Confirmation of the Disulfide Connectivity and Strategies for Chemical Synthesis of the Four-Disulfide-Bond-Stabilized Aspergillus giganteus Antifungal Protein, AFP

Emerging fungal infections require new, more efficient antifungal agents and therapies. AFP, a protein from Aspergillus giganteus with four disulfide bonds, is a promising candidate because it selectively inhibits the growth of filamentous fungi. In this work, the reduced form of AFP was prepared using native chemical ligation. The native protein was synthesized via oxidative folding with uniform protection for cysteine thiols. AFP's biological activity depends heavily on the pattern of natural disulfide bonds. Enzymatic digestion and MS analysis provide proof for interlocking disulfide topology (abcdabcd) that was previously assumed. With this knowledge, a semi-orthogonal thiol protection method was designed. By following this strategy, out of a possible 105, only 6 disulfide isomers formed and 1 of them proved to be identical with the native protein. This approach allows the synthesis of analogs for examining structure-activity relationships and, thus, preparing AFP variants with higher antifungal activity

Calcium binding of the antifungal protein PAF: Structure, dynamics and function aspects by NMR and MD simulations

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Calixarene-mediated assembly of a small antifungal protein

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Confirmation of the Disulfide Connectivity and Strategies for Chemical Synthesis of the Four-Disulfide-Bond-Stabilized Aspergillus giganteus Antifungal Protein, AFP

Emerging fungal infections require new, more efficient antifungal agents and therapies. AFP, a protein from Aspergillus giganteus with four disulfide bonds, is a promising candidate because it selectively inhibits the growth of filamentous fungi. In this work, the reduced form of AFP was prepared using native chemical ligation. The native protein was synthesized via oxidative folding with uniform protection for cysteine thiols. AFP's biological activity depends heavily on the pattern of natural disulfide bonds. Enzymatic digestion and MS analysis provide proof for interlocking disulfide topology (abcdabcd) that was previously assumed. With this knowledge, a semi-orthogonal thiol protection method was designed. By following this strategy, out of a possible 105, only 6 disulfide isomers formed and 1 of them proved to be identical with the native protein. This approach allows the synthesis of analogs for examining structure-activity relationships and, thus, preparing AFP variants with higher antifungal activity

New antimicrobial potential and structural properties of PAFB: a cationic, cysteine-rich protein from Penicillium chrysogenum Q176

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Az antipszichotikumok makrofágok funkcionális aktivitására gyakorolt hatása

Célom volt az atípusos antipszichotikumok hatásának vizsgálata humán monocita-makrofág differenciálódásra. Kísérleteim során használt gyógyszerek a Quetiapin, Aripiprasol, Ziprasidon, Risperidon, Clozapin és Olanzapin, a Haloperidol antipszichotikumok voltak. Vizsgáltam, hogy ezek a gyógyszerek befolyásolják-e a makrofágok fagocitózis képességét, citokin expresszióját és szuperoxid termelését. A monocitákat humán vérből izoláltam mágneses szeparálással, majd makrofág kolónia-stimuláló faktort (M-CSF) tartalmazó tápoldatban differenciáltattam, naponta ismétlődő, a klinikumban alkalmazott szérum koncentrációnak megfelelő antipszichotikum kezelés mellett. A fagocitózis képesség vizsgálatára humán vérből izolált apoptótikus neutrofil granulocitákkal végzett, áramlási citometriás meghatározáson alapuló fagocitózis mérést alkalmaztam. A makrofágok TNF-α szekrécióját, LPS stimulust követően szendvics ELISA módszerrel határoztam meg. A makrofágok PMA indukált szuperoxid termelését L-012 kemilumineszcenciás próba jelenlétében vizsgáltam. Eredményeim azt mutatják, hogy az in vivo kezelési koncentrációk mellett, az Olanzapin, Haloperidol és a Risperidon szignifikánsan csökkentette a makrofágok fagocitózis képességét apoptótikus neutrofilek iránt. Az LPS stimulust követően, a makrofág által szekretált TNF-α mennyiségét a gyógyszeres kezelés a legtöbb esetben csökkentette. Ugyanakkor a Haloperidol kivételével az antipszichotikumok fokozták a makrofágok PMA indukált szuperoxid termelését.MSc/MABiológu

Neutrofil granulociták génexpressziós mintázatának és proteom analízisének áttekintése

A neutrofil granulociták feladata a mikroorganizmusok fagocitózisa és elpusztítása,a fertőzések továbbterjedésének megakadályozásában fontos szerepet játszanak.BscBiológiag

Identification of microvascular and morphological alterations in eyes with central retinal non-perfusion.

PurposeTo evaluate the characteristics and morphological alterations in central retinal ischemia caused by diabetic retinopathy (DR) or retinal vein occlusion (RVO) as seen in optical coherence tomography angiography (OCTA) and their relationship to visual acuity.MethodsSwept-source optical coherence tomography (SSOCT) and OCTA (Topcon, Triton) data of patients with central involving retinal ischemia were analyzed in this cross-sectional study. The following parameters were evaluated: vessel parameters, foveal avascular zone (FAZ), intraretinal cysts (IRC), microaneurysms (MA), vascular collaterals in the superficial (SCP) and deep plexuses (DCP), hyperreflective foci (HRF), epiretinal membrane (ERM), external limiting membrane (ELM) and ellipsoid zone (EZ) disruption, as well as the disorganization of retinal inner layers (DRIL). Best-corrected visual acuity (BCVA), age, gender, disease duration and ocular history were also recorded.Results44 eyes of 44 patients (22 with RVO, 22 with DR) were analyzed. The mean age was 60.55 ± 11.38 years and mean BCVA 0.86 ± 0.36 (Snellen, 6m). No significant difference was found between DR subgroups (non proliferative vs. proliferative). Between RVO subgroups (CRVO vs. BRVO) a significant difference was found in term of collateral vessel of the DCP (p = 0.014). A pooled DR and RVO group were created and compared. Significantly more MAs (p = 0.007) and ERM (p = 0.007) were found in the DR group. Statistically significant negative correlation was demonstrated between FAZ and BCVA (p = 0.45) when analyzing all patients with retinal ischemia.ConclusionThis study has shown that the best predictor of visual outcome in center involved ischemic diseases is the size of FAZ. Besides the presence of MAs and ERM, all other OCT and OCTA parameters were present in a similar extent in DR and RVO group despite the completely different disease origins. Our results suggest that as soon as retinal ischemia in the macular region is present, it has a similar appearance and visual outcome independently of the underlying disease

DMSO-Induced Unfolding of the Antifungal Disulfide Protein PAF and Its Inactive Variant: A Combined NMR and DSC Study

PAF and related antifungal proteins are promising antimicrobial agents. They have highly stable folds around room temperature due to the presence of 3–4 disulfide bonds. However, unfolded states persist and contribute to the thermal equilibrium in aqueous solution, and low-populated states might influence their biological impact. To explore such equilibria during dimethyl sulfoxide (DMSO)-induced chemical unfolding, we studied PAF and its inactive variant PAFD19S using nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). According to the NMR monitoring at 310 K, the folded structures disappear above 80 v/v% DMSO concentration, while the unfolding is completely reversible. Evaluation of a few resolved peaks from viscosity-compensated 15N-1H HSQC spectra of PAF yielded ∆G = 23 ± 7 kJ/M as the average value for NMR unfolding enthalpy. The NMR-based structures of PAF and the mutant in 50 v/v% DMSO/H2O mixtures were more similar in the mixed solvents then they were in water. The 15N NMR relaxation dynamics in the same mixtures verified the rigid backbones of the NMR-visible fractions of the proteins; still, enhanced dynamics around the termini and some loops were observed. DSC monitoring of the Tm melting point showed parabolic dependence on the DMSO molar fraction and suggested that PAF is more stable than the inactive PAFD19S. The DSC experiments were irreversible due to the applied broad temperature range, but still suggestive of the endothermic unfolding of PAF

Calixarene-mediated assembly of a small antifungal protein

Synthetic macrocycles such as calixarenes and cucurbiturils are increasingly applied as mediators of protein assembly and crystallization. The macrocycle can facilitate assembly by providing a surface on which two or more proteins bind simultaneously. This work explores the capacity of the sulfonatocalix[n]arene (sclx(n)) series to effect crystallization of PAF, a small, cationic antifungal protein. Co-crystallization with sclx(4) , sclx(6) or sclx(8) led to high-resolution crystal structures. In the absence of sclx(n), diffraction-quality crystals of PAF were not obtained. Interestingly, all three sclx(n), were bound to a similar patch on PAF. The largest and most flexible variant, sclx(8) , yielded a dimer of PAF. Complex formation was evident in solution via NMR and ITC experiments, showing more pronounced effects with increasing macrocycle size. In agreement with the crystal structure, the ITC data suggested that sclx(8) acts as a bidentate ligand. The contributions of calixarene size/conformation to protein recognition and assembly are discussed. Finally, it is suggested that the conserved binding site for anionic calixarenes implicates this region of PAF in membrane binding, which is a prerequisite for antifungal activity.This research was supported by NUI Galway (Hardiman Scholarship to JMA), the Hungarian Science Fund (OKTA-ANN 110821 to GB), the European Regional Development Fund (GINOP-2.3.2-15-2016- 00008 to GB and GINOP-2.3.3-15-2016-00004) and Science Foundation Ireland (13/ERC/B2912 and 13/CDA/2168 to PBC). We acknowledge R. Pierattelli and the other organizers of the Chianti Workshop 2016 where this collaboration was initiated. Thanks also to SOLEIL synchrotron for beam time allocation, and the staff at beam line PROXIMA 2A for their assistance with data collection