Halomonas sp. strain ANAO-440 contains 3,866 predicted protein-coding sequences. This strain is capable of anaerobic arsenite oxidation and encodes an arxA-type arsenite oxidase within the arxB2AB1CD gene island. This genome sequence provides valuable information regarding the physiological diversity of Arx-dependent arsenite-oxidizing microorganisms.The draft genome sequence of  Halomonas sp. strain ANAO-440 is a heterotrophic arsenite-oxidizing halophile isolated from an alkaline saline lake located in the northern region of Khovsgol, Mongolia  and word size (30); the assembly resulted in 64 contigs with an N50 value of 193,648 bp and a maximum contig length of 449,532 bp. The draft genome sequence of strain ANAO-440 was 4,309,801 bp long with 534.0× genome coverage and a G+C content of 62.6%. Annotation was conducted using the NCBI Prokaryotic Genome Annotation Pipeline , such as Halomonas chromatireducens strain AGD 8-3 (GenBank accession number CP014226.1).Strain ANAO-440 was grown as previously described , and genarx gene island, arxB2ABCD, which was previously identified in Ectothiorhodospira sp. strain PHS-1 and A. ehrlichii MLHE-1  were present in the draft genome sequence, while lacking either the dissimilatory arsenate reductase gene arr or the aioA-type arsenite oxidase gene. The draft genome sequence of Halomonas sp. strain ANAO-440 provides valuable information regarding Arx-dependent arsenite oxidation in phylogenetically and physiologically diverse microorganisms.The draft genome sequence of ANAO-440 contained the i MLHE-1 . Howeverd MLHE-1  were absJAHXBV000000000, BioProject accession number PRJNA670823, BioSample accession number SAMN20309323, and SRA accession number SRR15204658.The draft genome sequence was deposited in GenBank under accession number


COVID-19 mRNA vaccines (Pfizer-BioNTech and Moderna) have been shown to be highly protective against COVID-19–associated hospitalizations  were screened for inclusion in this test-negative case-control assessment.Electronic health records were reviewed to obtain data on demographic characteristics, underlying medical conditions, presenting illness, SARS-CoV-2 test results, COVID-19 vaccination history, and clinical course during hospitalization. In the Atlanta and Houston VAMCs, COVID-19 vaccination status was further verified through a review of state immunization registries. Full vaccination was defined as receipt of both doses of an mRNA vaccine (Pfizer-BioNTech or Moderna) ≥14 days before the qualifying SARS-CoV-2 test. Participants who received only 1 dose of an mRNA COVID-19 vaccine, 2 mRNA doses with receipt of the second dose <14 days before the qualifying SARS-CoV-2 test, mixed mRNA vaccine products , or the Janssen (Johnson & Johnson) COVID-19 vaccine were excluded from the analysis. Available residual clinical respiratory specimens were collected from case-patients at all sites and sent to CDC for testing. Specimens were tested using CDC’s 2019-Novel Coronavirus RT-PCR Diagnostic PanelVaccine effectiveness (1 – adjusted odds ratio [aOR] × 100)**During February 1–August 6, 2021, a total of 1,494 hospitalized U.S. veterans met inclusion criteria. After excluding 319 ineligible persons , 388 case-patients and 787 controls were included in the analysis. Among these 1,175 patients, 1,093 (93.0%) were men, the median age was 68 years (interquartile range [IQR] = 59–75 years), 574 (48.9%) were Black, and 93 (7.9%) were Hispanic . PrevaleThe adjusted effectiveness of full vaccination in preventing COVID-19–associated hospitalization during the entire evaluation period  was 86.8% (95% CI = 80.4%–91.1%) . The adjAmong U.S. veterans hospitalized at five VAMCs, mRNA vaccines were 86.8% effective in preventing COVID-19–associated hospitalizations and remained highly effective during a period of Delta variant predominance. The mRNA vaccines were effective against COVID-19–associated hospitalization among all age groups, although lower effectiveness (79.8%) was observed among veterans aged ≥65 years. These findings support current evidence that COVID-19 mRNA vaccines are highly effective in preventing COVID-19–associated hospitalization  variant. Vaccine effectiveness in preventing COVID-19–related hospitalization was 80% among adults aged ≥65 years compared with 95% among adults aged 18–64 years.To protect against COVID-19–related hospitalization, all eligible persons should receive COVID-19 vaccination. Additional studies are needed to understand differences in COVID-19 vaccine effectiveness across age groups.


BMI-1, a polycomb ring finger oncogene, is highly expressed in multiple cancer cells and is involved in cancer cell proliferation, invasion, and apoptosis. BMI-1 represents a cancer stemness marker that is associated with the regulation of stem cell self-renewal. In this study, pharmacological inhibition (PTC596) or knockdown (siRNA) of BMI-1 reduced cancer stem-like cells and enhanced cancer cell death. Mechanistically, the inhibition of BMI-1 induced the downregulation of Mcl-1 protein, but not Mcl-1 mRNA. PTC596 downregulated Mcl-1 protein expression at the post-translational level through the proteasome-ubiquitin system. PTC596 and BMI-1 siRNA induced downregulation of DUB3 deubiquitinase, which was strongly linked to Mcl-1 destabilization. Furthermore, overexpression of Mcl-1 or DUB3 inhibited apoptosis by PTC596. Taken together, our findings reveal that the inhibition of BMI-1 induces Mcl-1 destabilization through downregulation of DUB3, resulting in the induction of cancer cell death. Cancer stem-like cells (CSCs), a small subpopulation of tumor cells, can initiate tumorigenesis through self-renewal and differentiation . The tumBMI-1 was initially identified as a transcription repressor belonging to the polycomb family . SubsequMany small-molecule BMI-1 inhibitors, such as PTC-209, PTC-028, and PTC596, suppress self-renewal ability, tumor growth, and cancer invasion . In addiIn this study, we investigated whether inhibition of BMI-1 using an inhibitor (PTC596) and siRNA induced apoptotic cell death through Mcl-1 destabilization caused by the decreased expression of DUB3.BMI-1 is associated with self-renewal of CSCs . To detePrevious studies have reported that various BMI-1 inhibitors induce cancer cell death ,18,19. WNext, we investigated the expression levels of apoptosis-related proteins using PTC596 and BMI-1 siRNA. PTC596 and BMI-1 siRNA only decreased Mcl-1 protein levels, whereas other proteins did not change A. HoweveWe investigated Mcl-1 ubiquitination by PTC596 and found that PTC596 induces ubiquitination of Mcl-1 protein A. The E3We found that PTC596 did not alter DUB3 mRNA expression A. TherefTo prove the common anti-cancer effect of PTC596, we examined its effects on various cancer cell lines. As expected, PTC596 increased the sub-G1 population and PARP cleavage in human hepatocellular carcinoma (SK-Hep1), prostate carcinoma (DU145), lung carcinoma (A549), and cervical cancer (HeLa) cell lines B. MoreovIn this study, we found that pharmacological inhibition and knockdown of BMI-1 increases apoptosis in cancer cells, but not in normal cells, and induces downregulation of Mcl-1 protein expression via the ubiquitination pathway. Downregulation of DUB3 by PTC596 was involved in apoptosis and Mcl-1 degradation. Therefore, our results suggest that the inhibition of BMI-1 induces DUB3-dependent Mcl-1 degradation, resulting in the enhancement of cancer apoptosis.PTC-209 was identified as the first small-molecule inhibitor by Kreso et al., and they also verified inhibition of the self-renewal ability of colorectal cancer-initiating cells . PTC-209Nishida et al. reported that PTC596 downregulates Mcl-1 expression in a proteasome-dependent manner in acute myeloid leukemia . HoweverIn a previous study, Maeda et al. reported that PTC596 induces Bax activation and downregulation of Bcl-2 and Bcl-xL expression . HoweverCollectively, our study illustrates that inhibition of BMI-1 induces apoptosis through DUB3-mediated Mcl-1 degradation. Therefore, PTC596 may be a potential drug for cancer therapy in the future.Human renal cancer cell carcinoma (Caki), human cervical cancer cell carcinoma (HeLa), human lung cancer cell carcinoma (A549), human prostate cancer cell carcinoma (DU145), human hepatocellular cancer cell carcinoma (SK-Hep1), and kidney normal cells (TCMK-1) were obtained from the American Type Culture Collection . Human mesangial cells were obtained from Lonza . Cell culture media were supplemented with 10% fetal bovine serum , 1% penicillin–streptomycin, and 100 μg/mL gentamycin . PTC596 was purchased from Selleck Chemicals . Sigma Chemical Co. supplied cycloheximide, MG132, and anti-actin . z-VAD-fmk was obtained from R&D Systems . Antibodies against PARP, anti-Bcl-xL, anti-Mcl-1, anti-BMI-1, and anti-cleaved caspase-3 antibodies were provided by Cell Signaling Technology . Lactacystin and anti-caspase-3 antibodies were purchased from Enzo Life Sciences . BD Biosciences  offered anti-XIAP, anti-Bax, and anti-Bim antibodies. Anti-Mcl-1, anti-cIAP1, anti-cIAP2, anti-Bcl-2, and anti-β-TrCP were obtained from Santa Cruz Biotechnology . Abnova supplied anti-USP9X antibody . Anti-USP1 and anti-FBW7 antibodies were purchased from Bethyl Laboratories . Novus Biologicals supplied anti-DUB3 antibodies . Anti-OTUD1 was purchased from Merck .TM Kit for ALDH assay (STEMCELLTM TECHNOLOGIES)  was used to measure stem cells, which were then analyzed by flow cytometry .To analyze the population of cancer stem-like cells, cancer cells were treated with PTC596 for 12 h. The ALDEFLUOR® easyCyte™ flow cytometer . To measure the fluorescence of the cancer stem cell marker CD44, harvested cells were suspended in PBS, incubated with APC-conjugated CD44 antibody  for 1 h at room temperature, and analyzed by flow cytometry. To investigate the alteration of protein expression, the total lysates were obtained in RIPA lysis buffer , then proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes   .® Real Time System III . The following primers were used to amplify the target genes: Mcl-1 (forward) 5′-ATG CTT CGG AAA CTG GAC AT-3′ and (reverse) 5′-TCC TGA TGC CAC CTT CTA GG-3′; USP1 (forward) 5′-TCT GTG CCT GCG TTG TTT GA-3′ and (reverse) 5′-CGT CCT TTG AAA TTG CCG GT-3′; DUB3 (forward) 5′-CCT CCC GAC GTA CTT GTG AT-3′ and (reverse) 5′-CAT GGA CTC CTG ATG TGT CG-3′; and actin (forward) 5′-CTA CAA TGA GCT GCG TGT G-3′ and (reverse) 5′-TGG GGT GTT GAA GGT CTC-3′.RNA was isolated using TRIzol reagent . cDNA was obtained using M-MLV reverse transcriptase . Blend Taq DNA polymerase  with primers was used to target genes: Mcl-1 (forward) 5′-GCG ACT GGC AAA GCT TGG CCT CAA-3′ and (reverse) 5′-GTT ACA GCT TGG ATC CCA ACT GCA-3′; USP1 (forward) 5′-TGA ACT TGC CAC TCA GC-3′ and (reverse) 5′-TTT CAC ACT TCC CCA AGT CC-3′; DUB3 (forward) 5′-CCG GGA GCA CTC TCA AAC AT-3′ and (reverse) 5′-TTG ACA CTC TGA GCT GCC TG-3′; and actin (forward) 5′-GGC ATC GTC ACC AAC TGG GAC-3′ and (reverse) 5′-CGA TTT CCC GCT CGG CCG TGG-3′. The amplified products were separated by electrophoresis on a 2% agarose gel and detected under ultraviolet light. For real-time PCR, SYBR Fast qPCR Mix  was used, and the reactions were analyzed on a Thermal Cycler Dice® RNAiMAX Reagent (Invitrogen). DUB3 plasmids was transfected using LipofectamineTM 2000 (Invitrogen) in Caki cells.BMI-1 siRNA was purchased from Santa Cruz Biotechnology . USP1 siRNA, DUB3 siRNA, and control siRNA were purchased from Bioneer . For siRNA knockdown, Caki cells were transfected using Lipofectamineg for 10 min at 4 °C. The supernatant was added to the primary antibody of the target protein overnight and then reacted with protein G agarose for 2 h. After centrifugation, the cells were washed twice with lysis buffer containing 1 mM PMSF and 5 mM NEM. The pellet was added to 2× sample buffer and incubated for 10 min at 95 °C. Ubiquitinated Mcl-1 was detected by Western blotting under denaturation conditions with HRP-conjugated anti-Ub .To detect the ubiquitination of Mcl-1, Caki cells were transfected with HA-ubiquitin plasmid and treated with MG132 (0.5 mM) and PTC596. The cells were harvested, washed with PBS containing 10 mM N-ethylmaleimide (NEM) , resuspended in 100 mL PBS/NEM containing 1% SDS, and boiled for 10 min at 95 °C. RIPA lysis buffer with NEM (5 mM) was added to the lysates, which were dissolved using a l mL syringe four times and centrifuged at 13,000× The data were analyzed by one-way ANOVA and post-hoc comparisons (Student–Newman–Keuls) using the Statistical Package for Social Sciences software .This study demonstrates that inhibition of BMI-1 decreases the cancer stem-like cell population and induces apoptotic cell death in cancer cells, but not in normal cells. PTC596 and BMI-1 siRNA induce Mcl-1 destabilization by downregulation of DUB3.


These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.HIV-1 latency posts a major obstacle for HIV-1 eradication. Currently, no desirable latency reversing agents (LRAs) have been implicated in the “Shock and Kill” strategy to mobilize the latently infected cells to be susceptible for clearance by immune responses. Identification of key cellular pathways that modulate HIV-1 latency helps to develop efficient LRAs. In this study, we demonstrate that the Wnt downstream β-catenin/TCF1 pathway is a crucial modulator for HIV-1 latency. The pharmacological activation of the β-catenin/TCF1 pathway with glycogen synthase kinase-3 (GSK3) inhibitors promoted transcription of HIV-1 proviral DNA and reactivated latency in CD4  However, the persistence of latent viral reservoirs posts an obstacle for cure . The latectively ,11. IdenGlycogen synthase kinase-3 (GSK3) is a ubiquitously expressed, highly conserved serine/threonine protein kinase found in all eukaryotes . GSK3 isGSK3 is a key negative regulator of canonical Wnt signalling, and in the turned-off status of Wnt signalling, GSK3 functionally associates with axin and adenomatous polyposis coli (APC) protein to phosphorylate β-catenin for the sequential proteasomal degradation; while the activation of Wnt signalling prevents GSK3-mediated β-catenin phosphorylation and stimulates the subsequent translocation of β-catenin to nucleus for regulating gene transcription . The nuc+ T cells, and 6-BIO-induced HIV-1 reactivation was evaluated in SIV-infected rhesus macaques and Peripheral blood mononuclear cells (PBMCs) isolated from cART-suppressed patients.In this study, we found that pharmacological suppression of GSK3 by small compound 6-BIO reactivated HIV-1 from latently infected CD4Macaca mulatta) and the related methods and experimental protocols have been approved by the Medical Ethics Review Committee of Institut Pasteur of Shanghai, Chinese Academy of Sciences (CAS), the Medical Ethics Review Committee of Zhengzhou Sixth People's Hospital, and the Institutional Animal Care and Use Committee of Guangzhou Institutes of Biomedicine and Health, CAS. Informed consent has been signed by HIV-1 patients. All experiments were performed in accordance with relevant national guidelines and regulations.The usages of human samples and rhesus macaques  were provided by Dr Shibo Jiang and Dr Lu Lu . HeLa-cell-derived TZM-bl indicator cells which contain LTR-driven luciferase reporter were a gift from Dr Paul Zhou . The HEK293T cells were kindly provided by Dr Li Wu . Cells were cultured in RPMI 1640 medium (Gibco) or Dulbecco's Modified Eagle Medium (DMEM) (Hyclone) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μg/ml of streptomycin (Invitrogen) at 37°C under 5% CO2.The HIV-1 latently infected CD46) were stimulated with different concentrations of 6-bromoindirubin-3′-oxime (6-BIO)  , or lithium chloride (LiCl)  (25 mM) for 24 h. Viral reactivation was detected by quantifying the cell-associated gag mRNA or titrating the produced viral particles in TZM-bl indicator cells by measuring luciferase activity. Viral reactivation in J-Lat 10.6 and A2 was detected by measuring GFP expression [For assessing HIV-1 reactivation from cells of ACH2, Jat-10.6 and Jat-A2 cells .The targeted sequences of shRNAs were listed in Supplementary Table 1. shRNA was cloned into the PLKO.1-puro shRNA expression vector. The packaging of shRNA lentiviruses was performed according to the PLKO.1 protocol (Addgene). Calcium phosphate-mediated transfection of HEK293T cells was used to generate shRNA lentiviruses as previously described .Total cellular RNA was extracted by TRIzol reagent (Invitrogen), and reverse transcribed into cDNA using the ReverTra Ace qPCR reverse transcription master mix with gDNA (genomic DNA) Remover (Toyobo). Real-time polymerase chain reaction (PCR) was performed using the Thunderbird SYBR qPCR Mix (Toyobo) with pre-denaturation at 95°C for 2 min, amplification with 40 cycles of denaturation , and annealing , on the ABI 7900HT Real-Time PCR system. The data were analysed by a SYBR green-based system (Toyobo), semi-quantified and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers were listed in Supplementary Table 1. Initial primers are targeted base pair 10–59 of the HIV-1 transcript; Proximal (Pro) primers are targeted base pairs 29–180 of the HIV-1 transcript; Intermediate (Int) primers are targeted base pair 836–1015 of the HIV-1 transcript; Distal (Dis) primers are targeted base pair 2341–2433 of HIV-1 transcript.The following antibodies were used for Chromatin Immunoprecipitation (ChIP) or immunoblotting: anti-TCF1, anti-beta-catenin , anti-Phospho-β-Catenin , anti-GSK3, anti-GSK3 phospho Y216 + Y279, anti-H3K4me3 , anti-H3K27ac , anti-H3K27me3 , anti-H3K9me3 , anti-GAPDH , and anti-Lamin B .g, the supernatant was boiled in reducing SDS sample loading buffer and subjected by SDS-PAGE. The indicated specific primary antibodies were used, followed by horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Sigma) as the secondary antibody. Nuclear and cytoplasmic protein fractions were purified using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturer’s introduction.For immunoblotting, cells were lysed for 1 h at 4°C in ice-cold lysis buffer . After centrifugation for 10 min at 12,000ChIP experiments were performed according to the protocol provided by EZ-ChIP Chromatin Immunoprecipitation kit (Millipore) as previously described . Cells w+ counts were analysed by flow cytometry (BD Biosciences). HIV-1 viral loads were quantified by PCR (Cobas Amplicor). HIV subtypes were determined by phylogenetic analysis of Gag-RT sequence with subtype reference (Supplementary Table 2).Peripheral blood from combination antiretroviral treatment (cART)-treated HIV-1 infected patients were collected from the cohort patients of Zhengzhou Sixth People's Hospital. Informed consent has been signed. These patients had undergone cART comprised of TDF (tenofovir disoproxil fumarate), 3TC (lamivudine), and EFV (efavirenz) for more than 3 years with an undetectable HIV-1 viral load in the plasma. CD4gag and tat-rev mRNA. Anti-CD3 PerCP-Cy5.5 (eBioscience), anti-CD4 PE-cy7 (BD Biosciences), N6 , and PE-streptavidin (BD Biosciences) were used for immunostaining. N6 antibody was provided by Jing-He Huang . Env positive cells were detected by Fortessa flow cytometer (BD Pharmingen) and analysed with the assistance of FlowJo 7.6.1 software.PBMCs from cART-treated HIV-1 infected patients were collected with Ficoll-Paque density gradient centrifugation . PBMCs were treated with 6-BIO  (1 μM) or phytohemagglutinin-P (PHA-P) (Sigma-Aldrich) (5 μg/mL) for 5 days and cultured in RPMI 1640 medium (Gibco) supplemented with 10% foetal bovine serum (Gibco) in the presence of recombinant interleukin-2 (IL-2) (RD Systems). HIV-1 reactivation was monitored by detecting the surface expression of HIV Envelope (Env) and the production of Macaca mulatta) were housed in the Experimental Animal Center of Guangzhou Institutes of Biomedicine and Health . SIVmac239  intravenously challenged macaques were treated with a potent and long-lasting nucleoside reverse transcriptase inhibitor FNC (2’-deoxy-2’-β-fluoro-4’-azidocytidine) (0.4 mg/kg) we developed recently for viral suppression  [gag mRNA were quantitated by real-time PCR as described previously [gag-specific primers (Supplementary Table 1). The copy number of viral RNA was calculated based on the standard curve of an in vitro-transcribed fragment of the SIVmac239 gag gene. The limitation for assay was 100 copies/mL plasma. The reverse transcribed cellular cDNAs were used as the template for PCR amplification by Phanta Max Super-Fidelity DNA Polymerase (Vazyme) with SIV-gag-specific 1st pair of primers (Supplementary Table 1); the diluted PCR products were further used as the qPCR template, and cell-associated gag mRNA number was determined by real-time qPCR with TB Green Premix Ex Taq II (TAKARA) with SIV-gag-specific 2nd pair of primers (Supplementary Table 1). The number of circulating CD3+, CD4+, and CD8+ T-lymphocytes were determined using BD TruCount tubes according to the manufacturer's instructions (BD Biosciences).Chinese rhesus macaques  according to the manufacturer’s protocol, and ribosomal RNA removed using QIAseq FastSelect-rRNA HMR Kits . Fragmented RNAs (average length approximately 200 bp) were subjected to first strand and second strand cDNA synthesis, followed by adaptor ligation and enrichment with a low-cycle according to the instructions of NEBNext UltraTM RNA Library Prep Kit for Illumina . The purified library products were evaluated using the Agilent 2200 TapeStation and Qubit2.0 . The libraries were paired-end sequenced  using Illumina HiSeq 3000 platform.featureCounts function in SubReads package v1.5.3 with default parameter. Next, counts matrix obtained from featureCounts was used as input for differential expression gene analysis with the bioconductor package DESeq2 v1.26 in Rv4.0. Gene counts more than 5 reads in a single sample or more than 50 total reads across all samples were retained for further analysis. Filtered counts matrix was normalized using the DESeq2 method to remove the library-specific artefacts. Principal component analysis was based on global transcriptome data using the build-in function prcomp in R software. The genes with log2 fold change >1 or < −1 and adjusted p value <0.05 corrected for multiple testing using the Benjamini-Hochberg method were considered significant. Transcription-factor enrichment analysis and functional enrichment analysis were performed using Metascape server tool (https://metascape.org/gp/index.html#/main/step1). Gene set enrichment analysis (GSEA) used the R package clusterProfiler v3.18.1.Raw RNA sequencing (RNA-seq) reads were filtered using Trimmomatic v0.36. The filtered reads were mapped to the human (hg38) reference genomes using HISAT v2.1 with corresponding gene annotations (GRCh38.p13) with default settings. Total counts per mapped gene were determined using p < 0.05, ** p < 0.01, *** p < 0.001.Graphpad Prism 8.0 was used for statistical analysis. For intra-group direct comparisons, Student’s unpaired two-tailed t test was performed to analyse significant differences. For comparisons of multiple groups, one-way ANOVAs were performed. Significance levels are indicated as * https://www.ncbi.nlm.nih.gov/bioproject/PRJNA747246).All raw RNA-seq data used in this study are available under BioProject accession code PRJNA747246 at the NCBI database  and GSK3β (47 kDa). The treatment of ACH2 cells with 6-BIO inhibited GSK3 kinase activity as demonstrated by the diminished phosphorylation of both α and β isoforms of GSK3 at Tyr216 and Tyr 279  is another well-known GSK3 kinase inhibitor by competition for magnesium, but LiCl does not alter GSK3 phosphorylation . In cons+ T cells.Taken together, these results demonstrate that the pharmacological inactivation of GSK3 kinase activity reactivates HIV-1 from latently infected CD4In the canonical Wnt signalling pathway, the nuclear β-catenin activates target genes through its interactions with transcription factors of TCF/LEF family members TCF1, TCF3, TCF4, and LEF1 . 6-BIO t+ T cells is achieved by activating the β-catenin/TCF1 axis.To ascertain this, we monitored the 6-BIO-induced HIV-1 reactivation when the endogenous expressions of GSK3α/β, β-catenin and TCF1 in ACH2 cells were knocked-down by infections with lentiviruses containing specific short hairpin RNAs (shRNA)  for 24 h. A dose-dependent manner of enhancement of viral infection was observed upon 6-BIO treatment, and 6-BIO treatment significantly increased the initiation and elongation of HIV-1 5′-LTR-driven transcription . Phytohemagglutinin-P (PHA-P) treatment was used as the positive stimulation control. Viral reactivation was detected with flow cytometry to analyse the HIV-1 Env expression on CD450 of SIVmac239, and then were treated with a novel nucleoside reverse transcriptase inhibitor FNC (2’-deoxy-2’-β-fluoro-4’-azidocytidine) we developed  (gag mRNAs (+ T, CD8+ T, and CD14+ monocytes, as shown by no increased surface expressions of CD25 and/or HLD-DR (+ and CD8+ T cells over the period of treatment (To further investigate the role of 6-BIO in a more controlled setting, we next evaluated the latency reversal activity of 6-BIO in four Chinese-origin rhesus macaques. These macaques were challenged with 5000TCID+ T cells. Given 6-BIO has been proposed for treatments of a wide range of diseases [The reversible repression of HIV-1 5′- LTR-mediated transcription represents the main mechanism for HIV-1 to maintain latency ,6,8,9. Idiseases , it prov+ T cells that form long-lived HIV-1 reservoir. Blockade of the interaction between β-catenin and co-activator CREB-binding protein, by a Wnt/β-catenin pathway small molecule inhibitor PRI-724, decreases the proliferation of stem cell memory- and central memory-CD4+ T in ART-suppressed SIVmac251-infected rhesus macaques, but unable to not significantly reduce the viral reservoir size [The canonical Wnt/β-catenin pathway is evolutionarily conserved and plays important roles in stem cell proliferation and pluripotency, neurodevelopment, embryonic development, and tumorigenesis . Dysreguoir size .+ T ACH2 cells show the higher expression of TCF1 and LEF1, therefore 6-BIO stimulates the β-catenin/TCF1 signalling for viral reactivation.The expression and function of the downstream TCF/LEF family members may have the cell-subtype selection. Most studies of Wnt/β-catenin signalling in relation to HIV-1 infection have focused on the regulation of TCF/LEF family member TCF4, which has been identified as a repressor of HIV-1 replication in astrocytes and monocytes . In astr+ T cells ACH2. The core DEGs related to the regulation of inflammation and cytokine signalling mainly included CCR1, IL-10, IL12RB2, IL18RAP, IL2RA, TNFSF8, etc. Non-specific activation of host cells can provide more susceptible target cells for HIV-1 infection [Transcriptome data also confirm 6-BIO-mediated activation of β-catenin/TCF1 signalling in CD4nfection . It seemOur study has some limitations. 6-BIO evaluation was performed in the limited number of 4 SIV-infected monkeys; the 6-BIO reactivation was started right after the SIV suppression in monkeys, and a longer duration for SIV suppression is more appropriate in reflecting HIV-1 latency in the long-term cART-suppressed patients. Besides of the monkey/SIV models, the humanized mouse models provide alternative tools for fully evaluating 6-BIO activity.+ T cell lines, resting CD4+ T cells from cART-suppressed patients and SIV-infected rhesus macaques, we demonstrate that the pharmacological suppression GSK3 kinase activity by 6-BIO reactivates HIV-1 from latently infected CD4+ T cells. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.Taken together, by using HIV-1 latently infected CD4Click here for additional data file.


However, knockdown of cdc42se1 could not significantly rescue PGC migration in maternal miR-202 mutant (MmiR-202) embryos, indicating that there are other target genes of miR-202-5p required for the regulation of PGC migration. Herein, we revealed the transcriptional profiles of wild type and MmiR-202 PGCs and obtained 129 differentially expressed genes (DEGs), of which 42 DEGs were enriched cell migration-related signaling pathways. From these DEGs, we identified two novel miR-202-5p target genes prdm12b and rab10. Furthermore, we found that disruption of rab10 expression led to significantly migratory defects of PGC by overexpression of rab10 siRNA, or WT, inactive as well as active forms of rab10 mRNA, and WT rab10 overexpression mediated migratory defects could be partially but significantly rescued by overexpression of miR-202-5p, demonstrating that rab10 is an important factor involved miR-202-5p mediated regulation of PGC migration. Taken together, the present results provide significant information for understanding the molecular mechanism by which miR-202-5p regulates PGC migration in zebrafish.MicroRNAs (miRNAs) play important roles in post-transcriptional repression in nearly every biological process including germ cell development. Previously, we have identified a zebrafish germ plasm-specific miRNA  Shortd nanos) ,9,10, lo xlsirt) ,12,13, p xlsirt) , as well-202-5p) ,16,17. T-202-5p) ,19,20.Dictyostelium discoideum or Entamoeba histolytica, zebrafish PGC migration utilizes the term of bleb, which is driven by hydrostatic pressure and cytoplasmic flow to emerge cells detaching from the actin cortex and forming a round protrusion in the cell front [Like ll front . PGC migll front . Then, tll front . The Cxcll front . During ll front , and an ll front . In addill front ,28. The ll front ,27. Therlet-7, the first identified miRNA in animals, is a critical factor in the determination of germ versus somatic cell fates in mammals [miR-430 plays an important role in the clearance of maternal mRNAs during maternal to zygotic transition [miR-430 regulation of Cxcl12 chemokine signaling confers genetic robustness in directing PGC migration towards the genital ridge [miR-202-5p has been reported as a gonad-specific miRNA in zebrafish [miR-202-5p is a component of maternal germ plasm [miR-202-5p results in an elevation in the level of cdc42se1, which decreases the expression of cdc42, ultimately, leading to severe migratory defects in PGCs [cdc42se1 can’t effectively rescue PGC migration in maternal miR-202 mutant (MmiR-202) embryos, suggesting that there might exist other miR-202-5p target genes contributing to the regulation of PGC migration through different signaling networks.MicroRNAs (miRNAs) are a kind of endogenous 18–25 nt non-coding RNAs, which pair with the mRNA of protein-coding genes to achieve post-transcriptional regulation in almost all biological processes including germ cell development ,30,31,32 mammals ,34,35,36ansition . The miRal ridge . Anotherebrafish , Atlantiebrafish , and medebrafish . In zebrrm plasm . Loss of in PGCs . HowevermiR-202-5p mediated regulation of PGC migration, we analyzed the transcriptional files of wild type (WT) and MmiR-202 PGCs by transcriptome sequencing. The transcriptome data revealed 129 differentially expressed genes (DEGs), which were widely distributed in cell migration related to signaling pathways such as extracellular matrix, cell polarity, cytoskeleton, cell adhesion, cell motility and small GTPase activity. From these DEGs, we identified two novel miR-202-5p target genes prdm12b and rab10. Furthermore, loss- and gain-of-function experiments indicated that the proper balance of rab10 was critical for PGC migration.To further understand the molecular mechanism underlying miR-202-5p mutant (MmiR-202) zebrafish line [gfp-nanos 3′utr mRNA in 1-cell embryos, and the GFP-labelled PGCs of WT or MmiR-202 embryos were isolated by fluorescence-activated cell sorting (FACS) at 24 h post fertilization (hpf). The collected PGCs were divided into three groups of WT and MmiR-202 PGC groups (300 PGCs each group) to construct six sequencing libraries. By Illumina sequencing, we obtained 546 M clean reads with about 91 M clean reads in each library, 89.2% of which were mapped to the zebrafish genome and annotated as 30,878 transcripts . To yield reliable results, only genes with FPKM ≥ 1 in at least three samples were subjected to analyze differentially expressed genes (DEGs). We obtained 129 DEGs including 46 up-regulated and 83 down-regulated DEGs in M WT PGCs A,B. Thes WT PGCs C. In summiR-202-5p mediated PGC migration, we performed bioinformatic analysis to predict functions of these DEGs and found that 42 DEGs were enriched in cell migration associated biological processes including extracellular matrix, cell polarity, cytoskeleton, cell adhesion, cell motility and small GTPase activity  of any DEG contained miR-202-5p binding sites by bioinformatic analysis. We found that the 3′UTR of rab10 and prdm12b contained the canonical seed sequences of miR-202-5p  of luciferase and transiently transfected them to HEK293T cells. Dual luciferases assay indicated that the relative activity of luciferase fusing WT 3′UTR of prdm12b or rab10 was significantly decreased in miR-202-5p mimics transfected HEK293T cells, whereas mutation of miR-202-5p binding sites significantly attenuated the repression . We observed a significant mislocalization of PGCs in rab10 siRNA injected embryos at 24 hpf  of rab10 siRNA were injected into 1-cell MmiR-202 embryos. In 300 pg control siRNA injected WT embryos, there were 6.21% embryos with an average number (0.91) of mislocalized PGC , miR-202-5p is co-localized with sox9 in primordial follicles. Loss of miR-202 impairs early folliculogenesis, decreases the number of mature follicles and leads to a dramatic reduction in female fecundity [miR-202-5p is a component of germ plasm. Loss of miR-202 seems to not affect zebrafish fertility but results in a significant defect in PGC migration [miR-202-5p uses to regulate PGC migration. miR-202-5p negatively regulates cdc42se1 to ensure the proper expression of cdc42 in PGCs. However, it remains vague whether there are other mechanisms for miR-202-5p regulating PGC migration by other target genes. In this study, we performed transcriptome sequencing of WT and MmiR-202 PGCs and obtained 146 DEGs. Furthermore, we found that rab10 acts as a downstream target gene of miR-202-5p and plays an important role in PGC migration. Therefore, we revealed a novel mechanism underlying miR-202-5p regulation of PGC migration.oduction , gametogoduction ,47, immuoduction , tumorigoduction ,49, and oduction ,50,51. Iogenesis ,52. In fogenesis . Chickenecundity . In zebrigration . PrevioumiR-202 PGCs. However, because of lacking the PGC-specific transgenic miR-202−/− line, it is difficult to collect MmiR-202 PGCs from the onset of PGC migration. The 3′UTRs of nos1, dnd and tdrd7 regulate differential localization between soma and PGCs [gfp-nanos 3′utr mRNA injected embryos, the PGC-specific fluorescence could be found at the late segmentation period, usually at 24 hpf. Therefore, we collected PGCs in gfp-nanos-3′utr mRNA injected WT and MmiR-202 embryos at 24 hpf to ensure the purity of isolated PGCs. The transcriptome results revealed that 42 DEGs in WT and MmiR-202 PGCs were significantly enriched in cell migration-associated biological processes  family are critical in regulation of DNA methylation and stem cell population maintenance. PRDM1 and PRDM14 are critical determinants of mammalian germ cell lineages from embryonic cells [prdm12b had no significant effect on PGC development in zebrafish (data not shown), suggesting PRDM family exert divergent roles in mammals and fish.Because miRNAs rarely encode functional protein products, they usually initiate hierarchical biological events by binding to the mRNAs of target genes to direct post-transcriptional repression. Therefore, identifying target genes will be helpful to understand how ic cells ,58. Howeic cells . We founrab10 binds with the male germ cell-specific Rab GTPase-activating protein (MGCRABGAP) and the MGCRABGAP-RAB complex localizes in the manchette structure of mature sperm, indicating a possible role in spermatogenesis [rab10 participates in PGC development remains unclear. Herein, we found that disruption of rab10 balance through siRNA mediated reduction, overexpression of WT rab10, inactive or active rab10 mutants led to PGC mislocalization, demonstrating that proper expression of rab10, especially for the balance of rab10 GDP- and GTP-bound states, is critical for PGC migration , and the methods were carried out in accordance with the approved guidelines.Tg kop: egfp-UTR-nos3 zebrafish were purchased from the China Zebrafish Resource Center. The maternal miR-202 mutant (MmiR-202) zebrafish has been generated in our previous study [The AB line and transgenic line us study . Fish we°C in Dulbecco’s modified Eagle’s medium (DMEM) supplied with 10% FBS  under a humidified atmosphere of air containing 5% CO2.HEK 293T cells were cultured at 37gfp-nos 3′utr mRNA was injected into 1-cell WT and MmiR-202 embryos. At 24 hpf, the embryos were digested to single-cell suspension by trypsin according to the zebrafish book . A total of 900 WT and 900 MmiR-202 PGCs were collected by FACS based on the GFP signal. The isolated PGCs were equally divided into three technical replicates of WT PGCs and MmiR-202 PGCs groups for Illumina sequencing on the MGIseq2000 platform .To label the PGCs with green fluorescence protein (GFP), 400pg Danio rerio GRCz10.89.gtf) with HISAT2 [miR-202 PGCs with DEGseq with BH method , and genes with q ≤ 0.05 and |log2_ratio| ≥ 1 were identified as differentially expressed genes. The biological signaling pathway of DEGs was analyzed by functional enrichment in GO terms  and KEGG pathways . GO terms with q < 0.05 were considered significantly enriched.After sequencing, clean reads were obtained with Perl scripts to clear raw read containing more than 5 adapter-polluted bases, the number of N bases accounting for more than 5%, as well as the phred quality value less than 19 accounting for more than 15%. Clean reads were mapped to the reference genome and annotation file downloaded from Ensembl  of rab10 siRNAs, as well as embryos injected with gfp mRNA or different dosages (50/100/200 pg each embryo) of rab10 mRNA were collected at 24 hpf, frozen in liquid nitrogen and stored in −80 °C. The collected embryos were homogenized in 1 mL of trizol reagent , and total RNA was extracted according to the manufacturer’s instructions.For RNA isolation, WT embryos, embryos injected control or miR-202 PGCs, the RNA of collected PGCs was reverse-transcribed with single-cell sequence-specific amplification kit to establish first the cDNA library . The extracted RNA of other embryos was reverse-transcribed into single-strand cDNA with primescript™ first-strand cDNA synthesis kit . To avoid the error caused by single reference genes in single-cell PCR validation, we used three common reference genes including ef1a, β-actin and rpl13a to normalize gene expression levels using 2−∆∆Ct method in every PCR examination. The results were representative of more than three independent experiments in triplicate. Each independent experiment was performed in triplicate.To examine the expression levels of predicted DEGs between WT and MqPCR analysis was performed on the LightCycler480 II  with the chamQ universal SYBR qPCR master mix  as previously described . Primer rab10 (ENSDARG00000046106) or prdm12b (ENSDARG00000007430) were cloned and fused to the ORF of luciferase and RFP in the psiCHECK2 and pCS2 plus (pCS2+) vectors, respectively.For luciferase or RFP reporters, the 3′UTRs of rab10 overexpression, full ORF and 3′UTR of rab10 was inserted to pCS2+ vector. To specifically overexpress rab10 in PGCs, the ORF of rab10 and gfp as well as the 3′UTR of nanos3 were inserted to pCS2+ vector to generate the pCS2-rab10-gfp-nos 3′utr plasmid.For universal miR-202-5p binding sites within the 3′UTRs of rab10 or prdm12b, and the inactive or active rab10 mutants were constructed with hieff muttm multi-site-directed mutagenesis kit  as previously described [All the point mutants including escribed ,45.For mRNA synthesis, recombinant pCS2+ plasmids were linearized with NotI and transcribed using SP6 mMESSAGE mMACHINE Kit  according to the manufacturer’s instructions.rab10, three dosages (50/150/300 pg each embryo) of rab10 siRNA (GCCAACATCAACATCGAGA) or 200 pg control siRNA  was respectively injected into 1-cell WT embryos to knockdown endogenous rab10, and the embryos were collected for qPCR analysis of rab10 expression. Subsequently, the rab10 siRNA was injected into 1-cell embryos of kop: egfp-UTR-nos3 line.To knockdown rab10, different dosages (50/100/200 pg each embryo) of rab10, rab10T23N and rab10Q68L mRNA were respectively injected into 1-cell embryos of Tg kop: egfp-UTR-nos3 line.To overexpress rab10 mRNA or rab10 siRNA, 50 pg rab10 siRNA or 100 pg rab10 mRNA were co-injected into 1-cell embryos of Tg kop: egfp-UTR-nos3 line, and PGC migration was observed at 24 hpf. Furthermore, 300 pg miR-202-5p mimics were co-injected to rescue PGC migration in rab10 mRNA injected embryos.To rescue PGC migration in embryos injected miR-202 PGCs, embryos were injected with different dosages (50/150/300 pg each embryo) of rab10 siRNA or 4000 pg cdc42se1 morpholino, respectively.To rescue migratory defects in Mrab10 or prdm12b were miR-202-5p target genes, 200 pg rfp-prdm12b-3′utr-wt, rfp-prdm12b-3′utr-mut, rfp-rab10-3′utr-wt or rfp-rab10-3′utr-mut mRNA was co-injected with control or miR-202-5p mimics. At 24 hpf, representative images of corresponding embryos were captured with the stereo fluorescence microscope . And the average signal intensity of RFP was measured by Image J (version 1.51).To confirm All the PGC migration was examined at 24 hpf with the stereo fluorescence microscope . All experiments were performed in more than three independent experiments in triplicate. Each independent experiment was performed in triplicate.psiCHECK2-3’UTR-prdm12b-WT (psiCHECK2-3’UTR-rab10-WT) or psiCHECK2-3’UTR-prdm12b-seed-Mut (psiCHECK2-3’UTR-rab10-seed-Mut) plasmids and 100nM control or miR-202-5p mimics with Lipofectamine 8000 . The cells were lysed 24 h post-transfection, and the luciferase activity was detected by the dual-luciferase reporter analysis system . The results were representative of more than three independent experiments, each performed in triplicate. Each independent experiment was performed in triplicate.At 70–80% confluence, HEK293T cells seeded in 24-well plates were transiently co-transfected 10 ng At 24 hpf, PGCs of different groups of embryos were observed with the stereoscopic fluorescence microscope. The delineation of normal and mislocalized phenotype were distinguished as following: embryos with less than 3 ectopic PGCs were regarded as normal, whereas embryos with more than 3 ectopic PGCs were regarded as mislocalization. At least four repeated groups of embryos (30–50 embryos in each group) were observed.t-test, and the phenotypes in different experimental groups were analyzed by one-way ANOVA. p < 0.5 was considered a statistically significant difference.The statistics were calculated and analyzed with SPSS version 20. The qPCR results were analyzed by Student’s


RAS family genes play essential roles in tumorigenesis, effective treatments targeting RAS-related tumors are lacking, partly because of an incomplete understanding of the complex signaling crosstalk within RAS-related tumors. Here, we performed a large-scale genetic screen in Drosophila eye imaginal discs and identified Misshapen (Msn) as a tumor suppressor that synergizes with oncogenic Ras (V12Ras) to induce c-Jun N-terminal kinase (JNK) activation and Hippo inactivation, then subsequently leads to tumor overgrowth and invasion. Moreover, ectopic Msn expression activates Hippo signaling pathway and suppresses Hippo signaling disruption-induced overgrowth. Importantly, we further found that Msn acts downstream of protocadherin Fat (Ft) to regulate Hippo signaling. Finally, we identified msn as a Yki/Sd target gene that regulates Hippo pathway in a negative feedback manner. Together, our findings identified Msn as a tumor suppressor and provide a novel insight into RAS-related tumorigenesis that may be relevant to human cancer biology.Although  RAS genes  identified in 1982 are the most frequently mutated oncogenes in various human cancers [RAS is a critical cancer driver and anti-RAS therapy is expected to be a promising direction for cancer treatment [RAS-related tumors [RAS-related tumors.The reatment ,4. Howevd tumors ,4, largeRAS-related tumors. Over the last few decades, Drosophila has been proven to be an excellent model organism for cancer research [Drosophila [Drosophila have identified that the disruption of cell polarity genes  collaborate with oncogenic Ras (V12Ras) or Notch to promote tumor overgrowth and invasion [Drosophila to dissect the mechanisms of RAS-related cooperative oncogenesis.A large-scale genetic screen is an effective unbiased method to systematically dissect the genetic bases in research ,6,7,8 anosophila ,10,11,12invasion . Thus, tDrosophila tumor models [Both c-Jun N-terminal kinase (JNK) and Hippo signaling pathways have been established in tumorigenesis. JNK pathway activation cooperates with Ras signaling and promotes tumor overgrowth and invasion . Simultar models ,16,17. Hr models ,19,20,21V12Ras-induced tumor overgrowth [misshapen  acts as a tumor suppressor that cooperates with oncogenic V12Ras to significantly promote tumor overgrowth and invasion by simultaneously activating JNK pathway and inactivating Hippo pathway. We found that Msn overexpression impeded Hippo-disruption-induced overgrowth by genetically acting downstream of protocadherin Fat (Ft). Additionally, we revealed that msn is a potential Yki/Sd target gene and it regulates Hippo pathway in a negative feedback manner. Together, these findings not only uncovered Msn as a novel regulator of RAS-related cooperative oncogenesis, but also provided a potential therapeutic target for RAS-related tumors.Recently, we performed a large-scale ethyl methanesulphonate (EMS)-induced genetic screen, aiming to uncover novel tumor suppressors that could synergistically promote ergrowth ,22. HereV12Ras to promote tumor growth and invasion, we performed a large-scale EMS-induced genetic screen on Drosophila eye-antennal imaginal discs utilizing the ey-FLP-based mosaic analysis with repressible cell marker (MARCM) technique [misshapen (CG16973) gene (labeled as 3208msn thereafter). Consistent with this, we found that null allele of msn (172msn) [V12Ras and exhibited similar degree of tumor overgrowth    also synergrowth E. More itype Msn . We furtectively . In accoshapen CG973 gene rd (VNC) D,K, a coosophila ,16,22, wergrowth F,F’, autergrowth , reducedergrowth I, and inergrowth J,L. In cergrowth . Consistergrowth M. Taken 3208msn/V12Ras tumors implies that JNK activation might be essential for tumorigenesis. Inconsequently, previous studies indicate that Msn acts as an MKKKK to activate the c-Jun N-terminal kinase (JNK) signaling pathway [msn alone had no significant change on puc transcription (msn synergized with V12Ras to induce intensive MMP1 and puc activation (Drosophila JNK homologue basket (DNbsk)  . It is ag region Q, which Drosophila gut have uncovered a genetic link between the STE20 kinase Msn and the Hippo pathway, indicating that Msn may act in parallel with mammalian homologue MST 1/2 (Hpo), the key component of Hippo pathway, to regulate mammalian homologue Lats 1/2 (Wts) and mammalian homologue YAP/TAZ (Yki) activity [−/−msn/V12Ras tumors might be caused by inactivation of Hippo signaling. We found that loss of msn alone in the developing eye disc showed neither changes in the endogenous expression of Hippo pathway target gene including Diap1  acts as a neoplastic tumor suppressor and would be eliminated by Hippo-mediated cell competition [−/−scrib clone sizes  caused small wing phenotype  to regulate Msn-mediated Hippo activation  under en-Gal4 significantly reduced the Msn transcription level, as demonstrated by a lacZ enhancer trap insertion within the msn locus (S168A) overexpression significantly upregulated msn transcription level  is overexpressed in various human cancers ,48,49,50 ex, wts ,20,21,51sn locus N–P. Convon level Q,R and cgulation S. Interegulation . To furt down sd T. Taken Drosophila has been widely used as a cancer model for the past few decades to dissect various human cancer biology questions, including cell–cell communication, tumor heterogeneity, clonal evolution, cancer cachexia, and antitumor drug resistance [Drosophila, especially the mosaic analysis with a repressible cell marker (MARCM) system [misshapen (CG16973) as a tumor suppressor that synergizes with V12Ras to drive tumor overgrowth by inactivating Hippo signaling in Drosophila  system ,53, make) system ,16,22. Iosophila . Drosophila Msn and its mammalian homologue MAP4K4/6/7 act in parallel and partially redundantly with canonical Hippo signaling component mammalian homologue MST 1/2 (Hpo) in regulating mammalian homologue Lats 1/2 (Wts) and mammalian homologue YAP/TAZ (Yki) [Misshapen (Msn) is a member of the STE20 kinase family; it was initially identified as a MAP kinase kinase kinase kinase (MKKKK) that activates the c-Jun N-terminal kinase (JNK) pathway ,26,27. MAZ (Yki) ,34,56,57AZ (Yki) ,20,21,58RAS family genes are considered to be one of the most highly mutated genes in various cancers [RAS-related tumors remains unsatisfactory, and the in vivo molecular mechanisms of RAS-related tumorigenesis are still not completely understood. Here, we identified msn as a tumor suppressor that cooperates with oncogenic V12Ras to promote tumor overgrowth and invasion by simultaneously activating JNK pathway and inactivating Hippo pathway. We found that Msn overexpression dramatically suppressed 1scrib/V12Ras-induced tumor overgrowth and invasion, a well-established Drosophila cancer model. Additionally, we revealed that msn acts downstream of Ft and regulates Hippo pathway in a negative feedback manner. It is worth noting that we could not exclude the possibility that −/−/RasV12msn tumors could also regulate other growth regulating pathways, including JAK-STAT signaling, which requires further investigation ; ey-Flp1; Act> y+ > Gal4, UAS-GFP; tub-Gal80, FRT80B (80B tester); ey-Flp5, Act > y+ > Gal4, UAS-GFP; FRT82B, tub-Gal80 (82B tester). Additional strains, including GMR-Gal4, en-Gal4, hh-Gal4, nub-Gal4, UAS-GFP, UAS-ft-IR, X1wts (#44251), 172msn (#5947), msn-LacZ (#11707), and UAS-Msn (#5946), were obtained from Bloomington Drosophila Stock Center; E69puc, UAS-V12Ras [UAS-DNbsk [1scrib [UAS-Wts, UAS-Yki, UAS-YkiS168A, UAS-yki-IR, and UAS-sd-IR were previously described [UAS-FtΔECD is a kind gift from Kenneth Irvine.All crosses were raised on standard escribed ,16,22 usS-RasV12 , UAS-bsk msn172 #47, msn-Lescribed . UAS-FtΔDrosophila chromosome 3 L in this screen. Male flies carrying FRT79E  were starved for 8 h and subsequently fed with 25 mM EMS solution overnight at room temperature. The mutagenized males were then mated to females of the genotype UAS-V12Ras; sb/TM6B. Single F1 males of the genotype UAS-V12Ras/CyO-GFP; ∗FRT79E/TM6B were crossed to Sp/CyO; sb/TM6B first and then crossed with the 79E tester line for validation of GFP-labeled tumor overgrowth phenotype under fluorescent microscope.We focused on Third-instar larvae eye-antennal discs were dissected in 1 × PBS, fixed in freshly made 4% paraformaldehyde, and stained as described previously  using thS2 cells were maintained in Schneider’s Drosophila Medium (Gibco #21720024) supplemented with 10% fetal bovine serum (Cellmax #SA112) and 1% penicillin–streptomycin (Gibco #15140122) at 28 °C.ΔECD-FLAG plasmids were co-transfected to S2 cells, performed using Effectene transfection reagent (Qiagen #301427) following the manufacturer’s instructions and harvested 72 h after transfection. Harvested S2 cells were lysed in 500 µL RIPA lysis buffer . After the samples were rotated for 1 h at 4 °C, lysates were centrifuged at 13000 rpm for 13 min at 4 °C. The lysate supernatant was incubated at 4 °C with 20 µL cleaned anti-FLAG M2 Affinity Gel (Sigma #A2220) for 3 h. The beads were washed with PBST four times, then mixed with SDS-PAGE Loading Buffer  (CWBIO #CW0027S) and heated to 95 °C for 7 min. After separation of proteins by SDS-PAGE, proteins were transferred to PVDF membrane (Merck Millipore #IPVH00010) which was blocked with 5% milk–PBST for 1 h, incubated in primary antibody overnight at 4 °C, washed with PBST, incubated with secondary antibody at room temperature for 1 h, and washed with PBST four times. Protein was detected using a Viber Fusion FX6 Spectra imaging system . The following antibodies for Western blotting were used: primary antibodies, rabbit-anti-FLAG  (EASYBIO #BE2005); rabbit-anti–HA (1:9000) (EASYBIO #BE2006); and secondary antibody anti-rabbit-HRP (1:8000) (Promega #W4011).Transfection, Co-IP, and Western blot analysis were performed as previously described with some modification . paw-GalDrosophila adult head of indicated genotypes was removed and total RNA was then extracted using TRIzol (Ambion). Total RNA was reverse-transcribed into cDNA with the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme); and quantitative PCR was performed with KAPA SYBR® FAST (KAPA BIOSYSTEMS) and quantified by the QuantStudio™ 5 Real-Time PCR System (ThermoFisher). RP49 was used as an internal control. The following primer sequences were used for real-time PCR. msn: F: 5′-TCCCTTGGACAGCAGCGATT-3′, R: 5′-AGTTCCATCGTTCCTAGCC-3′; rp49: F: 5′-TCCTACCAGCTTCAAGATGACC-3′, R: 5′-CACGTTGTGCACCAGGAACT-3′.p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).Clone and wing size were measured with ImageJ and Photoshop, respectively. Quantification of the data was presented in bar graphs created with GraphPad Prism 8. Data represent mean values + SD. We used Mann–Whitney U test for multiple comparisons to calculate statistical significance (*


Ephx2), resulting in the formation of toxic and pro-inflammatory vicinal diols (dihydroxy-FAs). Pharmacological inhibition of sEH is a promising strategy for the treatment of pain, inflammation, cardiovascular diseases, and other conditions. We tested the efficacy of a potent, selective sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Prophylactic TPPU treatment significantly ameliorated EAE without affecting circulating white blood cell counts. TPPU accumulated in the spinal cords (SCs), which was correlated with plasma TPPU concentration. Targeted lipidomics in EAE SCs and plasma identified that TPPU blocked production of dihydroxy-FAs efficiently and increased some EpFA species including 12(13)-epoxy-octadecenoic acid (12(13)-EpOME) and 17(18)-epoxy-eicosatrienoic acid (17(18)-EpETE). TPPU did not alter levels of cyclooxygenase (COX-1/2) metabolites, while it increased 12-hydroxyeicosatetraenoic acid (12-HETE) and other 12/15-lipoxygenase metabolites. These analytical results are consistent with sEH inhibitors that reduce neuroinflammation and accelerate anti-inflammatory responses, providing the possibility that sEH inhibitors could be used as a disease modifying therapy, as well as for MS-associated pain relief.Polyunsaturated fatty acids (PUFAs) are essential FAs for human health. Cytochrome P450 oxygenates PUFAs to produce anti-inflammatory and pain-resolving epoxy fatty acids (EpFAs) and other oxylipins whose epoxide ring is opened by the soluble epoxide hydrolase (sEH/ Thus, dietary PUFAs are essential for human beings. PUFAs affect membrane flexibility, fluidity, and stiffness -propyl}-pyrazol-1-yl)-benzenesulfonamide) to treat pain, cancer, hypertension, brain and heart diseases [Lipids are essential nutrients and major constituents of the central nervous system (CNS) ,2. FattyH17) or IFN-γ (TH1) [2 in EAE pathogenesis [2α (cPLA2α) in disease development [2 receptors (EP2 and EP4) [4 receptor (BLT1) in TH17 differentiation [1) expression on the cell surface as functional antagonists [Multiple sclerosis (MS) is a neurological disease that is pathologically characterized by the central nervous system (CNS)-specific demyelination and inflammation . Althoug-γ (TH1) , B cells-γ (TH1) ,19. We pogenesis ,21. EAE elopment , PGE2 reand EP4)  and the ntiation , and plantiation ,26. One ntiation . Based oagonists , resultiagonists . Moreoveagonists . These sIn this study, we tested the efficacy of an sEH inhibitor, TPPU (1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl) urea) in EAE. We also determined the lipid profiles in EAE SCs and plasma of TPPU-treated mice using a quantitative targeted lipidomics approach ,32,33,3435–55) on day 0 and prophylactically treated with TPPU  by oral administration starting from day 0. TPPU treatment significantly ameliorated EAE disease course as compared to controls  , siponimod , ozanimod , and ponesimod , which reduce the circulating pathogenic lymphocytes via S1P1 down-regulation [We first tested the efficacy of TPPU, a sEH-selective inhibitor, on EAE development. C57BL/6 female mice were subcutaneously immunized with complete Freund’s adjuvant containing myelin oligodendrocyte glycoprotein peptide (MOGy ANOVA) A. Cumulay ANOVA) B. TPPU ty ANOVA) B. We dety ANOVA) C. The why ANOVA) D. These Ephx2 in astrocytes [Next, the EAE SCs were stained with hematoxylin and eosin (H&E) and luxol fast blue (LFB)-cresyl violet to assess the degree of inflammation and demyelination . The vehtrocytes , suggestWe applied targeted lipidomics approaches to analyze lipid profiles in both plasma and SCs of EAE mice that were collected in the chronic phase of EAE. To investigate TPPU effects on lipid metabolism, we first analyzed total lipid levels in the COX, 5-LO, 12/15-LO, and CYP-sEH pathways by calculating the sum of metabolite levels in each pathway. AA metabolites produced by 12/15-LO were rich in EAE plasma (~1 µmol/L) and were up-regulated by TPPU . TPPU treatment did not affect fluxes in the COX and 5-LO pathways but showed a similar trend in the 12/15-LO pathway with that of plasma A. Levels10(TPPU/vehicle) < 0 in both plasma and SC) with a few exceptions such as 4,5-DiHDPE (10(TPPU/vehicle) > 0 in both plasma and SC; Ephx2 deficient mice.Differential lipid levels were computed for TPPU vs. control groups that were represented as a scatter plot . This pl5-DiHDPE . This wa5-DiHDPE , indicatIn the present study, we demonstrated the beneficial effect of TPPU in the EAE mice without changing the number of circulating lymphocytes, and also showed that it effectively reduced pro-inflammatory dihydro-FAs in SCs and blood. Currently available disease modifying therapies (DMTs) for MS treatment are mostly immunomodulatory drugs that decrease circulating T and B lymphocytes, and thus prevent pathogenic lymphocytes from penetrating the CNS . DMTs thsEH inhibitors stabilize most EpFAs studied to date to varying degrees . In geneAnti-inflammatory effects of 17(18)-EpETE have been proposed in several diseases including contact hypersensitivity  and non-1, which is produced from DHA by the actions of 15-LO and 5-LO, ameliorated EAE [Pharmacological and genetic sEH inhibition appears to alter FA fluxes towards the 12/15-LO pathway. This increased flux may be used for SPM production. Since specialized pro-resolving mediators  require 12/15-LO activity for their biosynthesis, sEH inhibition may enhance SPM production when substrate PUFAs are sufficiently provided. 12/15-LO deficiency aggravated EAE , supportated EAE . On the ated EAE , many eiated EAE . Therefoated EAE . TPPU is35–55 peptide emulsified in complete Freund’s adjuvant  containing 4 mg/mL Mycobacterium tuberculosis H37Ra  on day 0. Mice intraperitoneally received 250 ng pertussis toxin  on day 0 and day 2. Clinical scores were recorded daily. TPPU was synthesized as previously described [® PEG E 300 . Mice were treated with TPPU  from days 0 to 28. SCs were collected and frozen in liquid nitrogen for lipidomics analyses and stored at −80 °C. Blood was collected into K2EDTA-coated tubes  by cardiac puncture under deep anesthesia followed by hematological analyses . Plasma was collected and stored at −80 °C for lipidomics analyses.EAE induction was performed as in previous studies ,50. Brieescribed  and dissTM, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature. As previously described [Spinal cord samples were incubated overnight in 10% neutral buffered formalin  as previously described ,32. For Spinal cords were homogenized in ice-cold methanol containing 0.1% butylated hydroxytoluene and 0.1% acetic acid. Internal standard mixtures were spiked into the homogenates and stored at −80 °C for 20 h. Samples were processed by solid-phase extraction , reconstituted in 200 nM CUDA in methanol, and analyzed by modified LC-MS/MS .p-value of <0.05 was considered to be statistically significant.As appropriate, data were analyzed statistically using Prism software  including Student’s t test, Mann–Whitney U test, Fisher’s exact test, Tukey’s multiple comparison test, or two-way repeated measures ANOVA. A


Analysis of 1,213 monoclonal antibodies (mAbs) isolated from S-specific MBCs revealed a primarily de novo response that displayed increased somatic hypermutation, binding affinity, and neutralization potency over time, providing evidence for prolonged antibody affinity maturation. B cell immunodominance hierarchies were similar across donor repertoires and remained relatively stable as the immune response progressed. Cross-reactive B cell populations, likely re-called from prior endemic beta-coronavirus exposures, comprised a small but stable fraction of the repertoires and did not contribute to the neutralizing response. The neutralizing antibody response was dominated by public clonotypes that displayed significantly reduced activity against SARS-CoV-2 variants emerging in Brazil and South Africa that harbor mutations at positions 501, 484 and 417 in the S protein. Overall, the results provide insight into the dynamics, durability, and functional properties of the human B cell response to SARS-CoV-2 infection and have implications for the design of immunogens that preferentially stimulate protective B cell responses.A comprehensive understanding of the kinetics and evolution of the human B cell response to SARS-CoV-2 infection will facilitate the development of next-generation vaccines and therapies. Here, we longitudinally profiled this response in mild and severe COVID-19 patients over a period of five months. Serum neutralizing antibody (nAb) responses waned rapidly but spike (S)-specific IgG Longitudinal analysis of the human B cell response to SARS-CoV-2 infection reveals prolonged antibody evolution. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, has infected over 75 million people and claimed over 1.5 million lives in just 12 months. Although vaccines have been developed and deployed at an unprecedented pace, the protection afforded by these vaccines may be short-lived due to waning serum antibody titers and/or the emergence of SARS-CoV-2 strains that evade vaccine-induced immunity  responses targeting the SARS-CoV-2 spike (S) protein appear within two weeks following symptom onset and persist for several months  samples from a cohort of eight COVID-19 convalescent donors who experienced severe (n=3) or mild (n=5) disease. The first blood sample (Visit 1) was drawn a median of 35.5 days following the onset of symptoms to allow for an assessment of the early B cell response to SARS-CoV-2 infection. The second (Visit 2) and third (Visit 3) blood samples were collected a median of 95.5- and 153.5-days post-symptom onset, respectively, to evaluate the long-lived memory B cell response . ConsistWe next measured nAb activity in the serum samples using a previously described pseudovirus reporter assay based on vesicular stomatitis virus bearing SARS-CoV-2 S (VSV-SARS-CoV-2) . However+CD27+CD38+ B cells, had contracted to background levels by the first sampling time point in all donors . We detected SARS-CoV-2 S-specific IgG+ MBCs in all donors at Visit 1, and the frequencies of MBCs either increased or stabilized by Visit 3, providing evidence for continuous MBC formation over a period of several months  and a fluorescently labeled, prefusion stabilized SARS-CoV-2 S protein . Sequencing studies revealed high clonal diversity in the donor repertoires at all three time points studied, with 0-30% of clones belonging to expanded clonal lineages . In agreement with prior studies of early antibody responses to SARS-CoV-2 infection nfection . Across nfection . By Visinfection . FurtherTo investigate whether SARS-CoV-2 S-specific antibody immunodominance hierarchy shifts over time, we evaluated the antigenic sites targeted by the 1,213 mAbs isolated from all three time points using a panel of SARS-CoV-2 subunits and domains . Plates were immediately spun down at 1,000 × g for 30 s and stored at -80°C until use. Flow cytometry data was analyzed using FlowJo software.B cells were purified directly from human peripheral blood samples using the EasySep Direct Human B Cell Isolation Kit (Stem Cell Technologies Cat# 19674). Purified B cells were stained using anti-human CD19 , CD3 , CD8 , CD14 , CD16 , IgM , IgD , IgA , IgG , CD27 , CD71 , propidium iodide (PI), and a freshly-prepared mixture of PE- and APC-labeled SARS-CoV-2 S-2P protein tetramers (25 nM each). Class-switched B cells, defined as CD19+CD3−CD8− CD14−CD16−PI−CD27+CD38+ cells.Antibody-secreting cells (ASCs) were identified by staining B cells with anti-human CD19 , CD3 , CD8 , CD14 , CD16 , CD27 , CD38 , and propidium iodide (PI). ASCs were defined as CD19S. cerevisiae using the lithium acetate method for chemical transformation (6 yeast cells were mixed and incubated with 240 μl of polyethylene glycol (PEG) 3350 (50% w/v) , 36 μl of 1M lithium acetate , 10 μl of denatured salmon sperm DNA , 67 μl sterile water, 200 ng of the digested expression vectors and 10 μl each of unpurified VH and VL PCR products at 42°C for 45 min. Following transformation, the yeast were washed twice with sterile water, resuspended in selective media and plated. Finally, individual yeast colonies were picked for Sanger sequencing.Human antibody variable gene transcripts  were amplified by reverse transcription polymerase chain reaction (RT-PCR) using SuperScript IV enzyme (Thermo Scientific Cat# 18090050) followed by nested PCR using cocktails of variable region and IgM-, IgD-, IgA- and IgG-specific constant-region primers with HotStarTaq Plus DNA Polymerase (Qiagen Cat# 203646), as previously described  or LambdaFabSelect resin  for antibodies utilizing the kappa or lambda light chains, respectively. The Fabs captured on the resin surface were eluted using 200 mM acetic acid with 50 mM NaCl (pH 0.52.0) and neutralized with 1/8 (v/v) 2 M HEPES (pH 8.0).DApp) affinities were measured by BLI using a ForteBio Octet HTX instrument (Molecular Devices) as previously described (Apparent equilibrium dissociation constant (KD) affinities of Fabs, streptavidin biosensors (Molecular Devices) were used to immobilize biotinylated antigens  and then allowed to stand in PBSF for a minimum of 30 min. After a short (60 s) baseline step in PBSF, the antigen-loaded biosensors were exposed (180 s) to the Fab at 100 nM, then dipped (180 s) into PBSF to measure any dissociation of the Fabs from the biosensor surface. For binding responses > 0.1 nm, data were aligned, interstep corrected (to the association step), and fit to a 1:1 binding model using the ForteBio Data Analysis Software, version 11.1.To measure the monovalent equilibrium dissociation constant (KCompetition binding of mAbs to recombinant SARS-CoV-2 RBD with human ACE2 (hACE2) was evaluated using a ForteBio Octet HTX instrument (Molecular Devices) as previously described (S. cerevisiae (EBY100) cultures using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) according to the manufacturer’s protocol and recovered in selective SDCAA media.RBD-SD1 regions (spike residues 319 to 591) of SARS-CoV-2 and emerging SARS-CoV-2 variants  were expressed via yeast surface display for assessment of antibody binding. DNA sequences encoding SARS-CoV-2 Wuhan-1 (GenBank: MN908947.3), SARS-CoV-2 variants incorporating individual amino acid substitutions , or combinations of mutations  were obtained as gBlocks (IDT) and cloned into a yeast surface-display vector as previously described  into 96-well plates and washed with PBSF. Cells were then resuspended in 50 μL of Fab solution diluted in PBSF and incubated on ice for 2 hours. Cells were subsequently washed twice with PBSF and labeled with 50 μL of APC-conjugated monoclonal mouse anti-hemagglutinin tag (HA).11 antibody , PE-conjugated goat anti-human IgG polyclonal antibodies , and propidium iodide  for 15 min on ice. Cells were washed twice with PBSF before analyzing via flow cytometry on a BD FACS Canto II (BD Biosciences). Mean anti-human IgG PE fluorescence signal values were fitted as nonlinear regression curves in GraphPad Prism using the following equation: Y=Yminimum+ (X^Slope) * (Ymaximum-Yminimum)/(X^Slope + EC50^Slope)), where X is the Fab concentration and Y is the PE MFI. Concentrations displaying hook effects, defined as concentrations higher than those generating the maximum PE MFI signal, were excluded from analysis. The percent reduction in binding activity to the mutant RBD relative to the WT SARS-CoV-2 RBD was calculated using the following equation: (WT EC50/Variant EC50) * 100.Fabs were titrated on yeast surface displayed RBD variants to determine EC2 for 24 hours, after which luciferase activity was measured in cell lysates using the Bright-Glo system (Promega) with a Bio-Tek II plate reader. Percent neutralization was calculated as [100 - (mean RLU test wells/mean RLU positive control wells) × 100] and used to determine the 50% neutralization titers for serum (NT50) and half-maximal inhibitory concentrations for mAbs (IC50).Microneutralization assays were performed using a VSV-based pseudovirus system as previously described  were coated with 25 μl per well of SARS-CoV-2 S-2P, SARS-CoV-2 NTD, SARS-CoV-2 RBD, OC43 S, or HKU1 S proteins diluted to 5 μg/ml in PBS (pH 7.4) and incubated overnight at 4°C. Wells were washed 3 times with PBS then blocked with 5% (w/v) non-fat dried milk (NFDM) in PBS for 1 hour at 37°C. After removal of the blocking solution, serial dilutions of human serum in 5% NFDM-PBS were added to the wells and allowed to incubate for 1 hour at 37°C. Plates were washed three times with PBS then secondary cross-adsorbed anti-human IgG-HRP  detection antibody was added at 1:8000 dilution in 5% NFDM-PBS and incubated for 1 hour at 37°C. After washing three times with PBS, 25 μl per well of 1-Step Ultra TMB-ELISA Substrate Solution  was added to detect binding, followed by addition of an equal volume of stop reagent. Absorbance was measured at 450 nm using a Spectramax microplate Reader (Molecular Devices).450 > 0.8 were designated as binders.For mAb binding studies, 96-well ELISA plates were coated with 25 μl per well of SARS-CoV-2 RBD, NTD, S1 subunit or SARS-CoV S protein diluted to 5 μg/ml in PBS and incubated overnight at 4°C. Wells were washed 3 times with PBS and then blocked with 5% NFDM-PBS for 1 hour at 37°C. After removal of blocking buffer, test mAbs diluted to 100 nM in 5% NFDM-PBS were added and incubated for 1 hour at 37°C. Plates were then washed three times with PBS and then secondary cross-adsorbed anti-human Fab-HRP  detection antibody was added at 1:10000 dilution in 5% NFDM-PBS and incubated for 1 hour at 37°C. After washing three times with PBS, 25 μl/well of 1-Step Ultra TMB-ELISA Substrate Solution  was added to detect binding followed by addition of an equal volume of stop reagent. Absorbance was measured at 450 nm using a Spectramax microplate Reader (Molecular Devices). IgGs that displayed an ELISA binding OD


About 50% of all primary breast cancers show a low-level expression of HER2 (HER2-low), defined as immunohistochemically 1+ or 2+ and lack of HER2 gene amplification measured by in situ hybridization. This low HER2 expression is a promising new target for antibody–drug conjugates (ADCs) currently under investigation. Until now, little is known about the frequency and the prognostic value of low HER2-expression in metastatic breast cancer (MBC).The MBC-Registry of the Austrian Study Group of Medical Tumor Therapy (AGMT) is a multicenter nationwide ongoing registry for MBC patients in Austria. Unadjusted, univariate survival probabilities of progression-free survival (PFS) and overall survival (OS) were calculated by the Kaplan–Meier method and compared by the log-rank test. Multivariable adjusted hazard ratios were estimated by Cox regression models. In this analysis, only patients with known HER2 status and available survival data were included.P = 0.171) nor in the triple-negative subgroup , when compared to completely HER2-negative disease. Similar results were observed when HER2 IHC 2+ patients were compared to IHC 1+ or 0 patients.As of 11/15/2020, 1,973 patients were included in the AGMT-MBC-Registry. Out of 1,729 evaluable patients, 351 (20.3%) were HER2-positive, 608 (35.2%) were HER2-low and 770 (44.5%) were completely HER2-negative (HER2-0). Low HER2-expression was markedly more frequent in the hormone-receptor(HR)+ subgroup compared to the triple-negative subgroup (40% vs. 23%). In multivariable analysis, low HER2 expression did not significantly influence OS neither in the HR+ (HR 0.89; 95% CI 0.74–1.05; Low-HER2 expression did not have any impact on prognosis of metastatic breast cancer in this real-world population.The online version contains supplementary material available at 10.1186/s13058-021-01492-x. Amplification of human epidermal growth factor receptor 2 (HER2) is a well-established negative prognostic factor both in early and metastatic breast cancer (MBC). HER2-directed therapies, however, have changed the natural course of this disease. Nowadays, adequately treated HER2+ /hormone-receptor(HR)+ breast cancer belongs to the subtypes with the most favorable prognosis both in the early and the advanced stage , 2.In contrast to HER2 positivity, defined as immunohistochemically (IHC) 3+ or IHC 2+ and HER2 gene amplification measured by in situ hybridization (ISH) . In geneSeveral retrospective studies investigated the prognostic value of low HER2 expression in early breast cancer (EBC) –20. MostHere, we present data from a large nation-wide registry for MBC in Austria. Both the incidence and the prognostic value of low HER2 expression were investigated in dependence of the HR status.The MBC-registry of the Austrian Study Group of Medical Tumor Therapy (AGMT) is a multicenter nationwide ongoing retrospective and prospective registry for MBC patients in Austria.HER2+ was defined as IHC 3+ or IHC 2+ and ISH+ according to the American Society of Clinical Oncology/College of American Pathologists Clinical Practice (ASCO/CAP) guidelines . Table S6. HR+ model selection frequencies according Heinze G. et al. [17]. Table S7. HR- Model stability investigations according Heinze G. et al. [17]. Table S8. HR- Model selection frequencies according Heinze G. et al. [17].


In human glioblastomas, glioma pathogenesis-related protein1 (GliPR1) is overexpressed and appears to be an oncoprotein. We investigated whether GliPR1 knockdown in glioma cells by RNA interference exerts anti-glioma effects.Experiments used human glioblastoma cell lines transduced with GliPR1 shRNA . Transduction produced stringent doxycycline-dependent GliPR1 knockdown in clones  or stable GliPR1 knockdown in polyclonal cells . In vitro assessments included cellular proliferation and clonogenic survival. In vivo assessments in tumor-bearing nude mice included tumor growth and survival.P < 0.001).Using doxycycline-dependent GliPR1 knockdown, shGliPR1-transduced U87-MG clones demonstrated reductions in cellular proliferation in the presence versus absence of doxycycline. Using stable GliPR1 knockdown, polyclonal shGliPR1-transduced U87-MG, A172, and U343-MG cells consistently showed decreased clonogenic survival and induced apoptosis (higher proportion of early apoptotic cells) compared to control shLuc-transduced cells. In tumor-bearing nude mice, using doxycycline-dependent GliPR1 knockdown, subcutaneous and cranial transplantation of the U87-MG clone 980-5 (transduced with GliPR1 sh#301) resulted in reduced subcutaneous tumor volume and cerebral tumor area in doxycycline-treated mice versus those left untreated. Using stable GliPR1 knockdown, nude mice cranially transplanted with polyclonal U87-MG cells transduced with GliPR1 sh#258 had significantly prolonged survival compared to mice cranially transplanted with control shLuc-transduced cells (41 versus 26 days; GliPR1 knockdown in glioma cells decreased cellular proliferation, decreased clonogenic survival, and induced apoptosis in vitro, and reduced glioblastoma tumor growth and prolonged survival in vivo. These findings support that GliPR1 may have potential value as a therapeutic target.The online version contains supplementary material available at 10.1007/s11060-021-03737-3. Glioblastoma is the most frequent and aggressive primary brain tumor in adults and the prognosis remains poor . The therelated to testes-specific, vespid, and pathogenesis protein 1 (RTVP-1) 2)/2. All mice were sacrificed when respective control group became moribund. Brains were shock-frozen in 2-methylbutane, then sequential cryosections (10 μm) were stained with cresyl violet. Cerebral tumor area was determined in brain sections with the largest tumor extension using an Axioscope .In first experiments, effects of stringent doxycycline-dependent GliPR1 knockdown on tumor growth used the U87-MG 980-5 clone transduced with GliPR1 sh#301, with experiments carried out by EPO GmbH  using a published procedure  with min6 cells (in 10 µl PBS) were stereotactically transplanted into the right brain hemisphere. Kaplan-Meier survival curves were constructed, and median survival calculated using PRISM software . Brains were shock-frozen in 2-methylbutane, then sequential cryosections (10 μm) were stained with Haematoxylin and Eosin (H&E), Hoechst 33,342 , and for the proliferation marker Ki-67 .In a subsequent experiment, effects of stable GliPR1 knockdown on survival were investigated using polyclonal mCherry-tagged U87-MG cells transduced with GliPR1 sh#258 or control shLuc. Mice were randomized, balancing by weight and age. For cranial transplantation, 1 × 10t test for statistical analysis. In vivo experiments used Mann–Whitney U test for tumor growth and log-rank test for survival.In vitro experiments used Student’s Fig. S1Histology and immunohistochemistry of representative brain cryosections from tumor bearing mice after implantation of polyclonal mCherry-tagged U87-MG cells transduced with GliPR1 sh#258 or a control luciferase shRNA (shLuc). (a-b) Imaging for mCherry red fluorescent protein as surrogate marker for tumors (red) and counterstaining with Hoechst 33342 nucleic acid stain (blue). (c-d) Immunohistochemical staining for proliferation marker Ki-67 (brown). (e-f) Hematoxylin and eosin (H&E) staining shows high nuclear density in the tumor. Scale bar = 100 μm. Supplementary material 1 (PPTX 5689.6 kb)Below is the link to the electronic supplementary material.


Chatzikonstantinou et al.  conducten = 12). Both studies were approved by the local ethics committee.Blood samples of CLL registry (NCT02863692) patients were centrally evaluated after full COVID-19 vaccination. In total, 21/23 patients were included in the analyses (samples missing in 2/23). Vaccinated healthcare workers served as HC cohort   was detectable in the majority of CLL patients , including all seronegative individuals. However, CLL patients with detectable activity  had a response that was comparable to HC .SARS-CoV-2 receptor-binding domain (RBD) specific IgG antibodies, determined using Alinity ci SARS-CoV-2 IgG II Quant assay (Abbott), were detectable in 8/21 (38.1%) patients with SLL/CLL and 100% of HC (09) Fig. . No neutPLUS [ALP] kit [Mabtech]) and B cells (IgG ELISpot) analyses. Results are reported as spot-forming cells (SFC) per million PBMCs. T cell responses to SARS-CoV-2 peptide pool ([15-mers overlapping by 11 amino acids] spanning the entire spike protein) were considered positive if higher than twice the median response of pre-pandemic HC (48 SFC/106). The median number of SARS-CoV-2 specific T cells was 21.3. SFC (interquartile range [IQR] 0.0–145.0) for CLL patients as compared with 177.3 SFC [IQR 138.0–403.3] in HC .Peripheral blood mononuclear cells (PBMCs) were used for SARS-CoV-2 spike-specific T cells (Human IFNy ELISpot6). Overall, 1/14 SLL/CLL patients (7.1%) had detectable S-specific ASC (138 SFC) as compared with 100% in HC . The SARS-CoV-2 specific IgG titer of the ASC responding patient was with 11 360 BAU/ml the highest within the group of CLL patients. Looking at total IgG-secreting B cells, 13 patients without S-specific ASC did neither show any IgG-secreting B cells. Spots were too faint to be counted or detected at numbers below the cut-off.SARS-CoV-2 S1/2-specific antibody-secreting cells (ASC) were analyzed in 14/21 (66.7%) SLL/CLL patients. The cut-off value for positive responses were defined as the mean plus two standard deviations of the responses observed in pre-pandemic HC , as older individuals respond with lower antibody levels to vaccination. However, in the rather small fraction of SLL/CLL patients who responded to vaccination, similar titers of neutralizing antibodies were detectable in HC. Further, we only included one treatment-naïve patient and therefore cannot fully conclude the impact of CLL-directed treatment as compared with untreated CLL on cellular immunity. Previous trials reported inferior serologic immunogenicity in treatment-naïve patients as compared with patients previously treated , 5, 10. In conclusion, we demonstrate inferior T cell response to COVID-19 vaccines in SLL/CLL patients as compared with HC, supporting the importance of a third vaccine dose for those. The prime-boost regime, in particular the choice of best vaccine combination, is yet to determine. Our observation of discordant immune responses in the majority of patients indicates that the humoral response may not be reliable as the sole surrogate marker of protection in the patients with CLL and further B cell depleting malignancies, at least if negative.Supplement


Effective therapeutics are urgently needed to counter infection and improve outcomes for patients suffering from COVID-19 and to combat this pandemic. Manipulation of epigenetic machinery to influence viral infectivity of host cells is a relatively unexplored area. The bromodomain and extraterminal (BET) family of epigenetic readers have been reported to modulate SARS-CoV-2 infection. Herein, we demonstrate apabetalone, the most clinical advanced BET inhibitor, downregulates expression of cell surface receptors involved in SARS-CoV-2 entry, including angiotensin-converting enzyme 2 (ACE2) and dipeptidyl-peptidase 4 (DPP4 or CD26) in SARS-CoV-2 permissive cells. Moreover, we show that apabetalone inhibits SARS-CoV-2 infection in vitro to levels comparable to those of antiviral agents. Taken together, our study supports further evaluation of apabetalone to treat COVID-19, either alone or in combination with emerging therapeutics. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the novel coronavirus disease of 2019 (COVID-19) . UnfortuSARS-CoV-2 is typically transmitted through the inhalation of aerosols and respiratory droplets . InfectiACE2 gene expression, protein levels, and cellular binding of the SARS-CoV-2 spike protein in cell culture models and strikingly reduces infection with live SARS-CoV-2. DPP4/CD26, a potential cofactor for SARS-CoV-2 infection  supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (P/S). HepG2 (ATCC) and Huh-7  were cultured in medium recommended by the suppliers. Human Karpas-299 T cells  were cultured in RPMI-1640 supplemented with 10% FBS and P/S. Cryopreserved primary human hepatocytes (PHH) were plated as directed  and then treated with compounds in media containing 10% FBS (v/v) but without the dexamethasone recommended by Thermo Fisher. All cell lines were incubated at 37 °C in a humidified atmosphere enriched with 5% CO(CT cyclophilin−CT transcript of interest), and results were normalized to DMSO-treated samples.Cells were treated with BETi compounds or vehicle (DMSO) in complete medium for up to 96 hours. Following treatment, cells were harvested, and transcripts quantified via TaqMan real-time PCR as previously described ,31. BrieCalu-3 or Vero E6 cells treated with BETi for 48 h were lysed and sonicated as previously described . Cell ly® 647-conjugated human ACE2 antibody (R&D Systems #FAB933R). Live/dead  or zombie  viability dyes were included to gate viable cells. Cell surface ACE2 protein levels were measured on a BD FACSCelesta  and analyzed with FlowJo version 10 software (BD Biosciences). Cell surface ACE2 protein was stained with Alex FlourPost-BETi treatment, cells were detached with Accutase (Thermo Fisher), washed, and incubated with a recombinant SARS-CoV-2 spike protein receptor binding domain (RBD) fused to the human IgG1 Fc domain (R&D Systems #10499-CV-100) or control recombinant human IgG1 Fc protein (R&D Systems #10499-CV-100) for 30 min at room temperature. The cells were then washed and incubated with PE-conjugated goat anti-human Fc antibodies  for 30 min at 4°C. Spike RBD binding was measured via flow cytometry on a BD FACSCelesta (BD Biosciences) and analyzed with FlowJo software.3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays were used to determine BETi-induced cytotoxicity. Briefly, Calu-3 , Vero E6 , or Karpas-299  were treated with vehicle (DMSO) or increasing amounts of BETi for 48 h in 96-well plates, and then, the CellTiter 96® AQueous assay  was preformed according to the manufacturer’s instructions to determine cell proliferation. Absorbance signals from each well were acquired at 490 nm on a Tecan Infinite® M1000 Pro microplate reader . SARS-CoV-2 (strain BEI_USA-WA1/2020) was obtained from the BEI and propagated in Vero E6 cells. All live virus experiments were performed in the BSL3 laboratory at the University of Nebraska Medical Center . Briefly, Calu-3  were seeded in 96-well plates and cultured in complete medium overnight. Cells were pre-treated with BETi, camostat mesylate, or remdesivir for up to 48 h at 37°C. Treatments were washed off and cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 in complete media. Cells were fixed 48 h post-infection with 4% buffered paraformaldehyde  for 15 min at room temperature. The fixed cells were washed with phosphate buffered saline (PBS), permeabilized in 0.1% Triton X solution for 15 min, then blocked in 3% bovine serum albumin–PBS solution. The cells were incubated with anti-spike protein Rab  at 1:1000 in blocking solution overnight at 4°C, followed by incubation with 1:2000 diluted Alexa Fluor 488 conjugated secondary antibody (Invitrogen) for 1 h at room temperature. Cell nuclei were counterstained using Hoechst 33342 , and cytoplasmic membranes were stained with CellMask . Internal control conditions were included in each plate . Cells were imaged using a high content analysis system, Operetta CLS . Percentage inhibition of viral infection and cell viability  were calculated using Harmony 4.9 software (PerkinElmer Inc.). p < 0.05 was considered statistically significant.Statistical significance was determined through one-way ANOVA followed by Dunnett’s multiple comparison test. Comparisons were done vs. vehicle control, and ACE2 gene expression may be driven by BET proteins [ACE2 gene expression in SARS-CoV-2 permissible cell lines, including human lung epithelial cells; Calu-3, and monkey kidney epithelial cells; and Vero E6. In Calu-3 cells, 48 h of apabetalone treatment resulted in dose-dependent reduction in hACE2 transcripts by up to 90%, p < 0.001 (RhACE2 transcripts by ~40% (p < 0.001) and 80% (p < 0.001), respectively  reduced cell surface expression of ACE2 by ~40%,  = 0.047 E. Thus, DPP4) is a potential cofactor for SARS-CoV-2 entry into host cells, as its presence on the cell surface facilitates viral attachment [DPP4 mRNA . The comparator BETi, MZ1 and JQ1, were also efficacious. To model viral association with host cells, we measured the binding of recombinant SARS-CoV-2 spike protein receptor-binding domain fused with the human IgG1 Fc-epitope (spike RBD) to Calu-3 or Vero E6 cells. Apabetalone pre-treatment for 48 h dose-dependently attenuated the amount of spike RBD protein bound to Calu-3 cells by more than 80%  is a membrane-anchored protease linked to diabetes and implicated as a cofactor in SARS-CoV-2 uptake . DPP4 in T cells , in agrepression . The datIn patients with SARS-CoV-2, circulating monocytes and infiltrating macrophages increase in number, which may explain elevated levels of pro-inflammatory cytokines such as interleukin (IL)-6, IL-1, tumor necrosis factor-alpha (TNF-α), and IL-8 ,41,43, mApabetalone has undergone extensive clinical evaluation. To date, the drug has been administered to 1934 subjects in completed phase 1, 2, and 3 clinical studies, including 138 healthy volunteers, 576 patients with stable coronary artery disease, dyslipidemia, and/or pre-diabetes, on standard of care background therapy, 1212 patients with diabetes and acute coronary syndrome (ACS), and 8 patients with stage 4–5 chronic kidney disease . Overall, apabetalone is well tolerated by patients; adverse events were generally mild with little discernible difference between placebo- and active-treated subjects. The dose-limiting adverse event for apabetalone is a non-symptomatic elevation in serum transaminases  without adversely affecting serum bilirubin in any subjects. With a well-established safety profile, apabetalone is a novel, clinical trial ready candidate for the treatment of COVID-19 based on a dual mechanism of action that simultaneously lowers ACE2-mediated SARS-CoV-2 infection and combats hyper-inflammatory responses that underlie severe disease. Future clinical trials will evaluate apabetalone on top of standard of care to prevent advance of COVID-19 severity and improve outcomes.