Alpha-Synuclein Aggregation Pathway in Parkinson's Disease: Current Status and Novel Therapeutic Approaches

Following Alzheimer's, Parkinson's disease (PD) is the second-most common neurodegenerative disorder, sharing an unclear pathophysiology, a multifactorial profile, and massive social costs worldwide. Despite this, no disease-modifying therapy is available. PD is tightly associated with alpha-synuclein (alpha-Syn) deposits, which become organised into insoluble, amyloid fibrils. As a typical intrinsically disordered protein, alpha-Syn adopts a monomeric, random coil conformation in an aqueous solution, while its interaction with lipid membranes drives the transition of the molecule part into an alpha-helical structure. The central unstructured region of alpha-Syn is involved in fibril formation by converting to well-defined, beta-sheet rich secondary structures. Presently, most therapeutic strategies against PD are focused on designing small molecules, peptides, and peptidomimetics that can directly target alpha-Syn and its aggregation pathway. Other approaches include gene silencing, cell transplantation, stimulation of intracellular clearance with autophagy promoters, and degradation pathways based on immunotherapy of amyloid fibrils. In the present review, we summarise the current advances related to alpha-Syn aggregation/neurotoxicity. These findings present a valuable arsenal for the further development of efficient, nontoxic, and non-invasive therapeutic protocols for disease-modifying therapy that tackles disease onset and progression in the future

Sublimity and Freedom in Schiller's "Mary Stuart" and "The Maid of Orleans"

Rad ne sadrži sažetak

Antioxidative metabolism in white and green leaf tissues of variegated Pelargonium zonale and Plectranthus coleoides plants - visible light and uv-b radiation effects

Biljke izabrane kao eksperimentalni modeli za ovu disertaciju bile su panaširane muškatle (Pelargonium zonale) i tamjanike (Plectranthus coleoides). Zeleno-belo panaširano lišće predstavljalo je odličan sistem za proučavanje uticaja fotosinteze na antioksidativni i fenolni metabolizam, kao i za istraživanje metaboličkih interakcija između autotrofnog, „izvor“, tkiva, i heterotrofnog, „uvir“, tkiva unutar istog lista. U svom prirodnom staništu biljke su izložene visokom intenzitetu vidljive svetlosti i UV-B zračenju (280-315 nm). Visok intenzitet fotosintetski aktivnog zračenja (PAR, 400-700 nm) može da nadjača kapacitet fotosintetske potrošnje i procese energetskog rasipanja i da prouzrokuje inhibiciju fotosinteze, CO2 asimilacije i pojačanu akumulaciju reaktivnih kiseoničnih vrsta (eng. reactive oxygen species, ROS). Stoga su biljke razvile brojne mehanizme zaštite, koji uključuju i postojanje specijalizovanih enzimskih i neenzimskih antioksidanata. Cilj istraživanja ove teze bio je određivanje konstitutivnih komponenata antioksidativnog sistema i njihove distribucije u fotosintetski aktivnom i fotosintetski neaktivnom tkivu listova panaširanih vrsta. Pri optimalnim svetlosnim uslovima za rast, aktivnosti enzima askorbat-glutationskog (Asc-GSH) ciklusa, kao i Cu/Zn i Mn superoksid-dismutaze su bile veće u belom tkivu, dok su aktivnosti katalaze (CAT) i tilakoidne askorbat-peroksidaze (APX) bile veće u zelenom tkivu listova biljaka P. zonale. Biohemijskim i imunocitohemijskim analizama, pomoću transmisione elektronske mikroskopije, pokazano je da je Asc dvostruko više zastupljen u mezofilnim ćelijama zelenog tkiva (u peroksizomima, mitohondrijama i jedru), a GSH u mezofilnim ćelijama belog tkiva (u mitohondrijama, jedru i citosolu). S obzirom na doprinos fotosinteze u stvaranju reaktivnih kiseoničnih vrsta (eng. reactive oxygen species, ROS), praćen je odgovor enzimskih komponenata Asc-GSH ciklusa, CAT i peroksidaza III klase u belom i zelenom tkivu listova u uslovima stimulisane Melerove reakcije tokom izlaganja biljaka P. zonale visokom intenzitetu PAR-a i tretmanu parakvatom. Ovakav tretman je izazvao izraženiji odgovor enzimskih komponenti Asc-GSH ciklusa belog tkiva, u odnosu na zeleno, što je ukazalo da je belo tkivo dobro adaptirano protiv oksidativnog stresa. iii U drugom delu ove disertacije ispitivani su i specifični uticaji ambijentalnog UV-B zračenja (0.90 W m–2) i visokog intenziteta PAR-a (1350 µmol m–2 s –1) na fotosintetsku aktivnost i parametre izmene gasova (CO2 i H2O), kao i na antioksidativni i fenolni metabolizam panaširanih listova P. coleoides i P. zonale biljaka. Kako bi se izbegli mogući štetni efekti usled neprilagođenog UV-B/UV-A/PAR odnosa, kao i od nerealnih intenziteta i spektralnih karakteristika svih komponenti zračenja, eksperimenti su izvođeni u simulatorima sunčevog zračenja. Pokazano je da je efekat UV-B zračenja bio zavisan od biljne vrste, ali i od intenziteta pozadinskog PAR-a. U biljkama P. coleoides UV-B zračenje je stimulisalo brzinu asimilacije CO2 i provodljivost stoma, dok u biljkama P. zonale, UV-B zračenje nije uticalo na fotosintezu, već je preko trehaloznog signalnog puta dovelo do degradacije skroba i saharoze, i stimulisalo transport ugljenih hidrata iz zelenog u belo tkivo. Kako je u obe ispitivane biljne vrste UV-B zračenje dovelo do pojačane akumulacije glikozida hidroksicinamičnih kiselina i flavonoida (sa antocijanima), uglavnom u belom tkivu listova, predložena su dva mehanizma koji kompenzuju nedostatak fotosinteze i pojačane anaboličke zahteve u ovom tkivu. Visok intenzitet PAR-a je imao veći efekat na listove P. zonale u odnosu na listove P. coleoides biljaka. U biljkama P. zonale je stimulisao biosintezu fenilpropanoida i flavonoida sa orto-dihidroksi-supstituisanim B prstenom, a u sinergiji sa UV-B zračenjem, izazvao je porast aktivnosti APX, CAT i sadržaja Asc isključivo u zelenom tkivu listova biljaka P. zonale. Dobijeni rezultati ukazuju na postojanje različitih aklimatizacionih odgovora na UV-B zračenje u zelenom i belom tkivu biljaka P. zonale.In this thesis, variegated geranium (Pelargonium zonale) and Swedish ivy (Plectranthus coleoides) were used as model plants. Green-white variegated leaves are an excellent model system for investigating the effects of photosynthesis on antioxidative and phenolic metabolism, and “source”-“sink” interactions within the same leaf. In their natural habitat, plants are exposed to high light intensity and to UV-B radiation (280-315 nm). High levels of photosynthetically active radiation (PAR) however, might exceed the the photosynthetic assimilation capacity and energy dissipation processes and provoke inhibition of photosynthesis and enhanced accumulation of reactive oxygen species (ROS). Therefore, plants have developed numerous protection mechanisms, including specialized enzymatic and non-enzymatic antioxidants. The aim of this study was to determine constitutive components of antioxidative system and their distribution in photosynthetically active and non-active tissues of variegated plants. Under optimal light conditions, activities of ascorbate-glutathione (AscGSH) cycle enzymes, Cu/Zn and Mn superoxide dismutases were higher within white leaf tissue of P. zonale, while thylakoid ascorbate peroxidase (APX) and catalase (CAT) activities were higher in green leaf tissue. Biochemical and immunocytochemical analysis using transmission electron microscopy revealed two-fold higher Asc content in mesophyll cells of green leaf tissue (in peroxisomes, mitochondria, nucleus), while GSH was more abundant in mesophyll cells of white leaf tissue (in mitochondria, nucleus, cytosol). Since photosynthesis is the main source of ROS in the leaves, the second aim in this thesis was to analyze the response of Asc-GSH cycle components, CAT and class III peroxidases in green and white leaf tissues, under the conditions of stimulated Mehler reaction during the exposure of P. zonale to high PAR and paraquat. These treatments induced greater response of Asc-GSH cycle enzymes in white leaf tissue, compared with green one, indicative of the development of adaptive mechanisms to avoid oxidative stress. In the second half of this thesis, the specific effects of high PAR (1350 µmol m–2 s –1) and ambient UV-B irradiances (0.90 W m–2) on photosynthetic activity and gas v exchange parameters (CO2 and H2O), as well as on antioxidative and phenolic metabolism of variegated P. coleoides and P. zonale plants were studied. In order to avoid possible detrimental effects caused by unrealistic UV-B/UV-A/PAR ratios, quantity and spectral quality of radiation, the experiments were conducted in unique sun simulators. The results showed that ecologically relevant UV-B radiation-induced responses are species-specific and dependent on background light. Two mechanisms to compensate the absence of photosynthetic activity and increased anabolic demands in white, “sink” tissue were proposed. UV-B radiation stimulated CO2 assimilation rate and stomatal conductance in P. coleoides plants. On the other hand, in P. zonale, UV-B radiation, provoked starch and sucrose degradation via the trehalose signalling pathway, and subsequent sugar transport from green to white tissue, without affecting photosynthesis. UV-B radiation induced enhanced accumulation of hydroxycinnamic acid and flavonoid glycosides, in both variegated species, mostly in white leaf tissue. High PAR had stronger effects on the leaves of P. zonale plants, compared with P. coleoides plants. Additionally, high PAR stimulated specific accumulation of phenylpropanoids and flavonoids with ortho-dihydroxy B-ring substitution pattern, and synergistically with UV-B radiation, increased APX and CAT activities and Asc content, exclusively in green leaf tissue of P. zonale plants. Overall, these results show differential acclimatisation responses to UV-B radiation in green and in white tissue of P. zonale leave

Comparative assessment of erythrocyte sphingolipid levels as potential cardiovascular health markers in women from Libya and Serbia: a small-scale study

Aim: Cardiovascular diseases (CVDs) represent the major cause of morbidity and mortality worldwide including Libya, where they account for 43% of all deaths. Sphingolipids are involved in the pathology of numerous diseases including cardiovascular diseases and are proposed as potential biomarkers of cardiovascular health that could be more effective compared to traditional clinical biomarkers. The aim of this study was to determine the sphingolipid content in the erythrocyte membrane of Libyan migrant and Serbian resident women. In addition, to examine if sphingolipid levels could be used as a novel indicator of cardiovascular risk, we evaluated possible correlations with some well-established biomarkers of cardiovascular health. Materials and Methods: A total of 13 Libyan and 15 Serbian healthy women participated in the study. The high-performance version thin-layer chromatography (HPTLC) using the image analysis tool JustTLC was applied for quantification of erythrocytes’ sphingolipids. Results: Lower mean values of erythrocytes’ sphingolipids and cholesterol concentrations were found in the group of Libyan emigrants compared to Serbian resident women. Besides, in this group of apparently healthy women (n = 28), the sphingolipid content of erythrocytes was inversely related to the Omega-3 index (r =-0.492, p = 0.008) and directly linked to vitamin D status (r = 0.433, p = 0.021) and membrane cholesterol levels (r = 0.474, p = 0.011). Conclusion: The erythrocytes’ sphingolipid levels should be measured/assessed as an additional biomarker of CV health, by applying a simple and routine method. Still, further investigation in a larger population-specific context is warranted

An improved HPLC-DAD method for simultaneously measuring phenolics in the leaves of Tilia platyphyllos and Ailanthus altissima

Phenolic compounds are one of the most important groups of secondary metabolites in plants, with various physiological functions. Their diverse chemical structure and susceptibility to auto-oxidation, and their ability to act as both antioxidants and prooxidants in the presence of metal ions, are some of the main reasons why it is difficult to measure phenolic groups in plant tissues accurately. We present an optimized extraction and hydrolysis procedure which preserves the original chemical structure of phenolics. The presented HPLC method was improved to enable the simultaneous separation and quantification of 39 compounds from different phenolic subclasses (benzoic acids, hydroxycinnamic acids, flavones, flavonols, flavanones, flavanols, isoflavones, anthocyanidins; aglycones and glycosides). Recovery after extraction and complete hydrolysis of glycosides was more than 95% and 84%, respectively. The method was applied to the analysis and comparison of phenolic profiles in the leaves of two species, Ailanthus altissima (Mill.) Swingle and Tilia platyphyllos Scop. from an urban forest park and busy traffic area. The presence of cyanidin glycosides in A. altissima leaves was reported for the first time. Results indicated higher accumulation of phenolics, with two hydroxyl groups in the ortho- position, than flavonoids, with a monohydroxy substitution in the leaves of both species from a busy traffic area

In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship

Desiccation or extreme water loss leads to protein denaturation, aggregation, and degradation and impairs membrane lipid fluidity, resulting in loss of membrane integrity at the cellular level. The induction of late embryogenesis abundant proteins (LEAPs) is considered an essential component of desiccation tolerance strategy in so-called resurrection plants. This heterogeneous group of hydrophilic, non-globular proteins is characterised by a high structural plasticity that allows them to adopt a random conformation in aqueous solutions that transforms into α-helices during dehydration [1]. Therefore, LEAPs can interact with various ligands and partners, including ion sequestration and stabilisation of membranes and enzymes during freezing or drying [2]. Our new transcriptome database of an endemic resurrection species Ramonda serbica allowed us to identify 164 members of the LEA gene family. LEAPs of this sample data have an average primary sequence similarity and identity of 10% and 6%, respectively, but with a high variance (141 and 108), which means that the sample proteins can be classified based on domain homology. The averaging is based on multiple sequence alignment and the variance is estimated using pairwise sequence alignment scores. Accordingly, all identified LEAPs were clustered into six groups based on protein families (PFAM). Among these groups, LEAPs differ significantly in their secondary structure, disorder propensity and aggregation potential. Furthermore, we built homology models using PDB structures as templates. For each group, an ensemble of superimposed 3D homology models was analyzed. The information obtained from the representative structural models is key to understanding the function of LEAPs and the regulation of their intrinsic structural disorder-to-order transition during desiccation. This will pave the way for the identification of LEAPs endogenous partners and their targets in the cell and provide further insights into the protective mechanisms of desiccation tolerance.PDB50: A special symposium celebrating the 50th anniversary of Protein Data Bank, 4-5 May, 2021

Protein structural differences in Cytochrome c oxidase subunit 1 of two Heterogynis species as a new approach for species delimitation

Insects are the most diverse group in the animal kingdom, accounting for about two-thirds of all animals. Cytochrome c oxidase subunit 1 (COI) is the most commonly used marker gene for animal species delineation. However, the accuracy of this approach crucially depends on the degree of overlap between the intra- and interspecific variations. Recently, we have identified a new species, Heterogynis serbica sp. n. (Lepidoptera: Zygaenoidea, Heterogynidae) found on the Mt. Kopaonik, Republic of Serbia, Balkan Peninsula. This was done by integrating taxonomic approach using morpho-anatomical characteristics by comparative scanning electron microscopy (SEM), linear wing morphometry and COI-based DNA barcoding [1]. In this study, we have used a set of bioinformatics tools available online, to determine the differences in secondary and tertiary structure of the COI proteins from H. serbica sp. n. and H. zikici. We also compared the amino acid distribution and COI motif profiles between the two species. Our results provide strong evidence that protein structure of COI can help with COI-based DNA barcoding for taxon-specific purposes of species identification and delimitation studies. Millions of COI DNA sequences deposited in the public domain (which are still growing) carry huge potential for a comprehensive assessment of genetic variation in COI among insects by using here described analysis.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Induction of peroxidase isoforms in the roots of two Verbascum thapsus L. populations is involved in adaptive responses to excess Zn2+ and Cu2+

To investigate metal specific responses of root class III peroxidases (POD, EC 1.11.1.7), two populations of Verbascum thapsus L. were exposed to excess Zn2+ or Cu2+ for three weeks in hydroponic culture. One population originating from an uncontaminated area (NMET) and one from an industrial disposal area for jarosite residues from zinc refining (MET) were chosen to test the capacity of V. thapsus to adapt to excess metal in the soil. Exposure to 60 μM Zn2+ led to increased levels of protein carbonyl groups only in the roots of NMET, which was accompanied by higher increase of POD activity and NADH-oxidase activity compared with MET plants. New anionic and cationic POD isoforms were induced in the roots of both populations in response to Zn2+ treatment, while IAA-oxidase activity decreased. On the other hand, root growth was more affected by Cu2+ than Zn2+ in both populations, which was correlated with increased auxin-oxidase (IAA-oxidase) activity. Cu2+ induced an increased activity of anionic POD isoforms in the roots of both populations, yet the ratio of NADH-oxidative to peroxidative POD activity remained higher in NMET than in MET plants. Overall results show differential effects of Zn2+ and Cu2+ on POD activity in the roots of V. thapsus L. In addition, higher tolerance to Zn2+ in MET plants than in NMET indicated that these plants have developed an adaptive mechanism to cope with Zn2+ excess

Induction of peroxidase isoforms in the roots of two Verbascum thapsus L. populations is involved in adaptive responses to excess Zn2+ and Cu2+

To investigate metal specific responses of root class III peroxidases (POD, EC 1.11.1.7), two populations of Verbascum thapsus L. were exposed to excess Zn2+ or Cu2+ for three weeks in hydroponic culture. One population originating from an uncontaminated area (NMET) and one from an industrial disposal area for jarosite residues from zinc refining (MET) were chosen to test the capacity of V. thapsus to adapt to excess metal in the soil. Exposure to 60 μM Zn2+ led to increased levels of protein carbonyl groups only in the roots of NMET, which was accompanied by higher increase of POD activity and NADH-oxidase activity compared with MET plants. New anionic and cationic POD isoforms were induced in the roots of both populations in response to Zn2+ treatment, while IAA-oxidase activity decreased. On the other hand, root growth was more affected by Cu2+ than Zn2+ in both populations, which was correlated with increased auxin-oxidase (IAA-oxidase) activity. Cu2+ induced an increased activity of anionic POD isoforms in the roots of both populations, yet the ratio of NADH-oxidative to peroxidative POD activity remained higher in NMET than in MET plants. Overall results show differential effects of Zn2+ and Cu2+ on POD activity in the roots of V. thapsus L. In addition, higher tolerance to Zn2+ in MET plants than in NMET indicated that these plants have developed an adaptive mechanism to cope with Zn2+ excess

Viola odorata: Influence of supercritical fluid extraction on the efficiency of ultrasound- and microwave-assisted extraction of bioactive compounds

This study aimed at examining the effect of supercritical carbon dioxide (ScCO2) extraction on the subsequent extraction of the Viola odorata polar bioactive components. The raw material was firs submitted to ScCO2 extraction for the extraction of the lipophilic fraction. Then the exhausted raw material was subjected to ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) in order to extract the polar components. ScCO2 extraction was performed under the pressure of 300 bar and at a temperature of 40 °C for 4 hours. In order to see the effect of ScCO2, the UAE and MAE (50% aqueous ethanol solution as solvent) were conducted on both exhausted (residue-after the ScCO2) and unexploited raw materials. Also, the impact of the various UAE and MAE conditions was tested. The UAE was conducted on two different temperatures (40 and 50 °C) at the constant extraction time (40 and 20 min). MAE was conducted on two different extraction powers (470 and 800 W) and at the constant extraction time (10 min). The results were focused on the content of total phenols, total flavonoids and antioxidant activity of the obtained extracts. The yield during the ScCO2 process was 1.43% (w/w). It was noted that the extracts obtained by applying UAE and MAE after the ScCO2 were noticeably richer in the content of total phenols. UAE conditions of 40 °C and 20 min showed the highest yield of total phenols, recording 70.38 mg GAE/g DE, while the MAE at the power of 470 W achieved 11.89% higher yield of polyphenols in residue extracts. The antioxidant activity has also been in correlation with the concentration of polyphenols