Thoroughbreds are some of the most famous racehorses worldwide and are currently animals of high economic value. To understand genomic variability in Thoroughbreds, we identified genome-wide insertions and deletions (INDELs) and obtained their allele frequencies in this study. INDELs were obtained from whole-genome sequencing data of 101 Thoroughbred racehorses by mapping sequence reads to the horse reference genome. By integrating individual data, 1,453,349 and 113,047 INDELs were identified in the autosomal (1–31) and X chromosomes, respectively, while 18 INDELs were identified on the mitochondrial genome, totaling 1,566,414 INDELs. Of those, 779,457 loci (49.8%) were novel INDELs, while 786,957 loci (50.2%) were already registered in Ensembl. The sizes of diallelic INDELs ranged from −286 to +476, and the majority, 717,736 (52.14%) and 220,672 (16.03%), were 1-bp and 2-bp variants, respectively. Numerous INDELs were found to have lower frequencies of alternative (Alt) alleles. Many rare variants with low Alt allele frequencies (<0.5%) were also detected. In addition, 5955 loci were genotyped as having a minor allele frequency of 0.5 and being heterogeneous genotypes in all the horses. While short-read sequencing and its mapping to reference genome is a simple way of detecting variants, fake variants may be detected. Therefore, our data help to identify true variants in Thoroughbred horses. The INDEL database we constructed will provide useful information for genetic studies and industrial applications in Thoroughbred horses, including a gene-editing test for gene-doping control and a parentage test using INDELs for horse registration and identification. Thoroughbreds are some of the most famous racehorses worldwide. They had a founding population  around the 18th century, and have been bred as a closed group for approximately 400 years , and theThe horse genome was sequenced and assembled in 2007 as EquCab2.0, in which 2.33-Gb draft sequences were published . By mappInsertions and deletions (INDELs) are variants consisting of different allele sizes (one or more nucleotides) in the genome. When INDELs occur in amino acid-coding regions, they are generally known to result in a loss of function. Although the existence of INDELs in coding regions is deleterious, their roles and functions in the population are not fully elucidated.https://ftp.ensembl.org/pub/release-108/variation/gvf/equus_caballus/, accessed on 2 February 2023). However, although several Thoroughbred horses were used for identification of genome-wide variants in the previous studies, the number, frequency, type, and size of INDELs in Thoroughbred horses have not been well elucidated.INDELs are highly abundant in human and animal genomes, and approximately 2.4 million and 2.1 million INDELs were identified from 88 and 534 horses, respectively . In the https://www.internationalstudbook.com/, accessed on 2 February 2023) and the International Federation of Horseracing Authorities  has prohibited the use of genetically engineered horses. In addition, horses born from genetically engineered animals are recognised as engineered horses. Editing (insertion and deletion) of coding genes causes loss of function. For instance, knocking out the myostatin gene, which is known as a negative regulator of muscle growth, may affect body composition and racing performance [Gene doping is a practice in horseracing that has been prohibited to maintain integrity . One styformance ,13.Recently, a gene-editing test was developed to detect artificially edited sequences using the clustered, regularly interspaced, short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) . This teMicrosatellites have been recently used for parentage testing in horse registration. While about 12–20 markers have been used for construction of a panel, because of their multiple alleles, they have the disadvantage of high mutation rates due to slippage errors. Therefore, construction of a new panel using SNVs or INDELs that have low mutation rates is expected. However, it is difficult to find many polymorphic SNPs and INDELs.In our previous study , we presFASTQ (DDBJ: SAMD00573909 to SAMD00574009) of WGS data (150-bp pair-end reads) from 101 Thoroughbred horses  were used in this study . The 101https://bio-bwa.sourceforge.net/, accessed on 2 February 2023), Picard (version 2.13.2) , GATK HaplotypeCaller (version 4.0.8.1) , and SnpEff (version v4_0) . Quality control using QCleaner eliminates sequence reads for the following criteria: reads with a low-quality base (<20 Phred score), a quality value < 20 in 80% of their nucleotides, sequences of over five unknown nucleotides, only <32 bp length sequences, and those that are not mate pairs.Short INDELs were identified using the RESEQ pipeline . The pipeline was constructed using QCleaner (Amelieff Co.), Burrows–Wheeler Aligner   using the BWA with default parameters to obtain a BAM file. Duplicate reads were removed using the Picard tool. GATK HaplotypeCaller detected insertions and deletions, and then filtered using the VariantFiltration program based on the following criteria: cluster WindowSize: 10; MQ0 ≥ 4 and ((MQ0/(1.0 × DP)) > 0.1), DP < 10, QUAL < 30.0, QUAL ≥ 30 and QUAL < 50, QD < 1.5, HRun > 5, SB > −0.1. Detected INDELs were annotated using SnpEff. Finally, all the annotated information was provided in variant call format (VCF) files. An Integrative Genomics Viewer (Broad Institute) was used for visualising mapping data using BAM files and variant data using VCF files.https://www.r-project.org/, accessed on 2 February 2023) and its package  were used for statistical analyses of identified INDELs.The chromosome, position, reference (Ref) allele, alternative (Alt) allele, gene name, HGVSp, annotation, and annotation impact were collected from the VCF files of 101 horses and then integrated by Vcf2sql (Amelieff Co.). The allele frequency of diallelic INDELs on autosomal (1 to 31) chromosomes was calculated from the integrated data by Vcf2sql. Statistical software R  using statistical software R.Identified INDELs were compared with those registered in Ensembl Release 108 . To map genome-wide INDELs and SNVs on the horse ideogram, R Ideogram  was used.SNV data identified in the 101 horses were obtained from our previous study  and the The mapped region (×10) and coverage of sequence reads were represented as the averages of 2,438,085,403 bp  and 36.8 coverages (29.5–54.2), respectively . These dBy integrating filtered INDELs, 1,453,349 and 113,047 loci were identified in the autosomal (1–31) and X chromosomes, respectively . The majOf the 1,566,414 INDELs, 786,957 loci (50.2%) were already registered in the Ensembl (Release 108), while 779,457 loci (49.8%) were novel INDELs . Since 3As described below see , INDELs As 1,566,396 INDELs were detected in the Thoroughbred population through chromosomes 1–31 and X, one INDEL was detected every 1538 bp  on average.In 1,376,674 diallelic INDELs in autosomal and X chromosomes, 742,723 (54.0%) and 633,951 (46.0%) loci were detected as deletions and insertions, respectively B. The siThe allelic frequency distributions of 1,274,708 diallelic INDELs identified on autosomes were investigated in 101 Thoroughbred racehorses. The horizontal axis of the figure indicates the number (frequency) of Ref alleles in the population , meaning that the right edge shows a MAF of 0.00495 (<0.5%) for Alt allele, the centre shows MAF of 0.5, and the left edge shows a MAF of 0 . Many INDELs were detected as having a smaller frequency of Alt alleles. In particular, many rare variants with a low Alt allele frequency (<1%) in the population were detected .In the 1,274,708 diallelic INDELs, 141,686 loci had a MAF of 0.00495 for Alt alleles  were mainly distributed in fewer Alt allele counts in 101 Thoroughbred racehorses ; this isThe genomic regions of the 5955 INDELs with a MAF of 0.5 and heterozygous genotype in all horses were visualized on horse chromosomes , blue. INotably, 1137 of these INDELs were in the pericentromeric region  of ECA29. A similar trend was observed in SNVs with a MAF of 0.5 and heterozygous genotype in all horses . In addition, the other INDELs and SNVs with a MAF of 0.5 and heterozygous genotypes in all horses were widely distributed over short lengths . INDELs ASIP) gene was detected with a reference allele frequency of 0.248, while a short interspersed nuclear element (SINE) insertion was not detected in the myostatin (MSTN) gene region, based on our detection criteria.The number of INDELs detected in the functional region of the genome was also investigated . AlthougANKRD9), Rho GTPase-activating protein 45 (ARHGAP45), cache domain containing 1 (CACHD1), cardiotrophin 1 (CTF1), EMAP-like 4 (EML4), fibrosin-like 1 (FBRSL1), HDGF-like 2 (HDGFL2), junctophilin 2 (JPH2), mitochondrial ribosomal protein L15 (MRPL15), nuclear receptor corepressor 2 (NCOR2), SH3 and multiple ankyrin repeat domains 3 (SHANK3), serine/threonine kinase 11 (STK11), trinucleotide repeat-containing 18 (TNRC18), UPF1 RNA helicase and ATPase (UPF1), zinc finger protein 212 (ZNF212), zinc finger protein 282 (ZNF282), zinc finger protein 516 (ZNF516), and zinc finger protein 853 (ZNF853).Notably, 29 INDELs located in exons of 23 genes all had alternative alleles, causing frameshifts in the 101 horses . The disIn this study, 1,566,414 loci were identified as INDELs on autosomal and X chromosomes and mitochondrial genomes. It was confirmed that 779,457 loci (49.8%) were novel INDELs by comparing them with the Ensembl (Release 108). Our current and previous studies  identifiA recent study estimated a rate of 2.94 INDELs (1–20 bp) and 0.16 structural variants (>20 bp) per generation based on the WGS of 250 human families . In thisInterestingly, 5955 loci were genotyped with a MAF of 0.5 and heterogeneous genotypes in all the horses. Such genotype distribution is unlikely in a randomly collected population. Because these variants existed in continuous positions with high mapping coverage, fake variants may be detected by mapping of similar sequence reads in multiple regions. While short-read sequencing and its mapping to the reference genome is an easy way to detect variants, fake variants may be detected. Therefore, our data help to identify true variants in Thoroughbreds. As 2756 loci were registered in Ensembl (Release 108), further validation, such as designing their primers and probes, may need to use them as genetic markers.High INDEL density was observed on chromosomes 12 and 20. Previous studies observed a similar trend on the same chromosomes for SNVs and nonsynonymous substitutions ,16. AlthASIP gene was observed, and its genotypes were associated with coat colour [MSTN gene is associated with optimum race distance and muscle mass in Thoroughbred racehorses [MSTN had partial sequences soft-clipped by confirmation using the Integrative Genomics Viewer. Although INDELs over 400 bp were detected in this study; the SINEs in the MSTN region were not recognised as INDELs by our detection method, because similar sequences were widely distributed in the horse genome.INDELs in coding regions may be frameshift or non-frameshift variants. In this study, the same variant (C/CAGCAGAAAAGA) as a non-frameshift INDEL in the t colour ,22. In acehorses ,24. HoweFrameshift INDELs are generally deleterious and contribute to disease susceptibility, as evidenced by human genomics research that has identified many INDELs related to genetic diseases including cancer . NotablyIn human forensic sciences, INDELs have been noted as markers for individual identification testing ,27 becauGene doping in the horseracing industry is defined as administration of exogenous genes or therapeutic oligonucleotides to postnatal animals, and the creation of genetically modified animals. These have been prohibited by the IFHA and the ISBC. While the former can be target-specifically detected by quantitative PCR using a hydrolysis probe or sequencing using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry ,34, the The INDEL database we have constructed will provide useful information for genetic studies and industrial applications in Thoroughbred horses, including a gene-editing test for gene-doping control and a parentage test using INDELs for horse registration and identification.


COLQ) is the second-most common cause of CMS. However, data on pharmacological treatments are limited. Congenital myasthenic syndromes (CMS) refer to a series of inherited disorders caused by defects in various proteins. Mutation in the collagen-like tail subunit of asymmetric acetylcholinesterase  treated with β-adrenergic agonists showed positive effects. In addition, AChEIs should be avoided in CMS patients with COLQ mutations, as 90.5% (105/116) of patients treated with AChEIs showed either no or negative effects. A total of 42 studies including 164 patients with CMS due to 72 different COLQ mutations. (2) AChEIs should be avoided in patients with CMS with COLQ mutations. (1) β-adrenergic agonist therapy is the first pharmacological strategy for treating CMS with  N2-ACh receptor cation channels on the postjunctional membrane are activated, enabling skeletal muscle fiber contraction. ACh is cleared within several milliseconds of release, mainly by acetylcholinesterase (AChE) located on the postjunctional membrane . The collagen-like tail subunit of asymmetric AChE (COLQ) is crucial for anchoring AChE and the postjunctional membrane [The neuromuscular junction (NMJ) is a type of synaptic connection bridging motoneurons and skeletal muscle fibers that converts the electrical impulses of motoneurons into action potentials of muscle fibers . When elCongenital myasthenic syndromes (CMS) include a series of inherited disorders caused by defects in different proteins -5. CMS mCOLQ gene located on the short arm of chromosome 3 p24.2 is one of the most frequent causes of CMS [COLQ is approximately 0.25 per million total population [The prevalence of CMS in the total population is approximately 0.18 per 100,000 in Spain  and souts of CMS  and is ts of CMS . The prepulation .COLQ mutations.Significant research attention has been dedicated to developing CMS treatments. AChE inhibitors (AChEIs), such as pyridostigmine, enhance the effect of ACh quantum release by inhibiting decomposition . 3,4-dia1). The PRAD connects to the globular catalytic subunits, and the C-terminal region helps grapple to the basal lamina and initiates triple helix assembly while the collagen-like tail connects the two [2-ACh receptor cation channels and gene expression of synapses through its interaction with muscle-specific receptor tyrosine kinase (MuSK), thus demonstrating a synaptic regulation function [COLQ attaches the globular catalytic subunits of AChE to the synaptic basal lamina, thus anchoring and accumulating AChE in the extracellular matrix . AChE co the two , 17, 18. the two . In addifunction .COLQ have been identified thus far, and their locations and effects are listed in Fig. (2) and Supplementary Table S1. Frameshift or nonsense mutations can truncate the protein distal to PRAD [COLQ may lead to a deficiency in endplate AChE and thus insufficient ACh elimination ability. ACh in the synaptic space will only be eliminated by diffusion or by other esterases, such as butyrylcholinesterase, instead of the decomposition effect of endplate AChE. As a result, the interaction between ACh and AChR is more frequent and long-lasting [More than 70 mutations in  to PRAD , 22, whe to PRAD . These m-lasting . Excessi-lasting , 26.COLQ mutations were reported. One is the “classical” phenotype, which onsets at birth, induces moderate to very severe weakness, and does not respond to AChEIs [COLQ mutations have been identified, two more phenotypes have been recognized: later onset with graver symptoms or earlier onset with milder symptoms [Initially, two phenotypes of CMS with o AChEIs -29. The o AChEIs , 29, 30.symptoms .COLQ mutations.Different therapies have been applied for treating CMS in clinical settings. However, due to the interactions of various mechanisms, the same drug may have varying or even opposite effects on different CMS subtypes, which makes it necessary to analyze pharmacological treatments based on disease subtypes. Here, we reviewed related reports and attempted to determine the most appropriate pharmacological strategy for CMS caused by 22.1COLQ mutations,” “COLQ,” or “congenital myasthenic syndrome, COLQ”. The most recent search was performed on July 22, 2022. In addition, a manual search was performed to identify references in the obtained studies to identify other possible studies. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) [3). No randomized clinical trials (RCTs) were identified, and only observational case reports or studies were included in our analysis. The studies were read thoroughly to assess their eligibility for inclusion in the meta-analysis.The literature search was restricted to articles published in English. The search strategy was modified from that reported in a previous study -34. We s(PRISMA)  chart fo2.2COLQ mutations with no restriction regarding their age, sex, ethnicity, and treatment; and (3) genetic tests confirmed the COLQ mutation regardless of the details of the mutations. The exclusion criteria were as follows: (1) articles not reporting pharmacological treatment(s) and (2) articles that did not mention the treatment effect of a pharmacological therapy.Case reports and studies were included if they fulfilled the following criteria: (1) English-language articles; (2) patients with 2.3The following data were extracted directly from the articles: clinical information, gene mutations, pharmacological treatments, and follow-up data. Clinical data including sex, age at symptom onset, and age at the time of the case study were reported. If more than one patient had been reported in different studies or by the same group, the participants in the studies were compared. Data that appeared more than once were excluded from the analysis.2.4et al. [S2). The quality assessment included eight leading exploratory questions with a binary response (yes/no) to determine whether the items suggested the presence of bias.The quality of individual studies was assessed using the recently published “Tool for evaluating the methodological quality of case reports and case series” proposed by Murad et al.  based on2.5p < 0.05.Data analysis was performed using Statistical Package for Social Sciences  version 25. Statistical differences were estimated using the Kruskal-Wallis test or one-way analysis of variance followed by the Bonferroni post-hoc test. The analysis of the relationship between sex and treatment effect was estimated using the Mann-Whitney test (two groups). Simple linear regression was used to analyze the relationship between age of onset and treatment effect. Statistical significance was set at 33.1COLQ mutations [S3, including geographical settings, mutation, onset age, related risk factors, treatment, and effects. Six different pharmacological strategies were used, including AChE inhibitors (AChEIs), a type of drug that inhibits ACh hydrolysis; β-adrenergic receptor agonists (BAs); 3,4-DAP, a drug that increases ACh release from the nerve terminal by blocking voltage-gated potassium channels (VGKCs); fluoxetine (FLX); glucocorticosteroids (GCs); and other drugs (Table 1). Some patients underwent more than one kind of treatment. Because the effects of different strategies are described as negative, partial, positive, or remarkable, we categorized the treatment effect into four types: (-) no effect, negative or ambiguous response; (+) partial, incomplete, moderate, modest/mild effect, or initial positive response followed by ineffectiveness; (++) beneficial, positive, or clear effect; (+++) remarkable, dramatic, or satisfying effect.After searching, 42 studies meeting our inclusion and exclusion criteria were selected, including 164 patients with CMS due to 72 different utations , 37-61. 3.2COLQ mutations and 6 pharmacological treatment strategies. Additionally, four different treatment strategies were categorized as “other” because of their limited number. Among the five different strategies, β-adrenergic receptor agonists showed better effects than 3,4-DAP, AChEIs, and glucocorticosteroids .The selected studies reported on a total of 164 CMS patients diagnosed with S3. The β-adrenergic receptor agonists showed the best treatment effect, benefiting 74 of the 75 patients treated (98.7%) . In addition, AChEIs showed no effect on 105 of the 116 patients treated (90.5%) .Four different treatment strategies were categorized as “other” because of their limited number. Therefore, the group of others was not included in the statistical analysis. Their treatment effects are shown in Supplementary Table 3.3COLQ mutations were first divided into two phenotypes: the “classical” phenotype with early onset at birth and severe symptoms [CMS patients with symptoms -29 and asymptoms , 29, 30.symptoms . For our5).Data were evaluated according to phenotype and treatment effect where possible. Among the patients whose phenotypes were reported, 21 had the “classical” phenotype, 22 had the “late and mild” phenotype, 8 had the “late and severe” phenotype, and 7 had the “early and mild” phenotype. No significant difference in the treatment effect was found between the different phenotypes . For treating patients with the “late and mild” phenotype, β-adrenergic receptor agonists showed better effects than AChEIs and DAP, and FLX showed better effects than AChEIs and DAP . For treating patients with the “late and severe” phenotype, only β-adrenergic receptor agonists, AChEIs, and DAP were used, and β-adrenergic receptor agonists showed the best effect . Some patients underwent more than one kind of treatment. No significant difference was found in the treatment of patients with “early and mild” phenotypes owing to the limited data size.β-adrenergic receptor agonists showed a better effect than AChEI and DAP for treating patients with the “classical” phenotype. DAP showed a better effect than AChEIs .Among the enrolled patients, 123 were referred for sex and clear treatment effects . Male patients showed better treatment effects than female patients , the “early infancy” group , and the “later” group . Then, simple linear regression was used for analysis. Onset age was not found to have an influence on treatment effect in this study .Among all enrolled patients, clear information regarding onset age and treatment effect was provided for 122 patients. Firstly, we classified the patients into three groups according to their age when the symptoms onset: the “birth” group .A deficiency of endplate AChE caused by nset age , 27-30. 1, COLQ mutations can impair trimeric organization (MuSK-AChE-COLQ trimeric organization), truncate collagen domains, or influence the interaction between COLQ and MuSK. These factors can cause endplate AChE deficiency, which prolongs the half-life of ACh and leads to NMT blockade due to receptor desensitization [NMJs are highly specialized chemical synapses between motor neurons and muscle fibers that transform electrical impulses into ACh release. ACh binds to AChR at the endplate, causing the opening of sodium channels and muscle contraction . Typicaltization .SLC5A7, CHAT, SLC18A3, VAMP1, SYB1, SYT2, and MUNC13-1; synaptic mutations, such as LAMA5; and postsynaptic mutations, such as CHRNA1, FCCMS, MYO9A, PREPL, PLEC1, and CHRND. In addition, AChEIs can help some patients with defects in glycosylation, which is a common post-translational modification that participates in the formation, stabilization, and function of the NMJ [GFPT1, GMPPB, ALG2, ALG14, and DPAGT1, which hampers the solubility, folding, stability, assembly, and intracellular transport of peptides and induces NMT defects [COLQ mutations, AChEIs increase excess ACh. As a result, AChEIs do not have a significant positive effect and may have negative effects. Therefore, these drugs should be avoided once patients with CMS have been verified to have COLQ mutations.AChEIs, including pyridostigmine and neostigmine, inhibit ACh from breaking down to choline and acetate, thus prolonging the level and duration of action of the neurotransmitter ACh. As a frequently used drug for treating CMS, AChEIs benefit CMS patients with pre-synaptic mutations, such as  the NMJ , 64. Gly defects , 66. HowSNAP25, SYT2, MUNC13-1, LAMA5, COL13A1, CHRND, RAPSN, SLC25A1, or DPAGT1 mutations [4), some patients with COLQ mutations benefit from 3,4-DAP, which is contrary to the opinion that 3,4-DAP may worsen CMS in patients with COLQ mutations. However, the mechanism remains unclear. One possible explanation reported in previous research is an increase in muscle force caused by 3,4-DAP blocking delayed rectifier potassium channels, which prolongs the action potential by slowing repolarization and increasing intracellular Ca2+ levels [2+ directly enhances muscular strength without affecting the NMJ. Further studies are required to determine the underlying mechanisms.3,4-DAP blocks the presynaptic voltage-gated potassium channel complex, which prolongs nerve terminal depolarization and promotes ACh release from the nerve terminal into the synaptic cleft . It has utations . Interes+ levels . IncreasFLX is a selective serotonin reuptake inhibitor used to treat depression . It is aCOLQ mutations. These drugs are widely used in clinical practice. The beneficial effect of ephedrine in patients with myasthenia gravis has been recognized since the 1930s [DOK7 mutations. DOK7 is a muscle-intrinsic activator of MuSK. Mutations in DOK7 may lead to DOK7 deficiency, thus leading to NMJ defects [SLC5A7, LAMB2, COL13A1, CHRNE, DOK7, and MUSK [SYT2 [TOR1AIP1 [CHD8 [In our study, β-adrenergic receptor agonists performed best among all pharmacological methods for treating patients with CMS due to he 1930s . Physicihe 1930s , 72, buthe 1930s . Ephedri defects , 75. Altand MUSK , 66, 76,SK [SYT2 , TOR1AIPTOR1AIP1 , and CHDP1 [CHD8  mutationDOK7, thus stabilizing AChR clusters [Colq mutation showed gradual improvement in muscle strength and several postsynaptic morphological defects after salbutamol treatment, suggesting that β-adrenergic receptor agonists affect the endplate [Researchers have proved that salbutamol directly affects proteins located at the endplate and increases the number of AChR clusters in the C2C12 cell model of clusters . In a zeclusters . The mouendplate . One posendplate . Moreoveendplate . Chronicendplate , 86.In evidence-based medicine, systematic reviews of multiple RCTs provide the most reliable evidence to inform clinical practice. However, for disorders such as CMS with a notably low incidence rate and high subtype variation, carrying out RCTs is almost impossible, and there are no RCT on CMS thus far. Therefore, a meta-analysis of clinical knowledge regarding the efficacy of pharmacological treatments and other types of interventions based on observational studies and case reports is necessary. However, there were some limitations to our meta-analysis. (1) There might have been publication bias due to the possibility that only positive results were published. (2) Most reports that we reviewed lacked a quantitative assessment of treatment effectiveness. Instead, they were described qualitatively using words such as negative, partial, and positive. This may also lead to bias and flatten variations in reported treatment outcomes. (3) Insufficient information was provided regarding drug doses, so it was difficult to discern dose-effect relationships for the investigated treatments.COLQ mutations. We recommend that once a CMS patient is identified with a COLQ mutation, the clinician should stop administering AChEIs and provide β-adrenergic receptor agonists, such as salbutamol and ephedrine, for improved prognosis.Our meta-analysis provided evidence that β-adrenergic receptor agonists, including salbutamol and ephedrine, can be used as first-line pharmacological treatments for CMS patients with


Mitochondrial dysfunction, especially tricarboxylic acid (TCA) cycle arrest, is strongly associated with Alzheimer’s disease (AD), however, its systemic alterations in the central and peripheral of AD patients are not well defined. Here, we performed an integrated analysis of AD brain and peripheral blood cells transcriptomics to reveal the expression levels of nine TCA cycle enzymes involving 35 genes. The results showed that TCA cycle related genes were consistently down-regulated in the AD brain, whereas 11 genes were increased and 16 genes were decreased in the peripheral system. Pearson analysis of the TCA cycle genes with Aβ, Tau and mini-mental state examination (MMSE) revealed several significant correlated genes, including pyruvate dehydrogenase complex subunit (PDHB), isocitrate dehydrogenase subunits , 2-oxoglutarate dehydrogenase complex subunit (DLD), succinyl-CoA synthetase subunit (SUCLA2), malate dehydrogenase subunit (MDH1). In addition, SUCLA2, MDH1, and PDHB were also uniformly down-regulated in peripheral blood cells, suggesting that they may be candidate biomarkers for the early diagnosis of AD. Taken together, TCA cycle enzymes were systemically altered in AD progression, PDHB, SUCLA2, and MDH1 may be potential diagnostic and therapeutic targets. Alzheimer’s disease (AD) is the most common neurodegenerative disease, and its main clinical manifestations are cognitive impairment and progressive decline of memory ability . The maiNeurons have a high energy demand and are mainly operated by mitochondria, whose defects lead to synaptic dysfunction, neuroinflammation and neuronal death . Electro1 and peripheral blood cells  Gene Expression Comprehensive Database (GEO) . Specifi40 and phosphorylated Tau semi-quantitative analysis, of which 56 brain tissue samples with Aβ pathological grading, and 14 brain tissue samples with Tau pathological grading. In addition, to confirm the association between brain biomarkers and cognitive decline, we performed correlation analysis between mRNA expression of related genes and MMSE scores in AD patients . Graphpad prism software  was used for Pearson correlation analysis between gene mRNA expression and Aβ/Tau pathology, MMSE scores.To confirm the association between brain biomarkers and Aβ/Tau pathology, we performed correlation analysis between the mRNA expression of related genes and Aβ/Tau pathology scores in AD mouse brain. Specifically, immunofluorescence was applied to perform Aβ3 which included linear regression analysis adjusted for age and gender, and P-values < 0.05 was considered to be significant.Differentially expressed genes (DEGs) were identified using limma package in R,The TCA cycle consists 35 genes, forming 9 key enzyme complexes that collectively drive the conversion of pyruvate to acetyl-CoA and complete oxidation. Brain data were obtained from the Alzdata database (see text footnote 1) , for a tPyruvate dehydrogenase complex (PDHC) mainly promotes the conversion of pyruvate to acetyl-CoA, which then enters the tricarboxylic acid cycle . PyruvatAltogether, TCA cycle related genes were consistently down-regulated in the AD brain, PDHB, ACLY, IDH3B, IDH3G, DLD, SUCLA2, and MDH1 were significantly decreased in the four brain regions EC, HIP, TC and FC, which may be potential brain biomarkers .r| = 0.189–0.439, p < 0.05; r| = 0.653–0.847, p < 0.05; To determine the association between brain biomarkers and AD progression, we analyzed the expression of brain biomarkers in AD mice and correlated them with Aβ and Tau pathology . Pearsonr = 0.365–0.441, p < 0.05; To confirm the association between brain biomarkers and cognitive decline, we performed correlation analysis between mRNA expression of related genes and MMSE scores in AD patients  and GSE63061 , which were two large European blood cells RNA expression datasets. The expression of TCA cycle genes in the peripheral blood of AD patients were shown in The mRNA expression of TCA cycle genes in AD patients was systematically revealed by brain and peripheral blood cells transcriptome data, and combined with MMSE, Aβ/Tau correlation analysis to confirm that SUCLA2, MDH1, and PDHB genes may be candidate biomarkers that can effectively track the pathological and clinical manifestations of AD patients . By analEnergy metabolism disorder is one of the earliest symptoms in AD and MCI, and it may precede the appearance of clinical cognitive impairment . PositroPrevious studies have shown that TCA cycle related genes were significantly decreased in AD brain . GlucoseAlpha-ketoglutarate dehydrogenase complex (αKGDHc), a key rate-limiting enzyme in the TCA cycle, can convert alpha-ketoglutarate, coenzyme A and NAD + to succinyl-coenzyme A, NADH and CO2, and its activity is significantly reduced in AD . CholineSuccinyl-CoA ligase (SCL) catalyzes substrate level phosphorylation, and the related subunits are significantly reduced in AD, especially SUCLA2 . SuccinyMalate dehydrogenase is involved in two major metabolic processes, namely malate-aspartate shuttle and tricarboxylic acid cycle, which are essential for mitochondrial respiration and adenosine 5’-triphosphate (ATP) production . PreviouIn sum, we systematically analyzed the expression of TCA cycle related genes in the central and peripheral of AD, revealing molecules closely related to AD pathological development and clinical cognitive decline, providing valuable resources for further exploring the specific mechanisms between TCA cycle and AD progression. PDHB, SUCLA2, and MDH1 may be potential diagnostic and therapeutic targets.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.HY and DJ: experimental design and manuscript-writing. HY, DJ, and FW: experimental methods and data analysis. All authors contributed to the article and approved the submitted version.


Non-coding RNAs (ncRNAs) are a type of functional RNA molecules that are not translated into a protein. Increasing studies demonstrate that ncRNAs have a diversity of regulatory roles in ferroptosis, thus influencing the progression of cancers. In this study, we review the fundamental mechanisms and regulation network of ncRNAs on ferroptosis in various tumors, aiming to provide a systematic understanding of recently emerging non-coding RNAs and ferroptosis.As a special pattern of programmed cell death, ferroptosis is reported to participate in several processes of tumor progression, including regulating proliferation, suppressing apoptotic pathways, increasing metastasis, and acquiring drug resistance. The marked features of ferroptosis are an abnormal intracellular iron metabolism and lipid peroxidation that are pluralistically modulated by ferroptosis-related molecules and signals, such as iron metabolism, lipid peroxidation, system Xc Cell death has been reported to be closely related to cell growth and development, tissue repair, and various physiological processes . Cell de− protects the cell from ferroptosis via importing cystine and increasing the biosynthesis of GSH  . The accs of GSH . GPX4 ins of GSH . Nrf2 cas of GSH . Reactivs of GSH . Ferropts of GSH , ischemis of GSH , and cans of GSH . In reces of GSH ; Table 1via the Fenton reaction, inducing ferroptosis , and many instable molecules that require hydrogen atoms can combine with allylic hydrogen atoms in polyunsaturated fatty acids (PUFAs), thus forming lipid radicals  and solute carrier family 3 member 2  and possesses high selectivity for cystine and glutamate transport, especially for the anionic form. System Xc− can absorb one cystine and export one glutamate simultaneously  stress  increases system Xc− expression and glutamate exportation to inhibit ferroptosis  manner  “reader” YT521-B homology containing 2 (YTHDC2) repressed the system Xc− regulatory subunit SLC3A2 via decreasing the stability of homeobox A13 (HOXA13), which elevated SLC3A2 expression  stress . The exproptosis . Nrf2 siortation . In Zhan) manner . Moreovepression . In summ− dependent on cystine can increase GPX4 synthesis through the Rag-mTORC1-4EBP pathway, thus inhibiting ferroptosis , hydroxyl radicals (•OH), NO, and RO•  can bind to both Nrf2 and the actin cytoskeleton to retain Nrf2 in the cytoplasm , and Nrfroptosis . Moreovegenase 1 . Nrf2 cagenase 1 . TherefoP53, known as a tumor suppressor, is also associated with ferroptosis. Interestingly, the role of p53 on ferroptosis is still controversial. Jiang, L. et al. reported that p53 can increase susceptibility to ferroptosis by inhibiting SLC7A11 expression and abating cystine uptake . On the Different from messager RNA, non-coding RNAs (ncRNAs) cannot be directly translated into bioactive proteins but can regulate the transcription and translation of target transcripts. ncRNAs include microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), and chimeric RNA, but different ncRNAs have distinguishing regulatory mechanisms in ferroptosis.via binding to 3’-untranslated regions (3’UTR). MiR-27a-3p  are a type of RNAs, generally defined as transcripts of more than 200 nucleotides that are not translated into protein. LncRNAs regulate gene expression in a variety of ways at the epigenetic, chromatin remodeling, transcriptional, and translational levels. As for ferroptosis, lncRNAs usually interact with miRNAs in a competitive manner, leading to the inhibition of miRNAs. For instance, lncRNA H19 upregulates FTH1 by interacting with miR-19b-3p . LncRNA  miR-339 , and LIN miR-339 . In addi miR-339 . LncRNA  miR-339 . Chao Man of p53 . In shorCircular RNA (or circRNA) is a type of single-stranded RNA that, unlike linear RNA, forms a covalently closed continuous loop without 5’ caps or 3’ tails. Most current studies demonstrate that circRNAs function as a sponge for miRNAs, thus regulating ferroptosis-related genes. For example, circPVT1 sponges miR-30a-5p to target FZD3 ; circBCA6-methyladenosine (m6A) was reported to be involved with ncRNA-mediated ferroptosis. Methyltransferase METTL3 can be regulated by miR-4443, and METTL3 reduced ferroptosis suppressor protein 1 (FSP1) via m6A modification , a miR-19b-3p endogenous target, was activated to reduce intracellular iron followed by the repression of ferroptosis . LncRNA In lung adenocarcinoma (LUAD), many bioinformatics analyses predicted a large number of ferroptosis-related lncRNAs related to survival . Ferroptvia the miR-30a-5p/FZD3 axis. They found that knocking down circPVT1 increased ferroptosis through downregulating p-β-catenin, GPX4, and SLC7A11, while the inhibition of miR-30a-5p and overexpression of FZD3 reversed the phenotype through their upregulation  of CBS mRNA. Under decreased CBS levels, the methylation of the ACSL4 protein is reduced, leading to protein polyubiquitination and the degradation of ACSL4, which, in turn, decreases the proferroptosis phosphatidylethanolamine (PE)  . Huang, ine (PE) . Moreoveine (PE)   . Overallvia reducing transferrin, and while transferrin overexpression blocked miR-545-induced changes in ROS, MDA, and Fe2+ levels in HT-29 and HCT-116 cells, thereby inducing CRC ferroptosis  of Keap1, inhibiting Nrf2 ubiquitination and increasing Nrf2 protein stability, thus inhibiting ferroptosis and promoting chemoresistance . Yajun LPancreatic cancer is extremely malignant. There were 496,000 new cases and 466,000 deaths due to its badly poor prognosis . Xin QiuLiver cancer is the sixth most common cancer worldwide, with approximately 906,000 new cases and 830,000 deaths in 2020 . Lu, Y. via the interaction of the RNA-DNA sequence to increase the transcripts of GPX4, and while LINC01134 silencing leads to oxaliplatin sensitivity by inhibiting the total ROS, lipid ROS, and MDA levels and decreasing the GSH/GSSG ratio  by binding to the N-terminus to block the phosphorylation of HSPB1 at the Ser-15 site, thus activating the P38 MAPK pathway  new ovarian cancer cases and 207,000 deaths  globally in 2020 . Recent via sponging miR-4291 to upregulate ACSL4, thus promoting ferroptosis. Interestingly, they also found that I3RC (reverse complementary sequence in intron 3) can accelerate the circularization of circLMO1, and RNA-binding protein DExH-Box Helicase 9 (DHX9) negatively regulates circLMO1 (via the competing endogenous RNAs (ceRNAs) mechanism, and circEPSTI1 silence promotes the ferroptosis process mediated by SLC7A11  occurred worldwide in 2020 . LINC006via binding to the seed region of their 3’ UTR sequence. FANCD2 and CD44 silencing can increase intracellular iron accumulation and lipid ROS     . Taken t− subunit SLC1A5, which provides us with another dimension of thought concerning ferroptosis regulation and a new perspective on melanoma inhibition and chemotherapy sensitivity through ferroptosis.More than 324,000 new diagnosed melanoma cases and 57,000 deaths occurred worldwide in 2020 . Zhang, −, GPX4, ACSL4, Keap1/Nrf2 signals, and p53. Nevertheless, there are no ferroptosis-remarked ncRNAs yet. Some ncRNAs regulate ferroptosis in different malignant cancers; for example, miR-324-3p mediates ferroptosis in RCC and breast cancer simultaneously (As a distinctive PCD regulating cancer development, ferroptosis is characterized by an abnormal iron metabolism and lipid peroxidation. A large majority of studies suggest that ferroptosis is closely associated with malignant cancer development. Many familiar ferroptosis-remarked molecules have been confirmed, including system Xcaneously , and miRaneously . HoweverA large number of studies have shown that ncRNAs can affect the progression of liver cancer by mediating various ferroptosis-related classical molecules or signals, such as SLC7A11, GPX4, and ACSL4 . HoweverThere are few reports on the upstream regulation of ferroptosis-related ncRNAs until now. We believe the conventional transcription or splicing factors can potentially reveal a hidden repertoire as a mechanism of exploration. For example, HIF-1α can bind to the endogenous HRE site in the lncRNA PMAN promoter to increase lncRNA PMAN . ETS1 cavia the intervention of the expression of ncRNAs, thus affecting cancer cell proliferation, invasion, migration, and chemoresistance. However, there are possibly many limitations in the application of potential ncRNA-targeted therapeutic methods. First, the ncRNA-mediated ferroptosis process is finite in the regulation of tumorigenesis. Second, the individual differences in ncRNA gene expression and the individual sensibility to intervention compounds are also two unpredictable directions. Third, it is complicated for ncRNAs to regulate ferroptosis between cancer progression and chemoresistance. Consequently, more studies need to be performed to explore clinical compounds targeting ferroptosis-related ncRNAs.Targeting ncRNA-mediated ferroptosis may provide a prognosis reference and novel therapeutic methods to repress the development of malignant tumors. On one hand, an increasing number of studies suggest that ncRNAs can function as a biomarker to predict tumor progression and clinical prognosis. On the other hand, according to studies on ncRNA-regulated ferroptosis, the ferroptosis process can be altered


Pleomorphic liposarcoma is a rare malignant adipocytic tumor showing undifferentiated pleomorphic sarcoma morphology with various degrees of epithelioid features. It is sometimes difficult to distinguish from carcinoma metastasis. Immunohistochemical panel is very important for differential diagnosis; however, there is a risk that unexpected staining could lead to misinterpretation. We report a pleomorphic liposarcoma, epithelioid variant, in an 88-year-old man, with tricky-positive staining for GATA3. Histological examination revealed a tumor with epithelioid morphology. The tumor consists of solid sheets of epithelioid tumor cells with focal aggregates of pleomorphic lipoblasts. Immunohistochemically, the adipocytic tumor cell areas were positive for S100 protein, and the epithelioid tumor cells showed CAM 5.2 positivity. GATA3 was diffusely positive. The combination of CAM 5.2 and GATA3 staining suggested the possibility of metastatic cancer, but systemic clinical examinations did not detect any presence of a primary tumor, including urinary bladder, breasts, and salivary glands. The pathological diagnosis of pleomorphic liposarcoma, epithelioid variant, was made because of the presence of malignant lipoblasts. Our report may contribute for differential diagnosis of pleomorphic liposarcoma, epithelioid variant, with unexpected positive immunoreaction for GATA3. Immunohistochemical staining is widely used and the important tools supporting morphological diagnosis. Unexpected staining could lead to misinterpretation; therefore, pathologists should recognize immunohistochemical staining, including aberrant expression and nonspecific staining, as a pitfall. Here, we report a pleomorphic liposarcoma, epithelioid variant, in an 88-year-old man, with tricky-positive staining for GATA3, a commonly used marker of urothelial and breast-specific origin. Importantly, there are limited cases of GATA3 expression in pleomorphic liposarcomas.The specimen was formalin-fixed and paraffin-embedded (FFPE) and stained using hematoxylin and eosin. Immunohistochemistry (IHC) evaluation was performed with an automated immunostainer  as per the manufacturer's instructions using the following primary antibodies: cytokeratin (CK) AE1/AE3 , CAM 5.2 , GATA3 , S100 protein , CD34 , HMB45 , Melan A , MDM2 , CDK4 , p63 , CK7 , CK20 , CDX-2 , and TTF-1 .MDM2 FISH analysis using MDM2/CEP12 probe  to the MDM2 locus (12q15) and the chromosome 12 centromere (CEP12) is designed for formalin-fixed and paraffin-embedded tissue. FISH is performed on a tissue section considered a representative area, using manufacture's protocol. One hundred cells were evaluated with each probe and considered positive with at least 15 MDM2 signals per cell [An 88-year-old man presented with swelling in the right lower leg. He had a history of chondrosarcoma of the rib and ascending colon cancer. There was no evidence of family history including Li-Fraumeni syndrome. Routine laboratory test results were unremarkable. Magnetic resonance imaging revealed a 12.5 × 6 × 6 cm mass in the soleus muscle of the right lower leg, which showed heterogeneous signals suggestive of necrosis and hemorrhage Figures . The borGrossly, the tumor showed a solid gray tan-cut surface, nodular growth pattern, and central necrosis . HistoloThe patient developed multiple lung, liver, and bone metastases three months after surgery. The patient did not undergo any further treatment because of his age.Based on their clinical presentation and genetic background, liposarcomas are classified into six subtypes according to the current WHO Classification of Tumours, Soft Tissue and Bone Tumours 5th ed.) . Pleomorth ed. . In this study, we have identified two important pathological issues. First, pleomorphic liposarcoma, epithelioid variant, expresses GATA3. GATA3 is a zinc finger transcription factor associated with cell development and differentiation. GATA3 is known to play an important role in regulating genes involved in mammary-gland morphogenesis and luminal-cell differentiation, epidermal and follicular morphogenesis, and endothelial cells, especially in large vessels . ImmunohPatients with Li-Fraumeni syndrome have a history of multiple malignancies, including a broad spectrum of cancers and soft tissue sarcoma and leukemia. It is caused by germline mutations of the TP53 gene. In this case, patient has a history of multiple malignancies, including colorectal cancer and chondrosarcoma, but no hereditary tumor syndromes, such as Li-Fraumeni syndrome, have been noted.Here, we report a case of pleomorphic liposarcoma, epithelioid variant, diffusely positive for GATA3. When pleomorphic liposarcoma shows immunostaining with antibodies against cytokeratin and GATA3, it is challenging to distinguish liposarcoma from metastatic carcinoma, particularly urothelial carcinoma. In cases of sarcoma with epithelioid morphology, the expression of GATA3 should be evaluated carefully.


The objective of this investigation was to elucidate the association of miRNA-mediated genes in the regulation of dog testes development from immature to adult form by in-silico analysis. In silico analysis of differentially expressed (DE) testis miRNAs between healthy immature and mature dogs were performed using miRNet, STRING, and ClueGo programs. The determination of mRNA and protein expressions of predicted pivotal genes and their association with miRNA were studied. The predicted genes are involved in the governing of several key biological functions  in the testis as they evolve from immature to adult forms, mediated by several key signaling pathways , cytokines and hormones . Elucidation of DE-miRNA predicted genes’ specific roles, signal transduction pathways, and mechanisms, by mimics and inhibitors, which could perhaps offer diagnostic and therapeutic targets for infertility, cancer, and birth control.n = 4) and mature  dogs were utilized in this investigation. In silico analysis was performed using miRNet, STRING, and ClueGo programs. The determination of mRNA and protein expressions of predicted pivotal genes and their association with miRNA were studied. The results showed protein–protein interaction for the upregulated miRNAs, which revealed 978 enriched biological processes GO terms and 127 KEGG enrichment pathways, and for the down-regulated miRNAs revealed 405 significantly enriched biological processes GO terms and 72 significant KEGG enrichment pathways . The in-silico analysis of DE-miRNA’s associated genes revealed their involvement in the governing of several key biological functions  in the testis as they evolve from immature to adult forms, mediated by several key signaling pathways , cytokines and hormones . Elucidation of DE-miRNA predicted genes’ specific roles, signal transduction pathways, and mechanisms, by mimics and inhibitors, which could perhaps offer diagnostic and therapeutic targets for infertility, cancer, and birth control.High-throughput in-silico techniques help us understand the role of individual proteins, protein–protein interaction, and their biological functions by corroborating experimental data as epitomized biological networks. The objective of this investigation was to elucidate the association of miRNA-mediated genes in the regulation of dog testes development from immature to adult form by in-silico analysis. Differentially expressed (DE) canine testis miRNAs between healthy immature (2.2 ± 0.13 months;  MicroRNAs (miRNAs) play a key role in the differentiation, development, maintenance, and functions of various tissues. Spermatogenesis is a sequence of complex processes ,2, whichDicer is an endonuclease enzyme that belongs to the ribonuclease III (RNase III) family. It activates the RNA-induced silencing complex (RISC), which is essential for RNA interference. The RISC contains dsRNA binding proteins, including protein kinase RNA activator (PACT) and transactivation response RNA binding protein (TRBP) that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. Dicer plays an important role in spermatogenesis ,14. MiRNIt is conceivable that miRNAs can regulate meiosis and thus spermatogenesis  by regulThe objective of this investigation was to elucidate miRNA–mRNA interaction of differentially expressed (DE) miRNAs between immature and mature canine testis by constructing a protein–protein network and performing cluster gene analysis to elucidate key biological functions. n = 4 and adult: 11 ± 1.0 months; n = 4) undergoing elective castrations , membranes were washed in buffer containing 2% animal serum and 0.1% detergent. The membranes were then incubated in secondary antibodies [goat anti-mouse IgG-FITC for DNMTA1 and β-actin , and goat anti-rabbit IgG-FITC for PTEN  for 1 h at room temperature. The blots were then washed and scanned using the Pharos FX Plus system (Bio-Rad Laboratories). FITC fluorophore was excited at 488 nm and read at the emission wavelength of 530 nm. All possible negative controls, equivalent concentrations of nonspecific IgG or normal serum in place of the primary antibody, were included.Western blots for each testis tissue sample from mature and immature dogs were performed separately by methods described previously . BrieflyFor MiRNA-associated gene quantitative profiling, 32 upregulated and 12 downregulated miRNAs were included for in-silico analysis . Similarp < 1.0 × 10−16) and reveals 978 significantly enriched biological processes GO terms  and 127 significant  KEGG enrichment pathways (p < 1.11 × 10−14) and reveals 405 significantly enriched biological processes GO terms  and 72 significant  KEGG enrichment pathways  method for upregulated miRNAs and downregulated miRNAs were screened and presented in To interpret functionally nested gene ontology and pathway annotation networks for the predicted genes of adult dog testis, ClueGo nested network analysis was performed, and the results are presented in p < 0.05) in abundance in mature compared to immature dog testis . There was a positive association for the miRNA–mRNA pair (p < 0.05). The relative expressions of miRNA and associated hub gene mRNA in mature dog testis were also provided in The mRNA expressions for CDKN1A, EGFR, JUN, NOTCH1, and PIK3R1 were greater  proteins. The PTEN, DNMT1, and β-actin proteins were 47, ~180, and 42 kDa, respectively .In the present study using DE-miRNAs between immature and adult dog testis samples, we identified 613 genes involved in the regulation of testis development. The GO biological functional enrichment showed that genes were mainly enriched in regulation of cellular process, cellular response to stimulus, developmental process, cell population proliferation, cell death, cell differentiation, apoptotic process, and cell metabolic process. The KEGG pathway enrichment showed that genes were mainly enriched in cancer biology, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway, p53 signaling pathway, cellular senescence, hormone signaling pathway and human papillomavirus infection. Furthermore, we performed bioinformatics analysis to identify the potential key genes based on random selection algorithm, GO semantic similarity, PPI network, and cluster analysis. The results showed the involvement of differentially expressed genes in growth, sexual development, the maintenance of gluconeogenesis and lipid metabolism, cell proliferation, Sertoli and spermatogonial stem cells division and growth, cell cycle (cell cycle progression at G1-S phase), maturation, cell survival, and apoptosis. These key genes functioned as the essential molecules that might have mediated the testis development process between the immature and adult dogs.NOTCH1 and MYC regulates the cell cycle, cellular fate determination, cell proliferation, cell differentiation, and cellular apoptosis. MicroRNA-34b expression was associated with meiotic-specific cells from the murine testis [Micro RNAs critically regulate the proliferation and/or early differentiation of stem cell populations in testis . In mouse testis  and miR-e testis . In humae testis . The enhe testis . MicroRNe testis . Wnt/b-ce testis , and thee testis ,43, mTORe testis ,45,46, ae testis ,48. PTENe testis . Recent e testis . Furthere testis .EGF was identified as one of its target genes. Further, this upregulated miRNA in adult testis-associated hub genes CDH1 and MET interacted with EGF and EGFR. The ErbB signaling pathway-associated genes found were CDKN1A, EGFR, JUN, KRAS, MYC, and PIK3R1. The CDKN1A is the primary p53 target gene that mediates cell-cycle arrest [CDKNIA knockout mice plausibly affecting pubertal development [EGF mediates spermatogonial proliferation through its receptors ErbB1, ErbB2, and ErbB4 in the testis [EGF mediates spermatogonial proliferation through its receptors on Sertoli cells via activation of MAPK cascade and/or PI3K cascade by elevating the expressions of SCF, Ig-NRG1, and EGFRs (ERBBRs) [ERBB2 and ERBB3. Aberrant EGFR activation is a significant factor in the development and progression of multiple cancers [In the current study, cfa-miR-7a was upregulated in adult testis and e arrest ,52. LH se testis . It is p(ERBBRs) ,56. Furt(ERBBRs) ,58. Inte cancers  suggestiPIK3R1, and its signals are important for cell activities, including cell growth and division, migration, production of new proteins, transport of materials within cells, and cell survival. Studies suggest that PI3K signaling may be involved in the regulation of several hormones, including insulin. NF-kappaB and PI3K-Akt pathways are among PI3 K-associated pathways. PI3K-Akt pathway is an intracellular signal transduction pathway that promotes metabolism, proliferation, cell survival, growth, and angiogenesis in response to extracellular signals [FSH [PI3K by EGF occurs via the association of the p85 subunit of PI3K (PIK3R1) with the activated EGFR [BCL2L11, CCND1, CCNE1, CDK4, CDK6, CDKN1A, EGFR, FGFR1, JAK2, KDR, KRAS, MCL1, MET, MYC, PIK3R1, PTEN, and RELA.In the current study, the upregulated cfa-miR-29c-associated gene was  signals . This is signals . Furtherals [FSH . It shouted EGFR . PIK3R1/ted EGFR  but its VEGFA, which regulates neovascularization and cord formation, and potentially acts through the PI3K pathway during testis morphogenesis [VEGF signaling regulates germ cell proliferation and promotes testicular regeneration via direct action on germ cells and the enhancement of vascularization. Associated genes KDR, KRAS, and PIK3R1 in the VEGF signaling pathway in the current study, may regulate the testis morphogenesis and regeneration.Current investigation revealed that upregulated canine miR-15a and miR-378 were associated with hub gene ogenesis . FurtherEGFR, JUN, KRAS, and MMP2 in the regulation of the GnRH signaling pathway. The FSH regulates Sertoli cell proliferation during fetal and early postnatal life by activating cAMP/PKA/ERK1/2 and PI3K/Akt/mTORC1 dependent pathways, and by increasing the transcriptional activity of c-MYC and HIF2 and the expression of CCND1 [CCND1 is a hub gene for upregulated canine miRNAs 15a, 16, 19a and 20a, and MYC is a hub gene for upregulated miR-34c in mature testis in the current investigation, suggesting the involvement in the Sertoli cell proliferation as indicated in the previous studies. Insulin and IGF1 regulate testicular functions by activating PI3K/Akt and ERK1/2 signaling pathways. Mice lacking INSR and IGF1R in Sertoli cells showed a 72% reduction in testis size and a 79% reduction in daily sperm production [EGFR, JUN, KRAS, MMP2, PIK3R1, and RELA engage in the relaxin signaling pathway in the current study, could have contributed to Sertoli cell proliferation. Genes IGF1, AMH, hedgehog (DHH), and platelet-derived growth factor (PDGF) seem to regulate Leydig cell differentiation and function. The IGF1 stimulated differentiation and mitosis of Leydig cells [IGF1, DHH, and PDGF are Sertoli cells paracrine factors, it seems reasonable to speculate that thyroid hormone actions on Leydig cells might be, at least in part, mediated through Sertoli cells. Relaxin-induced Sertoli cell proliferation involves the activation of a Gi protein and the activation of EKR1/2 and PI3K/Akt pathways [FSH regulates Sertoli cell proliferation during the fetal and postnatal period via the SMAD pathway [NOTCH1 is identified as a hub gene for upregulated cfa-miR-34c in the current investigation. NOTCH1-IC forms a transcriptional complex with SMAD, a component of established TGF-β signaling, and regulates the expression of HES1 by binding to the promoter [NLK) was a hub gene for downregulated miR-181a, -181b. The NLK phosphorylates the NOTCH1 protein. NLK-mediated phosphorylation does not interfere with the nuclear localization of NOTCH1-IC but decreases the association of the Notch active transcription complex [Regulation of testis development by hormones has been described in . Clusterof CCND1 . The CCNoduction ,69. Relaoduction ,70. Assoig cells . Equallypathways ,70. Acti pathway ,74. The promoter . These spromoter . Interes complex . Inhibin complex ,67.TGFβs and activins, which signal through serine/threonine kinase receptor subunits to activate SMAD transcription factors. It has been shown that IL1α and IL1β increased DNA synthesis and Sertoli cell number in vitro and IL1α had a more potent effect than IL1β [TNFR1 has been detected in Sertoli cells and probably mediates TNFα biological actions, pro-inflammatory and immunoregulatory responses, and apoptosis [CXCL8, was identified as one of top 30 hub genes in the current study. It should be noted that PTEN (hub gene for upregulated miR-22 and downregulated miR-214) loss induces a selective upregulation of CXCL8 signaling that sustains cell growth and survival [CDK4, JUN, KRAS, PIK3R1, and RELA genes. This regulation could have contributed to the development and normal function of the testis.Cytokines play an important regulatory role in the development and normal function of the testis. They signal via the adapter protein MyD88 to activate NFκB; and han IL1β . TNFR1 hpoptosis . A chemosurvival . In the EGFR, ESR1, JUN, KRAS, MMP2, and PIK3R1 were involved in the estrogen signaling pathway in the current study. ESR was a hub gene for miR-18a. -19a, and -22 in the current study. Estrogens increase proliferation of Sertoli cells through ERα and GPER and on the other hand, at the end of the proliferative period it promotes cessation of proliferation and cell maturation through ERβ [ERα promotes cell proliferation through the activation of NFκB in a PI3K- and a ERK1/2-dependent manner and that this is accompanied by CCND1 induction [GPER activates Src/PI3K/Akt pathway which participates in E2-induced Sertoli cell proliferation via regulating the expression of S-phase kinase-associated protein 2 (SKP2) [ERβ promotes cell cycle exit and cell maturation through the activation of CREB in a PI3K-dependent manner and this leads to the expression of the Sertoli cell differentiation markers—CDKN1B, GATA1 (isoform GATA6 was a hub gene for down-regulated cfa-miR-181a and -181b), and DMRT1 [TRα1 and AIMP1 (p43) receptors. The mechanisms participating in these processes involve the regulation of CX43, c-MYC (MYC was identified as a hub gene for upregulated cfa-miR-34b and MYCN a hub gene for miR-101), P21CIP1 (CDKN1A a hub gene for upregulated cfa-miR-106a), and P27KIP1 (CDKN1B a hub gene for down-regulated cfa-miR-181a) [CCND1, ESR1, FOXO1, KRAS, MYC, NOTCH1, and PIK3R1 involved in the regulation of the thyroid signaling pathway.Androgen-dependent regulation of Sertoli cell proliferation is an indirect effect probably exerted through the secretion of a paracrine factor ,67. Direough ERβ ,67. ERα nduction . GPER ac2 (SKP2) . The ERβnd DMRT1 ,67. RoleiR-181a) ,84,85. CStudies observed positive  or negative  associations between miRNA and mRNA pairs . It shouCollectively, in the current study, GO analysis of differentially expressed genes showed that the down-regulated genes were significantly enriched in a large number of GO terms. Further, the GO analysis showed that most GO terms were downregulated, indicating that these DEGs may have played important roles in the development of the testis of immature dogs. However, the upregulated genes enriched in the GO terms were related to meiosis, protein ubiquitination, and fertilization of adult dogs. This suggests that a large number of genes with key roles in male reproduction traits are highly expressed in mature testis. Interestingly these genes are not expressed or expressed in low abundance in immature dog testis. Similar results were reported in a bull study . Our anaThe complex relationship between miRNA and gene interaction is a vital component of miRNA functional analysis in the testis development process. Abnormal expression of miRNAs and/or any regulation disturbance can lead to impaired germ cells, abnormal spermatogenesis, and even neoplasia. The present in-silico analysis showed the involvement of canine testicular miRNAs in structure and function. The DE-miRNAs between immature and adult canine testis and their associated genes involved in several regulatory pathways play a crucial role during the immature testis transition to the adult testis. A focused study of individual miRNA molecules may elucidate specific functions or problems, endorse the development of anti-oncogenic reagents and infertility/subfertility treatments, and bolster novel contraceptive technologies.


Mucosal head and neck squamous cell carcinoma (HNSCC) are the seventh most common cancer, with approximately 50% of patients living beyond 5 years. Immune checkpoint inhibitors (ICIs) have shown promising results in patients with recurrent or metastatic (R/M) disease, however, only a subset of patients benefit from immunotherapy. Studies have implicated the tumor microenvironment (TME) of HNSCC as a major factor in therapy response, highlighting the need to better understand the TME, particularly by spatially resolved means to determine cellular and molecular components. Here, we employed targeted spatial profiling of proteins on a cohort of pre-treatment tissues from patients with R/M disease to identify novel biomarkers of response within the tumor and stromal margins. By grouping patient outcome categories into response or non-response, based on Response Evaluation Criteria in Solid Tumors (RECIST) we show that immune checkpoint molecules, including PD-L1, B7-H3, and VISTA, were differentially expressed. Patient responders possessed significantly higher tumor expression of PD-L1 and B7-H3, but lower expression of VISTA. Analysis of response subgroups indicated that tumor necrosis factor receptor (TNFR) superfamily members including OX40L, CD27, 4-1BB, CD40, and CD95/Fas, were associated with immunotherapy outcome. CD40 expression was higher in patient-responders than non responders, while CD95/Fas expression was lower in patients with partial response (PR) relative to those with stable disease (SD) and progressive disease (PD). Furthermore, we found that high 4-1BB expression in the tumor compartment, but not in the stroma, was associated with better overall survival (OS) . Moreover, high CD40 expression in tumor regions , and high CD27 expression in the stroma  were associated with better survival outcomes. Taken together, this study supports the role of immune checkpoint molecules and implicates the TNFR superfamily as key players in immunotherapy response in our cohort of HNSCC. Validation of these findings in a prospective study is required to determine the robustness of these tissue signatures. Mucosal head and neck squamous cell carcinoma (HNSCC) is one of the cancers that severely impact patients’ quality of life and causes complications such as pain, psychosocial distress, and dysfunction , 2. TobaStudies have shown that the composition of the HNSCC tumor microenvironment (TME) and the interactions of immune cell types within the TME may be important determinants of treatment outcomes , 8. As aThis retrospective study has Human Research Ethics (HREC) approval from the Royal Brisbane and Women’s Hospital (RBWH) (LNR/2020/QRBW/66744) and The University of Queensland ratification. We identified n=41 HNSCC patients eligible for our study from the RWBH. Of the 41 patients, 20 had no available tissues, with 21 tissue blocks available for analysis. We collected formalin-fixed paraffin-embedded (FFPE) tissue samples from these 21 HNSCC patients, with samples collected prior to immunotherapy. Pathology Queensland prepared serial sections and hematoxylin and eosin (H&E) staining, while pathology reviews provided demarcation of tumour/stromal regions so that non-neoplastic epithelium could be avoided. Of the 21 tissues collected, n=17 were deemed of appropriate quality and tissue integrity for subsequent spatial analysis. Patients were treated with Pembrolizumab or Nivolumab and categorized based on response to therapy according to RECIST 1.1., including complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). The clinicopathological findings are shown in The FFPE tissue slides were processed and analyzed by the Nanostring GeoMx Digital Spatial Profiler (DSP) technology at Queensland University of Technology (QUT), Brisbane, Australia. Fluorescent morphology markers, including CD45, pan-cytokeratin (panCK), and SYTO-13 were used to visualize lymphocytes, tumor regions, and the nucleus, respectively. The slides were prepared and processed, using an immune-oncology panel Table 2,standR (v1.1.5). Filtering was first conducted to exclude slides and regions of interest (ROIs) with poor tissue quality due to poor staining or detachment. This resulted in having 17 patients tissue samples and 187 ROIs used for further analyses. Then, by using the function addPerROIQC from the standR package, marker LAG3 was filtered for being lowly expressed in over 90% of the ROIs. For data analysis, the log2-transformed count per million (logCPM) data were used to account for variation in library size. The relative log expression (RLE) and principal components analysis (PCA) of the logCPM data were used to assess the overall distribution, to identify any confounding factors from the experimental design and the detect presence of batch effects within the data. To remove unwanted technical variations observed in the RLE analysis, the data was normalized using size factor normalization methods in standR (v1.1.5).Quality control (QC) of the data was performed using Bioconductor R package edgeR (v3.34.0) (limma (3.48.0)  analysis was performed using R packages v3.34.0)  and limm(3.48.0) . Briefly(3.48.0) . The linSurvival analysis was performed by both Kaplan-Meier estimates using median protein expression for cohort stratification, and Cox proportional hazards model using continuous protein expression. Normalized replicate measurements in tumor/stroma compartment were averaged per patient for survival analysis. Survival analysis was performed using R package Survival  with theSingle-cell spatial phenotyping of the HNSCC FFPE slides was performed in collaboration with Akoya Biosciences  on the PhenoCycler-Fusion platform. Four tissue slides from four different patients were stained with a 45-plex antibody panel, including immune checkpoints, immune cell lineage, activation states, general tissue structure in a single step Table 3.This retrospective study spatially profiled HNSCC FFPE tissue samples from patients with metastatic HNSCC. The patients’ response to immunotherapy was categorized according to RECIST criteria. The study included patients with progressive disease , stable disease , partial response , and complete response . In terms of HPV status, n=7 patients were oropharyngeal p16 positive, n=3 patients were p16 negative, and n=7 patients were unknown Table 1.Using the Nanostring GeoMx DSP, we investigated the protein expression levels of 17 FFPE tissue samples from HNSCC patients. The tumor and stromal compartments were defined by masking high and low - panCK regions, measured against pathology H&E annotations, and the protein expression of each compartment was measured  plots and PCA to identify confounding experimental effects. Technical variation was captured by RLE plot 2B. The Differential expression was initially performed by grouping patients into those who responded to ICI treatment  (n=9) and those who did not (PD) (n=8). This analysis indicated higher expression of PD-L1, B7-H3, Bcl-2, BCLX, and BIM in regions of tumors that responded to ICI. VISTA, CD45RO, CD66b, and FOXP3 were identified to have lower expression levels in regions of ICI responsive tumors . Protein expression was used to stratify the patient cohort into groups above and below the median threshold  whole slide tissue images were obtained using the Akoya PhenoCycler-Fusion platform. The morphology markers included panCK (red), CD45 (green), DAPI (blue), and E-cadherin (pink) , including anti-PD-1 antibodies Pembrolizumab and Nivolumab, have shown promising results in patients with R/M HNSCC However, only 15 to 20% of patients benefit from single-agent ICI . The immrogation . In thisIt is well established that after T cell activation, T cells express inhibitory cell surface receptors, known as immune checkpoint proteins, such as program cell death-1 (PD-1), to prevent further activation. However, tumor cells exploit this process by expressing ligands for these receptors, for example programmed death-ligand 1 (PD-L1 or B7-H1) to bind PD-1, in order to cause T cell exhaustion, which results in T cell inactivation and reduced effector function . StudiesTNFR superfamily consists of 30 receptors and 19 ligands, all of which have been shown to be involved in cell signaling pathways, influencing the differentiation, proliferation, and survival of immune and non-immune cells , 34. In OX40L  is a type II glycoprotein that is expressed in a variety of cell types, including B cells, macrophages, and dendritic cells (DCs), and is involved in multiple T cell subtype activation . Studies4-1BB (CD137), a surface glycoprotein and costimulatory receptor expressed on activated T cells, was found to boost cytokine secretion, antiapoptotic molecule upregulation, and T cell effector function . Publish+ and CD8+ T cells, contributing to further tumor eradication and, eventually, survival improvement  can be found below: GSE217640 (GEO).This study has Human Research Ethics (HREC) approval from the Royal Brisbane and Women’s Hospital (RBWH) (LNR/2020/QRBW/66744) and The University of Queensland. The patients/participants provided their written informed consent to participate in this study.Idea concept, BH, KO’B, and AK. Methodology/experimental, HS, JM, NM, MW, MA, QN, RL, OB, BH, and AK. Data analysis, NL, JM, BC, NJ, CT, OB, and MD. Writing and critical review, all authors. All authors contributed to the article and approved the submitted version.


Aberrant activation of the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway is common in systemic lupus erythematosus (SLE), conferring immune-mediated properties in target tissues. Multiple cytokines activate different combinations of JAKs and STATs to alter the cell fate of target tissue and induce end-organ damage. Thus, the simultaneous blockade of several different cytokines by small molecules acting downstream intracellular signalling has gained traction. JAK inhibitors have been approved for the treatment of several rheumatic diseases, yet hitherto not for SLE. Nevertheless, JAK inhibitors including tofacitinib, baricitinib, and deucravacitinib have shown merit as treatments for SLE. Tofacitinib, a JAK1/3 inhibitor, reduced cholesterol levels, improved vascular function, and decreased the type I interferon signature in SLE patients. Baricitinib, a JAK1/2 inhibitor, demonstrated significant improvements in lupus rashes and arthritis in a phase 2 and a phase 3 randomised controlled trial, but the results were not replicated in another phase 3 trial. Deucravacitinib, a selective tyrosine kinase 2 (TYK2) inhibitor, yielded greater response rates than placebo in a phase 2 trial of SLE and will be investigated in larger phase 3 trials. TYK2 is activated in response to cytokines actively involved in lupus pathogenesis; this review highlights the potential of targeting TYK2 as a promising therapy for SLE. AlthougOver the last few decades, several biologic therapies have been developed to target specific inflammatory molecules and pathways. These drugs have shown encouraging results in rheumatic diseases, particularly rheumatoid arthritis (RA), psoriatic arthritis, and ankylosing spondylitis . Howevervia the phosphorylation of other proteins, particularly signal transducer and activator of transcription (STAT) proteins (Janus kinases (JAKs) are intracellular tyrosine kinases that mediate cytokine-mediated signalling proteins . There eproteins  -α, IFN-γ, interleukin (IL)-2, IL-6, IL-10, IL-12/IL-23, IL-19, IL-20, and IL-22 . These cTo date, two targeted biologic therapies have been approved for the treatment of SLE, i.e., belimumab and anifrolumab . HoweverIFN-α is a key cytokine in SLE and other immune-mediated diseases, and its signalling is mediated by the intracellular kinases JAK1 and TYK2  Figure . Tofacit3.3.1.3.1.1.New Zealand black/New Zealand white F1 (NZB/W-F1) and Murphy Roths Large/lymphoproliferation (MRL/lpr) murine models of SLE are commonly used to study pathogenetic aspects of lupus nephritis (LN). Experimental studies have shown that IFNs induce activation of STAT1 in glomerular mesangial cells , which iAnother experimental study tested the effectiveness of tofacitinib in the NZB/W-F1 model with regard to renal disease. Tofacitinib-treated mice showed renal histological improvements, decreased C3 and IgG glomerular deposits, significant reductions in the proportion of macrophages and T cells, and a decreased production of pro-inflammatory cytokines. The efficacy of tofacitinib was comparable to that of cyclophosphamide and mycophenolate mofetil . The potSTAT3 is believed to play a pivotal role in LN pathogenesis. Indeed, STAT3 knock-out lupus-prone mice develop milder renal disease with regard to immune component infiltration and deposits in the kidneys . In addiIFN signalling is an important mediator of cutaneous lupus . RuxolitInhibition of the JAK–STAT pathway is associated with a decrease in autoantibody titres. More specifically, lupus-prone mice treated with JAK2-STAT1 inhibitors, JAK1-STAT3 inhibitors, selective STAT3 inhibitors, and tofacitinib showed reduced serum levels of anti-dsDNA antibodies , 24, 27.3.1.2.P) has been shown to confer a protective effect against autoimmunity  tyrosine phosphatase was overexpressed in lupus B cells and indirectly activated the JAK–STAT pathway to induce IFN-α-mediated autophagy. Specifically, the activation of the JAK–STAT pathway was mediated by IFN-α-induced phosphorylation of TYK2 as a result of decreased phosphorylation of the E3 ubiquitin protein ligase c-cbl by STS-1. Thus, TYK2 seems to play an important role in the increased autophagy activity of SLE B cells .via TYK2 phosphorylation in the median prefrontal cortex . Recent studies in the field demonstrated that microglia cells are activated towards an inflammatory state involving the IL12/IL23p40 pathway in diffuse NPSLE . Specifil cortex . These f3.2.3.2.1.Specific mutations or polymorphisms in JAK or STAT genes have been associated with various disorders, including immunodeficiencies, haematological conditions, and autoimmune diseases . This isEx vivo studies in human SLE have shown that the JAK–STAT pathway upregulates the expression of IFN-regulated factor (IRF)-related genes in lupus T cells  is initiated by keratinocytes producing IFN-κ, which further promotes IFN responses and increases the keratinocyte sensitivity to UV light. This mechanism is dependent on the expression of TYK2 . Authorsne cells .4.4.1.Numerous translational studies conducted on murine models of lupus have demonstrated the robust therapeutic efficacy of tofacitinib, particularly for various manifestations such as cardiovascular disease. These findings led to the first phase 1 double-blinded safety trial, which yielded encouraging results . In thisA case series comprising ten SLE patients with active skin and/or musculoskeletal disease demonstrated adequate efficacy of tofacitinib at a dose of 5 mg twice daily throughout a follow-up period of up to 12 months . Seven o4.2.Baricitinib is an oral, selective inhibitor of JAK1/JAK2 that has been approved for the treatment of immune-mediated diseases, including COVID-19 . The firBased on the encouraging results of the phase 2 RTC of baricitinib, two phase 3 RTCs were designed to evaluate the potential efficacy of baricitinib in SLE, named SLE-BRAVE 1 and SLE-BRAVE 2, involving 760 and 775 SLE patients, respectively , 16. Par4.3.In a phase 1 clinical trial that involved both human samples and animal models of LN, the administration of an oral TYK2 inhibitor (BMS-986165) was shown to block multiple autoimmune pathways in human Th1, Th17, B cells, and myeloid cells, indicating a therapeutic potential . IncreasDesign of RCTs and key outcomes are summarised in 4.4.In a post-hoc analysis of the ORAL surveillance trial, it was found that RA patients with cardiovascular risk factors who were treated with tofacitinib had a higher likelihood of exhibiting a cardiovascular event, including myocardial infarction, stroke, and death due to cardiovascular disease . Additio5.Unfortunately, the SLE community has witnessed a multitude of failures of phase 2 and 3 clinical trials . HoweverAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.IP is supported by grants from the Swedish Rheumatism Association (R-969696), King Gustaf V’s 80-year Foundation (FAI-2020-0741), Swedish Society of Medicine (SLS-974449), Nyckelfonden (OLL-974804), Professor Nanna Svartz Foundation (2021-00436), Ulla and Roland Gustafsson Foundation (2021–26), Region Stockholm (FoUI-955483), and Karolinska Institutet.IP has received research funding and/or honoraria from Amgen, AstraZeneca, Aurinia, Elli Lilly, Gilead, GlaxoSmithKline, Janssen, Novartis, Otsuka, and Roche. The funders had no role in the design of the study, the analyses or interpretation of data, or the writing of the manuscript. DN declares that he has no conflicts of interest related to this work.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hypericum perforatum), Gingko biloba, Selaginella tamariscina, Torreya nucifera, and many other plants. Its pharmacological actions have been listed as antiviral, antibacterial, antioxidant, anti-inflammatory, antidiabetic, antidepressant, and neuroprotective. Molecular docking studies have found that amentoflavone binds strongly to the active site of the main protease (Mpro) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). As conventional antiviral medications are met with limited success against coronavirus disease-2019 (COVID-19) and vaccines are one of the only weapons against COVID-19 in the pharmaceutical armamentarium, traditional medicines are being considered for the forefront battle against COVID-19. Clinical studies with Hypericum and Gingko extract as additional or alternative drugs/supplements are registered. Here we review the potential of amentoflavone, an active agent in both Hypericum and Gingko extract as an adjunct therapy for COVID-19. Its anti-inflammatory, antioxidant, and sepsis preventive actions could provide protection against the “cytokine storm.” Compared with the herbal extracts, which induce cytochrome P450 (CYP) and uridine 5′-diphospho (UDP)-glucuronosyltransferases (UGT) activity producing a negative herb–drug interaction, amentoflavone is a potent inhibitor of CYP3A4, CYP2C9, and UGT. Further studies into the therapeutic potential of amentoflavone against the coronavirus infection are warranted.Amentoflavone is one of the flavonoids that are known for their antiviral effects and many of them are predicted to have inhibitory effects against severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome Coronavirus (MERS-CoV) enzymes 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro). Amentoflavone is a biflavonoid found in the herbal extracts of St. John's wort ( It reduprotease , 15.pro 3CLpro and thereby blocking the replication of the virus [Figure 1). The Mpro is currently the most studied drug target of the SARS-CoV virus as its structure and substrate specificity are conserved amongst SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 [pro proteolytic activity in vitro at an IC50 = 8.3 μM and Ki = 13.8 ± 1.5 μM) [50 is lower than that for the antiviral flavones, such as its parent molecule apigenin (280.8 μM), and lute-olin (20.2 μM) and quercetin (280.8 μM), and is currently under clinical trials as a drug for the treatment of COVID-19 . In addition, their study showed that amentoflavone binds strongly to the active site of 3CLpro having binding energy of −11.42 kcal/mol [Molecular docking studies predict amentoflavone to have potential to inhibit the SARS-CoV-2 by binding to its Mhe virus ,5,6,7 (FRS-CoV-2 . Fortuit 1.5 μM) . The IC5kcal/mol . This hakcal/mol , 19. Ameute-olin .2 μM andpro in any cellular system remain lacking.Such studies collectively emphasize the broad spectrum antiviral activity of amentoflavone, which can inhibit viral pathogenicity by targeting the viral proteins essential for its replication. As a functional FAS is essential for viral replication in general , the inhCOVID-19 is regarded primarily as an atypical pulmonary disease characterized by severe pneumonia with acute respiratory distress syndrome (ARDS). Emerging data shows that this disease also leads to hyperinflammatory state resulting in other complications such as liver, kidney, and heart injury, and septic shock . The cauFigure 2, [Ki = 8.06 μM) [Studies of the pharmacological functions of amentoflavone suggest it to be anti-inflammatory, antioxidative, and vasoprotective . The scaigure 2, , 26). Thigure 2, . Amentofigure 2, , 28. It igure 2, . Amentof8.06 μM) . This pr0−t) of amentoflavone glucuronides (410.9 ± 62.2 ng/mL h) was significantly higher than the parent compound (194.5 ± 16.9 ng/mL h). Moreover, the antioxidant property of amentoflavone may remain unaltered following glucuronidation [Figure 2). UGT1A1 and UGT1A3 are responsible for glucuronidation of amentoflavone and studies with microsomes in vitro in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) could not detect any oxidative metabolite of amentoflavone [However, certain characteristics of amentoflavone should be noted. Like other flavonoids, the absolute oral bioavailability of amentoflavone is extremely low (0.06% ± 0.04%) and it undergoes rapid glucuronidation and sulfation in vivo . The glunidation . Studiesnidation , 32, catnidation , and alsnidation   as both drug and dietary supplements to study their effects on depression, diabetes, cancer, acute coronary disease, inflammation, and Raynaud syndrome. Extracts of G. biloba contain only about 2% amentoflavone [G. biloba/EGb 761 has been attributed to bilobalide, ginkgolide A, B, quercetin, and kaempferol [As the uses of both conventional and traditional medications for COVID-19 are being explored, the possibility of herb–drug interactions should also be noted . CYP monoflavone , while Soflavone , and EGboflavone . The CYPempferol . NeverthBeing an inhibitor of both CYPs and UGT, the use of amentoflavone should be carefully reviewed to avoid the risk of toxicity due to increased bioavailability of the coad-ministered drugs. Administration of amentoflavone should also be calibrated against antithrombotic drugs like warfarin, rivaroxaban, or betrixaban to avoid bleeding. The possibility of administering amentoflavone as replacement of the conventional CYP 3A4 and 2C9, and UGT inhibitors, can also be considered. The currently approved antiviral drug remdesivir is an adenosine analog, which binds to the viral RdRp , and it Although our understanding of the antiviral properties and mechanism of action of amentoflavone and its glucuronides is far from complete, the strong binding affinity of amentoflavone to SARS-CoV 3CLpro and spike glycoprotein predicts it be an effective natural therapeutic drug candidate against SARS-CoV-2. Its strong antioxidant property along with anti-inflammatory, thrombin inhibition, and protection against lung injury could aid in fight against the SARS-CoV-2 pathogenic complications like hyperinflammatory and hypercoagulable states that lead to ALI and multiorgan failure.Selaginella sp. and that amounted to 65.31 mg (98% purity) from approximately 2.5 g of S. tamariscina [However, to provide a better understanding of its pharmacological effect against COVID-19 and using amentoflavone as an antiviral adjunct therapy against SARS-CoV-2, preclinical trials involving animal models need to be undertaken to confirm the antiviral activity of this biflavonoid, which, to our knowledge, has been shown only with assays in vitro. Although amentoflavone has been identified and isolated from many known medicinal plants and their herbal extracts that are used as dietary supplements, it should be noted that the content of amentoflavone in these extracts is very low. The highest content has been isolated from 5.31 mg 9% purity G. biloba and St. John's wort, quercetin is already an investigational drug and rutin is suspected to be an active ingredient in TCMs that have been allegedly effective in COVID-19 prevention [With the advent of COVID-19 vaccines and rise of new and more virulent SARS-CoV-2 mutants, there is an urgent need to undertake pharmaceutical research of natural compounds that show strong binding affinity to the coronavirus in silico. As natural ingredients of evention ; thus, a