Developing oxaliplatin and IL-15 Co-carried gels as drug depots to enable triple-interlocked combination therapy for colorectal cancer.

Chemo-immunotherapy, which involves the simultaneous use of chemotherapy drug and immunotherapeutic agent to achieve synergistic effects, plays a crucial role in cancer treatment. However, the immunosuppressive microenvironment, insufficient tumor specificity, and serious systemic side effects hinder their synergistic therapeutic effects and clinical applications. Herein, T cell and natural killer (NK) cell, which are the most important immune effector cells, were both activated to reverse the immunosuppressive microenvironment. To simplify drug carriers, oxaliplatin was selected as the chemotherapy drug which can both induce the ICD effect and activate T cells. IL-15 was selected to activate NK cells. To enhance the productivity of the carrier and reduce side effects, the easy-prepared thermosensitive hydrogel (OXL/IL-15 TG) was developed to co-load oxaliplatin-loaded liposomes (OXL) and IL-15. Colorectal cancer, suitable for in situ administration, was selected as model cancer. The resulting novel triple-interlocked combination therapy could directly kill the tumor cells, induces ICD effect and activate NK cells. After administration, OXL/IL-15 TG was formed serving as a drug depot, slowing releasing OXL and IL-15 non-interferencely. OXL around 165.47±7.04 nm was passively delivered to tumor tissue, killing tumor cells and inducing ICD effect. The results demonstrated that IL-15 stimulated the activation of NK cells. In tumor-bearing mice models, OXL/IL-15 TG exhibited a remarkable and noteworthy anti-tumor efficacy, and expanded survival rate. Notably, OXL/IL-15 TG led to an enhanced infiltration of CD3+CD8+ T cells and CD3-CD49+ NK cells within the tumor tissue. Overall, the triple-interlocked combination therapy provided a new idea for colorectal cancer therapy.

Polyoxometalate functionalized magnetic metal-organic framework with multi-affinity sites for efficient enrichment of phosphopeptides.

The reasonable design of metal-organic framework (MOF)-derived nanomaterial has important meaning in increasing the enrichment efficiency in the study of protein phosphorylation. In this work, a polyoxometalate (POM) functionalized magnetic MOF nanomaterial (Fe3O4@MIL-125-POM) was designed and fabricated. The nanomaterial with multi-affinity sites (unsaturated metal sites and metal oxide clusters) was used for the enrichment of phosphopeptides. Fe3O4@MIL-125-POM had high-efficient enrichment performance towards phosphopeptides (selectivity, a mass ratio of bovine serum albumin/α-casein/ß-casein at 5000:1:1; sensitivity, 0.1 fmol; satisfactory repeatability, ten times). Furthermore, Fe3O4@MIL-125-POM was employed to enrich phosphopeptides from non-fat milk digests, saliva, serum, and A549 cell lysate. The enrichment results illustrated the great potential of Fe3O4@MIL-125-POM for efficient identification of low-abundance phosphopeptides.

Cobalt Phthalocyanine-Modified Magnetic Metal-Organic Frameworks for Specific Enrichment of Phosphopeptides.

For mass spectrometry (MS)-based phosphoproteomics studies, sample pretreatment is an essential step for efficient identification of low-abundance phosphopeptides. Herein, a cobalt phthalocyanine-modified magnetic metal-organic framework (MOF) (Fe3O4@MIL-101-CoPc) was prepared and applied to enrich phosphopeptides before MS analysis. Fe3O4@MIL-101-CoPc exhibited an excellent magnetic response (74.98 emu g-1) and good hydrophilicity (7.75°), which were favorable for the enrichment. Fe3O4@MIL-101-CoPc showed good enrichment performance with high selectivity (1:1:5000), sensitivity (0.1 fmol), reusability (10 circles), and recovery (91.3%). Additionally, the Fe3O4@MIL-101-CoPc-based MS method was able to successfully detect 827 phosphopeptides from the A549 cell lysate, demonstrating a high enrichment efficiency (89.3%). This study promotes the application of postfunctionalized MOFs for phosphoproteomics analysis.

Development and psychometric evaluation of beliefs about the orthokeratology lens compliance scale (BOLCS): Based on the theory of planned behavior.

PURPOSE: Non-adherence to standard wear, care, and follow-up procedures is a major risk factor for contact lens-related complications. The effectiveness of orthokeratology largely depends on the wearer's adherence. However, a deficiency in scales capturing adherence beliefs pertinent to orthokeratology exacerbates the lack of guidance for effective intervention strategies. The purpose of this study is to develop and evaluate the psychometric properties of a new scale based on the Theory of Planned Behavior that assesses the level of adherence among ortho-k wearers. METHODS: This study involved three stages following the STROBE checklist: (1) developing initial scale items based on the Theory of Planned Behavior, a literature review, and a qualitative study; (2) evaluation of content and face validity; (3) psychometric testing on 296 participants. Item analysis, based on Classical Test Theory, assessed the overall consistency, reliability, and validity of the scale. RESULTS: The final 37-item Beliefs about the Orthokeratology Lens Compliance Scale (BOLCS) comprises 11 dimensions. The Cronbach's alpha coefficients for each dimension ranged from 0.560 to 0.798. The folded half reliabilities were 0.845, and the combined reliabilities ranged from 0.676 to 0.793, indicating strong reliability. Item-level CVI (I-CVI) and scale-level CVI/average (S-CVI/Ave) values, assessed by the panel, ranged from 0.71 to 1 and 0.954, respectively. Exploratory and confirmatory factor analyses supported a factor structure consistent with the theoretical model. CONCLUSIONS: The scale's construction adhered to a standardized process, yielding preliminary validation results with satisfactory reliability and validity.

Exploring the immune escape mechanisms in gastric cancer patients based on the deep AI algorithms and single-cell sequencing analysis.

Gastric cancer is a prevalent and deadly malignancy, and the response to immunotherapy varies among patients. This study aimed to develop a prognostic model for gastric cancer patients and investigate immune escape mechanisms using deep machine learning and single-cell sequencing analysis. Data from public databases were analysed, and a prediction model was constructed using 101 algorithms. The high-AIDPS group, characterized by increased AIDPS expression, exhibited worse survival, genomic variations and immune cell infiltration. These patients also showed immunotherapy tolerance. Treatment strategies targeting the high-AIDPS group identified three potential drugs. Additionally, distinct cluster groups and upregulated AIDPS-associated genes were observed in gastric adenocarcinoma cell lines. Inhibition of GHRL expression suppressed cancer cell activity, inhibited M2 polarization in macrophages and reduced invasiveness. Overall, AIDPS plays a critical role in gastric cancer prognosis, genomic variations, immune cell infiltration and immunotherapy response, and targeting GHRL expression holds promise for personalized treatment. These findings contribute to improved clinical management in gastric cancer.

The complete mitochondrial genome of Rhipicephalus haemaphysaloides and its phylogenetic analysis.

We conducted an analysis of the complete mitochondrial genome of Rhipicephalus haemaphysaloides, a tick species known for transmitting various bacteria and viruses. The mitochondrial genome of R. haemaphysaloides has a length of 14,739 bp and consists of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and 2 control regions. By utilizing the maximum likelihood method, we established the phylogenetic relationship among R. haemaphysaloides and other species within the Rhipicephalus genus of the Ixodidae family. This analysis revealed that R. haemaphysaloides and other Rhipicephalus species belong to the same clade, further affirming the taxonomic placement of R. haemaphysaloides within the Rhipicephalus genus. Furthermore, we compared the mitochondrial genomes of R. haemaphysaloides isolates from Changning, Yunnan Province, China, with isolates from Yangxin, Ganzhou, and Yingtan, Hubei Province, China. In summary, our investigation offers genetic proof endorsing the taxonomic categorization and phylogenetic placement of Ixodidae by assessing the entire mitochondrial genome of R. haemaphysaloides.

Enrichment of Phosphopeptides Based on Zirconium Phthalocyanine-Modified Magnetic Nanoparticles.

Highly selective extraction of phosphopeptides is necessary before mass spectrometry (MS) analysis. Herein, zirconium phthalocyanine-modified magnetic nanoparticles were prepared through a simple method. The Fe-O groups on Fe3O4 and the zirconium ions on phthalocyanine had a strong affinity for phosphopeptides based on immobilized metal ion affinity chromatography (IMAC). The enrichment platform exhibited low detection limit (0.01 fmol), high selectivity (α-/ß-casein/bovine serum albumin, 1/1/5000), good reusability (10 circles), and recovery (91.1 ± 1.1%) toward phosphopeptides. Nonfat milk, human serum, saliva, and A549 cell lysate were employed as actual samples to assess the applicability of the enrichment protocol. Metallo-phthalocyanine will be a competitive compound for designing highly efficient adsorbents and offers a new approach to phosphopeptide analysis.

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide.

OBJECTIVES: This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. METHODS: hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. RESULTS: Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). CONCLUSIONS: Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.

A fluorescence detection method for postharvest tomato epidermal defects based on improved YOLOv5m.

BACKGROUND: Tomato quality visual grading is greatly affected by the problems of smooth skin, uneven illumination and invisible defects that are difficult to identify. The realization of intelligent detection of postharvest epidermal defects is conducive to further improving the economic value of postharvest tomatoes. RESULTS: An image acquisition device that utilizes fluorescence technology has been designed to capture a dataset of tomato skin defects, encompassing categories such as rot defects, crack defects and imperceptible defects. The YOLOv5m model was improved by introducing Convolutional Block Attention Module and replacing part of the convolution kernels in the backbone network with Switchable Atrous Convolution. The results of comparison experiments and ablation experiments show that the Precision, Recall and mean Average Precision of the improved YOLOv5m model were 89.93%, 82.33% and 87.57%, which are higher than YOLOv5m, Faster R-CNN and YOLOv7, and the average detection time was reduced by 47.04 ms picture-1. CONCLUSION: The present study utilizes fluorescence imaging and an improved YOLOv5m model to detect tomato epidermal defects, resulting in better identification of imperceptible defects and detection of multiple categories of defects. This provides strong technical support for intelligent detection and quality grading of tomatoes. © 2024 Society of Chemical Industry.

GTPase activity of porcine Mx1 plays a dominant role in inhibiting the N-Nsp9 interaction and thus inhibiting PRRSV replication.

Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.

Safety, tolerability, pharmacokinetics and neutrophil elastase inhibitory effects of Sivelestat: A randomized, double-blind, placebo-controlled single- and multiple-dose escalation study in Chinese healthy subjects.

BACKGROUND AND OBJECTIVE: Neutrophil elastase has been identified as a potential therapeutic target for acute lung injury or acute respiratory distress syndrome, and Sivelestat is a selective, reversible and competitive neutrophil elastase inhibitor. This study was designed to investigate the safety, tolerability, pharmacokinetics and neutrophil elastase inhibitory effects of Sivelestat in healthy Chinese subjects. METHODS: A randomized, double-blind, placebo-controlled single- and multiple-dose escalation clinical trial was carried out. Briefly, healthy volunteers in twelve cohorts with 8 per cohort received 1.0-20.2 mg/kg/h Sivelestat or placebo in an intravenous infusion manner for two hours, and healthy volunteers in four cohorts received two hours intravenous infusion of 2.0-5.0 mg/kg/h Sivelestat or placebo with an interval of twelve hours for seven times. The safety and tolerability were evaluated and serial blood samples were collected for pharmacokinetics and neutrophil elastase inhibitory effects analysis at the specified time-point. RESULTS: A total of 128 subjects were enrolled and all participants completed the study except one. Sivelestat exhibited satisfactory safety and tolerability up to 20.2 mg/kg/h in single-dose cohorts and 5.0 mg/kg/h in multiple-dose cohorts. Even so, more attention should be paid to the safety risks when using high doses. The Cmax and AUC of Sivelestat increased in a dose dependent manner, and Tmax was similar for different dose cohorts. In multiple-dose cohorts, the plasma concentrations reached steady state 48 h after first administration and the accumulation of Cmax and AUC was not obvious. Furthermore, the Cmin_ss of 5.0 mg/kg/h dose cohort could meet the needs of clinical treatment. For some reason, the pharmacodynamics data revealed that the inhibitory effect of Sivelestat on neutrophil elastase content in healthy subjects was inconclusive. CONCLUSION: Sivelestat was safe and well tolerated with appropriate pharmacokinetic parameters, which provided support for more diverse dosing regimen in clinical application. CLINICAL TRIAL REGISTRATION: www.chinadrugtrials.org.cn identifier is CTR20210072.

Improving the Sensitivity and Linear Range of Photoionization Ion Mobility Spectrometry via Confining the Ion Recombination and Space Charge Effects Assisted by Theoretical Modeling.

Photoionization (PI) is an efficient ionization source for ion mobility spectrometry (IMS) and mass spectrometry. Its hyphenation with IMS (PI-IMS) has been employed in various on-site analysis scenarios targeting a wide range of compounds. However, the signal intensity and linear dynamic range of PI-IMS at ambient pressure usually do not follow the Beer-Lambert law predictions, and the factors causing that negative deviation remain unclear. In this work, a variable pressure PI-IMS system was developed to examine the ion loss effects from factors like ion recombination and space charge by varying its working pressure from 1 to 0.1 bar. Assisted by theoretical modeling, it was found that ion recombination could contribute up to 90% of signal intensity loss for ambient pressure PI-IMS setups. Lowering the pressure and increasing the electric field in PI-IMS helped suppress the ion recombination process and thus an optimal pressure Poptimal appeared for best signal intensity, despite the decreased net ion number density and the increased space charge effect. A simplified theoretical equation taking ion recombination as the primary ion loss factor was derived to link Poptimal with analyte concentration and electric field in PI-IMS, enabling a swift optimization of the PI-IMS performance. For example, compared to ambient pressure, PI-IMS at a Poptimal of 0.4 bar provided a signal intensity increment of more than 400% for 0.716 ppmv toluene and also expanded the linear dynamic range by more than two times. Revealing factors influencing the PI-IMS response would also benefit the applications of other chemical ionization sources in IMS or mass spectrometry (MS).

Two different autorefractors for vision screening in children and adolescents.

AIM: To compare the consistency of two autorefractors (Tianle RM-9000 and Topcon KR-800) for school-age myopia children, and to provide a basis for largescale data analysis and comparison. METHODS: The refractive error in 909 subjects (age 4-18y) were measured using both autorefractors without cycloplegia. The data were analyzed using Fourier decomposition and the correlation coefficients, intraclass correlation coefficients (ICC), and Bland-Altman limits of agreement (LoA) for each parameter were calculated. RESULTS: There was a strong correlation between the spherical equivalent (SE), sphere diopter (DS), and cylinder diopter (DC) readings of the Tianle RM-9000 and those of the Topcon KR-800, with correlation coefficient values of 0.98, 0.98 and 0.83 and ICC values of 0.99, 0.99 and 0.93, respectively. However, the correlation coefficients and ICC values of J0 and J45 were unreliable (R=-0.004, -0.034; both ICC<0.10). Bland-Altman analysis revealed that SE, DS, and DC measured by the Tianle RM-9000 were significantly biased toward myopia compared with the Topcon KR-800, and the mean differences were -0.072, -0.026, -0.091 D, respectively (all P<0.01). The minimum absolute value of the difference within the 95% LoA for SE, DS, and DC was 0.63 D, 0.50 D, 0.62 D, respectively; all these values were in the clinically acceptable range. For J0 and J45, the mean differences were close to zero (P=0.43, 0.84); however, the 95% LoA were relatively wide (J0 SD: 0.53; 95%CI: -1.00, 1.10; J45 SD: 0.52; 95%CI: -1.00, 1.00). CONCLUSION: The two autorefractors are consistent with each other, as the differences in SE, DS, and DC were within the clinically acceptable range. Readers can compare the data measured by either device in different studies and use the two devices in the same study to generate a dataset that can be analyzed together. However, the J0 and J45 vectors are unreliable and should not be used to assess astigmatism.

Zirconium-rich magnetic polyoxometalate-based metal-organic framework: Tailored for phosphopeptide analysis from lung cancer A549 cells.

Polyoxometalate-based metal-organic frameworks (POMOFs) have become a promising affinity material for separation and enrichment. The analysis of protein phosphorylation represents a challenge for the development of efficient enrichment materials. Here, a novel zirconium-rich magnetic POMOF was successfully designed and prepared for the enrichment of phosphopeptides. The binding affinity of the nanomaterial partly came from Fe-O clusters in the MOF. The Lewis acid-base interactions between V-O clusters and zirconium ions in V10O28-Zr4+ and phosphate groups in phosphopeptides further strengthened the enrichment ability. The zirconium-rich magnetic POMOF was employed to capture phosphopeptides from non-fat milk, human saliva, and serum. Additionally, 748 unique phosphopeptide peaks were detected from the tryptic digests of lung cancer A549 cell proteins with a high specificity (86.9 %). POMOFs will become an active competitor for the design of protein affinity materials and will provide a new approach for phosphopeptide analysis.

Itaconic Acid-Based Organic-Polymer Monolithic Column for Hydrophilic Capillary Electrochromatography and Its Application in Pharmaceutical Analysis.

Itaconic acid is an excellent hydrophilic monomer owing to the dicarboxylic group possessing strong polarity. This study reports on the preparation of a new organic-polymer monolithic column poly(itaconic acid-co-3-(acryloyloxy)-2-hydroxypropyl methacrylate) (poly(IA-co-AHM)) featuring excellent hydrophilic chromatography ability and its application in pharmaceutical analysis. The monolithic column was successfully synthesized by using the monomer itaconic acid and the cross-linker AHM through an in situ copolymerization method. Optical microscopy, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR) were employed for the characterization of the poly(IA-co-AHM) monolithic column, and all of these demonstrated that the prepared itaconic acid-based monolithic column exhibited satisfactory permeability and a homogeneous porous structure. Owing to the carboxylic groups of itaconic acid, a cathodic electroosmotic flow (EOF) was generated on the itaconic acid-based monolithic column among the pH ranges of the mobile phase from 4.0 to 9.0. Depending on the powerful hydrophilic interactions, different kinds of polar substances, including thioureas, nucleoside drugs, sulfonamides, and polypeptides, were separated efficiently by the itaconic acid-based monoliths poly(IA-co-AHM). The separations of polar compounds were successfully realized, even at a lower level of 50% acetonitrile content on this monolithic column. The highest column efficiencies corresponding to N,N'-dimethylthiourea and idoxuridine were 102 720 and 124 267 N/m, respectively. The poly(IA-co-AHM) monolithic column displayed excellent repeatability, whose relative standard deviations (RSDs) of the retention time and peak area were both lower than 5.0%. All experimental results demonstrated that the new itaconic acid-functionalized monolithic column was greatly appropriate to separate the polar compounds under the HILIC mode.

Development and application of a multiplex PCR method for the simultaneous detection of goose parvovirus, waterfowl reovirus, and goose astrovirus in Muscovy ducks.

A multiplex polymerase chain reaction (PCR) method was developed to detect and distinguish goose parvovirus (GPV), waterfowl reovirus (WRV), and goose astrovirus (GAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of these enteric viruses and were used to specifically amplify targeted fragments of 493 bp from the viral protein 3 (VP3) gene of GPV, 300 bp from the sigma A-encoding gene of WRV, and 156 bp from the capsid protein-encoding gene of GAstV. The results showed that the primers can specifically amplify target fragments, without any cross-amplification with other viruses, indicating that the method had good specificity. A sensitivity test showed that the detection limit of the multiplex PCR method was 1 × 103 viral copies. A total of 102 field samples from Muscovy ducks with clinically suspected diseases were evaluated using the newly developed multiplex PCR method. The ratio of positive samples to total samples for GPV, WRV, and GAstV was 73.53% (75/102) for multiplex PCR and was 73.53% (75/102) for routine PCR. Seventy-five positive samples were detected by both methods, for a coincidence ratio of 100%. This multiplex PCR method can simultaneously detect GPV, WRV, and GAstV, which are associated with viral enteritis, thereby providing a specific, sensitive, efficient, and accurate new tool for clinical diagnosis and laboratory epidemiological investigations.

Nano-Regulator Inhibits Tumor Immune Escape via the "Two-Way Regulation" Epigenetic Therapy Strategy.

Tumor immune escape caused by low levels of tumor immunogenicity and immune checkpoint-dependent suppression limits the immunotherapeutic effect. Herein, a "two-way regulation" epigenetic therapeutic strategy is proposed using a novel nano-regulator that inhibits tumor immune escape by upregulating expression of tumor-associated antigens (TAAs) to improve immunogenicity and downregulating programmed cell death 1 ligand 1 (PD-L1) expression to block programmed death-1 (PD-1)/PD-L1. To engineer the nano-regulator, the DNA methyltransferase (DNMT) inhibitor zebularine (Zeb) and the bromodomain-containing protein 4 (BRD4) inhibitor JQ1 are co-loaded into the cationic liposomes with condensing the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine (CpG) via electrostatic interactions to obtain G-J/ZL. Then, asparagine-glycine-arginine (NGR) modified material carboxymethyl-chitosan (CMCS) is coated on the surface of G-J/ZL to construct CG-J/ZL. CG-J/ZL is shown to target tumor tissue and disassemble under the acidic tumor microenvironment (TME). Zeb upregulated TAAs expression to improve the immunogenicity; JQ1 inhibited PD-L1 expression to block immune checkpoint; CpG promote dendritic cell (DC) maturation and reactivated the ability of tumour-associated macrophages (TAM) to kill tumor cells. Taken together, these results demonstrate that the nano-regulator CG-J/ZL can upregulate TAAs expression to enhance T-cell infiltration and downregulate PD-L1 expression to improve the recognition of tumor cells by T-cells, representing a promising strategy to improve antitumor immune response.

Preparation of high-efficiency titanium ion immobilized magnetic graphite nitride nanocomposite for phosphopeptide enrichment.

BACKGROUND: Protein phosphorylation has been implicated in life processes including molecular interaction, protein structure transformation, and malignant disease. An in-depth study of protein phosphorylation may provide vital information for the discovery of early biomarkers. Mass spectrometry (MS)-based techniques have become an important method for phosphopeptide identification. Nevertheless, direct detection remains challenging because of the low ionization efficiency of phosphopeptides and serious interference from non-phosphopeptides. There is a great need for an efficient enrichment strategy to analyze protein phosphorylation prior to MS analysis. RESULTS: In this study, a novel nanocomposite was prepared by introducing titanium ions into two-dimensional magnetic graphite nitride. The nanocomposite was combined with immobilized metal ion affinity chromatography (IMAC) and anion-exchange chromatography mechanisms for phosphoproteome research. The nanocomposite had the advantages of a large specific surface (412.9 m2 g-1), positive electricity (175.44 mV), and excellent magnetic property (35.7 emu g-1). Moreover, it presented satisfactory selectivity (α-casein:ß-casein:bovine serum albumin = 1:1:5000), a low detection limit (0.02 fmol), great recyclability (10 cycles), and high recovery (92.8%). The nanocomposite demonstrated great practicability for phosphopeptides from non-fat milk, human serum, and saliva. Further, the nanocomposite was applied to enrich phosphopeptides from a more complicated specimen, A549 cell lysate. A total of 890 phosphopeptides mapping to 564 phosphoproteins were successfully detected with nano LC-MS. SIGNIFICANCE: We successfully designed and developed an efficient analysis platform for phosphopeptides, which includes protein digestion, phosphopeptide enrichment, and MS detection. The MS-based enrichment platform was further used to analyze phosphopeptides from complicated bio-samples. This work paves the way for the design and preparation of graphite nitride-based IMAC materials for phosphoproteome analysis.

Cerium ions immobilized magnetic graphite nitride decorated with L-Alanyl-L-Glutamine as new chelator for enrichment of phosphopeptides.

Cerium ions immobilized magnetic graphite nitride material have been prepared using L-Alanyl-L-Glutamine as the new chelator. The resulting Fe3O4/g-C3N4-L-Ala-L-Gln-Ce4+, as an immobilized metal ion affinity chromatography (IMAC) sorbent, was reusable. This is due to the strong coordination interaction between L-Alanyl-L-Glutamine and cerium ions. After a series of characterizations, the magnetic nanocomposite showed high surface area, good hydrophilicity, positive electricity, and magnetic response. Fe3O4/g-C3N4-L-Ala-L-Gln-Ce4+ had high sensitivity (0.1 fmol), selectivity (α-/ß-casein/bovine serum albumin, 1:1:5000), and good recyclability (10 cycles). A total of 647 unique phosphopeptides mapped to 491 phosphoproteins were identified from A549 cell lysate by nano LC-MS analysis.