Synthesis of Dual-Emitting CdZnSe/Mn:ZnS Quantum Dots for Sensing the pH Change in Live Cells.

Intracellular pH is an important regulator of cell function, and its subtle changes may greatly affect cell activities and cause diseases. Reliable imaging of intracellular pH remains a huge challenge. Dual-emitting Mn2+-doped quantum dots (QDs) can be directly used as a ratiometric fluorescent probe without further modification, but they displayed low performance in terms of photoluminescence, stability, and intensity ratio regulation. Here, we report intrinsic dual-emitting CdZnSe/Mn:ZnS QDs with high photoluminescence efficiency, good stability, and biocompatibility. The emission intensity ratio was selectively regulated by Mn2+ doping. Because of aggregation-induced quenching of QDs, the exciton emission of CdZnSe/Mn:ZnS QDs (471 nm) was sensitive to pH, while the Mn2+-doped emission (606 nm) was passive to pH, which was probably due to little self-quenching in Mn2+-doped emission caused by weak Mn-Mn coupling interaction. Dual-emitting CdZnSe/Mn:ZnS QDs exhibited excellent pH-responsiveness in the range of pH 4.0 to 12.0 and were used for pH imaging in live HeLa cells. When the pH value of HeLa cells changed from 5.0 to 9.0, the emission changed from red to blue. Furthermore, these dual-emitting CdZnSe/Mn:ZnS QDs can provide a versatile platform for biosensors and biological imaging.

Two-Dimensional Protein Nanoarray as a Carrier of Sensing Elements for Gold-Based Immunosensing Systems.

Homogeneous and high-density immobilization of proteins on gold-based sensing surface without the loss of protein activity is of great significance for high-performance immunosensing but remains challenging. To realize more sensitive immunosensing, an improved method for protein immobilization on the gold surface is urgently required. Here, we propose a biological and mild approach by combining a genetically encoded SpyTag-SpyCatcher interaction system with a redesigned S-layer of bacteria. This method allows proteins of interest to be covalently linked with the S-layer in a biological manner and arranged orderly in a two-dimensional nanoarray on the gold surface. The activity of African swine fever virus proteins was significantly preserved after immobilization. In addition, our S-layer-based immobilization method exhibited an eightfold increase in detection sensitivity compared with the conventional chemical cross-linking for protein immobilization during serological tests. Together, our S-layer-based immobilization method provides an innovative approach for building a quality gold-based biosensing interface and should greatly contribute to the high-sensitivity sensing for a deeper understanding of pathogen infection and host immunity.

Identification and validation of STAT4 as a prognostic biomarker in acute myeloid leukemia.

Acute myelogenous leukemia (AML) is a common malignancy and is supposed to have the ability to escape host immune surveillance. The present study aimed to identify key genes in AML that may affect tumor immunity and to provide prognosis biomarkers of AML. The Cancer Genome Atlas (TCGA) dataset was screened for transcription factors (TFs) involved in immunity and influencing survival, combining Gene Expression Omnibus (GEO) data to validate the impact on patient survival. A prognostic signature was established using four transcription factors, and these genes play an important role in the immune system, with higher regulatory T cell (Treg) scores in high-risk patients compared with the low-risk group. Analysis of individual genes showed that STAT4 and Treg are closely related, which may be due to STAT4 transcribing related genes that affect immunity. STAT4 expression was positively correlated with the proportion of abnormal cells and promoted AML recurrence as verified by AML clinical patient samples. In addition, silencing of STAT4 significantly slowed down the proliferation capacity of HL60 cells. In conclusion, these findings suggest that STAT4 may be a potential biomarker for AML prognosis. As a key gene affecting the prognosis of AML patients, STAT4 has the potential to be a candidate diagnostic and prognostic biomarker for AML.

Construction of AlGaN/GaN high-electron-mobility transistor-based biosensor for ultrasensitive detection of SARS-CoV-2 spike proteins and virions.

The COVID-19 pandemic has highlighted the need for rapid and sensitive detection of SARS-CoV-2. Here, we report an ultrasensitive SARS-CoV-2 immunosensor by integration of an AlGaN/GaN high-electron-mobility transistor (HEMT) and anti-SARS-CoV-2 spike protein antibody. The AlGaN/GaN HEMT immunosensor has demonstrated the capability to detect SARS-CoV-2 spike proteins at an impressively low concentration of 10-22 M. The sensor was also applied to pseudoviruses and SARS-CoV-2 ΔN virions that display the Spike proteins with a single virion particle sensitivity. These features validate the potential of AlGaN/GaN HEMT biosensors for point of care tests targeting SARS-CoV-2. This research not only provides the first HEMT biosensing platform for ultrasensitive and label-free detection of SARS-CoV-2.

ProT-Diff: A Modularized and Efficient Strategy for De Novo Generation of Antimicrobial Peptide Sequences by Integrating Protein Language and Diffusion Models.

Antimicrobial peptides (AMPs) are a promising solution for treating antibiotic-resistant pathogens. However, efficient generation of diverse AMPs without prior knowledge of peptide structures or sequence alignments remains a challenge. Here, ProT-Diff is introduced, a modularized deep generative approach that combines a pretrained protein language model with a diffusion model for the de novo generation of AMPs sequences. ProT-Diff generates thousands of AMPs with diverse lengths and structures within a few hours. After silico physicochemical screening, 45 peptides are selected for experimental validation. Forty-four peptides showed antimicrobial activity against both gram-positive or gram-negative bacteria. Among broad-spectrum peptides, AMP_2 exhibited potent antimicrobial activity, low hemolysis, and minimal cytotoxicity. An in vivo assessment demonstrated its effectiveness against a drug-resistant E. coli strain in acute peritonitis. This study not only introduces a viable and user-friendly strategy for de novo generation of antimicrobial peptides, but also provides potential antimicrobial drug candidates with excellent activity. It is believed that this study will facilitate the development of other peptide-based drug candidates in the future, as well as proteins with tailored characteristics.

[Analysis of primary metabolites of ranolazine in dog urine by LC-MS(n)].

Ranolazine and metabolites in dog urine were identified by LC-MS(n). Dog urine samples were collected after ig 30 mg x kg(-1) ranolazine, then the samples were enriched and purified through solid-phase extraction cartridge. The purified samples were analyzed by LC-MS(n). The possible metabolites were discovered by comparing the full scan and SIM chromatograms of the test samples with the corresponding blanks. Seventeen phase I metabolites and fourteen phase II metabolites were identified in dog urine. Three metabolites were identified by comparing with the control article. The metabolites were formed via the following metabolic pathways: O-demethylation, O-dearylation, hydroxylation, N-dealkylation, amide hydrolysis, glucuronidation and sulfation. The LC-MS(n) method is suitable for the rapid identification of drug and its metabolites in biologic samples.

Structural insights into xylanase mutant 254RL1 for improved activity and lower pH optimum.

Xylanases degrade xylan to valuable end products. In our previous study, the alkaline xylanase S7-xyl from Bacillus halodurans S7 was engineered by rational design and the best mutant xylanase 254RL1 exhibited 3.4-fold improvements in specific activity at pH 9.0. Further research found that the enzyme activity at pH 6.0 was almost 2-fold than that at pH 9.0. To elucidate the reason of enhanced performance of 254RL1 at decreased pH optimum, we determined the X-ray crystal structure of 254RL1 at 2.21 Å resolution. The structural analysis revealed that the mutations enlarged the opening of the access tunnel and shortened the tunnel. Moreover, the mutations changed the hydrogen bond network around the catalytic residue and decreased the pKa value of acid-base catalyst E159 which reduced the pH optimum of the xylanase. The result provided the basis for the acid-alkaline engineering of the glycoside hydrolases.

Design and evaluation of an innovative floating and bioadhesive multiparticulate drug delivery system based on hollow structure.

In this study a gastric-retentive delivery system was prepared by a novel method which is reported here for the first time. An innovative floating and bioadhesive drug delivery system with a hollow structure was designed and prepared. The floating and bioadhesive drug delivery system was composed of a hollow spherical shell, a waterproof layer (Stearic acid), a drug layer (Ofloxacin), a release retarding film (the novel blended coating materials) and a bioadhesive layer (Carbomer 934P) prepared by using a liquid multi-layering process. A novel blended coating material was designed and investigated to solve the problem of the initial burst release of the formulation and the release mechanism of the novel material was analyzed in this study. The optimized formulation provided the sustained release characteristic and was able to float for 24h. The SEM cross-section images showed that the particulates were hollow with a spherical shell. X-ray images and pharmacokinetic studies (Frel = 124.1 ± 28.9%) in vivo showed that the gastric-retentive delivery system can be retained in the stomach for more than 6h. The floating and bioadhesive particulate drug delivery system based on a hollow structure with a dual function presented here is a viable alternative to other for gastroretentive drug delivery system.

Application of an ultra-performance liquid chromatography method with tandem mass spectrometry to pharmacokinetics, tissue distribution and excretion in the study of DAT-230, a novel tubulin-binding agent candidate, in rats.

A rapid, sensitive and high-throughput ultra-performance liquid chromatography method with tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of DAT-230 in rat plasma, urine, feces and tissues (heart, liver, spleen, lung, kidney, stomach, intestine and brain). The biological samples were prepared by protein precipitation, and separation was achieved on an ACQUITY™ UPLC BEH C18 column (50 mm × 2.1mm, 1.7 µm) with a mobile phase that consisted of methanol - 0.2% formic acid water (80:20, v/v) at a flow rate of 0.2 mL/min. The MS/MS ion transitions were monitored at m/z 372.17 → 357.17 for DAT-230 and m/z 406.08 → 345.16 for COH-203 (internal standard, IS). The calibration curve was linear in the range of 0.1-5200 ng/mL for all biological matrices (r(2) ≥ 0.996), and it had the same value for the lower limit of quantification. The validated method was successfully applied to the pharmacokinetics, tissue distribution and excretion study after intravenous administration of a 5mg/kg dose of DAT-230 to healthy Sprague-Dawley rats. The mean pharmacokinetic parameters of t1/2 and AUC0-12 were (1.1 ± 0.4)h and (861.0 ± 281.2) ng h/mL, respectively. Tissue distribution results indicated that DAT-230 exhibited rapid distribution and high liver, kidney, spleen, stomach and intestine uptake; these organs were indicated as the major target organs of the drug. In total, 5.3% of the administered DAT-230 was excreted in an unconverted form in urine, feces and bile, which implies that DAT-230 was excreted primarily in the form of metabolites.

A floating multiparticulate system for ofloxacin based on a multilayer structure: In vitro and in vivo evaluation.

The purpose of this research was to develop a novel gastroretentive multiparticulate system with floating ability. This system was designed to provide drug-loaded pellets coated with three successive coatings-the retarding film (ethyl cellulose), the effervescent layer (sodium bicarbonate) and the gas-entrapped polymeric membrane (Eudragit RL 30D). The floating pellets were evaluated for SEM, floating characteristic parameters, in vitro release and bioavailability in New Zealand rabbits. The zero-order release theory model is designed to interpret the release processes. Due to the swelling property, high flexibility and high water permeability, Eudragit RL 30D was used as a gas-entrapped polymeric membrane. The obtained pellets exhibit excellent floating ability and release characteristics. Analysis of the release mechanism showed a zero-order release for the first 8h because of the osmotic pressure of the saturated solution inside of the membrane, which was in accordance with that predicted. Abdominal X-ray images showed that the gastroretention period of the floating barium sulfate-labeled pellets was no less than 6h. The relative bioavailability of the floating pellets compared with reference tablets was 113.06 ± 23.83%. All these results showed that the floating pellets are a feasible approach for the gastroretentive drug delivery system.