PTPRN2 and DAXX in LMS, WT1-AS and TNXB in SS, VENTX and NTRK3 in pleomorphic RMS and MEST and the C14MC / miR-379/miR-656 in MFS. Our overall findings indicate the presence of objective epigenetic differences across primary and Met/Rec human tissue samples not previously reported.In this study we used the Illumina Infinium Methylation array to investigate in a cohort of matched archival human tissue samples (n = 32) from 14 individuals with soft tissue sarcomas if genome-wide methylation changes occur during metastatic and recurrent (Met/Rec) disease. A range of sarcoma types were selected for this study: leiomyosarcoma (LMS), myxofibrosarcoma (MFS), rhabdomyosarcoma (RMS) and synovial sarcoma (SS). We identified differential methylation in all Met/Rec matched samples, demonstrating that epigenomic differences develop during the clonal evolution of sarcomas. Differentially methylated regions and genes were detected, not been previously implicated in sarcoma progression, including at  Clusters rich in CpG sites, known as CpG islands, are involved in the control of transcription and gene expression, wherein unmethylated CpG islands allow transcription, whilst hypermethylated promoter regions repress gene expression4. Methylation alterations have been implicated in transformation of mesenchymal stem cells during sarcomagenesis6. Moreover, comprehensive DNA methylation profiling in soft tissue sarcomas (STSs) has previously shown that such epigenetic modifications are crucial in the biology and prognosis of several sarcoma types including its value as a tool to refine tumour classification15 (Ref added). Nonetheless, the contribution of genome-wide DNA methylation changes during progression of STSs has not been widely explored in matched metastatic/recurrent (Met/Rec) sarcoma samples. The Illumina Infinium MethylationEPIC BeadChip18 array interrogates over 850,000 methylation sites including CpG islands and has been previously validated on restored DNA from formalin-fixed paraffin-embedded (FFPE) tissue22 (References added). High correlation has been observed in these studies between matched fresh and FFPE tissue samples despite the lower quality of FFPE tissue. Thus it is now possible to interrogate archival FFPE clinical samples from patients who present with metastatic or recurrent disease during the natural course of sarcoma evolution.DNA methylation, a specific type of epigenetic modification, leads to changes in gene expression without modifications of the DNA sequence. It involves the addition of a methyl group to the carbon-5 position at cytosine residues on cytosine-phosphate-guanine (CpG) dinucleotides23). In the only study available with comprehensive genomic (but no epigenomic) data on specific sub-types of matched metastatic/recurrent sarcomas, Hofvander et al.24, demonstrated no major changes occurred at the genetic level during clonal evolution. The authors hypothesized that in the absence of obvious genomic differences, it is possible that epigenetic modifications play a major role in the metastatic cascade of sarcomas. DNA methylation changes were not identified to occur in a single study available of matched primary and recurrent well-differentiated liposarcomas25 but methylation analysis on other paired sarcoma types has not been published to date.Methylation status becomes a more attractive target of analysis given genome-wide studies have not revealed specific genetic drivers of sarcoma metastasis , whilst rare, is an aggressive sarcoma with poor prognosis and is also characterised by a complex karyotype20. In contrast Synovial sarcoma (SS) and Embryonal rhabdomyosarcoma (Emb-RMS) present in younger individuals, with the latter more commonly seen in a paediatric population. SS is characterised by the t translocation27 and Emb-RMS (sporadic) frequently harbours recurrent genetic alterations including LOH at 11p15 and polysomy 826. A shared issue across sarcomas regardless of their molecular signature and histological type, is a lack of biological predictors to determine which patients will remain free of disease and who will progress to develop advanced incurable tumours due to recurrence and/or metastatic disease. Furthermore, for those patients with refractory disease, there are no specific therapeutic targets available. In this study we sought to investigate in a limited number of FFPE sarcoma samples (n = 32) from 14 individuals  whether methylation changes at a genome-wide level arise in metastatic or recurrent disease. Our aim is to discover whether such changes play a role in the biology of recurrent/metastatic behaviour, which may enable the identification of predictive/prognostic biomarkers and/or potential therapeutic targets. Although our cohort is limited, our study is the first to address the contribution of methylation in progression of selected sarcoma types on paired human tissue samples.Leiomyosarcoma (LMS) and Myxofibrosarcoma (MFS) are examples of some of the most frequent sporadic sarcomas in the adult population26. The tumours were graded according to the French Federation of Cancer Centers Sarcoma Group (FNCLCC) system. Histopathological criteria for diagnosis included the use of immunohistochemical stains (IHC) and fluorescent in situ hybridization (FISH) depending on the tumour entity  Human Research Ethics Committee (HREC) reference 1312-417 M. Informed consent was not obtained as this is a retrospective cohort of de-identified archival tissue samples, which is compliant with HREC for de-identified human tissue samples. All methods included in this study were performed in accordance with the relevant guidelines and regulations. A retrospective database search was performed in the archives of Douglass Hanly Moir (DHM) Pathology laboratory to identify FFPE tissue blocks derived from surgical resections (biopsies excluded) of patients with selected sarcoma types , who developed metastatic (Mets) and/or recurrent (Rec) tumours within a 5-year period and whose residual archival tumour blocks were adequate for further molecular analysis . All the cases were originally diagnosed in the clinical setting by pathologists with expertise in sarcomas and/or gynaecological pathology following international guidelines as per the World Health Organization (WHO) classification and independently reviewed for this study to ensure diagnoses were in keeping with the current WHO 2020 classifications of soft tissue tumours.28. Infinium HD FFPE Restore kit  was used for DNA restoration.For DNA extraction, 1 mm tissue cores (n = 6–10 per case) were taken from FFPE tumour blocks using a tissue microarrayer (TMA) on samples with at least 50% of tumour cellularity. Specific details of DNA extraction, bisulphite conversion and restoration can be found in supplementary methods. Briefly, DNA was extracted using the ReliaPrep FFPE gDNA Miniprep System  and DNA integrity was assessed by real time PCR . Five-hundred ng of DNA was bisulphite-converted using the EZ DNA Methylation kit . The effectiveness of bisulphite conversion was assessed by methylation specific PCR following the method of Esteller et al29 and the ‘lumi’ Bioconductor package30. As tissue replicates were not feasible for this disease model we were restricted to direct comparisons of the tumour sites. To investigate the distribution of CpG sites, a beta-value difference of > 0.2 was selected to define probes with differential methylation between groups analysed as previously described20. Beta-values range from 0 to 1, where 0 indicates unmethylated and 1 indicates fully methylated31. The DMRforPairs32 package in R was used to identify differentially methylated regions (DMRs) of the genome. The DMRforPairs model is specifically designed to assess samples where replicates are not available. ‘Regions’ were defined as those with a minimum of 4 probes within a < 200 bp distance of each other with a median difference in M values > 1.4 between the samples of each Set. Regions were considered significant when the Benjamini Hochberg corrected p values were < 0.05. DMRs were sorted by adjusted P value and the top most significant region was provided. Variance analysis was used to rank probes by the greatest absolute difference and was calculated on the matrix of M-values in each pair of samples using the var command in R. The top 50 and 500 most variable probes with the largest difference in methylation were extracted from each sample and displayed on a heatmap  to view the methylation patterns among the top contributors. Functional analysis using the KEGG Pathway database for pathway mapping34 was used to identify the top 500 most variable probes in each set. Please refer to Suppl. Methods for full details.Genome-wide DNA methylation profiling was performed on restored DNA using the Illumina Infinium MethylationEPIC BeadChip , which interrogates over 850,000 methylation sites, including CpG islands. The BeadChips were scanned using an Illumina iScan and analyses were undertaken using the R statistical environment (v. 3.5.1)http://bioconductor.org/packages /release /bioc /html/conumee.html) was used to obtain CNV. Controls were taken from a publicly available EPIC submission of GIB  consisting 4 replicates. The CNV calling algorithm underlying Conumee -DNACopy- uses a threshold of < -0.3 and > 0.3 for copy loss and gain, respectively.This was performed by Array chromosomal genomic hybridization (aCGH) on 5 samples, but only 2 matched samples (SS) using SurePrint G3 Human CGH Microarrays, 8 × 60 K  and CytoGenomics Software  for analysis (Suppl. Methods). For all additional samples the program Conumee in R  presented in females with age ranging from 32 to 62 and primary site including uterus (n = 5), omentum  and kidney . Metastatic disease presented within 1 to 5 years of the initial diagnosis to the lung (n = 4), abdominal cavity (n = 2) or vagina  presented equally in males and females, age ranging 60 to 76 years and all were FNCLCC grade 3 with Rec/Met deposits presenting 1 to 2 years apart. Apart from one case with brain metastasis (set 2) the remaining 3 sets comprised recurrent tumours at the same anatomic site  originated in the spermatic cord of 17-(Emb-RMS) and 57-year old (Ple-RMS) males who did not receive therapy prior to surgery. Metastasis developed in the para-aortic lymph nodes (1 month: Emb-RMS) and brain  presented on the thigh of a 31 year-old woman, who developed multiple metachronous lung metastases (> 10 metastatic deposits resected on 6 separate operations) including recurrence within a 3 year period. No therapy was received prior to the resection of the primary tumour. She received chemotherapy and developed chemotherapy-induced cardiomyopathy and heart failure, and died 3 years after the primary diagnosis. No significant morphological differences, including complete absence of TILs, were identified across the different stages of progression Table .Figure 4Successful methylation profiles were obtained from all 32 samples. Fig. . Due to Variation in global methylation levels .Histopathological review on H&E-stained histological sections  clustered together indicating that these were more closely correlated with each other than to stages of progression  3; HLA-J, FAM50B and DAXX across two different DMRs on chr 6; PCDH20 on chr 13; HSPA2, PPP1R36, LTB4R and CIDEB across two different regions on chr 14; and DGRC6 and PRODH on chr 22. Although specific DMRs varied across the sets, consistently increased methylation levels were identified in Mets versus primary tumours for most of the identified DMRs  in both cases  and probes within these regions were differentially methylated between primary and metastatic LMSs: PTPRN2, KCNQ1 and MCF2L, all located to CpG islands, were the most commonly shared probes over all paired comparisons.Variance analysis (Suppl Table DGRC6 & PRODH at Chr 22 & set6 with LTB4R & CIDEB at Chr 14), all remaining samples did not show CNVs in the identified chromosomal regions. This excludes chromosomal aberrations as the underlying mechanism of differentially methylated genes in 5 of 7 LMS pairs . For set 2, hypermethylation of DMR in the metastatic deposit correlated with copy gain within the same chromosomal region and concordantly for set 6, copy loss in Mets 2 correlated with hypomethylation of the specific chromosome. However, CNV in both cases was only above the call threshold . For sFinally, enriched KEGG pathway analysis identified hypermethylated and hypomethylated pathways (Suppl. Tables MEST (chr 7), C14MC microRNA cluster , SNRPG and FAM136A (both in chr 2) and CCND2 (chr 12). The most variable probes identified through variance analysis were TBX15 (chr1), PLEC1 (chr8) and CDH15  showed minor changes in CNVs across the pairs . The most significant pathway identified by Reactome pathways analysis was the Receptor Tyrosine Kinase signalling followed by fibroblast growth factor family genes (FGFR3 and FGFR3c) and WNT signalling. Additional KEGG pathway analysis is shown in Suppl. Tables In Ple-RMS, the top DMR was located on chr 10q including the es Figs. ; Table 3es Figs. . The mosOLFML2B gene. KEGG pathway analysis is shown in Suppl. Tables In embryonal RMS, the top DMR was located on chr 20 including a 165 bp region, which showed decreased methylation in Mets compared to Primary . No genes were identified in this region and no underlying copy number variation was identified  and TNXB and SP2D6 (both within the same region on chr6) were in the top 10 differentially methylated genes between Primary versus Mets1, Mets2 and Rec. The primary sample revealed low methylation levels for both regions , which markedly dropped in Mets1, Mets2 and Rec  and in Mets4 (Chr12 containing RP11-366l20.2). These regions were not detected in the remaining samples  were similar in their level of methylation but methylation changes and DMRs were also identified across stages of progression. Interestingly, Rec showed more similar methylation levels and clustered more closely to Mets than to the Primary tumour Figs. . WT1-AS les Fig. . ThroughKEGG pathway analysis showed that enriched hypermethylated probes included insulin secretion and calcium signalling whilst pathways enriched with hypomethylated probes related to downregulation of cancer signalling pathways (Suppl. Tables Array CGH was performed on the primary SS and one of the metastatic deposits (Mets 2). This analysis confirmed, as expected, that SS is associated with simple karyotype as no significant chromosomal gains, losses or amplifications were detected neither in primary SS nor in Mets 2. Importantly, acquisition of complex genomic aberrations was not observed in Mets2 with the plots for both samples showing essentially identical appearance Fig. . Thiese In this study we performed for the first time, genome-wide methylation comparative analysis of matched selected sarcoma samples obtained at different time points during the evolution of the tumours. Differential methylation was consistently identified in Met/Rec disease in all our matched samples  reflecting the presence of objective epigenetic differences across primary and Met/Rec human tissue samples not previously reported.24, hypothesised that in the absence of genomic drivers, it is possible that methylation changes may play a major role in the metastatic cascade of sarcomas and our study aimed to address this hypothesis in spite of limitations due to FFPE-derived DNA and the small number of cases. Although sarcomas tend to develop late metastases, our cohort was enriched by sarcomas with aggressive behaviour as Met/Rec deposits occurred within 5 years after the initial presentation. We selected this biased cohort with the aim to identify key changes developing early during clonal evolution of aggressive sarcomas associated with recurrence and/or metastatic propensity.Although it would have been ideal to perform complementary genomic analysis, no significant gene mutations and/or gene drivers were reported to occur in selected matched sarcoma samples in the only study published to date using whole-exome sequencing and SNP arrays . In that study, Hofvander et al.12 in line with our data. In the context of LMS, this was uniformly seen in all cases of initial metastatic disease (Mets1) but not maintained in subsequent metastatic deposits (Mets2 and Mets3) for the two cases analysed. The reverse trend was identified for SS and Emb-RMS, which revealed hypomethylation in Met/Rec disease compared to the primary tumour. Finally, no specific methylation pattern was identified for MFS paired samples, which showed the largest variation in methylation levels compared to any other sarcoma type and which was independent of the content of TILs and FNLCC grade as most tumours (7 of 8 samples) lacked TILs and were grade 3.Global methylation patterns varied according to sarcoma type. Almost all LMSs and Ple-RMS showed DMRs associated with low levels of methylation in the primary tumour (unmethylated) but with increased methylation in paired metastatic samples. Interestingly, low methylation has been identified in primary sarcomas of high-risk sarcoma patients with poor prognosisDAXX and PTPRN2. DAXX (Death Domain–Associated) forms a dimer with ATRX  and dysfunction of this ATRX/DAXX complex is associated with alternative lengthening of telomeres (ALT), which occurs in a proportion of LMSs37. Moreover, loss of ATRX and/or DAXX has shown to predict aggressive clinical behaviour in LMSs and other smooth muscle tumours38. Accordingly, we identified hypomethylation of DAXX in Mets2 versus Mets1, which would correlate with gene loss / silencing in aggressive LMSs. PTPRN2 (Receptor-type tyrosine-protein phosphatase N2), identified through variance analysis as one of the most differentially methylated probes shared across most LMS samples, has been previously linked to primary LMS39. In a single study in breast cancer cells, PTPRN2 was shown to regulate actin dynamics, enhancing metastatic migration40 and based on this, we can hypothesise that PTPRN2 is also likely to act as a key regulator in the metastatic cascade of leiomyosarcomas but functional analyses are required to confirm this. Methylation modifications in other of the identified genes 44 have been described in carcinomas but their role in the pathogenesis of leiomyosarcoma progression has not been reported (apart from PCDH2 and PRODH45), and should be further explored. Finally, we also looked into overlapping genes in our list with other similar studies of primary LMSs. There are only 3 comparable studies using genome-wide methylation profiling45. The Cancer Genome Atlas Research Network, in their subset analysis on primary LMS9, identified that differences in methylation across these tumours were predominantly driven by anatomic location (uterine vs. soft tissue LMSs). Moreover, LMSs clustered in two groups with differential methylation signature, which resulted in differences in recurrence-free survival9. Although shared genes were not identified with our study, it is difficult to draw conclusions from this in view of the different study design and biological question. Miyata et al.45, on a methylation analysis of a limited number of normal myometrium, leiomyomas and leiomyosarcoma  identified global hypomethylation in LMSs, which is consistent with our findings, including overlapping genes . Interestingly, hypermethylation of protocadherin genes was significant in their study45 and also identified in some of our samples (PCDH20 and PCDH) but no previously identified in LMSs. We can hypothesise that some of these shared genes may be associated with progression or may represent key genes defining a malignant phenotype. Kommos et al.13, performed genome wide methylation for tumour classification in a range of uterine tumours including LMSs and carcinomas. Although specific genes were not discussed in their study (or in supplementary files) the authors showed a specific methylation signature in LMS, allowing classification according to histological type.We identified differentially methylated regions and genes in LMSs, which were unrelated to underlying CNV indicative of primary epigenetic events for these specific samples. From our gene list, two genes have been previously linked to LMS sarcomagenesis—MEST, C14MC, FAM136A, SNRPG, CCND2, TBX15, PLEC1 and CDH15 have not been previously reported in this sarcoma type. Gene silencing of MEST, a key regulator of IL-6/JAK/STAT3/Twist-1 pathway-mediated tumour metastasis, has been demonstrated in other sarcomas47 and concordantly, we observed hypomethylation for the DMR containing MEST in one of our sets. Downregulation of C14MC, also known as miR-379/miR-656 cluster, has been described in brain tumours48 and others49, and was significantly hypomethylated in the metastatic deposit in one of our cases. SNRPG, involved in mRNA splicing50, and other genes have been involved in carcinogenesis but methylation dysregulation in sarcomas has not been described before. As MFSs are known to be genetically unstable and characterised by the acquisition of multiple genomic and epigenomic aberrations during progression51, it is unlikely that individual genes and methylation modifications identified in these tumours are primary driving events. Our CNV analysis demonstrated low level of CNV for 2 of the 4 pairs but no evidence of gene amplification or homozygous deletions. Therefore, it is possible that copy gain/loss may account for some of the methylation differences in these samples but this is uncertain. Finally, we sought to identify common genes in our study with others published in primary MFS. Ogura et al.10, in the largest study of primary MFSs with epigenetic and genetic data (n = 41), identified 3 clusters with distinct methylation signatures. Although only one of their genes (PLEC) overlapped with ours, both our study and theirs, identified differential methylation of cell adhesion-related genes. Ogura et al.’s genes included CCND1 and CTNNB1 whilst genes from the same family in our cohort included CCND2 and CDH15 together with downregulation of cell adhesion pathways.The most variable genes differed among each MFS pairs. The roles of WT1-AS and TNXB became unmethylated in Rec/Mets. WT1-AS is a long non-coding RNA and the antisense transcript of Wilms’ tumour 1 gene (WT1). Downregulation of this gene enables cell proliferation, migration, and invasion leading to cancer progression in multiple cancer types, which has been correlated with advanced disease and poor clinical outcome56. TNXB encodes a tenascin which promotes epithelial mesenchymal transition (EMT) and is downregulated in cancer58, wherein methylation correlates with gene expression65. In agreement with the known downregulation of these genes in cancer, both TNXB and WT1-AS were unmethylated in Mets/Rec samples of SS, indicative of gene loss/downregulation during progression of SS, which has not been previously identified in this tumour type. Importantly, WT1-AS can negatively regulate transforming growth factor β (TGF-β) and TP53 and coincidently, TNXB also interacts with TGF-β, further facilitating EMT57. We can hypothesise that WT1-AS and TNXB epigenetic inactivation and downregulation in SS leads to activation of the TGF-β pathway. Accordingly, alterations in the TGF-β1/SMAD pathway and dysregulation of the EMT process, which enables cell invasion, migration, and proliferation, have been demonstrated in cell culture of SS cells66. Hence, TGF-β pathway inhibitors, already developed67, may potentially have effect in controlling metastatic/recurrent disease in SS. Further studies are required to confirm this. Additional genes  were also identified as unmethylated in individual stages of progression (Rec and Mets3) but were not shared across the samples. This suggests that differentially methylated subclones are likely to arise due to epigenetic dysregulation during the evolution of SS. Finally, by performing aCGH on two of the samples (Primary and Mets2), we demonstrated that the development of differentially methylated regions in metastases occurred in the absence of the acquisition of unbalanced chromosomal abnormalities as Mets2 maintained a simple karyotype identical to that of the untreated primary tumour , differentially methylated regions and genes were identified between the primary tumour and Rec/Met disease , being a potential target for immunotherapy (Reviewed in68). Interestingly, our case showed a high number of TILs in the primary tumour, which were completely absent in the brain metastasis. We can only hypothesise that VENTX downregulation may have played a role in altering the composition of the TME in this case and may be a potential therapeutic target in sarcomas associated with immune cell content. Although differences in the gene expression and methylation signatures of the TME are associated with significant prognostic differences in patients with STSs12, such differences in the TME are unlikely to explain the results in our remainder samples, as most of our cases (10 of 14 paired cases) were over-represented by so-called ‘cold tumours’, devoid of TILs. On the other hand, the role of UTF1 (undifferentiated embryonic cell transcription factor 1), which plays a role in stem-cell development69 and carcinogenesis72, and NTRK3 in RMS has not been described. NTRK3 methylation has been reported in other tumours73 and although tyrosine kinase inhibitors are widely available for NTRK-translocated tumours, the effect of these TRK inhibitors has not been explored in tumours with NTRK gain or loss due to methylation. In Emb-RMS, OLFML2B, Olfactomedin-like 2B, was the most variably methylated probe. This has not been previously investigated in this tumour type but it has been described as an oncogene in gastric carcinoma74.We analysed two unrelated types of rhabdomyosarcoma and although methylation changes have been consistently proven in Emb-RMS, these have not been studied before in Ple-RMS. In Ple-RMS, differentially methylated genes between Primary and Mets included Our findings also identified a level of correlation between variation in methylation levels and ‘histomorphological progression’ across matched pairs. Specifically, we identified that acquisition of morphological features  in Mets/Rec correlated with higher degree of global methylation. Concordantly, lack of morphological changes during stages of progression  correlated with lower variance in global methylation. As individual histomorphological features cannot be objectively quantitated, this conclusion is only observational.77). Nonetheless, most of the current understanding on the effects of irradiation on DNA methylation derives from in-vitro and in-vivo experimental systems with no significant data addressing the translational relevance in human tissue samples. Although we performed detailed morphological characterisation of the samples, currently there are no specific histological markers detected on the cancer/sarcoma cells, which allows distinction between changes secondary to treatment effect versus dedifferentiation or histological transformation/progression. With regards to the low number of samples included in our series, it is likely that our findings are significant in the leiomyosarcoma cohort as this has the largest number of cases, but for the other tumour types, we cannot exclude that genes and DMRs identified are the result of chance variation. Due to these limitations of the sample size, further validation in expanded sample cohorts is required to establish whether these specific markers can be of prognostic significance or therapeutic targets in advanced sarcomas.Our study has significant limitations, which include the low number of cases for each sarcoma type and the history of chemo/radiotherapy, mostly for treatment of subsequent metastatic/recurrent tumours Tables , 2. It iPTPRN2 and DAXX in LMS; WT1-AS and TNXB in SS; VENTX and NTRK3 in Ple-RMS and MEST and C14MC / miR-379/miR-656 cluster in MFS) that have been previously described in context of carcinoma progression and metastasis. Lines of further enquiry could aim to establish (1) if these potential candidate genes can be subjected to targeted therapy or (2) whether they may have utility as prognostic or predictive markers, allowing the identification of patients with sarcomas prone to develop metastasis or recurrent disease. As our cohort is enriched for sarcomas which developed early metastases and/or recurrence, it is possible that methylation for these genes may identify a subset of patients, who would benefit from more aggressive and early clinical management. In summary, we were able to demonstrate that methylation changes occur in all the sarcoma types analysed during progressive disease and appear to have an impact in the metastatic cascade.In summary, our study is the first to identify the role in sarcoma progression of differentially methylated genes (Supplementary Information.


To setup novel diagnostic method, to update current tests available in National Residue Control Plans (NRCPs) and in legal dispute when illicit practices on farm animals are suspected, the reliability of RGN profiling was assessed by histological and molecular techniques.Sex steroids administration in meat producing animals is forbidden within the EU to preserve consumers’ safety, but continuous monitoring to identify resurgence of their misuse is needed. Among biomarkers related to sex steroids abuse in veal calves the regucalcin (RGN) mRNA perturbations in testis have been described in RNAFormalin fixed paraffin embedded (FFPE) testis samples, chosen being the most effective preservation strategy adopted by histological NRCPs and allowing easier retrospective analysis if required by legal disputes, were analyzed from veal calves treated with nandrolone, 17β-estradiol and a cocktail of the two hormones. RGN levels were determined by quantitative Real Time PCR and Immunohistochemistry assays. Test performances were assessed and compared by multiple ROC curves.Both tests resulted sensitive and specific, allowing to enrich, in future field investigation, novel integrated diagnostic protocols needed to unveil sex steroid abuse.Developed RT-qPCR and IHC methods confirmed RGN as a useful and robust biomarker to detect illegal administration of sex steroid hormones in veal calves. The developed methods, successfully applied to ten years old FFPE blocks, could allow both retrospective analysis, when supplementary investigations are requested by authorities, and future implementation of current NRCPs. The use of sex steroids in food producing animals is forbidden within the EU to preserve consumers’ safety. Notwithstanding, use of prohibited designer drugs which are otherwise unmonitored by National Residue Control Plans (NRCP) was highlighted in the annual technical report of the European Food Safety Authority (EFSA) on drug residue analysis .Moreover, cocktails of drugs can also be used to escape official controls: the combined use at low concentration of different molecules gains sum effect and determines low residues levels, often hardly identifiable by routine monitoring also when performed by analytical methods like High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS).Illicit growth promoters’ cocktails are known to cause, together with increased meat production, several perturbations in different tissue biomarkers that can be exploited to setup diagnostic screening tools improving the efficacy of surveillance systems . RecentlHowever, their applicability as suitable diagnostic tools for daily testing of samples collected from slaughterhouses in the frame of NRCPs  could beConsidering recent implementations introduced for nucleic acid analysis on formalin fixed paraffin embedded (FFPE) tissues  the possFurthermore, when positive results for suspected use of steroids and/or steroids-like activity compounds are obtained by the untargeted histological screening approach applied in Italy according to the Residue Control Plans in place, further confirmatory tests are required.later reagents, preliminary storage at 4 °C for 24 h, removal of excess reagents before storage at −20 °C, etc.).The aim of this work was therefore to compare RGN mRNA with RGN protein levels on paraffin fixed testis tissue, in order to setup and combine these diagnostic tests for sex steroid hormones detection in veal calves in the frame of NRCPs. The new diagnostic protocol set up could also allow retrospective analyses on archival material when needed, without the complications and the extra costs expected for conventional RNA preservation procedures on collected samples  and analysed. Specifically, 40 testis blocks from calves similar in age (7 months old on average) treated with one of the four different protocols applied (see below) were tested.n = 10; 50 mg/head/week; four intramuscular injections), 17β-estradiol , a cocktail of both nandrolone and 17 β-estradiol  and 10 untreated control animalsThe experiment was authorised by the Italian Ministry of Health and the Ethics Committee of the University of Turin and was carried out according to European Economic Community Council Directive 86/ 609, recognised and adopted by the Italian Government (DLgs 27∕01∕1992 no. 116).All animals were treated from the sixth to the seventh month of age and slaughtered after a withdrawal period of at least 20 days. Treatment schedules and groups sizes were defined as follow: nandrolone  transcripts were designed to amplify the as short as possible amplicons and they were specifically targeted to the same genomic regions selected by Real Time PCR Taqman MGB assays were designed with Primer Express 3.0 Software (Applied Biosystems). To further enhance test sensitivity a one-step retro-transcription and quantitative Real Time PCR (RT-qPCR) strategy was chosen: this approach based on gene specific reverse transcription and multiplex Real Time PCR has been shown by previous studies as the one of the most fast and accurate strategies for gene expression analysis on FFPE material .Preliminary checks for primers, probes and secondary structures were performed by Primer Express 3.0 software and Mfold analysis .Total RNA extraction from collected FFPE testis was performed using the RNeasy FFPE kit  following the manufacturer’s recommendations.Quantity of extracted RNA and residual DNA was assessed respectively by Qubit BR-RNA and HS-DNA fluorometric assay  while RNA quality was checked by Bionalazyer 2100 using RNA 6000 Nano Chip . RNA samples selection for downstream RT-qPCR was made considering a RIN (RNA Integrity Number) cut of 1.4, characterized, according to previous studies , by a prThe RGN and PPIA cDNA synthesis and PCR amplification was performed multiplexing two Taqman assays in single 20 µL reactions, containing 11 µl of QuantiNova Probe RT-PCR Master Mix (Qiagen), 0.2 µL of 100x QuantiNova RT Mix (Qiagen), a final concentration of 0.8 µM for each primer, 0.2 µM of two TaqMan probes respectively, and 400 ng of total RNA. Primers and probes of the two assays are reported in The same modified PCR mixes adopted for standard curves amplification were also used to test RNA samples without retro-transcription step. These negative controls are needed to check unexpected amplifications signals from RGN/PPIA amplicons cross-contamination and/or presence of targeted pseudogenes in residual DNA.Paraffin sections (3 +/-2 µm) from all the 40 veal calves’ testes samples were deparaffinised and rehydrated in graded alcohols. Heat-induced (97 °C) citrate buffer (pH 6) antigen retrieval was applied in a water bath for 30 min. Slides were then incubated with 3% hydrogen peroxide for 30 min at room temperature to inhibit endogenous peroxidase activity. Sections were incubated with anti-RGN rabbit polyclonal antibody  diluted 1:100 for 60 min at room temperature. The EnVision System Kit  was used for identification purposes. Diaminobenzidine-hydrogen peroxide solution (Dako) was used as chromogen and applied for 4 min. Sections were slightly counterstained with hematoxylin, dehydrated, cleared and mounted. The specificity of the staining was assessed by the omission of the primary antibody.Digital images were obtained with a Nikon DS-Fi1 colour digital camera .A quantitative analysis of immunohistochemistry (IHC) was performed. Five digital images  were obtained with a Nikon DS-Fi1 colour digital camera (Nikon Instruments) for each slide and then a quantitative analysis of immunohistochemistry (IHC) was performed by NIS-Elements 4.5.Each image was analysed with the pixel classificatory, a tool that enables the user to group together and classify a range of similar pixels in an image. After the definition of the groups of pixels to be included in phase one on the bases of colour and dimension, the algorithm was saved. Afterward all images were automatically analysed and data on percentage of phase 1 expressed as percentages of positive tissue area versus total tissue area were exported on a data sheet for statistical analysis.p-value <0.05).Outlier detection for repeated measures was performed by the Grubbs’ test. For the analysis of FFPE samples by a quantitative approach alternative to ΔΔCq, the copy-ratio of RGN-PPIA was then calculated from quantified copy number of each target using a standard curve method. Main standard curves parameters such as linearity, slope, R2, Limit of Detection (LOD) and Limit of Quantification (LOQ) were determined as previously reported  for both relative quantification (RQ) by ΔΔCq  and stanreported , by bothreported . The LOQp <0.05.Regarding IHC determinations Shapiro–Wilk test was used to test for the normality of data. The one-way Kruskal–Wallis test and Dunn post hoc test were then performed to investigate differences among the groups when the data did not follow the normal distribution. Data were expressed as median and standard deviation. Significance was declared at Total RNA was extracted from all the 40 FFPE samples; RNA yields by fluorometric quantification ranged between 226 ng/µl and 2.41 µg/µl, while RIN ranged from 1.9 to 2.4.Regarding the gDNA yields fluorometric quantification ranged between 7.6 ng/µl and 300 ng/µl despite the DNAse incubation performed during extraction step: the unexpected high yields of residual gDNA on several tested samples were further checked by no-RT control reactions (only qPCR without RT step as previously stated), confirming presence of late Cq amplification plots from PPIA pseudogene copies that are present in bovine gDNA. However, a difference higher than 5 Cq between qPCR-only amplified RNAs and respective samples submitted to RT-qPCR was found. According to Relative Quantification analysis by the ΔΔCq method were then performed on all samples: comparative study confirmed that androgens cause a mean 2.62 fold reduction, estrogens cause a mean 3.52 fold reduction and the association of the two hormones causes a mean 6.16 fold reduction of RGN mRNA levels compared to untreated animals . ANOVA rParameters of daily standard curves loaded in each plate together with cDNA samples are reported in For PPIA/RGN copy-ratio calculation and ROC curve application, an optimal cut-off at 0.03118 was selected : at the p < 0.0001) in RGN expression was observed in all treated animals  compared to the control group. The results are reported in The IHC showed that RGN protein was predominantly expressed in Leydig cells and also in a variety of germ cells . AlthougROC curve was then calculated also for IHC test ; with a Graphical representation of both ROC curves was reported in In recent years FFPE materials, collected and stored for conventional histological and IHC analysis, are becoming more and more interesting for nucleic acid analysis . Long teTo further extend the applicability of FFPE samples profiling in current veterinary inspection and sampling activities, in order to improve detection of illicit treatments by growth promoters in meat production, a combined use of quantitative methods based on Immunohistochemistry and Polymerase Chain Reaction techniques was applied to RGN, an interesting biomarker responsive to sex steroids exposure recently described by RGN was first identified in 1978 as a calcium (Ca2+) binding protein . RGN expThe objective quantification of histological results is becoming essential to define strict and well established protocols. Recent studies showed indeed that quantitative data could allow a more precise correlation to clinical or biological data .The evaluation of IHC labelling for RGN in our study was therefore performed by automatic analysis to produce data that are more rigorous and on a continuous scale compared to qualitative and/or semiquantitative IHC approaches.IHC quantification data and cut-off setting by ROC curve showed satisfactory performances in terms of test specificity and sensitivity, allowing an automated and cheaper analysis when compared to RT-qPCR analysis performed on the same biomarker.later testis samples . Causes for this discrepancy are mostly related to the effects of FFPE on RNA fragmentation, which partially reduced the possibility to detect all starting copies of PPIA and RGN in an unknown sample, influencing therefore the copy-ratio values, as shown in different studies regarding mRNA quantification in FFPE specimens  was higher when compared to the cut-off value found by Starvaggi Cucuzza and co-authors  on RNAlapecimens .Regarding evaluation of suitable diagnostic protocols based on IHC and RT-qPCR, many statistical approaches are available to combine multiple diagnostic tests for screening purposes . HoweverBased on the preliminary results from this study, RT-qPCR confirms RGN as useful and robust biomarkers able, together ICH analysis, to detect illegal administration of sex steroid hormones in veal calves. Specifically, RT-qPCR was more sensitive than IHC test for detection of steroids abuse in veal calves by RGN expression analysis, but also more expensive and time consuming than automated quantitative IHC analysis. The availability of these different diagnostic tests to the labs involved in official controls would allow a convenient choice based on their operational capabilities.However, considering the potential field application of IHC and RT-qPCR for routine analyses in NRCPs, an efficient and balanced cost-benefit strategy to combine such tests will need to be further evaluated on field samples, with the aim to enlarge the numbers of screened animals/farms and, in the future, to limit confirmation analyses, usually based on more expensive analytical methods, only where needed.The study has shown a potential application of regucalcin (RGN) profiling at both transcriptional and protein level. Developed screening tests on formalin fixed paraffin embedded samples (FFPE) are suitable for monitoring activities at slaughterhouse planned according to the Italian histological monitoring plan to support official controls for sex steroid hormone misuse in calves. The flexibility of developed methods, successfully applied to ten years old FFPE blocks, could allow both retrospective analyses, when official investigations on growth promoters abuse are requested by authorities, and implementation/revision of current NRCPs.10.7717/peerj.10894/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.10894/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.10894/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.10894/supp-4Supplemental Information 4Click here for additional data file.


Decellularized natural bladder matrices (neobladders) represent an exciting means to regenerate the bladder following bladder cancer-associated cystectomy. In this study, we compare the evolution of decellularized matrices with recellularized matrices by seeding it with human adipose-derived mesenchymal stem cells (ADSC) after implantation following partial cystectomy in rats. We discovered significant anatomical differences since 10 days after neobladder implantation with the ADSC-containing matrices promoting a significant recovery of mature p63- and cytokeratin 7-positive urothelium. We also discovered significantly induced expression of the vimentin mesoderm marker in the submucosal layer in ADSC-seeded matrices. Interestingly, we found a higher expression of smooth muscle actin in transversal and longitudinal smooth muscle layers with ADSC-seeded matrices. Furthermore, ADSC also showed increased vascularization and nerve innervation of the neobladder as determined by the distribution of CD31 and S100β reactivity, respectively. We believe that ADSC and their paracrine-acting pro-regenerative secretome within decellularized matrices represent an efficient bladder substitution strategy; however, we require a fuller understanding of the mechanisms involved before clinical studies can begin. Acquired and congenital bladder abnormalities currently lack effective treatment strategies for organ substitution, and radical cystectomy remains as the current standard treatment for localized muscle-invasive bladder cancer . This prAs an alternative strategy, tissue engineering approaches can avoid these problems and achieve successful results in bladder reconstruction, including the application of decellularized matrices for bladder substitution . StudiesThe bladder performs repeated and coordinated contractions thanks to the specific characteristics of the smooth muscle fibers and their sophisticated organization in perpendicular layers, displaying connections with blood vessels and innervations through peripheral nerves through the complex matrix without compromising the storage capacity of the bladder or protection of the upper tract , 11. TheStem cell therapies have shown to be a promising approach to enhance the regeneration of tissue-engineered bladder tissue . AdiposeBladders were dissected from adult female Sprague-Dawley rats weighing 250–300 g. Immediately after dissection, bladders were washed three times in phosphate-buffered saline (PBS) containing 1% of streptomycin/penicillin  and then transferred to tris buffer  solution containing 1% sodium dodecyl sulfate  and incubated overnight at 4°C, shaking at 400 rpm. The bladders were then immersed in 0.5% Triton X-100 (Invitrogen) in Tris solution  for 4 h and then transferred to ammonia hydroxide solution (0.05%) in Tris buffer  for 2 h, shaking at 400 rpm. Bladders were repeatedly washed in sterile PBS containing S/P before being transferred to 70% ethanol for ten minutes and then stored in PBS-S/P at 4°C until transplantation or the re-cellularization process.6 ADSC ) was employed for bladder recellularization. Four hundred microliters of the ADSC suspension was injected into the bladder matrix submucosa using a 30G-Hamilton syringe , with the remaining 100 μl of ADSC suspension deposited into the lumen of the bladder matrix. The recellularized matrix bladders were individually cultured under sterile conditions, wholly covered with growth medium for 5 days in vitro (DIV). Bladders were manually rotated every 2 h during the first DIV and twice a day during the following days. Three bladders were employed for in vitro assays, including cell distribution and phenotypic analysis at 5 DIV. Phalloidin (P1951 de Sigma) staining for in vitro bladder fluorescence analysis was performed after overnight fixation of bladders with PFA  4%, and incubation for 1 h at room temperature at a 1/100 dilution. Whole bladders were mounted using Fluor Save Reagent  and the fluorescence signal of both Phalloidin and the red fluorescent cell linker was visualized by Confocal Microscopy .Human adipose tissue was obtained from surplus fat tissue during knee prosthesis surgery of four patients under sterile conditions. The human samples were anonymized, and the experimental procedure previously evaluated and accepted by the Regional Ethics Committee for Clinical Research with Medicines and Health Products following the Code of Practice 2014/01. As exclusion criteria, no samples were collected from patients with a history of cancer or infectious diseases at the time of the surgery . All human patients voluntarily signed an informed consent document for the use of the adipose samples. ADSC were isolated, expanded and characterized as previously described , 22. ADSn = 3/group). The key surgical steps are illustrated in Experimental animals were bred at the Animal Experimentation Unit of the Research Institute Príncipe Felipe , where the experimental protocol was previously approved by the Animal Care Committee in accordance with the National Guide to the Care and Use of Experimental Animals . Adult Sprague Dawley female rats were subdivided into two groups (i) for decellularized bladder matrix implantation and (ii) for 5 DIV ADSC recellularized bladder matrix implantation (in vitro assays (5 DIV) or from in vivo experimentation were fixed with 4% PFA for 4 h, then washed in PBS, and embedded in paraffin. Deparaffinized and hydrated sagittal slices of 4 μm tissue sections were stained with hematoxylin–eosin  or Masson Trichrome (MT) . For immunohistochemistry (IHQ), the paraffin-embedded sections were first deparaffinized, processed for antigen retrieval by incubation in citric acid-based un-masking solution (Vector Laboratories), permeabilized with a PBS solution containing 0.1% Triton X-100, and blocked with 5% goat serum in PBS for 1 h. The following primary antibodies were diluted in blocking solution and incubated for 60 min at room temperature at 1:100 dilutions: monoclonal mouse anti-P63 (IR662), anti-Cytokeratin 7 (IR619), anti-smooth muscle actin (SMA) (IR611), anti-Desmin (IR606), anti-Vimentin (IR630), anti-S100β (IR504), anti-CD31 (IR610) and anti-Ki67 (IR626) from Dako or anti-human mitochondria (MAB1273) from Chemicon. After being rinsed three times with PBS, cells were incubated with an HRP (horseradish persoxide)-conjugated goat anti-mouse IgG-HRP secondary antibody, for 40 min at room temperature, and the DAB  substrate kit (Envision Dako) performed in an automatic autostainer link 48 (Dako). Both, anatomical stainings and IHQ were scanned in a Panoramic 250 Flash II scanner  and images acquired with the Panoramic viewer software. Quantification of the images was performed with Image J, expressed in px2 or the percentage of positive cells and normalized to the total analyzed area.Bladder matrices, both decellularized or recellularized and from post hoc test was used. Statistical analyses were performed using GraphPad software. Differences were considered significant at *P values < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.Results were reported as the mean ± standard error of the mean as indicated for each set of data. For the comparisons between groups, statistical analysis of the results was performed by the one-way ANOVA, with appropriate corrections such as Tukey’s We decellularized native adult rat bladders via three consecutive washing steps in Tris buffer containing first SDS (1%), then Triton X-100 (0.5%) and ammonium hydroxide solution (0.05%) and then recellularized rat bladders with approximately 1 million human ADSC. We distributed 90% of these cells by direct injection into the matrix wall with a Hamilton syringe (30G) to cover the entire bladder, and then deposited the final 10% into the bladder lumen . We nextin vitro recellularized bladders demonstrated a concatenated alignment of the deposited ADSC in a single layer in the lumen with a flat cell shape attached to the external wall of the former urothelial stratum  within the matrices (stained in blue) since 10 days after implantation, although each displayed a different cell distribution and tissue organization . As showTo assess the nature and maturation stage of the regenerated cell layers found in the implanted neobladders, we performed immunohistochemical staining for p63  , cytokerWe failed to detect P63 staining in the decellularized bladder matrices, in accordance with the absence of any cell stratification at the former urothelial layer . AlthougSMA staining indicated the existence of infiltrating cells at the former detrusor and inner muscle layers in both decellularized and recellularized bladder matrices, indicating robust differentiation-inducing signaling from the extracellular matrix . HoweverWe employed vimentin, an intermediate filament present in the mesoderm-derived cells , to quanWe evaluated whether ADSC in the recellularized matrices increased the vascularization process by using the CD31 endothelial marker . The recSince the end of the 19th century, the search for an ideal urinary diversion strategy that complements cystectomy has been arduous and, unfortunately, unsuccessful . Currentin vivo implantation. We tested the capacity of human ADSC into a murine model of bladder cystectomy for further translational applications. We observed significantly improved tissue regeneration for the implanted ADSC-containing neobladders, immediately after almost complete cystectomy, when compared to implantation of the decellularized matrix bladder.Human ADSC integrated into the decellularized bladder matrix provide support and induce endogenous urinary bladder tissue regeneration after Decellularized matrices fabrication process has been perfected to better maintain the 3D architecture after matrix decellularization, and preserve the unique niche protein composition, mechanical properties and bioactive molecules to serve as perfect substitutes scaffold for tissue repair (reviewed in ). Chemicin vitro. Nevertheless, a simple concatenated layer of flat cells was located at the outermost layer of the forms transitional epithelium, exhibiting very low expression levels of cytokeratin 7, that displayed the primitive epithelial cell fate of seeded and migrating ADSC. Previous studies reported the need to culture ADSC with conditioning medium from primary urothelial cultures to induce a urothelial phenotype [Decellularized matrices have been shown to induce cell differentiation of their former tissue . Bladderhenotype ; here, wThe regeneration of the urothelium is the most challenging task in urinary tract cell therapy. Nevertheless, the combination of the proper matrix and the transplantation of undifferentiated ADSC could provide significant benefits to patients. The implantation of neobladders previously seeded with ADSC for 5 DIV led to a significant recovery in the stratified urothelium since 10 days post-implantation . HoweverSmooth muscle fibers formed in a longitudinal and transversal organization at the former detrusor location were found in the implanted recellularized bladder and not the bladder matrix . HoweverOf note, the implanted animals failed to control the vesical reflex until the end of the study after almost complete cystectomy. Macroscopic observation after neobladder dissection demonstrated a significant contracture (shortening of a muscle) with calcifications in the decellularized neobladders (data not shown), an issue identified as the major limitation during the regeneration process ; howeverin vitro recellularized with ADSC are suitable for bladder substitution. ADSC in the decellularized bladder matrices reconstitute the urothelium and smooth muscle layers’ organization with limited inflammatory invasion for a better functional vesical reflex regeneration.All in all, decellularized bladder matrices This research was funded by AEI RTI2018-095872-B-C21/ERDF and the Fundación de Urología Pedro Cifuentes. rbz049_Supplementary_DataClick here for additional data file.


Stony corals generate their calcium carbonate exoskeleton in a highly controlled biomineralization process mediated by a variety of macromolecules including proteins. Fully identifying and classifying these proteins is crucial to understanding their role in exoskeleton formation, yet no optimal method to purify and characterize the full suite of extracted coral skeletal proteins has been established and hence their complete composition remains obscure. Here, we tested four skeletal protein purification protocols using acetone precipitation and ultrafiltration dialysis filters to present a comprehensive scleractinian coral skeletal proteome. We identified a total of 60 proteins in the coral skeleton, 44 of which were not present in previously published stony coral skeletal proteomes. Extracted protein purification protocols carried out in this study revealed that no one method captures all proteins and each protocol revealed a unique set of method-exclusive proteins. To better understand the general mechanism of skeletal protein transportation, we further examined the proteins’ gene ontology, transmembrane domains, and signal peptides. We found that transmembrane domain proteins and signal peptide secretion pathways, by themselves, could not explain the transportation of proteins to the skeleton. We therefore propose that some proteins are transported to the skeleton via non-traditional secretion pathways. We continued the decalcification of the undissolved pellets with a second volume of 0.5 M acetic acid and allowed decalcification to proceed to completion, at which point the pH of the solution was measured at 5.5–6.5. We then combined both decalcification rounds  for each sample containing both the acid-soluble and -insoluble fractions, froze the total volumes at -80 °C, and dried them by overnight lyophilization. The dried pellets were stored at -80 °C until further processing. Because washes of the extracted proteins can reduce the representation of proteins, in this study we examined two protein concentrating and cleaning methods similar to those performed for previously published coral skeletal proteomes; (i) centrifugation ultrafiltration (CF methods  and edited. A brief summary of different cnidaria; cleaning, demineralization and extraction protocols for skeletal matrix proteins. . Figure 1. Scanning electron micrographs  showing that S. pistillata ground to 63 μm and cleaned as described are free of visible exogenous contaminating organic material. Soaking the cleaned ground skeleton in PBS while sonicating for an hour also did not reveal any protein banding on TGX Stain-free gels (c). Lanes in (c) are: 1a = Precision Plus Unstained ladder at auto-exposure (~5 seconds); 1b = the same lane as (1a) but exposed for 20 seconds; 1c = concentrated PBS soak exposed at 20 seconds; gels were activated under UV light for five minutes. No protein smearing or banding was observed in the concentrated PBS soak of the cleaned skeleton powder. Figure 2. Protein gel of organic matrix proteins extracted from cleaned S. pistillata skeleton powders following UV activation for five minutes (A) and then silver staining (B).


To evaluate association between bariatric surgery and changes in obstructive sleep apnea (OSA) severity and sleep architecture was as well as to asses continuous positive airway pressure (CPAP) effectiveness and compliance. We enrolled patients undergoing bariatric surgery. Polysomnography was performed in each patient preoperatively and 12 months after the procedure in a subgroup of patients diagnosed with OSA. STOP-BANG, Epworth Sleepiness Scale (ESS) and Berlin questionnaire scores were obtained pre- and postoperatively. CPAP compliance data was recorded during follow-up hospitalization. Among 44 patients with median age of 49.5 years, predominantly women (68.2%) pre- and postoperative polysomnography was performed. We observed significant improvement in STOP-BANG  and ESS  scores, apnea–hypopnea index , oxygen desaturation index  and sleep architecture parameters. CPAP compliance was poor with a median percentage of days with CPAP use accounting to 49.3%. Bariatric surgery is associated with a significant decrease in the number of sleep-related respiratory disturbances, as well as improvement of sleep efficiency. Postoperative CPAP therapy compliance was poor despite low rate of OSA resolution. This study suggests that patients with OSA undergoing bariatric surgery require postoperative reassessment. Its prevalence varies significantly depending on a country and ranges from 3.7% in Japan to 38.2% in the United States3. Obesity is a key risk factor for multiple diseases such as hypertension, diabetes, stroke, osteoarthritis and cancer, thus leading to premature disability and death4. It is considered the most important, best documented and one of the few modifiable risk factors for obstructive sleep apnea (OSA).Obesity represents a major global health challenge with estimated 650 million cases worldwide6. Untreated OSA is associated with a decline in quality of life, development of several cardiovascular comorbidities, increased risk of car accidents and premature mortality7. The gold standard for OSA treatment is continuous positive airway pressure (CPAP)5. Meta-analyses of randomized controlled trials proved the effectiveness of CPAP in attenuation of sleep apnea, reduction of OSA symptoms and OSA-associated comorbidity8.OSA is the most common manifestation of sleep breathing disorders and affects 4% males and 2% females in the general population9. It is indicated in patients with BMI > 40 kg/m2 or BMI > 35 kg/m2 accompanied by comorbidities i.e. heart failure, type 2 diabetes, hypertension and OSA10. Untreated OSA in obese individuals qualified for bariatric surgery is related to increased risk of complications in perioperative period11. Preoperatively initiated and effective CPAP therapy reduces the risk of complications associated with general anesthesia as well as the bariatric procedure itself12.Bariatric surgery is currently considered the most effective modality of obesity treatment, associated with long-term maintenance of body weight reductionDespite the beneficial effects of weight reduction on sleep breathing disorders confirmed by number of studies, there is still significant uncertainty whether bariatric surgery eliminates OSA and allows CPAP therapy discontinuation. This study was an attempt to elucidate above mentioned issues. The primary aim of this study was to assess the association between bariatric surgery and the severity of sleep-related disturbances.This was a single-centre observational study including obese patients undergoing primary bariatric surgery. Preoperative evaluation of OSA risk using validated screening tools and polysomnography were routinely performed in the Pulmonology Department. If a patient met obstructive sleep apnea diagnostic criteria and required PAP therapy, CPAP was introduced and titrated. After the index procedure in the 2nd Department of Surgery each patient diagnosed with OSA was referred to the Pulmonology Department for follow-up reassessment within 12 months after the index procedure including sleep questionnaires and polysomnography. Moreover, the CPAP compliance data from patients` CPAP devices was downloaded and analysed. If persistent sleep apnea requiring CPAP therapy was present in follow-up polysomnography, we performed a re-titration of therapeutic CPAP.Patients that finally did not undergo surgery, who did not meet the criteria of OSA (apnea–hypopnea index [AHI] < 5/h) or missed the follow up hospitalisation were excluded from final analysis. The data was collected between December 2014 and May 2018. The study complies with the Declaration of Helsinki. All patients gave informed consent to participate in the study and official approval for the study protocol from the Jagiellonian University Ethics Committee was obtained (KBET/126/B/2014).On admission to the Pulmonology Department clinical characteristics of study subjects including demographic data , physical examination  and comorbidities  was obtained. American Society of Anaesthesiologists Classification (ASA) score records were gathered from 2nd Department of General Surgery for individuals who eventually underwent bariatric surgery procedure.15. The STOP-BANG questionnaire includes four questions related to snoring (S), tiredness (T), observed apnea (O), high blood pressure (P) and additional anatomical and demographic queries; BMI (B), age (A), neck circumference (N) and male gender (G). For each question, answering “yes” scores 1, a “no” response scores 0, with cut-off score for increased OSA risk ≥ 313. The Berlin questionnaire is a self-administered questionnaire which consists of 10 questions in three categories related to the presence and severity of snoring, frequency of daytime sleepiness, and the presence of obesity or hypertension. In category one, high risk was defined as persistent symptoms in two or more questions related to snoring. In category two, high risk was defined as persistent daytime sleepiness, drowsiness while driving, or both. In category three, high risk was defined as a history of hypertension or a BMI higher than 30 kg/m2. High-risk subjects for OSA were those who were defined as high risk in at least two out of three categories14. ESS is a simple, self-administered questionnaire which is shown to provide a measurement of the subject's general level of daytime sleepiness during certain activities . Patient evaluated chance of dozing scoring from ‘’0′’ that indicates “no chance of dozing” to “3” that means high dozing chance. Result ≥ 10 indicates excessive daytime sleepiness that might be associated with OSA15.Patients were assessed using validated screening tools: STOP-BANG questionnaire, Berlin questionnaire and Epworth Sleepiness Scale (ESS)16. Each polysomnography was assessed by a pulmonologist certified in sleep medicine. Titration of therapeutic CPAP was performed using automatic CPAP machine (Philips Respironix REMstar Auto A-flex Series 60 device) coupled with polysomnography. CPAP recordings were analysed using Philips Encore Software.Polysomnography was performed using a 31-channel polysomnographic system (Phillips ALICE 6LDe) system. Detection of apneas and hypopneas as well as diagnosis of OSA and its severity assessment were performed in accordance with current American Academy of Sleep Medicine criteria17. A Veress needle was used to achieve pneumoperitoneum (15 mmHg). The routine procedure required insertion of four trocars during laparoscopic sleeve gastrectomy (LSG) and five trocars during laparoscopic Roux-en-Y gastric bypass (LRYGB). A sealer/divider or ultrasonic shears were used for dissection and coagulation. A 34-French gastric bougie was used to calibrate the gastric sleeve. Gastrectomy started 4–5 cm proximal to the pylorus with continuously applied linear staples straight to the angle of His. The staple line was reinforced by a running suture. The resected portion of the stomach was removed from the peritoneal cavity through the left flank trocar site during LSG. LRYGB required creation of a pouch by one horizontal 45-mm stapler followed by vertical stapling toward the angle of His, until the pouch was totally separated from the rest of the stomach. A gastrojejunal anastomosis was created with a linear stapler with hand-sewn closure of the remaining defect. The length of the alimentary and enzymatic limb was standardized in all patients as 150 and 100 cm, respectively. A jejunojejunal anastomosis was created using a linear stapler. A routine 10- or 12-mm port site closure was performed to prevent herniation. Perioperative care in our department is based on ERAS guidelines19.The surgical technique has been described in detail previouslyCategorical variables were presented as counts (%) and compared using chi-square test, while continuous variables were presented as median (interquartile range) and compared using Mann–Whitney U test unless otherwise specified. Differences between dependent continuous variables with non-normal distribution were assessed using Wilcoxon test. Correlation between two variables with non-normal distribution was assessed using Spearman`s Rank test. A two-sided p-value of 0.05 was considered statistically significant. It was a complete-case analysis. Patients with missing postoperative BMI values (n = 3) were excluded from analyses including this variable. Statistical analysis was performed using Statistica 13 software. Graphs were created with GraphPad Prism 8.3.Approval for the study protocol was obtained from the Jagiellonian University Ethics Committee was obtained (KBET/126/B/2014).All patients signed an informed consent.We enrolled 191 patients with preoperative polysomnography, of whom 43 (22.5%) patients eventually did not undergo bariatric surgery. Among the remaining 148 individuals who underwent bariatric surgery, 112 (75.7%) patients were diagnosed with obstructive sleep apnea and were referred to the pulmonology clinic for the follow-up hospitalization, of whom 44 patients (39.3%) were finally reassessed and included in the final analysis. Detailed information can be found in the study flowchart  years, 14 (31.8%) of whom were male. The most common comorbidities were hypertension , diabetes  and dyslipidemia . Majority of patients underwent laparoscopic sleeve gastrectomy , while laparoscopic Roux-en-Y gastric bypass was performed in the remaining 13 patients (29.5%). Detailed study participant characteristics are presented in Table .5–57.5 yPreoperative polysomnography revealed mild, moderate and severe obstructive sleep apnea in 1 (2.3%), 7 (15.9%) and 36 (81.8%) patients respectively.2  during polysomnography coupled with automatic CPAP compared to baseline polysomnography. Normalization of breathing disturbances (defined as AHI < 5/hour) was achieved in 25 (56.8%) patients. The median titrated therapeutic CPAP was 8.3 mbar (7.0–10.0).We observed a significant improvement in AHI , oxygen desaturation index  and mean SpOWe observed a significant improvement in majority of polysomnography parameters, e.g., AHI , ODI , mean hemoglobin oxygen saturation  and snoring . Detailed results are summarized in Table Bariatric surgery was associated with significant improvement of sleep efficiency  and significant decrease in number of respiratory related arousals  and total number of arousals . Detailed results are summarized in Table We performed an additional analysis of changes in disease severity before and after bariatric surgery. OSA severity category decreased in 17 (38.6%) patients, increased in 2 (4.5%) patients and did not change in 18 (40.9%) patients. Normalization of sleep disturbances was observed in 7 (15.9% patients).We observed significant decrease in both STOP-BANG  score and Epworth Sleepiness Scale  as well as decrease in proportion of patients assessed as high risk according to Berlin questionnaire (93.2% vs. 29.5%). Excessive daytime sleepiness, defined as ≥ 10 points in ESS, was present in 30 patients preoperatively (68.2%) and in 3 patients after bariatric surgery (6.8%). Differences in scales scores before and after surgery are presented in Fig. Compliance reports from patients’ CPAP devices were available in 36 patients. Median percentage of days with CPAP use accounted to 49.3% (15.8–76.0%), among which median percentage of days with CPAP use exceeding 4 h a day was 87.3% (35.9–97.3%). Effectiveness of therapy was reflected by median AHI during CPAP use accounting to 1.2 (0.4–2.3). We observed a significant reduction in therapeutic CPAP values  during re-titration with automatic CPAP coupled polysomnography.In this prospective observational study, including 148 patients undergoing elective bariatric surgery, OSA was diagnosed in over 75% individuals, with the majority of cases being severe. Based on postoperative polysomnography, performed in 44 individuals, we demonstrated that bariatric surgery does not sufficiently reduce OSA, despite certain improvement in disease symptoms as perceived by patients, which possibly translates to premature discontinuation of CPAP therapy. In this study we showed that patients with OSA who underwent bariatric surgery require postoperative reassessment in order to evaluate indications for CPAP therapy, its effectiveness and patients` compliance.23 and original studies29, including randomized controlled trials31, concluding that majority of OSA patients do not achieve OSA resolution after bariatric surgery. The postoperative AHI shown in presented study is generally consistent with available literature, already mentioning the problem of residual OSA, however its extent seems to be higher than previously reported. This is probably related to higher baseline AHI values and higher prevalence of severe OSA in our cohort, compared to previously published papers29.Polysomnography performed one year after index surgery demonstrated that bariatric surgery significantly reduces respiratory disturbances during sleep but does not entirely eliminate OSA. Normalization of respiratory disturbances during sleep was observed in only 16% of patients while severity of OSA increased, remained stable, and decreased without normalization in 5%, 39%, and 41% respectively. In the presented study we attempted to assess factors potentially associated with the success of bariatric surgery in terms of decrease in AHI. Our results suggest that the proportion of excess AHI lost after the index surgery is moderately associated with postoperative reduction in excess BMI and weakly negatively correlated with age, however we found no significant relation with sex and predominance of hypopneas or apneas. Further studies should focus on search for factors predicting failure of bariatric surgery in improving sleep disturbances, because it may optimize the selection of patients requiring particular attention of sleep medicine specialists in the follow-up. Impact of bariatric surgery on OSA was evaluated in number of systematic reviewsOne of the main advantages of our study is comprehensive evaluation of perioperative changes in all polysomnography parameters, while the majority of available data is based solely on perioperative changes in AHI, patient-reported OSA remission or sleep questionnaires results. There is limited data documenting the influence of bariatric surgery on sleep architecture, since vast majority of authors analysed only respiratory PSG parameters without appropriate evaluation of perioperative changes in sleep architecture. In the present study we found that postoperative weight loss improves sleep quality, which is reflected by a significant improvement of sleep efficiency and decrease in number of respiratory related arousals and total number of arousals.2 and might be independent from OSA presence32. The postulated EDS and obesity relationship could be explained by multiple pathophysiological mechanisms including overactivity of autonomic nervous system, hormonal and metabolic changes and obesity-related medical conditions35. Impact of weight loss on aforementioned pathways could partially clarify beneficial impact of bariatric surgery on EDS. Interestingly, there is data suggesting that the phenomenon of dramatic improvement of EDS related to weight reduction may occur regardless of OSA resolution35. The results of presented study might support the theory that EDS resolution does not fully correspond to change in apnea severity, since we demonstrated that bariatric surgery actually reduces EDS without satisfactory resolution of respiratory disturbances. Thus, diminished EDS related to bariatric surgery might rather be a result of more efficient sleep with less arousals. It has also been suggested that EDS in obese patients may depend on the level of nocturnal hypoxia32. In our study we demonstrated that bariatric surgery was associated with significant improvement of median and minimal SpO2 values and total time with SpO2 value < 90%. Moreover, the observed reduction of hypoxia duration and number of desaturation events was much more prominent than perioperative AHI decrease. These findings raise the question about the validity of OSA classification based only on AHI, thus omitting severity of hypoxia, which undoubtedly plays crucial role in pathogenesis of OSA symptoms and comorbidity. In our opinion, presented results suggest the need for routine postoperative PSG in order to precisely re-evaluate the severity of respiratory disturbances at least among patients diagnosed with severe OSA regardless of the postoperative weight loss or patient-reported alleviation of symptoms.In parallel to improvement in sleep architecture, we found that bariatric surgery was associated with marked alleviation of excessive daytime sleepiness (EDS) quantified with Epworth Sleepiness Scale, reduction of OSA symptoms based on STOP-BANG questionnaire as well as decrease in proportion of patients assessed as high risk according to Berlin questionnaire. It is estimated that EDS is present in about 30% of obese individuals with BMI > 35 kg/m37. This also applies to OSA patients treated with bariatric surgery. Moreover, patients with sleep apnea who underwent bariatric surgery tend to discontinue CPAP therapy more frequently than other OSA patients and this phenomenon must not be underestimated38. Some authors suggested that CPAP therapy discontinuation after bariatric surgery may have a negative impact on the obesity-related comorbidities and increase the risk of OSA-associated complications. The factor potentially influencing CPAP compliance may be a reduction of therapeutic CPAP values after bariatric surgery confirmed in postoperative CPAP titration performed simultaneously with PSG. Preoperatively titrated CPAP pressure may turn out to be too high after significant weight loss, leading to poor tolerance of CPAP therapy and its premature discontinuation. However, in the present study vast majority of patients were treated with automatic CPAP devices (APAP), thus eliminating the need to modify the therapeutic CPAP pressure during 12-month follow-up period in contrast to traditional fixed pressure CPAP devices. Despite this fact, the CPAP compliance remained poor. Thus, the question which PAP therapy is the most suitable for OSA patients undergoing bariatric surgery remains unanswered, due to lack of studies directly comparing compliance with different CPAP devices in this setting. That also clearly indicates that problem of CPAP cessation after BS is multifactorial and warrants further investigation.The present study also addressed the issue of premature postoperative CPAP cessation and poor CPAP compliance in patients with OSA undergoing bariatric procedures. We confirmed the effectiveness of CPAP therapy in this population with significant improvement of all crucial PSG parameters. Patients diagnosed with OSA were recommended to adhere to CPAP treatment preoperatively with indication to continue therapy for at least 12 months after bariatric surgery. The compliance reports obtained from patients’ CPAP devices at follow up visit were available in 36 cases. We proved high effectiveness of CPAP therapy reflected by median AHI accounting to 1.2/h (0.4–2.3/h) during CPAP. Unfortunately, compliance defined by median percentage of days with CPAP use accounted to less than 50%. We believe that actual CPAP compliance was even lower since great number of patients were excluded from the final analysis due to lack of postoperative PSG. There are published data indicating poor long-term CPAP compliance and problem of premature treatment discontinuation in general OSA populationThe undoubted advantage of this study is detailed evaluation of study cohort in tertiary centre sleep laboratory including fully supervised polysomnography (PSG), availability of comorbidity history, results of several types of sleep questionnaires, and CPAP therapy compliance data. However, there is one important limitation. It must be emphasized that from initially enrolled 148 individuals with preoperative PSG only 44 (40%) attended 12-month follow up hospitalization with postoperative PSG, which is an interesting observation itself. It might indicate a necessity for more frequent and probably more effectively enforced follow-up visits after bariatric surgery accompanied by CPAP compliance analysis and identification of possible problems influencing CPAP therapy . Simultaneously we should pay careful attention to patient re-education emphasizing the risk of OSA complications potentially associated with premature CPAP cessation and fact of high frequency of persistent asymptomatic OSA.This study suggests that bariatric surgery does not sufficiently reduce obstructive sleep apnea what warrants mandatory postoperative polysomnography in order to precisely re-evaluate at least all severe OSA patients regardless of the postoperative weight loss or patient-reported resolution of symptoms. Moreover, sleep questionnaires are probably not a reliable tool for evaluation of persistent OSA after bariatric surgery. Patients with OSA undergoing bariatric surgery are characterized by poor CPAP compliance what indicates the need for proper education and early follow-up visits with identification of potential factors influencing the therapy.


IgA nephropathy (IgAN) is a leading cause of chronic kidney disease and renal failure. The exact pathogenesis of IgAN is not well defined, but some genetic studies have led to a novel discovery that the immunoproteasome probably plays an important role in IgAN. The immunoproteasome is a proteasome variant that is expressed when cells are stressed or receive inflammatory signals. While immunoproteasome is suggested to be mainly involved in major histocompatibility complex-I (MHC-I) antigen presentation, recent studies indicate that it may assert broad functions in trafficking events that activate both innate and adaptive immunity. In this review, we first summarize new insights into its functions in immunity, and discuss how it underlies its associations with IgAN. We also highlight its potential as a therapeutic target for the future. IgA nephropathy (IgAN), the most common primary glomerulonephritis globally, is a leading cause of chronic kidney disease (CKD) and end stage kidney disease (ESKD) PSMB8 locus  associated with the susceptibility to IgAN. The PSMB8 locus encodes low molecular mass polypeptide 7 (LMP7) of the immunoproteasome, which is involved in antigen processing and presentation during infection. The typical exacerbation of hematuria in some patients following a mucosal infection suggests the participation of infectious agents in disease pathogenesis In one of the first large-scale GWAS conducted in IgAN, Gharavi, et al. UBE2L3, a gene involved in ubiquitin/proteasome pathway, were associated with susceptibility to both autoimmune diseases UBE2L3 has been further functionally validated involved in SLE by amplifying nuclear factor kappa B (NF-κB) activation and promoting plasma cell development In a shared genetics study between IgAN and systemic lupus erythematosus (SLE), we previously observed variants of Fig. 1). The proteasome consists of two parts including 19S regulatory particles and a 20S catalytic core particle. The 20S proteasome is composed of four heptameric rings with a barrel-shaped structure. Seven distinct α-subunits (α1-α7) form the two outer α-rings and the two inner rings possess seven different β-subunits (β1-β7) The 26S proteasome, the most common proteasome form in eukaryotic cells, is part of the ubiquitin-proteasome system (UPS) that is responsible for degrading damaged or misfolded proteins tagged with ubiquitin to maintain cellular homeostasis LMP2, LMP7, and MECL-1 genes From a clinical point of view, about 30% of patients with IgAN (mostly young subjects) have a clear history of disease exacerbation after upper respiratory or gastrointestinal infections Recent studies show both pathogenic and prognostic role of anti-glycan antibodies in IgAN, suggesting that IgAN is a type of autoimmune disease Fig. 2). Recent studies have elucidated roles of the immunoproteasome beyond antigen presentation. The enhanced immunoproteasome activation in IgAN may be associated with regulating inflammatory pathways, through a direct effect on NF-κB signaling and the production of proinflammatory cytokines As mentioned above, the TLR activation triggers a cascade of intracellular messages leading to activation of the NF-κB transcription pathway PSMB8 and PSMB9 genes encoding the LMP7 and LMP2 immunoproteasome subunits, respectively, have been discovered in autoinflammatory disorders PSMB8-associated mutations in LMP7, basal IL-6 production was significantly higher compared to healthy controls NF-κB is a pivotal transcription factor that promotes growth factors, proinflammatory cytokines, and cell survival signaling. Evidence from immunoproteasome-deficient mice has provided compelling support for the role of immunoproteasome in NF-κB activation. It was observed that cell lines lacking LMP2 had defects in processing NF-κB precursors (p100/p105) to the active transcription factors (p52/p50) PSMB8 gene, is necessary for Th17 differentiation and inhibits Treg differentiation PSMB8 gene, especially those individuals with high proteinuria PSMB8 gene expression As an immunological disorder, accumulating research evidence supports the involvement of multiple abnormal lymphocyte subsets in IgAN. Although a heterogeneous disease, a report observed higher proportions of circulatory Th2 and Th17 cells, lower Th1 and Treg cells in patients with IgAN Fig. 3). Although some of them home in the bone marrow and survive for longer periods, most plasma cells are short-lived after a few days of intense immunoglobulin (Ig) secretion In IgAN, some data suggest bacterial and viral antigens from antigens involved in upper respiratory and gastrointestinal mucosal infections could be found in the glomeruli in association with IgA deposits In general, the proteasome can degrade endoplasmic reticulum (ER) proteins which failed to fold properly It is speculated that the upregulation of the immunoproteasome in IgAN may partly balance the unfavorable proteasomal load/capacity ratio and prolong plasma cells survival. Similar to the constitutive proteasome, the immunoproteasome can also degrade ERAD substrates, misfolded and or damaged proteins to limit the accumulation of unwanted proteins and decrease cell apoptosis In recent years, studies have elucidated roles of the immunoproteasome beyond antigen presentation. The immunoproteasome participates in a variety of biological processes, including protein quality control, transcription, immune response, cell signaling, and apoptosis and so on Table 1Notably, several interesting trials testing the effect of proteasome inhibitor in patients with IgAN and significant proteinuria have been performed Fig. . However, clinical implications of the immunoproteasome in IgAN are still largely unexplored. There is much to be done in this area. For instance, areas of interest might include the following: (i) genetic analysis of the variation of immunoproteasome components and their correlation with immunoproteasome function; (ii) determining whether and how immunoproteasome impact the pathogenesis of IgAN; (iii) development of IgAN animal models that harbor mutations in the immunoproteasome gene; (iv) determining whether immunoproteasome can serve as a biomarker for clinical activity and aid in genetic screening of IgAN susceptible groups; (v) studying the potential of immunoproteasome as a therapeutic target in IgAN. Elucidating the exact pathological contribution of immunoproteasome dysfunction or dysregulation to the development of IgAN and the underlying causal mechanisms will undoubtedly bring valuable insights into the pathogenesis of IgAN and lead to the development of novel therapeutic approaches.In this review, we discuss the important role of immunoproteasome in several mechanisms respectively and it is likely that these mechanisms synergistically contribute to the susceptibility to IgAN and risk of disease progression. The dysfunction or dysregulation of the immunoproteasome may play a crucial role in IgAN pathogenesis and/or progression


Some of these sensors exhibited ultrahigh sensitivity to NH3 demonstrated by a detection limit of 140 ppb and excellent signal stability under variable humidity, making them among the best NH3 sensors.Organic heterostructures have emerged as highly promising transducers to realize high performance gas sensors. The key reason for such a huge interest in these devices is the associated organic heterojunction effect in which opposite free charges are accumulated at the interface making it highly conducting, which can be exploited in producing highly sensitive and faster response kinetics gas sensors. Metal phthalocyanines (MPc) have been extensively studied to fabricate organic heterostructures because of the large possibilities of structural engineering which are correlated with their bulk thin film properties. Accordingly, in this review, we have performed a comprehensive literature survey of the recent researches reported about MPc based organic heterostructures and their application in gas sensors. These heterostructures were used in Organic Field-Effect Transistor and Molecular Semiconductor—Doped Insulator sensing device configurations, in which change in their electrical properties such as field-effect mobility and saturation current in the former and current at a fixed bias in the latter under redox gases exposure were assessed to determine the chemosensing performances. These sensing devices have shown very high sensitivity to redox gases like nitrogen dioxide (NO Over the recent few decades, organic semiconductors have drawn tremendous attention in the development of electronic devices such as organic light-emitting diodes (OLEDs) , organic−) and holes (h+) free charge carriers at the interface because of the electron affinity and work function differences forming the space charge region (− or h+ . T. T3 sensresponse e in a ra 250 ppb g.3 sensing properties at different rh was investigated by Wannebroucq et al. [The effects of peripheral substitution with electron donating and accepting groups in sublayer MPc of MSDI device on its NHq et al. ,75. Base2 as top layer [3 (200 ppm)  was distinguishable, although, changes in rh from 70 to 30% interfered with sensor response towards NH3 and the baseline. The calibration curve of the n-MSDI sensor followed Langmuir type growth  a as sublop layer . The p-M200 ppm) b, while e growth d highlig3 sensing in a wide range of rh [3 and decrease in the recovery step in clean air  associated with LuPc2 was studied for NH3 sensing at different rh [3 sublayer, high sensitivity (LOD: 250 ppb) and stable response at variable rh between 10–70%.In a similar study, a MSDI based on a triphenodioxazine (TPDO) sublayer was studied for NHge of rh . The devlean air e. Notablerent rh . The dev3 sensing performance. In one of the device designs, aniline (ANI), tetrafluoroaniline (TFANI) and dimethoxyaniline (DMA) were electropolymerized on the ITO surface to deposit PANI, PTFANI and PDMA, respectively, and a high conducting LuPc2 was vacuum deposited over it [3  and LuPc2 [3 sensing performance of MSDI devices from the non-grafted one. The best NH3 sensing performance was obtained by the electrografting of DMBz molecule, starting from DMA precursor. The sensors response at different NH3 concentrations (10–90 ppm) and rh values are shown in 3 concentrations. Moreover, rh change has no significant interference in the sensor baseline and its RR values. The calibration curve  are accumulated at the interface in accordance with the workfunction difference of the constituents in the heterostructure. Consequently, the interface region of the heterostructure becomes highly conducting, which carrier dynamics can be altered by chemical doping by oxidizing and reducing gases proportionally to their concentrations, which is also the basis of their chemosensing applications.In summary, we have demonstrated that chemosensing devices incorporating MPc-based organic heterostructures have drawn significant research interest for redox gases detection over the past 10 years. The molecular engineering flexibility of the MPc structure, such as changes of metal atoms and substituents, which are also strongly correlated with their bulk electrical properties, make them an ideal material to develop heterostructures with tunable electrical characteristics that can be exploited in chemosensing devices. The key idea behind using MPc-based heterostructures in sensing devices is to benefit from the organic heterojunction effects in which free charges (e16Pc)/CuPc, PMMA/CuPc and PTCDI-C8/CuPc, were reported as chemosensors towards gases like NO2 and NH3. The advantage of using heterostructures was highlighted, which led to achieve larger changes in field-effect mobility and saturation current of the devices under exposure to these gases compared to OFET devices using a homogeneous structure with only one material. Compared to OFET chemosensors, MSDI device configurations have invited larger research interest in MPc heterostructures for chemosensing applications. Many studies, a majority from our group, have reported MSDI sensors incorporating monocyclic and bicyclic MPcs in heterostructures such as Cu(F16Pc)/LuPc2. These sensing devices displayed very high sensitivities and fast responses and recovery kinetics towards oxidizing gases (NO2 and ozone) as well as reducing gases (NH3) when used in conductometric or impedimetric transduction modes. Some of these research works focused on understanding the sensor working principles and it was reported that the semiconducting nature of the sublayer determined the n- or p-polarity of the sensor under exposure to redox gases. A survey of recent studies on MSDI devices found that attempts are being made to further improve the sensing performances by incorporating an additional organic-organic interface through electrochemical grafting or electropolymerization of substituted benzenes on the electrode surface. Indeed, by following this approach, NH3 sensors displaying high sensitivity, selectivity and stability in response at variable rh were realized having a LOD of 140 ppb, which is among the best NH3 sensing performances ever reported.MPc-based organic heterostructures were used mainly in OFET and MSDI device configurations of which MSDI were used only for the chemosensing applications while a majority of studies on OFET devices were focused on fundamental electrical property investigations. Nonetheless, some of these OFET devices incorporating MPc heterostructures, such as Cu, bias-dependent selectivity , miniatu3 sensing properties of chemiresistors based on these hybrid materials have shown improvements, demonstrated by experimental detection of 200 ppb of NH3 and long operation stability. Moreover, a recent study on graphene/metal oxide heterostructure have exhibited ultrafast response and recovery kinetics (response and recovery time as 21 s and 41 s) in NH3 detection [Regarding the newer design of organic heterostructure, creating an additional organic-organic junction by electrochemical grafting is an important and less explored strategy which should be further studied extensively using larger family of benzene molecules substituted with electron donating and accepting groups. To enhance the organic heterojunction effects, new and emerging organic nanomaterials such as graphene or its oxides should be associated with MPc. A few such studies have been reported in recent researches involving CuPc/reduced graphene oxide and CoPc/reduced graphene oxide ,81. The etection . Thus, g