HRAS-mutant tumors showed mixed responses dependent on cancer type. Co-occurring mutations may affect response. We aimed to uncover cooperative genetic events specific to HRAS-mutant tumors and study their effect on FTI sensitivity. Using targeted sequencing data from MSK-IMPACT and DFCI-GENIE databases we identified co-mutations in HRAS- vs KRAS- and NRAS-mutant cancers. HRAS-mutant cancers had a higher frequency of co-altered mutations (48.8%) in MAPK, PI3K, or RTK pathways genes compared to KRAS- and NRAS-mutant cancers . Class 3 BRAF, NF1, PTEN, and PIK3CA mutations were more prevalent in HRAS-mutant lineages. To study the effect of comutations on FTI sensitivity, HrasG13R was transfected into ‘RASless’  mouse embryonic fibroblasts (MEFs) which sensitized non-transfected MEFs to tipifarnib. Comutation in the form of Pten or Nf1 deletion or Pik3caH1047R or BrafG466E transduction led to relative resistance to tipifarnib in HrasG13R MEFs in the presence or absence of KrasWT. Combined treatment of tipifarnib with MEK inhibition sensitized cells to tipifarnib, including in MEFs with PI3K pathway comutations. HRAS-mutant tumors demonstrate lineage demonstrate lineage-dependent MAPK/PI3K pathway alterations that confer relative resistance to tipifarnib. Combined FTI and MEK inhibition is a promising combination for HRAS-mutant tumors.The clinical development of farnesyltransferase inhibitors (FTI) for  HRAS-mutant tumors is approximately 230,000 per year- representing 7% of all RAS-mutant tumors., lung, thyroid and melanoma., is a targeted therapy for t tumors.–12 FTIs membrane., 14 Althnot HRAS. A phase oprotein. In additoprotein. and urotoprotein. with HRAHRASG12V/TP53flox/flox) poorly differentiated thyroid cancers developed acquired resistance to FTIs associated with mutations in Nf1 and Gnas, which recapitulated FTI resistance when introduced into HRAS-mutant thyroid cancer cells. pathway mutations, MAP kinase (MAPK) pathway mutations and PI3 kinase (PI3K)/mTOR pathway mutations, and plotted based on cancer type and RAS-mutant type. Specific genes that were categorized are listed in Supplementary Table 1.Clonality of mutations was assessed using the FACETS (Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing) algorithm, which provides allele-specific copy number estimates at both the gene level and chromosome arm level. FACETS was used to infer cancer cell fraction associated with individual mutations in order to determine clonality status.KRASlox/lox/NRAS−/−/HRAS−/−/RERTert/ert) mouse embryonic fibroblasts (MEFs) were obtained from the Piro Lito lab  with permission from Dr. Mariano Barbacid .(G13R pReceiver-LV205 plasmid (Genecopia) using a lentiviral transfection technique with Opti-MEM, Mission® Lentiviral Packaging Mixture, and Fugune®6 Tranfection Reagent (Promega), HrasG13R-transfected colonies were identified following puromycin selection for at least one week. HRAS-expressing cells were selected and flow-sorted using the GFP signal from the bicistronic vector. Treatment with 600nM tamoxifen (4-OHT) for at least 2 weeks was used to flox-out the KRASlox/lox allele.“Rasless” . Cells weG13R allele. To knock out Nf1 a dual-guide RNA targeting RAS-binding domain in exon 21 and for Pten a dual-guide RNA against phosphatase domain in exon 5 were used. Respective dual-guide RNA with CAS9 and mCherry coexpression plasmids were custom designed and purchased from Vector builder (Supplementary Table 2). CRISPR-CAS9 plasmids were transfected using Fugene, transfected cells were selected by RFP flow sorting, single cell clones isolated, and confirmed for respective CRISPR knock outs. Gain-of-function BrafG466E (Class 3 BRAF) and Pik3caH1047R sequences were cloned into Cs-Mm34053-Lv213 (CMV-mutant gene-IRES2-mCherry-IRES-Blasticidin) (Addgene). They were then introduced using lentiviral transduction with 2 ug/mL blasticidin as the selection antibiotic for at least one week. Mass-transduced cells with the lowest 5% expression levels were selected as determined by flow cytometry using an IRES-mCherry signal as a reporter. Treatment with 4-OHT was used to delete KRAS as described previously assays were done by plating on Day 0 either 70,000 cells in 6-well plates in DMEM media with 15% fetal bovine serum (FBS) and penicillin/streptomycin/L-glutamine  or 100,000 cells for serum-starved (1% FBS) conditions. Cells were treated with the indicated drug concentrations on day 1 with subsequent drug and media change on day 3. Cells were harvested on day 6 by trypsinization and counted using a Vi-Cell series Cell Viability Analyzer (Beckman Coulter). For combination treatments, we plated 3000 cells/well in a 48-well plate in complete media and assayed responses with a crystal violet assay. Cells were treated on Day 1, with drug and media change on day 3. Cells were subsequently fixed and stained with crystal violet on day 6. The following day plates were imaged using Gelcount™ analyzer (Oxford Optronics) and subsequently resuspended in 10% acetic acid and counted using a microplate reader (Spectramax® iD5). IC50 and crystal violet assays were done in triplicate.Half maximal inhibitory concentration . SDS-PAGE and blocking were done as previously described(® 20 detergent (TBST) and blocked with a Goat anti-Rabbit (IRDye® 680RD) or Goat anti-Mouse (IRDye® 800CW) secondary antibody. Membranes were then imaged using a LI-COR Odyssey® DLx Imaging System.Cells treated in the indicated conditions were rinsed with cold PBS and lysed using RIPA (Millipore) supplemented with protease  and phosphatase inhibitors . Protein lysates were collected by spinning cells in a 4-degree centrifuge at 14,000 RPM for 20min. Protein concentrations were measured using the BCA kit (ThemoFisher Scientific) on a microplate reader , total ERK (#4695), pAKT-Ser473 (#9271), pAKT-Thr308 (#13038), total AKT (#2920), PTEN (#9559), PI3K-p110a (#4249) from Cell Signaling; Vinculin (V4139) from Sigma; BRAF (sc-5284) and KRAS (sc-517599) from Santa Cruz; HRAS (18295-1-AP) from ProteinTech; PI3K-p85 (06-195) from Millipore; Total RAS from ThermoScientific (#1862335). All antibodies were diluted to 1:1000.Tipifarnib (RR11577), AZD8186 (S7694), Alpelisib , and Pictilisib  were purchased from SelleckChem.RAS-mutant type. Differences in mutation frequencies between the various cohorts was determined utilizing Fisher’s exact test for binomial proportions. Significance was established as p<0.05.Genomic analysis of gene signature and gene level mutation frequencies were used to compare co-altered lesion prevalence by cancer type and in-vitro studies was performed using GraphPad Prism version 7.0., using two-tailed non-parametric t-tests. Significance was established as p<0.05. IC50 curves were generated on GraphPad using non-linear regression.Statistical analysis for the HRAS mutations were identified in the MSK-IMPACT and DFCI-Genie cohorts, with the number and relative frequency in the evaluated cancer types shown in HRAS codon mutations differed by cancer type with substitutions at Q61 being the most common, which was particularly enriched in thyroid and prostate cancers (p<0.001 for both) (Altogether 255 (1.4%) patients with canonical or both) , bottom.KRAS and NRAS were more prevalent than HRAS mutations (2516 (14.0%) and 701 (3.9%), respectively; p<0.001 vs HRAS for both), as shown in HRAS mutations were found in salivary gland and HNSCC cancers (both p<0.05). KRAS mutations were more prevalent in NSCLC (p<0.01) and NRAS mutations in thyroid and melanoma (both p<0.05).Overall HRAS-mutant cancers (48.4%) were found to have co-altered mutations along the RTK/MAPK/PI3K pathways compared to KRAS- (41.4%) and NRAS-mutant (38.4%) cancers . In addition, class 3 BRAF mutations were more prevalent in HRAS-mutant salivary gland (5%), and bladder (9%), and melanoma (11%) cancers, whereas none were present in the respective KRAS- and NRAS-mutant tumors . Class 2 BRAF mutations were also significantly more prevalent in HRAS (8%) compared to KRAS- and NRAS-mutant melanoma. Clonality analysis of HRAS-mutant/MAPK-co-altered tumors was limited to the MSK-IMPACT samples. With few exceptions HRAS mutations were clonal, whereas the clonality of the co-altered lesions was more variable  had significantly more co-alterations in PI3K/mTOR pathway genes compared to KRAS (32.2%) and NRAS (23.7%) (PTEN (21%) (p<0.05 vs KRAS- and NRAS-mutant NSCLC). PIK3CA mutations were found in 59% of HRAS-mutant salivary gland tumors but due to the limited number of KRAS- and NRAS-mutant tumors of this lineage this was not significantly different, but was more prevalent than in other HRAS-mutant cancers (p<0.05). Of note, EIF1AX mutations were found almost exclusively in HRAS-, KRAS-, and NRAS-mutant thyroid cancers (p<0.05), whereas STK11 mutations were found in greater proportion of HRAS-mutant NSCLC compared to the other HRAS-mutant cancer types. Clonality analysis for HRAS-mutant/PI3K-co-altered tumors and noteworthy for the majority of HRAS-mutant/PIK3CA (82.4%) and PTEN-mutant tumors (83.3%) being clonal ( (23.7%) ; p<0.05.g clonal .Kraslox/lox;Nras−/−;Hras−/−;RERTert/ert) MEFs were utilized to characterize the growth, signaling, and drug sensitivity of the most common co-altered mutations identified in the MSK-IMPACT and GENIE data (KrasWT expression when MEFs were exposed to 600nM tamoxifen (4OHT). (“Rasless” (NIE data . We confn (4OHT). Loss of reatment , with a reatment . Raslessor both) , left.HrasWT allele did not confer sensitivity to tipifarnib in the presence of a KrasWT allele (IC50: >3uM) but demonstrated exquisite sensitivity in the presence of tamoxifen , consistent with the on-target effect of the FTI (G13R (50: 324.7nM) as compared to parental and HrasWT-transfected cells even in the presence of KrasWT (p<0.001), whereas tamoxifen-induced KrasWT loss rendered HrasG13R-expressing cells highly sensitive to treatment .Introduction of an  the FTI , middle.TI , suggesting that KrasWT expression had a dampening effect on the proliferative drive induced by mutant Hras  . Crystal violet assays were performed to identify appropriate combination doses of tipifarnib and trametinib, with 100nM:10nM tipifarnib:trametinib determined to adequately inhibit growth in non-4OHT-treated HrasG13R- mutant MEFs . Trametinib and combination tipifarnib:trametinib treatment were highly effective at treating KrasWT-expressing HrasG13R cells, with cell viability relative to DMSO significantly lower than tipifarnib monotherapy at 120h  (KrasWT-expressing cells with trametinib monotherapy and tipifarnib:trametinib combination. The pERK rebound with combination tipifarnib:trametinib abated in 4OHT-treated cells.We next investigated whether the dampening effect of p<0.001) . In 4OHTp<0.001) , but rebBrafWT or BrafG466E cDNA into HrasG13R-expressing MEFs, cells were sorted for mCherry to select those in the 5th percentile of expression, which mimicked endogenous levels of Braf , whereas in 4OHT-treated cells they were highly sensitive to tipifarnib and not significantly different to each other (IC50 BrafG466E vs BrafWT: 5.9nM vs 6.9nM) . Trametinib monotherapy effectively inhibited the growth of HrasG13R/BrafG466E cells . Signalling was performed amongst various suspected homozygous clones with demonstrated increased pERK signal with NF1 loss-of-function    and 4J, : 0.8nM) –N. InterPten loss-of-function mutations in HrasG13R-transfected MEFs using sgRNA targeting of exon 5. Western blot analysis identified one clone with a homozygous Pten loss-of-function mutation and we observed concomitant increased pAkt-Ser473 signal  or Pictilisib (pan-PI3K inhibitor) monotherapy in the absence or presence of 4OHT  which demonstrated relative resistance to combination PI3K inhibition.We generated 3 signal . At base of 4OHT  and 5C. HrasG13R/PtenLOF MEFs was insensitive to the tipifarnib:AZD8186 or tipifarnib:pictilisib combinations .In the absence of 4OHT, growth assays of inations –E. When inations –G. WesteHrasG13R/PtenLOF cells given the sensitivity we observed in HrasG13R, HrasG13R/BrafG466E, and HrasG13R/Nf1lof Rasless-MEFs. Crystal violet assays were again employed to define optimal concentrations of drug . Despite the loss of function of Pten, trametinib monotherapy and combination tipifarnib:trametinib had significant anti- proliferative effects that were augmented by 4OHT  and picitilisib monotherapies As in previous experiments, we identified sensitive concentrations of drugs using crystal violet assays . Resistance to tipifarnib, alpelisib, and picitilisb monotherapy was not significantly overcome with combination tipifarnib:alpelisib or tipifarnib:pictilisib therapy –E. HowevKRAS expression on proliferation of HrasG13R-transfected MEFs with or without co-mutations. Cells were grown for 6 days in 15% . We found the combination of tipifarnib and alpelisib to have limited efficacy in the HrasG13R/Pik3caH1047R MEFs, which was modestly improved by deletion of Kras-WT. Instead the combination of the MEK inhibitor trametinib and tipifarnib was highly effective for HrasG13R MEFs harboring either Pik3caH1047R or deletion of Pten. Interestingly, Pten loss enhances MAPK activation and MEK inhibitor sensitivity in a mouse model of Her2/neu-driven breast cancer.(We found that comutations in the PI3K/mTOR pathway also imparted resistance to FTIs. Mutations in the PI3K pathway have been reported in patients with -1 trial. Combinatt cancer. These fiHRAS-mutant cancers. The combination of FT and MEK inhibitors was efficacious regardless of co-mutation status, which may help inform the design of future clinical trials.In summary, results from our study highlight the importance of co-altered mutations and of wild-type RAS in driving resistance to targeted therapy of


Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with a 5-year survival rate of 6% following a diagnosis, and novel therapeutic modalities are needed. Protease-activated receptor 1 (PAR1) is abundantly overexpressed by both tumor cells and multiple stroma cell subsets in the tumor microenvironment (TME), thereby offering a suitable immunotherapy target.A chimeric antigen receptor (CAR) strategy was applied to target PAR1 using a human anti-PAR1 scFv antibody fused to the transmembrane region with two co-stimulatory intracellular signaling domains of cluster of differentiation 28 (CD28) and CD137 (4-1BB), added to CD3ζ in tandem.The engineered PAR1CAR-T cells eliminated PAR1 overexpression and transforming growth factor (TGF)-β-mediated PAR1-upregulated cancer cells by approximately 80% in vitro. The adoptive transfer of PAR1CAR-T cells was persistently enhanced and induced the specific regression of established MIA PaCa-2 cancer cells by > 80% in xenograft models. Accordingly, proinflammatory cytokines/chemokines increased in CAR-T-cell-treated mouse sera, whereas Ki67 expression in tumors decreased. Furthermore, the targeted elimination of PAR1-expressing tumors reduced matrix metalloproteinase 1 (MMP1) levels, suggesting that the blocking of the PAR1/MMP1 pathway constitutes a new therapeutic option for PDAC treatment.Third-generation PAR1CAR-T cells have antitumor activity in the TME, providing innovative CAR-T-cell immunotherapy against PDAC.The online version contains supplementary material available at 10.1186/s12916-023-03053-9. Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignancy of both the digestive system and endocrine system with disappointing prognoses. The number of newly diagnosed PDAC cases worldwide is approaching five million each year. As the seventh leading cause of cancer-related deaths, PDAC also accounts for more than 4.6 million new deaths . Given t2+ signaling and facilitates pancreatic stellate cell proliferation, contributing to the development of PDAC , and cells were collected on days 1, 3, and 7 following treatment. PAR1 expression was measured by flow cytometry. (E) Results shown are the potential enhanced levels of PAR1 by the mean fluorescence intensity (MFI) over incubation times. (F) Standard 24-h cytotoxicity activities were determined using MTT assays with at least three replicates (n ≥ 3) with increasing effector/tumor (E/T) ratios of 1, 5, and 20 against the four normal cell lines following 60 ng/mL TGF-β stimulation for 7 days. Cytotoxic activities were compared with non-transduced CD3 T cell-treated and mock-transduced T cell-treated groups which served as the controls of PAR1CAR-T cells (* p < 0.05). Results shown are the mean ± SD of three independent experiments.Additional file 5:Figure S5. Significant cytotoxicity activities of PAR1CAR-T cells toward MIA PaCa-2 cells in different co-culture conditions by green fluorescent protein (GFP) labeling. GFP-labeled MIA PaCa-2 cells (MIA PaCa-2-GFP) co-cultured in different conditions including cancer-associated fibroblasts (CAFs), CAFs+CD4+CD25+regulatory T cells (Tregs), and CAF+CD4+CD25-T effector cells. Cell viability of cancer cells in response to PAR1CAR-T cells at an effector/tumor (E/T) ratio of 0.5 showed significant cytotoxicity activities toward MIA PaCa-2-GFP+CAFs  and MIA PaCa-2-GFP+CAFs+CD4+CD25+Tregs  compared to that of mock-transduced-T-cell controls following 24 h of treatment using GFP fluorescence imaging (left panel). The scale bar denotes 50 µm.Additional file 6. Uncropped gels/blots images.


Connective tissue disease-associated interstitial lung disease (CTD-ILD) is a severe manifestation of CTD that leads to significant morbidity and mortality. Clinically, ILD can occur in diverse CTDs. Pathologically, CTD-ILD is characterized by various histologic patterns, such as nonspecific interstitial pneumonia, organizing pneumonia, and usual interstitial pneumonia. Abnormal immune system responses have traditionally been instrumental in its pathophysiology, and various changes in immune cells have been described, especially in macrophages. This article first briefly overviews the epidemiology, clinical characteristics, impacts, and histopathologic changes associated with CTD-ILD. Next, it summarizes the roles of various signaling pathways in macrophages or products of macrophages in ILD, helped by insights gained from animal models. In the following sections, this review returns to studies of macrophages in CTD-ILD in humans for an overall picture of the current understanding. Finally, we direct attention to potential therapies targeting macrophages in CTD-ILD in investigation or in clinical trials, as well as the future directions regarding macrophages in the context of CTD-ILD. Although the field of macrophages in CTD-ILD is still in its infancy, several lines of evidence suggest the potential of this area. Interstitial lung diseases (ILDs) comprise a heterogeneous group of multiple entities characterized by damage to the lung parenchyma due to varying degrees of inflammation and fibrosis . ILDs ar5 ..2-integr(5) Glycolysis: Macrophages from fibrotic lungs assume the elevated expression of the glucose transporter GLUT1 , which aTRIB3 suppresses fibrotic changes in the lungs . In. In2O2 ges STAT6  50]. SA. SA2O2 ges STAT6  51]. S1. S12O2 ges STAT6  52]. Th. Th2O2 gSH2-containing inositol 5′-phosphatase (SHIP) promoter in macrophages, by which MBD2 represses SHIP expression and enhances PI3K/Akt signaling to promote the macrophage M2 program and the production of downstream TGF-β . Wh. WhSH2-c(13) TNF pathway: STAT1 is expressed in lung macrophages . STAT1 iIn addition to macrophages themselves, macrophage-derived secretory proteins and microRNAs also execute various biological functions to modulate the lung fibrotic process.(1) ADAM17: ADAM17 in association with membrane-bound IL-6Rα (mIL-6Rα) is increased in macrophages in fibrotic lungs. ADAM17 promotes the shedding of mIL-6Rα from the membranes of activated macrophages, thereby initiating IL-6 trans-signaling. IL-6 trans-signaling enhances fibroblast proliferation and extracellular matrix (ECM) production  and the (2) AT1R: Macrophages secrete a considerable number of exosomes bearing AT1R, which are taken up by fibroblasts and result in higher levels of AT1R, the activation of the profibrotic TGF-β/Smad2/Smad3 pathway, the production of α-collagen I, and augmented Ang II secretion by fibroblasts. Interestingly, Ang II increases the number of macrophage exosomes and AT1R secretion, leading to a positive feedback between Ang II and exosome production involved in lung fibrosis  62]..62].(3) CCL6: CCL6, also called C10, is expressed in the macrophages and also attracts macrophages. Upregulated CCL6 accounts for the increased susceptibility to PF in the mice model. The neutralization of CCL6 attenuates subsequent lung fibrosis  63]..63].(4) Fibronectin: Compared to normal macrophages, PF macrophages produce more fibronectin . Fibrone(5) Galectin-3: Galectin-3 is produced by macrophages and the levels of galectin-3 in CTD-ILD patients are higher than in control patients. Galectin-3 induces the production of TNF-α in macrophages, and galectin-3 expression in macrophages is also induced by TNF-α, creating a self-perpetuating cycle between TNF-α and galectin-3 to support inflammation. In addition, galectin-3 stimulates fibroblasts to induce proliferation . All the(6) IGF-I: Macrophages secrete IGF-I, which is increased in PF patients. Macrophage-derived IGF-I activates the prosurvival kinase Akt, which protects myofibroblasts from apoptosis, contributing to fibrosis  67]. Co. Co67]. (7) IL-1β: IL-1β induces inflammation with leukocyte influx and upregulates myofibroblast differentiation, resulting in augmented pulmonary collagen deposition  and lung(8) MIP-1α: Macrophages secrete MIP-1α, which promotes leukocyte accumulation . On the (9) miR-328: Macrophage-derived miR-328 inhibits the family with sequence similarity 13 member A (FAM13A). This induces lung fibroblast proliferation and the overexpression of collagen 1A, collagen 3A, and alpha-smooth muscle actin (α-SMA), which aggravate fibrosis  71]. On. On71]. + macrophages also accumulate in fibrotic lungs. Through interaction with DCC, macrophage-derived NTN1 impacts adrenergic nerve remodeling, which is required for lung fibrosis ((10) NTN1: NTN1fibrosis  73]..+ macrop(11) PDGF: Macrophages from animal models of PF secrete PDGF, which is a potent mitogen for fibroblasts, facilitating lung fibrosis  74]..74].(12) S100A4: M2-polarized macrophages secrete S100A4, which stimulates the proliferation and activation of fibroblasts and is increased in PF . The inh(13) TGF-β: In the PF model, macrophages secrete TGF-β . TGF-β i(14) Wnt: Wnt expression is noted in macrophages . Wnt/β-c(1) Pentraxin-2: Pentraxin-2, also called serum amyloid P (SAP) , reduces(2) miR-142-3p: Macrophages secrete antifibrotic miR-142-3p, which reduces the expression of transforming growth factor β receptor 1 (TGFβ-R1) and downstream profibrotic genes in PF  82]..82].The evidence mentioned above suggests that macrophages actively participate in the pathogenesis of ILD/PF. In theory, macrophages could also analogously take part in the course of CTD-ILD. This is supported by several observations.Although there are no animal models that directly address the molecular mechanisms through which macrophages contribute to DM-ILD, several studies provide indirect evidence implicating macrophages as the main player in DM-ILD. Pathological analyses show that the infiltration of CD163-positive macrophages into the alveolar spaces is more severe in fatal DM-ILD than in DM-ILD survivors , and incIn MPA-ILD, macrophage polarization is skewed toward M2, which recruits fibroblasts and transforms them into myofibroblasts, culminating in the formation of lung fibrosis . In the In RA-ILD, autoimmune/complement/interferon cascade genes are altered in macrophages, which might contribute to profibrotic inflammatory lung responses . SeveralIn RA-ILD, macrophages produce higher IL-6, which promotes the expansion of GM-CSF-producing T cells. GM-CSF is a strong inducer of neutrophil infiltration into the lung and contributes to the progression of RA-ILD in an animal model  89]. SD. SD89]. RGC32 deficiency in mice impairs the polarization of classically activated macrophages, attenuates the expression of inflammatory mediators in macrophages, including the fibrosis inducers iNOS and IL-1β, which are regulated by NFκB, and significantly ameliorates lung fibrosis  ABT-199: ABT-199, an inhibitor of Bcl-2, is a potential therapeutic agent that prevents fibrosis progression. Bcl-2 decreases caspase-3 activity, mediating the apoptosis resistance of macrophages, which facilitates lung fibrosis . Mice ha(2) Anti-IL-33: IL-33 is induced in macrophages during PF. Acting through ST2, IL-33 polarizes M2 macrophages to increase IL-13 and TGF-β1 production. This activates fibroblasts and stimulates fibroblast proliferation. Therefore, IL-33 enhances collagen synthesis by fibroblasts and amplifies the fibrosis of the lung. Accordingly, an anti-IL-33 antibody reduces lung fibrosis  97]..97].(3) Clevudine: Clevudine is a purine nucleoside analog indicated for hepatitis B virus (HBV) treatment. Interestingly, clevudine can inhibit the PI3K/Akt signaling and therefore suppresses M2 differentiation. Correspondingly, epithelial-to-mesenchymal transition (EMT) and M2-induced myofibroblast activation are alleviated , thus pr2O2 generation. Consequently, Jmjd3 expression is reduced, preventing macrophage M2 polarization and thereby attenuating lung tissue fibrosis ((4) Leflunomide: Leflunomide inhibits Cu,Zn-SOD-mediated Hfibrosis  49]..2O2 gene(5) GW4869: Macrophages secrete exosomes carrying AT1R, which are taken up by fibroblasts, activating collagen synthesis. GW4869, by inhibiting exosome secretion, attenuates fibrosis in a mouse model  62]..62].(6) IL-10: IL-10 suppresses TGF-β production in macrophages and subsequently decreases collagen production in fibroblasts , diminis(7) Methyl palmitate: Methyl palmitate inhibits the phosphorylation of inhibitory kappa B alpha (IκBα), resulting in decreased TNF-α and increased IL-10 production by macrophages . These c(8) Microcystin-leucine arginine (LR): The binding of microcystin-LR to glucose-regulated protein 78 kDa (GRP78) suppresses endoplasmic reticulum unfolded protein response (UPR) signaling, which inhibits M2 macrophage polarization  and lead(9) Niclosamide: S100A4 is secreted by M2 macrophages to enhance collagen production in lung fibroblasts. The inhibition of S100A4 production with niclosamide reduces collagen deposition and attenuates lung fibrosis  75]..75].(10) Nintedanib: By inhibiting CSF1R, nintedanib blocks the M2 differentiation of monocytes , which r(11) Pirfenidone: Pirfenidone has an indirect inhibitory effect on fibroblast proliferation by suppressing macrophage polarization toward the M2 phenotype , which i(12) PRI-724: β-catenin signaling promotes the TGF-β expression of macrophages, which contributes to collagen synthesis in fibroblasts. As a result, the β-catenin pathway inhibitor PRI-724 inhibits the production of TGF-β by alveolar macrophages and ameliorates collagen deposition in fibrotic lungs  79,104],10479,10(13) Recombinant human pentraxin-2 (rhPTX-2): Pentraxin-2, also called serum amyloid P (SAP), reduces M2 differentiation and subsequent lung fibrosis  80,81].,81.80,81(14) Schisandra: Schisandra decreases TGF-β, reduces the levels of Smad3 and Smad4, and upregulates Smad7, suppressing M2 macrophage polarization . Accordi(15) Ruxolitinib: In CTD-ILD, ruxolitinib, which inhibits JAK/STAT, prevents the upregulation of proinflammatory molecules  and profibrotic molecules  . Ruxolit(16) Tacrolimus: In the animal model of PF, tacrolimus inhibits signaling of JAK/STAT pathways and suppresses M2 polarization and M2-induced myofibroblast activation, thereby alleviating PF progression .clinicaltrials.gov (accessed on 25 March 2023), 39 clinical trials exploring the therapeutic effects of various agents for CTD-ILD have been planned or are already underway or completed (p = 0.06 by Fisher’s Exact Test). However, most available clinical trials examined agents which act on non-macrophage cells  rather than agents that act on macrophage cells . Therefore, it is hoped that future clinical trials will focus more on macrophage-directed agents.In practice, there is no standard treatment protocol for CTD-ILD. However, considering the autoimmune nature of CTDs, immunomodulatory drugs are the cornerstones in the pharmacologic treatment of CTD-ILD . Consideompleted . Of the Despite the significant morbidities and mortalities caused by CTD-ILD, publications detailing how macrophages contribute to ILD in the context of CTD are scarce. More research is necessary to elucidate the distinct roles of specific lung macrophage populations in the CTD-ILD, with the aim of providing new insights into the development of macrophage-directed therapeutic targets and diagnostic tools . Current


Pancreatic adenocarcinoma (PDAC) is well known for its rapid distant metastasis and local destructive behavior. Loss of Krüppel-like factor 10 (KLF10) contributes to distant migration of PDAC. The role of KLF10 in modulating tumorigenesis and stem cell phenotypes of PDAC is unclear.G12D; Pdx1-Cre) mice, a spontaneous murine PDAC model, was established to evaluate tumorigenesis. Tumor specimens of PDAC patients were immune-stained of KLF10 to correlate with local recurrence after curative resection. Conditional overexpressing KLF10 in MiaPaCa and stably depleting KLF10 in Panc-1 (Panc-1-pLKO-shKLF10) cells were established for evaluating sphere formation, stem cell markers expression and tumor growth. The signal pathways modulated by KLF10 for PDAC stem cell phenotypes were disclosed by microarray analysis and validated by western blot, qRT-PCR, luciferase reporter assay. Candidate targets to reverse PDAC tumor growth were demonstrated in murine model.Additional depletion of KLF10 in KC (LSL: KrasNotch-3 and -4 by competing with E74-like ETS transcription factor 3, a positive regulator, for promoter binding. Downregulation of Notch signaling, either genetically or pharmacologically, ameliorated the stem cell phenotypes of Panc-1-pLKO-shKLF10. The combination of metformin, which upregulated KLF10 expression via phosphorylating AMPK, and evodiamine, a non-toxic Notch-3 methylation stimulator, delayed tumor growth of PDAC with KLF10 deficiency in mice without prominent toxicity.KLF10, deficient in two-thirds of 105 patients with resected pancreatic PDAC, was associated with rapid local recurrence and large tumor size. Additional KLF10 depletion in KC mice accelerated progression from pancreatic intraepithelial neoplasia to PDAC. Increased sphere formation, expression of stem cell markers, and tumor growth were observed in Panc-1-pLKO-shKLF10 compared with vector control. Genetically or pharmacologically overexpression of KLF10 reversed the stem cell phenotypes induced by KLF10 depletion. Ingenuity pathway analysis and gene set enrichment analysis showed that Notch signaling molecules, including Notch receptors 3 and 4, were over-expressed in Panc-1-pLKO-shKLF10. KLF10 transcriptionally suppressed These results demonstrated a novel signaling pathway by which KLF10 modulates stem cell phenotypes in PDAC through transcriptionally regulating Notch signaling pathway. The elevation of KLF10 and suppression of Notch signaling may jointly reduce PDAC tumorigenesis and malignant progression.The online version contains supplementary material available at 10.1186/s12929-023-00937-z. Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies, typically presents with early distant metastasis and local invasion. PDAC is resistant to most cancer therapies, and despite recent progress in early detection and therapeutic efficacy, the overall patient survival rate remains low . TherefoOur previous studies have revealed that Krüppel-like factor 10 (KLF10), a zinc finger-containing transcription factor originally identified as an early response gene of TGFβ , was downregulated in two-thirds of PDAC patients due to epigenetic and post-translational regulation , 3. KLF1Notch-3 and -4 for transcription regulation. Upregulating KLF10 and/or suppressing Notch signaling, genetically or pharmacologically, delayed tumor growth of PDAC with KLF10 deficiency.In this study, we found that additional deletion of KLF10 in the murine model of spontaneous pancreatic cancer accelerated PDAC tumorigenesis. KLF10 deletion transcriptionally upregulated Notch signaling molecules to promote sphere formation, and tumorigenesis of PDAC. Mechanistically, KLF10 competed with E74-like ETS transcription factor 3 (ELF3) for promoter binding of 2 incubator; maintained for < 6 passages and histo-pathologically characterized through SCID mice xenograft studies. Metformin hydrochloride , dorsomorphin  and evodiamine (E3531) were purchased from Sigma-Aldrich. N-[N-]-(S)-phenylglycine t-butyl ester  was from MedChem Express.Human pancreatic cancer cell lines, Panc-1 (BCRC 60284) and MiaPaCa (BCRC 60139), were purchased from the Bioresource Collection and Research Center, Taiwan, and were authenticated by DNA fingerprinting. Panc-1 cells labeled with firefly luciferase plasmid vector (Panc-1-Luc) were kindly provided from Dr. Kelvin K. C. Tsai from Taipei Medical University. The mouse primary pancreatic adenocarcinoma (PDAC) cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 mg/mL streptomycin and 2 mM glutamine at 37 °C in a 5% COCells were washed in phosphate buffered saline (PBS) and collected by centrifugation. The cells were stained with primary antibodies including CD44-FITC , CD24-Alexa 647 , c-Met-Alexa 647 , CD326-BB515 , CD133-PE  at 4 °C for 30 min. After washing with PBS twice and re-suspended in 800 µL PBS, the cells were filtered through a 70 mm nylon mesh and carried out on a flow cytometer . The viable and single cells were gated for analyses. BD CellQuest pro (BD Biosciences) and FlowJo (BD Bioscience) software were used to analyze the data.G12D and p53Loxp/Loxp breeder mice, generated by Drs. Andrew M. Lowy, Tyler Jacks and Anton Berns . A larger tumor size  was found in patients with low expression of KLF10 -S-phenylglycine t-butyl ester  was used to suppress Notch signal transduction. NICD-3 and -4 as well as Hes 7, and c-Myc were down-regulated in Panc-1-pLKO-shKLF10 after DAPT treatment , Lag-1)-binding sites on DNA . KLF10 mNotch-3 and Notch-4 gene transcription, respectively. ELF3, playing an important oncogenic role in several tumors including PDAC, was reported to positively regulate Notch-3 gene transcription and modulate tumor-initiating cells in K-Ras-mediated lung adenocarcinoma [The Notch signaling pathway exerts both oncogenic and tumor suppressive functions, depending on the cellular context . Recent arcinoma . Furtherarcinoma . Levels arcinoma  or epigeNotch-1 alleles accelerated PanIN progression; whereas the deletion of Notch-2 prolonged survival and delayed PDAC development [In contrast to Notch-1 and -2, which were expressed widely in many tissues, Notch-3 was found most abundantly in vascular smooth muscle and pericytes, while Notch-4 was highly expressed on endothelial cells . The levelopment , 38, 39.elopment –43. Notcelopment . When exelopment . The oppEuodiae Fructus, has low toxicity and suppresses Notch-3 by activating DNA methyltransferase 3A induced Notch-3 methylation in non-small-cell lung cancer [In addition to γ-secretase inhibitor, we used evodiamine to suppress Notch-3 expression. Evodiamine, an alkaloid derived from g cancer . Despiteg cancer , the sigg cancer , 48. BecNotch-3 and Notch-4 genes promoters for transcription regulation. Loss of KLF10, observed in two-thirds of PDAC patients, favored Notch-3 and-4 expression and the further Notch signal transduction, resulting in the development of stem cell phenotype and tumorigenesis in PDAC. Elevating KLF10 expression and suppressing Notch signaling, either genetically or pharmacologically, ameliorated the malignant progression of PDAC with KLF10 deficiency.In conclusion, we demonstrated a novel signaling pathway of stem cell regulation in PDAC with KLF10 deficiency. We showed that KLF10 competes with ELF3 to bind to To fully exploit the therapeutic potential of our findings, additional work is needed to decipher the KLF10-modulated Notch signaling pathway molecules beyond Notch-3 and -4. The interactions between KLF10 family members and the Notch signaling pathways should also be explored to find potential counterbalances and dynamics in modulating stem cell phenotypes. We used metformin, DAPT, and evodiamine in the murine tumor model to demonstrate that KLF10, the Notch signaling pathway and Notch-3 are potential therapeutic targets in PDAC with KLF10 deficiency. The development of a KLF10 inducer and Notch signal inhibitor with high specificity and low toxicity is warranted to validate the importance of the KLF10/Notch signaling pathways in PDAC.A spontaneous murine model of PDAC with additional KLF10 depletion reveals accelerated malignant progression and identifies a mechanism of cancer stem cell phenotype involving Notch signaling activation, suggesting a potential therapeutic target.Additional file 1: Figure S1. Representative colony formation of primary murine PDAC cells from KPCmice as indicated.quantitative bar graphs of mean ± SE from cumulated data of three independent experiments. *p < 0.05,representative flow cytometry of CD133 and CD326 on murine PDAC cell lines from KPC and KKPC mice as indicated.quantitative bar graphs of mean ± SE from cumulated data of three independent experiments from each cell lines developed from KPCor KKPCmice as indicated. *p < 0.05,representative sphere formation of MiaPaCa cells of vector controlor with conditional KLF10 overexpressionwithout or with doxycyclinetreatment as indicated. Original magnification 100× and 400× of upper and lower panel, respectively.immunofluorescence stain of CD44on MiaPaCa-pLVX or MiaPaCa-pLVX-KLF10 without or with Dox treatment as indicated. DAPI was used for nuclei stain.quantitative bar graphs of mean ± SE from cumulated data of three independent experiments from MiaPaCa-pLVXor MiaPaCa-pLVX-KLF10 cells withoutor withDox treatment as indicated. *p < 0.05.Additional file 2: Figure S2. Representative immunofluorescence stain of fluorescence conjugated signal molecules as indicated on spheres of Panc-1-pLKO and Panc-1-pLKP-shKLF10. DAPIwas used for nuclei staining. Original magnification 400×.Representative immunofluorescence stain of KLF10, Notch-3 and -4 as indicated on spheres of MiaPaCa-pLVX or MiaPaCa-pLVX-KLF10 without or with Dox treatment. DAPIwas used for nuclei staining. Original magnification 400×.Representative immunoblots of Notch signal molecules expression of MiaPaCa-pLVX or MiaPaCa-pLVX-KLF10 without or with Dox treatment. β-actin was used as internal control.Additional file 3: Figure S3. Primer design ofNotch3/4 promoters for ChIP assay. Primer design for cloning ofNotch-3 andNotch-4 promoters of various deletions.Primer design for ELF3 and mutant S68ELF3.Representative GEO databases of correlation between ELF 3 versus Notch-3, Notch-4, and KLF10transcripts levels in pancreatic adenocarcinoma.Additional file 4: Figure S4. Representative sphere formation of Panc-1-pLKO and Panc-1-pLKO-shKLF10 without or with Notch-3 or Notch-4 depletion as indicated. Original magnification 100×Representative sphere formation of Panc-1-pLKO-shKLF10 without or with 5 µM DAPT treatment. Original magnification 100×.Additional file 5: Figure S5. Body weight of mice implanted orthotopically with Panc-1-pLKO-shKLF10 and treated with PBS, metformin, evodiamineor concomitant metformin and evodiamine. Each point represents mean ± SE from cumulated data of at least 5 mice.Additional file 6: Table S1. 10 pathway QPCR primer sequence.Notch pathway QPCR primer sequence.Additional file 7. Additional materials and methods.


Administration of a single dose of Ad5-nCoV vaccine to 10 of these PA cases elicited rapid and robust antibody responses as well as coordinated B-cell and cTfh responses, resulting in successful viral clearance. Vaccine-induced antibodies were able to neutralize various variants of concern and persisted for over 6 months, indicating long-term protection. Therefore, our study provides an insight into the immune status of PA infections and highlights vaccination as a potential treatment for prolonged SARS-CoV-2 infections.Persistent asymptomatic (PA) SARS-CoV-2 infections have been identified. The immune responses in these patients are unclear, and the development of effective treatments for these patients is needed. Here, we report a cohort of 23 PA cases carrying viral RNA for up to 191 days. PA cases displayed low levels of inflammatory and interferon response, weak antibody response, diminished circulating follicular helper T cells (cTfh), and inadequate specific CD4 A wide spectrum of clinical manifestations can be seen in COVID-19 patients ranging from asymptomatic, mild-to-moderate to severe-to-critical illness with multi-organ dysfunction and even death.4The devastating global pandemic of coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2.6 accounting for ~40–45% of SARS-CoV-2 infections.9 Notably, asymptomatic patients can also spread the virus efficiently10; furthermore, the duration of SARS-CoV-2 viral shedding is generally 3–46 days.13 However, some asymptomatic infections may persist with a longer duration of viral shedding as reported in our previous study;14 this persistence increases the risk of viral spread and may result in negative mental and physical consequences in these individuals as well as over-consumption of surveillance resources.15 Moreover, persistent infections may drive viral evolution; hence, the emergence of variants of concern (VOCs).16 Therefore, understanding the immune status of these persistently asymptomatic (PA) patients and developing treatments for the acceleration of viral clearance are critical to ending the pandemic.Increasing evidence has highlighted the prevalence of asymptomatic infections,17 Neutralizing antibody (nAb) titers were higher in convalescent patients with COVID-19 who received a single dose of mRNA vaccine than in naive individuals who received a third dose of mRNA vaccine.22 In addition, antigen-specific CD4+ and CD8+ T-cell responses induced by prior infections were enhanced by a single dose of mRNA vaccine.23 However, whether vaccinations can augment SARS-CoV-2-specific immune responses and accelerate viral clearance in patients with SARS-CoV-2 infection is unclear.Vaccination has been shown to be an effective way to not only build immunity in SARS-CoV-2-naive individuals, but also boost antiviral immunity in recovered people.+ and CD8+ T-cell responses, as well as circulating follicular helper T cells (cTfh). Ten of the 23 cases remained SARS-CoV-2 RNA-positive 18 weeks post infection and received a single dose of adenovirus type-5 vector-based COVID-19 vaccine  for therapeutic purposes. Rapid and robust antibody and coordinated B-cell and cTfh responses were induced by vaccination, resulting in successful viral clearance. Furthermore, vaccine-elicited antibodies exhibited neutralizing activities against various VOCs and persisted for over 6 months. Our study affords an insight into the immune status of PA SARS-CoV-2 infections. Our results may guide vaccination strategies for the prevention and treatment of prolonged infections.Here, we report a cohort of 23 PA patients with SARS-CoV-2 infection with a median viral shedding period of >100 days. All cases were young adults without immunodeficiency, but exhibited impaired immune activation against SARS-CoV-2, as indicated by lower levels of inflammation, interferon responses, antibody responses, specific CD4Twenty-three patients with PA SARS-CoV-2 infection (PA group) were enrolled in this study; 6 patients with non-prolonged asymptomatic infection (NA group), 19 patients with mild COVID-19 (mild group), 25 patients with severe COVID-19 (severe group), and 20 SARS-CoV-2-naive donors (naive group) were included as controls. PA patients had no comorbidities, while some patients in other groups had various comorbidities (Supplementary Table 24 Inflammatory cytokines interleukin (IL)-6 and IL-1β levels were remarkably elevated in the mild and severe groups but not in PA cases  remained in minor proportions during the infection  and central memory T cells  at an early stage. After 18 weeks of persistently low levels of viral burden, the proportion of both TEM and TCM decreased, while that of TEMRA increased , 2 (T2), 6 (T3), and 10 (T4) weeks post vaccination (wpv), corresponding to 18, 20, 24, and 28 wpo, respectively .ely Fig. . The dynORF1ab and N genes declined to undetectable levels within 2–10 weeks following Ad5-nCoV vaccination in PA patients  strain, B.1.351 (Beta), B.1.617 (Delta), BA.1.1 (Omicron), and BA.5 (Omicron) variants of SARS-CoV-2. Sera obtained from PA cases exhibited similar low neutralizing activity against WT, Beta, and Delta variants, and had no neutralizing effect on the Omicron variant (BA.1.1 and BA.5) before vaccination , 1111 (95% CI: 226–5453), 932 (95% CI: 258–3367), 81 (95% CI: 27–239), and 167 (95% CI: 81–343) up to 25 weeks post vaccination, respectively  was significantly increased in HD participants and exhibited a trend for an increase in PA cases, but this was not significant  for therapeutic purposes.36 Within the PA group, delayed viral clearance correlated with elevated levels of anti-inflammatory IL-10 and reduced levels of antiviral IFN-λ. IL-10 suppresses adaptive immune responses and impairs the control and clearance of pathogens in multiple infection models,37 suggesting that the upregulation of IL-10 levels might contribute to the failure in viral clearance. However, the in-depth mechanism of how IL-10 regulates host antiviral responses in SARS-CoV-2 infections needs to be further dissected.Here, we found relatively low levels of inflammatory, interferon, antibody, and antigen-specific T-cell responses in PA cases, indicating that the inadequate induction of immune responses might contribute to the persistence of infection. In line with our findings, a number of studies identified the pivotal roles of specific antibodies and T-cell responses in viral clearance regarding MERS-CoV, SARS-CoV, and symptomatic SARS-CoV-2 infections.38 However, whether vaccination could enhance immune responses in PA cases with an unresolved SARS-CoV-2 infection, helping to clear the virus or provide further protection against re-infection with various VOCs remained unclear. Here, we found that robust nAb responses were induced by vaccination in PA cases and strongly correlated with decreased viral loads; this result is consistent with the importance of nAb in viral control.33 Furthermore, these antibodies were able to persist at relatively high levels for over 6 months, whereas antibodies induced by two doses of mRNA or inactivated vaccines in SARS-CoV-2-naive participants were reported to decline at 6 months post vaccination.41 Notably, the nAbs could neutralize various SARS-CoV-2 VOCs circulating globally, including the currently dominant variant BA.5, implying protection against future emerging VOCs. One interesting question is why individuals who did not develop an appropriate immune reaction to the initial virus infection were capable to react robustly with a protective response to the vaccine. First, we speculate that this SARS-CoV-2 infection-vaccination scenario mimics the prime-boost immune process that could induce a robust immune response. Second, relatively low pro-inflammatory response in PA cases might be enhanced by the Ad5 vector and favor the induction of the immune response.44Convalescent patients with COVID-19 may benefit from vaccination whereby robust antibodies and specific T-cell responses can be elicited to protect the vaccinees against re-infection.+ T-cell responses, consistent with the improved specific T-cell responses in those who received three doses of mRNA SARS-CoV-2 vaccines.38 However, one dose of Ad5-nCoV vaccine had minimal impact on the magnitude of antigen-specific CD4+ T-cell responses in PA cases. Antigen-specific CD4+ T-cell subsets in both HD and PA groups were predominantly TEM; additionally, higher levels of specific CD4+ TEM prior to vaccination were correlated with stronger specific CD4+ T-cell responses post vaccination. Thus, the minimal increase in antigen-specific CD4+ T cells upon vaccination in PA cases might be explained by the minor proportion of TEM and TCM and greater proportion of terminally differentiated CD4+ TEMRA prior to vaccination.SARS-CoV-2-naive participants who had completed two doses of inactivated vaccine benefited from an additional dose of Ad5-nCoV vaccine with an enhancement of antigen-specific CD4This study has several limitations. First, the present findings are based on a small cohort and a lack of parallel persistent infection queues that received other types of COVID-19 vaccines or those who received no vaccine. Whether other types of vaccines, such as mRNA and inactivated viral vaccines, would provide similar protection is unclear. Although one of the 10 PA cases received Ad5-nCoV vaccination via muscular injection and the others via inhalation, this participant’s immune response was not distinct from the others, indicating that the vaccine administration route did not affect the efficacy of vaccination in PA cases; however, this requires confirmation in a larger cohort. Second, although a 25-week follow-up assessment revealed durable protective antibody responses in PA cases post vaccination, accurate evaluation of the longevity of protectiveness requires an extended assessment period. Whether vaccinated PA cases would benefit from a booster dose of the vaccine warrants further investigation. Third, we did not address whether earlier vaccination could provide more effective and rapid protection in PA cases.+ and CD8+ T-cell responses. Ad5-nCoV vaccination resulted in rapid and robust antibody responses and coordinated B-cell and cTfh responses in PA cases, contributing to subsequent viral clearance. Furthermore, vaccination-elicited antibodies exhibited broad-spectrum neutralizing activity against various VOCs and long-lived activity. Our study of the immune status in persistent asymptomatic SARS-CoV-2 infections may facilitate subsequent exploration of the mechanisms underlying persistent infection. Importantly, we demonstrate for the first time that vaccination is an effective treatment for prolonged SARS-CoV-2 infection and propose the notion of a therapeutic vaccine for COVID-19 treatment.In summary, our study reveals that prolonged SARS-CoV-2 infection may be attributed to impaired immune activation, which manifested as low levels of initial inflammation, interferon, and weak antibody response, markedly reduced cTfh, as well as inadequate specific CD4A total of 23 patients with persistent asymptomatic (PA) SARS-CoV-2 infection confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were enrolled in this study. For comparison, 6 asymptomatic patients with non-persistent SARS-CoV-2 infection (NA), 19 mildly ill patients (MP), and 25 severely ill patients (SP) were also included in this study  and nucleocapsid protein (N) genes . The results were considered positive when the cycle threshold (Ct) values of all genes were less than 40 cycles. Samples with Ct values of less than 40 cycles for only one gene were defined as inconclusive, and a second test was required.Nucleic acids were extracted from nasopharyngeal swabs by using the Prefilled Viral Total NA Kit-Flex and KingFisher Flex System  according to the manufacturer’s protocol. The presence of SARS-CoV-2 was identified using an RT-PCR kit targeting the open reading frame 1ab  was used to measure cytokine levels according to the manufacturer’s instructions. The levels of interleukin 1 beta (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and interferon-g (IFN-γ) were measured according to the manufacturer’s instructions. The levels of interferon-α (IFN-α) and interferon-λ (IFN-λ) were measured by using Human IFN-α ELISA KIT and Human IFN-λ ELISA KIT  according to the manufacturer’s protocols.+L) antibody (1:4000 dilution) . The cells were incubated at 37 °C. The reactions were developed using KPL TrueBlue Peroxidase substrates . The number of SARS-CoV-2 foci was calculated using an Elispot reader . The SARS-CoV-2 strains used in this study were isolated from COVID-19 patients in Guangdong, China, including wild-type , Beta (B.1.351), and Omicron (BA.1.1). The SARS-CoV-2 Delta  and Omicron  variants were obtained from the Guangdong Provincial Centre for Disease Control and Prevention, China. Experiments related to authentic SARS-CoV-2 were conducted at the Guangzhou Customs District Technology Center Biosafety level 3 Laboratory.The SARS-CoV-2 focus reduction neutralization test (FRNT) was performed in a certified Biosafety Level 3 (BSL-3) laboratory. A volume of 50 μL of plasma samples was serially diluted, mixed with 50 μL of SARS-CoV-2 virus  in 96-well plates, and incubated for 1 h at 37 °C. The mixtures were then transferred to 96-well plates seeded with Vero E6 cells  and incubated for 1 h at 37 °C to allow virus entry. The inocula were removed before adding the overlay media . The plates were then incubated at 37 °C for 24 h. The overlays were removed, and the cells were fixed with 4% paraformaldehyde solution for 30 min. Cells were permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit anti-SARS-CoV-N IgG  for 1 h at 37 °C before adding HRP-conjugated goat anti-rabbit IgG (HEnzyme-linked immunosorbent assay (ELISA) was used to analyze SARS-CoV-2-specific IgG and IgA antibodies against the SARS-CoV-2 spike (S), spike RBD (RBD), spike 1 (S1), and spike 2 (S2) proteins with a commercial antibody detection kit provided by the R&D Department of AtaGenix Laboratories Co., Ltd., Wuhan, China. ELISA was performed on the plasma samples according to the manufacturer’s instructions. Plates were read using a BioTek Epoch microplate reader  at an optical density (OD) of 450 nm. The standard curve of Ig concentration-OD450 was calculated using the standard substance in the kit that was detected simultaneously, and the Ig concentration in plasma was calculated.A set of 20-mer peptides encompassing four SARS-CoV-2 structures  and six putative accessory proteins  overlapping by 10 amino acids were synthesized and used to stimulate PBMCs. T-cell responses were measured using intracellular cytokine staining assays for interferon-γ (IFN-γ) and TNF.5–106 cells were stained with the indicated antibodies at 4 °C, and then labeled with cell viability staining dye  at room temperature (20–25 °C) in the dark. For in vitro intracellular cytokine staining, 105–106 cells were cultured in a well of 96-well round-bottom plates at 37 °C for 12 h in the presence of 40 µM SARS-CoV-2 peptide pool and brefeldin A . The cells were then labeled for cell surface markers, fixed/permeabilized with Cytofix/Cytoperm Solution (BD Biosciences), and stained with anti-intracellular cytokine/protein antibodies. All flow cytometric data were acquired on a BD FACSFortessa and analyzed using FlowJo software (BD Biosciences).The following anti-human monoclonal antibodies were used: BB515-CD45RA (HI100), BUV737-CD45RA (clone: HI100), PerCP-Cy5.5-CD4 (clone: RPA-T4), BB515-CD4 (clone: RPA-T4), PerCP-Cy5.5-CD8 (clone: SK1), BV786-CD8 (clone: RPA-T8), BV650-CD56 (clone: NCAM16.2), BV510-CD3 (clone: HIT3a), BUV496-CD3 (clone: UCHT1), PE-Cy7-CCR7 (clone: G043H7), APC-R700-CXCR5 (clone: RF8B2), BV711-CXCR5 (clone: J252D4), APC-IFN-γ (clone: B27), BV421-IFN-γ (clone: B27), PE-TNF (clone: MAb11), APC-Cy7-TNF (clone: MAb11), BV605-IL-2 (clone: MQ1-17H12), BV786-Ki-67 (clone: B56), PE-Granzyme B (clone: GB11), PE-Dazzle594-IL-4 (clone: MP4-25D2), ef660-IL-21 (clone: 3A3-N2), R718-IL17A (clone: N49-653), and PE-Cy5-IL-10 (clone: JES3-19F1). Antibodies were purchased from BD Biosciences, Thermo Fisher Scientific, or BioLegend. PBMCs were prepared from blood samples using Lympholyte-H (Cedarlane), according to the manufacturer’s instructions. The cells were stored in liquid nitrogen until further analysis. For surface staining, 10Fluorescent SARS-CoV-2-specific RBD probes were prepared by combining biotinylated proteins with fluorescently labeled streptavidin (SA). RBD probes were prepared at a ratio of 4 M of trimer to 1 M of SA. Two RBD probes, one labeled with phycoerythrin (PE) and one labeled with Alexa Fluor (AF)-647, were used in this panel to increase the specificity of detection of SARS-CoV-2-specific B cells. Cryopreserved PBMCs were thawed at 37 °C and stained with a flow cytometry panel consisting of viability dye (FVS440UV), IgM FITC, CD3 PE-CF594 (UCHT1), CD27 PE-Cy7 (M-T271), IgG APC-H7 (G18-145), CD138 BV421 (MI15), CD19 BV510 (SJ25C1), IgD BV650 (IA6-2), and CD38 BV786 (HIT2) . Cells were first stained with a cocktail of RBD probes for 60 min at 4 °C, washed with PBS, stained with the remaining antibody panel, and incubated for 15 min at room temperature. The cells were then resuspended in 2% FBS/PBS after fixing with Cytofix (BD Biosciences) for 30 min at 4 °C. All flow data were acquired on a BD FACSFortessa and analyzed using FlowJo software (BD Biosciences).Analysis and visualization were performed using R version 4.3.0 and Rstudio. Data were normalized, and packages including “FactoMineR”, “factoextra”, and “ggplot2” were used.U test was used to compare the differences between groups. Spearman’s correlation was used to assess the relationship between different factors. A P value <0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism software version 7.00. The Wilcoxon matched-pair signed-rank test was used to compare differences within the same group at different time points. The Mann–Whitney Supplementary file-clean version


MicroRNAs (miRNAs) are approximately 22 nucleotide-long non-coding RNAs that are encoded in the genome. miRNAs form base pairs with target mRNAs in the RNA-induced silencing complex and repress their expression through a mechanism called RNA silencing. Expression profiles of miRNAs differ between cells and tissues. In this study, we performed cytosine β-D-arabinofuranoside (AraC)-induced neuron-like differentiation of human NTERA2/D1 (NT2) cells and quantified endogenous miRNA levels using quantitative RT-PCR. In conclusion, pre-mir-106b and pre-mir-19b levels were decreased after AraC-induced neuron-like differentiation of NT2 cells, indicating the functional relevance of miRNAs in the differentiation of mammalian cells. MicroRNAs (miRNAs) are approximately 22 nucleotide-long non-coding RNAs that are encoded in the genomeNT2 cells derived from human testicular embryonic carcinoma cellsNT2 cells were treated with various concentrations of AraC  and cultured for up to 6 days . On dayIn conclusion, we found that the pre-mir-106b and pre-mir-19b levels were decreased after AraC-induced neuron-like differentiation of NT2 cells, indicating the functional relevance of miRNAs in the differentiation of mammalian cells.Cell culture2.NT2 cells were obtained from the ATCC (#CRL-1973) and were cultured in Dulbecco’s Modified Eagle’s medium (Wako) containing 10% fetal bovine serum (NICHIREI) and antibiotics  at 37°C in an atmosphere containing 5% COAraC-induced neuron-like differentiationNT2 cells were treated with AraC as described previously . Briefly, NT2 cells were seeded in a 12-well plate. Cells at 80–90% confluence were treated with 0, 0.01, 0.1, 1, 5, 10, and 20 μM of AraC . The medium containing AraC was replaced with fresh medium every 2 days.Quantitative RT-PCR (qRT-PCR)Total RNA was extracted using a FastGene RNA Premium Kit with FastGene miRNA enhancer (Nippon Genetics). The extracted RNA was used for complementary DNA (cDNA) synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using the KAPA SYBR Fast qPCR Master Mix ABI Prism Kit (Kapa Biosystems) and the QuantStudio Real-Time PCR system (Thermo Fisher Scientific). Primer sequences used in this study are listed in Reagents.PCR primers used in this study for qRT-PCR


Drosophila melanogaster testes. The product, namely, CDS-piRNAs, formed silencing complexes with Aubergine (Aub) in germ cells. Proximity proteome and functional analyses show that CDS-piRNAs and cluster/TE-piRNAs are distinct species occupying Aub, the former loading selectively relies on chaperone Cyclophilin 40. Moreover, Argonaute 2 (Ago2) and Dicer-2 activities were found critical for CDS-piRNA production. We provide evidence that Ago2-bound short interfering RNAs (siRNAs) and microRNAs (miRNAs) specify precursors to be processed into piRNAs. We further demonstrate that Aub is crucial in spermatid differentiation, regulating chromatins through mRNA cleavage. Collectively, our data illustrate a unique strategy used by male germ line, expanding piRNA repertoire for silencing of endogenous genes during spermatogenesis.PIWI-interacting RNA (piRNA) pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the underlying piRNA biogenesis mechanisms remain elusive. Here, we show that endogenous protein coding sequences (CDSs), which are normally used for translation, serve as origins of noncoding piRNA biogenesis in  A pathway where miRNA and siRNA produce piRNA expands the repertoire of small non-coding RNAs supporting sperm formation. In eukaryotes, diverse biological processes are controlled by silencing mechanisms relying on 20– to 30–nucleotide (nt) small noncoding RNAs. Small RNAs (sRNAs) interact with Argonaute (Ago) family proteins inside RNA-induced silencing complexes (RISCs), acting as sequence-dependent guide for selecting silencing targets. PIWI-interacting RNAs (piRNAs) comprise a group of sRNAs forming RISCs with PIWI-clade Ago members and accumulating in animal gonads. piRNA-directed mechanisms are crucial in defending germline genomes against transposable elements (TEs) and controlling endogenous gene expression ] and the cofactors (deadlock and cutoff) (stellate (ste), vasa (vas), and pirate (pira) can produce piRNAs via ping-pong, being targeted by trigger piRNAs derived from su(ste), AT-Chx, and petrel clusters, respectively , a representative host gene, 3′UTR-piRNAs bind Piwi and exert gene regulatory effects in gonadal soma (Drosophila germ cells.Similar to Silencing pathways exhibit adaptation to testis-specific RNA metabolism. This is demonstrated with Ago2 that is binding to short interfering RNAs (siRNAs) and crucial in RNA-based immunity against foreign pathogens  rather than in 3′UTRs and by their fates being loaded onto Aub but not Piwi. Furthermore, different from cluster/TE-piRNAs, neither Rhi-dependent cluster transcription nor Ago3-dependent ping-pong amplification is required for their production. Alternatively, their biogenesis relies on the activities of Cyp40 and Ago2. With regard to the latter, Ago2-bound siRNAs and miRNAs specify piRNA precursors and serve as triggers of secondary piRNA production. Generated piRNAs guide Aub for target mRNA cleavage, thereby directly involved in the control of endogenous gene expression for late spermatogenesis.In this study, we show that D. melanogaster. Analyses of deep sequencing (deep-seq) data, prepared in house and available in public (table S1), revealed that 23- to 29-nt piRNA-like reads accumulate to the exons of endogenous protein-coding genes to different degrees  and CG32000/anne are known to produce piRNAs interacting with Piwi  are known to be targeted by piRNAs from AT-ChX and petrel clusters, respectively; secondary piRNAs can be generated from their cleaved mRNAs, such as TEs and cluster transcripts . In addition, CDS-piRNAs were rarely derived from introns, and a fraction of reads contained exon-exon junction sequences  and compared with testicular datasets (fig. S2A). Curiously, ovarian Aub did not accumulate CDS-piRNAs mapping to genes identified in testes . MoreoveContrary to CDS-piRNAs, the transcript levels of many (10 of 14) host genes were comparable between ovaries and testes . TherefoDrosophila, an Hsp90 cochaperone Cyp40 is expressed in testicular germ cells and essential for sperm formation; however, its substrates/clients are elusive besides Ago2 . In denaturing gel electrophoresis, potentially biotinylated proteins were detected as distinct bands when Cyp40, but not green fluorescent protein (GFP) or nonfunctional Cyp40 variant (RKAA or ∆TPR) was used as bait (What testicular factors can activate CDS-piRNA production and Aub loading? sRNA loading step in RISC formation relies on Hsp70/Hsp90 chaperone machineries (des Ago2 . Cyp40 fdes Ago2  (35). mT as bait  (25).The screening identified dozens of proteins in the physical proximity of Cyp40. Consistent with our previous findings  = 0.71 ± 0.05; pira was one of exceptions; similar to cluster/TE-piRNAs, its piRNA level was unaffected /mus308 and ATP synthase beta . Their transcripts are known targets of siRNAs derived from CG4068 and hpRNA1, respectively, and cleaved by Ago2 . To this end, genuine CDS-piRNAs from 14 genes showed most severe decrease , suggesting their functional interaction in CDS-piRNA production. Conspicuously, gene-by-gene analysis revealed that muc14A exhibits a severe decrease in the piRNA level (muc14A is unique by containing ~300-nt repeats inside the coding region (fig. S4E). Accordingly, its atypical antisense-enriched CDS-piRNAs  were solely derived from muc14A but contained intragenic multimappers forming a hotspot (fig. S4F). To support the direct involvement of miR-316-3p in muc14A-piRNA production, a total of 18 target sites were identified and accumulated in muc14A antisense sequence (no site in sense sequence), although these sites would be largely cleavage incompetent due to the central mismatches (fig. S4F). Nonetheless, predicted target sites located nonrandomly and surrounded the piRNA hotspot, fitting to the idea that miR-316-3p–directed Ago2 can specify piRNA precursor region within target transcripts. Together, these data suggest that Ago2 uses both siRNAs and miRNAs for CDS-piRNA production.We further investigated how Ago2 activates CDS-piRNA production by examining if Ago2-bound miRNAs are involved. We previously reported that  mutants . Nonethemuc14A show phasing signatures (fig. S4G). This result suggests that si/miRNAs can direct Zuc-dependent, phased CDS-piRNA biogenesis.Multiple piRNAs can be generated from a single precursor transcript by phasing mechanism relying on Zucchini (Zuc)–mediated cleavage . These results suggest that CDS-piRNA pathway involving both Aub and Ago2 can support late spermatogenesis.As a step toward understanding the biological roles of CDS-piRNA pathway, we examined if and how  sterile . Those doduction  and knowaub (−/− ago2+/−aub), ago2 (+/− ago2−/−aub), or both  were obtained from progenies derived from the same parents, together with those of sibling heterozygous controls. Genes controlled by CDS-piRNA pathway could be misregulated in all mutant conditions. Consistent with previous studies, ATPsynbeta, a direct target of hpRNA1-derived siRNAs, was identified as one of overexpressed genes in ago2 mutant and dKO testes, but not in aub mutants , further supporting a role for Ago2 as processing trigger  < 0.0001; nejire (nej)/CBP and structure-specific recognition protein (ssrp), for which quantitative polymerase chain reaction (PCR) was performed to confirm the transcriptome output  signals on H4 were detectable in spermatid nuclei  enabling the transfer of precursors to Aub for efficient CDS-piRNA production. Responsible factors might exist among those identified in the physical proximity of Cyp40    (deficiency for cyp40), bam-Gal4/TM6, FLAG-HA-ago2 (yellow white (y w), 454ago2/TM3 , KOmiR-316/TM3 , Df(3 L)ED4978/TM6c  (deficiency for miR-316), Df(3 L)BSC558/TM6C  (deficiency for ago2), L811fsXdcr2 , HN2aub/CyO , N11aub/CyO t3, ago3/TM6B 02086, rhi/CyO , Df(2R)Exel7149/CyO  (deficiency for rhi), protamine-B-eGFP/CyO , nos-phiC31; P{y[+t7.7] = CaryP}attP40 , and nos-phiC31;; PBac{y[+]-attP-3B}VK00033 .Fly stocks are reared at room temperature (RT) on a molasses/yeast medium , twice with W2 buffer , twice with W3 buffer , and twice with W4 buffer [50 mM tris-HCl (pH 7.5) and 50 mM NaCl]. Proteins bound to magnet beads were denatured by boiling at 95°C for 5 min in 2× protein loading buffer  saturated with biotin, resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) in 5 to 20% precast gel (ATTO). After electrophoresis, proteins were visualized by using the Silver Stain MS Kit (Wako). Proteins in gel particles were digested with trypsin and analyzed by liquid chromatography tandem MS (LC-MS/MS) using Q Exactive (Thermo Fisher Scientific) and UltiMate 3000 Nano LC (Thermo Fisher Scientific) in CoMiT Omics Center . The mass spectrum data were analyzed by Mascot v2.5.1 (Matrix Science). Proteins were identified with threshold of 1 peptide minimal. Pseudo value of 0.5 was added to all identified proteins, and then peptide count values were normalized by using total peptide counts in a certain condition.mTurbo fusion proteins were expressed in germline-restricted manner under S ribosomal RNA (rRNA) was added to 1 μg of RNA solution, and 2S rRNA hybridizing with oligo DNA was digested with RNase H (NEB). RNA mixture depleted of 2S rRNA was loaded onto 8 M urea-polyacrylamide gel (12%) and size-separated in parallel with RNA ladder  in 0.5× tris-borate EDTA buffer. Gel area within the range of 20 to 30 nt was excised, and contained RNAs were eluted in 300 mM NaOAc (pH 5.2) solution by dilution overnight at 4°C with gentle rotation. RNA fragments were precipitated in the presence of 80% (v/v) ethanol and glycogen (40 μg/ml) . RNA pellet was rinsed twice with 80% (v/v) ethanol and then resuspended in RNase-free water.RNA was extracted from six hundred testes of ≤3 days old using miRNeasy (QIAGEN). Less than 200-nt fragments were collected using miRelute column (QIAGEN). A total of 2 pmol of oligo DNA (5′-AGTCTTACAACCCTCAACCATATGTAGTCCAAGCAGCACT-3′) containing complementary sequence to 22, 2 mM DTT, 10% (v/v) glycerol, 0.5% (v/v) Triton X-100, and complete tablet]. After centrifugation , to purify endogenous Aub proteins, the supernatant was incubated with anti-Aub antibodies (1:10) for 3 hours on ice then further incubated with the mixture of protein A Dynabeads and protein G Dynabeads (Thermo Fisher Scientific) overnight with gentle rotation. To purify GFP-Aub or GFP-Piwi, the supernatant was incubated with anti-GFP antibody-conjugated magnet beads [Medical and Biological Laboratories (MBL)] overnight with gentle rotation. The beads were washed five times with wash buffer . The bead-bound sRNAs were extracted with TRIzol LS reagent (Thermo Fisher Scientific) following the manufacturer’s protocol and precipitated in the presence of 50% (v/v) isopropanol and 20 μg of glycogen  overnight at −20°C. After centrifugation , the pellet was rinsed twice with 80% (v/v) ethanol and then resuspended in RNase-free water.Five hundred testes of ≤3 days old were homogenized using plastic pestle  in lysis buffer , resolved by SDS-PAGE, and transferred to 0.2-μm polyvinylidene difluoride membrane (Wako) using the semi-dry system . The membrane was blocked in 4% (w/v) skim milk  in 1× phosphate-buffered saline (PBS) supplemented with 0.1% (v/v) Tween 20 and further incubated with the following antibodies: anti-Aub antibody   bovine serum albumin  and fixed in 5.3% (v/v) paraformaldehyde  in 0.67× PBS buffer for 10 min. To observe DNA and ICs, testes were incubated with 1 μM 4′,6-diamidino-2-phenylindole and phalloidin Rhodamine X conjugated  in PBX buffer [1× PBS containing 0.2% (v/v) Triton X-100]. To observe ProtB-GFP, the emission signal of GFP was acquired. For immunostaining, fixed testes were washed with PBX and incubated with PBX containing 2% (w/v) BSA for 30 min for blocking. The primary antibody incubation was performed overnight at 4°C, and testes were washed with PBX at 25°C for 1 hour. The secondary antibody incubation was then performed at 25°C for 2 hours, and then testes were washed with PBX at 25°C for 1 hour. The following antibodies were used: anti-Vasa antibody , anti-guinea pig IgG–Alexa Fluor 555 , anti-H3K18Ac , anti-H3K27Ac , anti-H4K5Ac , anti-H4K8Ac , anti-H4K12Ac , anti-H4 , and anti-rabbit IgG-Alexa Fluor 555 . Images were acquired using confocal microscope LSM 900 (Zeiss). Images were processing by using Zen (Zeiss) and Pixelmator.RNA was extracted from ~20 testes of ≤3-day-old flies for each condition using TRIzol LS reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. Using DNase I (NEB)–treated RNA, cDNA was synthesized with 2.5 μM oligo(dT) adaptor using SuperScript III reverse transcriptase (Thermo Fisher Scientific). Quantivative reverse transcription PCR (qPCR) reaction was performed using KAPA SYBR FAST qPCR Master Mix (Roche) and gene-specific primers (table S6) in QuantStudio 5 Real-Time PCR system (ABI).y w virgin females for the first 3 days and with another six y w virgin females the following 2 days at 25°C. The total number of hatched eggs was counted.Single male was mated with six


Oryza sativa) exhibits tremendous aluminum (Al)-tolerance. The C2H2-transcription factor (TF) ART1 critically regulates rice Al tolerance via modulation of specific gene expression. However, little is known about the posttranscriptional ART1 regulation. Here, we identified an ART1-interacted gene OsNAC016 via a yeast two-hybrid (Y2H) assay. OsNAC016 was primarily expressed in roots and weakly induced by Al. Immunostaining showed that OsNAC016 was a nuclear protein and localized in all root cells. Knockout of OsNAC016 did not alter Al sensitivity. Overexpression of OsNAC016 resulted in less Al aggregation within roots and enhanced Al tolerance in rice. Based on transcriptomic and qRT-PCR evaluations, certain cell-wall-related or ART-regulated gene expressions such as OsMYB30 and OsFRDL4 were altered in OsNAC016-overexpressing plants. These results indicated that OsNAC016 interacts with ART1 to cooperatively regulate some Al-tolerance genes and is a critical regulatory factor in rice Al tolerance.Rice ( Aluminum (Al) is ubiquitously found within soil. In acidic soil, Al takes up an ionic form, which inhibits root growth and mineral absorption, thereby producing dwarf roots with impaired ability to absorb water and nutrients, thus, enhancing plant vulnerability to numerous stressors, including drought ,2. GivenALMT1) in wheat, and citrate transporters (HvAACT1 and SbMATE) in barley and sorghum [To remain viable under toxic Al or acidic soil conditions, plants established multiple approaches to internally and/or externally stimulate Al detoxification ,4. To in sorghum ,6,7.Japonica cultivars demonstrate very high Al tolerance in hydroponic conditions and in soil [Al resistance TF1) is reported to regulate at least 31 down-stream genes that participate in rice Al tolerance [STAR1, STAR2, Nrat1, OsALS1, OsCDT3, OsFRDL2, OsFRDL4, OsMGT1, OsEXPA10, and OsART2) using mutant approach [STAR1 and STAR2 associate to form an ABC transporter, which exports UDP-glucose and critically regulates Al detoxification through cell wall modification [Nrat1 produces a plasma-membrane-localized transporter protein that transports trivalent Al [OsALS1 produces a tonoplast-residing half-size Al-shuttling ABC transporter [Nrat1 and OsALS1 work synergistically to internally detoxify Al within rice. On the other hand, OsFRDL4 encoding a citrate transporter modulates root citrate release, which sequesters Al within the rhizosphere [ART1. Based on these findings, ART1 posttranscriptional or posttranslational modulation might be required for the activation of ART1.Rice  in soil . Dissimi in soil . A C2H2 olerance . Among tapproach ,16,17,18zosphere . MoreoveThe plant-specific NAC  TF family dominates the TF population within plants . At presOsNAC016-knockout and overexpressing lines under normal and Al treatment conditions. We also analyzed the root transcriptomes of OsNAC016-overexpressing and wild-type (WT) plants with or without Al treatment. The presented evidence revealed that OsNAC016 is involved in rice Al tolerance via the modulation of certain cell-wall-related genes and partial ART1-regulated genes.To gain insights into how ART1 is regulated at the posttranscriptional level, we screened for ART1-interacting proteins via yeast two-hybrid (Y2H) assay with truncated ART1 as bait and identified a NAC TF OsNAC016. We investigated the phenotypes of OsNAC016 encodes a 340-amino-acid protein harboring an NAM domain at its N terminus. To verify a physical association between OsNAC016 and ART1, we conducted a bimolecular fluorescence complementation (BiFC) assessment in rice protoplast cells. We observed the YFP signal in rice protoplasts co-expressing OsNAC016-nYFP and ART1-cYFP or ART1-nYFP and OsNAC016-cYFP, but not in those co-expressing control constructs. Furthermore, YFP signal nicely overlapped with the RFP signal of the nuclear marker Ghd7 [N. benthamiana leaves co-expressing MYC-tagged OsNAC016 and FLAG-tagged ART1 and found that OsNAC016 was coimmunoprecipitated with ART1 in planta . To probe the molecular mechanism of ART1 activation by Al, we utilized truncated ART1 (1–175 aa) lacking an activation domain as bait to screen for ART1-interacting proteins via yeast two-hybrid (Y2H) assay and identified eight candidate proteins . As seveker Ghd7   and another with a 307-bp deletion (osnac016-2), at varying sites within the first exon  treatment with 50 μM Al, Al levels within roots and the cell walls within root tips (0 to 1 cm from the apex) were drastically reduced in the overexpression lines, relative to the WT  rice roots treated with 0 or 40 μM Al for 8 h via an RNA-seq approach. Based on a threshold of twofold change, under normal conditions, we identified 1430 differentially expressed genes (DEGs) between the WT and OE line, and following Al exposure, 1441 DEGs between the lines (OsPAL6 (Os04g0518400), OsEXPA10 (Os04g0583500), OsMYB30 (Os02g0624300), OsCesA7 (Os10g0467800), OsBC1 (Os03g0416200), OsUgp1 (Os09g0553200) and OsUgp2 (Os02g0117700). Our RT-qPCR results corroborated our RNA-seq information, confirming the RNA-seq data reliability  and he lines . Among the lines . These 8he lines B, while he lines C. In theiability A–H. TherOsNAC016 might influence the ART1-modulated gene expressions. To verify this hypothesis, we assessed the expressions of 31 ART1-regulated downstream genes between WT and OE line in RNA-seq data  assay. A physical association between ART1 and OsNAC016 was further proved via BiFC and Co-IP experiments. OsNAC016 was primarily expressed within roots and slightly modulated via Al. Immunostaining showed that OsNAC016 was a nuclear protein and localized in all root cells. Knockout of OsNAC016 has a weak or no effect on the expressions of ART1 and ART1-modulated Al-tolerant genes under normal and Al stress. Furthermore, we observed no marked alterations in Al tolerance between WT and osnac016 mutants. One possibility is that there are redundant genes with similar function to OsNAC016, leading to no obvious Al sensitive phenotype in osnac016 mutants. This notion is supported by the presence of multiple homologs for OsNAC016 in the rice genome such as OsNAC58, OsNAC103, OsNAC122, and OsNAC131. On the other hand, overexpression of OsNAC016 resulted in less Al root aggregation and enhanced rice tolerance to Al stress. Based on transcriptomic sequencing and qRT-PCR assessment, the expressions of some genes related to cell wall or Al stress were altered in OsNAC016-overexpressing plants. These findings suggested that OsNAC016 contributes to the regulation of Al tolerance within rice.In this study, we obtained an ART1-interacted gene Secale sylvestre [Arabidopsis [OsNAC016 overexpressing lines showed reduced cell wall Al content and enhanced Al tolerance relative to the WT rice. The RNA-seq and qRT-PCR analyses demonstrated that the expression levels of some genes related to cell wall modification in the OsNAC016-overexpressing lines were significantly downregulated or upregulated relative to the WT rice under Al stress, including OsPAL6, OsMYB30, OsEXPA10, OsBC1, OsCesA7, OsUgp1 and OsUgp2. Among them, OsPAL6 and OsMYB30 are down-regulated while the other five genes are up-regulated in the OsNAC016-overexpressing lines. OsPAL6, a phenylalanine ammonia-lyase, forms the first major enzyme in the phenylpropanoid pathway, participating in lignin biosynthesis [4-coumarate: coenzyme A ligase 4CL4 and 4CL5 within rice [OsPAL6 and 4CL5 is positively regulated by OsMYB30 [OsMYB30 resulted in a lower cell wall Al aggregation and an enhanced Al tolerance within rice [OsEXPA10 encoding a cell wall expansin participated in root cell elongation through the modulation of cell-wall loosening. Knocking out OsEXPA10 led to a reduction in root length [OsBC3 caused a marked rise in cell wall pectin content [OsCesA7 resulted in a reduced cellulose and an increased cell wall hemicellulose content [OsUgp1 and OsUgp2 encode UDP-glucose pyrophosphorylase (UGPase), which accelerates the UTP and glucose-1-phosphate conversion to pyrophosphate and UDP-glucose [OsNAC016-overexpressing lines may be explained via modulation of the above-mentioned gene expressions, relative to the WT rice.The cell wall acts as an initial physical barrier that protects plant cells from harmful external environmental factors, such as, Al toxicity. Among the main Al-resistance strategies in plants is to decrease Al accumulation in the cell wall through modifying cell wall properties to alter its Al-binding capacity ,27,28,29hin rice ,31. The  OsMYB30 ,30,31. Khin rice . OsEXPA1t length . OsBC1 b content . General content . OsCesA7 content . Hemicel-glucose . UDP-gluOsNAC016 significantly increases the partial ART1-modulated gene expressions such as OsFRDL4, Os02g0770800, Os01g0919200, and Os01g0731600. Among the previously characterized ART1-modulated genes, OsFRDL4 expression was the most affected. OsFRDL4 is a plasma-membrane-localized citrate transporter that facilitates rice Al tolerance through releasing citrate from roots following Al stimuli. ART1 regulates OsFRDL4 expression by directly interacting with its promoter’s cis-element GGN(T/g/a/C)V(C/A/g)S(C/G) region [OsFRDL4  region . A recen) region . We perfOsNAC016 up-regulated many biological processes-related gene expressions such as peroxidase activity, antioxidant activity, and response to oxidative stress under normal conditions, implying that OsNAC016 may also play important regulatory roles in other stress responses. A recent study reported that OsNAC016 negatively regulated drought tolerance via down-regulating ABA- and drought-responsive genes in rice [OsSPX2 in rice [Additionally, transcriptome analysis revealed that overexpression of  in rice . More re in rice . These fOsNAC016 can lower root cell wall Al aggregation through modulation of some cell-wall-related and ART1-modulated gene expressions and thus improve Al tolerance in rice.In conclusion, OsNAC016 is the first protein identified as ART1-interacting protein. Overexpression of Oryza sativa cv Nipponbare), as well as 2 OsNAC016-knockout lines, and 2 OsNAC016 overexpression lines which were generated within the laboratory. Rice seeds underwent a 2-day submersion in deionized water without light at 28 °C. Germinated seeds were transferred to a floating net under a solution with 0.5 mM CaCl2 in an illumination incubator and used for various experiments.Herein, we used WT , basal roots (1–2 cm) and mature region (2–3 cm). To analyze the OsNAC016-mediated response to Al stress, rice seedlings (5 days old) were treated to varying Al concentrations  at pH 4.5 for 8 h or 30 μM Al over a wide range of experiment durations .Roots, stems, leaf blades, leaf sheaths and panicles were extracted from WT plants at the heading stage for organ-specific expression analysis of 2 and 50 μM Al solution (pH 4.5). For long-term Al treatment, seedlings (2-d-old) from individual lines underwent a 5-d treatment with a 0.5 mM CaCl2 and 20 μM Al solution (pH 4.5). Root lengths were recorded both prior to and after treatments using a ruler. Relative root elongation was computed using the following formular: (Al-treated root elongation)/(Al-untreated root elongation) × 100.For short-term Al treatment, seedlings (5-d-old) from individual lines underwent a 24-h treatment with a 0.5 mM CaClOsNAC016, OsNAC016 cDNA was amplified from the Nipponbare root cDNA via PCR and specific primers. The amplified cDNA fragment was ligated to the pCAMBIA1300-Ubi vector between the maize Ubiquitin 1 promoter and nopaline synthase terminator, generating the pCAMBIA1300-Ubi1:OsNAC016 construct. To create the CRISPR construct for OsNAC016, two sgRNA expression cassettes containing two different specific target sites for OsNAC016 were digested by BsaI and cloned into a pYLCRISPR/Cas9 vector [OsNAC016 construct. These constructs were next introduced into rice cv. Nipponbare via Agrobacterium tumefaciens strain EHA105-mediated transformation. The employed primer sequences are summarized in To construct the overexpression vector for 9 vector , produciHistone H3 was utilized. Relative gene expression employed the 2−ΔΔCT calculation method. The primers for qRT-PCR were shown in The Trizol reagent kit  was utilized for total RNA extraction as per kit directions. The Hiscript II Q RT SuperMix Kit  was then employed to eliminate genomic DNA and perform reverse transcription reactions. For qRT-PCR, ChanQTM SYBR Color qPCR Master Mix  was used in a StepOnePlus RT-PCR System . An internal standard of OsNAC016 into the pYL322-GFP vector upstream of GFP to create the pYL322-OsNAC016-GFP construct. Rice protoplasts were co-transfected with either pYL322-OsNAC016-GFP or a control vector along with a nuclear marker (OsGhd7-RFP), following the protocols described by Chen et al. [To assess OsNAC016 intracellular distribution, we inserted the coding sequence of n et al. . After 1OsNAC016 were amplified using specific primers Pro-NAC016-F/Pro-NAC016-R and 1300-NAC016-F/1300-NAC016-R, respectively. Subsequently, the promoter and CDS fragments were cloned into the HindIII and BamHI sites of the pCAMBIA1300-GFP vector. The subsequent construct (pCAMBIA1300-ProOsNAC016:OsNAC016-GFP) was introduced into Nipponbare to generate the transgenic lines harboring ProOsNAC016-OsNAC016-GFP. The employed primer sequences are summarized in The promoter (2000 bp) and CDS (1023 bp) sequences of ProOsNAC016:OsNAC016-GFP were embedded in a 5% agarose and sliced into sections (100-μm thickness) using a microslicer (VT1000 S Leica), which were then transferred to microscope slides for incubation with rabbit anti-GFP polyclonal antibodies, with subsequent incubation with secondary antibodies  at room temperature (RT). Cell nucleus was stained with DAPI. Fluorescent images were then taken via a confocal laser scanning microscope .To examine the OsNAC016 cellular localization, immunostaining was carried out using a GFP antibody following the protocol described by Yamaji and Ma in 2007 . In brieWe screened a yeast cDNA library, prepared from the 12-h 30 µM Al-treated roots of 5-day-old WT rice seedling using truncated ART1 CDS (1–525 bp) as bait and the Matchmaker Yeast Two-Hybrid System . To verify the truncated ART1 and target protein associations, the truncated ART1 and target gene coding sequences were inserted into pGBKT7 and pGADT7 vectors, respectively. The resulting bait and prey vectors were co-introduced into the yeast strain AH109 and cultured on an SD medium without Leu and Trp or lacking Leu, Trp, Ade, and His. The employed primer sequences are summarized in OsNAC016 and ART1 coding sequences were amplified from the Nipponbare root cDNA using corresponding primers, prior to cloning. The amplified products were cloned into the BiFC vectors pUC19-35S-Vn and pUC19-35S-Vc, producing the OsNAC016-cYFP, OsNAC016-nYFP, OsART1-cYFP and ART1-nYFP constructs. We prepared rice protoplast and conducted plasmid transformation into cells as reported previously [To prepare the BiFC constructs, the eviously . FollowiOsNAC016 and ART1 full-length coding sequences were ligated into the KpnI and BamHI sites of pCAMBIA1300-35S-FLAG and pCAMBIA1300-35S-MYC vectors, respectively. The pCAMBIA1300-35S:OsNAC016-FLAG and pCAMBIA1300-35S:ART1-MYC recombinant constructs were introduced into Agrobacterium strain (GV3101), prior to transformation into 4-week-old N. benthamiana leaves. Approximately 24 h later, total protein isolation was completed from infected leaves by Lysis extract NB1 buffer . Protein co-IP experiment was performed as reported by Xu et al. (2015) [The . (2015) . ProteinOsNAC016-overexpressing line seedlings were exposed for 8 h to a solution (pH 4.5) with 0.5 mM CaCl2 and 50 µM AlCl3. After 3 rinses in 0.5 mM CaCl2, 20 root segments (0–1 cm from the root tip) each replicate were extracted and then transferred to a 2 mL tube or ultrafree-MC centrifugal filter unit . To obtain root cell sap, we centrifuged root-containing units for 10 min at 3000× g at 4 °C. After that, the units were frozen overnight at −80 °C. The root cell sap solution was obtained via centrifugation of samples at 20,400× g for 10 min at RT. The residues in the units were washed three times with alcohol (70%) by a vortex. The cell sap was diluted with 5% nitric acid. Both cell walls and whole roots were digested by 65% nitric acid. The Al contents of cell sap, cell wall and whole root tip were assessed using ICP-MS .Five-day-old WT and two OsNAC016-overexpression line (OE) seedlings were subjected to a 0.5 mM CaCl2 solution (pH 4.5) with 0 or 50 µM AlCl3 for 8 h. The seedling roots were sampled for RNA-seq experiments. Following total root RNA extraction, we synthesized corresponding cDNA, and conducted cDNA library generation. cDNA library sequencing was conducted with the Illumina NovaSeq 6000 platform. Deseq2 was employed for gene expression comparison analysis between WT and OE lines. DEGs were identified using the following criteria: |log2(FoldChange)| ≥ 1 and padj ≤ 0.05. DEGs GO  analysis was done using hypergeometric tests, and p-values were utilized for determining significant enrichment of corresponding categories.Five-day-old WT and


Correction: BMC Public Health 23, 995 (2023)https://doi.org/10.1186/s12889-023-15906-2The original publication of this article  containeIncorrectThe concentration curve lies below the diagonal when CI is negativeCorrectThe concentration curve lies below the diagonal when CI is positive


In Jamaica, COVID-19 vaccines have been recommended for pregnant women since August 2021, yet data detailing COVID-19 vaccination behaviors among pregnant Jamaican women have been limited. Specifically, it is unknown if COVID-19 vaccination among pregnant Jamaican women has increased over time, similar to trends observed in the United States.A convenience sample of reproductive-aged Jamaican women were recruited from a teaching hospital during two distinct time periods (TP): (1) February 2022 and (2) May-July 2022. Participants self-identified as pregnant (versus nonpregnant) and completed a web-based survey that assessed self-reported COVID-19 vaccination and medical mistrust beliefs. We analyzed the relationship between TP and a predetermined set of variables using chi-square or Fisher’s exact tests. We used logistic regression to estimate adjusted odds ratios (aOR) and 95% confidence intervals (CI) for self-reported COVID-19 vaccination by TP, adjusting for age, education, and comorbidities.Of the 429 participants, 178 (41%) completed the survey in TP1 and 251 (59%) in TP2 (Table 1). Self-reported data, including trusted sources of COVID-19 vaccination and type of COVID-19 vaccine received, did not differ significantly between TP1 and TP2 (Table 1). Self-reported COVID-19 vaccination among pregnant women was 35% in TP1 vs. 60% in TP2 (aOR (95% CI): 2.78 (1.40 - 5.50). Pregnant women were less likely to agree with the medical mistrust belief, “I don’t trust the COVID-19 vaccine,” in TP2 compared with TP1 (aOR (95% CI): 0.43 (0.18 - 0.99). Self-reported vaccination and medical mistrust beliefs among nonpregnant women did not differ between TP1 and TP2.Self-reported COVID-19 vaccination among pregnant Jamaican women was significantly higher in TP2 compared with TP1, while medical mistrust related to COVID-19 vaccination was lower. To improve the efficacy of vaccination campaigns and enhance the communication of vaccine safety between pregnant women and their healthcare providers in the future, it is crucial to identify trends in COVID-19 vaccination and medical mistrust beliefs among pregnant women over time.Emily P. Hyle, MD MSc, UpToDate.com: Royalties