Effects of the anti-sucking device «SuckStop Müller¼ on calf behavior.

INTRODUCTION: When cross-sucking persists beyond calf-hood, it represents an important problem in dairy heifers and cows. It can cause teat injuries and severe mastitis and lead to significant economic losses. The «SuckStop Müller,¼ a novel anti-sucking device, is designed to give the calf a negative feedback when cross-sucking on a conspecific. The aim of this study was to assess whether wearing a SuckStop would result in behavioral changes other than cross-sucking and thus, impair welfare in the short- and longer-term. Sixteen group-housed calves were observed in groups of four on five days, before and after fitting the SuckStop: day -2, day -1, day 0, day 1, and day 9. Maintenance behaviors (e.g., feeding, drinking, lying) were recorded using instantaneous scan sampling. In addition, the frequency of contact behaviors (e.g., exploring the feeding fence, touching own body) was recorded by means of continuous focal animal observations. Contact behaviors were classified as «impaired¼ or «normal¼ depending on whether or not the calf flinched in response to the contact. Finally, the number of visits to the milk and concentrate feeders was extracted from the computer-controlled feeding system. Fitting a SuckStop resulted in a higher proportion of observations spent lying and less exploration behavior on day 0 and day 1 than on day -2, day -1, and day 9. On day 0 and day 1, 6,3 % of exploration behaviors were classified as impaired, compared to 0,4 % (day -2, day -1) before and 0,2 % (day 9) after fitting the SuckStop. On day 9, all calves had superficial ulcerations on the nasal septum. In four calves, these ulcerations were moderately severe, whereas all other calves had slight ulcerations. In summary, the calves habituated quickly to this novel anti-sucking device. Follow-up studies are necessary to assess the long-term relevance of tissue alterations in the nasal septum for calf welfare as well as the effect of the SuckStop on cross-sucking behavior.

INTRODUCTION: Lorsque la succion croisée persiste au-delà de l'âge de veau, elle représente un problème important chez les génisses et les vaches laitières. Elle peut provoquer des blessures aux trayons et des mastites graves et entraîner ainsi des pertes économiques importantes. Le «SuckStop Müller¼, un nouveau dispositif anti-succion, est conçu pour donner au veau un feedback négatif lorsqu'il suce un congénère. L'objectif de cette étude était de déterminer si le port d'un SuckStop pouvait entraîner des changements de comportement autres que la succion croisée et, par conséquent, nuire au bien-être à court et à long terme. Seize veaux logés en groupe ont été observés par groupes de quatre pendant cinq jours, avant et après la pose du SuckStop: jour ­2, jour ­1, jour 0, jour 1 et jour 9. Les comportements d'entretien (par exemple se nourrir, boire, se coucher) ont été enregistrés à l'aide d'un échantillonnage par balayage instantané. En outre, la fréquence des comportements de contact (par exemple, explorer le râtelier, toucher son propre corps) a été enregistrée au moyen d'observations continues de chaque animal. Les comportements de contact ont été classés comme «altérés¼ ou «normaux¼ selon que le veau a tressailli ou non en réponse au contact. Enfin, le nombre de visites aux distributeurs de lait et de concentré a été extrait du système d'alimentation contrôlé par ordinateur. L'installation d'un SuckStop a entraîné une proportion plus élevée d'observations de comportements couchés et moins de comportements d'exploration le jour 0 et le jour 1 que le jour ­2, le jour ­1 et le jour 9. Le jour 0 et le jour 1, 6,3 % des comportements d'exploration ont été classés comme déficients, contre 0,4 % (jour ­2, jour ­1) avant et 0,2 % (jour 9) après la pose du SuckStop. Au jour 9, tous les veaux présentaient des ulcérations superficielles sur la cloison nasale. Chez quatre veaux, ces ulcérations étaient modérément graves, tandis que tous les autres veaux présentaient de légères ulcérations. En résumé, les veaux se sont rapidement habitués à ce nouveau dispositif anti-suceur. Des études de suivi sont nécessaires pour évaluer la pertinence à long terme des altérations tissulaires de la cloison nasale pour le bien-être des veaux ainsi que l'effet du SuckStop sur le comportement de succion croisée.

Recent progress in the discovery of macrocyclic compounds as potential anti-infective therapeutics.

Novel therapeutic strategies are urgently needed for the treatment of serious diseases caused by viral, bacterial and parasitic infections, because currently used drugs are facing the problem of rapidly emerging resistance. There is also an urgent need for agents that act on novel pathogen-specific targets, in order to expand the repertoire of possible therapies. The high throughput screening of diverse small molecule compound libraries has provided only a limited number of new lead series, and the number of compounds acting on novel targets is even smaller. Natural product screening has traditionally been very successful in the anti-infective area. Several successful drugs on the market as well as other compounds in clinical development are derived from natural products. Amongst these, many are macrocyclic compounds in the 1-2 kDa size range. This review will describe recent advances and novel drug discovery approaches in the anti-infective area, focusing on synthetic and natural macrocyclic compounds for which in vivo proof of concept has been established. The review will also highlight the Protein Epitope Mimetics (PEM) technology as a novel tool in the drug discovery process. Here the structures of naturally occurring antimicrobial and antiviral peptides and proteins are used as starting points to generate novel macrocyclic mimetics, which can be produced and optimized efficiently by combinatorial synthetic methods. Several recent examples highlight the great potential of the PEM approach in the discovery of new anti-infective agents.

Engineering of complex polyketide biosynthesis--insights from sequencing of the monensin biosynthetic gene cluster.

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation.

A chain initiation factor common to both modular and aromatic polyketide synthases.

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named 'chain-length factor', is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.

Molecular basis of Celmer's rules: the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase.

BACKGROUND: Polyketides are compounds that possess medically significant activities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs. Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mechanisms that operate in natural PKS modules. RESULTS: The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2. The reduction products of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S, 3R) and (2R,3S) isomers of the corresponding beta-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only contains KR1 produced only the (2S,3R) isomer. CONCLUSIONS: In contrast with earlier results, KR1 selects only the (2S) isomer and reduces it stereospecifically to the (2S, 3R)-3-hydroxy-2-methyl acyl product. The KR domain of module 1 controls the stereochemical outcome at both methyl-and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester generated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereo-chemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, operate in modules 1 and 2 of the erythromycin PKS. These results should provide a more rational basis for designing hybrid PKSs to generate altered stereochemistry in polyketide products.

Dynamical studies of peptide motifs in the Plasmodium falciparum circumsporozoite surface protein by restrained and unrestrained MD simulations.

The immunodominant region on the circumsporozoite surface (CS) protein of the malaria parasite Plasmodium falciparum contains 37 repeated copies of a asparagine-alanine-asparagine-proline (NANP) motif NMR studies of linear synthetic peptides containing one, two or three repeat units provided evidence for nascent type I beta-turns within the NPNA cadence in aqueous solution. The beta-turns could be stabilised upon substituting proline for alpha-methylproline (p(Me)) in the dodecamer (NP(Me)NA)3, without loss of the ability to elicit antibodies cross-reactive with P. falciparum sporozoites. In this work, four 4 ns MD simulations of the dodecapeptide Acetyl-(NP(Me)NA)3, in water, using NOE distance restraints, using 3J-coupling constant restraints, using both these restraints and without restraints, were carried out to determine the conformations of this peptide in aqueous solution. An unrestrained MD simulation of the unmethylated Ac-(NPNA)3 peptide in water was also carried out to investigate the effect of the additional methyl groups on the structure and dynamics of the peptide. The application of NOE distance restraints and 3J-coupling constant restraints leads to contradictory results, probably due to different averaging time scales inherent to the measurement of these data, which exceed the 100 ps averaging applied in the simulations. The additional methyl groups lead to more compact structures, which display enhanced local fluctuations. The central tetrapeptide adopts a type I beta-turn, while the outer motifs display more conformational variability. The three motifs in the methylated dodecamer peptide, however, adopt frequently in the distance restrained MD simulation a compact structure such that the outer motifs appear to form a hydrophobic core by stacking of their two proline rings. This arrangement also suggests how a peptide containing multiple tandemly linked copies of a stable beta-turn NPNA motif might adopt a folded stem-like structure, which conceivably may be of biological relevance in the native CS protein.