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Rosuvastatin je lijek skupine statina koji se koristi za regulaciju visoke razine kolesterola u ljudskom tijelu. Isto tako, rosuvastatin i ostali statini pokazuju zaštitnu ulogu protiv oksidativnog stresa uzrokovanog slobodnim radikalima. Cilj ovog istraživanja bio je identificirati krajnje produkte koji su nastali radikalnom razgradnjom rosuvastatina. Kako bi se potaknula radikalna razgradnja, vodena otopina rosuvastatina ozračena je različitim dozama gama zračenja (50-1000 Gy) pri oksidativnim uvjetima. Rosuvastatin i srodni degradacijski produkti odvojeni su nanoC18 kolonom gradijentnom elucijom, a identifikacija je provedena pomoću vezanog sustava nanoUPLC i nanoESI-QTOF. Pomoću točno mjerenih masa, zajedno s algoritmom usporedbe izotopnih raspodjela, provedena je elementna analiza kojom je identificirano devet degradacijskih produkata. U ovom je istraživanju po prvi puta provedena gama-inducirana razgradnja rosuvastatina te detaljno opisana kemijska struktura, MS/MS fragmentacija te mehanizam nastanka pojedinog degradacijskog produkta. Priloženi rezultati doprinose razumijevanju razgradnog puta rosuvastatina i ostalih statina pri gama zračenju.

Energija nesumnjivo ima središnju ulogu u funkcioniranju organizama. Mnogi okolišni stresori, poput onečišćenja, hipoksije ili isušivanja, utječu na energijski status organizma. Energijski naboj adenilata (AEC) definiran je kao omjer adeninskih nukleotida: adenozin mono-, di- i trifosfata (AMP, ADP i ATP) [1]. Vrijednosti AEC od 0, 8 do 0, 9 pronađene su u organizmima koji žive u nezagađenom okolišu, dok vrijednosti ispod 0, 5 ukazuju na fiziološki kolaps. Cilj ovog istraživanja bio je odrediti AEC i omjer ATP/ADP te ih usporediti između različitih populacija slatkovodnog stigofilnog rakušca Synurella ambulans iz hipoheičke zone (HZ) rijeke Save (Medsave i Jarun) i helokrenog izvora Mala Bršljanica uzorkovanih zimi. Lokalitet Medsave se nalazi 3 km uzvodno od ispusta otpadnih voda grada Zaprešića, a Jarun 13 km nizvodno od njega, dok je Mala Bršljanica (13 km od Kutine) izolirani prirodni izvor, odabran kao referentna postaja. Vrijednosti AEC na Maloj Bršljanici bile su 0, 44, na Medsave 0, 41 i na Jarunu 0, 35. Zanimljivo je preživljavanje jedinki vrste S. ambulans pri tako niskim vrijednostima AEC-a, koje ukazuju na staničnu smrt. Ovakav niži energijski naboj (0, 3 - 0, 4) može se objasniti niskom učinkovitošću AMP deaminaze kod beskralješnjaka u odnosu na kralješnjake (0, 5 - 0, 6) [2]. Također, teški okolišni uvjeti u HZ-i mogu utjecati na energijski status jedinki vrste S. ambulans. Najviša vrijednost AEC zabilježena je na Maloj Bršljanici, lokaciji bez zabilježenog antropogenog utjecaja. Najniža vrijednost AEC zabilježena na Jarunu vjerojatno je posljedica otpadnih voda iz farmaceutske industrije i komunalnih otpadnih voda. Omjer ATP/ADP je odražavao AEC trend, s najvećom vrijednošću od 1, 06 na Maloj Bršljanici, 0, 91 na Medsave te najnižom vrijednosti od 0, 72 na Jarunu. Zaključno, istraženi parametri energijskog metabolizma vrste S. ambulans pokazali su da je AEC osjetljivi fiziološki biomarker odgovora organizma na okolišni stres. Energy undoubtedly plays a central role in the function of organisms. Many environmental stressors, such as pollution, hypoxia or desiccation, affect the organism’s energy status. The adenylate energy charge (AEC) was defined as the ratio of the adenine nucleotides: adenosine mono-, di-, and triphosphate (AMP, ADP, and ATP) [1]. AEC values of 0.8 - 0.9 were ascribed to organisms living in nonpolluted environments, while values below 0.5 were indicative of physiological collapse. This study aimed to determine AEC and ATP/ADP ratio and compare them between different populations of the freshwater stygophilous amphipod Synurella ambulans from hyporheic zone (HZ) of the Sava River (Medsave and Jarun) and helocrene spring Mala Bršljanica sampled in winter. Medsave is located 3 km upstream and Jarun 13 km downstream from the wastewater discharge of the town of Zaprešić, while Mala Bršljanica (13 km from Kutina) is isolated natural spring selected as reference site. AEC values at Mala Bršljanica were 0.44, 0.41 at Medsave and 0.35 at Jarun. Interestingly, S. ambulans can survive such low AEC values, predictive of cell death. This lower energy charge (0.3 - 0.4) may be explained by the low efficiency of AMP deaminase in invertebrates when compared to vertebrates (0.5 - 0.6) [2]. Also, harsh environmental conditions in the HZ may affect the energy status of S. ambulans. The highest AEC value was observed in Mala Bršljanica, location without known anthropogenic impact. The lowest AEC value noted at Jarun is possibly caused by wastewaters from the pharmaceutical industry and municipal wastewaters. The ATP/ADP ratio reflected the AEC pattern, with the highest value of 1.06 at Mala Bršljanica, 0.91 at Medsave, and the lowest value of 0.72 at Jarun. In summary, the investigated parameters of S. ambulans energy metabolism showed that AEC is a sensitive physiological biomarker of organism’s response to environmental stress.

Groundwater organisms are exposed to progressive pollution and multi-xenobiotic stressors that influence the organism’s physiology, metabolism and finally ecosystem functioning and biodiversity. A useful index of stress response at the cellular level is the adenylate energy charge (AEC). AEC is determined as the ratio of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) as follows AEC= ([ATP]+1/2[ADP])/([ATP]+[ADP]+[AMP]). The aim of the study was to quantify for the first time adenine nucleotides in stygophilous crustacean Synurella ambulans and compare the AEC at two sampling sites with various levels of pollution in four seasons. Separation and quantification of nucleotides was performed using ion-pair reversed phase HPLC, with UV detection at 260 nm. Amphipods were collected from the hyporheic zone (HZ) of the Sava River at Jarun-Zagreb and Medsave-Zaprešić (upstream of pharmaceutical wastewater outlet) sampling sites in December 2018 and April, July, October 2019. The Medsave population had significantly higher AEC values (0.41 ; 0.45) than the Jarun population (0.35 ; 0.31) in winter and spring sampling campaigns, indicating higher energy supply and thus lower metabolic stress. The possible impact of concentrations of metals/metalloids in interstitial water and in amphipods on seasonal and spatial variation of the AEC are discussed. AEC was shown as a useful index of environmental stress in S. ambulans since it can directly measure the change in available energy and thus the metabolic stress experienced by a given organism. In conclusion, it appears that the seasonal and spatial variations of the AEC values reflect the ecological state in the HZ. The financial support of the European Regional Development Fund for the Qua/Qua Protein project (KK.01.1.1.07.0023) and of Croatian Science Foundation for the AQUAMAPMET (IP-2014-09-4255) are highly acknowledged.

Energijski naboj adenilata (eng. adenylate energy charge, AEC) je pokazatelj energijskog statusa metabolizma stanice, za čiju procjenu je potrebno uzeti u obzir koncentracije sva tri nukleotida – adenozin-trifosfata (ATP), adenozin-difosfata (ADP) i adenozin-monofosfata (AMP) [1]. Promjene u ekološkim ili fiziološkim parametrima dovode do smanjenja AEC. Određivanje i kvantificiranje nukleotida provodi se tehnikama tekućinske kromatografije visoke djelotvornosti (HPLC), koje su korisne za izolaciju i kvantifikaciju nukleotida u ekstraktima bioloških tkiva. Cilj ovog istraživanja bio je ispitati različite eksperimentalne uvjete ekstrakcije adenilata na uzorcima slatkovodnog rakušca Gammarus fossarum iz Velikog potoka (planina Medvednica) te razviti najprikladniju metodu analize. U pripremi uzorka testirani su različiti tipovi homogenizatora: Potter- Elvehjemov, Ultraturax (Qiagen) i ultrazvučna kupelj, gdje se Potter- Elvehjemov homogenizator pokazao optimalnim rješenjem. Testirana je i homogenizacija u nekoliko različitih otopina: 50 mM NH4HCO3, 0, 5 M CCl3-COOH (trikloroctena kiselina, TCA) i 0, 5 M HClO4 (perklorna kiselina, PCA). Dobiveni kiseli ekstrakt biološkog materijala neophodno je neutralizirati jer u jako lužnatom ili kiselom mediju dolazi do hidrolize fosfatnih veza u adenilatima te su stoga ispitani različiti načini neutralizacije. Najbolji rezultati dobiveni su ekstrakcijom adenilata homogenizacijom u PCA te neutralizacijom s KOH (c=0, 3 M) i fosfatnim puferom pri uvjetima puferiranja smjese soli Na2HPO4 i KH2PO4 ; c=0, 2 M ; pH=8, 2. Također, testirana je primjena ekstrakcije na čvrstoj fazi (Strata X, Phenomenex) za pročišćavanje uzorka pri kojoj se pokazalo da ona ne doprinosi značajno poboljšanju rezolucije kromatograma adenilata, ali doprinosi uklanjanju onečišćenja u slučaju prisutnosti nepoželjnog matriksa. Adenilati su razdvojeni pomoću sustava HPLC na koloni ODS Hypersil C18 (5 m, 125  4 mm) izokratnim eluiranjem (150 mM KH2PO4/K2HPO4, 100 mM KCl i 10 mM tetrabutil amonij hidroksid, TBA kao ionskim parom ; pH=6) [2]uz primijenjeni protok od 1 mL/min. Priprema uzoraka za analizu predstavlja ključni korak te je stoga važno definirati sve uvjete izolacije adenilata iz biološkog materijala za pouzdano određivanje koncentracije AMP, ADP i ATP. Cijeli postupak pripreme uzorka neophodno je provesti u što kraćem vremenu na niskoj temperaturi (

Bolni otok zgloba može biti simptom mnogih bolesti u djece, među kojima se ističu dvije vrlo različite imunološki posredovane bolesti: reaktivni artritis (ReA) i juvenilni idiopatski artritis (JIA). Za razliku od ReA-a koji traje kraće i nakon kojeg dolazi do potpunog nestanka simptoma, u bolesnika s JIA-om dolazi do razvoja kronične bolesti koja može dovesti do trajnog oštećenja zglobova. Brojna istraživanja dala su naslutiti da crijevna mikrobiota ima vrlo važnu ulogu u oblikovanju imunološkog odgovora, zbog čega bi razlog različitom odgovoru na poznati ili nepoznati patogen u bolesnika s ReA-om i JIA-om mogao ležati u različitom sastavu mikrobiote. Cilj ovog istraživanja stoga je bio ispitati razliku u zastupljenosti podvrsta Escherichiae coli (E coli), jedne od najzastupljenijih bakterija unutar crijevne mikrobiote, prilikom prve pojave simptoma juvenilnog idiopatskog i reaktivnog artritisa. Ispitanici i metode. Uzorci stolice 14 bolesnika prikupljeni su prilikom prvog posjeta Zavodu za reumatologiju i imunologiju Kliničkog bolničkog centra Sestre milosrdnice u Zagrebu, a konačna dijagnoza bolesti postavljena je 22 Reumatizam 2019 ; 66(Suppl 1):15–30 Sažeci / Abstracts Usmena priopćenja / Oral communications tri mjeseca kasnije: u sedmero bolesnika postavljena je dijagnoza JIA-a, a u drugih sedmero ReA-a. Svi uzorci su analizirani masenom spektrometrijom na nanoLC-Synapt G2 Si instrumentu u Institutu Ruđer Bošković. Kako bi se identificirale najučestalije vrste E coli korišten je posebni računalni program, Protein Reader s ugrađenim Dust algoritmom koji je pretraživao NCBI nr bazu podataka koja sadrži zapise o više od 400 vrsta E coli. Medijan dobi bolesnika oboljelih od JIA-a bio je 7, 14, a bolesnika oboljelih od ReA-a 7, 11. Rezultati. Pojedine podvrste E coli (P0301867.1- 10, O104:H4, O103:H25, O111:H11, KTE and K) bile su tri puta učestalije u bolesnika s JIA-om, dok je u bolesnika s ReAom uočeno dvostruko više podvrsta E coli koje uzrokuju infektivne proljeve (DEC). Zaključak. Rezultati ovog istraživanja ukazali su na razliku u podvrstama E coli u stolici bolesnika s ReA-om i JIA-om u samim počecima bolesti. Budući da je E coli jedna od ključnih bakterija u crijevnoj mikrobioti, s više od 600 do sada prepoznatih podvrsta, razumno je za pretpostaviti kako uočene promjene mogu utjecati na rav notežu crijevne mikrobiote i oblikovanje imunosnog odgovora te pridonijeti razvoju kronične bolesti ili potpune regresije simptoma.

INTRODUCTION: Painful joint swelling is a symptom of many childhood diseases, most notably reactive arthritis (ReA) and juvenile idiopathic arthritis (JIA). The growing body of evidence suggests that gut microbiota could orchestrate the immune response and development of those diseases. This study aimed to assess the differences in the presence of Escherichia coli (E coli) subtypes in the stool of JIA and ReA patients at the first occurrence of symptoms. METHODS: Stool samples of 14 patients with joint swelling were collected during their first visit to Pediatric Rheumatology Clinic in Sestre milosrdnice University Hospital Center in Zagreb, Croatia. The samples were analyzed by mass spectrometry on nanoLC-Synapt G2 Si instrument. To identify the most abundant E coli subtypes, specialized software named Protein Reader with implemented Dust algorithm searched through NCBI nr database. RESULTS: Various E coli subtypes (P0301867.1-10, O104:H4, O103:H25, O111:H11, KTE and K) were three times more abundant in patients with JIA, while increased abundance of diarrheagenic E. coli (DEC) was detected in children with ReA. CONCLUSION: This pilot study has shown differences in the subtypes of E. coli present in the stool of children with ReA and JIA in the early stage of the disease. Since E coli is one of the paramount bacteria in gut microbiota, it is reasonable to assume that the differences described in this study can have a potential impact on the gut environment, contributing to the development of the chronic disease in JIA patients, or the resolution of symptoms in children with ReA.

Multidimensional chromatography followed by electrospray ionization mass spectrometry was used to characterize two different samples, mycoplasma in human urine samples and lipids from vegerable. Automated liquid handling platform is a strong tool for fast and precise chromatographic separation which can provide 6 fractions of 96 samples simultaneously in less then 120 minutes. Although a state-of-the-art technology, lack of suitable and nonreusable commercially available cartridges limits such automated platform in scientific research. A necessity for specific chromatography separation has led us to modify commercial chromatographic cartridges. Cartridges were detached and filled with more suitable stationary phase for sample separation and method procedures with sufficient recovery rate were developed for both chromatographic sample separation. Quaternary methylammonium (QMA) was used as a strong anion-exchange stationary phase for fractionation of peptides extracted from mycoplasma. Besides, polar silica sorbent was used as a stationary phase to retain molecules in nonpolar matrices, which can effectively separate compounds with small variations in structure, such as lipids. Afterward, six fractions of each sample were analysed using LC-MS system (nanoLC-ESIqTOF) and the raw data were processed using protein database search engine. Different stationary phases can be used for the development of a new multidimensional chromatography using liquid handling platform. Commercially available cartridges can be easily reused and loaded with desirable stacionary phase. Not less important, suchs automated platforms is no longer limited and can be used for various type of samples.

Multidimensional chromatography followed by MALDI- TOF mass spectrometry was used to identify species, sub-species and characterize proteins from different type of cultivated microorganisms. Automated liquid handling platform is a strong tool for fast and precise chromatographic separation which can provide 6 fractions of 96 samples simultaneously in less than 120 minutes. In-house developed cartridges and methods provided us 1-D, 2-D and even 3-D liquid handling chromatography methods dedicated to identify microorganisms to species and sub-species level. Cartridges are printed on 3-D printer and filled with suitable stationary phase for sample separation while method procedures are developed for tailored chromatographic tryptic peptide separation. Quaternary Methyl Ammonium (QMA) is used as a strong anion exchange stationary phase for fractionation of peptides derived from cell extracts followed by Strong Cation Exchange (SCX). All in all, 2-D liquid separation method suited for optimal peptide separation. Firstly, proteins were isolated from cells and digested with trypsin overnight to break down protein bonds into smaller peptides. Each sample of peptides was separated in 6 fractions using QMA stationary phases where obtained fractions were additionally separated in 6 fractions using SCX stationary phases. Later 36 fractions in total of each sample were analyzed using MALDI-TOF mass spectrometry and the raw data were processed using protein database search engine (ProteinReader, IRB). Different stationary phases can be used for the development of a new multidimensional chromatography methods using liquid handling platform. Commercially available cartridges can be easily replaced and loaded with desirable stationary phase where custom made method can be developed. Not less important, such automated platforms are no longer limited and can be used for various type of microbial samples.

Infections of the urinary tract are considered as one of the most prevalent bacterial infections in humans, with the highest rate of reoccurrence and antibiotic resistance. Therefore, rapid and accurate diagnosis is a key to a successful treatment and recovery. Urine is easily obtained, making itself a desirable sample for pathogen biomarker identification. Proteins found in urine are roughly divided on bacterial and human proteins. Proteome analysis of the named protein classes using mass spectrometry provides great sensitivity and selectivity for the detection of pathogen or human proteins. In the presented research, three urine samples: E. coli, K. pneumoniae and Mycoplasma spp. were analysed by standard clinical and mass spectrometry de novo sequencing method. E. coli and K. pneumoniae were confirmed by culture- based while Mycoplasma spp. was confirmed by PCR standardized clinical methods. On the other hand, mass spectrometry fast identification method includes urine proteome extraction protocol with bacterial cell lysis and protein isolation followed by Filter- Assisted Sample Preparation (FASP)1. After the protein lysis with trypsin and selective N- terminus peptide derivatization with CAF-/CAF+ reagent produced peptides are analysed in positive and negative ion mode. Tryptic peptide sequence identification in positive and negative MS/MS ion mode was carried out on MALDI-TOF/TOF mass spectrometer. E. coli and K. pneumoniae peptides were successfully identified after mass spectrometry analysis with in-house developed de novo sequencing Protein Reader software tool. Further on, analysis of small cell pathogen mycoplasma (1-2 m) was conducted on high resolution ESI mass spectrometry equipped with nano-LC separation system in order to distinguish human and pathogen peptides that were not separated after application of the same sample preparation protocol used on E. coli, K. pneumoniae urine samples. The results of later analysis showed a presence of mycoplasma in the sample suggesting the proposed protocol has potential for a routine identification of mycoplasma in human urine samples.

Urinary tract infections (UTIs) are the most common form of bacterial infections in the community and in hospitals, so fast and reliable microbial pathogen identification in human urine samples is required for clinical microbiology laboratory, especially for the world with more prevalent antibiotic resistance. In our study we analyzed 20 outpatient human urine samples with microbiologically proved presence of more than 105 CFU/ml with Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF). As a reference for bacterial diversity selected urine samples were determined using 16S rRNA gene sequencing. The direct approach, without step in which the microorganisms are isolated and grown, provided successful identification of bacteria at the genus and species levels, especially for monobacterial samples of Citrobacter koseri, Escherichia coli, Klebsiella spp., and Proteus spp. The results of this study demonstrates that we could identify different pathogen with MALDI-TOF/TOF from fresh or frozen, not cultivated human urine samples. In our further investigations double fractionation turned out to be a very good solution. Problems with hypothetical proteins during bioinformatics analysis with ProteinPlot will be figure out with in-house developed software based on natural language processing.

Majority of currently used LC-MS/MS techniques for protein identification is based on database matching of non-derivatized peptide signals recorded in positive ion mode. Although widely used, such an approach does not always provide satisfactory sequence coverage to unambiguously identify a protein sample. Protein de novo sequencing concept using CAF-/CAF+ reagent (chemically activated fragmentation negative/chemically activated fragmentation positive) enables fast, highly accurate, reliable and easy to use identification of microorganisms down to the species and subspecies level. CAF-/CAF+ (5-formylbenzene-1, 3-disulfonic acid*) is a chemical reagent for the derivatization of peptide samples prior to analysis by tandem mass spectrometry (MS/MS) that enables gathering of both positive and negative peptide ions datasets. Peptide sequences are read from both positive and negative MS/MS spectra and matched against the NCBInr database by developed software named ProteinReader. We present results that show identification of microorganisms down to the subspecies level using either MALDI- or ESI-TOF technique.

Currently used LC-MS/(MS) technique for protein identification is based on database matching of non-derivatized peptide signals recorded in the positive ion mode. Although widely used, such an approach does not always provide satisfactory sequence coverage to unambiguously identify a peptide/protein sample. We have introduced a peptide derivatization method that allows for spectra recording in both positive and negative ion mode in order to increase peptide sequence coverage. Peptides identified as overlapped amino acid sequences read from both ion modes assign peptides unambiguously after de novo sequencing and protein database identification. The protein-reading concept using CAF-/CAF+ reagent (chemically activated fragmentation negative/chemically activated fragmentation positive) enables fast, highly accurate, reliable and easy to use identification of microorganisms down to the species level. CAF-/CAF+ (5-formylbenzene-1, 3-disulfonic acid*) is a chemical reagent for the derivatization of peptide samples prior to analysis by MALDI (Matrix Assisted Laser Desorption Ionization) Tandem Mass Spectrometry (MS/MS). For the first time, mass spectrometry can exploit both positive and negative ion mode, either for species or proteins identification. The CAF-/CAF+ method enables de novo sequencing of derivatized peptides with negative and positive ion mode tandem mass spectrometry (MS/MS–and MS/MS+). Peptide sequences are read from MS/MS spectra and matched against the NCBInr database by developed software named ProteinReader and confirmed by the mass spectrometry data of elucidated peptide mass sequences derived from the annotated genome.

RATIONALE: One of the most challenging tasks of proteomics is peptide de novo sequencing. 4-Sulfophenyl isothiocyanate (SPITC) peptide derivatization enables acquisition of high-quality tandem mass spectra (MS/MS) for de novo sequencing, but unwanted non-specific reactions and reduced mass spectra (MS) signal intensities still represent the obstacles in highthroughput de novo sequencing. METHODS: We developed a SPITC peptide derivatization procedure under acidic conditions (pH LT = 5). Derivatized peptides were analyzed by matrix-assisted laser desorption/ionization (MALDI-MS) in negative ion mode followed by MS/MS in positive ion mode. A de novo sequencing tool, named DUST, adjusted to SPITC chemistry, was designed for successful high-throughput peptide de novo sequencing. This high-throughput peptide de novo sequencing was tested on Fusarium delphinoides, an organism with an uncharacterized genome. RESULTS: The SPITC derivatization procedure under acidic conditions produced a significantly improved MS dataset in comparison to commonly used derivatization under basic conditions. Signal intensities were 6 to 10 times greater and the over-sulfonation effect measured on lysine-containing peptides was significantly decreased. Furthermore, development of a novel DUST algorithm enabled automated de novo sequencing with the calculated accuracy of 70.6%. CONCLUSIONS: The SPITC derivatization and de novo sequencing approach outlined here provides a reliable method for high-throughput peptide de novo sequencing. High-throughput peptide de novo sequencing enabled protein mutation identification and identification of proteins from organisms with non-sequenced genomes. Copyright (C) 2016 John Wiley and Sons, Ltd.

Present state of knowledge about abiotic particles produced during phytoplankton bloom experiments as well as in the upper ocean is mostly based on information gathered by staining techniques. The most frequently followed microparticle class are transparent exopolymeric particles stainable by alcian blue (TEP). TEPs are described as gel particles in the size range from 3-5 μm to 100 μm. Particles retained on filters are stained with alcian blue (cationic dye that stains anionic polysaccharides) at pH 2.5. Such procedure may produce structural artifacts including aggregation and rearrangements. In order to gain information on native structure of particles it is essential to develop methods and techniques with potential to analyze particles in their natural aquatic environment without separation (filtration, centrifugation) and to avoid drying process. The Adriatic Sea is mainly phosphorus (P) limited, and this was the basis of the design of mesocosm bloom experiment "Rovinj 2003" to follow the dynamics of particle formation. We used a low (1 µM) and a high (6.3 µM) P addition, to attempt to achieve two different levels of biomass accumulation. At the same time, this was the unique opportunity to evaluate relation and relevance of presently available measurement techniques for monitoring marine particles. A more specific goal was to contrast particle formation in low P and high P nutrient enrichments using the advantages offered by electrochemical approach.

The eye’s lens is a biological marvel, a complex structure comprising about 1, 000 layers of perfectly clear cells. It has undergone several adaptations to fulfill its function to focus light originating from distant objects onto the retina where photoreception takes place. In order to focus to different distances the lens must accommodate its shape implicating that the lens is, in contrast to a solid glass lens, flexible. Atomic force microscopy (AFM) is a powerful tool to study the mechanical properties of biological objects with high spatial precision. Here we have used AFM featuring a spherical tip at the end of a soft cantilever to indent single fibre cells isolated from lens core and cortex. Quantitative characterization of each type of lens cells was performed by the determination of the Young’s modulus of elasticity. The elastic modulus of nuclear lens cells (4.69 kPa) was 20-fold higher than that of cortical lens cells (0.251 kPa) showing that single cells from the lens core and cortex can be unambiguously distinguished using the elastic modulus as criterion. Such measurements, carried out on the single cell level, will allow studying the effect of drugs and of aging on the mechanical properties of lens cells, and thus provide novel quantitative insights to accommodation and lens pathologies.

Significance of adhesion phenomena in single particle-electrode interaction became apparent since the discovery of adhesion signals of vesicles in seawater samples at a mercury electrode. By potentiostatic control of surface charge and tension, the adhesion forces can be fine tuned to study the interplay of complex forces involved in soft particle-electrode double-layer interactions. After studies of adhesion signals of nonpolar hydrocarbons and of negatively charged living cells (unicellular algae) we focus here on lipid vesicles (liposomes), the classical model in studying physical mechanisms of cell adhesion.

The static mercury electrode was used to detect directly and selectively vesicle-type particles (operationally defined as surface-active particles) in aqueous sample without any pretreatment in a phytoplankton bloom experiment Rovinj 2003.

Background and Purpose: Mesocosm bloom experiment "Rovinj 2003" was the unique opportunity to evaluate relation and relevance of presently available measurement techniques for monitoring marine particles. A more specific goal was to contrast particle formation in low P and high P nutrient enrichments using the advantages offered by electrochemical approach. Materials and Methods: A static mercury electrode (SME) immersed directly in seawater sample is conveniently used to count surface-active particles, SAP. The method is direct, rapid and reliable. Results: Concentration and size distribution of microparticles detected by electrochemical sensor is reported during the bloom in low P and high P nutrient enrichments. SAP concentration was significantly higher in low P nutrient enrichments. Conclusions: Electrochemical sensing of microparticles was successfully tested and results contrasted to alcian blue staining technique.

We present the amperometric response of single cell adhesion in a real time, from the initial attachment to a finite state of spread cell. The technique is based on measurement of double-layer charge displacement at the mercury drop electrode. The flow of compensating current reflects the dynamics of adhesive contact formation and subsequent spreading of a cell. The spike-shaped signals have the peak current in microampere range, duration in milliseconds, and displaced charge in the nanocoulomb range. The only hypothesis used in interpreting the signals is the validity of the electrical double-layer model. A surprising similarity to adhesion signals of droplets of liquid hydrocarbons (C12-C18) suggests that collective properties of cell exterior govern the dynamics of adhesion and rate of spreading, with the fluidity playing a major role. The characteristic potential range of adhesion can serve to study the interplay of complex surface forces involved in cell-electrode double-layer interactions.

The electrochemical techniques employing microelectrodes and high sensitivity time resolved recording of amperometric signals allow detection in real time of single events – oxidation or reduction of nano- and microparticles. In contrast to the voltammetry of solid microparticles where particles are already immobilized on the electrode surface, we shall treat the case where renewable electrode is exposed to the dispersion of organic microparticles. In that case the initial attachment is established by interplay of hydrodynamic and surface forces giving rise to specific signal for each particle attachment.

Nanoparticles of various materials are today implemented in wide variety of industrial, scientific and medical applications. Biocompatible nanoparticles are used for cell treatment ; as nano-biomarkers, for therapy and diagnostics. Nanoparticles can be synthesized conventionally either using wet chemistry methods or gas phase processes. Such nanoparticles are often characterized by impurities which are reaction products of additives and precursors. Laser ablation in liquids appeared to be a solution for that drawback. It is recognised as simple and versatile technique and there are almost no limitations in a selection of materials for the nanoparticle synthesis. The remarkable advantages of this technique over other techniques are absence of impurities in the final product (free of contaminating processes, chemical precursors not required), possibility of preparation of multicomponent nanoparticles, weak agglomeration, narrow size distribution of nanoparticles, etc. In our work we studied laser synthesized silver nanoparticles produced in water by ns Nd:YAG laser operating at 1064 nm with 100 mJ of output energy and 5 Hz of repetition rate. From AFM figures size- distribution of nanoparticles is obtained. Laser ablation is monitored by optical emission spectroscopy while colloidal solution is characterized by photospectrometry and photoluminescence. Our results show that average diameter of laser synthesized silver nanoparticles is around 10 nm and size distribution is relatively narrow. Our colloidal solution is very stable in time (few months) implying that nanoparticles are well dispersed and/or charged.

The basics of mass spectrometry will be considered, starting with instrumentation, followed by different techniques of ionization and fragmentation necessary to understand development of mass spectrometry during the past two decades. Particularly, this tutorial lecture will be focused on technical aspects of mass spectrometry:-ion sources-ion optics-ion separation-cells for fragmentation-ion detectors-collecting MS data-data processingTechniques for molecules and macromolecules separation and fractionation hyphenated to mass spectrometers, particularly mass spectrometry techniques for the large scale identification of proteins will be reviewed. Also, different methods for protein quantification by mass spectrometry will be described and discussed. Finally, bioinformatics principles to convert mass spectrometry data into peptide/protein sequences and to quantify them will be introduced.

Leucyl-tRNA synthetases (LeuRS) covalently couple tRNALeu with leucine, and thereby provide the pool of Leu-tRNALeu for ribosomal protein synthesis. LeuRS may also activate and transfer to tRNALeu structurally and chemically similar norvaline, a non–canonical amino acid that accumulates in Escherichia coli under micro-aerobic conditions. However, incorporation of norvaline into proteins is prevented by efficient intrinsic LeuRS hydrolytic activity toward norvalyl-tRNALeu within a dedicated post-transfer editing domain. In spite of the prevailing opinion that noncognate isoleucine mimics leucine well in the LeuRS synthetic reactions and thus requires editing to prevent errors in leucyl-tRNALeu synthesis, we now demonstrate that isoleucine is discriminated with high specificity within the synthetic site. Thermodynamic, structural and kinetic approaches establish that both very weak ground state binding and the decreased rate of the chemical step contribute to isoleucine discrimination. These results were complemented by in vivo experiments, where we show that while E. coli strain with editing deficient LeuRS grows normally in the presence of high isoleucine concentration, it displays growth defects under micro-aerobic conditions where norvaline accumulates. Our results reveal that LeuRS-mediated translational quality control represents the essential part of the major E. coli adaptive response necessary for survival in environments with low oxygen levels.

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Isoleucyl-tRNA synthetase (IleRS) catalyzes ATP- dependent covalent pairing of cognate isoleucine with cognate tRNAIle, thus providing accurate substrates for ribosomal protein synthesis. IleRS may also efficiently substitute isoleucine with structurally similar valine in the synthetic reaction. To prevent erroneous aminoacylation, IleRS developed extensive hydrolytic proofreading. This enzyme employs both the tRNA-dependent pre- transfer (hydrolysis of Val-AMP intermediate) and the post-transfer (hydrolysis of Val-tRNAIle) editing reactions within the synthetic and editing site, respectively[1]. Norvaline is a natural non- proteinogenic amino acid, structurally similar to isoleucine. It may accumulate in Escherichia coli under hypoxic conditions to milimolar concentrations. We have recently shown that norvaline is the major threat for error-free Leu- tRNALeu synthesis in E. coli[2]. To establish the mechanism of norvaline discrimination by IleRS, we utilized steady state and single-turnover kinetic analyses of the synthetic and editing pathways. We show that norvaline, similarly as valine, is not discriminated well in the synthetic reaction, and is efficiently activated and transferred to tRNAIle. However, the rapid post-transfer editing reaction precludes accumulation of Nva-tRNAIle, acting as the main defense against misincorporation of norvaline in place of isoleucine into cellular proteome. We further demonstrate that tRNA-dependent pre-transfer editing, that efficiently operates against Val- AMP, is also employed by IleRS to hydrolyze Nva- AMP. Our data thus establish that the tRNA- dependent pre-transfer editing activity, an idiosyncratic feature of IleRS proofreading, is characteristic of the IleRS:tRNAIle complex and is independent on the identity of non-cognate amino acid substrate.