[Mechanism of TRIM27 promoting inflammatory response in lung cancer cells].

Objective: To investigate the mechanism of tripartite motif-containing 27 (TRIM27) expression promoting inflammatory response in non-small lung cancer cells. Methods: Ten cases of lung cancer tissues and their matched normal tissue (the distance was 5 cm of the tumor marginal) from patients underwent resection in the People's Hospital of Pingyang Hospital Affiliated to Wenzhou Medical University were collected. The expression of TRIM27 was identified by using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. TRIM27 knockdown experiment included negative control (NC(TRIM27)) group, TRIM27 short interfering RNA (siRNA) group, NC(TRIM27)+ TNF-α group and TRIM27 siRNA+ TNF-α group. Interlukin-6 (IL-6) knockdown experiment included NC(IL-6) group and IL-6 siRNA group. The protein expressions of TRIM27, TNFR1, TNFR2 and some TNFR related inflammation factors were verified by qRT-PCR and WB. Results: The expression levels of TRIM27 in NSCLC tissues of different stages (stage Ⅰ: 2.81±0.58, stage Ⅱ: 3.32±1.38, stage Ⅲ: 3.67±1.24) was higher than that in the adjacent normal tissues (1.01±0.15, 0.92±0.10 and 1.05±0.12, P<0.05). The expression levels of TRIM27 mRNA in NC(TRIM27) group and NC(TRIM27)+ TNF-α group were 0.94±0.12 and 1.67±0.03, and the expression levels of TRIM27 protein were 0.31±0.02 and 0.38±0.01, respectively (P<0.05). The expression levels of IL-6 mRNA in NC(TRIM27)+ TNF-α group and TRIM27 siRNA+ TNF-α group were 11.35±0.12 and 5.62±0.15, respectively, and the expression levels of VCAM-1 mRNA were 18.75±0.17 and 9.35±0.11, respectively. STAT3 mRNA expression levels were 16.54±0.10 and 8.12±0.10, respectively, with statistical significance (P<0.05). The expression levels of IL-6 mRNA in NC(IL-6) group and IL-6 siRNA group were 1.10±0.07 and 0.52±0.16, respectively, and the expression levels of STAT3 mRNA were 1.01±0.01 and 0.48±0.12, respectively. The expression levels of TRIM27 mRNA were 1.03±0.01 and 0.30±0.11, respectively, with statistical significance (P<0.05). Conclusion: The upregulation of TRIM27 in NSCLC tissue and cells promotes the expression of TNF-α, and may activate inflammatory response by regulating TNF-α-induced IL-6/STAT3 signaling pathway.

Half-year follow-up of patients recovering from severe COVID-19: Analysis of symptoms and their risk factors.

OBJECTIVES: To understand the sequelae of COVID-19. METHODS: We followed up 1174 patients with severe coronavirus disease 2019 (COVID-19)who were recovered and discharged for 6 months. RESULTS: There were 175 cases with clear IgG results 6 months after discharge, of which 82 (46.9%) were IgG (+) and 16 (9.1%) were IgG (dim+). Four hundred and forty-one participants (55.4%) had some kind of sequelae. The most common symptoms were fatigue (25.3%), sleep disorder (23.2%) and shortness of breath (20.4%). In those who had sequelae, 262 (59.4%) had more than one symptom. Critical cases were more likely to have cough (20.5% vs 11.6%, p = 0.023) and hypomnesis (15.1% vs 8.0%, p = 0.041) than severe cases. Furthermore, univariate and multivariate logistic regression analyses revealed that women are more likely to have multiple symptoms (p = 0.002), fatigue (p = 0.009) and sleep disorder (p = 0.008), whereas critical illness was found as independent risk factor for hypomnesis (p = 0.045). CONCLUSION: Our study demonstrated the duration of antibody and sequelae of COVID-19 and compared the differences amongst different populations.

[CD44 regulates biological behavior and Ras signaling pathway in nasopharyngeal carcinoma stem cells].

Objective: To study the biological behavior of nasopharyngeal carcinoma stem cells and to explore the activation of Ras signaling pathway regulated by CD44. Methods: CNE2-SC and 5-8F-SC were nasopharyngeal carcinoma stem cells and obtained by serum-free suspension culture. Cell counting kit-8 (CCK-8) assay, colony formation assay, Transwell migration assay, cell adhesion array were used to investigate the growth, proliferation, migration and adhesion of nasopharyngeal carcinoma stem cells. Western blot test was used to detect the expressions of Ras signaling pathway related proteins and siRNA-mediated interference was used to determine the activation of Ras signaling pathway regulated by CD44. Results: The growth rates of CNE2-SC and 5-8F-SC cells were significantly lower than those of nasopharyngeal carcinoma cells at 24, 48 and 72 hours after inoculation (P<0.05). After 14 days of implantation, the colony formation rates of CNE2-SC (44.5±1.9)% and 5-8F-SC (47.4±1.8)% were higher than those of CNE2 (34.9±1.5)% and 5-8F (37.2±1.7)%, respectively(P<0.01). The migration cell number of CNE2-SC was (87.6±7.8), 3.97 times higher than that of CNE2 (P<0.01). The migration cell number of 5-8F-SC was (67.2±5.7), 3.07 times higher than 5-8F (P<0.01). The adhesion rates of CNE2-SC and CNE2 cells were (42.1±7.6)% and (8.9±2.0)%, respectively at 3 hours after inoculation and were (82.4±5.0)% and (12.1±2.2)% at 6 hours after inoculation, respectively. The adhesion rate of CNE2-SC cells was higher than that of CNE2 cells (all P<0.01). The adhesion rates of 5-8F-SC and 5-8F cells were (53.6±6.1)% and (7.3±1.5)% at 3 hours after inoculation, and (90.7±3.6)% and (11.0±1.2)% at 6 hours after inoculation, respectively. The adhesion rate of 5-8F-SC cells was higher than that of 5-8F cells (P<0.01). The expression levels of CD44, Ras and N-cadherin were significantly higher, while phosphatase and tensin homolog deleted on chromosome 10 (PTEN), E-cadherin in nasopharyngeal carcinoma stem cells were lower than those of the nasopharyngeal carcinoma cells. Furthermore, the levels of phosphorylated mitogen extracellular kinase1/2 (p-MEK1/2) and phosphorylated extracellular signal-regulated protein kinase1/2 (p-ERK1/2)were significantly increased in nasopharyngeal carcinoma stem cells (P<0.01). Correlation analysis showed that the protein expression levels of CD44 was highly positively correlated with RAS in nasopharyngeal carcinoma stem cells(r=0.985, P=0.002; r=0.962, P=0.038). Deletion of CD44 in CNE2-SC decreased the expression levels of HER-2, Ras and p-ERK1/2, p-Akt and phosphorylated protein kinase C-δ(p-PKCδ) (P<0.01). Conclusions: Despite compare to the nasopharyngeal carcinoma cell, nasopharyngeal carcinoma stem cells grows at a relatively slow rate, the capacities of clone formation, migration, adhesion are promoted. This may be related to the CD44-regulated abnormal activation of Ras signaling pathway.

[Comprehensive benefits of agroforestry snail control forests in Eryuan County, Yunnan Province].

OBJECTIVE: To investigate the efficiency of various agroforestry systems for snail control in plateau hilly schistosomiasis-endemic areas of Yunnan Province, so as to provide insights into the construction of agroforestry schistosomiasis control projects in plateau hilly regions. METHODS: The pilot areas of snail control forests with various agroforestry systems were built in snail-breeding farmlands in Eryuan County, Yunnan Province in 2010, and the economic benefits and snail control effect were investigated in 2018. In addition, a fuzzy comprehensive evaluation model was created to screen the agroforestry system with high comprehensive benefits. RESULTS: A total of 14 types of pilot areas of snail control forests with various agroforestry systems were built. Economic benefit analysis showed that the"walnut + garlic"pattern had the best economic benefit, with annual economic benefits of 270 000 Yuan/hm2, followed by the"walnut + chili"pattern (annual economic benefits of 120 000 Yuan/hm2) and the "walnut + vegetables"pattern (annual economic benefits of 105 000 Yuan/hm2). No snails were detected in 8 types of the agroforestry systems, including the"walnut + chili"pattern, the"walnut + tobacco"pattern and the"walnut + garlic"pattern; however, there were snail found with various densities in other types of systems. Fuzzy comprehensive evaluation showed that the"walnut + garlic"pattern had the best comprehensive control effect, followed by the"walnut + chili"pattern and the"walnut + tobacco" pattern, while the pure grassland pattern showed no effect on snail control. CONCLUSIONS: The agroforestry system is a preferential approach of forestry schistosomiasis control in plateau hilly schistosomiasis-endemic areas, which not only achieves snail control effects, but also promotes economic development and ecological construction in poor hilly areas.

FAIR data sharing: The roles of common data elements and harmonization.

The value of robust and responsible data sharing in clinical research and healthcare is recognized by patients, patient advocacy groups, researchers, journal editors, and the healthcare industry globally. Privacy and security concerns acknowledged, the act of exchanging data (interoperability) along with its meaning (semantic interoperability) across studies and between partners has been difficult, if not elusive. For shared data to retain its value, a recommendation has been made to follow the Findable, Accessible, Interoperable, Reusable (FAIR) principles. Without applying appropriate data exchange standards with domain-relevant content standards and accessible rich metadata that uses applicable terminologies, interoperability is burdened by the need for transformation and/or mapping. These obstacles to interoperability limit the findability, accessibility and reusability of data, thus diminishing its value and making it impossible to adhere to FAIR principles. One effort to standardize data collection has been through common data elements (CDEs). CDEs are data collection units comprising one or more questions together with a set of valid values. Some CDEs contain standardized terminology concepts that define the meaning of the data, and others include links to unique terminology concept identifiers and unique identifiers for each CDE; however, usually CDEs are defined for specific projects or collaborations and lack traceable or machine readable semantics. While the name implies that these are 'common', this has not necessarily been a requirement, and many CDEs have not been commonly used. The National Institutes of Health (NIH) CDEs are, in fact, a conglomerate of CDEs developed in silos by various NIH institutes. Therefore, CDEs have not brought the anticipated benefit to the industry through widescale interoperability, nor is there widespread reuse of CDEs. Certain institutes in the NIH recommend, albeit do not enforce, institute-specific preferred CDEs; however, at the NIH level a preponderance of choice and a lack of any overarching harmonization of CDEs or consistency in linking them to controlled terminology or common identifiers create confusion for researchers in their efforts to identify the best CDEs for their protocol. The problem of comparing data among studies is exacerbated when researchers select different CDEs for the same variable or data collection field. This manuscript explores reasons for the disappointingly low adoption of CDEs and the inability of CDEs or other clinical research standards to broadly solve the interoperability and data sharing problems. Recommendations are offered for rectifying this situation to enable responsible data sharing that will help in adherence to FAIR principles and the realization of Learning Health Systems for the sake of all of us as patients.

Multidimensional diffusion MRI with spectrally modulated gradients reveals unprecedented microstructural detail.

Characterization of porous media is essential in a wide range of biomedical and industrial applications. Microstructural features can be probed non-invasively by diffusion magnetic resonance imaging (dMRI). However, diffusion encoding in conventional dMRI may yield similar signatures for very different microstructures, which represents a significant limitation for disentangling individual microstructural features in heterogeneous materials. To solve this problem, we propose an augmented multidimensional diffusion encoding (MDE) framework, which unlocks a novel encoding dimension to assess time-dependent diffusion specific to structures with different microscopic anisotropies. Our approach relies on spectral analysis of complex but experimentally efficient MDE waveforms. Two independent contrasts to differentiate features such as cell shape and size can be generated directly by signal subtraction from only three types of measurements. Analytical calculations and simulations support our experimental observations. Proof-of-concept experiments were applied on samples with known and distinctly different microstructures. We further demonstrate substantially different contrasts in different tissue types of a post mortem brain. Our simultaneous assessment of restriction size and shape may be instrumental in studies of a wide range of porous materials, enable new insights into the microstructure of biological tissues or be of great value in diagnostics.

Precise label-free leukocyte subpopulation separation using hybrid acoustic-optical chip.

Leukocyte subpopulations contain crucial physiological information; hence, precise and specific leukocyte separation is very important for leukemia diagnosis and analysis. However, conventional centrifugation and immunofluorescence-based separation methods are inaccurate and inconvenient due to the overlapping cell size and density or complex marking processes. Herein, we report a new label-free technology for precise leukocyte subpopulation separation by synergy of acoustic and optical technologies. Standing surface acoustic wave (SSAW) solved the problem of gentle and precise focusing of cells in optical systems. In addition, SSAW was used for the separation of granulocytes, which have evident size distinction from other components. In case of lymphocytes and monocytes, which have overlap in size/density, optical force could distinguish them accurately based on the RI difference, with the convenience of acoustic pre-focusing. In this experiment, separation of three types of leukocyte subtypes with considerable throughput and purity was conducted, through which we obtained 99% pure lymphocytes, 98% pure monocytes, and 95% pure granulocytes. Experimental results prove that the device has robust ability in separating leukocyte phenotypes and have the advantages of being non-invasive, label-free and precise. In the future, this convenient hybrid method will be a potential powerful tool for auxiliary clinical diagnosis and analysis.

Investigation for the role of CTX-III and microRNA-98 in diagnosis and treatment of osteoarthritis.

OBJECTIVE: Osteoarthritis is a joint degeneration and proliferative inflammatory disease caused by obesity, joint deformities, trauma, and other factors. C-terminal collagen (CTX) is associated with cartilage degradation, and healthy cartilage state is one of the factors that affect osteoarthritis. microRNA-98 (miRNA-98) plays a role in inflammation. This study aims to investigate the levels of CTX-III and miRNA-98 in patients with osteoarthritis and their potential clinical usage. PATIENTS AND METHODS: Osteoarthritis was diagnosed according to the inclusion and exclusion criteria for osteoarthritis. Patients with osteoarthritis admitted to Jining No. 1 People's Hospital and healthy volunteers were included in this study. ELISA and Western blot analysis were used to detect levels of type III collagen CTX (CTX-III). Real time PCR was used to measure levels of miRNA-98 in the serum of both patients and healthy volunteers. RESULTS: Levels of CTX-III protein in osteoarthritis patients were significantly higher than that of healthy volunteers (p = 0.0013). Levels of miRNA-98 in the serum of osteoarthritis patients were significantly higher compared to that of healthy volunteers (p = 0.0065). After treatment, levels of CTX-III protein and serum miRNA-98 in patients with osteoarthritis were significantly decreased (p = 0.014, p = 0.021). Levels of CTX-III protein and serum miRNA-98 in patients with osteoarthritis were significantly higher compared to that of healthy volunteers (p = 0.0013). CONCLUSIONS: Both of the CTX-III and microRNA-98 are potential diagnostic indicators for the osteoarthritis.

Real-time detection and monitoring of the drug resistance of single myeloid leukemia cells by diffused total internal reflection.

Real-time detection and monitoring of the drug resistance of single cells have important significance in clinical diagnosis and therapy. Traditional methods operate a number of times for each individual concentration, and innovation is required for the design of more simple and efficient manipulation platforms with necessary higher sensitivity. Here, we have developed a novel diffused total internal reflection (TIR) method to perform drug metabolism and cytotoxicity analysis of trapped myeloid leukemia cells. Molm-13 cells, a type of acute myeloid leukemia cell, were chosen and injected into the device and fittingly captured by cell traps. Differing from previous studies, a series of different concentrations of azelaic acid (AZA) drug could be used from 0 mM to 50 mM through convection and diffusion processes in a single chip, with each concentration region featuring 50 cells, with a total of 549 cell trapping units. Thanks to the high sensitivity of the TIR method, only cells with the same drug concentration could be illuminated in the detection process. By adjusting the incident angle, we could exactly detect and monitor the drug resistance of the cells using different drug concentrations and the experimental resolution of the drug concentration was as small as 5 mM. Images of the membrane integrity and morphology of the cells in the bright field were measured and we also monitored the cell viabilities in the dark field over 2 hours. The effects of AZA on the Molm-13 cells were explored in different concentrations at the single cell level. Compared with the results of the traditional MTT assay method, the experimental results are more simple and accurate. A cell death of 5% at an AZA concentration of 5 mM was observed after 30 minutes, while a concentration of 40 mM corresponded to a 98% cell death. The designed method in this study provides a novel toolkit to control and monitor drug resistance at the single cell level more easily with higher sensitivity and we believe it has significant potential application in single cell quality assessment and medicine analysis in clinical practice.

Molecular analysis of MLH1 variants in Chinese sporadic colorectal cancer patients.

Single nucleotide polymorphisms (SNPs) in mismatch repair genes, especially in the MLH1 gene, are closely associated with susceptibility to hereditary nonpolyposis colorectal cancer. However, few relevant findings are available regarding the association between sporadic colorectal cancer (SCRC) and SNPs of MLH1 in Chinese patients. Therefore, the present study aimed to describe the pathogenic association between three important MLH1 polymorphisms and SCRC in the Chinese population. Peripheral blood samples from 156 SCRC patients and 311 healthy controls were collected. DNA was purified from peripheral blood, and the V384D, R217C, and I219V polymorphisms were evaluated using high-resolution melting analysis and direct sequencing. The association between the three important MLH1 polymorphisms and clinical pathological features of the SCRC patients was analyzed. In addition, PMS2-MLH1 protein interactions were determined by co-immunoprecipitation (Co-IP) to determine the protein functional alteration induced by these SNPs. Among the three polymorphisms, V384D was significantly associated with the risk of SCRC (OR = 31.36, P < 0.0001). The allele frequencies were 4.81 and 0.16% in the SCRC group. No association was found between SCRC and R217C, or between SCRC and I219V. Moreover, the allele frequency of R217C was significantly higher in the SCRC patients younger than 60 years than in those older than 60 years. Co-IP showed that the MLH1 R217C, V384D, and I219V variants had relative binding abilities with PMS2 of 0.59, 0.70, and 0.80, respectively, compared with the wild-type. These findings suggest that MLH1 V384D could be a promising genetic marker for susceptibility to SCRC.

Molecular cloning and expression analysis of MAT1 gene in black tiger shrimp (Penaeus monodon).

MAT1 (ménage à trois 1), an assembly factor and targeting subunit of the CDK-dependent kinase (CAK), can regulate the cell cycle, transcription, and DNA repair. This study was intended to investigate the role of MAT1 in the reproductive maturation of black tiger shrimp (Penaeus monodon). In this study, the P. monodon MAT1 (PmMAT1) gene was identified and characterized. The full-length cDNA of PmMAT1 was 1490 bp in length with an open-reading frame of 993 bp corresponding to 330 amino acids. The temporal expression of PmMAT1 in various tissues was measured by quantitative real-time PCR with the highest expression observed in ovaries. In the ovaries, the PmMAT1 gene was continuously but differentially expressed during the maturation stages. Comparative analyses of MAT1, CDK7, and cyclin H in the CAK complex of P. monodon indicated that the expression of CDK7 and cyclin H coincided with that of MAT1 during the ovary maturation stages. Serotonin (5-HT) injection promoted the expression level of PmMAT1 in the ovaries of shrimp at 6-48 h post-injection. These results indicate that PmMat1 plays a prominent role in the process of ovarian maturation.

MicroRNA-26b inhibits osteosarcoma cell migration and invasion by down-regulating PFKFB3 expression.

MicroRNAs regulate target gene expression and are involved in cell proliferation, apoptosis, differentiation, tumor invasion, and cancer stem cell regulation, among other processes. MicroRNA-26b (miR-26b) is closely related to tumor occurrence and development. In this study, we analyzed miR-26b expression in osteosarcoma tissue, its effect on Saos-2 osteosarcoma cell proliferation and invasion, and its relationship with 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression. Osteosarcoma tissue was obtained from surgical patients and normal tissue adjacent to the tumor was used as a control. Real-time polymerase chain reaction was applied to detect miR-26b expression in cancer tissue and normal tissue. A vector expressing miR-26b was constructed and transfected into Saos-2. An MTT assay, cell invasion assay, and scratch experiment were used to analyze the effect of miR-26b on Saos-2 cell proliferation, invasion, and migration abilities. Western blotting analysis was performed to investigate the role of miR-26b on PFKFB3 expression. miR-26b expression in normal tissue was 7.55-fold higher than in osteosarcoma tissue (t = 10.20, P = 0.006). Compared with control tissue, miR-26b significantly inhibited osteosarcoma proliferation, migration, and invasion (P < 0.05). Western blotting results revealed that PFKFB3 protein expression decreased in Saos-2 cells after transfection with miR-26b. miR-26b was down-regulated in osteosarcoma tissue. miR- 26b may inhibit osteosarcoma cell proliferation, migration, and invasion by regulating PFKFB3 protein expression. miR-26b may have a tumor suppressor role in tumor occurrence and development.

Characterization and expression analysis of a cyclin B gene from black tiger shrimp (Penaeus monodon).

The open reading frame of black tiger shrimp (Penaeus monodon) cyclin B (Pmcyclin B) was identified, based on cDNA sequence registered in GenBank (accession No. EF015590). The target sequence was 1206 bp, corresponding to 401 amino acids. Two conserved signature sequences of the cyclin B gene family were found in the Pmcyclin B deduced aa sequence. Temporal expression of Pmcyclin B in different tissues, including ovary, lymphoid organ, brain, blood, muscle, heart, gill, hepatopancreas, and intestine, were quantified by quantitative real time PCR. Messenger RNA expression levels of Pmcyclin B were greatest in the ovary, compared to other tissues (P < 0.05). Temporal expression of Pmcyclin B in the ovary at six different developmental stages was investigated by real-time PCR; no significant difference was observed (P < 0.05). Recombinant Pmcyclin B protein and its polyclonal antibody were successfully produced. Western blot analysis revealed differential expression of Pmcyclin B in ovaries in developmental stages II to IV; a positive signal (45 kDa) was observed in all ovarian stages assessed, but was most intense at stage III. Pmcyclin B protein was assessed by immunohistochemistry and was localized to the cytoplasm of prophase oocytes at stage II and enriched in the nuclei of pro-metaphase oocytes at stages III and IV. Results from this study indicate that Pmcyclin B is constitutively expressed and plays an important role in ovarian maturation in P. monodon.

Serum ferritin is associated with carotid atherosclerotic plaques but not intima-media Thickness in patients with abnormal glucose metabolism.

BACKGROUND: We investigated the association between serum ferritin and carotid artery lesions in populations with abnormal glucose metabolism. METHODS: We included 70 participants with abnormal glucose metabolism and 170 participants with normal glucose metabolism and measured their baseline serum ferritin levels. During follow-up carotid intima-media thickness and carotid plaque were evaluated. RESULTS: Serum ferritin levels were higher in the participants with abnormal glucose metabolism (p<0.01). We further divided the patients with abnormal glucose metabolism into subgroups with and without intima-media proliferation, and found that ferritin was excluded from the final equation in the logistic regression. Furthermore, age, waist circumference, ferritin, 2h-PG, and total cholesterol were significantly different between the subgroups with and without carotid plaque. When the above data were included in a logistic regression model, the p values obtained for age, ferritin, and 2h-PG were 0.004, 0.032, and 0.011, respectively. CONCLUSIONS: In the Chinese population, serum ferritin levels are significantly increased in patients with abnormal glucose metabolism. The carotid intima-media thickness showed no independent relationship with serum ferritin in patients with abnormal glucose metabolism. However, high serum ferritin is an important risk factor for carotid atherosclerosis in these patients.

Serological identification and bioinformatics analysis of immunogenic antigens in multiple myeloma.

Identifying appropriate tumor antigens is critical to the development of successful specific cancer immunotherapy. Serological analysis of tumor antigens by a recombinant cDNA expression library (SEREX) allows the systematic cloning of tumor antigens recognized by the spontaneous autoantibody repertoire of cancer patients. We applied SEREX to the cDNA expression library of cell line HMy2, which led to the isolation of six known characterized genes and 12 novel genes. Known genes, including ring finger protein 167, KLF10, TPT1, p02 protein, cDNA FLJ46859 fis, and DNMT1, were related to the development of different tumors. Bioinformatics was performed to predict 12 novel MMSA (multiple myeloma special antigen) genes. The prediction of tumor antigens provides potential targets for the immunotherapy of patients with multiple myeloma (MM) and help in the understanding of carcinogenesis. Crude lysate ELISA methodology indicated that the optical density value of MMSA-3 and MMSA-7 were significantly higher in MM patients than in healthy donors. Furthermore, SYBR Green real-time PCR showed that MMSA-1 presented with a high number of copy messages in MM. In summary, the antigens identified in this study may be potential candidates for diagnosis and targets for immunotherapy in MM.

Chromatographic purification and properties of a therapeutic human protein C concentrate.

Protein C deficiency (inherited and acquired) has a relatively high incidence rate in the general population worldwide. For many years, protein C deficient patients have been treated with fresh frozen plasma, prothrombin complex concentrates, heparin or oral anticoagulants, which all have clinical drawbacks. We report the production process of a highly purified human protein C concentrate from 1500 l of cryo-poor plasma by a four-step chromatographic procedure. After DEAE-Sephadex adsorption, protein C was separated from clotting factors II, VII and IX by DEAE-Sepharose FF and further purified, using a new strategy, by an on-line chromatographic system combining DMAE-Fractogel and heparin-Sepharose CL-6B. In addition, the product was treated against viral risks by solvent-detergent and nanofiltration on 15-nm membranes. The protein C concentrate was essentially free of other vitamin K-dependent proteins. Proteolytic activity was undetectable. Neither activated protein C, prekallikrein activator, nor activated vitamin K-dependent clotting factors were found resulting in good stability of the protein C activity. In vitro and in vivo animal tests did not reveal any sign of potential thrombogenicity. The final freeze-dried product had a mean protein C concentration of 58 IU/ml and a mean specific activity of 215 IU/mg protein, corresponding to over 12000-fold purification from plasma. Therefore, this concentrate appears to be of potential benefit for the treatment of protein C deficiency.

[Reconstruction of heel by reversed island fibular musculocutaneous flap].

OBJECTIVE: To explore the anatomical basis of blood supply and heel reconstruction by reversed island fibular musculocutaneous flap. METHODS: The blood supply of fibular musculocutaneous flap and the biomechanical characteristics of heel were studied by anatomical examination. One case with right heel full defect because of explosion injury was repaired by transfer of reversed island fibular vessels. The fibular flap was 14 cm in length with part of peroneus muscle and long flexor muscle of great toe. RESULTS: The lower part of fibular artery had plentiful anastomosis with anterior tibial artery and posterior tibial artery, which could provide ideal reversed blood supply. The rotatory point of vessel pedicle could be chosen according to the need of operation. The lowest site might be above 6 cm to lateral malleolus, and the vessel pedicle was 20 cm in length. The morphological feature of the reversed island fibular musculocutaneous flap was suitable to the biomechanical character of heel. The patient achieved satisfactory clinical result, the musculocutaneous flap survived well for 10 months of follow-up. CONCLUSION: The reversed island fibular musculocutaneous flap provide a new method for repairing the severe heel defect, especially in full defect of calcaneus and cuboid bone.

[Immunogold labeling electron microscopy showing vimentin filament anchored on nuclear pore complex].

The relationship between intermediate filament and nucleus is an important question to be solved. By combining sequential cell fractionation with immunoblotting, we showed that the intermediate filament protein in turkey erythrocyte is vimentin. Using pre-embedment immunogold labeling techniques together with sequential cell fractionation, we showed that cytoplasmic intermediate filaments in turkey erythrocyte are labeled specifically by rabbit anti-vimentin antibody-protein A-gold. Moreover, we showed that the cytoplasmic filaments anchored on nuclear pore complex were also labeled by rabbit anti-vimentin antibody-protein A-gold specifically. Our results demonstrated that cytoplasmic filaments anchored on nuclear pore complex was vimentin filament. The experiments indicated that vimentin filament may be anchored on nuclear pore complex by binding with Nup 180 and intermediate filament may be involved in nuclear transportation.

[Vimentin and Nup 180 in vitro binding assay].

In order to investigate relationship between vimentin and nuclear pore complex, we examined binding ability of vimentin, expressed in E. coli, with nucleoporin, isolated from rat liver nuclei, in vitro. Negative staining electron microscopy showed that the vimentin expressed in bacteria assembled 10 nm filament in vitro. SDS-PAGE and western blotting showed that Nup 180 bind to vimentin in binding assay in vitro. Combining immunogold labeling and negative staining electron microscopy techniques, we showed that Nup 180 bind on the 10 nm vimentin filaments. The experiment results indicated that vimentin filament may be anchored on nuclear pore complex in vivo by binding with Nup 180.

Purification of factor VIII/von Willebrand factor from human plasma on immobilized lentil lectin.

Human factor VIII/von Willebrand factor (FVIII/vWF) was shown to bind to immobilized lectins from Arachis, Ulex, Concanavalia, and Lens species. The protein/lectin interaction displayed higher affinities for the lectins from the last two species. However, the Lens culinaris lectin immobilized on Sepharose 4B (LCA-Sepharose) provided a more selective and flexible affinity system for the purification of FVIII/vWF than Concanavalia lectin. Chromatography on LCA-Sepharose of a purified FVIII containing a small proportion of vWF required a weak acidic medium (pH 6.3) and relatively slow kinetics (about 20 cm/h flow rate). The bound FVIII was specifically dissociated from LCA-Sepharose by methyl-alpha-D-mannopyranoside, and to a lesser extent by other monosaccharides such as D-glucose, methyl-alpha-D-glucopyranoside, D-mannose, and D-galactose. Application to whole plasma resulted in a capacity for FVIII/vWF of about 28 U/ml gel. Specific activities for eluted FVIII and vWF were 3 and 2.2 IU/mg protein, respectively, with respective FVIII:c and vWF:RCo recoveries of 57 and 40% from starting plasma. Coagulation factors II, X, VII, IX, V, and XI and fibrinogen were eliminated in the LCA matrix breakthrough fraction, improving the stability of the purified FVIII molecule. Purity of the LCA eluate was further enhanced by ion-exchange chromatography on DEAE-Fractogel TSK 650 M which reduced the amount of protein contaminants and provided a FVIII/vWF fraction with higher specific activity (45-80 IU/mg protein depending on the chromatographic conditions). The overall process yield was 45 and 25% for FVIII:c and vWF:RCo, respectively.