SARS-CoV-2 infections are frequently milder in children than adults, suggesting that immune responses may vary with age. However, information is limited regarding SARS-CoV-2 immune responses in young children.We compared receptor binding domain–binding antibody (RBDAb) titers and SARS-CoV-2–neutralizing antibody titers, measured by pseudovirus-neutralizing antibody assay in serum specimens obtained from children aged 0–4 years and 5–17 years and in adults aged 18–62 years at the time of enrollment in a prospective longitudinal household study of SARS-CoV-2 infection.P < 0.0001) and the highest RBDAb titers in 11 of 12 households with seropositive children and adults. Children aged 0–4 years had only 2-fold higher neutralizing antibody than adults, resulting in higher binding-to-neutralizing antibody ratios compared with adults .Among 56 seropositive participants at enrollment, children aged 0–4 years had more than 10-fold higher RBDAb titers than adults . Our understanding of the epidemiology of SARS CoV-2 infection in children has evolved since late 2019. Early in the pandemic, SARS-CoV-2 infections were diagnosed less frequently in children than in adults; this raised questions about whether children were less susceptible to infection. Studies have since documented that children can be infected at similar rates as adults . AlthougThe SARS-CoV-2 Epidemiology And Response in Children (SEARCh) study is a prospective household cohort study designed to address SARS-CoV-2 susceptibility, illness, transmission, and immunologic responses in children aged 0–4 years and their household members (see Methods). In this cross-sectional analysis of enrollment sera, we compare titers of SARS-CoV-2 binding and neutralizing antibody to WT SARS-CoV-2 and the Delta variant in adults, children aged 5–17 years, and children aged 0–4 years.P = 0.2006 for adults vs. all children). In 8 households with more than 1 member with prior suspected SARS-CoV-2 infection, all suspected infections were reported to have occurred within 1 calendar week of each other. None of the participants was hospitalized with COVID-19 disease before enrollment.Sera were collected from 682 SEARCh study participants in 175 households, including 332 (49%) adults aged 18–62 years, 96 (14%) children aged 5–17 years, and 254 (37%) children aged 0–4 years . We deteP < 0.0001) .Thirteen of 22 (59.1%) households had more than 1 member who was seropositive for RBDAb; all enrolled household members were seropositive in 5 of 22 households (22.7%)  was more than 10-fold higher in children aged 0–4 years than in adults (416 [IQR = 228–683] vs. 31 [IQR = 21–112]),  0.0001) ; childre (22.7%) . Childre (22.7%) .50) titers against WT SARS-CoV-2  . PsVNA ID50 neutralizing antibody titers against the Delta variant of SARS-CoV-2 were comparatively modest in all age groups  50% inhibitory dilution  . Childreuseholds . PsVNA I50 was highest in children aged 0–4 years (2.36 [IQR = 1.33–3.99]), followed by children aged 5–17 years (0.9 [IQR = 0.56–3.19]), and lowest in adults (0.35 [IQR = 0.15–0.90])  (80 (The median (IQR) B/N ratio using PsVNA ID adults) . Trends lts) .r2), and the conversion factor between this assay and the WHO standard is 1.0288. RBD antibody titers are expressed as BAU/mL. Sera that were RBD antibody positive were tested for neutralizing antibody using a pseudotyped reporter neutralization assay (PsVNA). Neutralization of WT (WA1.D614G) SARS-CoV-2 and of the Delta variant (B.1.617.2) of SARS-CoV-2 were measured at the Vaccine Research Center of the US NIH in a single-round-of-infection assay with lentivirus-based pseudotyped virus particles (pseudoviruses) as previously described  strain of SARS-CoV-2 using the Roche Elecsys Anti-SARS-CoV-2 S electrochemiluminescence immunoassay (Elecsys Anti-SARS-CoV-2) in the Department of Pathology, Johns Hopkins School of Medicine. The correlation between the WHO standard and the Roche Elecsys Anti-SARS-CoV-2 is 0.9996  in RStudio version 1.4.1717. A Prior to study participation, all participants or their parents/guardians provided informed consent, and children aged 7 years and older also provided assent. The study protocol was reviewed and approved by the Johns Hopkins Bloomberg School of Public Health Institutional Review Board (IRB00014200).RAK, VV, and FSD conceived and designed the study. EAS managed the study and acquired data. SDS and NDR performed and analyzed neutralizing antibody assays. MGQ, MKH, and MDK performed data analysis. All authors contributed intellectually and read, edited, and approved the final manuscript.


Acute myeloid leukemia (AML) is a malignant disorder derived from neoplastic myeloid progenitor cells characterized by abnormal proliferation and differentiation. Although novel therapeutics have recently been introduced, AML remains a therapeutic challenge with insufficient cure rates. In the last years, immune-directed therapies such as chimeric antigen receptor (CAR)-T cells were introduced, which showed outstanding clinical activity against B-cell malignancies including acute lymphoblastic leukemia (ALL). However, the application of CAR-T cells appears to be challenging due to the enormous molecular heterogeneity of the disease and potential long-term suppression of hematopoiesis. Here we report on the generation of CD33-targeted CAR-modified natural killer (NK) cells by transduction of blood-derived primary NK cells using baboon envelope pseudotyped lentiviral vectors (BaEV-LVs). Transduced cells displayed stable CAR-expression, unimpeded proliferation, and increased cytotoxic activity against CD33-positive OCI-AML2 and primary AML cells in vitro. Furthermore, CD33-CAR-NK cells strongly reduced leukemic burden and prevented bone marrow engraftment of leukemic cells in OCI-AML2 xenograft mouse models without observable side effects. Acute myeloid leukemia (AML) represents a devastating disease for which only limited therapeutic progress has been made in the last decades. Besides high-dose chemotherapy, allogeneic bone marrow transplantation currently constitutes the most effective therapy, which is not applicable for all patients, often not fully eliminates tumor cells, and harbors the significant risk of inducing a graft-versus-host disease. Overall, this results in poor prognosis especially for elderly patients –4. In otHere, we report on the generation of CD33-specific CAR-NK cells from peripheral blood using baboon envelope pseudotyped lentiviral vectors (BaEV-LV). CD33-CAR-NK cells showed stable CAR-expression, functional proliferation, and significantly increased killing capacity against CD33-positive AML cell lines as well as primary AML cells in vitro. Furthermore, CD33-CAR-NK cells effectively eliminated all bone marrow- and spleen-engrafted, as well as the majority of peripheral AML cells in an OCI-AML2 xenograft mouse model without detectable side effects.Frozen primary AML cells were thawed and cultivated in DMEM medium supplemented with 15% FBS, 50 µM ß-Mercaptoethanol, 1% Pen/Strep, 100 ng/mL stem cell factor (SCF), 10 ng/mL IL-3, 20 ng/mL IL-6, 10 ng/ml thrombopoietin (TPO) and 10 ng/ml FMS-like tyrosine kinase 3 ligand (FLT3L)). Cells were seeded with 700,000 cells in 3 ml per well of a six-well plate and 1 µg/ml f.c. DNAse was added. The usage of primary patient material was approved by the Ethics Commission of the University Hospital Frankfurt, Germany . All participants gave written informed consent in accordance with the Declaration of Helsinki.® Medium (Miltenyi Biotec) supplemented with 5% heat-inactivated (h.i.) human plasma (DRK-Blutspendedienst), 1% NK-MACS® Supplements (Miltenyi Biotec) and 1% Pen/Strep at concentrations of 1 × 106 cells/ml and cells split every 3–4 days. Depending on the experiment additionally, 80 ng/ml IL-1β (Miltenyi Biotec), 500 IU/ml IL-2 , 10 ng/ml IL-15  or 50 ng/ml IL-15  was added . Tr. Tr28]. PrkdcscidIl2rgtm1Wjl Tg1Eav/MloySzJ (NSG-SGM3) mouse model +) intravenously followed by a single administration of 1 × 107 CD33-CAR-NK cells at day 3 post tumor cell injection. Remarkably a nearly complete eradication of leukemic cells by day 21 post injection of AML cells could be observed in the majority of mice (4 out of 5 mice at day 21 (80%)) while only a deceleration of leukemic cell growth could be observed in mice treated with UTD-NK cells   Fig. . This trld) Fig. . BLI anald) Fig. . Additiold) Fig. . Chimerild) Fig. . Auxiliald) Fig. . Histolold) Fig. .Fig. 3ReOf note, following both single and repetitive CD33-CAR-NK cell injections, the animals did not show any clinical signs of cytokine release syndrome (CRS), weight reduction, altered appearance or behavior, or graft-versus-host-disease (GvHD), in line with histological analysis of lung, liver, and colon.In the rapidly moving field of CAR-immune cell therapies, recent studies have shown that primary CAR-NK cells are safe and can be highly effective . With thSupplemental Figures and legends


The discovery of immune checkpoints has been well known to provide novel clues for cancer treatments. Immunotherapy against the programmed cell death protein-1 (PD-1) /programmed death-ligand-1 (PD-L1), one of the most popular auxiliary treatments in recent years, has been applied in various tumor treatments, including non-small cell lung cancer (NSCLC). However, inevitable issues such as side effects and drug resistance emerge following the use of immune checkpoint inhibitors. The PI3K/AKT/mTOR pathway may participate in the regulation of PD-L1 expression. Abnormal PI3K/AKT/mTOR pathway activation results in increased PD-L1 protein translation, whereas PD-L1 overexpression can activate the PI3K/AKT/mTOR pathway inversely. Via downstream proteins, including 4E-BP1, STAT3, NF-κB, c-MYC, and AMPK in aberrant energy status, the PI3K/AKT/mTOR pathway can regulate PD-L1 post-transcription and translation. Besides, the regulation of the PI3K pathway by the PD-1/PD-L1 axis involves both tumor cells and the tumor immune microenvironment. Inhibitors targeting the PD-1/PD-L1 have been successfully applied in the treatment of gastrointestinal cancer and breast cancer. Meanwhile, drug resistance from alternative pathway activation also evidently affects clinical progress. To achieve a better therapeutic effect and quality of survival, the combination of multiple treatment modalities presents great research value. Here we reviewed the interaction between PD-1/PD-L1 and PI3K/AKT/mTOR pathway in the progression and treatment of NSCLC and summarized its clinical implications. The intracellular interactions between PD-1/PD-L1 and the PI3K/AKT/mTOR pathway indicate that PD-1/PD-L1 inhibitors have a wide range of potential applications. And we presented the mechanism for combining therapy with monoclonal antibody PD-1/PD-L1 and PI3K/AKT/mTOR inhibitors in this review, to broaden the therapies for NSCLC. And the high expression of Foxp3 mainly through inhibition of mTOR  increases the immunosuppressive effect as well NSCLC tumor cells usually express PD-L1 on the membrane and its receptor PD-1 is expressed on the membrane of CD8The anti-tumor immune function of T cells can be regained via anti-PD-1/PD-L1 drugs  has been reported to be involved in regulating the interaction between PI3K/AKT/mTOR pathway PD-1/PD-L1 activates the PI3K/AKT pathway not just in tumor cells but also in the immune microenvironment. It has been found in breast cancer that PD-1 on an element termed myeloid-driven suppressor cells (MDSC) immune microenvironment bound to PD-L1 on B cell can activate PI3K/AKT/NF-κB signaling pathway in B cell Several PI3K/AKT/mTOR pathway targeted therapeutic drugs mainly target related genes such as PIK3CA, AKT, TSC1/2, mTOR, PTEN, and so on AKT inhibitors can be divided into two types: AKT competitive inhibitors and AKT aliasing inhibitors. The former competitively inhibits the ATP binding site on AKT and prevents AKT activation. While the latter inhibits AKT activation and phosphorylation by altering the chemical structure of the AKT PH structural domain, thereby preventing AKT localization on the cell membrane. AKT competitive inhibitors are majorly listed below: Capivasertib (AZD5363) There are three generations of mTOR inhibitors Similar to other target agents, resistance can also happen when using PI3K/AKT/mTOR pathway targeted drugs + T cells. However, IFN-γ also activates AKT/mTOR pathway in cancer diseases, and induces PD-L1 expression, antagonizing its antitumor effect The effect of the PI3K/AKT/mTOR pathway on immune cells and the immune microenvironment is complicated and combined with multiple pathways. PI3K/AKT/mTOR inhibitors affect the PD-L1 expression in cancer cells + T cell activation and enhances cytotoxic effects. Activating PI3K-mTOR signaling in T cells in the tumor environment suppresses autoimmunity by inhibiting activation and differentiation of common T cells and specializing in CD4+Foxp3+ regulatory T cells (Tregs) PI3K/AKT/mTOR inhibitors also influence the antitumor effects of tumor immune cells infiltrating in cancers . The application of PD-L1 monoclonal antibodies enhances the antitumor immune effects of macrophages by inhibiting the AKT-mTOR pathway + T cell depletion by regulating the PI3K/AKT/mTOR pathway to play an antitumor immune role 99. At the same time, both PD-1/PD-L1 monoclonal antibodies and PI3K/AKT/mTOR pathway inhibitors may develop resistance through activation of the bypass pathway, and have drug toxicity and side effects when achieving significant cancer suppression + T cells As mentioned above, PI3K/AKT/mTOR pathway activation is closely related to PD-L1 expression and impacts the tumor immune microenvironment 95This review discussed the tumor immunosuppressive effect of PD-1/PD-L1 inhibitors and the fundamental scenario of immune checkpoint inhibition therapy with the application of monoclonal antibodies. Taking NSCLC as an example, the review explained the components of the PI3K/AKT/mTOR signaling pathway and described their functions in driving carcinogenesis and suppressing antitumor immunity respectively. We also introduced the relevant immunosuppressive agents including the role and situation of single agent use in anti-tumor represented by everolimus, as well as the feasibility of combining multiple targeted agents and multiple adverse medication effects. The viability of a therapeutic strategy combining PI3K/AKT/mTOR pathway inhibitors with PD-1/PD-L1 inhibition will be considered. To date, research into PI3K/AKT/mTOR signaling pathway inhibitors is still currently in progress, and it exhibits great positive significance to investigate the interaction between PD-L1 expression and PI3K/AKT/mTOR signaling pathway activation for addressing anticancer drug resistance, prolonging tumor patient survival, and improving patient prognosis.


Caenorhabditis elegans. In humans, the tumour suppressor protein Phosphatase and Tensin Homolog (PTEN) inhibits IIS, preventing excessive growth. PTEN variants are associated with disease, but how they affect PTEN function is not well understood. Here, we characterized variants of unknown significance (VUSs) implicated in autism spectrum disorder by studying homologous mutations in theC. elegans protein DAF-18 to infer how they play a role in human disease.We found that variants D66E and L115V are likely benign, H168Q is intermediate while variants H138R and T176I are likely pathogenic.Insulin and insulin-like growth factor signaling (IIS) is an anabolic pathway conserved among humans and Caenorhabditis elegans (C. elegans) . The insulin pathway is well conserved across metazoans, and thus using this model organismis advantageous as this allows us to characterize PTEN variants by studying itsC. elegans homolog DAF-18 . In 2019, Wong et al. characterized DAF-18 variants D66E, L115V, H138R, H168Q, and T176I, which correspond to PTEN variants D22E, L70V, H93R, H123Q, and T131I implicated in ASD . Their analysis focused on morphology and locomotion defects, while González-Cavazos et al. in 2019 studied the effects of these variants on dauer entry. Our work aimed not only to replicate the results found by González-Cavazos et al., 2019 but also to characterize the DAF-18 variants with three new assays: L1 arrest survival, Q-cell division in L1 arrest, and rate of pharyngeal pumping off-food.Phosphatase and Tensin Homolog (PTEN) acts as a key suppressor of the insulin signaling pathway and thus regulates cell growth and development . In humans, this protein still has numerous clinically important variants of unknown significance (VUS) which are implicated in disorders such as autism spectrum disorder (ASD) and are classified as neither pathogenic nor benign . Our research aims to classify five of these variants previously implicated in ASD using the model organismC. elegans enter dauer diapause under stressful conditions , which is typically accompanied by decreased insulin signaling . Measuring dauer entry is an effective way to indirectly assess the extent of activation of the insulin signaling pathway. In our work, we exposedC. elegansto high temperature (27 °C) for 48 hours, and dauer entry of wild type,daf-18 knockout (qu52), daf-18(sy879)[D66E], daf-18(sy887)[L115V], daf-18(sy881)[H138R],daf-18(sy885)[H168Q], anddaf-18(sy882)[T176I]strain were observed. Results published by González-Cavazos et al., 2019 using dauer pheromone to induce dauer showed that variantsdaf-18(sy879)[D66E],daf-18(sy887)[L115V] anddaf-18(sy885)[H168Q]were similar to wild type worms, whereasdaf-18(sy881)[H138R] anddaf-18(sy882)[T176I]variants were more dauer defective compared to wild type, withdaf-18(sy882)[T176I]showing a stronger phenotype thandaf-18(sy881)[H138R]. In our studies, we found that thedaf-18(sy879)[D66E] anddaf-18(sy887)[L115V] strains resembled wild type in terms of dauer entry, whereasdaf-18(sy885)[H168Q], anddaf-18(sy882)[T176I] were more dauer defective . Overall, our findings mostly agreed with published data, with the exception of thedaf-18(sy885)[H168Q] strain, though it did have more dauer entry than the dauer defectivedaf-18(qu52) control .daf-18(qu52) controls. Figure 1D shows thedaf-18(sy879)[D66E] is not significantly different from wild type which further classifies this variant as most benign. Thedaf-18(sy887)[L115V],anddaf-18(sy885)[H168Q] live longer in L1 arrest than thedaf-18(qu52)knockout and therefore are intermediate and not likely null alleles. Thedaf-18(sy881)[H138R] and daf-18(sy882)[T176I]worms have similar L1 arrest survival to that of thedaf-18(qu52) knockout strain, withdaf-18(sy881)[H138R] not being significantly different from thedaf-18(qu52) knockout . This fits in with the published data and further characterizes D66E, L115V, and H168Q variants as benign and H138R and T176I as pathogenic . This assay reveals potential phenotype uncoupling with thedaf-18(T176I) being more dauer defective than thedaf-18(sy881)[H138R] strain, while having a higher survival in L1 arrest (One-way ANOVA p=0.0283).Measuring L1 arrest survival is another way to assess activation of the insulin signaling pathway, with increased insulin signaling leading to shortened survival . We thus assessed the L1 arrest survival ability of the VUS strains compared to wild type anddaf-18(qu52) controls. Our experiments once again supporteddaf-18(sy879)[D66E], daf-18(sy887)[L115V],anddaf-18(sy885)[H168Q] variants displaying a more benign phenotype whiledaf-18(sy881)[H138R] anddaf-18(sy882)[T176I]variants were more pathogenic. Figure 1E shows that thedaf-18(sy881)[H138R] anddaf-18(sy882)[T176I]variants had some Q-cell division, which is indicative of an overactive IIS in L1 arrest and supports the deleterious effect of these variants. Notably, the phenotypes ofdaf-18(sy881)[H138R] anddaf-18(sy882)[T176I] variants were not as strong as thedaf-18(qu52) knockout control, which shows up to 100% Q-cell division in L1 arrest, suggesting that these variants are not completely null fordaf-18function.Another readout of inappropriate activation of IIS in L1 arrest is the presence of divided, fluorescently tagged neuroblast precursor “Q-cells” . We thus quantified Q cell division during L1 arrest of the VUS strains compared to wild type andC. elegans. Previous research shows that worms with an impaired IIS are unable to cease pumping while off food compared to wild type worms . We thus quantified the pharyngeal pumping rate of the five DAF-18 VUS strains when taken off the food. We found that thedaf-18 knockoutC. elegans had a higher pharyngeal pumping rate when taken off food , as published. Of the VUS strains,daf-18(sy879)[D66E], daf-18(sy887)[L115V], daf-18(sy885)[H168Q], anddaf-18(sy882)[T176I]were similar to wild type, whereasdaf-18(sy881)[H138R] variant resembled thedaf-18 knockout . This mostly supports our classification ofdaf-18(sy879)[D66E], daf-18(sy887)[L115V],anddaf-18(sy885)[H168Q] as benign anddaf-18(sy881)[H138R] anddaf-18(sy882)[T176I] as potentially pathogenic, except fordaf-18(sy882)[T176I], which could be due to phenotype uncoupling.Pharyngeal pumping can be used to measure food intake, which is increased during IIS activation indaf-18(sy882)[T176I] allele conferring defects in all but the pharyngeal pumping assay. Meanwhile, the strongest allele appeared to bedaf-18(sy881)[H138R], being significantly more deficient than wild type in all assays, and even being similar to thedaf-18(qu52) knockout when it came to L1 arrest survival and pharyngeal pumping. Future work involving structural analysis of these variants could complement our findings. Furthermore, assessing VUSs of PTEN could be done without first needing to find and create homologous DAF-18 variants through replacement ofdaf-18 with different variants of humanPTEN via CRISPR. Our research thus helped characterize PTEN variants and has laid the groundwork for analyzing future clinically significant variants.Together, the dauer entry, L1 arrest survival, Q-cell division in L1 arrest, and off-food pharyngeal pumping assays show that the D66E variant is most likely benign, being similar to wild type in all experiments. The L115V is also likely benign, being like wild type in 3 of the 4 assays. Meanwhile the H168Q variant appears to be intermediate in terms of its pathogenicity, showing phenotypic defects in half of the experiments. T176I is a stronger variant, with theC. elegansgrowth conditions:C. elegans strains were grown on standard nematode growth medium agar plates (NGM) seeded with 100ml saturated cultureEscherichia coli (E. coli) OP50 bacteria. Worms were kept in 20 °C at all times except during counting or unless stated otherwise.Dauer assay:100-150 synchronized L1 larvaewere plated on seeded NGM plates and kept at 27 °C for 48 hours to induce dauer . SDS selection (1% SDS for 30 minutes) was then used to identify dauer and non-dauer worms, as non-dauer worms are killed by the SDS solution. The dauervs.non-dauer (dead) animalswere then manually counted. Three repeats of dauervs.non-dauer count were done for each strain.L1 arrest survival assay: C. elegans were placed in L1 arrest though sodium hypochlorite treatment and kept in 1.5ml tubes in M9 buffer on a rotator at 20 °C. M9 concentration was adjusted on day 1 of L1 arrest to obtain 10-15 worms per 1ml. Aliquots of 10ml were taken daily to count alive versus deadC. elegans (n=100-150 on each day). Three repeats were done for each strain. Statistics were carried out using GraphPad Prism software version 9.Q-cell division assay: C. elegans were placed in L1 arrest using the methods described above. Q-cell division was scored manually throughout days 5-8 of L1 arrest by taking 10ml daily and counting the proportion ofC. elegans with divided Q-cells (n=100-150 on each day). Division was visualized though a tagging the promoter of a protein expressed in the touch neurons (Pmec-4::GFP), and recording presence of the Q-cell descendant neuron, AVM . Three repeats were done for each strain.Off-food pharyngeal pumping assay: 100-150 synchronized day 1 adultC. eleganswere washed three times with M9 buffer to remove bacteria. They were then plated onto NGM plates with no bacteria and pharyngeal pumping was manually measured (number of pumps per 10 seconds) after a 1-hour adjustment period.Strains:


Histone deacetylases (HDACs) regulate gene expression through the epigenetic modification of chromatin structure. HDAC6, unlike many other HDACs, is present in the cytoplasm. Its deacetylates non-histone proteins and plays diverse roles in cancer cell initiation, proliferation, autophagy, and anti-cancer drug resistance. The development of HDAC6-specific inhibitors has been relatively successful. Mechanisms of HDAC6-promoted anti-cancer drug resistance, cancer cell proliferation, and autophagy are discussed. The relationship between autophagy and anti-cancer drug resistance is discussed. The effects of combination therapy, which includes HDAC6 inhibitors, on the sensitivity of cancer cells to chemotherapeutics and immune checkpoint blockade are presented. A summary of clinical trials involving HDAC6-specific inhibitors is also presented. This review presents HDAC6 as a valuable target for developing anti-cancer drugs. Histone deacetylase 6 (HDAC6) deacetylates non-histone proteins, including cortactin, peroxiredoxin, and α-tubulin ,2,3. ForGlioblastoma is associated with high levels of the mesenchymal markers Snail, Slug, and N-cadherin. Glioblastoma cell lines show high levels of autophagic flux and activation of the sonic hedgehog (Shh) pathway . HDAC6 iHDAC6 promotes the growth of GBM cells by inhibiting the suppressor of mothers against decapentaplegic 2 (SMAD2) phosphorylation to downregulate  p21.The HDAC6 was reported to increase the level of the Ewing sarcoma breakpoint region 1 fusion gene (EWSR1-FLI1) . A high HDAC6 was shown to decrease the expression of tumor suppressor mammalian STE20-like kinase 1 (MST1) by deacetylating MST1 in breast cancer cells . HDAC6 iUpon epidermal growth factor (EGF) stimulation, HDAC6 was shown to interact with β-catenin at the caveolae membrane to deacetylate β-catenin (lysine residue 49) and inhibit the phosphorylation of β-catenin (Serine 45) . HDAC6 i+ EVs from human FEMX-I metastatic melanoma and Caco-2 colorectal carcinoma cells, leading to the downregulation of intracellular CD133 [Pentaspan membrane glycoprotein CD133 (Prominin-1), a marker of cancer stemness, can be used to predict the poor prognosis of patients with many different types of tumors. HDAC6 interacts with CD133 to inhibit CD133 degradation . CD133, ar CD133 . Tubacinar CD133 .A high level of transmembrane serine protease 4 (TMPRSS4) is associated with the poor prognosis of patients with non-small cell lung cancer (NSCLC), gastric cancer, colorectal cancer, prostate cancer, and other cancers. TMPRSS4 promoted tumor cell proliferation and metastasis by inducing specificity protein 1/3 (Sp1/3), activator protein-1 (AP-1), and NF-κB transcription factors . Sp1 is A high level of ubiquitin specific protease 10 (USP10) is associated with the poor overall survival of patients NSCLC with p53 mutations . USP10 wIn patients with colorectal cancer (CRC), expression levels of SET domain containing 7 (SET7), a histone lysine methyl transferase, in cancer tissues are lower than those in adjacent tissue. Decreases in the expression of SET7 can predict poor patient prognosis . SET7 anThe increased expression of HDAC6 was observed in anti-cancer drug-resistant cancer cells ,35. FiguMicroRNAs (miRNAs) are small non-coding RNAs that can downregulate the expression of genes involved in carcinogenesis ,37. The HDAC6 shows aberrant expression in diffuse large B-cell lymphoma (DLBCL). HDAC6 inhibition exerts anti-tumor effects both in vitro and in vivo . The decmiR-206 directly decreased the expression of glycolytic enhancer 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and suppressed the proliferation of ovarian cancer cells . miR-206TargetScan analysis predicted that miR-141 could target HDAC6 to protect against lung injury  Figure B. miR-43Unlike other HDACs, the development of HDAC6-specific inhibitors has been relatively successful ,47. NN-3LSD1 (lysine-specific demethylase 1) and HDAC6 selective dual inhibitors exerted synergistic effects in a xenograft model of multiple myeloma, exhibiting better effects than treatment with the single agent . The comThe overexpression of ATP binding cassette subfamily B member 1 (ABCB1) or ATP binding cassette subfamily G member 2 (ABCG2) decreased the sensitivity of human cancer cells to ACY-241 . Thus, HHDAC6 confers resistance to gefitinib by stabilizing EGFR . CAY1060Pan-HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, and sodium butyrate, increased the PTEN expression by inducing the acetylation of transcription factor p53-related p63 protein (ΔNp63α). The overexpression of ΔNp63α downregulated membrane-bound PTEN but enhanced the nuclear translocation of PTEN, leading to cisplatin resistance in oral cancer cells . Inhibitp = 0.001) and overall survival (p = 0.008) in patients with serous carcinoma [A high level of HDAC6 can predict the poor prognosis of glioblastoma patients ,74. Glioarcinoma . A low larcinoma .Cancer cells can activate immune checkpoint pathways to evade immune surveillance. Immune checkpoint molecules, such as programmed cell death-1 (PD-1) and PD-L1, allow cancer cells to proliferate by inhibiting cytolytic T cell (CTL) and natural killer (NK) cell activity ,78. EGFRHigh PD-L1 expression can predict the poor prognosis of patients with colorectal cancers . A high The elevated expression of PD-L1 conferred cisplatin resistance to NSCLCs . PD-L1 pThe PD-L1 level showed a positive correlation with the HDAC6 level in serous carcinoma . The sel+ T cells [+ T-cell phenotype in chronic lymphocytic leukemia [Tumor immunotherapy can exert anti-tumor effects by blocking the aggresome and proteasome pathways and reducing the number of M2 macrophages while increasing sensitivity to PD-L1 blockade . Since H T cells . Combinaleukemia . The comleukemia . Combinileukemia . Figure Autophagy either promotes or blocks tumorigenesis. Epigenetic regulation is involved in autophagy ,100,101.Autophagy promotes the degradation and recycling of damaged organelles regulating cellular homeostasis and energy metabolism ,104. HDAACY-1215 inhibited aggresome formation and autophagy, resulting in apoptotic cell death  Figure . The inhNutrient deprivation increased the expression of transactive response DNA binding protein-43 (TDP-43) in glioblastoma cell lines . TDP-43 TGF-β was shown to induce autophagy in cancer-associated fibroblasts and promote EMT and metastasis . Both thStem cell-like breast cancer cells are known to acquire resistance to metformin by increasing glycolysis . The antAutophagy is considered a cellular adaptive response against hypoxia, nutrient deprivation, and energy deprivation, indicating its cytoprotective role . ProtectR) displayed an increased expression level of CAGE (a cancer-associated gene) and autophagic flux compared to anti-cancer drug-sensitive parental gastric cancer cells (AGS cells) [A high level of ATG9A or ATG16L1 was reported to predict the poor overall survival and earlier relapse of patients with oral squamous cell carcinoma . A high S cells) . CAGE waS cells) ,147. TheProtective autophagy was shown to be responsible for resistance to AG1478, an inhibitor of EGFR tyrosine kinase, in ovarian cancer cells . ProtectTreatment with low-intensity focused ultrasound and microbubble (LIFU+MB) combined with paclitaxel (PTX) increased the apoptosis of paclitaxel-resistant ovarian cells by decreasing autophagy . Thus, tUnlike other HDACs, HDAC6 knockout did not induce a toxic effect . Thus, HInvestigations into the mechanism of the regulation of HDAC6 expression are necessary for a better understanding of HDAC6-promoted autophagy and anti-cancer drug resistance. However, extensive efforts have not been made to identify transcription factors that regulate HDAC6 expression. It will also be necessary to identify genes regulated by HDAC6. These genes might serve as targets for developing anti-cancer drugs. Many reports showed that miRNAs targeting HDAC6 regulated cancer cell proliferation . TargetSThe identification of novel HDAC6-binding proteins is necessary for developing anti-cancer drugs for cancer patients expressing high level of HDAC6. The identification of the domain of HDAC6 critical for binding to partner of HDAC6 is necessary for designing HDAC6-targeting anti-cancer drugs. Peptides that correspond to the binding domain of HDAC6 might regulate autophagy and overcome resistance to anti-cancer drugs. The tumor microenvironment (TME) consists of cancer cells, endothelial cells, cancer associated fibroblasts, and various innate and adaptive immune cells. These immune cells include B and T cells, dendritic cells, myeloid-derived suppressor cells (MDSCs), and macrophages (M1/M2). The TME is critical for cancer initiation and promotion. Cellular interactions involving cancer cells and immune cells can promote cancer cell progression . Exosoman = 17) [Currently, ACY-1215, ACY-241, KA2507, and JBI-802 are in clinical trials . Clinican = 17) . Among 1n = 17) . At the n = 17) . The oven = 17) . ACY-241n = 17) . Three (n = 17) . A combin = 17) .The screening of small molecules based on a full-length HDAC6 protein structure is needed to develop HDAC6-specific inhibitors. Concerted effects are needed to minimize cytotoxic effects, improve pharmacokinetics and pharmacodynamics, and improve the chemotherapeutic efficacy of HDAC6-specicfic inhibitors.Resistance to HDAC6 inhibitors will eventually develop. Thus, the identification of genes that confer resistance to HDAC6 inhibitors will be necessary. For this, the identification of genes regulated by HDAC6 inhibitors is necessary. Mechanisms of resistance to HDAC6 inhibitors may involve the overexpression of PD-L1 and PD-1, epigenetic regulation (acetylation and methylation), drug efflux, the activation of EGFR/PI3K/AKT signaling, the overexpression of oncogenes, and the downregulation of tumor suppressor genes.


This included FBXO34, CNTN2, and TMCC2 previously linked with COVID-19 severity using association studies. Overall, we report SNPs in 26 known and 12 novel candidate genes with strong molecular evidence implicating them in the pathophysiology of life-threatening COVID-19 and post-recovery sequelae. Of these few notable known genes include, HLA-DQB1, AHSG, ALOX5AP, MUC5AC, SMPD1, SPG7, SPEG,GAS6, and SERPINA12. These results enhance our understanding of the pathomechanisms underlying the COVID-19 clinical spectrum and may be exploited to prioritize biomarkers for predicting disease severity, as well as to improve treatment strategies in individuals of European ancestry.Host genetic variability plays a pivotal role in modulating COVID-19 clinical outcomes. Despite the functional relevance of protein-coding regions, rare variants located here are less likely to completely explain the considerable numbers of acutely affected COVID-19 patients worldwide. Using an exome-wide association approach, with individuals of European descent, we sought to identify common coding variants linked with variation in COVID-19 severity. Herein,  More than two years after its first outbreak, the coronavirus disease COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a prominent healthcare challenge and disruptor of social and economic activities worldwide. It shows a complex array of manifestations ranging from the complete absence of symptoms to severe clinical outcomes, such as acute respiratory distress syndrome (ARDS) 9 caused ,4,5,6,7.Solute Carrier Family 6 Member 20 (SLC6A20), Leucine Zipper Transcription Factor Like 1 (LZTFL1), C-C Motif Chemokine Receptor 9 (CCR9), C-X-C Motif Chemokine Receptor 6 (CXCR6), FYVE and Coiled-coil Domain Autophagy Adaptor 1 (FYCO1), and X-C Motif Chemokine Receptor 1 (XCR1) and the ABO blood group locus at 9q34.2, with respiratory failure in COVID-19 patients from Italy and Spain [2′-5′ Oligoadenylate Synthetase (OAS) cluster and Interferon Alpha and Beta Receptor Subunit 2 (IFNAR2) on chromosomes 12 and 21, respectively, that are involved in the antiviral type 1 interferon (IFN) response [Tyrosine Kinase 2 (TYK2), required for IFN, interleukin 12 (IL-12), IL-23 and T-helper 1/T-helper 17 cell-dependent immunity [Dipeptidyl Peptidase 9 (DPP9), involved in antigen presentation [ABO, SLC6A20, TYK2, DPP9, IFNAR2, and additionally reported a variant in Protein Phosphatase 1 Regulatory Subunit 15A (PPP1R15A) in influencing COVID-19 severity [This has been interrogated in a series of genome-wide association studies (GWASs) comparing COVID-19 patients and population controls. Ellinghaus et al., reported a significant association of variants in six candidate genes at chromosome 3p21.31, including response ,23, Tyroimmunity , and Dipentation , on chroentation . The COVseverity .REXO2), C11orf71, Nicotinamide N-Methyltransferase (NNMT1), and Cell Adhesion Molecule 1 Precursor (CADM1) at 11q23.3 and Cathepsin C (CTSC) at 11q14.2 [Von Willebrand Factor A Domain containing 8 , Phosphodiesterase 8B , CTSC (11q14.2), Thrombospondin Type 1 Domain containing 7B , Serine–Threonine kinase 39 , F-box Protein 34 , Ribosomal Protein L6 Pseudogene 27  and Methyltransferase 21C, AARS1 Lysine  that are expressed in the lung and associated with lung surface tension, airway obstruction, emphysema, T-cell mediated inflammation, and inflammatory cytokines [Since uninfected individuals or population controls may develop morbid COVID-19 subsequently upon contracting SARS-CoV-2 infection, we used asymptomatic COVID-19 patients as controls in a GWAS using a COVID-19 dataset generated by AncestryDNA , to unco 11q14.2 . Another 11q14.2 . This reToll Like Receptor 7 (TLR7), Toll Like Receptor Adaptor Molecule 1 (TICAM1), Interferon Regulatory Factor 3 (IRF3), and Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) are reported in severe cases of COVID-19, including in young patients without pre-existing comorbidities [Genetic determinants are known to modify the susceptibility to immune-mediated disorders and severe viral infections, such as from respiratory viruses, eg. Influenza virus A and SARS-CoV-1 ,33,34,35bidities ,40. Nevecohort 1 we compared non-hospitalized patients  to those that were hospitalized  and in cohort 2 we evaluated hospitalized COVID-19 patients on respiratory support  to those not requiring the same .Accordingly, we conducted a case–control based association study to query an exome dataset of 1,432,135 SNPs in 2692 COVID-19 patients of European ancestry from the GEN-COVID consortium, University of Sienna, Italy. We sought to identify common variations in the coding regions of the genome that may be associated with variable prognosis in SARS-CoV-2 infected patients. To this end we employed two strategies, first in A novel whole-exome dataset of COVID-19 patients was obtained from the GEN-COVID consortium, University of Siena, Italy (Sienna_COVID). It comprised of the data from 2960 COVID-19 patients and assessed 1,432,135 SNPs. The patients were graded on a scale of 0–5 based on their hospitalization status and the requirement of respiratory support .https://www.internationalgenome.org/data/, accessed on 22 April 2017). The merged dataset (Sienna_1K) comprised of 5464 individuals assessing 229,021 SNPs that were common between the two datasets. VCFtools v0.1.16 [The Sienna_COVID dataset was first merged with the genomic data of individuals from the 1000 Genomes Project  implemented in PLINK v1.9 using—pca command. The PC1 and PC2 are plotted in RStudio v1.4.1717 . To contN = 493), Hospitalized Without Respiratory Support , Hospitalized Requiring Oxygen Supplementation , Hospitalized Intubated , Hospitalized With CPAP-biPAP patients , and Dead .Among the 2692 patients considered, the number of individuals in each of the categories described here  are Non-cohort 1), and hospitalized patients on respiratory support (HWOS + HWCB + HI + D) versus those without it (NH + HWRS) (cohort 2), multiple regression-based case–control analyses were performed on the European_Sienna_COVID dataset in PLINK v1.9. Age, sex, and the first two principal components (PC1 and PC2) were employed as the covariates in the association analysis. To this end, COVID-19 patients were divided into two age groups: ≤50 years and >50 years. All covariates were included in a logistic regression model using --covar flag alongside the --logistic command. SNP-phenotype association was statistically delineated separately for all 1,432,135 SNPs to obtain Odd’s Ratio and respective p-value for each SNP after controlling for the covariates. The p-value < 0.001 was considered statistically significant. The −log10 p-values of all assessed SNPs for both cohort 1 and cohort 2 were plotted as Manhattan plots using ‘qqman’ package in R v3.5.2 [https://www.snp-nexus.org/v4/, accessed on 15 August 2022) web-based server for GRCh38/hg38 [To identify genetic variants with significant frequency variation between hospitalized (HWRS + HWOS + HWCB + HI + D) versus non-hospitalized (NH) patients  against those hospitalized  in cohort 1. And compared hospitalized COVID-19 patients on respiratory support  with those not requiring it  in cohort 2. Out of 1,432,135 SNPs employed in exome-wide genetic analysis, 229 (cohort 1) and 111 (cohort 2) SNPs showed significant association with the severity of COVID-19  after controlling for the covariates .The top 20 highly significant SNPs for p-value < 0.001) in cohort 1 were found to be associated with pathways , such as infectious diseases, NOTCH signaling, immunoregulatory interactions between lymphoid and non-lymphoid cells, and extracellular matrix (ECM) organization  in cohort 2, were linked with pathways involved in regulation of major histocompatibility complex (MHC) class II signaling, adaptive immune system modulation, carbohydrate metabolism, and RUNX transcription factor mediated regulation , and Transmembrane And Coiled-Coil Domain Family 2 (TMCC2) which have been previously linked with COVID-19 severity [FBXO34 showed significant association with the acuteness of SARS-CoV-2 infection primarily in the European ancestry [FBXO34 has also been linked with plasma protein levels in cardiovascular disease risk among Europeans, as well as with blood cell count [CNTN2 and TMCC2 occur in the same genomic region that is significantly associated with risk of poor COVID-19 outcomes in individuals with high European ancestry in Brazil [Our results included SNPs in the genes severity ,50. FBXOancestry . While iancestry . Moreovell count ,53,54,55cohort 1 we identified SNPs in the following genes, Killer Cell Immunoglobulin like Receptor, Two Ig Domains and Long Cytoplasmic Tail 1 (KIR2DL1) that is an inhibitory receptor upregulated in COVID-19 patients with acute respiratory distress reflecting reduced antiviral activity of natural killer cells in them [MBL Associated Serine Protease 2 (MASP2) that promotes complement cascade activation; variants in MASP2 resulting in its reduced expression are noted in asymptomatic elderly COVID-19 patients [Alpha-2 HS glycoprotein (AHSG) that modulates inflammation via attenuating macrophage activation and neutrophil degranulation and is significantly downregulated in severe COVID-19 [ATPase family AAA domain containing 3A (ATAD3A) that was upregulated in lymph nodes from COVID-19 autopsy cases [ATAD3A have also been linked to type I interferonopathy [-1,3-N-acetyl-glucosaminyltransferase 8 (B3GNT8) that encodes for a glycosyltransferase responsible for anchor point creation in poly-N-acetyl-lactosamine, glycan extensions that could modulate SARS-CoV-2 cellular invasion. Rare variants in B3GNT8 are linked with milder COVID-19 presentations [Arachidonate 5-Lipoxygenase Activating protein (ALOX5AP) encodes for 5-LOX activating protein (FLAP), an activating cofactor for the lipid mediator, Arachidonate 5-lipoxygenase (ALOX5) that generates leukotriene B4 (LTB4) associated with poor respiratory pathologies such as pneumonia, ARDS, and severe lung injury [ALOX5 activity and ALOX5AP expression are observed in bronchoalveolar lavage (BAL) neutrophils in critical COVID-19 patients [ALOX5AP, ALOX5, and plasma LTB4 are also noted in diabetic COVID-19 cases requiring intensive care [G Protein-Coupled Receptor Class C Group 5 Member C (GPRC5C) whose structural variants have been associated with poor prognosis in COVID-19 [Mitogen-activated Protein Kinase 7 (MAP2K7) encodes for an augmenter of the c-Jun kinase pathway during T-cell activation and is elevated in severe COVID-19 [Mucin 5 AC (MUC5AC) that encodes for a gel forming secreted glycoprotein, which is upregulated in the airway mucus of seriously ill SARS-CoV-2 infected patients [Receptor-interacting kinase 3 (RIPK3), a serine–threonine kinase implicated in non-caspase dependent apoptosis termed as necroptosis that leads to ARDS after trauma and sepsis [Ring Finger Protein 213 (RNF213), an E3 ubiquitin ligase and component of the proteasomal degradation machinery that is suppressed in monocytes from severe COVID-19 patients [Sphingomyelin phosphodiesterase 1 (SMPD1) that encodes for a sphinogomyelinase, which catalyzes the synthesis of ceramide, a bioactive lipid mediator involved in response to cellular damage. Upregulation of SMPD1 activity and ceramide have been noted in COVID-19 patients requiring intensive care [Spastic Paraplegia 7 (SPG7) shows increased expression upon SARS-CoV-2 infection [Specifically, in  in them . MBL Asspatients . Alpha-2COVID-19 . The mitsy cases ; pathogeonopathy . β-1,3-Nntations . Arachidg injury ,63. Congpatients . Increasive care . G ProteCOVID-19 . MitogenCOVID-19 ,68,69. Mpatients . Receptod sepsis ; increasd sepsis . Ring Fipatients . Sphingoive care . Spasticnfection . It enconfection . Furthernfection .cohort 2 our results included SNPs in the following genes, Kruppel Like Factor 6 (KLF6) that encodes for an inflammation regulator with high expression in the macrophages in BAL from grievously ill COVID-19 patients [Ubiquilin like (UBQLNL) that encodes for a regulator of proteostasis and showed enhanced expression in seriously ill COVID-19 patients [Fucosyltransferase 4 (FUT4), a marker for premature/immature neutrophils that showed elevated expression in severe SARS-CoV-2 infection and is likely associated with poor outcomes in sepsis [Growth-Arrest Specific 6 (GAS6) that encodes for a ligand functional in the restorative program initiated to counterbalance pro-inflammatory immune response [GAS6-AS1 encodes for a long non-coding RNA that is downregulated in SARS-CoV-2 infection in vitro and likely interacts with immune modulatory and SARS-CoV-2 interacting proteins such as Adenosine Deaminase RNA Specific (ADAR) and A-Kinase Anchoring Protein 8 Like (AKAP8L) [Serpin Family A Member 12 (SERPINA12) that is involved in the inhibition of kallikrein-dependent inflammation and is downregulated in critical cases of COVID-19 promoting uncontrolled inflammation and worsening of disease outcome [Integrin subunit alpha L (ITGAL), an integrin involved in monocyte migration across endothelium in anti-viral immune response was strongly activated in non-resident macrophages in severe COVID-19 [Striated Muscle Enriched Protein Kinase (SPEG) that is associated with cardiomyopathy and COVID-19 mortality [Mucin 4 (MUC4) that is downregulated in the blood of critically ill COVID-19 patients [In patients . Ubiquilpatients . Fucosyln sepsis ,80. Growresponse . GAS6 leresponse . Notablyresponse . Interes(AKAP8L) . Serpin  outcome ,85. InteCOVID-19 . Striateortality ,88. Mucipatients .human leukocyte antigens (HLA) at the MHC loci modifies the immune response to viral infections, including those caused by SARS-CoV-1 [HLA-DRB1, and HLA-DQB1 previously noted to be strongly repressed in a dominant population of dendritic cells [cohort 1 and 2, respectively. Reduced allele frequency of HLA-DRB1 has also been observed in severe COVID-19 [cohort 1 included HLA-DOB, involved in antigen processing and loading. Its alleles are overrepresented in symptomatic SARS-CoV-2 infected females in a Brazilian cohort [The MHC class I and II performs pivotal roles in the host adaptive immunity by modulating the antigen presentation on the cell surface for T-cell recognition . GeneticRS-CoV-1 , influenRS-CoV-1 , and MidRS-CoV-1 . We idenic cells , in acutic cells , in cohoCOVID-19 . In addin cohort .Lysine Acetyltransferase 2B (KAT2B), an epigenetic regulator of TGFKAT2B may modify disease progression and severity following SARS-CoV-2 infection. One Cut Homeobox 1 (ONECUT1) encodes for a transcription factor enriched in the liver and variants in it are associated with different forms of diabetes that is a known risk factor for severe outcomes in COVID-19 [Misshapen (Msn)/NIK related kinase 1 (MINK1) encodes for a component of the MAP kinase cascade that is involved in the phosphorylation and priming of the nucleotide-binding domain, leucine-rich-repeat containing family, pyrin-domain containing 3 (NLRP3) inflammasome [Spectrin alpha, Erythrocytic 1 (SPTA1) encodes for a structural protein in red blood cells (RBCs). In SARS-CoV-2 infected individuals increased levels and oxidation of SPTA1 peptides are noted reflecting structural aberration of RBCs [Transporter 2, ATP binding cassette Family B Member (TAP2) encodes for a component of the immunoproteasome that replaces the proteasome in haematopoietic cells, as part of the first line of defense against pathogens following pro-inflammatory cytokine IFN-TAP2 and other immunoproteasome components in lungs of COVID-19 patients and may result in worsening of disease prognosis [Unc-93 Homolog B1, TLR Signaling Regulator (UNC93B1) modulates activation of human plasmatoid predendritic cells, which play an important role in SARS-CoV-2 induced immune response [TGFInduced Factor Homeobox 1 (TGIF1), related to IFN signaling that is strongly upregulated following SARS-CoV-2 infection [Colony subunit factor 2 Receptor SubunitCSF2RB) that encodes the common subunit of the receptor for IL-3, IL-5, and granulocyte-monocyte stimulating factor (GM-CSF). Increased levels of various cytokines including GM-CSF occur in later stages of SARS-CoV-2 infection producing a self-amplifying cytokine loop that leads to ARDS and mortality [Hepatocyte Nuclear Factor 4 alpha (HNF4A) that encodes for an intestinal transcriptional regulator which promotes expression of Angiotensin 1 Converting Enzyme 2 (ACE2) and suppresses Transmembrane Serine Protease 2 (TMPRSS2), which are SARS-CoV-2 receptor and involved in viral protein priming, respectively, in the intestine [HNF4A in them.Other findings of interest included variants in novel candidate genes, so far not demonstrated to be directly associated with variability in COVID-19 presentation but with strong accessory evidence for the same. These include  disease . Interesrognosis . While tCOVID-19 ,101. Misammasome . Notablyammasome . Spectri of RBCs , which mmulation . Furtherrognosis . Unc-93 response . A detriresponse . Consistnfection . We alsoortality . Hepatocntestine . Gastrointestine , and maycohorts 1 and 2 identified SNPs in 17 and 9 genes, respectively, that are already associated with variability in SARS-CoV-2 infection severity  that is a component of the synaptonemal complex with an indispensable role in meiotic chromosome pairing and fertility in males [Dipeptidase 3 (DPEP3) that encodes a membrane-bound protein in testicular germ cells, which may be important for testicular function and is downregulated in convalescent male COVID-19 patients, likely contributing to compromised fertility in them [Desmoglein 2 (DSG2) that occurs at the intercalated discs coupling adjacent cardiomyocytes and is implicated in arrhythmogenic cardiomyopathy [Further, we also identified SNPs in genes, such as in males . Dipepti in them . Desmoglmyopathy . High lemyopathy . These dmyopathy ,118.Finally, we note the absence of information on the SARS-CoV-2 strains for the enrolled patients as a limitation of this approach. Different SARS-CoV-2 genetic strains are associated with distinct clinical outcomes . Since ‘Together with existing literature, our results improve the current understanding of genetic factors modulating the spectrum and gravity of the COVID-19 phenotype in individuals of European descent. The novel findings from this work warrant further validatory analysis and studies to facilitate a comprehensive molecular understanding of the COVID-19 pathology. Together the notable candidates from this work may be useful as biomarkers to inform on adverse prognosis and may facilitate the development and deployment of effective therapies, among COVID-19 patients of European ancestry.FBXO34, CNTN2, and TMCC2 discovered earlier in other case–control analyses studies. Overall we report SNPs in 26 genes with existing molecular links and 12 novel candidates with biological function or strong supporting evidences suggestive of a high possibility of involvement in modifying the COVID-19 clinical phenotype. These results not only broaden the current knowledge of molecular mechanisms underlying the COVID-19 pathophysiology but may be utilized in delineating a battery of biomarkers predictive of disease outlook in individuals with European ancestry.Using exome-wide genetic analysis with data from individuals of European descent we identified common variants in candidate genes that are significantly associated with the severity of COVID-19. Our findings include


We identified compounds that could inhibit CDK9 activity. One of such compound, 127, showed the strongest inhibitory activity against CDK9. Interestingly, it also inhibited CDK6 to a similar extent. We inspected the amino acid sequence and structural properties of the CDK9 hidden cavity to determine whether it is conserved in other CDKs, such as CDK6. The Ile61, comprising the center of the CDK9 hidden cavity, appears to be crucial for its kinase activity, thus indicating that the identification of the CDK9 hidden cavity may provide vital information for the development of novel CDK9 inhibitors.HIV-1 transcription is specifically augmented by a transcriptional activator complex composed of Tat, an HIV-1-encoded activator, and the host transcription elongation factor P-TEFb, which is composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1. Several observations suggest that P-TEFb is an attractive anti-HIV-1 drug target. However, the long-term cytotoxicity of CDK9 inhibitors hinders their widespread use in HIV-1 therapy. Thus, novel and safe inhibitors are sorely needed. By performing molecular dynamics simulations of the 3D structure of Tat/P-TEFb, we previously identified a unique cavity structure of CDK9, the CDK9 hidden cavity, that is specifically induced by Tat binding. Here, we attempted to identify compounds that fit this cavity and inhibit CDK9 activity by  Current combination antiretroviral therapy (cART) is considered effective in the treatment of HIV-1; however, lifelong treatment is required. In addition, chronic long-term infection with HIV-1 inevitably causes the emergence of resistant viruses and serious side effects [It is estimated that more than 39 million people worldwide are living with HIV-1, with approximately 1.8 million new infections and 940,000 deaths each year  RNA sequence –5. In thCDK9 was originally identified as a Tat-associated kinase (TAK) or cdc-2-like cyclin-dependent kinase PITALRE based on amino acid homology to other CDKs –14. The In addition to phosphorylation, several studies have indicated that CDK9 activity may be modulated by conformational changes. Analysis of the crystal structure of the P-TEFb/Tat complex suggests that Tat binding induces conformational changes in CDK9, leading to alterations in its substrate specificity . By perfin silico drug screening. We identified a chemical structure that is potentially useful for the development of specific inhibitors of HIV-1 replication.Recently, MD simulations of X-ray 3D structures have shown that local conformational changes occur within molecules, which may function as drug target cavities. For example, Filomia et al.  decoded in silico library screening based on putative druggable sites of the CDK9 hidden cavity using molecular docking against 5,431,536 drug-like [We carried out rug-like  compoundrug-like , 24 dockrug-like  Fig 1)..in silicCDK2/CycA2: Full-length human CDK2 (1–298 amino acids), accession number NP_001789.2 was co-expressed as an N-terminal GST-tagged protein with full-length GST-CyclinA2 (1–432 amino acids), accession number NP_001228.1 using the baculovirus expression system. Thereafter, GST-CDK2 was purified using glutathione sepharose chromatography.CDK2/CycE1: Full-length human CDK2 (1–298 amino acids), accession number NP_001789.2, was co-expressed as an N-terminal GST-tagged protein with full-length CyclinE1 (1–410 amino acids), accession number NP_001229.1 using the baculovirus expression system. Thereafter, GST-CDK2 was purified using glutathione sepharose chromatography.CDK3/CycE1: Full-length human CDK3 (1–305 amino acids), accession number NP_001249.1, was co-expressed as an N-terminal GST-fusion protein with full-length CyclinE1 (1–410 amino acids), accession number NP_001229.1 using the baculovirus expression system. Thereafter, GST-CDK3 was purified by using glutathione sepharose chromatography.CDK4/CycD3: Full-length human CDK4 (1–303 amino acids), accession number NP_000066.1 was co-expressed as N-terminal GST-fusion protein with full-length human GST-CyclinD3 (1–292 amino acids), accession number AAA51927.1 using the baculovirus expression system. Thereafter, GST-CDK4/CycD3 was subsequently purified using glutathione sepharose chromatography.CDK5/p25: Full-length human CDK5 (1–292 amino acids), accession number NP_004926.1, was co-expressed as an N-terminal GST-fusion protein with p25 (99–307 (end) amino acids), accession number NP_003876.1 using the baculovirus expression system. Thereafter, GST-CDK5 was purified by glutathione sepharose chromatography.CDK6/CycD3: Full-length human CDK6 (1–326 amino acids), accession number NP_001250.1, was co-expressed as an N-terminal GST-fusion protein with full-length human GST-CyclinD3 (1–292 amino acids), accession number AAA51927.1 using the baculovirus expression system. GST-CDK6/CycD3 was purified using glutathione sepharose chromatography and activated with His-CDK7/CycH/MAT1 complex. The activated GST-CDK6/CycD3 was subsequently purified using glutathione sepharose chromatography.CDK7/CycH/MAT1: Full-length human CDK7 (1–346 amino acids) of accession number NP_001790.1 was co-expressed as an N-terminal GST-fusion protein with full-length CyclinH (1–323 amino acids), accession number NP_001230.1, and full-length MAT1 (1–309 amino acids), accession number NP_002422.1 using the baculovirus expression system. Thereafter, GST-CDK7 was purified by using glutathione sepharose chromatography.CDK9/CycT1: Full-length human CDK9 (1–372 amino acids), accession number NP_001252.1 was co-expressed as N-terminal GST-fusion protein with full-length His-CyclinT1 (1–726 amino acids), accession number NP_001231.2 using the baculovirus expression system. GST-CDK9 was subsequently purified using glutathione sepharose chromatography.The test compound was dissolved in and diluted with dimethylsulfoxide (DMSO) to achieve 100-fold higher concentration. Then the solution was further 25-fold diluted with assay buffer  to make 4x compound solution. Reference compounds for the assay control were prepared similarly.http://www.carnabio.com/english/index.html). Briefly, 4x Substrate/ATP/Metal solution was prepared with kit buffer , and 2x kinase solution containing CDK/CycT1 complex was prepared with assay buffer. Five microliters of 4x compound solution, 5 μL of 4x Substrate/ATP/Metal solution, and 10 μL of 2x kinase solution were mixed and incubated in a well of polypropylene 384 well microplate for 1.5 or 5 hour(s) at room temperature. Reactions were stopped by the addition of 70 μL of Termination Buffer . The reaction mixture was applied to the LabChip™ system (PerkinElmer), and the product and substrate peptide peaks were separated and quantitated. The kinase reaction was evaluated by the product ratio calculated from peak heights of the product(P) and substrate(S) peptides using the formula (P/(P+S)). The readout value of reaction control (complete reaction mixture) was set as a 0% inhibition, and the readout value of background (Enzyme(-)) was set as a 100% inhibition, then the percent inhibition of each test solution was calculated. All experiments were performed in duplicate and had a positive control for the assay verification.Kinase inhibition profiles were determined using an off-chip mobility shift assay (MSA) provided by Carnabioscience   with rec127 was analyzed by MOE ligand analysis using Molecular Operating Environment (MOE) software 2020.0901 .Figures showing protein structures were prepared using PyMOL 2.5.2 . Sequences were aligned using Clustal W. The 2D interaction of the docking pose of CDK9/As previously reported , we perfin silico screening against the available chemical library (Namiki HTS library set ver. 201508) at a putative druggable site (hidden cavity) to identify a CDK9 inhibitor. The candidate compounds were narrowed down with progressively higher precision using three Glide docking programs: HTVS, SP, and XP , might be involved in the 127-mediated inhibition.As shown in ous CDKs . Among oous CDKs . Gln27 iin vitro. Compounds 1801, 1804, and 1805 significantly inhibited CDK9 , herpes simplex virus (HSV-1 and HSV-2), human cytomegalovirus (CMV), Epstein-Barr virus (EBV), human adenovirus, influenza A virus, dengue virus, and Kaposi sarcoma-associated virus (KSHV) . Most viS1 Fig127 are shown in boxes.Amino acid alignments of CDKs were performed using Clustal W. The key amino acid residues constituting the local structure surrounding (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 FileThe docking score was calculated using extra precision (XP) of Glide docking programs and shown in kcal/mol.(PDF)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(XLSX)Click here for additional data file.S6 File(XLSX)Click here for additional data file.


Cardiac fibrosis developed from fibrotic remodelling due to hypertension leads to heart failure. Immune cells are abundantly accumulated in fibrotic regions, but exact mechanisms how they contributed to cardiac fibrosis are not clear. Here we demonstrated that CD8 T cells are major contributor to cardiac fibrosis in hypertensive hearts. Stressed cardiomyocytes express STING-dependent RAE-1 that activates NKG2D + CD8 T cells to induce apoptosis of stressed cardiomyocytes. Preventing STING signalling in stressed cardiomyocytes attenuates cardiac fibrosis. Pharmacologically inhibiting cardiomyocayte-RAE-1 and CD8+ T cell-NKG2D axis may be a potential therapeutic strategy to prevent cardiac fibrosis in HFpEF patients. Cardiac fibrosis is central to heart failure (HF), especially HF with preserved ejection fraction (HFpEF), often caused by hypertension. Despite fibrosis causing diastolic dysfunction and impaired electrical conduction, responsible for arrhythmia-induced sudden cardiac death, the mechanisms are poorly defined and effective therapies are lacking. Here we show that crosstalk between cardiac cytotoxic memory CD8+ T cells and overly stressed cardiomyocytes is essential for development of non-ischemic hypertensive cardiac fibrosis.CD8 T cell depletion in hypertensive mice, strongly attenuated CF, reduced cardiac apoptosis and improved ventricular relaxation. Interaction between cytotoxic memory CD8+ T cells and overly stressed cardiomyocytes is highly dependent on the CD8+ T cells expressing the innate stress-sensing receptor NKG2D and stressed cardiomyocytes expressing the NKG2D activating ligand RAE-1. The interaction between NKG2D and RAE-1 results in CD8+ T cell activation, release of perforin, cardiomyocyte apoptosis, increased numbers of TGF-β1 expressing macrophages and fibrosis. Deleting NKG2D or perforin from CD8+ T cells greatly attenuates these effects. Activation of the cytoplasmic DNA-STING-TBK1-IRF3 signaling pathway in overly stressed cardiomyocytes is responsible for elevating RAE-1 and MCP-1, a macrophage attracting chemokine. Inhibiting STING activation greatly attenuates cardiomyocyte RAE-1 expression, the cardiomyocyte apoptosis, TGF-β1 and fibrosis.Our data highlight a novel pathway by which CD8 T cells contribute to an early triggering mechanism in CF development; preventing CD8+ T cell activation by inhibiting the cardiomyocyte RAE-1-CD8+ T cell-NKG2D axis holds promise for novel therapeutic strategies to limit hypertensive cardiac fibrosis. The accumulation of fibrotic tissue seen as a diffuse increase in collagen fibers accounts for most of the fibrotic remodeling of the heart during hypertension , 2, the via yet-to-be-defined innate TCR-independent mechanisms  mice were from Jackson Laboratory, Bar Harbor, Maine, USA; perforin knockout (pfp-/-) mice were from Mark Smyth, Peter MacCallum Cancer Centre, Melbourne, Australia, CD8β knockout mice were from David Davis, Australian National University, Canberra, Australia with approval from Dan Littman, New York School of Medicine, USA and wild-type C57BL/6 mice were from WEHI, Melbourne, Australia. Mixed chimeric mice whose CD8+ T cells were deficient in perforin or NKG2D were generated using our previously published procedure  were used to induce a short anesthesia for surgical procedures. Hypertension/pressure overload was induced by trans-aortic constriction (TAC) of the thoracic aorta reducing aortic lumen to 0.44mm as previously described by us . In ShamTau), and left ventricular end-diastolic pressure (LVEDP) as previously described ] whilst mice were ventilated 28 days post-surgeries using a 1.4F Millar microtipped transducer catheter . The Miller catheter was then inserted into the aorta and the aortic blood pressure recorded using a computer and AD Instrument software . The catheter was then advanced into the left ventricle to assess left ventricular diastolic function; –dp/dt (min), relaxation time constant  monoclonal antibody YTS 156.7 prepared from a hybridoma provided by Steve Cobbold, Oxford University, UK. Mice were administered 0.5 mg antibody/mouse/week i.p, for 4 weeks; IgG2b isotype was administered to control mice. CD4+ T cells were depleted using an anti-CD4 monoclonal antibody (GK1.5) 0.3mg/week i.p. and controls were given non-immune IgG2b. For STING inhibition mice were administered H-151 (0.21mg/day i.p.) commencing one day after surgery for 4 weeks; H-151 covalently blocks STING palmitoylation preventing TBK1 activation and STING phosphorylation , 29.via cervical dislocation.At the end of experiment, mice were euthanized using the gradual-fill method of carbon dioxide overdose followed by cervical dislocation. However, mice anesthetised for hemodynamic measurements were euthanized Flow cytometry was performed as previously described , using AThe left ventricle (LV) was dissected into three equal transverse LV slices and frozen in OTC at -70°C; 6 µm-thick sections were cut for histology/immunofluorescence stainings and 30 µm-thick sections for ROS assessment.For collagen deposition sections were stained with Picrosirius Red F3BA, (0.1% solution in saturated aqueous picric acid) and analysed using a computer –interfaced colour Imaging system . Ten randomly selected fields from each section were analysed and expressed as percent of the stained area , 14.Six µm sections were allowed to thaw then fixed in cold (-20°C) acetone for 20 min. Sections were sequentially incubated in 3% hydrogen peroxide in PBS (20 min), 10% normal serum/PBS (30 min) and biotin/avidin-blocking reagents (15 min each reagent) (Vector Laboratories). These sections were then incubated (1hr) with primary antibodies in serum: rat anti-mouse CD4 (BD: cat#550280), rat anti-mouse CD8 (BD: cat#550281), rat anti-mouse CD68 (BioRad: cat#MCA1957), rabbit anti-mouse TGFβ1 , or corresponding non-immune IgG’s. Then, sections were washed and incubated with the appropriate secondary antibody ] for 40 min, followed by incubation with streptavidin horseradish peroxidase complex (30 min) (Vector Laboratories) and counterstaining with haematoxylin. Antigens were visualised using 3, 3-diamonobenzidine (Sigma). Cell expressing antigens were quantitated by cell counting in ten randomly selected fields per section.Sections were fixed in acetone, washed and permeabilized in 0.1% Triton-X-100/PBS. After washings, sections were incubated in 10% normal serum/PBS before being incubated with primary antibodies in serum: rabbit anti-mouse CD8 , mouse anti-mouse NK1.1 (Biolegend: cat#108701), Armenian Hamster- anti-mouse TCR γ/δ (Biolegend: cat#108101), rat anti-mouse Perforin (LS Biosciences: cat#LS-C18632), biotin rat anti-mouse NKG2D , rabbit anti-mouse TGFβ1 , rat anti-mouse CD68 (BioRad: cat#MCA1957), rabbit anti-mouse troponin (Abcam: cat#ab125266), goat anti-mouse RAE-1 (R&D Systems: cat#AF1136), rabbit anti-mouse α-Actinin (Acris Antibodies: cat#BP210), rabbit anti-mouse phosphor-STING (Invitrogen: cat# PA5-105674), goat anti-mouse CX3CR1 (R & D Systems: cat#AF5825), mouse anti-mouse phospho p38MAPK (Biolegend: cat#690201), rabbit anti-mouse phospho-IRF3 , rabbit anti-mouse phospho-TBK1 (Imgenex: cat# IMX-5194), mouse anti-mouse NF-kB (Santa Cruz: cat# sc-109), rabbit anti-mouse MCP-1 (Abcam: cat #ab7202), rabbit anti-mouse α-Actinin Biotin (Bioss antibodies: cat# BS-10367R-Biotin) or corresponding isotype matched control IgG antibodies. Then sections were then washed and incubated with secondary antibodies/conjugates  for 30 min, washed in PBS and counterstained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI). For dual fluorescence stainings sections were first incubated with the first primary antibody/secondary antibody and after multiple washings were incubated with the second primary antibody and its corresponding secondary antibody. Antigens were assessed using an Olympus BX61 microscope with a FV11 CCD camera using cellSens imaging software (Olympus).In Situ Cell Death Detection Kit-POD (Roche: cat#11-684-817-910)]. Briefly, sections were incubated in 4% diethyl pyrocarbonate (DEPC)/100% ethanol at 4C, washed and fixed in acetone. Sections were permeabilized in 0.1% Triton-X-100 and incubated in 15% FCS/2% BSA/0.1M Tris-HCl buffer and after washing, they were incubated with a TUNEL reaction mixture at 37C (according to the manufacturer’s instructions), washed and counterstained with DAPI. TUNEL positive cells were visualized using Olympus BX61 microscope. For double immunofluorescence to identify apoptotic cardiomyocytes frozen sections were stained by TUNEL followed by rabbit anti-mouse α-Actinin . Then goat anti-rabbit Alexa 568  was used to detect Actinin. Nuclei were counterstained with DAPI and apoptotic cardiomyocytes visualised using a Nikon A1r confocal microscope. Ten randomly selected fields per section were analysed.Apoptotic cells were identified using a deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) system [Cytosolic DNA and cardiomyocytes were stained with Pico488 (picogreen), a highly sensitive fluorescent dye that can detect double stranded cytoplasmic DNA , 31 and via a 585 nm long-pass filter (Reactive oxygen species (ROS) were measured using dihydroethidium (DHE). Sections were incubated with DHE , protected from light at 37°C for 30 min. Images were obtained using a fluorescence microscope measuring fluorescence s filter .n represents mouse numbers in each experiment, as detailed in figure legends. Comparison between two groups were determined by two-tailed Student’s t-test, and three or more groups were compared by one-way ANOVA followed by Tukey-Kramer post hoc analysis. P < 0.05 was considered statistically significant.Randomized and coded animals and samples were processed and analyzed by blinded investigators. All data was analyzed statistically using Prism 8.0 . All results are expressed as man ± SEM. 3CR1. The majority of CD8+ T cells in fibrotic areas of TAC hearts expressed CX3CR1 (We used three different hypertensive mouse models to assess the importance of CD8+ T cells for development of cardiac fibrosis: 1) mice whose systolic blood pressure is elevated by trans-aortic constriction (TAC) inducing left ventricular pressure overload 2) mice with high-renin salt-independent 2K1C renovascular hypertension and 3) mice with salt-sensitive low-renin deoxycorticosterone (DOCA)-salt hypertension. We used highly specific anti-CD8β antibodies to deplete CD8αβ+ T (CD8+ T) cells . Their dd CX3CR1 . Given tAs CD4+ T cells contribute to development of cardiac fibrosis , we examvia innate mechanisms (-/-) mice in which CD8+ T cells were made deficient in perforin (+/+) mice received mixed bone marrow from CD8β-deficient (80%) and 20% from wild type mice  are greatly increased . Increasvia IRF3 , we invevia IRF3 , demonstvia IRF3 , 44. To via IRF3 , 47. TreTo confirm involvement of the STING, TBK-IRF3-NFkB signaling cascade we assessed the phosphorylation status of these molecules in relation to their intracellular location within cardiomyocytes. Phospho-TBK was apparent, localized in the perinuclear region , 49, a cvia interactions between NKG2D receptors expressed by the CD8+ T cells and RAE-1, the NKG2D ligand expressed by stressed cardiomyocytes. Bystander activation of CD8+ T cells is very common during viral infections  pore opening and mtDNA release  expressed by neighboring cardiomyocytes, fibroblasts, immune cells and parenchymal cells to initiate fibrosis ; fibrobl release , also in release . High RO release . Recentl release  but down release . Accumul release , 18, sugvia genetic deletion and have previously used such a strategy to define the role of CD8+ T cells in atherosclerosis  from the National Health and Medical Research Council of Australia.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Rationale: GLK (MAP4K3) activates PKCθ-IKKβ axis in T-cell activation and induces IL-17A-mediated autoimmune diseases. Attenuation of Treg differentiation and function by GLK could also contribute to autoimmune diseases.Methods: We analyzed the roles of GLK and IKKβ in Treg differentiation and function using T-cell-specific GLK transgenic mice and IKKβ conditional knockout mice. The mechanism of GLK/IKKβ-mediated attenuation of Treg differentiation/function was studied by chromatin-immunoprecipitation, reporter assays, in vitro kinase assays, protein-protein interaction assays, mass spectrometry, confocal microscopy, flow cytometry, and single-cell RNA sequencing (scRNA-seq) analysis.Results: We found that GLK signaling inhibited Foxp3 transcription by blocking the function of the transcription factor FoxO1. Mechanistically, GLK directly phosphorylated and activated IKKβ at Ser733 in a PKCθ-independent manner. The phospho-IKKβ Ser733 induced FoxO1 Ser319 phosphorylation and nuclear export, leading to Foxp3 downregulation. Consistently, scRNA-seq analyses showed that Foxp3 mRNA levels were inversely correlated with FoxO1 mRNA levels in GLK transgenic CD4+ T cells.Conclusions: GLK-IKKβ-FoxO1 signaling axis inhibits Foxp3 transcription, leading to reduction of Treg differentiation and suppressive activity, as well as induction of autoimmune disease. Here, we report that GLK downregulates Foxp3 transcription by inducing IKKβ-mediated FoxO1 phosphorylation.+CD25+Nrp-1+) cells in GLK Tg mice  cells displayed reduction of suppressive function in vitro  mice show normal Treg-cell development in the thymus, spleen, and peripheral blood in 5-week old mice e Figure A. We als+Foxp3+ T cells containing both nTreg and iTreg cells in colon lamina propria (cLP) was modestly decreased in Lck-GLK Tg mice . Exacerbated experimental autoimmune encephalomyelitis (EAE) symptoms and delayed recovery were manifested in MOG-immunized GLK Tg mice  cells were decreased in the central nervous system (CNS) of Lck-GLK Tg mice in the recovery phase  cells was decreased in GLK Tg T cells compared to that of wild-type T cells  cells, the intestinal immune system consists of 50-70% peripheral iTreg cells, which are rare in other secondary lymphoid systems e Figure C. After ransgene A. Consispurified B and co-in vitro differentiated iTreg cells in GLK-deficient T cells was comparable to that of wild-type T cells using an optimal condition with TGF-β , resulting in Lck-GLK Tg/IKKβ cKO mice. The frequency of splenic nTreg cells in Lck-GLK Tg/IKKβ cKO mice was comparable to those of wild-type or Lck-GLK Tg mice , respectively. Immunofluorescence imaging results showed that IKKβ and FoxO1 were co-localized in the cytoplasm of GLK transgenic iTreg cells, whereas these two proteins were co-localized in the nucleus of wild-type iTreg cells  assays showed an interaction between IKKβ and FoxO1 Figure A. ALPHA o Figure C. Furtheo Figure D. To exas Figure E. The PLs Figure F. In cons Figure F. These in vitro phosphorylated Flag-tagged FoxO1, followed by mass spectrometry (MS) analyses. Ser319, but not Thr24, was identified as the FoxO1 phosphorylation site by IKKβ  mutant proteins. IKKβ drastically induced FoxO1 phosphorylation at Ser319  mutant in HEK293T cells enhanced FoxO1 Ser319 phosphorylation Figure F. Overexs Figure H-I. Intes Figure . It is ps Figure J-K. Thes+ T cells, GLK transgene gradually decreased Foxp3 transcripts with the concomitant increase of FoxO1 expression  at the C-terminal region of IKKβ, mimicked by a mutant with 10 serine-to-glutamic acid residues, results in the inhibition of IKKβ kinase activity IKKβ plays complex roles in Treg development and activity. Treg cell development in the thymus is reduced in T-cell-specific IKKβ conditional knockout mice Taken together, our study reveals a critical mechanism of GLK-IKKβ signaling-inhibited Foxp3 transcription in T cells Figure . GLK oveAll mice were bred under specific pathogen-free facility at National Health Research Institutes (NHRI) and maintained according to institutional guidelines of the Institutional Animal Care and Use Committee of NHRI. All mouse lines were purchased and backcrossed as described previously HEK239T cells and human Jurkat T leukemia cells were obtained from American Type Culture Collection (ATCC). Human Jurkat TAg (J-TAg) cell clone was derived from the Jurkat T cell line by stably transfecting with the simian virus 40 large T antigen. HEK293T cells were cultured and maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum  plus penicillin  and streptomycin . Jurkat T cells and Jurkat (J-TAg) cells were maintained in RPMI-1640 (Thermo Fisher Scientific) supplemented with 10% FBS plus penicillin  and streptomycin . All cell lines were confirmed to be free from mycoplasma contamination.in vitro binding assay, purified FoxO1 proteins were isolated from Escherichia coli (BL21) and then subjected to GST pulldown assay.Anti-Flag , anti-HA , anti-phospho-serine , anti-vinculin , and anti-FoxO1 (Thr24) antibodies (#07-2126) were purchased from MilliporeSigma. Antibody specific for β-tubulin  was purchased from Thermo Fisher Scientific. Anti-phospho-FoxO1 (Ser319) antibody (#GTX50196) was purchased from GeneTex. Anti-actin (clone E814), anti-PKCθ (clone EPR1487(2), #ab110728), and anti-phospho-IKKβ (Ser180/181) antibodies (#ab55341) were purchased from Abcam. Anti-phospho-threonine/tyrosine (#9381), anti-phospho-FoxO1 (Ser256) (#PA5-17907), anti-phospho-FoxO1 (Ser319) (#2486), anti-FoxO1 , anti-IKKβ , anti-FoxO3a , and anti-phospho-FoxO3a (Ser253) (#9466) antibodies were purchased from Cell Signaling Technology. Anti-phospho-IKKβ (Ser733) antibody (#LS-C157829) was purchased from LSBio. Anti-GLK antibody (clone C3) was generated as described previously Large intestines were collected from mice. Large intestines were treated sequentially with 2 mM dithiothreitol  solution, 5 mM EDTA (Thermo Fisher Scientific) in HBSS solution, and then 100 U/ml collagenase Type I (MilliporeSigma) solution. Colon lamina propria (cLP) lymphocytes were collected and enriched by 44:67% Percoll (Cytiva) gradient.These experiments were performed as described preciously TM C tubes (Miltenyi Biotec), followed by homogenization using gentleMACSTM dissociator (Miltenyi Biotec). Lymphocytes were separated and enriched by 30:70% Percoll (Cytiva) gradient.The brain and spinal cord of mice were harvested and incubated with RPMI-1640 media in the gentleMACS+ T cells.Peripheral blood, the spleen, inguinal lymph nodes, and mesenteric lymph nodes were harvested from mice for purification of primary T cells. Peripheral blood samples were collected biweekly from wild-type and Lck-GLK Tg mice. Antibody cocktails against CD11b, B220, CD49b, CD235, TER-119, Nrp-1, and CD8  were used to deplete leukocytes from the organ and tissues, resulting in purified CD4+CD62L+) cells. Murine iTreg cells were generated in vitro in RPMI-1640 (Thermo Fisher Scientific) supplemented with TGF-β recombinant proteins , IL-2 recombinant proteins , anti-IFN-γ , and anti-IL-4  antibodies, and were cultured in the anti-CD3  plus anti-CD28  antibody-coated 48-well plate.Murine induced Treg (iTreg) cells were generated from splenic naïve T  by MACS magnetic-bead separation (Miltenyi Biotec). For purification of nTreg cells, nTreg cells were isolated from CD4+ T cells using biotin-conjugated anti-mouse Nrp-1 antibody  by MACS magnetic-bead separation (Miltenyi Biotec). For purification of the in vitro differentiated iTreg cells, iTreg cells were isolated from the in vitro differentiated CD4 cells using biotin-conjugated anti-mouse CD25 antibody  by MACS magnetic-bead separation (Miltenyi Biotec). The purity of iTreg (CD4+Foxp3+) cells was 90%  coated on 96-well round-bottom plates.Murine CD4 was 90% B. For dePrediluted antibodies were prepared for staining of cell surface markers. For intracellular staining, cells were permeabilized in Foxp3 Fix/Perm solution (BioLegend) and incubated with anti-Foxp3 antibodies diluted in Foxp3 Perm buffer (BioLegend). The antibodies used: anti-CD3-PerCP , anti-CD4-Pacific blue , anti-Foxp3-Alexa Fluor 488 , and anti-Nrp-1-APC . FACSCanto II (BD Biosciences) and FlowJo software (BD Biosciences) were used to collect and analyze the data.The sample preparation and chromatin immunoprecipitation (ChIP) assays were performed as described previously ® transfection system (Thermo Fisher Scientific). After 24 to 48 h, cells were lysed using lysis buffer . Results represent the mean ratios of the firefly luciferase activity to the renilla luciferase activity.The murine Foxp3 promoter (region -500/+100 bp relative to the Foxp3 transcription start site) was subcloned into pGL3 basic vector (Promega) containing firefly luciferase. The Foxp3-promoter/pGL3 reporter plasmid was co-transfected with renilla luciferase control plasmid (pRL-TK), FoxO1, FoxO1 (S319A), IKKβ, or IKKβ kinase-dead (K44M)-encoding plasmids alone or in different combinations into Jurkat T cells using NeonImmunoprecipitation, GST pulldown assays and immunoblotting analyses were performed as described previously 2+ ions, which trapped the phosphorylated proteins (such as p-FoxO1/p-FoxO3a/p-IKKβ) and increased their molecular weights. After electrophoresis, the Phos-tag gel was soaked in blotting buffer containing 1 mM EDTA (MilliporeSigma). The phosphorylated proteins and non-phosphorylated proteins on the Phos-tag gel were then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween® 20 (TBST) for 2 h, and then immunoblotted with anti-FoxO1, anti-FoxO3a, and anti-IKKβ antibodies to distinguish phosphorylated proteins from non-phosphorylated proteins.To determine phospho-FoxO1, phospho-FoxO3a, and phospho-IKKβ protein levels, Phos-tag SDS-PAGE ALPHA protein-protein interaction assays YFP-FoxO1 and CFP-IKKβ plasmids were co-transfected into HEK293T cells. When the pairs of YFP-fused FoxO1 and CFP-fused IKKβ proteins are within 8 nm, YFP signals will be stimulated by CFP signals. These fluorescence signals were detected by an EnVision 2104 Multilabel Plate Reader (PerkinElmer Life Sciences). The FRET efficiency Duolink In Situ Red Starter kit (MilliporeSigma) Murine T Cells were fixed in cold methanol for 1 min. After permeabilization with fixation/permeabilization buffer (BD Bioscience) for 1 h, the cells were blocked with 5% bovine serum albumin for 1 h. The cells were incubated with rabbit anti-FoxO1 and mouse anti-IKKβ primary antibodies (1:200 dilution) for 16 h and then incubated with anti-rabbit IgG-CF488 and anti-mouse IgG-CF594 secondary antibodies (1:500 dilution), respectively, for 1 h. The secondary antibodies were purchased from Biotium. The fluorescence signals were analyzed using Leica TCS SP5II confocal microscope TM Single-Cell Analysis System was used to generate the scRNA-seq library of T cells. SeqGeq version 1.6.0 (BD Biosciences) combined with R packaged Seurat plugin and ViolinBox plugin were used to analyze the cDNA quality, scRNA expression matrix, differentially expressed genes (DEGs), and gene ontology (GO) database.Murine T cells were purified from the spleen, inguinal lymph nodes, and mesenteric lymph nodes of wild-type and Lck-GLK Tg mice using negative selection. BD RhapsodyP values of less than 0.05 were considered statistically significant. Symbols of P values represent *P < 0.05, **P < 0.01, ***P < 0.001, and n.s.: not significant.The statistical significance between two unpaired groups was analyzed using two-tailed Student's t test. Supplementary figures and tables.Click here for additional data file.


Klebsiella quasipneumoniae MMCC7 is a multidrug- and heavy metal-resistant strain isolated from the feces of a pet shop eclectus parrot in Hong Kong. The complete genome, a single chromosome and circular plasmid , was determined by hybrid assembly. Klebsiella pneumoniae complex (KPC), which incorporates the two subspecies of Klebsiella quasipneumoniae, is the most clinically relevant—including notable hospital-acquired infections during the COVID-19 pandemic  housed at a pet shop in Wanchai, Hong Kong. After serial dilution in phosphate-buffered saline, a 100-μL aliquot of the 0.01% (wt/vol) extract was spread onto CHROMagar orientation medium (BD Biosciences) and incubated overnight. A single selected metallic-blue colony was passaged 10 times on Luria agar and finally incubated overnight in Luria broth    before gN50, 13,618 bp). Default parameters were used for all software unless otherwise specified.Paired-end short-read sequencing libraries were prepared using the NexteraXT DNA library preparation kit and sequenced via the Illumina MiSeq platform using v3 chemistry (2 × 300 bp). Adapter sequences were removed using Trimmomatic v0.32 , and reaThe complete genome sequence combined Illumina and MinION data sets using Unicycler v0.4.3 , yieldinKlebsiella quasipneumoniae supbsp. similipneumoniae 07A044T (GenBank accession number CP084787) . Plasmid pMMCC7, classified as IncFIB(K)_14[CladeI] using the KpVR Web-based tool (https://bioinfo-mml.sjtu.edu.cn/KpVR/index.php) (merRPTCA and copABCDRS operons (mrkABCDFJ operon coding type 3 fimbriae (blaTEM-1 (aph(3″)-Ib, aph(6)-Id  . It has  OKP-B-6 , as detedex.php) , contain operons , 30, andfimbriae . It alsoblaTEM-1 , aph(3″)ph(6)-Id , tetD, mK. quasipneumoniae MMCC7 are available through NCBI under BioProject number PRJNA759343, with GenBank accession numbers CP082785 (chromosome) and CP082786 (plasmid) and SRA numbers SRR15928120 (MinION) and SRR15928121 (MiSeq).The complete genome sequences and raw sequence data for