Apigenin did not reverse spatial memory recall impairment in GFAP-IL6 mice at 22 months of age. GFAP-IL6 mice may represent a suitable model for age-related neurodegenerative disease. Chronic apigenin supplementation significantly reduced microglia activation, but this did not correspond with spatial memory improvement in the Barnes Maze.Chronic neuroinflammation characterized by microglia reactivity is one of the main underlying processes in the initiation and progression of neurodegenerative diseases such as Alzheimer’s disease. This project characterized spatial memory during healthy aging and prolonged neuroinflammation in the chronic neuroinflammatory model, glial fibrillary acidic protein-interleukin 6 (GFAP-IL6). We investigated whether chronic treatment with the natural flavonoid, apigenin, could reduce microglia activation in the hippocampus and improve spatial memory. GFAP-IL6 transgenic and wild-type-like mice were fed with apigenin-enriched or control chow from 4 months of age and tested for spatial memory function at 6 and 22 months using the Barnes maze. Brain tissue was collected at 22 months to assess microgliosis and morphology using immunohistochemistry, stereology, and 3D single cell reconstruction. GFAP-IL6 mice showed age-dependent loss of spatial memory recall compared with wild-type-like mice. Chronic apigenin treatment decreased the number of Iba-1 Neuroinflammation is a fundamental process underlying the initiation and progression of neurodegenerative diseases such as Alzheimer’s disease (AD). It is characterized by microglial activation, reactive astrogliosis, expression of pro-inflammatory cytokines, and the release of reactive oxygen and nitrogen species. Although immune activation in the central nervous system (CNS) occurs as a protective reaction against microbial infection, acute injury or disease, uncontrolled and sustained neuroinflammation can create a neurotoxic environment that leads to neuronal injury, synaptic loss, and neuronal death. Importantly, while low level neuroinflammation is evident during healthy aging , this apAging is the main cause of microglial activation caused by an innate inflammatory response specific to the CNS . MicroglDespite decades of intensive research into neurodegenerative disorders, current pharmacological treatment is focused on symptom management, and no treatments limit disease progression or prevent disease occurrence . There iThere has been a growing interest in the use of natural polyphenols as multipotent agents to combat neurological disease. Epidemiological evidence demonstrates that dietary flavonoid intake is associated with a decreased risk of neurodegenerative diseases . FurtherApigenin  is a natural flavonoid of low molecular weight (MW 270.24), which is found in a variety of plants, fruits and vegetables, and has neuroprotective potential . Apigenivia inhibition of inducible NO synthase and cyclooxygenase-2 (Cox-2) in BV-2 murine microglial cells (via inhibition of interferon-γ (IFN-γ)-induced phosphorylation of signal transducer and activator of transcription 1 in murine microglia (The anti-inflammatory properties of apigenin are evident through a dose-dependent suppression of the inflammatory mediators nitric oxide (NO) and prostaglandin, al cells . Apigeniicroglia .APPxPS1) AD mouse model  transgenic mouse as a model of sustained microglia activation . In thispromoter . GFAP-ILpromoter , breachepromoter , b. We cn = 6–12/genotype and treatment group) were housed in the animal facility of the School of Medicine, Western Sydney University under a temperature-controlled environment (23°C and 60 ± 10% humidity), with a 12/12 h light–dark cycle. Mice were kindly donated by Prof Iain Campbell (University of Sydney)  and carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.Male and female WT (C57BL/6) and GFAP-IL6 heterozygous mice ( Sydney) . Mice weAnimals were randomly assigned to four groups: WT and GFAP-IL6 mice fed with control food pellets , and WT and GFAP-IL6 mice fed with apigenin containing pellets (400 ppm apigenin). Apigenin was supplied by Nutrafur S.A. . Apigenin was mixed with powdered standard mouse chow , and 15% water was added before the mixed diet was introduced to the pellet machine. In the pelleting machine, the temperature of the product did not exceed 43°C. After pelleting, the moisture content was reduced to stabilize the diet against microbial storage damage. This was done by loading the pellets into a shallow tray in an air dryer set at 45°C for 3 h. Over the drying cycle, the temperature of the pellets was increased slowly from 25 to 45°C. Mouse chow pellets were stored at room temperature in vacuum packed bags in the animal housing facility at the School of Medicine, Western Sydney University. Starting from 4 months of age, mice were fed with control chow or apigenin-containing chow and feeding continued until the end of the experiment. Food intake and body weight of the mice were recorded initially to determine tolerance to chow and dosage. The actual average daily Apigenin dosing of the mice (mg/kg bodyweight per day) was calculated to be 110 mg/kg. Behavioral testing was conducted at 6 and 22 months of age; mice were perfused for histology after testing at 22 months.The Barnes Maze (BM) is a navigational maze used to assess spatial learning and memory . The BM Test mice were trained to find the escape hole over 3 days. On the first day (habituation), mice were given one 20 s trial to explore the apparatus. At the end of the trial, the experimenter gently guided the mouse to the escape hole, and the mouse was left undisturbed in the escape hole with the lights and sound turned off for 2 min. During task acquisition (next 2 days), mice were given three 180 s trials daily separated by a 20–30 min intertrial interval, in which mice needed to find the escape hole. If the mouse did not find the escape hole within the trial, it was gently guided to the escape hole at the end of the trial. Once the mouse entered the escape hole, the lights and sound were switched off, and the mouse left undisturbed for 2 min on the first Acquisition day, and for 1 min on the second Acquisition day. Data for acquisition are presented as the average of the three trials per day.There was a 72-hour rest period between Acquisition and the Probe trial. During the single Probe trial, the escape hole was replaced with a false hole, and the mouse was allowed to explore the apparatus for 90 s.via primary latency (latency until first visit to the escape hole), path length (distance traveled to enter escape hole), errors , and speed. At Probe, attempted entries into the escape hole was compared to the average of entries into all other holes, as well as total distance traveled at Probe.Video footage was collected and analyzed with ANY-maze  v.4.99. BM performance during acquisition was assess For histological analysis [methods ], tissueImmunohistochemistry assays were performed on every sixth section . All was+ microglial cells in the hippocampus was counted using the Zeiss AxioImager M2 microscope equipped with MBF Biosciences StereoInvestigator (m = 1).The estimated number of Iba-1stigator . The con® software package. The super resolution images were obtained using Airyscan with 1.3× crop area, and Z-stacks were captured using a 63× objective and NA1.0 for reflective imaging, at a step size of 0.18–2 μm (unless otherwise specified). Reflective imaging was achieved using the 561 nm wavelength. The 3D reconstruction of microglia was performed using Neurolucida 360® (MBF Bioscience) software. Extended depth of focus images were obtained by collapsing the Z-stacked 3D images of resolution 1596 × 1596 pixels, to better identify the objects and provide greater accuracy. After importing the Z-stacks to Neurolucida 360 software, microglia were manually reconstructed using the user-guided mode, along the required planes, obtaining a reconstructed 3D image of each cell [methods  with an argon laser and processed using the Zen Blue[methods ].® built-in analysis software, Neurolucida Explorer, and each reconstructed cell was subject to multiple measures. As described recently by our group, the soma area, μm2; soma perimeter, μm; convex two-dimensional (2D) area of the branching field, μm2; convex perimeter of the branching field, μm; the total length of all processes, μm; the total number of processes from the soma; and the number of nodes (branch points) were measured using Branched Structure and Convex Hull analysis (r) centered at the cell soma, r = 0, with increase in size by a constant change in radius of 5 μm steps and counting the number of compartments crossing a given radius. Sholl diagrams were averaged and maximum value was obtained as the maximum number of crossings. This analysis determined the number of intersections, the process length (μm), the surface area of the cells (μm2), the process volume (μm3), the process diameter (μm), and a number of nodes of the cells for each radius.Morphometric data of each microglial cell was extracted with the Neurolucida 360analysis , a. Sholanalysis , a. Thispost hoc tests were performed when significant interactions were detected. To assess the percentage of escape hole entries, data was split by “genotype,” “age” and “diet,” and single sample t-tests performed .All behavioral and histological data was collapsed across sex, as we have not detected sex effects in GFAP-IL6 mice previously . Behaviopost hoc tests in Prism GraphPad (version 8). Sholl analysis data was compared using three-way ANOVA with the factors “genotype,” “diet,” and “radius.” Correlations of the Convex Hull area with the distinct morphological features of the microglia were analyzed using linear regression (R2) and correlation tests in Prism GraphPad (version 8). All behavioral and morphological data are presented as mean ± SEM.The distinct morphological features of hippocampal microglia were compared using two-way ANOVA with between factors “genotype” and “diet,” followed by Bonferroni To investigate how the accumulating effects of aging and chronic microglial activation impact on cognitive function and if cognitive decline could be ameliorated by apigenin supplementation, we performed the BM test on apigenin and standard diet-fed WT and GFAP-IL6 mice at 6 and 22 months of age. Repeated measures ANOVA revealed no significant effect of “diet” on the body weight of mice across a 4-month period .F = 15.5, p < 0.0001]; this was evident at 22 months, but not at 6 months   = 44.6, p < 0.0001; no interaction with “genotype” p > 0.05], and primary latency increased with age . Apigenin treatment did not modify primary latency in either genotype or age group . Together, this indicates a higher primary latency in GFAP-IL6 months at 22 months only; and this was not ameliorated by apigenin treatment.Glial fibrillary acidic protein-interleukin 6 mice exhibited a higher primary latency compared to WT controls [“genotype”  = 0.02] . In bothDuring acquisition, one path length data point for a WT apigenin-fed mouse was lost due to a technical error.F = 11.8, p < 0.0001] (p’s > 0.05). Path length was not different between the two ages  or genotypes . Apigenin treatment did not affect path length . Together, this suggests that on average both genotypes and age groups decreased their path length across acquisition, and apigenin treatment had no effect on path length.Average path length decreased across the 2 days of learning in all groups when data was averaged across all groups, suggesting improvement in learning over time [“days”  0.0001] . Note th 0.0001] , this waF = 0.1, p = 0.7; no interactions with “age” p > 0.05]. All groups decreased their errors across the 2 days of acquisition, indicating learning of the task   = 1.6, p = 0.2; no interactions with “genotype” p > 0.05]. Apigenin treatment did not affect errors in either genotype or age group . This suggests that both genotypes and ages decreased their errors across acquisition, and apigenin treatment had no effect on errors.Age did not increase errors in the BM [“age”  > 0.05] . GFAP-ILF = 19.2, p < 0.0001]  = 5.8, p = 0.02], and an “age” × “genotype” interaction trend  suggests GFAP-IL6 mice tended to be slower at 22 months than at 6 months, where this change was not evident in WT mice. Mean speed increased across testing days in all groups , but was unaffected by apigenin treatment . These results indicate GFAP-IL6 mice were slower than WT mice, and older mice were slower than younger mice.Overall, GFAP-IL6 mice were slower than WT mice [“genotype”  0.0001] . Older mF = 72.0, p < 0.0001]. GFAP-IL6 mice made fewer hole entries compared to WT mice , and aged mice also made fewer hole entries compared to younger mice, irrespective of genotype or diet . There was no effect of apigenin treatment on escape hole entries . A “location” × “genotype” interaction  indicates GFAP-IL6 mice exhibited poorer discrimination than WT mice, irrespective of age or diet. An “age” × “genotype” interaction  indicates GFAP-IL6 mice made fewer hole entries at 22 months than WT mice; this was not evident at 6 months. There was no “age” × “genotype” × “location” interaction (p > 0.05). Post hoc tests confirm that both genotypes entered the target hole more than other holes at 6 months, but this discrimination is only evident in WT mice at 22 months  = 8.0, p < 0.0001; no interactions with “age” or “diet” p’s > 0.05]  = 47.8, p < 0.0001; no interactions with “genotype” or “diet” p’s > 0.05]. Apigenin treatment did not affect distance traveled at probe .At Probe, GFAP-IL6 mice traveled less than WT controls [“genotype”  > 0.05] . Older mt-tests comparing escape hole entries to chance level  indicate all groups entered the escape hole more than chance at 6 months . However, at 22 months, only WT mice entered the escape hole more than chance .Single sample + microglia was performed in the hippocampus of control and apigenin-fed mice. Control GFAP-IL6 mice had 331,200 ± 19,400 Iba-1+ microglia, which was significantly higher than that of WT mice    = 17.5, p < 0.001]. However, “diet” alone seemed to have a marginally significant effect on microglia numbers . Post hoc tests confirmed that apigenin significantly reduced the number of Iba-1+ microglia in the GFAP-IL6 animals by 32.2% to 224,104 ± 22,030 (p < 0.005); this was similar to levels in WT standard diet-fed levels (p = 0.21).To determine effects of apigenin on hippocampal microglial populations, immunohistochemistry and stereological counting of Iba-1< 0.001] . A signid levels . Apigeni+ hippocampal microglial morphology, we used branched structure analysis with “diet” and “genotype” as variables. A main effect of “genotype” was observed in the measurements of soma area , soma perimeter  and number of nodes , with control GFAP-IL6 mice exhibiting larger soma area (42.98 ± 3.454 μm2), soma perimeter (27.04 ± 1.216 μm) and lower number of nodes (17.53 ± 2.131) compared to control WT mice  ] .F = 19.40, p < 0.0001], soma perimeter , and number of nodes , with apigenin-fed GFAP-IL6 mice having a reduced soma area (26.35 ± 1.542 μm2) and perimeter (21.12 ± 0.7796 μm) and a larger the number of nodes (29.87 ± 2.313) compared to control GFAP-IL6 mice  = 8.240, p = 0.0058] and soma perimeter .A main effect of “diet” was observed in the measurements of soma area , total process length , surface area , arbor volume , average arbor diameter , and nodes  per concentric radii, between GFAP-IL6 standard diet-fed and WT standard diet-fed mice. Investigating this further, Bonferroni multiple comparisons show a significant decrease in total process length at 25 μm radius , surface area at 25 μm , and number of nodes at 20 μm radius , for GFAP-IL6 standard diet-fed mice compared to WT standard diet-fed mice  = 2.13, p < 0.05], surface area , and nodes , per 5 μm radius in apigenin fed mice compared to standard diet-fed mice. Bonferroni multiple comparisons show a significant recovery after apigenin treatment for total process length at 10 μm radius , and the number of nodes at 10 μm  and 15 μm radius , for GFAP-IL6 apigenin-fed mice compared to GFAP-IL6 standard diet-fed mice  = 1.945, p < 0.05] observed for the number of nodes per radii, between all cohorts.Treatment with apigenin recovers complex microglia arborization patterns, and a significant “radius” × “diet” effect was observed with larger total length of processes [fed mice . Overall∗∗p = 0.0063) and WT apigenin diet (∗p = 0.0248) groups  groups .Neuroinflammation is hypothesized to be a major driving force behind neurodegenerative diseases. Currently, very few anti-neuroinflammatory compounds are available to treat neurodegenerative disease. Apigenin can supress inflammatory mediators in a dose-dependent manner in cell culture  and impr+ microglia and altered microglial morphology) in 22-month-old GFAP-IL6 mice compared with WT controls. Exploring microglia morphology could reflect altered brain physiology with a diverse impact on neuronal function. This aligns with previous studies showing apigenin treatment promotes multiple anti-inflammatory pathways, such as the prevention of NF-κB nuclear translocation .RC organized, analyzed, and drafted the behavioral results, and contributed significantly to drafting and editing the entire manuscript. RG analyzed and drafted the microglia morphology results and edited the manuscript. SS and CM fed and monitored the animal cohorts up to 6 months and ran the 6 months old behavioral tests. AF fed and monitored the animal cohorts from 6 to 22 months and ran the 22 months behavioral tests. HL and FU processed the brain tissue for immunohistochemistry. FU performed the immunohistochemical staining and contributed to microglia stereology counting. GM selected the animal model, co-designed the experiments, wrote the grant application to fund the study, supervised students, and edited the manuscript. TK provided the critical feedback on the manuscript. EG and GN co-designed the experiments, collected and organized the behavioral data, contributed to stereology, prepared the stereology analysis and figures, drafted and edited the manuscript, and co-supervised all students working on this project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ovarian cancer (OV) is an epithelial malignancy that intrigues people for its high mortality and lack of efficient treatment. Chemokine-like factor (CKLF)–like MARVEL transmembrane domain containing 6 (CMTM6) can be observed in various cancers, but its part in OV remains little known. Hence, the prognostic value and underlying mechanism of CMTM6 in OV were preliminarily evaluated. Here, we determined that CMTM6 expression was higher than that in normal controls. However, the upregulation of CMTM6 was associated with better prognosis. GSEA results suggested that CMTM6 is involved in the immune-related and metabolism-related pathways. GO/KEGG analysis of CMTM6 coexpressed genes was performed to survey the possible regulatory roles of CMTM6 in OV. Subsequently, CMTM6 expression was positively correlated with the infiltration levels of immune cells and the expression of diverse immune cell marker sets. Importantly, CMTM6 may influence prognosis partially by regulating immune infiltration in OV. Last, copy number variations (CNVs) and DNA methylation might prompt the abnormal CMTM6 expression in OV. In conclusion, CMTM6 can serve as a novel prognostic biomarker in patients with OV. The fatality rate of ovarian cancer (OV) is the highest among all female reproductive tract tumors . Most ofThe human chemokine-like factor (CKLF)–like MARVEL transmembrane domain-containing (CMTM) is a gene family consisting of nine members, CKLF and CMTM1–8 . Their evia modulating stability through ubiquitination  analysis, and survival analysis. Besides, further exploration was carried out to estimate the status of CMTM6 in the tumor microenvironment using TIMER 2.0, TISIDB, and GEPIA2 databases. The epigenetic alteration of CMTM6 is connected with its dysregulation. Collectively, the purpose of this article is to ascertain if CMTM6 could be a new potential prognostic biomarker for OV and hopefully to help filter OV patient’s suitable prognostic indicators for the therapy method.A pan-cancer analysis of CMTM6 was conducted by the GEPIA2 . The CMT2 at 37°C.Normal ovary cell line (IOSE80) and ovarian cancer cell lines  obtained from the American Type Culture Collection  were cultured in Roswell Park Memorial Institute (RPMI)1,640 medium  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cell lines were maintained in a humidified incubator in an atmosphere of 5% COTM Design and Analysis Software1.3.1 PCR System. The 2−ΔΔCt method was used for quantification, and fold change for targeted genes was normalized by internal control. The PCR reaction conditions were as follows: 95°C for 30 s, followed by 95°C for 5 s, and 60°C for 30 s: 40 cycles and then 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s again. The expression levels were normalized against those of the internal reference gene GAPDH. The primer sequences are listed as follows: CMTM6 forward 5′-ATGAAGGCCAGCAGAGACAG-3′, reverse 5′-GTGTACAGCCCCACTACGGA-3′; GAPDH forward 5′-CGCTCTCTGCTCCTCCTGTTC-3′; reverse: 5′-ATCCGTTGACTCCGACCTTCAC-3′.Total RNA of cell lines was extracted using RNAiso Plus reagent  and transcribed into cDNA with the reverse transcription kit (TaKaRa) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using cDNA primers specific for mRNA. All the real-time PCR reactions were performed using the QuantStudioThe cells were lysed for 30 min with RIPA buffer and then centrifuged at 12,000 g for 30 min at 4°C to collect the supernatants. After mixing with SDS sample buffer, the proteins were heated to 99°C for 10 min, separated on 12.5% SDS-polyacrylamide gels, transferred to PVDF membranes, and probed with antibodies against CMTM6  and actin  at 4°C overnight. A 1:2000 dilution of the HRP-linked anti-IgG  was used as secondary antibody, and blots were visualized by ECL , photographed using a cooled Tanon chemiluminescence gel imaging system , and band intensities were quantified using ImageJ software. The results were expressed as a ratio of band density to actin.To uncover the prognostic value of the CMTM6 gene in OV patients, survival analysis, for instance overall survival (OS) and disease-free survival (DFS), was conducted. Relevant data of OV patients were downloaded from TCGA datasets, which were visualized with Kaplan–Meier curves. The analyses were accomplished through the R packages “survival” and “survminer” . The p-vThe Kaplan–Meier plotter database was further wielded to estimate the prognostic value between gene expression and survival in cancers . The relIn some situations, GEPIA2 was also used to evaluate the prognostic value of CMTM6.p < 0.05 and normalized enrichment score (NES) > 1.GSEA is a powerful analytical method to predict whether a gene set is enriched in a specific biological state. The clusterProfiler package in R was used for GSEA to reveal the critical biological process and relevant signaling pathways affected by CMTM6 expression. The cutoff criteria were The top 100 coexpressed genes with CMTM6 were extracted from coexpression analysis datasets in the Oncomine database. Fold change≥2 and P-value≤0.001 were selected as thresholds.The GeneMANIA database is a resource-rich website containing gene information, analyzes gene lists, and prioritizes genes for functional assays with a high accuracy of the prediction algorithm . A figurvia the R package clusterProfiler (3.14.3)  analysis comprising cellular component (CC), molecular function (MF), and biological process (BP), as well as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of coexpressed genes were implemented (3.14.3) . P < 0.0TIMER2.0  is a comprehensive resource for systematical analysis of immune cell infiltrate across diverse cancer types . SpearmaTISIDB, well known as a web portal for tumor and immune system interaction, integrates multiple heterogenous data types . Here, TMeanwhile, the relationships between the expression of CMTM6 and gene markers of immune cells by the “correlation analysis” module of GEPIA2 were analyzed.UCSC Xena is available to inspect gene expression, copy number, methylation, and somatic mutation in TCGA OV patient cohort .The genomic alteration types, alteration frequency, and CMTM6 mRNA level vs. methylation in OV were analyzed through the “OncoPrint” and “Plots” module, and the survival situation of the altered group or not were analyzed by the “Comparison/Survival” module in cBioPortal .p < 0.05, and log-rank p value < 0.05 were thought to be statistical significant .Partial statistical analyses were performed by default by website recourse. The Wilcoxon rank sum test was adopted to compare the CMTM6 expression levels in tumor tissues and normal tissues based on TCGA and GTEx databases. All R packages were chosen by R software version 3.6.3. The box plots were generated with GraphPad Prism 9 software, data are presented as mean ± standard deviation, and one-way analysis of variance (ANOVA) was used to determine the differences of CMTM6 expression in cell lines. All cell experiments had been repeated at least three times. Only P value < 0.05, adjusted According to the GEPIA2 database query, we found that CMTM6 expression was upregulated in COAD (colon adenocarcinoma), GBM (glioblastoma multiforme), LAML (acute myeloid leukemia), LGG (brain lower grade glioma), OV (ovarian serous cystadenocarcinoma), PAAD (pancreatic adenocarcinoma), READ (rectum adenocarcinoma), STAD (stomach adenocarcinoma), THCA (thyroid carcinoma), and UCEC , especially in OV, compared with the corresponding normal tissues . ConsistThen, we dug the CMTM6 expression among groups of patients in line with different clinical parameters by using the UALCAN online tool, such as individual cancer stages, race, age, tumor grade, and TP53-mutation status. In terms of individual cancer stages, without increasing trend of CMTM6 level was witnessed as the aggravation of stages . RegrettThe data of cancer cell lines in the CCLE provided a large body of support about the expression of genes on many more cancer subtypes of various tissues of origin. With the use of the CCLE database, we discovered that CMTM6 was overexpressed in all cell lines of ovarian cancer . Afterwap = 0.032), and DFS (p = 0.043), compared with low expression of CMTM6.Based on the abovementioned consequences, we hypothesized that CMTM6 expression could well predict the survival of OV patients. To evaluate the role of CMTM6 in the OV patient survival, we classified the cancer cases from TCGA cohort into high-expression and low-expression groups. An unexpected observation in this regard is that the influence of CMTM6 overexpression in OV is related to preferable OS , but in ovarian endometrioid adenocarcinoma, high-expressed CMTM6 only exhibited decent OS . RegardiAs CMTM6 expression is positively correlated with survival of OV, we tried to identify signaling pathways modulated by CMTM6 expression level. The GSEA was conducted, and biological pathways were enriched in the natural killer cell–mediated cytotoxicity, chemokine signaling pathway, B cell receptor signaling pathway, and T cell receptor signaling pathway , which aTo explore the biological significance of CMTM6 in OV, we performed coexpression data mining to obtain the top 100 coexpressed genes of CMTM6 from a large cluster of 19,574 genes across Tothill Ovarian 295 samples in the Oncomine database . A PPI nr = 0.164, p = 9.38e-03), CD4+ T cell , neutrophils , and myeloid dendritic cells  by CMTM6 expression  of the Kaplan–Meier plotter disclose that the upregulation of the CCL20, CXCL5, CXCL8, and CXCL16 cohorts had a better prognosis in OV (Previous studies showed tumor-infiltrating lymphocytes (TILs) as independent predictors of prognosis in cancers . Furtherpression . Moreovedulators . The resdulators . Immunomdulators . With redulators . Regardidulators . To elucis in OV , and theis in OV . Followiis in OV . OverallTo enhance our understanding of CMTM6 cross-talk with immune regulation, we analyzed the relationships between CMTM6 expression and varied immune signatures using the correlation pattern of GEPIA2, including monocytes, TAMs, M1 macrophages, M2 macrophages, B cells, CD8+T cells, T cells , NK cells, neutrophils, and dendritic cells. Simultaneously, many T cell subgroups, such as Th1, Th2, Th17, Tfh, Treg, resting Tregs, effector Tregs, effector T cells, naïve T cells, effector memory T cells, resident memory T cells, and exhausted T cells, were analyzed as well , 4. We dp = 3.28e-7, r < 0, Spearman’s analysis; p = 3.406e-4, r < 0, Pearson’s analysis). Considering all this, CNV and DNA methylation might contribute to the elevated level of CMTM6 in OV.CNV refers to the insertion, deletion, duplication, translocation, and derived chromosomal structural variation of DNA fragments, which has been a pivotal factor in genetic variation and biodiversity . DNA metAmong gynecological oncology, ovarian cancer leads the way in death rates . OV is aCMTM6 is a widely expressed protein that plays a central role in membrane protein transport, transmembrane, and secreted protein . So far,We first identified high expression of CMTM6 in OV samples and cell lines as compared with normal samples and IOSE80 cell line, respectively. There is no apparent relevance between CMTM6 expression and grouped in advanced cancer stages and tumor grade nor with patient’s race, age, and TP53-mutation status. Of course, more clinical samples are needed to verify or overturn this phenomenon. It is worth special attention that the most clinically relevant finding was that high expression of CMTM6 was associated with good survival in data of TCGA. Furthermore, Kaplan–Meier plotter and PrognoScan database analyses also demonstrated that CMTM6 overexpression in OV patients had prolonged survival than those with low expression. The abovementioned observation strongly substantiates that CMTM6 is likely to be a latent prognostic marker in OV.GSEA analysis revealed the function of CMTM6 enriched in immune response–related pathways, such as natural killer cell–mediated cytotoxicity, chemokine signaling pathway, B cell receptor signaling pathway, and T cell receptor signaling pathway. NK cells, as antitumor immune cells, play an indispensable role in killing tumor cells and exerting immune function . FunctioTumor cells exist in a complex tumor environment (TME) . The essEpigenetic phenomena play an essential role in regulating genetic expression . As a rein vitro or in vivo experiments, as well as clinical validation, to explore the specific regulatory mechanisms between CMTM6 expression and genetic alterations, and momentous pathways selected by GSEA analysis in OV are imperative. In addition to this, to ensure greater reliability and representativeness of the results and assumptions, the samples should be expanded for further research. In the future, we intend to manipulate deep investigations in the role of CMTM6 in the occurrence and development of OV to support our conclusion that CMTM6 could be considered an available prognostic biomarker. Fortunately, a collection of plans has been formulated for some recent laboratory work.Our present study is not without limitations. Most notably, the majority of our ratiocinations were based on online integrative bioinformatics analysis tools. Hence, it is imperative to obtain more reliable data derived from Taken together, this study disclosed for the first time that increased CMTM6 expression in OV was an independent prognostic biomarker that prolonged survival, where immune-related and metabolic pathways favored this behavior. We hope that our findings will aid future research and help clinicians in the choice of appropriate measures for their patients and improve the long-term outcome of OV.


None of single NOS−/− or double NOSs−/− genotypes showed such features. These findings were observed in the triple n/i/eNOSs−/− mice as early as 4 weeks after birth. Cyclopaedic and quantitative comparisons of mRNA expression levels between the lungs of WT and triple n/i/eNOSs−/− mice by cap analysis of gene expression (CAGE) revealed that mRNA expression levels of three Wnt ligands and ten Wnt/β-catenin signaling components were significantly reduced in the lungs of triple n/i/eNOSs−/− mice. These results provide the first direct evidence that complete disruption of all three NOS genes results in spontaneous pulmonary emphysema in juvenile mice in vivo possibly through down-regulation of the Wnt/β-catenin signaling pathway, demonstrating a novel preventive role of the endogenous NO/NOS system in the occurrence of pulmonary emphysema.The roles of endogenous nitric oxide (NO) derived from the entire NO synthases (NOSs) system have yet to be fully elucidated. We addressed this issue in mice in which all three NOS isoforms were deleted. Under basal conditions, the triple n/i/eNOSs The number of COPD patients is expected to continue to increase due to an aging population and continued use of tobacco2. Bronchodilators, including anti-cholinergic agents and β2-adrenergic receptor stimulants are used in the treatment of symptomatic patients with COPD; however, since the precise pathogenesis of pulmonary emphysema has not been fully clarified, its definitive treatment has not yet been established.Pulmonary emphysema is a chronic lung condition that causes shortness of breath and is defined as the “abnormal permanent enlargement of alveolar air-space size distal to the terminal bronchioles, accompanied by destruction of the alveolar wall and without obvious fibrosis”. Chronic obstructive pulmonary disease (COPD), which includes pulmonary emphysema, is typically caused by long-term exposure to irritating gases or particulate matter, most often from tobacco smoke. Obstructive ventilatory disturbance due to COPD is usually progressive and sometimes shows exacerbation due to infection, which lowers life expectancy. In cross-sectional epidemiological studies worldwide, the prevalence of COPD in people over 40 years is around 10%l-arginine by three distinct isoforms of NO synthase (NOS), including neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS); all of which are expressed in human lung tissues under both physiological and pathological conditions5. nNOS is present in the airway epithelium, vascular endothelium, vascular smooth muscle cells (VSMCs), and nerve cells; iNOS is expressed in the airway epithelium, vascular endothelium, VSMCs, and alveolar macrophages; and eNOS is localized in the vascular endothelium, VSMCs, and type II alveolar epithelial cells8. There has been no previous study that characterizes the role of nNOS in pulmonary emphysema. Previous studies reported that pulmonary emphysema induced by elastase or tobacco smoke is mitigated in iNOS−/− mice to a greater extent than in wild-type (WT)9, whereas another study showed that pulmonary emphysema induced by elastase is comparable in iNOS−/− and WT mice10. Similar extents of elastase-induced pulmonary emphysema in eNOS−/− and WT mice have been indicated10.Nitric oxide (NO) plays important roles in maintaining homeostasis of the respiratory system. NO is synthesized from Nω-nitro-l-arginine methyl ester (l-NAME). It has been reported that long-term treatment with l-NAME alleviates tobacco smoke-induced pulmonary emphysema in mice11; however, it has also been shown that long-term treatment with the NO precursor l-arginine also reduces pulmonary emphysema, suggesting that the above-mentioned long-term effects of l-NAME are not caused primarily by the inhibition of NOSs11. The following lines of evidence support this notion: first, long-term vascular effects of l-NAME also are not caused by the inhibition of NOSs12; second, long-term treatment with l-NAME does not reduce eNOS activity13; third, multiple actions of l-NAME other than simple inhibition of NOSs have been reported15. Thus, due to lack of specificity of this inhibitor, the authentic roles of NOSs in the pathogenesis of pulmonary emphysema remains unclear. The most appropriate way to address this issue is to use mice that are deficient in the entire NOS system (i.e. triple n/i/eNOSs−/− mice)16. Here, we demonstrate that the triple n/i/eNOSs−/− mice spontaneously developed pulmonary emphysema.The in vivo roles of endogenous NO derived from the entire NOS system in pulmonary emphysema have been examined in pharmacological studies with non-selective NOSs inhibitors, such as −/−, iNOS−/−, eNOS−/−, double n/iNOSs−/−, n/eNOSs−/−, i/eNOSs−/−, and triple n/i/eNOSs−/− mice16. The mice were maintained on a regular diet . Sample size was empirically chosen. There was no exclusions of animals or data points.This study was approved by the Ethics Committee of Animal Care and Experimentation, the University of Occupational and Environmental Health, Kitakyushu, Japan. The study was carried out in compliance with the ARRIVE guidelines and in accordance with the American Veterinary Medical Association (AVMA) Guidelines. Experiments were performed in male wild-type (WT) C57BL/6J , single nNOS16.Blood samples were taken from the right axillary artery at the time of sacrifice and were collected using vacuum tubes containing sodium EDTA. The blood samples were immediately centrifuged at 3000 rpm at 4 °C for 15 min, and the supernatant plasma was stored at − 80 °C. Plasma NOx concentrations were measured by the Griess method using the ENO-20 NOx analysis system , as previously described2O. The tissues were embedded in paraffin and 3-μm-thick sections of the embedded lung tissues were stained with an hematoxylin and eosin solution or an elastic van Gieson (EVG) solution. Mean alveolar linear intercept length was determined by light microscopy at a total magnification of 100×, and 15 random microscopic images per lung tissue section were assessed by microscopic projection onto a reference grid, as previously reported10. The mean alveolar linear intercept length was calculated by dividing the total grid length by the number of intersections of alveolar wall gridlines. The alveolar destructive index was analyzed on five randomly selected frames per lung at 200× power, and was determined by means of 42-point grid as previously reported17. Structures lying under these points were classified as destroyed alveolar and/or duct space. Alveolar spaces were classed as destroyed if the wall of the alveolus was disrupted in 2 or more places or there were 2 or more disruptions of contiguous alveoli that were part of the structures opening onto a single duct system. Duct spaces were classed as destroyed if 2 or more isolated islands of lung parenchyma were observed in the lumen of a duct. Clearly abnormal morphology or classic emphysematous change were classified as destroyed, too. The alveolar destructive index was calculated as [D/D + N × 100]: D = number of destroyed points, N = number of normal points. EVG staining is useful for identifying the elastic fibers, and the content of the elastic fibers was estimated by measuring the lengths of EVG staining-positive elastic fibers (black color) in random 10 fields per lung tissue with a light microscope at a magnification of 400×, and the average values were compared.Mice were sacrificed at 8 weeks of age and the lungs were fixed with 15% formalin neutral buffer solution  at 25 cmH18, and intrapulmonary and surrounding extrapulmonary tissues  were automatically excluded18.Micro CT analysis was performed under general anesthesia with sevoflurane inhalation  using a micro CT system . Operating conditions of micro CT were as follows: 90 kV, 88 μA; chest CT: respiratory reconstruction mode; field of view: 25 mm; voxel size: 60 × 60 × 60 μm; and scan time: 4.0 min. Micro CT images of end-expiratory lung volumes and lung field CT values were calculated using an analyze 12.0 software . Lung parenchyma was defined as a region with X-ray attenuation values between − 1200 and − 300 HU (Hounsfield Unit) according to a previous studyg for 10 min at 4 °C and the supernatants were stored at − 80 °C. The cell pellet was diluted in PBS, and total cell number was counted with a hemocytometer after staining with Turk’s stain solution . Differential cell counts were determined using cell suspensions displayed on glass slides with a cytocentrifuge . The cells on the glass slides were dried, fixed and stained by the Diff-Quick method , and three hundred cells were identified under a microscope, as previously described19.Bronchoalveolar lavage was carried out by cannulating the trachea with a 20-gauge catheter. The lung was lavaged with three aliquots of 1.5 ml saline (0.9% NaCl). BALF was centrifuged at 700×20. To study the molecular mechanisms for spontaneous pulmonary emphysema in triple n/i/eNOSs−/− mice, gene ontology term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis21 were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/). Differences with P < 0.05 were regarded as statistically significant. The datasets of mRNA sequences were deposited in NCBI's Gene Expression Omnibus  and are accessible through GEO series accession number GSE64521.CAGE library preparation, sequencing, mapping and gene expression analysis were performed by DNAFORM . Briefly, quality and quantity of total RNA were checked by a bioanalyzer  to ensure that the RNA integrity numbers (RINs) were above 7.0 and the A260/280 and A260/230 ratios were above 1.7. First-strand cDNAs were reverse-transcribed from the RNAs, then second-stranded cDNAs were synthesized and selected by the Cap trapping method. After “bar code” tags were attached, CAGE libraries were constructed. Multiplexed 8 CAGE libraries were sequenced using single end reads of 75nt on an Illumina NextSeq 500 sequencer. The sequenced CAGE reads were mapped to the mouse mm9 genome using the BWA software program (version 0.7.12-r1039). The unmapped reads were then mapped by HISAT2 (version 2.0.5). The 5ʹ coordinates of CAGE-tags were input for RECLU clustering, with a maximum irreproducible discovery rate (IDR) was 0.1 and minimum count per million (CPM) value was 0.1. The R package edgeR in the RECLU pipeline was used to perform the differential analysis of the genesProtein expression levels of glycogen synthase kinase (GSK)-3β and lymphoid enhancer-binding factor 1 (LEF1) in the lung were measured by ELISA kits .22. The Database for Annotation, Visualization and Integrated Discovery (DAVID) (version 6.8) was used. A value of P < 0.05 was considered to be statistically significant.Results are expressed as mean ± SEM. For comparison of two groups, a Student’s t-test was performed. For comparison of more than two groups, one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons was carried out using the SPSS software. Statistical analysis of the CAGE sequencing data was performed by Danaform Inc.  as follows. The data of mRNA expression levels (gene counts) were corrected by size factors in order to normalize them by the normalization method of trimmed mean of M values (TMM). The corrected data were analyzed by the likelihood ratio test on a negative binomial distribution using edgeR as previously reported−/−, iNOS−/−, eNOS−/−, double n/iNOSs−/−, n/eNOSs−/−, i/eNOSs−/−, and triple n/i/eNOSs−/− mice at 8 weeks of age. The plasma NOx levels were significantly reduced in accordance with the number of disrupted NOS genes in a stepwise manner , with statistically significant differences (P < 0.05) and greater than 1.2-fold change; 2108 and 2609 mRNAs were significantly up- and down-regulated, respectively, in the lungs of the triple n/i/eNOSs−/− mice as compared with the WT mice. A volcano plot is presented in the Supplementary Fig. We performed CAGE sequencing in the lung tissues of the triple n/i/eNOSs−/− mice, statistically significant biological process terms included “Wnt signaling pathway,” “phosphorylation,” “cell proliferation,” “canonical Wnt signaling pathway,” etc.  signaling pathway,” etc. Fig. . In the −/− mice, we selected individual genes with statistically significant differences. Three Wnt ligand genes  were significantly reduced, and β-catenin also tended to decrease in the lungs of the triple n/i/eNOS−/− mice , FZD2, lipoprotein receptor-related protein 5 (LRP5), and LRP6, which are cell membrane receptors for the Wnt ligands; GSK-3β, Axin1, adenomatous polyposis (APC), dishevelled 1 (Dvl1), Dvl2, and Dvl3, which are cytoplasmic β-catenin regulators; and LEF1, which is a nucleic transcription factor , the triple n/i/eNOSs−/− mice, the cGMP-PKG signaling pathway was down-regulated. Consistent with our findings, it has been reported that cGC and cGMP levels and PKG activity are lowered in the lungs of mice with tobacco smoke-induced pulmonary emphysema25, that eNOS and sGC levels are reduced in the lungs of patients with COPD26, that long-term treatment with either sGC stimulators, inorganic nitrite, or l-arginine prevents pulmonary emphysema induced by tobacco smoke or elastase in mice or pigs28, and that long-term treatment with the sGC stimulator riociguat decreases airway resistance in patients with COPD and pulmonary hypertension29. Considering the defective eNOS and sGC levels and the beneficial effects of riociguat in the COPD patients, the present results may have clinical implications.NO synthesized by NOSs stimulates soluble guanylate cyclase (sGC) and generates the second messenger cGMP, which in turn activates PKG and exerts a variety of biological actions. In emphysematous lungs of the triple n/i/eNOSs−/− mice, but not in any single or double NOSs−/− mice. The plasma NOx levels were reduced in the order of single, double, and triple NOSs−/− mice, and were considerably preserved in the single NOS−/− and double NOSs−/− mice. Thus, in the single NOS−/− or double NOSs−/− mice, compensatory mechanisms by other NOSs that are not genetically disrupted may operate. Indeed, we previously reported compensatory up-regulation of other NOS isoforms in the single NOS−/− mice30.Spontaneous pulmonary emphysema was noted only in the triple n/i/eNOSs−/− mice. There was no difference in the BALF profile among the 8 genotypes studied. We then made cyclopaedic and quantitative comparisons of mRNA expression levels in the lungs of 4-week-old WT and triple n/i/eNOSs−/− mice by the CAGE sequencing with a next-generation sequencer. The CAGE sequencing is a recently developed gene expression analysis technique that is superior to RNA sequencing in terms of quantitativity. Conventional RNA sequencing has high quantitative performance that is comparable to real-time PCR (“gold standard” of mRNA quantitation methods) with 94% of quantitative concordance rate31; however, the RNA sequencing protocol includes a PCR amplification step of sample cDNAs, which incurs a reduction of quantitative performance. The CAGE sequencing does not require the PCR amplification step, thereby showing excellent quantitativity32. We thus employed the CAGE sequencing in the present study.We next examined the underlying mechanisms for spontaneous pulmonary emphysema in the triple n/i/eNOSs−/− mice was detected. mRNA expression levels of three Wnt ligand genes  and ten Wnt/β-catenin signaling component genes were decreased in the lungs of the triple n/i/eNOSs−/− mice. Specifically, down-regulated Wnt/β-catenin signaling component genes were FZD1, FZD2, and LRP5, which are the cell membrane receptors for the Wnt ligands and transmit Wnt signals into the cytoplasm; GSK-3β, Axin1, and APC, which regulate β-catenin amount in the cytoplasm; Dvl1, Dvl2, and Dvl3, which prevent β-catenin degradation by inhibiting its phosphorylation; and LEF1, which binds to β-catenin in the nucleus and acts as a transcription factor33. Protein expression levels of GSK-3β and LEF1 were also reduced in the lungs of the triple n/i/eNOSs−/− mice. Previous studies revealed the causal roles of down-regulation of the Wnt/β-catenin signaling in the pathogenesis of spontaneous pulmonary emphysema as follows: first, mice with tobacco smoke- and elastase-induced pulmonary emphysema show reductions of the Wnt ligands and Wnt/β-catenin signaling components34; second, activation of the Wnt/β-catenin signaling pathway by treatment with lithium chloride reverses the pulmonary emphysema, along with amelioration of the reductions of the Wnt ligands and Wnt/β-catenin signaling components34; third, human patients with a loss-of-function mutation in the Wnt4 gene exhibit lung dysgenesis , concomitant with reduced Wnt4 levels and enhanced β-catenin degradation35. Therefore, it is conceivable that down-regulation of the Wnt/β-catenin signaling pathway possibly mediates the development of spontaneous pulmonary emphysema in the triple n/i/eNOSs−/− mice. We did not confirm that spontaneous pulmonary emphysema in the triple n/i/eNOSs−/− mice was actually caused by down-regulation of this pathway.In the gene ontology term enrichment analysis and the KEGG pathway analysis, down-regulation of the Wnt signaling pathway in the lungs of the triple n/i/eNOSs36 and iNOS gene overexpression and knockdown attenuates and activates, respectively, the Wnt/β-catenin signaling in human colon and breast cancer cell lines37. Consistent with those lines of evidence, n/i/eNOSs deficiency resulted in down-regulation of the Wnt/β-catenin signaling in the lungs of mice. Thus, the NO/NOS system appears to positively regulate the Wnt/β-catenin signaling in general.Crosstalk between the NO/NOS system and the Wnt/β-catenin pathway has been reported in two previous studies: treatment with a NO donor improves down-regulated Wnt/β-catenin signaling in renal glomeruli of streptozotocin-induced diabetic rats24. Among the genes knocked out, foxf1 and TCF21 levels were reduced in the lungs of the triple n/i/eNOSs−/− mice, suggesting that the reduced foxf1 and TCF21 levels were possibly involved in the development of spontaneous pulmonary emphysema in the triple n/i/eNOSs−/− mice. It has been indicated that HDAC expression and activity are reduced in the lungs of patients with COPD, and that chronic treatment with a specific HDAC inhibitor, trichostatin A, causes pulmonary emphysema in rats38, while HDAC1 levels were decreased in the lungs of the triple n/i/eNOSs−/− mice. It has been reported that expression levels of sirtuin 1 (SIRT1), a gene related to longevity, are lower in the lungs of patients with COPD39, and that SIRT1 ablation and overexpression aggravated and ameliorated, respectively, pulmonary emphysema induced by tobacco smoke in mice40, while SIRT1 levels diminished in the lungs of the triple n/i/eNOSs−/− mice. Thus, it is likely that decreased HDAC1 and SIRT1 levels were also involved in the development of spontaneous pulmonary emphysema in the triple n/i/eNOSs−/− mice. Another possible mechanism is that elastic fibers might be more fragile in the triple n/i/eNOSs−/− mice.At least 10 strains of gene knockout mice that spontaneously develop pulmonary emphysema have thus far been reported−/− mice, but not in any single NOS−/− mice, as compared with the WT mice, suggesting a protective role of the NOS system in pulmonary fibrosis41. Hypoxia-induced pulmonary hypertension is deteriorated in the triple n/i/eNOSs−/− mice, and to a lesser extent, in the eNOS−/− mice, as compared with the WT mice, and hypoxia-induced pulmonary hypertension is also aggravated in the WT mice transplanted with triple n/i/eNOSs−/− bone marrow as compared with those with WT bone marrow, suggesting a protective role of the NOS system, specifically in bone marrow cells, in pulmonary hypertension19. However, ovalbumin-induced bronchial asthma is conversely more mitigated in triple n/i/eNOSs−/− than in the WT mice, suggesting an opposing detrimental role of the NOS system in bronchial asthma42. These results indicate that, even in the same lung, the roles of the NOS system are different in distinct disease states. In the present study, spontaneous pulmonary emphysema was found in the triple n/i/eNOSs−/− mice, suggesting a preventive role of the NOS system in the occurrence of pulmonary emphysema. It, thus, appears that the NOS system plays diverse roles in the pathogenesis of respiratory diseases.We have been studying the roles of the entire NOS system in respiratory diseases by the use of murine models of bleomycin-induced pulmonary fibrosis, hypoxia-induced pulmonary hypertension, and ovalbumin-induced bronchial asthma. Bleomycin-induced pulmonary fibrosis is exacerbated in the triple n/i/eNOSsWe were able to demonstrate that complete disruption of all three NOS genes resulted in the development of spontaneous pulmonary emphysema in juvenile mice in vivo possibly through down-regulation of the Wnt/β-catenin signaling pathway. Our findings should contribute to a better understanding of the molecular basis of pulmonary emphysema for which there is no definitive treatment.Supplementary Information.


In presence of indeterminate lesions by fine needle aspiration (FNA), thyroid cancer cannot always be easily diagnosed by conventional cytology. As a consequence, unnecessary removal of thyroid gland is performed in patients without cancer based on the lack of optimized diagnostic criteria. Aim of this study is identifying a molecular profile based on long noncoding RNAs (lncRNAs) expression capable to discriminate between benign and malignant nodules.n = 19) for cytologic suspicious thyroid nodules or to FNA biopsy (n = 135) for thyroid nodules suspicious at ultrasound. Three thyroid-specific genes , six cancer-associated lncRNAs , and two housekeeping genes (GAPDH and P0) were analyzed using Droplet Digital PCR (ddPCR).Patients were subjected to surgery (n = 11) but not in benign (n = 8) nodules after surgery, MALAT1, PVT1 and HOTAIR were selected as putative cancer biomarkers to analyze 135 FNA samples. Cytological and histopathological data from a subset of FNA patients (n = 34) were used to define a predictive algorithm based on a Naïve Bayes classifier using co-expression of MALAT1, PVT1, HOTAIR, and cytological class. This classifier exhibited a significant separation capability between malignant and benign nodules (P < 0.0001) as well as both rule in and rule out test potential with an accuracy of 94.12% and a negative predictive value (NPV) of 100% and a positive predictive value (PPV) of 91.67%.Based on higher co-expression in malignant (ddPCR analysis of selected lncRNAs in FNA biopsies appears a suitable molecular tool with the potential of improving diagnostic accuracy. Differentiated thyroid carcinoma, including papillary and follicular subtype, is the most common thyroid malignancy, representing more than 90% of all thyroid cancers. Thyroid nodules are diagnosed with increasing frequency in clinical practice . The firThe recent progress in thyroid cancer genetics and high-throughput technologies significantly improved molecular test for cancer diagnosis. Results from The Cancer Genome Atlas (TCGA) project provided a comprehensive genomic profile of thyroid cancer  and geneThe recently discovered lncRNAs emerging as critical regulators of several biological and cellular functions may represent a still unexploited tools with a potential diagnostic and prognostic values for numerous cancers , 8. In tIn recent years, many efforts have been made to design novel algorithms capable to discriminate between benign and malignant thyroid nodules based on US images  and alteHerein we set up an advanced mathematical model based on lncRNAs expression in FNA samples as additional novel tool for preoperative diagnosis of thyroid cancer. In particular, we analyzed, by Droplet Digital PCR (ddPCR), six cancer-associated lncRNAs  in both FNAs and surgical tissues. We defined a predictive algorithm based on naïve Bayes classifier for identifying patients with thyroid cancer with accuracy of 94.12% (sensitivity 100% and specificity 91.67%) using MALAT1, HOTAIR, and PVT1 expression in FNA samples and the cytological class. Our attempt, if confirmed in extended cohorts, might represent a reliable diagnostic tool.n = 19; n (F/M) = 17/2, age  = 51.1 ± 15.4 (F)/45.5 ± 13.4 (M)) based on cytologically suspicious thyroid nodules or to FNA biopsy  = 102/33, age  = 48.8 ± 13.5 (F)/53.3 ± 15.2 (M)) because of thyroid nodules ≥1 cm suspicious at US. Informed consent was obtained from each patient . All procedures were conducted according to the principles expressed in the Declaration of Helsinki, the institutional regulation, and Italian laws and guidelines. Surgical specimens were dissected from a qualified pathologist within 1 h of surgery and used for gene expression analysis. FNA biopsies were processed using the ThinPrep liquid-based cytology method as described in ref. [Patients were enrolled at the Endocrinology and Diabetes Center of Fondazione Policlinico Universitario A. Gemelli - IRCCS, Rome, Italy and subjected to surgery . cDNA preparation was performed using the high capacity kit (Applied Biosystems) according instruction as in ref. . DilutioTG (200 nM) 5′-CGGCCAATATCTTCGAGTACCA-3′ and 5′-GCTTCAGAAAGGCCGTTTCC-3′TPO 5′-CACTTGCCTGGCGAACAAAT-3′ and 5′-GGGTGGTCTCTGTTGTTGCA-3′NIS 5′-GTCCCCGGGCTTTTCCT-3′ and 5′-CATTGATGCTGGTGGATGCT-3′PVT1 5′-ACAGGCGTGTGCCCACAAA-3′ and 5′-CATGGTGAAACCCCGTCTCT-3′MEG3 5′-ATCCCTCACCCGGGTCTCT-3′ and 5′-CTTGGCAGCAGCTCAGCAT-3′MALAT1 (200 nM) as in ref. , HOTAIR LncRNA level was normalized to housekeeping gene p0 selected because of increased signal separation and lower rain between target and background droplets  and suspended in 50 μl of lysis buffer and processed according instruction . After centrifugation, 10 μl were subjected to retro-transcription with high capacity kit according protocol (Applied Biosystems). PreAmp step was performed using 2 μl of cDNA, Eva green mix reaction, and specific primers at 400 nM for 14 cycles at 95 °C for 15 s and at 58 °C for 4 min. One microliter preAmp (1:10 dilution) was used to perform ddPCR using EVA green . Representative detection of lncRNAs in FNAs is showed in Supplementary Fig. A naïve Bayes classifier has been used to discriminate between benign (denoted ben.) and malignant  thyroid nodules based on co-expression of a subset of lncRNAs. Such a conditional probability model provides an estimate of the probability that a thyroid nodule is either malignant or benign given the expression of MALAT1, HOTAIR, and PVT1, by computing the probabilitiesUsing Bayesian terminology, such a probability is proportional to the prior probability that a nodule is a tumor multiplied by the likelihood of the evidence. Considering the expressions of MALAT1, HOTAIR, and PVT1 as mutually independent random variables, conditional on the nodule being either malignant or benign, the two above probabilities are proportional to:The priors p and p(ben.), together with the likelihoods p(MALAT1|tum.), p(HOTAIR|tum.), p(PVT1|tum.), p(MALAT1|ben.), p(HOTAIR|ben.), and p(PVT1|ben.), have been estimated using a dataset containing the expressions of MALAT1, HOTAIR and PVT1 of 19 patients undergone to surgery. In particular, the priors have been estimated by evaluating the empirical occurrence of malignant and benign nodulesp, p, p, and p(MALAT1|ben.), p(HOTAIR|ben.), p(PVT1|ben.) have been estimated by fitting a kernel distribution with Epanechnikov bases functions and positive support to the data regarding the expressions of MALAT1, HOTAIR, and PVT1 in patients having malignant and benign nodules, respectively.On the other hand, the likelihoods Thus, the probability of a nodule being malignant or benign has been computed asAnother naïve Bayes classifier has been used to discriminate between benign and malignant nodules based on lncRNAs co-expression in FNA samples and of cytology. This conditional probability model provides an estimate of the probability that a thyroid nodule is either malignant or benign given the expression of MALAT1, HOTAIR, PVT1 determined through the FNA sample and its cytological class (denoted cyt.), that is it provides estimates for the probabilitiesp, p, p, and p(MALAT1|ben.), p(HOTAIR|ben.), p(PVT1|ben.) have been estimated as detailed above in the case of nodules that have been surgically removed.The priors p and p(ben.), together with the likelihoods p(MALAT1|tum.), p(HOTAIR|tum.), p(PVT1|tum.), p(cyt.|tum.), p(MALAT1|ben.), p(HOTAIR|ben.), p(PVT1|ben.), and p(cyt.|ben.) have been estimated using a dataset containing the expressions of MALAT1, HOTAIR, PVT1, and the cytological class of the nodule of 34 patients, which underwent surgery thus allowing for final histological analysis. In particular, the priors and the likelihoods p and p(cyt.|ben.) have been estimated by fitting a multivariate multinomial distribution to the cytological classes of patients having malignant and benign nodules, respectively, that isFinally, the likelihoods Thus, the probability of a nodule being malignant or benign has been computed asTo evaluate the effectiveness of the classifier, we used the bootstrap method: a statistical technique to estimate the performance of a classifier on data that have not been used for training , 31. Thit-test or chi-square test. Statistical analysis was performed using Matlab R2018b (RRID:SCR_001622) and/or Sigma Plot 13.0 (RRID:SCR_003210) statistical software. P values of < 0.05 were considered as significant.Data are expressed as mean ± SEM or as fold induction as indicated in figure legend. Significance was calculated using nonparametric paired two-tailed Student’s versus the contra-lateral tissue . On the contrary, no modulation was detected in benign nodule, according with literature. Of note, no MEG3 downregulation or H19 upregulation was observed in tumor lesions.Based on previously described lncRNAs deregulation in thyroid cancer, we selected a subgroup of lncRNAs to investigate their expression in thyroid cancer tissue samples. Fresh post-surgery explants were used to set up detection by ddPCR of a panel of transcripts: three thyroid-specific genes (thyroglobulin (TG), thyroperoxidase (TPO), and sodium/iodide symporter (NIS)), six cancer-associated lncRNAs , and two housekeeping genes (GAPDH and P0)  expressed significantly higher expression of MALAT1, HOTAIR, and PVT1 compared to benign nodules (n = 8). At univariate analysis, MALAT1, HOTAIR, and PVT1 resulted with significantly higher expression in malignant (n = 11) than in benign (n = 8) nodules . Furthermore, the proposed classifier has an accuracy of 94.74% since it correctly classified 18/19 patients. The sensitivity of the classifier is 100% since all the benign nodules have been correctly classified, whereas its specificity is 90.91% since 10/11 malignant nodules are correctly classified. These results indicate that the proposed naïve Bayes classifier offers a good potential for ruling out the presence of malignant thyroid nodules.Next, we attempt to develop a diagnostic molecular taxonomy based on lncRNAs level in which higher MALAT1, HOTAIR, and PVT1 co-expression might indicate a malignant lesion. In particular, the expression of MALAT1, HOTAIR, and PVT1 in the above 19 thyroid nodules has been used to fit kernel distributions with Epanechnikov bases functions and positive support to determine the likelihoods p, p, p, and p(MALAT1|ben.), p(HOTAIR|ben.), and p(PVT1|ben.). Figure n = 35) were excluded . Figure n = 100). A subset of these patients underwent to surgery (n = 34) with the following distribution: 6 TIR3A, 10 TIR3B, 7 TIR4 and 11 TIR5. The cancer prevalence was 24 out of 34 patients , 4 TIR3B (40%), 6 TIR4 (85.7%), and 11 TIR5 (100%) resulted as papillary thyroid carcinoma). Considered as single variables, MALAT1, HOTAIR and PVT1 showed a trend toward higher expression in malignant lesions as compared to benign nodules, without reaching statistical significance , p(HOTAIR|tum.), p(PVT1|tum.), p(cyt.1|tum.), p(MALAT1|ben.), p(HOTAIR|ben.), p(PVT1|ben.), and p(cyt.|ben.). Figure Based on the above results, MALAT1, HOTAIR and PVT1 were also analyzed by ddPCR on an independent cohort 135 patients undergoing FNAs Fig. . TG and nce Fig. . Howevern = 34) and the same dataset has been used for both training and testing, it exhibited a good statistical significance (P < 0.0001).Using a dataset containing the MALAT1, HOTAIR, and PVT1 expression and the cytological class of the above 34 patients, the designed naïve Bayes classifier has been used to compute the probabilities that a thyroid nodule is malignant or benign. Hence, each thyroid nodule has been classified as malignant if the probability p is greater than or equal to 0.5648, or as benign otherwise. Figure Jmax = 0.9167. This threshold has been used to generate the confusion matrix depicted in the right subplot of Fig. The ROC curve has been used to determine the optimal threshold value 0.5648 by determining the threshold corresponding to the point that is closest to , so to maximize the Youden’s J statistic, 4 synthetic dataset by evaluating the accuracy on both the training samples and on the out-of-bag samples. Figure In order to evaluate the performance of the classifier based on the expressions of MALAT1, HOTAIR, PVT1 and on the cytological class for unprecedented data, the bootstrap method has been used over 10Clinical evaluation, US, and cytology are the main tools to rule out malignancy. According to ATA 2015 guidelines, all patients presenting thyroid nodules with suspicious US features and intermediate/high-risk nodule as assessed by cytological staging may undergo surgery , 5. HoweIn thyroid cancer, lncRNAs are key regulators of several biological processes  and are We first analyzed, by ddPCR expression of several cancer-associated lncRNAs  in both FNAs and surgical specimens and selected MALAT1, HOTAIR, and PVT1 as cancer biomarkers. The classifier for surgically removed thyroid nodules has been trained and tested using the expressions of MALAT1, HOTAIR and PVT1 of 19 patients, whereas the classifier for nodules removed via FNA has been trained and tested using the expressions of MALAT1, HOTAIR, PVT1 and the cytological class of other 34 patients. We developed a diagnostic test using a mathematical model based on a naïve Bayes classifier in which MALAT1, HOTAIR, and PVT1 co-expression in FNAs, applied with cytology classification, may function as both rule in and rule out test with an accuracy of 94.12% (sensitivity 100% and specificity 91.67%). Overall, the malignant prediction with our test seems to fit results better than with ThyroSeq v3 , along with the high cancer prevalence in the FNA sample set , perspectively our findings are promising to improve the diagnostic accuracy of cytology. In future work, the proposed diagnostic approach, which combined molecular data from lncRNA expression and a mathematical model, might apply to other validation dataset to further evaluate its accuracy.An added value of our study lies in the use of ddPCR, a technique characterized by lower cost, large diffusion, and rapid execution, as compared to NGS, with a potential wide clinical prospective. Further studies are needed to confirm our findings on larger cohorts, both in comparison and integration of data.In conclusion, quantification of selected lncRNAs in FNA biopsies and application of our combined mathematical and molecular approach represent a novel diagnostic test that, if confirmed on larger scale, might improve diagnostic accuracy contributing to advice decision-making on surgical treatment.Supplementary Figures


To assess patient and clinician acceptability of handheld 6-lead ECG, for obtaining information about cardiac rhythm and electrical intervals, in acute general adult mental health ward inpatients who refuse traditional 12-lead ECG.In a previous audit of patients admitted to four acute general adult mental health wards, we found that 1 in 4 patients refused 12-lead ECG for at least two weeks, with 1 in 6 refusing throughout their entire stay. ECG refusers were significantly more likely to have a psychotic illness than non-refusers and were thus more likely to benefit from medications that carry a risk of prolonging the QT interval. Less invasive, handheld, 6-lead ECG, which includes measurement of lead II  is available on the NHS supply chain. Whilst not providing the full range of information that 12-lead ECG is able to provide, handheld 6-lead ECG might be an acceptable alternative in patients who would otherwise never have any form of ECG performed.We developed a Standard Operating Procedure for use of handheld 6-lead ECG and provided training for junior doctors on the four wards that were the subject of our original audit. These doctors were then able to offer the device to patients on their wards who refused 12-lead ECG. Doctors completed a short feedback form each time a handheld ECG was offered.So far, handheld 6-lead ECGs have been offered to 17 patients who refused 12-lead ECGs. Mean age (± SD) was 36.1 (± 12.6) years, and 4 of these patients were female. 13 patients (76%) accepted a handheld ECG. One of these attempts failed due to patient agitation. Attempts took a mean of 7 (± 5.4) minutes. 54% of recordings were described as “very easy” by clinicians, whereas 15%, 23% and 8% were described as “somewhat easy”, “intermediate”, and “somewhat difficult”, respectively. Clinician difficulties focussed on patient movement with impact on electrode contact and trace quality. Where answered (N = 10), 90% of patients stated they would recommend a handheld ECG to others. Patients liked the speed of the process, that it felt “less scary”, and that it was less invasive and did not involve removing clothing.Our initial findings from this pilot suggest that handheld 6-lead ECG may be acceptable, both to clinicians and patients, as a means of obtaining information on cardiac rhythm and electrical intervals for patients who refuse 12-lead ECGs.


RV + Qbest LV1 + Qbest LV2). The best “pseudo MPP”  and best BVp (QRV + Qbest LV) were also calculated. A theoretical longevity was estimated for each configuration at the threshold without a safety margin. Results: A total of 235 configurations were tested in 15 consecutive patients. “Pseudo-MPP” was feasible in 80% of patients with 3.1 ± 2.6 vectors available per-patient and LVproximal-LVdistal (most distant electrodes) vectors were available in 47% of patients. Each MPP pacing spike electrical charge was comparable to “pseudo-MPP” . Theoretical longevity was 6.2 years for MPP, 5.6 years for “pseudo-MPP” and 13.7 years for BVp. Conclusions: “Pseudo MPP” using intentional anodal capture with a quadripolar left ventricular lead, mimicking conventional multisite pacing, is feasible in most of CRT patients, with comparable energy consumption. Further studies on their potential clinical impact are needed.Background: Up to 40% of patients are CRT non-responders. Multisite pacing, using a unique quadripolar lead, also called multipoint/multipole pacing (MPP), is a potential alternative. We sought to determine the feasibility of intentional anodal capture using a single LV quadripolar lead, to reproduce MPP without the need of a specific algorithm . Methods: Consecutive patients implanted with a commercially available CRT device and a quadripolar LV lead in our department were prospectively included. The electric charge  of RV and LV pacing spikes were calculated for all available LV pacing configurations at the threshold. The best MPP was defined as the configuration with the lowest consumption (Q Cardiac resynchronization therapy (CRT) in heart failure showed a diminution of mortality and morbidity ,3,4,5,6.The initial concept of LV multisite pacing (MSp) emerged in this context and showed that pacing using 2 LV leads was associated with improved LV reverse remodeling, as compared with standard biventricular pacing (BVp) , especiaAnodal capture results, during bipolar pacing, from a high density current from the cathode allows for the capturing of the myocardium near the anode. Depolarization arises from both the anode and the cathode of the used electrical bipole. Intentional anodal capture during bipolar pacing by the LV quadripolar lead, so-called “pseudo-MPP” , may havWe sought to determine the feasibility of the intentional anodal capture using a single LV quadripolar lead, to reproduce MPP without the need for a specific algorithm.® , Boston Scientific®  and Medtronic®  were used.Consecutive patients implanted with a CRT device and a quadripolar LV lead in our department were prospectively included. Commercially available MPP-capable CRT device from AbbottEnrolled patients provided oral informed consent and data were collected in accordance with institutional guidelines on ethics.Electrical and ECG tests were performed after implantation. Threshold and impedance were manually measured in each programmable LV configuration. The pacing duration was 0.4 milliseconds. “Pseudo-MPP” stimulation was identified by a QRS morphology resulting from the fusion of the unipolar-cathode paced QRS complex and the unipolar-anode paced QRS complex. Its threshold was identified using an LV true bipolar vector decremental threshold test . VectorsRV + Qbest LV1 + Qbest LV2). The best “pseudo MPP”  and best BVp (QRV + Qbest LV) were also calculated. A theoretical longevity was estimated for best BVp, best MPP and best “pseudo-MPP” configurations in order to compare the battery drain. We considered permanent (100%) RV and LV pacing at 60 beats per minute in the VVI mode without atrial pacing, without any electrical shock, at the threshold without a safety margin, with a pacing duration of 0.4 ms, and a usable capacity of 1000 mAh.Using the data of the threshold tests, the electric charge  of the RV and LV pacing spikes were calculated for all available LV-pacing configurations at the threshold at 0.4 ms (without any safety margin). The first LV vector for MPP was named LV1, the second was named LV2. The best MPP was defined as the configuration with the lowest consumption . Qualitative variables are described using counts and percentages and continuous quantitative variables as mean ± standard deviation. Comparisons between MPP, pseudo-MPP and BVp were performed using non-parametric tests. Statistical significance was assumed at p < 0.05. All statistical analyses were performed using JMP®, 5 with a Boston® and 5 with a Medtronic® device).A total of 235 configurations were tested in 15 consecutive patients  vectors was available in 47% of patients . The mean “pseudo-MPP” threshold was 5.2 ± 0.9 V. Only two vectors with PNS during the “pseudo-MPP” test were excluded.Pacing characteristics are described in p = 0.15). The theoretical longevity was 6.2 years for MPP, 5.6 years for “pseudo-MPP” and 13.7 years for BVp  “pseudo-MPP” is feasible in 80% of patients, in almost half of the patients when using the most distant electrodes; (2) “pseudo-MPP” and MPP energy consumptions are comparable.Dell’Era et al. enrolled 30 CRT patients and tested, during the implantation procedure, the anodal capture on quadripolar leads from the three main manufacturers, using a Medtronic pacing-sensing analyzer .Conversely, we evaluated the feasibility of “pseudo-MPP” after implantation using the implanted CRT devices algorithms. They found an anodal capture in 93% of the patients, with a maximum pacing impulsion of 10 V at 0.5 ms, a much higher value than the one programmable in “real life”, explaining the lower 80% rate in our study. The mean anodal capture threshold was 3.9 ± 2.4 V at 0.5 ms (5.2 ± 0.9 V at 0.4 ms in our study) and the mean cathodal capture threshold was 1.9 ± 1.6 V at 0.5 ms (2.2 ± 0.8 V at 0.4 ms in our study), which was found to be comparable in both studies.“Pseudo MPP” has several advantages. It allows for MPP in MPP incompatible devices. Anodal capture is also associated with absence of intraventricular delay (0 ms), which could be useful in devices with a minimum programmable delay (5 ms for Abbott and 10 ms for Medtronic for example), and may achieve a better resynchronization .We do not provide data on any hemodynamics improvement. Morishima et al. showed an acute improvement on dP/dT max during “pseudo-MPP” comparable to MPP . The benefits of MPP and more so of “pseudo-MPP” remain unclear, and acute hemodynamics improvement does not predict the long-term response and/or clinical outcomes.Anodal capture is time consuming, necessitating a 12-lead electrocardiogram and a manual threshold test, which may be difficult to perform routinely. Very limited changes in the QRS morphology during “pseudo-MPP” threshold tests were observed in a few cases, with a difficult determination of the true “pseudo-MPP” threshold and probably limited effects on the depolarization wavefront and resynchronization.“Pseudo-MPP” and MPP are associated with rapid battery depletion as compared with conventional BVp. Indeed, the battery drain was about twice the difference versus BVp. A prospective study of the selected patients would be interesting in order to detect potential clinical impact. However, high battery drain would be associated with frequent generator changes and associated risks (infectious for example). In our opinion, only non-responder patients to CRT could ethically be included by comparing the best BVp versus MPP and “pseudo-MPP”. “Pseudo MPP”, using intentional anodal capture with a quadripolar left ventricular lead, mimicking conventional multisite pacing, is feasible in most CRT patients, with a comparable energy consumption. Further studies on the potential clinical impact are needed.


Retinal alterations in inherited metabolic diseases associated with neurodegeneration are poorly studied. The objective was to study retinal thickness, specifically the components of the ganglion cell complex (GCC)—nerve fiber layer (NFL), ganglion cell layer (GCL), and inner plexiform layer (IPL)—using spectral-domain optical coherence tomography (SD-OCT) in two different diseases with potential dopaminergic depletion, phenylketonuria (PKU) and Gaucher disease type 3 (GD3).Retinal layers in 19 patients with PKU, 15 patients with GD3, and 93 healthy individuals were measured using peripapillary ring scan and macular SD-OCT. Linear mixed models were computed including an adjustment for age, sex, and spherical equivalent. We calculated Spearman’s rank correlations between retinal layer measurements and clinical and/or laboratory parameters.p = 0.002), and outer ring (p = 0.012), sparing the fovea (p = 0.12) in PKU, while in GD3, all subfields were thinned . In both conditions, thinning was most evident in the NFL, GCL, and IPL, while OPL (outer plexiform layer) was thickened. Peripapillary retinal nerve fiber layer measurements remained normal. GCL and IPL in PKU correlated with tyrosine serum concentration.Thinning of total retinal thickness was found in the macular inner ring (Thinning of the NFL, GCL, and IPL, with thickened OPL, are both found in PKU and in GD3. Low dopamine concentrations in the retina might promote these effects. However, these data do not give evidence that retinal measurements can be used as a biomarker for disease severity in patients with GD3. Optical coherence tomography (OCT), a non-invasive technique with a high resolution, enables to describe retinal structures almost at a cellular level in vivo. OCT layers correlate well with histological findings of the retina , 2. The Ganglion cell complex (GCC) thinning has recently been reported for the first time in the inherited metabolic diseases phenylketonuria and GaucOur aim was to study segmental retinal layers, specifically the components of the ganglion cell complex (GCC)—nerve fiber layer (NFL), ganglion cell layer (GCL), and inner plexiform layer (IPL)—by means of spectral-domain (SD) optical coherence tomography (OCT) in two different diseases with potential dopaminergic depletion, phenylketonuria (PKU) and Gaucher disease type 3 (GD3). The second aim was to identify whether retinal layer measurements correlate with established disease features and if they may be used as a biomarker for disease severity. This should be of special interest for patients with GD3.This study included 19 patients aged 6 to 46 years with mild, or classical, phenylketonuria or tetrahydrobiopterin-deficient hyperphenylalaninemia, 15 patients aged 6 to 44 years with Gaucher disease type 3, and 93 controls aged 6 to 75 years. The patients’ inclusion criteria were genetical and/or biochemical prove of their disease. Participants under 6 years of age were excluded. Controls were included if they had no ocular disease and age-appropriate visual function, as well as no relevant systemic disease . Controls were recruited stratifying for age (seven subgroups with at least 10 participants were built for an even age distribution).The study was approved by the Medical Ethical Committee of the State Chamber of Medicine of Rhineland Palatinate in Mainz, Germany (reference number 837.373.14). All persons or their parents/guardians gave their written informed consent prior to inclusion in the study. The research adhered to the tenets of the Declaration of Helsinki.The examination included non-cycloplegic auto-refraction measurements , best-corrected visual acuity testing, slit lamp biomicroscopy, and fundus examination, as well as orthoptic examination, which were published elsewhere –23. SpheImaging of the optic nerve head and the macula was carried out using spectral-domain (SD) optical coherence tomography (OCT)  with automatic real-time function for image averaging. We acquired a peripapillary OCT and a macular OCT. The peripapillary retinal nerve fiber layer (pRNFL) was imaged with a diameter of 12° (corresponding to 3.47 mm in the standard eye), and a standard corneal curvature of 7.7 mm. For the macular OCT, 49 horizontal single scans were acquired. After semi-automated segmentation of the retinal layers as provided by the OCT software , all scans were assessed regarding their quality by a board-certified ophthalmologist (SH). Those with segmentation errors were corrected, or excluded in cases of poor image quality. In cases of poor data in only one sector, this sector was excluded prior to analysis; if more sectors were affected, then the complete OCT dataset of this eye was excluded.Mean retinal thickness of 9 macular layers was used for the analysis: total retinal layer thickness of the macula, nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), outer retinal layer (ORL) , and retinal pigment epithelium (RPE).The 6-mm macular scan measurements were classified according to the ETDRS segments (“Early Treatment Diabetic Retinopathy Study” subfields). Central zone, inner ring, and outer ring with diameters of 1, 3, and 6 mm, respectively, were included in the analysis. The average of all points within the central zone (1 mm diameter) was defined as foveal thickness, the inner ring (1 to 3 mm) as parafoveal thickness, and the outer ring (3 to 6 mm) as perifoveal thickness.first, current phenylalanine serum concentrations of each individual (mean 693 µmol/l ± 384 µmol/l); second, current tyrosine serum concentrations of each individual (mean 105 µmol/l ± 60 µmol/l); third, disease treatment ; and fourth, disease type  [first, the modified severity scoring tool (mSST), which is based on twelve domains including horizontal gaze palsy, cranial nerve palsy, seizures and age at first seizures, cognitive ability, ataxia, tremor, spasticity, rigidity, dysphagia, dysarthria, and spinal alignment [Second, we considered the phenotype severity , of which intermediate phenotype was associated with homozygous L444P mutation [Third/fourth, we included horizontal/vertical peak velocity of reflexive saccades in GD3 .Typical variables described for disease stage in PKU and GD3 were determined and obtained from the patient’s records. For PKU, we used  µmol/l) , classiclignment . Second,mutation . Third/fMedians, interquartile ranges, minimums, and maximums were calculated for all continuous variables. For variables distributed normally, means and standard deviations were computed. For dichotomous variables, absolute and relative frequencies were computed.p-values should be regarded as continuous parameters that reflect the level of evidence from our explorative analysis and are therefore reported exactly.To analyze the differences of retinal thickness with respect to PKU and GD3, we used linear mixed models to control for the inclusion of one and two eyes of a study participant (as random effect). A further adjustment for age, sex, and spherical equivalent was included in the statistical analysis. Full thickness measurements are more susceptible to these parameters, than single layer measurements, which is why we focused on single layer correlation analysis as follows. Spearman’s rank correlation was conducted to correlate thinned or thickened layers  with disease-specific variables of PKU  and GD3 . The other retinal layers, which were not different to controls, or which were underrepresented in the specific subfield (fovea), were not further analyzed. Correlation coefficient rho of ≥ 0.5 was regarded a moderate correlation, correlation coefficient of ≥ 0.3 was considered a weak correlation, and < 0.3 was considered a no correlation. Statistical analysis was performed using R version 4.0.4. All All patients examined were included in the study. From the 19 PKU patients (mean age 20 ± 12 years), 15 GD3 patients (mean age 20 ± 10 years), and 93 controls (mean age 32 ± 17 years), we excluded 6, 8, and 8 eyes respectively for the pRNFL analysis due to poor image quality, and 0, 2, and 6 eyes respectively for the macular OCT analysis.The OCT measurements indicate a significant thinner total retinal thickness in the inner and outer ring, but not in the fovea. This pattern affected the NFL, GCL, IPL, and ONL, while OPL was thickened. The differences were most evident in the NFL, GCL, and IPL (see Table p = 0.0008 (< 0.001); inner ring: rho = 0.51, p = 0.025), as well as IPL did . ONL correlated inversely with current phenylalanine serum concentration . Early-treated PKU patients had rather thick GCL  and thick IPL  compared to late-treated patients.GCL correlated with current tyrosine serum concentration  compared to the controls, even after adjusting for sex, age, and spherical equivalent Table . The retp = 0.046; inner ring: rho =  − 0.39, p = 0.11), and OPL correlated inversely with horizontal peak velocity . In GD3, no correlations were found for foveal total retinal thickness, and GCL, and none with vertical peak velocity or phenotype severity (mild/intermediate/severe).NFL outer ring correlated inversely with mSST , but the difference did not remain significant after adjusting for sex, age, and spherical equivalent  and a group without (n = 37), independently of the type of GD. Their results were that GCC thickness differed between young patients with parkinsonian features and those without parkinsonian features. However, between GD1 (n = 14) and GD3 (n = 34), GCC thickness did not differ significantly, although all GD patients together (93.1 (± 7.0) µm) differed from the controls (98.7 (± 9.6) µm) [n = 11) or GBA mutation carriers (intermediate level of glucocerebrosidase activity) in a study conducted by McNeill et al. [In line with previous investigations, we confirm significant retinal thinning in the NFL (outer ring), GCL (outer and inner ring), and IPL (inner ring) after adjusting for sex, age, and refraction, and thickening of the OPL in GD3. Our data thus attest reduced GCC as reported by Tantawy et al., who divided a cohort of GD patients aged 11 to 29 years into a group with parkinsonian features (9.6) µm) . Similarl et al. . GD1 patl et al. , 20, altl et al. . In GD1,l et al. . In GD3,The pathways of retinal damage including that of retinal ganglion cells are not fully understood in both diseases. Changes in neurotransmitter metabolism are discussed to play a role. Retinal dopamine deficiency is discussed to play a role in primary retina degeneration and secondary loss of dopamine-regulated neurons . BesidesAs follow-up data are missing, we cannot exclude that in GD3 metabolic imbalances during embryonic development already influence a normal retina development.While Serfozo et al. found that average pRNFL was reduced—without prove of reduction in the quadrants—in early-treated phenylketonuria , we did glaucoma of normal pressure or a nervous degeneration [. While no other studies have investigated pRNFL in GD3, Weill et. al reported abnormal pRNFL scans in one-third of GD1 patients with significant thinning of the average, superior, and inferior pRNFL [Peripapillary RNFL reduction as detected by Matos et al. in a GD3 patient is not confirmed by our data. In this case, the authors attributed their findings to either neration . While nor pRNFL . This paor pRNFL . From ouThis study has several limitations. First, we did not have complete long-term blood biomarker data of the PKU group  for the analysis. We also could not provide data of parkinsonian motor signs  , or prodThis OCT study with PKU and GD3 patients confirmed that retinal thickness is reduced, to elucidate, at the level of the NFL, GCL, IPL, and ONL, while OPL is thickened in PKU patients. The same is held true for GD3 although with fewer significance. Individual follow-up examinations are required for evaluation and detection of a progression of retinal neurodegeneration. This is important, because current therapies for these conditions might interfere with progressive morphological changes of the retina.


In individual cores, AOA also exhibit similar cell-specific power requirements, regardless of the AOA population size or sediment depth/age. Such quantitative insights establish a relationship between the power supply and the total abundance of AOA, and therefore lay a foundation for a first-order estimate of the standing stock of AOA in global marine oxic sediments.Energy/power availability is regarded as one of the ultimate controlling factors of microbial abundance in the deep biosphere, where fewer cells are found in habitats of lower energy availability. A critical assumption driving the proportional relationship between total cell abundance and power availability is that the cell-specific power requirement keeps constant or varies over smaller ranges than other variables, which has yet to be validated. Here we present a quantitative framework to determine the cell-specific power requirement of the omnipresent ammonia-oxidizing archaea (AOA) in eight sediment cores with 3–4 orders of magnitude variations of organic matter flux and oxygen penetration depth. Our results show that despite the six orders of magnitude variations in the rates and power supply of nitrification and AOA abundances across these eight cores, the cell-specific power requirement of AOA from different cores and depths overlaps within the narrow range of 10 The deep sedimentary biosphere underneath the seafloor is estimated to host a similar number of microbes as found in the oceans , 2. The 2 oxidation   and its activity coefficient γi, which was computed as a function of temperature and ionic strength by using an extended version of the Debye−Hückel equation [Gr with sediment depth. Since NH3 is a more likely substrate of ammonia-oxidizing archaea [3 rather than NH4+ in this calculation. We estimated NH3 concentrations from the porewater NH4+ concentrations based on the pH-sensitive NH3/NH4+ equilibrium [−)−1.The Gibbs free energy was calculated for nitrification , taken as the modeled nitrification rates. The resulting Ps, in the unit of kJ m−3 yr−1, was converted to the unit of Watt m−3, given that 1 Watt is equal to 1 Joule per second (1 J/s).Following ref. , the powamoA gene following the procedure described in [Total DNA in sediment horizons of the four AMOR cores was extracted as described in . AOA aburibed in , with qPribed in . Total cribed in , 23, assribed in , 23, butribed in , 31).For sediments from the NPG, SPG, and Arabian Sea where AOA abundance data are not available, we estimated the AOA abundances from the total cell counts, based on the power-law distribution of the relative abundance of AOA with sediment depth derived from the compilation of the amplicon sequencing datasets from AMOR , and NPNitrosopumilus maritimus SCM1 and NAOA6 incubated under various phosphate-limited conditions, [N. adriaticus NF5 and N. piranensis D3C reported in [N. cobalaminigenes HCA1, N. oxyclinae HCE1, and N. ureiphilus PS01 reported in [−1 h−1 [−)−1) for the calculation. For comparison, we also included the cell-specific power requirement of AOB Nitrosomonas marina, 2.8 × 10−14 W cell−1, calculated by [Cell-specific power requirement of AOA was calculated by dividing the power supply of nitrification by the AOA abundance estimated from the total cell numbers, assuming all the AOA cells are equally active  at a particular site and contribute equally to the nitrification activity. To assess how low the power requirements of the sedimentary AOA are, we compared their cell-specific power requirements with values of various AOA strains cultured in the laboratory, including ditions, , N. adriorted in , as wellorted in . We calc [−1 h−1 , 51) andlated by  based onlated by .2+) from deeper anoxic sediments ; (ii) nitrate concentration in the pore water increases beneath the sediment−water interface. Similar nitrate profiles were extensively observed in abyssal sediments (>2000 m water depth) [All sediment cores considered in this study Fig.  were retr depth) , 55–60 a2, NO3−, DIC, and TOC generally match well with the measured ones  would eliminate denitrification in the oxic zone, but make the simulated nitrate profile deviate substantially from the measured one , we adopted values of 4–30 µM for kO2 Table , the oxyone Fig. . Denitriorg m−2 yr−1) in SAST and the lowest (1.4 × 10−4 mol Corg m−2 yr−1) in SPG_U1370 (Table 2 concentration drops below 1 µM) varies over three orders of magnitude at different locations . CoGr, in the unit of kJ (mol e−)−1, was calculated for all cores based on the modeled pore-water profiles. Although in some cores  the modeled NH4+ concentrations in the oxic sediments are up to one order of magnitude higher than the detection limit −1 −1; [Given that nitrite, the intermediate compound of nitrification, was not observed to accumulate in any of the oxic sediments but only detected in some anoxic layers , we assmit Fig. , such ovmit Fig. . Our Gib)−1 Fig. . The calg. −)−1; ), suggesGr. Power supply of nitrification generally decreases with depth in individual cores . In they = 22.3*exp(−2x), R2 = 0.64, n = 40; Fig. AOA are the most abundant archaea in marine oxic sediments, as shown in some of the investigated sediment sites  were estimated in SAST, intermediate in the AMOR cores, and the lowest in the SPG_U1370 (1.0 × 102−1.0 × 105 cells cm−3)  Fig. . AOA abuls cm−3, ), but arThe calculated AOA abundance also shows a log−log decreasing relationship with sediment depth across the eight cores Fig. , resembl−19–10−17 W cell−1  and methanogens (with a median of 1.50 × 10−20 W cell−1) in global anoxic sediments [We calculated the cell-specific power requirement of AOA by assuming all the AOA cells have similar power requirements. This assumption is supported by the observation that AOA communities in the oxic sediments of NP_U1383E  and GC08l−1 Fig. , regardlum BS‐1; ) but are cell−1; ). This m cell−1; ) which wediments , consistediments . This diediments . While mediments , 16, our−15−8.8 × 10−14 W cell−1, based on the data reported in [Nitrosomonas marina grown under chemostat conditions  cell−1, )  could be very close to the BPR of AOA, which was thought to support the maintenance operation, like repair and replacement of damaged biomolecules, cross-membrane transport of ions, and nutrients and/or energy substrates [Our calculation of cell-specific power requirements provides a realistic constraint on BPR of AOA in the energy-limited subseafloor sediments. We observed that AOA inhabiting marine oxic sediments have similar power requirements spanning from the top centimeters to 42 mbsf Fig.  regardlebstrates , 16. The−18–10−17) in the deeper part of the oxic zones ., 89.60, l seabed , 90.Our study provides quantitative insights into the energetics of AOA in marine oxic sediments, from the top millimeters to 42 meters below seafloor. By examining eight sediment cores with markedly different organic matter fluxes and OPDs, we showed that the reaction rate and power supply of nitrification exhibit a vertical decreasing trend in individual cores and also six-orders-of-magnitude variations between different cores. AOA abundance showed a log−log decreasing relationship with depth in marine oxic sediments, suggesting that this functional group is controlled by power ultimately derived from the degradation of organic matter.The cell-specific power requirement of AOA is two to five orders of magnitude lower than the reported values for AOA and AOB obtained under laboratory conditions. The cell-specific power requirement of AOA varies in a narrow range throughout the examined sediment depths, and is similar across the five contrasting sites, providing a realistic constraint on the BPR of microbial life inhabiting subseafloor sediments. Combining with the abundant existing sediment geochemical data, our quantification of cell-specific power requirements of AOA may lay a foundation for a first-order estimate of the standing stock of AOA in the global marine oxic sediments.Supplementary Information