Heterologous survey of 130 DNA transposons in human cells highlights their functional divergence and expands the genome engineering toolbox.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

Author Correction: STEEP mediates STING ER exit and activation of signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

STEEP mediates STING ER exit and activation of signaling.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

In vivo CRISPR gene editing in patients with herpetic stromal keratitis.

In vivo CRISPR gene therapy holds large clinical potential, but the safety and efficacy remain largely unknown. Here, we injected a single dose of herpes simplex virus 1 (HSV-1)-targeting CRISPR formulation in the cornea of three patients with severe refractory herpetic stromal keratitis (HSK) during corneal transplantation. Our study is an investigator-initiated, open-label, single-arm, non-randomized interventional trial at a single center (NCT04560790). We found neither detectable CRISPR-induced off-target cleavages by GUIDE-seq nor systemic adverse events for 18 months on average in all three patients. The HSV-1 remained undetectable during the study. Our preliminary clinical results suggest that in vivo gene editing targeting the HSV-1 genome holds acceptable safety as a potential therapy for HSK.

Changes of Facial Lipidomics by Intense Pulsed Light Treatment Based on LC-MS.

BACKGROUND: Intense pulsed light (IPL) has been widely used to improve cutaneous photoaging in recent years. Several studies began to explore the changes of skin barrier function after treatment, but the changes of skin surface lipids (SSL), especially specific lipid content and types are still unclear. METHODS: A total of 25 female volunteers were included in our study, and each of them received three full-face treatments with one month apart. Before the first treatment and 1 month after the last treatment, we collected clinical photos and skin stratum corneum samples from individuals. A 5-level scale was used to evaluate the efficacy of IPL treatment, liquid chromatography-mass spectrometry (LC-MS), and Orthogonal Partial Least Squares Discrimination Analysis (OPLS-DA) were used to analyze the changes of SSL. RESULTS: Two patients got no improvement after treatment, 6 patients had poor improvement and mild improvement was achieved in 9 patients, 5 and 3 patients reported moderate and significant improvement. The overall "effective" rate was 68 % and the "significant effective" rate was 32 %. The results showed 18 lipid subclasses and 487 lipid molecules were identified. The change of total lipid volume was not statistically significant (P = 0.088>0.05), but lipid subclass analysis showed the amount of Triglyceride (TG), Phosphatidic Acid (PA), Phosphatidylglycerol (PG) and Lysophosphatidylglycerol (LPG) were significantly increased (P < 0.05). There were 55 kinds of lipid molecules with significant difference after treatment (P < 0.05), and 51 of them belong to TG. The analysis of chain saturation of TG showed that the quantity of TG with 0, 1 and 2 unsaturated bonds increased significantly (P < 0.05). CONCLUSIONS: IPL treatment does not have a significant effect on the overall amount of lipids while the amount of TG, PA, PG, LPG were significantly increased. These lipid changes may potentially improve the skin barrier function, but more high-quality and comprehensive studies are still needed. BULLET POINT: Lipidomics analysis based on LC-MS; Changes of skin surface lipid after IPL treatment; the relationships between skin surface lipid and skin barrier functions. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The Efficacy of Fractional Carbon Dioxide Laser in Surgical Scars Treatment: A system Review and Meta-analysis.

BACKGROUND: Surgical scars seriously affect a patient's quality of life, and they have a strong impact on individuals. Many studies have reported the results of using fractional carbon dioxide (CO2) laser to treat surgical scars and have generally found it to be effective. OBJECTIVES: We conducted a meta-analysis with the objective of evaluating and proving the efficacy of fractional CO2 laser therapy for surgical scars. METHODS: We performed a search of databases including PubMed, Web of Science, Embase and the Cochrane Library. The outcomes of the meta-analysis were overall scores on the Vancouver Scar Scale (VSS) and its four dimensions (pigmentation, vascularity, pliability and height). Statistical analysis was performed using RevMan 5.4 software. RESULTS: A total of ten studies were included in this meta-analysis, including six randomized controlled trials (RCTs) and four nonrandomized controlled trials (N-RCTs). In the meta-analysis of RCTs and N-RCTs, similar results were obtained, and fractional CO2 laser irradiation significantly decreased VSS scores (P < 0.00001). In addition, fractional CO2 laser irradiation also had a significant effect on scores on the pigmentation (P = 0.08), vascularity (P = 0.001), flexibility (P = 0.005) and height (P = 0.008) dimensions. Except for mild pain during treatment and temporary erythema after treatment, most patients had no obvious adverse reactions. CONCLUSION: Our study found that fractional CO2 laser exhibits excellent efficacy and safety in terms of surgical scar treatment. Thus, we hope it becomes more widely available to patients with surgical scars. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors   www.springer.com/00266 .

Frameshift and wild-type proteins are often highly similar because the genetic code and genomes were optimized for frameshift tolerance.

Frameshift mutations have been considered of significant importance for the molecular evolution of proteins and their coding genes, while frameshift protein sequences encoded in the alternative reading frames of coding genes have been considered to be meaningless. However, functional frameshifts have been found widely existing. It was puzzling how a frameshift protein kept its structure and functionality while substantial changes occurred in its primary amino-acid sequence. This study shows that the similarities among frameshifts and wild types are higher than random similarities and are determined at different levels. Frameshift substitutions are more conservative than random substitutions in the standard genetic code (SGC). The frameshift substitutions score of SGC ranks in the top 2.0-3.5% of alternative genetic codes, showing that SGC is nearly optimal for frameshift tolerance. In many genes and certain genomes, frameshift-resistant codons and codon pairs appear more frequently than expected, suggesting that frameshift tolerance is achieved through not only the optimality of the genetic code but, more importantly, the further optimization of a specific gene or genome through the usages of codons/codon pairs, which sheds light on the role of frameshift mutations in molecular and genomic evolution.

Attenuation of cGAS-STING signaling is mediated by a p62/SQSTM1-dependent autophagy pathway activated by TBK1.

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62-deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy-associated vesicles. Thus, DNA sensing induces the cGAS-STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.

Intense pulsed light treatment for inflammatory skin diseases: a review.

Although intense pulsed light (IPL) has been commonly used in the field of medical cosmetics in recent years, the exact outcomes of IPL in the treatment of inflammatory skin diseases remain unclear. To assess the clinical evidence for the use of IPL in the treatment of various inflammatory skin diseases and propose evidence-based recommendations, we searched for relevant publications in the PubMed and Web of Science databases and provided updated information. The inflammatory skin diseases treated with IPL consisted of acne vulgaris, rosacea, psoriasis, hidradenitis suppurativa (HS), atopic dermatitis (AD), Riehl's melanosis, lupus erythematosus, cutaneous sarcoidosis, pilonidal cysts, and pigmented actinic lichen planus (PALP). The efficacy of IPL treatment for these inflammatory skin diseases was described and evaluated. Forty-two studies were included to provide this assessment. The evidence suggests that IPL can effectively and safely improve acne vulgaris and rosacea (recommendation grade B). For other described inflammatory skin diseases, IPL can be used as a tentative or supplementary treatment (recommendation grade C and D). The main complications include transitory erythema, edema, and pain, with the possibility of hyperpigmentation, blisters, and a burning sensation in some individuals.

Efficacy and safety of pulsed dye laser for the treatment of surgical scars: a systematic review and meta-analysis.

Various clinical trials have explored whether the pulsed dye laser (PDL) method is safe to treat scars, especially surgical scars. However, comprehensive evidence confirming the exact outcomes of PDL for treating surgical scars is lacking. The efficacy and safety of PDL in the treatment of surgical scars were determined through a review of several studies. The PubMed, Embase, Cochrane Library, and Web of Science databases were searched, and the main clinical outcomes were Vancouver Scar Scale (VSS) scores in terms of pigmentation, vascularity, pliability, and height. Review Manager 5.4 software was used for statistical analyses of the data; we chose a standardized mean difference (SMZ) to present the results with 95% confidence interval (CI). Overall, seven randomized controlled trials were used for this meta-analysis, all of these papers used 585 nm or 595 nm PDL with 7 mm or 10 mm spot size and a fluence of 3.5 to 10 J/cm2 for treating surgical scars; besides, the pulse duration ranged from 450 µs to 10 ms. We found that PDL significantly resulted in decreased VSS scores (P = 0.02) in four aspects: pigmentation (P = 0.0002), vascularity (P < 0.00001), pliability (P = 0.0002), and height (P = 0.0002). Moreover, scar improvement was similar when using 585 nm and 595 nm PDL in terms of pigmentation (P = 0.76), vascularity (P = 0.34), pliability (P = 0.64), and height (P = 0.57). Furthermore, our review indicated that PDL has no obvious adverse effects for most people, except transitory erythema and purpura. The meta-analysis showed that both 585 nm and 595 nm PDL therapy can effectively reduce the VSS score, suggesting that PDL can be a safe and effective method for the treatment of surgical scars.

DeepAntigen: a novel method for neoantigen prioritization via 3D genome and deep sparse learning.

MOTIVATION: The mutations of cancers can encode the seeds of their own destruction, in the form of T-cell recognizable immunogenic peptides, also known as neoantigens. It is computationally challenging, however, to accurately prioritize the potential neoantigen candidates according to their ability of activating the T-cell immunoresponse, especially when the somatic mutations are abundant. Although a few neoantigen prioritization methods have been proposed to address this issue, advanced machine learning model that is specifically designed to tackle this problem is still lacking. Moreover, none of the existing methods considers the original DNA loci of the neoantigens in the perspective of 3D genome which may provide key information for inferring neoantigens' immunogenicity. RESULTS: In this study, we discovered that DNA loci of the immunopositive and immunonegative MHC-I neoantigens have distinct spatial distribution patterns across the genome. We therefore used the 3D genome information along with an ensemble pMHC-I coding strategy, and developed a group feature selection-based deep sparse neural network model (DNN-GFS) that is optimized for neoantigen prioritization. DNN-GFS demonstrated increased neoantigen prioritization power comparing to existing sequence-based approaches. We also developed a webserver named deepAntigen (http://yishi.sjtu.edu.cn/deepAntigen) that implements the DNN-GFS as well as other machine learning methods. We believe that this work provides a new perspective toward more accurate neoantigen prediction which eventually contribute to personalized cancer immunotherapy. AVAILABILITY AND IMPLEMENTATION: Data and implementation are available on webserver: http://yishi.sjtu.edu.cn/deepAntigen. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Central venous catheters misplaced in paraspinal veins: A systematic literature review based on case reports.

BACKGROUND: Paraspinal vein misplacement is an uncommon complication of central venous catheterization via lower extremities, most of which have been reported in case reports. AIM: To determine the clinical characteristics of paraspinal vein misplacement. DESIGN: This was a systematic review. METHODS: A systematic literature search in the PubMed, EMBASE, Scopus, and Web of Science databases was performed from their inception to 18 June 2019. Case reports and small case series describing central venous catheter misplacement in the paraspinal vein were included. Data on the catheterization procedure, catheter tip position, complications, and radiographic features of misplacement were extracted. RESULTS: Thirty studies with a total of 36 patients were included. The ascending lumbar vein accounted for the majority of misplacements (n = 30), followed by the lumbar vein (n = 4), iliolumbar vein (n = 1), and vertebral venous plexus (n = 1). Six patients had eventful catheterization procedures. Twenty-six patients experienced misplacement-induced complications, of whom seven died. The most common complications included cerebrospinal fluid abnormalities, neurological symptoms, and deteriorated respiration. Among the entire case cohort, the onset of complications was the primary sign that alerted medical staff to misplacement (n = 23). The typical radiographic characteristics were posterior deviation of the catheter course overlapping with the spine on lateral X-rays and a bend, kink, or hump in the catheter course on anteroposterior X-rays at the L4 to L5 levels. CONCLUSIONS: Nurses should be aware of this particular complication if a patient who has undergone catheterization via a lower extremity presents deterioration of neurological function and respiration. RELEVANCE TO CLINICAL PRACTICE: Lateral X-ray radiography is an effective method to verify misplacement and is recommended as routine practice during catheterization via lower extremities.

An overview of development in gene therapeutics in China.

After setbacks related to serious adverse events 20 years ago, gene therapy is now coming back to the central stage worldwide. In the past few years, gene therapy has shown astonishing efficacy against genetic diseases and cancers. In history, China carried out the world's second gene therapy clinical trial in 1991 for hemophilia B and approved the world's first gene therapy product-Gendicine-in 2003. In recent years, numerous efforts have been made on gene editing. Here, we reviewed the past of gene therapy in China and highlighted recent advances. We also discussed the regulations and future perspectives of gene therapy in China.

DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors.

DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.

Diagnostic performance of angiography-derived fractional flow reserve and CT-derived fractional flow reserve: A systematic review and Bayesian network meta-analysis.

OBJECTIVE: Accumulating evidence has demonstrated that fractional flow reserves (FFRs) derived from invasive coronary angiograms (CA-FFRs) and coronary computed tomography angiography-derived FFRs (CT-FFRs) are promising alternatives to wire-based FFRs. However, it remains unclear which method has better diagnostic performance. This systematic review and meta-analysis aimed to compare the diagnostic performances of the two approaches. METHODS: The Cochrane Library, PubMed, Embase, Medline (Ovid), the Chinese China National Knowledge Infrastructure Database (CNKI), VIP, and WanFang Data databases were searched for relevant studies that included comparisons between CA-FFR and CT-FFR, from their respective database inceptions until January 1, 2023. Studies where both noninvasive FFR (including CA-FFR and CT-FFR) and invasive FFR (as a reference standard) were performed for the diagnosis of ischemic coronary artery disease and were designed as prospective, paired diagnostic studies, were pulled. The diagnostic test accuracy method and Bayesian hierarchical summary receiver operating characteristic (ROC) model for network meta-analysis (NMA) of diagnostic tests (HSROC-NMADT) were both used to perform a meta-analysis on the data. RESULTS: Twenty-six studies were included in this NMA. The results from both the diagnostic test accuracy and HSROC-NMADT methods revealed that the diagnostic accuracy of CA-FFR was higher than that of CT-FFR, in terms of sensitivity (Se; 0.86 vs. 0.84), specificity (Sp; 0.90 vs. 0.78), positive predictive value (PPV; 0.83 vs. 0.70), and negative predictive value (NPV; 0.91 vs. 0.89) for the detection of myocardial ischemia. A cumulative ranking curve analysis indicated that CA-FFR had a higher diagnostic accuracy than CT-FFR in the context of this study, with a higher area under the ROC curve (AUC; 0.94 vs. 0.87). CONCLUSIONS: Although both of these two commonly used virtual FFR methods showed high levels of diagnostic accuracy, we demonstrated that CA-FFR had a better Se, Sp, PPV, NPV, and AUC than CT-FFR. However, this study provided only indirect comparisions; therefore, larger studies are warranted to directly compare the diagnostic performances of these two approaches.

Identification of restrictive molecules involved in oncolytic virotherapy using genome-wide CRISPR screening.

Oncolytic viruses (OVs) offer a novel approach to treat solid tumors; however, their efficacy is frequently suboptimal due to various limiting factors. To address this challenge, we engineered an OV containing targets for neuron-specific microRNA-124 and Granulocyte-macrophage colony-stimulating factor (GM-CSF), significantly enhancing its neuronal safety while minimally compromising its replication capacity. Moreover, we identified PARP1 as an HSV-1 replication restriction factor using genome-wide CRISPR screening. In models of glioblastoma (GBM) and triple-negative breast cancer (TNBC), we showed that the combination of OV and a PARP inhibitor (PARPi) exhibited superior efficacy compared to either monotherapy. Additionally, single-cell RNA sequencing (scRNA-seq) revealed that this combination therapy sensitized TNBC to immune checkpoint blockade, and the incorporation of an immune checkpoint inhibitor (ICI) further increased the survival rate of tumor-bearing mice. The combination of PARPi and ICI synergistically enhanced the ability of OV to establish durable tumor-specific immune responses. Our study effectively overcomes the inherent limitations of OV therapy, providing valuable insights for the clinical treatment of TNBC, GBM, and other malignancies.

Modified lentiviral globin gene therapy for pediatric ß0/ß0 transfusion-dependent ß-thalassemia: A single-center, single-arm pilot trial.

ß0/ß0 thalassemia is the most severe type of transfusion-dependent ß-thalassemia (TDT) and is still a challenge facing lentiviral gene therapy. Here, we report the interim analysis of a single-center, single-arm pilot trial (NCT05015920) evaluating the safety and efficacy of a ß-globin expression-optimized and insulator-engineered lentivirus-modified cell product (BD211) in ß0/ß0 TDT. Two female children were enrolled, infused with BD211, and followed up for an average of 25.5 months. Engraftment of genetically modified hematopoietic stem and progenitor cells was successful and sustained in both patients. No unexpected safety issues occurred during conditioning or after infusion. Both patients achieved transfusion independence for over 22 months. The treatment extended the lifespan of red blood cells by over 42 days. Single-cell DNA/RNA-sequencing analysis of the dynamic changes of gene-modified cells, transgene expression, and oncogene activation showed no notable adverse effects. Optimized lentiviral gene therapy may safely and effectively treat all ß-thalassemia.

Genomic analysis of human brain metastases treated with stereotactic radiosurgery reveals unique signature based on treatment failure.

Stereotactic radiosurgery (SRS) has been shown to be efficacious for the treatment of limited brain metastasis (BM); however, the effects of SRS on human brain metastases have yet to be studied. We performed genomic analysis on resected brain metastases from patients whose resected lesion was previously treated with SRS. Our analyses demonstrated for the first time that patients possess a distinct genomic signature based on type of treatment failure including local failure, leptomeningeal spread, and radio-necrosis. Examination of the center and peripheral edge of the tumors treated with SRS indicated differential DNA damage distribution and an enrichment for tumor suppressor mutations and DNA damage repair pathways along the peripheral edge. Furthermore, the two clinical modalities used to deliver SRS, LINAC and GK, demonstrated differential effects on the tumor landscape even between controlled primary sites. Our study provides, in human, biological evidence of differential effects of SRS across BM's.

Dendritic-cell-targeting virus-like particles as potent mRNA vaccine carriers.

Messenger RNA vaccines lack specificity for dendritic cells (DCs)-the most effective cells at antigen presentation. Here we report the design and performance of a DC-targeting virus-like particle pseudotyped with an engineered Sindbis-virus glycoprotein that recognizes a surface protein on DCs, and packaging mRNA encoding for the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or for the glycoproteins B and D of herpes simplex virus 1. Injection of the DC-targeting SARS-CoV-2 mRNA vaccine in the footpad of mice led to substantially higher and durable antigen-specific immunoglobulin-G titres and cellular immune responses than untargeted virus-like particles and lipid-nanoparticle formulations. The vaccines also protected the mice from infection with SARS-CoV-2 or with herpes simplex virus 1. Virus-like particles with preferential uptake by DCs may facilitate the development of potent prophylactic and therapeutic vaccines.

A lentiviral vector-based genetic sensor system for comparative analysis of permeability and activity of vitamin D3 analogues in xenotransplanted human skin.

Vitamin D3 analogues are widely used topical and oral remedies for various ailments such as psoriasis, osteoporosis and secondary hyperparathyroidism. In topical treatment, high skin permeability and cellular uptake are key criteria for beneficial effects due to the natural barrier properties of skin. In this study, we wish to establish an in vivo model that allows the comparison of permeability and activity of vitamin D3 analogues in human skin. We generate a bipartite, genetic sensor technology that combines efficient lentivirus-directed gene delivery to xenotransplanted human skin with vitamin D3-induced expression of a luciferase reporter gene and live imaging of animals by bioluminescence imaging. Based on the induction of a transcriptional activator consisting of the vitamin D receptor fused to the Gal4 DNA-binding domain, the vitamin D3-responsive sensor facilitates non-invasive and rapid assessment of permeability and functional properties of vitamin D3 analogues. By topical application of a panel of vitamin D3 analogues onto 'sensorized' human skin, the sensor produces a drug-induced readout with a magnitude and persistence that allow a direct comparative analysis of different analogues. This novel genetic tool has great potential as a non-invasive in vivo screening system for further development and refinement of vitamin D3 analogues.