Separation and forensic identification of sperm from cell mixtures using anti-hLCN6 monoclonal antibody coupled magnetic beads / 生物工程学报

Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻⁹ mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 10⁴/mL. When the number of sperm cells was 10³/mL, 10⁴/mL and 10⁵/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.

Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene

Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.

Yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of DNA

ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.

PD-L1 Expression on Circulating CD34+ Hematopoietic Stem Cells Closely Correlated with T-cell Apoptosis in Chronic Hepatitis C Infected Patients

BACKGROUND AND OBJECTIVES: Lack of understanding of the interplay between hematopoietic stem cells (HSCs) and the immune system has severely hampered stem cell research. Programmed death-1 (PD-L1) has been reported on parenchymal cells in patients with chronically inflamed livers and found to play an essential role in T cell homeostasis regulation. However, the bidirectional interaction between HSCs and lymphocytes remains elusive. Here, we aimed to get more insight into circulating CD34+ HSCs PD-L1 expression and T cell apoptosis in chronic HCV infected patients. METHODS: CD34+ HSCs were isolated and purified by immunomagnetic separation. PD-L1 expression was analyzed by quantitative PCR and flow cytometry. Furthermore, co-culture experiments between CD34+ HSCs and T-lymphocytes were established. T-cell lymphocyte apoptosis in peripheral blood and in cultures was detected. RESULTS: CD34+ HSCs constitutively express low levels of PD-L1. Its expression is up-regulated in chronic HCV infected patients. Moreover, PD-L1 expression on circulating CD34+ HSCs enhanced T cell apoptosis in peripheral blood and co-culture. CONCLUSION: Our results suggest novel bidirectional interplay between HSCs and lymphocytes mediated by PD-L1 expression on CD34+ HSCs. PD-L1 expression correlated with T-cell lymphocyte apoptosis. This may contribute to immunomodulatory properties of HSCs which improves its use for allogeneic transplantation.

Community-based surveillance of Cryptosporidium in the indigenous community of Boliwong, Philippines: from April to December 2017 / 한국역학회지

OBJECTIVES: For the first time, Boliwong, an indigenous community in the Philippines, was surveyed for the prevalence of Cryptosporidium from April to December 2017. METHODS: Cryptosporidium oocysts were detected in samples from the river, creek, and water pumps via immunomagnetic separation techniques, and from human and animal concentrated faecal samples using the modified Ziehl-Neelsen technique. RESULTS: Seven of the 24 water samples (29.2%) were positive for Cryptosporidium, with the highest concentration (0.8 oocyst/L) detected in the creek. Of 35 fecal samples from different animal groups, 8 (21.6%) were positive for Cryptosporidium oocysts. The highest intensity of oocyst shedding was detected in dogs (χ2=8.00). Of the 137 human fecal samples, 39 (28.5%) were infected with Cryptosporidium. In this study, 3 risk factors were found to be associated with infection: (1) location (crude odds ratio [cOR], 16.39; 95% confidence interval [CI], 2.11 to 127.41; p=0.008), (2) drinking water from the natural spring (cOR, 0.29; 95% CI, 0.11 to 0.82; p<0.05), and (3) using an open pit as a sanitary toilet facility (cOR, 2.44; 95% CI, 1.14 to 5.20; p<0.05). When the cOR was adjusted, using an open pit as a sanitary toilet facility remained a significant risk factor of infection (adjusted OR, 0.41; 95% CI, 0.19 to 0.90; p<0.05). CONCLUSIONS: There is a potentially emerging Cryptosporidium zoonosis in Boliwong, Lagawe, Philippines. It is recommended that the toilet facilities and the water system in the community be rehabilitated to avoid any possible disease outbreak. Health education is also needed in the community to maintain proper hygiene and sanitation practices.

Community-based surveillance of Cryptosporidium in the indigenous community of Boliwong, Philippines: from April to December 2017 / 한국역학회지

OBJECTIVES: For the first time, Boliwong, an indigenous community in the Philippines, was surveyed for the prevalence of Cryptosporidium from April to December 2017.METHODS: Cryptosporidium oocysts were detected in samples from the river, creek, and water pumps via immunomagnetic separation techniques, and from human and animal concentrated faecal samples using the modified Ziehl-Neelsen technique.RESULTS: Seven of the 24 water samples (29.2%) were positive for Cryptosporidium, with the highest concentration (0.8 oocyst/L) detected in the creek. Of 35 fecal samples from different animal groups, 8 (21.6%) were positive for Cryptosporidium oocysts. The highest intensity of oocyst shedding was detected in dogs (χ2=8.00). Of the 137 human fecal samples, 39 (28.5%) were infected with Cryptosporidium. In this study, 3 risk factors were found to be associated with infection: (1) location (crude odds ratio [cOR], 16.39; 95% confidence interval [CI], 2.11 to 127.41; p=0.008), (2) drinking water from the natural spring (cOR, 0.29; 95% CI, 0.11 to 0.82; p<0.05), and (3) using an open pit as a sanitary toilet facility (cOR, 2.44; 95% CI, 1.14 to 5.20; p<0.05). When the cOR was adjusted, using an open pit as a sanitary toilet facility remained a significant risk factor of infection (adjusted OR, 0.41; 95% CI, 0.19 to 0.90; p<0.05).CONCLUSIONS: There is a potentially emerging Cryptosporidium zoonosis in Boliwong, Lagawe, Philippines. It is recommended that the toilet facilities and the water system in the community be rehabilitated to avoid any possible disease outbreak. Health education is also needed in the community to maintain proper hygiene and sanitation practices.

Cancer Stem Cells are Depolarized Relative to Normal Stem Cells Derived from Human Livers

ABSTRACT Introduction and aim. The inability to distinguish cancer (CSCs) from normal stem cells (NSCs) has hindered attempts to identify safer, more effective therapies for hepatocellular carcinoma (HCC). The aim of this study was to document and compare cell membrane potential differences (PDs) of CSCs and NSCs derived from human HCC and healthy livers respectively and determine whether altered GABAergic innervation could explain the differences. Material and methods. Epithelial cell adhesion molecule (EpCAM) positive stem cells were isolated from human liver tissues by magnetic bead separations. Cellular PDs were recorded by microelectrode impalement of freshly isolated cells. GABAA receptor subunit expression was documented by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence. Results. CSCs were significantly depolarized (-7.0 ± 1.3 mV) relative to NSCs (-23.0 ± 1.4 mV, p < 0.01). The depolarized state was associated with different GABAA receptor subunit expression profiles wherein phasic transmission, represented by GAGAA α3 subunit expression, was prevalent in CSCs while tonic transmission, represented by GABAA α6 subunit expression, prevailed in NSCs. In addition, GABAA subunits α3, β3, γ3 and δ were strongly expressed in CSCs while GABAA π expression was dominant in NSCs. CSCs and NSCs responded similarly to GABAA receptor agonists (ΔPD: 12.5 ± 1.2 mV and 11.0 ± 3.5 mV respectively). Conclusion. The results of this study indicate that CSCs are significantly depolarized relative to NSCs and these differences are associated with differences in GABAA receptor subunit expression. Together they provide new insights into the pathogenesis and possible treatment of human HCC.

Culture of rat corpus cavernosal endothelial cells using modified immunomagnetic beads and cloning / 中华男科学杂志

Objective@#To search for the methods of isolating, purifying and culturing corpus cavernosal endothelial cells (CCECs) from SD rats, observe their growth characteristics, and providing seed cells for the study of erectile dysfunction (ED).@*METHODS@#The corpus cavernosal tissue from the SD rat was digested with 0.1% elastase, followed by purification of CCECs with immunomagnetic beads. After further amplification, monoclonal CCECs were sorted out with the cloning cylinder and their morphological and proliferative characteristics were observed. The von Willebrand factor (VWF) in the CCECs was identified by immunofluorescence staining, the CD31 molecule detected by immumohistochemistry, the purity of the CCECs determined by flow cytometry, and the proliferation of the cells measured with CCK-8 and growth curves.@*RESULTS@#After 7 days of purification and culture, the CCECs were fused into a monolayer under the inverted phase-contrast microscope, arranged like flagstones. The growth curves showed that the CCECs were in latency with a low growth rate at 1－2 days, in the logarithmic growth phase with a rapid rate at 3－4 days, and into the platform phase around the 6th day. VWF was positively expressed in the CCECs with much green fluorescence, and so was CD31 with a large number of brownish particles. The positive rate of the CCECs which were labelled with the VWF purified with magnetic beads combined with cloning cylinders was up to (91.9±3.75)%.@*CONCLUSIONS@#High-purity rat CCECs can be cultured successfully using immunomagnetic beads combined with cloning cylinders, with stable proliferation and passage in the endothelial cell medium.

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR

Abstract The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900 -PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.

Método imunomagnético associado ao meio MesenCult® na obtenção de células mononucleares da medula óssea de coelhos negativas para o anticorpo monoclonal CD45 / Immunomagnetic method associated with MesenCult® medium to obtain bone marrow mononuclear cells from rabbits negative for CD45 monoclonal antibody

O objetivo detse artigo é de descrever um protocolo de isolamento das células mononucleares da medula óssea de coelhos, seguido de purificação celular por depleção negativa com o anticorpo monoclonal CD45 e posterior expansão em meio de cultura MesenCult®. Dez coelhos machos adultos, da raça Nova Zelândia, com idade média de 1,0±0,2 anos e peso médio 3,5±0,24kg, foram utilizados para padronização da metodologia. O isolamento das células mononuclares da medula óssea foi realizado pelo gradiente de densidade Ficoll-paque® e a purificação e obtenção das células- pela depleção negativa com o anticorpo monoclonal CD45 em base imunomagnética. A população celular obtida foi expandida posteriormente em meio de cultura MesenCult®. No isolamento pelo gradiente de icoll-Paque® foi obtido um rendimento médio de 7,31x106 células/mL. Após purificação e obtenção das possíveis células-tronco mesenquimais pela base imunomagnética, houve um decréscimo do rendimento para 2,28x106 células/mL, mas o processo de expansão foi incrementado pelo cultivo celular. Os resultados indicaram que as células obtidas da fração mononuclear da medula óssea, cultivadas in vitro foram capazes de gerar células aderentes 24 horas após o cultivo, com predominância de células fibroblastóides sugestivas de células-tronco mesenquimais. Concluiu-se que a obtenção de células-tronco mesenquimais pode ser alcançada após purificação das células mononucleares da medula óssea de coelhos pelo método imunomagético, o meio de cultura MesenCult® proporciona um ambiente adequado para a rápida expansão in vitro e o número de passagens exerce influência negativa sobre as características morfológicas das células.

The objective of this study was to describe guidelines for the isolation of bone marrow mononuclear cells from rabbits, followed by cell purification by negative depletion with CD45 monoclonal antibody, and further expansion in MesenCult® medium. Ten adult male New Zealand White rabbits, age average of 1.0±0.2 years and weighting 3.5±0.24kg, were used to obtain a standardized method. The mononuclear cells of the bone marrow were isolated with Ficoll-paque® density gradient centrifugation, and the cell purification and acquisition was completed by negative depletion with CD45 monoclonal antibody in immunomagnetic base. The cell population obtained was expanded in MesenCult® medium. Through isolation with Ficoll-paque® density gradient was possible to obtain an average yield of 7.31x106 cells/mL. After purification and acquisiton of potential mesenchymal stem cells by the immunomagnetic base, there was a yield decrease to 2.28x106 cells/mL; however the expansion process was increased in cell culture. The results indicated that cells obtained from the mononuclear fraction of bone marrow and cultivated in vitro were capable to generate adherent cells 24 hours after culture, with predominance of fibroblastoid cells suggestive of mesenchymal stem cells. It can be concluded that mesenchymal stem cells can be achieved with purified rabbit bone marrow mononuclear cells through the immunomagnetic method, as the MesenCult® medium provides a suitable environment for a quick in vitro expansion, and the number of passages exerts negative influence on the morphological characteristics.

Application and prospect of circulating tumor cells detection in colorectal cancer / 中华胃肠外科杂志

About 30%-50% of colorectal cancer patients would develop recurrence and metastasis. At present, there is still a lack of effective evaluation method for recurrence, metastasis and prognosis. In recent years, a great progress about circulating tumor cells (CTC) in diagnosis and treatment of colorectal cancer has been made. The most common CTC detection methods include immunocytochemistry, flow cytometry, PCR, immunomagnetic separation, optical fiber array scanning and CTC chip. Based on present studies, researchers reach the consensus that CTC is clinically valuable in the following aspects: detection of occult metastasis, monitor of disease progress and evaluation of response to treatment. With recent development of clinical specialization, multi-disciplinary treatment (MDT), gene detection and targeted therapy, individualized treatment may greatly improve overall survive and disease-free survival of colorectal cancer patients. However, the methods above depend on tumor tissues that are always impractical to obtain for late stage and non-surgery patients. Moreover, the size of specimen is always small, making gene expression and mutation detection difficult. CTC detection may solve such problems based on molecular biology with high plausibility and repeatability. Therefore, CTC detection can be used as a new diagnosis tool. It is believed that CTC detection will play an important role in early diagnosis, evaluating recurrence, metastasis, making individualized treatment and predicting prognosis.

Estudio comparativo del aislamiento de exosomas derivados de células madre mesenquimales humanas para uso clínico / Comparative study of isolated human mesenchymal stem cell derived exosomes for clinical use / Estudo comparativo do isolamento de exossomos derivados de celulas-tronco mesenquimais humanas para uso clinico

Los exosomas son microvesículas derivadas de la exocitosis que son liberados al espacio extracelular. Las funciones de los exosomas dependen en gran medida de la célula de la cual provienen. Los exosomas derivados de células madre mesenquimales (Mesenchymal Stem Cells, MSCs), al igual que las células de origen, poseen un enorme potencial terapéutico que favorece la regeneración tisular y reduce la inflamación. Los prometedores resultados preclínicos que emplean estos exosomas han abierto las puertas a la futura aplicación clínica de estas vesículas. El diseño de nuevos protocolos que permitan el aislamiento de exosomas para su aplicación clínica es una necesidad actual teniendo en cuenta el enorme interés que ha surgido a raíz de los prometedores resultados en ensayos preclínicos. El objetivo de este trabajo ha sido comparar, en términos de rendimiento, tamaño y pureza, diferentes métodos de aislamiento de exosomas a partir de MSCs humanas. Los resultados obtenidos demuestran que el uso de filtros concentradores para sobrenadantes de cultivos podría ser una alternativa a los protocolos convencionales basados en la ultracentrifugación. Los resultados de este trabajo permiten orientar en el diseño de protocolos para la obtención de exosomas derivados de MSCs en grado clínico...

Association between respiratory and postural adaptations and self-perception of school-aged children with mouth breathing in relation to their quality of life

Objective: To investigate the respiratory and postural adaptations associated with mouth and nasal breathing and to evaluate the associations of such adaptations in mouth breathers' self-perceived quality of life. Method: Cross-sectional study with mouth breathers (initial n=116 and final n=48) and nasal breathers (initial n=131 and final n=24) from elementary school, aged between 7 and 14 years. Chest expansion, using cirtometry, the breathing pattern and the use of accessory muscles, by means of clinical evaluations and photogrammetry, and flexibility tests were evaluated in both groups. Subsequently, the mouth breathers were asked to complete the quality of life questionnaire. Statistical tests: Chi-square, odds ratio, Mann-Whitney, and binomial tests were first applied followed by logistic regressions. Results: Thoracic breathing (p=0.04), using of accessory muscles (p=0.03) and reductions in flexibility (p=0.001) increased the chances of an individual being a mouth breather when compared to nasal breathers. Subsequently, using of accessory muscles decreased the chances of snoring among mouth breathers (p=0.03); the presence of shoulder asymmetry reduced the chances of experiencing quiet sleep (p=0.05) and increased the chances of coughing or being tired when playing or running (p=0.008). Finally, forward head position reduced the chances of waking up at night (p=0.04) and experiencing shortness of breath (p=0.05). Conclusions: Respiratory and postural adaptations increased the chances of individuals persisting with mouth breathing. Additionally, these adaptations could be associated with mouth breathers' self-perceived quality of life. .

Detection of Molecular Cytogenetic Aberrations by Fluorescence in Situ Hybridization in Different Bone Marrow Samples of Multiple Myeloma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect the molecular cytogenetic abnormalities in different bone marrow samples of multiple myeloma by using fluorescence in situ hybridization (FISH) technology. The bone marrow cells from 48 cases of MM were taken for sorting the plasma cells using CD138 magnetic beads, and the biopsy tissue from 10 cases of MM was taken and embedded in parafin, then the 2 kinds of samples were detected by using FISH. D13s319/RB1, 1q21/P53, IgH, IgH/FGFR3, IgH/MAF probes were detected in 58 patients with new diagnosed multiple myeloma (MM) by FISH technology.</p><p><b>RESULTS</b>Fluorescent signals were both seen in 2 different types of bone marrow samples and cytogenetic aberrations were detected in 30/58 (51.7%) patients, 29.3% (17 out of 58) cases had both D13S319 and RB1 deletion. The positive rates of P53 deletion, 1q21 amplification and IgH rearrangement were 12%, 27.6% and 20.7%, respectively. Only 7 cases (23.3%) had one cytogenetic abnormality, other 23 (76.7%) cases all had 2 to 5 kinds of different abnormalities.</p><p><b>CONCLUSION</b>More than half of MM patients have cytogenetic change, and most of them are complex chromosomal abnormality. The different kinds of samples can expand the useful extension of FISH technology and acquire more cytogenetic information for clinician.</p>

Isolation and characterisation of human gingival margin-derived STRO-1/MACS(+) and MACS(-) cell populations / 国际口腔科学杂志·英文版

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS⁺) and STRO-1-negative (MACS⁻) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS⁺ and MACS⁻ cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS⁺ and MACS(-) cell fractions showed plastic adherence. MACS⁺ cells, in contrast to MACS⁻ cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS⁺ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS⁻ cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS⁺ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS⁻ cells demonstrated slight osteogenic potential. Unstimulated MACS⁺ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS⁻ cells demonstrated higher expression of osteonectin (P<0.05; Mann-Whitney). The present study is the first to compare gingival MACS⁺ and MACS⁻ cell populations demonstrating that MACS⁺ cells, in contrast to MACS⁻ cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS⁺ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS⁺ cells are a unique renewable source of multipotent stem/progenitor cells.

Immunomagnetic separation combined with RT-qPCR for determining the efficacy of disinfectants against human noroviruses

Little is known about the effectiveness of disinfectants against human noroviruses [NoV] partially because human NoV cannot be routinely cultured in laboratory. The objective of this study was to develop a NoV monoclonal antibody-conjugated immunomagnetic separation [IMS] procedure combined with real-time reverse transcription polymerase chain reaction [RT-qPCR] assays to study the in vitro efficacy of disinfectants against human NoV. Monoclonal antibodies against Norwalk virus [NV, GI.1] and NoV GII.4 were produced using unique NoV capsid proteins, and the antibodies were conjugated to magnetic Dynalbeads. The immunomagnetic beads were used to simultaneously capture intact NoV in samples and effectively remove PCR inhibitors. We examined the efficacy of ethanol, sodium hypochlorite, nine commercially available disinfectants, and one prototype disinfectant using the IMS/RT-qPCR. The sensitivity of this procedure was approximately 100 virus particles for both the NV and GII.4 viruses. The average log reductions in in vitro activities varied between disinfectants. The prototype disinfectant produced an average 3.19-log reduction in NV and a 1.38-log reduction in GII.4. The prototype disinfectant is promising of inactivating NoV. This method can be used to evaluate in vitro activity of disinfectants against human NoV. The IMS/RT-qPCR method is promising as an effective method to remove PCR inhibitors in disinfectants and enable the evaluation of the efficacy of disinfectants

The establishment of a novel method of nano-immunomagnetic separation and Real-time PCR for detecting Vibrio cholerae from seafood / 中华预防医学杂志

<p><b>OBJECTIVE</b>A novel method of Nano-Immunomagnetic Separation (Nano-IMS) plus Real-time PCR was established for detecting Vibrio cholerae.</p><p><b>METHODS</b>The Nano-Immunomagnetic Beads were created by using the monoclonal antibody of Vibrio cholerae, which was named Nano-IMB-Vc. Nano-IMB-Vc has specific adsorption of Vibrio cholerae, combined with Real-time PCR technology, a method for rapid detection of Vibrio cholerae was established. The capture specificity of Nano-IMB-Vc was tested by using 15 bacteria strains. The specificity of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria strains. The sensitivity of Nano-IMS plus Real-time PCR were tested in pure culture and in artificial samples and compared with NMKL No.156.</p><p><b>RESULTS</b>The capture ratio of Nano-IMB-Vc was reached 70.2% at the level of 10(3) CFU/ml. In pure culture, the sensitivity of Nano-IMS plus Real-time PCR was reached at 5.4×10(2) CFU/ml. The specific of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria. The results showed that 102 strains of Vibrio cholerae test results were all positive, and the rest of the 101 strains of non-target bacteria test results were negative. No cross-reaction was founded. Add 1 CFU vibrio cholerae per 25 g sample, it could be detect with Nano-IMS plus Real-time PCR method after 8 hours enrichment.</p><p><b>CONCLUSIONS</b>The Nano-IMS plus Real-time PCR method of Vibrio cholerae established in this study has good specificity and sensitivity, which could be applied to the rapid detection of Vibrio cholerae.</p>

Detection of circulating tumor cells in patients with hepatocellular carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the detection efficiency of circulating tumor cells (CTCs) in patients with hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Immunomagnetic negative enrichment by nanometer magnetic beads and label-free capture with Captor(TM) system were used to isolate and enrich CTCs from peripheral blood of HCC patients, and epithelial and HCC markers were applied to identify CTCs by immunofluorescence staining. CTCs were detected in 50 HCC patients before and after hepatectomy to test the method for isolation, enrichment and identification. The dynamic changes of pre- and post-operative CTCs' numbers were compared. The clinical data were analyzed using SPSS 19.0 software.</p><p><b>RESULTS</b>Negative enrichment methods by nanometer magnetic beads and label-free capture using Captor(TM) system were both suitable for CTCs isolation and enrichment in HCC patients. The positive detection rate of CTCs in HCC patients via negative enrichment was 96.0% (48/50), the preoperative median number of CTCs was 16 per 7.5 ml blood, and the postoperative median number was 17 per 7.5 ml blood.</p><p><b>CONCLUSIONS</b>Both negative enrichment and Captor(TM) system are suitable for isolation and enrichment of CTCs in HCC patients. There is a significant difference in the numbers of CTCs before and after operation, and dynamic detection of CTCs will provide helpful prognostic information for HCC patients in clinics.</p>

Research advances on DNA extraction methods from peripheral blood mononuclear cells / 中国实验血液学杂志

DNA extraction is a basic technology of molecular biology. The purity and the integrality of DNA structure are necessary for different experiments of gene engineering. As commonly used materials in the clinical detection, the fast, efficient isolation and extraction of genomic DNA from peripheral blood mononuclear cells is very important for the inspection and analysis of clinical blood. At present, there are many methods for extracting DNA, such as phenol-chloroform method, salting out method, centrifugal adsorption column chromatography method (artificial methods), magnetic beads (semi-automatic method) and DNA extraction kit. In this article, a brief review of the principle for existing DNA blood extraction method, the specific steps and the assessment of the specific methods briefly are summarized.

Rapid detection of Listeria monocytogenes by immunomagnetic separation combined with selective medium / 生物工程学报

Listeria monocytogenes is a pathogenic bacterium, therefore, it is essential for food safety monitoring to establish a rapid and specific detecting method. In this study, immunomagnetic beads and selective medium were combined to detect Listeria monocytogenes at different concentrations (10(1)-10(5) CFU/mL). Other three types of Listeria spp., Staphylococcus aureus and Vibrio parahaemolyticus were also detected to conduct the cross-reaction analysis. Meanwhile, contaminated milk samples were prepared to explore the limit of detection of immunomagnetic beads combining with selective medium. Results showed that Listeria monocytogenes with the concentration of 10(3) CFU/mL and above was successfully detected. Milk samples were detected within 6 hours, with a detection limit of 0.7 CFU/mL. The method developed is capable of detecting milk samples within 30 h, which is 38 h faster compared with national standard method with the same sensitivity.