ABL-class and JAK-STAT signaling pathway activating alterations have been associated with both a poor post-induction minimal residual disease (MRD) response and an inferior outcome in B-cell acute lymphoblastic leukemia (B-ALL). However, in most of the studies patients received non-uniform treatment.BCR-ABL1-negative B-ALL patients who had been uniformly treated according to MRD-directed NOPHO ALL-2008 protocol. Targeted RNA sequencing and FISH analysis were performed in cases without canonical B-ALL genomic alterations (high hyperdiploids and low hypodiploids included).We performed a population-based analysis of 160 (122 pediatric and 38 adult) Lithuanian p = 0.003). JAK-STAT pathway fusions were present in 4/160 (2.5%) cases. Of note, P2RY8-CRLF2 fusion was absent in both pediatric and adult B-ALL cases. Patients with ABL-class or JAK-STAT pathway fusions had a poor MRD response and were assigned to the higher risk groups, and had an inferior event-free survival (EFS) / overall survival (OS) compared to patients without these fusions. In a multivariate analysis, positivity for ABL-class and JAK-STAT fusions was a risk factor for worse EFS (p = 0.046) but not for OS (p = 0.278) in adults.We identified ABL-class fusions in 3/160 (1.9%) B-ALL patients, and exclusively in adults (P2RY8-CRLF2 gene fusion in the Lithuanian BCR-ABL1 negative B-ALL cohort. Future (larger) studies are warranted to confirm an inferior event-free survival of ABL-class/JAK-STAT fusion-positive adult patients in MRD-directed protocols.We report a low overall frequency of ABL-class and JAK-STAT fusions and the absence of The online version contains supplementary material available at 10.1186/s12885-020-07781-6. BCR-ABL1 (Philadelphia chromosome-like (Ph-like) or BCR-ABL1-like B-ALL) [B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive and genetically heterogeneous disease which mostly affects children and young adults . A numbee B-ALL) , 5.CRLF2 gene and IKZF1 gene deletions are also common in the Ph-like B-ALL subgroup resulting in a worse outcome in some series [Whole transcriptome/exome sequencing and fluorescence in situ hybridization (FISH) analysis of Ph-like B-ALL samples revealed high frequency of genomic alterations disrupting the normal function of kinase and cytokine receptor signaling pathways of which ABL-class kinase and JAK-STAT signaling pathway genes were the most commonly affected . ABL-clae series , 17.In most studies, a Ph-like B-ALL was clinically characterized as a high-risk group with inferior overall survival in intensively treated children and adults –20. In cIn this study we performed a comparative genomic and clinical data analysis of pediatric and adult B-ALL patients to characterize the incidence of ABL-class and JAK-STAT signaling pathway activating alterations in the Lithuanian population and to determine their clinical significance in a MRD-risk directed treatment setting.BCR-ABL1-negative B-ALL in Lithuania and enrolled into a NOPHO ALL-2008 clinical trial [From July 2008 to December 2017, pediatric (1─17 year-olds) and adult (18─45 year-olds) B-ALL patients who had been diagnosed with ETV6-RUNX1, t/TCF3-PBX1, 11q23/KMT2A gene rearrangements, iAMP21, dic, high hyperdiploidy (51–67 chromosomes) and low hypodiploidy (31–39 chromosomes). Cases without canonical B-ALL translocations were selected for RNA sequencing. Patients with ploidy shifts (high hyperdiploidy or low hypodiploidy) were also selected for RNA sequencing as they could harbor additional kinase and cytokine receptor signaling pathway alterations [The diagnosis of ALL was based on a bone marrow biopsy or aspirate showing ≥20% leukemic blasts. The protocol specific genomic analysis was limited to canonical B-ALL genomic alterations – t/erations .KMT2A gene rearrangements, iAMP21, dic or low hypodiploidy were present. The MRD analysis was performed on days 15, 29 and 79 (only for standard risk (SR) and intermediate risk (IR) patients) or after each block (in high-risk (HR) arm until MRD-negativity). If MRD was ≥0.1% on day 79 (SR and IR) or after block B1 (HR), patients were referred to an allogeneic hematopoietic stem cell transplant (SCT) after receiving at least one additional block of therapy and having MRD below < 0.1%  (HR-SCT arm).Patients were stratified to the three risk groups defined in the NOPHO ALL-2008 protocol detailed elsewhere [The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Vilnius Regional Bioethics Committee. The patients provided written informed consent. The waiver for obtaining written informed consent from patients who could not be reached despite our best efforts was approved by the Vilnius Regional Bioethics Committee according to national regulations.Total RNA was purified from fresh bone marrow samples at the time of diagnosis using silica-membrane-based purification protocols and stored at − 80 °C. All bone marrow samples had blast counts ≥70%. Before RNA sequencing (RNA-Seq), purified RNA concentration and quality was assessed using NanoDrop2000 spectrophotometer.ABL1, ABL2, PDGFRB, CSF1R) and JAK-STAT  signaling pathways. At least 4 million paired-end reads were obtained for each sample. NGS data analysis was performed using the Illumina BaseSpace Informatics Suite (Illumina). TopHat/STAR aligners and a Manta fusion caller were used to detect novel and recurrent gene fusions. At least one partner gene was required to detect novel gene fusion. Only high-confidence fusions were called that met threshold filters: split and paired unique reads (≥3), fusion contig align (≥16 bp in length), coverage after fusion (≥100 bp), break end homology (≤10 bp).RNA-Seq libraries were constructed using the Illumina TruSight Pan-Cancer sequencing kit , following the manufacturer’s protocol and recommendations. The libraries were sequenced using Illumina MiSeq genome analyzer (Illumina). A total of 1385 cancer-related genes were analyzed including frequently mutated genes of ABL-class .BCR-ABL1 n = 5, KMT2A gene rearrangements n = 5, TCF3-PBX1 n = 2, ETV6-RUNX1 n = 1). All in-frame gene fusions were identified, therefore the RNA-Seq method was used for B-ALL study patients.Prior to the study, the RNA-Seq method was first validated using thirteen B-ALL control samples with previously identified gene fusions (CRLF2 gene (CRLF2 (Xp22/Yp11) Break / IGH Fusion,TC; Leica Biosystems, Wetzlar, Germany) was performed to identify P2RY8-CRLF2 and IGH-CRLF2 gene fusions. FISH analysis for ABL1 , ABL2 , JAK2 (JAK2 (9p24) Break; Leica Biosystems) and PDGFRB (PDGFRB (5q32) Break; Leica Biosystems) genes were used to confirm respective gene fusions detected by the RNA-Seq method. FISH analysis was performed according to manufacturer protocols.Due to technical limitations of RNA-Seq, additional FISH analysis of Standard RT-PCR followed by a gel electrophoresis and Sanger sequencing were used to confirm in-frame fusions that were not confirmed with an additional FISH analysis. Fusion specific PCR primers were constructed for each case , which covers the entire genome with an average spacing of 9.6 kb. This coverage allows an average resolution of 31 kb which was used for detection of PAR1 region deletion.p-value < 0.05 was considered to indicate statistical significance. Statistical analyses were performed using SPSS software version 20.Differences in the prevalence of parameters between the groups were determined using the Mann-Whitney test or Independent-Samples T-test for continuous variables depending on their distributions. For categorical analyses, either a Chi-square or a Fisher exact test was used. Univariate and multivariate Cox proportional hazard regression models were used to evaluate the effect of non-canonical B-ALL genomic alterations on survival. The Kaplan-Meier method was used to estimate the time to event distributions , the Log-Rank test was used to compare differences in survival curves. The overall survival (OS) was computed from the date of diagnosis until the date of death or last known follow-up date. The event-free survival (EFS) was defined as the time from diagnosis to the event of resistant disease, relapse, induction death, death in remission, second malignancy or date of the last follow-up if a patient had no events. A BCR-ABL1 negative B-ALL patient population in Lithuania during the July 2008 – December 2017 period were included into this study /iAMP21 aberrations  cases and were more common among children than adults . In contrast, KMT2A gene rearrangements were detected in 11/160 (6.9%) B-ALL cases (4 children vs. 7 adults). Importantly, RNA-Seq analysis identified two additional KMT2A gene rearrangements (del(11q23)/KMT2A-CBL, del(11q23)/KMT2A-ATP5L) that had been missed by FISH. iAMP21 aberration was exclusive to the pediatric group  while dic was present in 1/160 (0.6%) adult patient.In total, 99/160 (61.9%) cases harbored at least one canonical B-ALL genomic alteration Table , Fig. 1.KMT2A gene rearrangements identified by RNA-Seq, remaining cases without canonical B-ALL gene fusions and cases with high hyperdiploidy or low hypodiploidy (n = 101) were selected for RNA-Seq and CRLF2 gene break FISH analysis to identify other kinase and cytokine receptor activating lesions /EBF1-PDGFRB fusions. All ABL-class fusions were exclusive to adults (p = 0.003).In-frame fusions of ABL-class genes were detected in 3/101 (3%) patients Table . One caseukemias . The remBCR-JAK2 fusion in one pediatric patient. FISH analysis revealed three cases with t /IGH-CRLF2 gene fusion, of which two were adults. BCR-JAK2 fusion was detected in one case with high hyperdiploidy which further confirms that JAK-STAT pathway gene fusions can be detected in this entity. Both adult cases with IGH-CRLF2 fusions had additional gene mutations. One IGH-CRLF2-positive case harbored FLT3-TKDD835Y and NRASG13R gene mutations while another case had CRLF2F232C gene mutation. RNA-seq and FISH analysis detected no P2RY8-CRLF2 fusions in our study patients. SNP Array analysis was available for 81/101 (80.2%) of the sequenced B-ALL patients. No deletions of PAR1 region were identified in any of the cases confirming the absence of P2RY8-CRLF2 fusion.JAK-STAT pathway fusions were identified in 4/101 (4%) cases. ABL-class and JAK-STAT pathway fusions were mutually exclusive. RNA-Seq identified t/PAX5 gene rearrangements: t/PAX5-GREB1L, t/PAX5-NCOA5, dic/PAX5-ETV6, dic/PAX5-FOXP1 and dic/PAX5-NOL4L. One case with PAX5-GREB1L fusion had an additional JAK1F838V mutation, while in the other two PAX5-rearranged cases KRASG12V and NRASG12C mutations were identified. Three cases had fusions involving ZNF384 gene  of which the TCF3-ZNF384 positive case had an additional NRASG13R mutation. Though ETV6-RUNX1 is the most common alteration in childhood B-ALL, data analysis revealed a novel fusion of ETV6 and RUNX2 genes in one pediatric patient while another patient harbored a t/CUX1-NUMT1 fusion.We identified other gene fusions in 10/101 (9.9%) pediatric B-ALL cases. Five cases had JAK1 (n = 2), JAK2 (n = 7), CRLF2 (n = 1) .Gene mutation analysis of RNA-Seq data revealed ten other JAK-STAT pathway mutations in a total of 9/101 (8.9%) cases: JAK1 n = , JAK2 (nNRAS gene mutations were the most common (n = 29) while the incidence of KRAS (n = 17) and PTPN11 (n = 7) gene mutations was lower. One case had mutations in NRAS and PTPN11 genes while another case had two different NRAS gene mutations (G12S and G12A) in the same sample. Two other cases had both KRAS and NRAS gene mutations in the same sample. Ras pathway mutations were detected in 16/35 (45.7%) high hyperdyploid and 2/5 (40%) low hypodiploidy cases.A total of fifty-three Ras pathway gene point mutations were detected in 48/101 (47.5%) cases (Table FLT3-TKD activating mutations of codons D835 (n = 5) and I836 (n = 3) were found in 8/101 (7.9%) cases. Most of the FLT3-TKD positive cases (75%) were present with Ras pathway gene mutations. Other FLT3 gene kinase domain point mutations were present in 5/101 (5%) patients , CREBBP (n = 5) and TP53 (n = 3) genes were the most recurrent in this group of patients. RNA-Seq and FISH methods failed to identify ALL-related gene fusions and mutations in 14/101 (13.9%) cases.Other gene mutations were identified in 23/101 (22.8%) patients. Mutations in BCR-ABL1-negative B-ALL cases) were positive for either ABL-class or JAK-STAT pathway fusions (AJS-positive group) (Table n = 153)  adult and two of 75 (2.7%) pediatric B-ALL cases without canonical B-ALL alterations (5/38 (13.2%) adult and 2/122 (1.6%) pediatric n = 5) vs. AJS negative (n = 33) B-ALL cases in adults since the number of AJS-positive pediatric cases was too small for detailed analysis. Four AJS-positive adult patients were evaluated for residual disease on induction day 15 (one patient died during induction). All four AJS-positive patients (100%) had ≥5% blasts on day 15 compared to 9 (29%) AJS-negative patients (p = 0.019) (Table p = 0.052).We further compared the outcome of AJS positive (9) Table . After dp = 0.009) and OS was 14 vs. 36 months (p = 0.098) in AJS-positive vs. AJS-negative adults, respectively    cases with ABL-class fusions  [In our study, ABL-class fusions were identified in 3/101 (3%) B-ALL cases without canonical B-ALL alterations or 3/160 (1.9%) 9%) Fig. . In compangement  while ABangement . Heatleyve B-ALL . ABL-claR n = 1) . In a siR n = 1) . The incWe identified JAK-STAT pathway fusions in 4/101 (4%) cases without canonical B-ALL alterations. JAK-STAT pathway fusions occur in approximately 3–5% of childhood and in up to 15% of adult B-ALL ; howeverCRLF2 rearrangements in B-ALL is approximately 5% and the rate gets higher in cases without canonical B-ALL alterations (10–30%) and in patients with Down syndrome (> 50%) [IGH-CRLF2 fusions were identified in 3/101 (2.9%) B-ALL cases without canonical B-ALL alterations or in 3/160 (1.9%) BCR-ABL1-negative B-ALL cases. Notably, we did not detect any P2RY8-CRLF2 gene fusions in our cohort by either RNA-Seq or CRLF2 gene break FISH. In comparison, a Swedish study identified CRLF2 gene rearrangements in 16 of 189 (8.5%) pediatric BCR-ABL-negative B-ALL patients, of which P2RY8-CRLF2 fusion was the most common  [P2RY8-CRLF2 gene fusion is the result of deletion of PAR1 region in either X or Y chromosomes. To further confirm the absence of P2RY8-CRLF2 fusion, we used SNP Array data of 81/101 (80.2%) sequenced patients and did not detect any deletions in the corresponding PAR1 region. In addition, another study has shown that P2RY8-CRLF2 fusion can be identified using the RNA-Seq method and an analysis algorithm similar to ours [P2RY8-CRLF2 fusion was missed due to technical reasons in our study patients. CRLF2 gene rearrangements are common in populations with Hispanic ancestry and are associated with increased risk of relapse in both children and adults [IGH-CRLF2 and the absence of P2RY8-CRLF2 gene fusions in our cohort. In other B-ALL studies, approximately half of CRLF2-rearranged patients also harbored additional JAK1 or JAK2 gene mutations [FLT3-TKDD835Y and NRASG13R gene mutations and another case was positive for a CRLF2F232C activating mutation. CRLF2F232C gene mutation is known to promote constitutive dimerization and cytokine-independent growth which results in gene overexpression in a similar manner as with CRLF2 gene rearrangement [Overall, the frequency of  (> 50%) , 31, 32. to ours , making d adults , 34–36. utations , howeverangement .FLT3, JAK-STAT and Ras pathway genes had no prognostic significance on either EFS or OS among all age groups.Other gene fusions were found in 10/101 (9.9%) patients and were exclusive to the pediatric group Fig. . RNA-SeqKMT2A gene rearrangements that had been missed by FISH analysis at diagnosis. Both samples had blast counts of > 90%. We re-ran the FISH analysis; however, the secondary results were also negative. Notably, both fusions were formed as a result of the aberrations of the 11q23 chromosome region. The close proximity of fused genes and relatively small size of chromosome aberrations could have potentially caused false negative FISH results. Patients with KMT2A gene rearrangements have lower survival rates and generally require therapy intensification in first remission [KMT2A-ATP5L fusion was also recently described as an unfavorable prognostic marker in young adults with Ph-like ALL [RNA-Seq analysis revealed two adult B-ALL cases with like ALL . Our finPh-like B-ALL has been associated with a worse outcome in several studies , 19, 39.p = 0.046) but not OS (p = 0.278) in adults  countries and Iceland, we had access to and studied Lithuanian patients only. The number of ABL-class or JAK-STAT pathway fusion-positive patients was low and a few events occurring by chance could have significantly affected the statistics of clinical outcome. As already mentioned, our results are not directly comparable to the previous studies of Ph-like B-ALL. Nevertheless, the results of our study are clinically relevant. We report the results of a relatively large and homogenous group of pediatric and adult patients coming from the Baltic part of Europe and show that the incidence of ABL-class or JAK-STAT pathway fusions in this geographic area is lower as compared to the US or other European regions , 18, 30.BCR-ABL1-negative B-ALL patients. Specifically, P2RY8-CRLF2 gene fusion was not detected using three different genome interrogation methods. The poor MRD response and an inferior clinical outcome in patients harboring either ABL-class or JAK-STAT pathway fusions should be confirmed in future prospective studies.ABL-class and JAK-STAT pathway fusions are uncommon in the population-based cohort of Lithuanian Additional file 1: Table S1, Table S2.


The autophagy-lysosome pathway is a major protein degradation pathway stimulated by multiple cellular stresses, including nutrient or growth factor deprivation, hypoxia, misfolded proteins, damaged organelles, and intracellular pathogens. Recent studies have revealed that transcription factor EB (TFEB) and transcription factor E3 (TFE3) play a pivotal role in the biogenesis and functions of autophagosome and lysosome. Here we report that three translation inhibitors  can facilitate the nuclear translocation of TFEB/TFE3 via dephosphorylation and 14-3-3 dissociation. In addition, the inhibitor-mediated TFEB/TFE3 nuclear translocation significantly increases the transcriptional expression of their downstream genes involved in the biogenesis and function of autophagosome and lysosome. Furthermore, we demonstrated that translation inhibition increased autophagosome biogenesis but impaired the degradative autolysosome formation because of lysosomal dysfunction. These results highlight the previously unrecognized function of the translation inhibitors as activators of TFEB/TFE3, suggesting a novel biological role of translation inhibition in autophagy regulation. Macroautophagy, hereafter referred to as autophagy, is a normal degradative pathway that exists in all eukaryotic cells ,2. Autop+-ATPase (V-ATPase), an ATP-driven proton pump located on the lysosomal transmembrane [The progression and resolution of autophagy critically depends on the lysosomal function, as lysosomes play a role in the breakdown and recycling of cellular compartments. Lysosome is a single membrane-bound compartment that is filled with more than 60 resident acid hydrolases: proteases, phosphatases, lipases, nucleases, and glycosidases ,9. Most membrane ,11. Lysomembrane ,13. Cathmembrane ,15. TherThe induction and formation of autophagosome during autophagy are followed by late stages such as autophagosome-lysosome fusion and cargo degradation for completion of autophagy ,5,11. Th2+-calmodulin-dependent protein phosphatase calcineurin is activated, without further phosphorylation of TFEB and TFE3, resulting in the prevention of binding to 14-3-3 and rapid accumulation of TFEB and TFE3 in the nucleus [Autophagy was considered as a pathway exclusively regulated by cellular processes in enucleated cells forming autophagosomes . However nucleus ,38,39,40 nucleus ,42,43. T nucleus ,27,28.Aglaia. It is an inhibitor of eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase. It preferentially represses translation by clamping eIF4A onto purine-rich regions within mRNA 5′ leaders, followed by inhibition of 43S pre-initiation complexes (PICs), leading to the premature translation from uORF and the inhibition of downstream ORF translation [Protein biosynthesis is one of the major metabolic processes, which are crucial for maintaining cellular functions including autophagy. Therefore, eukaryotic translation is an attractive target to destroy fast-growing tumor cells and impair and/or delay the spread of fast-replicating viral pathogens ,46,47,48nslation ,58. Furtnslation . Among tnslation , althougnslation ,62. HoweIn the present study, we analyzed the impact of translation inhibition using three chemicals  on TFEB localization and autophagy pathways. Such chemical-mediated translation inhibition promoted TFEB dephosphorylation and nuclear translocation. TFEB nuclear localization was modulated by a phosphatase calcineurin but not a kinase mTORC1. In addition, LTM and RocA significantly increased the expression of a number of genes downstream of TFEB required for autophagosome formation, lysosome biogenesis, and lysosome function. Further, we demonstrated that treatment with LTM and RocA facilitated autophagosome biogenesis but prevented degradative autolysosome formation.To analyze changes in TFEB cellular localization under diverse experimental conditions, we established a mouse embryonic fibroblast (MEF) cell line expressing a human TFEB-fused enhanced green fluorescent protein (EGFP) at the C-terminal and a control MEF cell line expressing EGFP only . As it iNext, we determined whether the inhibition of protein synthesis induced TFEB nuclear translocation in the MEF cell line, by treating MEF-TFEB-EGFP cell lines with glutarimide-containing natural products cycloheximide (CHX) and lactimidomycin (LTM) in a dose –D and tiAlthough CHX and LTM are specific inhibitors of protein synthesis blocking the ribosome at the translocation stage, we cannot exclude the possibility that TFEB and TFE3 nuclear translocation might be induced as an unknown side effect. Therefore, a different type of translation inhibitor RocA with distinct mechanism of action was selected to reproduce our observation. RocA, an inhibitor of eukaryotic initiation factor 4A (eIF4A), preferentially represses translation by clamping eIF4A onto purine-rich regions within mRNA 5′ leaders. It then interferes with the scanning of 43S pre-initiation complexes (PICs) ,58. FurtFirst, in dose-dependent experiments  RocA clearly showed its translational inhibitory effect . As expeTo exclude the nuclear translocation of TFEB in response to three translation inhibitors as a cell type-specific event, we assessed endogenous TFEB nuclear translocation in both mouse immortalized hepatocytes and human HeLa cells . In agreIt has been proposed that the cellular localization and activity of TFEB are mainly controlled by its phosphorylation status ,29,31 alThe phosphorylation status of TFEB is regulated by multiple kinases including mTORC1 and phosphatase calcineurin ,38,42,71We next assessed whether LTM and RocA-induced TFEB/TFE3 nuclear translocation upregulated the transcription of autophagy-related genes. MEF-TFEB-EGFP cells were treated with LTM for 8 h or RocA for 8 and 16 h. Following 8 h of treatment, both inhibitors induced nuclear translocation of more than 80% TFEB and drastic translation inhibition B,C,E,F. Although we observed a significant transcriptional increase in translation inhibition-mediated autophagy-related genes in LTM and RocA-treated cells, the results did not indicate activation of autophagic pathways, such as autophagosome formation. Therefore, to assess whether translation inhibition can promote autophagy, an autophagosome marker protein microtubule-associated proteins 1 light chain 3B (hereafter referred to as LC3B) was analyzed. The conversion of LC3B(I) to LC3B(II), a key molecule involved in autophagosome, is a key event in autophagosome formation . As show+-ATPases and a blocker of autophagosome-lysosome fusion [Increased levels of LC3B(II) proteins and LC3A/B-positive autophagosomes following LTM and RocA treatment represent either autophagy activation or suppression of late steps in the autophagy pathway ,72. To ae fusion ,72. Durie fusion . In the e fusion ,75 may p+EGFP− LC3B puncta) but reduced autophagosomes (mRFP+EGFP+ LC3B puncta) (left panels and graph in +EGFP− LC3B puncta) was not increased although the number of autophagosomes (mRFP+EGFP+ LC3B puncta) was increased  ,72. We pautophagic and lysosomal genes without a new protein synthesis. In addition, we observed that LTM and RocA promoted autophagosome formation. However, the translation inhibition prevented the degradative autolysosome formation and induced lysosomal dysfunction. Therefore, translation inhibition might be one of the components of TFEB/TFE3 activation although it eventually induces dysfunction of autophagosome maturation probably because of inhibition of protein synthesis  (751-036-151), peroxidase-conjugated AffiniPure rabbit anti-goat IgG (H + L) (305-035-003), peroxidase AffiniPure goat anti-rat IgG (H + L) (112-035-003), Alexa Fluor 594-conjugated AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG (H + L) (711-586-152), Alexa Fluor 594-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (H + L) (115-586-003), Alexa Fluor 647 AffiniPure F(ab’)2 fragment goat anti-mouse IgG (H + L) (115-606-146) , goat anti-rabbit Alexa Fluor 488 (A-11034), and goat anti-rat Alexa Flour 647 (A-21247) . Rabbit polyclonal anti-TFE3 was a gift from Professor Hiderou Yoshida, Department of Molecular Biochemistry, Graduate school of Life Science, University of Hyogo, Hyogo, Japan.The following antibodies were used in this study: rabbit polyclonal anti-GFP ; mouse monoclonal anti-GFP (B-2) (sc-9996), mouse monoclonal anti-pan 14-3-3 (sc-133232) , rabbit polyclonal anti-TFEB , rabbit polyclonal anti-TFEB , mouse monoclonal anti-phospho-(Ser) 14-3-3 binding motif (#9606), rabbit polyclonal anti-LC3A/B (#4108), rabbit monoclonal anti-mTOR (#2983), rabbit polyclonal anti-phosphoSer2448-mTOR (#2971), rabbit polyclonal anti-p70 S6 kinase (#2971), mouse monoclonal anti-phospho Thr389-p70 S6 kinase (#9206) , rabbit polyclonal anti-LC3B (L7543), rat monoclonal anti-LAMP1 (1D4B-C), rat monoclonal anti-LAMP2 (ABL-93-C) , goat polyclonal anti-Cathepsin B (AF965), goat polyclonal anti-Cathepsin L (AF1515) , mouse monoclonal anti-β-Actin (A5441) , rabbit polyclonal anti-histone H3 , mouse monoclonal anti-puromycin , peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) (111-035-003), peroxidase-conjugated AffiniPure F(ab’)The following chemicals were used in this study: cycloheximide , rocaglamide A  and bafilomycin A1  , lactimidomycin , LysoTracker Red DND-99 , puromycin (sc-108071) and cyclosporin A  , Torin2 , FK506 , 4′,6-diamidino-2-phenylindole dihydrochloride , and blasticidin S HCl (R210-01) . For TFEB-EGFP or EGFP expression, human TFEB fused with the enhanced green fluorescent protein (EGFP) expressing pLUB-hTFEB-EGFP-IRES-Bla or EGFP expressing pLUB-EGFP-IRES-Bla plasmids was constructed. The pLUB-IRES-Bla plasmid was constructed by changing the CMV promoter of pLVX-IRES-Bla with the ubiquitin C promoter of pUB-EGFP . The Ssp1-EcoRI fragment containing ubiquitin C promoter from pUB-EGFP was inserted into pLVX-IRES-Bla treated with ClaI-Klenow-EcoRI. To construct pLUB-hTFEB-EGFP-IRES-Bla, the NotI-Klenow-EcoRI fragment containing hTFEB-EGFP from pEGFP-N1-TFEB  was inserted into pLUB-IRES-Bla treated with SmaI-EcoRI. To construct pLUB-EGFP-IRES-Bla, the NotI-Klenow-EcoRI fragment containing EGFP of pEGFP-N1  was inserted into pLUB-IRES-Bla treated with BamHI-Klenow-EcoRI.To generate a tandem fluorescent reporter expressing mRFP-EGFP-LC3B, pEGFP-LC3B was constructed by inserting the cDNA fragment encoding LC3B from pmRFP-LC3B  treated with BglII and BamHI into the pEGFP-C1  treated with the same restriction enzymes. The coding sequence fragment of mRFP was amplified from pmRFP-LC3B vector via polymerase chain reaction (PCR) with the following primers: 5′-GAGAGCTAGCGGCCACCATGGCCTCCTCCGAGGAC-3′ and 5′-GAGAACCGGTCCACCGGCGCCGGTGGAGTGGCG-3′. The tandem fluorescent reporter pmRFP-EGFP-LC3B was constructed by inserting the PCR product of the mRFP sequence treated with NheI and AgeI into pEGFP-LC3B treated with the same restriction enzymes.2.Mouse embryonic fibroblast (MEF) cells were described previously  and cultTo generate TFEB-EGFP or EGFP stably expressing MEF cell lines, lentiviral particles containing pLUB-hTFEB-EGFP-IRES-Bla or pLUB-EGFP-IRES-Bla constructs were produced in Lenti-X-293T cells using LentiX packaging Single Shot Protocol-At-A-Glance Kit (Clontech). Lentivirus production was verified using Lenti-X GoStix™ . MEF cells were transduced with each virus supernatant for 48 h. These infected cells were layered at one cell per well into 96-well cell culture plates via serial dilutions. These cells were cultured in the culture media containing 5 μg/mL blasticidin S HCl  for 10 days. The expression of TFEB-EGFP or EGFP was monitored by western blot with anti-GFP antibody and anti-TFEB. Green fluorescence-positive colonies were examined under confocal microscopy using an FV1200-OSR microscope  as described in the next section. Among several positive clones identified, we selected those that showed high expression of TFEB-EGFP or EGFP and regulated nuclear translocation of TFEB-EGFP upon treatment with well-known autophagy inducers such as Earle’s balanced salt solution (EBSS) or Torin2.Cells were plated on collagen-coated glass coverslips in 6-well dishes and cultured overnight. These cells were treated with the indicated chemicals for indicated times, followed by rinsing twice with phosphate-buffered saline (PBS). Cells were then fixed with 4% paraformaldehyde in PBS for 15 min, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. For puromycin, TFEB or TFE3 staining, cells were blocked with 3% bovine serum albumin (BSA) in PBS for 1 h and incubated with the indicated primary antibodies overnight at 4 °C. Especially, for TFE3 staining in immortalized hepatocytes, cells were blocked with 2.5% normal horse serum  for 1 h and incubated with the primary antibody against TFE3 overnight at 4 °C.To visualize LC3A/B puncta or the colocalization of punctate LC3A/B and LAMP1, the cells on the coverslip were fixed with 100% methanol for 10 min at −20 °C. These cells were blocked with 3% BSA in PBS containing 0.05% Tween-20 for 1 h and incubated with the indicated primary antibodies overnight at 4 °C.Cells incubated with the indicated primary antibodies were further incubated with fluorescence-conjugated secondary antibodies for 1 h at room temperature, followed by nuclear staining with DAPI (Invitrogen). Finally, coverslips were mounted on ProLong Gold mounting medium (Invitrogen) and cells were visualized via confocal laser microscopy using a FV1200-OSR microscope . The puromycin intensity was measured using the mean fluorescence intensity tool in FV10-ASW-4.2 software . The colocalization of LC3A/B and LAMP1 was measured using Pearson’s correlation coefficient calculator tool in the FV10-ASW-4.2 software .5 cells. On the next day, the cells were treated with the indicated chemicals in a phenol-red free culture medium  for the indicated times. In the last 30 min of the chemical treatment, 100 nM LysoTracker Red DND-99 (L7528)  was added to the cell culture media. The live images of lysosomes were visualized under an FV1200-OSR microscope . The LysoTracker Red intensity was measured using the mean fluorescence intensity tool in FV10-ASW-4.2 software .For lysosome staining, MEF TFEB-EGFP cells and HeLa cells were plated on collagen-coated 35-mm glass bottom confocal dishes  at a density of 1.2 × 10TransIT-LT1 transfection reagent  according to the manufacturer’s instructions for 30 h, followed by treatment with Torin2, LTM or RocA for the indicated times. These cells were additionally incubated with puromycin (10 μg/mL for 10 min) to label actively translating peptides. Cells were then prepared as described above.For the autophagic flux assay using a tandem fluorescent probe, the pmRFP-EGFP-LC3B was transfected into the HeLa cells using Mirus Bio g for 15 min at 4 °C and supernatants were collected. The protein concentration was determined using Pierce™ BCA Protein Assay Kit . Next, the same amount of protein lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene difluoride or nitrocellulose membranes. The membranes were blocked with 5% non-fat skim milk in 1× Tris-Buffered Saline-Tween 20  at room temperature for 1 h. Membranes were incubated with the indicated primary antibodies at 4 °C overnight and then with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Targeted proteins were visualized with SuperSignal™ West Pico PLUS Chemiluminescent substrate  and detected using an Azure Biosystems C300 .Cells were lysed in Nondiet P40 lysis buffer . Cell lysates were centrifuged at 13,000× 5 cells. The next day, cells were treated with CHX, LTM or RocA for the indicated times. These cells were collected in completed growth media and then washed once with PBS. Cell pellets were dissolved in 250 μL immunoprecipitation (IP) lysis buffer  supplemented with half protease and phosphatase inhibitor cocktail at 1× final concentration . Lysed cells were passed through a 26 G needle eight times. Cell lysates were incubated on ice for 20 min and centrifuged at 13,000× g for 15 min at 4 °C to collect soluble fractions. To prepare the antibody-bead complex, 2 μg/mL of GFP antibody  was rotated with 30 μL of slurry containing protein A/G agarose plus beads  in 1 mL of IP binding buffer  supplemented with half protease and phosphatase inhibitor cocktail at 1× final concentration  for 3 h at 4 °C. Then, 1 mg protein lysate was diluted in 1 mL of the IP binding buffer and transferred to the GFP antibody-protein A/G agarose bead complex and incubated under rotation for an additional 3 h at 4 °C. After incubation, beads were washed twice in 1 mL of the IP binding buffer. Samples were eluted with 45 μL of 1.5× SDS sample loading buffer, boiled at 100 °C for 5 min, and separated by SDS-PAGE.TFEB-EGFP expressing MEF (MEF-TFEB-EGFP) cells were plated onto 100 mm culture dishes at a density of 7 × 105 cells. The next day, cells were treated with CHX, LTM, or RocA for the indicated times. Cells were collected in the completed growth media, followed by washing once with cold PBS. Cell pellets were lysed in ice-cold hypotonic buffer  for 40 min on ice. Cell lysates were centrifuged at 13,000× g for 15 min. Supernatants were collected as cytoplasmic fractions. Cell pellets obtained as nuclear fractions were washed with the hypotonic buffer to completely remove the residual cytoplasmic fractions. The cell pellets were resuspended in nuclear extraction buffer . The protein concentration was measured, and the lysate samples were analyzed using western blot.The MEF-TFEB-EGFP cells were plated on 100 mm culture dishes at a density of 7 × 10The total RNAs were isolated from MEF-TFEB-EGFP cells treated with indicated chemicals for indicated time using a QIAzol lysis reagent . The cDNAs were synthesized with a high-capacity cDNA RT kit  for quantitative polymerase chain reaction (qPCR). The qPCRs were carried out with a TOPreal™ qPCR 2X PreMIX (SYBR Green with High ROX)  using a StepOnePlus Real Time System . The specificity of each primer pair was confirmed using the melting curve analysis. The copy number relative to β-actin mRNA was calculated as previously described . Primer t-tests and p < 0.05 was considered statistically significant.All data are presented as means ± SEM. The data were analyzed using GraphPad Prism 5 . The statistical significance of differences between groups was evaluated via the unpaired two-tailed Student’s In this study, we demonstrated the previously unrecognized function of a well-known translation inhibitor CHX and two new translation inhibitors, LTM and RocA, in autophagy, which can be used to destroy rapidly growing tumor cells and impair or delay the spread of fast-replicating viral pathogens. Further studies on autophagy regulation by these inhibitors might reveal more detailed molecular mechanisms of cell death induced by them, which can be used to develop cancer or antiviral drugs. In addition, as our findings suggest a new biological function of translation inhibition in autophagy regulation, it is worthwhile to find a cellular translation regulation factor that may share molecular mechanism(s) of TFEB/TFE3 activation with translation inhibitors. Such investigation may introduce a new cellular mechanism of translation inhibition-mediated autophagy regulation.


The human cytoplasmatic pool of tRNA for the 20 proteinogenic amino acids and selenocysteine is composed of 48 isoacceptor families –those tRNA with different anticodons– divided into 253 different isodecoder species –those tRNAs that share the same anticodon but present sequence variations in other positions , 2. All The advances in high-throughput sequencing technologies have demonstrated that tRNAs are differentially transcribed and expressed among tissues . Such inThe importance of tRNAs in cell physiology is emphasized by the discovery that these molecules and their derived fragments are distorted in pathological processes, including cancer . Cancer-in vitro [Alterations in DNA methylation constitute a frequent mechanism by which tumor cells inactivate the expression of many coding and non-coding genes to acquire their malignant features . This epin vitro . HoweverTo identify DNA methylation defects in tDNA, we retrieved the Infinium HumanMethylation450 (HM450) methylation microarray probes’ identity that mapped into the loci corresponding to the 416 high confidence tDNA that are found in the human nuclear genome [The data mining of HM450 microarray-derived data of thousands of samples from various cancer types and normal tissues available at The Cancer Genome Atlas (TCGA) and of a panel encompassing approximately 1000 cell lines revealed global differences in tDNA methylation in TCGA primary tumors and cancer cell lines compared to TCGA normal samples, mostly tending towards hypermethylation in malignant samples  when considering all the samples: tRNA-Arg-TCT-4-1, tRNA-Ile-AAT-8-1, and tRNA-Val-CAC-2-1  416 high confidence tDNAs annotated in the GRCh37/hg19 human genome (top). 138 of them are included in the HM450 DNA methylation microarray. 95 of these 138 are located further than 2 kb from any other RNAPII-transcribed gene. The withdrawal of cross-reactive CpGs yielded the 71 different tDNAs that can be efficiently interrogated with this approach (below). The distribution by amino acid and anticodon of the 416 total tDNA genes (top) and those of the 71 that are represented in the HM450 DNA methylation microarray (below) are provided. (B) Average methylation of the 71 tDNA genes according to the HM450 DNA methylation microarray data in normal (left) and tumor (middle) TCGA samples and in cell lines (right). FDR-adjusted p-values correspond to the Mann-Whitney U-test used to compare the methylation average between TCGA normal and tumor samples. ns, not significant; * FDR < 0.05; ** FDR < 0.01; *** FDR < 0.001.Additional file 2: Figure S2. The methylation status of tRNA-Arg-TCT-4-1, tRNA-Ile-AAT-8-1, and tRNA-Val-CAC-2-1 is associated with their expression levels in TCGA tumors. (A) Volcano plot representing the Spearman’s correlation coefficient (ρ) and -log10 FDR of each computed Spearman’s correlation from Figure 1B. Statistically significant associations are shown in blue. (B) Barplot summarizing the tissue frequency of all the statistically significant negative associations between methylation and expression from panel A. Additional file 3: Figure S3. Bisulfite genomic sequencing confirms the methylation status of tRNA-Arg-TCT-4-1 andtRNA-Ile-AAT-8-1 according to the HM450 microarray data. (A) DNA methylation status of tRNA-Arg-TCT-4-1 (cg12798524) and tRNA-Ile-AAT-8-1 (cg21339923) in DND41 and SW48 according to the HM450 microarray data. b-values corresponding to single CpGs are shown for each tDNA. (B) Bisulfite genomic sequencing of tRNA-Arg-TCT-4-1 (top) and tRNA-Ile-AAT-8-1 (below) genes in DND41 and SW48 cell lines. The tDNA gene genomic sequence is indicated with a blue bracket. The orange rectangles correspond to the A and B boxes of the tDNA. The TSS is marked with a black arrow. CpG dinucleotides are represented as short vertical lines, and their methylation status is denoted with black (methylated) or white (unmethylated) squares. The CpG included in the HM450 microarray is marked with a red asterisk.Additional file 4: Figure S4. Impact on growth and apoptosis of the DNA methylation inhibitor 5’-azacytidine(AZA) in DND41 and SW48 cancer cell lines. (A) MTT assay growth assay show a reduced growth of treated cells in both DND41 and SW48 cells lines upon the use of the demethylating agent. Data at each time points are the mean ± SD of four biological replicates. Statistical differences were determined using an unpaired two-tailed Student’s t-test at the 96 hours final time point. *** p < 0.001 (B) Apoptosis quantification by Annexin V incorporation reveals a significant percentage of cells affected by the treatment in DND41 and SW48 cell lines. Data shown represent the mean ± SD of four biological replicates analyzed by unpaired two-tailed Student’s t-test. * p < 0.05; *** p < 0.001.Additional file 5: Figure S5. The demethylation of tRNA-Arg-TCT-4-1 gene is associated with an increased tRNA expression in KIRP and UCEC TCGA tumors. (A) Frequency of tRNA-Arg-TCT-4-1 hypomethylation in tumors derived from TCGA according to the tissue of origin. (B) tRNA-Arg-TCT-4-1 expression is higher in those KIRP (top) and UCEC (below) TCGA tumors where this gene is hypomethylated. Statistical differences in tRNA expression between groups of samples were determined using a two-sided Mann-Whitney U-test; *** p < 0.001. Samples were considered hypomethylated or hypermethylated when tRNA-Arg-TCT-4-1 β-value was lower or higher than 0.33, respectively. Additional file 6: Figure S6.Generation of a tRNA-Arg-TCT-4-1 knockout model in HEC1 cell line using the CRISPR/Cas9 system. (A) Overview of the CRISPR/Cas9 system targeting tRNA-Arg-TCT-4-1 gene. Two different sgRNA (in green and orange) were simultaneously transfected to introduce a deletion of the gene of interest. (B) Genomic PCR using primers flanking tRNA-Arg-TCT-4-1 (black arrows in the overview representation) shows a deletion in two HEC1 knockout cells. (C) qRT-PCR confirms the silencing of tRNA-Arg-TCT-4-1 in the selected HEC1 knockout cells. Data correspond to the mean ± SD of three biological replicates analyzed using the unpaired two-tailed Student’s t-test. *** p < 0.001. Additional file 7: Supplementary methodsAdditional file 8: Table S1. Summary of the results obtained from the Univariate Cox regression analyses performed to compare TCGA patients’ prognosis according to their tDNA methylation levels. Blue-colored cells indicate statistical significance (FDR ≤ 0.05).Additional file 9: Table S2. Detailed list of primers used. All sequences are provided from 5’ to 3’. For tRNA qRT-PCR primers, the commercial reference is provided.


Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), is a contagious viral disease affecting various birds and poultry worldwide. In this project, differentially expressed (DE) circRNAs, miRNAs and mRNAs were identified by high-throughput RNA sequencing (RNA-Seq) in chicken thymus at 24, 48, 72 or 96 h post LaSota NDV vaccine injection versus pre-inoculation group. The vital terms or pathways enriched by vaccine-influenced genes were tested through KEGG and GO analysis. DE genes implicated in innate immunity were preliminarily screened out through GO, InnateDB and Reactome Pathway databases. The interaction networks of DE innate immune genes were established by STRING website. Considering the high expression of gga-miR-6631-5p across all the four time points, DE circRNAs or mRNAs with the possibility to bind to gga-miR-6631-5p were screened out. Among DE genes that had the probability to interact with gga-miR-6631-5p, 7 genes were found to be related to innate immunity. Furthermore, gga-miR-6631-5p promoted LaSota NDV replication by targeting insulin induced gene 1 (INSIG1) in DF-1 chicken fibroblast cells. Taken together, our data provided the comprehensive information about molecular responses to NDV LaSota vaccine in Chinese Partridge Shank Chickens and elucidated the vital roles of gga-miR-6631-5p/INSIG1 axis in LaSota NDV replication. ND is a major threat to poultry industry and public health throughout the world because of its high incidence and mortality rate4. NDV (a single-stranded RNA virus), also called avian paramyxovirus serotype 1 virus, is a member of the Avulavirus genus and Paramyxoviridae family5. NDV can be divided into 3 major pathotypes  according to their virulence6. Lentogenic strain can trigger mild respiratory infection, while velogenic strain can cause hemorrhagic intestinal lesions, respiratory and nervous disorders and high mortality7. The vaccination of NDV is an effective approach to control ND8. Currently, inactivated and live attenuated NDV strains are frequently used as the vaccines to protect poultry and birds against virulent NDV infection9. Live vaccines are known for their strong protective efficacy by virtue of their capacity to efficiently induce a series of robust immune responses10. Among these live attenuated NDV vaccine schemes, LaSota strain is the most widely utilized formulation in multiple countries10. However, “vaccine failure” (e.g. improper or incomplete immunization) is a frequently occurred problem that can give rise to the death of poultry post virulent NDV infection8.Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), is a devastating transmissible disease that can affect human and more than 250 avian species 16. High-throughput RNA sequencing (RNA-seq), a revolutionized technology with low background signal, can provide the comprehensive transcriptome information of cells, tissues and circulatory system under specific conditions18. Over the past decades, RNA-seq, function annotation/enrichment and bioinformatics prediction analysis has been used to determine the expression profiles of transcripts  and speculate/decipher the functions and regulatory networks of these biomolecules22.Recently, non-coding RNAs including circular RNAs (circRNAs) and microRNAs (miRNAs) have attracted much attention from researchers due to their regulatory potential on gene expression in animals including human and poultryTo better manage the problem of “vaccine failure”, it is imperative to have a deep insight into thymic transcriptome alterations in response to NDV LaSota vaccine treatment and related regulatory networks. In this project, RNA-seq technology was used to identify differentially expressed (DE) circRNAs, miRNAs, and mRNAs at different time intervals  post NDV LaSota vaccine inoculation in the thymic tissues of Chinese Partridge Shank Chickens. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis was performed to characterize the functions of DE genes and identify crucial biological processes/pathways/terms in response to vaccine treatment. Additionally, GO, Reactome pathway, and InnateDB databases were utilized to screen out DE genes implicated in innate immunity. Furthermore, bioinformatics analysis was conducted to identify DE circRNAs or mRNAs that had the possibility to interact with a core miRNA. Also, the influence of this central miRNA on NDV replication along with its downstream targets were examined by in vitro cell experiments.In this text, differential expression profiles of circRNAs, miRNAs and mRNAs were examined by RNA-seq in chicken thymic tissues at 24 (B), 48 (C), 72 (D) or 96 (E) h after LaSota NDV vaccine inoculation compared to pre-inoculation (A) group, and corresponding results were presented in Supplementary Tables 2FoldChange|> 1, P value < 0.05) in B versus (vs) A, C vs A, D vs A, or E vs A groups, respectively  in B vs A, C vs A, D vs A, or E vs A group, respectively  . We noticed that 86 items could be found among 96 screened innate immune genes with known names (7 IDs without known names) in STRING website (https://string-db.org/cgi/network.pl?taskId=da9LIhlXqdF5). The image of interaction network was presented in Fig. Next, the interactions among proteins encoded by DE innate immune genes were explored through STRING: functional protein association networks  post NDV vaccine inoculation, we further investigated potential interaction pairs of circRNAs and gga-miR-6631-5p. As presented in sheet 1 of Supplementary Table Recent studies showed that some circRNAs could exert their regulatory roles through serving as miRNA sponges16. Prediction analysis showed that 1431 genes had the potential gga-miR-6631-5p binding sites  post NDV vaccine treatment, we supposed that gga-miR-6631-5p might play vital roles in the responses to LaSota NDV. RT-qPCR assay also validated that gga-miR-6631-5p was highly expressed in LoSota-infected DF-1 cells compared to uninfected cells  were selected for further investigations. RT-qPCR assay revealed that gga-miR-6631-5p overexpression led to the remarkable down-regulation of INSIG1, HSPBP1, SERPING1 and PSMD11 mRNA levels, but had no much effect on E2F1 mRNA expression Fig. A. In viells Fig. F. In addlls Fig. F. In a wIn the present study, we demonstrated that 14, 16, 3 or 8 circRNAs were differentially expressed in thymic tissues of chickens at 24 h, 48 h, 72 h or 96 h after NDV LaSota vaccine inoculation relative to pre-inoculation group. Moreover, 5, 8, 10 or 12 DE miRNAs were identified in B vs A, C vs A, D vs A, or E vs A group, respectively. Furthermore, 1386, 1514, 1196, or 728 mRNAs were found to be differentially expressed in B vs A, C vs A, D vs A, or E vs A group, respectively.25, more innate immunity-related genes were screened out through InnateDB and Reactome Pathway databases. Integration analysis showed that a total of 103 innate immune genes annotated by GO, Reactome pathway, and InnateDB databases were differentially expressed in response to NDV infection. Among these innate immunity genes, 4 genes  were low expressed and 8 genes  were highly expressed in all the groups of B vs A, C vs A, D vs A, and E vs A. AREG, a member of the epidermal growth factor (EGF) family, has been found to be expressed by multiple innate immune cell populations  and adaptive immune cell populations  besides epithelial and mesenchymal cells26. Moreover, AREG was reported to be involved in mediating pathogen resistance, orchestrating tissue repair and homeostasis, inhibiting local inflammation and regulating immune responses26. Qiao et al. demonstrated that GATA4 loss inhibited inflammatory responses and NF-κB activation induced by lipopolysaccharide in human dental pulp cells27. Also, Kang et al. presented that GATA4 was involved in the regulation of inflammatory responses28.Additionally, 32 DE genes were found to be implicated in innate immune responses based on the GO annotation data. Considering the conservation of innate immune responses among different organisms30, suggesting the vital roles of phagosome pathway in immune responses. It has been well documented that RNA virus can be recognized by Toll-like receptors, RIG-I-like receptors and NOD-like receptors, whose activation in response to viral RNAs can initiate innate immune responses such as pro-inflammatory cytokine secretion and type I interferon (IFN) production33. Moreover, Fournier et al. pointed out that the intranasal application of NDV MTH68 strain could induce pro-inflammatory cytokine secretion in mouse bronchial lavage fluid and activate RIG-I and IFN pathways in mouse dendritic cells34. In addition, previous studies showed that RIG-I and IFN pathways played crucial roles in protecting host cells from NDV infection36.Furthermore, the interaction network of proteins encoded by DE innate immune genes was established through STRING website. KEGG enrichment analyses showed that these DE innate immune genes mainly participated in the regulation of 9 KEGG pathways including nucleotide-binding oligomerization domain-containing (NOD)-like receptor signaling pathway, retinoic acid-inducible gene I (RIG-I)-like receptor signaling pathway, cytokine-cytokine receptor interaction and phagosome pathway. Some researchers proposed that phagosome might function as the link organelle between innate and adaptive immunity37. Recently, accumulating evidence shows that circRNAs can function as miRNA sponges (ceRNAs) to relieve the inhibitory activity of miRNAs on target mRNAs by sequestering miRNAs39. Considering the abnormal expression of gga-miR-6631-5p across all the time points post NDV vaccine infection, circRNAs or mRNA targets that had a possibility to interact with gga-miR-6631-5p were predicted using the miRanda software. Combined with expression data of circRNAs/mRNAs and prediction data of circRNAs/mRNAs and gga-miR-6631-5p, 6 DE circRNAs and 64 DE genes in B vs A group, 8 DE circRNAs and 73 DE genes in C vs A group, 1 DE circRNA and 64 DE genes in D vs A group, and 2 DE circRNAs and 33 DE genes in E vs A group were found to have the possibility to bind to gga-miR-6631-5p. Among these DE gga-miR-6631-5p targets, 5 genes  in B vs A group, 5 genes  in C vs A group, 4 genes  in D vs A group and 3 genes  in E vs A group were found to be implicated in innate immune responses.Despite the discovery of multitudinous circRNAs over the past decades, the functions and regulatory mechanisms of most circRNAs remain unknown40. TBK1 was a vital kinase in the induction of antiviral innate immune responses41 and STING was a negative regulator in the replication of multiple RNA viruses such as Vesicular Stomatitis virus and Sindbis virus42. In addition, Ran et al. pointed out that STING knockdown suppressed the expression of interferon regulatory factor 7 (IRF7), type I interferon (IFN)-α, and IFN-β in chicken embryo fibroblasts43.Moreover, our data revealed that gga-miR-6631-5p expression was markedly up-regulated in DF-1 cells infected with LaSota NDV virus compared with uninfected cells. Enforced expression of gga-miR-6631-5p facilitated LaSota NDV replication through targeting INSIG1 in DF-1 cells. INSIG1 has been found to be a bridge in the activation of TANK-binding kinase 1 (TBK1) by promoting K27-linked polyubiquitination of stimulator of interferon genes (STING) and INSIG1 loss curbed STING-mediated antiviral gene induction44, spleen45 or trachea46 from Leghorn and Fayoumi chickens and chicken embryo fibroblasts47 mainly explored the influence of NDV vaccine treatment on immune cells, molecular pathways, and gene expression. In our project, we investigated the expression profile alterations of circRNAs, miRNAs and mRNAs in thymic tissues of Chinese Partridge Shank Chickens at 24, 48, 72, 96 h post NDV infection versus pre-inoculation group. In addition, we further analyzed the expression patterns of 712 innate immune genes annotated by InnateDB database and 319 innate immune genes annotated by Reactome Pathway database in thymic tissues of Chinese Partridge Shank Chickens. A total of 103 innate immune genes annotated by GO, Reactome pathway, and InnateDB databases were identified to be differentially expressed in response to NDV infection in our study. Moreover, we established the potential interaction network of proteins encoded by these dysregulated innate immune genes through String website. Additionally, some possible circRNAs/gga-miR-6631-5p and gga-miR-6631-5p/genes pairs were constructed based on bioinformatics prediction analysis. Furthermore, our data revealed that gga-miR-6631-5p promoted LaSota NDV replication by targeting INSIG1 in DF-1 cells. These data might contribute to the investigation on interactions of NDV LaSota vaccine and Chinese Partridge Shank chickens and the development of new strategies for improving LaSota vaccine efficacy, which has vital values in NDV vaccine immunization work for Chinese native chickens.The previous transcriptome analyses in embryosIn summary, our data provided the time-specific comprehensive information of circRNAs, miRNAs and mRNAs in response to NDV LaSota vaccine infection in thymic tissues of Chinese Partridge Shank chickens and elucidated the vital roles of gga-miR-6631-5p/INSIG1 axis in LaSota NDV replication. Also, multiple innate immune genes implicated in NDV LaSota vaccine infection were screened out.5 50% egg-infectious dose (EID50) of LaSota NDV vaccine  through an ocular route. Chickens were anesthetized and euthanized by the intravenous injection of pentobarbital sodium solution (30 mg/kg body weight), followed by the collection of thymic tissue samples from three chickens at the indicated time point  after NDV vaccine injection. Animal experiments were conducted at Animal Laboratory Center of Henan University of Animal Husbandry and Economy. All in vivo experiments were carried out in compliance with the ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357).All experimental procedures were approved by Institutional Animal Care and Use Committee of Henan University of Animal Husbandry and Economy. Specific pathogen-free (SPF) Chinese Partridge Shank Chickens (30-days-old) free of NDV antibody were reared in the rooms at the biosafety level II facility. These chickens were inoculated with or without 10Total RNA was extracted from thymic tissues using Trizol Reagent  and quantified using NanoDrop 2000 spectrophotometer (Thermo Scientific). Moreover, the quality of isolated RNA was analyzed on Agilent Bioanalyzer 2100 . High-quality RNA samples  were selected for subsequent experiments. cDNA libraries for mRNAs and circRNAs were constructed using the TruSeq Stranded Total RNA Library Prep Kit . Small RNA libraries were conducted using the NEBNext Multiplex Small RNA Library Prep Set for Illumina  based on the manufacturer’s protocols. After quantified and qualified by Agilent Bioanalyzer 2100 (Agilent Technologies), signal strand cDNA libraries were sequenced on IlluminaHiSeq 2500 instrument (Illumina) through Shanghai Personal Biotechnology Co. Ltd. .Raw sequencing data were analyzed and filtered to obtain high-quality clean data, which were then aligned to the reference genome of Gallus_gallus.Gallus_gallus-5.0 (assembly GCA_000002315.3) using Tophat2.P value was less than 0.05.Expression analysis was conducted using the method of fragments per kilobase of transcript per million mapped reads (FPKM). Genes were considered as DE when the absolute value of fold change was greater than 2 and http://www.kegg.jp/), GO (http://www.geneontology.org/), Enzyme Commission (EC) (http://enzyme.expasy.org/), UniProt Knowledgebase (http://www.uniprot.org/help/uniprotkb/), Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (eggNOG) (http://eggnog.embl.de/version_3.0/) databases. Innate immune genes were annotated by InnateDB (https://www.innatedb.ca/) and Reactome Pathway database (http://www.reactome.org/).Annotation analysis for dysregulated genes were conducted using KEGG (www.kegg.jp/kegg/kegg1.html) enrichment analysis was performed using KOBAS database (http://kobas.cbi.pku.edu.cn/kobas3). In GO and KEGG enrichment analysis, p value was obtained by hypergeometric test and then corrected using the multiple hypothesis test of Benjamini and Hochberg (FDR correction method). P value after false discovery rate (FDR) correction < 0.05 represented that the terms were significantly enriched. KEGG enrichment analysis was performed as previously described50.GO and KEGG 51.Venn analysis was performed using the jvenn website (https://string-db.org/).The interactions among proteins encoded by DE innate immune genes were analyzed through STRING: functional protein association networks  were maintained in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum (Thermo Scientific) at 37 °C in a 5% COThe INSIG1-specific small interference RNAs (si-INSIG1) and its negative control si-con were designed and synthesized from Shanghai GenePharma Co., Ltd. . The gga-miR-6631-5p mimic and NC mimic control, gga-miR-6631-5p inhibitor (in-miR-6631-5p) and its negative control in-NC were purchased from Thermo Scientific Co., Ltd. Cell transfection was performed using Lipofectamine 3000 Reagent (Thermo Scientific) following the instructions of manufacturer.−ΔΔCT. GAPDH or 5S ribosomal RNA (5S rRNA) functioned as the endogenous inference to normalize the expression of mRNAs or gga-miR-6631-5p, respectively. Quantitative PCR primers were presented as follows: 5’-ACACTCCAGCTGGGGAAGAGAATGCTGTGG-3’ (forward) and 5’-TGGTGTCGTGGAGTCG-3’ (reverse) for gga-miR-6631-5p; 5’-CACCACAGCCACAGGATTAC-3’ (forward) and 5’-GAAGTCCCCAAAGTCACAGTC-3’ (reverse) for E2F transcription factor 1 (E2F1); 5’-CAGGAGGAGTGACCGTAGGA-3’ (forward) and 5’-CTTTCACACTGGCACCATGC-3’ (reverse) for insulin induced gene 1 (INSIG1); 5’-CACATCCTGGGAGCACTCTG-3’ (forward) and 5’-GTACTCTCCTCAGTGGGGGT-3’ (reverse) for HSPA (Hsp70) binding protein 1 (HSPBP1); 5’-GTGGAATTTGTGGCAGCACC-3’ (forward) and 5’-CTCCTTCTGCTCCCTCGTTG-3’ (reverse) for serpin family G member 1 (SERPING1); 5’-TCGATCTGTGCTTGGAGTGC-3’ (forward) and 5’-GTACCGCTTGGTGTCGAAGT-3’ (reverse) for proteasome 26S subunit, non-ATPase 11 (PSMD11); 5’-GGTAACGCCCGATCT-3’ (forward) and 5’-CGGTATTCCCAGGAGG-3’ (reverse) for 5S rRNA; 5’-GCGAGATGGTGAAAGTCGGA-3’ (forward) and 5’-TTCCCGTTCTCAGCCTTGAC-3’ (reverse) for GAPDH.RNA was isolated and quantified as described above. cDNA first stands were synthesized from RNA template using the iScript cDNA Synthesis Kit  and random primer (for mRNAs) or stem-loop gga-miR-6631-5p RT primer (5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCAGAACC-3’). Next, real-time quantitative PCR reaction was performed using ITaq Universal SYBR Green Super mix (Bio-Rad) and quantitative primers. Relative expression levels of gga-miR-6631-5p and mRNAs were calculated using the method of 2Partial fragment of INSIG1 3’UTR covering gga-miR-6631-5p binding sites was constructed into psiCHECK-2 vector by Hanbio Biotechnology Co., ltd.  and the recombinant plasmid was termed as INSIG1-wt reporter. INSIG1-mut reporter containing mutant gga-miR-6631-5p binding sites was also constructed by Hanbio Biotechnology Co., ltd. DF-1 cells were co-transfected with gga-miR-6631-5p mimic or NC mimic control and INSIG1-wt or INSIG1-mut reporter. At 48 h upon transfection, luciferase activities were measured through Dual-Luciferase Reporter Assay Kit .50 assay using Reed-Muench method.DF-1 cells were transfected with corresponding miRNA mimic, miRNA inhibitor or siRNA, alone or in combination. At 24 h after transfection, DF-1 cells were infected with LaSota NDV at the multiplicity of infection (MOI) of 1. At 12, 24, 36, 48 h after infection, virus titers in cell supernatant were determined through TCIDP < 0.05 as statistically significant. Difference between groups was compared using Student’s T test. Results were presented as mean ± standard deviation (SD).Statistics analysis was performed on GraphPad Prism 7 software  with All methods were carried out in accordance with relevant guidelines and regulations.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.Supplementary Information 12.Supplementary Information 13.Supplementary Information 14.Supplementary Information 15.Supplementary Information 16.Supplementary Information 17.Supplementary Information 18.Supplementary Information 19.Supplementary Information 20.Supplementary Information 21.Supplementary Information 22.Supplementary Information 23.Supplementary Information 24.Supplementary Information 25.Supplementary Information 26.


Therefore, we derived and validated sex-specific thresholds for serum 25(OH)D predictive of poor physical function using 5 cohorts comprising 16,388 community-dwelling older adults (60.9% women). Using a cohort-stratified random two-thirds sample, we found incident slow gait was best discriminated by 25(OH)D<24.0 versus 25(OH)D>=24.0 ng/mL among women  and 25(OH)D<21.0 versus 25(OH)D >=21.0 ng/mL among men . Estimates from the remaining one-third validation sample were similar. Empirically identified and validated sex-specific 25(OH)D thresholds from multiple well-characterized cohorts of older adults may yield more biologically meaningful definitions in important sub-populations. Such thresholds may serve as candidate reference concentrations or inform design of vitamin D intervention trials in older adults.


Signal Transduction and Targeted Therapy 10.1038/s41392-021-00764-4, published online 20 October 2021Correction to: 1, the authors noticed one inadvertent mistake occurred in Fig. Since the publication of this reviewThe original review article has been corrected.


The waning efficacy of SARS-CoV-2 vaccines, combined with the continued emergence of variants resistant to vaccine-induced immunity, has reignited debate over the need for booster vaccine doses. To address this, we examined the neutralizing antibody response against the spike protein of five major SARS-CoV-2 variants, D614G, Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), in health care workers (HCWs) vaccinated with SARS-CoV-2 mRNA vaccines. Serum samples were collected pre-vaccination, three weeks post-first vaccination, one month post-second vaccination, and six months post-second vaccination. Minimal neutralizing antibody titers were detected against Omicron pseudovirus at all four time points, including for a majority of patients who had SARS-CoV-2 breakthrough infections. Neutralizing antibody titers against all other variant spike protein-bearing pseudoviruses declined dramatically from one to six months after the second mRNA vaccine dose, although SARS-CoV-2 infection boosted vaccine responses. Additionally, mRNA-1273-vaccinated HCWs exhibited about two-fold higher neutralizing antibody titers than BNT162b2-vaccinated HCWs. Together these results demonstrate possible waning of antibody-mediated protection against SARS-CoV-2 variants that is dependent on prior infection status and the mRNA vaccine received. They also show that the Omicron variant spike protein can almost completely escape from neutralizing antibodies elicited in recipients of only two mRNA vaccine doses. SARS-CoV-2-specific neutralizing antibodies induced by two doses of mRNA vaccine wane after six months but increase after breakthrough infection. Since its emergence in late 2019, the Coronavirus Disease 2019 (COVID-19) pandemic has led to over 323 million confirmed cases and over 5 million deaths as of January 18, 2022  have emerged. Soon after initial identification, SARS-CoV-2 acquired a predominant D614G mutation in its spike protein. This mutation leads to enhanced transmissibility, likely due to increased stability of the spike protein, as well as increased viral titers in the nasopharynx and increased infectivity , 26 days (IQR = 22.5-28) post-second vaccine dose (Post 2nd), and 194 days (IQR = 190-197.75) post-second vaccine dose (Six Months). According to the pre-determined titer of pseudotyped viruses, we adjusted the volumes of each so that equivalent amounts of infectious virus were used in neutralization assays. HCW serum samples were 4-fold serially diluted, then pseudotyped virus was added for one hour at final dilutions of 1:80, 1:320, 1:1280, 1:5120, 1:20480, and no serum control. For a valid comparison, samples from the same individual collected at different time points were tested side by side on the same plate. HEK293T-ACE2 cells were infected with the virus-serum mixture, and Gaussia luciferase activity was assayed 48 hours and 72 hours after infection. Neutralizing titer 50% (NT50) values were determined by a nonlinear least-squares regression model.We used our previously reported  against D614G (7 of 48), Alpha (17 of 48), Beta (22 of 48), and Delta (14 of 48) following the first dose of vaccine   between these values for all variants except Omicron  every 22 weeks compared with Beta  every 37 weeks   against all five viruses compared to the anti-N protein negative HCWs (n = 43)  for all variants (p = 0.033)  exhibited a 22.6-fold higher mean NT50 value below the limit of detection against Omicron, a comparison between mRNA-1273 and BNT162b2 was not informative  (50 titers (p < 0.001) compared to females against all five variants over the post-vaccination time points (fig. S1). Indeed, studies on sex-based differences in response to COVID-19 mRNA-vaccination have yielded conflicting results , which could be due to the relatively young pool of individuals in this cohort  compared to older adults in a prior mRNA vaccine efficacy study , 26 days (IQR = 22.5 to 28) post-second vaccine dose (Post 2nd), and 194 days (IQR = 190 to 197.75) post-second vaccine dose (6 Months). HCWs received their second vaccine dose between January and February of 2021. No other demographic information was collected on these HCWs. HCW COVID-19 status was determined by anti-N protein ELISA. Of the 48 HCWs examined, one was anti-SARS-CoV-2 N protein positive by ELISA for their pre-vaccination sample. Additionally, four HCWs became anti-N protein positive for their post-first vaccine dose sample, three became positive for their post-second vaccine dose sample, and four became positive for their six-month vaccine sample. This indicated that these 12 individuals were infected by SARS-CoV-2 at different phases after vaccination. HCW serum samples were then assessed for nAb titers against SARS-CoV-2 variant spike protein-pseudotyped lentiviruses. Individual-level NT50 values are presented in data file S1.De-identified vaccinated HCW’s serum samples were collected under approved IRB protocols (2020H0228 and 2020H0527). These 48 HCWs ranged in age from 22 to 61 years  and included 26 male and 22 female HCWs. HCWs were vaccinated with either the Moderna mRNA-1273 vaccine (n = 22) or the Pfizer/BioNTech BNT162b2 vaccine (n = 26). Serum samples were collected from HCWs at four time points, with a median time point of 222 days (IQR = 215 to 225.75) pre-first vaccine dose (Pre), 21 days (IQR = 19.25 to 23) post-first vaccine dose  promoter, both oriented in an antisense orientation relative to the HIV-1 genome. This Gaussia luciferase reporter gene contains a sense orientation intron, which prevents the expression of Gaussia luciferase in the virus-producing cells. However, after the intron is spliced from full-length virus genomes upon integrated into target cells, target cells can produce Gaussia luciferase, which is secreted in mammalian cell culture  and HEK293T-ACE2 cells  were maintained in Dulbecco’s Modified Eagles Medium  supplemented with 10% (v/v) fetal bovine serum  and 1% (v/v) penicillin/streptomycin . Cells were maintained at 37°C and 5% COGaussia luciferase activity in the media of infected cells was measured. Twenty μL of cell culture media and 20 μL of Gaussia luciferase substrate  were combined in white polystyrene 96-well plates. Luminescence was immediately measured by a BioTek Cytation5 plate-reader.Pseudotyped lentivirus was produced by co-transfection of HEK293T cells with pNL4-3-HIV-1-inGluc and pcDNA3.1 vector expressing the spike protein of interest  in a 2:1 ratio using polyethylenimine (PEI) transfection. Virus was collected at 24 hours, 48 hours, and 72 hours after transfection, then was pooled and stored at -80°C. To determine relative titers of harvested virus, the pseudotyped virus for each of the SARS-CoV-2 variants was used to infect HEK293T-ACE2 cells. Then, 48 hours and 72 hours after infection, Gaussia luciferase activity in cell culture media was then assayed 48 hours and 72 hours after infection, as described above. Neutralizing titer 50% (NT50) for each serum sample was determined by non-linear regression with least-squares fit in GraphPad Prism 5 (GraphPad Software).Virus neutralization assays were performed as previously reported  with Bonferroni’s multiple testing correction data in  or one-w data in . For cor


Evodia rutaecarpa (Wu-Chu-Yu) is a well-known Chinese medicine, and rutaecarpine (Rut) is a main bioactive component with substantial beneficial properties including vasodilation. To address a research gap, we investigated the inhibitory mechanisms of Rut in washed human platelets and experimental mice. At low concentrations (1–5 μM), Rut strongly inhibited collagen-induced platelet aggregation, whereas it exerted only a slight or no effect on platelets stimulated with other agonists . Rut markedly inhibited P-selectin expression; adenosine triphosphate release; [Ca2+]i mobilization; hydroxyl radical formation; and phospholipase C (PLC)γ2/protein kinase C (PKC), mitogen-activated protein kinase, and phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3β (GSK3β) phosphorylation stimulated by collagen. SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor) did not reverse Rut-mediated antiplatelet aggregation. Rut was not directly responding to vasodilator-stimulated phosphoprotein phosphorylation. Rut significantly increased the occlusion time of fluorescence irradiated thrombotic platelet plug formation. The findings demonstrated that Rut exerts a strong effect against platelet activation through the PLCγ2/PKC and PI3K/Akt/GSK3β pathways. Thus, Rut can be a potential therapeutic agent for thromboembolic disorders.The role of activated platelets in acute and chronic cardiovascular diseases (CVDs) is well established. Therefore, antiplatelet drugs significantly reduce the risk of severe CVDs.  Evodia rutaecarpa (the dried fruit of this plant is known as “Wu-Chu-Yu” in China), have long been in traditional Chinese medication for the treatment of gastrointestinal disorders, headache, amenorrhea, and postpartum hemorrhage -inositol monophosphate, suggesting that the inhibition of platelet aggregation by Rut is mediated through the inhibition of phospholipase C (PLC) activation i) also plays an important role in platelet aggregation. Treatment with 2.5- and 5-μM Rut reduced the collagen-induced increase in [Ca2+]i by approximately 30% and 50%, respectively  (The release of granular contents (in particular P-selectin from α-granules and adenosine diphosphate (ADP)/adenosine triphosphate (ATP) and Cahe cells . As indit manner B. In add; n = 4) C.3). DAG activates protein kinase C (PKC), triggering an approximately 47-kDa protein that is predominantly phosphorylated (pleckstrin or p47) and causes granule secretion, the main role of IP3 is to induce calcium mobilization i at the time of early phase of platelet activation amino]ethyl]5-isoquinolinesulfonamide dihydrochloride (H-89); 2,3,9,10,11,12-hexahydro-10R-methoxy-2,9-dimethyl-1-oxo-9S, 12R-epoxy-1H-diindolopyrrolobenzodiaz8 cells/mL) were prepared as described previously i mobilization, the supernatant was incubated with Fura 2-AM (5 µM) for 1 h. Platelet suspensions (3.6 × 108 cells/mL) were prepared as described in the previous section, which were adjusted to 1 mM Ca2+. The relative intracellular Ca2+ concentration was measured using a Hitachi Spectrometer F-7000  at the excitation wavelengths of 340 and 380 nm and the emission wavelength of 500 nm i, which in turn eventually inhibit platelet aggregation. The results of the study may exhibit the potential of Rut in therapeutic application for thrombotic diseases.Balanced diet and healthy lifestyle are key variable risk factors for the primordial prevention of CVDs. The results of this study offer new understandings into the role of Rut in human platelet activation. In particular, Rut effectively inhibited platelet activation by hindering the PI3K/Akt/GSK3β and MAPK pathways through a cyclic nucleotides/VASP-independent mechanism. Rut inhibits the P-selectin expression, ATP release, and [Ca


Correction to: J Exp Clin Cancer Res 40, 287 (2021)https://doi.org/10.1186/s13046-021-02081-8Following publication of the original article , the autAdditional file 1: Supplementary Tables 1-8Additional file 2: Supplementary Figures 1-9The correction does not have any effect on the results or conclusions of the paper. The original article has been corrected.


SARS-CoV-2 surface glycoprotein mutations per processed protein.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface glycoproteins deposited to the NCBI GenBank from Europe, by the mid of April 2021 (12.04.2021) were analysed. At least one amino acid mutation relative to YP_009724390.1 referent SARS-CoV-2 surface glycoprotein: Wuhan-Hu-1 complete genome (NCBI accession: NC_045512) was found in 788 SARS-CoV-2 surface glycoproteins. Data was computed by NCBI Cobalt multiple alignment tool  Knowing SARS-CoV-2 spike protein mutations is useful for automated prediction of the change in binding affinity towards neutralizing antibodies. Many crystallographic structures of neutralizing antibodies in complex to SARS-CoV-2 spike protein domains , receptor-binding domain (RBD)) are available at Protein Data Bank (•http://biosig.unimelb.edu.au/dynamut/) http://biosig.unimelb.edu.au/duet/) http://marid.bioc.cam.ac.uk/sdm2) http://biosig.unimelb.edu.au/mcsm_ppi2/) Provided data can be exploited to analyse SARS-CoV-2 surface glycoprotein phylogenesis. Also suitable for mutagenetic studies. Changes in protein stability, protein flexibility and protein to protein interactions upon computed alterations, can be computationally inspected with available tools, such as: DynaMut (•Evolutionary properties of SARS-CoV-2 surface glycoprotein can be monitored based on provided data. Conclusions for enhanced (reduced) variants’ infectivity due to SARS-CoV-2 spike protein mutations can be suggested.1Appendix A available in Data shown in Appendix A in •Country: origin of SARS-CoV-2 surface glycoproteins;•Date of accession: date of GenBank accession;•Number of SARS-CoV-2 spike proteins with unknown amino acids (at least one): if any, their number is reported here;•Number of SARS-CoV-2 spike proteins (no unknown amino acid): refers to SARS-CoV-2 surface glycoproteins with at least one mutation relative to YP_009724390.1 referent SARS-CoV-2 surface glycoprotein ;•Date of computation: the date when alignment was performed;•Sequences with unknown amino acid: listed by accession identifiers.Metadata provides information on:•Seq: ID: NCBI accession identifier of SARS-CoV-2 spike protein;•Changes: List of computed mutations in SARS-CoV-2 spike protein.Following metadata section, mutations report is provided. Mutations report lists SARS-CoV-2 spike proteins with at least one amino acid change relative to YP_009724390.1 spike reference. For each SARS-CoV-2 spike protein there is sperate entry provided. There are two attributes per entry:Annotation Statistics on the type and number of mutations is also provided.Microsoft Excel Workbook SARS-CoV-2SpikeProteinMutations.xlsx, available in this article, contains data on mutations found in 788 SARS-CoV-2 spike proteins. SARS-CoV-2 spike proteins that matched exactly the referent protein (YP_009724390.1) are not reported in the dataset. None of the spike proteins that contained unknown amino acid and there were 28 such  was alig2Cobalt's multiple alignment output for each European country was processed by C# application developed in Visual Studio 2019 and thus dataset presented in this article was generated. The list of mutations was recorded in string format. C# program performed residues’ comparison between YP_009724390.1 reference and each SARS-CoV-2 spike protein to identify mutations. For that reason, string variable Changes was used. Each time new SARS-CoV-2 spike protein was about to be processed, (string) Changes was set up to empty string “”. As mutations were detected, they were tracked to (string) Changes in: FOREACH European countryRUN Cobalt for YP_009724390.1 AND SARS-CoV-2 spike proteinsFROM Cobalt output:FOREACH SARS-COV-2 spike protein:(string) Changes=””FORIF(YP_009724390.1[i]!=SARS-COV-2 spike protein[i])Changes+=YP_009724390.1[i]+”->”+SARS-COV-2 spike protein[i]+” p: ”+i.ToString+” ”File.WriteMutations reports available in dataset reported in this article, served as a source to compute SARS-CoV-2 spike protein variants reported in Appendix A in If the computed key is not contained in the dictionary, then entry:  is added to the dictionary, For this reason, the previous code was upgraded up to variants’ tracking ability at continental level. The upgrade is suggested by the code in italic bellow. The whole process is clearly illustrated on Dictionary<string, int> Variants=empty DictionaryFOREACH European countryRUN Cobalt for YP_009724390.1 AND SARS-CoV-2 spike proteinsFROM Cobalt output:FOREACH SARS-COV-2 spike protein:(string) Changes=””(string) key=””FORIF(YP_009724390.1[i]!=SARS-COV-2 spike protein[i])Changes+=YP_009724390.1[i]+”->”+SARS-COV-2 spike protein[i]+” p: ”+i.ToString+” ”key+=YP_009724390.1[i]+i.ToString+SARS-COV-2 spike protein[i]File.WriteIF(Variants.ContainsKey(key))Variants[key].value++ElseVariants.AddDone Stojanov: Conceptualization, Methodology, Data Collection, Visualization, Writing-Original draft preparation.The author declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Ustilago shanxiensis CBS 10075, which was isolated from a wilting leaf in China, produces mannosylerythritol lipid (MEL) biosurfactants. Here, we report the draft genome sequence of U. shanxiensis CBS 10075, which was 21.7 Mbp in size, with a GC content of 52.55%, comprising 65 scaffolds.The basidiomycetous yeast  Ustilago shanxiensis CBS 10075 (renamed from Pseudozyma shanxiensis CBS 10075) is a basidiomycetous yeast that was isolated from a withered leaf of Quercus mongolica Fisch in China . U. shanxiensis CBS 10075 produces MEL-C compounds as a mixture of 4-O-[noyl)-β-d-mannopyranosyl]-d-erythritol and 4-O-[noyl-2′-O-butanoyl)-β-d-mannopyranosyl]-d-erythritol. The MELs produced by U. shanxiensis CBS 10075 are more hydrophilic than those typically produced by yeasts such as Moesziomyces antarcticus (renamed from Pseudozyma antarctica)  of the U. shanxiensis genome was prepared using a NEBNext Ultra DNA library preparation kit for Illumina , and sequencing was performed using the MiSeq platform . Sequencing data comprising a total of 10,427,528 paired-end reads were generated; each read was 250 bp in length. A mate-paired library  was then prepared using a Nextera mate pair sample preparation kit (Illumina), generating 9,469,570 mate-paired reads. The quality of paired-end and mate-paired reads was checked by FastQC version 0.11.2. Genome sequence assembly using the ALLPATHS-LG version R46449 assembler  composed of 271 contigs generated from the paired-end and mate-paired reads, with 81× and 64.5× sequence coverage, respectively. The U. shanxiensis CBS 10075 draft genome size was 21.7 Mbp . The length of the longest scaffold was 2,733 kbp. Five MEL biosynthesis genes were predicted using AUGUSTUS version 2.5.5 (UshEMT1 (an erythritol-mannosyl-transferase), UshMAC1 and UshMAC2 (acyl-coenzyme A [CoA]-dependent acyltransferases), UshMAT1 (an acetyltransferase), and UshMMF1 (a putative MEL transporter). This cluster of MEL biosynthesis genes was first reported in Ustilago maydis . The genome sequence of U. shanxiensis CBS 10075 will improve our understanding of the molecular mechanisms driving MEL production and will enable the development of MELs tailored for a range of industries.The CBS 10075 genome sequence contained a conserved MEL biosynthesis gene cluster comprising five genes in scaffold 6, namely, o maydis . Recentlis genes  that shoU. shanxiensis CBS 10075 genome has been deposited in DDBJ/EMBL/GenBank under accession numbers BPMX01000001 to BPMX01000065. The DDBJ Sequence Read Archive (DRA) accession number is DRR306489. The protein identification numbers are as follows: UshMAC2, GIZ99647; UshEMT1, GIZ99648; UshMAC1, GIZ99649; UshMMF1, GIZ99650; and UshMAT1, GIZ99651.The nucleic acid sequence of the


Scientific Reportshttps://doi.org/10.1038/s41598-021-98589-2, published online 23 September 2021.Correction to: The original version of this Article contained an error in the Acknowledgments section.“This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MEST) (No. NRF-2020R1A2C2014422).”now reads:“This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MSIT) (No. NRF-2020R1A2C2014422).”The original Article has been corrected.


Cancer-associated fibroblasts in the stromal tumor microenvironment play a key role in cancer progression, invasion, metastasis, and therapy resistance. Cancer-associated fibroblasts communicate with tumor cells through diverse factors, such as growth factors, hedgehog proteins, cytokines, and chemokines, regulating signaling activity in paracrine as well as paracrine-reciprocal ways. Furthermore, cancer-associated fibroblasts, not only tumor cells, secrete exosomes that drive pre-metastatic niche formation and metastasis.Pancreatic cancer is currently the fourth leading cause of cancer deaths in the United States, and the overall 5 year survival rate is still only around 10%. Pancreatic cancer exhibits a remarkable resistance to established therapeutic options such as chemotherapy and radiotherapy, in part due to the dense stromal tumor microenvironment, where cancer-associated fibroblasts are the major stromal cell type. Cancer-associated fibroblasts further play a key role in cancer progression, invasion, and metastasis. Cancer-associated fibroblasts communicate with tumor cells, not only through paracrine as well as paracrine-reciprocal signaling regulators but also by way of exosomes. In the current manuscript, we discuss intercellular mediators between cancer-associated fibroblasts and pancreatic cancer cells in a paracrine as well as paracrine-reciprocal manner. Further recent findings on exosomes in pancreatic cancer and metastasis are summarized. Pancreatic cancer is currently the fourth leading cause of cancer deaths in the United States, and its incidence continues to increase in both females and males . PancreaPdx1-Cre; G12D/+lox-stop-lox-Kras; R172H/+Trp53) (CCN2) gene (encoded for CTGF) expression (CCN2 gene expression (SLC39A4 gene) activates STAT3, which subsequently induces ZEB1. ZEB1 in turn activates the expression of integrin ITGA3 and ITGB1. Integrin α3β1 signaling inhibits the expression of gemcitabine transporter equilibrative nucleoside transporter 1   plasticity. EMT plasticity is a dynamic and reversible transition between EMT and mesenchymal-to-epithelial transition (MET) . In. InPdx1-R172H/+)  [11]. CTR172H/+)  13]. In. InPdx1-pression  [13]. ZEpression  12]. ZE. ZEPdx1-pression  14]. Zi. ZiPdx1-SLC29A1)  15]. ZI. ZIPdx1-on (MET) . It has Ptf1a-Cre; G12D/+lox-stop-lox-Kras; lox/+Trp53) . SH. SHPtf1- targets  18]. IG. IGPtf1-KPC mice . LDE225,KPC mice . Taken t-Rosa26) . Furtherpression . It has molox/−) . The rolActa2 (coding α-SMA), several genes such as Col1a1, Col5a1, Col6a1, Ctgf, and Vim are upregulated in myCAFs . Bl. BlRip3 lox mice . On the tastasis . Pancrealox/lox) . The rea+ T cells to reduce their infiltration of the juxtatumoral compartment (less than 100 µm from the tumor) of pancreatic cancer [+ T cells in the juxtatumoral compartment exhibit prolonged postsurgical survival [+ T cells in juxtatumoral compartments in KPC mice [Nfkb1 in PSCs reduces CXC12 secretion, increases infiltration of CD8+ T cells, inhibits tumor growth, and improves host survival, evaluated by orthotopic co-injection experiments with primary tumor cells from KPC mice and PSCs [+ T cell tumor infiltration and decreases tumor cell density  [Cxcr4 in KPC mice  attenuates fibrogenesis and decreases α-SMA and PDGFR-α-positive cells but increases tumor size [Cxcr4 loss are inconsistent. CXCL12 binds CXCR7 with higher affinity than CXCR4 [Cxcr4 deficiency can increase activity of CXCR7 signaling in pancreatic cancer has not been analyzed.Activated PSCs sequester CD8c cancer . Pancreasurvival . AdminisKPC mice . T cellsKPC mice . Lack ofand PSCs . CXCL12 and PSCs . Treatmeand PSCs . Another2826486) . It has mor size . Furtheran CXCR4 . In CXCLan CXCR4 . CXCR7 pan CXCR4 . WhetherFsp-Cre; lox/loxFak) increases breast and pancreatic cancer growth [Fak-depletion in CAFs promote secretion of several chemokines CCL6, CCL11, and CCL12. These chemokines activate chemokine receptors CCR1/CCR2 on cancer cells  and 6-phosphogluconate dehydrogenase (6PGD) in the oxidative branch of pentose-phosphate pathway (PPP) [Ptf1a-Cre; G12D/+lox-stop-lox-Kras; tm3(DTR/GFP)AyrFoxp3) induces reprogramming the fibroblast population, leading to a reduced number of α-SMA+ myCAFs; an increase in Ccl3, Ccl6, and Ccl8 expression; increased myeloid cell infiltration; and accelerated pancreatic carcinogenesis [Ptf1a-Cre; G12D/+lox-stop-lox-Kras; tm3(DTR/GFP)AyrFoxp3 mice [Depletion of focal-adhesion kinase (FAK) in the FSP1-positive cells in mice (r growth . Low FAKr growth . Fak-deper cells . The enhay (PPP) . Paracriay (PPP) . Treg fray (PPP) . To thatogenesis . CCR1 isogenesis . CCR1 blAyr mice .KRAS mutation ratio (≥5%) in circulating exosomal DNA is associated with shorter progression-free survival and overall survival of pancreatic cancer patients [+ circulating exosomes than in healthy donors, and elevated level of GPC1+ circulating exosomes is also observed in Ptf1a-Cre; G12D/+lox-stop-lox-Kras; lox/loxTgfbr2 mice [EV signature) with EGFR, EPCAM, MUC1, GPC1, and WNT2 shows an 86% sensitivity and 81% specificity [Exosomes are a subset of cell-released, membranous-structured extracellular vesicles that also include microvesicles, microparticles, ectosomes, and apoptotic bodies . Exosomepatients . Due to patients . Additiopatients . A Dickkpatients . High expatients . Blockadpatients . Glypicapatients . Pancrealox mice . GPC1 allox mice . Multipacificity . Anothercificity . Treatmecificity . GPC1 iscificity . GPC1 ancificity . It has cificity . GW4869 cificity . Inhibitcificity .Prkd1 accelerates pancreatic tumorigenesis, drives lung metastasis, and enhances secretion of extracellular vesicles  [Prkd1-deficiency in pancreatic cancer cells increases α6β4 loading into extracellular vesicles that requires CD82 [G12D suppresses pancreatic cancer progression and improves survival of KPC and Ptf1a-Cre; G12D/+lox-stop-lox-Kras; lox/loxTgfbr2 mice [Tumor-driven exosomes can prepare the pre-metastatic niche in distant organs. Integrin subtypes expressed on tumor-driven exosomes can predict the site of metastasis . It has lox/lox) . Prkd1-dres CD82 . Exosomeres CD82 . Exosomeres CD82 . Exosomelox mice .Recent studies have demonstrated that a large number of factors, including growth factors, hedgehog molecules, inflammatory cytokines, and chemokines derived from both cancer-associated fibroblasts and tumor cells, act as key autocrine, paracrine, and reciprocal mediators in signal transduction networks. Cancer-associated fibroblasts play an important role, not only in the tumor microenvironment but also in secretion, regulation of exosomes, and in the preparation of the pre-metastatic niche in distant organs. Exosomes can be used as a carrier of therapeutic reagents for pancreatic cancer. A broad and better understanding of the interactions between cancer-associated fibroblasts and tumor cells is important for developing novel therapeutic strategies that improve the outcome of pancreatic cancer patients.


NL4-3 significantly decrease or eliminate viral infectivity due to defective fusion and increased gp120 shedding. To identify compensatory Env mutations that restore viral infectivity, we infected a CD4+ T-cell line with PR mutants pseudotyped with wild-type (WT) HIV-1 Env or vesicular stomatitis virus envelope glycoprotein (VSV-G). We found that PR mutant-infected CD4+ T-cells produced infectious viruses at 7 days postinfection (dpi). Sequencing of the env cDNA from cells infected with the recovered HIV-1 revealed that the S534P mutant reverted to serine or threonine at residue 534. Interestingly, the combined PR-mutant HIV-1 (S534P/T536A or S534P/T536A/T538A) recovered its infectivity and reverted to S534, but maintained the T536A or T538A mutation, suggesting that HIV-1 replication in CD4+ T-cells can tolerate T536A and T538A Env mutations, but not S534P. Moreover, VSV-G-pseudotyped HIV-1 mutants with a fusion-defective Env also recovered infectivity in CD4+ T-cells through reverted Env mutations. These new observations help define the Env residues critical for HIV-1 infection and demonstrate that Env-defective HIV-1 mutants can rapidly regain replication competency in CD4+ T-cells.HIV-1 envelope glycoprotein (Env) interacts with cell surface receptors and induces membrane fusion to enter cells and initiate infection. HIV-1 Env on virions comprises trimers of the gp120 and gp41 subunits. The polar region (PR) in the N-terminus of gp41 is composed of 17 conserved residues, including seven polar amino acids. We have reported that the PR is crucial for Env trimer stability and fusogenicity. Mutations of three highly conserved residues  in the PR of HIV-1IMPORTANCE Our previous mutagenesis study revealed that serine at position 534 of HIV-1 Env is critical for viral infectivity. We found that HIV-1 Env containing serine to proline mutation at position 534 (S534P) are incapable of supporting virus-cell and cell-cell fusion. To investigate whether these mutant viruses can recover infectivity and what amino acid changes account for recovered infectivity, we infected CD4+ T-cells with Env-mutant HIV-1 pseudotyped with WT HIV-1 Env or VSV-G and monitored cultures for the production of infectious viruses. Our results showed that most of the pseudotyped viruses recovered their infectivity within 1-week postinfection, and all the recovered viruses mutated proline at position 534. These observations help define the Env residues critical for HIV-1 replication. Because Env-defective HIV-1 mutants can rapidly regain replication competency in CD4+ T-cells, it is important to carefully monitor viral mutations for biosafety consideration when using HIV-1-derived lentivirus vectors pseudotyped with Env. NL4-3 can efficiently infect target cells (+ T-cell line with HIV-1 PR mutants pseudotyped with WT HIV-1 Env or vesicular stomatitis virus envelope glycoprotein (VSV-G).HIV-1 envelope glycoprotein (Env) comprises a surface unit (gp120) and a transmembrane unit gp41). HIV-1 Env forms trimers incorporated into virions to mediate receptor recognition and fusion-based entry 13. The gp1. HIV-1 5–et cells . To examNL4-3  as described  levels were quantified and equal amounts of WT or mutant HIV-1 were used to infect TZM-bl cells that express luciferase upon productive HIV-1 infection . Supernaenv genes of the recovered viruses and sequenced 800–900 nt covering the PR. To eliminate noninfectious virus particles produced by Hut/CCR5 cells, the collected M1/Env, M3/Env, and M4/Env viruses were used to infect fresh Hut/CCR5 cells. Infected Hut/CCR5 cells were collected at 3 dpi for cellular DNA extraction, and full-length env genes were cloned from integrated proviral DNA. For each recovered virus, six clones of the env gene were sequenced. Interestingly, the PR sequences of recovered viruses are identical to that of WT Env  and WT Env are identical aside from the PR point mutations sites (To identify sequence changes in Env of the reversion mutants, we cloned full-length f WT Env . Moreovens sites , it is penv sequence changes (Next, to exclude the possibility of recombination between mutant and WT Env, VSV-G-pseudotyped mutant viruses were generated in HEK293T cells by transfection, and then used to infect Hut/CCR5 cells to determine replication kinetics and  changes . These V changes . At 3 dp changes . Howeverenv gene recombination in virus producer HEK293T cells when generating Env-, but not VSV-G-pseudotyped HIV-1.To determine the infectivity of VSV-G-pseudotyped viruses in Hut/CCR5 cells from 3 to 15 dpi, supernatants of Hut/CCR5 cells were collected every 2 days to quantify viral infectivity using TZM-bl cells with equal viral p24 input. Consistent with Env-pseudotyped HIV-1 infection of Hut/CCR5 cells , M1/VSV-env gene of replication-competent HIV-1 recovered from M1/VSV-G, M3/VSV-G and M4/VSV-G, fresh Hut/CCR5 cells were infected with the recovered viruses. Cellular DNA was extracted at 3 dpi for cloning of full-length env genes from integrated proviral DNA. For each recovered virus, 6 clones of the env gene were sequenced. All of the recovered viruses mutated proline at position 534 (To identify sequence changes in the tion 534 . All buttion 534 . These rtion 534  showing tion 534 . Our pretion 534 .Our structural analysis  showed t+ T-cells. Our results also highlight the importance of carefully monitoring viral mutations for biosafety consideration when using HIV-1-derived lentivirus vectors pseudotyped with Env (In summary, our new observations further define the Env residues critical for HIV-1 infection and demonstrate that Env-defective HIV-1 mutants can rapidly regain replication competency in CD4with Env , 14.