Comparison of enzyme-linked immunosorbent assay systems using rift valley fever virus nucleocapsid protein and inactivated virus as antigens

Background

Rift Valley Fever (RVF) is a mosquito-borne viral zoonosis. To detect RVF virus (RVFV) infection, indirect immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme linked immunosorbent assays (ELISAs) which utilize recombinant RVFV nucleocapsid (RVFV-N) protein as assay antigen, have reportedly been used, however, there is still a need to develop more sensitive and specific methods of detection.

Methods

RVFV-N protein was expressed in Escherichia coli (E. coli) and purified by histidine-tag based affinity chromatography. This recombinant RVFV-N (rRVFV-N) protein was then used as antigen to develop an IgG sandwich ELISA and IgM capture ELISAs for human sera. Ninety six serum samples collected from healthy volunteers during the RVF surveillance programme in Kenya in 2013, and 93 serum samples collected from RVF-suspected patients during the 2006–2007 RVF outbreak in Kenya were used respectively, to evaluate the newly established rRVFV-N protein-based IgG sandwich ELISA and IgM capture ELISA systems in comparison with the inactivated virus-based ELISA systems.

Results

rRVFV-N protein-based-IgG sandwich ELISA and IgM capture ELISA for human sera were established. Both the new ELISA systems were in 100% concordance with the inactivated virus-based ELISA systems, with a sensitivity and specificity of 100%.

Conclusions

Recombinant RVFV-N is a safe and affordable antigen for RVF diagnosis. Our rRVFV-N-based ELISA systems are safe and reliable tools for diagnosis of RVFV infection in humans and especially useful in large-scale epidemiological investigation and for application in developing countries.

     

Development of human health damage factors for PM<Subscript>2.5</Subscript> based on a global chemical transport model

Purpose

Health damage from ambient fine particulate matter (PM_2.5) shows large regional variations and can have an impact on a global scale due to its transboundary movement. However, existing damage factors (DFs) for human health in life cycle assessments (LCA) are calculated only for a few limited regions based on various regional chemical transport models (CTMs). The aim of this research is to estimate the human health DFs of PM_2.5 originating from ten different regions of the world by using one global CTM.

Methods

The DFs express changes in worldwide disability-adjusted life years (DALYs) due to unit emission of black carbon and organic carbon (BCOC), nitrogen oxides (NO _x ), and sulfur dioxide (SO₂). DFs for ten regions were calculated as follows. Firstly, we divided the whole world into ten regions. With a global CTM (MIROC-ESM-CHEM), we estimated the concentration change of PM_2.5 on the world caused by changes in the emission of a targeted precursor substance from a specific region. Secondly, we used population data and epidemiological concentration response functions (CRFs) of mortality and morbidity to estimate changes in the word’s DALYs occurring due to changes in the concentration of PM_2.5. Finally, the above calculations were done for all ten regions.

Results and discussion

DFs of BCOC, NO _x , and SO₂ for ten regions were estimated. The range of DFs could be up to one order of magnitude among the ten regions in each of the target substances. While population density was an important parameter, variation in transport of PM_2.5 on a continental level occurring due to different emission regions was found to have a significant influence on DFs. Especially for regions of Europe, Russia, and the Middle East, the amount of damage which occurred outside of the emitted region was estimated at a quarter, a quarter, and a third of their DFs, respectively. It was disclosed that the DFs will be underestimated if the transboundary of PM_2.5 is not taken into account in those regions.

Conclusions

The human health damage factors of PM_2.5 produced by BCOC, NO _x , and SO₂ are estimated for ten regions by using one global chemical transport model. It became clear that the variation of transport for PM_2.5 on a continental level greatly influences the regionality in DFs. For further research to quantify regional differences, it is important to consider the regional values of concentration response function (CRF) and DALY loss per case of disease or death.

     

Large‐scale culture of a megakaryocytic progenitor cell line with a single‐use bioreactor system

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single‐use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large‐scale production and feasibility of meeting clinical‐grade standards. The current work evaluated the capacity of a single‐use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (k_La) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h^?1, respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7‐fold with a total cell number of 1.2 ± 0.2 × 10⁹ cells L^?1. In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single‐use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362–369, 2018     

Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light-induced release of CO-mediated inhibition

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl- p -phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO-mediated inhibition. The action spectra for photoreactivation of CO-inhibited cytochrome c oxidase in isolated mitochondria and CO-blocked respiration in TMPD-treated eggs were found to be similar to the absorption spectrum of CO-bound cytochrome aa ₃. In PMS-treated eggs and fertilized eggs, the maximum photoreactivation of CO-inhibited respiration occurred at a light fluence rate higher than that for maximum photoreactivation of CO-inhibited respiration in TMPD-treated eggs, with peaks at the same wavelengths as those in the absorption spectrum of reduced cytochrome b. A similar phenomenon was seen for NADH cytochrome c reductase in mitochondria. Thus, cytochrome c oxidase and NADH cytochrome c reductase, whose activities are not altered by fertilization, seem to be functional, even in unfertilized eggs. In unfertilized eggs, difference spectra indicated that PMS and sperm augmented cytochrome b reduction and that TMPD accelerated cytochrome c reduction without cytochrome b reduction. Therefore, it is likely that depression of electron transport to cytochrome b , which is augmented by PMS and sperm, is responsible for the low respiratory rate in unfertilized eggs.     

Optimum medium for large-scale culture of Tetraselmis tetrathele

The prasinophyte Tetraselmis tetrathele is an alga commonly used as livefood for aquatic animal larvae. The alga is usually cultured in Guillard& Ryther medium (Guillard F) or a fertilizer enriched seawater mediumfor the large-scale culture of Nannochloropsis. However, Guillard F is toocomplicated to use for large-scale culture, and some fertilizers are impureand insoluble. A new enriched seawater medium for the large-scale culture ofT. tetrathele (ES-T.T.) was formulated by modifying the Guillard F medium.NaNO₃, NaH₂PO₄, Fe-EDTA andMnCl₂were selected as essential additives for the medium bysystematically removing each additive of Guillard F. Results from axenicculture experiments, using an artificial seawater medium, the requiredamount of these additives was estimated to be, 150 mg NaNO₃,10 mg NaH₂PO₄ · 2H₂0, 15 mgFe-EDTA and 360 µg MnCl₂ · 4H₂O,per liter of seawater. The productive rate of T. tetrathele in ES-T.T. washigher than in a fertilized medium in a 100 liter outdoor cultureexperiment. In 10 liter indoor culture experiments, no significantdifference was detected in production rates between ES-T.T. and Guillard F.Therefore, ES-T.T. is simple and effective to use as a large-scale culturemedium for T. tetrathele.     

Larval morphometry of the Japanese flounder,Paralichthys olivaceus

The larval development of the Japanese flounder,Paralichthys olivaceus, was surveyed using two types of morphometric analyses, modified allometry and polar coordinate analysis by principal component analysis (PCA). In the former, centroid size was used as a growth index instead of total length (TL), such enabling the determination of more detailed changes in each character than ordinary allometry based upon TL. Polar coordinate analysis disclosed two remarkable inflexions during the larval development ofP. olivaceus. Postlarvae ofP. olivaceus were found to undergo four developmental phases. From the point of view of metamorphosis, the phases were named drifting larva, premetamorphic larva, metamorphic larva and postmetamorphic larva, respectively. These phases were also tested by other characters related to flounder metamorphosis.     

Developmental expression of extracellular matrix components in intramuscular connective tissue of bovine semitendinosus muscle

 We have investigated the expression patterns of extracellular matrix components in intramuscular connective tissue during the development of bovine semitendinosus muscle by means of indirect immunofluorescence techniques. Types I, III, V, and VI collagen and fibronectin were located in the endomysium and the perimysium. Type IV collagen, laminin, and heparan sulfate proteoglycans (PGs) were exclusively located in the endomysium, and dermatan sulfate PGs existed only in the perimysium. The localization of these components in the intramuscular connective tissue of semitendinosus muscle remained unchanged throughout prenatal and postnatal growth of cattle, suggesting that they are essential for forming and maintaining structures of the endomysium and perimysium in bovine semitendinosus muscle. On the other hand, decorin was undetectable in the endomysium of neonates, although other matrix components were already expressed. It was expressed slightly in the endomysium of 2-month-old calves, and clearly detectable in the endomysium of cattle more than 6 months old. Chondroitin sulfate PGs were barely detectable in the perimysium of fetuses and neonatal calves, and progressively appeared during postnatal development of the muscle. It seems likely that these PGs are closely related to the postnatal development of the endomysium and perimysium. Accepted: 30 October 1996