Simultaneous quantification of total and free testosterone in human serum by LC-MS/MS.

Testosterone (TTe) and free testosterone (FTe) are clinically important indicators for the diagnosis of androgen disorders, so accurate quantitative determination of them in serum is clinically of paramount significance. Currently, there is no available method suitable for routine and simultaneous measurement of TTe and FTe. Here, we developed a new UPLC-MS/MS method to quantify serum TTe and FTe simultaneously and accurately. Rapid equilibrium dialysis was used to obtain FTe in serum followed by derivatization with hydroxylamine hydrochloride. With these strategies, TTe and FTe could be measured in single injection. After optimizing the extraction and derivatization conditions, the performance of LC-MS/MS was evaluated and applied to quantify the levels of TTe and FTe in clinical samples from 42 patients. The assays were linear for TTe within the range of 0.2-30 ng/mL and for FTe within the range of 1.5-1000 pg/mL. This improved method provided a limit of quantification for TTe of 0.2 ng/mL and for FTe of 1.5 pg/mL. The intra- and inter-run CVs were less than 4.3% and 3.6% for TTe and less than 8.2% and 6.7% for FTe, respectively. The intra- and inter-run accuracies for both TTe and FTe were in the range of 96.1-108.1%. Interference, carryover effect, and matrix effect were in acceptable range. In conclusion, our new LC-MS/MS method is simple to perform and can serve as a reliable method for simultaneous determination of TTe and FTe in clinical practice, providing important information for diagnosis, treatment, and monitoring of androgen-related diseases.

α­thalassemia deletion [­SEA (Southeast Asian)] and a compound heterozygote for the Chinese Gγ+(Aγδß)0/ßCD17­thalassemia mutation: A case report.

In the present study, an α­thalassemia deletion [­SEA (Southeast Asian)] and a compound heterozygote for the Chinese Gγ+(Aγδß)0/ßCD17­thalassemia mutation in a 15­year­old girl was identified by gap­PCR, PCR­reverse dot­blot hybridization and multiplex ligation­dependent probe amplification. Molecular analysis indicated that the proband's father carried a hemoglobin subunit ß (HBB) heterozygous mutation in codon 17 (CD17; c.52A>T), the mother was a double heterozygous carrier of the Chinese Gγ+(Aγδß)0­thalassemia mutation combined with an ­SEA deletion, and the proband inherited both mutations from her mother and father, thus carrying the Chinese Gγ+(Aγδß)0/ßCD17­thalassemia combined with the­SEA deletion in a compound heterozygous state. The proband was diagnosed as severe thalassemia intermedia and experienced a clinical phenotype aggravation (severe anemia and splenomegaly) from no obvious clinical symptoms to being dependent on monthly blood transfusions.

N-glycomic profiling reveals dysregulated N-glycans of peripheral neuropathy in type 2 diabetes.

Given the increasing morbidity of diabetes mellitus type 2 (T2DM) with peripheral neuropathy (PN), efficient screening for T2DM-PN is of great significance. Altered N-glycosylation is closely associated with T2DM progression, whereas its association with T2DM-PN remains uncharacterized. In this study, N-glycomic profiling was performed to identify the N-glycan features between T2DM patients with (n = 39, T2DM-PN) and without PN (n = 36, T2DM-C). Another independent set of T2DM patients (n = 29 for both T2DM-C and T2DM-PN) were utilized to validate these N-glycomic features. There were 10 N-glycans varied significantly between T2DM-C and T2DM-PN (p < 0.05 and 0.7 < AUC < 0.9), of which T2DM-PN was associated with increased oligomannose and core-fucosylation of sialylated glycans, and decreased bisected mono-sialylated glycan. Notably, these results were validated by an independent set of T2DM-C and T2DM-PN. This is the first profiling for N-glycan features in T2DM-PN patients, which reliably differentiates them from T2DM controls, thus providing a prospective profile of glyco-biomarkers for the screening and diagnosis of T2DM-PN.

Flowing on-line preparation of deglycosylation, labeling and purification for N-glycan analysis.

The current in-solution analysis of N-glycans suffers from several disadvantages including tedious de-glycosylation time and multi-step pre-treatment procedures. Here, an ultra-simple flowing on-line analysis of labeled N-glycans for high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed for eliminating the deficiencies. This on-line analysis consisted of an immobilized enzyme reactor (IMER) of PNGase F for efficient release of N-glycans, labeling of released N-glycans and following purification of derivatives on microfluidic chip. Notably, efficient preparations for all type of N-glycans were completed within ∼30 min. To our best knowledge, this is the first time to integrated the whole preparation of N-glycan deglycosylation, labeling and purification only by a simple fluidic flow with our developed device. Good reproducibility and stability were achieved with the relative standard deviation (RSD) less than 10%. Furthermore, the glycome studies with human serum revealed a good adaptability for biological samples. Our work provides an efficient N-glycomic strategy that can be applied to further multilayered clinical analysis.

An Empirical Dilatancy Model for Coarse-Grained Soil under the Influence of Freeze-Thaw Cycles.

In the era of high-speed trains, it is very important to ensure the safety and stability of rail tracks under adverse conditions including seasonal freezing and thawing. Freeze-thaw cycles (FTCs) affecting the engineering performance of coarse-grained soil (CGS) is one of the major reasons for track deterioration. The reported results of a number of static freeze-thaw triaxial tests on the shear behaviour of CGS are analysed herein. It was observed that confining pressure (σ3) and FTCs have a significant influence on the shear behaviour of CGS. In this paper, an empirical mathematical model has been proposed to capture the dilatancy of CGS subjected to FTCs during shearing. The empirical constants a, b, and c proposed in the model are a function of σ3 and FTCs. The results of the model have been compared with the laboratory experiments and are found to be in good agreement.

Sample-Centric Feature Generation for Semi-Supervised Few-Shot Learning.

Semi-supervised few-shot learning aims to improve the model generalization ability by means of both limited labeled data and widely-available unlabeled data. Previous works attempt to model the relations between the few-shot labeled data and extra unlabeled data, by performing a label propagation or pseudo-labeling process using an episodic training strategy. However, the feature distribution represented by the pseudo-labeled data itself is coarse-grained, meaning that there might be a large distribution gap between the pseudo-labeled data and the real query data. To this end, we propose a sample-centric feature generation (SFG) approach for semi-supervised few-shot image classification. Specifically, the few-shot labeled samples from different classes are initially trained to predict pseudo-labels for the potential unlabeled samples. Next, a semi-supervised meta-generator is utilized to produce derivative features centering around each pseudo-labeled sample, enriching the intra-class feature diversity. Meanwhile, the sample-centric generation constrains the generated features to be compact and close to the pseudo-labeled sample, ensuring the inter-class feature discriminability. Further, a reliability assessment (RA) metric is developed to weaken the influence of generated outliers on model learning. Extensive experiments validate the effectiveness of the proposed feature generation approach on challenging one- and few-shot image classification benchmarks.

N-Glycomic profiling reveals dysregulated glycans related to oral cancer using MALDI-MS.

Aberrant protein N-glycosylation is one of the hallmarks of malignancy, while its role in oral cancer (OC) progression is still poorly understood making it a perfect biomarker for early-stage cancer. In this work, we describe a MALDI-MS-based N-glycomic analysis with multivariate data analysis approach for exploration and classification of the patients with OC. Various statistical analyses were used to determine differential N-glycome profiles. There were 8 glycan structures significantly increased (p < 0.05) in all OC-derived  microvesicles (MVs). Importantly, the increases in these glycans were significantly greater in the early stage of OC than those in the advanced stage of OC. Additionally, N-glycomic profiles from the serum were also performed, demonstrating a significant change of 11 glycan structures (p < 0.05). Our data highlighted the effectiveness approach of MALDI-MS-based MV N-glycomic profiling on OC screening, and presented strong potential for further identifying targeted glycoproteomics signatures of the cancer.

Fmoc N-hydroxysuccinimide ester: A facile and multifunctional role in N-glycan analysis.

N-glycans that are fluorescently tagged by glycosylamine acylation have become a promising way for glycan biomarker discovery. Here, we describe a simple and rapid method using Fmoc N-hydroxysuccinimide ester (Fmoc-OSu) to label N-glycans by reacting with their corresponding intermediate glycosylamines produced by microwave-assisted deglycosylation. After optimizing reaction conditions, this derivatization reaction can be effectively achieved under 40 °C for 1 h. Moreover, the comparison of fluorescent intensities for Fmoc-OSu, Fmoc-Cl and 2-AA labeling strategies were also performed. Among which, the fluorescent intensities of Fmoc-OSu labeled glycan derivatives were approximately 5 and 13 times higher than that labeled by Fmoc-Cl and 2-AA respectively. Furthermore, the developed derivatization strategy has also been applied for analyzing serum N-glycans, aiming to screen specific biomarkers for early diagnosis of lung squamous cell cancer. More interestingly, the preparation of free reducing N-glycan standards have been achieved by the combination of HPLC fraction of Fmoc labeled glycan derivatives and Fmoc releasing chemistry. Overall, this proposed method has the potential to be used in functional glycomic study.

Microwave irradiation-assisted high-efficiency N-glycan release using oriented immobilization of PNGase F on magnetic particles.

Peptide-N-glycosidase F (PNGase F) is the most frequently used enzyme to release N-glycan from glycoproteins in glycomics; however, the releasing process using PNGase F is tedious and can range in duration from hours to overnight. Recently, efforts have been made to accelerate this enzymatic reaction, and they include the use of microwave irradiation, ultrahigh pressure, enzyme immobilization, and other techniques. Here, we developed a novel method combining the oriented immobilization of PNGase F on magnetic particles and microwave-assisted enzymatic digestion techniques to achieve highly efficient release of N-glycans. The oriented immobilization of PNGase F on magnetic particles utilizes the affinity of its co-expressed His-tag towards iminodiacetic acid-Nickel modified magnetic particles. Compared with non-oriented immobilization, the oriented immobilization of PNGase F exhibits several advantages including tolerance to high temperature (52 °C) and the ability to retain strong activity after more than five reuses. When used in combination with microwave irradiation, efficient N-glycan removal from ribonuclease B was achieved within 5 min. The proposed strategy was also used to release glycan from fetuin and human serum and has proven to provide a promising deglycosylation method for the characterization of protein glycosylation.

Development of a filter-aided extraction method coupled with glycosylamine labeling to simplify and enhance high performance liquid chromatography-based N-glycan analysis.

Efficient sample pretreatment of N-glycans from glycoproteins is essential but challenging due to the limitations of existing tedious and laborious methods in N-glycomics. This study aimed to establish a filter-aided extraction method coupled with glycosylamine AQC labeling for a simple and rapid direct HPLC-FLD-based analysis of N-glycans. The developed method was demonstrated to be simpler and more sensitive compared to previous HILIC SPE purification method coupled with glycosylamine labeling. It has been validated with wild-type N-glycans from human transferrin and RNase B and then was successfully applied to investigate N-glycan profiles of the transferrin in human serum and a monoclonal antibody (mAb). Results showed good applicability of the method for complex samples. Additionally, this method is compatible with the replicate determination of N-glycan samples to assess the high-throughput analysis of glycan variability in mAb sample.

Bacteriophage T4 capsid as a nanocarrier for Peptide-N-Glycosidase F immobilization through self-assembly.

Enzyme immobilization is widely applied in biocatalysis to improve stability and facilitate recovery and reuse of enzymes. However, high cost of supporting materials and laborious immobilization procedures has limited its industrial application and commercialization. In this study, we report a novel self-assembly immobilization system using bacteriophage T4 capsid as a nanocarrier. The system utilizes the binding sites of the small outer capsid protein, Soc, on the T4 capsid. Enzymes as Soc fusions constructed with regular molecular cloning technology expressed at the appropriate time during phage assembly and self-assembled onto the capsids. The proof of principle experiment was carried out by immobilizing ß-galactosidase, and the system was successfully applied to the immobilization of an important glycomics enzyme, Peptide-N-Glycosidase F. Production of Peptide-N-Glycosidase F and simultaneous immobilization was finished within seven hours. Characterizations of the immobilized Peptide-N-Glycosidase F indicated high retention of activity and well reserved deglycosylation capacity. The immobilized Peptide-N-Glycosidase F was easily recycled by centrifugation and exhibited good stability that sustained five repeated uses. This novel system uses the self-amplified T4 capsid as the nanoparticle-type of supporting material, and operates with a self-assembly procedure, making it a simple and low-cost enzyme immobilization technology with promising application potentials.

Circulating Plasma miRNAs as Potential Biomarkers of Non-Small Cell Lung Cancer Obtained by High-Throughput Real-Time PCR Profiling.

BACKGROUND: Because of limited stability and sensitivity, circulating miRNAs as noninvasive biomarkers have not so far been used for early diagnosis and prognosis of non-small cell lung cancer (NSCLC) in clinic. Therefore, it is imperative to find more reliable biomarker(s). METHODS: We performed one of most sensitive qRT-PCR assays, S-Poly(T) Plus, to select differently expressed miRNAs from genome-wide miRNA profiling. miRNA candidates were validated through a three-phase selection and two validation processes with 437 NSCLC cases and 415 controls. RESULTS: A unique set of 7 and 9 miRNAs differed significantly in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) samples compared with those in controls, of which, there were 5 universal biomarkers for NSCLC (ADC or SCC). Ten of 11 miRNAs could discriminate early stage (stage I) of NSCLC from healthy individuals. Risk score was obtained from the validation set-1 and was tested using the ROC curves with a high area under ROC curve of 0.89 in ADC and 0.96 in SCC. Ultimately, potential biomarkers and the risk score were verified by the validation set-2 with a sensitivity of 94% and a specificity of 91.6% in ADC, and a sensitivity of 98.5% and a specificity of 51.5% in SCC, respectively. CONCLUSIONS: Taken together, 7 miRNAs and 9 miRNAs may provide noninvasive biomarkers for diagnosis and prognosis in ADC and SCC, respectively. IMPACT: On the basis of our sensitive and accurate method, we hope that these candidate miRNAs may have strong impact on the early lung cancer diagnosis.

Cellulose microspheres-filled pipet tips for purification and enrichment of glycans and glycopeptides.

The pretreatment of low-abundance glycans/glycopeptides prior to mass spectrometry (MS) analysis is of great significance for glycosylation identification of proteins. Herein, a simple one-step purification and enrichment procedure of glycans and glycopeptides was achieved using cellulose microspheres-filled pipet tips (CM-PTs). CM-PTs were fabricated by conveniently filling cellulose microspheres synthesized by sol-gel transition method into 100 µL pipet tips. The whole enrichment and purification process were completed within two minutes by pipetting up and down for dozens of times (30-40), followed by the direct analysis of MALDI TOF-MS. Upon the assessment of model glycoproteins, including human immunoglobulin G (IgG) and horse radish peroxidase (HRP), the CM-PT method exhibited good selectivity (IgG/BSA = 1:100), high sensitivity (1 ng µL-1 of IgG), large binding capacity (125 mg g-1, IgG/cellulose microspheres) and excellent reproducibility in glycopeptide enrichment. The capability for purifying glycans of CM-PTs was also investigated, presenting high binding capacity (8 mg g-1, maltoheptaose/cellulose microspheres) and good recoveries (81.3-89.8%). The developed in-pipet-tip system shows a great potential in efficient and high-throughput analysis of protein glycosylation.

Isomer-specific profiling of N-glycans derived from human serum for potential biomarker discovery in pancreatic cancer.

Glycosylation is one of the most important post-translational modifications of protein. Recently, global profiling of human serum glycomics has become a noninvasive method for cancer-related biomarker discovery and many studies have focused on compositional glycan profiling. In contrast, structure-specific glycan profiling may provide more potential biomarkers with higher specificity than compositional profiling. In this work, N-glycans released from human serum were neutralized with methylamine and reduced by ammonia-borane complex prior to profiling using nanoLC-ESI-MS with porous graphitized carbon (PGC) and relative abundances of over 280 isomers were compared between pancreatic cancer (PC) cases (n = 32) and healthy controls (n = 32). Statistical analysis identified 25 specific-isomeric biomarkers with significant differences (p-value < 0.05). ROC and PCA analysis were performed to assess the potential biomarkers which were identified as being significantly altered in cancer. The AUCs of the significantly changed specific-isomers were ranging from 0.712 to 0.949. In addition, with the combination of all potential biomarkers, a higher AUC of 0.976 with sensitivity (93.5%) and specificity (90.6%) was obtained. Overall, the proposed strategy coupled to relative quantitative analysis of isomeric glycans make it possible to discover new biomarkers for the diagnosis of PC. SIGNIFICANCE: Pancreatic cancer (PC) has a poor prognosis with a five-year survival rate <5%. Therefore, a strategy for accurate diagnosis of PC is indeed required. In this paper, a dual-derivatized strategy for structure-specific glycan profiling has been used and according to our best knowledge, this is the first application of this strategy for PC biomarker discovery, in which the separation, identification and relative quantification of isomeric glycans can be simultaneously obtained. In addition, by in-depth analysis of isomeric glycans, the full description of the stereo- and region- diversity of glycans can also be achieved, which might provide more potential information for PC biomarker discovery.

Determination of N-glycans by high performance liquid chromatography using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as the glycosylamine labeling reagent.

Robust, efficient identification and accurate quantification of N-glycans are of great significance in N-glycomics analysis. Here, a simple and rapid derivatization method, based on the combination of microwave-assisted deglycosylation and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) labeling, was developed for the analysis of N-glycan by high performance liquid chromatography with fluorescence detection (HPLC-FLD). After optimizing various parameters affecting deglycosylation and derivatization by RNase B, the time for N-glycan labeling was shortened to 50 min with ∼10-fold enhancement in detection sensitivity comparing to conventional 2-aminobenzoic acid (2-AA) labeling method. Additionally, the method showed good linearity (correlation coefficients > 0.991) and reproducibility (RSD < 8.7%). These advantages of the proposed method were further validated by the analysis of complex samples, including fetuin and human serum. Investigation of serum N-glycome for preliminary diagnosis of human lung cancer was conducted, where significant changes of several N-glycans corresponding to core-fucosylated, mono- and disialylated glycans have been evidenced by a series of statistical analysis.

Relative quantitation of neutral and sialylated N-glycans using stable isotopic labeled d0/d5-benzoyl chloride by MALDI-MS.

Quantitative analysis of glycans is an emerging field in glycomic research. Herein we present a rapid and effective dual-labeling strategy, in the combination of isotopic derivatization of N-glycosylamine-based glycans by d0/d5-benzoyl chloride and methylamidation of sialic acids, to relatively quantify both neutral and sialylated N-glycans simultaneously by MALDI-MS. The derivatization efficiencies were increased by microwave-accelerated deglycosylation which not only largely reduce the time of glycoprotein deglycosylation but also inhibit the hydrolysis of intermediate glycosylamines produced by PNGase F digestion. Three model glycoproteins, including RNase B, bovine fetuin and IgG from human serum, were applied to validate this technique. Results showed that the glycans from microgram level of glycoprotein can be successfully quantified with high reproducibility and the whole time of analytical procedure was shortened to 4 h. Furthermore, this proposed method was applied for the comparative analysis of N-glycans from serum of healthy donors and multiple myeloma patients. It was found that five N-glycans may be as the potential biomarkers for rapid detection of early multiple myeloma, indicating the feasibility of this strategy in monitoring subtle quantitative differences of serum glycomics.

Microwave-assisted deglycosylation for rapid and sensitive analysis of N-glycans via glycosylamine derivatization.

A rapid and sensitive N-glycan profiling strategy for MALDI-MS incorporating the use of deglycosylation with microwave assistance and the co-derivatization of glycosylamine labeling with tris(2,4,6-trimethoxyphenyl)phosphonium acetic acid N-hydroxysuccinimide ester (TMPP-Ac-OSu) and methylamidation has been developed in this work. Notably, highly efficient release and tagging of N-glycans from ribonuclease B was achieved in less than 90 min, providing up to 35-fold enhancement of MALDI-MS sensitivity with comparison to underivatized N-glycans. After further validation with other two standard glycoproteins (ovalbumin and bovine fetuin), the proposed strategy was applied to human serum for preliminary pathological analysis of N-glycans between healthy and lung cancer individuals. As a result, significant differences (T test p value <0.01) of 6 glycan structures were determined from 54 detected N-glycan structures with only 50 nL of loading amount and further confirmed through PCA and ROC (AUC) analyses between two sample sets. Subsequently, the trend of each lung cancer stage and controls in expression of the selected glycans was implemented with T test and box-plots. Accordingly, these structures can be used as potential lung cancer glycan-based biomarkers and for further definition of cancer progression highlighting the ability of proposed method to rapidly and efficiently analyze N-glycome present in human serum. Graphical abstract MALDI-TOF MS analysis of N-glycans by microwave-assisted deglycosylation and glycosylamine derivatization.

Identification of reference genes for circulating microRNA analysis in colorectal cancer.

Quantitative real-time PCR (qPCR) is the most frequently used method for measuring expression levels of microRNAs (miRNAs), which is based on normalization to endogenous references. Although circulating miRNAs have been regarded as potential non-invasive biomarker of disease, no study has been performed so far on reference miRNAs for normalization in colorectal cancer. In this study we tried to identify optimal reference miRNAs for qPCR analysis across colorectal cancer patients and healthy individuals. 485 blood-derived miRNAs were profiled in serum sample pools of both colorectal cancer and healthy control. Seven candidate miRNAs chosen from profiling results as well as three previous reported reference miRNAs were validated using qPCR in 30 colorectal cancer patients and 30 healthy individuals, and thereafter analyzed by statistical algorithms BestKeeper, geNorm and NormFinder. Taken together, hsa-miR-93-5p, hsa-miR-25-3p and hsa-miR-106b-5p were recommended as a set of suitable reference genes. More interestingly, the three miRNAs validated from 485 miRNAs are derived from a single primary transcript, indicting the cluster may be highly conserved in colorectal cancer. However, all three miRNAs differed significantly between healthy individuals and non-small cell lung cancer or breast cancer patients and could not be used as reference genes in the two types of cancer.

Profiling of differentially expressed microRNAs in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a kind of primary cardiomyopathy characterized by the fibro-fatty replacement of right ventricular myocardium. Currently, myocardial microRNAs have been reported to play critical role in the pathophysiology of cardiovascular pathophysiology. So far, the profiling of microRNAs in ARVC has not been described. In this study, we applied S-Poly (T) Plus method to investigate the expression profile of microRNAs in 24 ARVC patients heart samples. The tissue levels of 1078 human microRNAs were assessed and were compared with levels in a group of 24 healthy controls. Analysis of the area under the receiver operating characteristic curve (ROC) supported the 21 validated microRNAs to be miRNA signatures of ARVC, eleven microRNAs were significantly increased in ARVC heart tissues and ten microRNAs were significantly decreased. After functional enrichment analysis, miR-21-5p and miR-135b were correlated with Wnt and Hippo pathway, which might involve in the molecular pathophysiology of ARVC. Overall, our data suggested that myocardial microRNAs were involved in the pathophysiology of ARVC, miR-21-5p and miR-135b were significantly associated with both the myocardium adipose and fibrosis, which was a potential disease pathway for ARVC and might to be useful as therapeutic targets for ARVC.

An improved method for detecting circulating microRNAs with S-Poly(T) Plus real-time PCR.

We herein describe a simple, sensitive and specific method for analysis of circulating microRNAs (miRNA), termed S-Poly(T) Plus real-time PCR assay. This new method is based on our previously developed S-Poly(T) method, in which a unique S-Poly(T) primer is used during reverse-transcription to increase sensitivity and specificity. Further increased sensitivity and simplicity of S-Poly(T) Plus, in comparison with the S-Poly(T) method, were achieved by a single-step, multiple-stage reaction, where RNAs were polyadenylated and reverse-transcribed at the same time. The sensitivity of circulating miRNA detection was further improved by a modified method of total RNA isolation from serum/plasma, S/P miRsol, in which glycogen was used to increase the RNA yield. We validated our methods by quantifying miRNA expression profiles in the sera of the patients with pulmonary arterial hypertension associated with congenital heart disease. In conclusion, we developed a simple, sensitive, and specific method for detecting circulating miRNAs that allows the measurement of 266 miRNAs from 100 µl of serum or plasma. This method presents a promising tool for basic miRNA research and clinical diagnosis of human diseases based on miRNA biomarkers.