Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target

Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family''s role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s'' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative structural (gene and protein structure-level) and evolutionary analysis of a fungal P450 family.     

Synthesis of Ester-Linked Ursolic Acid-Based Hybrid Compounds: Potential Antibacterial and Anticancer Agents

The molecular hybridization of two or more drugs into a single molecule is an effective drug design approach to reduce pill burden and improve patient treatment adherence. Ursolic acid-based hybrid compounds were synthesized and characterized followed by molecular docking studies. In vitro studies against various bacterial strains and human cancer cells (MDA-MB-231, HeLa, and MCF-7) were performed. Compounds 14 – 19 , 21 , 34 , 31 , and 30 demonstrated significant antibacterial activities with MIC values of 15.625 μg/ml. Compounds 29 and 34 were more cytotoxic than ursolic acid, with IC₅₀ values of 46.99 and 48.18 μg/ml. Compounds 29 and 34 in the docking studies presented favourable binding interactions and better docking energy against the Epidermal Growth Factor Receptor (EGFR) than the parent compound, ursolic acid. The findings revealed that the ursolic acid scaffold is a promising precursor for the development of molecules with promising anticancer and antimicrobial activities. However, more studies are needed to fully understand their mode of action.     

Comparative analysis of the Metarhizium anisopliae secretome in response to exposure to the greyback cane grub and grub cuticles

Metarhizium anisopliae is a well-characterized biocontrol agent of a wide range of insects including cane grubs. In this study, a two-dimensional (2D) electrophoresis was used to display secreted proteins of M. anisopliae strain FI-1045 growing on the whole greyback cane grubs and their isolated cuticles. Hydrolytic enzymes secreted by M. anisopliae play a key role in insect cuticle-degradation and initiation of the infection process. We have identified all the 101 protein spots displayed by cross-species identification (CSI) from the fungal kingdom. Among the identified proteins were 64-kDa serine carboxypeptidase, 1,3 beta-exoglucanase, Dynamin GTPase, THZ kinase, calcineurin like phosphoesterase, and phosphatidylinositol kinase secreted by M. ansiopliae (FI-1045) in response to exposure to the greyback cane grubs and their isolated cuticles. These proteins have not been previously identified from the culture supernatant of M. anisopliae during infection. To our knowledge, this the first proteomic map established to study the extracellular proteins secreted by M. ansiopliae (FI-1045) during infection of greyback cane grubs and its cuticles.     

Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury

Previous studies indicate that deficiency of endothelial nitric oxide (NO) synthase (eNOS)-derived NO exacerbates myocardial reperfusion injury. We hypothesized that overexpression of eNOS would reduce the extent of myocardial ischemia-reperfusion (MI/R) injury. We investigated two distinct strains of transgenic (TG) mice overexpressing the eNOS gene (eNOS TG). Bovine eNOS was overexpressed in one strain (eNOS TG-Kobe), whereas the human eNOS gene was overexpressed in the other strain (eNOS TG-RT). Non-TG (NTG) and eNOS TG mice were subjected to 30 min of coronary artery occlusion followed by 24 h of reperfusion, and the extent of myocardial infarction was determined. Myocardial infarct size was reduced by 33% in the eNOS TG-Kobe strain (P < 0.05 vs. NTG) and by 32% in the eNOS TG-RT strain (P < 0.05 vs. NTG). However, postischemic cardiac function (cardiac output, fractional shortening) was not improved in the eNOS TG-Kobe mouse at 24 h of reperfusion [P = not significant (NS) vs. NTG]. In additional studies, eNOS TG-Kobe mice were subjected to 30 min of myocardial infarction and 7 days of reperfusion. Fractional shortening and the first derivative of left ventricular pressure were measured in eNOS TG-Kobe and NTG mice, and no significant differences in contractility were observed (P = NS) between the eNOS TG mice and NTG controls. Left ventricular end-diastolic pressure was significantly (P < 0.05 vs. NTG) reduced in the eNOS TG-Kobe strain at 7 days of reperfusion. The cardioprotective effects of eNOS overexpression on myocardial infarct size were ablated by Nomega-nitro-l-arginine methyl ester (300 mg/kg) pretreatment. Thus genetic overexpression of eNOS in mice attenuates myocardial infarction after MI/R but fails to significantly protect against postischemic myocardial contractile dysfunction in mice.     

The application of the pedigree approach to the distributions foreseen in ecoinvent v3

Purpose

Data used in life cycle inventories are uncertain (Ciroth et al. Int J Life Cycle Assess 9(4):216–226, 2004). The ecoinvent LCI database considers uncertainty on exchange values. The default approach applied to quantify uncertainty in ecoinvent is a semi-quantitative approach based on the use of a pedigree matrix; it considers two types of uncertainties: the basic uncertainty (the epistemic error) and the additional uncertainty (the uncertainty due to using imperfect data). This approach as implemented in ecoinvent v2 has several weaknesses or limitations, one being that uncertainty is always considered as following a lognormal distribution. The aim of this paper is to show how ecoinvent v3 will apply this approach to all types of distributions allowed by the ecoSpold v2 data format.

Methods

A new methodology was developed to apply the semi-quantitative approach to distributions other than the lognormal. This methodology and the consequent formulas were based on (1) how the basic and the additional uncertainties are combined for the lognormal distribution and on (2) the links between the lognormal and the normal distributions. These two points are summarized in four principles. In order to test the robustness of the proposed approach, the resulting parameters for all probability density functions (PDFs) are tested with those obtained through a Monte Carlo simulation. This comparison will validate the proposed approach.

Results and discussion

In order to combine the basic and the additional uncertainties for the considered distributions, the coefficient of variation (CV) is used as a relative measure of dispersion. Formulas to express the definition parameters for each distribution modeling a flow with its total uncertainty are given. The obtained results are illustrated with default values; they agree with the results obtained through the Monte Carlo simulation. Some limitations of the proposed approach are cited.

Conclusions

Providing formulas to apply the semi-quantitative pedigree approach to distributions other than the lognormal will allow the life cycle assessment (LCA) practitioner to select the appropriate distribution to model a datum with its total uncertainty. These data variability definition technique can be applied on all flow exchanges and also on parameters which play an important role in ecoinvent v3.

     

The osmotic/calcium stress theory of brain damage: Are free radicals involved?

This overview presents data showing that glucose use increases and that excitatory amino acids (i.e., glutamate, aspartate), taurine and ascorbate increase in the extracellular fluid during seizures. During the cellular hyperactive state taurine appears to serve as an osmoregulator and ascorbate may serve as either an antioxidant or as a pro-oxidant. Finally, a unifying hypothesis is given for seizure-induced brain damage. This unifying hypothesis states that during seizures there is a release of excitatory amino acids which act on glutamatergic receptors, increasing neuronal activity and thereby increasing glucose use. This hyperactivity of cells causes an influx, of calcium (i.e. calcium stress) and water movements (i.e., osmotic stress) into the cells that culminate in brain damage mediated by reactive oxygen species.Special issue dedicated to Dr. Frederick E. Samson     

Inhibitory Effect of Nasal Intermittent Positive Pressure Ventilation on Gastroesophageal Reflux

Non-invasive intermittent positive pressure ventilation can lead to esophageal insufflations and in turn to gastric distension. The fact that the latter induces transient relaxation of the lower esophageal sphincter implies that it may increase gastroesophageal refluxes. We previously reported that nasal Pressure Support Ventilation (nPSV), contrary to nasal Neurally-Adjusted Ventilatory Assist (nNAVA), triggers active inspiratory laryngeal closure. This suggests that esophageal insufflations are more frequent in nPSV than in nNAVA. The objectives of the present study were to test the hypotheses that: i) gastroesophageal refluxes are increased during nPSV compared to both control condition and nNAVA; ii) esophageal insufflations occur more frequently during nPSV than nNAVA. Polysomnographic recordings and esophageal multichannel intraluminal impedance pHmetry were performed in nine chronically instrumented newborn lambs to study gastroesophageal refluxes, esophageal insufflations, states of alertness, laryngeal closure and respiration. Recordings were repeated without sedation in control condition, nPSV (15/4 cmH₂O) and nNAVA (~ 15/4 cmH₂O). The number of gastroesophageal refluxes recorded over six hours, expressed as median (interquartile range), decreased during both nPSV (1 (0, 3)) and nNAVA [1 (0, 3)] compared to control condition (5 (3, 10)), (p < 0.05). Meanwhile, the esophageal insufflation index did not differ between nPSV (40 (11, 61) h^-1) and nNAVA (10 (9, 56) h^-1) (p = 0.8). In conclusion, nPSV and nNAVA similarly inhibit gastroesophageal refluxes in healthy newborn lambs at pressures that do not lead to gastric distension. In addition, the occurrence of esophageal insufflations is not significantly different between nPSV and nNAVA. The strong inhibitory effect of nIPPV on gastroesophageal refluxes appears identical to that reported with nasal continuous positive airway pressure.     

Inhibition of (3H)vitellogenin uptake by isolated amphibian oocytes injected with cytoplasm from progesterone-treated mature oocytes.

Fully grown meiotically immature (germinal vesicle stage) amphibian oocytes incorporate radioactive protein ([³H]vitellogenin) following in vitro culture. In vitro exposure of such oocytes to exogenous progesterone induces germinal vesicle breakdown and inhibits incorporation of vitellogenin. In the present studies, we have investigated the effects of cytoplasm taken from mature and immature oocytes on incorporation of vitellogenin and nuclear breakdown following microinjection of this material into immature oocytes. Vitellogenin incorporation was markedly suppressed in oocytes which underwent nuclear breakdown following injection with cytoplasm from mature oocytes. Incorporation of vitellogenin into oocytes which did not mature after injection with cytoplasm taken from mature oocytes resembled that seen in oocytes injected with immature cytoplasm. The degree of suppression of vitellogenin incorporation following cytoplasmic injections was similar to that seen in uninjected oocytes treated with progesterone. Oocytes injected with cytoplasm obtained from immature oocytes did not undergo either nuclear breakdown or changes in vitellogenin incorporation. The results suggest that cytoplasm obtained from mature oocytes contains a factor(s) which alters directly or indirectly the capacity of the oocyte cell membrane to incorporate vitellogenin. Enucleated immature oocytes also incorporated [³H]vitellogenin, and injection of such oocytes with mature, but not immature, oocyte cytoplasm suppressed vitellogenin incorporation. Suppressive effects of injected cytoplasm thus appear to be mediated through physiological changes in the recipient oocyte cytoplasm rather than the nuclear component.     

Characterization and Molecular Profiling of PSEN1 Familial Alzheimer's Disease iPSC-Derived Neural Progenitors

Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer''s disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes.