Interrogation of phosphor-specific interaction on a high-throughput label-free optical biosensor system–Epic® system

The Epic^® system, a high-throughput label-free optical biosensor system, is applied for the biochemical interrogation of phosphor-specific interactions of the 14-3-3 protein and its substrates. It has shown the capability not only for high-throughput characterization of binding rank and affinity but also for the exploration of potential interacting kinases for the substrates. A perspective of biochemical applications for diagnostics and biomarker discovery, as well as cell-based applications for endogenous receptors and viral infection characterization, are also provided.     

“Aqua-space®”, a New Headspace Method for Isolation of Natural Floral Aromas Using Humidified Air as a Carrier Gas

A new method called “Aqua-space^®” was developed for the isolation of the natural fragrances of plants. Living flowers were enclosed in a space under simulated natural conditions, and humidified air was pumped into the space as a fragrance carrier. In a comparison among three isolation methods, i.e., Aqua-space^®, headspace, and solvent extraction, the Aqua-space^® method proved to be the most effective in retaining natural fragrances with abundant oxygenated components key to floral fragrances.     

Biomedical applications of ion mobility-enhanced data-independent acquisition-based label-free quantitative proteomics

Mass spectrometry-based proteomics greatly benefited from recent improvements in instrument performance and the development of bioinformatics solutions facilitating the high-throughput quantification of proteins in complex biological samples. In addition to quantification approaches using stable isotope labeling, label-free quantification has emerged as the method of choice for many laboratories. Over the last years, data-independent acquisition approaches have gained increasing popularity. The integration of ion mobility separation into commercial instruments enabled researchers to achieve deep proteome coverage from limiting sample amounts. Additionally, ion mobility provides a new dimension of separation for the quantitative assessment of complex proteomes, facilitating precise label-free quantification even of highly complex samples. The present work provides a thorough overview of the combination of ion mobility and data-independent acquisition-based label-free quantification LC-MS and its applications in biomedical research.     

ARGX-110, a highly potent antibody targeting CD70, eliminates tumors via both enhanced ADCC and immune checkpoint blockade

Overexpression of CD70 has been documented in a variety of solid and hematological tumors, where it is thought to play a role in tumor proliferation and evasion of immune surveillance. Here, we describe ARGX-110, a defucosylated IgG1 monoclonal antibody (mAb) that selectively targets and neutralizes CD70, the ligand of CD27.

ARGX-110 was generated by immunization of outbred llamas. The antibody was germlined to 95% human identity, and its anti-tumor efficacy was tested in several in vitro assays. ARGX-110 binds CD70 with picomolar affinity. In depletion studies, ARGX-110 lyses tumor cells with greater efficacy than its fucosylated version. In addition, ARGX-110 demonstrates strong complement-dependent cytotoxicity and antibody-dependent cellular phagocytosis activity. ARGX-110 inhibits signaling of CD27, which results in blocking of the activation and proliferation of Tregs. In a Raji xenograft model, administration of the fucosylated version of ARGX-110 resulted in a prolonged survival at doses of 0.1 mg/kg and above. The pharmacokinetics of ARGX-110 was tested in cynomolgus monkeys; the calculated half-life is 12 days.

In conclusion, ARGX-110 is a potent blocking mAb with a dual mode of action against both CD70-bearing tumor cells and CD70-dependent Tregs. This antibody is now in a Phase 1 study in patients with advanced malignancies expressing CD70 (NCT01813539).     

Phytoremediation: green technology for the removal of mixed contaminants of a water supply reservoir

The Iraí Reservoir, a water supply in Brazil, is constantly impacted by anthropogenic activities such as waste inputs from agriculture, hospitals and urbanization, resulting toxic cyanobacterial blooms causing economic, social and environmental problems. This study assessed the concentration of some common contaminants of the Iraí Reservoir, namely paracetamol, diclofenac and microcystin-LR and tested whether a laboratory scale Green Liver System^® would serve as a suitable technology to remove these contaminants. Further, the study investigated whether the pollutants caused adverse effects to the macrophytes using catalase as a biomarker for oxidative stress and investigated whether biotransformation (glutathione S-transferase) was a main route for detoxification. Egeria densa, Ceratophyllum demersum and Myriophyllum aquaticum were exposed to a mixture of the three contaminants for 14?days in a concentration range similar to those detected in the reservoir. The plants removed 93% of diclofenac and 100% of MC-LR after 14?days. Paracetamol could not be detected. Catalase and glutathione S-transferase enzyme activities remained unaltered after the 14-day exposure, indicating that the mixture did not cause oxidative stress. The study showed that the aquatic macrophytes used are suitable tools to apply in a Green Liver System^® for the remediation of mixed pollutants.     

Degradation of Polychlorinated Biphenyls by UV-Catalyzed Photolysis

Photolysis of five polychlorinated biphenyl (PCB) congeners [2,4,4′-trichlorobiphenyl (PCB 28), 2,2′,5,′5-tetrachlorobiphenyl (PCB 52), 2,2′,4,5,5′-pentachlorobiphenyl (PCB 101), 2,2′,4,4′,5,′5-hexachlorobiphenyl (PCB 153) and 2,2′,3,4,4′,5,′5-heptachlorobiphenyl (PCB 180)] individually and in combination were carried out in the solvents methanol, ethanol, and 2-propanol. The disappearance of parent congener generally increased with UV intensity. The solvents had significant or limited effect on the removal of PCBs depending on the congener used. Because 2-propanol was highly toxic and methoxylated products were formed when methanol was used, ethanol was selected as the optimum solvent. The results of photolysis of the PCB mixture showed that PCB 52 was formed and accumulated after 4 h of photolysis. The addition of sodium hydroxide increased the rate of photolysis of the PCB mixture. One hundred percent removal can be obtained of the PCB in mixture in 90 min under optimized conditions. Gas chromatography–mass spectrometry was used to determine the intermediates of the photolysis of PCBs under optimized conditions. For the PCB congeners and mixture studied, the major photolytic intermediates were less chlorinated congeners, and biphenyl was the major product with minor amounts of hydroxylated PCBs, ethylated, dimethylated, and methylated biphenyls. Biphenyl could be further degraded by a prolonged photolysis. Toxicity of the PCB mixture during photolysis was monitored by the Microtox^® test. It was found that the toxicity increased at the early stage of photolysis, and gradually decreased as the reaction proceeded. After 90 min, the EC₅₀ of the reaction mixture was similar to that of the untreated sample.     

Construction of a 19F-lectin biosensor for glycoprotein imaging by using affinity-guided DMAP chemistry

In this study, assisted by affinity-guided DMAP strategy, we developed a novel ¹⁹F-modified lectin as a biosensor for specific detection and imaging of glycoproteins. Exploited the large chemical shift anisotropy property of ¹⁹F nuclei, glycoproteins detected by our ¹⁹F-biosensor are signatured by broadened peaks in ¹⁹F NMR, hence enabled the distinction between glycoproteins and small molecule saccharides. Such signal on/off switching was also applied to glycoprotein imaging by ¹⁹F MRI.     

Multiplex proteomic approaches to sepsis research: case studies employing new technologies

Sepsis is a multifactorial disease that provides unique challenges to the critical care physician. Diagnosis is hampered by the lack of a quantitative in vitro diagnostic test, instead, it relies on a series of clinical measures. The complex nature of the disease, with involvement of several physiologic systems, suggests a need to simultaneously monitor many clinical parameters. Novel proteomic technologies now exist that enable the multiplex measurement of multiple protein analytes from the same sample. Integration of these analytical measures with patient clinical data may provide the foundation for a better understanding of disease diagnosis, disease progression and the selection of optimal therapeutic regimen. The future challenge is the translation of these multiplex approaches from investigative research to clinical diagnostics for the greatest impact on patient treatment decisions.     

Proteasome inhibitor MG132 blocks viral DNA replication and assembly of human cytomegalovirus

This study provides evidence that proteasomal activity is required at multiple steps in human cytomegalovirus replication. Electron microscopy revealed that no viral particles were assembled in the presence of proteasome inhibitor MG132. Immunofluorescence and Western blot analyses using MG132 demonstrated that immediate early gene expression was suppressed at low but not high MOI. In contrast, expression of late proteins was completely blocked independent of MOI. Additionally, pulsed-field gel electrophoresis demonstrated that MG132 interferes with cleavage of HCMV DNA. Bromodeoxyuridine incorporation studies showed that de novo viral DNA synthesis is reduced in the presence of MG132. Furthermore, in contrast to previous hypotheses we demonstrated that neither the ND10 components PML and hDaxx nor NFkappaB activation represent the target for MG132.     

Up-regulation of ras-GAP genes is reversed by a MEK inhibitor and doxorubicin in v-Ki-ras-transformed NIH/3T3 fibroblasts

Ras-GTPase-activating proteins (Ras-GAPs) have been implicated both as suppressors of Ras and as effectors in regulating cellular activities. To study whether Ras-GAPs have roles in tumor cell survival or not, mRNA levels of ras-related genes were measured in v-Ki-ras-transformed (DT) and the parental NIH/3T3 cells, using real-time PCR. mRNA levels of p120-Gap, Gap1(m), and PIK3CA were increased in DT cells compared with NIH/3T3 cells. p120-Gap and PIK3CA genes were induced by addition of serum or epidermal growth factor to serum-starved DT cells. Three anti-cancer drugs, an ERK kinase (MEK) inhibitor PD98059, a topoisomerase II poison doxorubicin (adriamycin), and a histone deacetylase inhibitor trichostatin A, selectively blocked the overexpression of p120-Gap and Gap1(m) genes in DT cells. These drugs also caused reversion of DT cells to the adherent shape associated with growth arrest. Our results suggest that p120-Gap and Gap1(m) genes provide important biomarkers for cancer therapies.     

Paediatric bed fall computer simulation model development and validation

Falls from beds and other household furniture are common scenarios stated to conceal child abuse. Knowledge of the biomechanics associated with short-distance falls may aid clinicians in distinguishing between abusive and accidental injuries. Computer simulation is a useful tool to investigate injury-producing events and to study the effect of altering event parameters on injury risk. In this study, a paediatric bed fall computer simulation model was developed and validated. The simulation was created using Mathematical Dynamic Modeling^® software with a child restraint air bag interaction (CRABI) 12-month-old anthropomorphic test device (ATD) representing the fall victim. The model was validated using data from physical fall experiments of the same scenario with an instrumented CRABI ATD. Validation was conducted using both observational and statistical comparisons. Future parametric sensitivity studies using this model will lead to an improved understanding of relationships between child (fall victim) parameters, fall environment parameters and injury potential.     

Analysis of the interactions between HIV-1 and the cellular prion protein in a human cell line

The cellular prion protein (PrP(c)) is highly conserved in mammals and expressed widely in different tissues but its physiological role remains elusive. Recently, the human PrP(c) was shown to possess nucleic acid binding and chaperoning properties similar to human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, a key viral factor in virus structure and replication. These findings prompted us to determine if PrP(c) could influence HIV-1 replication. We used the human 293T cell line as a model system, since only a very low level of PrP(c) accumulates in these cells. Expression of PrP at a high level resulted in a specific decrease of HIV-1 Env and Vpr expression. Despite similar levels of intracellular Gag, virus production was reduced by eightfold and infectivity by three- to fourfold in the presence of PrP(c). A PrP(c) mutant lacking the glycosylphosphatidylinositol (GPI) anchor peptide did not impair HIV-1 production, suggesting that PrP(c) trafficking is critical for this inhibitory effect. Coexpressing HIV-1 and PrP(c) in these cells also caused a fraction of PrP(c) to become partially proteinase K-resistant (PrP(res)), further illustrating the interactions between HIV-1 and PrP(c).     

Development and characterization in vitro of a catalase-based biosensor for hydrogen peroxide monitoring

There is increasing evidence that hydrogen peroxide (H₂O₂) may act as a neuromodulator in the brain, as well as contributing to neurodegeneration in diseased states, such as Parkinson's disease. The ability to monitor changes in endogenous H₂O₂ in vivo with high temporal resolution is essential in order to further elucidate the roles of H₂O₂ in the central nervous system. Here, we describe the in vitro characterization of an implantable catalase-based H₂O₂ biosensor. The biosensor comprises two amperometric electrodes, one with catalase immobilized on the surface and one without enzyme (blank). The analytical signal is then the difference between the two electrodes. The H₂O₂ sensitivity of various designs was compared, and ranged from 0 to 56 ± 4 mA cm⁻² M⁻¹. The most successful design incorporated a Nafion^® layer followed by a poly-o-phenylenediamine (PPD) polymer layer. Catalase was adsorbed onto the PPD layer and then cross-linked with glutaraldehyde. The ability of the biosensors to exclude interference from ascorbic acid, and other interference species found in vivo, was also tested. A variety of the catalase-based biosensor designs described here show promise for in vivo monitoring of endogenous H₂O₂ in the brain.     

Conserved water-mediated recognition and dynamics of NAD+ (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10?ns simulation of the IMP–NAD⁺ complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD⁺. Three conserved water molecules (W1, W, and W1′) in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD⁺) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP–NAD⁺-enzyme complexes and their recognition to NAD⁺, some covalent modification at carboxamide group of di-nucleotide (NAD⁺) has been made by substituting the –CONH₂group by –CONHNH₂ (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.     

Phospho-specific recognition by 14-3-3 proteins and antibodies monitored by a high throughput label-free optical biosensor

Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs. Using a resonant waveguide grating biosensor, the Epic mark System, we have developed a generic assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with a solution-based fluorescence anisotropy assay, our results support that the high throughput resonant waveguide grating biosensor system has favorable technical profiles in detecting protein-protein interactions that recognize phosphorylated motifs. Hence it provides a new generic HTS platform for phospho-detection.     

A snapshot of viral evolution from genome analysis of the tectiviridae family

The origin, evolution and relationships of viruses are all fascinating topics. Current thinking in these areas is strongly influenced by the tailed double-stranded (ds) DNA bacteriophages. These viruses have mosaic genomes produced by genetic exchange and so new natural isolates are quite dissimilar to each other, and to laboratory strains. Consequently, they are not amenable to study by current tools for phylogenetic analysis. Less attention has been paid to the Tectiviridae family, which embraces icosahedral dsDNA bacterial viruses with an internal lipid membrane. It includes viruses, such as PRD1, that infect Gram-negative bacteria, as well as viruses like Bam35 with Gram-positive hosts. Although PRD1 and Bam35 have closely related virion morphology and genome organization, they have no detectable sequence similarity. There is strong evidence that the Bam35 coat protein has the "double-barrel trimer" arrangement of PRD1 that was first observed in adenovirus and is predicted to occur in other viruses with large facets. It is very likely that a single ancestral virus gave rise to this very large group of viruses. The unprecedented degree of conservation recently observed for two Bam35-like tectiviruses made it important to investigate those infecting Gram-negative bacteria. The DNA sequences for six PRD1-like isolates (PRD1, PR3, PR4, PR5, L17, PR772) have now been determined. Remarkably, these bacteriophages, isolated at distinctly different dates and global locations, have almost identical genomes. The discovery of almost invariant genomes for the two main Tectiviridae groups contrasts sharply with the situation in the tailed dsDNA bacteriophages. Notably, it permits a sequence analysis of the isolates revealing that the tectiviral proteins can be dissected into a slowly evolving group descended from the ancestor, the viral self, and a more rapidly changing group reflecting interactions with the host.     

MALDI-TOF mass spectrometry in the clinical mycology laboratory: identification of fungi and beyond

MALDI-TOF mass spectrometry (MS) is becoming essential in most clinical microbiology laboratories throughout the world. Its successful use is mainly attributable to the low operational costs, the universality and flexibility of detection, as well as the specificity and speed of analysis. Based on characteristic protein spectra obtained from intact cells – by means of simple, rapid and reproducible preanalytical and analytical protocols – MALDI-TOF MS allows a highly discriminatory identification of yeasts and filamentous fungi starting from colonies. Whenever used early, direct identification of yeasts from positive blood cultures has the potential to greatly shorten turnaround times and to improve laboratory diagnosis of fungemia. More recently, but still at an infancy stage, MALDI-TOF MS is used to perform strain typing and to determine antifungal drug susceptibility. In this article, the authors discuss how the MALDI-TOF MS technology is destined to become a powerful tool for routine mycological diagnostics.     

Laboratory evaluation of a new strain CCM 8367 of Isaria fumosorosea (syn. Paecilomyces fumosoroseus) on Spodoptera littoralis (Boisd.)

Laboratory experiments were conducted to assess the efficiency of the new strain CCM 8367 of Isaria fumosorosea on different stages of the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.), including soil treatments with pre-pupal and pupal stages. Treatments of larval instars showed a high susceptibility with 3rd, 5th and last instars to suspension of this fungus with concentration 5?×?10⁷ spores/ml. Larval mortality was over 90%. There were no significant differences (P?=?0.7929, F?=?0.2346) between instar treatments. The commercial fungus, PreFeRal® strain Apopka 97 of I. fumosorosea, which was used in comparison with this new strain caused mortality rates of between 63.33 and 90%. Statistically, differences between the effects of CCM 8367 strain and Apopka 97 were highly significant on the last instar (P?=?0.0064, F?=?6.479) and extremely significant on the 3rd instar (P?<?0.0001, F?=?13.29). No significant differences were recorded between the two strains on the 5th instar (P?=?0.0597, F?=?3.233). Fungal treatments with the late stage insects (end of the last instar or pupal stage) in soil yielded interesting results: the mortality rate on end of final instar larvae was 16.66% when treated with Apopka 97 and 83.33% when treated with CCM 8367. Soil containing pupae of S. littoralis that were inoculated with CCM8367 resulted in a high number of malformed adults, and the mortality rate was 64.52% (32.27% of malformed adults died and 32.25% of pupae fully infected by fungus). Only 3.23% of samples produced morphologically normal adults in this test. The results conclude that the strain of I. fumosorosea CCM 8367 has strong insecticidal effects on S. littoralis and has the potential to be implemented as a novel biocontrol agent.     

Effect of ethylisopropyl amiloride,a Na+-H+ exchange inhibitor,on cardioprotective effect of ischaemic and angiotensin preconditioning

The present study is designed to investigate the role of Na⁺-H⁺ exchanger in the cardioprotective effect of ischaemic and angiotensin (Ang II) preconditioning. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Left ventricular developed pressure (LVDP) and dp/dt were recorded to evaluate myocardial contractility. Four episodes of ischaemic or Ang II preconditioning markedly reduced LDH and CK release in coronary effluent and decreased myocardial infarct size. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a Na⁺-H⁺ exchange inhibitor, produced no marked effect on ischaemic preconditioning and Ang II preconditioning induced cardioprotection. On the other hand, EIPA administration prior to global ischaemia produced a similar reduction in myocardial injury as was noted with ischaemic preconditioning or Ang II preconditioning. On the basis of these results, it may be concluded that inhibition of Na⁺-H⁺ exchanger protects against ischaemia-reperfusion induced myocardial injury whereas activation of Na⁺-H⁺ exchanger may not mediate the cardioprotective effect of ischaemic and Ang II preconditioning.