Medulloblastoma, the most common pediatric malignant brain tumor, continues to have a high rate of morbidity and mortality in childhood. Recent advances in cancer genomics, single-cell sequencing, and sophisticated tumor models have revolutionized the characterization and stratification of medulloblastoma. In this review, we discuss heterogeneity associated with four major subgroups of medulloblastoma  on the molecular and cellular levels, including histological features, genetic and epigenetic alterations, proteomic landscape, cell-of-origin, tumor microenvironment, and therapeutic approaches. The intratumoral molecular heterogeneity and intertumoral cellular diversity clearly underlie the divergent biology and clinical behavior of these lesions and highlight the future role of precision treatment in this devastating brain tumor in children. Medulloblastoma (MB) is the most common malignant brain tumor of childhood, which is classified as an embryonal tumor located in the cerebellum. With an incidence rate of 0.156 cases per 100,000 population, MB ranks second behind leukemia in incidence, but carries a much worse overall prognosis . Of thesTP53 mutations, and Group 3 have the worse prognosis while the WNT driven MBs are associated with better prognosis [Multiple studies using genetic and transcriptional profiling of MB samples identified four distinct molecular subgroups: wingless (WNT), sonic hedgehog (SHH), Group 3, and Group 4 ,7,8,9,10rognosis ,12. Due rognosis ,14,15. Trognosis .Recently, single-cell RNA sequencing (scRNA-seq) based studies provided new insights on molecular and cellular heterogeneity, which underlie the divergent biology and clinical behavior in MB ,18,19. TIn 2012, researchers reached a consensus that classified MB into four different subgroups based on their molecular characterization, namely WNT, SHH, Group 3, and Group 4 . Of thesCTNNB1, deletion of chromosome 6, and strong immunohistochemical nuclear staining for β-catenin [Of the different subgroups, the WNT MB has the best prognosis and accounts for 10% of all patients with MB. Patients with WNT MB are expected to have > 90% survival . Named b-catenin . While W-catenin .PTCH1, SMO, SUFU, and amplifications of GLI1 and GLI2 [SUFU, are at increased risk to develop SHH MB in infancy [MYCN amplification are more significant in young children [GLI2 amplification in adults often means a much worse prognosis than in young children [TP53 mutations and MYCN/GLI2 amplifications. SHHβ and SHHγ occur in young children, whereas SHHβ tumors have a worse overall survival with frequent metastases compared to SHHγ tumors that are enriched for the MBEN histology. SHHδ tumors primarily occur in adults, have a favorable prognosis and have a high frequency of TERT promoter mutations.SHH MB is the dominant subgroup in both young children (≤3 years old) and adults (≥16 years old) . This suand GLI2 ,28. It i infancy . This SH infancy . Additio infancy . Clinica infancy . Desmopl infancy . Cytogenchildren . Chromoschildren . Recentlchildren . SHHα suMYC amplifications, GABRA5 overexpression, and SMARCA4 mutations [MYC amplification is more frequent in Group 3γ; Group 3β tumors have a higher frequency of OTX2 gain, DDX31 loss, and high GFI1/GFI1B expression [Of the subgroups, Group 3 has the worst prognosis in MB, with a 5-year survival ranging from 39% to 58%, depending on age of the patient and treatment regimen . One potutations . Due to utations ,24. A reutations . Group 3pression .PRDM6 (17%) and GFI1/GFI1B (5–10%), somatic mutations of KDM6A (9% ), ZMYM3 (6%), KMT2C (6%) and KBTBD4 (6%), and amplifications of MYCN (6%), OTX2 (6%), and CDK6 (6%) [MYCN and CDK6 amplification in group 4α, SNCAIP duplication in group 4β, and CDK6 amplification in group 4γ [Group 4 MB is the most common form of MB and accounts for 35–40% of all MBs . SimilarDK6 (6%) . Cytogen/GFI1B 5–%, somatiDK6 (6%) . RecentlDK6 (6%) . Clinicagroup 4γ .Epigenetic regulation plays an important role in MB development, which mainly includes DNA methylation, histone modifications, ATP-dependent chromatin remodeling, and genomic structural variations. Importantly, epigenetic regulators serve as oncogenes or tumor suppressors in a context-dependent manner across the distinct subtype of MB ,34,35.DNA methylation is a well-characterized epigenetic mechanism, typically occurring on CpG islands of gene promoter regions, resulting in transcriptional repression during normal development and tumorigenesis . Based oMLL2/KMT2D and MLL3/KMT2C, two histone-lysine N-methyltransferases that regulate H3K4 methylation, were identified in 16% of MB [MLL2 mutations were slightly enriched in WNT and SHH subgroups, while MLL3 were found only in Group 3 and Group 4 MBs [KDM6A/UTX, a H3K27me demethylase binding to MLL2/3 complex, is the most frequently mutated gene and co-occurs with ZMYM3 mutations in Group 4 MBs [CREBBP and EP300, encoding histone acetyltransferases (HATs) CBP and p300 respectively, were found in MB [ALK in WNT, SMO and NTRK3 in SHH, LMO1, LMO2 and MYC in Group 3, and ETV4 and PAX5 in Group 4 MBs [MYC expression in Group 3 MB, suggesting therapeutic potential for this subgroup by using BET inhibitors  [GPS2, BCOR and LDB1, which is associated with histone deactylases (HDACs), are frequently mutated and active in SHH MB, suggesting effective response of HDACs inhibitors in the treatment of this subgroup tumors [Histone modifications play a crucial role in controlling chromatin structure and gene transcription, which include histone methylation, phosphorylation, acetylation, and ubiquitination. Genomics studies of MB provided strong evidence that alterations of histone modifiers result in deregulating the epigenetic machinery, particularly in modifications of lysine methylation and/or acetylation, which fundamentally contributes to MB development in the distinct subgroups ,40,41. Sup 4 MBs . In contup 4 MBs ,43,44. Hup 4 MBs ,44. In and in MB ,46; thesnd in MB . Based ond in MB . These sup 4 MBs . Anotherg., JQ1) ,50. In cp tumors .SMARCA4/BRG1 are the most common in WNT and Group 3 MBs than those in SHH and Group 4 MBs [CHD7 is frequently mutated in Group 3 and Group 4 MBs [CHD7 mutations have reduced EZH2 expression levels [ATP-dependent chromatin remodeling complexes, such as switch/sucrose non-fermentable (SWI/SNF) and chromodomain helicase DNA-binding (CHD), can utilize the energy from ATP hydrolysis to reorganize chromatin structure for regulation of gene expression. Recurrent mutations in SWI/SNF family members including up 4 MBs ,42,44. Oup 4 MBs ,52,53, iGFI1B or its paralogue GFI1 in Group 3 and Group 4 MBs [GFI1B and GFI1 locus or surrounding genomic regions, including deletions, inversions, duplications, and interchromosomal translocations. Topologically, these SVs juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, resulting in transcriptional activation of these oncogenes and malignant transformation in Group 3 and Group 4 MBs [Structural variations (SVs), including all structural and quantitative chromosomal rearrangements, not only contribute to the genetic diversity of the human genome, but also modulate basic mechanisms of gene regulation by altering higher-order chromatin organization . Importaup 4 MBs . Interesup 4 MBs .Genomic characterization of MB has identified the genetic and epigenetic heterogenicity but struggles to define functional biological processes involved in tumorigenesis. Proteomic and phosphoproteomic analysis could provide insight into discovering active oncogenic signaling pathways and mechanisms in MB. Using quantitative (phospho)-proteomics, Forget et al. defined highly divergent posttranscriptional pathway regulation in MB subgroups in a total of 41 flash-frozen primary MB tumors, particularly in Group 3 and Group 4 MBs . SpecifiThe four main histologic types of MB  recognized by the WHO are characterized based on their histological morphology, with separations made on the grade of nodularity, desmoplasia, and anaplasia ,3,60. OfAlthough MBs are thought to originate from primitive and undeveloped cells in the brain, the cell of origin for MB subgroups remains controversial. Most recently, three single-cell RNA sequencing (scRNA-seq) studies have provided a clearer picture of MB putative subtype-specific origins, highlighting the molecular and cellular diversity of MB across all subgroups, with the potential insights into understanding of tumor development and treatment response ,18,19. + glial lineage progenitors were identified to play a pivotal role in tumor initiation, therapy-resistance and recurrence [+ stem cells, which give rise to a variety of differentiated progeny including GNPs and UBCs [Based on investigation of associations between genotype and MB cell type, Gibson et al. discovered that WNT MBs arise outside the cerebellum from the lower rhombic lip (LRL) and embryonic dorsal brainstem, whereas SHH MBs are thought to originate from the cerebellar hemispheres . Furthercurrence . Interescurrence . In contcurrence ,18. Grouand UBCs ,18. In cand UBCs ,18. As fand UBCs . TogetheThe tumor microenvironment (TME) plays an important role in terms of tumor progression, evolution, and overall prognosis. TME encompasses the various signaling molecules, supporting cells, immune system cells, blood vessels, extracellular matrices, and nutrients that contribute to tumor progression and therapy response ,65. EmerThe blood–brain barrier (BBB), an anatomic structure consisting of a variety of cell types including endothelial cells (ECs), astrocytes and pericytes, is also an important factor in maintaining TME. Of interest, there is often a functional BBB that prevents the tumor from being exposed to potential chemotherapies found in the blood stream. However, WNT MB, compared to other MB subgroups, was identified to have a paucity of functional BBB, making this subset of tumors potentially more susceptible to chemotherapies that may not cross the BBB . Mechani+ T cells; PD-L1 expression is highest in WNT and SHH MBs, but lowest in Group 4 MB; Group 3 and Group 4 MBs have the largest number of cytotoxic lymphocytes and ECs [+ CD8+ T cells, resulting in a survival benefit in the Group 3 animals only after the treatment with PD-L1 and CTLA4 inhibitors [Infiltration of various immune cells in TME is of great interest because these infiltrating leukocytes either interfere with tumor progression or promote tumor growth, underlying response and efficacy of immunotherapy. Recent studies based on the quantification of gene expression signatures uncovered dramatical diversity of immune TME among the MB subgroups ,69. Of i and ECs . Importahibitors . Therefohibitors . NonetheClinically, a physical examination aligning with neuroimaging, biopsy, and cerebrospinal fluid tests is in general being used for MB diagnosis. In 2016, the World Health Organization (WHO) classification of the Central Nervous System (CNS) tumors initiated an integrative approach including molecular parameters in combination with histology for MB diagnoses . FurtherThe therapies for MB treatment are currently based on the patient’s risk factors, and consist of surgery, radiotherapy (RT), and chemotherapy (CT) ,75. AlthCraniospinal irradiation (CSI) is usually a follow-up treatment after surgery for children older than age 3 years. Based on patient risk, the treatment dose is 23.4 Gray (Gy) for standard-risk patients and 36–39 Gy for high-risk patients ,79,80,81With increasing knowledge in MB genomics and biology, precision medicine is an emerging approach to clinical care that takes into account tumor genetic make-up and individual variations. Based on molecular classification of MB, we summarized the completed and ongoing clinical trials in PTCH1 or SMO can benefit from SMO inhibitor, vismodegib [SUFU mutation, MYCN and GLI2 amplifications, may not benefit from vismodegib treatment, and patients may also develop irreversible growth plate fusions after vismodegib treatment, which all limit widespread clinical application [SHH MB with recurrent mutations in smodegib ,85. Howelication ,87,88,89MYC or MYCN amplification, metastasis, and young age (<3 years of age). A multicenter clinical trial is ongoing in the comprehensive evaluation of current treatment options by integrating molecular subgroup and risk stratification status into the trial design (NCT01878617), in which Group 3/Group 4 MBs are prioritized for more rigorous treatment with pemetrexed and gemcitabine [MYC plays an important oncogenic role in many cancers, it is challenging to be directly targeted by small molecules and antibodies due to a lack of an enzymatic active site and its nuclear location [MYC expression and thereby induce cell death [MYC/MYCN amplification (NCT03936465). Furthermore, ongoing trials aim to evaluate the inhibition of checkpoint kinases  alone or in combination with CT drugs in brain tumors including recurrent and refractory SHH, Group 3/Group 4 MBs . Group 3 and Group 4 MBs have heterogenous clinical characteristics and outcomes associated with citabine . While Mlocation . Given tll death , a pediaRecent advances in cancer genomics, single-cell sequencing, and sophisticated tumor models have revolutionized our understanding of the biology of MB. It is becoming increasingly clear that MB is a heterogeneous disease with a high degree of diversity on various molecular and cellular levels. Four major subgroups of MB  display tremendous subtype-specificity in genetic and epigenetic alterations, proteomic landscape, cell-of-origin, tumor microenvironment, current therapies, and clinical trial design. Undoubtedly, these findings shed unprecedented light on the development of tailored treatment for children with MB. However, the side effects of current therapies are still a major obstacle to successful MB treatment. In the future, greater emphasis needs to be placed on the molecular characterization of MB in the clinic, as identification of the individual subgroups at diagnosis could help shape the treatment and care of the patients, and potentially improve the overall survival. Furthermore, as further elucidation of the activated pathways becomes known, precise and effective therapies targeting the driver mutations in subtype-specificity can be made available to the patients with this devastating brain tumor in children.


Furthermore, 21 of the differentially expressed genes reported in our previous RNA-Seq study were predicted as target genes for some of the 71 miRNAs. Gene ontology and KEGG pathway analyses showed that these targets were enriched for functions such as metabolism of protein and fat, and development of mammary gland, which indicating the critical role of these miRNAs in regulating the formation of milk protein and fat. With dual luciferase report assay, we further validated the regulatory role of 7 differentially expressed miRNAs through interaction with the specific sequences in 3′UTR of the targets. In conclusion, the current study investigated the complexity of the mammary gland transcriptome in dairy cattle using small RNA-seq. Comprehensive analysis of differential miRNAs expression and the data from previous study RNA-seq provided the opportunity to identify the key candidate genes for milk composition traits.A total of 31 differentially expressed genes in the mammary glands were identified in our previous study using RNA sequencing (RNA-Seq), for lactating cows with extremely high and low milk protein and fat percentages. To determine the regulation of milk composition traits, we herein investigated the expression profiles of microRNA (miRNA) using small RNA sequencing based on the same samples as in the previous RNA-Seq experiment. A total of 497 known miRNAs  and 49 novel miRNAs among the reads were identified. Among these miRNAs, 71 were found differentially expressed between the high and low groups ( MicroRNAs (miRNAs), which are a class of non-coding small RNA (sRNA) molecules with the length of 18-24 nucleotides, are important regulators of gene expression. They can play important roles in a wide range of biological processes, including animal and plant development, cell differentiation, proliferation, apoptosis, and metabolism . In animThe bovine mammary gland is a complex organ which grows and develops after calving and is able to produce more than 30,000 kg of milk in a complete lactation cycle . BecauseIn a previous study from our lab , 31 diffhigh group vs. low group) same as our previous RNA-Seq experiment (high group) had high PP (3.6% and 3.8%) and FP (3.9% and 4.5%); the other two cows (low group) showed low PP  and FP .In the current study, the mammary gland epithelium samples of four lactating Chinese Holstein cows . Total RNA was extracted from one piece of mammary gland epithelium samples from each cow and quality was controlled according to the protocols described by The preparation of small RNA library, including quality control and sequencing, was performed by Novogene . The preparation of library was performed on 3 μg total RNA per sample using an IlluminaTruSeq™Small RNA Sample Preparation Kit . The samples were indexed using four codes in order to facilitate sequencing of these samples on one flow cell channel. Quality control in library preparation showed that adapter-adapter contamination was <5% and 85% of the sequences were miRNAs. The samples were subsequently sequenced on the Illumina Hiseq2000 platform and 50-bp single-end reads were obtained.The sequencing data were obtained in the format of Illumina FASTQ (Illumina). The procedure of data filtering included removing low quality reads, reads containing poly-N stretches, reads with 5′primer contaminants, reads with 3′primers or the insert tag, and reads with poly-A, T, G, or C stretches. Thereafter, the sRNA tags within a certain range (18-30 nt) were retained for the successive steps. The Q20, Q30, and GC-content of the cleaned reads were calculated to evaluate the quality of data. Then, the sRNA tags were mapped to the bovine genome assembly (UMD3.1.66) using Bowtie , no mismThe expression of miRNA was measured as counts per million (CPM) using the following formula: normalized expression = mapped read count/total reads × 1000000 , and DESFurthermore, two cows in the same group were used to eliminate the background noise of individual-specific transcription by applying a pairwise approach, which enabled acquisition of more relevant data from the two groups.TargetScan 6.2 and MiRanda  were useGene ontology (GO) functional enrichment analysis was used for the candidate target genes of the miRNAs. GOseq with the Wallenius non-central hyper-geometric distribution , which c–ΔΔCT method and normalized against the U6 gene  for each sample.Expression levels of selected miRNAs were confirmed by quantitative real-time PCR (qRT-PCR) using the DyNAmo SYBR Green PCR kit  on a LightCycler480 . qRT-PCR of target mRNAs was performed using specific miRNA stem-loop primers  and all TRIB3, M-SAA3.2, PTHLH, and VEGFA, were PCR amplified using DNA collected from the bovine mammary gland samples applied for sequencing in this study as a template, and connected into pmirGLO Dual-Luciferase miRNA Target Expression Vector   of four predicted target genes for the identified miRNAs, Promega) , respect deleted , 3. AfteTRIB3, M-SAA3.2, PTHLH, and VEGFA) expression, the full-length TRIB3, M-SAA3.2, PTHLH and VEGFA 3′UTRs and the corresponding mutant version (the seed sequences were deleted) were transfected into human embryonic kidney HEK293 cells , respectively. These cells were cultured at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4.5 g/liter glucose, 5% fetal bovine serum (Invitrogen), 2 mmol/liter glutamine, and antibiotics. Before transfection, HEK293 cells were plated into 24-well plates at 1.0 × 105 cells/well 24 h. 30 ng empty pmirGLO vector, pmirGLO-TRIB3/M-SAA3.2/PTHLH/VEGFA-3′UTR with 50 μl opti-MEM (Invitrogen) and 30 nM  mimic miRNA, inhibitor miRNA, control miRNA (GenePharma) were co-transfected into each well with 1 μl Lipofectamine 2000 (Invitrogen). 30 ng mutants of the TRIB3/M-SAA3.2/PTHLH/VEGFA 3′UTR with 50 μl opti-MEM (Invitrogen) and 30 nM  mimic miRNA, control miRNA (GenePharma) were co-transfected into each well with 1 μl Lipofectamine 2000 (Invitrogen). Relative firefly luciferase activities  were measured 24 h after transfection with the Dual-Luciferase Reporter Assay Kit (Promega) on TECAN Infinite 200 multifunctional microplate reader (TECAN). All experiments were performed in triplicate so that data averaged from three independent experiments.To further explore the repressing mechanism of miRNAs on the expression of 4 target genes . Distribution of the length for reads showed that most of the generated reads had 21 (>24%), 22 (>30%), and 23 (>13%) nucleotides (2) between the two individuals within the high and low groups for milk PP and FP was calculated based on the CPM mapped fragment of each cow and was shown to be 0.988 and 0.980, respectively. This indicated that the similarity of the two biological replicates within each group was sufficiently high , 12,745,512 (high milk PP and FP), 9,744,027 (low milk PP and FP), and 9,682,136 (low milk PP and FP) high-quality cleaned reads were obtained from the four sRNA libraries , κ-casein (CSN3), α-lactalbumin (LALBA), diacylglycerol O-acyltransferase 2 (DGAT2), growth hormone receptor (GHR), signal transducer and activator of transcription 5B (STAT5B), and stearoyl-coenzyme A desaturase (SCD) etc. This finding implied that the identified mammary miRNAs in this study were involved in metabolism of milk protein and lipid through the regulation of key genes affecting these traits.Among the uniquely aligned reads across the four samples and six downloaded miRNA libraries , 24,320,p < 0.05, FDR q < 0.05), with 35 were up-regulated and 36 were down-regulated in the high milk PP and FP group compared with the low group , and the expression levels of miR-21-3p, miR-29c, miR-106b and miR-190a were lower in the high group than in the low group (p < 0.05). Whereas, Novel-13, Novel-2, Novel-22, Novel-32, Novel-4 and Novel-42 did not display significant differences on miRNA levels between the two groups (p > 0.05)  . Such exp < 0.01, FDR q < 0.01). Under the GO biological process category, the enriched terms related to lipid and protein metabolisms and cell growth were included such as protein binding, protein localization, protein transport, protein complex, regulation of protein metabolic process, lipid biosynthetic process, programmed cell death, protein targeting, lipid metabolic process, amino acid transport, regulation of protein kinase activity, and cellular response to mechanical stimulus  annotation analysis was performed. It was found that these targets have a wide range of diverse functions, among which most were involved in protein and lipid metabolism, mammary gland development and differentiation, and immune functions , adipocytokine, mammalian target of rapamycin (mTOR), glycosphingolipid biosynthesis, glycerophospholipid metabolism, hypoxia inducible factor-1 (HIF-1), and phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) signaling pathways .For the 71 top half differentially expressed miRNAs, 5,634 target genes were obtained and the targets were highly enriched in biological process consisting of synthesis and metabolism of protein and energy metabolism, as well as pathways mainly related to synthesis and metabolism of lipid and protein including glutathione metabolism, NF-kappa B signaling pathway, mTOR signaling pathway, fatty acid degradation, fatty acid metabolism and protein processing in endoplasmic reticulum .TRIB3), serum amyloid A1 (SAA1), serum amyloid A3 (SAA3), mammary serum amyloid A3 (M-SAA3.2), vascular endothelial growth factor A (VEGFA), parathyroid hormone-like hormone (PTHLH) and ribosomal protein L23A (RPL23A). In addition, KEGG pathway analysis using KOBAS, showed that two of the 21 target genes, DNA-damage-inducible transcript 3 (DDIT3) and nuclear receptor subfamily 4, group A, member 1 (NR4A1), were involved in the MAPK signaling pathway that plays critical role in protein synthesis and metabolism and fatty acid metabolism pathway  and cyclin-dependent kinase inhibitor 1A (CDKN1A), were involved in the mTOR, HIF-1, PI3K-Akt, p53 and duct acid secretion signaling pathways, which are mostly related to synthesis and metabolism of protein and fat , while the inhibitor of miR-2904 yielded the same luciferase level as negative control (p > 0.05) , and the depressed expression of such miRNAs did not change the luciferase level in HEK293 cells transfected with their inhibitors (p > 0.05), respectively , respectively  . Howeverectively . When thectively . When thad libitum or deprived of food affecting milk composition reported by The current study is the first comparative profiles of the mRNA and miRNA transcriptome in the mammary gland epithelium of dairy cows to the best of our knowledge. In this study, we generated an extensive miRNA expression profile of the mammary glands from lactating cows with extremely high and low milk PP and FP, and identified a total of 497 known bovine miRNAs and 49 novel bovine miRNAs. In previous studies, bovine miRNAs were identified using computational and direct cloning approaches . Li et aGHR, which is a major gene for milk composition traits , VEGFA, parathyroid hormone-like hormone (PTHLH), cation transport regulator homolog 1 (CHAC1), and NR4A1. Therefore, miR-15a was considered may affect milk composition by regulating the expression of these genes, although miR-15a was not one of the differentially expressed miRNA identified in this study. It was reported that miR-23b inhibited the expression of the transforming growth factor-beta (TGF-β) signaling  were predicted to be targets of some of the identified miRNAs, which implied that they may play critical regulatory roles in mammary gland development and milk composition.miR-15a has been reported to be critical in cell development , cell cyn traits . In the ignaling . In the ignaling . In addiSAA1), serum amyloid A1 (SAA3), and mammary serum amyloid A3.2 (M-SAA3.2) were predicted to be regulated by miR-146b ; VEGFA was regulated by miR-125a, miR-125b, miR-106b, and miR-2904; and ribosomal protein L23a(RPL23A), tribbles homolog 3 (TRIB3), and PTHLH were regulated by miR-378c, miR-2904, and miR-106b, respectively. Moreover, Cai et al., performed RNA sequencing with mammary gland tissue samples from six Chinese Holstein cows with three extremely high and three low milk protein percentage phenotypes and miR-2904, miR-339b, miR-146b, miR-339a, miR-29c, miR-106b, miR-190a, miR-21-3p, miR-15a, miR-486, miR-135, miR-101a, miR-152 and miR-139 were found differentially expressed, which were also identified in our study and targeted on four differentially expressed genes . These seven genes represent the most promising candidates may affect milk PP and FP in dairy cattle (BRMS1) has already been implicated, and inhibited both migration and invasion related to metastasis , miR-21-3p, miR-224, miR-31, miR-345-5p, and miR-3431. mTOR signaling is known as playing a fundamental role in adipogenesis (STAT5A) in mammary epithelial cells, and the phosphorylation of STAT5 is known to play important roles in the regulation of milk protein gene expression and mammary development  or some unknown biological mechanisms. Actually, target prediction was only the first step for studies on interaction between miRNA and their targets. The miRNAs and targets with reverse expression patterns will be considered as the key components for further validation.Considering that microRNAs regulate gene expression by targeting specific sequences in the 3′UTR of their cognate genes , the regIn this study, only two biological replicates, which were the same as in our previous RNA-seq investigation , were usTRIB3, M-SAA3.2 and PTHLH were significantly down-regulated by miR-2904, miR-29c/miR-146b/miR-339a and miR-339b/miR-106b/miR-190a through binding the specific target sequences in 3′UTR of these genes, respectively.Using sRNA sequencing, 497 known bovine miRNAs and 49 novel bovine miRNAs were identified in the mammary glands of lactating dairy cows. Among all these miRNAs, 71 were differentially expressed between cows with the high and low milk PP and FP. Combined with our previous RNA-Seq data, 21 differentially expressed genes were predicted as the targets for some of the 71 differentially expressed miRNAs. Biological processes related to protein metabolism, fat metabolism, and mammary gland development were enriched for some of the identified miRNAs, which indicated that they may play critical roles in regulating of milk protein and fat traits in dairy cattle. Expression of the SRR3631014, SRR3631016, SRR3631053, and SRR3631054).The datasets GENERATED for this study can be found in NCBI SRA Accession Numbers:  at China Agricultural University who have reviewed and approved the experiments. Samples were collected specifically for this study following standard procedures with the full agreement of the Beijing Sanyuanlvhe Dairy Farming Center who owned the animals.XC and MY performed the RNA-related experiments, data analysis, and wrote the manuscript. SZ, CW, and QZ participated in the experimental design and helped interpret the results. XG participated in the interpretation of the results, writing, and revision of the manuscript. DS conceived and designed the experiments, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


KIR polymorphism associates with infection control and multiple autoimmune and pregnancy syndromes. Here we analyze KIR diversity of 241 individuals from five groups of Iranians. These five populations represent Baloch, Kurd, and Lur, together comprising 15% of the ethnically diverse Iranian population. We identified 159 KIR alleles, including 11 not previously characterized. We also identified 170 centromeric and 94 telomeric haplotypes, and 15 different KIR haplotypes carrying either a deletion or duplication encompassing one or more complete KIR genes. As expected, comparing our data with those representing major worldwide populations revealed the greatest similarity between Iranians and Europeans. Despite this similarity we observed higher frequencies of KIR3DL1*001 in Iran than any other population, and the highest frequency of HLA-B*51, a Bw4-containing allotype that acts as a strong educator of KIR3DL1*001+ NK cells. Compared to Europeans, the Iranians we studied also have a reduced frequency of 3DL1*004, which encodes an allotype that is not expressed at the NK cell surface. Concurrent with the resulting high frequency of strong viable interactions between inhibitory KIR and polymorphic HLA class I, the majority of KIR-A haplotypes characterized do not express a functional activating receptor. By contrast, the most frequent KIR-B haplotype in Iran expresses only one functional inhibitory KIR and the maximum number of activating KIR. This first complete, high-resolution, characterization of the KIR locus of Iranians will form a valuable reference for future clinical and population studies.Natural killer (NK) cells are innate lymphocytes that eliminate infected and transformed cells. They discriminate healthy from diseased tissue through killer cell Ig-like receptor (KIR) recognition of HLA class I ligands. Directly impacting NK cell function,  Natural killer (NK) cells are essential for human immunity to infection and cancer, and for successful reproduction , 2. To dKIR locus spans 150–350 kbp of chromosome 19q13.4  and two pseudogenes (KIR2DP1 and KIR3DP1)  , 11. KIRKIR3DP1) . KIR havelection . The KIR alleles . KIR-A hisorders . Additioplotypes , 19.KIR affects cell surface expression, ligand specificity, ligand binding strength, and intracellular signaling   , diverge (Yucpa) –29, Euro (Yucpa) , 31, Eas (Yucpa) , and Oce (Yucpa) . Togetheorldwide . HLA-B*5 KIR3DL1 , 36. In KIR locus diversity of three indigenous Iranian populations was determined to high-resolution by analyzing genomic DNA from 241 healthy unrelated donors  and recombinant alleles. Novel allele sequences were analyzed by visual inspection: reads specific to the relevant gene were isolated by bioinformatics filtering, aligned to the closest reference allele using MIRA 4.0.2 . The populations used were Japanese (N = 115) (N = 61) (N = 131) (N = 49) (N = 378) (N = 79)  , Yucpa A(N = 61) , GhanaiaN = 131) , Māori f(N = 49) , EuropeaN = 378) , and Kho(N = 79) .KIR genes of 241 individuals from five populations, representing three groups of Iranians; the Lurs, Kurds and Baloch. The Lurs comprised individuals from the cities of Khoramabad, Lordegan and Yasuj. Only two KIR genes were present as two copies (2N) in every individual, KIR3DL3 at the centromeric end of the KIR locus and KIR3DL2 at the telomeric end  as well as KIR2DL5, the most frequent allele observed is absence of the gene . Also encoded are KIR2DL1*004, an attenuated receptor  and a full-length KIR2DL4 (*005). This combination of common centromeric and telomeric KIR-B haplotypes can therefore provide the maximum number of activating KIR. The most frequent KIR-A haplotype encodes four inhibitory receptors specific for polymorphic HLA class I and carries KIR2DS4*003, which is not-expressed because of a 22 bp deletion in exon 5  compared to the other six populations as well as to all other populations analyzed to date  than Kurd , Lorestan  and Yasuj . KIR3DL1*004 is retained in the cytoplasm and unable to bind HLA-Bw4+ HLA-A or -B on target cells . Contributing to these differences is the high frequency of HLA-B*51 in the Baloch  than Europe   , 59. We  disease . It is udiseases –63. AmonKIR-A and -B haplotypes are present in all human populations, where they are maintained by balancing selection, likely because KIR-A haplotypes favor infection control, particularly viral infections, and KIR-B haplotypes favor successful fetal implantation (KIR-A haplotypes express all possible inhibitory receptors specific for HLA class I, whereas KIR-B haplotypes express fewer inhibitory receptors but more activating receptors. Thus, whereas the inhibitory KIR help prime NK cells to be able to be responsive to HLA class I loss during infection, the activating KIR can both promote fetal trophoblast invasion and recognize specific pathogen-derived peptides to control certain infections (KIR-A and KIR-B haplotypes is extreme. The common KIR-A haplotypes express no activating KIR, due to the high frequency of the truncated KIR2DS4 variant, and the common KIR-B haplotypes provide the maximum number of activating receptors. In this regard, the KIR-B haplotypes differ considerably from those of Africans and were likely obtained since the out of Africa migration, through adaptive introgression with ancient humans (antation , 16. Accfections , 65. In t humans .KIR locus at allelic resolution in Iranian populations and place it in the context of the HLA ligands recognized by KIR. Iran is a culturally diverse country and the ethnic groups we have studied comprise ~15% of the population (KIR gene content diversity is observed across Iran (In summary, we describe the pulation . Becauseoss Iran –71, it wAll relevant data are included in the article/The studies involving human participants were reviewed and approved by the Medical Research Ethics Committee of Shiraz University of Medical Sciences. The patients/participants provided their written informed consent to participate in this study.PP, EA, AG, and PN conceived and designed experiments. EA, NN-G, and ST performed lab experiments. CA, PN, JAT, LG, NN-G, and ST analyzed data. AG, PP, WM, JH, JT, and LM provided materials. CA, PN, and PP wrote the paper. All authors approved the final submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


KRAS and the tumor suppressors TP53, CDKN2A, and SMAD4. Although the presence of few drivers, several signaling pathways are involved in the oncogenesis of this cancer type, some of them with promising targets for precision oncology. Pancreatic cancer is recognized as one of immunosuppressive phenotype cancer: it is characterized by a fibrotic-desmoplastic stroma, in which there is an intensive cross-talk between several cellular  and acellular  components. In this review; we aim to describe the current knowledge of the genetic/biological landscape of pancreatic cancer and the composition of its tumor microenvironment; in order to better direct in the intrinsic labyrinth of this complex tumor type. Indeed; disentangling the genetic and molecular characteristics of cancer cells and the environment in which they evolve may represent the crucial step towards more effective therapeutic strategiesThe threatening notoriety of pancreatic cancer mainly arises from its negligible early diagnosis, highly aggressive progression, failure of conventional therapeutic options and consequent very poor prognosis. The most important driver genes of pancreatic cancer are the oncogene  Pancreatic cancer (PC) is perhaps the most lethal among solid tumors worldwide; although incidence varies between different countries, PC is the seventh leading cause of global cancer-related deaths in industrialized countries, the fourth in Italy, and the third in the United States of America ,2,3. LonAs highlighted above, such PC unique aggressiveness is due to both specific genetic and molecular traits (acquired through either a stepwise or a chromothripsis-mediated simultaneous loss of multiple tumor suppressors and gain of multiple oncogenes), but to complex interactions with a generally obstructive and immunosuppressive tumor microenvironment (TME), which confer a highly malignant phenotype, as well  5,6].,6.5,6].Although combination chemotherapy has to some extent improved cure rates, survival, and quality of life in both early and advanced PC, anti-angiogenic drugs, molecularly targeted agents and immune checkpoint inhibitors have proven ineffective, so far, in PC ,8. Even KRAS (Kirsten rat sarcoma), TP53 (tumor suppressor protein 53), CDKN2A (cyclin dependent kinase inhibitor 2A), and SMAD4 (SMAD family member 4), and molecular pathways) has been elucidated to an unprecedented extent; on the other hand, it is now clear that an intense crosstalk between stroma and cancer cells, mediated by soluble factors, leads to extensive fibrosis and deposition of extracellular matrix (ECM) proteins, resulting in poor perfusion and hypoxic conditions which, in turn, favor the evolution of aggressive clones  and KRAS, which are altered in up to 60% and to 80% of cases, respectively ,11273,11+ myofibroblasts correlated with poor survival of PC patients, thereby leading to the hypothesis that fibrosis constitutes a “protective” TME, rather than a tumor promoter. Alpha-SMA+ myofibroblasts knock-out mice display multiple adverse outcomes, such as reduced immunosurveillance and gemcitabine resistance; chemoresistance could be reverted by a combination of gemcitabine plus anti-CTLA-4 treatment, which resulted in increased animal survival [On the other hand, in other reports low levels of α-SMAsurvival .+ and CD4+ T cells) [Another important element in PC stroma is represented by endothelial cells and the mutual influence between endothelial cells and PC microenvironment is now well established. Indeed, the mere presence of conditioned medium derived from hypoxic PSC increases angiogenesis by inducing endothelial cells proliferation and migration in vitro and in vivo . ConversT cells) . MoreoveT cells) .In PC, cellular stromal components are also characterized by different immune cells which influence each other with endothelial cells to define an immunosuppressive TME.+ and CD11c+), they exhibit pro-inflammatory properties; conversely, M2-polarized macrophages express CD163 and CD204 and exert tumor-promoting functions [Macrophages infiltrate PC attracted by vascular endothelial growth factor (VEGF)/VEGFR2 interactions  and affeunctions ,122. In unctions ,124. In unctions . In lineunctions . In partunctions . Consistunctions .+ neutrophils and CCR2+ macrophages results in an increased antitumor immune response and response to the chemotherapy treatment with the FOLFIRINOX regimen, due to the reduction of infiltrating myeloid cells in the tumor [Similar to macrophages, neutrophils represent another group of myeloid-derived circulating cells belonging to innate immunity. Despite their role in TME is still debate, the increasing knowledge of this cell type led to differentiate tumor associated neutrophils in N1 (with anti-tumor properties) and N2 (with pro-tumor properties), according with the type of cytotoxicity exerted and the ability to activate different immune cells . Particuhe tumor .+ T cells are classified in T helper CD4+, Treg CD4+/CD25+/Forkhead box P3  sub-populations are more represented than the tumor-killing Th1 (T-bet+) and their levels correlate with poor prognosis [+ cells characterized by the production of IL-17 (Th17) has been shown to play a pro-tumor role in PC, where both tumor-infiltrating Th17 CD4+ cells and circulating levels of Il-17 and IL-23 correlated with advanced stage and worse OS [+, CD8+, macrophages and dendritic cells—DC) [Among immune cells, T lymphocytes are the most represented population and significantly influences the clinical outcome of PC patients . Accordixic CD8+ . CD4+ T etter OS ,132. On rognosis ,134. Tasrognosis . Anotherworse OS . The welworse OS ,136. In ells—DC) . Accordiells—DC) ,138. Howells—DC) .+ are cytotoxic T cells which may curtail the proliferation of tumor cells through two mechanisms: macrophages and interferon (IFN)-γ, activation [+ T cells are rarely detectable in PC and their presence is associated with better prognosis  prevents MDSC recruitment and tumor development [MDSC are a heterogeneous group of immature cells classified in polymorphonuclear granulocytic MDSC and mononuclear monocytic MDSC, although they lack specific surface markers . MDSC ar8+ cells ,82. Indecells [+ ,146. Morcells [+ . Due to elopment .+ and CD8+ T cells [+/CD8+ tumor infiltration and an improved patient prognosis [Antigen-presenting DC cells exert their effects in antitumor response mainly through MHC-mediated activation of CD4 T cells . Due to  T cells ,150. Levrognosis ,151,152.rognosis . Althougrognosis ,155.PC is histologically characterized by the abundance of ECM which, in addition to mechanical support, influences both the cellular and molecular composition of pancreatic stroma . This thCollagens are one the best characterized and most abundant components of PC’s ECM and 28 types with at least a triple-helical domain are currently identified . In TME,2+ metalloprotein and is the first proteoglycan shown to be involved in the regulation of cell growth, by inhibiting TGF-β, ERBB2 dimerization and receptor tyrosine kinase signaling [p = 0.0006). In the same paper, they also demonstrated that lumican increases apoptotic signals via hypoxia-inducible factor (HIF)1-α blockade in PC cell lines in vitro [Many of the ECM-characterizing proteins are glycoproteins, a large group of molecules divided in four families, according to cellular and subcellular localization, genetic homology and protein activity . Proteogignaling ,171,172.ignaling ,173. Könignaling . Biglycaignaling . More spignaling . Yamamotignaling . Li and in vitro . Hence, in vitro .+ cytotoxic T cell infiltration in PC; FAK inhibition limited tumor progression, markedly reduced tumor fibrosis, decreased the numbers of tumor-infiltrating immunosuppressive cells, and rendered the previously unresponsive KPC mouse model responsive to T cell immunotherapy and programmed cell death protein (PD)-1 antagonists. [Fibronectin is the most abundant acellular component within PC tumor stroma and is mainly produced by aPSC ,177,178.gonists. .Cytokines are a family of soluble factors, classified into growth factors, chemokines, angiogenic factors and interferons and involved in tumor immune-landscape, by exerting pro-  and anti-inflammatory (IL-10 and TGF-β) functions . In TME,p = 0.056), especially when combined with high IL-6 levels  [IL-1β is a pro-inflammatory cytokine synthetized by monocytes, tissue macrophages, and PC cells; through the binding to the type 1 IL-1R, shared with IL-1α, the cleaved form of IL-1β activates NF-κB in PC cells . Etoposio below) .p = 0.0001) [IL-6 is another proinflammatory cytokine, which activates JAK 2 and signal transducers and activators of transcription (STAT)1 and STAT3, through the IL-6 presentation to the signal-transducer gp130 by IL-6 receptor . Once ac 0.0001)  and IL-6 0.0001) .p = 0.012) and shorter OS in PC patients (p < 0.001) [Initially identified as a neutrophil-activating peptide, IL-8 is now a well-established proinflammatory chemokine, which signals through the binding to the CXCR1/2 receptors, thereby activating several G-protein-mediated signaling cascades involved in conferring several pro-tumoral properties to cancer cells . Indeed,< 0.001) . Further< 0.001) .Another pleiotropic pro-inflammatory cytokine often overexpressed in PC is MIF. Although MIF was initially identified as a specific product of activated T lymphocytes, it is currently known that macrophages represent the main source of this soluble factor: TNF-α activation regulates MIF production and viceversa in an auto-amplification loop . MIF int+ T cells and activated B cells are the main source of IL-10 production in TME. The IL-10R1 is involved in the activation of specific IL-10 functions and the heterodimerization of IL-10R1 and IL-10R2 chains is required for outside-in cell signaling [+ T-cell response induced by mesothelin, an immunogen involved in promoting metastasis and tumor proliferation and a potential therapeutic target in PC [Monocytes, macrophages, Th2 CD4ignaling . As an iignaling . IL-10 iet in PC . Consistet in PC . IL-10 let in PC ,102.KRAS mutant contexts [+ differentiation in co-culture with PC cells, TGF-β blockade improves antigen specific response and decreases T cells apoptosis; as a consequence, DC vaccination in combination with TGF-β blockade results in increased DFS in murine tumor models [TGF-β plays an essential, but also complex and controversial, role in PC. TGF-β maintains homeostasis in normal tissue; however, being genetically unstable entities, cancer cells have the capacity to corrupt this suppressive influence. As a consequence, through the binding to the TGF-βRI and TGF-βRII heterotetrameric complexes, pathological forms of TGF-β signaling promote tumor growth and metastasis by activating downstream canonical Smad-dependent, as well as Smad-independent, intracellular pathways ,204,205.contexts . TGF-β ir models . In PC, r models ,106.Similar to TGF-β, TNF-α plays a controversial role in PC stage progression and therapeutic response. Indeed, TNF-α exerts its activity as both a pro-tumoral and an anti-tumoral soluble factor . This duThe overall five-year survival rate for PC patients remains below 10%  and although median OS in advanced disease has improved with modern chemotherapy it remains below one year . ApproxiSo far, results from clinical trials testing agents targeting cell autonomous signaling aberrations and angiogenesis have been quite disappointing in PC. Clinical trials investigating inhibitors of the EGFR family of receptors, of the MAPK and of the Hedgehog signaling pathways have failed to demonstrate an improvement in OS in PC patients ,216,217.BRCA mutations and several case studies reported responses to poly-ADP-ribose-polymerase (PARP) inhibitors [So far, alterations in the DNA damage repair machinery appear to be the most promising target(s) in PC . Five tohibitors ,222. Whihibitors ,223. Addhibitors .More recently, attempts at modulating TSI with agents targeting different TME components, have also failed: pegvorhyaluronidase alfa, an agent targeting tumor hyaluronan, napabucasin, an agent targeting cancer cell stemness, recombinant pegylated IL-10, and a combination of anti-CTLA-4 and anti-PD-L1 antibodies have all recently shown lack of efficacy in clinical trials in advanced PC patients.p < 0.01; for IP-10 mOS 95% CI high vs. low, 3.6 (2.5–7.7) vs. 12.5 (7.1–17.0) months, HR 95% CI = 2.78 (1.55–4.99) p < 0.001) [p < 0.01; for IP-10, 10.9 (5.5–12.4) vs. 3.6 (2.5–4.0) months, HR 95% CI = 0.4 (0.23–0.67) p < 0.0015) [+ lymphocytes around PC [The largest effort of integrated genomic analysis in understanding the molecular pathology of PC has recently confirmed the TGF-β as the most recurrently mutated signal transduction pathway in PC . The the< 0.001) . On the  0.0015) . These r 0.0015) ,243,244.round PC . Based oround PC . AlthougPC remains arguably the deadliest of solid tumors. Advances in the understanding of the genomic and transcriptomic complexity and heterogeneity of this disease, as well as elucidation of basic biological mechanisms underlying PC transformation and progression to a highly malignant phenotype, are of paramount importance to drive further clinical research and improve survival. However, clinical experiments clearly show that, with few exceptions, targeting individual players within the extremely complex and multifaceted crosstalk between the cancer cell genetic background, soluble factors, and cellular and acellular components of a usually highly desmoplastic stroma represents an oversimplification endowed with little potential for clinical efficacy.KRAS mutations are associated to an increased production of the GM-CSF, which causes the expansion of immunosuppressive Gr1+CD11b+ myeloid cells in mouse PC models; GM-CSF inhibition, in turn, could prevent MDSC recruitment and tumor development [Research efforts should thus concentrate on dissecting the currently elusive Ariadne’s string that links early occurring genetic aberrations of transformed epithelial cells, with their ability to secrete specific soluble factors  which, in turn, will shape the molecular and cellular composition of the TME; TME, on the other hand, will respond to tumor growth (and presumably to the selective pressure applied by treatments) by activating different sets of soluble factors which, in turn, will influence tumor cell behavior and reshape the TME. In depth understanding examples of how the tumor genetic background can influence TME composition and response to treatments include, for example, evidence that loss of the PTEN tumor suppressor results in immunotherapy resistance, by directly inducing the T-cell inhibitor PD-L1 in several malignancies, including in mouse pancreatic cancer models ,248,249.elopment .+ cytotoxic T cell infiltration; inhibition of intracellular FAK signaling reduces tumor fibrosis, decreases the numbers of tumor-infiltrating immunosuppressive cells, and restores PC sensitivity T cell immunotherapy and PD-1 antagonists [TME, on the other hand, may activate tumor cell signaling pathways which, in turn, cause profound TME remodeling: for example, FAK by PC cell’s integrin binding to fibronectin and collagens induces fibrosis and poor CD8agonists .Cytokines secreted by either PC cells or stromal elements in the TME have pleiotropic, and sometimes unpredictable, effects: for example, both CAF and aPSC act in an autocrine and paracrine fashion in order to modulate the activity of the other cellular types in TME, such as endothelial and immune cells . Cancer Finally, TME components  provide clinical proof of concept supporting the proposed biological mechanism; (2) identify relevant biomarkers; (3) lay solid foundations for success in phase III clinical trials.


Injection of rats with STZ induced hyperglycemia. Rats with STZ-induced diabetes, with or without AFB1 intoxication, had significantly elevated activities of serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, and levels of serum urea, creatinine, cholesterol, 8-oxo-2′-deoxyguanosine, interleukin-1β, interleukin-6, and tumor necrosis factor-α. In addition, these rats exhibited increased lipid peroxidation and reduced glutathione concentration and activities of superoxide dismutase, catalase, and glutathione peroxidase enzymes in the hepatic and renal tissues. In contrast, administration of FUC to diabetic rats, with or without AFB1 intoxication, ameliorated the altered serum parameters, reduced oxidative stress, DNA damage, and inflammatory biomarkers, and enhanced the antioxidant defense system in the hepatic and renal tissues. These results indicated that FUC ameliorated diabetes and AFB1-induced hepatorenal injuries through alleviating oxidative stress, DNA damage, and inflammation.The current study was carried out to evaluate the ameliorative effect of fucoidan against aflatoxicosis-induced hepatorenal toxicity in streptozotocin-induced diabetic rats. Sixty-four Wister albino male rats were randomly assigned into eight groups (8 rats each) that received normal saline, fucoidan (FUC) at 100 mg/kg/day orally for 4 weeks, streptozotocin (STZ) at 50 mg/kg/i.p. single dose, STZ plus FUC, aflatoxin B In developing countries, DM ranks as the 5of death . Diabetecretion) . Extensicretion) . Strepto animals .Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nominus as secondary metabolites. Humans are exposed to aflatoxins through ingestion of contaminated food [1 (AFB1) [1 8,9-epoxide by microsomal cytochrome P450. The resulting metabolite binds to DNA, RNA, and proteins, resulting in hepatic and renal damage [1 stimulates mRNA expression of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6) [1 exposure depend on the dose and duration of treatment [1 [1-disordered T1DM elevated energy-producing mechanisms, gluconeogenesis, lipid, and oxidative phosphorylation, reduced major urinary protein 1, insulin sensitivity indicator, and subsequently elevated blood glucose level [1 and diabetes in human subjects [Aflatoxins are produced by ted food . Storage1 (AFB1) . Its toxl damage . Exposurse level . There isubjects . In addisubjects .Saccharina japonica, Undaria pinnatifida, and Sargassum hemiphyllum, and some animal species as sea cucumber [In vitro and in vivo studies showed that FUCs have various biological activities such as hypoglycemic, nephroprotective, antioxidant, anti-inflammatory, anticoagulant, and antiviral effects [Fucoidans (FUCs) are highly sulfated polysaccharides, isolated from the cell walls of various species of brown seaweeds, such as cucumber . In vitr effects , 15. Man effects . The cur1 were purchased from Sigma Chemical Co. . Fucoidan  was obtained from Absunutrix Lyfetrition (USA). The kits, used for determination of blood glucose and serum metabolites levels, were obtained from BioDiagnostics Co. . ELISA kits, used to measure the serum levels of inflammatory cytokines, were obtained from R&D , while the kits for 8-OhdG measurement were purchased from Cayman Chemical .Streptozotocin and aflatoxin Bad libitum. Rats were maintained under these environmental conditions for one week for adaption before the beginning of the experiment. The experimental design was approved by the Research Ethical Committee of the Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt .Sixty-four Wister albino male rats of 180 to 200 g weights were bought from the Egyptian Organization for Biological Products and Vaccines. Rats were kept at 25 ± 2°C and 12 h light/dark cycle in a well-ventilated room. Rats were given an access to food and water Rats were randomly assigned into eight different experimental groups (8 rats each).The control rats were given normal physiological saline.The second group rats were given FUC at 100 mg/kg/day orally [y orally  between The third group rats were administered STZ at 50 mg/kg/i.p.  after 12 h fasting at the beginning of the experiment [periment .The fourth group rats were administered STZ as the third group and FUC as the second group.The fifth group rats were given AFB1 at 50 μg/kg/i.p. during the fifth and sixth weeks [th weeks .The sixth group rats were administered AFB1 as the fifth group and FUC as the second group.The seventh group rats were administered STZ as the third group and AFB1 as the fifth group.The eighth group rats were administered STZ as the third group, AFB1 as the fifth group, and FUC as the second group.The experiment design is illustrated in 2HPO4/NaH2PO4) (pH 7.4), containing 0.1 mM EDTA then centrifuged for 30 minutes at 5000 rpm. The supernatant was collected and maintained at -80°C for subsequent analysis.Blood samples were collected at the end of the experiment. Blood samples were left to clot at room temperature for 30 min and then centrifuged at 2500 rpm for 15 min, and sera samples were separated and stored at -20°C till biochemical assessment. The rats were later sacrificed by decapitation and the liver and kidney tissues were collected and washed with normal physiological saline solution. Then, tissue samples were homogenized in ice-cold buffer containing 50 mM sodium phosphate-buffered saline , urea, and creatinine were evaluated spectrophotometrically  , nitric The concentration of serum 8-oxo-2′-deoxyguanosine (8-OhdG) was determined by using 8-OhdG competitive assay kit  that detects free 8-OHdG and DNA-pound 8-OhdG.β, IL-6, and TNF-α were determined, by here using commercially available ELISA kits obtained from R&D  according to the manufacturers' instructions.The serum levels of IL-1P ≤ 0.05.All data were expressed as the means ± SEM, using SPSS software . Data were analyzed by here using one-way ANOVA followed by Duncan's post hoc test to test the significant differences between experimental groups. The differences among groups were considered statistically significant at 1 intoxication) with FUC (4th and 8th groups) significantly decreased blood glucose levels compared to diabetic, nontreated rats (3th and 7th groups). On the other hand, both AFB1 and FUC  had no significant effects on fasting blood glucose levels  was associated with significant increases in serum activities of ALT, AST, and ALP (that was most prominent in the combination group). In contrast, treatment of diabetic rats with or without AFB1 intoxication with FUC  normalized the activities of serum AST, ALT, and ALP. FUC alone had no significant effect on the activities of serum AST, ALT, and ALP compared to control rats  was associated with significant increases in serum urea and creatinine levels (that was most prominent in the combination group). However, treatment of diabetic rats with or without AFB1 intoxication with FUC  significantly reduced serum urea and creatinine levels, compared with the 5th and 7th groups. Treatment with FUC alone was not associated with significant changes in serum urea and creatinine levels compared with the control rats  was associated with significantly increased serum cholesterol levels in comparison to control rats. However, treatment of diabetic or nondiabetic rats intoxicated with AFB1, with FUC , normalized serum cholesterol levels, compared with the 3rd, 5th, and 7th groups. Treatment with FUC alone did not cause significant changes in serum cholesterol levels compared with the control rats  was associated with significant increases in hepatic and renal tissue concentrations of MDA and NO in comparison to control rats. However, treatment of diabetic and nondiabetic rats intoxicated with AFB1 with FUC  normalized MDA and NO concentrations in both hepatic and renal tissues  reversed the effects of both diabetes and AFB1 intoxication on the aforementioned parameters. Treatment with FUC alone significantly elevated GSH concentration and GSH-Px, SOD, and CAT activities in the hepatic and renal tissues compared with the control group  reduced serum 8-OhdG, IL-1β, IL6, and TNF-α levels compared to nontreated rats . FUC itself had no significant effects on the serum 8-OhdG, IL-1β, IL6, and TNF-α levels in comparison to control rats  and hence the production of inflammatory cytokines [The current study showed that injection of rats with STZ induced hyperglycemia, probably due to the irreversible cytotoxic effects of STZ on the ficiency . Oxidatificiency , 43. Simn et al.  and Zaban et al. . This maal cells , 45. To T Tables  and 3. TT Tables . In addiic acids . Diabetend TNF-α  leading ytokines . A formepression . In addiytokines .1 as evidenced by reduced blood glucose levels, activities of serum AST, ALT, and ALP, and serum levels of urea, creatinine, 8-OHDG, IL-1B, IL-6, and TNF-α. These findings were parallel with those of Wang et al. who concluded that FUC reduces STZ-induced hyperglycemia and kidney damage in rats [κB, protein kinase B, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase [α, IL-1β, and IL-6 in mice [1-induced hepatorenal damages through suppressing oxidative stress-induced DNA damage and proinflammatory cytokine production.On the contrary, treatment of diabetic rats with FUC ameliorated the hepatorenal toxic effects of DM and/or AFB in rats . The glu in rats . Similar in rats –57 and l in rats . These e in rats , 55. The in rats , 60, and in rats . In addin kinase . Moreove in mice . Further in mice . Therefo1-induced hepatorenal injuries are probably mediated by oxidative stress, DNA damage, and inflammation. However, treatment with FUC ameliorated DM and AFB1-induced hepatorenal injuries, mostly due to its antioxidant and anti-inflammatory effects.In conclusion, DM and AFB


Patients with COVID-19 have revealed a massive outbreak around the world, leading to widespread concerns in global scope. Figuring out the transmission route of COVID-19 is necessary to control further spread. We analyzed the data of 43 patients in Baodi Department Store (China) to supplement the transmission route and epidemiological characteristics of COVID-19 in a cluster outbreak. Incubation median was estimated to endure 5.95 days (2–13 days). Almost 76.3% of patients sought medical attention immediately uponillness onset. The median period ofillness onsetto hospitalization and confirmation were 3.96 days (0–14) and 5.58 days (1–21), respectively. Patients with different cluster case could demonstrate unique epidemiological characteristics due to the particularity of outbreak sites. SRAS-CoV-2 can be released into the surrounding air through patient’s respiratory tract activities, and can exist for a long time for long-distance transportation. SRAS-CoV-2 RNA can be detected in aerosol in different sites, including isolation ward, general ward, outdoor, toilet, hallway, and crowded public area. Environmental factors influencing were analyzed and indicated that the SARS-CoV-2 transportation in aerosol was dependent on temperature, air humidity, ventilation rate and inactivating chemicals (ozone) content. As for the infection route of case numbers 2 to 6, 10, 13, 16, 17, 18, 20 and 23, we believe that aerosol transmission played a significant role in analyzing their exposure history and environmental conditions in Baodi Department Store. Aerosol transmission could occur in some cluster cases when the environmental factors are suitable, and it is an indispensable route of COVID-19 spread.


Calcium reabsorption in the distal nephron of the kidney is functionally coupled to sodium transport. Apart from Ca2+ transport systems, CaSR signaling affects relevant distal Na+-(K+)-2Cl− cotransporters, NKCC2 and NCC. NKCC2 and NCC are activated by a kinase cascade comprising with-no-lysine [K] kinases (WNKs) and two homologous Ste20-related kinases, SPAK and OSR1. Gain-of-function mutations within the WNK-SPAK/OSR1-NKCC2/NCC pathway lead to renal salt retention and hypertension, whereas loss-of-function mutations have been associated with salt-losing tubulopathies such as Bartter or Gitelman syndromes. A Bartter-like syndrome has been also described in patients carrying gain-of-function mutations in the CaSR gene. Recent work suggested that CaSR signals via the WNK-SPAK/OSR1 cascade to modulate salt reabsorption along the distal nephron. The review presented here summarizes the latest progress in understanding of functional interactions between CaSR and WNKs and their potential impact on the renal salt handling and blood pressure.The kidney is essential for systemic calcium homeostasis. Urinary calcium excretion can be viewed as an integrative renal response to endocrine and local stimuli. The extracellular calcium-sensing receptor (CaSR) elicits a number of adaptive reactions to increased plasma Ca In this context, early epidemiologic studies demonstrated increased incidence of hypertension in individuals with restricted dietary calcium intake , calcitriol and calcitonin, enabling adaptive responses to systemic shifts of calcium homeostasis ,2,3,4. Tiew, see ). Intracransport . Via theransport ,10,11. Sransport ,15. The ransport ,17,18. T) in DCT ). NKCC2 ) in DCT ,21. NCC ) in DCT ,23 (for ) in DCT ). The exsegments ). Loss-osegments ,27. Simisegments . Patientsegments . Alternasegments . Excessisegments ,32). Thesegments ). Their segments . Gain-ofsegments . Similarsegments . Enhancesegments ,34. Aparsegments ,37. Dephsphatase ,39,40. Csphatase . Therefo review, ). Moreov review, ,44,45. TSince the cloning of mammalian CaSR, numerous efforts have been undertaken to determine its renal distribution, but the obtained results remain in part controversial. Initial analysis of CaSR expression sites in the rat kidney revealed presence of the CaSR mRNA in the glomerulus and along the entire nephron ,46. An a2+ and Mg2+ cations via claudin 16 (CLDN16) and CLDN19, which form heterodimeric paracellular divalent cation channels  . The pa review, ). CaSR m2+ from intracellular stores, and decrease of intracellular cAMP concentration .2+]o stimulate biosynthesis of cytochrome P450 and phospholipase A2-derived eicosanoids, which suppress the ROMK function [+ transport in TAL is also sensitive to [Ca2+]o and CaSR activity i exerts inhibitory effects on the catalytic WNK activity, CaSR-induced decrease of basolateral K+ efflux may lead to [Cl−]i accumulation and suppress the WNK-SPAK/OSR1 pathway i in response to CaSR activation may exert further effects on WNKs. Binding of Ca2+ to the highly conserved locus of mammalian WNK kinases termed “acidic motif” has been proposed to directly modulate their activity i in response to CaSR activation may facilitate dephosphorylation of KLHL3 by calcineurin, thereby promoting its interaction with WNKs and their degradation i on WNK4 activity [2+]i in DCT would then activate truncated WNK4 variants via Ca2+-binding to their acidic domain without competitive inhibitory effects mediated by C-terminal interaction with CaM [−]i i ,96,97. ICaM [−]i ,98,99. ICaM [−]i ,56. Kir4CaM [−]i ,101. Fro3) cells ,56, furtnditions ,56,89.2+ reabsorption, which is an active process mediated by the transient receptor potential vanilloid member 5 (TRPV5) at the luminal side, calbindin D28K as intracellular carrier, as well as the basolateral NCX1 and plasma membrane Ca2+-ATPase i and activation of V-ATPase [2+ [2+ concentrations .2+ excretion to the needs of the body. To achieve this purpose, CaSR signaling regulates a wide range of electrolyte transporters and channels, which are directly or indirectly involved in the Ca2+ reabsorption along the nephron and collecting duct system. With respect to salt-handling, global effects of CaSR hyperactivity due to gain-of-function mutations in the human CaSR gene recapitulate a Bartter-like syndrome with moderate salt wasting but rather severe hypocalcemia compared to the classical Bartter syndrome variants [2+ metabolism in endocrine disorders . Calcim review, ,121). Ho


Several signalling pathways, including the JAK/STAT signalling pathway, have been identified to regulate the differentiation of human bone marrow skeletal  stem cells (hBMSCs) into bone-forming osteoblasts. Members of the JAK family mediate the intracellular signalling of various of cytokines and growth factors, leading to the regulation of cell proliferation and differentiation into bone-forming osteoblastic cells. Inhibition of JAK2 leads to decoupling of its downstream mediator, STAT3, and the subsequent inhibition of JAK/STAT signalling. However, the crucial role of JAK2 in hBMSCs biology has not been studied in detail. A JAK2 inhibitor, Fedratinib, was identified during a chemical biology screen of a small molecule library for effects on the osteoblastic differentiation of hMSC-TERT cells. Alkaline phosphatase activity and staining assays were conducted as indicators of osteoblastic differentiation, while Alizarin red staining was used as an indicator of in vitro mineralised matrix formation. Changes in gene expression were assessed using quantitative real-time polymerase chain reaction. Fedratinib exerted significant inhibitory effects on the osteoblastic differentiation of hMSC-TERT cells, as demonstrated by reduced ALP activity, in vitro mineralised matrix formation and downregulation of osteoblast-related gene expression, including ALP, ON, OC, RUNX2, OPN, and COL1A1. To identify the underlying molecular mechanisms, we examined the effects of Fedratinib on a molecular signature of several target genes known to affect hMSC-TERT differentiation into osteoblasts. Fedratinib inhibited the expression of LIF, SOCS3, RRAD, NOTCH3, TNF, COMP, THBS2, and IL6, which are associated with various signalling pathways, including TGFβ signalling, insulin signalling, focal adhesion, Notch Signalling, IL-6 signalling, endochondral ossification, TNF-α, and cytokines and inflammatory response. We identified a JAK2 inhibitor (Fedratinib) as a powerful inhibitor of the osteoblastic differentiation of hMSC-TERT cells, which may be useful as a therapeutic option for treating conditions associated with ectopic bone formation or osteosclerotic metastases. Janus kinase (JAK)/signalling transducers and activators of transcription (STAT) signalling has been identified as the downstream signalling of a wide array of growth factors, hormones, and cytokines (such as interferon-γ and interleukin-6 family members) that regulate different cellular processes, including cell proliferation, differentiation, cellular senescence, and apoptosis ,5,6,7,8.hBMSCs are multipotent stem cells that reside in the bone marrow and are capable to differentiate into mesodermal cells, including bone-forming osteoblasts ,12. VariJAK/STAT signalling plays an important role in bone development. It has been shown that inhibition of JAK2 disconnects it from the downstream mediator STAT3, leading to the inhibition of hMSCs differentiation into osteoblasts ,23. Small molecule inhibitors targeting specific intracellular signalling pathways are currently used in vitro as a biochemical tool to identify the molecular mechanisms involved in stem cell differentiation into osteoblast lineages ,25,26,27Previously, we reported the results of a small molecule library screen that recognised various small molecule inhibitors with diverse effects on the osteoblastic differentiation of hMSC-TERT cells using Alkaline phosphatase (ALP) activity quantification as a read-out . Among tFedratinib-treated hMSC-TERT cells (3 µM) exhibited a significant reduction in ALP production, as evidenced by reduced cytochemical staining intensity B compareTo confirm our findings, we tested the effect of an additional two JAK2 inhibitors, LY2784544 and XL019, on the osteoblastic differentiation of hMSC-TERT cells. Both treatments yielded significant reductions in ALP production as evidenced by reduced cytochemical staining intensity E,H compaTo confirm that Fedratinib targeted the JAK2 signalling pathway, hMSC-TERT cells were treated with Fedratinib at the same concentration (3 μM), and 24 h later, the expression of JAK2, STAT5, STAT3, AKT, and ERK, which are downstream readouts of the JAK2 signalling pathway, was assessed using qRT-PCR. The data presented in We have previously reported several small molecule inhibitors with inhibitory effects on the osteoblastic differentiation of hMSC-TERT cells as shown by global gene expression profiling ,20,22. VhBMSCs are multipotent cells that can differentiate into bone-forming osteoblastic cells during bone formation and remodelling ,12. UndeFedratinib is an oral JAK2 inhibitor  that hasWe observed that Fedratinib treatment reduced osteoblastic differentiation, in vitro mineralisation, and the expression of osteoblast-related genes. The role of the JAK/STAT signalling pathway in triggering osteoblastic differentiation has been widely described ,23,31,32Osteoblastic differentiation, bone development, mineralisation, and fracture repair can be mediated by several intracellular signalling pathways, such as the JAK/STAT pathway ,31. The JAK2 phosphorylation stimulates several signalling pathways, including STAT3, STAT5, the phosphoinositide-3 kinase (PI3K)/AKT pathway and the mitogen activated protein kinase pathway involving ERK kinases, which control cell survival, proliferation and differentiation . STAT5 aGlobal gene expression profiling of hMSC-TERT cells after treatment with previously reported small molecules that inhibit osteoblast differentiation ,20,22 idWe used the hMSC-TERT cell line as a model of hBMSCs in all experiments included in this study. The hMSC-TERT cell line was generated by overexpressing the human telomerase reverse transcriptase gene (hTERT). The hMSC-TERT cell line retains all the distinctive features of primary hBMSCs, including infinite self-renewal and potency, and expresses all gene markers ,46.D-glucose, 110 mg/L 10% sodium pyruvate, 4 mM L-glutamine, 10% fetal bovine serum (FBS), 1% non-essential amino acids, and 1% penicillin-streptomycin, as previously defined [2 incubators at 37 °C and 95% humidity. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM), a basal medium supplemented with 4500 mg/L  defined . All reaL-ascorbic acid , 10 mM b-glycerophosphate , 10 nM calcitriol , and 10 nM dexamethasone (Sigma-Aldrich), in addition to 10% FBS, and 1% penicillin-streptomycin, as previously defined [Cells were maintained to reach 85% confluence before replacing the medium with osteoblast induction medium containing DMEM supplemented with 50 mg/mL  defined . The steCell viability analysis was conducted using the alamarBlue method according to the manufacturer’s recommendations  as previously defined . To obtaCell apoptosis was measured by a fluorescence-based assay using the acridine orange/ethidium bromide (AO/EtBr) dyes as previously defined , in cellThe BioVision ALP activity colorimetric assay kit  was used to measure the ALP activity, as previously defined, with some modifications . On day On day 10 of osteoblastic differentiation, ALP staining was conducted on cells maintained in 6-well plates as previously defined . Cells wAlizarin red staining was conducted on day 21 of osteoblastic differentiation using the 2% Alizarin Red S Staining Kit , as previously defined . Cells wOn day 10 of osteoblastic differentiation, total RNA was extracted from cell pellets using the total RNA Purification Kit  according the manufacturer’s instructions, as previously defined . The conQuantitative real time-polymerase chain reaction (RT-PCR) was conducted on an Applied Biosystems ViiA™ 7 Real-Time PCR System  using fast SYBR Green. The primers used in the present study are listed in t-test was used to assess statistical significance, and p-values < 0.05 were set as being statistically significant.Statistical analysis and graphing were carried out using Microsoft Excel 2010 and the GraphPad Prism 6.0 software , respectively. The results are presented as the mean ± SEM of at least two independent experiments. Unpaired, two-tailed Student’s Fedratinib has been approved by the FDA for the treatment of myelofibrosis. Our data suggest the plausible use of Fedratinib to treat bone diseases characterised by increased bone formation and mineralisation such as sclerotic bone metastases, craniosynostosis, and heart valve calcification. However, the clinical efficiency of Fedratinib in these conditions requires further studies.


The anaplastic lymphoma kinase (ALK) fusion has been identified to be a driver gene in lung cancer, and serves as important diagnostic and therapeutic targets. Owing to the advanced sequencing technologies, new partner genes of ALK have been constantly detected.A 55-year-old Chinese woman went to our hospital because of cough and expectoration for 1 year. The patient had no fever, chest pain and hemoptysis.She was diagnosed with lung adenocarcinoma. Because she had no operational condition, combination chemotherapy with docetaxel and cisplatin (CP) for 4 cycles was adopted. However, computed tomography (CT) scan indicated progression disease (PD). To explore possibility of targeted therapy, the tumor samples were subjected to next-generation sequencing (NGS), and a rare double ALK fusion variant EML4-ALK and CDK15-ALK was identified.The patient subsequently received crizotinib treatment, and achieved partial response (PR). No significant drug related adverse reactions were found during crizotinib treatment. The progression-free survival achieved 23 months.Together, we identified a rare double ALK fusion variant, EML4-ALK and CDK15-ALK, in a patient with lung adenocarcinoma. The patient benefited from crizotinib treatment, which could provide a certain reference for the patients with such gene alteration. Anaplastic lymphoma kinase (ALK) fusions are detected in many cancers with the highest detection rate in NSCLC. ALK fusion is an important driver gene in NSCLC, fusion partners of which have been discovered gradually, including EML4, CMTR1, KLC1, TNIP2 and CUX1.–7 However, ALK double fusion in NSCLC is still rare even though ALK fusion is detected in about 4% of lung adenocarcinoma. Here we report a rare double ALK fusion variant, EML4-ALK and CDK15-ALK in a patient with lung adenocarcinoma who responded well to crizotinib.Lung cancer is a malignant disease with high morbidity and mortality. Lung adenocarcinoma originates from the epithelium and belongs to non-small cell lung cancer (NSCLC).2The patient was a 55-year-old female with no smoking history and no family history of cancer. In February 2017, she went to our hospital because of cough and expectoration for 1 year. The patient had no fever, chest pain and hemoptysis. Chest enhanced CT showed a mass lesion in the upper lobe of the right lung and multiple enlarged lymph nodes in the mediastinum. The pathological diagnosis of the biopsy was low differentiated adenocarcinoma, stage IIIB. Because the patient had no operational condition, combination chemotherapy with docetaxel and cisplatin (CP) for 4 cycles was adopted. CT re-examination showed that the tumor was enlarged and the assessment was progression disease (PD) according to RECIST 1.1 , and the results showed that this patient carried two ALK fusions simultaneously, EML4-ALK (V3) and CDK15-ALK Fig. . EML4-AL3 EML4-ALK fusion is the classic and most common fusion mutation in NSCLC. In 2007, SODA et al. found the EML4-ALK fusion gene for the first time in NSCLC, which could lead to the occurrence of cancer. Thus, the ALK fusion has been considered as a very important lung cancer driver gene.In this report, we identified a rare double ALK fusion, EML4-ALK and CDK15-ALK, in the patient with lung adenocarcinoma. CDK15-ALK fusion was a recently identified fusion. CDK15 gene encodes cyclin dependent kinase, and involves in the resistance to tumor necrosis factor induced apoptosis. However, different ALK fusion variants have different responses to crizotinib. Based on the different cleavage sites, EML4-ALK fusion gene variants can be divided into several subtypes, V1, V2, V3a/b/c, V4, V5a/b, V6a/b, V7, V8a/b/d and V10. Among them, subtypes of V1 (32-33.3%), V2 (8-11.1%) and V3a/b (32-44%) are the most common mutation subtypes, while other EML4-ALK fusion variants only account for 5.6% to 12%.,12ALK inhibitors are widely used in cancer-targeted therapy now. Crizotinib is a small-molecule tyrosine kinase inhibitor (TKI) which actively inhibits ALK, MET, and ROS1. Crizotinib showed significant activity in ALK-driven tumors and was FDA approved for ALK-positive advanced NSCLC. Yoshida et al found that the objective response rate (ORR) and disease control rate (DCR) of the EML4-ALK variant V1 responding to Crizotinib were 74% and 95% respectively, while other ALK fusions were 63% and 63%. Another clinical study showed that the 2-year PFS of NSCLC patients with V3a/b was 26.4%, significantly lower than that other subgroups. It suggested that EML4-ALK variants 3a/b may be a major source of ALK inhibitor resistance in the clinic. In addition, in vitro experiments, the protein encoded by EML4-ALK V3 fusion showed longer half-life and stronger carcinogenic signals than other mutant subtypes, and this may be another reason for the worse prognosis of the V3 patients, which is not related to the drug resistance. Christopoulos et al demonstrated that EML4-ALK V3 fusion is a high-risk feature for ALK+ NSCLC. In this study, the patient carried EML4-ALK V3 mutation and CDK15-ALK fusion, but she still showed a good response to crizotinib treatment. This is a very lucky thing, but how long this response last needs further observation. Thus, continuous studies on the form of ALK fusion, the correlation between them and the sensitivity of targeted drugs have great clinical significance. Moreover, with accumulation of related information, it is necessary to include molecular testing beyond FISH in the future diagnostic guidelines for ALK+ NSCLC and to develop more effective strategies for management of higher-risk, V3-positive cases.Several studies have explored the potential association of EML4-ALK fusion with therapeutic response, but the results are inconclusive. Lei et al did not observe any significant difference in the efficacy of crizotinib between patients with the EML4-ALK fusion variant V3, V1 and other less frequent variants V2.In summary, we represented a rare double ALK fusion composed of a classic fusion variant of EML4-ALK and a newly discovered CDK15-ALK in a lung adenocarcinoma. This report fully demonstrated the complexity and diversity of ALK fusion. Also, the curative effect of crizotinib in the treatment of the patient provided a certain therapeutic reference for the patients with such gene alterations.Conceptualization: Jun Guo, Da JiangData curation: Junping Shi, Ming Yao, Yi JinInvestigation: Dengxiang Liu, Weiling Liu, Kai WangMethodology: Jun Guo, Junping Shi, Ming YaoValidation: Yi Jin, Dengxiang LiuWriting – original draft: Jun Guo, Da JiangWriting – review & editing: Jun Guo, Junping Shi, Ming Yao, Yi Jin, Dengxiang Liu, Weiling Liu, Kai Wang, Da Jiang