Characterization and application of fusogenic liposomes

Conventional drug delivery strategies use the endocytic pathway to introduce biomolecules like proteins, DNA, or antibiotics into living cells. The main disadvantage of endocytic uptake is the quick intercellular degradation of the cargo. Compared to this, a more promising alternative for efficient molecular delivery is the induction of membrane fusion between liposomes and mammalian cells. Therefore special liposomes with extraordinary high fusion efficiency, so-called fusogenic liposomes (FLs), have been developed for such purposes. Due to the complete fusion of the liposomal membrane and the cellular plasma membrane, the cargo molecules can be effectively released into the cell cytoplasm, avoiding their degradation. In the last decade, applications relying on FLs became more and more relevant, however, the exact fusion mechanism is still to be elucidated. Therefore the aims of this work have been to investigate those liposomes and their fusogenicity with living mammalian cells dependent on lipid composition as well as environmental conditions to elucidate the most important factors inducing fusogenic structures within the liposomes. For structural characterization of the liposomes dynamic light and neutron scattering as well as solid state-NMR, freeze-fracture-STEM, Cryo-TEM, and differential scanning calorimetry were applied. Fusion efficiency was investigated by fluorescence microscopy and flow cytometry using Chinese hamster ovary (CHO) cells as an in vitro mammalian cell model. The first results showed that fusogenic liposomes (FLs) need cationic lipids with inverted conical molecular shapes and aromatic components at a distinct concentration as well as a neutral lipid for the best fusion induction. Neutral lipids with long and unsaturated chains and a small head group (e.g., PEs) do not change the liposomal fusion ability while those with saturated short chains and a big head group (e.g., PCs) do, and in most extreme cases revert the uptake mechanism to endocytosis. Additionally, a new application of fusogenic liposomes was established. For the first time, cationic liposomes with high fusion ability were successfully used as carrier particles for the delivery of the radionuclide 131I into mammalian breast cancer cells in vitro. The FLs reached the cancer cells with high efficiency and delivered their cargo into the cell cytoplasm. The control treatment of human red blood cells did not give positive results on fusion, and in this case, the delivery of the cargo was neglectable. These results considered FLs as an appropriate tool for applications in nuclear medicine. Further results showed that as the structural reorganization of the liposomal membrane supply the total required driving force to overcome the energy barrier of the different fusion intermediate steps, like in the case of FLs, changes of the fusion conditions such as temperature, osmolality or ionic concentration of the buffer did not influence the fusion success. In the case of the endocytic liposomes (ELs), buffer conditions played a crucial role in successful fusion, however, fusion efficiency remains infinitesimal under physiological conditions. To elucidate the correlation between efficient membrane fusion and liposomal characteristics, structural investigations of FLs with the best fusion efficiency were also carried out. Here, the simultaneous presence of lipid bilayers and small micelles of around 50 to 100 nm in diameter with high surface curvatures were found. Based on the obtained results, a theoretical mechanism of membrane fusion between FLs and cellular membranes could be proposed. The positively charged lipid is necessary for establishing contact between the two membranes. The micelles are formed by the neutral, phosphoethanolamine, lipids. The lipid bilayer enclosing inverted micelles has a high positive membrane curvature, which is especially favorable for the positively charged lipid molecules. Such curvature stress usually promotes the fusion-stalk formation and subsequent membrane fusion; therefore, the proposed fusion mechanism is called a modified stalk mechanism. Moreover, traces of other three-dimensional (3D) phases with high membrane curvature such us sponge-, inverted hexagonal-, and cubic phases could not be excluded. The present structures are probably metastable precursors, such as a rhombohedral phase, that reduce bilayer stability, which is leading to the pore formation occurring. In comparison to this, ELs formed only lamellar phases shown as non-fusogenic under physiological conditions. These results give rise to the hypothesis that the predominant presence of 3D-like and 3D phases with high membrane curvatures is the most important criterion for efficient membrane fusion induction

Quality assessment, antimicrobial activity organic sunflower honey and use of Maldi-tof mass spectrometry for the identification bacteria isolated from honey

The aim of this study was to investigate the physicochemical parameters of quality, microbiological safety and antimicrobial potential of four samples of organic sunflower honey from the Banat area (northeastern Serbia). Humidity, ash, pH, free acidity, electrical conductivity, hydroxymethylfurfural content, sugar content and diastase activity were measured. Microbiological analysis revealed the presence of total aerobic mesophilic bacteria, coliforms, aerobic endospores, lactic acid bacteria (LAB) and a good number of molds and yeasts. The isolate identification was carried out using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The antimicrobial effect of organic sunflower honey was investigated on five ATCC strains of bacteria: Staphilococcus aureus ATCC 29213; Escherichia coli ATCC 25922; Salmonella enterica ATCC 10708; Yersinia enterocolitica ATCC 23715; and Bacillus subtilis ATCC 23857. The results of the study showed that all honey samples meet international quality standards for all physicochemical parameters. Microbiological analysis of Sunflower honey confirmed the total bacterial counts for all samples ranged from1.80 to 1.85 x10-2cfu/g-1, whereby no presence Clostridium spp., coliform bacteria, as well as molds was detected. Investigation of the antimicrobial activity of honey samples revealed that all bacteria showed clear zones of inhibition in honey concentrations of 40-100%, which is a satisfactory result for flower honey

Фулеропиролидини са ортогонално флексибилним супституентима – синтеза и електрохемијске особине

A large series of disubstituted fulleropyrrolidines was synthesized and analyzed by cyclic voltammetry. The three main groups of target com­pounds differ by a flexible N-chain, while their further diversity was achieved by the introduction of various rigid, aryl substituents at the pyrrolidine carbon. Some dialkyl analogues were also designed for comparison, A standard [3+2]-cycloaddition of in situ generated azomethine ylides to C60 afforded a variety of disubstituted fulleropyrrolidines. Furthermore, a set of dumbbell-shaped di(fulleropyrrolidine) derivatives containing rigid fumaryl or isophthaloyl diamide platform was prepared with the aim of investigating a long-range effect of the second fulleropyrrolidine moiety on their electrochemical pro­perties. All compounds were fully characterized by comparative analysis of spectral data, while examination of electrochemical properties was performed on representative samples, distinguished by main structural subunits. All com­pounds expressed quite similar electron-accepting ability, lower than C60, but higher in comparison to structurally similar N-methylfulleropyrrolidine.Велика серија дисупституисаних фулеропиролидина је синтетисана и анализирана цикличном волтаметријом. Три главне групе циљних једињења разликују се по флекси- билном бочном низу на атому азота, а додатна диверсификација постигнута је увођењем различитих крутих, арил-супституената на пиролидинском атому угљеника. За поре- ђење су синтетисани и одређени диалкил-аналози. Сет дисупституисаних фулеропиро- лидина добијен је стандардном [3+2]-циклоадицијом ин ситу генерисаних азометинских илида на C60. Осим тога, у циљу испитивања даљинског утицаја фулеропиролидинске структуре на електрохемијске особине, синтетисани су и дифулеренски деривати код којих су терминално позициониране фулеренске подјединице премошћене фумарилном или изофталоилном крутом платформом. Сва једињења су у потпуности окарактерисана упоредном анализом спектралних података, док је сет одабраних једињења, са разли- кама у главним структурним подјединицама, искоришћен за испитивање електрохемиј- ских особина. Сва испитивана једињења показала су врло сличну способност прихватања електрона, нижу у односу на C60, а вишу од структурно сличног N-метилфулеропиро- лидина

PREGLED METODA ISPITIVANJA ŽIVOTNE SPOSOBNOSTI SEMENA

Vigour is defined as the potential of seeds to germinate rapidly and uniformly under adverse and optimum conditions. These criteria imply that vigorous seeds will establish early field stands which provide competitive advantages against weeds and microflora. These advantages could lead to higher crop yields. Vigour tests are divided in several categories. Physical tests measure seed characteristics such as size, weight, and density. Physiological tests use some parameter of germination or seedling growth - speed of germination, standard germination, accelerated ageing, cold test, Hiltner test. Biochemical tests observe chemical reactions involved in cellular maintenance - tetrazolium (dehidrogenase), respiration, GADA (glutamic acid decarboxylase activity), protein, conductivity (leaching), enzyme activity. Many of the vigour test methods need to be standardize for specific crop species in the future.Ţivotna sposobnost semena definiše se kao potencijal semena da brzo i ujednaĉeno klija u povolјnim i u nepovolјnim uslovima. Ona izraţava obim oštećenja koja se vremenom nakuplјaju u semenu. Razliĉiti metodi ispitivanja ţivotne sposobnosti semena izvode se tako da u relativno kratkom roku, jednostavno i pouzdano daju sliku kvaliteta semena i dopune rezultate dobijene ispitivanjem klijavosti. Partije semena koje imaju visoku klijavost ali slabo nicanje u polјu imaju nisku ţivotnu sposobnost. Razlike u ţivotnoj sposobnosti semena takoĊe se odraţavaju na brzinu klijanja i razvoja klijanaca bilo u povolјnim bilo nepovolјnim uslovima. Direktna ispitivanja ţivotne sposobnosti mere razlike u pogledu nekog vida klijanja ili rasta, najĉešće pod stresnim uslovima. Jedna od prednosti ovih ispitivanja je što se njima vrednuju faktori koji utiĉu na ţivotnu sposobnost kao što su starenje, mehaniĉke povrede i morfološke osobine semena. Drugo je što ĉesto koriste uslove sliĉne onim u polјu i kratko traju te su jednostavna za tumaĉenje. Fiziološka ispitivanja odnose se na uticaj starenja semena na klijanje ili poĉetne etape razvoja klijanca. Ova ispitivanja su modifikovana ispitivanja klijavosti. Ţivotna sposobnost najjednostavnije se procenjuje kao brzina klijanja a na osnovu energije klijanja (prvo brojanje) pri standardnom ispitivanju klijavosti. S druge strane, brzina klijanja moţe se vrednovati izraĉunavanjem srednjeg vremena klijanja, srednje vrednosti klijanja i maksimalne vrednosti klijanja (seme drvenastih vrsta) te na osnovu ispitivanja rasta klijanca (suva masa klijanca, duţina izdanka, duţina korenĉića). Ispitivanja ţivotne sposobnosti semena u nepovolјnim (stresnim) kontrolisanim uslovima simuliraju dejstvo jednog (više) spolјnih faktora koji deluju u polјu. U ovaj vid ispitivanja spadaju hladni i Hiltnerov test. Testovi starenja zasnovani su na izglanju semena visokoj temperaturi i/ili vlaţnosti vazduha u odreĊenom vremenskom periodu. Najpoznatija ispitivanja iz ove grupe obuhvataju ubrzano starenje semena i kontrolisano propadanje semena. Indirektna ispitivanja usmerena su na jedan aspekt ţivotne spsobnosti, pre nego na ocenu efekata više faktora na klijanje ili rast mladog klijanca. Biohemijska ispitivanja spadaju u indirektna ispitivanja ţivotne sposobnosti i obiĉno prate nivo jedne od posledica starenja. U ovu grupu ispitivanja spadaju merenje elektroprovodlјivosti sadrţaja osloboĊenog iz semena (pasulј, soja, bob); zatim procena enzimske aktivnosti u semenu koja se boji tetrazolijumom (ţita, soja, pamuk, grašak, detelina) ili merenje aktivnosti dekarboksilaze glutaminske kiseline (soja, pasulј). Struĉna literatura poznaje brojne metaboliĉke markere pogodne za biohemijska ispitivanja ţivotne sposbnosti što zalazi u genomiku, proteomiku i druge uske discipline koje mogu pruţiti korisne, merlјive parametre ţivotne sposobnosti semena. Direktne i indirektne metode ispitivanja ţivotne sposobnosti dalјe će se razvijati kako u pogledu osobina koje prate tako i u pogledu bilјnih vrsta na koje se primenjuju te je od posebnog znaĉaja njihova sistematizacija i standardizacija

Machine learning chemometric model for Raman spectroscopy based honey quality assessment

INTRODUCTION: According to Codex Alimentarius (2001), “Honey is the natural sweet substance, produced by honeybees from the nectar of plants or from secretions of living parts of plants, or excretions of plant-sucking insects on the living parts of plants, which the bees collect, transform by combining with specific substances of their own, deposit, dehydrate, store and leave in honeycombs to ripen and mature”. Honey is mostly made up of sugars, as well as enzymes, amino acids, organic acids, vitamins, aromatic compounds, minerals and carotenoids. It contains a lot of flavonoids and phenolic acids, which have a lot of biological effects and functions such as natural antioxidants, anti-inflammatory and antimicrobial properties. Its composition is particularly variable, depending on its botanical and geographical origins Because of its exclusive flavor and high dietary value, natural honey is more expensive than other sweeteners. This is the reason why honey is a target of adulteration. The problem is that counterfeiting honey is relatively easy, but detection is difficult. Further, the authenticity of honey is a global important problem for commercial producers and consumers. Accordingly, a fast and non-destructive method of detecting counterfeits is needed. OBJECTIVES: The aim of this paper is to verify the possibility of Raman spectroscopy and Support Vector Machine (SVM) for classification of two different honeys and their fake duplicates. For this purpose, meadow and acacia honeys were selected. METHOD / DESIGN: Spectra of homemade and counterfeits honey were recorded using XploRA Raman spectrometer (Horiba Jobin Yvon). Raman scattering was excited by laser at a wavelength of 785 nm equipped with a 600 lines/mm grating; spectra were recorded by applying exposure time 10 s and accumulated from 10 times scans, using 100% filter. Spectral resolution was 3 cm−1 and autocalibration was done each time before recording of spectra by 520.47 cm−1 line of silicon. In order to assess a possible sample inhomogeneity, thirty Raman spectra in the region from 200-3400 cm-1 were recorded for each sample. All spectra were baseline-corrected, normalized and smoothed. After that PCA (Principal component analysis) was conducted and obtained PCs (first two PCs) served as a features for support vector machine (SVM) classification method. Data were divided into training model (70 %) and training data (30 %). Pre-processing was done by Unscrambler X 10.4 software (CAMO software, Norway). In order to determine the best shape of the hyperplane and decision boundary, several kernel function were used: linear, radial basis and polynomial function. The SVM was conducted by Python and Scikit-learn package. RESULTS: Support vector machine showed high accuracy in classification of different honey samples. Accordingly, the best discrimination power showed SVM with polynomial function (100%), followed by radial basis (96.67%) and linear (81.82%)

Raman spectroscopy coupled with chemometric modeling approaches for authentication of different paprika varieties at physiological maturity

Five Balkan paprika varieties at physiological maturity were investigated by means of Raman spectroscopy in order to discriminate the differences which stemmed from their genetic variability since the plants were grown under the same experimental conditions. The spectra were obtained using the 532 nm wavelength. In an effort to find the best classification power, several pre-processing methods were applied: 1) baseline correction, unit vector normalization; 2) baseline correction, unit vector normalization and first Savitzky-Golay derivative; 3) baseline correction, unit vector normalization and second Savitzky-Golay derivative; 4) baseline correction, unit vector normalization and third Savitzky-Golay derivative. All of the pre-processing methods were followed by making PCA-LDA (Principal Component Analysis-Linear Discriminant Analysis), QDA (Quadratic Discriminant Analysis), and PLS-DA (Partial Least Square - Discriminant Analysis) classification models. QDA showed the best discrimination power (83.87–100% and 89.47–100% for the training and the test data, respectively), then PCA-LDA (0.00–100 and 0.00–100% for the training and the test data, respectively) and PLS-DA (19.35–100% and 0.00–100.00% for the training and the test data, respectively). The results pointed out the applicability of chemometric modeling associated with Raman spectroscopy in the assessment of nutritionally similar samples, such as the studied red paprika varieties

Effectiveness of Bradyrhizobium japonicum inoculation on nodulation dynamics in Glycine max (L.) roots

Bradyrhizobium japonicum bacteria on the intensity of nodulation during nine phenophases (R1-R7) in relation to the isoflavone content in soybean seeds. Nine domestic soybean varieties were sown in three replicates, with Bradyrhizobium japonicum inoculated and non-inoculated seeds. The different morphological characteristics of nodules, the number and mass of nodules were studied during plants’ ontogenesis. The content of individual isoflavones in the sowing seed material was determined using high performance liquid chromatography mass spectrometry (HPLC-MS). The most abundant isoflavone was acetylgenistin in the variety Galeb (2741.4 μg/g), and the highest content of all isoflavones was recorded in the variety Gorštak (8117.7 μg/g). The variety Sava of the inoculated treatment exhibited the highest average values of the nodule mass (21.1 mg) and the highest number of nodules (23.3) in the phenophase, in which 10% of pods reached full length (R4). The average values of mass and number of nodules were calculated for the entire vegetation period, being higher in the inoculated treatment. There was no positive correlation between the content of isoflavones in seeds and the number and mass of nodules. The impact of quantity of isoflavone on nodulation intensity in soybeans was not significant,, which could be related to already saturated soil with nitrogen-fixing bacteria

Challenges and possibilities of organic seed production with the emphasis on control of pathogens

The aim of this paper is to show the state of production of seed intended for the establishment of organic crops in Serbia. Current regulations in many countries require that the seed and planting material used for organic farming must be produced according to the rules applicable to organic crops. Organic seed production, compared to conventional, carries a higher risk of weed seed contamination and of seed-borne diseases which make it more difficult to obtain high quality seeds. The early harvest is one of the possible measures to improve the seed health and there are various forms of seed treatments (natural plant extracts and substances of natural origin). Results within the field of seed production are closely related to conservation of plant landraces and exchange of their seeds that are necessary for organic food producers in Serbia

Analiza sadržaja mineralnih materija u plodovima Lycium barbarum L. (Solanaceae) gajenim u Srbiji

Fruits of Lycium barbarum L. (Solanaceae), known as wolfberries or goji berries, have been long used in traditional medicine and nutrition in Asian countries. However, in recent years, they have become popular in Europe, including Serbia. However, there are limited data on the chemical composition of goji berries cultivated in our country. This study aimed to estimate and perform a comparative analysis of the mineral content of goji berries from six locations in Serbia. After microwave digestion, macro- (K, Na, P, S, Mg, Ca) and microelement (Fe, Zn, Cu, Cr, Mn, Mo) contents in analyzed samples were determined by the optical emission spectrometry with induced coupled plasma (ICP-OES). As expected, mineral concentrations varied significantly among goji berry samples from different locations (p < 0.05). In general, the decreasing orders of minerals in goji berries were: K > P > S > Na > Mg > Ca > Zn > Fe > Cu > Mn > Cr > Mo. According to the Recommended Daily Allowance (RDA) values, dried goji berries (30 g) provide a rich source of Cr and Cu, while their contribution to the daily intake of other minerals was below 15% RDA.Plodovi Lycium barbarum L. (Solanaceae), poznatiji kao vučje bobice ili godži, imaju dugu istoriju upotrebe u tradicionalnoj medicini i ishrani stanovnika azijskih zemalja, a poslednjih godina su sve popularniji u Evropi, uključujući i Srbiju. Međutim, nema mnogo podataka o hemijskom sastavu plodova godži gajenih u našoj zemlji. Cilj ovog rada je bio da se izvrši komparativna analiza mineralnog sastava plodova godži prikupljenih sa šest različitih lokacija u Srbiji. Nakon prethodne mikrotalasne digestije, sadržaj makro- (K, Na, P, S, Mg, Ca) i mikroelemenata (Fe, Zn, Cu, Cr, Mn, Mo) u ispitivanim uzorcima suvih plodova godži, određen je primenom optičke emisione spektrometrije sa indukovano spregnutom plazmom (ICP-OES). Kao što je i očekivano, dobijeni rezultati ukazuju na statistički značajne razlike u sadržaju mineralnih materija u plodovima godži prikupljenih sa različitih lokacija (p < 0,05). Generalno, mineralne materije su zastupljene po sledećem opadajućem redosledu koncentracija: K > P > S > Na > Mg > Ca > Zn > Fe > Cu > Mn > Cr > Mo. Utvrđeni sadržaj makro- i mikroelemenata u jednoj porciji (30 g suvih plodova godži), u odnosu na nutritivne referentne vrednosti (NRV), ukazuje da su plodovi godži bogat izvor Cr i Cu, dok je doprinos jedne porcije plodova godži dnevnom unosu ostalih mineralnih materija ispod 15% NRV.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

SERBIAN SEED INDUSTRY AND UNIVERSITY AFTER TRANSITION

In this paper we discuss the cooperation of the Serbian agricultural institutes, faculties and seed industry which are deserved for the development of seed science and technology and its applications in the sector of agriculture. It is particularly emphasized the connection between research and educational institutions in the field of biotechnology and the importance of their alienation. In addition, it raised a question concerning the reorganization of the existing universities and research institutes in Serbia after the Bologna reform.Univerzitet i polјoprivredni instituti u drugoj polovini ХХ stoleća izveli su uspešan prenos znanja iz razvijenih zemalјa u SFRJ. Pre dvadesetak godina zapoĉela je tranzicija drţava istoĉne i jugoistoĉne Evrope što je dovelo do znaĉajnih promena polјoprivrede i obrazovanja. Mnoga velika srpska polјoprivredna preduzeća nestala su sa privredne scene a njihovo mesto zauzele su strane kompanije. Promene su zahvatile i oblast visokog obrazovanja te se danas oseća potreba da se aktualizuje veza izmeĊu naših univerziteta, istraţivaĉkih istitucija u oblasti polјoprivrede i industrije semena kao vaţnog dela javnog ţivota. Naime, sredinom prošlog veka na prostoru SFRJ obrazovana je mreţa polјoprivrednih istraţivaĉkih instituta i preduzeća koja su postigla znaĉajne uspehe u oblasti selekcije i proizvodnje semena. U senci ovog uspeha ostala je ĉinjenica da su polјoprivredni instituti od univerziteta preuzeli znaĉajan deo istraţivanja što je ograniĉilo univerzitete, budući da je veći deo laboratorija i opreme bio u institutima. Univeziteti su zajedno sa institutima imali sve: studente, nastavnike, laboratorije, istraţivaĉku opremu, ali oni su tada, kao i danas, bili organizaciono odvojeni te ni jedni ni drugi nisu mogli da ostvare veće rezultate. U uslovima društvene svojine ovaj naĉin rada bio je odrţiv. Danas je takvo razdvajanje smetnja njihovom razvoju te se polјoprivredni instituti prilagoĊavaju privredi u kojoj dominira privatna svojina. Shodno promenama u društvu, postojeća drţavna univerzitetska mreţa pretrpela je promene primenom bolonjske reforme kojom je srpsko visoko obrazovanje upodoblјeno eropskom školskom prostoru. Na osnovu dosadašnjeg iskustva naziru se tri scenarija ureĊenja univerziteta. Prvi je oĉuvanje postojeće organizacije (pluralizam uz tihe, evolutivne promene), drugi je ukidanje privatnih univerziteta (radikalno rešenje), treći je podela postojećih univerziteta na manje celine prema nauĉnim oblastima (tehniĉki, biotehnološki i td). Moguća regionalizacija Srbije nametnuće pitanje poloţaja univerziteta u odnosu na lokalnu samoupravu (gradove) tj. da li su univerziteti isklјuĉivo nacionalne (drţavne) institucije? Buduće promene visokog obrazovanja morale bi da poĊu od toga da će se jedan deo instituta pridruţiti univerzitetima dok će ostali oĉuvati svoju pravnu samostalnost i postati ono što su uvek i bili – preduzeća koja se bave proizvodnjom i prodajom semenske robe. Ova transformacija olakšala bi ureĊenje odnosa izmeĊu polјoprivrednih instituta (semenskih preduzeća), univerziteta i banke bilјnih gena. To je od posebnog znaĉaja budući da u postojećim istraţivaĉkim programima instituta, koje finansira vlada, nema jasne orijentacije na semenarstvo. Ne ĉudi što struĉna terminologija u oblasti semenarstva slabo razvijena a što bi se moglo pobolјšati udruţenim delovanjem predstavnika akademske zajednice i semenske industrije i podizanjem nivoa domaćih struĉnih ĉasopisa u ovoj oblasti. Pred industrijom semena i univerzitetom stoje brojna pitanja koja su od znaĉaja za dalјi razvoj semenarstva i polјoprivrede