Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.

CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

Fibroblast growth factor 21 alleviates diabetes-induced cognitive decline.

Diabetes mellitus (DM) causes damage to the central nervous system, resulting in cognitive impairment. Fibroblast growth factor 21 (FGF21) exhibits the potential to alleviate neurodegeneration. However, the therapeutic effect of intracerebroventricular (i.c.v) FGF21 infusion on diabetes-induced cognitive decline (DICD) and its potential mechanisms remain unclear. In this study, the impact of FGF21 on DICD was explored, and 1H nuclear magnetic resonance (NMR)-based metabolomics plus 13C NMR spectroscopy in combine with intravenous [1-13C]-glucose infusion were used to investigate the underlying metabolic mechanism. Results revealed that i.c.v FGF21 infusion effectively improved learning and memory performance of DICD mice; neuron loss and apoptosis in hippocampus and cortex were significantly blocked, suggesting a potential neuroprotective role of FGF21 in DICD. Metabolomics results revealed that FGF21 modulated DICD metabolic alterations related to glucose and neurotransmitter metabolism, which are characterized by distinct recovered enrichment of [3-13C]-lactate, [3-13C]-aspartate, [4-13C]-glutamine, [3-13C]-glutamine, [4-13C]-glutamate, and [4-13C]- γ-aminobutyric acid (GABA) from [1-13C]-glucose. Moreover, diabetes-induced neuron injury and metabolic dysfunctions might be mediated by PI3K/AKT/GSK-3ß signaling pathway inactivation in the hippocampus and cortex, which were activated by i.c.v injection of FGF21. These findings indicate that i.c.v FGF21 infusion exerts its neuroprotective effect on DICD by remodeling cerebral glucose and neurotransmitter metabolism by activating the PI3K/AKT/GSK-3ß signaling pathway.

Aberrant auditory metabolite levels and topological properties are associated with cognitive decline in presbycusis patients.

Presbycusis has been reported as related to cognitive decline, but its underlying neurophysiological mechanism is still unclear. This study aimed to investigate the relationship between metabolite levels, cognitive function, and node characteristics in presbycusis based on graph theory methods. Eighty-four elderly individuals with presbycusis and 63 age-matched normal hearing controls underwent magnetic resonance spectroscopy, functional magnetic resonance imaging scans, audiological assessment, and cognitive assessment. Compared with the normal hearing group, presbycusis patients exhibited reduced gamma-aminobutyric acid and glutamate levels in the auditory region, increased nodal characteristics in the temporal lobe and precuneus, as well as decreased nodal characteristics in the superior occipital gyrus and medial orbital. The right gamma-aminobutyric acid levels were negatively correlated with the degree centrality in the right precuneus and the executive function. Degree centrality in the right precuneus exhibited significant correlations with information processing speed and executive function, while degree centrality in the left medial orbital demonstrated a negative association with speech recognition ability. The degree centrality and node efficiency in the superior occipital gyrus exhibited a negative association with hearing loss and speech recognition ability, respectively. These observed changes indicate alterations in metabolite levels and reorganization patterns at the brain network level after auditory deprivation.

The arginine methyltransferase Prmt1 coordinates the germline arginine methylome essential for spermatogonial homeostasis and male fertility.

Arginine methylation, catalyzed by the protein arginine methyltransferases (PRMTs), is a common post-translational protein modification (PTM) that is engaged in a plethora of biological events. However, little is known about how the methylarginine-directed signaling functions in germline development. In this study, we discover that Prmt1 is predominantly distributed in the nuclei of spermatogonia but weakly in the spermatocytes throughout mouse spermatogenesis. By exploiting a combination of three Cre-mediated Prmt1 knockout mouse lines, we unravel that Prmt1 is essential for spermatogonial establishment and maintenance, and that Prmt1-catalyzed asymmetric methylarginine coordinates inherent transcriptional homeostasis within spermatogonial cells. In conjunction with high-throughput CUT&Tag profiling and modified mini-bulk Smart-seq2 analyses, we unveil that the Prmt1-deposited H4R3me2a mark is permissively enriched at promoter and exon/intron regions, and sculpts a distinctive transcriptomic landscape as well as the alternative splicing pattern, in the mouse spermatogonia. Collectively, our study provides the genetic and mechanistic evidence that connects the Prmt1-deposited methylarginine signaling to the establishment and maintenance of a high-fidelity transcriptomic identity in orchestrating spermatogonial development in the mammalian germline.

A noninvasive multianalytical approach establishment for risk assessment and gastric cancer screening.

Effective screening and early detection are critical to improve the prognosis of gastric cancer (GC). Our study aims to explore noninvasive multianalytical biomarkers and construct integrative models for preliminary risk assessment and GC detection. Whole genomewide methylation marker discovery was conducted with CpG tandems target amplification (CTTA) in cfDNA from large asymptomatic screening participants in a high-risk area of GC. The methylation and mutation candidates were validated simultaneously using one plasma from patients at various gastric lesion stages by multiplex profiling with Mutation Capsule Plus (MCP). Helicobacter pylori specific antibodies were detected with a recomLine assay. Integrated models were constructed and validated by the combination of multianalytical biomarkers. A total of 146 and 120 novel methylation markers were found in CpG islands and promoter regions across the genome with CTTA. The methylation markers together with the candidate mutations were validated with MCP and used to establish a 133-methylation-marker panel for risk assessment of suspicious precancerous lesions and GC cases and a 49-methylation-marker panel as well as a 144-amplicon-mutation panel for GC detection. An integrated model comprising both methylation and specific antibody panels performed better for risk assessment than a traditional model (AUC, 0.83 and 0.63, P < .001). A second model for GC detection integrating methylation and mutation panels also outperformed the traditional model (AUC, 0.82 and 0.68, P = .005). Our study established methylation, mutation and H. pylori-specific antibody panels and constructed two integrated models for risk assessment and GC screening. Our findings provide new insights for a more precise GC screening strategy in the future.

Cyclophilin A regulates the apoptosis of A549 cells by stabilizing Twist1 protein.

Cyclophilin A (CypA, also known as PPIA) is an essential member of the immunophilin family. As an intracellular target of the immunosuppressive drug cyclosporin A (CsA) or a peptidyl-prolyl cis/trans isomerase (PPIase), it catalyzes the cis-trans isomerization of proline amidic peptide bonds, through which it regulates a variety of biological processes, such as intracellular signaling, transcription and apoptosis. In this study, we found that intracellular CypA enhanced Twist1 phosphorylation at Ser68 and inhibited apoptosis in A549 cells. Mechanistically, CypA could mediate the phosphorylation of Twist1 at Ser68 via p38 mitogen-activated protein kinase (also known as MAPK14), which inhibited its ubiquitylation-mediated degradation. In addition, CypA increased interaction between Twist1 and p65 (also known as RELA), as well as nuclear accumulation of the Twist1-p65 complex, which regulated Twist1-dependent expression of CDH1 and CDH2. Our findings collectively indicate the role of CypA in Twist1-mediated apoptosis of A549 cells through stabilizing Twist1 protein.

Combined analysis of the microbiome, metabolome and transcriptome of silkie chickens in response to avian pathogenic E. coli (APEC).

Avian colibacillosis is a bacterial disease caused by avian pathogenic Escherichia coli (APEC) that results in great losses in the poultry industry every year. Individual Silkie chickens of the same breed that are given the same feed in the same feeding conditions have different levels of resistance or susceptibility to APEC. Differences in gut microbes, gut metabolites, and gene expression in the spleen of APEC-resistant and APEC-susceptible chickens were compared, and multiple omics associations were analyzed to explore the mechanism of resistance to APEC in Silkie chickens. Compared with those in the APEC-susceptible group, the APEC-resistant group showed significantly increased abundances of many gut microorganisms, including Bacillus, Thermoactinomyces, Arthrobacter, and Ureibacillus, which were positively correlated with norvaline, l-arginine, and valyl-glycine levels. Intestinal tryptophan, indole, and indole derivative-related differentially abundant metabolites played an active role in combatting APEC infection. In the spleen, "response to stimulus" was the most significantly enriched GO term, and "cytokine‒cytokine receptor interaction" was the most significantly enriched KEGG pathway. The arginine biosynthesis and PPAR signaling pathways were the KEGG pathways that were significantly enriched with differentially abundant metabolites and differentially expressed genes. This study provides new insight into the prevention and treatment of APEC infection in Silkie chickens and lays a foundation to study the mechanism of APEC infection in poultry.

Deep Learning Model for Grading and Localization of Lumbar Disc Herniation on Magnetic Resonance Imaging.

BACKGROUND: Methods for grading and localization of lumbar disc herniation (LDH) on MRI are complex, time-consuming, and subjective. Utilizing deep learning (DL) models as assistance would mitigate such complexities. PURPOSE: To develop an interpretable DL model capable of grading and localizing LDH. STUDY TYPE: Retrospective. SUBJECTS: 1496 patients (M/F: 783/713) were evaluated, and randomly divided into training (70%), validation (10%), and test (20%) sets. FIELD STRENGTH/SEQUENCE: 1.5T MRI for axial T2-weighted sequences (spin echo). ASSESSMENT: The training set was annotated by three spinal surgeons using the Michigan State University classification to train the DL model. The test set was annotated by a spinal surgery expert (as ground truth labels), and two spinal surgeons (comparison with the trained model). An external test set was employed to evaluate the generalizability of the DL model. STATISTICAL TESTS: Calculated intersection over union (IoU) for detection consistency, utilized Gwet's AC1 to assess interobserver agreement, and evaluated model performance based on sensitivity and specificity, with statistical significance set at P < 0.05. RESULTS: The DL model achieved high detection consistency in both the internal test dataset (grading: mean IoU 0.84, recall 99.6%; localization: IoU 0.82, recall 99.5%) and external test dataset (grading: 0.72, 98.0%; localization: 0.71, 97.6%). For internal testing, the DL model (grading: 0.81; localization: 0.76), Rater 1 (0.88; 0.82), and Rater 2 (0.86; 0.83) demonstrated results highly consistent with the ground truth labels. The overall sensitivity of the DL model was 87.0% for grading and 84.0% for localization, while the specificity was 95.5% and 94.4%. For external testing, the DL model showed an appreciable decrease in consistency (grading: 0.69; localization: 0.66), sensitivity (77.2%; 76.7%), and specificity (92.3%; 91.8%). DATA CONCLUSION: The classification capabilities of the DL model closely resemble those of spinal surgeons. For future improvement, enriching the diversity of cases could enhance the model's generalization. TECHNICAL EFFICACY: Stage 2.

Photoactive rhodamine-based photosensitizer eliminates Staphylococcus aureus via superoxide radical photosensitization.

Due to the antibiotics abuse, bacterial infection has become one of the leading causes of human death worldwide. Novel selective antimicrobial agents are urgently needed, with the hope of maintaining the balance of the microbial environment. Photo-activated chemotherapeutics have shown great potential to eliminate bacteria with appealing spatiotemporal selectivity. In this work, we reported the structural modification to enhance the triplet excited state property of Rhodamine B, synthesizing a rhodamine-based photosensitizer RBPy. Upon light activation, RBPy exhibited much stronger photosensitization ability than the parent compound Rhodamine B both in solution and in bacteria. Importantly, RBPy can selectively inactivate Staphylococcus aureus and inhibit biofilm formation with high biocompatibility. This work provides a new strategy to develop rhodamine-based photoactive chemotherapeutics for antimicrobial photodynamic therapy.

Pallidum volume as a predictor for the effectiveness of mindfulness-based cognitive therapy and psycho-education in unmedicated patients with obsessive-compulsive disorder.

BACKGROUND: Mindfulness-based cognitive therapy (MBCT) has been documented to be effective in treating obsessive-compulsive disorder (OCD). However, the neurobiological basis of MBCT remains largely elusive, which makes it clinically challenging to predict which patients are more likely to respond poorly. Hence, identifying biomarkers for predicting treatment outcomes holds both scientific and clinical values. This prognostic study aims to investigate whether pre-treatment brain morphological metrics can predict the effectiveness of MBCT, compared with psycho-education (PE) as an active placebo, among patients with OCD. METHODS: A total of 32 patients with OCD were included in this prognostic study. They received magnetic resonance imaging (MRI) brain scans before treatment. Subsequently, 16 patients received 10 weeks of MBCT, while the other 16 patients underwent a 10-week PE program. The effectiveness of the treatments was primarily assessed by the reduction rate of the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) total score before and after the treatment. We investigated whether several predefined OCD-associated brain morphological metrics, selected based on prior published studies by the ENIGMA Consortium, could predict the treatment effectiveness. RESULTS: Both the MBCT and PE groups exhibited substantial reductions in Y-BOCS scores over 10 weeks of treatment, with the MBCT group showing a larger reduction. Notably, the pallidum total volume was associated with treatment effectiveness, irrespective of the intervention group. Specifically, a linear regression model utilizing the pre-treatment pallidum volume to predict the treatment effectiveness suggested that a one-cubic-centimeter increase in pallidum volume corresponded to a 22.3% decrease in the Y-BOCS total score reduction rate. CONCLUSIONS: Pallidum volume may serve as a promising predictor for the effectiveness of MBCT and PE, and perhaps, other treatments with the shared mechanisms by MBCT and PE, among patients with OCD.

The effect of choice on memory across development.

Numerous studies have demonstrated the role of making choices as an internal motivator to improve performance, and recent studies in the domain of memory have focused on adults. To chart the developmental trend of the choice effect on memory, we conducted a series of seven experiments involving children, adolescents, and young adults. Participants (N = 512) aged 5 to 26 years performed a choice encoding task that manipulated the opportunities to choose and then took a memory test. Using different types of experimental materials and corroborated by a mini meta-analysis, we found that the choice effect on memory was significant in childhood and early adolescence but not significant in late adolescence and early adulthood. The developmental changes were statistically significant, particularly evident during the transition from early to late adolescence. These findings suggest that the internal value of choice decreases across development and contributes to our understanding of developmental differences in the role of choice in memory.

ETNPPL modulates hyperinsulinemia-induced insulin resistance through the SIK1/ROS-mediated inactivation of the PI3K/AKT signaling pathway in hepatocytes.

Hyperinsulinemia is a critical risk factor for the pathogenesis of insulin resistance (IR) in metabolic tissues, including the liver. Ethanolamine phosphate phospholyase (ETNPPL), a newly discovered metabolic enzyme that converts phosphoethanolamine (PEA) to ammonia, inorganic phosphate, and acetaldehyde, is abundantly expressed in liver tissue. Whether it plays a role in the regulation of hyperinsulinemia-induced IR in hepatocytes remains elusive. Here, we established an in vitro hyperinsulinemia-induced IR model in the HepG2 human liver cancer cell line and primary mouse hepatocyte via a high dose of insulin treatment. Next, we overexpressed ETNPPL by using lentivirus-mediated ectopic to investigate the effects of ETNPPL per se on IR without insulin stimulation. To explore the underlying mechanism of ETNPPL mediating hyperinsulinemia-induced IR in HepG2, we performed genome-wide transcriptional analysis using RNA sequencing (RNA-seq) to identify the downstream target gene of ETNPPL. The results showed that ETNPPL expression levels in both mRNA and protein were significantly upregulated in hyperinsulinemia-induced IR in HepG2 and primary mouse hepatocytes. Upon silencing ETNPPL, hyperinsulinemia-induced IR was ameliorated. Under normal conditions without IR in hepatocytes, overexpressing ETNPPL promotes IR, reactive oxygen species (ROS) generation, and AKT inactivation. Transcriptome analysis revealed that salt-inducible kinase 1 (SIK1) is markedly downregulated in the ETNPPL knockdown HepG2 cells. Moreover, disrupting SIK1 prevents ETNPPL-induced ROS accumulation, damage to the PI3K/AKT pathway and IR. Our study reveals that ETNPPL mediates hyperinsulinemia-induced IR through the SIK1/ROS-mediated inactivation of the PI3K/AKT signaling pathway in hepatocyte cells. Targeting ETNPPL may present a potential strategy for hyperinsulinemia-associated metabolic disorders such as type 2 diabetes.

MiR-3960 inhibits bladder cancer progression via targeting of DEXI.

PURPOSE: MicroRNAs (miRNAs) are dominant cargo in exosomes and act as master regulators of cell function, inhibiting mRNA translation and affecting gene silencing. Some aspects of tissue-specific miRNA transport in bladder cancer (BC) and its role in cancer progression are not fully understood. MATERIALS AND METHODS: A microarray was used to identify miRNAs in mouse bladder carcinoma cell line MB49 exosomes. Real-time reverse transcription polymerase chain reaction was used to examine the expression of miRNAs in BC and healthy donor serum. Western blotting and immunohistochemical staining were used to examine the expression of dexamethasone-induced protein (DEXI) in patients with BC. CRISPR-Cas 9 was used to knock out Dexi in MB49, and flow cytometry was performed to test cell proliferation ability and apoptosis under chemotherapy. Human BC organoid culture, miR-3960 transfection, and 293T-exosome-loaded miR-3960 delivery were used to analyze the effect of miR-3960 on BC progression. RESULTS: The results showed that miR-3960 levels in BC tissue were positively correlated with patient survival time. Dexi was a major target of miR-3960. Dexi knockout inhibited MB49 cell proliferation and promoted cisplatin- and gemcitabine-induced apoptosis. Transfection of miR-3960 mimic inhibited DEXI expression and organoid growth. In parallel, 293T-exosome-loaded miR-3960 delivery and Dexi knockout significantly inhibited subcutaneous growth of MB49 cells in vivo. CONCLUSION: Our results demonstrate the potential role of miR-3960-mediated inhibition of DEXI as a therapeutic strategy against BC.

Mechanism of co-operation of mutant IL-7Rα and mutant NRAS in acute lymphoblastic leukemia: role of MYC.

Acute lymphoblastic leukemia (ALL) is an aggressive leukemia which can be derived from either T-cell or B-cell precursors. With current treatments, the survival rate is high, but the treatments are highly toxic with severe side effects. Individual mutations in IL7Rssand RAS pathways have been previously shown to be prevalent in ALL and especially in relapsed patients. The relationship of IL-7R77and RAS was investigated by transducing immature mouse thymocytes with the combination of these mutants. The resultant ALL cells were analyzed to identify the regulators and the oncoproteins that are upregulated or downregulated by the combination of IL7Rα with NRAS. Leukemia cells showed a significant increase in IL7Rw-mediated BCL2 expression, and an increase in MYC protein levels, was mainly induced by NRAS signaling. MYC was both necessary and sufficient to replace mutant NRAS and drugs targeting the MYC pathway showed a therapeutic benefit in IL-7R7/NRAS T-ALL. We suggest that MYC protein stability can be regulated by PLK-1 kinase, which was increased mainly by the NRAS signal. These studies identify novel pathways of oncogenesis and new targets for intervention that could lead to better therapeutic development.

Isolation and characterization of a novel phage belonging to a new genus against Vibrio parahaemolyticus.

BACKGROUND: Vibrio parahaemolyticus is a major foodborne pathogen that contaminates aquatic products and causes great economic losses to aquaculture. Because of the emergence of multidrug-resistant V. parahaemolyticus strains, bacteriophages are considered promising agents for their biocontrol as an alternative or supplement to antibiotics. In this study, a lytic vibriophage, vB_VpaM_R16F (R16F), infecting V. parahaemolyticus 1.1997T was isolated, characterized and evaluated for its biocontrol potential. METHODS: A vibriophage R16F was isolated from sewage from a seafood market with the double-layer agar method. R16F was studied by transmission electron microscopy, host range, sensitivity of phage particles to chloroform, one-step growth curve and lytic activity. The phage genome was sequenced and in-depth characterized, including phylogenetic and taxonomic analysis. RESULTS: R16F belongs to the myovirus morphotype and infects V. parahaemolyticus, but not nine other Vibrio spp. As characterized by determining its host range, one-step growth curve, and lytic activity, phage R16F was found to highly effective in lysing host cells with a short latent period (< 10 min) and a small burst size (13 plaque-forming units). R16F has a linear double-stranded DNA with genome size 139,011 bp and a G + C content of 35.21%. Phylogenetic and intergenomic nucleotide sequence similarity analysis revealed that R16F is distinct from currently known vibriophages and belongs to a novel genus. Several genes (e.g., encoding ultraviolet damage endonuclease and endolysin) that may enhance environmental competitiveness were found in the genome of R16F, while no antibiotic resistance- or virulence factor-related gene was detected. CONCLUSIONS: In consideration of its biological and genetic properties, this newly discovered phage R16F belongs to a novel genus and may be a potential alternate biocontrol agent.

Effect of interesting respiratory rehabilitation training for the treatment of refractory Mycoplasma pneumoniae pneumonia in children.

BACKGROUNDS: Refractory Mycoplasma pneumoniae pneumonia (RMPP) cause damage of pulmonary function and physical therapy assisting medical treatment is needed. OBJECTIVE: The aim of this study was to investigate the effect of interesting respiratory rehabilitation training on pulmonary function in children with RMPP. METHODS: A total of 76 children with diagnoses of RMPP in our hospital from January 2020 to February 2021 were enrolled in this prospective study. According to the random number table method, they were divided into the control group and the study group, with 38 cases in each group. The control group were given conventional treatment, and the study group received interesting respiratory rehabilitation training in the basis of conventional treatment. The antipyretic time, disappearance time of pulmonary shadow and cough, length of hospital stay, pulmonary function (first second of expiratory volume (FEV1), forced vital capacity (FVC), FEV1/FVC) at 1 day before and after intervention, serum interleukin-6 (IL-6), C-reactive protein (CRP), tumor necrosis factor (TNF-α), and quality of life (Pediatric Quality of Life Inventory, PedsQL 4.0 scale) were observed in the two groups. RESULTS: The antipyretic time, disappearance time of pulmonary shadow and cough, length of hospital stay in the study group were shorter than those in the control group (P < 0.05). One day before intervention, there was no significant difference in FVC, FEV1, FEV1/FVC IL-6, CRP, and TNF-α between the two groups (P > 0.05). One day after intervention, FVC, FEV1 and FEV1/FVC in the study group were better than those in the control group (P < 0.05), and the levels of IL-6, CRP, and TNF-α in the study group were lower than those in the control group with significant difference (P < 0.05). One day before intervention, there were no significant differences in physiological function, emotional function, social function, and school function between the two groups (P > 0.05). After intervention, physiological function, emotional function, social function, and school function of the study group were better than those of the control group (P < 0.05). CONCLUSION: The interesting respiratory rehabilitation training can effectively improve the pulmonary function of children with RMPP, with strong flexibility, which is worthy of clinical application.

Mitochondrial dysfunction and oxidative stress in diabetic wound.

Diabetic wounds nowadays have become a major health challenge with the changes of the disease spectrum. Mitochondria are closely associated with stubborn nonhealing diabetic wounds for their vital role in energy metabolism, redox homeostasis, and signal transduction. There is significant mitochondrial dysfunction and oxidative stress in diabetic wounds. However, the contribution of mitochondrial dysfunction in oxidative stress induced nonhealing diabetic wound is still not fully understood. In this review, we will briefly summarize the current knowledge of the reported signaling pathways and therapeutic strategies involved in mitochondrial dysfunction in diabetic wounds. The findings provide further understanding of strategies that focus on mitochondria in diabetic wound treatment.

Novel photoactivated Indole-pyridine chemotherapeutics with strong antimicrobial and antibiofilm activity toward Staphylococcus aureus.

The challenge of antibiotic resistance worldwide has brought an urgent need to explore novel drugs for bacterial infections. Antimicrobial photodynamic therapy has been proven to be a potential antimicrobial modality but is limited by biofilms. In this study, we synthesized three cationic photosensitizers with strong photoinduced antimicrobial and antibiofilm activities toward gram-positive Staphylococcus aureus. The indole-pyridine compounds illustrated multiple type I/II photosensitization and coenzyme NAD(P)H photocatalytic activity upon excitation. A mechanistic study showed that intracellular reactive oxygen generation and NAD(P)H oxidation caused membrane damage, leading to protein/nucleus acid leakage. This research provides insights into the development of novel chemotherapeutics with synergetic photodynamic and photocatalytic reactivity.

Long-term results of radiofrequency ablation for locally recurrent papillary thyroid carcinoma.

OBJECTIVE: To evaluate the long-term efficacy and safety of ultrasound-guided radiofrequency ablation (RFA) for treating locally recurrent papillary thyroid cancer (PTC). METHODS: This retrospective study involved 32 patients with pathologically confirmed locally recurrent PTC. The ablation zone was assessed by contrast-enhanced ultrasound (CEUS) after RFA. At baseline, 6 and 12 months and every 6 months or 12 months thereafter, the following results were recorded: recurrence rate, largest diameter, volume, volume reduction rate (VRR) of recurrent lesions, serum thyroglobulin (Tg) level and complications. RESULTS: 58 recurrent lesions in 32 patients were successfully ablated with RFA. The mean follow-up time was 73.19 ± 12.68 months (range, 60 to 98 months). At the last follow-up, almost all ablated lesions disappeared completely, and only one lesion showed scar-like changes. Nine (28.13%) patients developed new locally recurrent tumors; they were successfully treated with repeat RFA sessions. No new recurrent lesions were found during the follow-up. The largest diameter and volume of recurrent lesions decreased from 13.71 ± 6.48 mm and 520.43 ± 627.85 mm3 to 0 each at the end of observation period (p < .001). The average VRRs at 6, 12, 24, 36, 48, 60 months and last follow-up after RFA were 54.17%, 72.90%, 82.28%, 89.30%, 92.57%, 96.60%, 96.88%, 98.14% and 100% respectively. The median of serum Tg level was decreased from 1.48 ng/mL to 0.00 ng/mL (p < .05). No complications were reported during the follow-up. CONCLUSIONS: US-guided RFA is an effective and safe option for treating locally recurrent PTC in selected patients, with favorable long-term outcomes.

Exosomal miR-125b-5p derived from adipose-derived mesenchymal stem cells enhance diabetic hindlimb ischemia repair via targeting alkaline ceramidase 2.

INTRODUCTION: Ischemic diseases caused by diabetes continue to pose a major health challenge and effective treatments are in high demand. Mesenchymal stem cells (MSCs) derived exosomes have aroused broad attention as a cell-free treatment for ischemic diseases. However, the efficacy of exosomes from adipose-derived mesenchymal stem cells (ADSC-Exos) in treating diabetic lower limb ischemic injury remains unclear. METHODS: Exosomes were isolated from ADSCs culture supernatants by differential ultracentrifugation and their effect on C2C12 cells and HUVECs was assessed by EdU, Transwell, and in vitro tube formation assays separately. The recovery of limb function after ADSC-Exos treatment was evaluated by Laser-Doppler perfusion imaging, limb function score, and histological analysis. Subsequently, miRNA sequencing and rescue experiments were performed to figure out the responsible miRNA for the protective role of ADSC-Exos on diabetic hindlimb ischemic injury. Finally, the direct target of miRNA in C2C12 cells was confirmed by bioinformatic analysis and dual-luciferase report gene assay. RESULTS: ADSC-Exos have the potential to promote proliferation and migration of C2C12 cells and to promote HUVECs angiogenesis. In vivo experiments have shown that ADSC-Exos can protect ischemic skeletal muscle, promote the repair of muscle injury, and accelerate vascular regeneration. Combined with bioinformatics analysis, miR-125b-5p may be a key molecule in this process. Transfer of miR-125b-5p into C2C12 cells was able to promote cell proliferation and migration by suppressing ACER2 overexpression. CONCLUSION: The findings revealed that miR-125b-5p derived from ADSC-Exos may play a critical role in ischemic muscle reparation by targeting ACER2. In conclusion, our study may provide new insights into the potential of ADSC-Exos as a treatment option for diabetic lower limb ischemia.