Potent and broad HIV-1 neutralization in fusion peptide-primed SHIV-infected macaques.

An antibody-based HIV-1 vaccine will require the induction of potent cross-reactive HIV-1-neutralizing responses. To demonstrate feasibility toward this goal, we combined vaccination targeting the fusion-peptide site of vulnerability with infection by simian-human immunodeficiency virus (SHIV). In four macaques with vaccine-induced neutralizing responses, SHIV infection boosted plasma neutralization to 45%-77% breadth (geometric mean 50% inhibitory dilution [ID50] ∼100) on a 208-strain panel. Molecular dissection of these responses by antibody isolation and cryo-electron microscopy (cryo-EM) structure determination revealed 15 of 16 antibody lineages with cross-clade neutralization to be directed toward the fusion-peptide site of vulnerability. In each macaque, isolated antibodies from memory B cells recapitulated the plasma-neutralizing response, with fusion-peptide-binding antibodies reaching breadths of 40%-60% (50% inhibitory concentration [IC50] < 50 µg/mL) and total lineage-concentrations estimates of 50-200 µg/mL. Longitudinal mapping indicated that these responses arose prior to SHIV infection. Collectively, these results provide in vivo molecular examples for one to a few B cell lineages affording potent, broadly neutralizing plasma responses.

Antibody Lineages with Vaccine-Induced Antigen-Binding Hotspots Develop Broad HIV Neutralization.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires.

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.

Vaccination induces maturation in a mouse model of diverse unmutated VRC01-class precursors to HIV-neutralizing antibodies with >50% breadth.

Vaccine elicitation of broadly neutralizing antibodies (bnAbs) is a key HIV-research goal. The VRC01 class of bnAbs targets the CD4-binding site on the HIV-envelope trimer and requires extensive somatic hypermutation (SHM) to neutralize effectively. Despite substantial progress, vaccine-induced VRC01-class antibodies starting from unmutated precursors have exhibited limited neutralization breadth, particularly against viruses bearing glycan on loop D residue N276 (glycan276), present on most circulating strains. Here, using sequential immunization of immunoglobulin (Ig)-humanized mice expressing diverse unmutated VRC01-class antibody precursors, we elicited serum responses capable of neutralizing viruses bearing glycan276 and isolated multiple lineages of VRC01-class bnAbs, including two with >50% breadth on a 208-strain panel. Crystal structures of representative bnAbs revealed the same mode of recognition as known VRC01-class bnAbs. Structure-function studies further pinpointed key mutations and correlated their induction with specific immunizations. VRC01-class bnAbs can thus be matured by sequential immunization from unmutated ancestors to >50% breadth, and we delineate immunogens and regimens inducing key SHM.

Glycan Masking Focuses Immune Responses to the HIV-1 CD4-Binding Site and Enhances Elicitation of VRC01-Class Precursor Antibodies.

An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-2∗02, the germline VRC01 VH segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G+ B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design.

In situ formed depot of elastin-like polypeptide-hirudin fusion protein for long-acting antithrombotic therapy.

Thrombosis, induced by abnormal coagulation or fibrinolytic systems, is the most common pathology associated with many life-threatening cardio-cerebrovascular diseases. However, first-line anticoagulant drugs suffer from rapid drug elimination and risk of hemorrhagic complications. Here, we developed an in situ formed depot of elastin-like polypeptide (ELP)-hirudin fusion protein with a prodrug-like feature for long-term antithrombotic therapy. Highly secretory expression of the fusion protein was achieved with the assistance of the Ffu312 tag. Integration of hirudin, ELP, and responsive moiety can customize fusion proteins with properties of adjustable in vivo retention and controllable recovery of drug bioactivity. After subcutaneous injection, the fusion protein can form a reservoir through temperature-induced coacervation of ELP and slowly diffuse into the blood circulation. The biological activity of hirudin is shielded due to the N-terminal modification, while the activated key proteases upon thrombus occurrence trigger the cleavage of fusion protein together with the release of hirudin, which has antithrombotic activity to counteract thrombosis. We substantiated that the optimized fusion protein produced long-term antithrombotic effects without the risk of bleeding in multiple animal thrombosis models.

Differential effects of itacitinib, fedratinib, and ruxolitinib in mouse models of hemophagocytic lymphohistiocytosis.

ABSTRACT: Hemophagocytic lymphohistiocytosis (HLH) comprises a severe hyperinflammatory phenotype driven by the overproduction of cytokines, many of which signal via the JAK/STAT pathway. Indeed, the JAK1/2 inhibitor ruxolitinib has demonstrated efficacy in preclinical studies and early-phase clinical trials in HLH. Nevertheless, concerns remain for ruxolitinib-induced cytopenias, which are postulated to result from the blockade of JAK2-dependent hematopoietic growth factors. To explore the therapeutic effects of selective JAK inhibition in mouse models of HLH, we carried out studies incorporating the JAK1 inhibitor itacitinib, JAK2 inhibitor fedratinib, and JAK1/2 inhibitor ruxolitinib. All 3 drugs were well-tolerated and at the doses tested, they suppressed interferon-gamma (IFN-γ)-induced STAT1 phosphorylation in vitro and in vivo. Itacitinib, but not fedratinib, significantly improved survival and clinical scores in CpG-induced secondary HLH. Conversely, in primary HLH, in which perforin-deficient (Prf1-/-) mice are infected with lymphocytic choriomeningitis virus (LCMV), itacitinib, and fedratinib performed suboptimally. Ruxolitinib demonstrated excellent clinical efficacy in both HLH models. RNA-sequencing of splenocytes from LCMV-infected Prf1-/- mice revealed that itacitinib targeted inflammatory and metabolic pathway genes in CD8 T cells, whereas fedratinib targeted genes regulating cell proliferation and metabolism. In monocytes, neither drug conferred major transcriptional impacts. Consistent with its superior clinical effects, ruxolitinib exerted the greatest transcriptional changes in CD8 T cells and monocytes, targeting more genes across several biologic pathways, most notably JAK-dependent proinflammatory signaling. We conclude that JAK1 inhibition is sufficient to curtail CpG-induced disease, but combined inhibition of JAK1 and JAK2 is needed to best control LCMV-induced immunopathology.

Outcomes in patients with ETV6::RUNX1 or high-hyperdiploid B-ALL treated in the St. Jude Total Therapy XV/XVI studies.

Children with ETV6::RUNX1 or high-hyperdiploid B-acute lymphoblastic leukemia (B-ALL) have favorable outcomes. The St. Jude (SJ) classification considers these patients low-risk, regardless of their National Cancer Institute (NCI) risk, except when there is slow minimal residual disease (MRD) response or central nervous system/testicular involvement. We analyzed outcomes in children (aged 1-18.99 years) with these genotypes in the SJ Total XV and XVI studies (2000-2017). Patients with ETV6::RUNX1 (n = 222) or high-hyperdiploid (n = 296) B-ALL had 5-year event-free survival (EFS) of 97.7% ± 1.1% and 94.7% ± 1.4%, respectively. For ETV6::RUNX1, EFS was comparable for NCI standard-risk and high-risk patients (97.8% ± 1.2% and 97.5% ± 2.6%, respectively; P = 0.917) and for SJ low-risk and standard-risk patients (97.4% ± 1.2% and 100.0%; P = 0.360). Thirty-seven of 40 NCI high-risk patients who received SJ low-risk therapy had excellent EFS (97.3% ± 2.8%). For high-hyperdiploid B-ALL, EFS was worse for NCI high-risk patients than for standard-risk patients (87.6% ± 4.5% and 96.4% ± 1.3%; P = 0.016). EFS was similar for NCI standard-risk and high-risk patients classified as SJ low-risk (96.0% ± 1.5% and 96.9% ± 3.2%; P = 0.719); however, EFS was worse for NCI high-risk patients than for NCI standard-risk patients receiving SJ standard/high-risk therapy (77.4% ± 8.2% and 98.0% ± 2.2%; P = 0.004). NCI high-risk patients with ETV6::RUNX1 or high-hyperdiploid B-ALL who received SJ low-risk therapy had lower incidences of thrombosis (P = 0.013) and pancreatitis (P = 0.011) than those who received SJ standard/high-risk therapy. Contemporary MRD-directed therapy yielded excellent outcomes, except for NCI high-risk high-hyperdiploid B-ALL patients with slow MRD response, who require new treatment approaches. Among NCI high-risk patients, 93% with ETV6::RUNX1 and 54% with high-hyperdiploid B-ALL experienced excellent outcomes with a low-intensity regimen. These trials were registered at www.clinicaltrials.gov as #NCT00137111 and #NCT00549848.

Microenvironment Engineering of Heterogeneous Catalysts for Liquid-Phase Environmental Catalysis.

Environmental catalysis has emerged as a scientific frontier in mitigating water pollution and advancing circular chemistry and reaction microenvironment significantly influences the catalytic performance and efficiency. This review delves into microenvironment engineering within liquid-phase environmental catalysis, categorizing microenvironments into four scales: atom/molecule-level modulation, nano/microscale-confined structures, interface and surface regulation, and external field effects. Each category is analyzed for its unique characteristics and merits, emphasizing its potential to significantly enhance catalytic efficiency and selectivity. Following this overview, we introduced recent advancements in advanced material and system design to promote liquid-phase environmental catalysis (e.g., water purification, transformation to value-added products, and green synthesis), leveraging state-of-the-art microenvironment engineering technologies. These discussions showcase microenvironment engineering was applied in different reactions to fine-tune catalytic regimes and improve the efficiency from both thermodynamics and kinetics perspectives. Lastly, we discussed the challenges and future directions in microenvironment engineering. This review underscores the potential of microenvironment engineering in intelligent materials and system design to drive the development of more effective and sustainable catalytic solutions to environmental decontamination.

Low-sugar universal mRNA vaccine against coronavirus variants with deletion of glycosites in the S2 or stem of SARS-CoV-2 spike messenger RNA (mRNA).

Since the outbreak of Severe Acute Respiratory Syndrome Virus-2 (SARS-CoV-2) in 2019, more than 15 million spike protein sequences have been identified, raising a new challenge for the development of a broadly protective vaccine against the various emerging variants. We found that the virus, like most other human viruses, depends on host-made glycans to shield the conserved epitopes on spike protein from immune response and demonstrated that deletion of the glycan shields exposed highly conserved epitopes and elicited broadly protective immune responses. In this study, we identified 17 conserved epitopes from 14 million spike protein sequences and 11 of the conserved epitopes are in the S2 domain, including the six most conserved epitopes in the stem region. We also demonstrated that deletion of the glycosites in the spike messenger RNA (mRNA) S2 domain or the stem region exposed the highly conserved epitopes and elicited broadly protective immune responses, particularly CD-8+ T cell response against various SARS-CoV-2 variants, and other human coronaviruses including MERS, SARS viruses, and those causing common cold.

Structure-based design of a single-chain triple-disulfide-stabilized fusion-glycoprotein trimer that elicits high-titer neutralizing responses against human metapneumovirus.

The Pneumoviridae family of viruses includes human metapneumovirus (HMPV) and respiratory syncytial virus (RSV). The closely related Paramyxoviridae family includes parainfluenza viruses (PIVs). These three viral pathogens cause acute respiratory tract infections with substantial disease burden in the young, the elderly, and the immune-compromised. While promising subunit vaccines are being developed with prefusion-stabilized forms of the fusion glycoproteins (Fs) of RSV and PIVs, for which neutralizing titers elicited by the prefusion (pre-F) conformation of F are much higher than for the postfusion (post-F) conformation, with HMPV, pre-F and post-F immunogens described thus far elicit similar neutralizing responses, and it has been unclear which conformation, pre-F or post-F, would be the most effective HMPV F-vaccine immunogen. Here, we investigate the impact of further stabilizing HMPV F in the pre-F state. We replaced the furin-cleavage site with a flexible linker, creating a single chain F that yielded increased amounts of pre-F stabilized trimers, enabling the generation and assessment of F trimers stabilized by multiple disulfide bonds. Introduced prolines could increase both expression yields and antigenic recognition by the pre-F specific antibody, MPE8. The cryo-EM structure of a triple disulfide-stabilized pre-F trimer with the variable region of antibody MPE8 at 3.25-Å resolution confirmed the formation of designed disulfides and provided structural details on the MPE8 interface. Immunogenicity assessments in naïve mice showed the triple disulfide-stabilized pre-F trimer could elicit high titer neutralization, >10-fold higher than elicited by post-F. Immunogenicity assessments in pre-exposed rhesus macaques showed the triple disulfide-stabilized pre-F could recall high neutralizing titers after a single immunization, with little discrimination in the recall response between pre-F and post-F immunogens. However, the triple disulfide-stabilized pre-F adsorbed HMPV-directed responses from commercially available pooled human immunoglobulin more fully than post-F. Collectively, these results suggest single-chain triple disulfide-stabilized pre-F trimers to be promising HMPV-vaccine antigens.

A TGF-ß-dominant chemoresistant phenotype of hepatoblastoma associated with aflatoxin exposure in children.

BACKGROUND AND AIMS: Hepatoblastoma (HB) is the most common liver cancer in children, posing a serious threat to children's health. Chemoresistance is the leading cause of mortality in patients with HB. A more explicit definition of the features of chemotherapy resistance in HB represents a fundamental urgent need. APPROACH AND RESULTS: We performed an integrative analysis including single-cell RNA sequencing, whole-exome sequencing, and bulk RNA sequencing in 180 HB samples, to reveal genomic features, transcriptomic profiles, and the immune microenvironment of HB. Multicolor immunohistochemistry staining and in vitro experiments were performed for validation. Here, we reported four HB transcriptional subtypes primarily defined by differential expression of transcription factors. Among them, the S2A subtype, characterized by strong expression of progenitor ( MYCN , MIXL1 ) and mesenchymal transcription factors ( TWIST1 , TBX5 ), was defined as a new chemoresistant subtype. The S2A subtype showed increased TGF-ß cancer-associated fibroblast and an immunosuppressive microenvironment induced by the upregulated TGF-ß of HB. Interestingly, the S2A subtype enriched SBS24 signature and significantly higher serum aflatoxin B1-albumin (AFB1-ALB) level in comparison with other subtypes. Functional assays indicated that aflatoxin promotes HB to upregulate TGF-ß. Furthermore, clinical prognostic analysis showed that serum AFB1-ALB is a potential indicator of HB chemoresistance and prognosis. CONCLUSIONS: Our studies offer new insights into the relationship between aflatoxin and HB chemoresistance and provide important implications for its diagnosis and treatment.

A nanopore-based cucumber genome assembly reveals structural variations at two QTLs controlling hypocotyl elongation.

The Xishuangbanna (XIS) cucumber (Cucumis sativus var. xishuangbannanesis) is a semiwild variety that has many distinct agronomic traits. Here, long reads generated by Nanopore sequencing technology helped assembling a high-quality genome (contig N50 = 8.7 Mb) of landrace XIS49. A total of 10,036 structural/sequence variations (SVs) were identified when comparing with Chinese Long (CL), and known SVs controlling spines, tubercles, and carpel number were confirmed in XIS49 genome. Two QTLs of hypocotyl elongation under low light, SH3.1 and SH6.1, were fine-mapped using introgression lines (donor parent, XIS49; recurrent parent, CL). SH3.1 encodes a red-light receptor Phytochrome B (PhyB, CsaV3_3G015190). A ∼4 kb region with large deletion and highly divergent regions (HDRs) were identified in the promoter of the PhyB gene in XIS49. Loss of function of this PhyB caused a super-long hypocotyl phenotype. SH6.1 encodes a CCCH-type zinc finger protein FRIGIDA-ESSENTIAL LIKE (FEL, CsaV3_6G050300). FEL negatively regulated hypocotyl elongation but it was transcriptionally suppressed by long terminal repeats retrotransposon insertion in CL cucumber. Mechanistically, FEL physically binds to the promoter of CONSTITUTIVE PHOTOMORPHOGENIC 1a (COP1a), regulating the expression of COP1a and the downstream hypocotyl elongation. These above results demonstrate the genetic mechanism of cucumber hypocotyl elongation under low light.

The genomic landscape of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21.

Intrachromosomal amplification of chromosome 21 defines a subtype of high-risk childhood acute lymphoblastic leukemia (iAMP21-ALL) characterized by copy number changes and complex rearrangements of chromosome 21. The genomic basis of iAMP21-ALL and the pathogenic role of the region of amplification of chromosome 21 to leukemogenesis remains incompletely understood. In this study, using integrated whole genome and transcriptome sequencing of 124 patients with iAMP21-ALL, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL based on the patterns of copy number alteration and structural variation. This large data set enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which were differentially expressed compared with non-iAMP21-ALL ones, including multiple genes implicated in the pathogenesis of acute leukemia (CHAF1B, DYRK1A, ERG, HMGN1, and RUNX1). Using multimodal single-cell genomic profiling, including single-cell whole genome sequencing of 2 cases, we documented clonal heterogeneity and genomic evolution, demonstrating that the acquisition of the iAMP21 chromosome is an early event that may undergo progressive amplification during disease ontogeny. We show that UV-mutational signatures and high mutation load are characteristic secondary genetic features. Although the genomic alterations of chromosome 21 are variable, these integrated genomic analyses and demonstration of an extended common minimal region of amplification broaden the definition of iAMP21-ALL for more precise diagnosis using cytogenetic or genomic methods to inform clinical management.

Correction of Ito in human induced pluripotent stem Cell-derived cardiomyocyte carrying DPP6 mutation in early repolarization syndrome by CRISPR/Cas9 genome editing.

Early repolarization syndrome (ERS) is defined as occurring in patients with early repolarization pattern who have survived idiopathic ventricular fibrillation with clinical evaluation unrevealing for other explanations. The pathophysiologic basis of the ERS is currently uncertain. The objective of the present study was to examine the electrophysiological mechanism of ERS utilizing induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 genome editing. Whole genome sequencing was used to identify the DPP6 (c.2561T > C/p.L854P) variant in four families with sudden cardiac arrest induced by ERS. Cardiomyocytes were generated from iPSCs from a 14-year-old boy in the four families with ERS and an unrelated healthy control subject. Patch clamp recordings revealed more significant prolongation of the action potential duration (APD) and increased transient outward potassium current (Ito) (103.97 ± 18.73 pA/pF vs 44.36 ± 16.54 pA/pF at +70 mV, P < 0.05) in ERS cardiomyocytes compared with control cardiomyocytes. Of note, the selective correction of the causal variant in iPSC-derived cardiomyocytes using CRISPR/Cas9 gene editing normalized the Ito, whereas prolongation of the APD remained unchanged. ERS cardiomyocytes carrying DPP6 mutation increased Ito and lengthen APD, which maybe lay the electrophysiological foundation of ERS.

α-Synuclein reduces acetylserotonin O-methyltransferase mediated melatonin biosynthesis by microtubule-associated protein 1 light chain 3 beta-related degradation pathway.

Previous studies have demonstrated that α-synuclein (α-SYN) is closely associated with rapid eye movement sleep behavior disorder (RBD) related to several neurodegenerative disorders. However, the exact molecular mechanisms are still rarely investigated. In the present study, we found that in the α-SYNA53T induced RBD-like behavior mouse model, the melatonin level in the plasma and pineal gland were significantly decreased. To elucidate the underlying mechanism of α-SYN-induced melatonin reduction, we investigated the effect of α-SYN in melatonin biosynthesis. Our findings showed that α-SYN reduced the level and activity of melatonin synthesis enzyme acetylserotonin O-methyltransferase (ASMT) in the pineal gland and in the cell cultures. In addition, we found that microtubule-associated protein 1 light chain 3 beta (LC3B) as an important autophagy adapter is involved in the degradation of ASMT. Immunoprecipitation assays revealed that α-SYN increases the binding between LC3B and ASMT, leading to ASMT degradation and a consequent reduction in melatonin biosynthesis. Collectively, our results demonstrate the molecular mechanisms of α-SYN in melatonin biosynthesis, indicating that melatonin is an important molecule involved in the α-SYN-associated RBD-like behaviors, which may provide a potential therapeutic target for RBD of Parkinson's disease.

P300 Interacted With N-Myc and Regulated Its Protein Stability via Altering Its Post-Translational Modifications in Neuroblastoma.

MYCN amplification is an independent risk factor for poor prognosis in neuroblastoma (NB), but its protein product cannot be directly targeted because of protein structure. Thus, this study aimed to explore novel ways to indirectly target N-Myc by regulating its post-translational modifications (PTMs) and therefore protein stability. N-Myc coimmunoprecipitation combined with HPLC-MS/MS identified 16 PTM residues and 114 potential N-Myc-interacting proteins. Notably, both acetylation and ubiquitination were identified on lysine 199 of N-Myc. We then discovered that p300, which can interact with N-Myc, modulated the protein stability of N-Myc in MYCN-amplified NB cell lines and simultaneously regulated the acetylation level and ubiquitination level on lysine-199 of N-Myc protein in vitro. Furthermore, p300 correlated with poor prognosis in NB patients. Taken together, p300 can be considered as a potential therapeutic target to treat MYCN-amplified NB patients, and other identified PTMs and interacting proteins also provide potential targets for further study.

Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility.

DNA double-strand breaks (DSBs) are functionally linked to genomic instability in spermatocytes and to male infertility. The heavy metal cadmium (Cd) is known to induce DNA damage in spermatocytes by unknown mechanisms. Here, we showed that Cd ions impaired the canonical non-homologous end-joining (NHEJ) repair pathway, but not the homologous recombination (HR) repair pathway, through stimulation of Ser2056 and Thr2609 phosphorylation of DNA-PKcs at DSB sites. Hyper-phosphorylation of DNA-PKcs led to its premature dissociation from DNA ends and the Ku complex, preventing recruitment of processing enzymes and further ligation of DNA ends. Specifically, this cascade was initiated by the loss of PP5 phosphatase activity, which results from the dissociation of PP5 from its activating ions (Mn), that is antagonized by Cd ions through a competitive mechanism. In accordance, in a mouse model Cd-induced genomic instability and consequential male reproductive dysfunction were effectively reversed by a high dosage of Mn ions. Together, our findings corroborate a protein phosphorylation-mediated genomic instability pathway in spermatocytes that is triggered by exchange of heavy metal ions.

Glycosite-deleted mRNA of SARS-CoV-2 spike protein as a broad-spectrum vaccine.

Development of the messenger RNA (mRNA) vaccine has emerged as an effective and speedy strategy to control the spread of new pathogens. After vaccination, the mRNA is translated into the real protein vaccine, and there is no need to manufacture the protein in vitro. However, the fate of mRNA and its posttranslational modification inside the cell may affect immune response. Here, we showed that the mRNA vaccine of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with deletion of glycosites in the receptor-binding domain (RBD) or especially the subunit 2 (S2) domain to expose more conserved epitopes elicited stronger antibody and CD8+ T cell responses with broader protection against the alpha, beta, gamma, delta, and omicron variants, compared to the unmodified mRNA. Immunization of such mRNA resulted in accumulation of misfolded spike protein in the endoplasmic reticulum, causing the up-regulation of BiP/GRP78, XBP1, and p-eIF2α to induce cell apoptosis and strong CD8+ T cell response. In addition, dendritic cells (DCs) incubated with S2-glysosite deleted mRNA vaccine increased class I major histocompatibility complex (MHC I) expression. This study provides a direction for the development of broad-spectrum mRNA vaccines which may not be achieved with the use of expressed proteins as antigens.