Using the ratio of an established IFN-γ signature and IMS led to consistently better prediction of the ICI therapy outcome compared to a collection of nine published GEP signatures from the literature on a newly generated internal validation cohort (n = 55) and three published datasets of metastatic melanoma treated with anti-PD-1 (n = 54) and anti-CTLA-4 (n = 42), as well as in patients with gastric cancer treated with anti-PD-1 (n = 45), demonstrating the potential utility of IMS as a predictive biomarker that complements existing GEP signatures for immunotherapy.Immune checkpoint inhibitor (ICI) treatments produce clinical benefit in many patients. However, better pretreatment predictive biomarkers for ICI are still needed to help match individual patients to the treatment most likely to be of benefit. Existing gene expression profiling (GEP)-based biomarkers for ICI are primarily focused on measuring a T cell-inflamed tumor microenvironment that contributes positively to the response to ICI. Here, we identified an immunosuppression signature (IMS) through analyzing RNA sequencing data from a combined discovery cohort ( In addition, the efficacy of ICIs has been observed to be significantly lower for East Asian melanoma patients than for Caucasian patients6.Historically, advanced melanoma has a poor prognosis, with a 5-year survival rate of less than 10%.9. Although the potential predictive power of PD-L1 expression for the clinical benefit of anti-PD-1 therapy remains controversial for advanced melanoma patients11, higher TMB has been correlated with a superior clinical response13, improved survival15, and durable benefit16 in advanced melanomas. In Asian melanoma patients, acral18 and mucosal melanomas18 are the predominant subtypes and generally have a low point mutation burden. Consequently, it is not clear whether TMB is an effective predictor for advanced Asian melanoma patients.Published data suggest that tumor mutational burden (TMB) and PD-L1 expression may predict the clinical benefit of anti-PD-1 therapy in multiple cancer types19. A recent meta-review showed that predictive models built on inflamed GEP signatures achieved a moderate area under the receiver operator curve (AUC) value of 0.65 for the summary receiver operation characteristic (sROC) curve generated from 10 different solid tumor types in 8135 patients20.In addition to TMB and PD-L1 expression, prediction models based on GEPs have also been proposed. Most GEP signatures consider T cell inflamed microenvironments characterized by the upregulation of IFN-γ signaling, antigen presentation, and immune checkpoint-related genes when predicting response to ICIs across multiple cancer types. However, these features are necessary but not always sufficient for a cancer patient to receive clinical benefit from ICI treatmentsHere, we argued that immune suppressive elements in the tumor microenvironment (TME) should be considered in combination with an inflamed GEP signature to more accurately predict ICI therapy outcomes. The main objective of this study was to develop immunosuppressive GEP signatures that, when used in combination with inflamed GEP signatures, could better stratify patients based on their potential benefits from ICI therapy. We started by analyzing RNA-seq data from baseline biopsy samples of melanoma patients treated with anti-PD-1 therapy and identified a set of 18 genes that played an “antagonistic” role against a pro-inflammatory TME and lead to negative outcomes in the discovery cohort consisting of multiple datasets. Our results reveal that key genes of the identified IMS are related to hallmark activities of cancer-associated fibroblasts (CAFs), macrophages and epithelial to mesenchymal transition (EMT), and the balance between the IFN-γ signature and the IMS plays an important predictive role in both immunotherapy-naive primary tumors from The Cancer Genome Atlas (TCGA) database and ICI-treated patients.21 of advanced melanoma treated with an anti-PD-1 ICI with response and RNA-seq data, which we used as our discovery cohort . We then identified 18 genes of which the expression levels, after adjusting for IFN-γ signature score, are consistently associated with negative response to ICI in the discovery cohort as our IMS  and tumor-associated macrophages (TAMs) (CD16323 and SIGLEC125), as well as their associated cytokines or chemokines that lead to an immunosuppressive microenvironment  and stromal activities that lead to tumor proliferation, invasion and immune escape such as EMT or extracellular matrix (ECM) degradation . Therefore, high infiltration of CAFs and myeloid cells and their related stromal activities may be the reasons behind the lack of response from patients with an inflammatory TME. To further validate this hypothesis, we performed digital cell composition analysis using xCell30 on a combined melanoma dataset consisting of the three datasets in the discovery cohort and a TCGA melanoma dataset (n = 516) and in TCGA melanoma dataset only (n = 309), and analyzed the distribution of different immune cell types with respect to the IFN-γ signature and IMS scores.The identified IMS shows a strong presence of genes related to CAFs , monocytes  and macrophages   but not the IFN-γ− subgroups. On the other hand, higher IFN-γ signature scores were associated with increased infiltration of CD8+ T cells, CD4+ T cells and B cells. However, the association of IMS scores and abundances of these cells within the microenvironment is not significant. Similar association can be observed on cell type abundance results from TCGA melanoma dataset only  Fig. . Stratif01) Fig. . Fibrobl Fig. . Sn = 11,043; Fig. r = 0.53 (p < 0.0001); however, IMS scores were poorly explained by IFN-γ signature scores (R2 = 0.28), suggesting that these two signatures are not fully overlapping and might contribute complementary information regarding the TME. A similar conclusion can be made on the correlation of the IMS and IFN-γ scores on selected cancer types from TCGA  and overall survival (OS) varied according to cancer type  in the Cox model for cancer types with this information available on TCGA and found that the association of the IFN-γ/IMS ratio and survival remains significant for BRCA, BLCA, SKCM and UVM after adjusted for tumor stage  and on selected cancer types  to anti-PD-1 therapies for cancer types with efficacy performance data available32 to get an educated guess of the applicability of these scores as potential biomarkers for immunotherapy. A positive correlation of ORR with the IFN-γ signature score , CESC34, BLCA35, lung squamous cell carcinoma (LUSC)36 and liver hepatocellular carcinoma (LIHC), have shown clinical sensitivity to ICI therapies  and predicted nonresponder groups (n = 26), IFN-γ/IMS successfully captured 67.71% of nonresponders (23 out of 35) with only 3 exceptions  and 2 patients with stable disease (SD) were misclassified as nonresponders), achieving a classification accuracy of 88.5% (p = 0.0006) for this group. On the other hand, of the 29 patients classified as predicted responders by IFN-γ/IMS, 17 (13 patients with a PR/CR and 4 patients with SD) actually responded. Overall, a higher IFN-γ/IMS ratio was associated with a better ORR  possibly due to limited sample size, and relatively short follow-up period of this cohort. In the public dataset of 54 preclinical metastasis melanoma treated with anti-PD-1 (Liu1913), IFN-γ/IMS achieved an AUC of 0.66 , Beijing, China. In this dataset, IFN-γ/IMS achieved a prediction accuracy of AUC = 0.81 40 and metastatic gastric cancer patients treated with anti-PD-1 therapy 41. The resulting AUCs were 0.75  can be found in these top 23 frequent IMS genes from the randomized test. Further analysis of these genes on a public single-cell RNA-seq (scRNA) dataset from melanoma42 indicated that most of these genes are highly expressed on CAF  production by cancer cells helps to induce CAFs, and ablating INHBA decreases the CAF phenotype both in vitro and in vivo49. CAFs hinder antitumour immunity by secreting immunosuppressive cytokines such as IL-10 and TGF-β, reducing the function and viability of cytotoxic T lymphocytes51 and attracting immunosuppressive myeloid cells, including TAMs, via CCL252. Notably, Siglec-1/CD163 is associated with the activation of macrophages towards an immunosuppressive phenotype, and accordingly, the expression of both CAF (FAP) and TAM (SIGLEC1) markers is associated with poor clinical outcomes across multiple tumor types56. CAF-mediated EMT, which is strongly correlated with the expression of AXL, TWIST2 and ADAM12 from the IMS, can result in biomechanical and biochemical changes that facilitate tumor immune escape, invasion, and metastasis57. The dense collagen matrix produced by CAFs may also present a physical barrier to the infiltration of T lymphocytes58 or treatments reaching the cancer cells59. Indeed, the association between a lack of response to ICIs and upregulated EMT-related genes has been observed in multiple cancers60, and inhibiting CAF/TAM-related pathways and extracellular collagen and hyaluronan can induce T cell accumulation and improve the outcome of ICIs64, reinforcing the role of those stromal-related activities in limiting the efficacy of immune checkpoint blockade immunotherapy.Interestingly, the genes identified in our IMS through the above computational method were highly consistent with several important biological activities related to innate or acquired resistance to ICIs. CAFs are a nonredundant, immunosuppressive component of the TME65. Typically, this is done with a set of housekeeping genes that are expected to maintain constant expression levels under different experimental conditions66. However, it has become increasingly clear that housekeeping gene expression levels may vary considerably in some conditions68. When that happens, the normalization process itself can lead to increased intersample “noise” that covers meaningful differences in target genes if the chosen housekeeping genes fluctuate randomly or erroneous results if there is a directional change in the housekeeping genes between experimental groups69. The calculation of the IFN-γ/IMS ratio provides a self-normalization method that directly measures the balance between contradicting biological processes within the tumor microenvironment, thus providing a selection process that is more robust to the confounding factor of intersample gene expression variations to identify genes that contribute negatively to the outcome of immunotherapy.Using the ratio of opposing immune signatures instead of the absolute value of individual signatures as a predictive biomarker brings another advantage. It is well known that to compensate for potential technical variation, raw gene expression data from RNA-seq must be normalized so that meaningful biological comparisons can be made70, as well as the difference in the cellular composition of primary and metastatic tumors. The IFN-γ/IMS ratio was primarily designed as a predictive biomarker for anti-PD-1 therapy for melanoma. Therefore, the OS results reported in this paper should not be overinterpreted since OS is determined not only by response but also by events not considered in the current model such as acquired therapy resistance, adverse event, or presence of other targetable mutations, which need to be further evaluated towards a more meaningful prognostic biomarker. Finally, our analysis is retrospective in nature, and validation of the findings in additional datasets is warranted.Our study has limitations. Since the current ICI clinical trials have generated gene expression data for only a limited number of pretreatment samples, which were insufficient to train robust prediction models, we did not systematically optimize the weights of individual genes in the IFN-γ/IMS ratio calculation. With more RNA-seq data available from subsequent studies, we expect that further optimization of the combined biomarker will yield even better predictive accuracy. In this study, we did not attempt to specify a universally applicable cutoff point for IFN-γ/IMS for different datasets due to potential batch effects from different RNA-seq procedures conducted at multiple sites. Rather, we demonstrated a trend that shows an increase in benefits with increasing IFN-γ/IMS ratios. Nevertheless, we envision that a relevant cutoff would need to be aligned to specific assay designs and clinical situations as the cutoff value may be affected by the sampling bias from intra-tumor heterogeneity17 and shows decreased response to PD-1 monotherapy11. Although recent research found that the efficacy could be greatly improved by PD-1 based combination therapy in mucosal melanoma cohort71, the molecular mechanisms and predictive biomarkers are yet to be explored. Nevertheless, results in this study indicate that the IMS signature plays a very similar role in profiling the immune suppressive microenvironment of acral or mucosal melanoma although it was derived from cutaneous melanoma. In this regard, it is anticipated that IMS or future immunosuppressive signatures gleaning through deeper understanding of the immunosuppressive mechanisms of cancer would enable the development of more effective stratification models or therapeutic combinations to increase the efficacy and cost-effectiveness of immunotherapies for the benefits of cancer patients.In conclusion, the IMS studied in this paper exemplifies the potential of using GEP signatures for modeling the adverse TME, and using IMS in combination with an existing inflammatory GEP signature enables better identification of patients who could respond favorably to ICIs. Currently, clinical trials are assessing the efficacy of combining anti-PD-1 therapy with medicines that target at normalization of immune suppressive TME including the CSF1R inhibitor Cabrilizumab for the treatment of resectable biliary tract cancer (NCT03768531), CCR2 inhibitor plozalizumab for the treatment of melanoma (NCT02723006), the FAP inhibitor RO6874281 for the treatment of metastatic head and neck, esophageal or cervical cancers (NCT03386721) and metastatic melanoma (NCT03875079), and the TGF-β inhibitor galunisertib (LY2157299) for the treatment of advanced-stage NSCLC or hepatocellular carcinoma (NCT02423343) and metastatic pancreatic cancer (NCT02734160). However, due to the diversity of the immune evasion mechanisms in inflammatory tumors, specific immunosuppressive mechanisms utilized by each individual tumor would still need to be fully understood and gauged to better direct patients to different combination therapy options. PUCH samples are mostly acral or mucosal, and are not biologically similar to samples from the TCGA or other melanoma datasets used in this study, which are mostly cutaneous. Acral or mucosal melanoma in primary site often has deeper Breslow thickness, higher incidence of ulceration, and is more prone to visceral metastasis. Compared with cutaneous melanoma. acral or mucosal melanoma has lower TMBIn this study, we obtained 55 formalin-fixed, paraffin-embedded (FFPE) pretreatment tumor tissues from melanoma patients treated with anti-PD-1 monotherapy at PUCH, Beijing, China, between March 2016 and March 2019 Table . Diagnos72, and gene expression was quantified using RSEM73. Coding region reads were counted to calculate fragments per kilobase of transcript per million mapped reads (FPKM) values at the gene level and log2-transformed before analysis to avoid extremely skewed gene expression distributions.Total RNA was extracted from unstained FFPE tumor samples by the All Prep-DNA/RNA-Micro Kit (Qiagen) following the standard manufacturer’s protocol. Reverse transcription and second-strand cDNA synthesis were subsequently performed. Barcoded RNA libraries were generated and captured by a customized whole-exome panel. All libraries were sequenced on the Illumina NovaSeq 6000 platform with 2 × 150 bp paired-end reads. The mean sequencing coverage across all samples was ∼100× (3.5 G). RNA-seq reads were mapped to the human reference genome GRCh37 using STARn = 51)15, Hugo16 (n = 28)14, Gide19 (n = 41)21, VanAllen15 (n = 42)40, Liu19 (n = 54)13, and Kim18 (n = 45)41 datasets  were used. The immunotherapy outcomes provided in the original publications following RECIST guidelines (PR/CR/SD/PD) were used in our analysis. The gene expression data of VanAllen15 and Liu19 were downloaded from respective references as provided by the authors. For Riaz17, Hugo16, Gide19 and Kim18, the RNA-seq raw data was obtained and processed by the above-mentioned pipeline to generate the gene expression data.We collected the RNA-seq data of melanoma patients from six immunotherapy studies with gene expression profiles for pretreatment tumors and complete clinical information, including the Riaz17  genes  with low variance across a set of banked tumor samples from a variety of cancer types. The log2-transformed expression of each gene was normalized by subtracting the arithmetic mean of the log2-transformed expressions of the housekeeping genes.t-test to capture genes that were significantly uplifted in the nonresponse group (PD) versus the response group (PR/CR) after they were normalized by the IFN-γ signature scores. Due to the large dimensionality of the data, we restricted our search to the 770 cancer immune-related genes curated in Nanostring’s IO 360 panel. In addition, to avoid potential batch effects from different datasets, the one-sided Student’s t-test was performed on each individual dataset independently and the resulting p-values from the three datasets in the discovery cohort were then combined using Pearson’s method45 to avoid artefacts due to single significance from individual dataset. Finally, the genes were ranked based on their Pearson combined p-values, and the top 18 genes were identified as our IMS genes.To identify the IMS genes, we first normalized the gene expression levels of a patient with respect to his/her IFN-γ signature score. We then performed a one-sided Student’s 80 44. Similarly, the IMS score was calculated as the arithmetic mean of the log2-transformed, housekeeping gene-normalized expression levels of the 18 IMS genes listed in Supplementary Table For the IFN-γ signature in this paper, we used the arithmetic mean of the log2-transformed, house-keeping gene-normalized expression levels of the 10-gene “preliminary” IFN-γ signature  was performed to reduce the dimensionality of the scRNA-seq profiles. Then t-SNE projections were generated using the first 25 principal components. Both PCA and t-SNE analysis were performed by RunPCA and RunTSNE functions in the Seurat package (version 3.1.0) with default parameters.Briefly, scRNA-seq data of 31 melanoma tumors were downloaded from GEO database (GSE115978)p-values were used unless otherwise specified, and a p-value less than 0.05 was considered significant. For boxplots, center mark is median and whiskers are minimum/maximum unless specified otherwise.Associations between categorical measurements and patient groups, such as the predictive accuracy of different biomarkers/panels, were evaluated using Fisher’s exact test. Differences in continuous measurements were tested using the two-tailed Mann–Whitney U-test. Correlations between two groups of continuous variables were evaluated using Pearson correlation analysis. Multivariable Cox proportional hazard regression models were used to identify prognostic factors, and the results were reported as hazard ratios (HRs) with 95% confidence intervals. The Kaplan–Meier method was utilized to estimate OS and PFS curves, and difference between groups were assessed using the log-rank test. Two-sided To validate the robustness of the proposed approach in identifying the IMS GEP signature, we performed a randomized permutation test where three datasets were randomly selected from the seven datasets Table  as the dhttp://www.graphpad.com), and the Python language and programming environment were used for the remainder of the statistical analysis. The abundances of multiple cell types in whole tissue samples were estimated using xCell30.PRISM was used for basic statistical analysis and plotting (All the procedures including the collection, processing and analysis of tumor samples in this study were approved by the Institutional Review Board of the PUCH. Written informed consent was obtained from all participants.Further information on research design is available in the Supplementary InformationReporting Summary


Pericardial adipose tissue is associated with excess cardiovascular risk factors and may itself be a marker for and modulator of cardiovascular disease, possibly through paracrine activity as pericardial adipose tissue is metabolically highly active. In many research studies pericardial fat thickness (PFT) is measured using transthoracic echocardiography from the parasternal long-axis view, from which PFT over the right ventricular (RV) free wall is determined. However, the ability of echocardiography to measure PFT is limited by acoustic window issues and potential confounding with pericardial effusion. Pericardial fat is readily seen on routine CMR imaging for ventricular function, but there is no standard approach to assessment of pericardial adipose burden by CMR. For simplicity and to facilitate comparison with the literature, we elected to measure RV free wall PFT by CMR from the 4-chamber view. In this abstract we sought to determine whether measurement of PFT at its maximum versus at a predefined location  would change stratification of subjects by PFT.2) over the mid-level right ventricle (4chMID) and at maximal thickness in the same orientation (4chMAX). Sex-specific ranking of subjects by PFT was performed for 4chMID and for 4chMAX. Changes between 4chMID and 4chMAX ranks were summarized as median [interquartile] differences. Additionally, subjects were then stratified by quintiles of PFT. We assessed concordance of individual subject rankings by Spearman correlation (rs) and tabulated number of across-quintile changes for each sex.500 adults  were randomly selected from the Framingham Offspring Cohort CMR study database. Subjects were imaged on a 1.5-T scanner  using a cardiac array coil for RF signal reception. PFT was measured at end-diastole from the 4-chamber view using a cine SSFP sequence  and women (rs = 0.763), both p < 0.0001. The median [interquartile] rank-differences were 31  for men and 30  for women. Figure PFT could be measured successfully for all subjects, with good reproduciblity between readers . Men had significantly greater PFT than women for both 4chMAX  and 4chMID . There was good correlation for ranking of PFT by 4chMAX and 4chMID measurements for both men (rMen have greater PFT than women, but concordance in ranking between 4ChMAX and 4ChMID PFT was similar for both sexes. As ranking of subjects was similar regardless of whether maximal or mid-RV PFT was used, it may be reasonable to select a standardized measurement method, i.e. mid-RV level PFT, for consistency. Further work is needed to determine whether PFT measured by CMR has additional prognostic value over standard clinical predictors of cardiovascular risk, but PFT can be determined reliably and reproducibly using routine SSFP sequences for assessment of ventricular function, so that PFT can be determined without increasing examination time in a standard clinical CMR examination.


Methylation of mitochondrial tRNAs (mt-tRNA) at the 9th position (“p9 site”) is known to impact translational efficiency and downstream mitochondrial function; however, direct assessment of mt-RNA methylation is challenging. Recent RNA sequence-based methods have been developed to reliably identify post-transcriptional methylation. Though p9 methylation has been studied in healthy human populations and in the context of cancer, it has not yet been analyzed in neurodegenerative disease, where mitochondrial dysfunction is a prominent and early hallmark of disease progression.Mitochondrial p9 methylation was inferred from multi-allelic calls in RNA-seq data. Gene-based association studies were performed in FUMA. Correlations between nuclear gene expression and p9 methylation were tested using Spearman’s rho. Fisher’s Exact test was used in PANTHER and IPA to test for overrepresentation and enrichment of biological processes and pathways in the top nuclear genes correlated with p9 methylation.Variable methylation was observed at 11 p9 sites in post-mortem cerebellar tissue of elderly subjects who were either healthy or diagnosed with Alzheimer’s disease (AD), progressive supranuclear palsy (PSP) or pathological aging (PA). Similarities in degree of methylation were observed between AD and PSP. Certain nuclear encoded genes were identified as significantly associated with p9 methylation. Expression of 5300 nuclear encoded genes was significantly correlated with p9 methylation, with AD and PSP subjects exhibiting similar expression profiles. Overrepresentation and enrichment testing using the top transcripts revealed enrichment for a number of molecular processes, terms and pathways including many of which that were mitochondrial-related.With mitochondrial dysfunction being an established hallmark of neurodegenerative disease pathophysiology, this work sheds light on the potential molecular underpinnings of this dysfunction. Here we show overlap in cerebellar pathophysiology between common tauopathies such as Alzheimer’s disease and progressive supranuclear palsy. Whether p9 hypermethylation is a cause or consequence of pathology remains an area of focus. Mitochondrial organelles play essential roles in oxidative phosphorylation, apoptosis, calcium homeostasis and a number of cell-signalling pathways. Many lines of evidence have implicated mitochondrial dysfunction in aging and neurodegenerative disease. Though the genetic etiology of neurodegeneration is highly heterogeneous, there is an extensive literature implicating mitochondrial dysfunction as a common and early factor in the pathophysiology of a number of neurodegenerative diseases . Much ofMitochondria contain their own 16,569 base pair genome consisting of 13 mRNA genes encoding electron transport chain subunits and 2 rRNA genes, which are ‘punctuated’ by 22 tRNA genes . One of Though various methods exist for measuring RNA methylation including in situ hybridization, methylation-specific antibodies and methylation-sensitive enzymes, many methods lack specificity and/or have limited experimental use, due to the high concentration of input RNA required . SeveralMRPP3) . I. I25]. INevertheless, it is important to acknowledge the technical limitations associated with traditional RNA sequencing workflows. Here we sought to investigate post-transcriptional methylation of mt-tRNAs using total RNA sequence data. Importantly, these RNA molecules are highly folded and small in nature, often making them difficult to capture. Traditional total RNA sequencing protocols, such as that used here often 1) do not enrich for, and 2) usually detail removal of, small RNAs. This means that mature mt-tRNA reads may have been under-represented in this dataset. Therefore, any conclusions made here may not be based on the entire mature mt-tRNA pool. Future studies investigating p9 methylation should aim to use small RNA enrichment protocols, as well as perform cDNA size selection prior to RNA sequencing.SLC28A3) located on chromosome 9 were found to be significantly associated with methylation at p9 site 585. The strength of this result was further substantiated by the clustering of neighbouring SNPs in linkage disequilibrium within the gene region  mapping to solute carrier family 28 member 3  on chromosome 15 (likely due to the restrictive window size of ±10 kb that was used) reached the suggestive threshold for association with p9 methylation at position 585. SENP8 is known to catalyze pathways associated with neddylation, a post-transcriptional modification analogous to ubiquitination; these pathways are also responsible for the endocytic degradation of APP. Dysfunction of these pathways has been observed in AD leading to characteristic accumulation of APP and Aβ [A single SNP (rs2034879) that mapped to several genes , dynamiP and Aβ , althougTRAIP and IP6K1 located on chromosome 3. TRAIP encodes TRAF interacting protein, an E3 ubiquitin ligase that plays a key role in cell survival and apoptosis. TRAIP serves important roles in cell cycle checkpoints by regulating spindle assembly, appropriate chromosome distribution during cell division, and DNA damage responses [IP6K1 encodes an inositol phosphokinase responsible for synthesis of 5-diphosphoinositol phentakisphosphate (5-IP7) from hexakisphosphate (IP6). IP6K1 serves as an important upstream regulator for various metabolic processes , apoptosis and global transcription [IP6K1 gene region have been previously identified, using tag SNP analysis, as being associated with AD [IP6K1−/−) yeast models have shown decreased mitochondrial respiration, yet increases in ATP, a paradox that is thought to be due to enhanced glycolysis and depletion of metabolic processes requiring ATP [Methylation at six of the 11 p9 sites  was significantly associated with a single SNP (rs9872864) mapping to two genes esponses . Cell cyesponses . IP6K1 ecription . It has cription  and vari with AD . IP6K1 a with AD . Knockouring ATP . Furtherring ATP  demonstrWhile several of the genes associated here are loosely connected to mitochondrial function, we did not identify any loci that may directly affect mt-tRNA processing. It is interesting to note that 5 out of the 6 genes identified in the gene-based GWAS had CADD scores > 10 (based on the top leading SNP or maximum CADD score of variants within the gene region). In brief, Combined Annotation Dependent Depletion (CADD) scoring is a predictive machine learning tool that allows for estimation of the deleteriousness and/or pathogenicity of causal genetic variants . In geneMRPP3, a key player in tRNA processing in the mitochondria. We did not replicate the MRPP3 association which is not entirely surprising since their study was conducted in normal adults (40–69 years) without any particular pathological conditions versus our study of aged subjects with neurodegenerative disease. It may follow that the variability in MRPP3 associated with p9 methylation levels described in Hodgkinson et al., is presumably normal, whereas p9 methylation in our cohort is presumably a result of pathological process(es). The resulting genotype associations may be pointing to novel gene variants that are upstream effectors of mitochondrial function. Conversely, it is important to acknowledge the caveat that gene-based association testing was performed together on all individuals of this cohort; the small sample size of each diagnostic category  left us underpowered to detect significant associations when analyzing by each group separately. Given that we identified p9 hypermethylation in both AD and PSP, it is possible that genes identified here as significantly associated with p9 methylation may be related to individual pathologies and not necessarily post-transcriptional RNA processes.Hodkinson et al. , reporteWe identified 5300 genes significantly correlated with p9 methylation. Using principle component analysis, we identified clustering of gene expression profiles by disease state with AD and PSP, and NC and PA grouping together respectively. Of the top gene expression hits, we identified over-represented GO terms related to a number of cellular components and processes using PANTHER. Interestingly, there was over-representation of mitochondrial components.DNA repair and cellular response to DNA damage stimulus) and canonical pathways  appeared to correlate with functions of one of the top genes (i.e.TRAIP) identified in the gene-based GWAS. Though some of the GO terms and canonical pathways are rather ambiguous, it does imply that p9 methylation is associated with altered transcription of gene-sets important for mitochondrial function.In order to identify tissue-specific changes, we performed further enrichment analysis in IPA using a cerebellum tissue reference and all of the transcripts (5300 unique genes) significantly correlated with p9 methylation. Again, several of the enriched pathways identified were linked to mitochondrial function and homeostasis, as well as DNA damage/repair. We also identified a number of pathways related to RNA processing. In addition, some of the GO terms (e.g. This is the first work analyzing mitochondrial p9 methylation in the context of neurodegeneration. Here we report an association between p9 methylation and nuclear-encoded gene expression, as well as mitochondrial-related regulation; although, the implications of these associations are unclear. We observed comparable post-transcriptional mt-tRNA hypermethylation and nuclear gene expression profiles in the cerebellum of Alzheimer’s disease and progressive supranuclear palsy patients. Although technical limitations in this analysis do not allow us to conclude on methylation status of the mature tRNA pool, the results here are likely indicative of molecular similarities underlying the mitochondrial dysfunction already known to occur within the cerebellum of individuals diagnosed with either of these tauopathies.A basal level of p9 methylation is required for proper tRNA folding and stability  and the Though at present, it is unclear whether mt-tRNA methylation is a cause or consequence of pathology, these results point to a potential role for mitochondrial post-transcriptional methylation in the pathophysiology of common tauopathies such as Alzheimer’s disease and progressive supranuclear palsy. Given this, post-transcriptional mt-tRNA methylation may serve as an exciting area of investigation for the development of future treatment strategies for neurodegenerative disease.Additional file 1. Additional figures.Additional file 2. Gene-based GWAS results.Additional file 3. PANTHER over-representation test results.Additional file 4. Boxplot by diagnosis.


Pasta made from 100% durum wheat semolina was used as a control. Soy protein isolate inclusion into banana pasta increased total phenolic content (TPC) and antioxidant capacities, while egg white protein decreased the TPC and antioxidant capacities with the increasing level of addition. Starch digestibility was affected by the type of protein addition. Egg white protein lowered starch digestibility compared to soy protein isolate. Protein inclusion in banana pasta also altered protein digestibility, amino acid profiles and protein digestibility-corrected amino acid score (PDCAAS). Soy protein isolate increased protein digestibility of gluten-free pasta compared to egg white protein. Protein enrichment gave better amino acid profiles of banana pasta compared to semolina pasta with egg white protein and performed a better PDCAAS compared to soy protein isolate. These results showed that soy protein isolate and egg white protein addition enhanced nutritional qualities and digestibility properties of gluten-free banana pasta.The effects of egg white protein and soy protein isolate addition on the nutritional and digestibility of gluten-free pasta based on banana flour were studied. The level of protein additions (soy protein or egg white protein) were 0, 5, 10 and 15% of banana flour ( The effort to provide suitable pasta for celiac and gluten intolerance-related patients has led to the production of gluten-free (GF) pasta. There are many varieties of GF pasta made from rice and other GF flours available, but these products often have poor cooking quality and technological difficulties compared to conventional wheat pasta as well as having an inferior nutritional quality especially with regard to minerals and bioactive compounds ,2. CommeOne GF flour that has great potential to be developed as a GF pasta ingredient because of its nutritional properties is banana flour. Banana flour is derived from unripe bananas which have a high phenolic content and high antioxidant capacity . Banana Banana flour has been applied in either wheat-based or GF pasta manufacture as an alternative base material, improving the pasta and making it comparable with wheat only counterparts ,9,10,11.Some of the research into banana pasta stated the nutritional values of pasta products, but none investigated how the ingredients affected the nutritional and digestibility properties of the final product. Sarawong at al.  reportedMost commercial GF pasta use hydrocolloids and emulsifier to improve cooking quality which led to the consumer association with artificial flavour ,15. AnotBanana flour was obtained from Food Compass Limited . Soy protein isolate (with a protein content of 91%) was obtained from Bulk Powder Limited . Egg white protein powder (80% protein content) was bought from Nothing Naughty Limited . Durum semolina  was used as a control.w/w). Semolina was also used to make control pasta. A pasta-making machine fitted with a spaghetti die  was used to produce the pasta.Banana flour was mixed with soy protein isolate or egg white protein powder in different levels: 0%, 5%, 10% and 15% addition of flour (w/v) and mixed for 20 min based on previous research [All dry components (300 g) were mixed in the pasta maker for 4 min. The pH of the mixture was kept at 7 for comparison purposes with the control pasta. Boiling water was added at 70%  of the sample extracts was determined spectrophotometrically using Folin-Ciocalteau’s (F-C) reagent according to the method described by Lim and Murtijaya  and Khan3·7H2O at 10:1:1 (v/v/v). A standard of iron (II) sulphate (FeSO4·7H2O) (0–200 μmol) or sample extract (250 μL) was added to 2.5 mL of the FRAP reagent and the absorbance at 593 nm was recorded immediately after the addition of the sample and again after 2 h incubation at 37 °C [2+/g sample.FRAP was assessed according to Khanizadeh et al.  with sliat 37 °C . The res•+ was prepared by reacting colourless ABTS stock solution (7 mM in water) with 2.45 nM potassium persulfate and allowing the reaction to stand for 16 hours in the dark at room temperature. On the day of analysis, the ABTS•+ solution was diluted with PBS (pH 7.4) to an absorbance of 0.70 (±0.02) at 734 nm and 3 mL transferred to a cuvette. After the addition of 300 μL Trolox (0–200 μmol) or sample extract, the mixture was well mixed, allowed to stand for 6 min and the absorbance was read at 734 nm. The results were expressed as Trolox equivalents (TE).The ABTS radical scavenging assay was based on the method of Cai et al. . ABTS•+ Cooked pasta (5 g) were cut with a knife to obtain a 2–5 mm size. The reducing sugars released over 120 min were evaluated for each pasta type as described by Foschia et al. .Protein content was determined by using element analyser model Vario MAX CN to determine percentage of total nitrogen. The value of total nitrogen content was multiplied by 6.25 to get crude protein content value.In-vitro protein digestibility of cooked pasta was performed using a multi-enzyme technique following the method used by Desai et al. . ProteinAmino acid profiles were determined using Agilent 1100 series  high-performance liquid chromatography following the methodology used by Heems et al. . The samp < 0.05) was performed to determine significant difference between treatments.All experiments were conducted in triplicate. The analysis of variance (ANOVA) was conducted in Minitab statistical software . Statistical differences in all nutritional characteristics were determined by one-way ANOVA for all treatments and general linear model analysis of variance (ANOVA) with 2 factors (type of protein and level of protein) with semolina pasta excluded. Tukey’s comparison test  of raw materials and pasta samples are shown in High TPC in soy protein isolate also contributed to enhancing TPC of banana pasta. The TPC of banana pasta increased by increasing fortification level of soy protein isolate. Soy protein isolate alongside with other soy products has been reported to contain high total phenolic content ranging from 104.3 to 243.1 mg GAE/100 g . DiffereAntioxidant activities including ferric reducing/antioxidant power (FRAP) and ABTS radical scavenging capacity of raw materials and pasta sample can be seen in Soy protein isolate addition provided higher FRAP and ABTS values compared to egg white protein. A higher soy protein level gave higher antioxidant capacities especially for ABTS values. On the contrary, higher egg white protein incorporation in banana pasta reduced antioxidant activities. This can be explained by lower FRAP and ABTS values in egg white protein, while soy protein isolate has high value similar to banana flour. Soy protein utilization in many food formulations has been established because of its functional and nutritional properties . Soy proThe effect of protein incorporation on starch digestibility can be seen in Some research projects have reported that protein inclusion in pasta based on semolina flour arrived at lower in-vitro starch digestibility properties compared to semolina pasta ,32,33. NThe amount of protein is not the only aspect to consider, but also the quality of protein itself . Oat pro-) and amino (-NH3+) groups. Free amino groups deionize in water and protons (H+) are liberated at neutral pH (8.0). The free H+ released into the solution decreased in pH value. The phenolic present in pasta could react with amino acid creating protein cross-link that inhibits further enzymatic protein degradation [Protein digestion was observed by pH change over time during multi-enzymes  incubation of pasta samples which can be seen in radation .Protein content, protein digestibility and protein availability of pasta samples are shown in Amino acid profiles (mg per g protein) of the pasta samples can be seen in Amino acid scores and protein digestibility-corrected amino acid scores can be seen in Protein digestibility-corrected amino acid score of cooked pasta samples ranged between 0.07 to 1.00 . Banana The addition of egg white protein and soy protein isolate into banana pasta altered the total phenolic content and antioxidant capacities compared to semolina pasta and pure banana pasta. Soy protein isolate increased TPC, ferric reducing/antioxidant power and ABTS radical scavenging capacity of banana pasta by the increasing level of fortification. On the contrary, egg white protein lowered phenolic content and antioxidant capacity values with increasing egg white protein addition.Glycaemic properties of banana pasta were also affected by the type of protein addition. Egg white protein inhibited reducing sugars released in fortified banana pasta, while soy protein isolate did not have significant impact on starch digestion properties. Protein enrichment in banana pasta also altered protein digestibility. Protein digestibility increased along with the increased protein inclusion in banana pasta. Amino acid profiles of enriched banana pasta also enhanced and met daily requirement for adult human with optimum protein digestibility-corrected amino acid score. These results show that soy protein isolate and egg white protein addition into gluten-free pasta formulation based on banana flour improved the nutritional and digestibility properties of banana pasta better than those of semolina pasta.


Radiation-induced sarcoma of the breast is an iatrogenic malignancy that occurs secondary to radiotherapy, which is most commonly given following breast conservation surgery. It has an incidence of 3.2 per 1,000 patients at 15 years and is associated with a poor prognosis. We report a 62-year-old female with a history of bilateral breast conservation surgery and radiotherapy 5 years ago presenting with bilateral angiosarcoma. This case report highlights the importance of considering radiation-induced angiosarcoma of the breast as a differential diagnosis in a patient with recurrent breast neoplasms. The challenges in the management with recent evidence on new treatment modalities are discussed. The management of breast cancer, the commonest cancer worldwide in women, has evolved during the last several decades where breast conservative treatment with systemic therapy has replaced radical surgery, improving quality of life and overall survival , 2. HoweA 57-year-old Sri Lankan Sinhalese woman was diagnosed with bilateral stage IIB (T3N0M0) invasive ductal carcinoma, which was of Nottingham grade 2 and oestrogen receptor (ER), progesterone receptor (PR) positive, and human epidermal growth factor receptor-2 (HER-2) negative. She underwent bilateral wide local excision and level II axillary lymph node dissection with negative resection margins followed by standard bilateral adjuvant radiotherapy  and 5 years of endocrine therapy with tamoxifen. Five years following the initial diagnosis, she presented with a rapidly enlarging right breast lump with skin erosion and bleeding of 2 weeks duration . UltrasoMacroscopic analysis of the specimens showed solid, irregular, haemorrhagic lesions. The right-side lesion involved the nipple areolar complex with surface ulceration measuring 60 × 50 × 30 mm and another lesion was found in the central quadrant measuring 30 × 25 × 25 mm. Microscopic analysis of all lesions showed similar features with anastomosing channels of vascular spaces lined by atypical cells with markedly pleomorphic vesicular nuclei and moderate eosinophillic cytoplasm . MitosesImmunohistochemical analyses showed strong and diffuse cytoplasmic and membrane positivity for CD 31 and occasional cytoplasmic and membrane positivity for CD 34 . Tumour Contrast-enhanced computed tomography scan showed no evidence of metastatic disease. She was started on chemotherapy and was disease-free at 15 months follow up.In 1907, Borman et al. describes the first case of angiosarcoma of the breast, whereas the first case of radiation-induced secondary angiosarcoma was described Body et al. in 1987 . With thThe reported patient presented with a rapidly enlarging right breast lump with skin erosion and spontaneous bleeding, which is an unusual presentation. Several case reports describe skin discoloration, thickening, and dimpling as the clinical presentation which may be subtle, delaying the presentation and diagnosis , 7. LyouManagement of radiation-induced angiosarcoma requires an aggressive surgical approach with complete resection with wide margins as an attempt for cure. Morgan et al. analysed 33 patients and found that a standard mastectomy may not be adequate . The autTargeted therapy such as vascular endothelial growth factor (VEGF) inhibitors are being investigated as novel treatment options. This is because over-expression of VEGF is considered as a vital mechanism in tumour genesis . A phaseAlthough radiation-induced angiosarcoma is described in literature, the development of bilateral angiosarcoma of the breast within a short interval following radiotherapy is extremely rare . The incAlthough radiation-induced angiosarcoma of the breast is an aggressive tumour with poor prognosis, the benefits of radiation therapy for primary breast cancer clearly outweighs the overall risk of developing a sarcoma . TherefoRadiation-induced angiosarcoma is a rare and aggressive disease which poses diagnostic and therapeutic challenges. The reported patient had an unusual presentation with a rapidly growing fungating breast mass with spontaneous bleeding and bilateral disease. Radical surgical excision with adjuvant chemotherapy is offered with potential for cure.


Few studies have longitudinally investigated the association between objectively measured sleep and time to develop dementia. This study leverages polysomnography (PSG) sleep data extracted from the VA national electronic health records (VA-EHR) to assess the association between sleep and time to develop dementia. We identified 61,165 PSG reports from the VA-EHR from 2000 to 2019 using CPT codes. Patients who developed dementia were identified using all-cause dementia ICD-9/10 codes documented on two separate visits starting one year after the PSG study until the end of 2019 in a 1-year sliding period . Using the first appearance of ICD-9/10 code as dementia onset time, patients were clustered into 3 groups of early-, mid-, and late time to develop dementia . Natural language processing was used to extract sleep efficiency (SE) and sleep onset latency (SOL). Univariate analysis was used to compare the groups. After adjusting for age, SE was significantly higher in the late (76%) vs early (69%) group and SOL was significantly shorter in late (21m) versus early (33m) group. SE was higher and SOL was shorter in patients who developed dementia later compared to those who developed dementia earlier. Greater sleep continuity in late dementia onset group suggests that sleep may be a modifiable risk factor that could potentially delay the onset of dementia.


Lung cancer accounts for approximately 11% of all cancer cases, however the 5-year survival rate is often below 20%. Consequently, lung cancer is the leading cause of cancer related mortality worldwide , has been extensively reviewed  pathway. TGF-β regulates a multitude of cellular pathways including the DDR, cellular proliferation and the epithelial-mesenchymal transition (EMT). It plays both positive and negative roles in cancer development and progression. In established tumors, high TGF-β expression can drive metastasis, tumor heterogeneity and chemoresistance . The remaining 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.