RNA‐based sequencing is considered ideal for detecting pathogenic fusion‐genes compared to DNA‐based assays and provides valuable information about the relative expression of driver genes. However, RNA from formalin‐fixed paraffin‐embedded tissue has issues with both quantity and quality, making RNA‐based sequencing difficult in clinical practice. Analyzing stamp‐derived RNA with next‐generation sequencing (NGS) can address the above‐mentioned obstacles. In this study, we validated the analytical specifications and clinical performance of our custom panel for RNA‐based assays on the Ion Torrent platform.To evaluate our custom RNA lung panel, we first examined the gene sequences of RNA derived from 35 NSCLC tissues with diverse backgrounds by conventional methods and NGS. Next, we moved to the clinical phase, where clinical samples  were used to examine variants. In the clinical phase we conducted an NGS analysis while simultaneously applying conventional approaches to assess the feasibility and validity of the panel in clinical practice.In the prerun phase, all of the variants confirmed with conventional methods were detected by NGS. In the clinical phase, a total of 80 patients were enrolled and 80 tumor specimens were sequenced from February 2018 to December 2018. There were 66 cases in which the RNA concentration was too low to be measured, but sequencing was successful in the vast majority of cases. The concordance between NGS and conventional methods was 95.0%.RNA extraction using stamp specimens and panel sequencing by NGS were considered applicable in clinical settings.Significant findings of the studyNext‐generation sequencing using RNA from stamp specimens was able to detect driver gene changes in non‐small cell lung cancer including fusion genes with the same accuracy as conventional methods.What this study addsUsing RNA from stamp specimens obtained from biopsy increases the number of candidate cases for RNA sequencing in clinical settings. ALK, ROS1 and NTRK) is now recommended as a standard test for the evaluation of advanced NSCLC.EGFR mutations), RNA‐based sequencing provides valuable information concerning the relative expression of driver genes.Various targeted agents have been developed for non‐small cell lung cancer (NSCLC), and fusion gene evaluation  and Sanger sequencing. Furthermore, a highly sensitive PCR method (the cycleave PCR method and the fragment method) was used to verify the results obtained by both conventional PCR and the Ion Torrent platform.This study was approved by the Aichi Cancer Center Hospital's Institutional Review Board. All patients provided their written, informed consent. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki.Tumor samples were collected from Aichi Cancer Center Hospital, Japan. The current study consisted of two phases; a prerun phase and a clinical phase. The aim of the first stage (prerun phase) was to evaluate the accuracy of the custom panels. For the validation of the custom RNA lung panel, we examined the gene sequences of RNA derived from 35 NSCLC tissues with diverse backgrounds by conventional methods and NGS. Next, we moved to the next clinical phase, where clinical samples  were used to examine variants. In the clinical phase we conducted an NGS analysis while simultaneously applying conventional approaches to assess the feasibility and validity of the panel in clinical practice. A total of 80 patients were enrolled in the clinical phase of the study from February 2018 to December 2018.RNA used in the prerun phase was derived from various samples, including formalin‐fixed paraffin‐embedded (FFPE) sections and stamp‐slides. Normal tissue was not analyzed. For FFPE sections, tumor sections  were microscopically dissected, deparaffinized with xylene and washed twice in ethanol. After drying at 37°C, the samples were digested with proteinase K at 56°C. A total of three stamp slides were used. RNA extraction was performed applying the RNeasy Mini Kit (Qiagen), as per the manufacturer's instructions. RNA was quantified with the Qubit 2.0 using the RNA HS Assay Kit (Thermo Fisher Scientific). The lowest detectable concentration was 4 ng/μL.In the clinical phase, a total of four stamp‐slides were made using the direct smear method, and one slide was subjected to May‐Giemsa staining to confirm the presence of cancer cells Fig . When apEGFR were amplified by PCR and analyzed. The primers and cycling conditions for PCR amplification were modified from methods published previously.After the synthesis of cDNA, exons 18 to 21 of We used a chimeric DNA‐RNA‐DNA probe labeled with a fluorescent dye and quencher at each end.To detect the deletion in exon 19 of the EGFR gene, a common fragment analysis was used. Sample DNA was amplified with a FAM‐labeled primer set, and PCR products were electrophoresed. PCR amplified the shorter segment of DNA, creating a new peak in an electropherogram when a deletion mutation was present.MET exon 14 skipping. To confirm the presence of a sufficient number of reads in the NGS assay, primers for five housekeeping genes  were included in the primer mix, and the resultant reads were mapped. Even if no fusion genes were found, the assay itself was considered successful when a sufficient number of reads originating from these housekeeping genes was mapped.A custom panel for lung cancer (Ion Ampliseq 84 328 675–1) was designed by a research team at Thermo Fisher Scientific and manufactured as single primer pools by Thermo Fisher Scientific. This panel covers somatic mutation hot spots in five cancer‐associated genes and is capable of detecting gene translocation events . In the clinical phase, when the analysis was successfully performed by the conventional method, we conducted an NGS assay. An Ion Torrent adapter‐ligated library was generated using the custom panel described above. The concentration and size of the library were determined using the Applied Biosystems StepOne Real‐Time PCR system and Ion Library TaqMan Quantitation kit (both from Thermo Fisher Scientific). Sample emulsion PCR, emulsion breaking and enrichment were performed using the Ion Chef system (Thermo Fisher Scientific), according to the manufacturer's instructions. An input concentration of one DNA template copy per ion sphere particle (ISP) was added to the emulsion PCR master mix, and the emulsion was generated using the Ion Chef System (Thermo Fisher Scientific). Template‐positive ISPs were enriched, and sequencing was performed using semiconductor chips on the Ion Torrent S5 (Thermo Fisher Scientific). In this study, we used semiconductor chips of Ion 530 Chip Kit, which was later we changed to the Ion 540 Chip Kit, in order to collect an adequate number of sequenced reads.Data from the S5 runs were initially processed using the Ion Torrent platform‐specific pipeline software program Torrent Suite v4.0 (Thermo Fisher Scientific) to generate sequence reads, trim adapter sequences and filter and remove poor signal‐profile reads. We prepared a reference sequence to match the custom primer and mapped reads to the reference using Torrent Suite. The total number of reads mapped is an indicator of whether or not the NGS run was successful. The total mapped panel reads were counted, and 20 000 reads was considered sufficient for a further analysis; however, with a small sample size, clinical samples sometimes fail to achieve that level. In such cases, we proceeded with the analysis and searched for variants.P < 0.01. The second filter was employed by visually examining mutations using the CLC Genomics Workbench software program version 10.0.0 (Qiagen), as well as by filtering out possible strand‐specific errors.Initial variant calling from the Ion AmpliSeq sequencing data was generated with a plug‐in “variant caller” program. To eliminate erroneous base calling, three filtering steps were used to generate final variant calling. The first filter was set at an average depth of total coverage of >50, each variant coverage of >15 and In the detection of driver gene alteration other than gene rearrangement, DNA‐based NGS was performed only when discrepancies occurred in the results with conventional methods and RNA‐based sequencing. We used AmpliSeq Cancer Hotspot panel to validate the genetic alterations, described previously.ROS1 rearranged, eight ALK rearranged, and four were RET rearranged NSCLC  and immunohistochemistry; 11 cases were The clinical characteristics of the evaluated cases are shown in Table In the prerun phase, the median number of leads was 595 910, ranging from 3183 to 1 710 173. All of the variants confirmed with conventional methods were detected by NGS.Next, we started the clinical phase. From February 2018 to December 2018, a total of 80 patients were enrolled, and 80 tumor specimens were sequenced. The clinical characteristics are shown in Table EGFR). The deletion was not detectable by RNA‐based NGS and was only detected by DNA‐based NGS. Although this case had the EGFR‐activating mutation, the maximum effect of afatinib was stable disease, lasting as short as three months, so some biological mechanisms other than the EGFR pathway may have contributed to the cancer progression. This failure of RNA‐based NGS may be related to the paucity of EGFR mRNA with the activating mutation, considering that the EGFR‐tyrosine kinase inhibitor afatinib showed a minor effect on tumors in the patient. In the second case, although a very weak signal was detected by the cycleave method, the KRAS status was determined to be wild‐type. On NGS (both RNA‐based and DNA‐based), KRAS Gly13Glu was detected. In the third case, KRAS Gly12Cys was also negative by the conventional cycleave method but was detected by NGS (both RNA‐based and DNA‐based). From the stamp‐derived RNA in the fourth case, the CD74‐NRG1 fusion gene was detected only by NGS (RNA‐based) and later was confirmed using cDNA‐based Sanger sequencing.The concordance between NGS and conventional methods was 95.0%. Of the four inconsistencies Table , a reanaALK or ROS1) have proven effective against fusion gene‐derived tumors and been approved. Given their ability to detect structural rearrangements and provide information about the expression, most groups have favored the use of RNA‐based approaches for fusion gene detection. However, one major concern associated with the efficient examination of gene rearrangement is the technical difficulty of obtaining high‐quality RNA.Fusion gene detection is mandatory for the initial evaluation of advanced NSCLC, as various agents that target fusion gene kinase , RNA could be reverse‐transcribed into cDNA, and the resultant cDNA was successfully tested with NGS. Previously, Miki In some cases, DNA‐based sequencing is still required to assess the tumor mutational burden (TMB). The TMB was recently shown to contribute to the immune recognition of cancer and was suggested to be a predictor of the response to cancer immunotherapy.When applying the method described above, it is necessary to carefully examine the handling flow of biomaterial at the facility. For example, a rapid on‐site cytologic examination is a critical step that needs to be performed before NGS. At the NGS stage, library preparation and chip loading are both conducted by the Ion Chef semi‐automatically, which helps smooth the transition to the resultant bioinformatic tasks.ALK, ROS1, RET and NRG1 fusion genes based on the ratio of the 3′ end and 5′ end expression. A disproportionally high expression of the 3′ end of the gene compared to the 5′ end implies an oncogenic fusion event involving the gene.One major limitation associated with our system is that previous knowledge of all possible fusion patterns is necessary in order to prepare an ideal panel for examination. To overcome this limitation, this assay includes a method for detecting ROS1 and RET). NTRK oncogenic fusion is an emerging target for the subset of solid malignancies including NSCLC. Several clinical trials have shown the safety and efficacy of TRK fusion kinase inhibitors, and highly potent pan‐TRK inhibitors have already been approved by regulatory agencies.NTRK fusion gene.Another limitation associated with our study is that we were unable to evaluate all rearrangements using NGS with RNA from stamp cytology because of the rarity of some gene rearrangements  is unknown from the study. Moreover, assuming that a touch imprint sample is used, it is unknown how many slides are best used for RNA extraction. The larger the number, the more RNA can be extracted, but the handling on site becomes complicated.The vast majority of current diagnostic genomic practices are being challenged by massive parallel sequencing technology. Panel‐based RNA assays as a tool for the initial genomic evaluation of known driver gene alteration can deliver useful results that compete with existing traditional methods in both feasibility and accuracy.The authors report no conflict of interest.


Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCoV-BJ70 and CRCoV-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that rNPs of CCoV and CRCoV were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the rNP from CRCoV could cross-react with mice antisera against human coronavirus , while rNP of CCoV had cross-reactivity with only anti-sera against viruses belonging to the same group (HCoV-229E and NL63). In summary, CCoV and CRCoV rNPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.To characterize the antigenicity of nucleocapsid proteins (NP) derived from canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) in China, the N genes of CCoV (CCoV-BJ70) and CRCoV (CRCoV-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs (rNPs) were expressed in


Immune checkpoint inhibitors-induced myocarditis presents unique clinical challenges. Here, we assessed post-marketing safety of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), programmed cell death-1 (PD-1), and programmed death-ligand 1 (PD-L1) inhibitors by mining the real-world data reported in two international pharmacovigilance databases.We analyzed immune checkpoint inhibitors (ICIs)-associated fatal adverse drug events (ADEs) reports from the US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) collected from July 1, 2014 to December 31, 2019 and data from EudraVigilance (EV) database accessed on February 29, 2020. Three different data mining approaches were used to detect the signal of fatal myocarditis caused by ICIs.Based on 7613 ICIs-related ADEs reported to the EV database and 5786 ICIs-associated ADEs submitted to the FAERS database, the most frequently reported ADE was ipilimumab-related colitis. For myocarditis, nivolumab-associated myocarditis was the most common. Among the five fatal toxic effects associated with ICIs, the lethality rate of myocarditis was the highest. Therefore, we further analyzed ICI-associated myocarditis and found that elderly patients and male patients were more likely to develop ICIs-related myocarditis. The results of signal detection showed that the risk signal of avelumab-related myocarditis detected by reporting odds ratio (ROR) method and proportional reporting ratios (PRR) method was the highest, whereas the signal strength of ipilimumab-related myocarditis detected by Bayesian confidence propagation neural networks (BCPNN) method was the strongest.The findings of this study indicated the potential safety issues of developing myocarditis when using ICIs, which were consistent with the results of previous clinical trials and could provide a reference for clinical workers when using ICIs.The online version contains supplementary material available at 10.1186/s12885-020-07741-0. Immune checkpoint inhibitors (ICIs), such as cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), programmed cell death-1 (PD-1), and programmed death-ligand 1 (PD-L1) inhibitors that can inhibit the co-inhibitory immune checkpoint pathways have completely changed the treatment landscape of various malignant tumors –4. TheseThe US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) is one of the international spontaneous reporting systems, that is designed to support the FDA’s post-marketing safety surveillance program for drugs approved by the FDA. This database includes all information on adverse drug events (ADEs) and medication errors collected by the FDA. EudraVigilance (EV) is another international spontaneous reporting pharmacovigilance database for ADEs maintained by the European Medicines Agency. Data from these databases can be acquired by the public and utilized to provide evidence for the safe use of the drugs, especially for newly marketed drugs and uncommon ADRs. The objective of this study was to analyze the ICIs-associated myocarditis and to determine the signal of ICIs-associated myocarditis by mining the data reported in the spontaneous reporting systems.https://fis.fda.gov/extensions/FPD-QDE-FAERS/FPD-QDE-FAERS.html), and EV (http://www.adrreports.eu/en/search.html) was accessed and queried on February 29, 2020. In this study, the processing of data downloaded from FAERS database followed the customized strategy described previously   and the risk of ADRs , 13. ThiDespite the limitations, the findings of this study indicated the potential safety issues of developing myocarditis when using ICIs, which was consistent with the results of previous clinical trials and could provide a reference for clinical workers when using ICIs.The results of this study showed that the application of ICIs was associated with an increase in fatal toxic effects, especially myocarditis, which was consistent with previous studies. It was suggested that clinicians should pay attention to these fatal ICIs-associated ADRs and take preventive measures when treating tumor patients with immunotherapy based on ICIs. The findings of this study also provided objective evidence for post-marketing safety of ICIs, thereby ensuring the safe use of these drugs and improving the prognosis of patients with cancer.Additional file 1: Table S1. A 2 × 2 Contingency Table for Disproportionality Analysis. Table S2. Signal Values of ICIs-Associated ADRs.


RNA epigenetics is perhaps the most recent field of interest for translational epigeneticists. RNA modifications create such an extensive network of epigenetically driven combinations whose role in physiology and pathophysiology is still far from being elucidated. Not surprisingly, some of the players determining changes in RNA structure are in common with those involved in DNA and chromatin structure regulation, while other molecules seem very specific to RNA. It is envisaged, then, that new small molecules, acting selectively on RNA epigenetic changes, will be reported soon, opening new therapeutic interventions based on the correction of the RNA epigenetic landscape. In this review, we shall summarize some aspects of RNA epigenetics limited to those in which the potential clinical translatability to cardiovascular disease is emerging. Translational epigenetics is a relatively new branch of molecular biology that investigates regulatory processes occurring molecularly “above” the primary DNA sequence associated with physiological and pathophysiological conditions. Specifically, of applied translational epigenetics interest are those mechanisms that introduce functional changes into DNA, RNA, and sometimes proteins, without introducing changes into their primary sequence, and with essential implications in organismal function and disease. Hence, translational epigenetics pays attention to the effect of chemical modifications on DNA , histoneAmong the different RNA species, there are non-coding RNAs (ncRNAs), which are ribonucleic acid sequences that do not codify for proteins. They recently became of interest for their important regulatory function and perspective diagnostic-therapeutic potential. Conventionally, ncRNAs are classified by their length: 200 nucleotides are the cut off between long (lncRNAs) and short non-coding RNAs (sncRNAs). The latter group includes microRNAs (miRNAs) . However1-methyladenosine, m1A) [6-methyladenosine, m6A) [6A occurs at the 5′-AGG(m6)AC-3′ consensus sequence of some mRNAs [6A of RNA can be oxidatively demethylated into N6-hydroximethyladenosine (hm6A) and N6-formyladenosine (f6A) which may modulate RNA–protein interaction affecting gene regulation [7G) [Although several reviews have been written about the regulatory role of ncRNAs ,15,16,17ne, m1A)  and 6  . In partme mRNAs , their sme mRNAs  and theime mRNAs . Interesgulation . These pgulation   . This moion [7G)  and recaion [7G)   ,33,34,35Cardiovascular diseases (CVDs) are a pandemic problem that in 2017 caused around 17.7 million deaths worldwide . These d6A RNA methyltransferase METTL3 seems to play a crucial role in eccentric cardiomyocyte remodeling [6A mRNA in mouse neonatal but not adult cardiac cells [6A in this context, METTL3 knockout animals were exposed to pressure overload. Gene-deleted mice were resistant to cardiac hypertrophy indicating that the control of RNA methylation is important to prevent the development of heart failure likely by regulating RNA processing [Recently, the American Heart Association identified chronic heart failure as the most critical damaging condition for the heart in the aging population . Myocardmodeling . One METmodeling . The worac cells . To explocessing  , resulting in lower protein levels than healthy controls [6A , which is involved in lysosomal biogenesis [A recent observation suggested a role for mmyocytes  subjecteogenesis , and repogenesis . This progenesis . Interesogenesis   is known to methylate the transcript encoding the intercellular adhesion molecule 1 (ICAM-1), increasing the corresponding protein’s synthesis and enhancing leukocytes adhesion to the endothelial layer . NoteworHyperhomocysteinemia (HHcy) is a metabolic alteration often associated with inflammation and frequently detected in cardiovascular and chronic diseases. Of interest, the effect of HHcy seems mediated by Nsun2, which, in T cells, methylates the mRNA encoding for interleukin (IL)-17A . The metInflammatory signals are at the basis of all chronic diseases, and they are often triggered by dysmetabolic conditions associated with aging, a process widely defined as inflammaging ,52,53,54Moreover, myeloid cells from MALAT1-deficient mice displayed a higher adhesiveness to atherosclerotic lesions and lower levels of MALAT1 expression in human plaques could be related to worse prognosis caused by the infiltration of inflammatory CD45 + cells . In addiIn a series of in vitro experiments performed by using human umbilical vein endothelial cells in the presence of oxidized low-density lipoproteins and platelet-derived growth factor, miR-125b was downregulated whereas podocalyxin (PODXL), a member of the cluster of differentiation 34 of sialomucins, resulted as upregulated . InteresIn the context of regulatory processes, it emerged clearly that positive and negative feedback loops are present during the process of RNA maturation in which methylation might play a role at different levels. For example, the expression of miRNAs is frequently regulated by methylation of the corresponding DNA locus. The transcription of miR-200 family members requires, in fact, DNA demethylation at the promoter level and is enhanced by reducing the activity of DNMTs . In a raHHcy is a known risk factor for ischemic stroke , in whicIn summary, the methylation of H19 locus has effects on H19 expression and function and interferes with the activity of DNA methylases determining changes in the expression level of other genes with potential consequences on cardiovascular homeostasis see .Other ncRNA species are regulated by methylation at the genomic level with consequences on cardiovascular homeostasis. In vivo, the miR-145 expression has an impact on atherosclerosis and pulmonary hypertension. High levels of this miR protect blood vessels from plaque formation and hypertension ,75. HencAdditionally, miR-145 regulates ubiquitin-like containing PHD and RING finger domains 1 (UHRF1), which interact with DNA methyltransferase 1 (DNMT1), suggesting an indirect link between miRNA modulation and DNA methylation . DNMT1 wSeveral studies have demonstrated the role of methylation in the epigenetic regulation of lncRNAs expression during cardiac development and repair. Here we provide a few recent examples of this very recent aspect discussing whether they could be developed as therapeutic targets.An example is given by the lncRNA named cardiomyocyte proliferation regulator (CPR) that, if downregulated, induces cell cycle activation and decreases scar formation after cardiac injury . CPR regAlthough the field of epigenetics is rushing forward, most of the epigenetic studies on CVDs and/or chronic diseases are still focused on DNA methylation , histone6A and m5C come from cancer biology leaving cardiovascular diseases relatively unexplored [Recent literature highlighted the controversial role of RNA methylation, occurring mainly on adenosines, and resulting in increased mRNA instability, repressed protein synthesis ,45, or iexplored ,84,85.At present, several lncRNAs and miRNAs have been investigated in differential pathophysiological contexts including hypoxia , cardiac


FMR1 gene, resulting in a progressive development of tremors, ataxia, and neuropsychological problems. This highly disabling disease is quite common in the general population with an estimation of about 20 million PM carriers worldwide. The chances of developing FXTAS increase dramatically with age, with about 45% of male carriers over the age of 50 being affected. Both the gene and pathogenic trigger, a mutant expansion of CGG RNA, causing FXTAS are known. This makes it an interesting disease to develop targeted therapeutic interventions for. Yet, no such interventions are available at this moment. Here we discuss in silico, in vitro, and in vivo approaches and how they have been used to identify the molecular determinants of FXTAS pathology. These approaches have yielded substantial information about FXTAS pathology and, consequently, many markers have emerged to play a key role in understanding the disease mechanism. Integration of the different approaches is expected to provide crucial information about the value of these markers as either therapeutic target or biomarker, essential to monitor therapeutic interventions in the future.Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative monogenetic disorder affecting carriers of premutation (PM) forms of the  FMR1) responsible for the disease finds its origin on the X chromosome and codes for the Fragile X Mental Retardation Protein called FMRP. This protein is essential in the neuronal development and synaptic plasticity of the brain. The 5′ untranslated region (UTR) of the FMR1 gene carries a variable number of CGG repeats, which is between 4 and 55 in healthy individuals. Due to the instability of the repeat the CGG sequence can expand to 55–200 repeats over generations (Fragile X-associated Tremor and Ataxia syndrome (FXTAS) is a late onset neurodegenerative disease that affects Fragile X premutation (PM) carriers. The Fragile X gene  toxic aggregation of Repeat-Associated Non-AUG (RAN) polyglycine peptides translated from the FMR1 5′ UTR  to interact with specific RBPs. was assessed through the catRAPID algorithm . In addition, strong binding propensities were found for an additional set of 92 proteins that are known to aggregate in stress granules , MBNL1 and HNRNP-G  RNA involved in neuronal translational . Differeargeting .The RNase H protection approach is used to detect DNA and RNA fragments in cell lysates. RNase H cleaves the target RNA molecule at a specific site hybridized with a DNA probe. In these specific sites, if a protein is bound, the hybridization is blocked, therefore no cleavage by RNase H will occur.FMR1 5′ UTR region favors formation of a three-stranded nucleic acid structure, composed of a DNA:RNA called R-loop formation, with the nascent RNA assembling with the template DNA strand (FMR1 R-loop formation As expected for R-loop formation. Treatment with purified recombinant human RNases H1 and H2 eliminated DNA–RNA interaction (It has been observed that transcription through the GC-rich A strand . Using Deraction .in situ hybridization (FISH) co-localization techniques require knowing in advance which RNA and protein interaction will be studied. This technique is commonly used in FXTAS research as it allows verifying where CGG repeats localize with other molecules. By means of FISH coupled to immunofluorescence it has been shown that CGG expansions and TRA2A significantly co-localize in COS7 cells , PM carriers and FXTAS. PM carriers of the expanded CGG repeat have a 2 to 8-fold elevation of FMR1 mRNA levels when compared to healthy CGG repeat individuals  model. This mouse model was developed in Rotterdam at the Erasmus MC in the Netherlands. This model was generated by replacing the endogenous murine Fmr1 8CGG repeat with a human 98CGG repeat containing the human FMR1 flanking regions. The Fmr1 mouse promoter was left unchanged. This was done by homologous recombination in embryonic stem (ES) cells  cells . Upon pa present . The CGG KI mice  have bee KI mice . Althougf memory . Hippocamplexity . Recentlin depth . Indeed,patients .nih KI) with a CGG repeat length between 118-120CGGs by generating a CGG repeat flanked by SfiI sites and then ligating this CGG repeat into the target construct with exon 1 of the mouse Fmr1 gene in the correct orientation minimalizing changes to the mouse regions flanking the CGG repeat, the gene itself and the promoter  or in context of the enhanced green fluorescent protein (L7-CGG90-EGFP) specifically in Purkinje neurons using the pcp2/L7 promoter in the cerebellum  in France created two transgenic mouse models  containing the full-length human tability . These mtability . These Yoduction . Importa effects .in vivo expression of expanded CGG RNA leads to severe toxicity in the liver by affecting reactive-oxygen species (ROS) signaling  and the other line expressing the reverse tetracycline-controlled transactivator protein (rtTA), which can be activated using doxycycline or doxycycline-derivative in drinking water or food. The TRE-element was coupled to an expanded CGG repeat in frame with eGFP while the rtTA was coupled to an hnRNP promoter. Both mouse lines were crossbred generating double transgenic offspring with ubiquitous expression of 90CGG repeat RNA -rtTA driver was used to activate expression of normal length CGG RNA or expanded CGG repeat RNA fused to eGFP . The PrPdut KI mouse model of FXTAS recapitulates most aspects seen in FXTAS, these mice have relatively few numbers of astrocytes with intranuclear inclusions. Therefore an astroglial-specific mouse model was necessary. This transgenic mouse line with a C57BL/6j background was generated through pronuclear injection using a astrocyte-specific Gfa2 promoter to induce expression of an expanded 99CGG repeat fused to an eGFP marker gene only in astrocytes ethyl)-6H-pyridocarbazol-2-ium  compound has been prioritized as a candidate for its ability to bind to loops produced by unpaired G-G nucleotides in CGG hairpins (1a and structurally similar compounds reduce RAN translation of FMR1 in cultured cells and patient-derived neurons (Candidates identified in hairpins . The 1a hairpins . In agre neurons . Three s neurons . These s neurons .in silico and in vitro section. RH and SH wrote the in vivo section.GT and TB-O wrote the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer EL declared a past co-authorship with one of the authors RH.


Protein and DNA have been considered as the major components of chromatin. But beyond that, an increasing number of studies show that RNA occupies a large amount of chromatin and acts as a regulator of nuclear architecture. A significant fraction of long non-coding RNAs (lncRNAs) prefers to stay in the nucleus and cooperate with protein complexes to modulate epigenetic regulation, phase separation, compartment formation, and nuclear organization. An RNA strand also can invade into double-stranded DNA to form RNA:DNA hybrids (R-loops) in living cells, contributing to the regulation of gene expression and genomic instability. In this review, we discuss how nuclear lncRNAs orchestrate cellular processes through their interactions with proteins and DNA and summarize the recent genome-wide techniques to study the functions of lncRNAs by revealing their interactomes in vivo. Over the past decade, intensive studies have demonstrated that long non-coding RNAs (lncRNAs) are key regulators in embryonic development , DNA damThe first study showed that RNA constitutes a significant fraction of chromatin date back over 50 years ago , 42. LatRNA activator (SRA) noncoding RNA that is associated with CTCF, reduced CTCF-mediated insulator activity [IGF2/H19 imprinting control region and increased IGF2 expression. It was also reported that a lncRNA, Jpx, activates Xist RNA expression by evicting CTCF from binding to Xist promoter [It has been reported that the CCCTC-binding transcription factor (CTCF), which promotes chromatin loop formation, binds to thousands of RNAs . Depletiactivity  at the Ipromoter . These rIt was proposed that lncRNA transcription guides genome organization . For exain cis and the Xi (inactive X chromosome) became Xa (active X chromosome) like conformation [Xist RNA is one of the well-studied lncRNAs involved in epigenetic regulation and gene silencing. It is a 17-kb lncRNA that is expressed from the inactive X-chromosome (Xi) , coatingormation . Lee et ormation , 57 to pormation , 59. Remormation  in Xi. TEnhancer RNAs were first discovered by two early studies through the genome-wide sequencing such as RNA-seq or ChIP-seq , 62. TheDrosophila, where homologous chromosomes were closely paired in somatic nuclei [, is highly expressed in undifferentiated ES cells, and antagonizes Xist action. During ES differentiation in female cells, transient homologous pairing occurs, and the Xist RNA expression increases to initiate chromosome-wide silencing. It was reported that Tsix RNA mediates the homologous pairing between two X chromosomes, thus breaking epigenetic symmetry between the two X chromosomes, as well as driving the random choices for the selection of inactive or active X chromosomes [Oct4 and various cytokine genes [Homologous chromosome pairing rarely occurs in somatic cells under normal growth conditions with only a few exceptions. Transvection was first observed in 1908 in c nuclei . It is ac nuclei . Tsix, aomosomes , 73. Moromosomes . Not limne genes –77. Althne genes , 78, 79,, TERC, LTR ERV-9 [To map the RNA-associated chromatin in vivo, Chu et al. and Simon et al. utilized biotinylated antisense DNA oligo probes to capture a specific RNA that is associated with chromatin , 8 Tabl, named CTR ERV-9 , 81. CHAand a large set of non-coding RNAs that are bound to active promoters, enhancers, and super-enhancers in a tissue-specific manner [Recently, several methods were developed to reveal all interactions between RNA and DNA in the nucleus, including MARGI-seq (Mapping RNA-genome interactions), ChAR-seq (Chromatin-Associated RNA sequencing), and GRID-seq  –13. The c manner –13. All-As discussed previously, long non-coding RNAs may serve as scaffolds to bring two or several distant DNA loci into spatial proximity. To understand how a specific RNA interacts with chromatin in a 3D space, Mumbach et al. developed a method, named HiChIRP, which combines Hi-C and ChIRP . They inRNA binding proteins (RBPs) are associated with a large number of human disorders, such as autoimmune and neurologic diseases –84. Rema(identification of direct RNA interacting proteins), RAP-MS (RNA antisense purification coupled with mass spectrometry) and ChIRP-MS (comprehensive identification of RNA-binding proteins by mass spectrometry) to determine Xist RNA interacting proteins [Recently, researchers have elaborated on the methods for pulling down RNA-proteins complexes via antisense oligos that are complementary to targeted RNA sequences. Therefore, this type of capture is not limited by the requirement of antibodies against the proteins of interest for immunoprecipitation. In 2015, three groups established RNA-centric capture methods, including iDRiP-MS proteins –20 , RBR-ID (proteomic identification of RNA-binding regions) introduces 4SU (4-thiouridine) into RNAs, crosslinks RNA-proteins by UV light, and compares the mass spectrometry of 4SU treated and non-treated samples . An RNA-Unlike DNA, RNA is a single strand of nucleic acids and is capable of folding into 3D structures that range from simple helical elements to complex tertiary structures and quaternary ribonucleoprotein assemblies . The cha3) to probe RNA secondary structures in living cells for all four bases. They used a cell-permeable SHAPE reagent, NAI-N3, which adds an azide group to NAI (2-methylnicotinic acid imidazolide) to label flexible regions of RNA. After RNA isolation, a biotin moiety is selectively added to NAI-N3-modified RNA by copper-free click chemistry, allowing the biotin-streptavidin purification of modified RNA after RNA fragmentation. By comparison of in vivo and in vitro icSHAPE, they observed that all RNAs are less folded in vivo, suggesting that RNA structures largely depend on intracellular environments. Moreover, they found that regulatory RNAs, such as lncRNAs and primary microRNA (miRNA) precursors, preserve their structures better than mRNAs in vivo. To selectively enrich some specific RNA molecules, COMRADES (cross-linking of matched RNAs and deep sequencing) combines RNA capture and CLICK chemistry to probe RNA structures and RNA-RNA interactions [To profile genome-wide RNA structures in vivo, Ding et al. developed a method, called DMS-seq  Table 1. In the ractions . In the In addition, an RNA molecule can base-pair with other RNA molecules to form RNA duplexes (such as miRNA and its target RNA) bound by RBPs. To identify miRNA targets, Tollervey et al. developed a method, named CLASH  , 31 to iTCF21 promoter, thus facilitating GADD45A binding, local DNA demethylation and TCF21 expression [, which forms an R-loop structure with the central intron of GATA3-AS1 and tethers the MLL H3K4 methyltransferase to GATA3 gene locus, thereby regulating GATA3 expression [R-loops are three-stranded nucleic acid structures in which a strand of RNA hybridizes with a strand of DNA, while the other strand of DNA loops out. The R-loop structure was first described in 1976 in the study  in whichpression , 91. Anopression . Furtherpression . The deppression .in cis during transcription when a nascent RNA hybridizes to the DNA template behind the moving RNA polymerase [in trans, which means that an RNA transcript at one locus hybridizes with homologous DNA at another locus [It is generally believed that R-loops form lymerase . Howeverer locus , and theer locus , 96. BecTo map R-loops in a genome-wide scale, Ginno et al. developed a method, named DRIP-seq (DNA-RNA immunoprecipitation coupled to high-throughput sequencing), which utilizes an antibody S9.6)  to speci  to specCellular organelles such as mitochondria and Golgi apparatuses composed of lipid bilayer membrane structures, which help the formation of compartments and separate biological processes within a cell. In contrast, membraneless structures are formed through a process known as liquid-liquid phase separation (LLPS) and are made of RNA-protein droplets . One of How do RNAs promote phase separation synergistically with protein-protein interactions? The in vitro studies reported that RNA repeats, such as trinucleotide repeat and G-quadruplex, can undergo a phase transition to form either a condensed liquid or a gel-like state  through The Human Genome and ENCODE Projects have shown that above 90% of the genome is transcribed , 72 into


Porites and Agaricia). We find one modern reef, however, whose community composition remains within the HRV showing that it has not transitioned to a novel state. Reef-matrix cores extracted from this reef reveal that the coral community has remained in this state for over 800 years, suggesting long-term stability and resistance to the region-wide shift to novel states. Without these data to provide historical context, this potentially robust and stable reef would be overlooked since it does not fulfil expectations of what a Caribbean coral reef should look like in the absence of humans. This example illustrates how defining past variation using the fossil record can improve our understanding of modern degradation and guide conservation.Many Caribbean coral reefs are heavily degraded, yet their pre-human, natural states are often assumed or estimated using space-for-time substitution approaches. Here we use an 11-hectare suite of fossilised mid-Holocene (7.2–5.6 ka) fringing reefs in Caribbean Panama to define natural variation in hard coral community structure before human-impact to provide context to the states of the same reefs today. We collected bulk samples from four trenches dug into the mid-Holocene fossil reef and surficial bulk samples from 2–10 m depths on five adjacent modern reefs extending over 5 km. Analysis of the abundances of coral taxa in fossil bulk samples define the Historical Range of Variation (HRV) in community structure of the reefs. When compared to the community structure of adjacent modern reefs, we find that most coral communities today fall outside the HRV, identifying them as novel ecosystems and corroborating the well-documented transition from acroporid-dominated Caribbean reefs to reefs dominated by stress-tolerant taxa ( Consequently, modern benchmarks used to inform and evaluate the success of conservation actions rely on space-for-time substitution—an approach that uses spatial patterns across modern ecosystems as a proxy for temporal changes within ecosystems. The space-for-time substitution approach is a mainstay across conservation ecology, and has previously proved effective in coral reef ecology8, but the assumption that spatial and temporal variation are equivalent may not necessarily be true9 especially in habitats that are naturally highly variable on small spatial and temporal scales, like coral reefs10. Conditions from one reef therefore cannot necessarily be used to define another’s baseline. Additionally, with so few reconstructions of the state of reefs before human impact, baselines rely on limited definitions of reef states that likely fail to capture the high spatial and temporal complexity of coral reefs11. Accordingly, to prioritize conservation activities, contextualise restoration goals, and reveal the drivers of ecosystem change, it is important to better quantify spatial and temporal variation on Caribbean reefs, both past and present.Caribbean coral reefs began to deteriorate long before they were first surveyed13 that uses paleontological and historical records to define past spatial and temporal variation in ecosystem states with the aim of helping to determine if current states and rates of change fall within or outside the natural range of variation15. Coral reefs are potentially well-suited to the application of this approach given their high community complexity, and their propensity to preserve a wide array of reef community members24 and past environmental signals25 as they accrete. In this study, we apply the HRV approach to quantify variation in pre-human coral communities in a suite of lagoonal fringing coral reefs in western Caribbean Panama  is an approach developed in the field of terrestrial ecology18 and have persisted since27. Today, the reefs fringing the bay are primarily composed of stress-tolerant and “weedy” corals like branching Porites and Agaricia29, but it hasn’t been demonstrated if this represents the natural state of reefs in the bay. Coral cover is generally moderate to low and fish communities are depauperate of adults30. Almirante Bay is well protected from wave action and currents, and sits outside the hurricane belt. High rainfall, deforestation and changing land use, including a ~140-year banana industry31, cause relatively higher suspended particles and dissolved nutrients conditions compared to other Caribbean reef systems33. The bay today experiences periodic hypoxia that sometimes causes widespread death of corals and other aerobic sessile organisms35.The fringing coral reefs, seagrass and mangrove systems of Almirante Bay Fig.  became e18, a date which we confirm in this study (see later).Two large-scale construction projects led to the excavation of ~50 hectares of a fossil fringing coral reef in the eastern portion of Almirante Bay Figs.  and 2. P36. Paleotemperature reconstructions of the Caribbean at this time are equivocal, suggesting either mid-Holocene warming37 or cooling38 with greater seasonality39 relative to today. Most of these reconstructions relate to regions to the north and eastern Caribbean region. Estimates from the southwestern Caribbean of Panama where our study is based are limited, but available models indicate similar climatic conditions to today39. Other available evidence suggests the fossil reef formed in a setting similar to the reefs of today. Post-glacial sea level rise had stabilised close to modern day levels as the fossil reef accreted41, a result corroborated by our elevation models, similarities between coral (see later) and the structure of fish communities in the reefs as observed through fossil otolith assemblages21. Reefs in the region today sit to the south of the hurricane belt and this appears to have also been the case for at least the last 7 ka42. The aspect and exposure of the fossil reef was the same as it is today given that the Island of Colón, which today forms the Almirante bay  and locate major fossil habitats  of reef framework covering a stratigraphic depth of ~15 cm from each of the four trenches we excavated Figs.  and 2B. 50 – a purpose-built reference collection – and other guides51. The relative mass of each coral taxon was square-root transformed and used to compare the assemblage structure of each sample using Bray-Curtis dissimilarities, which were ordinated using non-metric multidimensional scaling (NMDS) to place samples in multivariate ecological space based on the relative abundance of coral taxa skeletal weights50. PERMANOVA was used to test for significant differences in dissimilarities among groups. Corals of unknown taxa were removed from all analyses. These corals are listed as “unknown” in Table 50. “Unknown” corals totalled 8.26% of all coral weights in mid-Holocene samples and 10.02% in modern samples  was computed using the isoMDS function in the software package MASS53 in R which was then plotted using ggplot54 in R55. The function adonis  in the software package vegan52 was used to test for significant differences in dissimilarities among groups. To confirm that differences between communities were valid, and not an artefact of differences between groups in how much each community deviates from the centroid of the group we used beta-dispersion with the betadisper function [Multivariate homogeneity of groups dispersions (variances)] in the software package vegan52. All analyses were performed in R (R core team).In both mid-Holocene and modern bulk samples, all coral skeletal remains in the >2 mm fraction were identified to species or genus using the Caribbean Coral Skeleton Identification Guidees Table , suggestAcropora palmata and U-Th on two branches of A. cervicornis  at Punta Caracol reef. See supplementary materials for further information on radiometric dating methods.We used both U-Th and radiocarbon radiometric dating to provide temporal context to the community composition data in both the fossil and modern reefs. To determine how old and for how long the mid-Holocene reef grew, we used calibrated radiocarbon dating on eight pieces of is Table  and comb18  Table , a time -5.6 ka T58, presumably because the branching coral acts as a rigid framework that limits infaunal and epifaunal bioturbation which can otherwise cause considerable vertical mixing in other settings60. These findings, along with the high frequency of fragile branching corals found in life position in the fossil reef  ranged in age from 1926 to 2012 AD , the abundances of coral taxa in modern and fossil bulk samples yield meaningful and comparable ecological signals that relate to the relative contribution of different taxa in a time-averaged living community.Death assemblages sampled through bulk-sampling are not directly comparable to traditional reef survey methods such as live coral cover. Variable rates of carbonate production and potential ephemeral growth patterns of different coral taxa will offset the relative abundances between traditional survey methods and abundances in skeletal records. In some cases, however, the contribution to skeletal framework in death assemblages can be remarkably constant across different taxa and different growth modes, and accretion rates are instead controlled more by local environmental and biotic conditions on the reefWe used the relative abundances of coral taxa in the fossil samples to define the HRV of the reefs in the mid-Holocene and compare that HRV to the community composition of modern adjacent reefs. Ordination of the data demonstrates that, overall, the modern coral communities in Almirante Bay are distinct from the fossil-defined HRV Fig. . The ecoAcropora cervicornis), which was rare or absent on many of the modern reefs in our study  or lettuce leaf coral (Agaricia spp.)  Fig. , corroboA. cervicornis  might have played a role in maintaining community stability on the reef. A. cervicornis, like most acroporid corals, is a species that has traded disease and predation-resistance for fast-growth and high-rates of clonal reproduction23. Previous work has shown that the population of A. cervicornis at the Punta Caracol reef has particularly low genetic diversity relative to other conspecific populations in the Bocas del Toro region, a sign that it has been isolated from other populations and established principally through clonal propagation71. Could a resilient, genetically isolated population of A. cervicornis have persisted for more than 800 years at Punta Caracol through clonal propagation? We propose that historical information, such as the data presented here, can be used to help make more informed choices when selecting resilient genets to translocate coral colonies, improve connectivity and recruitment78 and promote recovery79.Determining why Punta Caracol is an exceptional reef that has resisted the shift to novel states could reveal useful information that can be repurposed for the conservation of other reefs. Surprisingly though, we see no clear differences in the reef’s abiotic characteristics  or biotic characteristics ion Fig.  This impion Fig.  undermin80, a notion that persists even though reefs exhibit considerable variation in these measures today2 and did so in the past16. This assumption is reinforced by space-for-time substitution studies that take reefs that are perceived to be healthy and presume their states are the baselines for reefs perceived to be degraded. Our study highlights the pitfalls of the use of space-for-time substitution approaches in coral reefs. Not even an experienced reef ecologist is likely to think that Punta Caracol resembles a pre-human impact Caribbean reef because living coral cover and fish biomass, often used as a comparative metric of reef health, are not particularly high on this reef 32. Yet despite failing to fulfil expectations of what many might consider coral reefs should look like in the absence of humans, we demonstrate here, through the use of the HRV approach, that the coral community at Punta Caracol has persisted for centuries in a state that retains at least some elements of their prior condition before human impact.There is a growing trend in conservation biology to accept that the majority of ecosystems today have already shifted into irreversible novel states and, consequently, that conservation resources should not be squandered trying to coerce restoration to unachievable historical conditions. For example, the near-ecological extinction of megafauna and fish from overharvesting and the chronic and intensifying effects of successive coral bleaching make returning Caribbean coral reefs to their past states almost unimaginable. While generally true, this paradigm involves the assumption that historical conditions of Caribbean coral communities were universally of high coral cover and high fish biomass81, the unclear directionality of future ecosystem shifts70 and the complex impact of unidentified future stressors82. Conservationists interested in supporting coral reefs must therefore hedge their bets by conserving variability itself to provide nature with the raw material for the selection of resilience83. Resources for conservation will always be limited and decisions must be made about where priority should be given. We suggest that such decisions be made not only based on current states and future predictions but also through the use of HRV data to extend time scales, establish location-specific baselines and uncover elements of resilience that might otherwise be overlooked.Defining the HRV of an ecosystem using the fossil record can therefore provide powerful, location-specific context for modern ecosystem change Fig. , which cAll data supporting the findings of this study are available within the paper and its Supporting Information files.Supplementary Information.


Aim: To refine and assess the validity of an algorithm to detect lying down from raw data of thigh-worn accelerometers. Axivity-AX3 accelerometers were placed on the thigh and upper back (reference) on adults in a development dataset (n = 50) and a validation dataset (n = 47) for 7 days. Sedentary time from the open Acti4-algorithm was used as input to the algorithm. In addition to the thigh-rotation criterion in the existing algorithm, two criteria based on standard deviation of acceleration and a time duration criterion of sedentary bouts were added. The mean difference (95% agreement-limits) between the total identified lying time/day, between the refined algorithm and the reference was +2.9  min in the development dataset and +6.5  min in the validation dataset. The refined algorithm can be used to estimate lying time in studies using different accelerometer brands.Body postural allocation during daily life is important for health, and can be assessed with thigh-worn accelerometers. An algorithm based on sedentary bouts from the proprietary ActivePAL software can detect lying down from a single thigh-worn accelerometer using rotations of the thigh. However, it is not usable across brands of accelerometers. This algorithm has the potential to be refined.  The time spent in various body postures and physical activities are important for health . AssessmAlgorithms for detection of postures and activities from thigh worn accelerometers use information from the accelerometer to calculate movements of the thigh and the angle of the thigh towards the line of gravity . In prinLyden et al. have presented an algorithm that instead uses information about rotation of the thigh to detect lying down episodes from a single thigh-worn accelerometer . The ideThe Prospective Physical Activity, Sitting and Sleep consortium (ProPASS) aims to pool and harmonize studies of thigh-worn accelerometer data . A prereThe aim was to integrate, and refine the method to identify lying down time, suggested by Lyden et al., with the open Acti4 algorithm and to validate this method in a dataset with additional back-worn accelerometer as the reference.Participants to this study were recruited from the ongoing Halland County Knee Osteoarthritis (HALLOA) cohort longitudinal study. The inclusion criteria for HALLOA was (1) be 30–65 years old; (2) have knee pain; (3) no history of anterior cruciate ligament (ACL) injury; (4) no inflammatory rheumatic disease; (5) not have previously confirmed knee osteoarthritis. In addition, they should be in work to enter the present study. The population consisted of people who work day-shifts, night-shifts and also 24-h shifts. The 50 first participants, with valid accelerometer data, that joined the study were used in a development dataset. Then, the next 47 participants, with valid accelerometer data, from the same study were used in a validation dataset . The criThe development and validation dataset were found be comparable regarding distributions of gender, age, BMI, height, KOOS-Pain and KOOS-ADL using a non-parametric Mann Whitney U test .All participants were equipped with two tri-axial accelerometers  by a research assistant at the study centre. The accelerometers were positioned at (1) the upper back ; (2) the right thigh  and only remove them if they experienced itching from the tape or any other discomfort. If removal of accelerometers occurred, or if the accelerometers fell off, they were instructed to note the time and contact the research assistant for further instructions.The two accelerometers were time synchronized with a custom made Matlab algorithm. The algorithm synchronized the accelerometer signals by conducting a robust-fit linear regression of the time shift found by the cross-correlation of the two accelerometer signals under free living conditions. Before further analysis, accelerometer signals were calibrated automatically according to the method presented by Van Hees et al. . In ordeValid accelerometer data was defined as cases with both successful time synchronization and placement correction.backINC) above 45 degrees (backθ), towards the horizontal plane. Positive angles of backθ indicated stomach lying or leaning forward. Then if the angle between the sagittal axis coming out of the front of the thigh and the horizontal plane (thighθ), were more than 45 degrees during these stomach lying episodes, these were reclassified to sitting as follows:As reference, all lying bouts were detected with the use of both the thigh and back accelerometer. The sedentary bouts of sitting and lying were first detected by the validated non-proprietary Acti4 algorithm for the thigh-worn accelerometer ,16. The  degrees . The res degrees . This waIn order to filter out false positive lying episodes due to when the person was leaning sideways the following equation was used.Two algorithms (A and B) were developed based on the sedentary classifications from the Acti4 algorithm. Algorithm A is a direct implementation of the lying detection according to the method described by Lyden et al. . AlgoritIt was hypothesised that sitting with crossed legs may lead to false positive lying detection . We hypoSince the algorithm will not identify lying if the participants not rotates the thigh in the bed , we hypoWe also hypothesised that true lying episodes are longer than such false positive cases. The distributions of the bout lengths indicated that sitting bouts were normally considerably shorter lying bouts . TherefoDuring the development of the algorithm, we also found situations where the rotation angle crossed the threshold, but then very shortly after the crossing, went back below the threshold again. Therefore, momentary crossings with less than 5 s duration were not considered in Algorithm B.Preliminary values for the included thresholds for SDVM_1, MinLieT and SDVM_2 were chosen based on the information from the distributions. Then a simple optimization procedure (by changing only a single threshold at a time) of the three thresholds was performed to achieve the maximum sensitivity plus specificity (see 2.6 statistics). The resultant thresholds were SDVM_1 = 0.01 g, MinLieT = 20 min, and SDVM_2 = 0.004 g respectively. In addition, by both investigating the distributions of the thigh rotation angles  and usinBoth Algorithm A and B were validated by applying those algorithms in the validation dataset and comparing the results from them with the reference method. The comparison was carried out using the sensitivity, specificity and a Bland-Altman analysis as described in 2.6 Statistics.The sensitivity and specificity as well as accuracy were calculated by comparing the classifications of lying down, between the reference method and both Algorithm A and B, for each second (Equations (3)–(5)). Since lying was fairly prevalent in the both datasets  in addition to sensitivity and specificity, accuracy was also deemed to be a valid performance index of the algorithms:TP = number of true lying-down seconds with respect to reference;FP = number of false lying-down seconds with respect to reference;TN = number of true non-lying-down seconds with respect to reference;FN = number of false non-lying-down seconds with respect to reference.The difference in lying down time duration, between the Algorithm A and the reference as well as Algorithm B and reference were calculated for each day. The mean differences in lying down time duration and standard deviation (SD) over all days were calculated.For each participant, the averaged lying down time per day was also calculated by dividing the total lying down time by the each participants measured time (days). Then the mean values and SD for those differences were also calculated.The 95% limits of agreement (LOA) was calculated by taking the mean differences ±1.96 times the standard deviation of the difference . Only daIn order to investigate if the participants’ height, BMI, KOOS-pain or KOOS-ADL could influence the accuracy of the algorithm, correlations between the differences in detected lying time between Algorithm B, and the reference per subject and day were performed with Pearson correlation. IBM SPSS Statistics for Windows, version 21  was used.Manual inspections were performed to identify potential patterns in the errors. The 5% of the days in which lying down time differed the most, for Algorithm B in the validation dataset were inspected.In total there were 407 and 339 valid measured days in the development dataset and test dataset respectively. Averaged measured days per participant was 7.7 (SD ± 1.2) in the development dataset and 7.2 (SD ± 2.8) in the validation dataset.The mean lying down times across all measured days was in the development dataset 7.7 (SD ± 3.1) hours and 7.7 (SD ± 3.1) hours in the validation dataset according to the reference.Both Algorithm A and B overestimated the time of lying down compared to the reference method in both the development and validation datasets. The mean lying down time difference per day for Algorithm A compared to the reference was +25.5 min (SD ± 71), in the development dataset and +26.9 min (SD ± 81) in the validation dataset. The mean lying down time difference per day for Algorithm B compared to the reference method was +2.9 min (SD ± 70) in the development dataset and +6.5 min (SD ± 79) in the validation dataset. The time differences  and 95% limits of agreements are shown in The mean lying down time difference per participant  for Algorithm A compared to the reference was +32 min (SD ± 51) in the development dataset and +32.9 min (SD ± 51) in the validation dataset. The mean lying down time difference per participant for Algorithm B compared to the reference method was +5.4 min (SD ± 49) in the development dataset and +10.9 min (SD ± 58) in the validation dataset. The time differences per participant and 95% limits of agreements are shown in There were no correlations between the difference in detected time by Algorithm B and the reference method and the participants height Out of the 18 days, that corresponded to the days with the 5% highest difference between lying down time according to Algorithm B and the reference, 13 days were derived from unique participants. In 17 of these days the major differences in classifications occurred in daytime during leisure, defined from the participants work diary.In this study, we developed two non-proprietary algorithms (A and B) aimed at detecting lying down time from raw tri-axial accelerometric data of a single thigh worn accelerometer. Algorithm A was a direct implementation of an earlier presented method by Lyden et al. The difference between Algorithm A and the Lyden method is that the Lyden method is based on definitions of sedentary bouts from the proprietary ActivePAL software  and AlgoAlgorithm A overestimated lying down time with 27 min per day compared to the reference in free living conditions (which corresponded to 6% of the average lying time). The Lyden method has been validated before, using a similar, but not identical, reference method, finding it to underestimate lying down time by 25 min per day in free living conditions . These dAnother difference is that the lying down detection is based on sedentary bouts detected by the proprietary ActivePAL software in the Lyden method and the Acti4 algorithm in our developed algorithms. Because of the proprietary nature of ActivPAL software, a comparison of these methods at the algorithm-level is not possible. Both the ActiPAL software and Acti4 algorithm are shown to be robust and valid methods to detect sedentary bouts ,16,18,19The refined Algorithm B overestimated lying down time with +6.5 min per day compared with the reference, corresponding to 1.4% of the average lying time. Over 24 h this overestimation correspond to negligible 0.4% of the day. The precision was somewhat higher than observed for the Lyden method before .For Algorithm B, 5% of the observed days were days where the lying estimation differed more than 173 min from the reference, corresponding to 12% of the time per day. When we manually inspected these 5% worst classified days we found that for almost all of these days the misclassifications had occurred during leisure within daytime. One probable explanation is that the misclassification occurred when the participants were half lying down . This would then indicate an ambiguity in the definitions of is sitting and lying rather than a limitation in the lying down algorithm.In very large dataset, errors may be of minor importance as long as the error is random and not biased by any systematic error due to personal characteristics or health related issues of interest. Neither, height, BMI, knee pain nor reduced ability to perform activities in daily life due to knee pain, affected the estimations of lying time in the present material. This indicates that there are no systematic errors due to these health related variables.The studied population represented a variation in occupations and work-hours–including people who work day-shift, night-shift and also 24-h shift. It should be noted that if the algorithms should be used on other populations (ex. children) validity of the algorithms needs to be re-evaluated.One general limitation with algorithms to detect lying based on information from rotations of thigh worn accelerometer is that if the person never rotates their thigh during lying period the algorithms fail to identify these situations. In Algorithm B we therefore introduced a criteria that if someone has been sedentary, with none or very small, movements that sedentary bout should be identified as lying. However, the acceleration threshold that was introduced in the Algorithm B for this purpose is very low and might, in theory, be below the minimum acceleration that can be measured by some earlier version of common devices with 8-bit resolution or less.The present study offers non-proprietary algorithms for any accelerometer device that can output raw tri- axial accelerometer data. We have used the Axivity AX3 in this validation study but there are no reasons to believe that the results would be different with any other devices with similar acceleration range and resolution. This has been a demand from researchers and specifically from the ProPASS consortium that need such a tool for processing pooled raw thigh accelerometer data from cohorts around the world. Both Algorithm A and the improved Algorithm B be can be used together with the Acti4 algorithm when raw accelerometer data from different brands shall be pooled and processed in a harmonized way. Acti4 has already been empirically evaluated to be suitable for that purpose .Automatically identifying bedtime, without the use of a diary, would highly decrease the cost and burden with both collecting and processing accelerometer data in large observational studies. So far, algorithms that automatically identifies bedtime from thigh accelerometers has been based on evaluation of the length and time patterns of sedentary bouts over the day ,21. We bIn this paper two non-proprietary algorithms to identify lying down time from thigh worn accelerometers have been presented and validated. One of the algorithms is a direct implementation of the earlier presented method by Lyden et al., and the other is a refined version of the first.The study confirms the earlier presented accuracy of the method by Lyden, but now in a much larger dataset with a better reference. Both algorithms accurately identified 94% of the lying time. The improved version decreased bias in lying time detection from 27 min/day to 6.5 min/day compared the direct implementation of the earlier presented method by Lyden et al.The algorithms can be used to estimate lying time in studies with pooled raw data from thigh worn accelerometers of different accelerometer brands.


Therapeutic neovascularization can facilitate blood flow recovery in patients with ischemic cardiovascular disease, the leading cause of death worldwide. Neovascularization encompasses both angiogenesis, the sprouting of new capillaries from existing vessels, and arteriogenesis, the maturation of preexisting collateral arterioles into fully functional arteries. Both angiogenesis and arteriogenesis are highly multifactorial processes that require a multifactorial regulator to be stimulated simultaneously. MicroRNAs can regulate both angiogenesis and arteriogenesis due to their ability to modulate expression of many genes simultaneously. Recent studies have revealed that many microRNAs have variants with altered terminal sequences, known as isomiRs. Additionally, endogenous microRNAs have been identified that carry biochemically modified nucleotides, revealing a dynamic microRNA epitranscriptome. Both types of microRNA alterations were shown to be dynamically regulated in response to ischemia and are able to influence neovascularization by affecting the microRNA’s biogenesis, or even its silencing activity. Therefore, these novel regulatory layers influence microRNA functioning and could provide new opportunities to stimulate neovascularization. In this review we will highlight the formation and function of isomiRs and various forms of microRNA modifications, and discuss recent findings that demonstrate that both isomiRs and microRNA modifications directly affect neovascularization and vascular remodeling. Ischemic cardiovascular disease (CVD) is the leading cause of death in worldwide and was responsible for approximately 17.8 million deaths in 2017 ,4. ThereA potential strategy to treat patients with ischemia is to stimulate neovascularization, which is the body’s natural repair mechanism to restore blood flow to ischemic tissues. Postnatal neovascularization is comprised of angiogenesis, the sprouting of new capillaries from existing vessels, and arteriogenesis, the maturation of preexisting collateral arterioles into fully functional arteries. Both angiogenesis and arteriogenesis are highly multifactorial processes that involve multiple types of vascular and immune cells. In order to improve neovascularization as a whole, therapeutic strategies which simultaneously target both angiogenesis and arteriogenesis are needed ,6. During the last decade, microRNAs have emerged as multifactorial regulators of neovascularization ,8,9. MicAfter the occlusion of a large artery, blood flow to the downstream tissues is hampered, causing ischemia. Blood flow towards the ischemic tissue can be restored by a process called arteriogenesis. Arteriogenesis is the growth and maturation of collateral arteries from a pre-existing arteriole network, which connects all major arteries in the body . ArterioThe process of angiogenesis, on the other hand, is the sprouting of a new capillary from the existing vasculature in order to redistribute local blood flow towards ischemic areas. Unlike arteriogenesis, angiogenesis is driven by the hypoxia caused by ischemia, and revolves around resolving local ischemia rather than restoring arterial blood flow after the occlusion of a vessel. Angiogenesis is initiated when an angiogenic stimulus, produced by hypoxic cells, activates the vascular endothelial layer. Activated ECs will start to proliferate and migrate towards the stimulus, such as vascular endothelial growth factor (VEGF), resulting in a new capillary . Next toSince both angiogenesis and arteriogenesis are highly multifactorial processes, the simultaneous stimulation of both processes requires a multifactorial regulator, like microRNAs ,8.MicroRNAs are endogenous, small non-coding RNA molecules that inhibit translation of mRNAs. The microRNA’s target selection is predominantly determined by the microRNA’s ‘seed sequence’, nucleotides 2–8 at the 5′-end of a microRNA ,26, whicThe biogenesis of microRNAs starts with the transcription of the microRNA containing gene, yielding a primary microRNA (pri-miR) which then undergoes several steps of maturation to form the mature and functional microRNA  27]. Fi. Fi27]. MicroRNA biogenesis is strictly regulated, even at a microRNA-specific level, by numerous factors, including DNA methylation, activity modulation of key maturation proteins and many RNA-binding proteins ,31,32,33In 2007, the importance of microRNAs in neovascularization was demonstrated for the first time when several studies showed that Dicer-dependent microRNAs were required for angiogenesis ,36,37. SFor example, Bonauer et al. showed that miR-92a is highly expressed in human ECs and functions as negative regulator of angiogenesis . InhibitBoth miR-126-3p and -5p are also highly expressed in ECs where they promote angiogenesis by stimulating EC proliferation and VEGF signaling and regulating leukocyte adhesion ,47,48,49MiR-21-5p regulates proliferation and apoptosis of vascular wall smooth muscle cells ,59 and pAdditionally, it is noteworthy that several groups of genomically clustered microRNAs have been identified that are able to broadly regulate neovascularization in response to ischemia: Knockout of the miR-17/92 gene cluster (located on chromosome 14 in mice and on human chromosome 13) increased both angiogenesis and arteriogenesis ,62, whilTypically, microRNAs have been defined as a single sequence of RNA nucleotides, and are listed as such in the principle public microRNA database, miRbase . HoweverIsomiRs are microRNA sequence variants that have one or more nucleotides added or deleted at their 5′- and/or 3′-ends compared to the canonical microRNA sequence. O-methylation (2′OMe).RNA nucleotide modifications are biochemical modifications of the standard RNA nucleotides, which are performed by enzymes present in all living organisms. Recent studies have demonstrated that these RNA nucleotide modifications have a functional regulatory role and form what has been named the ‘epitranscriptome’ . While mBelow, we will discuss those studies that demonstrate that isomiRs and microRNA A-to-I editing and m6A can be actively regulated and play a directing role in neovascularization, as well as other modifications (including 2′OMe) that are likely to have a similar role.IsomiRs were discovered when microRNA sequencing studies observed that many microRNAs had sequence variants with one or more nucleotides added or deleted from the 5′- and/or 3′-ends compared to the ‘canonical’ microRNA sequence ,66. WhilIsomiRs are primarily generated by cleavage variations of either DROSHA or DICER during microRNA biogenesis  68,73].,73.68,73In general, 3′-isomiRs are more abundant than 5′-isomiRs, however, a number of microRNAs do have prevalent 5′-isomiRs ,76,77,78Due to the prevalence of isomiRs, most microRNAs that are known regulators of neovascularization have isomiRs. In fact, the microRNA loci with the most known isomiRs are miR-21-5p  and miR-IsomiR expression profiles can vary based on cell type and in response to biological stimuli, including stimuli associated with neovascularization ,76,78,92We have recently performed a focused study on the 5′-isomiR of miR-411-5p from the vasoactive 14q32 microRNA cluster in order to collect direct evidence that isomiRs are actively regulated during ischemia-induced neovascularization . We founA-to-I editing is the biochemical modification of adenosines into inosines by deamination. Unlike adenosine, inosine preferentially binds to cytidine and is therefore generally interpreted as guanosine by the cellular machinery . This foADARs specifically target double stranded RNA structures, including those found in pri-miRs . The ediMicroRNA editing is a widespread phenomenon which also affects many vasoactive microRNAs, as demonstrated recently in a study by Li et al. The authors mapped microRNA A-to-I editing at an unprecedented scale and found 2711 potential pri-miR editing sites within approximately 80% of all human pri-miRs . MicroRNIn the field of cancer research, several microRNA editing events were shown to have a functional effect on cell migration and/or proliferation ,120,121,We have recently demonstrated that microRNA-editing can also directly regulate neovascularization. We showed that A-to-I-editing of miR-487b-3p, another microRNA from the vasoactive 14q32 microRNA cluster, is increased in ischemic muscle tissues undergoing neovascularization after induction of hindlimb ischemia . MiR-4876-methyladenosine (m6A) is perhaps the most prevalent RNA nucleotide modification in eukaryotic cells and is present in more than 25% of human transcripts [The modification of adenosine to Nnscripts ,125. m6Anscripts , supportnscripts ,128. Strnscripts ,130. m6Anscripts ,132,133.nscripts ,135,136.nscripts ,138, mosnscripts ,140,141.While most m6A research has focused predominantly on mRNAs, several studies have demonstrated that m6A is important for microRNA biogenesis. Alarcon et al. demonstrated that pri-miRs are marked by the METTL3-dependent m6A . Pri-miRTwo recent studies have demonstrated the importance of m6A in cardiovascular homeostasis. Dorn et al. demonstrates that METTL3-dependant m6A helps modulate cardiac homeostasis and hypertrophic stress responses in mice . The ovePri-miR m6A marks were shown to be required for the appropriate processing of most pri-miRs to mature miRNAs, including vasoactive microRNAs ,143. FurCombined, the abundance of m6A, its importance in microRNA biogenesis and functioning, and the dysregulation of m6A during ischemia and cardiovascular disease, suggest that m6A of microRNAs could play an important role in ischemic cardiovascular disease and neovascularization.As mentioned above, numerous other RNA nucleotide modifications exist, however, their presence and function in small RNAs (16–28 nucleotides long), which consist mostly of microRNAs , remainsBelow, we will report the key findings of these studied RNA modifications and highlight which properties could potentially affect microRNA function. Furthermore, the discussed nucleotide modifications and their potential effects on microRNAs are summarized in Pseudouridine (Ψ) is one of the most abundant RNA modifications ,156. Ψ iO-methylation (2′OMe) can reside on all four ribonucleosides and is widely conserved [It is known that 2′-onserved ,162. Furonserved ,163,164.onserved ,161,165.onserved ,167. Intonserved . Howeveronserved ,170,171.Recent methodological advances have demonstrated that m1A is a transcriptome-wide modification ,173. SevWhile best known as a DNA modification in the epigenome, m5C can be installed on RNAs too by members from the NSUN family (nucleolar protein/sun RNA methyltransferase family) and by DNMT2 (DNA methyltransferase-2) and is therefore also part of the epitranscriptome ,180,181.In tRNAs and rRNAs, m2G is a relatively common RNA modification, however, which m2G writers are responsible in humans remains unclear ,188. IntThe epitranscriptome is dynamically regulated. This is abundantly clear for m6A modifications due to the discovery of both m6A writers and erasers ,148. NotSeveral studies have also indicated that certain RNA nucleotide modifications regulate each other. As mentioned previously, 2′OMe was found to protect adenosine residues from A-to-I editing ,166,167.During the past decade, both isomiRs and the epitranscriptome have emerged as novel and dynamic layers of regulation of gene expression. Both types of microRNA alterations have been shown to modulate key physiological responses, including neovascularization by affecting the microRNA’s biogenesis, stability and function. MicroRNAs have already been established as multifactorial regulators of both angiogenesis and arteriogenesis ,8,9, andThe first therapeutic small RNA (Patisiran) was granted FDA approval in 2018 and the first phase 2 clinical trials with microRNA-oriented RNA therapeutics are currently ongoing, highlighting that microRNA therapeutics are on their way to clinical practice . Over thAlternatively, tissue- and pathology-specific regulation of the microRNA alterations could potentially be used as a biomarker for cardiovascular disease, considering that isomiR expression profiles were found sufficient to distinguish between cancer subtypes .Finally, while the abundance and function of many nucleotide modifications have not been studied in microRNAs yet, it is likely that most, if not all, will prove clinically relevant. The unique properties of certain nucleotide modifications, like for example m6A, could be exploited to enhance the specificity of microRNA-therapeutics when targeting microRNAs carrying such modifications. It is important to note that further advances in technology and methodology are required to expand our knowledge of the microRNA epitranscriptome ,206. How


An outbreak of novel coronavirus-related pneumonia COVID-19, that was identified in December 2019, has expanded rapidly, with cases now confirmed in more than 211 countries or areas. This constant transmission of a novel coronavirus and its ability to spread from human to human have prompted scientists to develop new approaches for treatment of COVID-19. A recent study has shown that remdesivir and chloroquine effectively inhibit the replication and infection of severe acute respiratory syndrome coronavirus-2  in vitro. In the United States, one case of COVID-19 was successfully treated with compassionate use of remdesivir in January of 2020. In addition, a clinically proven protease inhibitor, camostat mesylate, has been demonstrated to inhibit Calu-3 infection with SARS-CoV-2 and prevent SARS-2-spike protein (S protein)-mediated entry into primary human lung cells. Here, we systemically discuss the pharmacological therapeutics targeting RNA-dependent RNA polymerase (RdRp), proteinase and S protein for treatment of SARS-CoV-2 infection. This review should shed light on the fundamental rationale behind inhibition of SARS-CoV-2 enzymes RdRp as new therapeutic approaches for management of patients with COVID-19. In addition, we will discuss the viability and challenges in targeting RdRp and proteinase, and application of natural product quinoline and its analog chloroquine for treatment of coronavirus infection. Finally, determining the structural-functional relationships of the S protein of SARS-CoV-2 will provide new insights into inhibition of interactions between S protein and angiotensin-converting enzyme 2 (ACE2) and enable us to develop novel therapeutic approaches for novel coronavirus SARS-CoV-2. Since its discovery in December 2019, the novel coronavirus-related pneumonia COVID-19 has continued to disseminate, with the current case count close to 1,214,466 cases, and more than 67,767 deaths according to the World Health Organization (WHO) as of 7 April 2020 . In addiRespiratory viral infection is a global health concern because the virus is contagious and may cause life-threatening respiratory infection and severe pneumonia in humans . CurrentThe coronavirus (CoV) family has a large homogeneous “spike protein”. This spike protein (S protein) is responsible for interacting with the host cells, such as the pulmonary and parabronchial epithelial cell, and helps the coronavirus get through the epithelial cell membrane . In addiAlthough genome replication and transcription are well known to be regulated by the viral RdRp, several host factors have been implicated in this process. RNA chaperones are usually nonspecific nucleic acid binding proteins, which have long disordered structures that promote RNA molecules to adjust conformational changes. For example, coronavirus nucleoproteins (N protein) have RNA chaperone activity and function as an RNA chaperone, which could help template switching ,33,34. IIn addition, several virus proteins and host factors are essential for the assembly and release of coronavirus. Homotypic interaction of M protein serves as the scaffold for the virus assembly and morphogenesis in the infected cells, specifically, both the interaction between the membrane (M) and S protein and the interaction between M and N protein promote the recruitment of structural components to the assembly location of host cells ,38. For There are two subunits of S protein, including the S1 subunit with a receptor-binding domain that engages with the host cell receptor ACE2, and the S2 subunit involved in regulating fusion between the viral and host cell membranes . The S pResearchers have posted the genome sequence information of SARS-CoV-2 isolated from pneumonia patients on the USCS Genome Browser ,46. ThisCurrently, there is no effective drug for treating COVID-19 although there was one case reported as having been treated successfully with compassionate use of remdsivir in the US. Recent studies have found that small molecules of remdesivir and chloroquine effectively suppress the replication of SARS-CoV-2 in vitro . AccordiRemdesivir  was initially developed to examine its effect on inhibition of Ebola virus (EBOV) replication . RdRp caRemdesivir has been recently developed as a potential antiviral drug candidate against a wide array of RNA virus such as SARS-CoV and MERS-CoV5 infections in both in vitro cell experiment and preclinical studies with mice and nonhuman primate (NHP) models . Recent Due to the COVID-19 pandemic and global health concern, it is urgent to develop effective broad-spectrum virus replication inhibitors to manage patients with COVID-19. Drug targets among coronaviruses include the main protease 3CL(pro) and papain-like protease(PLpro). These proteinase play essential roles in processing polyproteins and viral replication. The structures of the unliganded SARS-CoV-2 M(pro) and its complex with an alpha-ketoamide inhibitor have been recently revealed . CompariNo therapies specific or effective for human coronavirus SARS-CoV-2 have been approved by Food and Drug Administration (FDA). β-D-N4-hydroxycytidine  was initially synthesized as an orally bioavailable ribonucleoside analog with broad-spectrum antiviral activity against various RNA viruses such as Ebola . Recent Under the current outbreak of COVID-19, it is necessary to repurpose natural products to manage patients with COVID-19. Recent studies have shown that hydroxychloroquine can improve the outcomes of COVID-19 patients in small clinical trials although it should be used with caution on humans due to its toxicity. There are a large number of natural products with known safety profiles, such as isoflavones and artemisinin. Recent study has shown that several isoflavones and related flavonoid compounds have potent antiviral properties. In this regards, natural products that have been repurposed for broad-spectrum anti-viral therapy can offer safe and inexpensive platforms for discovery of efficient and novel agents for treatment of SARS-CoV-2. It will be of significance if these FDA-approved drugs could be repurposed for treatment of COVID-19. Recent studies found that naturally occurring flavonoids exhibit a broad-spectrum of antiviral effects against RNA virus such as polio-virus type 1, parainfluenza virus type 3 (Pf-3), and respiratory syncytial virus (RSV) by inhibiting their replication . ComputaRecent study revealed that the protein sequence of the drug binding pocket of the enzymes is highly homogeneous between SARS-CoV and SARS-CoV-2 . Much prRevealing the structural-functional relationships of the S protein of SARS-CoV-2 will provide new insights into inhibition of interactions between S protein and angiotensin-converting enzyme 2 (ACE2) to develop novel therapeutic approaches for coronavirus. More studies are focused on investigating the mechanism of coronavirus invasion into host cells. Similar to SARS–CoV and MERS-CoV, the novel coronavirus SARS-CoV-2 is armed with a large “spike protein”, which is used to interact with host cells and then gain entry through the cell membrane ,110. RecAngiotensin-converting enzyme 2 (ACE2) is required for coronavirus invasion into host cells. The viral spike glycoprotein utilizes ACE2 as a host protein receptor and mediates merging of the viral and host membranes. This allows viral entry into host epithelial cells and host species tropism . Given tThe outbreak of COVID-19, and previous devastating SARS and MERS-CoV, highlight the importance for developing effective approaches for treatment of human coronavirus infections (SARS coronavirus anti-infectives). This will decrease risk of disease dissemination, ameliorate disease progression, and bring down the need for intensive supportive care. Furthermore, treatments for moderate cases to decrease the time span of illness and infectivity may also be of significance for preventing COVID19 from becoming more wide-spread. Recent study demonstrated that there was no observed benefit with lopinavir–ritonavir treatment in hospitalized adult patients with severe COVID-19 . Future Because there is no specific antiviral treatment for COVID-19 infection, the widely investigated small molecule compound remdesvir could be a potential antiviral agent, based on pre-clinical studies in SARS-CoV and MERS-CoV infections. Remdesivir is a 1′-cyano-substituted adenosine nucleotide analogue prodrug that can be metabolized into its active form to exhibit broad-spectrum antiviral activity against coronavirus as shown in There are no therapeutics proven effective for the treatment of severe illness caused by SARS-CoV-2, so a phase 3 randomized, double-blind, placebo-controlled study was designed to examine the efficacy of remdsivir in adult patients with severe COVID-19 respiratory disease. The clinical trial is sponsored by Capital Medical University and China-Japan Friendship Hospital. The inclusion criteria include: (1) confirmation of COVID-19 by using laboratory RT-PCR; (2) less than 12 days since symptom; (3) lung involvement confirmed with CT chest imaging; (4) patients with a SaO2/SPO2 ≤ 94% in room air condition. The exclusion criteria include: severe liver disease and severe renal impairment; and patients with any experimental treatment for COVID-19 . 452 participants will be recruited and the clinical trial outcome will be released in May 2020. In adults presenting with hypoxic respiratory failure or acute respiratory distress syndrome (ARDS) from COVID-19, invasive mechanical ventilation, a conservative fluid strategy over a liberal fluid strategy, and intermittent boluses of neuromuscular blocking agents (NMBA) to facilitate protective lung ventilation are necessary for supporting treatment . For sevRecent studies have shown that SARS-CoV-2 receptor ACE2 and transmembrane protease serine 2 (TMPRSS2) are primarily expressed in bronchial transient secretory cells, which provides the rationale that supporting treatment on lung should be emphasized . The expSeveral host factors regulate the replication of coronavirus and induce dramatic changes in the host cellular structure and function, simultaneously. Induction of critical signaling proteins is crucial for the pathogenesis of CoV and is also involved in the activation of the innate immune response during CoV infection. Neutrophils are the first immune cells recruited to sites of viral infection. Recent studies demonstrate that excessive recruitment of neutrophils and formation of neutrophil extracellular traps results in acute lung injury of influenza pneumonitis . In addiCoronavirus infection promotes the activation of inflammasome in host cells and activates innate immunity ,139. In Recent study indicate that there is a substantial number of undocumented infections and this facilitates the rapid dissemination of SARS-CoV2 . These f