Fluorescence Enzyme Research Articles

Production of therapeutic iduronate-2-sulfatase enzyme with a novel single-stranded RNA virus vector.

The Sendai virus vector has received a lot of attention due to its broad tropism for mammalian cells. As a result of efforts for genetic studies based on a mutant virus, we can now express more than 10 genes of up to 13.5 kilo nucleotides in a single vector with high protein expression efficiency. To prove this benefit, we examined the efficacy of the novel ribonucleic acid (RNA) virus vector harboring the human iduronate-2-sulfatase (IDS) gene with 1,653 base pairs, a causative gene for mucopolysaccharidosis type II, also known as a disorder of lysosomal storage disorders. As expected, this novel RNA vector with the human IDS gene exhibited its marked expression as determined by the expression of enhanced green fluorescent protein and IDS enzyme activity. While these cells exhibited a normal growth rate, the BHK-21 transformant cells stably expressing the human IDS gene persistently generated an active human IDS enzyme extracellularly. The human IDS protein produced failed to be incorporated into the lysosome when cells were pretreated with mannose-6-phosphate, demonstrating that this human IDS enzyme has potential for therapeutic use by cross-correction. These results suggest that our novel RNA vector may be applicable for further clinical settings.

Physiological Mechanism of Drought-Resistant Rice Coping With Drought Stress

Drought stress is one of the major threats to rice production. The weakening of leaf photosynthesis due to drought is the main reason for the reduction of grain yield, but its mechanism is still obscure. The objectives of this study were to reveal the physiological mechanism of drought stress affecting photosynthetic capacity and grain yield. Pot experiments were conducted with drought-tolerant cultivars Hanyou113 (HY113) and Zhonghan3 (ZH3) and drought-sensitive cultivar Huanghuazhan (HHZ) under four water management treatments (traditional flooding (CK), mild drought stress (LD), moderate drought stress (MD) and severe drought stress (HD)) at heading stage in 2013 and 2014. Compared with CK, grain yield was significantly reduced by 14.9%, 30.8%, and 12.8% in HY113, HHZ, and ZH3 under LD, 32.9%, 33.7%, and 22.9% in HY113, HHZ and ZH3 under MD and 53.6%, 45.6%, and 30.7% in HY113, HHZ, and ZH3 under HD, respectively. The photosynthetic rate (Pn) decreased by 49.0% from 20.0 to 10.2 µmol m−2 s−1 in HY113, and 67.6% from 23.4 to 7.58 µmol m−2 s−1 in HHZ, and 39.3% from 23.4 to 14.2 µmol m−2 s−1 in ZH3 under HD. The Pn of HHZ was similar to that of ZH3 under CK conditions. During the drought periods from LD to HD at heading stage, the leaf water potential (LWP) reduced 31.9%, 54.8%, and 15.7% in HY113, HHZ, and ZH3, respectively. The non-photochemical quenching (NPQ) of HY113, HHZ, and ZH3 flag leaves increased by 150%, 97.6%, and 218%, respectively. The effective quantum yield of PSII photochemistry (ΦPSII) of flag leaves reduced by 20.3%, 11.9%, and 22.1% in HY113, HHZ, and ZH3, respectively. The enzymatic activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased by 11.4%, 18.0%, and 21.8% in HY113, and 13.2%, 14.3%, and 30.9% in HHZ, and 13.4% 21.7%, and 17.6% in ZH3 under MD on average across two seasons. The yield reduction of drought-resistant cultivars (HY113, ZH3) was smaller than that of conventional cultivars (HHZ). Maintaining leaf water potential (LWP), Pn, photosystem II (PSII) original light energy conversion efficiency, non–photochemical quenching coefficient (NPQ), and increasing in the ratio of photochemical reaction energy in fluorescence and antioxidant enzyme activity is the physiological basis to achieve a relatively high photosynthesis. These traits could be the target for breeder to develop drought-tolerant varieties.

Mixed red and blue light promotes tomato seedlings growth by influencing leaf anatomy, photosynthesis, CO2 assimilation and endogenous hormones

The light frequency of red (R) and blue (B) plays a crucial role within plant growth cycle. Whereas, the relationship between the metabolic pathways involved in photosynthesis and carbohydrate accumulation in tomato seedling and hormone regulation still needs further investigation. This research comprehensively analyzed changes in leaf anatomy, stomatal traits, pigment content, gas exchange parameters, chlorophyll fluorescence, Rubisco and Calvin cycle-related enzyme expression and activities, as well as endogenous hormones biosynthesis in tomato (Solanum lycopersicum L.) seedlings, which radiated by four light qualities, including monochromatic white light (W), B, R and mixed light of R and B (RB). The findings showed the biomass accumulation as well as CO2 assimilation were greatly increased in seedlings under B and especially under RB light. Their leaves were also thicker, and stomatal conductance, levels of pigment, photosynthetic electron transport capacity, and the photosynthetic rate were enhanced. In addition, enzyme expression level and activity related to the Calvin cycle were up-regulated. Moreover, RB-induced growth was accompanied by a significant inhibition in gibberellin (GA) concentration as well as an increment in auxin (indole-3-acetic acid (IAA)) in stem and root. Overall, photosynthesis of RB-treated seedlings was positively regulated by leaf anatomy and photosynthetic electron transportation as well as maintaining the activation states of the Calvin cycle, and then inducing changes in carbohydrate accumulation. In addition, both GA and IAA were found to be involved causally in the increased shoot and root biomass of tomato seedlings under RB treatment.

In vitro effects of 95% khat ethanol extract (KEE) on human recombinant cytochrome P450 (CYP)1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5.

Khat, a natural amphetamine-like psychostimulant plant, are widely consumed globally. Concurrent intake of khat and xenobiotics may lead to herb-drug interactions and adverse drug reactions (ADRs). This study is a continuation of our previous study, targeted to evaluate the in vitro inhibitory effects of khat ethanol extract (KEE) on human cytochrome (CYP) 1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, and CYP3A5, major human drug metabolizing enzymes. In vitro fluorescence enzyme assays were employed to assess CYPs inhibition with the presence and absence of various KEE concentrations. KEE reversibly inhibited CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 but not CYP1A2 with IC50 values of 25.5, 99, 4.5, 21, 27, 17, and 10μg/mL respectively. No irreversible inhibition of KEE on all the eight CYPs were identified. The Ki values of CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 were 20.9, 85, 4.8, 18.3, 59.3, 3, and 21.7μg/mL, respectively. KEE inhibited CYP2B6 via competitive or mixed inhibition; CYP2E1 via un-competitive or mixed inhibition; while CYP2A6, CYP2C8, CYP2C19, CYP2J2 and CYP3A5 via non-competitive or mixed inhibition. Caution should be taken by khat users who are on medications metabolized by CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, and CYP3A5.

Total IgE assessment in newborn umbilical cord blood

In this minireview we present important data on umbilical cord blood IgE antibodies in neonates and total IgE as a predictive biomarker for the development of allergen sensitization and atopic diseases later in life. Discussions regarding the methods for determining total IgE in serum or plasma from cord blood samples are focused on the main immunoassays used in different studies and clinical practice. The fluorescence enzyme immunoassay with anti-IgE covalently coupled to a capsulated cellulose polymer solid-phase is nowadays currently used to measure total IgE in human serum or plasma. The umbilical cord blood total IgE levels are quantitatively determined by using its low range assay. Specific and practical aspects regarding cord blood sampling, including specimen collection from the umbilical cord vessels and careful preparation of serum and plasma, alongside with knowledge of the principles of the immune methods used are important to avoid preanalytical and analytical errors and for obtaining accurate results of this risk biomarker for allergy.

Peanut components measured by ISAC: comparison with ImmunoCap and clinical relevance in peanut allergic children

BackgroundSpecific IgE (sIgE) against the peanut component Arachis hypogaea (Ara h) 2 has been shown to be the most important allergen to discriminate between peanut allergy and peanut tolerance. Several studies determined sIgE cut off values for Ara h 2, determined by singleplex measurements. However, cut off values for Ara h 2 from multiplex arrays are less well defined. The aim of this study was to evaluate the correlation between Ara h 2 sIgE determined by singleplex versus multiplex measurements and to assess the diagnostic value of the different peanut components included in Immuno Solid-phase Allergen Chip (ISAC) multiplex analysis in children with a suspected peanut allergy.MethodsIn this retrospective study we analyzed Ara h 2 sIgE values with singleplex Fluorescence Enzyme Immunoassay (FEIA, ImmunoCap) and multiplex microarray (ISAC) measurements in 117 children with a suspected peanut allergy. Also, other peanut components measured by ISAC were analyzed. Double blinded placebo controlled oral food challenges were used as golden standard.ResultsAmong all studied peanut components FEIA Ara h 2 sIgE showed the highest area under the curve (AUC, 0.922), followed by ISAC Ara h 6 and Ara h 2 sIgE with AUCs of respectively 0.906 and 0.902. Best cut off values to diagnose peanut allergy were 4.40 kU/l for FEIA Ara h 2 sIgE and, 7.43 ISU and 8.13 ISU for respectively Ara h 2 and Ara h 6 sIgE in ISAC microarray. Ara h 2 sIgE determined in FEIA and ISAC showed a good correlation (r = 0.88; p < 0.01).ConclusionAra h 6 and Ara h 2 sIgE in multiplex ISAC are both good predictors of clinical peanut allergy in Dutch children, and their performance is comparable to the use of Ara h 2 in singleplex FEIA. The simultaneous measurement of different peanut components using ISAC is an advantage and clinically useful to detect peanut allergic children that are Ara h 2 negative but sensitized to other peanut proteins such as Ara h 6.

DogCatcher allows loop-friendly protein-protein ligation

SummaryThere are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/SpyCatcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures.

Increase in phosphorus concentration reduces the toxicity of copper in wheat roots (Triticum aestivum L.)

The increase in the availability of phosphorus (P) can reduce the toxicity of copper (Cu) to plants because it favors the formation of complexes and precipitates, reducing the bioavailability of this metal. The objective of this study was to investigate the capacity of P supplementation to decrease the phytotoxicity of excess Cu in the growth and in physiological parameters of wheat plants. Plants of wheat (Triticum aestivum L.) were cultivated on sand substrate and, for 30 days, submitted to the control treatments - Hoagland & Arnon solution 100% (H&A); H&A + 47 mg P L−1 (H&A + P); H&A + 28 Cu mg L−1 (H&A + Cu); H&A + 47 mg P L−1 + 28 mg Cu L−1 (H&A + P + Cu). Plant growth, tissue nutrient concentration, photosynthetic pigment concentration, chlorophyll a fluorescence and superoxide dismutase enzyme activity in the leaves were evaluated. We observed that the treatments H&A + P and H&A + Cu provided higher height, while H&A + P + Cu provided higher dry matter yield of shoots and roots in plants. A higher Cu content in the leaves was observed in the plants cultivated with addition of 28 mg Cu L−1, however the application of Cu did not cause loss of photochemical energy and energy dissipation in the PSII antenna complex. The addition of 47 mg P L−1 provided a higher rate of electron transport in wheat plants. Therefore, wheat plants were tolerant to increasing the concentration of Cu in the culture medium and the addition of P attenuated the toxic effects caused by Cu, mainly in the roots of plants.

Comparison of antinuclear antibody profiles obtained using line immunoassay and fluorescence enzyme immunoassay.

ObjectiveLIA-ANA-Profile-17S is a multiplex line immunoassay that simultaneously detects 17 antinuclear antibodies (ANAs) against extractable nuclear antigens (ENAs). We evaluated the utility of LIA-ANA-Profile-17S as a supplement to ANA indirect immunofluorescence (IIF) and EliA ENA (a fluorescence enzyme immunoassay) for diagnosis of ANA-associated rheumatic diseases.MethodsSera were collected from 245 patients referred for an ANA IIF test. LIA-ANA-Profile-17S results were compared with those of EliA ENA. The kappa coefficients, agreement rates, and diagnostic performance of these tests were assessed for systemic lupus erythematosus (SLE) and Sjögren’s syndrome (SjS).ResultsWe observed almost perfect interassay agreement for antibodies against Ro52/Ro60, CENP-B, and Scl-70 (kappa = 0.91, 0.97, and 1.00, respectively); strong agreement for anti-SS-B/La antibody (kappa = 0.81); and relatively low agreement for other antibodies, including those against dsDNA, Sm, RNP, and Jo-1. For SLE diagnosis, LIA-ANA-Profile-17S showed lower sensitivity and similar specificity compared with EliA ENA. The sensitivity and specificity of these two assays were similar for SjS diagnosis.ConclusionsThe specificity of LIA-ANA-Profile-17S was enhanced when combined with ANA IIF and was comparable with that of EliA ENA. LIA-ANA-Profile-17S showed relatively good agreement with EliA ENA. In combination with ANA IIF, these assays showed enhanced diagnostic performance.

The Effects of Triclosan on Physiological and Photosynthetic Characteristics of Chlorella vulgaris

Triclosan has been widely used as addition ingredient in personal care and medical antibacterial products, and the increasing amounts of triclosan discharged in aquatic environments pose a potential risk to aquatic ecological systems. In this study, we investigated the effects of exposure to varying triclosan concentrations on the growth, chlorophyll fluorescence and antioxidant enzyme activity of Chlorella vulgaris. The results showed that low-concentration triclosan (&lt;0.75 mg/L) can stimulate the growth of Chlorella vulgaris, whereas 1.05 mg/L triclosan exhibited significant inhibition. Low-concentration triclosan (&lt;0.75 mg/L) could improve the tolerance and utilization ability of Chlorella vulgaris in relation to strong light. We observed a significant increase in the malondialdehyde content of Chlorella vulgaris exposed to 1.05 mg/L triclosan. The intracellular superoxide dismutase and catalase (CAT) activities of Chlorella vulgaris exposed to triclosan were higher than the control groups, and the increase in this activity was positively correlated with the concentration of triclosan. The results also showed that excessive H2O2 may in turn damage the CAT structure and eventually inactivate CAT activity when Chlorella vulgaris is exposed to 1.05 mg/L triclosan. This study provided a theoretical basis which can be used to evaluate the ecological risk of triclosan in the aquatic environment.

Laboratory Examination of Syphilis

Syphilis, is sexually transmitted disease caused by spirochete Treponema pallidum subsp.pallidum. It have many diverse clinical manifestations that occur in distinct stages. Early diagnosis and management are the main things to prevent transmission and complication. Direct test or morphological observation is the definitive diagnosis of syphilis. This can be done through animal inoculation test, dark field microscopy, direct fluorescence antibody (DFA), and nucleid acid amplification test (NAAT). While the indirect test is a nontreponemal serologic test consist of Wasserman test, venereal disease research laboratory (VDRL), toluidine red unheated serum test (TRUST), unheated serum reagin (USR), rapid plasma reagin (RPR) and treponemal serologic test, such as T. pallidum passive particle agglutination (TPPA), T. pallidum haemagglutination assay (TPHA), fluorescent treponemal antibody absorption (FTA-Abs), enzyme immunoassay (EIA) and rapid test. The algorithm of serologic test can be divided into traditional or reverse.

Laboratory Examination of Syphilis

Syphilis, is sexually transmitted disease caused by spirochete Treponema pallidum subsp.pallidum. It have many diverse clinical manifestations that occur in distinct stages. Early diagnosis and management are the main things to prevent transmission and complication. Direct test or morphological observation is the definitive diagnosis of syphilis. This can be done through animal inoculation test, dark field microscopy, direct fluorescence antibody (DFA), and nucleid acid amplification test (NAAT). While the indirect test is a nontreponemal serologic test consist of Wasserman test, venereal disease research laboratory (VDRL), toluidine red unheated serum test (TRUST), unheated serum reagin (USR), rapid plasma reagin (RPR) and treponemal serologic test, such as T. pallidum passive particle agglutination (TPPA), T. pallidum haemagglutination assay (TPHA), fluorescent treponemal antibody absorption (FTA-Abs), enzyme immunoassay (EIA) and rapid test. The algorithm of serologic test can be divided into traditional or reverse.

Binding mechanism and functional evaluation of quercetin 3-rhamnoside on lipase

The interaction between lipase and quercetin 3-rhamnoside was studied by fluorescence spectroscopy, enzyme kinetics, and molecular dynamics simulation. The results showed that quercetin 3-rhamnoside had a strong quenching effect on the intrinsic fluorescence of lipase. The binding constant decreased with increasing temperature, and the number of binding sites approached 1. Thermodynamic parameters indicated that hydrogen bonding and van der Waals forces are the dominant forces when the interaction occurs. Circular dichroism spectroscopy and infrared spectroscopy proved that the ligand perturbed the structure of lipase. Enzyme kinetics results showed that quercetin 3-rhamnoside inhibited lipase, and the inhibitory effect was dose-dependent. Molecular dynamics simulation further explained the interaction mechanism and inhibitory effect. This study confirmed the inhibitory effect of quercetin 3-rhamnoside on lipase explained their binding mechanism, which will contribute to guiding the development of fat-reducing functional foods.

Evaluation of a novel particle-based multi-analyte technology for the detection of anti-fibrillarin antibodies

Systemic sclerosis (SSc) is a heterogeneous autoimmune disease associated with several anti-nuclear antibodies (ANA), including those in the classification criteria (anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA Pol III). However, the presence of less common antibodies such as anti-fibrillarin (U3-RNP) that generate a clumpy nucleolar pattern by HEp-2 indirect immunofluorescence assay (IFA, ICAP AC-9) are considered disease specific and are with clinical subsets of SSc, therefore playing a role in diagnosis and prognosis. A specific and sensitive anti-fibrillarin assay would be an important addition to serological diagnosis and evaluation of SSc. The goal of this study was to evaluate a new particle-based multi-analyte technology (PMAT) for the measurement of anti-fibrillarin antibodies. A total of 149 patient samples were collected including 47 samples from France (Lyon and Paris, n = 32) and Italy (Careggi Hospital, Florence, n = 15) selected based on AC-9 HEp-2 IFA staining (> 1:640, clumpy nucleolar pattern) and 102 non-SSc controls (inflammatory bowel disease (IBD) n = 20, Sjögren’s syndrome (SjS) n = 20, infectious disease (ID) n = 7, systemic lupus erythematosus (SLE) n = 17, rheumatoid arthritis (RA) n = 17, and healthy individuals (HI) n = 21). All samples were tested on the anti-fibrillarin PMAT assay (research use only, Inova Diagnostics, USA). Additionally, the 47 anti-fibrillarin positive samples were also tested on PMAT assays for detecting other autoantibodies in ANA-associated rheumatic diseases (AARD). Anti-fibrillarin antibody data performed by fluorescence enzyme immunoassay (FEIA, Thermo Fisher, Germany) was available for 34 samples. The anti-fibrillarin PMAT assay was positive in 31/32 (96.9%, France) and 12/15 (80.0%, Italy) of samples preselected based on the AC-9 IIF pattern (difference p = 0.09). Collectively, the PMAT assay showed 91.5% (95% confidence interval (CI): 80.1–96.6%) sensitivity with 100.0% (95% CI: 96.4–100.0%) specificity in non-SSc controls. Strong agreement was found between PMAT and FEIA with 100.0% positive qualitative agreement (34/34) and quantitative agreement (Spearman’s rho = 0.89, 95% CI: 0.77.9–0.95%, p < 0.0001). Although most anti-fibrillarin positive samples were mono-specific (69.8%), some expressed additional antibodies (namely Scl-70, centromere, dsDNA, Ro52, Ro60, SS-B, Ribo-P, DFS70, and EJ). In conclusion, this first study on anti-fibrillarin antibodies measured using a novel PMAT assay shows promising results where the new PMAT assay had high level of agreement to FEIA for the detection of anti-fibrillarin antibodies. The availability of novel AFA assays such as PMAT might facilitate the clinical deployment, additional studies, standardization efforts, and potentially consideration of AFA for next generations of the classification criteria.

Basic principles of immunohistochemistry and epithelial immunohistochemical markers

Dr. Albert Coons et al in 1941 first initiated immunohistochemistry to identify cellular or tissue constituents by means of antigen and antibody interactions. The site of antibody binding can be identified either by direct labeling of antibody or by use of secondary labeling method. Immunohistochemistry (IHC) involves the use of labeled antibodies to localize the antigens in the tissues through antigen-antibody interactions and are visualized either by fluorescent dye, enzyme, radioactive element or colloidal gold. IHC usage has an obvious advantage over the traditional one, which uses special enzyme staining technique that identify only limited number of proteins, enzymes and certain tissue structure. IHC can be commonly employed to distinguish the tissue of epithelial and mesenchymal differentiation, cellular changes which are normally not visible in regular staining techniques, and earliest changes in transformed tissues. IHC is relatively rapid and simple method to better determine the specific neoplastic tissue origin, the behavior or progression of neoplasm. Hence IHC have gained importance in the medical research and has been considered as a crucial & widely used technique in research laboratories as well as in clinical diagnostics. In present review IHC markers for epithelial origin that can be utilized to diagnose the tumor or to determine the prognosis of the tumor.. Keywords: Immunohistochemistry, Antigen, Antibody labeling, Epithelial origin markers.

The detection of antithyroid antibodies in cerebrospinal fluid using fluorescent enzyme immuno assay (FEIA) is suitable for biological diagnosis of Hashimoto's encephalopathy

Hashimoto encephalopathy (HE) is a rare condition often underdiagnosed. The clinical picture is heterogeneous with numerous neurological signs and is associated with the presence of high levels of anti-thyroperoxidase (TPO) and / or anti-thyroglobulin (TG) antibodies in the blood and cerebrospinal fluid (CSF). The determination of anti-TPO and anti-TG antibodies in CSF is performed in only few laboratories. The aim of our study was to adapt the EliATM fluoroenzymatic immuno assay (FEIA) to the detection of these autoantibodies in the CSF, and to compare the results with our previously published ELISA test (Blanchin S. 2007). For the FEIA technique, the detection threshold, and the quantification threshold have been determined for anti-TPO and anti-TG antibodies. FEIA results were concordant with ELISA at 75% and 100% for anti-TPO and anti-TG antibodies, respectively. Coefficients of variation (CV) of the intra-assay and inter-assay results were calculated as well as the uncertainties of measurement. The anti-TPO and anti-TG antibodies detection in CSF using FEIA technique correlate with the previously published ELISA and show good analytical performances. The availability of PhadiaTM 250 analyzer in a large number of laboratories will allow an easier biological detection. We hope that this test will respond to physician needs and help for HE diagnosis.

Foliar applications of urea and melatonin to alleviate waterlogging stress on photosynthesis and antioxidant metabolism in sorghum seedlings

Waterlogging represents a serious abiotic stress for plants that retards crop growth and reduces production. Therefore, exploration of effective ways to alleviate the impacts of waterlogging has important theoretical and practical applications. This study investigated the responses of seedlings of two sorghum cultivars with different waterlogging tolerances to exogenous foliar treatment with melatonin (MT) and nitrogen (N) under waterlogged conditions. The chlorophyll fluorescence, photosynthetic capabilities, and antioxidant enzyme activities of these seedlings were measured after treatments with waterlogging and either exogenous application of MT, N, or both. The results showed that waterlogging severely limited sorghum growth and reduced dry matter accumulation in both cultivars. However, the negative effects of waterlogging were mitigated by spaying MT and N on leaves, especially of the waterlogging sensitive cultivar. Notably, exogenous foliar spraying of MT and N enhanced the chlorophyll content and improved the maximal quantum yield of photosystem II photochemistry (Fv/Fm), resulting in higher rates of photosynthesis. In addition, exogenous spraying of MT and N markedly decreased the malondialdehyde (MDA) content by upregulating antioxidant enzyme activities. The results also show that the combined effects of MT and N were better than the application of either alone and that exogenous foliar spraying of MT and N significantly increased the soluble protein content of leaves of sorghum. Overall, these results indicate the beneficial roles of MT and N in alleviating waterlogging stress in sorghum and show that their application may represent a strategy to mitigate waterlogging stress during agricultural production.

Development of a sensitive and stable chemiluminescent immunoassay for detection of birch pollen allergic specific IgE based on recombinant Bet v1 protein

BackgroundQuantitative detection of allergens is of great significance for clarifying the cause, treatment, and prevention of allergy disease. Birch pollen is one of the most common inhalational allergens and Bet v1 is the major component allergen of birch allergen. This study aims to develop a stable and sensitive chemiluminescence immunoassay (CLIA) for the detection of birch pollen allergic specific IgE (sIgE) based on recombinant Bet v1 (rBet v1) protein.MethodsrBet v1 protein was expressed in Escherichia coli and purified. Then rBet v1 was applied to detect sIgE in human serum. The performance of the established CLIA was evaluated and compared with Phadia rBet v1 fluorescence enzyme immunoassay (FEIA) system.ResultsThe developed CLIA for sIgE to rBet v1 detection shows excellent performance. The assay showed a linear range from 0.1 to 100 IU/mL, with a low detection limit of 0.06 IU/mL. A total of 164 samples were evaluated by CLIA and compared with the results of FEIA. The positive, negative, and total coincidence rate was 90.6% (87/96), 91.2% (62/68), and 90.9% (149/164), respectively. The r-value of Spearman's rank correlation analysis was 0.935 (P < 0.001). The use of high levels of bilirubin (50 mg/dL), hemoglobin (400 mg/dL) and lipid (2000 mg/dL) didn't interfere with the results.ConclusionsThe proposed CLIA exhibits excellent performance for the detection of rBet v1 specific IgE. It can be a reliable tool for the early diagnosis of hypersensitivity.

Inexpensive Near-Infrared Fluorimeters: Enabling Translation of nIR-Based Assays to the Field.

The practical impact of analytical probes that transduce in the near-infrared (nIR) has been dampened by the lack of cost-effective and portable nIR fluorimeters. Herein, we demonstrate straightforward designs for an inexpensive microplate reader and a portable fluorimeter. These instruments require minimally complex machining and fabrication and operate with an open-source programming language (Python). Complete wiring diagrams, assembly diagrams, and scripts are provided. To demonstrate the utility of these two instruments, we performed high-throughput and field-side measurements of soil samples to evaluate the effect of soil management strategies on extracellular proteolytic, cellulolytic, and lignin-modifying activities. This was accomplished with fluorescent enzyme probes that utilized uniquely sensitive transducers exclusive to the nIR spectrum, single-walled carbon nanotubes. We also used the portable fluorimeter to evaluate spatial variations of proteolytic activity within individual field plots, while minimizing the effects of soil storage and handling. These demonstrations indicate the utility of these fluorimeters for translating analytical probes that operate in the nIR beyond the laboratory and into actual use.

Boceprevir, Calpain Inhibitors II and XII, and GC-376 Have Broad-Spectrum Antiviral Activity against Coronaviruses.

As the COVID-19 pandemic continues to unfold, the morbidity and mortality are increasing daily. Effective treatment for SARS-CoV-2 is urgently needed. We recently discovered four SARS-CoV-2 main protease (Mpro) inhibitors including boceprevir, calpain inhibitors II and XII, and GC-376 with potent antiviral activity against infectious SARS-CoV-2 in cell culture. In this study, we further characterized the mechanism of action of these four compounds using the SARS-CoV-2 pseudovirus neutralization assay. It was found that GC-376 and calpain inhibitors II and XII have a dual mechanism of action by inhibiting both viral Mpro and host cathepsin L in Vero cells. To rule out the cell-type dependent effect, the antiviral activity of these four compounds against SARS-CoV-2 was also confirmed in type 2 transmembrane serine protease-expressing Caco-2 cells using the viral yield reduction assay. In addition, we found that these four compounds have broad-spectrum antiviral activity in inhibiting not only SARS-CoV-2 but also SARS-CoV, and MERS-CoV, as well as human coronaviruses (CoVs) 229E, OC43, and NL63. The mechanism of action is through targeting the viral Mpro, which was supported by the thermal shift-binding assay and enzymatic fluorescence resonance energy transfer assay. We further showed that these four compounds have additive antiviral effect when combined with remdesivir. Altogether, these results suggest that boceprevir, calpain inhibitors II and XII, and GC-376 might be promising starting points for further development against existing human coronaviruses as well as future emerging CoVs.