusion of undiagnosed TPF.Persistent or progressive vestibulocochlear symptoms after a penetrating trauma should raise suspicion of TPF. Worsening symptoms, a scar on the posterior aspect of the TM, or stapes dislocation or pneumolabyrinth on CT can help in the early identification of TPF. Preoperative mild bone-conduction hearing loss or localized pneumolabyrinth can be favorable factors for satisfactory hearing outcomes. Although the impact of stapes injury type or its repair method on hearing prognosis remains uncertain, complete stapes repositioning may be considered, even for an invaginated stapes. Surgical repair of the fistula should be considered immediately once TPF is suspected. Even for TPF with moderate or severe sensorineural hearing loss, surgical hearing restoration should be expected within approximately 10 days of injury.


Pycnoporus cinnabarinus, offers a highly interesting spectrum of bioactivity, partly due to the formation of the orange–red pigment cinnabarin. However, apart from a few studies addressing its diverse potential biotechnological applications, there is still a large gap of knowledge concerning the influence of light on the formation of cinnabarin. The aim of this work was to investigate the effect of different irradiations on the cinnabarin content, the growth, and the morphology of three different P. cinnabarinus strains. We used highly standardized irradiation conditions and cultivation techniques in combination with newly developed methods for the extraction and direct quantification of cinnabarin.The demand for natural pigments in general, and for fungi-derived pigments in particular, is constantly rising. Wood-decomposing fungi represent a promising source for natural pigments and they are usually easy to cultivate in pure culture. One of them, i.e., Ee = 1.5 ± 0.18 W m−2) had considerable effects on the growth and colony appearance of all three P. cinnabarinus strains tested. The cinnabarin content determined was, thus, dependent on the irradiation wavelength applied, allowing strain-specific thresholds to be defined. Irradiation with wavelengths below this strain-specific threshold corresponded to a lower cinnabarin content, at least at the intensity applied. The orange–red pigment appeared by light microscopy as incrusted extracellular plaques present on the hyphal walls. Highly efficient vegetative propagation occurred by arthroconidia, and we observed the tendency that this asexual reproduction was (i) most frequent in the dark but (ii) never occurred under UV-A exposure.Red, green, blue, and UV-A irradiation (mean irradiance P. cinnabarinus. This confirms that it is advisable to consider the wavelength of the light used in future biotechnological productions of natural pigments. This study highlights a differential photo-dependence of growth, morphology, and cinnabarin formation in The online version contains supplementary material available at 10.1007/s43630-023-00493-3. Fungi, including fungal endophytes ).pigments . In addisleeanus .Pycnoporus  is a genus of bracket fungi forming bright vermilion, console-shaped annual fruiting bodies on dead wood. The main pigment of these conspicuously colored fruiting bodies is cinnabarin, an amino substituted phenoxazine-3-one derivative +, [M + K]+, [M + Na]+; instrument: Orbitrap, MS2 mass accuracy (ppm): 5 ppm, candidates stored: 10; min candidates per ion stored: 1; use DB formulas only: all. ZODIAC was used with default settings. The CSI:FingerID fingerprint prediction +, [M + K]+, [M + Na]+; general: score threshold activated, search DBs: all. Lastly, the CANOPUS compound class prediction was performed + at m/z = 287.0649. All relevant spectra for the classic chemical characterization : δ = 9.60 , 8.74 , 7.55 , 7.53 , 7.49 , 6.63 , 5.50 , 4.89 . UV/Vis (MeOH): λmax = 202, 233, 430, and 446 nm. IR (cm−1) = 3267w, 2916s, 2849s, 1672w, 1655m, 1591s, 1580s, 1466m, 1302w, 1187w, 1081m, 974m, 912w, 857w, 787m, 689m, 608w, 562w, 506w, 450w,434w.Cinnabarin (CAS 146-90-70) was obtained as a red solid from the acetone extract of ine with . Furthertion Fig –S6 and ttion Fig . 1H-NMR c = 100 µg/mL) were used to prepare 14 calibration levels  by dilution in DMSO. The resulting solutions were immediately analyzed by HPLC–DAD as described above. Integration of the target peak was done with Origin 2020 and the calibration curve was calculated using a linear regression in MS Excel 365 .Two separately weighed stock solutions in DMSO  R2 = 0.9997, (iii) linear range = 2.28 E−4 - 0.11 mg mL−1, (iv) LOD = 4.96 E−4 mg mL−1, and (v) LOQ = 1.50 E−3 mg mL−1.The following calibration curve parameters were determined for cinnabarin based on the ICH guideline : (i) regP. cinnabarinus and its red–orange pigmentation. To determine the optimal temperature for growth in the subsequent irradiation experiments, triplicates were cultivated at ten different temperatures and kept in darkness to exclude the factor light  revealed MEA as the best match for the growth of ght Fig. .Fig. 1 RPycnoporus cinnabarinus strains were highly temperature tolerant up to T = 42 °C  was not reached at T = 25 °C  Thm = 12 to 55 mg per Petri dish.Irradiation with different wavelengths showed no consistent effect on the formation of fungal biomass for the three strains Fig. . After 7The highest cinnabarin content relative to biomass was found for strain IBF20170021, which at the same time showed the lowest growth compared to the other two strains Figs. . The oppOverall, the cinnabarin content correlated clearly with the irradiation wavelength in a species-specific pattern, i.e., blue light and UV light increased the cinnabarin content in all three strains compared to cultures grown in the dark. However, the resulting cinnabarin quantity was highly strain specific Fig. .Cinnabarin was constitutively present in all strains and its levels were generally low in cultures grown in the dark or under red light. As indicated before, the content depended on the irradiation condition and differed between the strains. Increased cinnabarin contents were detected above a certain, strain-specific photon energy. Below this energy, the content was statistically indistinguishable from the one under dark conditions. In two strains (IBF20160231 and IBF20180012), the content was slightly increased under green irradiation with respect to dark or red conditions. However, the difference was insignificant. In contrast, in strain IBF20170021, green irradiation increased the detectable cinnabarin content by a factor of 4 to 5 compared to dark or red conditions. Irradiation with high energetic blue or UV-A light increased the cinnabarin content in all strains even more.To answer the question, if the boost of the cinnabarin content under blue or UV-A irradiation was in the same order of magnitude for all strains, the contents were expressed relatively to the cinnabarin content of the respective strain under dark conditions Fig. . This noPycnoporus cinnabarinus exhibited quite characteristic features in pure culture, and several of the micro-morphological characteristics were photo-induced. As these cultures were obtained from fruiting body tissues, the initial mycelial growth was represented by dikaryotic, hyaline generative hyphae with clamp connections at the septa. The diameter of generative hyphae ranged from 2.5 to 4.0 µm.Generative hyphae differentiated either into unbranched, thick-walled skeletal hyphae (diameter 2.5–4.0 µm), or into arthroconidia. Skeletal hyphae are usually unbranched and thick-walled hyphae without clamp connections. They are formed by hyaline generative hyphae gradually transforming into pigmented, thick-walled hyphae. When exposed to natural light conditions, hyphae of all transitional morphological stages produce conspicuous, extracellular, incrusted pigments forming irregular bands and crusts on the hyphal surface. These epihyphal pigments could be dissolved in 3% KOH and were highly concentrated in older cultures, where they were also present as very dark reddish brown, amorphous matter situated between the hyphae.The observed intensity of hyphal pigment production Fig.  and coloP. cinnabarinus pure cultures . This was also true for more or less circular shaped colonies, which sometimes formed a considerable amount of aerial hyphae in random areas. Thus, the culture diameter did not provide the required accuracy as an estimate for fungal growth or biomass.−2), and (ii) embedded in a Petri dish without deionized water for autoclaving. This procedure resulted in only slightly wrinkled cellophane discs, which could be applied flat on the surface of the solidified medium without trapping air bubbles. This is important, as air bubbles can cause inhomogeneous nutrient availability, resulting in reduced fungal growth.We, therefore, used dry weight as estimation for the fungal biomass, allowing us to normalize the cinnabarin content more adequately. Different methods for estimating fungal biomasses have been mentioned in the literature, such as (i) melting or cooking of agar plates , (ii) thEe = 1.5 ± 0.18 W m−2) was chosen for all wavelengths. This specific value was selected based on the maximal irradiance of the LEDs with the lowest power, i.e., the green one. In addition to this low irradiance, a 3.7-fold higher irradiance  was selected for the UV-A irradiation to better simulate the conditions at higher altitudes. The LIGHT BOX was developed specifically for a highly standardized photo-treatment of solid-state cultivations. As fungal growth on and in solid substrates is common in natural habitats—and therefore generally physiologically advantageous for the formation of secondary metabolites [Light is a well-known factor in triggering fungal growth and secondary metabolite formation . It is, abolites —, solid abolites .λpeak (FWHM) = 635 (18) nm, 519 (38) nm, 452 (19) nm, and 369 (13) nm, i.e., red, green, blue, and UV-A) were applied. For comparability of the impact, the irradiance was standardized to Ee = 1.5 ± 0.18 W m−2. Surplus, near-visible UV-A with an elevated irradiance of Ee = 5.6 ± 1.8 W m−2 was tested to elucidate a possible dosage dependency of UV-A irradiation and content of pigments.To distinguish possibly different effects of distinct irradiations on growth and content of cinnabarin, different spectra  resulted in an increased content of pigments. This indicates a possible protective function of cinnabarin against damage caused by high-energy light, or against changes in the habitat occurring synchronously along with these light dynamics.With UV-A and blue light, both, the formation of biomass and the content of cinnabarin, were enhanced in all three strains in the same order of magnitude, with respect to dark conditions Fig. . One expXanthoria parietina [In addition, the aggregation of pigments and the exact location in relation to the hyphae Fig.  give a farietina , UV protDuring vegetative growth within the woody substrate, hyphae are usually not exposed to light. However, irradiation becomes a prominent environmental factor as soon as hyphae reach areas close to the substrate surface. Precisely under these conditions, the cinnabarin content increases. Thus, the possible functions might be as diverse as the changed conditions: for example, an elevated fitness (i) in competitive interactions with other microorganisms  or macro-organisms (such as fungivore repellents in fungus–fungivore interactions), (ii) in reproductive mechanisms , and (iii) for hyphal growth.P. sanguineus, the cinnabarin content was increased by opposite irradiation conditions. In detail, the production of extrolites was highest in the absence of light and only partially inhibited by red light, whereas it was reduced by > 85% under green and blue light irradiation [Compared to the formation of extrolites  in the related species adiation . On the The transition from vegetative growth to a reproductive survival strategy is usually induced by changes in the availability—or the properties—of the substrate and the surrounding habitat. For example, vegetative growth and highly efficient vegetative reproduction might be preferred within the nourishing substrate, i.e., in the dark . In contP. cinnabarinus, the formation of such dikaryotic vegetative spores Below is the link to the electronic supplementary material.


The relationship between obesity and hearing loss among the middle-aged and older population remained unclear. Moreover, few studies have focused on the impact of gender on this association.This cohort study extracted the data from the China Health and Retirement Longitudinal Study, a national survey of adults aged 45 years or over. Waist circumference was categorized into three groups: normal, pre-central obesity, and central obesity. We classified BMI into four categories: underweight, normal weight, overweight, and obese. The primary endpoint was the incidence of self-reported hearing loss.Of the 14,237 participants, 1972 incidents of hearing loss were identified during a median 6.9 years of follow-up. The cumulative incidence of hearing loss was 13.9% (95% CI 13.3% -14.4%). Our study showed that central obesity was significantly associated with hearing loss , and this relationship was more prominent in males . Among male participants, the underweight group was at the highest risk of hearing loss . Compared with the normal weight group, the adjusted HR for hearing loss in the obese groups was 0.69 (95%CI 0.51–0.94) among men. Among female participants, only the overweight group had a lower risk of hearing loss than the normal weight group .Being overweight and obese were significantly associated with a decreased risk of hearing loss, whereas being underweight was associated with an increased risk of hearing loss. With the changes in modern lifestyles, being overweight and obese are becoming a global concern. According to the most recent national survey data, over half of the adults in China are either overweight (34.3%) or obese 16.4%) [6.4% [1].Hearing loss is a highly prevalent disabling chronic condition, with more than 466 million people worldwide affected by this condition . EmerginRecently, accumulating evidence has been supporting the “obesity paradox,” or the possible health benefits of being overweight. A survey by JAMA reports that diabetes patients who are overweight or obese had a lower mortality rate . SimilarIn total, there currently exist gaps in the knowledge of the associations of BMI levels and waist circumference with the incidence of hearing loss. This study was therefore aimed at exploring the associations of BMI levels and waist circumference with hearing loss, over a median 6.9 years of follow-up, utilising data from a community-based longitudinal study of Chinese elderly derived from the China Health and Retirement Longitudinal Study (CHARLS) for its large sample size, containing mid-aged and elderly participants and the thorough and detailed information on the exposure and outcome.Established during 2011–2012, the CHARLS cohort enrolled 17,708 adults aged over 45 years from 450 study communities across China. The baseline data collected included: sociodemographic information, lifestyle behaviors, health status, and functioning, physical measurements , and blood samples . Details of the design and survey methods have been described elsewhere .For the present analysis, 24,805 adults from the baseline survey  and refreshment samples (n = 7097) who engaged in periodic follow-up were identified for this present analysis. Refreshment samples are new sample members recruited as the study progresses to better represent younger participants . ParticiWaist circumference was measured to the nearest 0.1 cm using soft measure tape at the navel level at baseline. We categorized waist circumference into three groups: normal , pre-central obesity , and central obesity  .2), normal weight (18.5-<24.0 kg/m2), overweight (24.0-<28.0 kg/m2), and obese (≥ 28 kg/m2).Height and weight were measured by trained investigators with the participants standing bare feet on a stadiometer (Seca™ 213) and weight scale (Omron™ HN-286), respectively. BMI was calculated as body weight in kilograms divided by the squared height in meters to measure general adiposity. We classified BMI into four categories based on Chinese criteria : underweThe primary endpoint was the incidence of self-reported hearing loss. We defined hearing loss as a response of “Poor” to the question “Would you say your hearing is excellent, very good, good, fair, or poor?”, or a response of “Yes” to the question “Do you have one of the following disabilities: hearing problem” in the follow-up interviews. Self-reported hearing loss has been verified as a valid tool previously –24.hukou status , household expenditure, smoking , alcohol consumption , diabetes (yes or no), and hypertension (yes or no). Diabetes was determined by meeting any of the following 4 criteria: (1) ≧ 126 mg/dL fasting glucose; (2) ≧ 200 mg/dL non-fasting glucose; (3) a self-reported physician diagnosis of diabetes; (4) treatment with hypoglycaemic medication [Similar to previous studies , 25, 26,dication . Hypertedication .2 tests between sexes, respectively. Person-years at risk were calculated from the baseline date until a report of hearing loss, death, loss to follow-up, or the end of follow-up(the date of wave 4 survey). The incidences of hearing loss were calculated by dividing the number of events by person-time at risk. A cox proportional hazards regression analysis was performed with the presence of hearing loss as a dependent variable and waist circumference/BMI as an independent variable to estimate the hazard ratio (HR) and 95% confidence interval (CI). Considering the potentially increasing mortality rate with age, competing risk regression models (death as competing risk) using the Fine and Gray model were performed to estimate subdistribution hazard ratios (SHRs) and 95%CIs [Characteristics of the study population are described as means (SDs) for continuous variables and percentages for categorical variables, with differences evaluated using Student’s t-tests and Pearson χd 95%CIs . MultivaAll analyses were performed separately for men and women using STATA statistical software, version 15.0 (StataCorp LLC). All p-values were two-tailed, and the level of statistical significance was defined as P < 0.05.A total of 14, 237 individuals were included in the analysis, including 6,993 men and 7,244 women, with a mean (SD) age of 57.35 (9.19) years for men and 57.22 (9.32) years for women (P = 0.390). Compared with women, men were more likely to smoke tobacco  and consume alcohol . Female participants had a higher prevalence of diabetes , a higher proportion of central obesity , and a higher measure of BMI  , 885 incident cases of hearing loss among men and 1087 cases among women were documented. The incidence rate of hearing loss per 1000 person-years for men was 24.57 (95%CI 23.00-26.25) and 28.36(95%CI 26.72–30.09) for women. The rate ratio of incident hearing loss for men compared with women was 0.87 .In multivariable-adjusted cox regression models, compared with the normal group, central obesity was significantly associated with decreased incidence of hearing loss in men  but not in women . A similar but not statistically significant trend was observed in the pre-central obese group Table .Among men, compared with the normal weight group, the adjusted HR (95%CI) for the risk of hearing loss was 0.69 (95%CI 0.51–0.94) for the obese group, whereas underweight was inversely associated with hearing loss . Corresponding data for women exhibited a similar trend, although the association seemed less evident than for men. The respective hazard ratios among women were 0.87 (95%CI 0.71–1.07) and 1.17 (95%CI 0.91–1.49) (Table 2), underweight male participants with healthy waist circumference had a higher risk  of developing hearing loss, whereas overweight and obese male participants with central obesity (waist circumference ≥ 90 cm) had a lower risk for hearing loss with HRs 0.75 (95%CI 0.60–0.95) and 0.70 (95%CI 0.51–0.97), respectively. However, we did not observe a significant association among female participants  and BMI 18.5–24.0 kg/m.0 kg/m2,During the follow-up, we documented 467 deaths among men and 284 deaths among women. Overall, the strength of associations in the competing risk models slightly reduced compared with the main analysis. The association between waist circumference and hearing loss remained significant among men but not women Table . SimilarIn this study, central obesity was significantly associated with a decreased risk of hearing loss in males but not in females over a 6.9-year period. Compared with the normal weight group, participants in the overweight and obese group had a lower risk of hearing loss, whereas those who were underweight had a higher risk of hearing loss.In our study, being underweight was significantly associated with an increased risk of hearing loss. This pattern was especially evident for underweight participants with normal waist circumference. This finding was consistent with a recent population-based study conducted among 161,052 Korean subjects, in which the rates of hearing loss in the underweight groups were significantly higher than the normal weight group (24.9% vs. 20.4%) . This reWhile our study demonstrated a possible protective role obesity had on hearing loss, previous studies find the opposite relationship. For example, several previous studies suggested that obesity was an independent risk factor for hearing loss –9. A recOf note, the effect of central obesity on hearing loss was solely found in males rather than in females, showing a sex-related difference in the “obesity paradox”. Similarly, previous studies reported that the obesity paradox in heart failure outcomes appears to be more pronounced in males than in females , 52. NevStrengths of this study include its nationally representative sample, large sample size, longitudinal design, validated methods to quantify BMI and waist circumference, and high participation rate. However, this study contains some limitations. First, the assessment of hearing loss was based on self-report, which may result in an underestimation of the hearing loss incidence. Nevertheless, other studies have shown that self-reported hearing loss was a valid measurement among Chinese  and otheIn conclusion, our study suggested that being overweight and obese were significantly associated with a decreased risk of hearing loss in the middle-aged and elderly over a 6.9-year period. Being underweight was observed to be positively associated with hearing loss. Further work is needed to explore the underlying mechanisms. The results from our study suggested that for the middle-aged and elderly, gaining an appropriate amount of weight may reduce the risk of hearing loss and have a positive effect on hearing function.


Papio ursinus). We successfully identified giving and receiving grooming with high precision (81% and 91%) and recall (87% and 79%). Giving grooming was associated with a distinct rhythmical signal along the surge axis. Receiving grooming had similar acceleration signals to resting, and thus was more difficult to assign. We applied our machine learning model to n = 680 collar data days from n = 12 baboons and found that grooming rates obtained from accelerometers were significantly and positively correlated with direct observation rates for giving but not receiving grooming. The ability to collect continuous grooming data in wild populations will allow researchers to re-examine and expand upon long-standing questions regarding the formation and function of grooming bonds.Quantification of activity budgets is pivotal for understanding how animals respond to changes in their environment. Social grooming is a key activity that underpins various social processes with consequences for health and fitness. Traditional methods use direct  observations to calculate grooming rates, providing systematic but sparse data. Accelerometers, in contrast, can quantify activity budgets continuously but have not been used to quantify social grooming. We test whether grooming can be accurately identified using machine learning (random forest model) trained on labelled acceleration data from wild chacma baboons ( The obssensu ‘biological markets' : 0.85g [0.62g–1.09g]) showed no overlap with any other behaviour, and foraging and travelling had medians that fell outside the interquartile ranges of all other behaviours . Conversely, the three ‘inactive' behaviours  showed substantial overlap in VeDBAs ranges . Median VeDBAs for resting (0.031 g) was slightly higher than for receiving grooming (0.027 g) , which probably led to the overestimation of receiving grooming during the night (see ‘Activity budgets').Smoothed VeDBA (VeDBAs) was the most important variable for distinguishing among behaviours b. VeDBAsb). The interquartile ranges for static acceleration channels overlapped for all behaviours, but the interquartile range for receiving grooming was consistently the largest followed by resting , suggesting that a large range of postures is adopted during these behaviours.Static acceleration along the heave and surge axes (both provide information on posture) was also important, with stZ and stX ranked second and fifth, and pitch (forward/backward rotation) ranked fourth b. The ing [0.00007g–0.0002g]; figure 1b). This suggests that giving grooming occurs on a regular low-amplitude frequency .Three of the power spectrum densities (PSDs), i.e. PSD2X, PSD1Z, PSD2Z were in the top 10 most important variables. Notably, PSD2X was important for identifying giving grooming with a median that did not fall within the interquartile ranges of other behaviours (median [first and third quartile]: 0.0001. 3.2c). Resting had the lowest precision (79%) and recall (77%) and was mostly confused with giving or receiving grooming (c). Walking, foraging and running had high precision and recall . The slightly lower recall for walking compared with other active behaviours was primarily due to instances misclassified as foraging . Receiving grooming had 91% precision and 79% recall, while giving grooming had 81% precision and 87% recall c. Restingrooming c. Walkinforaging , probabl. 3.3−1, total collar days = 680; electronic supplementary material, table S8). Baboons spent on average (mean ± s.d.) 21.4 ± 9.0% of their time resting, 18.8 ± 6.3% giving and 30.0 ± 8.1% receiving grooming . When restricting the collar data to direct observation hours (07.00–17.00) the baboons spent 19.0 ± 9.6% of their time resting, 18.7 ± 6.6% giving grooming and 15.2 ± 6.5% receiving grooming . See electronic supplementary material, tables S8 and S9 for active behaviours .Activity budgets were calculated by applying the random forest model to the dataset (24 h db) associated with resting overlapped more with receiving grooming during the night than during the day, which was the case .Based on the results of the accelerometer-identified activity budgets, which suggested receiving grooming may be confused with resting (particularly during the night), we calculated whether VeDBAs , but not for receiving grooming . Rates from accelerometer and focal data were significantly correlated for resting  and running  but not for foraging  and walking . Focal sampling resulted in lower rates of receiving grooming  but not significantly different rates of giving grooming  compared with accelerometer data. Focal sampling showed higher rates of foraging , but lower rates of walking  and running . There was no significant difference in resting rates between the two methods .Overall behavioural budgets (during observation hours of 07.00–17.00) calculated using focal and accelerometer data (based on random forest models) revealed comparable activity budgets  budgets for each individual. Third, we compared rates of grooming obtained from focal data (direct observation) with rates obtained from accelerometer data (using the random forest model). Below, we discuss each objective and associated findings in turn. We also discuss the implications of this methodological advance for the study of social grooming and future avenues for its application.This study aimed to quantify grooming from accelerometer data using machine learning. We first used random forest models to identify receiving and giving grooming (and other behaviours) from accelerometer data collected from This study is the first to identify grooming with high precision and recall for both actors  and receivers  using tri-axial accelerometer data. Compared with previous work on male baboons only , the focX-axis (surge) which falls outside the interquartile ranges of any other behaviours . This surge (back-and-forth) motion makes sense when considering the typical grooming rhythm, in which the actor repeatedly moves their hands forwards and across the recipient in front of them. Power spectra are typically used for identifying locomotion which produces repeated oscillations [Felis catus) is also associated with differently paced cyclic patterns along the surge axis [Canis dingo), ‘self-grooming’ was classified as a ‘medium' activity class (repetitive head movement) associated with higher overall dynamic body activity (ODBA) compared with resting behaviours [Acceleration profiles for giving grooming show that sufficient movement takes place to produce a distinctive cyclic pattern, with a median PSD2 along the llations ,69,96,97rge axis . In a sthaviours . Moreovehaviours ,68.b). By contrast, the relatively small but repetitive movements during giving grooming were discernible in the PSDs (figure 1b).Taken together, the present and previous studies suggest that the act of giving grooming, while stationary, can still produce a distinct acceleration pattern that is discernible from other stationary behaviours . Our study also highlights the value of performing further waveform analyses to obtain descriptive statistics of how signal varies across time (e.g. to detect repetitive patterns), rather than relying on measures of general body activity. For instance, previous work on captive rhesus macaques using omni-directional accelerometers  successfully differentiated ‘active' from ‘inactive' behaviours , but thefigure 1b).For receiving grooming, the acceleration profile presents a challenge due to its resemblance to resting (the difference between sitting versus sitting while being groomed is inevitably subtle). Previous studies testing the use of tri-axial accelerometry to differentiate between non-active or slowly executed behaviours suggest that there are limits to what fine-scale changes in movement can be detected using accelerometers alone ,99. As bfigure 1There are several reasons why resting may generate more overall body movement than receiving grooming. First, based on the labelled dataset, daytime resting is a behavioural state that is relatively brief compared with receiving grooming . Thus, resting does not necessarily reflect uninterrupted periods of relaxation (which would presumably be associated with very low VeDBAs), but rather takes place as a relatively brief pause between activities. Consequently, when taking a moving average across a 3 s window (as was done to obtain VeDBAs), the calculation will take into account a second of the behaviour that precedes and follows resting. Quick transitions can result in misclassifications of behaviours , and, cog (b). Essentially, this reduces the distinction to: was the baboon ‘still' or ‘very still'? Based on the results from the random forest model, ‘still' corresponds to ‘resting’ and ‘very still' corresponds to ‘receiving grooming' in a relatively reliable way within the training data (approx. 23 h), but the assumption may not hold across all contexts for the full study period , which should be borne in mind when interpreting receiving grooming budgets (see below). Future studies, or further exploration of this dataset, could investigate ways to distinguish receiving grooming and resting with more certainty. Using GPS-identified dyads may help, i.e. if two baboons are spatially close and are classified as giving and receiving grooming, respectively, this may further help confirm receiving grooming. However, baboons often groom in small sub-groups [Leptonychotes weddellii [Halochoerus grypus [The above notwithstanding, receiving grooming and resting are similar from an accelerometry perspective with a difference in VeDBAs of less than 0.1g b. Essentb-groups , and it b-groups ,106. Foreddellii ) and bets grypus ), respecs grypus  and wriss grypus . Self-dis grypus  and can s grypus ,111, thus grypus , but this grypus . Collar-Suricata suricatta [Desmondus rotundus [Equus caballus [The ability to quantify grooming continuously (as made possible by using collars) opens a wealth of potential questions for investigation, for instance in the fields of socio-endocrinology , socioecuricatta , Desmondrotundus , Equus ccaballus ), groomib). This raises the question whether receiving grooming is more prone to confusion with resting during the night compared with the day, which may be the case if resting during the night is more still (due to sleeping) compared with resting during the day . Indeed, other studies treat resting and sleeping as separate behaviours due to the difference in energetic demands [Our second objective—calculating activity budgets based on random forest models—revealed an important aspect of the evaluation of random forest model performance. The accelerometer-identified activity budgets across 24 h suggest that overall baboons spent on average 30% of time engaged in receiving grooming, 19% giving grooming and 21% resting. When restricting the time window to direct observation times (07.00–17.00), baboons spent on average 15% receiving grooming, 19% giving grooming and 18% resting. The steep increase in receiving grooming (±15%) when including night hours, compared with a modest increase in resting (±2%), strongly suggests that resting during the night is misclassified as receiving grooming . As disfigure 1 demands ,125 and  demands .The median/range VeDBAs calculations for resting during day versus night in the present study are in line with this, showing more overlap in VeDBAs between resting and receiving grooming at night . As a consequence, when the random forest model is trained using a biased testing dataset (e.g. daytime resting but no night-time sleeping), it can appear to function well in the first phase  but, once applied to a new independent dataset, make predictions that are comparable to random guesses . When coAs a third and final objective, this study offered a unique opportunity to compare acceleration-based activity budgets with direct focal observations . Usuallyb), while giving grooming (figure 4c) and resting (figure 4a) were comparable between the two methods. These findings suggest focal data are returning both different and relatively lower individual rates for receiving grooming, but not the other two stationary behaviours. The lack of correlation might be due to the relatively small range in individual rates of receiving grooming  visible in figure 4b), compared with giving grooming and resting ranges  in the focal data, which could make it harder to detect individual differences in receiving grooming and thus correlate to accelerometer-identified rates. Receiving grooming was also the least frequent of the three stationary behaviours, both in focal and collar identified rates .The relative breakdown of behaviours was comparable between the two methods, with baboons spending most of the day foraging, followed by resting, giving grooming, receiving grooming, walking and running . Moreover, active behaviours such as walking and running are probably underestimated as the focal individual is more easily lost ; C Chris, individOverall, this study presents a step towards the quantification of social grooming in unprecedented detail, but its applicability largely depends on the specific research aim(s) and the feasibility of using collars. Deploying collars requires ethical , logisti