Development and multicenter performance evaluation of fully automated SARS-CoV-2 IgM and IgG immunoassays.

Objectives: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread globally. The laboratory diagnosis of SARS-CoV-2 infection has relied on nucleic acid testing; however, it has some limitations, such as low throughput and high rates of false negatives. Tests of higher sensitivity are needed to effectively identify infected patients. Methods: This study has developed fully automated chemiluminescent immunoassays to determine IgM and IgG antibodies to SARS-CoV-2 in human serum. The assay performance has been evaluated at 10 hospitals. Clinical specificity was evaluated by measuring 972 hospitalized patients and 586 donors of a normal population. Clinical sensitivity was assessed on 513 confirmed cases of SARS-CoV-2 by RT-PCR. Results: The assays demonstrated satisfied assay precision with coefficient of variation of less than 4.45%. Inactivation of specimen did not affect assay measurement. SARS-CoV-2 IgM showed clinical specificity of 97.33 and 99.49% for hospitalized patients and the normal population respectively, and SARS-CoV-2 IgG showed clinical specificity of 97.43 and 99.15% respectively. SARS-CoV-2 IgM showed clinical sensitivity of 82.54, 92.93, and 84.62% before 7 days, 7-14 days, and after 14 days respectively, since onset of symptoms, and SARS-CoV-2 IgG showed clinical sensitivity of 80.95, 97.98, and 99.15% respectively at the same time points above. Conclusions: We have developed fully automated immunoassays for detecting SARS-CoV-2 IgM and IgG antibodies in human serum. The assays demonstrated high clinical specificity and sensitivity, and add great value to nucleic acid testing in fighting against the global pandemic of the SARS-CoV-2 infection.

Effect of sustained training on glycolysis and fatty acids oxidation in swimming muscles and liver in juvenile tinfoil barb Barbonymus schwanenfeldii (Bleeker, 1854).

The present study examines the effect of sustained exercise on glycolysis and fatty acids oxidation in the swimming muscles and liver in juvenile tinfoil barb (Barbonymus schwanenfeldii). The subjects were divided into one control group (water current speed of 0.0 bl s-1, body length per second) and two training groups (1.0 and 2.0 bl s-1), respectively. Results showed that the glycolysis was stimulated by high-speed training in the white muscle, accompanied by significantly increased activities of hexokinase, pyruvate kinase, and reduction in glycogen contents in training groups (P < 0.05). On the contrary, the extreme fatty acids oxidation was observed in the red muscle in high-speed training group, showed significant reduction in crude lipid content with a significant increase in the activities of hormone-sensitive lipase, ß-hydroxyacyl-CoA dehydrogenase, and cytochrome C oxidase in 2.0 bl s-1 group (P < 0.05). In addition, the saturated fatty acids in the red muscle and monounsaturated fatty acids in the white muscle were used preferentially during sustained training, respectively. Furthermore, the glycogen content within the liver was also significantly reduced with increasing training intensities (P < 0.05). These results suggested that glycogen and fatty acids were all used as a fuel to support sustained swimming in two functional muscles in B. schwanenfeldii, but higher glycolysis and fatty acids oxidation were seen in white and red muscles during high-speed swimming, respectively. Furthermore, the hepatic glycogen played an important role in the supply of energy in sustained training periods in B. schwanenfeldii.

Effects of ration levels on growth and reproduction from larvae to first-time spawning in the female Gambusia affinis.

Somatic growth and reproduction were examined in individual laboratory-grown female Gambusia affinis fed with high (H), medium (M) and low (L) ration levels from birth to the first-time spawning. Results showed that the body length and weight, condition factor (CF), wet weight gain (WG(w)), specific growth rate in wet weight (SGR(w)) and ration levels in terms of energy (RL(e)) decreased significantly (p < 0.05) with decreasing ration levels from birth to first-time spawning. On the contrary, the food conversion efficiency in terms of energy (FCE(e)) increased significantly (p < 0.05) with the decreasing ration levels from birth to first-time sexual maturity. Furthermore, higher percentages of energy intake from food were allocated to somatic and gonad growth in M and L groups compared to the H group before sexual maturity; In addition, the time for first-time spawning in groups M and L was longer than that of the H group. As a result, the gonad-somatic index (GSI) and oocytes/embryos weight in M and L groups were similar to that of the H group, although the ovary weight and oocytes/embryos numbers were all lower than that of the H group. Also, similar growth performances were observed in second-generation offspring, which were produced by female parents fed with different ration levels. These findings suggest that the female G. affinis could produce a number of healthy offspring under conditions of food restriction, and that this could be achieved by increasing the energy allocated to gonad development, reducing fecundity and delaying spawning time. These life strategies ensured that G. affinis could survive and thrive in adverse environmental conditions and exhibit characteristics of invasive fish species.

Purification and characterization of mutant miniPlasmin for thrombolytic therapy.

BACKGROUND: Previous animal studies by us and others have indicated that catheter-administered plasmin or its des-kringle derivatives may be more appropriate alternatives to plasminogen activators for treating thrombolytic diseases, since it has a very short serum half-life and therefore does not result in hemorrhaging. We have previously produced recombinant miniPlasmin (mPlasmin) that was proven suitable for treating peripheral arterial occlusion in animal models. However, our previous results showed that non-specific cleavage at position K698 of mPlasmin during activation hindered the further development of this promising therapeutic candidate. In order to minimize or eliminate the non-specific cleavage problem, we performed saturation mutagenesis at the K698 position to develop a mutant form of mPlasmin for thrombolytic therapy. METHODS: We changed K698 to 16 other amino acids, with preferred E. coli codons. Each of these mutants were expressed in E. coli as inclusion bodies and then refolded, purified, and subsequently characterized by detailed kinetic assays/experiments/studies which identified highly active mutants devoid of non-specific cleavage. RESULTS: Activation studies indicated that at those conditions in which the wild type enzyme is cut at the non-specific position K698, the active mutants can be activated without being cleaved at this position. CONCLUSIONS: From the above results, we selected two mutants, K698Q and K698N, as our lead candidates for further thrombolytic drug developments. The selected mutants are potentially better therapeutic candidates for thrombolytic therapy.

1,10-Phenanthroline-based periodic mesoporous organosilica: from its synthesis to its application in the cobalt-catalyzed alkyne hydrosilylation.

1,10-Phenanthroline (Phen) is a typical ligand for metal complexation and various metal/Phen complexes have been applied as a catalyst in several organic transformations. This study reports the synthesis of a Phen-based periodic mesoporous organosilica (Phen-PMO) with the Phen moieties being directly incorporated into the organosilica framework. The Phen-PMO precursor, 3,8-bis[(triisopropoxysilyl)methyl]-1,10-phenanthroline (1a), was prepared via the Kumada-Tamao-Corriu cross-coupling of 3,8-dibromo-1,10-phenanthroline and [(triisopropoxysilyl)methyl]magnesium chloride. The co-condensation of 1a and 1,2-bis(triethoxysilyl)ethane in the presence of P123 as the template surfactant afforded Phen-PMO 3 with an ordered 2-D hexagonal mesoporous structure as confirmed by nitrogen adsorption/desorption measurements, X-ray diffraction, and transition electron microscopy. Co(OAc)2 was immobilized on Phen-PMO 3, and the obtained complex showed good catalytic activity for the hydrosilylation reaction of phenylacetylene with phenylsilane.

[Application of magnetic immunofluorescence assay based on microfluidic technology to detection of Epstein-Barr virus].

Early diagnosis of Epstein-Barr virus (EBV) can reduce the risk of major illnesses. Disadvantages of EBV antibody detection methods that are commonly used clinically include lengthy assay time, need for a lot of reagent, and low efficiency. Compared with traditional detection methods, microfluidics technology offers high throughput, low reagent consumption, less bio-contamination, and a higher degree of automation. Advantages of magnetic immunofluorescence technology include high detection efficiency and a strong signal. The combined advantages of the two methods can compensate for the shortcomings of traditional methods. In the present study, polymethyl methacrylate (PMMA) as the raw material was subjected to laser cutting and vacuum hot pressing to quickly obtain chips. Magnetic beads labeled with antigen and fluorescent microspheres labeled with anti-human antibody were then rapidly lyophilized into microspheres by freeze-drying and embedded into the chips. After incubation and cleaning, the last step was detection. Image J software was used to analyze the mean fluorescence intensity and obtain negative or positive test results. To determine the precision of the chip, high- and low-value samples of each item were retested 10 times. The mean values were calculated to obtain the relative standard deviation (RSD) for several common pathogens. Furthermore, the coincidence rate of clinical samples was tested using a chemiluminescence immunoassay (CLIA) to determine the potential clinical application value. The RSD of the precision test for each item was <10%, indicating good precision. The precision of the accelerated stability test was not verified. Specificity test results revealed no cross-reaction with some common pathogen antibodies, indicating good specificity. It remains to be verified whether the antibodies detected by this method cross-react with other herpes simplex viruses, such as types 1 and 2, Kaposi's sarcoma-associated virus, and human herpes virus type 6 and 7. Of the 121 clinical samples tested, statistical analysis of the data indicated good agreement with the chemiluminescence immunoassay in clinical trials. EB viral capsid antigen (EB VCA) IgG positive coincidence rate was 95.77% (68/71), the negative coincidence rate was 86% (43/50) (Kappa=0.828, P<0.05), the limit of detection (LOD) was 1.92 U/mL, and the linear range was 1.92 to 200 U/mL. The EB VCA IgA positive coincidence rate was 92% (46/50), negative coincidence rate was 92.96% (66/71) (Kappa=0.847, P<0.05), LOD was 2.79 U/mL, and the linear range was 2.79 to 200 U/mL. The positive coincidence rate of EB nuclear antigen 1 (EB NA1) IgG was 92.96% (66/71), the negative coincidence rate was 92% (46/50) (Kappa=0.847, P<0.05), the LOD was 3.13 U/mL, and the linear range was 3.13 to 200 U/mL. The positive coincidence rate of EB NA1 IgA was 90% (45/50), the negative coincidence rate was 91.55% (65/71) (Kappa=0.813, P<0.05), the LOD was 1.53 U/mL, and the linear range was 1.53 to 200 U/mL. Compared with the traditional enzyme-linked immunosorbent assay, the novel method featured a shorter detection time, reduced use of reagent, high degree of automation, and less bio-contamination. Compared with CLIA, advantages of the novel method include multi-item combined detection, long luminescence time, and simple use as a basic health service. Compared with silicon and ceramic microfluidic chips, advantages of the selected PMMA material include low processing cost, short processing time, simple processing technology, and easy industrialization. A refinement that can still be made include the use of molding instead of laser cutting technology, which can further shorten the chip processing time. In summary, a microfluidic detection platform was initially built to provide a rapid, sensitive, simple, highly automated, and easy to be used by basic health service for the quantitative combined detection of EBV VCA and EB NA1 IgG and IgA.

Oxidation-resistant and thermostable forms of alpha-1 antitrypsin from Escherichia coli inclusion bodies.

Native α1-antitrypsin (AAT) is a 52-kDa glycoprotein that acts as an antiprotease and is the physiological inhibitor of neutrophil serine proteases. The main function of AAT is to protect the lung from proteolytic damage induced by inflammation. AAT deficiency (AATD) is a codominant autosomal disorder caused by pathogenic mutations in SERPINA1 gene, leading to reduced levels of serum AAT. The deficiency is known to increase the risk of pulmonary emphysema and chronic obstructive pulmonary disease as a consequence of proteolytic imbalance induced by inflammation, associated in many instances with cigarette smoking and other environmental hazards. Currently, the available therapy for lung disease associated with AATD is serum purified human AAT injected into patients on a weekly basis. It would be advantageous to replace serum-derived AAT with a recombinant version which is stable and resistant to oxidation. We have expressed AAT in Escherichia coli as inclusion bodies and developed a highly efficient refolding and purification process. We engineered a series of mutant forms of AAT to achieve enhance thermostability and oxidation resistance. Moreover, we synthesized an active form of AAT via cysteine-pegylation to achieve a markedly extended half-life in vivo. The resulting molecule, which retains comparable activity to the wild-type form, is expected to be an improved therapeutic agent for treating hereditary emphysema. In addition, the molecule may also be used to treat other types of emphysema caused by smoking, cystic fibrosis, pulmonary hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease.

Differences in swimming ability and its response to starvation among male and female Gambusia affinis.

To explore the differences in the swimming ability and environmental adaptive abilities between male and female Gambusia affinis, we assessed the differences in burst swimming speeds (Uburst), critical swimming speeds (Ucrit) and their related fin areas, and consumption of energy substances after starvation at 0 (control group), 15, 30, 45, and 60 days, respectively. The results showed that the pectoral and caudal fin areas did not differ significantly between male and female G. affinis However, the dry mass, condition factors, and absolute contents of glycogen, lipids, and proteins were significantly elevated in females in the control group (P<0.05), whereas Uburst and Ucrit were significantly low (P<0.05). After starvation of 60 days, the rate of consumption of lipids was significantly low in the females (P<0.05). Although Uburst and Ucrit decreased linearly with increased duration of starvation, the coefficient of linear equation between Ucrit and starvation time was significantly lower in females than males (P<0.05). These findings indicated that low body mass and condition factors reduce the relative bear load and moving resistance that causes high swimming performance in male G. affinis High contents of energy substances and low rate of consumption of lipids result in stable Ucrit in females during hunger.

Structural conservation and food habit-related liver expression of uncoupling protein 2 gene in five major Chinese carps.

The full-length cDNA of grass carp (Ctenopharyngodon idellus) and silver carp (Hypophthalmichthys molitrix) uncoupling protein 2 (UCP2) was obtained from liver. The grass carp UCP2 cDNA was determined to be 1152 bp in length with an open reading frame that encodes 310 amino acids. Five introns (Intron 3, 4, 5, 6 and 7) in the translated region, and partial sequence of Intron 2 in the untranslated region of grass carp UCP2 gene were also obtained. Gene structure comparison between grass carp and mammalian (human and mouse) UCP2 gene shows that, the UCP2 gene structure of grass carp is much similar to that of human and mouse. Partial UCP2 cDNA sequences of bighead carp (Aristichthys nobilis) and mud carp (Cirrhinus molitorella), were further determined. Together with the common carp (Cyprinus carpio) UCP2 sequence from GenBank (AJ243486), multiple alignment result shows that the nucleotide and amino acid sequences of the UCP2 gene, were highly conserved among the five major Chinese carps that belong to four subfamilies. Using beta-actin as control, the ratio UCP2/beta-actin mRNA (%) was determined to be 149.4 +/- 15.6 (common carp), 127.4 +/- 22.1(mud carp), 96.7 +/- 12.7 (silver carp), 94.1 +/- 26.8 (bighead carp) and 63.7 +/- 16.2 (grass carp). The relative liver UCP2 expression of the five major Chinese carps, shows a close relationship with their food habit: benthos and detritus-eating fish (common carp and mud carp) > planktivorious fish (silver carp and bighead carp) > herbivorous fish (grass carp). We suggest that liver UCP2 might be important for Chinese carps to detoxify cyanotoxins and bacteria in debris and plankton food.

[Effects of starvation on the consumption of energy sources and swimming performance in juvenile Gambusia affinis and Tanichthys albonubes].

To explore the consumption of energy sources and swimming performance of juvenile Gambusia affinis and Tanichthys albonubes after starvation, contents of glycogen, lipid and protein, burst swimming speeds (Uburst), and critical swimming speeds (Ucrit) at different starvation times (0, 10, 20, 30 and 40 days) were evaluated. The results showed that, at 0 day, contents of glycogen and lipid were significantly lower in G. affinis than those in T. albonubes, whereas no significant difference in content of protein between two experimental fish was found. Swimming speeds in G. affinis were significantly lower than those in T. albonubes for all swimming performances. After different starvation scenarios, content of glycogen both in G. affinis and T. albonubes decreased significantly in power function trend with starvation time and were close to zero after starvation for 10 days, whereas the contents of lipid and protein were linearly significantly decreased. The slope of line regression equation between content of lipid and starvation time in G. affinis was significantly lower than that in T. albonubes, whereas there was a significantly higher slope of line equation between content of protein and starvation time in G. affinis. 40 days later, the consumption rate of glycogen both in G. affinis and T. albonubes were significantly higher than that of lipid, while the consumption rate of protein was the least. Consumption amounts of glycogen in all experimental fish were the least, G. affinis consumed more protein than lipid, and T. albonubes consumed more lipid than protein. Uburst and Ucrit decreased significantly linearly with starvation time for all experimental fish. Slope of linear equation between Uburst and starvation time was not significantly different between G. affinis and T. albonubes. However, the straight slope between Ucrit and starvation time was significantly lower in G. affinis than that in T. albonubes. These findings indicated that there was close relationship between the consumption of energy sources and swimming performance in starvation. Although the store amounts of energy sources and swimming performance were lower in G. affinis than those in T. albonubes, G. affinis mainly used protein during starvation. The result of more stable lipid content and Ucrit in G. affinis in starvation compared with that in T. albonubes indicated that G. affinis had a fair endurance to starvation, which helped them to adapt to the poor nutrition environment in stream habitat.

[Effect of marine culture on the quality of coastal water in Guangdong Province in the summer].

The contents of nitrogen, phosphorus and plankton in aquatic areas and non-sea-farming areas, which were found in Shantou, Huiyang, Zhuhai, Yangjiang and Zhanjiang in Guangdong Province respectively, were measured in 2000 in the summer with the aim of estimating the environmental problem of marine culture. The concentration of total nitrogen(TN), particle total nitrogen(PTN), total phosphorus(TP), particle total phosphorus(PTP) in aquatic areas, which were 0.506-1.244 mumol/L, 0.367-1.066 mumol/L, 0.112-0.232 mumol/L and 0.054-0.157 mumol/L respectively, were higher than non-sea-farming areas, but marine culture had no effect on the concentration of dissolvable total nitrogen(DTN) and dissolvable total phosphorus(DTP), TN:TP ratios and DTN:DTP ratios. The most phytoplankton in both aquatic areas and non-sea-farming areas was Chaetoceros, and that of zooplankton was Copepoda (including adult and larva). Marine culture affected the diversity of plankton and the population densities of some species, but not to the number of plankton kinds and the total individuals of all phytoplankton or all zooplankton.