Increasing the Assembly Efficacy of Peptidic ß-Sheets for a Highly-Sensitive HIV Detection.

Our recent publication illustrates the critical role of phenylalanine-mediated aromatic-aromatic interactions in determining the assembly of peptidic ß-sheets. However, the effect of phenylalanine number on regulating the assembly efficacy of peptidic ß-sheets remains poorly understood. We herein evaluate the assembly efficacy of ß-sheets of a series of oligopeptides which contain 0, 1, 2, or 3 phenylalanine in their molecular backbones. In our assembly system, two phenylalanine (2F) is the minimum number for driving the assembly of ß-sheets of oligopeptides. Oligopeptides with three phenylalanine (3F) show significantly increased assembly efficacy of ß-sheets compared to that with 2F. These results suggest a positive correlation between the phenylalanine number and assembly efficacy of ß-sheets. By improving the assembly efficacy of ß-sheets, we further develop a highly sensitive HIV analytical system in which the specific binding of ß-sheets with Congo Red induces enhanced fluorescence. For HIV p24 detection, the 3F-based analytical system (0.61 pg/mL) shows a significantly lower limit of detection (LOD) than the 2F-based analytical system (2.44 pg/mL), both of which are more sensitive than commercial ELISA (5 pg/mL) used in the clinic. This work not only illustrates the effect of phenylalanine number on regulating the assembly efficacy of ß-sheets but also provides a guideline for the construction of a highly sensitive analytical system of disease diagnosis.

Molecular Design of ß-Sheet Peptide for the Multi-Modal Analysis of Disease.

Intermolecular forces constrain peptide conformation. However, the role of each intermolecular force in constraining peptide conformation remains poorly understood. In this work, we show that aromatic-aromatic interactions drive peptides into ß-sheets, and the hydrophobic effect determines the assembly speed of peptides. By using intermolecular forces to artificially control the assembly of ß-sheets, a multi-modal analytical system was developed that allows five readouts and dual qualitative-quantitative analysis, and satisfies both point-of-care testing (POCT) and laboratory-based testing. For Mycoplasma Pneumoniae diagnosis, this system eradicates misdiagnosis (from 30 % to 0 %) and broadens the linear range by three-fold, both of which are critical for guiding therapy. This work not only illustrates exact roles of intermolecular forces in driving the formation of ß-sheets, but also provides a guideline for the construction of a multi-modal analytical system for disease diagnosis.

Comparison between a new platelet count drop method PL-11, light transmission aggregometry, VerifyNow aspirin system and thromboelastography for monitoring short-term aspirin effects in healthy individuals.

Platelet function has been described by many laboratory assays, and PL-11 is a new point-of-care platelet function analyzer based on platelet count drop method, which counts platelet before and after the addition of agonists in the citrated whole blood samples. The present study sought to compare PL-11 with other three major more established assays, light transmission aggregometry (LTA), VerifyNow™ aspirin system and thromboelastography (TEG), for monitoring the short-term aspirin responses in healthy individuals. Ten healthy young men took 100 mg/d aspirin for 3-day treatment. Platelet function was measured via PL-11, LTA, VerifyNow and TEG, respectively. The blood samples were collected at baseline, 2 hour, 1 day during the aspirin treatment and 1 day, 5 ± 1 days, 8 ± 1 days after the aspirin withdrawal. Moreover, 90 additional healthy subjects were recruited to establish a reference range for PL-11. Platelet function of healthy subjects decreased significantly 2 hours after 100 mg/d aspirin intake and began to recover during 4-6 days after the aspirin withdrawal. Correlations between methods were PL-11 vs. LTA (r = 0.614, p < 0.01); PL-11 vs. VerifyNow (r = 0.829, p < 0.01); PL-11 vs. TEG (r = 0.697, p < 0.001). There was no significant bias between PL-11 and LTA at baseline (bias = 1.94%, p = 0.804) using Bland-Altman analysis, while the data of PL-11 were significantly higher than LTA (bias = 24.02%, p < 0.001) during the aspirin therapy. The reference range for PL-11 in healthy young individuals was from 66.8 to 90.5% (95%CI). When aspirin low-responsiveness was defined as LTA > 20%, the cut-off values for each method were, respectively: PL-11 > 50%, VerifyNow > 533 ARU, TEG > 60.2%. The results of different platelet function assays were uninterchangeable for monitoring aspirin response and correlations among them were also varied. Correlations among PL-11 and other three major assays suggested the ability of PL-11 to assess the treatment effects of aspirin. But a large cohort study is needed to confirm the cut-off value of aspirin response detected by PL-11.

[Clinical significance of new urine red blood cell parameter in different kinds of glomerulonephritis].

OBJECTIVE: To investigate the clinical value of new kinds of urinary erythrocyte morphology parameter in discriminating different pathology types of glomerulonephritis. METHODS: All of the 52 urine samples were from glomerulonephritis patients who had been diagnosed by renal biopsy results. The change of the percentage of acanthocytes, the size of RBC, the shape of RBC between the primary glomerulonephritis (39 cases) and secondary glomerulonephritis (13 cases) urine were detected by AVE-764 fully automatic urine cell analyzer. RESULTS: Acanthocytes could be found in both primary glomerulonephritis and secondary glomerulonephritis. Of the patients whose acanthocytes percentages above 10%, 94.1% had primary glomerulonephritis and 5.9% had secondary glomerulonephritis. The picture of size-shape phase were classified as strip-type, inverted triangle-type and hanging tail-type. 95.2% Strip-type cases were from primary glomerulonephritis patients. Triangle-typenormally cases were all from primary glomerulonephritis patients. Hanging tail-type cases were all from secondary glomerulonephritis. CONCLUSION: High acanthocytes percentage is most common in primary glomerulonephritis, going with the size and shape of RBC can be useful in the differential diagnosis of different pathology types of glomerulonephritis.

The effects of HIV Tat DNA on regulating the immune response of HIV DNA vaccine in mice.

BACKGROUND: HIV trans-activator protein (Tat) is the crucial factor to control HIV transcription, and is usually considered as an important immunogen for the design of HIV vaccine. Recent studies reported some special bio-activities of Tat protein on immunoregulation. However, to date, few studies have focused on exploring the effects of Tat expression plasmid (pTat) on regulating the immune responses induced by HIV DNA vaccines. In this study, our main objective is to investigate the immunoregulation mediated by pTat in mice. METHODS: Four gene-coding plasmids (pTat, pGag, pEnv and pPol) were constructed, and the gene expression was detected by western blot method. The effects of pTat on regulating the immune responses to antigens Gag, Env, Pol were assessed by enzyme-linked immunospot and enzyme-linked immunosorbent assay. The data was analysed by one-way analysis of variance. RESULTS: After two immunizations, mice vaccinated with antigen expressing plasmid (pGag, pEnv or pPol) plus pTat exhibited significantly stronger IFN-gamma response than that vaccinated with the corresponding antigen alone. Moreover, mice receiving two injections of antigen plus pTat exhibited the same strong IFN-gamma response as those receiving three injections of antigen alone did. Furthermore, addition of pTat not only induced a more balanced Th1 and Th2 response, but also broadened IgG subclass responses to antigens Gag and Pol. CONCLUSION: pTat exhibited the appreciable effects on modulating immune responses to HIV antigens Gag, Env and Pol, providing us interesting clues on how to optimize HIV DNA vaccine.

Application and development of review criteria on Sysmex XT-2100 in Chinese at a large third-grade class-A hospital.

BACKGROUND: Hematology analysis is an essential component of patient assessment and used in screening, diagnosis, and the planning of care. The objective of the study was to find out the suitable hematology review criteria for large scale general hospitals in China via the analysis of experimental data from Sysmex XE-2100 hematology analyzer. METHODS: A total 1486 blood samples were detected with the Sysmes XE-2100. Based on hematology review criteria suggested by international consensus group and a positive smear finding new optimal review rules were determined. RESULTS: With the International Article 41 Review Rules, the true positive ratio (TP), the false positive ratio (FP), the true negative ratio (TN), and the false negative ratio (FN) was 14.0% (208/1486), 31.49% (468/1486) 52.42% (779/1486), and 2.09% (31/1486), respectively. With the help of Laboman 4.2 software (the Sysmex Corporation), 19 rules for review of automated CBC and WBC differential were set up. With our review rules, the TP, FP, TN, and FN was 13.86% (206/1486), 25.17% (374/1486), 58.75% (873/1486) and 2.22% (33/1486), respectively. The review rules were validated, the FN was 0.96%, blasts and immature cells were not omitted. CONCLUSIONS: The review criteria can be developed in light of the rules of the International Consensus Group for Hematology Review, but should be improved depending on different laboratory's requirements.

Clinical laboratory urine analysis: comparison of the UriSed automated microscopic analyzer and the manual microscopy.

BACKGROUND: Several automated urine sediment analyzers have been introduced to clinical laboratories. Automated microscopic pattern recognition is a new technique for urine particle analysis. We evaluated the analytical and diagnostic performance of the UriSed automated microscopic analyzer and compared with manual microscopy for urine sediment analysis. METHODS: Precision, linearity, carry-over, and method comparison were carried out. A total of 600 urine samples sent for urinalysis were assessed using the UriSed automated microscopic analyzer and manual microscopy. RESULTS: Within-run and between-run precision of the UriSed for red blood cells (RBC) and white blood cells (WBC) were acceptable at all levels (CV < 20%). Within-run and between-run imprecision of the UriSed testing for cast, squamous epithelial cells (EPI), and bacteria (BAC) were good at middle level and high level (CV < 20%). The linearity analysis revealed substantial agreement between the measured value and the theoretical value of the UriSed for RBC, WBC, cast, EPI, and BAC (r > 0.95). There was no carry-over. RBC, WBC, and squamous epithelial cells with sensitivities and specificities were more than 80% in this study. CONCLUSIONS: There is substantial agreement between the UriSed automated microscopic analyzer and the manual microscopy methods. The UriSed provides for a rapid turnaround time.

Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateral meristem.

BACKGROUND: Somatic embryogenesis is a major process for plant regeneration. However, cell communication and the gene regulatory network responsible for cell reprogramming during somatic embryogenesis are still largely unclear. Recent advances in single-cell technologies enable us to explore the mechanism of plant regeneration at single-cell resolution. RESULTS: We generate a high-resolution single-cell transcriptomic landscape of hypocotyl tissue from the highly regenerable cotton genotype Jin668 and the recalcitrant TM-1. We identify nine putative cell clusters and 23 cluster-specific marker genes for both cultivars. We find that the primary vascular cell is the major cell type that undergoes cell fate transition in response to external stimulation. Further developmental trajectory and gene regulatory network analysis of these cell clusters reveals that a total of 41 hormone response-related genes, including LAX2, LAX1, and LOX3, exhibit different expression patterns in the primary xylem and cambium region of Jin668 and TM-1. We also identify novel genes, including CSEF, PIS1, AFB2, ATHB2, PLC2, and PLT3, that are involved in regeneration. We demonstrate that LAX2, LAX1 and LOX3 play important roles in callus proliferation and plant regeneration by CRISPR/Cas9 editing and overexpression assay. CONCLUSIONS: This study provides novel insights on the role of the regulatory network in cell fate transition and reprogramming during plant regeneration driven by somatic embryogenesis.

Development of microscopic review criteria by comparison urine flow cytometer, strip and manual microscopic examination.

BACKGROUND: Microscopic examination is essential for urine analysis, but a time-consuming procedure. This study was undertaken to evaluate an automated urinalysis system - the Sysmex UF-1000i (URISYS 2400) for the analysis of urine constituents including chemistry components and particles. The objective was to screen urine samples and determine the screening criteria which would minimize the number of specimens reviewed with the microscope yet ensuring correct results. METHODS: A total of 1300 urine samples were sent for urinalysis using the automated system and compared with results obtained from manual microscopy using the Fuchs-Rosenthal counting chamber. RESULTS: Using Pearson statistics, we observed correlation between the UF-1000i and manual microscopy: for red blood cells (RBCs) r was 0.949, for white blood cells (WBCs) r was 0.882, for epithelial cells (EC) r was less than 0.76, for casts r was less than 0.7, while correlation between the URISYS 2400 and manual microscopy: for red blood cells r was 0.772 and for white blood cells r was 0.771. With the help of Uriaccess (an expert system provided by the Sysmex Corporation), 37 rules for microscopic review were set up. The review rules were validated, the review rate was less than 30% and the false-positive and false-negative results were acceptably low. CONCLUSIONS: UF-1000i is capable of reproducible measurement of urine particles within the clinically relevant range and shows its advantage over URISYS 2400. It is an optimal strategy for urine sample screening using the combination of the two methods.

[Changing characteristic of blood coagulation factors and their correlation with blood coagulation status in different hepatic diseases].

OBJECTIVE: To investigate the correlation between pro coagulation factors and anti-coagulation factors synthesized by the liver, and the correlation between fibrin degradation products (FDP) and D-dimer (D-D) concentration and coagulation proteins synthesized by extra-hepatic tissues, in different liver diseases; to explore the relationship between coagulation and bleeding in hepatic diseases. METHODS: Chronic hepatitis B (CHB) patients, CHB-related liver cirrhosis patients, CHB-related liver failure patients and healthy (normal) controls were selected for study and provided blood samples for analysis. The activity of coagulation factors (F) II, V, VII, VIII, IX, X, XI, and XII was detected using the one-stage clotting method. Coagulogram analysis, including activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT), was conducted by the solidification method. Antithrombin III (AT-III) and protein C (PC) activities were measured by chromogenic substrate assay. FDP concentration was detected using immunoturbidimetry. Tissue factor pathway inhibitor (TFPI), thrombomodulin (TM), von Willebrand factor (vWF), and tissue factor (TF) concentrations were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: With the exception of FVIII, coagulation factors and anticoagulant proteins synthesized by the liver were decreased and the coagulogram was extended for all patients. Likewise, the FDP and D-D concentrations were increased in blood. CHB patients, however, presented with increased levels of FVIII, TFPI, TM, vWF, and TF. Pairwise comparison indicated statistical differences existed among CHB, CHB-related liver cirrhosis, and liver failure patients: TFPI: 239.3+/-206.4, 315.0+/-258.6, and 319.5+/-298.1 -- higher than normal control: 104.0+/-87.1, F = 5.453, P less than 0.05; vWF: 70.3+/-29.5, 105.5+/-58.0, and 179.3+/-61.7 -- higher than normal control: 21.9+/-7.2, F = 20.104, P less than 0.05; TF: 85.9+/-85.7, 234.2+/-202.9, and 344.7+/-214.6 -- higher than normal control: 12.8+/-8.1, F = 8.619, P less than 0.05; FVIII: 157.2+/-53.4, 206.9+/-86.9, and 335.7+/-117.7 -- higher than normal control: 105.5+/-46.2, F = 13.418, P less than 0.05. CONCLUSION: In parallel to the progression of liver diseases, pro coagulation and anti-coagulation elements synthesized by the liver were reduced. In contrast, fibrinolysis activity was enhanced, which is expected to lead to an imbalance between blood clotting and anti-clotting factors. This may be an important cause for the bleeding that occurs in end-stage liver disease. Expressions of TFPI, TM, vWF, and TF significantly change in the early stage of liver diseases, as compared to normal (healthy) levels, and may represent a sensitive indicator of vascular injury.

Two dimensional nanosheets as immunoregulator improve HIV vaccine efficacy.

Two-dimensional (2D) nanosheets as carriers have shown promising potential for surface-displaying or loading various drugs. Nevertheless, developing sheet-like materials themselves into an immunoregulator has never been realized so far. In this study, we take advantage of the immunoregulatory effects of rare earth elements themselves and develop water-soluble erbium-dysprosium 2D nanosheets (2D NSs). Such 2D NSs can target lymph nodes and activate macrophages to improve vaccine efficacy in mice significantly. Transcriptome analysis further reveals that six critical molecules (Msr1, Ccr2, Serpinb9, Klrk1, Klrd1, Klrc1) closely correlate with 2D NS-mediated immunoregulation in vivo. For the first time, the present work realizes a proof-of-concept for designing immunoregulatory 2D NSs and shows a promising potential of 2D NSs for improving the immunoprophylaxis/immunotherapy of vaccines.

Erythropoiesis changes with increasing age in the elderly Chinese.

INTRODUCTION: Erythropoiesis slowly decreases with increasing age, which may be reflected in red blood cell (RBC) parameters. This multicentre collaborative study aimed to investigate the changes in erythropoiesis with increasing age in a healthy Chinese population. METHODS: A total of 14,591 healthy individuals (6,713 aged at least 60 y and 7,878 aged below 60 y) from seven cities across China were enrolled. K2-EDTA anticoagulant blood samples were analysed. The results are presented as median and 2.5-97.5th percentile. RESULTS: RBC parameters showed some differences between the two groups divided by the age of 60 in the Chinese population. The median, 2.5th and 97.5th percentile values of RBC, haemoglobin (HGB) and haematocrit (HCT) in patients aged ≥ 60 y were significantly lower than in those ˂ 60 y. The values of mean cell volume (MCV), mean cell haemoglobin (MCH) and red cell distribution width (RDW) were higher in the group aged ≥ 60 y. Men had significantly higher RBC, HGB, HCT, MCV, MCH and RDW indices than women. The prevalence of anaemia gradually increased with age in men and was higher than that in women after 50. The median haemoglobin and MCV in Nanning and Guangzhou were lower than those in other regions. CONCLUSION: RBC parameters varied with increasing age and differed between males and females, indicating that erythropoiesis decreases in the elderly Chinese population. Subsequent studies should be conducted for age- and sex-specific reference intervals in healthy elderly Chinese populations.

Protein analysis of extracellular vesicles to monitor and predict therapeutic response in metastatic breast cancer.

Molecular profiling of circulating extracellular vesicles (EVs) provides a promising noninvasive means to diagnose, monitor, and predict the course of metastatic breast cancer (MBC). However, the analysis of EV protein markers has been confounded by the presence of soluble protein counterparts in peripheral blood. Here we use a rapid, sensitive, and low-cost thermophoretic aptasensor (TAS) to profile cancer-associated protein profiles of plasma EVs without the interference of soluble proteins. We show that the EV signature (a weighted sum of eight EV protein markers) has a high accuracy (91.1 %) for discrimination of MBC, non-metastatic breast cancer (NMBC), and healthy donors (HD). For MBC patients undergoing therapies, the EV signature can accurately monitor the treatment response across the training, validation, and prospective cohorts, and serve as an independent prognostic factor for progression free survival in MBC patients. Together, this work highlights the potential clinical utility of EVs in management of MBC.

Assessment of validity of INR system for patients with liver disease associated with viral hepatitis.

International Normalized Ratio (INR), which standardizes prothrombin time (PT) during oral anticoagulation, has been extended to standardize PT in liver diseases and is included in all prognostic models of survival, the classification of CHILD-Pugh or Meld. However, the mechanisms of PT prolongation in liver diseases differ from those involved in oral anticoagulation. Our aim was to assess the validity of the INR system for patients with liver disease associated with viral hepatitis. We prospectively collected blood samples from 61 patients with liver disease associated with viral hepatitis; control patients were on warfarin (n = 20). PTs were measured on a STA-R coagulometer with six thromboplastin reagents, and INRs were calculated using instrument-specific ISIs. Simultaneously, we selected 15 pairs of patients in the study population and in the control population such that INR values for each patient pair are almost equal. For these 15 pairs of patients, we performed factor assays and measured the coagulant activities of factors II, V, VI, and X and fibrinogen. Analysis of results for the control population confirms the validity of the INR system for patients on oral anticoagulants in that there was no significant difference between the reported INRs for the six different thromboplastin reagents. Conversely, for the study population, there was a significant difference between the INR results using the different reagents. Results for fibrinogen and factors V, VII, and X showed significant differences between the two groups; however, control and patient results for factor II were not statistically different. The INR system is not valid for comparison of patients with liver disease associated with viral hepatitis because different reagents do not yield the same INR for the same sample.

[Effects of extracellular calcium concentration on platelets aggregation, coagulation indices and thromboelastography].

OBJECTIVE: To detect the effect of extracellular Ca2+ concentrations on test results of coagulation-related parameters. METHODS: Blood samples of outpatient medical volunteers were collected and then different doses of calcium chloride added. The rate of platelet aggregation (n = 42), prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (APTT) (n = 21) and parameters of thromboelastography (n = 30) were detected according to the standard protocols by plasma turbidimetry, coagulation and recalcification respectively. RESULTS: When the plasma Ca2+ concentration was in the range of 0.1 - 33.7 mmol/L, the rate of platelet aggregation gradually increased with a increasing concentration of Ca2+. And the rates induced by adenosine diphosphate (ADP) and arachidonic acid (AA) were (51.8 +/- 9.6)% - (94.7 +/- 4.8)% and (64.4 +/- 12.2)% - (93.2 +/- 5.5)% respectively. When the Ca2+ concentration was 39.0 mmol/L, the rate decreased markedly [ADP (9.1 +/- 5.3)%, AA (11.1 +/- 4.5)%, both P < 0.01]. When the Ca2+ concentration was in the range of 0.1 - 33.7 mmol/L, the values of PT gradually increased with a increasing concentration of Ca2+. The values of TT changed in "V"-type and became minimum when the calcium concentration was 4.4 mmol/L. The values of APTT decreased with higher calcium concentrations and could not be determined when the concentration increased above 0.5 mmol/L. When the Ca2+ concentration was in the range of 0.4 - 27.3 mmol/L, the values of reaction time and coagulation time of thromboelastography changed in "V"-type and became nearly minimal at the Ca2+ concentration of about 2.1 mmol/L. The values of alpha angle and maximum amplitude changed in "V"-type and became maximal at the Ca2+ concentration of 2.1 mmol/L. CONCLUSIONS: The effect of Ca2+ concentration on the testing results of coagulation-related parameters is significant. A high calcium ( > or = 39 mmol/L) can inhibit the platelet aggregation, coagulation factor activity and blood coagulation. The Ca2+ concentration of 2.1 mmol/L seems to be the optimal concentration for thromboelastography by recalcification method.

[Identification of asthenozoospermia-associated proteins in human seminal plasma by shotgun proteomic strategy].

OBJECTIVE: To identify asthenozoospermia-associated proteins in seminal plasma by the shotgun proteomic strategy. METHODS: Six seminal plasma samples were collected by Percoll respectively from healthy fertile and asthenozoospermia volunteers, balanced, mixed, and then the mixture was separated by SDS-PAGE. The proteins in the gel were enzymolyzed, extracted and identified by the shotgun proteomic strategy. The identified proteins with the unique peptide count > or =2 or the unique peptide count=1 but the total count > or =4 were compared between the two groups. RESULTS: A total of 172 differential proteins were identified, of which, 89 were exclusively from the asthenozoospermia and 83 exclusively from the healthy fertile men. According to the molecular function, these differential proteins were mainly the types of signal transduction and catalytic activity. CONCLUSION: Functionally, 10 of the proteins are particularly important, which include annexin VI isoform 2, isoform 1 of interleukin-6 receptor subunit beta precursor, Mr 400,000 protein, cytosolic dynein heavy chain, alpha-actinin-4, receptor-type tyrosine-protein phosphatase eta precursor, vitamin D-binding protein precursor, protein S100-A11, protein S100-A9 and ANXA4.

[Identification of differential proteins in the seminal plasma of healthy fertile and non-obstructive azoospermia men by shotgun proteomic strategy].

OBJECTIVE: To identify differential proteins in the seminal plasma of healthy fertile men and non-obstructive azoospermia patients by the shotgun proteomic strategy. METHODS: Six seminal plasma samples from 3 healthy fertile and 3 non-obstructive azoospermia volunteers were collected by Percoll isolation, balanced-mixed, and followed by separation of the mixture by SDS-PAGE. The proteins were subjected to in-gel enzymolysis and isolation of peptide fragments, and then identified by the shotgun proteomic strategy. Then comparative analyses were made between the two groups on the identified proteins with the unique peptide count > or = 2 and = 1 but with the peptide count > or = 4. RESULTS: A total of 213 differential proteins were identified, 133 in the non-obstructive azoospermia patients and 80 in the healthy fertile men. According to the molecular function, these differential proteins mainly fell into the types of signal transduction, cytoskeleton and catalytic activity, especially oxidoreductase activity in the latter type. Eighteen of the differential proteins were found to be of particular significance, including dynein heavy chain, fatty acid synthase, and tubulin alpha-6 chain. CONCLUSION: The differential proteins identified in this study were many in number and various in function, which not only demonstrated the value of the shotgun proteomic strategy in protein identification, but also suggested the complicated pathogenesis and varied types of non-obstructive azoospermia. The samples must be selected strictly based on their gene and histological types. Non-obstructive azoospermia was shown to be related with the M phase of the mitotic cell cycle at the protein level, but its specific mechanism remains unknown.

Surface-modified mesoporous nanofibers for microfluidic immunosensor with an ultra-sensitivity and high signal-to-noise ratio.

How to balance the sensitivity and signal-to-noise ratio of immunosensor remains many challenges during various diseases diagnosis. Here we develop a new microfluidic immunosensor based on surface-modified mesoporous nanofibers, and simultaneously realize an ultra-sensitivity and high signal-to-noise ratio for the detection of multiple biomarkers. In the current study, we fabricated titanium dioxide (TiO2)-based mesoporous electrospinning nanofibers, and modified nanofiber surface with both octadecylphosphonic acid (OPA) and poly(ethylene oxide)-poly(propylene oxide) triblock copolymer (PEO-PPO-PEO). Such nanofibers as solid substrate are covered on microfluidic channels. The porosity of our nanofibers dramatically increased the adsorption capability of antibodies, realizing an ultra sensitivity of biomarker detection. PEO-PPO-PEO modification can significantly block non-specific absorptions, obtaining a satisfied signal-to-noise ratio. For the detection of HIV p24 and interleukin 5 (IL-5), our immunosensor increased 6.41 and 6.93 fold in sensitivity and improved 504.66% and 512.80% in signal-to-noise ratio, in compared with gold standard immunoassay (ELISA) used in the clinic. Our immunosensor also broaden the linear range for the detection of HIV p24 (0.86-800 pg/ml) and IL-5 (0.70-800 pg/ml), in compared with ELISA which is 5.54-500 pg/ml for HIV p24 and 4.84-500 pg/ml for IL-5. Our work provided a guideline for the construction of advanced point-of-care immunosensor with an ultra-sensitivity and high signal-to-noise ratio for disease diagnosis.

[Identification of proteins in the seminal plasma of healthy fertile men by shotgun proteomic strategy].

OBJECTIVE: To identify proteins in the seminal plasma of healthy fertile men. METHODS: Three seminal plasma samples were collected from healthy fertile volunteers by Percoll isolation, and then the balanced mixture of the seminal plasma was separated by SDS-PAGE. The proteins in the gel band underwent enzymoloysis, and was extracted and identified by shotgun proteomic strategy. RESULTS: A total of 331 proteins were identified, with the molecular weight (MW) ranging from 8 000 to 572 068 and the isoelectric point (pI) from 4.36 to 11.05. Based on the molecular function and biological process of the proteins, 51 (15.4%) were classified as transport proteins, 11 (3.32%) as cell movement proteins, 63 (19.03%) as signal transduction proteins, 147 (44.4%) as proteases, 38 (11.5%) as enzyme regulator proteins, 21 (6.3%) as programmed cell death proteins, 12 (3.62%) as structural proteins and 59 (17.8%) as proteins with unknown molecular function. CONCLUSION: Shotgun proteomic strategy is a good method for protein identification. Annexin A, Annexin-associated proteins and the Ras-related protein Rab were the major members of the signal transducer proteins identified. Ca2+ and G protein signal pathways may play a most important role in the extracellular signal transduction into cells, but the interactions between these proteins remain unknown. The great quantity of enzymes and enzyme regulator proteins identified in the seminal plasma may be closely related with the maintenance of sperm motility and metabolism.

Low-cost thermophoretic profiling of extracellular-vesicle surface proteins for the early detection and classification of cancers.

Non-invasive assays for early cancer screening are hampered by challenges in the isolation and profiling of circulating biomarkers. Here, we show that surface proteins from serum extracellular vesicles labelled with a panel of seven fluorescent aptamers can be profiled, via thermophoretic enrichment and linear discriminant analysis, for cancer detection and classification. In a cohort of 102 patients, including 6 cancer types at stages I-IV, the assay detected stage I cancers with 95% sensitivity (95% confidence interval (CI): 74-100%) and 100% specificity (95% CI: 80-100%), and classified the cancer type with an overall accuracy of 68% (95% CI: 59-77%). For patients who underwent prostate biopsies, the assay was superior to the analysis of prostate-specific antigen levels (area under the curve: 0.94 versus 0.68; 33 patients) for the discrimination of prostate cancer and benign prostate enlargement, and also in the assessment of biochemical cancer recurrence after radical prostatectomy. The assay is inexpensive, fast, and requires small serum volumes (<1 µl), and if validated in larger cohorts may facilitate cancer screening, classification and monitoring.