Functional rejuvenation of aged neural stem cells by Plagl2 and anti-Dyrk1a activity.

The regenerative potential of neural stem cells (NSCs) declines during aging, leading to cognitive dysfunctions. This decline involves up-regulation of senescence-associated genes, but inactivation of such genes failed to reverse aging of hippocampal NSCs. Because many genes are up-regulated or down-regulated during aging, manipulation of single genes would be insufficient to reverse aging. Here we searched for a gene combination that can rejuvenate NSCs in the aged mouse brain from nuclear factors differentially expressed between embryonic and adult NSCs and their modulators. We found that a combination of inducing the zinc finger transcription factor gene Plagl2 and inhibiting Dyrk1a, a gene associated with Down syndrome (a genetic disorder known to accelerate aging), rejuvenated aged hippocampal NSCs, which already lost proliferative and neurogenic potential. Such rejuvenated NSCs proliferated and produced new neurons continuously at the level observed in juvenile hippocampi, leading to improved cognition. Epigenome, transcriptome, and live-imaging analyses indicated that this gene combination induces up-regulation of embryo-associated genes and down-regulation of age-associated genes by changing their chromatin accessibility, thereby rejuvenating aged dormant NSCs to function like juvenile active NSCs. Thus, aging of NSCs can be reversed to induce functional neurogenesis continuously, offering a way to treat age-related neurological disorders.

A nanobody interaction with SARS-COV-2 Spike allows the versatile targeting of lentivirus vectors.

While investigating methods to target gene delivery vectors to specific cell types, we examined the potential of using a nanobody against the SARS-CoV-2 Spike protein receptor-binding domain to direct lentivirus infection of Spike-expressing cells. Using four different approaches, we found that lentiviruses with surface-exposed nanobody domains selectively infect Spike-expressing cells. Targeting is dependent on the fusion function of the Spike protein, and conforms to a model in which nanobody binding to the Spike protein triggers the Spike fusion machinery. The nanobody-Spike interaction also is capable of directing cell-cell fusion and the selective infection of nanobody-expressing cells by Spike-pseudotyped lentivirus vectors. Significantly, cells infected with SARS-CoV-2 are efficiently and selectively infected by lentivirus vectors pseudotyped with a chimeric nanobody protein. Our results suggest that cells infected by any virus that forms syncytia may be targeted for gene delivery by using an appropriate nanobody or virus receptor mimic. Vectors modified in this fashion may prove useful in the delivery of immunomodulators to infected foci to mitigate the effects of viral infections.IMPORTANCEWe have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.

Secondary failure of lentiviral vector gene therapy in a cerebral adrenoleukodystrophy patient with an ABCD1 whole-gene deletion.

A 9-year-old boy with adrenoleukodystrophy due to ABCD1 whole-gene deletion was diagnosed with active cerebral adrenoleukodystrophy characterized by demyelination and gadolinium enhancement on brain MRI. He underwent hematopoietic cell transplant (HCT) with autologous CD34+ cells transduced with an ABCD1-expressing lentiviral vector (eli-cel [elivaldogene autotemcel]) as part of the ALD-104 clinical trial. Fifty days after HCT, the patient's MRI showed gadolinium resolution; the whole-blood vector copy number (VCN) was 0.666 copies/mL. Six months following HCT, an MRI showed re-emergence of gadolinium enhancement; the VCN had decreased to 0.029 copies/mL. Polyclonal antibodies to the ABCD1 gene product were detectable 9 months after transplant, showing reactivity to peroxisomes, suggesting an immune response; however, no antibody binding to human CD34+ cells could be shown. The patient underwent a successful allogeneic HCT 12 months after gene therapy with resultant gadolinium resolution, cerebral disease stabilization, and the disappearance of antibodies. The coincident VCN loss and appearance of antibody to the ABCD1 gene product is of interest, and we postulate that it is related to the patient's whole ABCD1 gene deletion. We suggest close monitoring of loss of gene therapy efficacy due to immune response in patients with full deletions who are considering gene therapy.

A CRISPR Resource for Individual, Combinatorial, or Multiplexed Gene Knockout.

We have combined a machine-learning approach with other strategies to optimize knockout efficiency with the CRISPR/Cas9 system. In addition, we have developed a multiplexed sgRNA expression strategy that promotes the functional ablation of single genes and allows for combinatorial targeting. These strategies have been combined to design and construct a genome-wide, sequence-verified, arrayed CRISPR library. This resource allows single-target or combinatorial genetic screens to be carried out at scale in a multiplexed or arrayed format. By conducting parallel loss-of-function screens, we compare our approach to existing sgRNA design and expression strategies.

Lentiviral Gene Therapy for Cystic Fibrosis: A Promising Approach and First-In-Human Trial.

Cystic fibrosis is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. While cystic fibrosis is a multi-organ disease, the leading causes of morbidity and mortality are related to progressive lung disease. Current understanding of the effects of the broad spectrum of CFTR mutations on CFTR function has allowed for the development of CFTR modulator therapies. Despite the remarkable impact that these therapies have had, there remains a significant proportion of people with cystic fibrosis (estimated at 10-15% of the global cystic fibrosis population) who are genetically ineligible for, or intolerant to, current CFTR-targeting therapies and whose therapeutic needs remain unmet. Inhaled genetic therapies offer the prospect of addressing the unmet pulmonary treatment need in people with cystic fibrosis, with several approaches, including gene addition therapy (the focus of this review), RNA-based therapies, antisense oligonucleotides and gene editing, being explored. Various non-viral and viral vectors have been investigated for cystic fibrosis gene addition therapy for mutation-agnostic restoration of CFTR function in the lungs. Lentiviral vectors offer the prospect of highly efficient and long-lasting gene expression, and the potential to be safely and, in contrast to other commonly used viral vectors, effectively re-dosed. A third-generation lentiviral vector pseudotyped with Sendai virus F and HN envelope proteins (rSIV.F/HN) has been developed for the treatment of cystic fibrosis. Promising preclinical results support the progression of this vector carrying a full-length CFTR transgene (BI 3720931) into a first-in-human clinical trial expected to begin in 2024.

V-CARMA: A tool for the detection and modification of antigen-specific T cells.

T cells promote our body's ability to battle cancers and infectious diseases but can act pathologically in autoimmunity. The recognition of peptides presented by major histocompatibility complex (pMHC) molecules by T cell receptors (TCRs) enables T cell-mediated responses. To modify disease-relevant T cells, new tools to genetically modify T cells and decode their antigen recognition are needed. Here, we present an approach using viruses pseudotyped with peptides loaded on MHC called V-CARMA (Viral ChimAeric Receptor MHC-Antigen) to specifically target T cells expressing cognate TCRs for antigen discovery and T cell engineering. We show that lentiviruses displaying antigens on human leukocyte antigen (HLA) class I and class II molecules can robustly infect CD8+ and CD4+ T cells expressing cognate TCRs, respectively. The infection rates of the pseudotyped lentiviruses (PLVs) are correlated with the binding affinity of the TCR to its cognate antigen. Furthermore, peptide-HLA pseudotyped lentivirus V-CARMA constructs can identify target cells from a mixed T cell population, suppress PD-1 expression on CD8+ T cells via PDCD1 shRNA delivery, and induce apoptosis in autoreactive CD4+ T cells. Thus, V-CARMA is a versatile tool for TCR ligand identification and selective T cell manipulation.

Phylogenetic Reconstruction and Functional Characterization of the Ancestral Nef Protein of Primate Lentiviruses.

Nef is an accessory protein unique to the primate HIV-1, HIV-2, and SIV lentiviruses. During infection, Nef functions by interacting with multiple host proteins within infected cells to evade the immune response and enhance virion infectivity. Notably, Nef can counter immune regulators such as CD4 and MHC-I, as well as the SERINC5 restriction factor in infected cells. In this study, we generated a posterior sample of time-scaled phylogenies relating SIV and HIV Nef sequences, followed by reconstruction of ancestral sequences at the root and internal nodes of the sampled trees up to the HIV-1 Group M ancestor. Upon expression of the ancestral primate lentivirus Nef protein within CD4+ HeLa cells, flow cytometry analysis revealed that the primate lentivirus Nef ancestor robustly downregulated cell-surface SERINC5, yet only partially downregulated CD4 from the cell surface. Further analysis revealed that the Nef-mediated CD4 downregulation ability evolved gradually, while Nef-mediated SERINC5 downregulation was recovered abruptly in the HIV-1/M ancestor. Overall, this study provides a framework to reconstruct ancestral viral proteins and enable the functional characterization of these proteins to delineate how functions could have changed throughout evolutionary history.

A highly sensitive reporter system to monitor endogenous YAP1/TAZ activity and its application in various human cells.

The activation of yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) has been implicated in both regeneration and tumorigenesis, thus representing a double-edged sword in tissue homeostasis. However, how the activity of YAP1/TAZ is regulated or what leads to its dysregulation in these processes remains unknown. To explore the upstream stimuli modulating the cellular activity of YAP1/TAZ, we developed a highly sensitive YAP1/TAZ/TEAD-responsive DNA element (YRE) and incorporated it into a lentivirus-based reporter cell system to allow for sensitive and specific monitoring of the endogenous activity of YAP1/TAZ in terms of luciferase activity in vitro and Venus fluorescence in vivo. Furthermore, by replacing YRE with TCF- and NF-κB-binding DNA elements, we demonstrated the applicability of this reporter system to other pathways such as Wnt/ß-catenin/TCF- and IL-1ß/NF-κB-mediated signaling, respectively. The practicality of this system was evaluated by performing cell-based reporter screening of a chemical compound library consisting of 364 known inhibitors, using reporter-introduced cells capable of quantifying YAP1/TAZ- and ß-catenin-mediated transcription activities, which led to the identification of multiple inhibitors, including previously known as well as novel modulators of these signaling pathways. We further confirmed that novel YAP1/TAZ modulators, such as potassium ionophores, Janus kinase inhibitors, platelet-derived growth factor receptor inhibitors, and genotoxic stress inducers, alter the protein level or phosphorylation of endogenous YAP1/TAZ and the expression of their target genes. Thus, this reporter system provides a powerful tool to monitor endogenous signaling activities of interest (even in living cells) and search for modulators in various cellular contexts.

Overexpression of multidrug resistance-associated protein 1 protects against cardiotoxicity by augmenting the doxorubicin efflux from cardiomyocytes.

Doxorubicin is a commonly used anti-cancer drug used in treating a variety of malignancies. However, a major adverse effect is cardiotoxicity, which is dose dependent and can be either acute or chronic. Doxorubicin causes injury by DNA damage, the formation of free reactive oxygen radicals and induction of apoptosis. Our aim is to induce expression of the multidrug resistance-associated protein 1 (MRP1) in cardiomyocytes derived from human iPS cells (hiPSC-CM), to determine whether this will allow cells to effectively remove doxorubicin and confer cardioprotection. We generated a lentivirus vector encoding MRP1 (LV.MRP1) and validated its function in HEK293T cells and stem cell-derived cardiomyocytes (hiPSC-CM) by quantitative PCR and western blot analysis. The activity of the overexpressed MRP1 was also tested, by quantifying the amount of fluorescent dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. Finally, we demonstrated that hiPSC-CM transduced with LV.MRP1 were protected against doxorubicin injury. In conclusion, we have shown that we can successfully overexpress MRP1 protein in hiPSC-CM, with functional transporter activity leading to protection against doxorubicin-induced toxicity.

Enhancing Endogenous Hyaluronic Acid in Osteoarthritic Joints with an Anti-Inflammatory Supramolecular Nanofiber Hydrogel Delivering HAS2 Lentivirus.

Osteoarthritis (OA) management remains challenging because of its intricate pathogenesis. Intra-articular injections of drugs, such as glucocorticoids and hyaluronic acid (HA), have certain limitations, including the risk of joint infection, pain, and swelling. Hydrogel-based therapeutic strategies have attracted considerable attention because of their enormous therapeutic potential. Herein, a supramolecular nanofiber hydrogel is developed using dexamethasone sodium phosphate (DexP) as a vector to deliver lentivirus-encoding hyaluronan synthase 2 (HAS2) (HAS2@DexP-Gel). During hydrogel degradation, HAS2 lentivirus and DexP molecules are slowly released. Intra-articular injection of HAS2@DexP-Gel promotes endogenous HA production and suppresses synovial inflammation. Additionally, HAS2@DexP-Gel reduces subchondral bone resorption in the anterior cruciate ligament transection-induced OA mice, attenuates cartilage degeneration, and delays OA progression. HAS2@DexP-Gel exhibited good biocompatibility both in vitro and in vivo. The therapeutic mechanisms of the HAS2@DexP-Gel are investigated using single-cell RNA sequencing. HAS2@DexP-Gel optimizes the microenvironment of the synovial tissue by modulating the proportion of synovial cell subpopulations and regulating the interactions between synovial fibroblasts and macrophages. The innovative nanofiber hydrogel, HAS2@DexP-Gel, effectively enhances endogenous HA production while reducing synovial inflammation. This comprehensive approach holds promise for improving joint function, alleviating pain, and slowing OA progression, thereby providing significant benefits to patients.

Generation of SARS-CoV-2 spike receptor binding domain mutants and functional screening for immune evaders using a novel lentivirus-based system.

The emergence of rapid and continuous mutations of severe acute respiratory syndrome 2 (SARS-CoV-2) spike glycoprotein that increased with the Omicron variant points out the necessity to anticipate such mutations for conceiving specific and adaptable therapies to avoid another pandemic. The crucial target for the antibody treatment and vaccine design is the receptor binding domain (RBD) of the SARS-CoV-2 spike. It is also the site where the virus has shown its high ability to mutate and consequently escape immune response. We developed a robust and simple method for generating a large number of functional SARS-CoV-2 spike RBD mutants by error-prone PCR and a novel nonreplicative lentivirus-based system. We prepared anti-RBD wild type (WT) polyclonal antibodies and used them to screen and select for mutant libraries that escape inhibition of virion entry into recipient cells expressing human angiotensin-converting enzyme 2 and transmembrane serine protease 2. We isolated, cloned, and sequenced six mutants totally bearing nine mutation sites. Eight mutations were found in successive WT variants, including Omicron and other recombinants, whereas one is novel. These results, together with the detailed functional analyses of two mutants provided the proof of concept for our approach.

The evolving landscape of gene therapy strategies for the treatment of osteoarthritis.

OBJECTIVES: Significant advances have been made in our understanding of osteoarthritis (OA) pathogenesis; however, no disease-modifying therapies have been identified. This review will summarize the gene therapy landscape, its initial successes for OA, and possible challenges using recent studies and examples of gene therapies in clinical trials. DESIGN: This narrative review has three major sections: 1) vector systems for OA gene therapy, 2) current and emerging targets for OA gene therapy, and 3) considerations and future directions. RESULTS: Gene therapy is the strategy by which nucleic acids are delivered to treat and reverse disease progression. Specificity and prolonged expression of these nucleic acids are achieved by manipulating promoters, genes, and vector systems. Certain vector systems also allow for the development of combinatorial nucleic acid strategies that can be delivered in a single intraarticular injection - an approach likely required to treat the complexity of OA pathogenesis. Several viral and non-viral vector-based gene therapies are in clinical trials for OA, and many more are being evaluated in the preclinical arena. CONCLUSIONS: In a post-coronavirus disease 2019 (COVID-19) era, the future of gene therapy for OA is certainly promising; however, the majority of preclinical validation continues to focus heavily on post-traumatic models and changes in only cartilage and subchondral bone. To ensure successful translation, new candidates in the preclinical arena should be examined against all joint tissues as well as pain using diverse models of injury-, obesity-, and age-induced disease. Lastly, consideration must be given to strategies for repeat administration and the cost of treatment owing to the chronic nature of OA.

Targeting the long non-coding RNA MIAT for the treatment of fibroids in an animal model.

Our previous studies indicated that there is overexpression of MIAT in fibroids and MIAT is a sponge for the miR-29 family in these tumors. The objective of the present study was to determine if the knockdown of MIAT in fibroid xenografts will increase miR-29 levels and reduce the expression of genes targeted by this miRNA such as collagen and cell cycle regulatory proteins in a mouse model for fibroids. Ovariectomized CB-17 SCID/Beige mice bearing estrogen/progesterone pellets were implanted subcutaneously in the flank with equal weight of fibroid explants which had been transduced by lentivirus for either control (empty vector) or MIAT knockdown for four weeks (n=7). Knockdown of MIAT in fibroid xenografts resulted in a 30% reduction of tumor weight and a marked increase in miR-29a, -b, and -c levels in the xenografts. There was reduced cell proliferation and expression of cell cycle regulatory genes CCND1, CDK2, and E2F1 and no significant changes in apoptosis. The xenografts with MIAT knockdown expressed lower mRNA and protein levels of FN1, COL3A1, and TGF-ß3, and total collagen protein. Targeting MIAT, which sponges the pro-fibrotic miR-29 family, is an effective therapy for fibroids by reducing cell proliferation and thereby, tumor growth and accumulation of ECM, which is a hallmark of these benign gynecologic tumors.

Hmga1-overexpressing lentivirus protects against osteoporosis by activating the Wnt/ß-catenin pathway in the osteogenic differentiation of BMSCs.

Postmenopausal osteoporosis is associated with bone formation inhibition mediated by the impaired osteogenic differentiation potential of bone marrow mesenchymal stem cells (BMSCs). However, identifying and confirming the essential genes in the osteogenic differentiation of BMSCs and osteoporosis remain challenging. The study aimed at revealing the key gene that regulated osteogenic differentiation of BMSCs and led to osteoporosis, thus exploring its therapeutic effect in osteoporosis. In the present study, six essential genes related to the osteogenic differentiation of BMSCs and osteoporosis were identified, namely, fibrillin 2 (Fbn2), leucine-rich repeat-containing 17 (Lrrc17), heat shock protein b7 (Hspb7), high mobility group AT-hook 1 (Hmga1), nexilin F-actin-binding protein (Nexn), and endothelial cell-specific molecule 1 (Esm1). Furthermore, the in vivo and in vitro experiments showed that Hmga1 expression was increased during the osteogenic differentiation of rat BMSCs, while Hmga1 expression was decreased in the bone tissue of ovariectomized (OVX) rats. Moreover, the expression of osteogenic differentiation-related genes, the activity of alkaline phosphatase (ALP), and the number of mineralized nodules were increased after Hmga1 overexpression, which was partially reversed by a Wnt signaling inhibitor (DKK1). In addition, after injecting Hmga1-overexpressing lentivirus into the bone marrow cavity of OVX rats, the bone loss, and osteogenic differentiation inhibition of BMSCs in OVX rats were partially reversed, while osteoclast differentiation promotion of BMSCs in OVX rats was unaffected. Taken together, the present study confirms that Hmga1 prevents OVX-induced bone loss by the Wnt signaling pathway and reveals that Hmga1 is a potential gene therapeutic target for postmenopausal osteoporosis.

Peptide ligands targeting the vesicular stomatitis virus G (VSV-G) protein for the affinity purification of lentivirus particles.

The recent uptick in the approval of ex vivo cell therapies highlights the relevance of lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification-currently reliant on filtration and anion-exchange or size-exclusion chromatography-suffers from long process times and low yield of transducing particles, which translate into high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50%-60% of viral genomes; 40%-50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·109 TU per mL of resin, or 5·1011 vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality.

Lentiviral vector determinants of anion-exchange chromatography elution heterogeneity.

The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G]) did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).

Fate control engagement augments NK cell responses in LV/hu-IL-12 transduced sarcoma.

INTRODUCTION: NK cells are an untapped resource for cancer therapy. Sarcomas transduced with lentiviruses to express human IL-12 are only cleared in mice bearing mature human NK cells. However, systemic inflammation limits IL-12 utilization. Fate control a.k.a. "suicide mechanisms" regulate unchecked systemic inflammation caused by cellular immunotherapies. Despite increasing utilization, there remains limited data on immune consequences or tumor-directed effects of fate control. OBJECTIVES: We sought to engage the mutant thymidylate kinase (mTMPK) metabolic fate control system to regulate systemic inflammation and assess the impact on NK cell effector functions. METHODS: Primary human sarcoma short-passage samples and cell lines were transduced with LV/hu-IL-12_mTMPK engineering expression of IL-12 and an AZT-associated fate control enzyme. We assessed transduced sarcoma responses to AZT engagement and subsequent modulation of NK cell functions as measured by inflammatory cytokine production and cytotoxicity. RESULTS: AZT administration to transduced (LV/hu-IL-12_mTMPK) short-passage primary human sarcomas and human Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines, abrogated the robust expression of human IL-12. Fate control activation elicited a specific dose-dependent cytotoxic effect measured by metabolic activity (WST-1) and cell death (Incucyte). NK effector functions of IFN-γ and cytotoxic granule release were significantly augmented despite IL-12 abrogation. This correlated with preferentially induced expression of NK cell activation ligands. CONCLUSIONS: mTMPK fate control engagement terminates transduced sarcoma IL-12 production and triggers cell death, but also augments an NK cell-mediated response coinciding with metabolic stress activating surface ligand induction. Fate control engagement could offer a novel immune activation method for NK cell-mediated cancer clearance.

Analysis of the Expression of PRDX6 in Patients with Hepatocellular Carcinoma and its Effect on the Phenotype of Hepatocellular Carcinoma Cells.

Objectives: This research aimed to study the expression of PRDX6 mRNA in hepatocellular carcinoma (HCC) and its effect on the prognosis of HCC. Moreover, the effect of PRDX6 gene knockdown on the proliferation, migration, and invasion of HepG2 cells mediated by lentivirus was also examined. This study offers a theoretical and experimental basis for further research on the mechanism of PRDX6 in liver cancer and new methods for clinical diagnosis and treatment. Methods: RNA sequence data of 369 HCC patients were screened through the TCGA database, and the expression and clinical characteristics of PRDX6 mRNA were analyzed based on high-throughput RNA sequencing data. HepG2 cells were divided into WT, sh-NC and sh-PRDX6 groups. Real-time PCR and Western blot were used to detect the expression levels of the PRDX6 gene and protein, respectively. CCK8 method was used to detect the proliferation activity of HepG2 cells, scratch healing test was used to detect the migration ability, Transwell chamber was used to detect the invasion ability, and Western blot was used to detect the expression levels of PI3K/Akt/mTOR signaling pathway and Notch signaling pathway-related proteins. Results: The expression of PRDX6 was significantly correlated with the gender, race, clinical stage, histological grade, and survival time of HCC patients (P < 0.05). Compared with that in WT and sh-NC groups, the expression level of PRDX6 protein in HCC patients was significantly lower (P < 0.01), the proliferation activity of HCC cells was significantly decreased (P < 0.05), and the migration and invasion ability was significantly decreased (P < 0.05) in the sh-PRDX6 group. The expression levels of PI3K, p-Akt, p-mTOR, Notch1, and Hes1 proteins in the sh-PRDX6 group were significantly lower than those in WT and sh-NC groups (P < 0.05). Conclusion: The expression of PRDX6 may be closely related to the prognosis of HCC. Lentivirus-mediated PRDX6 knockdown can inhibit the proliferation, migration and invasion of HCC cells, which may be related to its regulating the PI3K/Akt/mTOR and Notch1 signaling pathways. PRDX6 is expected to be a new target for the diagnosis and treatment of liver cancer.

Divergent low-density lipoprotein receptor (LDLR) linked to low VSV G-dependent viral infectivity and unique serum lipid profile in zebra finches.

The low-density lipoprotein receptor (LDLR) is key to cellular cholesterol uptake and is also the main receptor for the vesicular stomatitis virus glycoprotein (VSV G). Here we show that in songbirds LDLR is highly divergent and lacks domains critical for ligand binding and cellular trafficking, inconsistent with universal structure conservation and function across vertebrates. Linked to the LDLR functional domain loss, zebra finches show inefficient infectivity by lentiviruses (LVs) pseudotyped with VSV G, which can be rescued by the expression of human LDLR. Finches also show an atypical plasma lipid distribution that relies largely on high-density lipoprotein (HDL). These findings provide insights into the genetics and evolution of viral infectivity and cholesterol transport mechanisms in vertebrates.

Characterization and expression of highly active recombinant human glucuronyl C5-epimerase in mammalian cells.

Heparin, a highly sulfated and epimerized form of heparan sulfate, is a linear polysaccharide with anticoagulant activity widely used in the clinic to prevent and treat thrombotic diseases. However, there are several noteworthy drawbacks associated with animal-sourced heparin during the preparation process. The in vitro enzymatic synthesis of heparin has become a promising substitute for animal-derived heparin. The synthesis of bioengineered heparin involves recombinant expression and preparation of polymerases, sulfotransferases, and an epimerase. D-glucuronyl C5-epimerase (HSepi) catalyzes D-glucuronic acids immediately adjacent to N-sulfo-glucosamine units to L-iduronic acid. Preparation of recombinant HSepi with high activity and production yield for in vitro heparin synthesis has not been resolved as of now. The findings of this study indicate that the catalytic activity of HSepi is regulated using post-translational modifications, including N-linked glycosylation and disulfide bond formation. Further mutation studies suggest that tyrosine residues, such as Tyr168, Tyr222, Tyr500, Tyr560, and Tyr578, are crucial in maintaining HSepi activity. A high-yield expression strategy was established using the lentiviral-based transduction system to produce recombinant HSepi (HSepi589) with a specific activity of up to 1.6 IU/mg. Together, this study contributes to the preparation of highly active HSepi for the enzymatic synthesis of heparins by providing additional insights into the catalytic activity of HSepi.