A study of the moving rate of positive results for use in a patient-based real-time quality control program on a procalcitonin point-of-care testing analyzer.

OBJECTIVE: To establish an applicable and highly sensitive patient-based real-time quality control (PBRTQC) program based on a data model constructed with patients' results of a procalcitonin point-of-care testing (POCT) analyzer. METHODS: Patients' results were retrospectively collected within one year. The Excel software was used to establish quality control (QC) programs of the moving average (MA) and the moving rate of positive results (MR). A Monte Carlo simulation was used to introduce positive and negative biases between 0.01 and 1 ng/ml at random points of the testing data set. Different parameters were used to detect the biases, and the detection efficiency was expressed using the median number of patient samples affected until error detection (MNPed). After comparing the MNPeds of different programs, MA and MR programs with appropriate parameters were selected, and validation plots were generated using MNPeds and maximum number of the patient samples affected (MAX). ß curves were generated using the power function of the programs, the performances were compared with that of the conventional QC program. RESULTS: Neither the conventional QC nor MA program was sensitive to small bias, While MR program can detect the minimum positive bias of 0.06 ng/ml and negative of 0.4 ng/ml at an average daily run size of 10 specimens, with FRs < 1.0%, ßs < 1%. CONCLUSION: The MR program, which is more sensitive to small biases than conventional QC and MA programs, with low FR and ß. As such, it can be used as a PBRTQC program with high performance.

Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus.

An outbreak of African swine fever (ASF) in China in 2018 caused substantial economic losses to the swine industry. To accurately diagnose clinical infection with ASF virus (ASFV), we developed a TaqMan probe-based duplex real-time PCR that simultaneously detected two discontinuous genes in the virus genome, thereby preventing the inaccurate results obtained with only one reaction. Two sets of ASFV gene-specific primers, along with two fluorescent TaqMan probes were designed to target conserved regions of the B646L and B438L genes. This method had high sensitivity and specificity, with a limit of detection of 10 copies/µL, and it did not cross-react with the genomes of other viral pathogens that affect pigs (i.e., CSFV, PRRSV, PEDV, PRV, PPV and PCV2). Overall, 180 clinical samples from ASFV-infected pig farms were used to compare this method with a commercial kit, which yielded excellent consistency (98.3%). This new diagnostic method should greatly improve the efficiency of ASFV surveillance and reduce economic losses, providing benefits for both animal and public health.

Magnetic Noise Enabled Biocompass.

The discovery of magnetic protein provides a new understanding of a biocompass at the molecular level. However, the mechanism by which magnetic protein enables a biocompass is still under debate, mainly because of the absence of permanent magnetism in the magnetic protein at room temperature. Here, based on a widely accepted radical pair model of a biocompass, we propose a microscopic mechanism that allows the biocompass to operate without a finite magnetization of the magnetic protein in a biological environment. With the structure of the magnetic protein, we show that the magnetic fluctuation, rather than the permanent magnetism, of the magnetic protein can enable geomagnetic field sensing. An analysis of the quantum dynamics of our microscopic model reveals the necessary conditions for optimal sensitivity. Our work clarifies the mechanism by which magnetic protein enables a biocompass.

Simultaneous Preconcentration and Desalting of Organic Solutes in Aqueous Solutions by Bubble Bursting.

Significant efforts are being made to develop more practical and versatile approaches for the preconcentration and purification of complex chemical samples. Inspired by the naturally occurring enrichment of organic compounds in sea aerosols, in this study we demonstrate the potential of induced bubble bursting as an approach for the preconcentration of organic solutes in various aqueous solutions. Apart from the preconcentration of organics, notable decrease in the concentration of metal salt components was discovered for the first time. On the basis of a series of model experiments, the phenomenon has been attributed to intermolecular competition at the surface interface of rising bubbles. Overall, our results indicate the high versatility and simplicity of the bubble bursting approach for the simultaneous preconcentration and desalting of organic solutes in aqueous solutions for mass spectrometry, chromatography, optical detection, and other fields of analysis.

Combination of aqueous two-phase flotation and inverse transition cycling: Strategies for separation and purification of recombinant ß-glucosidase from cell lysis solution.

This work was developed to solve the problems of the restriction of non-specific adsorption and time-dependent denaturation in the purification of recombinant proteins by multistage chromatographic procedures. A novel purification method (ATPF-ITC) which combining aqueous two-phase flotation (ATPF) with inverse transition cycling (ITC) was established and used to efficiently purify recombinant ß-glucosidase (GLEGB) from cell lysis solution. First, GLEGB would preferentially adsorb on the nitrogen bubble interface relied on the hydrophobic property of the graphene-binding (GB) tag and enter into the top phase of ATPF. Second, GLEGB was achieved further purification by one-round ITC method based on the thermosensitive of the elastin-like polypeptide (ELP) tag. Consequently, the enzymatic activity recovery of GLEGB was 124.92% ± 0.83%, and the purification factor reached 24.26 ± 0.22. The purification results remained stable after six polymer cycles, and the process of ATPF-ITC had no negative effect on the structure of recombinant protein.

A high efficiency method combining metal chelate ionic liquid-based aqueous two-phase flotation with two-step precipitation process for bromelain purification.

This work was to develop a cost-effective and sustainable method which included metal chelate ionic liquid-based aqueous two-phase flotation (IL-based ATPF) and a two-step precipitation process for purifying bromelain from pineapple. Firstly, the metal chelate IL-based ATPF with a copper chelate-functionalized thermosensitive block copolymer (L64-IDA-Cu(II)) as trapping agent was used as the primary purification to obtain the L64-IDA-Cu(II)-bromelain complex. Secondly, the two-step precipitation process based on the thermosensitive properties of the L64-IDA-Cu(II) was mainly carried out to achieve the further purification of bromelain. According to a series of optimal experiments, the enzymatic activity recovery of final bromelain was 95.22 ±â€¯0.04%, and the purification factor reached 6.56 ±â€¯0.03. The results of recycling of ILs and the trapping agent were satisfactory. Furthermore, the conclusions of comparison with other methods proved the superiority of this method. This novel recycling purification method has a goodindustrial prospect in future.

Horseradish peroxidase immobilized on the magnetic composite microspheres for high catalytic ability and operational stability.

In the present study, a novel and efficient immobilization for horseradish peroxidase (HRP) had been developed by using 6-arm magnetic composite microsphere (Fe3O4@PAA-6-arm-PEG-NH2) containing 6-arm polyethylene glycol (6-arm-PEG-NH2) and Fe3O4. The morphology and chemical properties of Fe3O4@PAA-6-arm-PEG-NH2 were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectra (FTIR), X-ray powder diffraction (XRD), vibrating sample magnetometer (VSM) and thermogravimetric analysis (TGA). The immobilization conditions and loading capacity of the carrier toward HRP were also investigated. According to the results, the optimum immobilization conditions were as follows: glutaraldehyde concentration of 0.10 mol L-1, the carrier concentration of 7 mg L-1, the temperature of 35 °C and immobilization time of 180 min. The loading capacity of the immobilized HRP arrived to 139.82 mg g-1 (RSD = 0.87%). Compared with free HRP, the stability of the immobilized HRP had been enhanced in a wide range of temperature and pH. The activity of the immobilized HRP retained 71.05% of its initial activity after 60 days storage. It was also found that the activity of the immobilized HRP retained 61.06% of its initial activity after eight times of successive reuse. The immobilized HRP could hydrolyze phenol to 94.4% within 10 min. It proved that the immobilized HRP could improve the performance of HRP, which had a good prospect in biological application.

Synergetic effect of Ni2+ and 5-acrylamidobenzoboroxole functional groups anchoring on magnetic nanoparticles for enhanced immobilization of horseradish peroxidase.

In this work, a novel Ni2+&AABOB bifunctional core-shell magnetic composite nanoparticles (MNPs), Fe3O4@p(AABOB-co-AIM-Ni2+), were synthesized for the immobilization of horseradish peroxidase (HRP). Two kinds of interaction are simultaneously incorporated to improve the stability and reusability of immobilized HRP: one is the coordination between Ni2+ and amide group in HRP, and the other is the interaction of the wulff-type boronic acid group with cis-diol-containing HRP which belongs to a type of glycoproteins. Accordingly, the proposed approach achieved high enrichment capacity (174.75 mg/g, protein/beads), and the activity of immobilized HRP still achieved 80% after reuse for 5 cycles. The degradation efficiency of phenol in the presence of immobilized HRP reached 93% within 10 min, which was significantly higher than that of free HRP only about 50% within 20 min. In addition, the immobilized HRP can be easily separated from the reaction solution by an external magnetic field.

[Determination of trace amounts of oxytetracycline in surface water samples by bubble enrichment-high performance liquid chromatography].

A method was established to determine the trace amounts of oxytetracycline (OTC) in surface water samples by bubble enrichment-high performance liquid chromatography (HPLC). A novel sample pretreatment method, bubble enrichment, was used to enrich trace amounts of oxytetracycline in aqueous solutions, and the effects of bubble enrichment conditions on the enrichment efficiency was investigated. Enrichment factor of oxytetracycline had a maximum of 37.06, RSD was 4.8% (n=11), and LOD was 0.038 mg/L under the optimized conditions of bubble enrichment and chromatography. The method was applied to the determination of oxytetracycline in surface water samples, with an average recovery of 101.9%. It is obvious that bubble enrichment has a positive effect on the enrichment of oxytetracycline, and hence, can be combined with chromatography to realize rapid, sensitive, and accurate detection of oxytetracycline in surface water samples. Additionally, the bubble enrichment method used in this paper does not require any organic solvent for the pretreatment of samples, and furthermore, the device is simple, inexpensive, and easy to operate; therefore, it is a very green sample pretreatment method with great research and promotion value. It is expected to be applicable to the analysis of other trace substances in complex samples.

Detection of creatinine in exhaled breath of humans with chronic kidney disease by extractive electrospray ionization mass spectrometry.

Exhaled breath contains chemicals that have a diagnostic value in human pathologies. Here in vivo breath analysis of creatinine has been demonstrated by constructing a novel platform based on extractive electrospray ionization mass spectrometry (EESI-MS) without sample pretreatment. Under optimized experimental conditions, the limit of creatinine detection in breath was 30.57 ng L(-1), and the linear range of detection was from 0.3 µg L(-1) to 100 µg L(-1). The concentration range of creatinine in the exhaled breath of 50 volunteers with chronic kidney disease was from 42 pptv to 924 pptv, and the range of the relative standard deviations was from 9.3% to 19.2%. The method provides high sensitivity, high specificity and high speed for semi-quantitative analysis of creatinine in exhaled human breath.

Selection, proliferation and differentiation of bone marrow-derived liver stem cells with a culture system containing cholestatic serum in vitro.

AIM: To explore the feasibility of direct separation, selective proliferation and differentiation of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. METHODS: Whole bone marrow cells of rats cultured in routine medium were replaced with conditioning selection media containing 20 mL/L, 50 mL/L, 70 mL/L, and 100 mL/L cholestatic sera, respectively, after they attached to the plates. The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures. Then the selected BDLSC were induced to proliferate and differentiate with the addition of hepatocyte growth factor (HGF). The morphology and phenotypic markers of BDLSC were characterized using immunohistochemistry, RT-PCR and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay. RESULTS: Bone marrow cells formed fibroblast-like but not hepatocyte-like colonies in the presence of 20 mL/L cholestatic serum. In 70 mL/L cholestatic serum, BDLSC colonies could be selected but could not maintain good growth status. In 100 mL/L cholestatic serum, all of the bone marrow cells were unable to survive. A 50 mL/L cholestatic serum was the optimal concentration for the selection of BDLSC at which BDLSC could survive while the other populations of the bone marrow cells could not. The selected BDLSC proliferated and differentiated after HGF was added. Hepatocyte-like colony-forming units (H-CFU) then were formed. H-CFU expressed markers of embryonic hepatocytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors (HNF-1alpha and HNF-3beta). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selected medium containing cholestatic serum can select BDLSC from whole bone marrow cells. It will be a new way to provide a readily available alternate source of cells for clinical hepatocyte therapy.

Passage of bone-marrow-derived liver stem cells in a proliferating culture system.

AIM: To explore the feasibility of passage of bone-marrow-derived liver stem cells (BDLSCs) in culture systems that contain cholestatic serum. METHODS: Whole bone marrow cells of rats were purified with conditioning selection media that contained 50 mL/L cholestatic serum. The selected BDLSCs were grown in a proliferating culture system and a differentiating culture system. The culture systems contained factors that stimulated the proliferation and differentiation of BDLSCs. Each passage of the proliferated stem cells was subjected to flow cytometry to detect stem cell markers. The morphology and phenotypic markers of BDLSCs were characterized using immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay. RESULTS: The conditioning selection medium isolated BDLSCs directly from cultured bone marrow cells. The selected BDLSCs could be proliferated for six passages and maintained stable markers in our proliferating system. When the culture system was changed to a differentiating system, hepatocyte-like colony-forming units (H-CFUs) were formed. H-CFUs expressed markers of embryonic hepatocytes (alpha-fetoprotein, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors 1alpha and -3beta). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: BDLSCs can be selected directly from bone marrow cells, and pure BDLSCs can be proliferated for six passages. The differentiated cells have hepatocyte-like phenotypes and functions. BDLSCs represent a new method to provide a readily available alternate source of cells for clinical hepatocyte therapy.

Modified technique of pancreaticojejunal anastomosis with invagination following pancreaticoduodenectomy: a cohort study.

BACKGROUND: Leakage from pancreatic anastomoses remains the single most important morbidity after pancreaticoduodenectomy and contributes to prolonged hospitalization and mortality. This observational cohort study reported the surgical outcome of a modified invagination technique of pancreaticojejunostomy after pancreaticoduodenectomy. METHODS: Between December 2001 and December 2007, a total of 52 consecutive patients underwent elective pancreaticoduodenectomy for benign or malignant pathologies of the pancreas or the periampullary region in a tertiary referral center. All patients underwent our modified invagination technique of pancreaticojejunostomy regardless of the characteristics of the pancreatic stump. Data were collected prospectively. RESULTS: The mean hospital stay was 12.6 +/- 3.2 days. The incidence of overall surgical complications was 9.6%. No patient developed pancreatic fistula. One patient (1.9%) died of respiratory failure on postoperative day 7. CONCLUSIONS: We reported our pancreaticojejunostomy anastomosis technique with a pancreatic fistula rate of 0% and low intra-abdominal complication rate. The favorable results of this technique warrant further investigation in large prospective cohort studies and prospective randomized controlled studies.