CRISPR/Cas9 System with Dual gRNAs Synergically Inhibit Hepatitis B Virus Replication.

BACKGROUND: In recent years, a gene-editing technology known as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been developed and is progressively advancing into clinical trials. While current antiviral therapies are unable to eliminate the Hepatitis B virus (HBV), it stands as a prime target for the CRISPR/Cas9 technology. The objective of this study was to enhance the efficacy of CRISPR/Cas9 in suppressing HBV replication, lowering HBsAg and HBeAg levels, and eliminating covalently closed circular DNA (cccDNA). METHODS: To enhance the anti-HBV effectiveness of CRISPR/Cas9, our study delved into a dual-guide RNA (gRNA) strategy. After evaluating the antiviral activities of multiple gRNAs that effectively impeded HBV replication, we identified three specific gRNAs-namely 10, 4, and 21. These gRNAs were selected for their targeting of distinct yet conserved regions within the HBV genome. RESULTS: In HBV-stable cell lines, namely HepAD38, and HBV infection models of HepG2-NTCP cells, our investigation revealed that the co-application of gRNA-10 with either gRNA-4 or gRNA-21 within the CRISPR/Cas9 system demonstrated heightened efficacy in impeding HBV replication, reducing the levels of HBsAg, HBeAg, and cccDNA levels, along with a more pronounced promotion of HBsAg clearance when compared to the use of a single gRNA. CONCLUSIONS: The CRISPR/Cas9 system employing dual gRNAs has proven highly effective in both suppressing HBV replication and facilitating HBsAg clearance. This promising outcome suggests that it holds potential to emerge as a novel approach for achieving the functional cure of patients with HBV infection.

The MuSK agonist antibody protects the neuromuscular junction and extends the lifespan in C9orf72-ALS mice.

The disassembly of the neuromuscular junction (NMJ) is an early event in amyotrophic lateral sclerosis (ALS), ultimately leading to motor dysfunction and lethal respiratory paralysis. The hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most common genetic mutation, and the dipeptide repeat (DPR) proteins have been shown to cause neurodegeneration. While no drugs can treat ALS patients efficiently, new treatment strategies are urgently needed. Here, we report that a MuSK agonist antibody alleviates poly-PR-induced NMJ deficits in C9orf72-ALS mice. The HB9-PRF/F mice, which express poly-PR proteins in motor neurons, exhibited impaired motor behavior and NMJ deficits. Mechanistically, poly-PR proteins interacted with Agrin to disrupt the interaction between Agrin and Lrp4, leading to attenuated activation of MuSK. Treatment with a MuSK agonist antibody rescued NMJ deficits, and extended the lifespan of C9orf72-ALS mice. Moreover, impaired NMJ transmission was observed in C9orf72-ALS patients. These findings identify the mechanism by which poly-PR proteins attenuate MuSK activation and NMJ transmission, highlighting the potential of promoting MuSK activation with an agonist antibody as a therapeutic strategy to protect NMJ function and prolong the lifespan of ALS patients.

Epstein-Barr virus positive gastric cancer: the pathological basis of CT findings and radiomics models prediction.

PURPOSE: To analyze the clinicopathologic information and CT imaging features of Epstein-Barr virus (EBV)-positive gastric cancer (GC) and establish CT-based radiomics models to predict the EBV status of GC. METHODS: This retrospective study included 144 GC cases, including 48 EBV-positive cases. Pathological and immunohistochemical information was collected. CT enlarged LN and morphological characteristics were also assessed. Radiomics models were constructed to predict the EBV status, including decision tree (DT), logistic regression (LR), random forest (RF), and support vector machine (SVM). RESULTS: T stage, Lauren classification, histological differentiation, nerve invasion, VEGFR2, E-cadherin, PD-L1, and Ki67 differed significantly between the EBV-positive and -negative groups (p = 0.015, 0.030, 0.006, 0.022, 0.028, 0.030, < 0.001, and < 0.001, respectively). CT enlarged LN and large ulceration differed significantly between the two groups (p = 0.019 and 0.043, respectively). The number of patients in the training and validation cohorts was 100 (with 33 EBV-positive cases) and 44 (with 15 EBV-positive cases). In the training cohort, the radiomics models using DT, LR, RF, and SVM yielded areas under the curve (AUCs) of 0.905, 0.771, 0.836, and 0.886, respectively. In the validation cohort, the diagnostic efficacy of radiomics models using the four classifiers were 0.737, 0.722, 0.751, and 0.713, respectively. CONCLUSION: A significantly higher proportion of CT enlarged LN and a significantly lower proportion of large ulceration were found in EBV-positive GC. The prediction efficiency of radiomics models with different classifiers to predict EBV status in GC was good.

Comparison of outcomes between preoperative and postoperative systemic treatment in patients with hepatocellular carcinoma: a SEER database-based study.

Background: Significant advancements in systemic treatment for hepatocellular carcinoma have been made in recent years. However, the optimal timing of systemic treatment before or after surgery remains unknown. This study aims to evaluate the impact of sequencing systemic treatment and surgical intervention on the long-term prognosis of hepatocellular carcinoma patients. Methods: In our study, we analyzed data from patients diagnosed with primary liver cancer (2004-2015) extracted from the SEER database. Patients who underwent both systemic treatment and surgical intervention were selected, divided into preoperative and postoperative systemic therapy groups. The primary endpoint of the study is overall survival(OS), and the secondary endpoint is cancer-specific survival (CSS). Propensity score matching (PSM) reduced the influence of confounding factors, while Kaplan-Meier curves and a multivariable Cox proportional hazards model accounted for variables during survival analysis. Results: A total of 1918 eligible HCC patients were included, with 1406 cases in the preoperative systemic treatment group and 512 cases in the postoperative systemic treatment group. Survival analysis showed that both the preoperative group demonstrated longer median overall survival (OS) and median cancer-specific survival (CSS) before and after PSM. After conducting multivariate COX regression analysis with stepwise adjustment of input variables, the postoperative systemic treatment group continued to exhibit a higher risk of all-cause mortality (HR: 1.84, 95% CI: 1.55-2.1) and cancer-specific mortality (HR: 2.10, 95% CI: 1.73-2.54). Subgroup analysis indicated consistent results for overall survival (OS) across different subgroups. Conclusions: Hepatocellular carcinoma patients from the SEER database who received preoperative systemic therapy had superior OS and CSS compared to those who received postoperative systemic therapy.

Visible-Light-Promoted Selenylation/Cyclization of o-Alkynyl Benzylazides/o-Propargyl Arylazides: Synthesis of Seleno-Substituted Isoquinolines and Quinolines.

A simple and efficient visible-light-promoted selenylation/cyclization of o-alkynyl benzylazides/o-propargyl arylazides have been realized for the practical synthesis of seleno-substituted isoquinolines and quinolines. This strategy provides the synthesis of valuable seleno-substituted isoquinoline and quinoline derivatives via the construction of one C(sp2)-Se bond and one C-N bond within one process.

Analysis of the efficacy and influencing factors of preoperative P-SOX neoadjuvant chemotherapy regimen for progressive gastric cancer-construction of a clinical prediction model.

Preoperative neoadjuvant chemotherapy is one of the most common treatments for patients with advanced gastric cancer that cannot be completely removed by surgery. Nab-paclitaxel is a nano-formulation of paclitaxel that has been shown to be effective in treating stomach cancer. In addition, oxaliplatin + S-1 (SOX) has been a first-line chemotherapy regimen for gastric cancer, and it has the effect of tumor downstaging, improving the R0 resection rate, and reducing the postoperative recurrence rate, but the side effects are significant. During the application of oxaliplatin, obvious gastrointestinal reactions such as nausea and vomiting can be observed. There may also be blood system side effects such as leukopenia and thrombocytopenia, as well as serious adverse reactions such as peripheral neuropathy. Therefore, we reduced the amount of oxaliplatin in SOX and added nab-paclitaxel on the basis of this, in order to increase the efficacy while reducing the side effects of SOX regimen. We selected 192 patients with advanced gastric cancer admitted to the Department of Gastrointestinal Oncology of Qinghai University Hospital from July 2019 to February 2022, and all were treated with nab-paclitaxel plus oxaliplatin + S-1 neoadjuvant chemotherapy regimen, and underwent further surgery after chemotherapy. The tumor regression grade (TRG grade) and response evaluation criteria of solid tumor 1.1 (RECIST1.1) were taken as the dependent variables. According to TRG classification, 120 patients were effective (grade 0, 1, 2 = 62.50%, age: 55.63 ± 9.02 years), 72 patients were ineffective (grade 3 = 37.50%, 55.82 ± 9.21 years), and the effective rate of chemotherapy was 62.50%. According to RECIST1.1, 116 patients were effective (CR + PR = 60.42%, mean age 55.84 ± 9.02 years), 76 patients were ineffective (SD + PD = 39.58%, 55.47 ± 9.19 years), and the effective rate was 60.42%. The factors p < 0.2 in univariate logistic regression analysis were included in multivariate logistic regression analysis, and p < 0.05 was the statistical difference, and statistically significant factors were screened out for modeling and plotted the nomogram. Among them, in the tumor regression grade, the final factors related to effective chemotherapy are the degree of differentiation, cT. stage, tumor diameter, chemotherapy cycle, and the final factors related to effective chemotherapy in the solid tumor response evaluation criteria are the degree of differentiation, cT. stage, tumor diameter. Therefore, we conclude that the regimen of nab-paclitaxel combined with oxaliplatin and S-1 has certain positive significance in the treatment of advanced gastric cancer.

C9orf72 functions in the nucleus to regulate DNA damage repair.

The hexanucleotide GGGGCC repeat expansion in the intronic region of C9orf72 is the most common cause of Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeat expansion-generated toxic RNAs and dipeptide repeats (DPRs) including poly-GR, have been extensively studied in neurodegeneration. Moreover, haploinsufficiency has been implicated as a disease mechanism but how C9orf72 deficiency contributes to neurodegeneration remains unclear. Here, we show that C9orf72 deficiency exacerbates poly-GR-induced neurodegeneration by attenuating non-homologous end joining (NHEJ) repair. We demonstrate that C9orf72 localizes to the nucleus and is rapidly recruited to sites of DNA damage. C9orf72 deficiency resulted in impaired NHEJ repair through attenuated DNA-PK complex assembly and DNA damage response (DDR) signaling. In mouse models, we found that C9orf72 deficiency exacerbated poly-GR-induced neuronal loss, glial activation, and neuromuscular deficits. Furthermore, DNA damage accumulated in C9orf72-deficient neurons that expressed poly-GR, resulting in excessive activation of PARP-1. PARP-1 inhibition rescued neuronal death in cultured neurons treated with poly-GR peptides. Together, our results support a pathological mechanism where C9orf72 haploinsufficiency synergizes with poly-GR-induced DNA double-strand breaks to exacerbate the accumulation of DNA damage and PARP-1 overactivation in C9orf72 ALS/FTD patients.

DDX1 is a prognostic biomarker and correlates with immune infiltrations in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading lethal malignant tumors worldwide. DEAD-box (DDX) family helicases are implicated in numerous human cancers. However, the role of DDX1 in HCC has not yet been fully elucidated. We downloaded gene expression data and clinical information data of HCC from The Cancer Genome Atlas and International Cancer Genome Consortium (ICGC) database and conducted subsequent analyses using the R package and online portal. The results revealed that HCC tissues had higher DDX1 expression compared with either paired or unpaired normal tissues. The increased DDX1 expression was closely related to the advanced pathological grade and histologic grade of HCC. Further analysis suggested that patients with high DDX1 expression contributed to poor prognosis The Cox regression analysis revealed that the expression level of DDX1 was an independent prognostic factor for HCC. In addition, an ICGC cohort was used for external validation. The cBio-Portal, MethSurv, and UALCAN database were used for evaluating the genomic mechanism. Moreover, the Tumor Immune Estimation Resource dataset and QUANTISEQ algorithm revealed that DDX1 expression positively correlates with immune infiltrating cells. We also identified the DDX1-related differentially expressed genes (DEGs) and explored their biological functions by GO, KEGG, and GSEA analyses, which indicated that DDX1 may regulate the progression of HCC. In general, increased DDX1 expression predicts a poor prognosis and drives the progression of HCC.

Synthesis of Selenyl-Substituted Quinoline Derivatives via Substrate-Controlled Three-Component Domino Reactions.

A simple and efficient method for the preparation of selenyl-substituted quinoline derivatives through a CSp3-H selenylation of in situ-generated 3-acetyl quinoline has been developed. This protocol is easy to handle, scalable, and good functional group tolerant, providing a rapid method to 3-selenoacetyl quinoline and 3-diselenoacetyl quinoline derivatives.

Dual Gas-responsive Fluorescent Diblock Copolymer Synthesized via RAFT Polymerization.

Stimulus-responsive polymers with luminescence properties have a wide range of applications in the fields of controlled drug release, fluorescent probes, and biological stents. In this paper, carbon dioxide (CO2)/oxygen (O2) dual-responsive fluorescent diblock copolymers were synthesized by the reversible addition-fragmentation chain transfer (RAFT) polymerization method with two fluorescent monomers synthesized as its luminescence source, DEAEMA (CO2 responsive monomer) and tFMA (O2 responsive monomer). An experimental study demonstrated that the synthesized stimulus-responsive fluorescent polymer had a high sensitivity to CO2; the double-responsive fluorescent diblock copolymer could form and achieve the reversal of polymer micelles in the aqueous solution when it was sequentially subjected to the introduction of CO2 and O2.

High-throughput sequencing reveals omnivorous and preferential diets of the rotifer Polyarthra in situ.

Knowledge of in situ diet of widespread rotifers is crucial for accurately understanding the trophic position, ecological function, and adaptability to environmental changes in aquatic ecosystems. However, it is challenging to achieve the in situ diet information due to the lack of efficient and comprehensive methods. Here, we investigated the diet composition of Polyarthra in a subtropical lake using high-throughput sequencing (HTS) of a rRNA metabarcode for Polyarthra and ambient water samples. After eliminating Polyarthra sequences, a total of 159 operational taxonomic units (OTUs) from taxa in 15 phyla were detected from Polyarthra gut content samples. Most of the OTUs belong to Chlorophyta, followed by unclassified Fungi, Chrysophyta, Dinoflagellata, Ciliophora, Bacillariophyta, Cryptophyta, Arthropoda, Cercozoa, Mollusca, Apicomplexa, Haptophyta, Amoebozoa, Chordata and other eukaryotes. Our results showed that Polyarthra mainly grazed on Chlorophyta, which may result from the high relative abundance of Chlorophyta in ambient waters. In contrast, Chrysophyceae and Synurophyceae were enriched in Polyarthra's gut, indicating that this rotifer prefers these taxa as food. Moreover, correlation analysis showed that total nitrogen, transparency, depth, Chlorophyll-a and total phosphorus were key factors for the variation of the eukaryotic community in the Polyarthra gut contents. When the concentration of nutrients in the water environment decreased, Polyarthra shifted from herbivorous feeding to more carnivorous feeding. Thus, Polyarthra is generally omnivorous but preference for Chrysophytes and Synurophytes, and it responds to the environmental changes by adopting a flexible feeding strategy. This could partly explain why the widespread rotifers have apparently wide tolerance toward spatial and environmental changes.

Initial Experience With Diffusion-Weighted Magnetic Resonance Imaging for the Evaluation of Endometrial Fibrosis.

OBJECTIVE: This study aimed to determine the feasibility of diffusion-weighted imaging for detecting endometrial fibrosis in patients with intrauterine injury. METHODS: This prospective study included 34 patients with endometrial fibrosis and 34 healthy controls. All participants underwent T2-weighted and diffusion-weighted magnetic resonance imaging with b values of 0 and 1000 s/mm2 during the periovulatory phase with a dominant follicle. The endometrial apparent diffusion coefficient (ADC) and uterine anatomical parameters (endometrial thickness [EMT], length of the uterine cavity [LUC], and junctional zone thickness [JZT]) were measured and compared. Performance of the uterine endometrial ADC and anatomical parameters in diagnosing endometrial fibrosis was evaluated. RESULTS: Patients with endometrial fibrosis showed a lower endometrial ADC, lower EMT, shorter LUC, and higher JZT than did healthy controls (all, P < 0.001). Endometrial ADC value and uterine anatomical parameters showed good performance in diagnosing endometrial fibrosis, with the areas under the receiver operating characteristic curves of 0.976, 0.870, 0.883, and 0.864, respectively. The area under the curve of ADC was significantly higher than those of EMT (z = 1.973, P = 0.0485), LUC (z = 2.059, P = 0.0395), and JZT (z = 2.484, P = 0.0130). Intraobserver and interobserver agreements of endometrial ADC value measurements were excellent for both patients (intraclass correlation coefficient = 0.987 and 0.983, respectively) and healthy women (intraclass correlation coefficient = 0.986 and 0.989, respectively). CONCLUSIONS: Our preliminary results suggest that diffusion-weighted imaging has the potential to be a noninvasive imaging tool for the quantitative assessment of endometrial fibrosis.

NF-κB and neutrophil extracellular traps cooperate to promote breast cancer progression and metastasis.

Aberrant NF-κB activation and neutrophil extracellular traps (NETs) are associated with breast cancer progression. How NF-κB and NETs modulate each other in breast cancer development remains unclear. Here, we found that NETs induced by phorbol 12-myristate 13-acetate promote breast cancer cell progression. In turn, cancer cells-derived factors, such as IL-8 and granulocyte colony-stimulating factor, stimulate neutrophils to form NETs. Mechanistically, NETs increased the interaction of NF-κB essential modifier (NEMO) with IκB kinase (IKK)α/ß and enhanced NF-κB activation. We then employed a cell-permeable peptide corresponding to the NEMO-binding domain (NBD) of IKKα/ß, termed NBD peptide, which disrupts NETs-mediated NEMO interaction with IKKα/ß and abolished NF-κB activation in vitro. NBD peptide also reduced IL-8 level and NETs formation, and suppressed primary tumor growth and/or lung metastasis in human breast cancer mouse xenograft models and mouse spontaneous breast cancer model. Blockade of NET formation using a peptidylarginine deiminase 4 (PAD4) pharmacologic inhibitor decreased NF-κB activation and tumor metastasis. Collectively, these data suggest that NF-κB associates with NETs to form a positive loop facilitating breast tumor progression and metastasis, and that selective inhibition of NF-κB and PAD4-dependent NETs provides an effective therapeutic approach for treating breast cancer.

Molecular diagnosis of Kallmann syndrome with diabetes by whole exome sequencing and bioinformatic approaches.

BACKGROUND: Kallmann syndrome (KS) is a hypogonadotropic hypogonadism accompanied by anosmia or hyposmia. It is associated with the low secretion of gonadotropins which can lead to other abnormal endocrine metabolism disorders such as diabetes. Through genetic and molecular biological methods, more than 10 KS pathogenic genes have been found. AIM: To identify the existing mutation sites of KS with diabetes and reveal the relationship between genotype and phenotype. METHODS: We studied KS pathogenesis through high-throughput exome sequencing on four diabetes' patients with KS for screening the potential pathogenic sites and exploring the genotype-phenotype correlation. Clinical data and peripheral blood samples were collected from the patients. White blood cells were separated and genomic DNA was extracted. High-throughput sequencing of all exons in the candidate pathogenic genes of probands was performed, and the results obtained were analyzed. RESULTS: Sequencing revealed mutations in the KLB p.T313M, ANOS1 p.C172F, and IGSF10 gene (p.Lys1819Arg and p.Arg1035Thr) at different sites, which may have been associated with disease onset. CONCLUSION: The diagnosis of KS is challenging, especially in early puberty, and the clinical manifestations reflect physical delays in development and puberty. Timely diagnosis and treatment can induce puberty, thereby improving sexual, bone, metabolic and mental health.

The role of MYB proto-oncogene like 2 in tamoxifen resistance in breast cancer.

Despite the efficacy of tamoxifen in preventing disease relapse, a large portion of breast cancer patients show intrinsic or acquired resistance to tamoxifen, leading to treatment failure and unfavorable clinical outcome. MYB proto-oncogene like 2 (MYBL2) is a transcription factor implicated in the initiation and progression of various human cancers. However, its role in tamoxifen resistance in breast cancer remained largely unknown. In the present study, by analyzing public transcriptome dataset, we found that MYBL2 is overexpressed in breast cancer and is associated with the poor prognosis of breast cancer patients. By establishing tamoxifen-resistant breast cancer cell lines, we also provided evidence that MYBL2 overexpression contributes to tamoxifen resistance by up-regulating its downstream transcriptional effectors involved in cell proliferation (PLK1, PRC1), survival (BIRC5) and metastasis (HMMR). In contrast, inhibiting those genes via MYBL2 depletion suppresses cancer progression, restores tamoxifen and eventually reduces the risk of disease recurrence. All these findings revealed a critical role of MYBL2 in promoting tamoxifen resistance and exacerbating the progression of breast cancer, which may serve as a novel therapeutic target to overcome drug resistance and improve the prognosis of breast cancer patients.

Evaluation of Chronic Pancreatitis With T1 ρ MRI: A Preliminary Study.

BACKGROUND: Chronic pancreatitis (CP) can result in persistent damage to the endocrine and exocrine tissues of the pancreas. There is an unmet need for quantitative methods to evaluate CP noninvasively. PURPOSE: To investigate the utility of T1 ρ magnetic resonance imaging (MRI) for the assessment of CP. STUDY TYPE: Prospective. POPULATION: Twenty patients with CP and 24 healthy volunteers. FIELD STRENGTH/SEQUENCE: 3T MRI including T1 ρ sequence (spin lock time = 0, 1, 10, 20, 40, 60 msec). ASSESSMENT: Pancreatic T1 ρ values and anterior-posterior (AP) diameters in the head, body, and tail were measured in all participants. Regions of interest with circle (ROIcircle ) and free-hand (ROIFH ) were drawn for T1 ρ value measurements. STATISTICAL TESTS: Mann-Whitney U-test; Wilcoxon Signed Ranks test; receiver operating characteristic (ROC) curve; and Bland-Altman analysis. RESULTS: The T1 ρ values of pancreatic tail and the mean T1 ρ values for ROIcircle and the T1 ρ values of pancreatic tail for ROIFH in patients with CP were significantly higher than those in healthy volunteers (all P < 0.05). Pancreatic head AP diameter significantly increased, while pancreatic tail AP diameter significantly decreased in patients with CP compared with healthy volunteers (both P < 0.05). The areas under the ROC curves (AUCs) of pancreatic tail T1 ρ values with ROIcircle and tail AP diameter in diagnosing CP were 0.744 and 0.798, respectively. A combination of pancreatic tail T1 ρ values with ROIcircle and tail AP diameter achieved good performance for diagnosing CP (AUC = 0.838). DATA CONCLUSION: T1 ρ MRI might be a potential technique for the noninvasive evaluation of CP. Level of Evidence 2 Technical Efficacy Stage 2 J. MAGN. RESON. IMAGING 2021;53:577-584.

Comparison of autofluorescence and white-light bronchoscopies performed with the Evis Lucera Spectrum for the detection of bronchial cancers: a meta-analysis.

BACKGROUND: Many recent studies have reported that autofluorescence bronchoscopy (AFB) has a superior sensitivity and decreased specificity in the diagnosis of bronchial cancers when compared with white-light bronchoscopy (WLB). We specifically analyzed the diagnostic performances of autofluorescence imaging video bronchoscopy (AFI) performed with the Evis Lucera Spectrum from Olympus, which is a relatively novel approach in detecting and delineating bronchial cancers, and compared it to the older WLB method. METHODS: We searched the PubMed, Embase, Web of Science, and CNKI databases from inception to July 12th, 2018 for trials in which patients were diagnosed with lung cancer via concurrent or combined use of AFI and WLB. The included studies were required to have a histologic diagnosis as the gold standard comparison, and a sufficient amount of data was extracted to assess the diagnostic capacity. A 2×2 table was constructed, and the area under the receiver-operating characteristic curve (AUC) of AFI and WLB was estimated by using a stochastic model for diagnostic meta-analysis using STATA software. RESULTS: A total of 10 articles were eligible for the meta analysis, comprising 1,830 patients with complete data included in the analysis. AFI showed a superior sensitivity of 0.92 (95% CI, 0.88-0.95) over WLB's 0.70 (95% CI, 0.58-0.80) with P<0.01, and a comparable specificity of 0.67 (95% CI, 0.51-0.80) compared with WLB's 0.78 (95% CI, 0.68-0.86) with P=0.056. Egger's test P value (0.225) demonstrated that there was no publication bias. CONCLUSIONS: Our research showed that in the evaluation of bronchial cancers, AFI was superior to conventional WLB. With its higher sensitivity, AFI could be valuable for avoiding misdiagnosis.

Alginate Lyase Guided Silver Nanocomposites for Eradicating Pseudomonas aeruginosa from Lungs.

Pseudomonas aeruginosa (P. aeruginosa) infections lead to a high mortality rate for cystic fibrosis or immunocompromised patients. The alginate of the biofilm was believed to be the key factor disabling immune therapy and antibiotic treatments. A silver nanocomposite consisting of silver nanoparticles and a mesoporous organosilica layer was created to deliver two pharmaceutical compounds (alginate lyase and ceftazidime) to degrade the alginate and eradicate P. aeruginosa from the lungs. The introduction of thioether-bridged mesoporous organosilica into the nanocomposites greatly benefited the conjunction of foreign functional molecules such as alginate lyase and increased their hemocompatibility and drug-loading capacity. Silver nanocomposites with a uniform diameter (∼39 nm) exhibited a high dispersity, good biocompatibility, and high ceftazidime-loading capacity (380.96 mg/g). Notably, the silver nanocomposites displayed a low pH-dependent drug release and degradation profiles (pH 6.4), guaranteeing the targeted release of the drugs in the acidic niches of the P. aeruginosa biofilm. Indeed, particles loaded with alginate lyase and ceftazidime exhibited high inhibitory and degradation effects on the biofilm of P. aeruginosa PAO1 based on the specific catalytic activity of the enzyme to the alginate and antibacterial function of their loaded ceftazidime and silver ions. It should be noted that the enzyme-decorated nanocomposites succeeded in eradicating P. aeruginosa PAO1 from the mouse lungs and decreasing the lung injuries. No deaths or serious side effects were observed during the experiments. We believe that the silver nanocomposites with high biocompatibility and organic group-incorporated framework have the potential to be used to deliver multiple functional molecules for antibacterial therapy in clinical application.

A rapid and sensitive method for quantification of ibrutinib in rat plasma by UPLC-ESI-MS/MS: validation and application to pharmacokinetic studies of a novel ibrutinib nanocrystalline.

Ibrutinib has an excellent effect in the treatment of mantle cell lymphoma so it has attracted much attention. A novel ibrutinib nanocrystalline was exploited in our study to improve the bioavailability. A fast and reliable UPLC-MS/MS method was established for the accurate quantification of ibrutinib in rat plasma. The chromatographic separation was achieved by an Agilent zorbax SB-C18 rapid solution HD column (2.1 × 50 mm, 1.8 µm). The mobile phase consisted of deionized water (containing 10 mm ammonium acetate and 0.1% formic acid) and pure acetonitrile. Isocratic elution (water-acetonitrile 10:90, v/v) was adopted and the flow rate was 0.4 mL/min. Column temperature was set to 40°C. Vilazodone was used as the internal standard in this analytical method. Multiple reaction monitoring mode with positive electrospray ionization was selected to detect ibrutinib and vilazodone. Acetonitrile was used to precipitate protein to extract plasma samples. There was no endogenous interference for both ibrutinib and vilazodone and the linear range of this method was 1-2000 ng/mL. The recoveries were 98.4, 97.4 and 102.7% at low, medium and high concentrations. Accordingly, the matrix effect was 96.6, 111.1 and 99.6%. The pharmacokinetic difference between ibrutinib crude and a novel ibrutinib nanocrystalline in rats was investigated by this validated method successfully. The peak concentration and area under the concentration-time curve showed significant differences in gender and the bioavailability was improved after oral administration of ibrutinib nanocrystalline.

Geranylgeranyl diphosphate synthase 1 knockout ameliorates ventilator-induced lung injury via regulation of TLR2/4-AP-1 signaling.

OBJECTIVE: To investigate the role of geranylgeranyl diphosphate synthase 1 (GGPPS1) in ventilator-induced lung injury along with the underlying mechanism. METHODS: A murine VILI model was induced by high-tidal volume ventilation in both wild-type and GGPPS1 knockout mice. GGPPS1 expression was detected in the bronchoalveolar lavage fluid (BALF) supernatants of acute respiratory distress syndrome (ARDS) patients and healthy volunteers, as well as in lung tissues and BALF supernatants of the VILI mice using enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription polymerase chain reaction (qRT-PCR), western bolt and immunohistochemical (IHC). The wet/dry ratio, total BALF proteins, and lung injury score were analyzed. The percentage of neutrophils was detected by flow cytometry and IHC. Inflammatory cytokine levels were measured by ELISA and qRT-PCR. The related expression of Toll-like receptor (TLR)2/4 and its downstream proteins was evaluated by western blot. RESULTS: GGPPS1 in BALF supernatants was upregulated in ARDS patients and the VILI mice. Depletion of GGPPS1 significantly alleviated the severity of ventilator induced lung injury in mice. Total cell count, neutrophils and inflammatory cytokines (interleukin [IL]-6, IL-1ß, IL-18 and tumor necrosis factor-α) levels in BALF were reduced after GGPPS1 depletion. Moreover, addition of exogenous GGPP in GGPPS-deficient mice significantly exacerbated the severity of ventilator induced lung injury as compared to the PBS treated controls. Mechanistically, the expression of TLR2/4, as well as downstream proteins including activator protein-1 (AP-1) was suppressed in lung tissues of GGPPS1-deficient mice. CONCLUSION: GGPPS1 promoted the pathogenesis of VILI by modulating the TLR2/4-AP-1 signaling pathway, and GGPPS1 knockout significantly alleviated the lung injury and inflammation in the VILI mice.