A new multifunctional fluorescent"turn-on"biosensor was constructed based on a guanine-rich hairpin probe and G-quadruplex/hemin DNAzyme.The probe was labeled with quencher and fluorescein at 5′and 3′terminus,respectively.In the absence of the target,the probe form hairpin structure and the fluorescence of the fluorescein was quenched by the quencher.However,in the presence of the target,the hairpin structure transformed to G-quadruplex and the fluorescence was recovered.By monitoring the fluorescence of the probe before and after combining with the target,both hemin and hemoglobin were detected rapidly and conveniently with high sensitivity.The limit of detection for hemin and hemoglobin were estimated to be 283 pmol/L and 109 pmol/L,respectively.The specific interaction between the DNA probe and hemin were investigated via absorption and fluorescence spectra.Furthermore,L-cysteine was detected with naked eyes based on the peroxidase-like activity of the new G-quadruplex/hemin DNAzyme.The limit of detection for L-cysteine was estimated to be 3.6 μmol/L,which was lower than the normal level of intracellular L-cysteine(30-200 μmol/L).The method developed here was rapid,simple and sensitive for detection of hemin,hemoglobin and L-cysteine,demonstrating great potential in bioanalysis,drug screening,and disease diagnosis.

Objective:To evaluate the performance of an artificial intelligent (AI)-based automated digital cell morphology analyzer (hereinafter referred as AI morphology analyzer) in detecting peripheral white blood cells (WBCs).Methods:A multi-center study. 1. A total of 3010 venous blood samples were collected from 11 tertiary hospitals nationwide, and 14 types of WBCs were analyzed with the AI morphology analyzers. The pre-classification results were compared with the post-classification results reviewed by senior morphological experts in evaluate the accuracy, sensitivity, specificity, and agreement of the AI morphology analyzers on the WBC pre-classification. 2. 400 blood samples (no less than 50% of the samples with abnormal WBCs after pre-classification and manual review) were selected from 3 010 samples, and the morphologists conducted manual microscopic examinations to differentiate different types of WBCs. The correlation between the post-classification and the manual microscopic examination results was analyzed. 3. Blood samples of patients diagnosed with lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndrome, or myeloproliferative neoplasms were selected from the 3 010 blood samples. The performance of the AI morphology analyzers in these five hematological malignancies was evaluated by comparing the pre-classification and post-classification results. Cohen′s kappa test was used to analyze the consistency of WBC pre-classification and expert audit results, and Passing-Bablock regression analysis was used for comparison test, and accuracy, sensitivity, specificity, and agreement were calculated according to the formula.Results:1. AI morphology analyzers can pre-classify 14 types of WBCs and nucleated red blood cells. Compared with the post-classification results reviewed by senior morphological experts, the pre-classification accuracy of total WBCs reached 97.97%, of which the pre-classification accuracies of normal WBCs and abnormal WBCs were more than 96% and 87%, respectively. 2. The post-classification results reviewed by senior morphological experts correlated well with the manual differential results for all types of WBCs and nucleated red blood cells (neutrophils, lymphocytes, monocytes, eosinophils, basophils, immature granulocytes, blast cells, nucleated erythrocytes and malignant cells r>0.90 respectively, reactive lymphocytes r=0.85). With reference, the positive smear of abnormal cell types defined by The International Consensus Group for Hematology, the AI morphology analyzer has the similar screening ability for abnormal WBC samples as the manual microscopic examination. 3. For the blood samples with malignant hematologic diseases, the AI morphology analyzers showed accuracies higher than 84% on blast cells pre-classification, and the sensitivities were higher than 94%. In acute myeloid leukemia, the sensitivity of abnormal promyelocytes pre-classification exceeded 95%. Conclusion:The AI morphology analyzer showed high pre-classification accuracies and sensitivities on all types of leukocytes in peripheral blood when comparing with the post-classification results reviewed by experts. The post-classification results also showed a good correlation with the manual differential results. The AI morphology analyzer provides an efficient adjunctive white blood cell detection method for screening malignant hematological diseases.

Objective: To explore HIV-1 drug resistance and influencing factors among people living with HIV/AIDS before antiretroviral therapy in Liangshan Yi Autonomous Prefecture (Liangshan). Methods: Between January 1 and June 30, in both 2017 and 2018, a cross-sectional survey was conducted in Liangshan HIV-1 pol sequences were gathered and analyzed according to WHO Guidelines on HIV drug resistance surveillance of 2014. Both HyPhy 2.2.4 and Cytoscape 3.6.1 software were used to analyze the drug resistant strains of HIV-1 transmission network. Results: A total of 464 people living with HIV/AIDS was recruited. The proportion of HIV-1 CRF07_BC subtype was 88.6% (411/464), with HIV-1 drug resistance rate was 9.9% (46/464). The HIV-1 drug resistance rates of non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors(NRTI) and protease inhibitors (PI) were 6.7% (31/464), 1.9% (9/464) and 0.4% (2/464) respectively. New recombinant strains of HIV-1 URF_01BC subtype was independently clustered according to the drug resistant mutation sites. Results from the multivariate logistic analysis showed that injected drug users group had higher risk on HIV-1 drug resistance (aOR=3.03, 95%CI:1.40-6.54) than heterosexual group among people living with HIV/AIDS. Conclusions HIV-1 drug resistance rate had already been in a high level before antiretroviral therapy was in place. The newly identified recombinant strains of HIV-1 URF_01BC subtype were independently clustered according to the drug resistant mutation sites. It was necessary to strengthen the prevention of the HIV-1 drug resistant strains transmission.

BACKGROUNDPlasmacytoid dendritic cells (pDCs) and cytokines play an important role in occurrence and recovery of hepatitis B virus (HBV) infection. The aim of this study was to explore the frequency and function of pDC and serum cytokine network profiles in patients with acute or chronic HBV infection.

METHODSThe healthy individuals (HI group), hepatitis B envelope antigen (HBeAg)-positive chronic HBV patients in immune tolerance (IT) phase (IT group), HBeAg-positive chronic HBV patients (CHB group), and acute HBV patients (AHB group) were enrolled in this study. The frequency of cluster of differentiation antigen 86 (CD86) + pDC and the counts of CD86 molecular expressed on surface of pDC were tested by flow cytometer. The quantitative determinations of cytokines, including Fms-like tyrosine kinase 3 ligand (Flt-3L), interferon (IFN)-α2, IFN-γ, interleukin (IL)-17A, IL-6, IL-10, transforming growth factor (TGF)-β1 and TGF-β2, were performed using Luminex multiplex technology.

RESULTSIn this study, there were 13 patients in HI group, 30 in IT group, 50 in CHB group, and 32 in AHB group. Compared with HI group, HBV infected group (including all patients in IT, CHB and AHB groups) had significantly higher counts of CD86 molecular expressed on the surface of pDC (4596.5 ± 896.5 vs. 7097.7 ± 3124.6; P < 0.001). The counts of CD86 molecular expressed on the surface of pDC in CHB group (7739.2 ± 4125.4) was significantly higher than that of IT group (6393.4 ± 1653.6, P = 0.043). Compared with IT group, the profile of cytokines of Flt-3L, IFN-γ, and IL-17A was decreased, IFN-α2 was significantly increased (P = 0.012) in CHB group. The contents of IL-10, TGF-β1, and TGF-β2 in AHB group were significantly increased compared with IT and CHB groups (all P < 0.05).

CONCLUSIONSThis study demonstrated that the function of pDC was unaffected in HBV infection. The enhanced function of pDC and IFN-α2 might involve triggering the immune response from IT to hepatitis active phase in HBV infection. Acute patients mainly presented as down-regulation of the immune response by enhanced IL-10 and TGF-β.

Exposure to free silica induces silicosis and myofibroblasts are regarded as primary effector cells. Fibrocytes can differentiate into myofibroblast. Therefore, the present study was designed to investigate whether fibrocytes participate in silicosis. The rat model of silicosis was established. Hematoxylin-eosin stainings and Masson stainings were used to evaluate the histopathology and collagen deposition. Flow cytometry and immunofluorescence were performed to detect the number of fibrocytes and their contribution to myofibroblasts. Results showed that fibrocytes participate in silicosis. Trend analysis of different sources of myofibroblasts during silicosis indicated that fibrocytes and lung type II epithelial cell-derived myofibroblasts play an important role in the early stage of silicosis, while resident lung fibroblast-derived myofibroblasts play a predominant role during the fibrosis formative period.
Animals ; Disease Models, Animal ; Lung ; cytology ; Myofibroblasts ; drug effects ; pathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Silicon Dioxide ; toxicity ; Silicosis ; etiology ; pathology

Background: Cytokines play an important role in occurrence and recovery of hepatitis B virus (HBV) infection. The aim of this study was to investigate the changes of cytokines concentration and its correlation to alanine aminotransferase (ALT), HBV deoxyribonucleic acid (HBV-DNA), hepatitis B envelope antigen (HBeAg), and HBV surface antigen (HBsAg) in the development of chronic hepatitis B (CHB). Methods: Thirteen healthy individuals (HI), 30 chronic HBV-infected patients in immune tolerant (IT) phase, and 55 CHB patients were enrolled between August 2015 and May 2017. The peripheral blood samples were collected from all individuals. The levels of interferon (IFN)-α2, interleukin (IL)-10, transforming growth factor (TGF)-β1, HBV-DNA, HBsAg, and HBeAg and liver function were measured. The quantitative determinations of cytokines levels, including IFN-α2, IL-10, and TGF-β1 were performed using Luminex multiplex technology. The correlation of cytokines to ALT, HBV-DNA, HBsAg, and HBeAg was analyzed by linear regression analysis. Results: IFN-α2 levels were similar between HI and IT groups (15.35 [5.70, 67.65] pg/ml vs. 15.24 [4.07, 30.73] pg/ml, Z = -0.610, P = 0.542), while it elevated significantly in CHB group (35.29 [15.94, 70.15] pg/ml vs. 15.24 [4.07, 30.73] pg/ml; Z = -2.522, P = 0.012). Compared with HI group (3.73 [2.98, 11.92] pg/ml), IL-10 concentrations in IT group (5.02 [2.98, 10.11] pg/ml), and CHB group (7.48 [3.10, 18.00] pg/ml) slightly increased (χ = 2.015, P = 0.365), and there was no significant difference between IT and CHB group (Z = -1.419, P = 0.156). The TGF-β1 levels among HI (3.59 ± 0.20 pg/ml), IT (3.62 ± 0.55 pg/ml), and CHB groups (3.64 ± 0.30 pg/ml) were similar (χ = 2.739, P = 0.254). In all chronic HBV-infected patients (including patients in IT and CHB groups), the elevation of IFN-α2 level was significantly associated with ALT level (β= 0.389, t = 2.423, P = 0.018), and was also negatively correlated to HBV-DNA load (β = -0.358, t = -2.308, P = 0.024), HBsAg (β = -0.359, t = -2.288, P = 0.025), and HBeAg contents (β = -0.355, t = -2.258, P = 0.027). However, when both ALT level and cytokines were included as independent variable, HBV-DNA load, HBsAg, and HBeAg contents were only correlated to ALT level (β = -0.459, t = -4.225, P = 0.000; β = -0.616, t = -6.334, P = 0.000; and β = -0.290, t = -2.433, P = 0.018; respectively). Conclusions IFN-α2 elevation was associated with ALT level in patients with chronic HBV infection. However, in CHB patients, only ALT level was correlated to HBV-DNA, HBsAg and HBeAg contents.
Adult ; Alanine Transaminase ; blood ; Antigens, Surface ; Case-Control Studies ; Cytokines ; blood ; DNA, Viral ; Female ; Hepatitis B ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B e Antigens ; Hepatitis B virus ; Hepatitis B, Chronic ; blood ; immunology ; Humans ; Male ; Young Adult

AlM:To investigate the factors and solutions of Tibetan Plateau excimer laser in situ keratomileusis ( LASlK ) for myopia overcorrection.METHODS: The relevant information, 32 cases ( 58 eyes) in 126 cases (252 eyes) had obvious overcorrection after LASlK were analyzed.RESULTS: Two months after surgery, 32 cases ( 58 eyes) overcorrection (23. 0%), uncorrected visual acuity of 0. 5 ~0. 8, overcorrection range of +1. 50 ~ +2. 25DS, subjective inserts were ≥ 1. 0; Five case ( 7 eyes ) overcorrection 6mo after surgery (2. 8%), uncorrected visual acuity 0. 8~1. 0-2 , overcorrection range is +0. 75 ~+1. 25DS, subjective inserts were≥1. 0. Corneal thickness of overcorrection was 500~563μm, preoperative refraction was -5. 00 ~ -7. 50D, astigmatism -1. 50 ~ -2. 75DC, preoperative best corrected visual acuity ≥1. 0.CONCLUSlON: Overcorrection and long recovery time after LASlK in Tibet, possibly with local factors altitude, temperature, humidity, surgical parameters and situation.

Objective To investigate the tissue remodeling and cell alignment of TDBM scaffolds seeded with rabbit tenocytes under the cycle dynamic tensile force or static tension-free culture in vitro. Methods TDBM were made by ourselves, and uniaxial cyclic tendon stretching device was designed and manufactured on our own. Primary tenocytes were isolated from the Achilles tendon of three-day-old New Zealand white rabbits and seeded into scaffolds, and were cultured collectively in DMEM in vitro. Samples were divided into two groups:dynamic tension-loaded group, and static tension-free group. Fresh natural tendons were used to be positive control. The experiment's time was six weeks. The scaffold-cell complexes were harvested at 3 and 7 days of culture for Inverted microscope and scanning electron micrograph (SEM) analysis. The morphological characters of the samples, including the general view, HE and Masson's dyeing, were observed at 2, 4 and 6 weeks. In addition, the gene expression of the I-type collagen and III-type collagen of the samples was detected by using Real time PCR at every week. Set fresh natural tendon as control. Results The inverted microscope and SEM showed that it was nice compatible condition between the tendon cells and TD-BM scaffold. In addition, the tendon of tension-loaded group revealed a structure of longitudinally aligned collagen fi bers and dense structure of collagen fibers arranged in orderly form a unique corrugated structure. Tenocytes layer located between the col-lagen fibers and aligned longitudinally along the force axis, with increased matrix deposition after the 3th week showed by RT-PCR. The cell/matrix ratio decreases. When cultured to 6 weeks, the tissue structure was very similar to that of fresh natural ten-don pattern. By contrast, HE and Masson's staining revealed the collagen fibro-tissue structure in tension-free groups with disorga-nized matrix structure and randomly distributed cells. Collagen fibers were gradually degraded with time. In tension-loaded group, Real-time PCR showed that gene expression of I-type collagen and III-type collagen gradually increase, but in tension-free group, the relative gene expression of I-type collagen and III-type collagen was highest at 3rd week, and from that time the relative expres-sion gradually decrease. Conclusion Under the dynamic stress, the TDBM scaffolds seeded with rabbit tenocytes can promote extra-cellular matrix biosynthesis and tendon structure remodeling, which is a promising method for tendon tissue engineering.

OBJECTIVETo investigate the mechanisms underlying bone marrow damage by iron overload in pancytopenic patients with positive BMMNC-Coombs test (IRP).

METHODSTwenty-one iron overloading, 26 non-iron overloading IRP patients and 10 normal controls were enrolled in this study. The expressions of ROS, Bcl-2, Caspase-3 and apoptosis of BMMNC were analyzed by flow cytometry (FCM). Antioxidants were added to iron overloading IRP BMMNC, and then the changes of indices above were detected by FCM. The number and apoptosis of T lymphocytes of IRP patients were also detected.

RESULTSROS and apoptosis of BMMNC, myelocytes, erythrocytes and stem cells of iron overloading IRP patients were significantly higher than that of non-iron overloading IRP ones and normal controls (P < 0.05). The expressions of Bcl-2 on BMMNC, erythrocytes and stem cells of iron overloading IRP patients were significantly lower than those of non-iron overloading IRP ones (P < 0.05). The levels of Caspase-3 on myelocytes, erythrocytes and stem cells of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP ones and normal controls (P < 0.05). After treatment with antioxidants, the expressions of ROS, Caspase-3 and apoptosis of iron overloading IRP BMMNC significantly decreased, but opposite for Bcl-2. The percentages of CD4(+) lymphocytes [ ( 40.86 ± 8.74）%] and CD4(+)/CD8(+) (1.44 ± 0.36) in PB of iron overloading IRP patients were significantly higher than that of non-iron overloading IRP ones [(35.96 ± 7.03)% and 1.14 ± 0.37] and normal controls [(28.00 ± 6.73)% and 0.79 ± 0.21], respectively (P < 0.05), as opposite for CD8(+) lymphocytes (P < 0.05). The apoptosis of CD8(+) lymphocytes [(27.35 ± 10.76)%] and the ratio of CD8(+) apoptosis/CD4(+) apoptosis (2.51 ± 0.81) in BM of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP ones [(15.47 ± 8.99)%] and normal controls (1.39 ± 0.47), respectively (P < 0.05). The apoptosis of erythrocytes and stem cells coated with auto-antibodies in BM of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP and normal controls.

CONCLUSIONMechanisms underlying bone marrow damage by iron overload might be through the follows: ①The increased ROS induced by excessive iron deposition affected the expressions of Caspase-3 and Bcl-2, which caused more BMMNC apoptosis; ②The abnormal number and ratio of T lymphocytes caused by iron overload aggravated the abnormality of immunity of IRP; ③Iron overload may increase the damage to erythrocytes and stem cells coated with auto-antibodies.

Adolescent ; Adult ; Aged ; Bone Marrow ; pathology ; Case-Control Studies ; Caspase 3 ; metabolism ; Coombs Test ; Female ; Humans ; Iron Overload ; Male ; Middle Aged ; Pancytopenia ; immunology ; pathology ; physiopathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Reactive Oxygen Species ; metabolism ; Young Adult

OBJECTIVETo study the quantity and function of bone marrow (BM) T follicular helper (Tfh) cells of the cytopenia patients with positive bone marrow mononuclear cells (BMMNC)- Coombs test (also known as immuno-related pancytopenia, IRP), and explore the role of Tfh cells in the pathogenesis of IRP.

METHODSForty- three untreated IRP patients, 47 recovered IRP patients and 25 healthy donors were enrolled in this study. The percentages of Tfh cells, Tfh-related molecules ICOS, CD40L, IL-21 and Bcl-6 in BM were investigated by flow cytometry and semiquantitive RT-PCR.

RESULTSThe ratio of CD4⁺CXCR5⁺/CD4⁺ cells of untreated IRP patients [(28.79 ± 19.70)%] was significantly higher than that of recovered IRP patients [(21.15 ± 12.81)% ] and normal controls ([ 13.42 ± 6.72)% ](P<0.05). The ratio of CD4⁺CXCR5⁺ICOS⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.05 ± 4.71)% ] was significantly higher than that of recovered IRP patients [(2.96 ± 2.89)% ] and normal controls [(2.99 ± 2.23)% ] (P<0.05). The ratio of CD4⁺CXCR5⁺CD40L⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.87 ± 4.14)%] and recovered IRP patients [(6.52±5.47)%] were significantly higher than that of normal controls [(2.93 ± 2.92)%] (P<0.05). The ratio of intracytoplasmic CD4⁺CXCR5⁺IL-21⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(8.20 ± 7.41)% ] and recovered IRP patients [(6.30 ± 6.03)% ] were significantly higher than that of normal controls [(3.43 ± 3.40)%] (P<0.05). The relative expressions of Bcl-6 mRNA in BMMNC were 0.625 ± 0.248, 0.485 ± 0.253, 0.306 ± 0.210 in three groups, respectively. The differences between untreated IRP patients, recovered IRP patients and normal controls were significant (P<0.05).

CONCLUSIONThere exists increased quantity and hyperfunction of Tfh cells in the IRP patients, they may play important role in the pathogenesis of IRP. Tfh cells and their related effector molecules could be a potential therapeutic target for the disease.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Child ; Child, Preschool ; Coombs Test ; Female ; Flow Cytometry ; Humans ; Interleukins ; metabolism ; Lymphocyte Count ; Male ; Middle Aged ; Pancytopenia ; blood ; diagnosis ; etiology ; T-Lymphocytes, Helper-Inducer ; cytology ; metabolism ; Young Adult