Prospective Clinical Application Research Articles

Real-time plasmid transmission detection pipeline.

Plasmid-mediated spread of antimicrobial resistance is a major challenge for clinical microbiology, and monitoring of potential plasmid transmissions is essential to combat further dissemination. Whole-genome sequencing is often used to surveil nosocomial transmissions but usually limited to the detection of clonal transmissions (based on chromosomal markers). Recent advances in long-read sequencing technologies enable full reconstruction of plasmids and the detection of very similar plasmids, but so far, easy-to-use bioinformatic tools for this purpose have been missing. Here, we present an evaluation of an innovative real-time plasmid transmission detection pipeline. It is integrated into the GUI-based SeqSphere+ software, which already offers core-genome multi-locus sequence typing-based pathogen outbreak detection. It requires very limited bioinformatics knowledge, and its database, automated analyses, and alert system make it well suited for prospective clinical application.

Deep learning-based automatic ASPECTS calculation can improve diagnosis efficiency in patients with acute ischemic stroke: a multicenter study.

The Alberta Stroke Program Early CT Score (ASPECTS), a systematic method for assessing ischemic changes in acute ischemic stroke using non-contrast computed tomography (NCCT), is often interpreted relying on expert experience and can vary between readers. This study aimed to develop a clinically applicable automatic ASPECTS system employing deep learning (DL). This study enrolled 1987 NCCT scans that were retrospectively collected from four centers between January 2017 and October 2021. A DL-based system for automated ASPECTS assessment was trained on a development cohort (N = 1767) and validated on an independent test cohort (N = 220). The consensus of experienced physicians was regarded as a reference standard. The validity and reliability of the proposed system were assessed against physicians' readings. A real-world prospective application study with 13,399 patients was used for system validation in clinical contexts. The DL-based system achieved an area under the receiver operating characteristic curve (AUC) of 84.97% and an intraclass correlation coefficient (ICC) of 0.84 for overall-level analysis on the test cohort. The system's diagnostic sensitivity was 94.61% for patients with dichotomized ASPECTS at a threshold of ≥ 6, with substantial agreement (ICC = 0.65) with expert ratings. Combining the system with physicians improved AUC from 67.43 to 89.76%, reducing diagnosis time from 130.6 ± 66.3 s to 33.3 ± 8.3 s (p < 0.001). During the application in clinical contexts, 94.0% (12,591) of scans successfully processed by the system were utilized by clinicians, and 96% of physicians acknowledged significant improvement in work efficiency. The proposed DL-based system could accurately and rapidly determine ASPECTS, which might facilitate clinical workflow for early intervention. The deep learning-based automated ASPECTS evaluation system can accurately and rapidly determine ASPECTS for early intervention in clinical workflows, reducing processing time for physicians by 74.8%, but still requires validation by physicians when in clinical applications. The deep learning-based system for ASPECTS quantification has been shown to be non-inferior to expert-rated ASPECTS. This system improved the consistency of ASPECTS evaluation and reduced processing time to 33.3 seconds per scan. 94.0% of scans successfully processed by the system were utilized by clinicians during the prospective clinical application.

Mechanistic prediction and validation of Brevilin A Therapeutic effects in Lung Cancer

BackgroundTraditional Chinese medicine (TCM) has been found widespread application in neoplasm treatment, yielding promising therapeutic candidates. Previous studies have revealed the anti-cancer properties of Brevilin A, a naturally occurring sesquiterpene lactone derived from Centipeda minima (L.) A.Br. (C. minima), a TCM herb, specifically against lung cancer. However, the underlying mechanisms of its effects remain elusive. This study employs network pharmacology and experimental analyses to unravel the molecular mechanisms of Brevilin A in lung cancer.MethodsThe Batman-TCM, Swiss Target Prediction, Pharmmapper, SuperPred, and BindingDB databases were screened to identify Brevilin A targets. Lung cancer-related targets were sourced from GEO, Genecards, OMIM, TTD, and Drugbank databases. Utilizing Cytoscape software, a protein-protein interaction (PPI) network was established. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene set enrichment analysis (GSEA), and gene-pathway correlation analysis were conducted using R software. To validate network pharmacology results, molecular docking, molecular dynamics simulations, and in vitro experiments were performed.ResultsWe identified 599 Brevilin A-associated targets and 3864 lung cancer-related targets, with 155 overlapping genes considered as candidate targets for Brevilin A against lung cancer. The PPI network highlighted STAT3, TNF, HIF1A, PTEN, ESR1, and MTOR as potential therapeutic targets. GO and KEGG analyses revealed 2893 enriched GO terms and 157 enriched KEGG pathways, including the PI3K-Akt signaling pathway, FoxO signaling pathway, and HIF-1 signaling pathway. GSEA demonstrated a close association between hub genes and lung cancer. Gene-pathway correlation analysis indicated significant associations between hub genes and the cellular response to hypoxia pathway. Molecular docking and dynamics simulations confirmed Brevilin A’s interaction with PTEN and HIF1A, respectively. In vitro experiments demonstrated Brevilin A-induced dose- and time-dependent cell death in A549 cells. Notably, Brevilin A treatment significantly reduced HIF-1α mRNA expression while increasing PTEN mRNA levels.ConclusionsThis study demonstrates that Brevilin A exerts anti-cancer effects in treating lung cancer through a multi-target and multi-pathway manner, with the HIF pathway potentially being involved. These results lay a theoretical foundation for the prospective clinical application of Brevilin A.

Efficacy of real-time artificial intelligence-aid endoscopic ultrasonography diagnostic system in discriminating gastrointestinal stromal tumors and leiomyomas: a multicenter diagnostic study

Gastrointestinal stromal tumors (GISTs) represent the most prevalent type of subepithelial lesions (SELs) with malignant potential. Current imaging tools struggle to differentiate GISTs from leiomyomas. This study aimed to create and assess a real-time artificial intelligence (AI) system using endoscopic ultrasonography (EUS) images to differentiate between GISTs and leiomyomas. The AI system underwent development and evaluation using EUS images from 5 endoscopic centers in China between January 2020 and August 2023. EUS images of 1101 participants with SELs were retrospectively collected for AI system development. A cohort of 241 participants with SELs was recruited for external AI system evaluation. Another cohort of 59 participants with SELs was prospectively enrolled to assess the real-time clinical application of the AI system. The AI system's performance was compared to that of endoscopists. This study is registered with Chictr.org.cn, Number ChiCT2000035787. The AI system displayed an area under the curve (AUC) of 0.948 (95% CI: 0.921-0.969) for discriminating GISTs and leiomyomas. The AI system's accuracy (ACC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) reached 91.7% (95% CI 87.5%-94.6%), 90.3% (95% CI 83.4%-94.5%), 93.0% (95% CI 87.2%-96.3%), 91.9% (95% CI 85.3%-95.7%), and 91.5% (95% CI 85.5%-95.2%), respectively. Moreover, the AI system exhibited excellent performance in diagnosing ≤20mm SELs (ACC 93.5%, 95% CI 0.900-0.969). In a prospective real-time clinical application trial, the AI system achieved an AUC of 0.865 (95% CI 0.764-0.966) and 0.864 (95% CI 0.762-0.966) for GISTs and leiomyomas diagnosis, respectively, markedly surpassing endoscopists [AUC 0.698 (95% CI 0.562-0.834) for GISTs and AUC 0.695 (95% CI 0.546-0.825) for leiomyomas]. We successfully developed a real-time AI-assisted EUS diagnostic system. The incorporation of the real-time AI system during EUS examinations can assist endoscopists in rapidly and accurately differentiating various types of SELs in clinical practice, facilitating improved diagnostic and therapeutic decision-making. Science and Technology Commission Foundation of Shanghai Municipality, Science and Technology Commission Foundation of the Xuhui District, the Interdisciplinary Program of Shanghai Jiao Tong University and the Research Funds of Shanghai Sixth people's Hospital.

#2368 GHRH levels and AhR expression in CKD patients before and after kidney transplantation

Abstract Background and Aims Chronic kidney disease (CKD) is an irreversible decline in renal function where kidney transplantation (KT) is the optimal therapeutic intervention. We aim to investigate the role and activation of the uremic toxin receptor aryl hydrocarbon receptor (AhR) in the microvasculature, particularly its implication in the blood-brain barrier impairment. We will also explore the potential role of growth hormone-releasing hormone (GHRH) as a biomarker reflecting the resolution of uremia-induced inflammation, early vascular ageing (EVA) phenotype, and potential enhancement in central nervous system (CNS) function post-KT. Method Resistance arteries from KT patients and non-CKD controls were isolated and expression of AhR was analysed by immunohistochemistry. The study used plasma samples from 60 KT patients, and levels of GHRH were measured at baseline and two years after transplantation using ELISA. Results Following KT, our initial findings indicate a decrease in levels of DBP, Lp(a), creatinine, homocysteine, phosphate, and troponin T among KT patients. Conversely, there was an increase in albumin, HBA1c, calcium and NfL post-transplantation. The expression of AhR in the resistance arteries of KT patients (2.6%) was notably lower compared to non-CKD controls (9.7%). Plasma concentrations of GHRH were significantly higher (p &amp;lt; 0.0001) in CKD-5 patients two years post-KT (2.94 ng/mL, IQR 2.60-3.43) compared to baseline (2.32 mg/L, IQR 1.84-3.24). This elevated trend in GHRH levels post-KT was observed in both sexes, with males (p &amp;lt; 0.0001) and females (p 0.0084) showing higher levels—males (3.0 ng/mL, IQR 2.6-3.5) and females (2.8 ng/mL, IQR 2.4-3.5)—compared to baseline level of males (2.4 ng/mL, IQR 1.8-3.2) and females (2.2 ng/mL, 1.9-3.3). No significant difference in GHRH levels was observed when assessing extreme EVA phenotypes based on the presence or absence of calcification or fibrosis. Conclusion Contrary to our initial expectation, the preliminary results indicate a downregulation in AhR expression within resistance vessels of CKD patients, implying modifications in AhR signaling within the uremic environment. The observed alteration in GHRH levels presents a promising avenue for further investigation focusing on its possible application as a biomarker for assessing CNS status in CKD patients undergoing KT. Prospective clinical application could involve the administration of GHRH agonists as a protective measure against cognitive impairment, considering its deficiency in CKD. Such supplementation holds promise to prevent further cognitive morbidity. The modulation in the levels of this hormone has the potential to significantly influence the treatment trajectory of CKD patients and enhance clinical outcomes.

Human Adipose-Derived Mesenchymal Stem Cells Colonize and Promote Healing of Leprosy Ulcer by Inducing Neuro-Vascularization.

Leprosy ulcer is a chronic and recurrent disease resulting from nerve injury. While existing treatments partially facilitate ulcer healing, they exhibit limited ability to address localized nerve repair, leading to a risk of recurrence. Moreover, there is a dearth of animal models to evaluate the preclinical efficacy and safety of novel therapeutic approaches. Over the years, adipose-derived mesenchymal stem cells have been extensively employed in regenerative medicine as an optimal cell therapy source for fostering skin ulcer healing. They have also demonstrated the capacity to enhance nerve regeneration in in vitro experiments and clinical trials. In this study, we established a NU/NU mouse foot pad leprosy ulcer model, transplanted human adipose-derived stem cells (hADSCs) into leprosy ulcers via local injection, and conducted subsequent follow-up. Our findings revealed that hADSCs persisted in the leprosy ulcer and facilitated the healing process. In this respect, gross observation and histological analysis revealed increased granular formation, collagen synthesis, and re-epithelialization in the local ulcer area. RNA-Seq data revealed that the upregulated differential genes resulting from the transplantation intervention were not only enriched in pathways related to re-epithelialization and collagen synthesis but also contributed to local nerve regeneration. Furthermore, immunofluorescence assays revealed the increased expression of angiogenesis markers-CD31 and VEGFa, cell proliferation markers-Ki67 and TGF-β, and nerve regeneration markers-β3-tubulin, SOX10, NGF, and NT-3. These results underscore the potential of hADSCs in promoting the healing of leprosy ulcers and offer valuable preclinical data for their prospective clinical application.

Exhaled breath and urinary volatile organic compounds (VOCs) for cancer diagnoses, and microbial-related VOC metabolic pathway analysis: a systematic review and meta-analysis.

The gradual evolution of the detection and quantification of volatile organic compounds (VOCs) has been instrumental in cancer diagnosis. The primary objective of this study was to assess the diagnostic potential of exhaled breath and urinary VOCs in cancer detection. As VOCs are indicative of tumor and human metabolism, our work also sought to investigate the metabolic pathways linked to the development of cancerous tumors. An electronic search was performed in the PubMed database. Original studies on VOCs within exhaled breath and urine for cancer detection with a control group were included. A meta-analysis was conducted using a bivariate model to assess the sensitivity and specificity of the VOCs for cancer detection. Fagan's nomogram was designed to leverage the findings from our diagnostic analysis for the purpose of estimating the likelihood of cancer in patients. Ultimately, MetOrigin was employed to conduct an analysis of the metabolic pathways associated with VOCs in relation to both human and/or microbiota. The pooled sensitivity, specificity and the area under the curve for cancer screening utilizing exhaled breath and urinary VOCs were determined to be 0.89, 0.88, and 0.95, respectively. A pretest probability of 51% can be considered as the threshold for diagnosing cancers with VOCs. As the estimated pretest probability of cancer exceeds 51%, it becomes more appropriate to emphasize the 'ruling in' approach. Conversely, when the estimated pretest probability of cancer falls below 51%, it is more suitable to emphasize the 'ruling out' approach. A total of 14, 14, 6, and 7 microbiota-related VOCs were identified in relation to lung, colorectal, breast, and liver cancers, respectively. The enrichment analysis of volatile metabolites revealed a significant enrichment of butanoate metabolism in the aforementioned tumor types. The analysis of exhaled breath and urinary VOCs showed promise for cancer screening. In addition, the enrichment analysis of volatile metabolites revealed a significant enrichment of butanoate metabolism in four tumor types, namely lung, colorectum, breast and liver. These findings hold significant implications for the prospective clinical application of multiomics correlation in disease management and the exploration of potential therapeutic targets.

Integrative proteomics and metabolomics explore the effect and mechanism of Qiyin granules on improving nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) has emerged as a prominent global health concern, representing a substantial burden within the spectrum of chronic liver diseases. Despite its escalating prevalence, a definitive therapeutic strategy or efficacious pharmacological intervention for NAFLD has yet to receive official approval to date. While Fu Fang Qiyin granules have exhibited efficacy in addressing NAFLD, the intricacies of their underlying mechanism of action remain inadequately elucidated. In this study, we substantiated the ameliorative impact of Qiyin on highfat diet (HFD)induced NAFLD in rat models. The results of metabonomics showed that 108 potential biomarkers in serum and urine related to amino acid metabolism, energy metabolism, and pyrimidine metabolism, have returned to normal levels compared to the model group. Hepatic transcriptomics further indicated that Qiyin potentially confers protective effects against NAFLD by mediating liver inflammation and fibrosis through lumican (LUM) and decorin (DCN). In summation, our investigation provides compelling evidence affirming the therapeutic promise of Qiyin for NAFLD. It elucidates the underlying mechanistic pathways, furnishing a compelling rationale for its prospective clinical application.

PL02.2.A RAPID, COMPREHENSIVE REPORTING OF MOLECULAR DIAGNOSTICS WITH RAPID-CNS2: A PROSPECTIVE VALIDATION COHORT

Abstract BACKGROUND The WHO classification of CNS tumours 2021 recommends reporting a wide range of molecular alterations for WHO-compatible diagnoses. Conventional molecular diagnostic workflows warrant considerable investment and long turnaround times limited by batching. Nanopore sequencing enables long read sequencing, real-time targeting and simultaneous base modification detection with compact devices. Rapid-CNS2- a rapid, comprehensive adaptive sampling based sequencing pipeline with a turnaround time of 5 days for CNS tumours was previously described. This study presents an optimised Rapid-CNS2 pipeline and prospective validation cohort that successfully reports hard-to-detect variants. MATERIAL AND METHODS We performed targeted adaptive sampling-based sequencing on 156 samples comprising a mix of archival and current diagnostic cases from the Department of Neuropathology, University Hospital Heidelberg. We tested variable conditions including minimum DNA input, target size, flowcell reuse and sequencing times. An analysis pipeline for detection of single nucleotide variants (SNV), indels, copy number alterations (CNA), fusions, complex structural variants (SV), target gene methylation and methylation classification incorporating multi-GPU optimization was deployed. Results were compared to NGS panel sequencing and EPIC array analyses. RESULTS Integrated diagnoses for all cases were in accordance with those issued using molecular alterations reported by conventional analyses. Rapid-CNS2 showed improved resolution over NGS panel sequencing for CNAs. Complete concordance was achieved for MGMT promoter methylation status and methylation classification. High accuracy was obtained for detection of SNV/Indels. Long reads spanning the breakpoint identified clinically relevant fusions. Subclonal SVs including EGFR vIII missed by NGS panel sequencing were detected with high confidence. The optimized workflow shortened the turnaround time to 30 hours from sample receipt to report and minimum input DNA required was reduced to 500ng. Due to the versatility of adaptive sampling, the analyses can readily be limited to a smaller set of targets or methylation and CNAs only, further reducing the turnaround time. CONCLUSION We present a thorough validation and prospective clinical application of Rapid-CNS2 with cases spanning a range of brain tumours and metastases. The improved custom neurooncology adaptive sampling-based sequencing pipeline enables simultaneous copy-number, mutational, structural variant and methylation analysis with flexible target selection, no additional library preparation, competitive costs and next-day reporting of RESULTS

Synthetic Ligament-Assisted Functional Soft-Tissue Reconstruction in Tumor-Related Proximal Femoral Replacements

BackgroundSoft-tissue functional reconstruction is important for restoring hip function in proximal femoral replacement for tumor resection. This study evaluated the functional outcome and postoperative complications of a specific synthetic ligament for soft-tissue functional reconstruction in proximal femoral replacement for tumor resection. MethodsThis cohort included 80 patients (40 men and 40 women, mean age, 40 years (range, 10 to 79)) who had benign invasive tumors (n = 13), primary malignant bone tumors (n = 40), or bone metastases (n = 27). Patients’ medical records, imaging files, surgical details, and postoperative pathological diagnoses were collected. Tumor staging was performed with the Enneking staging criteria. Lower limb and hip functions were assessed using the Musculoskeletal Tumor Society scoring system and Harris Hip score, respectively. ResultsMean postoperative Musculoskeletal Tumor Society and Harris Hip scores were 27 (range, 24 to 30) and 90 (range, 76 to 97), respectively, indicating satisfactory range of motion and stability. Trendelenburg gait was observed in 3 osteosarcoma patients (3.8%), and 6 patients showed unequal lower-limb lengths. Reoperations occurred in 5 cases, including 3 cases of deep vein thromboses and 1 case each of giant cell granuloma and periprosthetic infection. There were 27 patients who had bone metastases who did not require reoperation. ConclusionsThe synthetic ligament reconstruction of the hip with proximal femoral replacement for tumor resection was associated with improved outcomes. Its implementation exhibits the potential to reduce postoperative incidences of hip dislocation and periprostheses infection, thereby warranting its prospective clinical application.

Recent Advances of Proteomics in Management of Acute Kidney Injury.

Acute Kidney Injury (AKI) is currently recognized as a life-threatening disease, leading to an exponential increase in morbidity and mortality worldwide. At present, AKI is characterized by a significant increase in serum creatinine (SCr) levels, typically followed by a sudden drop in glomerulus filtration rate (GFR). Changes in urine output are usually associated with the renal inability to excrete urea and other nitrogenous waste products, causing extracellular volume and electrolyte imbalances. Several molecular mechanisms were proposed to be affiliated with AKI development and progression, ultimately involving renal epithelium tubular cell-cycle arrest, inflammation, mitochondrial dysfunction, the inability to recover and regenerate proximal tubules, and impaired endothelial function. Diagnosis and prognosis using state-of-the-art clinical markers are often late and provide poor outcomes at disease onset. Inappropriate clinical assessment is a strong disease contributor, actively driving progression towards end stage renal disease (ESRD). Proteins, as the main functional and structural unit of the cell, provide the opportunity to monitor the disease on a molecular level. Changes in the proteomic profiles are pivotal for the expression of molecular pathways and disease pathogenesis. Introduction of highly-sensitive and innovative technology enabled the discovery of novel biomarkers for improved risk stratification, better and more cost-effective medical care for the ill patients and advanced personalized medicine. In line with those strategies, this review provides and discusses the latest findings of proteomic-based biomarkers and their prospective clinical application for AKI management.

Clinical significance of CD166 and HER-2 in different types of gastric cancer.

Cluster of differentiation 166 (CD166), a cancer stem cell (CSC) marker, and human epidermal growth factor receptor 2 (HER-2) are expressed in a diversity of malignancies and is associated with tumor progression. Although studies regarding the importance of CSC markers and HER-2 in gastric cancer (GC) have rapidly developed, their clinicopathological, prognosis, and diagnosis value still remain unsatisfying in GC. Therefore, the present study aims to investigate the clinical, prognostic, and diagnostic significance of CD166 and HER-2 in different histological types of GC. Bioinformatic analysis was applied to determine the clinical importance of CD166 and HER-2 expression based on their tissue localization in primary GC tumors and the normal adjacent samples. The expression patterns, clinical significance, prognosis, and diagnosis value of CD166 and HER-2 proteins in tissue microarrays (TMAs) of 206 GC samples, including Signet Ring Cell (SRC) and intestinal types and also 28 adjacent normal tissues were evaluated using immunohistochemistry (IHC). The results indicated that the expression of CD166 (membranous and cytoplasmic) and HER-2 were significantly up-regulated in tumor cells compared to adjacent normal tissues (P = 0.010, P < 0.001, and P = 0.011, respectively). A statistically significant association was detected between a high level of membranous expression of CD166 and lymphovascular invasion (P = 0.006); We also observed a statistically significant association between high cytoplasmic expression of CD166 protein and more invasion of the subserosa (P = 0.040) in the SRC type. In contrast, there was no correlation between the expression of HER-2 and clinicopathologic characteristics. Both CD166 and HER-2 showed reasonable accuracy and high specificity as diagnostic markers. Our results confirmed that increased membranous and cytoplasmic expression of CD166 showed clinical significance in the SRC type and is associated with the progression of the disease and more aggressive tumor behaviors. These findings can be used to assist in designating subgroups of patients that require different follow-up strategies, and also, they might be utilized as the prognostic or diagnostic biomarkers in these types of GC for prospective clinical application.

Small diameter expanded polytetrafluoroethylene vascular graft with differentiated inner and outer biomacromolecules for collaborative endothelialization, anti-thrombogenicity and anti-inflammation

Without differentiated inner and outer biological function, expanded polytetrafluoroethylene (ePTFE) small-diameter (<6 mm) artificial blood vessels would fail in vivo due to foreign body rejection, thrombosis, and hyperplasia. In order to synergistically promote endothelialization, anti-thrombogenicity, and anti-inflammatory function, we modified the inner and outer surface of ePTFE, respectively, by grafting functional biomolecules, such as heparin and epigallocatechin gallate (EGCG), into the inner surface and polyethyleneimine and rapamycin into the outer surface via layer-by-layer self-assembly. Fourier-transform infrared spectroscopy showed the successful incorporation of EGCG, heparin, and rapamycin. The collaborative release profile of heparin and rapamycin lasted for 42 days, respectively. The inner surface promoted human umbilical vein endothelial cells (HUVECs) adhesion and growth and that the outer surface inhibited smooth muscle cells growth and proliferation. The modified ePTFE effectively regulated the differentiation behavior of RAW264.7, inhibited the expression of proinflammatory mediator TNF-α, and up-regulated the expression of anti-inflammatory genes Arg1 and Tgfb-1. The ex vivo circulation results indicated that the occlusions and total thrombus weight of modified ePTFE was much lower than that of the thrombus formed on the ePTFE, presenting good anti-thrombogenic properties. Hence, the straightforward yet efficient synergistic surface functionalization approach presented a potential resolution for the prospective clinical application of small-diameter ePTFE blood vessel grafts.

PH-Responsive Hexa-Histidine Metal Assembly (HmA) with Enhanced Biocatalytic Cascades as the Vehicle for Glucose-Mediated Long-Acting Insulin Delivery.

Diabetes has been listed as one of the three major diseases that endanger human health. Accurately injecting insulin (Ins) depending on the level of blood glucose (LBG) is the standard treatment, especially controlling LBG in the long-term by a single injection. Herein, the pH-responsive hexa-histidine metal assembly (HmA) encapsulated with enzymes (GOx and CAT) and Ins (HmA@GCI) is engineered as the vehicle for glucose-mediated insulin delivery. HmA not only shows high proteins loading efficiency, but also well retained proteins activity and protect proteins from protease damage. Within HmA, the biocatalytic activities of enzymes and the efficiency of the cascade reaction between GOx and CAT are enhanced, leading to a super response to the change of LBG with insulin release and efficient clearance of harmful byproducts of GOx (H2 O2 ). In the treatment of diabetic mice, HmA@GCI reduces LBG to normal in half an hour and maintains for more than 5 days by a single subcutaneous injection, and nearly 24 days with four consecutive injections. During the test period, no symptoms of hypoglycemia and toxicity to tissues and organs are observed. These results indicate that HmA@GCI is a safe and long-acting hypoglycemic agent with prospective clinical application.

Statistical breathing curve sampling to quantify interplay effects of moving lung tumors in a 4D Monte Carlo dose calculation framework

PurposeThe interplay between respiratory tumor motion and dose application by intensity modulated radiotherapy (IMRT) techniques can potentially lead to undesirable and non-intuitive deviations from the planned dose distribution. We developed a 4D Monte Carlo (MC) dose recalculation framework featuring statistical breathing curve sampling, to precisely simulate the dose distribution for moving target volumes aiming at a comprehensive assessment of interplay effects. MethodsWe implemented a dose accumulation tool that enables dose recalculations of arbitrary breathing curves including the actual breathing curve of the patient. This MC dose recalculation framework is based on linac log-files, facilitating a high temporal resolution up to 0.1 s. By statistical analysis of 128 different breathing curves, interplay susceptibility of different treatment parameters was evaluated for an exemplary patient case. To facilitate prospective clinical application in the treatment planning stage, in which patient breathing curves or linac log-files are not available, we derived a log-file free version with breathing curves generated by a random walk approach. Interplay was quantified by standard deviations σ in D5%, D50% and D95%. ResultsInterplay induced dose deviations for single fractions were observed and evaluated for IMRT and volumetric arc therapy (σD95% up to 1.3 %) showing a decrease with higher fraction doses and an increase with higher MU rates. Interplay effects for conformal treatment techniques were negligible (σ<0.1%). The log-file free version and the random walk generated breathing curves yielded similar results (deviations in σ< 0.1 %) and can be used as substitutes for interplay assessment. ConclusionIt is feasible to combine statistically sampled breathing curves with MC dose calculations. The universality of the presented framework allows comprehensive assessment of interplay effects in retrospective and prospective clinically relevant scenarios.

Antibacterial Effect of Copper Sulfide Nanoparticles on Infected Wound Healing.

Background: It is widely acknowledged that pathogenic germs delay wound healing to some extent. To explore factors influencing the wound healing process, the current study was conducted to evaluate the antibacterial effect of topical application of copper sulfide nanoparticles (CuS NPs) in vitro and on infected wound healing process in the rat model. Materials and Methods: In this study, the morphology and size of CuS NPs were detected. Staphylococcus aureus and Escherichia coli were used so that the antibacterial ability of CuS NPs could be evaluated better. In addition, a 2-cm circular full-thickness wound infected with a solution of 107 colony forming units (CFU) Staphylococcus aureus was created on the back of each rat. The rats were divided into four groups including the control group, the 100 mcg/mL CuS NPs group, the 250 mcg/mL CuS NPs group, and the 500 mcg/mL CuS NPs group. Tissue bacterial count and histologic assessment were evaluated. Results: The results indicated that CuS NPs had antibacterial activity against Staphylococcus aureus and Escherichia coli. Moreover, they could decrease the incidence of bacterial colonization and promote wound healing through re-epithelialization and collagen deposition. Furthermore, CuS NPs could maintain Cu2+ continuous release and inhibit the viability of Staphylococcus aureus through lipid peroxidation. Conclusions: This study found that CuS NPs have fine antibacterial properties, and particularly, the 500 mcg/mL CuS NPs had better effects, without increase of side effects. They could promote infected wound healing, the prospective clinical application of which was further confirmed in the treatment of wound infection.

Photobiomodulation: An Effective Approach to Enhance Proliferation and Differentiation of Adipose-Derived Stem Cells into Osteoblasts.

Osteoporosis is regarded as the most common chronic metabolic bone condition in humans. In osteoporosis, bone mesenchymal stem cells (MSCs) have reduced cellular function. Regenerative medicine using adipose-derived stem cell (ADSC) transplantation can promote the growth and strength of new bones, improve bone stability, and reduce the risk of fractures. Various methods have been attempted to differentiate ADSCs to functioning specialized cells for prospective clinical application. However, commonly used therapies have resulted in damage to the donor site and morbidity, immune reactions, carcinogenic generation, and postoperative difficulties. Photobiomodulation (PBM) improves ADSC differentiation and proliferation along with reducing clinical difficulties such as treatment failures to common drug therapies and late initiation of treatment. PBM is a noninvasive, nonthermal treatment that encourages cells to produce more energy and to undergo self-repair by using visible green and red and invisible near-infrared (NIR) radiation. The use of PBM for ADSC proliferation and differentiation has been widely studied with multiple outcomes observed due to laser fluence and wavelength dependence. In this article, the potential for differentiating ADSCs into osteoblasts and the various methods used, including biological induction, chemical induction, and PBM, will be addressed. Likewise, the optimal laser parameters that could improve the proliferation and differentiation of ADSC, translating into clinical success, will be commented on.

In vivo regeneration of rat laryngeal cartilage with mesenchymal stem cells derived from human induced pluripotent stem cells via neural crest cells.

The laryngotracheal cartilage is a cardinal framework for the maintenance of the airway for breathing, which occasionally requires reconstruction. Because hyaline cartilage has a poor intrinsic regenerative ability, various regenerative approaches have been attempted to regenerate laryngotracheal cartilage. The use of autologous mesenchymal stem cells (MSCs) for cartilage regeneration has been widely investigated. However, long-term culture may limit proliferative capacity. Human-induced pluripotent stem cell-derived MSCs (iMSCs) can circumvent this problem due to their unlimited proliferative capacity. This study aimed to investigate the efficacy of iMSCs in the regeneration of thyroid cartilage in immunodeficient rats. Herein, we induced iMSCs through neural crest cell intermediates. For the relevance to prospective future clinical application, induction was conducted under xeno-free/serum-free conditions. Then, clumps fabricated from an iMSC/extracellular matrix complex (C-iMSC) were transplanted into thyroid cartilage defects in immunodeficient rats. Histological examinations revealed cartilage-like regenerated tissue and human nuclear antigen (HNA)-positive surviving transplanted cells in the regenerated lesion. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells. These results indicated that the transplanted C-iMSCs promoted thyroid cartilage regeneration and some of the iMSCs differentiated into chondrogenic lineage cells. Induced MSCs may be a promising candidate cell therapy for human laryngotracheal reconstruction.

The NEDD8-activating enzyme inhibition with MLN4924 sensitizes human cancer cells of different origins to apoptosis and necroptosis

ObjectivesMLN4924 is an inhibitor of NEDD8-activating enzyme (NAE) that interferes with the cullin-RING ubiquitin ligase complexes formation and the nuclear factor kappa B (NF-κB) activation. Here, we investigated the cytotoxic effect of MLN4924 and its ability to sensitize a broad range of cancer cells of different origins to tumour necrosis factor-α (TNF)-induced cell death alongside unravelling its mechanism of action. Materials and methodsCell viability and caspases processing were determined after MLN4924 treatment either alone or with zVAD-fmk (pan caspase inhibitor), necrostatin-1 (nec-1, RIPK1 inhibitor) and necrosulfonamide (NSA, MLKL inhibitor). Moreover, MLN4924 ability to potentiate TNF-induced cell death was evaluated in 24 cell lines of different cancer origins. The impact of NAE inhibition with MLN4924 on TNF-induced apoptosis and necroptosis was evaluated using zVAD-fmk and nec-1, respectively. ResultsMLN4924 alone was able to induce cell death in different cell lines that was attributed to apoptosis induction. Also, MLN4924 sensitized different cancer cell lines to TNF-induced cell death. MLN4924/TNF-induced cell death was apoptosis and necroptosis dependent that may be attributed to MLN4924 inhibition of NF-κB pathway activation. ConclusionsTargeting NAE and NF-κB pathway with MLN4924 represents a substantial approach to enhance the sensitivity of diverse types of cancer cells. Moreover, the broad in vitro screening of MLN4924 anticancer activity provides a valuable guidance for elucidating the susceptible cancer types for the prospective clinical application of MLN4924.

Tumour dormancy in inflammatory microenvironment: A promising therapeutic strategy for cancer-related bone metastasis.

Cancer metastasis is a unique feature of malignant tumours. Even bone can become a common colonization site due to the tendency of solid tumours, including breast cancer (BCa) and prostate cancer (PCa), to metastasize to bone. Currently, a previous concept in tumour metabolism called tumour dormancy may be a promising target for antitumour treatment. When disseminated tumour cells (DTCs) metastasize to the bone microenvironment, they form a flexible regulatory network called the "bone-tumour-inflammation network". In this network, bone turnover as well as metabolism, tumour progression, angiogenesis and inflammatory responses are highly unified and coordinated, and a slight shift in this balance can result in the disruption of the microenvironment, uncontrolled inflammatory responses and excessive tumour growth. The purpose of this review is to highlight the regulatory effect of the "bone-tumour-inflammation network" in tumour dormancy. Osteoblast-secreted factors, bone turnover and macrophages are emphasized and occupy in the main part of the review. In addition, the prospective clinical application of tumour dormancy is also discussed, which shows the direction of future research.