Caratterizzazione del legame di molecole di interesse farmaceutico alla sieroalbumina umana mediante biocromatografia e dicroismo circolare

Negli ultimi anni, un crescente numero di studiosi ha focalizzato la propria attenzione sullo sviluppo di strategie che permettessero di caratterizzare le proprietà ADMET dei farmaci in via di sviluppo, il più rapidamente possibile. Questa tendenza origina dalla consapevolezza che circa la metà dei farmaci in via di sviluppo non viene commercializzato perché ha carenze nelle caratteristiche ADME, e che almeno la metà delle molecole che riescono ad essere commercializzate, hanno comunque qualche problema tossicologico o ADME [1]. Infatti, poco importa quanto una molecola possa essere attiva o specifica: perché possa diventare farmaco è necessario che venga ben assorbita, distribuita nell’organismo, metabolizzata non troppo rapidamente, ne troppo lentamente e completamente eliminata. Inoltre la molecola e i suoi metaboliti non dovrebbero essere tossici per l’organismo. Quindi è chiaro come una rapida determinazione dei parametri ADMET in fasi precoci dello sviluppo del farmaco, consenta di risparmiare tempo e denaro, permettendo di selezionare da subito i composti più promettenti e di lasciar perdere quelli con caratteristiche negative. Questa tesi si colloca in questo contesto, e mostra l’applicazione di una tecnica semplice, la biocromatografia, per caratterizzare rapidamente il legame di librerie di composti alla sieroalbumina umana (HSA). Inoltre mostra l’utilizzo di un’altra tecnica indipendente, il dicroismo circolare, che permette di studiare gli stessi sistemi farmaco-proteina, in soluzione, dando informazioni supplementari riguardo alla stereochimica del processo di legame. La HSA è la proteina più abbondante presente nel sangue. Questa proteina funziona da carrier per un gran numero di molecole, sia endogene, come ad esempio bilirubina, tiroxina, ormoni steroidei, acidi grassi, che xenobiotici. Inoltre aumenta la solubilità di molecole lipofile poco solubili in ambiente acquoso, come ad esempio i tassani. Il legame alla HSA è generalmente stereoselettivo e ad avviene a livello di siti di legame ad alta affinità. Inoltre è ben noto che la competizione tra farmaci o tra un farmaco e metaboliti endogeni, possa variare in maniera significativa la loro frazione libera, modificandone l’attività e la tossicità. Per queste sue proprietà la HSA può influenzare sia le proprietà farmacocinetiche che farmacodinamiche dei farmaci. Non è inusuale che un intero progetto di sviluppo di un farmaco possa venire abbandonato a causa di un’affinità troppo elevata alla HSA, o a un tempo di emivita troppo corto, o a una scarsa distribuzione dovuta ad un debole legame alla HSA. Dal punto di vista farmacocinetico, quindi, la HSA è la proteina di trasporto del plasma più importante. Un gran numero di pubblicazioni dimostra l’affidabilità della tecnica biocromatografica nello studio dei fenomeni di bioriconoscimento tra proteine e piccole molecole [2-6]. Il mio lavoro si è focalizzato principalmente sull’uso della biocromatografia come metodo per valutare le caratteristiche di legame di alcune serie di composti di interesse farmaceutico alla HSA, e sul miglioramento di tale tecnica. Per ottenere una miglior comprensione dei meccanismi di legame delle molecole studiate, gli stessi sistemi farmaco-HSA sono stati studiati anche con il dicroismo circolare (CD). Inizialmente, la HSA è stata immobilizzata su una colonna di silice epossidica impaccata 50 x 4.6 mm di diametro interno, utilizzando una procedura precedentemente riportata in letteratura [7], con alcune piccole modifiche. In breve, l’immobilizzazione è stata effettuata ponendo a ricircolo, attraverso una colonna precedentemente impaccata, una soluzione di HSA in determinate condizioni di pH e forza ionica. La colonna è stata quindi caratterizzata per quanto riguarda la quantità di proteina correttamente immobilizzata, attraverso l’analisi frontale di L-triptofano [8]. Di seguito, sono stati iniettati in colonna alcune soluzioni raceme di molecole note legare la HSA in maniera enantioselettiva, per controllare che la procedura di immobilizzazione non avesse modificato le proprietà di legame della proteina. Dopo essere stata caratterizzata, la colonna è stata utilizzata per determinare la percentuale di legame di una piccola serie di inibitori della proteasi HIV (IPs), e per individuarne il sito(i) di legame. La percentuale di legame è stata calcolata attraverso il fattore di capacità (k) dei campioni. Questo parametro in fase acquosa è stato estrapolato linearmente dal grafico log k contro la percentuale (v/v) di 1-propanolo presente nella fase mobile. Solamente per due dei cinque composti analizzati è stato possibile misurare direttamente il valore di k in assenza di solvente organico. Tutti gli IPs analizzati hanno mostrato un’elevata percentuale di legame alla HSA: in particolare, il valore per ritonavir, lopinavir e saquinavir è risultato maggiore del 95%. Questi risultati sono in accordo con dati presenti in letteratura, ottenuti attraverso il biosensore ottico [9]. Inoltre, questi risultati sono coerenti con la significativa riduzione di attività inibitoria di questi composti osservata in presenza di HSA. Questa riduzione sembra essere maggiore per i composti che legano maggiormente la proteina [10]. Successivamente sono stati eseguiti degli studi di competizione tramite cromatografia zonale. Questo metodo prevede di utilizzare una soluzione a concentrazione nota di un competitore come fase mobile, mentre piccole quantità di analita vengono iniettate nella colonna funzionalizzata con HSA. I competitori sono stati selezionati in base al loro legame selettivo ad uno dei principali siti di legame sulla proteina. In particolare, sono stati utilizzati salicilato di sodio, ibuprofene e valproato di sodio come marker dei siti I, II e sito della bilirubina, rispettivamente. Questi studi hanno mostrato un legame indipendente dei PIs ai siti I e II, mentre è stata osservata una debole anticooperatività per il sito della bilirubina. Lo stesso sistema farmaco-proteina è stato infine investigato in soluzione attraverso l’uso del dicroismo circolare. In particolare, è stato monitorata la variazione del segnale CD indotto di un complesso equimolare [HSA]/[bilirubina], a seguito dell’aggiunta di aliquote di ritonavir, scelto come rappresentante della serie. I risultati confermano la lieve anticooperatività per il sito della bilirubina osservato precedentemente negli studi biocromatografici. Successivamente, lo stesso protocollo descritto precedentemente è stato applicato a una colonna di silice epossidica monolitica 50 x 4.6 mm, per valutare l’affidabilità del supporto monolitico per applicazioni biocromatografiche. Il supporto monolitico monolitico ha mostrato buone caratteristiche cromatografiche in termini di contropressione, efficienza e stabilità, oltre che affidabilità nella determinazione dei parametri di legame alla HSA. Questa colonna è stata utilizzata per la determinazione della percentuale di legame alla HSA di una serie di poliamminochinoni sviluppati nell’ambito di una ricerca sulla malattia di Alzheimer. Tutti i composti hanno mostrato una percentuale di legame superiore al 95%. Inoltre, è stata osservata una correlazione tra percentuale di legame è caratteristiche della catena laterale (lunghezza e numero di gruppi amminici). Successivamente sono stati effettuati studi di competizione dei composti in esame tramite il dicroismo circolare in cui è stato evidenziato un effetto anticooperativo dei poliamminochinoni ai siti I e II, mentre rispetto al sito della bilirubina il legame si è dimostrato indipendente. Le conoscenze acquisite con il supporto monolitico precedentemente descritto, sono state applicate a una colonna di silice epossidica più corta (10 x 4.6 mm). Il metodo di determinazione della percentuale di legame utilizzato negli studi precedenti si basa su dati ottenuti con più esperimenti, quindi è necessario molto tempo prima di ottenere il dato finale. L’uso di una colonna più corta permette di ridurre i tempi di ritenzione degli analiti, per cui la determinazione della percentuale di legame alla HSA diventa molto più rapida. Si passa quindi da una analisi a medio rendimento a una analisi di screening ad alto rendimento (highthroughput- screening, HTS). Inoltre, la riduzione dei tempi di analisi, permette di evitare l’uso di soventi organici nella fase mobile. Dopo aver caratterizzato la colonna da 10 mm con lo stesso metodo precedentemente descritto per le altre colonne, sono stati iniettati una serie di standard variando il flusso della fase mobile, per valutare la possibilità di utilizzare flussi elevati. La colonna è stata quindi impiegata per stimare la percentuale di legame di una serie di molecole con differenti caratteristiche chimiche. Successivamente è stata valutata la possibilità di utilizzare una colonna così corta, anche per studi di competizione, ed è stata indagato il legame di una serie di composti al sito I. Infine è stata effettuata una valutazione della stabilità della colonna in seguito ad un uso estensivo. L’uso di supporti cromatografici funzionalizzati con albumine di diversa origine (ratto, cane, guinea pig, hamster, topo, coniglio), può essere proposto come applicazione futura di queste colonne HTS. Infatti, la possibilità di ottenere informazioni del legame dei farmaci in via di sviluppo alle diverse albumine, permetterebbe un migliore paragone tra i dati ottenuti tramite esperimenti in vitro e i dati ottenuti con esperimenti sull’animale, facilitando la successiva estrapolazione all’uomo, con la velocità di un metodo HTS. Inoltre, verrebbe ridotto anche il numero di animali utilizzati nelle sperimentazioni. Alcuni lavori presenti in letteratura dimostrano l’affidabilita di colonne funzionalizzate con albumine di diversa origine [11-13]: l’utilizzo di colonne più corte potrebbe aumentarne le applicazioni.

Allestimenti di reattori multistadio per la digestione anaerobica dei reflui agro-industriali e confronto con processo convenzionale

This study fits into the context of activities aim at waste bioremediation and valorization through the production of energy according to principles of environmental sustainability. The experimental work was carried out at the laboratories of the Department of Civil Engineering, Environmental and Materials (DICAM) of the Faculty of Engineering. The main objective was to enhance the treatment of high organic loading waste, such as manure and cheese whey, through advanced anaerobic digestion systems in order to obtain biogas rich in methane. On the basis of the premise that the environmental conditions pertaining in most anaerobic wastewater digesters are not optimal for both fermentative and methanogenic microorganisms, the research was particularly focused on the implementation of two-phase anaerobic digesters. In fact a two-phase process permits selection and enrichment of different bacteria in each digester by independently controlling the digester operating conditions. Thus, the first phase (acidogenesis) can be operated to optimize acidogenic growth and the second phase (methanogenesis) to optimize methanogenic growth. (Ince O. , 1998). Before reactors’ set up, , some lab scale experiments were carried out to identify the best manure and whey ratio and the best conditions of temperature, pH, hydraulic retention time of acidogenesis an methanogenic phases.

Algorithmen zur labelfreien quantitativen Proteomanalyse auf Basis datenunabhängig-akquirierter LC-MS-Daten

Moderne ESI-LC-MS/MS-Techniken erlauben in Verbindung mit Bottom-up-Ansätzen eine qualitative und quantitative Charakterisierung mehrerer tausend Proteine in einem einzigen Experiment. Für die labelfreie Proteinquantifizierung eignen sich besonders datenunabhängige Akquisitionsmethoden wie MSE und die IMS-Varianten HDMSE und UDMSE. Durch ihre hohe Komplexität stellen die so erfassten Daten besondere Anforderungen an die Analysesoftware. Eine quantitative Analyse der MSE/HDMSE/UDMSE-Daten blieb bislang wenigen kommerziellen Lösungen vorbehalten. rn| In der vorliegenden Arbeit wurden eine Strategie und eine Reihe neuer Methoden zur messungsübergreifenden, quantitativen Analyse labelfreier MSE/HDMSE/UDMSE-Daten entwickelt und als Software ISOQuant implementiert. Für die ersten Schritte der Datenanalyse (Featuredetektion, Peptid- und Proteinidentifikation) wird die kommerzielle Software PLGS verwendet. Anschließend werden die unabhängigen PLGS-Ergebnisse aller Messungen eines Experiments in einer relationalen Datenbank zusammengeführt und mit Hilfe der dedizierten Algorithmen (Retentionszeitalignment, Feature-Clustering, multidimensionale Normalisierung der Intensitäten, mehrstufige Datenfilterung, Proteininferenz, Umverteilung der Intensitäten geteilter Peptide, Proteinquantifizierung) überarbeitet. Durch diese Nachbearbeitung wird die Reproduzierbarkeit der qualitativen und quantitativen Ergebnisse signifikant gesteigert.rn| Um die Performance der quantitativen Datenanalyse zu evaluieren und mit anderen Lösungen zu vergleichen, wurde ein Satz von exakt definierten Hybridproteom-Proben entwickelt. Die Proben wurden mit den Methoden MSE und UDMSE erfasst, mit Progenesis QIP, synapter und ISOQuant analysiert und verglichen. Im Gegensatz zu synapter und Progenesis QIP konnte ISOQuant sowohl eine hohe Reproduzierbarkeit der Proteinidentifikation als auch eine hohe Präzision und Richtigkeit der Proteinquantifizierung erreichen.rn| Schlussfolgernd ermöglichen die vorgestellten Algorithmen und der Analyseworkflow zuverlässige und reproduzierbare quantitative Datenanalysen. Mit der Software ISOQuant wurde ein einfaches und effizientes Werkzeug für routinemäßige Hochdurchsatzanalysen labelfreier MSE/HDMSE/UDMSE-Daten entwickelt. Mit den Hybridproteom-Proben und den Bewertungsmetriken wurde ein umfassendes System zur Evaluierung quantitativer Akquisitions- und Datenanalysesysteme vorgestellt.

Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge

Farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in synthesis, metabolism, and transport of bile acids and thus plays a major role in maintaining bile acid homeostasis. In this study, metabolomic responses were investigated in urine of wild-type and Fxr-null mice fed cholic acid, an FXR ligand, using ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS). Multivariate data analysis between wild-type and Fxr-null mice on a cholic acid diet revealed that the most increased ions were metabolites of p-cresol (4-methylphenol), corticosterone, and cholic acid in Fxr-null mice. The structural identities of the above metabolites were confirmed by chemical synthesis and by comparing retention time (RT) and/or tandem mass fragmentation patterns of the urinary metabolites with the authentic standards. Tauro-3alpha,6,7alpha,12alpha-tetrol (3alpha,6,7alpha,12alpha-tetrahydroxy-5beta-cholestan-26-oyltaurine), one of the most increased metabolites in Fxr-null mice on a CA diet, is a marker for efficient hydroxylation of toxic bile acids possibly through induction of Cyp3a11. A cholestatic model induced by lithocholic acid revealed that enhanced expression of Cyp3a11 is the major defense mechanism to detoxify cholestatic bile acids in Fxr-null mice. These results will be useful for identification of biomarkers for cholestasis and for determination of adaptive molecular mechanisms in cholestasis.

Tandem mass spectrometry identification and LC-MS quantification of intact cytokinin nucleotides in K-562 human leukemia cells

We describe here a new reversed-phase high-performance liquid chromatography with mass spectrometry detection method for quantifying intact cytokinin nucleotides in human K-562 leukemia cells. Tandem mass spectrometry was used to identify the intracellular metabolites (cytokinin monophosphorylated, diphosphorylated, and triphosphorylated nucleotides) in riboside-treated cells. For the protein precipitation and sample preparation, a trichloroacetic acid extraction method is used. Samples are then back-extracted with diethyl ether, lyophilized, reconstituted, and injected into the LC system. Analytes were quantified in negative selected ion monitoring mode using a single quadrupole mass spectrometer. The method was validated in terms of retention time stabilities, limits of detection, linearity, recovery, and analytical accuracy. The developed method was linear in the range of 1-1,000 pmol for all studied compounds. The limits of detection for the analytes vary from 0.2 to 0.6 pmol.

In vivo evaluation in cynomolgus monkey brain and metabolism of [¹⁸F]fluorodeprenyl: a new MAO-B pet radioligand

In this study, we evaluated the in vivo characteristics of a new monoamine oxidase type B (MAO-B) radioligand, [¹⁸F]fluorodeprenyl, by positron emission tomography (PET) in two cynomolgus monkeys. The brain uptake of [¹⁸F]fluorodeprenyl was more than 7% (600% SUV) of the total injected radioactivity and similar to that of [¹¹C]deprenyl, an established MAO-B radioligand. The highest uptake was observed in the striatum, one of the MAO-B-rich regions, with a peak at approximately 2-3 min after injection, followed by lower uptake in the thalamus and the cortex and lowest uptake in the cerebellum. Brain uptake of [¹⁸F]fluorodeprenyl was largely inhibited by preadministration of the MAO-B inhibitor, L-deprenyl, whereas clorgyline, a MAO Type A blocker, had no significant inhibitory effect, thus demonstrating selectivity for MAO-B. [¹⁸F]Fluorodeprenyl showed relatively slow metabolism with the presence of two radiometabolite peaks with similar retention time as the labeled metabolites of [¹¹C]deprenyl. These results suggest that [¹⁸F]fluorodeprenyl is a potential PET radioligand for visualization of MAO-B activity.

Interaction of the receptor FGFRL1 with the negative regulator Spred1

FGFRL1 is a member of the fibroblast growth factor receptor family. It plays an essential role during branching morphogenesis of the metanephric kidneys, as mice with a targeted deletion of the Fgfrl1 gene show severe kidney dysplasia. Here we used the yeast two-hybrid system to demonstrate that FGFRL1 binds with its C-terminal, histidine-rich domain to Spred1 and to other proteins of the Sprouty/Spred family. Members of this family are known to act as negative regulators of the Ras/Raf/Erk signaling pathway. Truncation experiments further showed that FGFRL1 interacts with the SPR domain of Spred1, a domain that is shared by all members of the Sprouty/Spred family. The interaction could be verified by coprecipitation of the interaction partners from solution and by codistribution at the cell membrane of COS1 and HEK293 cells. Interestingly, Spred1 increased the retention time of FGFRL1 at the plasma membrane where the receptor might interact with ligands. FGFRL1 and members of the Sprouty/Spred family belong to the FGF synexpression group, which also includes FGF3, FGF8, Sef and Isthmin. It is conceivable that FGFRL1, Sef and some Sprouty/Spred proteins work in concert to control growth factor signaling during branching morphogenesis of the kidneys and other organs.

Purification and characterization of a 34-kDa, heat stable glycoprotein from Synadenium grantii latex: action on human fibrinogen and fibrin clot

Latex glycoprotein (LGP) from Synadenium grantii latex was purified by the combination of heat precipitation and gel permeation chromatography. LGP is a heat stable protein even at 80 degrees C showed a sharp single band both in SDS-PAGE as well as in native (acidic) PAGE. LGP is a monomeric protein appears as single band under reducing condition. It is a less hydrophobic protein showed sharp single peak in RP-HPLC with retention time of 13.3 m. The relative molecular mass of LGP is 34.4 kDa. CD spectrum of LGP explains less content of alpha-helix (7%), and high content of beta-pleated sheets (48%) and random coils (46%). The N-terminal sequence of LGP is D-F-P-S-D-W-Y-A-Y-E-G-Y-V-I-D-R-P-F-S. Purified LGP is a fibrinogen degrading protease hydrolyses all the three subunits in the order of Aalpha, Bbeta and gamma. The hydrolytic pattern is totally different from plasmin as well as thrombin. LGP reduces recalcification time from 165 to 30 s with citrated human plasma but did not show thrombin like as well as factor Xa-like activity. Although LGP induces procoagulant activity, it hydrolyses partially cross-linked fibrin clot. It hydrolyses all the subunits of partially cross-linked fibrin clot (alpha- chains, beta-chain and gamma-gamma dimer). LGP is a serine protease, inhibited by PMSF. Other serine protease inhibitors, aprotinin and leupeptin did not inhibit the caseinolytic activity as well as fibrinogenolytic activity. We report purification and characterization of a glycoprotein from Synadenium grantii latex with human fibrino(geno)lytic activity.

THE POTENTIAL OF INDUSTRIAL WASTE AND AGRICULTURAL FEEDSTOCK TOWARDS SUSTAINABLE BIOFUELS PRODUCTION: TECHNO-ECONOMIC AND ENVIRONMENTAL IMPACT PERSPECTIVES

This Ph.D. research is comprised of three major components; (i) Characterization study to analyze the composition of defatted corn syrup (DCS) from a dry corn mill facility (ii) Hydrolysis experiments to optimize the production of fermentable sugars and amino acid platform using DCS and (iii) Sustainability analyses. Analyses of DCS included total solids, ash content, total protein, amino acids, inorganic elements, starch, total carbohydrates, lignin, organic acids, glycerol, and presence of functional groups. Total solids content was 37.4% (± 0.4%) by weight, and the mass balance closure was 101%. Total carbohydrates [27% (± 5%) wt.] comprised of starch (5.6%), soluble monomer carbohydrates (12%) and non-starch carbohydrates (10%). Hemicellulose components (structural and non-structural) were; xylan (6%), xylose (1%), mannan (1%), mannose (0.4%), arabinan (1%), arabinose (0.4%), galatactan (3%) and galactose (0.4%). Based on the measured physical and chemical components, bio-chemical conversion route and subsequent fermentation to value added products was identified as promising. DCS has potential to serve as an important fermentation feedstock for bio-based chemicals production. In the sugar hydrolysis experiments, reaction parameters such as acid concentration and retention time were analyzed to determine the optimal conditions to maximize monomer sugar yields while keeping the inhibitors at minimum. Total fermentable sugars produced can reach approximately 86% of theoretical yield when subjected to dilute acid pretreatment (DAP). DAP followed by subsequent enzymatic hydrolysis was most effective for 0 wt% acid hydrolysate samples and least efficient towards 1 and 2 wt% acid hydrolysate samples. The best hydrolysis scheme DCS from an industry's point of view is standalone 60 minutes dilute acid hydrolysis at 2 wt% acid concentration. The combined effect of hydrolysis reaction time, temperature and ratio of enzyme to substrate ratio to develop hydrolysis process that optimizes the production of amino acids in DCS were studied. Four key hydrolysis pathways were investigated for the production of amino acids using DCS. The first hydrolysis pathway is the amino acid analysis using DAP. The second pathway is DAP of DCS followed by protein hydrolysis using proteases [Trypsin, Pronase E (Streptomyces griseus) and Protex 6L]. The third hydrolysis pathway investigated a standalone experiment using proteases (Trypsin, Pronase E, Protex 6L, and Alcalase) on the DCS without any pretreatment. The final pathway investigated the use of Accellerase 1500® and Protex 6L to simultaneously produce fermentable sugars and amino acids over a 24 hour hydrolysis reaction time. The 3 key objectives of the techno-economic analysis component of this PhD research included; (i) Development of a process design for the production of both the sugar and amino acid platforms with DAP using DCS (ii) A preliminary cost analysis to estimate the initial capital cost and operating cost of this facility (iii) A greenhouse gas analysis to understand the environmental impact of this facility. Using Aspen Plus®, a conceptual process design has been constructed. Finally, both Aspen Plus Economic Analyzer® and Simapro® sofware were employed to conduct the cost analysis as well as the carbon footprint emissions of this process facility respectively. Another section of my PhD research work focused on the life cycle assessment (LCA) of commonly used dairy feeds in the U.S. Greenhouse gas (GHG) emissions analysis was conducted for cultivation, harvesting, and production of common dairy feeds used for the production of dairy milk in the U.S. The goal was to determine the carbon footprint [grams CO2 equivalents (gCO2e)/kg of dry feed] in the U.S. on a regional basis, identify key inputs, and make recommendations for emissions reduction. The final section of my Ph.D. research work was an LCA of a single dairy feed mill located in Michigan, USA. The primary goal was to conduct a preliminary assessment of dairy feed mill operations and ultimately determine the GHG emissions for 1 kilogram of milled dairy feed.

Bench-scale evaluation of the activated sludge process for treatment of a high -strength chemical plant wastewater

A bench-scale treatability study was conducted on a high-strength wastewater from a chemical plant to develop an alternative for the existing waste stabilization pond treatment system. The objective of this study was to determine the treatability of the wastewater by the activated sludge process and, if treatable, to determine appropriate operating conditions, and to evaluate the degradability of bis(2-chloroethyl)ether (Chlorex) and benzene in the activated sludge system. Four 4-L Plexi-glass, complete mixing, continuous flow activated sludge reactors were operated in parallel under different operating conditions over a 6-month period. The operating conditions examined were hydraulic retention time (HRT), sludge retention time (SRT), nutrient supplement, and Chlorex/benzene spikes. Generally the activated sludge system treating high-strength wastewater was stable under large variations of organic loading and operating conditions. At an HRT of 2 days, more than 90% removal efficiency with good sludge settleability was achieved when the organic loading was less than 0.4 g BOD$\sb5$/g MLVSS/d or 0.8 g COD/g MLVSS/d. At least 20 days of SRT was required to maintain steady operation. Phosphorus addition enhanced the performance of the system especially during stressed operation. On the average, removals of benzene and Chlorex were 73-86% and 37-65%, respectively. In addition, the low-strength wastewater was treatable by activated sludge process, showing more than 90% BOD removal at a HRT of 0.5 days. In general, the sludge had poor settling characteristics. The aerated lagoon process treating high-strength wastewater also provided significant organic reduction, but did not produce an acceptable effluent concentration. ^

Chemical characterization and quantification of the brown algal storage compound laminarin

In search of a meaningful stress indicator for Fucus vesiculosus we found that the often used quantitative determination procedures for the polysaccharide laminarin (beta-1,3-glucan) result in different kind of problems, uncertainties and limitations. This chemical long-term storage form of carbon enables perennial brown algae in seasonally fluctuating ecosystems to uncouple growth from photosynthesis. Because of this high ecological relevance a reliable and precise method for determination and quantification of laminarin is needed. Therefore, a simple, cold water extraction method coupled to a new quantitative liquid chromatography-mass spectrometrical method (LC-MS) was developed. Laminarin was determined in nine out of twelve brown algal species, and its expected typical molar mass distribution of 2000-7000 Da was confirmed. Furthermore, laminarin consisted of a complex mixture of different chemical forms, since fifteen chemical laminarin species with distinct molecular weights were measured in nine species of brown algae. Laminarin concentrations in the algal tissues ranged from 0.03 to 0.86% dry weight (DW). The direct chemical characterization and quantification of laminarin by LC-MS represents a powerful method to verify the biochemical and ecological importance of laminarin for brown algae. Single individuals of Laminaria hyperborea, L. digitata, Saccharina latissima, F. serratus, F. vesiculosus, F. spiralis, Himanthalia elongata, Cystoseira tamariscifolia, Pelvetia canaliculata, Ascophyllum nodosum, Halidrys siliquosa and Dictyota dichotoma were collected in fall (18.11.2013) during spring low tide from the shore of Finavarra, Co. Clare, west coast of Ireland (53° 09' 25'' N, 09° 06' 58'' W). After sampling, the different algae were immediately transported to the lab, lyophilized and sent to the University of Rostock. Laminarin was extracted with cold ultrapure water from the algal samples. Before extraction they were ground to < 1 mm grain size with an analytical mill (Ika MF 10 Basic). The algal material (approx. 1.5 g DW) was extracted in ultrapure water (8 mL) on a shaker (250 rpm) for 5 h. After the addition of surplus ultrapure water (4 mL) and shaking manually, 1 mL of the sample was filter centrifuged (45 µm) at 14,000 rpm (Hettich Mikro 22 R). The slightly viscous supernatant was free of suspended material and converted into a microvial (300 µL) for further analysis. The extracts were analyzed using liquid chromatography-mass spectrometry (LC-MS) analysis (LTQ Velos Pro ion trap spectrometer with Accela HPLC, Thermo Scientific). Laminarin species were separated on a KinetexTM column (2.6 µm C18, 150 x 3 mm). The mobile phase was 90 % ultrapure water and 10 % acetonitrile, run isocratically at a flow rate of 0.2 mL min-1. MS was working in ESI negative ion mode in a mass range of 100 - 4000 amu. Glucose contents were determined after extraction using high-performance liquid chromatography (HPLC). Extracted samples were analyzed in an HPLC (SmartLine, Knauer GmbH) equipped with a SUPELCOGELTM Ca column (30 x 7,8 mm without preColumn) and RI-detector (S2300 PDA S2800). Water was used as eluent at a flow rate of 0.8 mL min-1 at 75 °C. Glucose was quantified by comparison of the retention time and peak area with standard solutions using ChromGate software. Mannitol was extracted from three subsamples of 10-20 mg powdered alga material (L. hyperborea, L. digitata, S. latissima, F. serratus, F. vesiculosus, F. spiralis, H. elongata, P. canaliculata, A. nodosum, H. siliquosa) and quantified, following the HPLC method described by Karsten et al. (1991). For analyzing carbon and nitrogen contents, dried algal material was ground to powder and three subsamples of 2 mg from each alga thalli were loaded and packed into tin cartridges (6×6×12 mm). The packages were combusted at 950 °C and the absolute contents of C and N were automatically quantified in an elemental analyzer (Elementar Vario EL III, Germany) using acetanilide as standard according to Verardo et al. (1990).

C1-C8 hydrocarbons in DSDP Leg 64 Holes sediments

Sediments from the Baja California Continental Margin Transect - Sites 474 and 476 - showed small amounts of C2-C8 hydrocarbons and functionalized compounds (alkenes) typical of organic-rich, Recent, cold (<30°C) marine sediments. In contrast, some samples from Sites 477, 478, 479, and Hole 481A in the Guaymas Basin, an active spreading center, showed the characteristics of thermally generated hydrocarbons. These include an increase (sometimes exponential) in amount and diversity of C2-C8 hydrocarbons and a decrease in alkenes in more thermally mature sediments. The results indicate that the injection of basaltic sills has minimal effect on C2-C8 hydrocarbon generation except in the immediate vicinity of the sill. The absence of light hydrocarbons close to the hottest sills suggests that the compounds distill away as they are formed in these areas of very active hydrothermal circulation. A sample of young sediment exposed to very high temperatures (>300°C) from deeper thermal sources at the hottest site, 477, showed a very limited hydrocarbon distribution, including primarily ethane, benzene, and toluene, together with smaller amounts of propane and butane.

Pore water acetate and hydrogen, helium and methane gas concentrations in ODP Leg 204 sediments

Acetate and hydrogen concentrations in pore fluids were measured in samples taken at seven sites from southern Hydrate Ridge (SHR) offshore Oregon, USA. Acetate concentrations ranged from 3.17 to 2515 µM. The maximum acetate concentrations occurred at Site 1251, which was drilled on a slope basin to the east of SHR at depths just above the bottom-simulating reflector (BSR) that marks the boundary of gas hydrate stability. Acetate maxima and localized high acetate concentrations occurred at the BSR at all sites and frequently corresponded with areas of gas hydrate accumulation, suggesting an empirical relationship. Acetate concentrations were typically at a minimum near the seafloor and above the sulfate/methane interface, where sulfate-reducing bacteria may consume acetate. Hydrogen concentrations in pressure core samples ranged from 16.45 to 1036 parts per million by volume (ppmv). In some cases, hydrogen and acetate concentrations were elevated concurrently, suggesting a positive correlation. However, sampling of hydrogen was limited in comparison to acetate, so any relationships between the two analytes, if present, were difficult to discern.

(Table 1) Organic carbon contents and vitrinite reflection in sediments from DSDP Holes 63-467, 63-471, and 64-481A

A number of C25 and C30 highly branched isoprenoid (HBI) sulphur compounds (E.G., thiolanes, 1-oxo-thiolanes, thiophenes, and benzo[b]thiophenes) with 2,6,10,14-tetramethyl-7-(3-methylpentyl)pentadecane and 2,6,10,14,18-pentamethyl-7-(3-methylpentyl)nonadecane carbon skeletons were identified in sediments, ranging from Holocene to Upper Cretaceous. These identifications are based on mass spectral characterisation, desulphurisation, and, in some cases, by comparison of mass spectral and relative retention time data with those of authentic standards. The presence of unsaturated C25 and C30 HBI thiolanes in a Recent sediment from the Black Sea (age 3-6 ka) strongly supports their formation during early diagenesis. The co-occurrence of HBI polyenes (C25 and C30) and unsaturated HBI thiolanes (C25 and C30) possessing two double bonds less than the corresponding HBI polyenes, in this Recent sediment, testifies to the formation of unsaturated HBI thiolanes by a reaction of inorganic sulphur species with double bonds of the HBI polyenes. Furthermore, a diagenetic scheme for HBI sulphur compounds is proposed based on the identification of HBI sulphur compounds in sediment samples with different maturity levels.

Nutritional impact on health and performance in intensively reared rabbits

The present work summarizes research related to the definition of nutrient recommendations for feeds used in the intensive production of rabbit's meat. Fibre is the main chemical constituent of rabbit diets that typically contain 320 to 360 and 50 to 90 g/kg of insoluble and soluble fibre, respectively. Instead, the dietary contents of cereal grains (∼120 to 160 g/kg), fat (15 to 25 g/kg) and protein concentrates (150 to 180 g/kg) are usually low with respect to other intensively reared monogastric animals. Cell wall constituents are not well digested in rabbits, but this effect is compensated by its stimulus of gut motility, which leads to an increasing rate of passage of digesta, and allows achieving an elevated dry matter intake. A high feed consumption and an adequate balance in essential nutrients are required to sustain the elevated needs of high-productive rabbits measured either as reproductive yield, milk production or growth rate in the fattening period. Around weaning, pathologies occur in a context of incomplete development of the digestive physiology of young rabbits. The supply of balanced diets has also been related to the prevention of disorders by means of three mechanisms: (i) promoting a lower retention time of the digesta in the digestive tract through feeding fibre sources with optimal chemical and physical characteristics, (ii) restricting feed intake after weaning or (iii) causing a lower flow of easily available substrates into the fermentative area by modifying feed composition (e.g. by lowering protein and starch contents, increasing its digestibility or partially substituting insoluble with soluble fibre), or by delaying age at weaning. The alteration in the gut microbiota composition has been postulated as the possible primary cause of these pathologies.