Penicillin-binding protein 3 of Streptococcus pneumoniae and its application in screening of β-lactams in milk

The soluble form of penicillin-binding protein 3 (sPBP3^∗) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP–Amp) to the sPBP3^∗ by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3^∗ exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.     

Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins

Bt Cry toxin is a kind of bio-toxins that used for genetically modified crops (GMC) transformation widely. In this study, total 15 positive clones could bind the Bt Cry toxins which isolated from a human domain antibody library by 5 rounds affinity selection. According to analyzing of PCR amplification and enzyme-linked immunosorbent assay (ELISA), the most positive phage domain antibody (named F5) gene was cloned into the pET26b vector and expressed in E. coli BL21. The purified antibody was used to develop an indirect competitive ELISA (IC-ELISA) for Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins, respectively. The working range of detection for standard curves in IC-ELISA were 0.258–1.407 μg/mL, the medium inhibition concentration (IC₅₀) were 0.727–0.892 μg/mL and detection limit (IC₁₀) were 0.029–0.074 μg/mL for those Bt Cry toxins. The affinity of F5 domain antibody with Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins were 1.21–5.94 × 10⁷ M⁻¹. The average recoveries of the 5 kinds of Bt Cry toxins from spiked wheat samples were ranged from 81.2%–100.8% with a CV at 2.5%–9.4%. The results showed that we successfully obtained the broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins in agricultural product samples.     

DNA melting analysis: application of the "open tube" format for detection of mutant KRAS

High-resolution melting (HRM) analysis is a very effective method for genotyping and mutation scanning that is usually performed just after PCR amplification (the “closed tube” format). Though simple and convenient, the closed tube format makes the HRM dependent on the PCR mix, not generally optimal for DNA melting analysis. Here, the “open tube” format, namely the post-PCR optimization procedure (amplicon shortening and solution chemistry modification), is proposed. As a result, mutation scanning of short amplicons becomes feasible on a standard real-time PCR instrument (not primarily designed for HRM) using SYBR Green I. This approach has allowed us to considerably enhance the sensitivity of detecting mutant KRAS using both low- and high-resolution systems (the Bio-Rad iQ5–SYBR Green I and Bio-Rad CFX96–EvaGreen, respectively). The open tube format, though more laborious than the closed tube one, can be used in situations when maximal sensitivity of the method is needed. It also permits standardization of DNA melting experiments and the introduction of instruments of a “lower level” into the range of those suitable for mutation scanning.     

Contribution of phosphoglucosamine mutase to the resistance of Streptococcus gordonii DL1 to polymorphonuclear leukocyte killing

Phosphoglucosamine mutase (GlmM; EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the biosynthetic pathway leading to the formation of the peptidoglycan precursor uridine 5'-diphospho- N -acetylglucosamine. We have recently identified the gene ( glmM ) encoding the enzyme of Streptococcus gordonii , an early colonizer on the human tooth and an important cause of infective endocarditis, and indicated that the glmM mutation in S. gordonii appears to influence bacterial cell growth, morphology, and sensitivity to penicillins. In the present study, we assessed whether the glmM mutation also affects escape from polymorphonuclear leukocyte (PMN)-dependent killing. Although no differences in attachment to human PMNs were observed between the glmM mutant and the wild-type S. gordonii , the glmM mutation resulted in increased sensitivity to PMN-dependent killing. Compared with the wild type, the glmM mutant induced increased superoxide anion production and lysozyme release by PMNs. Moreover, the glmM mutant is more sensitive to lysozyme, indicating that the GlmM may be required for synthesis of firm peptidoglycans for resistance to bacterial cell lysis. These findings suggest that the GlmM contributes to the resistance of S. gordonii to PMN-dependent killing. Enzymes such as GlmM could be novel drug targets for this organism.     

Identification of the Streptococcus gordonii glmM gene encoding phosphoglucosamine mutase and its role in bacterial cell morphology, biofilm formation, and sensitivity to antibiotics

Phosphoglucosamine mutase (EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, an essential step in the biosynthetic pathway leading to the formation of peptidoglycan precursor uridine 5'-diphospho-N-acetylglucosamine. The gene (glmM) of Escherichia coli encoding the enzyme has been identified previously. We have now identified a glmM homolog in Streptococcus gordonii, an early colonizer on the human tooth and an important cause of infective endocarditis, and have confirmed that the gene encodes phosphoglucosamine mutase by assaying the enzymatic activity of the recombinant GlmM protein. Insertional glmM mutant of S. gordonii did not produce GlmM, and had a growth rate that was approximately half that of the wild type. Morphological analyses clearly indicated that the glmM mutation causes marked elongation of the streptococcal chains, enlargement of bacterial cells, and increased roughness of the bacterial cell surface. Furthermore, the glmM mutation reduces biofilm formation and increases sensitivity to penicillins relative to wild type. All of these phenotypic changes were also observed in a glmM deletion mutant, and were restored by the complementation with plasmid-borne glmM. These results suggest that, in S. gordonii, mutations in glmM appear to influence bacterial cell growth and morphology, biofilm formation, and sensitivity to penicillins.     

A single mutation within a Ca binding loop increases proteolytic activity,thermal stability,and surfactant stability

We improved the enzymatic properties of the oxidatively stable alkaline serine protease KP-43 through protein engineering to make it more suitable for use in laundry detergents. To enhance proteolytic activity, the gene encoding KP-43 was mutagenized by error-prone PCR. Screening identified a Tyr195Cys mutant enzyme that exhibited increased specific activity toward casein between pH 7 and 11. At pH 10, the mutant displayed 1.3-fold higher specific activity for casein compared to the wild-type enzyme, but the activity of the mutant was essentially unchanged toward several synthetic peptides. Furthermore, the Tyr195Cys mutation significantly increased thermal stability and surfactant stability of the enzyme under oxidizing conditions. Examination of the crystal structure of KP-43 revealed that Tyr195 is a solvent exposed residue that forms part of a flexible loop that binds a Ca^2 + ion. This residue lies 15–20 Å away from the residues comprising the catalytic triad of the enzyme. These results suggest that the substitution at position 195 does not alter the structure of the active center, but instead may affect a substrate–enzyme interaction. We propose that the Tyr195Cys mutation enhances the interaction with Ca^2 + and affects the packing of the Ca^2 + binding loop, consequently increasing protein stability. The simultaneously increased proteolytic activity, thermal stability, and surfactant stability of the Tyr195Cys mutant enzyme make the protein an ideal candidate for laundry detergent application.     

Development of analytical methods for some penicillins in bovine milk by ion-paired chromatography and confirmation by thermospray mass spectrometry

Analytical methods for the determination of cloxacillin, ampicillin/hetacillin, and amoxicillin in bovine milk were developed. The methods involved ultrafiltration of milk diluted with methanol, acetonitrile, and water on a 10 000-dalton cut-off filter. Separation of penicillins from other milk components was accomplished by ion-paired chromatography using a microbore column. The penicillins were detected using ultraviolet photodiode array (UV-PDA) detection and confirmed by thermospray liquid chromatography—mass spectrometry (LC—MS). The thermospray spectra of these compounds exhibited [M + H]⁺ and [M + Na]⁺ ions along with several fragment ions. The limits of detection for these antibiotics were estimated to be 50 to 100 ppb for LC with UV-PDA detection and 100–200 ppb for thermospray LC—MS detection.     

Localized surface plasmon resonance based gold nanobiosensor: Determination of thyroid stimulating hormone

An immunoassay method based on the peak shift of the localized surface plasmon resonance (LSPR) absorption maxima has been developed for the determination of the thyroid stimulating hormone (TSH) in human blood serum. The anti-TSH antibody was adsorbed on the synthesized gold nanoparticles by electrostatic forces. The efficiency of the nanobiosensor was improved by optimizing the factors affecting the probe construction such as the pH and the antibody to gold nanoparticles ratio. Dynamic light scattering was applied for the characterization of the constructed probe. The amount of peak shift of the LSPR absorption maxima was selected as the basis for determination of TSH antigen. The linear dynamic range of 0.4–12.5 mIU L⁻¹ and the calibration sensitivity of 1.71 L mIU⁻¹ were obtained. The human control serum sample was analyzed for TSH by constructed nanobiosensor and the acceptable results were obtained.     

Colorectal Cancer Patients with Low Abundance of KRAS Mutation May Benefit from EGFR Antibody Therapy

Epidermal growth factor receptor monoclonal antibody was approved for treatment of metastatic colorectal cancer patients carrying KRAS wild type DNA. However, recent studies showed that patients with KRAS G13D mutation may benefit from EGFR antibody therapy. In this study we tried to explore whether the abundance of KRAS mutation could affect the efficacy of EGFR antibody therapy. We firstly established a PNA-PCR method which could calculate the percentage of KRAS mutation in total DNA and proved its ability on 47 colorectal cancer samples bearing KRAS mutations. Then we analyzed the correlation between the abundance of KRAS mutations and efficacy of EGFR antibody therapy in another 35 metastatic colorectal cancer patients. We proved that PNA-PCR assay could calculate the abundance of KRAS mutation and the percentage of mutant DNA in tumor cells varied a lot (10.8%∼98.3%) on the 47 colorectal cancer patients. The efficacy of EGFR antibody correlated with the abundance of KRAS mutations: in the KRAS mutation less than 30% group, the disease control rate was 44.4% (4/9); the disease control rate of 30∼80% group was 5.6% (1/18) and the >80% group was 12.5% (1/8) (P = 0.038). In summary, our study showed that PNA-PCR method could easily detect the percentage of KRAS mutation in tumor cells and colorectal cancer patients with low abundance of KRAS mutation might benefit from EGFR antibody therapy.     

Microtiter plate quantification of mutant and wild-type huntingtin normalized to cell count

Huntington’s disease is caused by a gain-of-function neurotoxic mutation in normally neuroprotective huntingtin. Sensitive assays are required to discriminate mutant huntingtin from wild-type huntingtin. We have developed a normalized 384-plate assay for determination of mutant and wild-type huntingtin. Based on a single pipetting step, the sensitive assay uses two antibody pairs for simultaneous mutant and wild-type huntingtin time-resolved fluorescence resonance energy transfer detection combined with PicoGreen quantification of double-stranded DNA. The assay can be used for discovery of drugs reducing mutant huntingtin over wild-type huntingtin and for assessing the value of huntingtin as a disease progression marker, and it is adaptable to other proteins of interest.     

Label-free electrochemical immunosensor based on Nile blue A-reduced graphene oxide nanocomposites for carcinoembryonic antigen detection

In this article, a novel, label-free, and inherent electroactive redox immunosensor for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and Nile blue A (NB) hybridized electrochemically reduced graphene oxide (NB–ERGO) is proposed. The composite of NB–graphene oxide (NB–GO) was prepared by π–π stacking interaction. Then, chronoamperometry was adopted to simultaneously reduce HAuCl₄ and nanocomposites of NB–GO for synthesizing AuNPs/NB–ERGO. The immunosensor was fabricated by capturing CEA antibody (anti-CEA) at this nanocomposite modified electrode. The immunosensor determination was based on the fact that, due to the formation of antigen–antibody immunocomplex, the decreased response currents of NB were directly proportional to the concentrations of CEA. Under optimal conditions, the linear range of the proposed immunosensor was estimated to be from 0.001 to 40 ng ml⁻¹ and the detection limit was estimated to be 0.00045 ng ml⁻¹. The proposed immunosensor was used to determine CEA in clinical serum samples with satisfactory results. The proposed method may provide promising potential application in clinical immunoassays with the properties of facile procedure, stability, high sensitivity, and selectivity.     

A universal antibody-derived targeting agent

We report bacterial expression of a single-chain antibody (ScFv) reactive against the haptens 4-hydroxy-3 nitrophenylacetic acid (NP) and 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) that is suitable for targeting to mammalian cells in vitro in a novel two-step targeting strategy. Hapten-derivatized primary antibodies of known specificity, bound to target cells, can capture the ScFv. Specificity resides in the interaction of the primary targeting antibody with the target and the interaction of the ScFv for NP/NIP, since the ScFv does not bind cells and nonderivatized antibodies bound at cells cannot capture the ScFv. The ScFv described here can therefore be considered as a universal agent for delivery of drugs, toxins, or radionuclides to any cell type for which a previously characterized antibody exists.     

The importance of the hydrophilic region of PsbL for the plastoquinone electron acceptor complex of Photosystem II

The PsbL protein is a 4.5 kDa subunit at the monomer–monomer interface of Photosystem II (PS II) consisting of a single membrane-spanning domain and a hydrophilic stretch of ~ 15 residues facing the cytosolic (or stromal) side of the photosystem. Deletion of conserved residues in the N-terminal region has been used to investigate the importance of this hydrophilic extension. Using Synechocystis sp. PCC 6803, three deletion strains: ?(N6–N8), ?(P11–V12) and ?(E13–N15), have been created. The ?(N6–N8) and ?(P11–V12) strains remained photoautotrophic but were more susceptible to photodamage than the wild type; however, the ?(E13–N15) cells had the most severe phenotype. The Δ(E13–N15) mutant showed decreased photoautotrophic growth, a reduced number of PS II centers, impaired oxygen evolution in the presence of PS II-specific electron acceptors, and was highly susceptible to photodamage. The decay kinetics of chlorophyll a variable fluorescence after a single turnover saturating flash and the sensitivity to low concentrations of PS II-directed herbicides in the Δ(E13–N15) strain indicate that the binding of plastoquinone to the Q_B-binding site had been altered such that the affinity of Q_B is reduced. In addition, the PS II-specific electron acceptor 2,5-dimethyl-p-benzoquinone was found to inhibit electron transfer through the quinone-acceptor complex of the ?(E13–N15) strain. The PsbL Y20A mutant was also investigated and it exhibited increased susceptibility to photodamage and increased herbicide sensitivity. Our data suggest that the N-terminal hydrophilic region of PsbL influences forward electron transfer from Q_A through indirect interactions with the D–E loop of the D1 reaction center protein. Our results further indicate that disruption of interactions between the N-terminal region of PsbL and other PS II subunits or lipids destabilizes PS II dimer formation. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Enhanced transport of plant‐produced rabies single‐chain antibody‐RVG peptide fusion protein across an in cellulo blood–brain barrier device

The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in planta for targeted delivery across the blood–brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system. This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralizing single‐chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognizes the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv‐RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv‐RVG fusion. Both ScFv and ScFv‐RVG fusion molecules had potent neutralization activity against RABVin cellulo. The ScFv‐RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant‐produced ScFv‐RVG^P fusion could translocate across the cells. This study indicates that the plant‐produced ScFv‐RVG^P fusion protein was able to cross the in celluloBBB and neutralize RABV.     

Fatal mitochondrial myopathy,lactic acidosis,and complex I deficiency associated with a heteroplasmic A→G mutation at position 3251 in the mitochondrial tRNALeu(UUR) gene

A girl, who died at 14 years of age from a rapidly progressive mitochondrial myopathy, was found to be heteroplasmic for a mutation in the mitochondrial tRNALeu(UUR) gene at position 3251. A large proportion of muscle fibres contained accumulations of abnormal mitochondria but no cytochrome c oxidase deficient fibres were present. Polarographic and enzymatic measurements on isolated muscle mitochondria revealed a profound isolated complex I deficiency. A high percentage of mutant mtDNA was found in muscle (94%), fibroblasts (93%), brain (90%), liver (80%), and heart (79%). The family was not available for investigation. For genotype to phenotype correlation studies, we investigated the proportion of mutated mtDNA in single muscle fibres of normal appearance and muscle fibres with accumulations of mitochondria. The proportion of mutant mtDNA was 28% (range < 0.3%–86%) in normal-appearing fibres and 61% (range 15%–88%) in abnormal fibres. The difference in the proportion of mutant mtDNA was highly significant (P < 0.001) between the two groups of fibres.     

Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library

Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08–6.44 ng mL⁻¹ and the medium inhibition of control (IC₅₀) was 0.73 ng mL⁻¹. It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%–96.6% and with a coefficient of variation (CV) among 2.0%–8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples.     

Simultaneous determination of aminomethylbenzoic acid,cefminox sodium and etamsylate in human urine by capillary electrophoresis

A sensitive and selective capillary electrophoresis method is developed, for the first time, for effective separation and simultaneous determination of aminomethylbezoic acid (PAMBA), cefminox sodium (CMNX) and etamsylate (ETM). The electrophoresis conditions were investigated and optimized. A 25 mM phosphate solution (pH 8.5) was used as a buffer and the peak area was determined with UV detection at 216 nm wavelength under 18 kV separation voltage. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 3.13–150 μg/mL for PAMBA, 6.25–150 μg/mL for CMNX and 3.13–150 μg/mL for ETM, with the correlation coefficients of >0.999. The limit of detection (LOD) for PAMBA, CMNX and ETM was 1.04, 2.08 and 1.04 μg/mL, respectively. Their recoveries in human urine were in the range from 90.2% to 101% with the RSD (n = 5) of 0.7–3.1%. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for analysis of the test drugs in human urine at clinically relevant concentrations.     

Development of a localized surface plasmon resonance-based gold nanobiosensor for the determination of prolactin hormone in human serum

A localized surface plasmon resonance immunoassay has been developed to determine prolactin hormone in human serum samples. Gold nanoparticles were synthesized, and the probe was prepared by electrostatic adsorption of antibody on the surfaces of gold nanoparticles. The pH and the antibody-to-gold nanoparticle ratio, as the factors affecting the probe functions, were optimized. The constructed nanobiosensor was characterized by dynamic light scattering. The sensor was applied for the determination of prolactin antigen concentration based on the amount of localized surface plasmon resonance peak shift. A linear dynamic range of 1–40 ng ml⁻¹, a detection limit of 0.8 ng ml⁻¹, and sensitivity of 10 pg ml⁻¹ were obtained. Finally, the nanobiosensor was applied for the determination of prolactin in human control serum sample.     

Differences in preterm and term milk fatty acid compositions may be caused by the different hormonal milieu of early parturition

Introduction

The hormonal milieus of pregnancy and lactation are driving forces of nutrient fluxes supporting infant growth and development. The decrease of insulin sensitivity with compensatory hyperinsulinemia with advancing gestation, causes adipose tissue lipolysis and hepatic de novo lipogenesis (DNL).

Subjects and methods

We compared fatty acid (FA) contents and FA-indices for enzyme activities between preterm (28–36 weeks) and term (37–42) milks, and between colostrum (2–5 days), transitional (6–15) and mature (16–56) milks. We interpreted FA differences between preterm and term milks, and their changes with lactation, in terms of the well known decrease of insulin sensitivity during gestation and its subsequent postpartum restoration, respectively.

Results

Compared with term colostrum, preterm colostrum contained higher indices of DNL in the breast (DNL-breast) and medium chain saturated-FA (MCSAFA), and lower DNL-liver and monounsaturated-FA (MUFA). Preterm milk also had higher docosahexaenoic acid (DHA) in colostrum and transitional milk and higher arachidonic acid (AA) in mature milk. Most preterm-term differences vanished with advancing lactation. In both preterm and term milks, DNL-breast and MCSAFA increased with advancing lactation, while DNL-liver, MUFA, long chain SAFA and AA decreased. DHA decreased in term milk. MUFA was inversely related to MCSAFA in all samples, correlated inversely with PUFA in colostrum and transitional milks, but positively in mature milk. MCSAFA correlated inversely with PUFA in mature milk.

Conclusion

Higher maternal insulin sensitivity at preterm birth may be the cause of lower MUFA (a proxy for DNL-liver) and higher MCSAFA (a proxy for DNL-breast) in preterm colostrum, compared with term colostrum. Restoring insulin sensitivity after delivery may be an important driving force for milk FA-changes in early lactation.     

Enzyme-Linked Immunosorbent Assay of Ampicillin in Milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10–1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. Limit of detection for ampicillin in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).