Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK

Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic streptococci (βHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 βHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI–NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10⁴ IU mg^?1). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.     

Intraguild predation by the generalist predator Orius majusculus on the parasitoid Encarsia formosa

Intraguild predation of Orius majusculus (Reuter) (Heteroptera: Anthocoridae) on Encarsia formosa (Gahan) (Hymenoptera: Aphelinidae), both natural enemies of Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae), was studied under laboratory conditions. The experiments quantified prey consumption by 5th instar nymphs and adults of O. majusculus offered unparasitised 3rd, early 4th or 4th instar B. tabaci nymphs or parasitised nymphs containing 2nd or 3rd larval instar or pupal parasitoids. In addition, prey preference of the two stages of O. majusculus for parasitised or unparasitised whitefly nymphs was studied using nine different prey combinations. Both predator stages readily preyed upon on both unparasitised and parasitised B. tabaci. In no-choice experiments, predation on 3rd instar E. formosa by adult predators was the highest, while predator nymphs preyed most on unparasitised 3rd instar B. tabaci and 2nd instar parasitoids. Predation of predator stages was lowest on 4th instar B. tabaci and E. formosa pupae. In all prey combinations, both stages of O. majusculus showed a significant preference for parasitised over unparasitised whitefly nymphs except for the combination of 5th instars of O. majusculus with early 4th instar whiteflies and E. formosa pupae. The results indicate that intraguild interactions between O. majusculus and E. formosa may have negative effects on biological control of B. tabaci.     

Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.     

Aspergillus fumigatus Invasion Increases with Progressive Airway Ischemia

Despite the prevalence of Aspergillus-related disease in immune suppressed lung transplant patients, little is known of the host-pathogen interaction. Because of the mould’s angiotropic nature and because of its capacity to thrive in hypoxic conditions, we hypothesized that the degree of Aspergillus invasion would increase with progressive rejection-mediated ischemia of the allograft. To study this relationship, we utilized a novel orthotopic tracheal transplant model of Aspergillus infection, in which it was possible to assess the effects of tissue hypoxia and ischemia on airway infectivity. Laser Doppler flowmetry and FITC-lectin were used to determine blood perfusion, and a fiber optic microsensor was used to measure airway tissue oxygen tension. Fungal burden and depth of invasion were graded using histopathology. We demonstrated a high efficacy (80%) for producing a localized fungal tracheal infection with the majority of infection occurring at the donor-recipient anastomosis; Aspergillus was more invasive in allogeneic compared to syngeneic groups. During the study period, the overall kinetics of both non-infected and infected allografts was similar, demonstrating a progressive loss of perfusion and oxygenation, which reached a nadir by days 10-12 post-transplantation. The extent of Aspergillus invasion directly correlated with the degree of graft hypoxia and ischemia. Compared to the midtrachea, the donor-recipient anastomotic site exhibited lower perfusion and more invasive disease; a finding consistent with clinical experience. For the first time, we identify ischemia as a putative risk factor for Aspergillus invasion. Therapeutic approaches focused on preserving vascular health may play an important role in limiting Aspergillus infections.     

Development of Novel Prime-Boost Strategies Based on a Tri-Gene Fusion Recombinant L. tarentolae Vaccine against Experimental Murine Visceral Leishmaniasis

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB^-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB^-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB^-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL.     

Revealing substitution effects on the strength and nature of halogen-hydride interactions: a theoretical study

A quantum chemistry study was carried out to investigate the strength and nature of halogen bond interactions in HXeH···XCCY complexes, where X = Cl, Br and Y = H, F, Cl, Br, CN, NC, C₂H, CH₃, OH, SH, NH₂. Examination of the electrostatic potentials V(r) of the XCCY molecules reveals that the addition of substituents has a significant effect upon the most positive electrostatic potential on the surface of the interacting halogen atom. We found that the magnitude of atomic charges and multipole moments depends upon the halogen atom X and is rather sensitive to the electron-withdrawing/donating power of the remainder of the molecule. An excellent correlation was found between the most positive electrostatic potentials on the halogen atom and the interaction energies. For either HXeH···ClCCY or HXeH···BrCCY complexes, an approximate linear correlation between the interaction energies and halogens multipole moments are established, indicating that the electrostatic and polarization interactions are responsible for the stability of the complexes. According to energy decomposition analysis, it is revealed that the electrostatic interactions are the major source of the attraction in the HXeH···XCCY complexes. Furthermore, the changes in the electrostatic term are mainly responsible for the dependence of interaction energy on the halogen atom.

Molecular dynamics and QM/MM-based 3D interaction analyses of cyclin-E inhibitors

Abnormal expression of cyclin-dependent kinase 2 (CDK2)/cyclin-E is detected in colorectal, ovarian, breast and prostate cancers. The study of CDK2 with a bound inhibitor revealed CDK2 as a potential therapeutic target for several proliferative diseases. Several highly selective inhibitors of CDK2 are currently undergoing clinical trials, but possibilities remain for the identification and development of novel and improved inhibitors. For example, in silico targeting of ATP-competitive inhibitors of CDKs is of special interest. A series of 3,5-diaminoindazoles was studied using molecular docking and comparative field analyses. We used post-docking short time molecular dynamics (MD) simulation to account for receptor flexibility. The three types of structures, i.e., the highest energy, lowest energy and the structure most resembling the X-ray structure (three complexes) were identified for all ligands. QM/MM energy calculations were performed using a DFT b3lyp/6–31 g* and MM OPLS-2005 force field. Conceptual DFT properties such as the interaction energy of ligand to protein, global hardness (η), HOMO density, electrostatic potential, and electron density were calculated and related to inhibitory activity. CoMFA and CoMSIA were used to account for steric and electrostatic interactions. The results of this study provide insight into the bioactive conformation, interactions involved, and the effect of different drug fragments over different biological activities.     

Dynamic Behavior of Rat Phosphoenolpyruvate Carboxykinase Inhibitors: New Mechanism for Enzyme Inhibition

As an enzyme acting at the junction of gluconeogenic pathway, phosphoenolpyruvate carboxykinase (PEPCK) controls substrate flow from Krebs cycle toward glucose production. Therefore, it would be advantageous to design effective inhibitors to inactivate PEPCK in diabetes mellitus and other abnormalities caused by insulin resistance. Such inhibitors may compensate the metabolic consequences of ex-activity of PEPCK at these conditions. Understanding the mechanism by which inhibitors exert their effect on enzyme activity is of great interest for designing stronger inhibitors. In the present work, molecular dynamic simulations were used to study enzyme-inhibitor interactions. Our results indicate that inhibitors of PEPCK with their short chains interact with enzyme active site through non-covalent interactions of electrostatic and hydrogen bond nature. The data also show that inhibitors neither reach a stable state in their binding site nor make static complex with the enzyme active site. Instead, they interact with functional groups of active site residues in a dynamic fashion. In this way, oxalate and sulfoacetate carrying two negative groups of higher charge density and optimum spacing from each other, show more dynamic behavior (lower stability in their binding site) and more inhibitory effects than other inhibitors used (phosphonoformate, phosphoglycolate and 3-phosphonopropionate).