1,720,974 research outputs found
Photodynamic therapy and cationic antimicrobial peptides: strategies to overcome bacterial resistance to antibiotics
No full textThe worldwide rise of antibiotic resistance stimulates the search of new strategies for killing bacteria, whose mechanisms of action are different from those of antibiotics. Two promising strategies in this respect are the use of cationic antimicrobial peptides (CAMPs) and photodynamic therapy (PDT). Both approaches have been considered and some aspects elucidated during my PhD. Therefore, studies along two different research lines, having the same final aim and merging in the last part of the project, have been carried out. The first research line focused on PDT, in particular I investigated how several experimental factors affect the phototoxic activity against bacteria of cationic porphyrins, and I identified some of their molecular targets in Staphylococcus aureus. The second research line focused on the mechanism of action of a CAMP, apidaecin 1b, that was then conjugated to a photoactive porphyrin to obtain a new and broad-spectrum antimicrobial agent.
PDT utilizes a visible light absorbing molecule, called photosensitizer (PS), that in the presence of oxygen generates cytotoxic reactive species that kill bacterial cells previously loaded with the PS.
I studied the effects of some experimental factors that could affect the binding of the PS to the bacterial cell, and, as a consequence, the efficiency of bacteria photoinactivation. In particular, the effects of the cell washing after incubation with the PS, and of the presence of cations in the incubation medium, were considered. For these studies a dicationic porphyrin was used, and its ability to photoinactivate S. aureus and E. coli was measured after irradiation of the bacteria with increasing doses of blue light. Illumination was carried out with the unbound PS left in the suspension, as well as after one or four washings of the cells. The washings produced a completely different effect in the two microorganisms: slightly decreased the photokilling of S. aureus and strongly increased the photokilling of E. coli. The increased photokilling of E. coli was explained with a re-localisation, occurring during the time needed for the washing procedure, of the porphyrin molecules in inner cellular sites critical for survival. This is very likely to occur, considering the short (5 min.) incubation time of the cells with the PS. The presence of a monovalent cation (Na+) in the cell medium during incubation and illumination did not affect negatively the photoinactivation of S. aureus, that was slightly increased by the presence of Ca2+ and Mg2+. On the contrary, the phoinactivation of E. coli was slightly decreased in the presence of both mono- and divalent cations, and the effect was more evident in cells illuminated without washings. In all cases, the bacteria photoinactivation with this dicationic porphyrin was little affected by the presence of cations, in comparison to other cationic porphyrins.
A classical proteomic approach, that included two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis, was used to identify some proteins that are potential primary molecular targets of PDT. Towards this aim, the change of the proteomic profile of S. aureus after PDT treatments was investigated using two different cationic porphyrins. For each of these two porphyrins, that strongly differ in their photoinactivation activity, I have selected two PDT treatments on the base of the different survival of treated bacteria: a sublethal one, which allows a bacterial survival of 60 to 80%, and a stronger one, which allows only a bacterial survival of about 1%. 2-DE maps from PDT-treated and untreated samples were compared using the Proteomweaver software for 2-DE maps analysis. All proteins showing a significant difference in their intensity levels between untreated and PDT treated 2-DE maps were identified by mass spectrometry (MALDI-TOF/TOF). All identified proteins were then divided into functional classes on the basis of their cellular function, in order to identify the pathways that were more damaged by PDT. Among the 265 proteins identified by the Proteomweaver analysis, 70 changed their intensity levels after PDT treatments; most of these are involved in the response to oxidative stress, in the energy metabolism and in the uptake of sugar. The comparison of the effects of PDT treatments between the two porphyrins revealed that several proteins were modified by both of them, and generally showing the same trend. Interestingly, the modifications of the proteomic patterns induced by PDT treatments are not consistent with a response of S. aureus cells to oxidative stress induced by other oxidizing agents (hydrogen peroxide, superoxide or diamide), therefore suggesting a selective targeting of specific proteins. These findings provide new insight in the understanding of the mechanism of action of PDT, and may be useful to design new photosensitizing agents with improved antibacterial activity.
In the second research line I studied the antibacterial activity, and the ability to translocate into the bacterial cell, of the insect antimicrobial peptide apidaecin 1b. The improved understanding of the translocation mechanism of apidaecin was then used to investigate the possibility to use apidaecin itself as a cargo delivering molecule, by conjugating it with a porphyrin, in an attempt to obtain a new broad-spectrum antimicrobial agent.
Apidaecin 1b is a small (18 residues) peptide that is extensively studied because of its unique properties, which include the ability to inhibit some Gram (-) bacteria growth and a high capability to translocate into cells with a non pore-forming mechanism. Moreover, no toxicity against eukaryotic cells has been reported even at concentrations that totally inhibit bacterial growth. In order to study its mechanism of action, each of the three arginine residues (or all of them together) were replaced with the corresponding peptoid residue, N-(3-guanidinopropyl) glycine (NArg), and the minimal inhibitory concentrations (MICs) of all of these peptide-peptoid hybrids were evaluated against E. coli, P. aeruginosa and S. aureus. No antimicrobial activity was detected for apidaecin and its peptide-peptoid hybrids against S. aureus and P. aeruginosa. On the contrary, against E. coli apidaecin showed a MIC value of 8-16 μM, while its peptide-peptoid hybrids substituted in arginine 4 and 12 showed a little decrease of antimicrobial activity and those substituted in arginine 17 or in all three arginines completely lost their activity. These results suggested that the arginine in position 17 is particularly important in the translocation mechanism into the bacterial cell. Therefore, apidaecin and its peptide-peptoid hybrids were labelled with fluorescein, in order to monitor by means of fluorescence microscopy and flow cytometry their ability to tightly bind to bacterial cells. The fluorescein-labelling completely abolished the antimicrobial activity of all our compounds and inhibited to a great extent their binding and translocation into bacterial cells. This effect was mainly due to the additional mass brought by fluorescein to apidaecin and its hybrids. Anyway, it was observed that the peptide-peptoid hybrid substituted in arginine 17 did not show any binding to bacterial cells. This finding confirmed the importance of this residue for antimicrobial activity, and, in general, the importance of the C-terminal of apidaecin in the binding and translocation into the bacterial cells.
The conjugate (called T-api), resulting from the coupling of an anionic porphyrin (monocarboxy-tetraphenylporfine, cTPP) with the N-terminal of apidaecin 1b, did not show any antimicrobial activity in the dark. On the contrary, following activation with blue light, T-api caused a significant reduction of bacteria survival with an efficacy strongly dependent on the bacteria species. The phototreatment was extremely successful against E. coli and S. aureus, while the photokilling efficiency against P. aeruginosa was much lower but sufficient to induce a significant reduction of survival. Irradiation experiments were also performed by treating bacteria with cTPP alone, apidaecin 1b alone or the two agents together but not conjugated. None of these treatments affected the survival of both E. coli and P. aeruginosa, while S. aureus survival was decreased by cTPP, but to a lesser extent in comparison to T-api.
Thus, it was demonstrated that the conjugation of an antimicrobial peptide with a photoactive molecule can lead to the formation of antimicrobial agents very effective and displaying a broader activity in comparison to the single components.cIl forte aumento, a livello mondiale, del fenomeno della resistenza agli antibiotici richiede lo sviluppo di nuove strategie antimicrobiche, basate su meccanismi d’azione diversi da quelli degli antibiotici, per combattere le infezioni batteriche. Due delle strategie più promettenti in quest’ottica sono la terapia fotodinamica (PDT) e l’utilizzo di peptidi cationici antimicrobici (CAMPs). Durante il mio dottorato di ricerca ho studiato alcuni aspetti peculiari di entrambe queste strategie, in due linee di studio che alla fine sono state riunite. Nella prima linea di ricerca, focalizzata sulla PDT con porfirine cationiche, ho studiato quale impatto abbiano alcune condizioni sperimentali sull’efficienza di fotosensibilizzazione di batteri, ed ho identificato alcuni dei bersagli molecolari della loro azione su Staphylococcus aureus. Nella seconda linea di ricerca ho studiato il meccanismo d’azione di un particolare CAMP, l’apidaecina 1b, che ho successivamente coniugato con un fotosensibilizzatore allo scopo di creare un nuovo, più efficiente, agente antimicrobico.
La PDT utilizza molecole capaci di assorbire la luce visibile, dette fotosensibilizzatori (PS), che, quando illuminate in presenza di ossigeno molecolare, generano specie reattive dell’ossigeno, che hanno un forte effetto citotossico su cellule batteriche precedentemente incubate col PS stesso.
Nella prima fase di studio ho valutato l’effetto di alcune condizioni sperimentali sulla capacità del PS di legarsi alla cellula batterica e, di conseguenza, sull’efficienza di fotoinattivazione di batteri. In particolare sono stati valutati gli effetti di lavaggi effettuati dopo l’incubazione dei batteri con il PS, nonché della presenza di diversi cationi nel mezzo di incubazione. In questo studio è stata utilizzata una porfirina dicationica, la cui efficienza nella fotoinattivazione di Staphylococcus aureus ed Escherichia coli è stata misurata mediante irradiamento con dose crescenti di luce blu. L’irradiamento è stato effettuato sia lasciando nella sospensione batterica il PS non legato alle cellule che rimuovendolo mediante uno o quattro lavaggi. Questi lavaggi hanno prodotto effetti completamente opposti nei due microrganismi oggetto di studio: da un lato si è riscontrato un forte aumento dell’efficienza di fotosensibilizzazione di E. coli, dall’altro un decremento di quella di S. aureus. L’aumento dell’efficienza di fotosensibilizzazione in E. coli è probabilmente dovuto al fatto che, nel tempo necessario per effettuare i lavaggi, la frazione di porfirina legata alle cellule batteriche riesce a raggiungere siti cellulari più sensibili alla PDT. Il fatto che si sia utilizzato un tempo di incubazione molto breve (5 minuti) rende molto plausibile questa ipotesi. L’aggiunta di un catione monovalente (Na+) nel mezzo di irradiamento non ha causato alcuna variazione dell’efficienza di fotosensibilizzazione di S. aureus, che invece è stata fortemente incrementata da quella di cationi bivalenti (Ca2+ e Mg2+). Al contrario, la fotosensibilizzazione di E. coli è stata sensibilmente diminuita in presenza di cationi (sia mono che bivalenti), con un effetto più marcato in assenza di lavaggi. In ogni caso, utilizzando questa porfirina dicationica gli effetti prodotti sia dai lavaggi che dalla presenza di cationi sono stati minori di quelli riscontrati in precedenza con altri fotosensibilizzatori.
Per l’identificazione di alcune delle proteine che sono bersaglio della PDT è stato scelto un approccio di tipo proteomico, comprendente la separazione con elettroforesi bidimensionale dei lisati batterici e l’identificazione di proteine con tecniche di spettrometria di massa. Al fine di ottenere un’analisi il più possibile dettagliata, sono stati valutati i cambiamenti nel profilo proteomico di S. aureus causati dalla PDT con due diverse porfirine cationiche. Per ciascuna di queste porfirine, che differiscono notevolmente nell’attività fotosensibilizzante, sono stati selezionati, sulla base della differente mortalità indotta in sospensioni di S. aureus, due trattamenti fotodinamici: uno subletale, che consente una sopravvivenza dal 60 all’80% dei batteri, e l’altro più forte, che consente la sopravvivenza di circa l’1% dei batteri. Le mappe bidimensionali ottenute da lisati proteici di batteri non sottoposti a PDT sono quindi state confrontate, mediante l’utilizzo dell’apposito software Proteomweaver, con quelle ottenute da lisati di batteri sottoposti ai diversi trattamenti fotodinamici. Tutte le proteine delle mappe bidimensionali che, a seguito dell’analisi, hanno mostrato di essere state significativamente modificate dai trattamenti fotodinamici, sono quindi state identificate tramite spettrometria di massa (MALDI-TOF/TOF). Sulla base delle loro funzioni nella cellula, le proteine identificate sono quindi state assegnate a diverse classi funzionali, al fine di scoprire quali funzioni cellulari venissero maggiormente colpite dalla PDT. Tra le 265 proteine globalmente identificate dall’analisi con Proteomweaver, 70 hanno mostrato significative variazioni di intensità dovute ai trattamenti fotodinamici; tra queste, la maggioranza era composta da proteine implicate nella risposta allo stress ossidativo, nel metabolismo energetico e nella captazione di zuccheri. Comparando gli effetti della PDT tra le due porfirine, si è scoperto che i livelli di intensità di molte proteine sono stati modificati da entrambe, ed in genere nella stessa direzione. Particolarmente interessante è stata la scoperta che le tipologie di modifica del profilo proteomico di S. aureus, causate dai trattamenti fotodinamici, non sono compatibili con le risposte ad agenti ossidanti (come per esempio perossidi o superossidi) da parte della cellula batterica; questo suggerisce che la PDT ha come bersagli specifiche proteine. I risultati ottenuti sono di particolare importanza perché, approfondendo la conoscenza del meccanismo d’azione della PDT, potrebbero aiutare nel disegno di nuovi fotosensibilizzatori più efficienti di quelli attualmente in uso.
Nella seconda linea di ricerca ho studiato alcune proprietà legate all’attività antimicrobica ed alla capacità di ingresso nella cellula batterica di un peptide cationico antimicrobico, l’apidaecina 1b. I risultati ottenuti da questo studio hanno quindi permesso di utilizzare l’apidaecina stessa come vettore di altre molecole, coniugandola con una porfirina al fine di ottenere un nuovo agente antimicrobico, con un maggior spettro d’azione rispetto ai suoi singoli costituenti.
L’apidecina 1b è un piccolo (soli 18 amminoacidi) peptide che viene molto studiato per via di alcune sue particolari capacità, tra cui una buona inibizione della crescita di batteri Gram (-) e, soprattutto, un’eccezionale abilità nell’entrare nelle cellule mediante un meccanismo che non comporta la formazione di pori nelle membrane. Inoltre, è stato dimostrato che l’apidaecina non presenta tossicità per cellule eucariotiche a concentrazioni che sono invece letali per i batteri. Allo scopo di studiare il meccanismo d’azione dell’apidaecina sono stati sintetizzati degli ibridi peptide-peptoide dell’apidaecina stessa, nei quali ognuno dei tre residui di arginina è stato sostituito con residui di N-(3-guanidinopropyl)-glicina, e si sono quindi valutate le MIC (minima concentrazione inibente) di ognuno degli analoghi nei confronti di diversi batteri. Né l’apidaecina né i suoi ibridi peptide-peptoide hanno mostrato attività antimicrobica nei confronti di Staphylococcus aureus e Pseudomonas aeruginosa. Invece, in Escherichia coli si è potuta osservare una lieve diminuzione del valore della MIC, rispetto al peptide naturale, con gli ibridi sostituiti nelle posizioni 4 e 12, mentre l’attività antimicrobica veniva completamente persa nell’ibrido sostituito nella posizione 17.
Questi risultati suggeriscono che l’arginina in posizione 17 possa giocare un ruolo particolarmente importante nel meccanismo di traslocazione dell’apidaecina all’interno della cellula. Quindi, sia l’apidaecina che i suoi ibridi peptide-peptoide sono stati marcati mediante legame con una molecola di fluoresceina, allo scopo di monitorarne la capacità di legame con la cellula batterica mediante tecniche di microscopia di fluorescenza e citometria di flusso. Sfortunatamente, la marcatura con la fluoresceina ha causato la perdita dell’attività antimicrobica e di gran parte della capacità di legarsi ed entrare nella cellula batterica sia dell’apidaecina che dei suoi ibridi. Questo effetto è dovuto principalmente alla massa aggiuntiva portata dalla fluoresceina ai peptidi. Tuttavia, si è osservato che, a differenza degli altri, l’ibrido peptide-peptoide con la sostituzione dell’arginina 17 non presentava alcuna capacità di legarsi alle cellule batteriche. Questo risultato ha confermato sia l’importanza di questa arginina per la capacità antimicrobica dell’apidaecina che, in generale, l’importanza del dominio C-terminale sulla capacità di legame e di ingresso nella cellula batterica.
Il coniugato (che è stato chiamato T-api), ottenuto dall’unione di una porfirina anionica (monocarbossi-tetrafenil porfirina, cTPP) con il dominio N-terminale dell’apidaecina 1b, non ha mostrato alcuna attività antimicrobica al buio. Tuttavia, in seguito all’irradiamento con luce blu, T-api si è dimostrato un efficiente fotosensibilizzatore, con un’efficienza fortemente dipendente dalle diverse tipologie di batteri considerate. In particolare, il trattamento fotodinamico con T-api è stato estremamente efficace con E. coli e S. aureus, e leggermente meno efficace, ma comunque sufficiente per indurre un’apprezzabile mortalità, in P. aeruginosa. Esperimenti analoghi, eseguiti trattando i batteri con la porfirina oppure l’apidaecina da sole, oppure con le due insieme ma non coniugate, non hanno causato alcuna mortalità in E. coli e P. aeruginosa. In S. aureus, invece, si è riscontrata una marcata mortalità in seguito al trattamento con cTPP e luce, ma comunque minore di quella ottenuta con T-api.
Quindi, ho dimostrato che la coniugazione di un peptide antimicrobico con una molecola fotosensibilizzante può portare alla sintesi di agenti antimicrobici estremamente efficaci, e con uno spettro d’azione superiore rispetto a quelli dei suoi singoli componenti
Antimicrobial peptide-porphyrin conjugates: new tools in photodynamic therapy
No full textAntimicrobial PhotoDynamic Therapy (APDT) represents a very promising strategy, particularly for the treatment of localized infectious diseases.[1] PDT involves the use of a non-toxic photosensitizer (PS), f. i. porphyrins, phthalocyanines, in combination with harmless visible light of appropriate wavelength to excite the PS. In the presence of the oxygen, the excited PS transfer energy or electrons to the ground state molecular oxygen, producing reactive oxygen species (ROS), e. g. singlet oxygen and hydroxyl radical, that affecting the integrity and function of different cellular components, e.g. proteins, nucleic acids and lipids, cause cell death. Advantages of APDT over traditional antibiotics include a broad spectrum activity, also against antibiotic-resistant species and the lack of development of resistance mechanisms due to the multi-target process. Gram-positive bacteria can be efficiently killed by light after their incubation with a number of PS. On the contrary Gram-negative bacteria are less susceptible to photodynamic killing and only cationic PS can bind efficiently to this type of bacteria and induce their photoinactivation. An alternative approach to improve the susceptibility of Gram-negative bacteria against neutral PS, involves the covalent attachment of the PS to a polymer molecule containing basic amino groups. Recently we have shown that the conjugation of a neutral porphyrin to the cationic antimicrobial peptide apidaecin 1b (T-api), belonging to the Pro-Arg rich peptide family, afforded a new antibacterial agent with a broader spectrum activity than two individual components or a mixture of them.[2] In the present study we extend our investigation to two new PS-apidaecin conjugates in which either a porphycene (G), a structural porphyrin isomer, or a cationic porphyrin (Y) are covalently linked to the peptide N-terminal end. The bactericidal activity of two new conjugates, in the dark or under light activation, was compared to that of the photosentizers alone and of the coniugate with the neutral porphyrin. PS-apidaecin conjugates did not exhibit any toxicity against both Gram negative (E.coli) and Gram positive (S.aureus) bacteria in the dark. On the contrary, following activation with red light (36 J/cm2), the porphycene conjugate (G-api) caused a significant (3-4 log10) reduction of E. coli survival, comparable to that of T-api, but it was less effective than the porphyrin counterpart against S. aureus. The cationic porphyrin and its apidaecin conjugate (Y-api), following activation with blue light (13 J/cm2), caused complete killing of both Gram-negative and –positive bacteria at lower concentrations than G-api and T-api
Porphyrin−Apidaecin Conjugate as a New Broad Spectrum Antibacterial Agent
No full textThe conjugation of the cationic antimicrobial peptide, apidaecin Ib, to the anionic photosensitizer, 5(4′-carboxyphenyl)-10,15,20-triphenylporphyrin (cTPP), afforded a new antibacterial agent effective, under light activation, against both Gram-positive and Gram-negative bacteria. At low concentrations (1.5−15 μM) the conjugate was able to reduce the survival of Escherichia coli cells by 3−4 log10, and most notably, it resulted photoactive also against hard-to-treat Pseudomonas aeruginosa, although at higher concentration (60 μM). Under similar conditions, the photosensitizer alone was only photoactive against Staphylococcus aureus while the unconjugated peptide was inactive against all the bacterial strains tested. This study shows the possibility of obtaining new broad-spectrum apidaecin−photosensitizer conjugates with potent antibacterial activity
PORPHYRIN-TYPE PHOTOSENSITISERS FOR KILLING BACTERIA AND CANCER CELLS WITH PHOTODYNAMIC THERAPY
No full textThe photosensitising properties displayed by many porphyrin-type molecules have been widely exploited for therapeutic purposes in the so called photodynamic therapy (PDT). In PDT, cells and tissues are first loaded with a photosensitizing drug which is then activated by illumination with suitable wavelengths of visible light to generate reactive oxygen species, mainly singlet oxygen, that kill cells through oxidative damages. Both prokaryotic and eukaryotic cells can be killed by PDT by selecting photosensitizers with physico-chemical properties favoring interaction/internalization with the particular cell type. We have been studying porphyrins and some derivatives to explore their potential usefulness for photodynamic therapy of tumours as well as for antimicrobial photodynamic therapy. In the last years we are exploring strategies that are able to improve efficiency and selectivity of PDT in killing cancer cells as well as bacteria. In the attempt of increasing the efficiency in killing bacteria, porphyrins have been conjugated to cationic antimicrobial peptides that are known to exhibit selectivity against bacterial cells. When compared to the corresponding free porphyrins, the conjugates showed very different outcomes, in terms of efficiency of bacteria photokilling, that were determined by the type of bacteria (Gram-positive or Gram-negative), the hydro/lipo-philicity and/or electric charge of the porphyrin that affect the strength of interaction of the conjugates with the bacterial cells. Our results showed that an anionic and hydrophobic porphyrin, devoid of photoactivity against the Gram-negative E. coli, acquired the ability to efficiently kill this bacterium when conjugated to the peptides. The effect of conjugation to cationic antimicrobial peptides of a tri-cationic porphyrin was less obvious very likely because, as other positively charged PS, this porphyrin exhibited by itself some photoactivity against both Gram-positive and –negative bacteria, which is preserved or even reduced by the conjugates [1]. In spite of the selectivity displayed by antimicrobial cationic peptides against bacterial cells, the porphyrin-peptide conjugates resulted very toxic by photoactivation also against eukaryotic cells. We found that normal human fibroblasts were completely killed by illumination with blue light after incubation with 5 and 3 μM of the free anionic or cation porphyrin respectively. The same effect was found with the porpyrin-peptides conjugates at concentrations 2-3 orders of magnitude lower than the free porphyrins. Therefore it appears that cationic antimicrobial peptides may represent useful carriers for improving internalization of drugs in mammalian cells, as already found for cell penetrating peptides. Based on this we are exploring the potential of the peptides to improve the delivery of PSs also to cancer cells.
[1] Dosselli R., Tampieri C., Ruiz-Gonzalez R., De Munari S., Ragàs X., Sánchez-García D., Agut M., Nonell S., Reddi E., Gobbo M. J. .Med. Chem. 2013, 56, 1052
Antimicrobial photodynamic therapy: photophysical and phototoxic activities of conjugates of apidaecin with different photosensitizers
No full textAntimicrobial PhotoDynamic Therapy (APDT) represents a very promising strategy, particularly for the treatment of localized infectious diseases.[1] PDT involves the use of a non-toxic photosensitizer (PS) that forms reactive oxygen species (ROS) when irradiated with visible light of appropriate wavelength. ROS (e. g. 1O2) can cause irreversible damages to a variety of cellular components, resulting in cytotoxicity. Advantages of APDT over traditional antibiotics include a broad spectrum activity, also against antibiotic-resistant species and the lack of development of resistance mechanisms due to the multi-target process. Whereas Gram-positive bacteria can be efficiently killed by light after incubation with a number of PS, Gram-negative bacteria are less susceptible to photodynamic killing. Only cationic PS can bind efficiently to Gram-negative bacteria and induce their photoinactivation. Recently we have shown that the conjugation of a cationic antimicrobial peptide (apidaecin 1b ) to a neutral porphyrin afforded a new antibacterial agent with a broader spectrum activity than two individual components or a mixture of them.[2] Here we present the results obtained with two new PS-apidaecin conjugates in which either a porphycene (G), a structural porphyrin isomer, or a cationic porphyrin (Y) are covalently linked to the peptide N-terminal end. By a combination of spectroscopic and time resolved photophysical techniques, we have monitored the ability of conjugates to produce the main cytotoxic species (1O2) in different solvents and in E. coli (the prototypical Gram-negative bacterium), obtaining information on the possible localization of the PS in the cell. The photokilling activity of the conjugates was markedly concentration-dependent, and the most potent one, Y-apidaecin, caused complete killing of bacteria at 10 μM (E. coli) and 1.5 μM (S. aureus) concentrations.
References
1. Hamblin, M. R.; Hasan, T. Photochem. Photobiol. Sci. 2004, 3, 436-450
2. Dosselli, R.; Gobbo, M.; Bolognini, E.; Campestrini, S.; Reddi E. ACS Med. Chem. Lett. 2010, 35-38
Sperm storage by males causes changes in sperm phenotype and influences the reproductive fitness of males and their sons
Full text linkRecent studies suggest that environmentally induced effects on sperm phenotype can influence offspring phenotype beyond the classic Mendelian inheritance mechanism. However, establishing whether such effects are conveyed purely through ejaculates, independently of maternal environmental effects, remains a significant challenge. Here, we assess whether environmentally induced effects on sperm phenotype affects male reproductive success and offspring fitness. We experimentally manipulated the duration of sperm storage by males, and thus sperm age, in the internally fertilizing fish Poecilia reticulata. We first confirm that sperm ageing influences sperm quality and consequently males reproductive success. Specifically, we show that aged sperm exhibit impaired velocity and are competitively inferior to fresh sperm when ejaculates compete to fertilize eggs. We then used homospermic (noncompetitive) artificial insemination to inseminate females with old or fresh sperm and found that male offspring arising from fertilizations by experimentally aged sperm suffered consistently impaired sperm quality when just sexually mature (four months old) and subsequently as adults (13 months old). Although we have yet to determine whether these effects have a genetic or epigenetic basis, our analyses provide evidence that environmentally induced variation in sperm phenotype constitutes an important source of variation in male reproductive fitness that has far reaching implications for offspring fitness
Pattern of inbreeding depression, condition dependence, and additive genetic variance in Trinidadian guppy ejaculate traits
No full textIn polyandrous species, a male's reproductive success depends on his fertilization capability and traits enhancing competitive fertilization success will be under strong, directional selection. This leads to the prediction that these traits should show stronger condition dependence and larger genetic variance than other traits subject to weaker or stabilizing selection. While empirical evidence of condition dependence in postcopulatory traits is increasing, the comparison between sexually selected and ‘control’ traits is often based on untested assumption concerning the different strength of selection acting on these traits. Furthermore, information on selection in the past is essential, as both condition dependence and genetic variance of a trait are likely to be influenced by the pattern of selection acting historically on it. Using the guppy (Poecilia reticulata), a livebearing fish with high levels of multiple paternity, we performed three independent experiments on three ejaculate quality traits, sperm number, velocity, and size, which have been previously shown to be subject to strong, intermediate, and weak directional postcopulatory selection, respectively. First, we conducted an inbreeding experiment to determine the pattern of selection in the past. Second, we used a diet restriction experiment to estimate their level of condition dependence. Third, we used a half-sib/full-sib mating design to estimate the coefficients of additive genetic variance (CVA) underlying these traits. Additionally, using a simulated predator evasion test, we showed that both inbreeding and diet restriction significantly reduced condition. According to predictions, sperm number showed higher inbreeding depression, stronger condition dependence, and larger CVA than sperm velocity and sperm size. The lack of significant genetic correlation between sperm number and velocity suggests that the former may respond to selection independently one from other ejaculate quality traits. Finally, the association between sperm number and condition suggests that this trait may mediate the genetic benefits of polyandry which have been shown in this species
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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