Low tCys (≤25th percentile) was associated with a decrease of 0.28 Z-scores for HAZ, 0.10 Z-scores for WHZ, and 0.21 Z-scores for WAZ compared to being >25th percentile. In young Indian children from low-to-middle socioeconomic neighborhoods, a low plasma total cysteine concentration was associated with an increased risk of poor anthropometric status.High-quality protein has been associated with child growth; however, the role of the amino acid cysteine remains unclear. The aim was to measure the extent to which plasma total cysteine (tCys) concentration is associated with anthropometric status in children aged 6–30 months living in New Delhi, India. The study was a prospective cohort study including 2102 children. We calculated Z-scores for height-for-age (HAZ), weight-for-height (WHZ), or weight-for-age (WAZ) according to the WHO Child Growth Standards. We used multiple regression models to estimate the association between tCys and the anthropometric indices. A high proportion of the children were categorized as malnourished at enrolment; 41% were stunted (HAZ ≤ −2), 19% were wasted (WHZ ≤ −2) and 42% underweight (WAZ ≤ −2). Plasma total cysteine (tCys) was significantly associated with HAZ, WHZ and WAZ after adjusting for relevant confounders ( Child undernutrition is a significant public health problem in developing countries. It is estimated that globally, 150.8 million children under the age of 5 are stunted and 50.5 million are wasted ,3,4 as wYoung children grow rapidly and have additional nutritional demands to enhance optimal growth and development . These dAnimal studies have shown that cysteine supplementation in mice increased growth plate thickness through upregulation of insulin-like growth factor-I (IGF-1), and led to improvements in different bone parameters . Thus, tFurther, high cysteine levels have repeatedly been associated with adult obesity based on evidence from cellular, animal, and epidemiological studies ,18,19,20In this secondary analysis, we aimed to assess to what extend plasma total cysteine (tCys) concentration is associated with poor anthropometric status in children aged 6–30 months living in low-to-middle socioeconomic neighborhoods of New Delhi, India.www.clinicaltrials.gov), which took place from 1998 until 2000 in New Delhi, India. It evaluated the effect of daily zinc supplementation on the incidence of acute lower respiratory tract infections and pneumonia [We used data from a randomized controlled trial .Lactobacillus casei was used to measure plasma cobalamin concentrations [Lactobacillus leichmannii was used for plasma folate [12  and corrects for age and folate status. This is suggested to increase the precision of the diagnosis of vitamin B12 deficiency [Anthropometric measurements and non-fasting venous blood samples were taken at enrollment and the end of the study, i.e., exactly four months after enrollment. Weight was measured with electronic scales (seca), reading to the nearest 100 g. Length (or height for children ≥ 24 months) was measured with locally manufactured length boards with a precision of 0.1 cm. Heavy clothing and nappies were removed before measurements. Blood samples (≈5 mL) were collected between 900 h and 1200 h in heparinized polypropylene tubes (Sarstedt) by a study physician. The samples were centrifuged, and plasma was stored immediately in polypropylene vials (Eppendorf) at −20 °C for subsequent analysis. A microbiological assay with a chloramphenicol-resistant strain of trations , while aa folate . Measurea folate . The beta folate ,28,29. Fa folate . The comficiency . We inclficiency .Upon enrolment, the study physician also interviewed the caretaker including questions on the birthdate of the child, breastfeeding practices, years of education of the parents, family size and household income.Statistical analyses were done with STATA 15.0 statistical software  and R version 3.5.1 .We calculated Z-scores for height-for-age (HAZ), weight-for-height (WHZ) and weight-for-age (WAZ) according to the WHO Child Growth Standards . Stuntint-test, a Wilcoxon rank-sum test or a Chi2-Test, respectively.Baseline characteristics are reported as mean (SD), median (IQR) or proportion, as appropriate. We dichotomized values for plasma cysteine at the 25th percentile because established reference values for plasma cysteine concentrations do not exist. Differences in means, medians or proportions between children with a tCys concentration above the 25th percentile compared to those on the 25th percentile or below were tested with an unpaired 12 status (3cB12). In addition, we assessed an interaction between breastfeeding status and age. All potential confounders were assessed in bivariable models, and all variables significant at a level of 0.2 were taken into a multivariable model simultaneously. All variables not significant at a level of 0.05 were taken out one by one and removed if the coefficient for tCys did not change considerably (>15%). After all non-significant variables were removed from the multivariable model, all variables initially not taken into the model, were entered one by one again and retained if now significant (p < 0.05).We built generalized estimating equation (GEE) models with anthropometric indices as outcome and tCys concentration at enrollment as exposure. This approach uses growth data from both time points (enrollment and 4 months later) and accounts for interdependence between multiple measurements in the same child . For conWe used generalized additive models (GAM) to depict a potential non-linear relationship between tCys concentration and anthropometric indices . We adjusted for all confounders identified in the GEE model (of the Gaussian family) and deleted values <2.5th percentile and ≥97.5th percentile for tCys to avoid overfitting at the extremes.12 concentration.Plasma total cysteine concentrations and anthropometric measurements at both time points were available for 2102 children . The demp < 0.001). With one unit increase in cysteine concentration (in µmol/L), the Z-scores for all anthropometric indicators increased by 0.003–0.005 Z-scores  at baseline was significantly associated with HAZ, WHZ, and WAZ after adjusting for relevant confounders , tCys concentration did not differ from children with this information. Those children without information on tCys (n = 216), had slightly higher mean Z-scores for all anthropometric indicators at baseline . Density plots can be seen in the Information for this analysis was available for 85% of all the children enrolled in the original trial. In children with no available information on anthropometry  concentration was positively associated with height-for-age (HAZ), weight-for-height (WHZ) and weight-for-age (WAZ). Children with low tCys concentrations (≤25th percentile) had a ~20% higher risk of being stunted, or underweight. The risk of wasting did not reach statistical significance.The low intake of high-quality protein intake, as well as amino acid, is associated with poor childhood growth ,12,13,14The role of cysteine in kwashiorkor, a form of severe malnutrition, has been the focus in some studies ,22,23 seOther relevant literature on cysteine comes from studies on growth restrictions in utero. In animal studies, supplementing pregnant piglets with N-acetylcysteine (NAC), a precursor converted to cysteine in the liver, prevented intra-uterine growth restriction (IUGR) . The admThe GAM plots in our work  show a gData for the study come from a randomized controlled trial on zinc supplementation which limits the generalizability. Information for this analysis was available for 85% of all the children enrolled in the original trial. Although children without information on tCys had slightly higher mean Z-scores for all anthropometric indicators, all other characteristics in our analysis did not differ significantly; thus, we do not expect that missing data on tCys would bias our results considerably. The strength of our study was that we could base our analysis of high-quality data from a community-based cohort. This investigation was designed as an observational study, and although we considered several relevant confounders, we cannot exclude residual confounding. Half of the children were supplemented with zinc in this study. Although in the main trial the zinc group had a significantly reduced incidence of diarrhea and respiratory tract infections compared to those receiving placebo ,45, oralThe 10% variation in the cysteine assay is a potential limitation; however, this non-differential misclassification in the exposure decreased the precision of our effect measure estimates and could attenuate the observed associations between tCys and growth . We usedThe low intake of high-quality protein intake, as well as amino acid, is associated with poor childhood growth ,12,13,14We did not include morbidity data in our analyses and cannot assess if cysteine concentration is associated with ponderal and linear growth via inflammation, mucosal damage, or other morbidities. A large part of the pediatric populations in low- and middle-income countries are affected by environmental enteric dysfunction (EED), characterized by structural and functional changes in the small intestines, leading to chronic inflammation and malabsorption of nutrients. EED is increasingly recognized as a risk factor for malnutrition ,53,54,55In our community-based population, young Indian children with a low plasma total cysteine concentration had an elevated risk of a poor anthropometric status. Based on our results, we cannot conclude that cysteine deficiency causes poor growth. Whether or not the association between cysteine and growth is due to metabolic processes related to nutritional status or due to residual confounding is not known. The role of cysteine in malnutrition should be estimated in more comprehensive studies, including several biomarkers, dietary intake, and preferably in populations representing the whole range of nutritional status during various critical life-cycle windows.


Neuroendocrine prostate cancer (NEPC) can arise de novo, but much more commonly occurs as a consequence of a selective pressure from androgen deprivation therapy or androgen receptor antagonists used for prostate cancer (PCa) treatment. The process is known as neuroendocrine transdifferentiation. There is little molecular characterization of NEPCs and consequently there is no standard treatment for this kind of tumors, characterized by highly metastases rates and poor survival. For this purpose, we profiled 54 PCa samples with more than 10-years follow-up for gene and miRNA expression. We divided samples into two groups (NE-like vs. AdenoPCa), according to their clinical and molecular features. NE-like tumors were characterized by a neuroendocrine fingerprint made of known neuroendocrine markers and novel molecules, including long non-coding RNAs and components of the estrogen receptor signaling. A gene expression signature able to predict NEPC was built and tested on independently published datasets. This study identified molecular features , at the time of surgery, that may anticipate the NE transformation process of prostate adenocarcinoma. Our results may contribute to improving the diagnosis and treatment of this subgroup of tumors for which traditional therapy regimens do not show beneficial effects. Prostate cancer (PCa) is the most frequently diagnosed cancer in men and the second leading cause of cancer death worldwide . The majOne underestimated aspect of prostate cancer progression regards the cross-talk between androgen and estrogen signaling pathways. It has been demonstrated that ESR2 is down-regulated in epithelial cells during development of prostate cancer . ESR1 isThis study aimed to identify molecular features, at the time of prostate cancer surgery, able to anticipate the NE transformation process of prostate adenocarcinoma, to improve the diagnosis and treatment of this subgroup of tumors for which traditional therapy regimens do not show beneficial results. We exploited a cohort of 54 prostate cancer samples for which multi-omics analysis was performed on protein-coding, long non-coding and micro RNAs. We deeply investigated the molecular aspects differentiating tumors with the highest levels of expression of Chromogranin A (CHGA), one of the most valuable markers of NEPC , togetheTo our knowledge, the role of estrogen signaling in NEPC has not yet been investigated. Here we showed that the estrogen signaling, and in particular ESR1, increased in NE-like samples from our cohort and in a model of prostate cancer organotypic slice culture (OSC). Data were validated on other publicly available datasets, providing an opportunity for further studies and for clinical intervention.Finally, we identified a molecular signature able to predict the neuroendocrine transformation of prostate adenocarcinomas in two independent datasets, and that can be exploited to improve the diagnosis and the therapeutic approach for NEPCs.n = 22). The remaining samples were classified as classical prostate adenocarcinomas   together with high levels of Chromogranin A  were classified as neuroendocrine-like tumors  .The long-lasting follow-up of our cohort (more than 10 years) was exploited to investigate biochemical recurrence and clinical features of NE-like and AdenoPCa samples. The Gleason score was statistically different between the two groups, with higher scores in NE-like tumors, while biochemical relapse (BCR), grade, age at surgery and initial PSA did not show any statistically significant difference .Gene and microRNA (miRNA) expression profiling with Agilent 8x60k arrays was performed to identify protein-coding RNAs (pcRNA), long non-coding RNAs (lncRNA) and miRNA differentially expressed in NE-like versus AdenoPCa samples. Data reduction analysis using either whole mRNA or miRNA profiles was not able to divide NE-like from AdenoPCa tumors , pointinp-value < 0.05) for the selection of differentially expressed genes, since we were looking even for small differences at the molecular level in two groups of samples that were undistinguishable from a histopathological point of view. Globally, 295 pcRNAs, 24 lncRNAs, and 6 miRNAs were up-regulated, while 206 pcRNAs, 15 lncRNAs, and 1 miRNA were down-regulated in NE-like vs. AdenoPCa samples  were down-regulated in NE-like versus AdenoPCa tumors D.David and Metacore tools were used to perform functional classification on the lists of differentially expressed genes in NE-like vs. AdenoPCa samples in terms of gene ontology, pathway maps, molecular networks, diseases, and drug targets. Interestingly, Metacore analysis revealed that many elements related to the estrogen pathway emerged from the study of up-regulated genes A.ESR1 was more expressed in NE-like tumors when compared to AdenoPCa samples , together with another estrogen receptor gene, GPER1 , while ESR2 did not show changes in expression . AlthougAlso diseases such as neuroectodermal tumors and prostatic neoplasms showed statistically significant enrichment for up-regulated genes. On the other hand, the androgen receptor signaling cross-talk network was enriched among down-regulated genes A.Functional analysis of up-regulated genes in NE-like vs. AdenoPCa samples using David showed enrichment in biological processes typically controlled by neuropeptides, such as modulation of ion currents, synaptic vesicles and catecholamines secretion. Endocrine features were also present , together with positive control of ERBB/EGFR pathway A. ConcerNetwork analysis based on manually curated information from the literature and with differentially expressed pcRNAs, ncRNAs and miRNAs, performed by using Metacore, identified direct interactions between different molecular features used as input. Twenty-four hubs with 5 or more connections were identified. ESR1 and AR were the most connected proteins in the network, with 88 and 82 edges, respectively B, and wiTranscription factor interactome analysis pointed out several enriched transcription factors that may putatively control a significant number of up- or down-regulated genes in the NE-like vs. AdenoPCa comparison. Again, ESR1 and AR were among the most statistically significant transcription factors for up-regulated genes, while ESR2 and AR for down-regulated genes C. This rFinally, the drug target identification function, available within Metacore, was able to identify 14 known up-regulated and 8 known down-regulated drug target genes among the list of DEGs .Pre-ranked Gene set enrichment analysis (GSEA) unraveled a statistically significant association between up-regulated genes in NE-like tumors and up-regulated genes of the late “NE tumor” gene signature by Mauri et al.  and the −6), with a positive correlation with CHGA and a negative correlation with AR  treated with estradiol (E2), which showed an up-regulation of elements related to neuronal and endocrine functions . We then exploited VCaP cells from the GSE43988 GEO series , where ESimilar results were obtained by analyzing prostasphere-forming cells of human LNCaP cells treated with estradiol (GSE37531): also in this case, we noticed an overrepresentation of processes and pathways related to neuroendocrine functions . Interestingly, for all the three independent datasets analyzed, an enrichment of up- and down-regulated transcriptional targets for both AR and ESR1 emerged, suggesting a possible interplay between these two proteins in controlling the neuroendocrine transdifferentiation process of prostate cancer .To test the hypothesis that estrogen signaling somehow participates in neuroendocrine prostate tumor transdifferentiation, while androgen signaling is suppressed, we analyzed the overlap between i) up-regulated genes in NE-like vs. AdenoPCa, ii) up-regulated genes in PCa OSC treated with estradiol (E2) and iii) down-regulated genes in PCa OSC treated with dihydrotestosterone (DHT) E. Chromop-value = 0.0008 and 0.02, respectively)  in the study from Beltran and colleagues [p-value = 4.37 × 10−5).Among the list of differentially expressed lncRNAs, HOTAIR and MALAT1 emerged as up-regulated in NE-like vs. AdenoPCa tumors  . HOTAIR p-value < 0.05) were involved in processes such as ion transport, regulation of hormone levels, vesicle organization, secretion, hormone metabolic process and cell death . On the other hand, genes negatively correlated with HOTAIR  were enriched in terms of metabolic/catabolic processes, mRNA processing, cell cycle, response to stress, autophagy, IGF-1 Signaling Pathway, Prostate cancer, HIF-1 signaling pathway, Estrogen signaling pathway . We exploited the external dataset GSE61270 [Interestingly, HOTAIR expression was positively correlated with CHGA and other neuroendocrine and CRPC markers and negatively correlated with AR and other genes involved in prostate carcinogenesis A,B. GlobGSE61270 , in whicp-value = 0.0026) and concomitant positive correlation with ESR1  in our cohort of prostate cancer tissues. Since miRNAs and lncRNAs mainly orchestrate precise gene regulatory networks, we decided to investigate more in detail the connections existing between AR, ESR1, HOTAIR and miR-31-5p. A direct interaction network, based on scientific evidence, emerged between the four molecules, with a mutual regulatory model between miR-31, which inhibits the expression of AR [MiRNA analysis led to a few deregulated miRNAs: 6 up-regulated  and 1 down-regulated (miR-99a-5p) in NE-like vs. AdenoPCa comparison. Interestingly, miR-31-5p was the miRNA showing the most negative correlation with AR  in NE-like vs. AdenoPCa samples of our cohort was firstly used for unsupervised hierarchical clustering on RNA-Seq data from Beltran  consistiTo identify a gene signature able to predict the neuroendocrine transformation of prostate adenocarcinomas, penalized logistic regression was applied to our dataset, using Ridge penalty and starting from 8 transcripts overexpressed in NE-like vs. AdenoPCa from our cohort, in Mauri’s Late NEPC  and in GNeuroendocrine transdifferentiation of prostate cancer is still poorly understood and is thought to affect approximately 25% of the tumors of men who die of prostate cancer . This trWe analyzed the expression profiles of coding, long non-coding and microRNAs in a cohort of freshly frozen localized prostate tumors from patients who underwent radical prostatectomy between 2003 and 2005 and for whom we have a follow-up of more than ten years. By coupling Chromogranin A mRNA expression with the response to hormonal therapy, survival, and perineural invasion information, samples were divided into NE-like and AdenoPCa. Multi-omics comparison of the two groups revealed that NE-like tumors were characterized by the overexpression of a neuroendocrine fingerprint, composed by known neuroendocrine, neuronal and CRPC markers, and novel molecules, including long non-coding RNAs such as MALAT1, as well as the estrogen receptor signaling. We found an up-regulation of HOTAIR in NE-like samples, an evidence recently corroborated by two independent studies ,30. PosiThe function of ESR1 in NEPC has not yet been investigated, while its role on prostate tissue is ascertained. For this reason, there is an increasing interest in the use of SERMs (selective estrogen receptor modulators) as a potential singular or adjunctive agents for the treatment of PCa , with maFinally, to identify an NEPC expression signature able to predict NE transdifferentiation in localized prostate cancer at the time of diagnosis, we exploited expression profiles of several independent cohorts of human tumors or mouse xenografts and trained a classifier on our dataset by fitting a logistic regression model. The predictive score was then tested both on Beltran dataset and on patient-derived xenograft tumors, where the development of NEPC from conventional prostatic adenocarcinoma was correctly predicted. Our classifier of course included CHGA, an unquestioned NEPC marker. It also contained SMPD3 that plays a role in the metastatic bone formation and has been found deregulated in bone-forming osteoblasts upon treatment with osteolytic prostate cancer cell-conditioned medium . SCG2 enIn conclusion, a score that combines the expression of eight transcripts deregulated in several NEPC was derived and could be applied to predict NEPC transdifferentiation in localized tumors, at the time of diagnosis. These results may be complemented with other tools that are in progress to achieve sensitive and earlier detections, such as serum chromogranin A detection, analysis of NEPC-specific circulating tumor cells and of circulating tumor DNA ,55.Prostate cancer tissue samples containing at least 70% neoplastic cells were obtained from 54 patients who had undergone radical prostatectomy at the Hospital of Biella . All patients gave informed consent and the study was approved by the local Ethical Committee (study ID: SERPROS C.E. 149/11).2, 10−7M) for 6 hours before harvesting. This study was authorized by the ethical committee of Fondazione Policlinico Gemelli-Università Cattolica of Rome, Italy . All procedures were conducted according to the principles expressed in the Declaration of Helsinki, the institutional regulation and Italian laws and guidelines.Organotypic slice cultures (OSCs) were generated as described in Aiello et al. (SREP 2016). Medium was replaced daily and after 3/4 days slices were treated with or without 17β-estradiol  was amplified using the Amino Allyl MessageAmp aRNA Kit  to obtain amino allyl antisense RNA (aaRNA) following the method developed by Eberwine and coworkers. Briefly: mRNA was reverse transcribed in cDNA single strand; after the second strand synthesis, cDNA was in vitro transcribed in aaRNA including amino allyl modified nucleotides (aaUTP). Both dsDNA and aaRNA underwent a purification step using columns provided with the kit. Labeling was performed using NHS ester Cy3 or Cy5 dyes , able to react with the modified RNA. Labeling efficiency and mRNA quantity were checked using NanoDrop ND-1000 Spectrophotometer .Equal amounts of differentially labeled specimens from sample and reference were put together, fragmented and hybridized to oligonucleotide glass arrays with sequences representing 27,958 Entrez Gene RNAs and 7,419 lincRNAs . All steps were performed using the In Situ Hybridization kit-plus (Agilent Technologies) and following the 60-mer oligo microarray processing protocol (Agilent Technologies). Then, slides were washed following the Agilent wash procedure. Slides were scanned with the dual-laser Agilent scanner G2505C. For each sample, a dye-swap replicate was performed. Raw data elaboration was carried out with Bioconductor using R statistical language. The LIMMA  package was used to analyze gene expression profiles of prostate cancer tissues. Raw intensity values were background subtracted  and normalized using the loess method, for the within array normalization, and Aquantile, for the between array normalization. Duplicated probes were averaged. Then, Cy3 and Cy5 channels were separated using the separate channel analysis of two-color data, available within the Limma package. For gene expression profiling of OSCs, RNA quality and quantity were assessed using Agilent 2100 bioanalyzer (Agilent Technologies) and NanoDrop ND-1000 spectro-photometer , respectively. Gene expression profiling was carried out using the two-color labeling method by means of Low Input Quick Amp Labeling Kit (Agilent Technologies): labeling, hybridization, slide washing and scanning were performed following the manufacturer’s protocols. Briefly, mRNA from 100 ng of totRNA was amplified, labeled with Cy3 or Cy5 and purified with columns. 300 ng of differentially labeled specimens were hybridized on Agilent Human Gene Expression v3 8x60K microarrays. Then, slides were washed and scanned using the Agilent Scanner version C .n = 48) using the 1-color labeling method and following the manufacturer’s protocols (Agilent Technologies). Briefly, 100 ng of total RNA was dephosphorylated and denatured; then a ligation and labeling step with Cy3 was performed. Samples were hybridized to oligonucleotide glass arrays  with sequences representing probes for 866 human and 89 human viral miRNAs from the Sanger database version 12.0 . Slides were scanned with the dual-laser Agilent scanner G2505C. Raw intensity values were background subtracted  and normalized using the quantile method using the limma package.miRNA expression profiling was carried out on primary prostate tumors . MeV version 4.9.0 was used for unsupervised hierarchical clustering performed on the global gene expression profiling from Beltran dataset. Pearson distance as similarity metrics and average linkage as the linkage method were used.prcomp function, available within the stats package in R.The principal component analysis was performed on the whole gene and miRNA expression data with the https://david.ncifcrf.gov/). Over-represented biological processes, molecular functions, and cellular components of the Gene Ontology (GO) were investigated with the functional annotation tool available within DAVID 6.8  was used for network and pathway analysis for transcription factor interactome analysis and for drug target identification. In particular, a network consisting of direct interactions was built between AR and ESR1 and modulated pcRNAs, lncRNAs, and miRNAs. Enrichr was used to test if particular chromosomal locations were enriched in the group of DEGs in NE-like vs. AdenoPCa samples . J-CircoPre-ranked GSEA was used to evaluate significant enrichment in predefined sets of genes. The following datasets were downloaded from the Gene Expression Omnibus (GEO) database : GSE5998Beltran’s dataset was downloaded from cBioPortal website  as Z-scocor.test function, available within the R stats package, was used to assess linear dependence between variables through Pearson correlation.Fisher’s exact test was used to perform the statistical comparison of proportions, using the software language R. The glmnet R package. The best tuning parameter was chosen by ten-fold cross-validation. Then, we built a score by summing the Ridge coefficient estimates of the variables multiplied by their values. The area measured the accuracy of the classifier was measured by the area under the ROC curve (AUC). To build a classifier able to discriminate AdenoPCa from NEPC in Beltran’s dataset, a penalized estimation was used, starting from 8 transcripts that were differentially expressed in NE-like vs. AdenoPCa from our cohort, in Mauri’s Late NEPC and in GSE59984 PDX models of NEPC. In particular, we fitted a logistic regression model with the Ridge penalty on the 8 selected genes, using the Our data contributed to describing molecular features of localized prostate tumors, at the time of surgery, that may anticipate neuroendocrine transformation of prostate adenocarcinomas. This was achieved by expression profiling analysis of coding, long non-coding and microRNAs in a cohort of freshly frozen localized PCas from patients with over 10 years of follow-up. In particular, the subgroup of PCas with highest levels of Chromogranin A and worst clinical outcome and/or perineural invasion at the time of diagnosis showed overexpression of a neuroendocrine fingerprint, composed by known neuroendocrine, neuronal and CRPC markers, and novel molecules, including long non-coding RNAs such as MALAT1. Interestingly, HOTAIR and ESR1 were overexpressed and AR underexpressed in NE-like samples, evidence that was also validated on independent datasets. Finally, we built a score by combining the expression of eight transcripts derived by our and two independent studies and applied it to predict NEPC transdifferentiation on two other published cohorts. These results provide an opportunity for further research, clinical intervention, and early detection of localized prostate adenocarcinomas that are prone to develop neuroendocrine transdifferentiation.


The current SARS-CoV-2 outbreak leads to a growing need of point-of-care thoracic imaging that is compatible with isolation settings and infection prevention precautions. We retrospectively reviewed 17 COVID-19 patients who received point-of-care lung ultrasound imaging in our isolation unit. Lung ultrasound was able to detect interstitial lung disease effectively; severe cases showed bilaterally distributed B-Lines with or without consolidations; one case showed bilateral pleural plaques. Corresponding to CT scans, interstitial involvement is accurately depicted as B-Lines on lung ultrasound. Lung ultrasound might be suitable for detecting interstitial involvement in a bedside setting under high security isolation precautions. Dynamic detection of air within the bronchi or perfusion detected by color Doppler may help differentiate these signs further [SARS-CoV-2 is a coronavirus that emerged in December 2019 in Wuhan city, Province Hubei, China , 4. Poin further . LUS is  further . A recen further . Due to APLIO 300 TUS-A300 ultrasound system  with a 3.5 MHz convex probe or a NEMIO SSA-550a ultrasound system  with a 3.75 MHz convex probe. We assessed at least 4 intercostal spaces in both the anterior and lateral chest wall. In all but two patients, who were in critical condition and could not sit upright, we assessed at least 6 intercostal spaces in the posterior chest wall. In the supine patient, the probe was placed perpendicularly to the skin in each intercostal space, starting at the parasternal line, and moved laterally to obtain views of large portions of the lung surface. Posterior lung scans in the sitting patient started at the paravertebral line, moving laterally. All scans were strictly performed in longitudinal orientation (with the index of the probe facing towards the patients’ head). We assessed the appearance of A-Lines, B-Lines, consolidations, and pleural abnormalities. Either TW (attending physician) or DM  performed LUS and discussed the obtained images with the other ultrasound examiner to reach a consensus. Both TW and DM have had 3 years of experience in LUS, focusing on LUS in pulmonary infections.We retrospectively reviewed LUS examinations that we performed in our isolation unit in patients who tested positive in polymerase chain reaction (PCR) of oral and nasal swabs for SARS-CoV-2. All patients received thorough scanning of their anterior, lateral, and posterior chest wall using either an If available, we reviewed corresponding conventional imaging that was performed in these patients for comparison. CXR was performed in posterior-anterior projection and lateral projection in stable patients, or in anterior-posterior projection as bedside radiograph in critically ill patients. Chest CT was performed without contrast agent for assessment of pulmonary SARS-CoV-2 involvement, or with contrast agent in critically ill patients where PE posed a probable differential diagnosis. Conventional imaging was evaluated by residents in our department of radiology and by their supervising attending physicians, who reviewed and discussed the images together with physicians from the infectious disease department.We also reviewed the necessity of oxygen supplementation, mechanical ventilation, and auscultation findings in these patients.We performed LUS in 17 COVID-19 patients , no more than 2 b-Lines in any intercostal space, no irregular pleural line, and no consolidation.In 3 patients, we found small consolidations  with or without adjacent comet tail artifacts, but no ICS with 3 or more B-Lines.One patient had a small pleural effusion with a sagittal extent  of 4 mm unilaterally, but no ICS with 3 or more B-Lines and no consolidation.tissue-like and shred signs posterolaterally with numerous B-Lines anteriorly.In 9 patients, we found 3 or more B-Lines  within the lungs. In 5 patients with inconspicuous CXR who did not have CT performed, LUS revealed subtle pulmonary pathologies in 3 patients. One of these was a case of bilateral pleural plaques seen on ultrasound  in affected intercostal spaces where CT indicated these pathologies. In patients who had a high degree of interstitial pathology indicated by B-lines, consolidations also tended to be more extensive. We would like to point out that 4 patients had very small consolidations that were not revealed on CXR. The patients were asymptomatic, their LUS only revealed A-Lines apart from small any kind of abnormality in 6 out of 7 cases where CT was abnormal.LUS showed abnormalities corresponding to chest CT in 5 out of 7 cases. LUS showed This limited retrospective analysis demonstrates our experience with LUS in COVID-19 patients. This descriptive case series should indicate the potential of point-of-care LUS compared to radiographic imaging, particularly compared to CXR under IPC precautions.We detected three or more B-Lines in at least two ICS in 9 symptomatic COVID-19 patients and never in asymptomatic patients who tested positive for SARS-CoV-2. One patient with respiratory distress who showed interstitial involvement on CT did not have three or more B-Lines per ICS on LUS. As interstitial involvement progresses to ground glass opacities on CT, B-Lines become more numerous and confluent  LUS in COVID-19 reportedly  reveals As reported before , pleuraltissue-like sign with absent perfusion (as demonstrated on color or power Doppler) [Another pulmonary pathology frequently seen in COVID-19 is pulmonary embolism (PE) [Doppler) , 17. LUSDoppler) , 17, offDoppler) . UltrasoDoppler) . The useDoppler) , 21 whicIn non-COVID patients, lung ultrasound could also discern the severity of acute respiratory distress syndrome (ARDS) , to whicConclusively, asymptomatic patients rarely show interstitial involvement (more than three B-Lines per ICS) on lung ultrasound, and the minute consolidations or pleural abnormalities that we did find in asymptomatic patients may or may not be attributable to COVID-19. Patients in respiratory distress show signs of interstitial disease, usually several regions with multiple B-Lines per ICS in asymmetric patterns and varying consolidations.This brief report is obviously very limited, and as we did not perform all imaging modalities in each patient, we cannot conclude or prove which is superior. We could not systematically analyze LUS due to limited sample size and because we inconsistently performed conventional imaging or invasive diagnostics, based on the patients’ differing disease severity. LUS cannot access the entire lung surface, and one should keep in mind that LUS will not detect abnormalities at all if they do not reach the pleura. Furthermore, ultrasound is generally considered operator-dependent and requires a learning period. We do however see indications that LUS suitable for detecting the subtle interstitial and alveolar pathologies of viral SARS-CoV-2 pneumonia. LUS might be considered especially for use under IPC measures necessary for COVID-19. As cases are expected to rise, we anticipate more extensive use of LUS in COVID-19 as it can be done at bedside under quarantine precautions, accelerates diagnostic imaging , and can


This study is aimed at identifying the impact of a team-based train-the-trainer program (TTT-P) to enhance healthcare professional (HCP) skills in patient education during medical rehabilitation. Focusing on patient-reported outcomes, a prospective, sequential two-cohort study was conducted in the fields of psychosomatic and oncological rehabilitation. Two hundred fifteen patients were evaluated before (Cohort 1) and 196 post implementation of TTT-P (Cohort 2). Patients of both cohorts completed validated questionnaires on self-management (heiQ®), general self-efficacy , and quality of life (WHOQOL-Bref) at the beginning, at the end, and at the 6-month follow-up to analyze short- and intermediate-term effects. Analyses were conducted separately for the psychosomatic and oncological setting. Results showed that TTT-P had no impact on patient outcomes in both rehabilitation settings. Patients did report positive outcomes as a result of the whole inpatient rehabilitation programs, though effects at follow-up were mostly small to medium size. Concerning self-management competencies, cancer patients gained less benefit during rehabilitation than psychosomatic patients. In conclusion, TTT-P did not result in measurable improvements at the patient level, likely because of the limited nature of the intervention. However, these populations of rehabilitants took benefit from participating in a multimodal rehabilitation program, of which patient education is one part. Chronic illnesses have great impact on the patients' lives, regarding both physical and mental health. Patient self-management has been proposed to be an effective strategy to manage the chronic effects of illness, i.e., in cancer . Self-maDelivering quality patient education demands specific knowledge on medical conditions and their treatment, models of health psychology, and factors determining health-related behavior . PatientPatient education provided by HCP of different disciplines plays an important role within German medical rehabilitation , 18. AccThough train-the-trainer approaches are widely used and may be considered to be an effective strategy to enhance HCP skills in patient education, the impact of such an approach on patient outcomes has rarely been examined. However, a narrative review indicated that improvements of patient outcomes could be gained by implementation of train-the-trainer programs . Our stuWe conducted a multicentered, prospective, sequential pre-post cohort study in two rehabilitation clinics in Germany, one specialized in oncological rehabilitation and one in psychosomatic inpatient rehabilitation. Patients treated before implementation of TTT-P (Cohort 1) were compared to those treated post implementation (Cohort 2). Cohort 1 in the psychosomatic sample included patients admitted from 26 April 2014 to 5 October 2014 and Cohort 2 those admitted from 27 January 2015 to 30 December 2015. Cohort 1 in the oncological sample included patients admitted from 23 September 2014 to 5 December 2014, and Cohort 2 those admitted from 26 October 2015 to 24 November 2016. In both settings, a waiting period elapsed after HCP had received TTT-P to avoid overlap of patients of Cohort 1 at the onset of Cohort 2. Patients were unaware to which group they belonged.Patients were consecutively recruited by the treating rehabilitation physician or psychologist during the first clinical encounter. Inclusion criteria were an ICD-10 diagnosis of cancer or psychosomatic/psychiatric disorder (depending on the rehabilitation setting) and being aged ≥18 years. Exclusion criteria were cognitive impairment, insufficient German, or severe psychological/physical stress (assessed by the treating physician/psychologist).Data were collected at the beginning of the rehabilitation program, at the end, and at the 6 months post. During rehabilitation, patient questionnaires were handed out at the first and last clinical encounters with physicians/psychologists. The follow-up questionnaire was mailed together with a return envelope including a single reminder after four weeks. Medical data were collected from the treating physician/psychologist by means of a short standardized form and from routine data. In each clinic, a person was determined who was responsible for equipping physicians/psychologists with study material, monitoring data collection during rehabilitation, and managing data collection at the follow-up.The study was approved by the ethics committees of the General Medical Councils of Hamburg, Northrhine and Westphalia-Lippe. Written informed consent was obtained from all study participants.The TTT-P received by HCP is aimed at enhancing skills requested for quality patient education. At an interval of approximately three months, it comprised two 2-day seminars (in-house) and was provided by an external organization specialized in TTT-P for patient educators in the health sector. Thus, it was carried out independently from the research group evaluating the intervention. It followed an interprofessional, team-based approach and was designed for a general health education program for patients receiving inpatient medical rehabilitation, irrespective of indications. Content of the TTT-P was modules addressing (a) leading and moderating groups, (b) activating and motivating patients, and (c) use of didactical methods.Overall, 29 HCP participated in the TTT-P, of which 11 worked in the psychosomatic rehabilitation clinic and 18 in the oncological rehabilitation clinic. Regarding disciplines, most HCP were psychosocial staff (44%), followed by physicians (35%), nursing staff and physical therapists (9%), and others (13%). In 63% of HCP, the work experience in the current occupational field was >5 years. Before implementation of the TTT-P, 13% reported little experience in patient education, whereas 29% had 1 to 5 years of experience, 21% > 5 to 10 years, and 38% > 10 years. The weekly workload covering patient education measures during working hours was mean 17.1% . The same HCP continued to educate patients after implementation of the TTT-P.Patients received a rehabilitation program with high treatment intensity. Clinics are staffed with multidisciplinary HCP including physicians, nursing staff, physical therapists/sport teachers, psychologists/other psychosocial therapists, social workers, and nutritionists. Oncological rehabilitation on average lasts 3 weeks and is mostly initiated after completion of primary treatment. It comprises medical treatment, physical training, psychological support/therapy, and social counseling . PsychosThe primary outcome was self-management competencies at the end of the rehabilitation program (short-term effects). Secondary outcomes were general self-efficacy and QoL (short-term effects) and self-management competencies, self-efficacy, and QoL at the 6-month follow-up (intermediate-term effects). We used validated and reliable self-report questionnaires for measurement of outcomes.The German version of the Health Education Impact Questionnaire (heiQ®) was applied to measure self-management , 26. TheFor measurement of self-efficacy, the general self-efficacy (GSE) scale  was applQoL was assessed using the WHOQOL-Bref , which iTo gain insights into beneficial aspects of patient education, patients answered an open-ended question at the end of the rehabilitation program: “Which aspects of patient education did you perceive as most helpful? Please describe.” Participants could note as many aspects as wanted.Information on sociodemographic variables was collected from patients, and medical data were obtained from the treating physicians/psychologists. The dose of patient education during rehabilitation  was der2 .Due to diverging sample characteristics, effects of the TTT-P were analyzed separately for the psychosomatic and oncological rehabilitation settings. Short-term effects (end of the rehabilitation program) and intermediate-term effects (6-month follow-up) were evaluated using analysis of variance (ANOVA) with repeated measures. Effect sizes for time, group , and interaction effects (time∗group) were calculated by partial etaTranscripts of aspects that were perceived as helpful in the context of patient education were first coded and analyzed using qualitative content analysis to identify key themes. In total, 141 psychosomatic patients and 97 cancer patients responded to the question. All transcripts of beneficial aspects were coded for the presence or absence of each of the five themes identified. Group differences between frequencies of emerging themes  were analyzed using chi-square tests. All analyses were performed using the statistical package SPSS version 18.0 .Overall, 411 patients  completed the first, 334 (81.3%) second, and 232 (69.5%) third assessment . Data coOf 220 psychosomatic patients, 49% belonged to Cohort 1 and 51% to Cohort 2. Across cohorts, mean age was in the midforties, one-third were male, and approximately half of the patients were diagnosed with affective disorders. Of 191 cancer patients, 57% belonged to Cohort 1 and 42% to Cohort 2. In both, half of the patients were male, mean age was about 60 years, and approximately 40% had cancer of the digestive organs. Details on patient characteristics are reported in p = .001; oncological: 62.6 vs. 58.6 years, p = .017). Further, respondents of the oncological sample had reported less depressive symptoms on the Hospital Anxiety and Depression Scale (HADS) than nonrespondents .On average, respondents in both settings were older than nonrespondents at the first assessment .Among the psychosomatic sample, the delivered dose of procedures from the KTL category “information, motivation, and education” was average 1.4 ± 0.7 hours per week with 100% of patients receiving such interventions. Cohorts were comparable with regard to the received dose of interventions .Among the oncological sample, the delivered dose of therapeutic procedures from the respective KTL category was average 3.9 ± 1.9 hours per week with 100% of patients receiving such interventions. Although a slightly higher dose of interventions was documented for patients post TTT-P, no significant group difference was observed . Cancer patients improved slightly but significantly in “positive and active engagement in life,” “health-directed activities,” and “skill and technique acquisition” .Patients of each setting reported improved self-management at the end of the rehabilitation program. Most self-management competency scores increased significantly among psychosomatic patients, except for “health service navigation”  but declined in cancer patients . Psychosomatic patients reported improved QoL outcomes across all scales  and cancer patients for all scales except for “environment” .During the rehabilitation program, general self-efficacy significantly improved in psychosomatic patients . In cancer patients, changes were only significant in two out of eight scales: “positive and active engagement in life” and “skill and technique acquisition” . We found significant time effects for general self-efficacy in psychosomatic patients , but not in the oncological sample. Changes of QoL scores revealed to be significant for all scales in psychosomatic patients . With exception of “environment,” this also applied for cancer patients .Regarding self-management competencies, changes over time were significant across all scales but “health service navigation” in psychosomatic patients  compared with Cohort 1 . PatientPatient education plays an important role in medical rehabilitation programs. Its goal is to help patients improve their knowledge, skills, and motivation and to engage them in coping with their disease . HCP whoOur study showed that rehabilitation outcomes did not differ before (Cohort 1) and post (Cohort 2) implementation of the TTT-P, neither at the end of the rehabilitation program nor at the 6-month follow-up. Thus, our hypotheses that patients receiving rehabilitation after the HCPs' training would report better self-management competencies, self-efficacy, and QoL could not be validated. The only difference between cohorts was found in the qualitative analysis of aspects of patient education that were perceived as helpful. Here, “information” was more often mentioned by patients of Cohort 2, but this finding only applied to psychosomatic patients.One possible explanation is that patient education is a small part of the 3-5-week lasting, multicomplex rehabilitation programs that patients received. The main goals of medical rehabilitation programs are improvement of the patient's medical condition, development of coping strategies and reintegration into social and working life with the ICF  serving Apart from these findings, our results show that patient outcomes improved short term (from the beginning to the end of the rehabilitation program), which might indicate the effectiveness of the rehabilitation programs. Patients of both settings reported better self-management competencies, but in psychosomatic patients, stronger and more systematic effects of time were found. Short-term QoL also significantly improved in both settings, with exception of “environment” in cancer patients. Fostering self-management competencies and enabling patients' reintegration into social life, despite disabilities and side effects limiting the patients' QoL, are an important goal of rehabilitation programs . TherefoSome limitations of the study should be noted. First, systematic collection of data on nonparticipants was not feasible due to organizational reasons, and a selection bias towards patients with better physical and/or mental health and those with higher motivation is possible. Thus, data has to be interpreted carefully with respect to generalization, while satisfying response rates within the study sample strengthen robustness of data. Second, we conducted a sequential pre-post cohort study design with each rehabilitation clinic serving as its own control. While having advantages , the design might have caused pitfalls such as selection bias, and a parallel-group study design using a matched comparison group of patients from nonintervention rehabilitation clinics might be considered for future research.Third, the intervention was embedded with patient education, which is part of all rehabilitation programs in Germany, and differences of the patient education procedures before and after the TTT-P were not measured. Fourth, knowledge is missing if to which effect HCP could use trained skills, i.e., because of large patient groups, time limitations, or other impeding factors, or if they used them already before. Further, we do not know how many patients were educated by different disciplines and/or HCP; thus, possible imbalances and its effects cannot be assessed. These missing but viable data limit the ability to answer our research question, and future research should address these methodological shortcomings.In conclusion, our results underscore the possible effects of comprehensive, multidisciplinary medical rehabilitation programs on patients' self-management competencies, self-efficacy, and QoL, particularly in the short term. We addressed the question if effectiveness of a train-the-trainer approach could be measured at the level of patient outcomes, basing on the requirement that measuring effectiveness of any clinical activity needs to incorporate measures of the direct impact on the patient's health . However


RNA) at expressed biallelic loci, and corelates it to gene expression from the corresponding cell.Recently, pioneering expression quantitative trait loci (eQTL) studies on single cell RNA sequencing (scRNA-seq) data have revealed new and cell-specific regulatory single nucleotide variants (SNVs). Here, we present an alternative QTL-related approach applicable to transcribed SNV loci from scRNA-seq data: scReQTL. ScReQTL uses Variant Allele Fraction  loci.Our approach employs the advantage that, when estimated from multiple cells, VAFScReQTL is relevant to the rapidly growing source of scRNA-seq data and can be applied to outline SNVs potentially contributing to cell type-specific and/or dynamic genetic interactions from an individual scRNA-seq dataset.Availability:https://github.com/HorvathLab/NGS/tree/master/scReQTLThe online version contains supplementary material available at 10.1186/s12864-020-07334-y. In recent years, single cell RNA-seq (scRNA-seq) has become an increasingly accessible platform for genomic studies . By enabA popular method to study SNVs effects on GE is eQTL (Expressed Quantitative Trait Loci), which is based on testing for a correlation between the number of alleles bearing the variant nucleotide at the position of interest, and the level of local (cis) or distant (trans) GE . eQTLs hRecently, pioneering eQTL studies on scRNA-seq data have emerged. By utilizing the advantages of the single cell resolution, these studies have revealed many new regulatory SNVs, including those with cell-specific or transient effects , 13–16. RNA) at biallelic SNV loci. To correlate VAFRNA to GE from single cells, we estimate VAFRNA in the individual cell alignments, and correlate VAFRNA with GE from the individual cells using a linear regression model [RNA estimation from RNA-seq data [RNA and GE from bulk RNA-sequencing data – ReQTL (RNA-eQTL) [Here, we explore an alternative approach to assess the correlation between GE and expression of SNVs located within transcribed genes from scRNA-seq data. The expression of the SNVs is estimated as the proportion of variant-bearing RNA molecules  . We termWe applied scReQTL on publicly available scRNA-seq generated on the 10 × Genomics Chromium platform using 3′-based protocol on 26,640 human adipose-derived mesenchymal stem cells (ADSCs), obtained from three healthy donors. This scReQTL analysis includes approximately 4 billion scRNA-seq reads. ScReQTL analysis was performed after classification of the cells by cell type, and only SNVs covered by a minimum of 10 unique sequencing reads per cell were included in the analysis. Across the three samples, we identified 1272 unique scReQTLs. scReQTLs common between individuals or cell types were consistent in terms of the directionality of the relationship and the effect size. In addition, scReQTLs were substantially enriched in known gene-gene interactions and significant genome-wide association studies (GWAS) loci.Methods. Below, we describe the workflow elements that we identified as specific and essential for the scReQTL analysis.An example of scReQTL workflow using publicly available tools is presented in Fig. RNA assessment, and SNV-GE correlation by cell type.The scReQTL workflow includes three major components: scRNA-seq data processing, VAFProcessing includes barcode and UMI modeling, alignment, GE estimation and cell type classification, and can employ a variety of publicly available tools. In the exemplified workflow, we process the barcodes using UMItools, and align using STAR the alignments are then deduplicated based in UMIs [RNA estimations can be sensitive to allele mapping bias, SNV-aware alignment recommended. We perform SNV-aware alignment for the list of positions of interest to be used as input for scReQTL analyses. Here, we perform SNV-aware alignment against the biallelic positions called by GATK in the corresponding pooled alignments applying a two-pass 2-pass STAR-WASP as previously described [ in UMIs , 27. Becescribed , 28, 29.escribed , 31; in GE estimation is performed on the SNV-aware alignments, using FeatureCounts to assess the raw gene counts [e counts , followee counts , 33. TheCell type identification is performed using SingleR . The expVAFRNA is assessed from the individual cell alignments at the positions of interest using SCReadCounts [var and nref, respectively), calculates VAFRNA (VAFRNA = nvar / (nvar + nref)) and outputs the values in an SNV-barcode matrix. The SNV-barcode matrices are in a format analogous to the GE-barcode matrices and can be directly used in the ReQTL analyses. To address stochasticity of sampling, estimations of VAFRNA require a threshold of minimal number of unique sequencing reads (minR). Our previous research shows that current scRNA-seq datasets can contain hundreds of SNV sites covered by minimum of 10 sequencing reads (minR > 10) and thousands of SNV sites with minR > 5 [RNA estimated at sites with minR > 10; from here on, we refer to these loci as informative. We note that for minR we are referring to sequencing reads with unique UMIs which are derived from unique mRNA molecules. The VAFRNA distribution of the qualifying SNVs is then examined to identify the most variable VAFRNA loci (see Methods). VAFRNA distributions before and after filtering of uninformative (minR< 10) and non-variable VAFRNA are shown on Fig. adCounts . For eacminR > 5 . In the SNV-GE correlations (scReQTLs) are then computed for each donor, stratified by cell type (see Methods). To qualify for scReQTLs analysis an SNV locus is required to have informative and variable VAFRNA estimations from at least 20 cells per analysis. The variable VAFRNA were correlated to the normalized GE values of the variable genes using linear regression model as implemented in Matrix eQTL [rix eQTL ; quantilrix eQTL . BrieflyRNA loci retained for scReQTL analysis in the three donors (by cell type) is shown in Table The number of variable genes and VAFAmong the unique scReQTLs, 7 were identified in more than one cell type or sample (Supplementary Table TNNT3) and one was located in an exon of the non-coding RNA LINC01119. In contrast, cis-eQTLs are frequently located in the promoter and other regulatory sequences, often transcriptionally silent and therefore not detected by the ReQTL/scReQTL approach.Next, we investigated the relationship between cis- and trans-scReQTLs. Of the significant scReQTLs, only 6 represented cis-correlations, representing 4 distinct SNV-gene pairs,  eQTLs reported in the GTEx database , (b) ReQhttps://www.gtexportal.org). First, we identified the SNVs and genes used as an input for scReQTLs, and participating in known eQTLs: a total of 111 input SNVs and 2024 input genes participated in at least one eQTL reported in GTEx. Across the 49 tissues, scReQTL identified 32 correlations  and AKR1C2 (negative); these scReQTLs were consistent across cell types  and adipose visceral (215 samples) - following the published protocol . Using t5 samplesThe different sets of SNV-gene pairs identified by scReQTL and ReQTL (as well as eQTL) suggests different regulatory relationships captured by scReQTLs. Bulk ReQTLs and eQTLs show a high overlap between each other and are both based on abundance of variant alleles across multiple individuals with different genotypes. In contrast, scReQTL operates in a setting of identical genotypes, where it is likely to capture RNA-based genetic interactions, possibly with transient and/or cell-specific effects.RNA of an SNV located in one of the genes and expression level of the other), we assessed if these gene pairs were enriched in known gene-gene interactions. We downloaded the known gene-gene (human) interactions from the STRING database [p < 10e-4, permutation test using 10,000 permutations, Fig. IFIT1 and IFITM2, AURKA and PLK, and CKS2 and CDC20 . Finally, we assessed the predicted functional and/or pathogenic scores of the scReQTL SNVs using 17 models including SIFT, Polyphen2, LRT, MutationTaster, MutationAssessor, FATHMM, PROVEAN, VEST3, CADD, DANN, fathmm-MKL, MetaSVM, MetaLR, integratedFit, GERP++, phyloP, and phastCons, as implemented in ANNOVAR [Next, we assessed if the scReQTLs SNVs are enriched in specific clinical phenotypes obtained from the ClinVar database . Fifteen ANNOVAR ; this daApplication of scReQTLs requires consideration of several factors. First, because scReQTLs assess expressed SNV loci, they cannot capture variants in transcriptionally silent genomic regions. In addition, SNV loci with expression levels below the required minimum number of RNA-seq reads (minR) are not included in the scReQTL analyses. Furthermore, when assessed with the platform used in this study - 10x Genomics Chromium v3 chemistry – the analyzed SNVs are confined to those located within the length of the sequencing read  from the 3′ end of the transcript. For the above reasons, scReQTLs accessible SNVs represent a subset of the expressed SNVs and are not designed to cover the full set of SNVs in the genome/transcriptome.Second, when a genetically regulated gene is captured by scReQTL analysis, the scReQTLs may highlight SNVs that are co-allelic to the actual causative SNV(s). This is the case for SNVs positioned outside the transcribed regions or outside the coverage of the sequencing library.RNA estimation, which can be affected by technical parameters, including allele mapping bias [Third, scReQTLs are based on VAFing bias , 46. Theing bias , 29.RNA estimation is the selection of cutoff for minimal number of reads, minR. When selecting minR for an analysis, a major factor is the balance between the confidence of VAFRNA estimation (high minR) and the inclusivity of SNVs . Our previous research shows that for current 10× Genomics scRNA-seq datasets, minR > 5 provides a reasonable balance between VAFRNA confidence and SNV inclusivity, while at lower cutoffs (i.e. minR = 3) stochasticity of sampling can affect the VAFRNA estimation [> 10. In addition, to assess the scReQTL at lower minR cut-offs, for a subset of the dataset (N7) we performed repeated analyses varying the value of minR in one-step increments between 5 and 9 and analyzed the outputs. The first observation is that, as expected, minR inversely correlates with the number of input SNV loci and the number of significant scReQTLs , as well as loci with over 75% of the VAFRNA values in the ranges 0–0.25 or 0.75–1 . As we show in our previous research [RNA distribution changes substantially with different minR, which affects the subset of variants retained after filtering. Importantly, each of the distinct sets of significant SNV-gene pairs obtained at different minR showed very strong enrichment in known gene-gene interactions, which supports confident scReQTL observations with VAFRNA cutoffs of 5 and above  out of the 20 cells VAFRNA is in the range 0.25–0.4 or 0.6–0.75 . From the binomial distribution we compute that this gives 0.0206 probability of observing variable loci by chance assuming a 50% probability of success on each of at least 15 out of 20 trials. This probability decreases with increasing number of cells. We consider the maximal 0.02 chance threshold for wrongly assigning variability of the loci to be reasonably conservative and acceptable. In addition, we showed in our recent study [Another threshold to consider is the minimal number of cells to be used for scReQTL, in this study set to 20. Using a minimum of 20 cells for assessing SNV-GE correlations is a result of setting a threshold for filtering loci with non-variable VAFnt study  that 20 nt study ). For 10Furthermore, scReQTLs can be affected by the accuracy of the variant call, including assessment of presence or absence of an SNV, and assignment of a biallelic state. The presented pipeline uses scRNA-seq data only, where we call SNVs from pooled scRNA-seq data and select for scReQTL analysis highly confident heterozygous sites based on mapping and Phred quality, genomic position , and previously validated rsIDs. To confidently assign biallelic state, we select SNVs with a minimum of 50 unique reads supporting each allele from the pooled scRNA-seq. By default, this selection excludes heterozygous SNVs with strong non-random monoallelic expression. Therefore, while the above approach is suitable for datasets with no matched DNA, when available, DNA-estimated genotypes can be helpful to interpret the context of the scReQTL findings.RNA is estimated for all the input sites, and SNVs not present in the assessed sample  are removed during the VAFRNA filtering step  will vary depending on conditions, disease states and stochastic factors. Therefore, scReQTLs are expected to be transient and their interpretation requires consideration of the dynamics of the variables underlying the correlation.Finally, VAFchanisms . Due to RNA at expressed heterozygous SNVs in place of the genotypes, to correlate allele prevalence to gene-expression levels. By using VAFRNA across multiple cells of the same sample, scReQTLs introduce several new analytical aspects.Single-cell RNA-seq eQTL analyses define an emerging research niche that brings major benefits for the understanding of functional genetic variation including the identification of cell-type and condition-specific correlations –16, 48. RNA, similarly to GE, is both dynamic and cell-type-specific [RNA to the most variable genes. Third, as compared to the discrete genotype values , VAFRNA can obtain continuous values spread along the entire VAFRNA range , allowing for more precise computation of the proportion of each allele represented in the RNA in a given cell. Fourth, scReQTL operates in the context of (largely) identical genotypes, which narrows the observed effects to RNA-mediated interactions. Finally, scReQTL does not necessarily require matched DNA , and therefore can be applied on scRNA-seq data alone. Related to that, scReQTL analyses can be performed using pre-defined SNVs of interest, such as RNA-editing sites and sets of dbSNP.First, and perhaps most importantly, as scReQTL is implemented on multiple single cells from the same sample, it can be applied to assess the effects of SNVs in a single sample or individual. This is particularly applicable for rare SNVs which are challenging to study via population-based approaches. We envision that this scReQTL feature can benefit studies on functionality of infrequent and de novo mutations causing rare phenotypes, as well as somatic mutations in cancer. Second, scReQTLs increase the dynamicity of the SNV-gene correlations, as VAFspecific . In part> 10, which retained between 308 and 721 input SNVs per sample, in our prior research we show that at minR > 5 the number of SNVs is higher by an order of magnitude [> 10, cis-acting SNVs are located in genes with high expression, which likely contain a high proportion of stably expressed genes, including with house-keeping functions. Notably, the identified trans-scReQTLs are significantly enriched in known gene-gene correlations  and, in the case of 3′-based scRNA-seq protocols, by the length of the sequencing read. Therefore, scReQTLs can analyze only a proportion of the transcribed SNVs. This limitation is expected to be gradually reduced with the progress of the sequencing technologies. Additional attenuation of this constraint is possible through reducing the value of minR used in the analysis. Indeed, while in this study we apply minR agnitude . Second,Finally, at present, a direct comparison between scReQTLs and single cell eQTLs is limited, to a large extend due to a narrow overlap between the sets of SNVs matching the requirements for scReQTL inputs; these SNVs are located mostly in the expressed 3′-end of the gene, as compared to the genome-wide DNA-genotyped loci used in the sc-eQTLs. Additional contributing factors are the different cell sources as well as the different capturing protocols (i.e. SmartSeq vs 10xGenomics). With the advances of the scRNA-seq technologies, and the extension of single-cell QTL-based approaches to more tissues and cell types, comparisons between sc-eQTLs and scReQTLs are expected to provide meaningful information on underlying mechanisms in a cell-type specififc context.> 10) we identified 1272 distinct scReQTLs. These scReQTLs include a considerable number of correlations which involve SNVs previously highlighted by GWAS and are significantly enriched in known gene-gene interactions. These results demonstrate that scReQTLs can be used to identify novel genetic interactions, including those which are specific to a given cell-type.Our scReQTL analysis includes approximately 4 billion RNA-seq reads from 26,640 human ADSCs, obtained from three healthy donors. We chose the 10xGenomics platform due to its growing popularity, high throughput, and the support for unique molecular identifiers (UMI) for the removal of PCR-related sequencing bias. Using stringent cutoff for SNV coverage  nucleotides in the pooled alignments. SNV loci were annotated using SeattleSeq v.13.00 (dbSNP build 153), and loci positioned in repetitive or intergenic regions were removed. The SNV lists were further filtered based on the following requirements: QUAL  > 100, MQ  > 60, QD  > 2, and FS (Fisher’s exact test estimated strand bias) = 0.000. The filtered SNV lists (per donor) were then used as an input for a second, SNV-aware alignment using STAR-WASP [The cell barcodes and UMIs were extracted using UMI-tools from the pooled (per donor) raw sequencing reads . The pooTAR-WASP .sctransform function as implemented in Seurat v.3.0 [sctransform function integrates the previous Seurat functions NormalizeData, ScaleData, and FindVariableFeatures. The cell-feature distributions were than plotted to identify and filter out outliers and low-quality cells, which we defined as [To estimate gene expression, we first apply FeatureCount on the individual alignments to assess the row gene counts per cell . We thenat v.3.0 , 33, whifined as  cells wiTo define individual cell types from the ADSCs, we used SingleR version 1.0.5 . SingleRRNA is assessed from the individual alignments as we have previously described [var and nref, respectively), calculates VAFRNA (VAFRNA = nvar / (nvar + nref), and filters out positions not covered by the user-defined minimum number of reads (minR); minR is constant across the genome [RNA from a minimum of 20 cells from the same cell type (per donor). The VAFRNA distribution is then examined and loci with non-variable VAFRNA are filtered out. Loci were considered non-variable if: (1) over 75% of the VAFRNA values are in the range of 0.5 ± 0.1 , and (2) over 75% of the VAFRNA values are in the ranges 0–0.25 or 0.75–1 .VAFescribed , using tescribed . At eache genome . For theSNV-GE correlations (scReQTLs) were computed for each donor, across the cells of each type separately. To qualify for scReQTLs analysis, an SNV locus is required to have informative and variable VAFRNA estimations (minR> 10) from at least 20 cells per analysis. The variable VAFRNA were correlated to the normalized GE values of the most variable genes using a linear regression model as implemented in Matrix eQTL [rix eQTL . The toprix eQTL . Brieflyrix eQTL . All thep-value of 0.05 was considered significant, unless otherwise stated. For estimation of significant scReQTL, we applied FDR as implemented in the Matrix eQTL package. Specifically, once Matrix eQTL discovers a set of significant gene-SNP pairs, it estimates a corresponding q-value (FDR) for each of them using Benjamini–Hochberg procedure under the assumption that the tests are independent or positively correlated [Throughout the analysis we used the default statistical tests (with built-in multiple testing corrections) implemented in the used software packages , where rrelated , 51. ForAdditional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.Additional file 7.


The newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted the world dramatically, forcing the medical community to quickly and effectively find ways to manage coronavirus disease 2019 (COVID-19). The COVID-19 pandemic has shown many similarities to the human immunodeficiency virus pandemic in 1981, from the fear of treating patients for a virus we have little knowledge of, to analyzing how the levels of CD4+ are affected in both diseases. Declining numbers of CD4+ levels are classically seen with HIV patients, however, given the immune response of our bodies, these levels have also been seen to decrease during an active COVID-19 infection. Besides, there is speculation that people living with HIV are at a higher risk for mortality if infected with SARS-CoV-2. Therefore, the interaction of these two viruses can create a syndemic culture, and thus, the need to monitor and treat patients with human immunodeficiency virus and COVID-19 cautiously. At the end of December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified in Wuhan City, Hubei Province, China, as the cause of the outbreak of pneumonia of unknown etiology . This isA 59-year-old African American gentleman presented to our facility with progressive dry cough, worsening shortness of breath, loss of appetite, nausea, diarrhea, anuria and generalized weakness. The patient had a past medical history of chronic hypertension, end-stage renal disease receiving renal replacement therapy, and HIV diagnosed in 1994 treated with oral antiretroviral therapy. He had recently tested positive for COVID-19, by a reverse transcription polymerase chain reaction (RT-PCR) nasopharyngeal swab sample. A computed tomography (CT) of the chest depicted bilateral ground-glass opacities in both upper and lower lobes  60%. Throughout the course of his stay, the patient’s absolute CD4+ and CD8+ lymphocyte count showed a progressive decline  and taking antiviral therapy could have immunomodulatory effects by decreasing the production of inflammatory cytokines . The risk factor severity for each person with HIV could also contribute to increased immune response and mortality. This includes those with age greater than 65 years, hypertension, diabetes mellitus, chronic obstructive pulmonary disease, and kidney disease . Our patConversely, in one retrospective study of 21 COVID-19 patients with HIV, it was noted that outcomes were worse because they required intensive care unit (ICU) care compared to the patients who did not have HIV . This suThere is still not enough information regarding the outcome of a patient who has co-infection with HIV and COVID-19. In our case, although the levels of CD4+ lymphocytes were declining, it was not associated with mortality. We also noticed that while these levels were declining throughout his hospital stay, once his COVID-19 infection was resolved, the levels of CD4+ lymphocytes also improved. We feel that a study in the population with HIV and COVID-19 co-infection is warranted to better understand the pathophysiology to help manage our patients more effectively.


P = 0.022, in PSC-BPPV; 70.0% vs. 24.0%, P = 0.020, in HSC-BPPV). We thought that they actually had hidden PSC-BPPV and the otoconial debris may still in the PSC; this untreated PSC-BPPV might cause the remnant symptoms. In conclusion, vertical nystagmus in the BLT may indicate the presence of PSC-BPPV. Moreover, vertical nystagmus during the BLT may occur in patients with hidden PSC-BPPV who complain of remnant vertigo symptoms. Vertical nystagmus shown in the BLT may not include the possibility of central vertigo.The ‘Bow and Lean Test’ (BLT) was developed for proper diagnosis of horizontal semicircular canal benign paroxysmal positional vertigo (HSC-BPPV). Occasionally, down- and/or up-beating vertical nystagmus is observed during the BLT. This study analyzed patients who exhibited vertical nystagmus in the BLT to comprehend the clinical significance of this sign. Of 2872 patients with vertigo between 2010 and 2015, 225 patients who showed vertical nystagmus in the BLT were enrolled. All patterns of vertical nystagmus were described based on their types of BPPV. After performing therapeutic maneuvers for BPPV, remnant symptoms in the BLT findings were investigated. Of the 225 patients with vertical nystagmus, 163 were posterior semicircular canal BPPV (PSC-BPPV). Down-beating in the bowing position and no nystagmus in the leaning position (‘Down/–’) was the most common type (190 of 225 patients). In addition, the nystagmus occurred in the form of ‘–/Up’, ‘Down/Up’, and ‘–/Down’. The pattern of vertical nystagmus may be related to the position of otoconia in the canals. The location of the otoconia enables the diagnosis of hidden PSC-BPPV. Even after treatment for BPPV, patients with vertical nystagmus in the BLT tended to complain remnant vertigo symptoms (44.8% vs. 23.9%,  Briefly, the BLT is based on the direction of nystagmus when a patient’s head bows and leans in a sitting position. In canalolithiasis, the affected ear is in the same direction as the nystagmus when bowing; in cupulolithiasis, the direction of nystagmus leads to the affected ear at the leaning position1. The BLT provides a much simpler and more precise method to identify the affected ear than confirmation of the lesion side through the comparison of nystagmus intensity in the conventional head roll test. By determining the affected ear more accurately, the BLT could ultimately improve the final remission rate of HSC-BPPV. In a follow-up study, patients who underwent both the BLT and head roll test demonstrated a final remission rate of 83.1%; however, the remission rate was 67.4% in patients diagnosed with HSC-BPPV using the head roll test alone2.Since the Bow and Lean Test (BLT) was first introduced5. Down-beating nystagmus from central lesions such as cerebellar disorder may be easily recognized from BPPV, having different characteristics of no latency, long duration, no fatigue, and no fixation. However, the clinical significance of vertical nystagmus in the BLT has not been clarified.Notably, when the BLT was performed to diagnose the affected ear in patients with HSC-BPPV, up- or down-beating vertical nystagmus was observed in some patients. In fact, vertical nystagmus itself could be observed in the head hanging position, such as the Dix-Hallpike test, for the diagnosis of posterior semicircular canal BPPV (PSC-BPPV). Up-beating nystagmus in the Dix-Hallpike test suggests PSC-BPPV, whereas down-beating nystagmus is generally considered indicative of central nervous system abnormalities including cerebellar disorders or for the relatively rare (3% of BPPV) anterior semicircular canal BPPV (ASC-BPPV)Therefore, this study was designed to interpret vertical nystagmus in the BLT and analyze its clinical significance. Furthermore, we evaluated the usefulness of the BLT for showing vertical nystagmus in PSC-BPPV, especially in terms of the localization of otoconia.Table Canalith repositioning procedures, such as the Epley maneuver, Barbeque maneuver with or without vibration, or reverse Epley maneuver, were the treatment for BPPV. The number of the repositioning maneuver required for complete remission in the groups are shown in Fig. Among 1848 non-BPPV patients, vertical nystagmus in the BLT was observed in 17 patients (0.9%)  of the 96 BPPV patients showed normal findings in this evaluation; other findings were the following: senile changes such as mild diffuse atrophy or vessel stenosis in 18 (18.8%), acute infarction in 1 (1.0%), brain tumor in 2 (2.1%), and traumatic brain hemorrhage in 12 (12.5%) patients.Of the 96 BPPV patients, 78 were diagnosed with PSC-BPPV. Of these 78 patients, most had favorable progress after treatment of PSC-BPPV. Only four patients  complained of residual dizziness, although none had persistent vertical nystagmus. Fifteen patients were diagnosed with HSC-BPPV. Twelve patients had normal findings; the remaining three had vessel stenosis, a brain tumor (meningioma), and mild diffuse atrophy. Only the 86-year-old patient with brain atrophy had continuous down-beating nystagmus in the BLT, with residual dizziness after canalith repositioning maneuvers. In the other two patients, nystagmus disappeared after treatment. Three patients had mixed type BPPV, but all three had normal brain examination findings. All 17 non-BPPV patients also underwent brain examinations, but none had abnormal findings that could cause central vertigo. Therefore, down-beating nystagmus in the BLT is less commonly related to central lesions than previously suspected.1, we have performed this test in all patients suspected of BPPV. However, we have often encountered patients with up- or down-beating vertical nystagmus in the BLT; thus, we performed this study to investigate the mechanism and clinical application of this vertical nystagmus. Of the 1024 patients diagnosed with BPPV, vertical nystagmus in the BLT was observed in approximately 20% , between August 2010 and July 2015, 1024 were diagnosed with BPPV, and 1848 were not (non-BPPV). Patients who showed vertical nystagmus in the BLT on the first day of their hospital visit, regardless of the final diagnosis, were included in this study. Two hundred and eight (20.3%) of the 1024 BPPV patients were included in the BPPV group, and 17 (0.9%) of the 1848 non-BPPV patients were included in the non-BPPV group  using a computerized video eye-movement recorder . PSC-BPPV was diagnosed, where nystagmus beat vertically upward and torsionally downward in the Dix-Hallpike test. When the torsional movement was counterclockwise during the right side test, the right ear was affected; clockwise torsional movement beating during the left side test indicated that the left ear was affectedet al.1. The BLT evaluated the direction of nystagmus when patients bowed their heads over 90° to 120° (bowing nystagmus) and leaned their heads backward over 45° to 60° (leaning nystagmus) in the sitting position. For canalolithiasis, the affected ear was determined to be in the same direction as the bowing nystagmus and in the direction opposite to the leaning nystagmus. In patients who did not show bowing or leaning nystagmus, or patients who showed vertical nystagmus, the affected ear was the side with more intense nystagmus in canalolithiasis and with less intense nystagmus in cupulolithiasis. Regardless of the results of the Dix-Hallpike test and head roll test, the BLT was performed in all 2872 patients. Notably, the BLT was the first maneuver performed, because the Dix-Hallpike test and head roll test could cause changes in otoconial location.HSC-BPPV was first confirmed by geotropic or apogeotropic paroxysmal nystagmus provoked by the head roll test when the head was turned from the supine to both lateral positions. Geotropic movement of the eyeball was indicative of canalolithiasis, whereas apogeotropic movement suggested cupulolithiasis. The affected ear was determined by using the BLT protocol introduced by Choung Patients with BPPV were treated with the canalith repositioning procedures based on the location of otoconia; the Epley maneuver, Barbeque maneuver, Barbeque maneuver with vibration, and reverse Epley maneuver were applied to patients with PSC-BPPV, canalolithiasis of HSC-BPPV, cupulolithiasis of HSC-BPPV, and ASC-BPPV, respectively. Non-BPPV patient were individually managed based on the possible causes of vertigo.In the 225 patients included in this study, all aspects of vertical nystagmus in the BLT were classified in accordance with each patient’s diagnosis Table . The finAll statistical analyses were performed using IBM SPSS Statistics for Windows , and p values less than 0.05 were considered statistically significant.


European Society for Endocrinology recently published a statement on COVID-19 and endocrine diseases ; however, thyroid diseases were not mentioned specifically. We have therefore reviewed the current literature on thyroid diseases (excluding cancer) and COVID-19, including data from the previous coronavirus pandemic caused by the SARS-associated coronavirus (SARS-CoV), a member of the same family Coronaviridae leading to severe acute respiratory syndrome (SARS). At the moment there are no data suggesting that thyroid patients are at higher risk of COVID-19, but this requites further research and data analysis.The novel coronavirus disease COVID-19 is produced by SARS-CoV-2. WHO has declared COVID-19 as a public health emergency, with the most susceptible populations (requiring ventilation) being the elderly, pregnant women and people with associated co-morbidities including heart failure, uncontrolled diabetes, chronic obstructive pulmonary disease, asthma and cancer. However, such general guidance does not provide information regarding COVID-19 risks in patients with suffering from pre-existing thyroid problems, and furthermore, we do not know whether patients with COVID-19 (symptomatic or without symptoms), who have not previously had thyroid issues develop endocrine thyroid dysfunction after infection. The  WHO declared coronavirus COVID-19 disease (caused by SARS-CoV-2) a public health emergency, with the most susceptible populations being the elderly, pregnant women and people with associated co-morbidities including heart failure, diabetes, asthma or cancer. In many countries, severe measures have been put in place concentrating on self-isolation and ‘lockdown’ to slow down disease spread and prevent loss of life .However, such general guidance does not provide information regarding COVID-19 risks in patients with pre-existing thyroid problems. We also do not know whether COVID-19 patients, symptomatic or without symptoms, develop de novo thyroid dysfunction after infection.https://www.gmc-uk.org/ethical-guidance/ethical-hub/remote-consultations) [Autoimmune thyroid disease (AITD) remains very common, and patients with both under- and overactive thyroid turn to their physicians for guidance if they belong to a high-risk group. In the UK there are on-going discussions with employers which may have a significant impact on workers being required to self-isolate (and work from home when possible), or to continue to come to the workplace, especially in the group of patients on anti-thyroid medications who were warned about the possible side-effect of agranulocytosis and advised that, should they have fever or a sore throat, they should seek immediate medical attention. Difficulties in accessing specialist care result in further anxiety. Patients are also not sure if the benefit from blood monitoring to adjust thyroid medications overwhelms the risk of COVID-19 exposure. Currently in the UK, patients with COVID-19 symptoms are advised to self-isolate: so, if such patients suffer also from Graves’ disease, they are unable to attend for ‘routine’ blood tests. A new way of care delivery via remote consultations is being increasingly used (over the telephone or video) and has been endorsed (when appropriate) by the UK General Medical Council (tations) .Endocrine and diabetes societies have responded to COVID-19 by producing guidance in cortisol-deficient patients , patients treated with glucocorticoids for underlying inflammatory conditions, and in patients with diabetes , 8. HoweEuropean Society for Endocrinology recently published a statement in the journal Endocrine with the following recommendations to adequately protect the patient and ask for COVID-19 testing if exposed, to avoid unnecessary routine appointments in person but to put in place online/email/phone consultation services. For patients with diabetes the recommendation was made to closely monitor glycaemic control and strictly adhere to ‘sick day rules’ in case of COVID-19 infection. For hypo-adrenal patients, close clinical monitoring and the need for increased replacement therapy  was recommended [The To the best of our knowledge, there are no papers published so far specifically on thyroid/AITD and COVID-19. However, some analogies and assistance may be drawn from the rheumatology experience which also deals with autoimmune diseases.Thyroid abnormalities may represent an isolated alteration, but they also may be the harbinger of a future autoimmune polyglandular syndrome . They maThe question regarding the risk of the potential use of anti-thyroid medication during the COVID-19 pandemic is more difficult to answer, and a precautionary approach might be wise, while awaiting further information. Non-chemotherapy idiosyncratic drug-induced neutropenia (IDIN) is a relatively rare but potentially fatal disorder that occurs in susceptible individuals, with an incidence of 2.4–15.4 cases per million population/year (summarised by Curtis) . AffecteSome knowledge regarding the thyroid can be gathered from a previous coronavirus pandemic. The severe acute respiratory syndrome (SARS) epidemic started in November 2002 and spread worldwide. SARS is an infectious condition caused by SARS-CoV, a member of the same family Coronaviridae . SeveralIn a group of prospectively recruited 61 SARS survivors (with no pre-existing endocrine conditions and investigated at 3/12 after recovery), a reversible hypophysitis or direct hypothalamic effect cased by the virus was suggested. Four (6.7%) SARS patients were reported to be biochemically hypothyroid, including three cases with central hypothyroidism  and one with primary hypothyroidism: 24 (39.3%) patients had evidence of hypocortisolism and 2 (3.3%) of them also had transient subclinical thyrotoxicosis , 18.Low serum triiodothyronine and thyroxine levels, commonly found in patients with SARS, have also been reported. In 48 SARS patients, serum fT3 and fT4 levels were decreased in the acute phase in 94% and 46%, respectively, whereas during the convalescent phase of the disease in 90% and 38%, respectively. The degree of decrease in fT3 correlated with disease severity. Serum thyroid-stimulating hormone (TSH) concentration in patients with SARS was also significantly decreased in comparison to control group, again suggesting central hypothyroidism , 19. Hownot provide evidence of a clear benefit of treatment using thyroid hormones in the euthyroid sick syndrome, although on-going clinical trials are focusing on new management strategies to explore whether restoration of normal serum thyroid hormone levels improves patients prognosis and clinical outcomes [The reduced TSH level reported by Wang et al.  in patieoutcomes . At presIn a recently published series of COVID-19 critically ill COVID-19 patients admitted to the ICU, no pre-existing thyroid problems were reported , and it seems that no new thyroid issues have been reported during the COVID-19 illness in this patients cohort . While iwww.euthyroid.com).In conclusion, there are no data available currently to suggest that patients with AITD remain at higher risk of COVID-19, and it seems likely that most asymptomatic and mildly affected patients should be treated as in normal times. While the risks of anti-thyroid drugs remain, they are probably not directly germane to such patients who should be managed in the usual manner. For many, indeed most patients, some delay in routine blood testing should not cause concern, and patients should be reassured. In patients severely affected by COVID-19, changes in thyroid function may relate to a ‘sick euthyroid’ syndrome, but there may be specific thyroid-related damage which requires further investigation. Further information regarding COVID-19 and thyroid patients has just been published in ETA Public Health Board statement (