OliTag-seq enhances in cellulo detection of CRISPR-Cas9 off-targets.

The potential for off-target mutations is a critical concern for the therapeutic application of CRISPR-Cas9 gene editing. Current detection methodologies, such as GUIDE-seq, exhibit limitations in oligonucleotide integration efficiency and sensitivity, which could hinder their utility in clinical settings. To address these issues, we introduce OliTag-seq, an in-cellulo assay specifically engineered to enhance the detection of off-target events.

Mean arterial pressure to norepinephrine equivalent dose ratio for predicting renal replacement therapy requirement: a retrospective analysis from the MIMIC-IV.

Background: This study aimed to assess the predictive value of the ratio of mean arterial pressure (MAP) to the corresponding peak rate of norepinephrine equivalent dose (NEQ) within the first day in patients with shock for the subsequent renal replacement therapy (RRT) requirement.

Methods: Patients were identified using the Medical Information Mart for Intensive Care IV (MIMIC-IV) database. The relationship was investigated using a restricted cubic spline curve, and propensity score matching(PSM) was used to eliminate differences between groups.

Efficacy of opioids for traumatic pain in the emergency department: a systematic review and Bayesian network meta-analysis.

To systematically assess and rank the efficacy of opioid medications for traumatic pain in the emergency department in terms of pain relief, adverse events and rescue analgesia. Four databases were systematically searched until 26 September 2022: PubMed, Embase, Cochrane Library, and Web of Science. Outcomes were pain relief, adverse events (dizziness, hypotension, pruritus, sedation), and rescue analgesia.

1,3-Pentadiene-Assistant Living Anionic Terpolymerization: Composition Impact on Kinetics and Microstructure Sequence Primary Analysis.

The combination of a living anionic technology and a unique alternating strategy provided an exciting opportunity to prepare novel and well-defined poly(1,3-pentadiene--syrene--1,1-diphenylethylene) resins consisting of three alternating sequences of modules ( zwitterions). "A" being Styrene (St)/1,3-pentadiene (PD), "B" being diphenylethylene (DPE)/PD, "A" being DPE/St, respectively, A wide composition range of new polyolefin resins, i.e.

Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared with SpCas9 in Genome Editing.

A series of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) systems have been engineered for genome editing. The most widely used Cas9 is SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus. However, a comparison of their detailed gene editing outcomes is still lacking.

Improved and Flexible HDR Editing by Targeting Introns in iPSCs.

Highly efficient gene knockout (KO) editing of CRISPR-Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele.

Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.

Background: After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences.

Results: We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs).

HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs.

Genome-edited human induced pluripotent stem cells (iPSCs) hold great promise for therapeutic applications. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockout and homology-directed repair (HDR)-edited iPSC lines, particularly for silent genes. This is partially due to chromatin compaction, inevitably limiting Cas9 access to the target DNA.

Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types.

Unique anabolic action of stem cell gene therapy overexpressing PDGFB-DSS6 fusion protein in OVX osteoporosis mouse model.

In the present study we sought to improve the efficacy and safety of our Sca1 PDGFB stem cell gene therapy for osteoporosis in ovariectomized (OVX) mouse model. This therapy is administered by marrow transplantation. We established the promise of this approach by previously showing that this therapy in normal mice increase bone density, increased endosteal cortical and trabecular bone formation, caused de novo trabecular bone formation, increased cortical thickness and improve bone strength.

Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse.

Background: Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.

Impact of Fibronectin Knockout on Proliferation and Differentiation of Human Infrapatellar Fat Pad-Derived Stem Cells.

Fibronectin plays an essential role in tissue development and regeneration. However, the effects of fibronectin knockout (FN1-KO) on stem cells' proliferation and differentiation remain unknown. In this study, CRISPR/Cas9 generated FN1-KO in human infrapatellar fat pad-derived stem cells (IPFSCs) was evaluated for proliferation ability including cell cycle and surface markers as well as stemness gene expression and for differentiation capacity including chondrogenic and adipogenic differentiation.

Liver Kinase B1 Fine-Tunes Lineage Commitment of Human Fetal Synovium-Derived Stem Cells.

Liver kinase B1 (LKB1), a serine/threonine protein, is a key regulator in stem cell function and energy metabolism. Herein, we describe the role of LKB1 in modulating the differentiation of synovium-derived stem cells (SDSCs) toward chondrogenic, adipogenic, and osteogenic lineages. Human fetal SDSCs were transduced with CRISPR associated protein 9 (Cas9)-single-guide RNA vectors to knockout or lentiviral vectors to overexpress the LKB1 gene.

High-Level Precise Knockin of iPSCs by Simultaneous Reprogramming and Genome Editing of Human Peripheral Blood Mononuclear Cells.

We have developed an improved episomal vector system for efficient generation of integration-free induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells. More recently, we reported that the use of an optimized CRISPR-Cas9 system together with a double-cut donor increases homology-directed repair-mediated precise gene knockin efficiency by 5- to 10-fold. Here, we report the integration of blood cell reprogramming and genome editing in a single step.

HOXA4, down-regulated in lung cancer, inhibits the growth, motility and invasion of lung cancer cells.

The involvement of HOXA4 in colorectal cancer and epithelial ovarian cancer has been reported. Although it has been reported that the Hoxa4 gene is involved in the patterning of the mouse lung during embryonic development, little is known about the biological functions of HOXA4 in lung cancer. In the current study, HOXA4 expression was down-regulated in lung cancer tissues when compared with non-cancerous tissues.

Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.

Background: Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs).

Results: Here, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs.

Leptin and IL-8: two novel cytokines screened out in childhood lead exposure.

Lead is a toxic heavy metal with many recognized adverse health side effects. The central nervous system is the main target of lead toxicity. Although many studies on lead toxicity were conducted, the mechanism of lead toxicity remains uncertain.

Validity and reliability of the Taiwanese version of the Pittsburgh Sleep Quality Index in cancer patients.

Background: Sleep disturbance remains a common symptom among cancer patients. Assessment of sleep disturbance in cancer patients is hindered by infrequent use of standardized tools for sleep management.

Objectives: The purposes of this study were to validate the Taiwanese version of the Pittsburgh Sleep Quality Index (PSQI-T) and to determine detection cut-off points in cancer patients.

[Regulation effects of grafting on cinnamic acid and vanillin in eggplant root exudates].

Cinnamic acid and vanillin are the allelochemicals commonly existed in eggplant root exudates. With pot culture experiment, the regulation effects of grafting on the cinnamic acid and vanillin in eggplant root exudates were studied, and the results showed that grafting decreased the amount of the two substances, especially of vanillin, in eggplants root system. The maximum reduction amount of cinnamic acid reached 68.

[Inhibitory effects of natural plant extracts on Verticillium albo-atrum].

This paper studied the inhibitory effects of 54 kinds of ethanol-extracted plant solutions on Verticillium albo-atrum. The results showed that 15 kinds of these extracts could inhibit the growth of verticillium albo-atrum mycelium, with an inhibitory rate more than 50%, among which, the inhibitory rate of Asarum sieboldii, Coptis chinensis, Magnolia officinalis, Acacia catechu, Sophora flavescens, Glycyrrhiza uralensis, Cnidium monnieri, Platycodon grandiflorum and Allium. cepa.

[Allelopathy of decomposing pepper stalk on pepper growth].

With decomposing pepper stalk as test material, this paper studied its allelopathy on the growth of pepper plants. The results showed that after 60 days of decomposition, the decomposed pepper stalk could decrease the plant height, stem diameter, dry weights of above-and underground biomass, leaf area, and chlorophyll content of pepper plants by 0.0374 - 0.