PTCH1 mutation as a potential predictive biomarker for immune checkpoint inhibitors in gastrointestinal cancer.

Immune checkpoint inhibitors (ICIs) have become prominent therapies for gastrointestinal cancer (GC). However, it is urgent to screen patients who can benefit from ICIs. Protein patched homolog 1 (PTCH1) is a frequently altered gene in GC. We attempt to explore the association between PTCH1 mutation and immunotherapy efficacy. The Memorial Sloan Kettering Cancer Center (MSKCC) cohort (n = 236) with GC (esophageal, gastric and colorectal cancers) patients receiving ICIs was used for discovery and the Peking University Cancer Hospital (PUCH) GC cohort (n = 92) was used for validation. Overall survival (OS) and tumor mutational burden (TMB) of the PTCH1 mutant-type (PTCH1-MUT) and PTCH1 wild-type (PTCH1-WT) groups were compared. Furthermore, GC data were collected from The Cancer Genome Atlas to assess the potential mechanisms. In the MSKCC cohort, PTCH1-MUT group showed significantly better OS (P = 0.017) and higher TMB. Multivariate analysis showed that PTCH1 mutation was associated with better OS. In the PUCH cohort, PTCH1-MUT group showed significantly longer OS (P = 0.036) and progression-free survival, and higher durable clinical benefit and TMB. Immune cell infiltration analysis revealed that PTCH1-MUT group had significantly higher distributions of CD8 T cells, CD4 T cells, NK cells, mast cells and M1 cells. The PTCH1-MUT group showed significantly higher expression of most immune-related genes. Gene set enrichment analysis showed that the PTCH1-MUT group had enriched INF-γ response, INF-α response, glycolysis and reactive oxygen species pathway gene sets. PTCH1 mutation may represent a potential biomarker for predicting ICIs response in GC. Nevertheless, prospective cohort studies should be performed to further validate our results.

Efficacy of Rituximab in the Treatment of Pemphigus Vulgaris: A Meta-analysis.

Background: Pemphigus vulgaris (PV) accounts for about 80% of all patients with pemphigus, and is the type with the most serious condition and the worst prognosis among autoimmune bullous diseases. Glucocorticoid and immunosuppressor are the main treatment method for PV. Methods: The computer retrieves four databases obtain controlled trials on the effects of Rituximab in patients with pemphigus vulgaris. After a rigorous literature quality evaluation, data analysis was performed using RevMan 5.3 software. Results: 7 studies were ultimately included in this meta-analysis. 6 studies reported the Remission rate of the test group and the control group, which was significantly higher (OR:2.26; 95% Cl: 1.80,2.82; P < .01) than the control group. Meta-analysis showed that the improvement of the Recurrence rate was significantly lower than the control group (OR:0.36; 95% Cl: 0.20,0.67; P < .01). Meta-analysis showed that the Adverse reactions was no significant statistical significance than the control group (OR:0.82; 95% Cl: 0.53,1.28; P = .383). Conclusion: The results of this study suggest that Rituximab may be effective in patients with pemphigus vulgaris, which will bring light for patients and doctors. And the above conclusions need to be verified by more high-quality studies.

Quantitative N-Glycomic and N-Glycoproteomic Profiling of Peach [Prunus persica (L.) Batsch] during Fruit Ripening.

Being part of the human diet, peach is an important fruit consumed worldwide. In the present study, a systematic first insight into the N-glycosylation of peach fruit during ripening was provided. First, N-glycome by reactive matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated that 6 of 24 N-glycans of peach were differentially expressed. Second, a comparative N-glycoproteome was characterized via 18O-tagged N-glycosylation site labeling followed by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS). Totally 1464 N-glycosites on 881 N-glycoproteins were identified, among which 291 N-glycosites on 237 N-glycoproteins were expressed differentially with a fold change value of 1.5 or 0.67. The enrichment analysis of GO and KEGG revealed that four pathways including other glycan degradation, phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and protein processing in endoplasmic reticulum were mainly enriched, in which several important N-glycoproteins with dynamic change during fruit ripening were further screened out. Our findings on a large scale for N-glycosylation analysis of peach fruit during ripening may provide new molecular insights for comprehending N-glycoprotein functions, which should be of great interest to both glycobiologists and analytical chemists.

Radical-Induced Dissociation for Oligonucleotide Sequencing by TiO2/ZnAl-Layered Double Oxide-Assisted Laser Desorption/Ionization Mass Spectrometry.

De novo sequencing of oligonucleotides remains challenging, especially for oligonucleotides with post-transcriptional or synthetic modifications. Mass spectrometry (MS) sequencing can reliably detect and locate all of the modification sites in oligonucleotides via m/z variance. However, current MS-based sequencing methods exhibit complex spectra and low ion abundance and usually require coupled instrumentation. Herein, we demonstrate a method of oligonucleotide sequencing using TiO2/ZnAl-layered double oxide (LDO)-assisted laser desorption/ionization (LDI)-MS based on radical-induced dissociation (RID). ·CH2OH radicals can be produced on the surface of a TiO2/ZnAl-LDO matrix via ultraviolet light, inducing an attack on the active site of the oligonucleotide phosphate skeleton to create typical "a-, a-B-, c·-, d-, w-, and y"-type fragments. Compared with the spectra obtained via collision-based methods, such as collision-induced dissociation and higher-energy collisional dissociation, the LDI-MS spectra based on RID exhibit single-charged signals, fewer types of fragments, and a lower proportion of unknown noise peaks. We demonstrate full sequence coverage for a 6-mer 2'-O-methyl-modified oligonucleotide and a 21-mer small interfering RNA and show that RID can sequence oligonucleotides with modifications. Importantly, the mechanism responsible for the RID of the oligonucleotide phosphate skeleton was investigated through offline experiments, demonstrating consistent results with density functional theory calculations.

Newly revealed variants of SERPINA3 in generalized pustular psoriasis attenuate inhibition of ACT on cathepsin G.

Generalized pustular psoriasis (GPP) is an autoinflammatory skin disease whose pathogenesis has not yet been fully elucidated. Alpha-1-antichymotrypsin(ACT) is a protein encoded by the SERPINA3 gene and an inhibitor of cathepsin G. One study of a European sample suggested that the loss of ACT function caused by SERPINA3 mutation is implicated in GPP. However, the role of SERPINA3 in the pathogenesis of GPP in other ethnic populations is unclear. To explore this, seventy children with GPP were performed next-generation sequencing to identify rare variants in the SERPINA3 gene. Bioinformatic analysis and functional tests were used to determine the effects of the variants, and a comprehensive analysis was performed to determine the pathogenicity of the variants and whether they are associated with GPP. One rare deletion and three rare missense variants were identified in the SERPINA3 gene in GPP. The deletion variant c.1246_1247del was found to result in a mutant protein with an extension of 10 amino acids and a C-terminal of 20 amino acids that was completely different from the wild-type. This mutant was found to impede secretion of ACT, thus failing to function as an inhibitor of cathepsin G. Two missense variants were found to reduce the ability of ACT to inhibit cathepsin G enzymatic activity. The association analysis suggested that the deletion variant is associated with GPP. This study identified four rare novel mutations of SERPINA3 and demonstrated that three of these mutations result in loss of function, contributing to the pathogenesis of pediatric-onset GPP in the Asian population.

POTEE mutation as a potential predictive biomarker for immune checkpoint inhibitors in lung adenocarcinoma.

Precise selection of patients who could benefit from immune checkpoint inhibitors (ICIs) is an important challenge for immunotherapy in lung cancer. POTEE (POTE Ankyrin Domain Family Member E) is a member of one primate-specific gene family which have been identified as cancer-related antigens and potential target for immunotherapy of cancer. Here, we investigated the correlation between POTEE mutation and the clinical outcome of ICIs treatment in non-small cell lung cancer (NSCLC). We merged three NSCLC cohorts (n = 165) to assess predictive value of POTEE mutation of immunotherapy efficacy in NSCLC. The prognostic analysis and the potential molecular mechanism exploration were conducted based on the data from The Cancer Genome Atlas (TCGA) database. In the merged cohort, patients with POTEE-mutation (POTEE-Mut) had a significantly higher objective response rate (ORR) (100% vs 27.7%; P < 0.001) and longer progression-free survival (PFS) (P = 0.001; HR 0.08; 95% CI 0.01 - 0.54) compared to patients with POTEE wild-type (POTEE-WT) in NSCLC. Also, patients with POTEE-Mut showed higher ORR (100% vs 27.2%; P < 0.001) and longer PFS (P = 0.001; HR 0.07; 95% CI 0.01 - 0.52) in lung adenocarcinoma (LUAD). POTEE mutation was significantly associated with higher tumor mutational burden (TMB) and higher neoantigen load (NAL), but not with PD-L1 expression in LUAD. Gene set enrichment analyses (GSEA) analysis revealed prominent enrichment of signatures related to DNA repair in POTEE-Mut group (P < 0.001) in LUAD. Our results indicate that POTEE mutation could serve as a potential predictive biomarker for ICIs in LUAD. However, prospective cohort studies are still needed for further validation.

Liquid biopsy using cell-free DNA in the early diagnosis of hepatocellular carcinoma.

Hepatocellular carcinoma ranks fourth in cancer-related causes of death worldwide and second in China. Patients with hepatocellular carcinoma (HCC) at the early stage have a better prognosis compared to HCC patients at the late stage. Therefore, early screening for HCC is critical for clinical treatment decisions and improving the prognosis of patients. Ultrasound (US), computed tomography (CT), and serum alpha fetoprotein (AFP) have been used to screen HCC, but HCC is still difficult to be diagnosed in the early stage due to the low sensitivity of the above methods. It is urgent to find a method with high sensitivity and specificity for the early diagnosis of HCC. Liquid biopsy is a noninvasive detection method using blood or other bodily fluids. Cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) are important biomarkers for liquid biopsy. Recently, HCC screening methods using the application of cfDNA and ctDNA have become the hot spot of early HCC diagnostics. In this mini review, we summarize the latest research progress of liquid biopsy based on blood cfDNA in early screening of HCC.

Toward a New Era of Coral Reef Monitoring.

Coral reefs host some of the highest concentrations of biodiversity and economic value in the oceans, yet these ecosystems are under threat due to climate change and other human impacts. Reef monitoring is routinely used to help prioritize reefs for conservation and evaluate the success of intervention efforts. Reef status and health are most frequently characterized using diver-based surveys, but the inherent limitations of these methods mean there is a growing need for advanced, standardized, and automated reef techniques that capture the complex nature of the ecosystem. Here we draw on experiences from our own interdisciplinary research programs to describe advances in in situ diver-based and autonomous reef monitoring. We present our vision for integrating interdisciplinary measurements for select "case-study" reefs worldwide and for learning patterns within the biological, physical, and chemical reef components and their interactions. Ultimately, these efforts could support the development of a scalable and standardized suite of sensors that capture and relay key data to assist in categorizing reef health. This framework has the potential to provide stakeholders with the information necessary to assess reef health during an unprecedented time of reef change as well as restoration and intervention activities.

AtHDA6 functions as an H3K18ac eraser to maintain pericentromeric CHG methylation in Arabidopsis thaliana.

Pericentromeric DNA, consisting of high-copy-number tandem repeats and transposable elements, is normally silenced through DNA methylation and histone modifications to maintain chromosomal integrity and stability. Although histone deacetylase 6 (HDA6) has been known to participate in pericentromeric silencing, the mechanism is still yet unclear. Here, using whole genome bisulfite sequencing (WGBS) and chromatin immunoprecipitation-sequencing (ChIP-Seq), we mapped the genome-wide patterns of differential DNA methylation and histone H3 lysine 18 acetylation (H3K18ac) in wild-type and hda6 mutant strains. Results show pericentromeric CHG hypomethylation in hda6 mutants was mediated by DNA demethylases, not by DNA methyltransferases as previously thought. DNA demethylases can recognize H3K18ac mark and then be recruited to the chromatin. Using biochemical assays, we found that HDA6 could function as an 'eraser' enzyme for H3K18ac mark to prevent DNA demethylation. Oxford Nanopore Technology Direct RNA Sequencing (ONT DRS) also revealed that hda6 mutants with H3K18ac accumulation and CHG hypomethylation were shown to have transcriptionally active pericentromeric DNA.

MicroRNA-483-5p accentuates cisplatin-induced acute kidney injury by targeting GPX3.

The ability of cisplatin (cis-diamminedichloroplatinum II) toxicity to induce acute kidney injury (AKI) has attracted attention and concern for a long time, but the molecular mechanism of action for cisplatin is not clear. MicroRNA-483 is involved in several diseases, such as tumorigenesis and osteoarthritis, but its renal target and potential role in AKI are unknown. In this study, we explored the pathogenic role and underlying mechanism of miR-483-5p in cisplatin-induced AKI, using transgenic mice, clinical specimen, and in vitro cell line. We found that miR-483-5p was significantly upregulated by cisplatin in a cisplatin-induced mouse model, in serum samples of patients who received cisplatin therapy, and in NRK-52E cells. Overexpression of miR-483-5p in mouse kidneys by stereotactic renal injection of lentiviruses mediated miR-483-5p or generation of conditional miR-483-overexpressing transgenic mice accentuated cisplatin-induced AKI by increasing oxidative stress, promoting apoptosis, and inhibiting autophagy of tubular cells. Furthermore, our results revealed miR-483-5p directly targeted to GPX3, overexpression of which rescued cisplatin-induced AKI by inhibiting oxidative stress and apoptosis of tubular cells, but not by regulating autophagy. Collectively, miR-483-5p is upregulated by cisplatin and exacerbates cisplatin-induced AKI via negative regulation of GPX3 and contributing oxidative stress and tubular cell apoptosis. These findings reveal a pathogenic role for miR-483-5p in cisplatin-induced AKI and suggest a novel target for the diagnosis and treatment of AKI.

Heat-Assisted Dual Neutral Spray Ionization for High-Performance Online Desalting in Mass Spectrometric Analysis.

In mass spectrometry (MS), nonvolatile salts contaminate the transmission system and cause ion suppression, hampering MS analysis. When MS is combined with liquid chromatography (LC) that uses a salty mobile phase, the problems become more intractable due to long analysis time. Here, a novel heat-assisted dual neutral spray ionization (HADSI) method was developed, which projected sample solution spray and solvent spray onto a heated plate to achieve online desalting and high ionization. The experimental parameters of HADSI were optimized, which indicated that the plate temperature was crucial for ionization and desalination. Eight drug compounds dissolved in various commonly used buffers were directly analyzed using HADSI-MS, even though the concentration of PBS buffer reached 500 mmol/L. The established method showed considerable sensitivity in the positive ion mode with the limits of detection at the level of nmol/L, and good linearity (R2 > 0.99) was achieved for all the analyzed compounds. The repeatability and intra- and interday precisions of the method were evaluated, demonstrating the feasibility and reliability of the analysis of salty samples by HADSI-MS. Further, the method was demonstrated to tolerate the long-time analysis of high-salt LC eluates and the device was easy to maintain. Finally, a crude roxithromycin product was separated by LC and then analyzed by HADSI-MS, and seven unknown impurities and nine known impurities were successfully detected. Our results indicated that HADSI-MS may have potential applications in academic and industrial fields.

The clinical application value of phase angle of six parts in nutritional evaluation of tumor patients.

OBJECTIVES: The purpose of this study was to explore the clinical application value of phase angle (PA) of six parts in the nutritional evaluation and construct a prediction model for diagnosing malnutrition of tumor patients. METHODS: A total of 1129 patients with malignant tumors were analyzed retrospectively. The age, sex, tumor location and body mass index (BMI) of the patients were collected, and PA of six parts was measured. The Patient Subjective Global Assessment (PG-SGA) was used to evaluate the nutritional status of each patient. RESULTS: According to the PG-SGA, 66.5% (n = 750) of the patients were evaluated as malnourished. Patients under the age of 65 had higher PA values. The PA value of men was higher than that of women (except PA-RL). In different disease groups, the PA-RA and PA-TR values were significantly different. In our study, PA value increases with BMI and decreases with PG-SGA (except PG-SGA 0-1 group). Multivariate regression analysis indicates that the age (HR = 1.051, 95% CI 1.037-1.066, P < 0.001), BMI (HR = 0.885, 95% CI 0.849-0.924, P < 0.001), and PA-WB (HR = 0.615, 95% CI 0.546-0.692, P < 0.001) were independent significant predictors associated with malnutrition. The AUC of the prediction model is 0.7631 (p < 0.001), indicating that the model including age, BMI, and PA-WB has certain diagnostic value for the diagnosis of malnutrition. CONCLUSION: The PA-WB is an independent prognostic factor of malnutrition. The prediction model constructed by age, BMI, and PA-WB can be used as a useful tool for nutritional evaluation of tumor patients. TRIAL REGISTRATION: Clinical Trial No.: ChiCTR2100047858.

Unraveling allosteric landscapes of allosterome with ASD.

Allosteric regulation is one of the most direct and efficient ways to fine-tune protein function; it is induced by the binding of a ligand at an allosteric site that is topographically distinct from an orthosteric site. The Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) was developed ten years ago to provide comprehensive information related to allosteric regulation. In recent years, allosteric regulation has received great attention in biological research, bioengineering, and drug discovery, leading to the emergence of entire allosteric landscapes as allosteromes. To facilitate research from the perspective of the allosterome, in ASD 2019, novel features were curated as follows: (i) >10 000 potential allosteric sites of human proteins were deposited for allosteric drug discovery; (ii) 7 human allosterome maps, including protease and ion channel maps, were built to reveal allosteric evolution within families; (iii) 1312 somatic missense mutations at allosteric sites were collected from patient samples from 33 cancer types and (iv) 1493 pharmacophores extracted from allosteric sites were provided for modulator screening. Over the past ten years, the ASD has become a central resource for studying allosteric regulation and will play more important roles in both target identification and allosteric drug discovery in the future.

Constitutional delay of growth and puberty in female mice is induced by circadian rhythm disruption in utero.

Constitutional delay of growth and puberty (CDGP) refers to the late onset of puberty. CDGP is associated with poor psychosocial outcomes and elevated risk of cardiovascular and osteoporotic diseases, especially in women. The environmental factors that contribute to CDGP are poorly understood. Here, we investigated the effects of chronic circadian disturbance (CCD) during the fetal stage on the pubertal development of female mice. Compared to non-stressed female (NS-F) mice that were not exposed to CCD in utero, adolescent CCD female (CCD-F) mice exhibited phenotypes that were consistent with CDGP, including lower body weight, reduced levels of circulating gonadal hormones, decreased expression of gonadal hormones and steroid synthesis-related enzymes in the ovary and hypothalamus, irregular estrus cycles, and tardive vaginal introitus initial opening (VO) days (equivalent to the menarche). Phenotypic differences in the above-noted parameters were not observed in CCD-F mice once they had reached adulthood. The expression of genes involved in fatty acid metabolism was perturbed in the ovary and hypothalamus of CCD-F mice. In addition, the ovaries of these animals exhibited altered diurnal expression profiles of circadian clock genes. Together, our findings not only suggest that CCD during fetal development may result in delayed puberty in female mice, they also offer insights on potential mechanisms that underlie CDGP.

Effects of Lycium barbarum L. Polysaccharides on Vascular Retinopathy: An Insight Review.

Vascular retinopathy is a pathological change in the retina caused by ocular or systemic vascular diseases that can lead to blurred vision and the risk of blindness. Lycium barbarum polysaccharides (LBPs) are extracted from the fruit of traditional Chinese medicine, L. barbarum. They have strong biological activities, including immune regulation, antioxidation, and neuroprotection, and have been shown to improve vision in numerous studies. At present, there is no systematic literature review of LBPs on vascular retinal prevention and treatment. We review the structural characterization and extraction methods of LBPs, focus on the mechanism and pharmacokinetics of LBPs in improving vascular retinopathy, and discuss the future clinical application and lack of work. LBPs are involved in the regulation of VEGF, Rho/ROCK, PI3K/Akt/mTOR, Nrf2/HO-1, AGEs/RAGE signaling pathways, which can alleviate the occurrence and development of vascular retinal diseases in an inflammatory response, oxidative stress, apoptosis, autophagy, and neuroprotection. LBPs are mainly absorbed by the small intestine and stomach and excreted through urine and feces. Their low bioavailability in vivo has led to the development of novel dosage forms, including multicompartment delivery systems and scaffolds. Data from the literature confirm the medicinal potential of LBPs as a new direction for the prevention and complementary treatment of vascular retinopathy.

Effects of ectomycorrhizal fungi (Suillus variegatus) on the growth, hydraulic function, and non-structural carbohydrates of Pinus tabulaeformis under drought stress.

BACKGROUND: A better understanding of non-structural carbohydrate (NSC) dynamics in trees under drought stress is critical to elucidate the mechanisms underlying forest decline and tree mortality from extended periods of drought. This study aimed to assess the contribution of ectomycorrhizal (ECM) fungus (Suillus variegatus) to hydraulic function and NSC in roots, stems, and leaves of Pinus tabulaeformis subjected to different water deficit intensity. We performed a continuous controlled drought pot experiment from July 10 to September 10, 2019 using P. tabulaeformis seedlings under 80, 40, and 20% of the field moisture capacity that represented the absence of non-drought, moderate drought, and severe drought stress, respectively. RESULTS: Results indicated that S. variegatus decreased the mortality rate and increased height, root biomass, and leaf biomass of P. tabulaeformis seedlings under moderate and severe drought stress. Meanwhile, the photosynthetic rates, stomatal conductance, and transpiration rates of P. tabulaeformis were significantly increased after S. variegatus inoculation. Moreover, the inoculation of S. variegatus also significantly increased the NSC concentrations of all seedling tissues, enhanced the soluble sugars content, and increased the ratios of soluble sugars to starch on all tissues under severe drought. Overall, the inoculation of S. variegatus has great potential for improving the hydraulic function, increasing the NSC storage, and improving the growth of P. tabulaeformis under severe drought. CONCLUSIONS: Therefore, the S. variegatus can be used as a potential application strain for ecological restoration on arid regions of the Loess Plateau, especially in the P. tabulaeformis woodlands.

Lignin as a MALDI matrix for small molecules: a proof of concept.

Driven by the interest in metabolomic studies and the progress of imaging techniques, small molecule analysis is booming, while it remains challenging to be realized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Herein, lignin, the second most abundant biomass in nature, was applied as a dual-ion-mode MALDI matrix for the first time to analyze small molecules. The low ionization efficiency and strong optical absorption properties make lignin a potential MALDI matrix in small molecule analysis. A total of 30 different small molecules were identified qualitatively and six kinds of representative molecules were detected quantitatively with a good linear response (R2 > 0.995). To verify the accuracy of our quantitative method in MALDI, myricitrin, a major bioactive component in Chinese bayberry, was analyzed in different cultivars and tissues. The myricitrin content in real samples detected by MALDI was highly consistent (R2 > 0.999) with that detected by high-performance liquid chromatography, thus indicating the applicability of the lignin matrix. Further characterization by ultraviolet and nuclear magnetic resonance spectroscopy was carried out to explain the possible mechanism of lignin as a matrix and provide more theories for a rational matrix design.

Conformational Selection Mechanism Provides Structural Insights into the Optimization of APC-Asef Inhibitors.

Metastasis is the major cause of death in colorectal cancer and it has been proven that inhibiting an interaction between adenomatous polyposis coli (APC) and Rho guanine nucleotide exchange factor 4 (Asef) efficaciously restrain metastasis. However, current inhibitors cannot achieve a satisfying effect in vivo and need to be optimized. In the present study, we applied molecular dynamics (MD) simulations and extensive analyses to apo and holo APC systems in order to reveal the inhibitor mechanism in detail and provide insights into optimization. MD simulations suggested that apo APC takes on a broad array of conformations and inhibitors stabilize conformation selectively. Representative structures in trajectories show specific APC-ligand interactions, explaining the different binding process. The stability and dynamic properties of systems elucidate the inherent factors of the conformation selection mechanism. Binding free energy analysis quantitatively confirms key interface residues and guide optimization. This study elucidates the conformation selection mechanism in APC-Asef inhibition and provides insights into peptide-based drug design.

Parallel On-Target Derivatization for Mass Calibration and Rapid Profiling of N-Glycans by MALDI-TOF MS.

Glycosylation is an important post-translational modification of proteins, and abnormal glycosylation is involved in a variety of diseases. Accurate and rapid profiling of N-glycans by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is still technically challenging and hampered mainly by mass drift of instrument, manual identification of spectrum peaks, and poor cocrystallization with traditional matrices besides low ionization efficiency of analytes. In the present study, a parallel on-target derivatization strategy (POTDS), on the basis of two rationally combined matrices, i.e., 3-hydrazinobenzoic acid plus DHB (DHB/3HBA) and quinoline-3-carbohydrazide plus DHB (DHB/Q3CH), was proposed for mass calibration and rapid detection of reducing N-glycans. Both DHB/3HBA and DHB/Q3CH show high derivatization efficiency and can improve the ionization efficiency of reducing N-glycans significantly. For mass calibration, in combination with dextrans, DHB/3HBA and DHB/Q3CH prove to be highly sensitive matrices facilitating both MS and MS2 calibration for N-glycans in dual polarities. For rapid identification, the regular mass difference observed for each N-glycan labeled with Q3CH and 3HBA respectively can eliminate the occurrence of false positives and promote automated identification of N-glycans in complex samples. For relative quantitation, the acid-base pair of DHB/Q3CH generates a concentrated cocrystallization of glycan-matrix mixtures at the edge of the droplet uniformly, exhibiting good linearity (R2 > 0.998) and accuracy (RSD ≤ 10%). Furthermore, the established POTDS was successfully utilized to assess N-glycans of serum from HCC patients, revealing potential for biomarker discovery in clinical practice.

Exploring Interactions of Aptamers with Aß40 Amyloid Aggregates and Its Application: Detection of Amyloid Aggregates.

The exhaustive investigating interactions between recognition probes and amyloid aggregates, especially simultaneous recognition events, are challenging and crucial for the design of biosensing probes and further diagnosis of amyloid diseases. In the present work, the interactions of aptamers (Apts) with ß-amyloid (Aß) aggregates were explored thoroughly by single-molecule force spectroscopy (SMFS). Indeed, it was found that the interaction of aptamer1 (Apt1)-amyloid aggregates was different from that of aptamer2 (Apt2)-Aß40 aggregates at the single-molecule level. Especially, the interaction force of Apt1-Aß40 fibril showed a double distinguishing Gaussian fitting. The only unimodal distribution of the force histogram was displayed for the interactions of Apt2-Aß40 oligomer, Apt2-Aß40 fibril, and Apt1-Aß40 oligomer. More intriguingly, two Apts could bind to amyloid aggregates simultaneously. With the assistance of two Apts recognition, a novel sensitive dual Apt-based surface plasmon resonance (SPR) sensor using Au nanoparticles (AuNPs) was developed for quantifying Aß40 aggregates. The dual Apt-based SPR sensor not only avoided the limitation of steric hindrance and epitope but also employed simple operation as well as inexpensive recognition probes. A detection limit as low as 0.2 pM for Aß40 oligomer and 0.05 pM for Aß40 fibril could be achieved. Moreover, the established sensor could be successfully applied to detect Aß40 aggregates in artificial cerebrospinal fluid (CSF) and undiluted real CSF. This work could provide a strategy to monitor a simultaneous recognition event using SMFS and broaden the application of Apts in the diagnosis of neurodegenerative diseases.