Pazopanib attenuated bleomycin-induced pulmonary fibrosis via suppressing TGF-ß1 signaling pathway.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease with a high mortality rate and limited treatment efficacy. Nintedanib, a tyrosine kinase inhibitor, is clinically used to treat pulmonary fibrosis. At present, only nintedanib is on the market for the treatment of pulmonary fibrosis. Pazopanib is a drug for the treatment of renal cell carcinoma and advanced soft tissue sarcoma. Methods: In this study, we explored whether pazopanib can attenuate bleomycin (BLM)-induced pulmonary fibrosis and explored its antifibrotic mechanism. In vivo and in vitro investigations were carried out to investigate the efficacy and mechanism of action of pazopanib in pulmonary fibrosis. Results: In vivo experiments showed that pazopanib can alleviate pulmonary fibrosis caused by BLM, reduce the degree of collagen deposition and improve lung function. In vitro experiments showed that pazopanib suppressed transforming growth factor-ß1 (TGF-ß1)-induced myofibroblast activation and promoted apoptosis and autophagy in myofibroblasts. Further mechanistic studies demonstrated that pazopanib inhibited the TGF-ß1/Smad and non-Smad signaling pathways during fibroblast activation. Conclusions: In conclusion, pazopanib attenuated BLM-induced pulmonary fibrosis by suppressing the TGF-ß1 signaling pathway. Pazopanib inhibits myofibroblast activation, migration, autophagy, apoptosis, and extracellular matrix (ECM) buildup by downregulating the TGF-ß1/Smad signal route and the TGF-ß1/non-Smad signal pathway. It has the same target as nintedanib and is a tyrosine kinase inhibitor.

Evaluation of Acarbose Bioequivalence in Healthy Chinese Populations Using Novel Pharmacodynamic End Points.

Acarbose is a widely used α-glucosidase inhibitor for the management of postprandial hyperglycemia in patients with type 2 diabetes mellitus. Recent pilot studies on acarbose bioequivalence (BE) have successfully identified additional pharmacodynamic (PD) parameters as valid end points. Nevertheless, there was a scarcity of published pivotal studies using novel PD parameters. The purpose of the study is to investigate the acarbose BE using the new PD parameters. The study was conducted with an open, randomized, 2-period crossover design. A total of 64 healthy Chinese volunteers received either the reference (R) or test (T) acarbose at a dose of 2×50 mg orally, followed by a 1-week washout period. After sucrose treatment (baseline) and sucrose/acarbose co-administration, serum glucose, and insulin concentrations were assessed. The rectifying approach yielded geometric mean ratios of 102.9% for maximum serum glucose concentration with deduction of glucose concentration at 0 hour and 105.3% for the area under the serum glucose concentration-time curve profile 0-2 hours after coadministration of sucrose and acarbose with deduction of baseline (AUC0-2 h,r ). The 90% confidence intervals of maximum serum glucose concentration with deduction of glucose concentration at 0 hour and the area under the serum glucose concentration-time curve profile 0-2 hours after coadministration of sucrose and acarbose with deduction of baseline all fell within the acceptance limits. The incidence of adverse events after the T or R drug was comparable, and healthy subjects were well tolerated. The findings of our investigation clearly show that the PD parameters of the rectifying method exhibit enhanced suitability and sensitivity when assessing acarbose BE in healthy participants. The T and R drugs were bioequivalent using the novel PD parameters, and both drugs demonstrated good safety and tolerability.

Detect-seq, a chemical labeling and biotin pull-down approach for the unbiased and genome-wide off-target evaluation of programmable cytosine base editors.

Programmable cytosine base editors show promising approaches for correcting pathogenic mutations; yet, their off-target effects have been of great concern. Detect-seq (dU-detection enabled by C-to-T transition during sequencing) is an unbiased, sensitive method for the off-target evaluation of programmable cytosine base editors. It profiles the editome by tracing the editing intermediate dU, which is introduced inside living cells and edited by programmable cytosine base editors. The genomic DNA is extracted, preprocessed and labeled by successive chemical and enzymatic reactions, followed by biotin pull-down to enrich the dU-containing loci for sequencing. Here, we describe a detailed protocol for performing the Detect-seq experiment, and a customized, open-source, bioinformatic pipeline for analyzing the characteristic Detect-seq data is also provided. Unlike those previous whole-genome sequencing-based methods, Detect-seq uses an enrichment strategy and hence is endowed with great sensitivity, a higher signal-to-noise ratio and no requirement for high sequencing depth. Furthermore, Detect-seq is widely applicable for both mitotic and postmitotic biological systems. The entire protocol typically takes 5 d from the genomic DNA extraction to sequencing and ~1 week for data analysis.

An efficient photo Fenton system for in-situ evolution of H2O2via defective iron-based metal organic framework@ZnIn2S4 core-shell Z-scheme heterojunction nanoreactor.

The fabrication of an efficient photoFenton system without the addition of H2O2 is still a challenge and is cost-effective and favorable for practical applications. In this work, a core@shell Z-scheme heterojunction nanoreactor was successfully fabricated, in which hierarchical two-dimensional (2D) ZnIn2S4 nanosheets are coated on defective iron-based metal-organic frameworks (MOFs) (NH2-MIL-88B(Fe)), realizing efficient in-situ evolution of H2O2 and constructing an optimal heterogeneous Fenton platform. The degradation rates of defective NH2-MIL-88B(Fe)@ZnIn2S4 (0.4 g L-1) for bisphenol A and ofloxacin under visible light irradiation within 180 min reached 99.4% and 98.5%, respectively, and the photocatalytic hydrogen production efficiency was approximately 502 µmol h-1 g-1. The excellent photoFenton performance was attributed to the introduction of ligand defects into the MOF, which can adjust the band structure to enhance the light absorption capacity, and the in-situ generation of H2O2 accelerating the Fe3+/Fe2+ conversion. In addition, the formation of the core@shell nanoreactor Z-scheme heterojunction structure promoted spatial charge separation. This strategy offers new ideas for constructing efficient photocatalysis and photoFenton systems.

Mitochondrial base editor induces substantial nuclear off-target mutations.

DddA-derived cytosine base editors (DdCBEs)-which are fusions of split DddA halves and transcription activator-like effector (TALE) array proteins from bacteria-enable targeted C•G-to-T•A conversions in mitochondrial DNA1. However, their genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or TAS-independent. TAS-dependent off-target sites in the nuclear DNA are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by paired TALE proteins positioned in close proximity. TAS-independent off-target sites on nuclear DNA are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with binding sites for the transcription factor CTCF and are enriched in topologically associating domain boundaries. We engineered DdCBE to alleviate such off-target effects. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base-editing tools.

A novel signal enhancement strategy for the detection of DNA oxidative damage biomarker 8-OHdG based on the synergy between ß-CD-CuNCs and multi-walled carbon nanotubes.

OBJECTIVE: To propose a novel signal enhancement strategy based on the synergy between ß-CD-CuNCs and multi-walled carbon nanotubes (MWCNTs) for the detection of DNA oxidative damage biomarker 8-Hydroxy-2'-deoxyguanosine (8-OHdG). METHODS: The sensor was constructed with the ß-CD-CuNCs-MWCNTs-nafion film and successfully used for the quantitative detection of 8-OhdG in the presence of biomolecules such as ascorbic acid (AA) and uric acid (UA). To investigate the surface morphology of the modified electrode, Transmission Electron Microscopy (TEM), Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) were performed on bare and modified electrodes. RESULTS: According to Differential Pulse Voltammetry (DPV) results, there was a linear relationship between peak current and concentration of 8-OhdG, ranging from 1.0×10-7 to 1.0×10-6 mol/L (R2=0.9926) and 1.0×10-6 to 2.0×10-5 mol/L (R2=0.9933). The detection limit (S/N=3) was 33 nmol/L. CONCLUSIONS: The proposed sensor had been successfully applied to the determination of 8-OHdG in human urine samples with high recovery rates.

Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors.

Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, their genome-wide specificity remains poorly understood. Here we report Detect-seq for the evaluation of CBE specificity. It enables sensitive detection of CBE-induced off-target sites at the genome-wide level. Detect-seq leverages chemical labeling and biotin pulldown to trace the editing intermediate deoxyuridine, thereby revealing the editome of CBE. In addition to Cas9-independent and typical Cas9-dependent off-target sites, we discovered edits outside the protospacer sequence (that is, out-of-protospacer) and on the target strand (which pairs with the single-guide RNA). Such unexpected off-target edits are prevalent and can exhibit a high editing ratio, while their occurrences exhibit cell-type dependency and cannot be predicted based on the sgRNA sequence. Moreover, we found out-of-protospacer and target-strand edits nearby the on-target sites tested, challenging the general knowledge that CBEs do not induce proximal off-target mutations. Collectively, our approaches allow unbiased analysis of the CBE editome and provide a widely applicable tool for specificity evaluation of various emerging genome editing tools.

Integrative analysis of long extracellular RNAs reveals a detection panel of noncoding RNAs for liver cancer.

Rationale: Long extracellular RNAs (exRNAs) in plasma can be profiled by new sequencing technologies, even with low abundance. However, cancer-related exRNAs and their variations remain understudied. Methods: We investigated different variations (i.e. differential expression, alternative splicing, alternative polyadenylation, and differential editing) in diverse long exRNA species (e.g. long noncoding RNAs and circular RNAs) using 79 plasma exosomal RNA-seq (exoRNA-seq) datasets of multiple cancer types. We then integrated 53 exoRNA-seq datasets and 65 self-profiled cell-free RNA-seq (cfRNA-seq) datasets to identify recurrent variations in liver cancer patients. We further combined TCGA tissue RNA-seq datasets and validated biomarker candidates by RT-qPCR in an individual cohort of more than 100 plasma samples. Finally, we used machine learning models to identify a signature of 3 noncoding RNAs for the detection of liver cancer. Results: We found that different types of RNA variations identified from exoRNA-seq data were enriched in pathways related to tumorigenesis and metastasis, immune, and metabolism, suggesting that cancer signals can be detected from long exRNAs. Subsequently, we identified more than 100 recurrent variations in plasma from liver cancer patients by integrating exoRNA-seq and cfRNA-seq datasets. From these datasets, 5 significantly up-regulated long exRNAs were confirmed by TCGA data and validated by RT-qPCR in an independent cohort. When using machine learning models to combine two of these validated circular and structured RNAs (SNORD3B-1, circ-0080695) with a miRNA (miR-122) as a panel to classify liver cancer patients from healthy donors, the average AUROC of the cross-validation was 89.4%. The selected 3-RNA panel successfully detected 79.2% AFP-negative samples and 77.1% early-stage liver cancer samples in the testing and validation sets. Conclusions: Our study revealed that different types of RNA variations related to cancer can be detected in plasma and identified a 3-RNA detection panel for liver cancer, especially for AFP-negative and early-stage patients.

Effect of temperatures and alternating anoxic/oxic sequencing batch reactor (SBR) operating modes on extracellular polymeric substances in activated sludge.

In order to investigate the effect of temperatures and operating modes on extracellular polymeric substances (EPS) contents, three sequencing batch reactors (SBRs) were operated at temperatures of 15, 25, and 35 °C (R15 °C, R25 °C, and R35 °C, respectively), with two SBRs operated under alternating anoxic/oxic conditions (RA/O and RO/A, respectively). Results showed that higher contents of tightly bound EPS (TB-EPS) and total EPS appeared in R15 °C, while loosely bound EPS (LB-EPS) dominated in R35 °C. In all three kinds of EPS (LB-EPS, TB-EPS and total EPS) assessed, protein was the main component in R15 °C and R25 °C, while polysaccharides dominated in R35 °C. Moreover, compared with RO/A, RA/O was favorable for the production of the three kinds of EPS. Furthermore, three kinds of EPS and their components were augmented during the nitrification process, while they declined during the denitrification process under all conditions except for R35 °C.

A homogeneous photoelectrochemical hydrogen sulfide sensor based on the electronic transfer mediated by tetrasulfophthalocyanine.

Hydrogen sulfide (H2S) can regulate a variety of physiological functions, and the development of sensitive H2S detection methods is a requirement. In this study, a homogeneous photoelectrochemical (PEC) sensor for H2S detection was constructed based on the energy level matching of iron(iii) phthalocyanine-4,4',4'',4'''-tetrasulfonic acid ([Fe(iii)PcS4]+) and n-GaN. The photocurrent of n-GaN could be suppressed by monomeric [Fe(iii)PcS4]+ since the photogenerated electrons in the conduction band (CB) of n-GaN could be injected into the LUMO of [Fe(iii)PcS4]+. Under weak alkaline conditions, monomeric [Fe(iii)PcS4]+ can be converted to [Fe(i)PcS4]- after reacting with H2S with high selectivity. The LUMO of [Fe(i)PcS4]- was higher than the CB of n-GaN, so the photogenerated electrons in the LUMO of [Fe(i)PcS4]- could be injected back into the CB of n-GaN. The electron-hole pair recombination could be hindered, which resulted in the recovery of the system photocurrent. In the H2S concentration range of 10.0 nM-50.0 µM, a linear relationship was obtained between the photocurrent and the logarithm of H2S concentration with a detection limit of 3.40 nM. The proposed method avoids tedious electrode modifying procedures required in conventional PEC sensors and it was applied to detect extracellular H2S in rat brains coupled with microdialysis.

A fluorescence signal amplification strategy for modification-free ratiometric determination of tyrosinase in situ based on the use of dual-templated copper nanoclusters.

A fluorescence resonance energy transfer (FRET)-based in situ fluorescence signal amplification strategy is described for the determination of tyrosinase (TYR). In this assay, a dual-templated copper nanocluster (CuNCs) stabilized by bovine serum albumin (BSA) and glycylglycine (Gly-Gly) was used as an energy donor. Metyrosine was employed as a TYR substrate because its enzyme catalytic product (methyldopa) was able to function as a monomer molecule to form fluorescent polymethyldopa (PMeDP) with the assistance of BSA/Gly-Gly CuNCs. In this process, PMeDP can combine with BSA/Gly-Gly CuNCs without extra modification and then acts as an energy receptor, which leads to a remarkable FRET from BSA/Gly-Gly CuNCs to PMeDP. Interestingly, the fluorescence intensity of PMeDP was strengthened greatly in the FRET-based sensor compared to the separate excitation, which provided good sensitivity for TYR sensing. Illuminated under a UV light source, the fluorescence signal change is observed from dark violet to bright green. Therefore, the present sensing system affords a reliable ratiometric assay for TYR determination. Also, the ratio of fluorescence intensity between PMeDP (λem at 505 nm, F505) and BSA/Gly-Gly CuNCs (λem at 415 nm, F415) was used for quantitative determination of TYR. The sensing system was easily operated in aqueous media with an exciting detection limit of 44.0 U L-1. This sensing strategy has been applied to the screening of inhibitors. Graphical abstract Schematic representation of the strategy for the determination of tyrosinase.

Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis.

Staphylococcus species, categorized into Staphylococcus aureus and non-aureus staphylococci (NAS), are frequent causes of mastitis in dairy cattle around the world. Current treatments using antimicrobials are under increasing scrutiny due to rising prevalence of multi-drug resistance in S. aureus. Objectives of this study were to determine: (1) genetic diversity of Staphylococcus species isolated from clinical mastitis in cows from large Chinese dairy farms; and (2) prevalence and distribution of antimicrobial resistance genes (ARG) in these isolates. Staphylococcus aureus (n = 96) were isolated from 26 herds located in 12 provinces of China, whereas NAS (n = 112) were isolated from 59 herds located in 18 provinces of China. The NAS were identified at the species level using a partial 16S rRNA sequencing method, whereas random amplification of polymorphic DNA (RAPD) PCR was done to determine genetic relationships of isolates. Finally, PCR was used to detect resistance and biofilm formation genes. Staphylococcus chromogenes (33%) was the most common NAS species, followed by Staphylococcus sciuri (17%) and Staphylococcus epidermidis (8%). Staphylococcus aureus was grouped in 12 genotypes, of which 2 types represented 56% of isolates. Staphylococcus chromogenes (n = 37) clustered into 8 RAPD types, with 2 prevalent types containing 73% of isolates. The most prevalent ARG in S. aureus isolates was blaZ (95%), followed by tetM (33%), tetK (31%), ermT (26%), and aacA-aphD (23%). The mecA and vanA were detected in 16 and 4% of isolates, respectively. In NAS, blaZ (100%), mecA (73%), tetK (79%), tetM (96%), mphC (63%), and msrA (54%) were frequently detected. Antimicrobial resistance genes mecA, tetK, tetL, tetM, dfrG, ermB, msrA, mphC, aadD, and aphA3 were more commonly detected in NAS than in S. aureus. Biofilm formation genes (icaA and icaD) were frequently detected in staphylococci isolated from bovine clinical mastitis. The existence of predominant RAPD types in S. aureus and S. chromogenes isolates across Chinese dairy farms indicated that specific genotypes had disseminated within herds and become more udder-adapted. High prevalence of ARG, especially in NAS, highlighted the risk of selection of multi-drug resistant staphylococci with potential as a reservoir of ARG.

[Influence of Operating Modes for the Alternating Anoxic/Oxic Process on Biological Nitrogen Removal and Extracellular Polymeric Substances of Activated Sludge].

Nitrogen removal, extracellular polymeric substances (EPS), and the chemical composition (protein (PN), polysaccharide (PS), and DNA) by the aerobic/anoxic (O/A) and the anoxic/aerobic (A/O) modes were studied in a sequencing batch reactor (SBR) fed with domestic wastewater. The results showed that the removal rates of NH4+-N were 97.5% and 98.0% in the two operating modes, respectively, and a removal efficiency of NH4+-N with high efficiency and stability was obtained. The nitrification rate was positively correlated with the nitrogen loading ratio. The influence of operating modes for the alternating anoxic/oxic mode on extracellular polymeric substances of activated sludge was evaluated. The EPS constituent in the A/O mode was slightly higher than the O/A mode. The operating mode had no effect on the contents of PN, PS, and DNA in tightly bound EPS (TB-EPS) and TB-EPS. However, PN and PS in loosely bound EPS (LB-EPS) and LB-EPS in the A/O mode were 1.38 to 1.56 times those of the O/A mode. In the two operating modes, PSs were the main constituents in the TB-EPS and EPS, while PNs were the main constituents in LB-EPS. The EPS content had a good linear correlation with the sludge settling performance.

Preparation and evaluation of 1-deoxynojirimycin sustained-release pellets vs conventional immediate-release tablets.

1-Deoxynojirimycin sustained-release pellets, which exhibit known release and absorption profiles, are used for the treatment of diabetes mellitus. In this study, a fluidised bed coater was employed to prepare new, drug-loaded pellets. In the dissolution test, it was found that 1-DNJ pellets exhibited a sustained release effect after being coated with hydroxypropyl methyl cellulose phthalate-55 S. For sustained-release pellets and immediate-release pellets, there was significant difference in the mean cumulative drug concentration profile in different media evaluation. In the bioavailability study, the ratio of mean relative bioavailability of the SR pellets to the IR tablets was calculated by the DAS from the AUC0-24 h of 1-DNJ and was found to be 117.3%. This suggested that the behaviour in vivo of the 1-DNJ SR pellets was superior to the IR tablets, which indicated the designed preparation method of the 1-DNJ SR pellets was acceptable for achieving sustained release of 1-DNJ with enhanced bioavailability.

[Effect of Temperature on Nitrogen Removal Performance and the Extracellular Polymeric Substance (EPS) in a Sequencing Batch Reactor (SBR)].

In this paper, the long-term effects of temperature on the nitrogen removal performance and the extracellular polymeric substance (EPS) in a sequencing batch reactor (SBR) treating synthetic wastewater was investigated under three temperature conditions (15℃, 25℃, 35℃). The results showed that high temperatures (35℃) could promote the establishment of short-cut nitrification processes and improve nitrogen removal performance greatly. Temperature had a significant impact on the EPS and its composition. With an increased temperature, the EPS and tightly bound EPS (TB-EPS) content decreased, while, loosely bound EPS (LB-EPS) increased slowly. TB-EPS became dominant in the EPS (the percentage of TB-EPS/EPS was 69.0%-79.5%), however, the ratio of TB-EPS/LB-EPS decreased from 3.8 (15℃) to 3.6 (25℃), and then to 2.2 (35℃) with a gradual increase in temperature. Moreover, protein (PN) and DNA in the EPS, TB-EPS, and LB-EPS decreased with an increasing temperature. Carbohydrates (PS) in the EPS and LB-EPS increased as temperature increased, nevertheless, PS in TB-EPS decreased. Furthermore, 25℃ was identified as the breaking-point temperature in the variation of PN, DNA and PS concentrations. At 15℃ and 25℃, PN was the main component in TB-EPS and LB-EPS. PS has the second highest concentration and DNA the least. However, PS were the dominant component at 35℃, with PN having the second highest concentration, and DNA having a subtle concentration. Moreover, at 15℃ and 25℃, the EPS content increased in the nitrification process and reduced in the denitrification process.

[Synergetic Inhibitory Effect of Free Ammonia and Aeration Phase Length Control on the Activity of Nitrifying Bacteria].

Three sequencing batch reactors (SBRs) labeled with R(Ahead), R(Exact) and R(Exceed) were employed to investigate the synergetic inhibition effect of free ammonia (FA) and length of aeration phase on the activity of ammonia-oxidizing bacteria ( AOB) and nitrite- oxidizing bacteria (NOB) after shortcut nitritation was achieved in the systems. The experiments were conducted under the conditions of three FA concentrations (0.5, 5. 1, 10.1 mg · L⁻¹) combined with three kinds of aeration time (t(Exact): the time when ammonia oxidation was completed; t(Ahead): 30 min ahead of the time when ammonia oxidation was completed; t(Exceed): 30 min exceeded when the time ammonia oxidation was completed). It was found that short-cut nitrification could be successfully established in three reactors with a FA level of 10.1 mg · L⁻¹. Meanwhile, the speed of achieving nitritation was in the sequence of R(Ahead) > R(Exact) > R(Exceed) with operational cycles of 56, 62 and 72, respectively. Compared to AOB, NOB in the three reactors was observed to be more sensitive to FA, resulting in AOB activity higher than NOB activity throughout the whole experimental period. Moreover, there was great difference in the activity coefficient ( η) between AOB and NOB. The activity coefficients of AOB were in the order of η(RExact) > η(RExceed) > η(RAhead) with the values of 104.4%, 100% and 85.8%, respectively. Nevertheless, the activity coefficients of NOB were in the order of η(RExceed) > η(RExact) > η(RAhead) with the values of 71.2%, 64.9% and 50.2%, respectively.