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Here, we have tested the N-terminal derived peptides that correspond to the eight human canonical RNases RN against planktonic cells and biofilms of C. RN3 and RN7 peptides displayed the most potent inhibitory effect with a mechanism of action characterized by cell-wall binding, membrane permeabilization and biofilm eradication activities. Both peptides are able to eradicate planktonic and sessile cells, and to alter their gene expression, reinforcing its role as a lead candidate to develop novel antifungal and antibiofilm therapies.

Candida albicans is the most common fungal pathogen that threatens hospitalized and immunocompromised patients. Together with candidiasis in skin and mucosal infections, fungal septic shock cases are also reported in elderly or immunosuppressed patients [ 1 ]. Most of the serious symptoms of candidiasis are associated with biofilm formation occurring on the surfaces of host tissues and medical devices, enhancing C.

Moreover, the most representative feature of Candida biofilms is its increasing tolerance to conventional antifungal therapy. The protection of the biofilm embedded cells against noxious agents, such as antifungals and the host immune system arsenal, favors the thriving of persister cells and hinders the treatment of biofilm-associated infections [ 2 , 3 ].

It has been estimated that the eradication of biofilm communities requires concentrations of antifungals times higher compared to their planktonic stage [ 4 , 5 ]. This fact drastically reduces the availability of antifungal drugs appropriate to tackle C. Frequently, drug doses need to be increased over the safety level [ 2 ]. Moreover, we are currently facing an increase in yeast pathogenesis along with the emergence of drug-resistant strains, urging the need for innovation of new effective antifungal agents.

Antimicrobial proteins and peptides AMPs are considered one of the main ancestral host defense systems. Largely distributed throughout the different organism tissues, AMPs play an essential role as part of the human innate immune system, constantly protecting the body against microbial invasion and diseases [ 6 , 7 , 8 ].

Because of their wide distribution, physicochemical features, biological activities and rapid antimicrobial action against a broad spectrum of microbes, AMPs have recently attracted significant attention as encouraging antibiotic candidates [ 9 , 10 , 11 , 12 , 13 ].

Furthermore, due to their direct action at the microbial membrane and multifaceted intracellular killing mechanisms, the use of AMPs as antibiotics should reduce the appearance of resistant pathogens, a scenario that has been favored by the abuse of conventional antibiotics [ 14 , 15 ]. The emergence of resistance mechanisms associated with fungal infections has also encouraged the characterization of natural host defense peptides as lead candidates for the design of novel drugs.

In particular, several synthetic AMPs were successfully tested against C. Among the human peptides that participate in the host innate immunity against fungal infections, we find cathelicidins, histatins and defensins, that combine direct action to the pathogen cell together with indirect immune-modulation activities [ 17 ]. In our research group, we have explored the use of human secretory RNases as antimicrobial agents against Gram-negative and Gram-positive bacteria [ 18 ].

Following structure-functional studies, an RNase derived peptide was proposed as a novel antibiofilm agents against Gram-negative bacteria [ 19 , 20 ]. Likewise, in the search for novel antifungal agents, we recently reported that human antimicrobial RNases are also effective against C. Interestingly, the fungicidal activity of the RNases is initiated by the binding to the pathogen cell wall, followed by their translocation and intracellular targeting [ 21 ].

In previous attempts to define the antimicrobial activity of the hRNases, we designed the derived peptides corresponding to the N-terminal of all them. The results demonstrated that the main antimicrobial activities are retained at the first 45 residues of the parental ribonucleases. A comparison between each N-terminal derived peptide and the related parental protein confirmed that their abilities to disrupt model membranes and inherent antimicrobial activities were retained [ 23 , 24 , 25 ].

In this work, we have explored the mechanism of action of the 1—45 N-terminus peptides of human canonical RNases hRNases against Candida planktonic cells and the action toward established and early-biofilms. The promising results place hRNase-derived peptides in the spotlight as therapeutic lead compounds and reinforce the value of our own innate immune response arsenal in the fight against fungal infections.

The hRNase-derived peptides were designed, taking as reference the 1—45 segment in hRNase3 sequence, based on previous work in our laboratory that defined the protein minimal domain retaining full antimicrobial properties against Gram-positive and —negative bacteria. The N-terminal residues of the hRNases represent a conserved evolutionary region that retains the protein host-defense properties [ 23 , 24 , 25 ].

Figure 1 and Table 1 summarizes the main physiochemical, structural and biological properties of the selected peptides. The eight N-terminal peptides RN1—8 , comprising equivalent structural regions of the human canonical RNases N-termini [residues 1—45 of hRNases 2, 6, 7 and 8; residues 1—48 of hRNases 1 and 4; and residues 1—47 of hRNase 5;] were selected for synthesis to test their antimicrobial capabilities against C.

As previously, the two cysteine residues present at the N-terminal region were replaced by isosteric serine residues [ 23 , 25 ]. Previous structural analysis and predictive studies indicated that the peptides adopted an helix in a membrane-like environment [ 23 ] Figure 1 and Figure S1.

The substitution of Arg versus Lys in peptide structures sought to increase the cell penetration capacity, as reported previously [ 26 , 27 ]. It is noteworthy to underline the high pI and degree of hydrophobicity of the peptides Table 1 , adopting an amphipathic structure Figure S1 and showing values within the standard range of most-known AMPs [ 28 ], which are considered essential for their functional activity [ 29 ].

Table 1 also includes the relative estimated values for the antimicrobial activity of the hRNase parental proteins, as referenced in our previous work [ 30 ]. A Sequence alignment of hRNases. Conserved regions are boxed; highly conserved amino acids are colored in white over a red background, whereas moderately conserved amino acids regions are colored in red. The secondary structure of hRNase3 is displayed as a reference on top of the alignment.

The N-terminal antimicrobial domain is highlighted in blue and the agglutination-promoting region in orange. Previous work in our laboratory using C. Here, we report the antifungal activity of the eight N-terminal peptides corresponding to the eight human canonical RNases. MFC values are summarized in Table 2. Five peptides 1, 3, 4, 6 and 7 were found active against C. On the other hand, peptides 2, 5 and 8 were inactive. A comparison of the bactericidal and candidacidal activities of hRNases Table 1 and Table 2 highlighted a positive correlation, confirming that the antimicrobial activity is preserved at the N—terminus.

Minimal fungicidal concentration MFC was determined as the lowest concentration of peptide that killed at least Likewise, previous characterization of the peptide hemolytic activities [ 23 ] also confirmed that no significant toxicity is observed at more than 10x the achieved fungicidal concentration. The peptides RN3K and RN7R were also tested in their antifungal activities, but the concentrations to reach an antimicrobial activity were higher in relation to their wild type peptide Supplementary Table S1.

Another specific feature of the mechanism of action of some AMPs is the ability to agglutinate bacteria, a property proposed to facilitate the removal of the infection focus. Considering that hRNase3 and its N-terminal domain [ 31 ] displayed a significant bacterial agglutinating activity, we also assayed the RNases 3 and 7 action on Candida cell cultures. Accordingly, we confirmed that hRNase3 is able to agglutinate C.

Following up, in order to check whether hRNase-derived peptides also share this property against C. In any case, after incubation of C. In addition, when comparing MAC results between C. Interestingly, a comparison of the MAC values indicate a higher agglutinating activity for C.

In previous works, we assayed hRNase3 interaction to different bacterial cell wall components such as lipopolysaccharide LPS and peptidoglycans [ 32 , 33 ], as well as heterosaccharides present at the eukaryote extracellular matrix [ 34 , 35 , 36 ]. As reported in our previous work on antimicrobial hRNases, one of the first steps to counteract the bacterial growth relies on the protein cell wall binding and membrane destabilization activity [ 31 , 33 ].

In an effort to characterize the antifungal mechanism of hRNase-derived peptides RN , we assessed their capacity to depolarize and permeabilize C. Results showed that overall most of the peptides active against C. The data agrees with previous depolarization and leakage values in both Gram-negative and Gram-positive bacteria species. For a better characterization of the mechanism, we compared the kinetics of the membrane destabilization and killing process by hRNase-derived peptides Table 3.

RN3 showed the best values in terms of cell survival, depolarization and permeabilization, followed by RN7 and RN6. Previously, RNase derived N-terminal peptides were reported to have the ability to bind anionic liposomes and trigger the vesicles lysis [ 23 ]. Moreover, as it has been reported for other AMPs [ 37 ], a positive correlation had been observed regarding the lytic activity of the parental RNases in both synthetic lipid vesicles and bacterial cells [ 31 , 32 , 38 ].

The present study on C. ED 50 depolarization values were calculated with peptide concentrations from 0. All values are averaged from three replicates of two independent experiments. On the other hand, cationic peptides can display a high binding affinity toward anionic polymeric structures, such as the bacterial and fungal cell envelopes. Two main layers can be distinguished in the C. These polysaccharides confer strength and favor the cell shape. Here, we selected hRNase3 and its N-terminal derived peptide as the most antimicrobial active member of the family, to assess the binding ability to both C.

Binding of hRNase3 peptide to C. The supernatant soluble fractions were prepared as described in the Experimental Procedures. We also based our selection in the physicochemical properties of these peptides that could facilitate the nucleic acid binding ability. Our previous work has demonstrated the effective internalization of hRNases3 and 7 within C.

The in-vitro DNA binding ability was determined by monitoring the electrophoretic mobility of plasmidic DNA on an agarose gel. Gel retardation assay. In order to get further insight into the mechanism of action of RN3 and RN7 derived peptides against C. We measured the viability and the biofilm mass following peptide treatment by resazurin reduction and crystal violet staining.

Additionally, we evaluated by confocal microscopy the biofilm viability and overall structure. Biofilms formation is often associated with antifungal resistance, as compared to planktonic cells, and requires drug concentrations of about 30— times to achieve equivalent reduction of the cell metabolic activity [ 42 ]. Here, we assayed the peptide concentrations corresponding to 5, 10 and 20 x MFC fold. For amphotericin B and colistin we took as an internal reference MFC values of 0.

The antibiofilm activity of the hRNase-derived peptides was assessed using PrestoBlue TM reduction assay, which enables quantitation of the number of living cells in 24h-old biofilms after treatment. The antibiofilm activity of all peptides was compared at an equivalent MFC fold concentration. B The biofilm was stained by crystal violet, then the dye was solubilized in ethanol, its absorbance measured at nm and the percentage of biofilm reduction was compared to untreated wells control.

All experiments were done in triplicate. Interestingly, RN3 peptide showed a similar activity to the one displayed by colistin. However, the RN3 peptide did not achieve the antibiofilm activity values registered for amphotericin B, which is currently considered one of the most effective antifungal agents [ 44 ]. Unfortunately, the main problem encountered in the clinical use of amphotericin B is its high cytotoxicity and limited pharmacological application, due to its adverse side-effects, such as fever, vomiting, anemia and nephrotoxicity [ 46 ].

Amphotericin B was used as a control of inhibition. Both peptides drastically reduced the C. Interestingly, the samples treated with the peptides, and RN3 in particular, showed a morphological change from yeast to hyphae stage that was not observed in the control biofilm Figure 5.

The untreated biofilm control was composed of non-branched pseudo and yeast forms. We can hypothesize that as a mechanism of defense against the anticandidal treatment, the sessile cells within the biofilm try to evade the antimitotic action by hyphae formation, limiting thereby the drug penetration into the biofilm. A Visualization by confocal microscopy of mature C.

Right-up detailed zone in control and RN3 images correspond to the overlapping of SYTO9 and PI, showing an increment of hypha formation and reduction of hyphal-viability. Following up, we investigated whether the peptide treatment could alter the gene expression profile of Candida cells. The first approach to determinate the changes in the gene expression was quantified in Candida planktonic cells using a peptide concentration of 0. High expression of 18s might respond to a cellular stress caused by the peptides addition.

Gene expression profile in planktonic cells of C. The mean values are averaged from two independent experiments performed in duplicates. Lastly, we evaluated the effect of the RN3 and RN7 peptides on the expression of some genes related to the biofilm formation. The potential molecular mechanism behind the ability of these RNase derived peptides to prevent the growth of C. Following the incubation of C. Our results demonstrated suppression in the expression level of the hyphae-specific gene that encodes for surface adhesins ALS3 and the CYR1 and EFG1 transcriptional regulation genes.

The results also indicate that the hRNase derived peptides treatment can also affect the biofilm formation by reducing the levels of adhesion proteins, interfering with hyphae formation and hindering the proper production of extracellular matrix components, which are essential for biofilm formation. The relative levels of gene expression are presented as fold changes in peptide-treated groups RN3 peptide in dark grey and RN7 peptide in light grey with respect to the untreated controls in black.

All gene transcript levels were normalized against ACT1 gene expression pointed line. A high number of antimicrobial peptides have been proposed as alternative antibiotics to combat microbial resistance. In contrast, there is still a scarce availability of peptide-based antifungal drugs [ 2 , 47 ] and only very few are in clinical trial [ 11 , 48 , 49 ].

The virulence of C. Noteworthy, Andes and coworkers observed the presence of host proteins within the extracellular matrix of fungal biofilms, with an abundance of leukocyte byproducts [ 50 ]. Indeed, proteins from our innate immune system embody the principal source of lead candidates to develop novel antimicrobial agents. Within this context, our laboratory has explored the antimicrobial mechanisms of action of the hRNases secreted by innate cells [ 22 , 51 , 52 ].

Several members of the vertebrate-specific RNase A superfamily have shown antimicrobial features apparently unrelated to their catalytic activity [ 30 , 53 , 54 ]. In an attempt to deepen the understanding of their biological role it has been proposed that RNases might have emerged as a host-defense family in vertebrate evolution [ 22 , 55 , 56 ]. Our previous studies on RNase A family members have revealed that the antimicrobial activity is retained by their N-terminal-derived peptides [ 19 , 20 , 23 , 24 ].

In particular, the action of the N-terminus of hRNase3 was characterized against Gram-positive and Gram-negative bacteria. Complementarily, Supplementary Figure S1 shows how the helical secondary structure would provide to the N-termini peptide a characteristic amphipathic nature. A comparative evolutionary study across vertebrate RNases lineages suggested that the N-terminal region has been conserved to carry out a host defense function [ 23 ].

Structural comparison of the structure of the hRNase3 N-terminus solved by NMR [ 34 ] and the comparative CD analysis of the eight hRNases respective peptides indicated that without sharing a high sequence identity, all the peptides adopt an equivalent structure [ 23 ]. Noteworthy, as reported for many other AMPs, the eight peptides were mostly unstructured when free in an aqueous solution and increased their alpha-helical content in the presence of a membrane like environment [ 23 ].

In order to expand knowledge of the mechanism of action of hRNases against C. This comparative study of the N-terminal region of the eight human hRNases against an eukaryotic pathogen such as C. The present results corroborate that the antimicrobial activity of hRNases against fungi is also preserved at their N-terminal domain. The data reveal a high antifungal activity, with MFC values at sub micromolar range for most tested derivative peptides, being the RNase N-terminal derived peptides 3, 6 and 7 the most effective ones Table 2.

These results are in agreement with previous studies where the parental proteins were tested against C. In addition, the current data using C. Another specific feature of the N-terminal peptides is the cell agglutinating ability, proposed as a mechanism to restrain the pathogens at the infection focus [ 54 , 58 ]. Previous studies highlighted the bacterial agglutination for both hRNase3 and its N-terminal peptide [ 23 , 54 , 59 ]. Interestingly, higher agglutination activities are observed for Candida cells in comparison to the tested Gram-negative bacteria [ 23 , 59 ].

In particular, an outstanding high activity is observed here for the RN3 peptide. Previous work identified aggregation prone domains at the N-terminus of RNases, showing a correlation between positive agglutination values and the sequences aggregation propensity [ 23 , 54 ]. Besides, the bacteria cell agglutination was also observed to be enhanced by the LPS interaction [ 58 ]. We report here positive binding of hRNase3 and its derived peptide for both Candida cells and their main cell wall component Figure 2.

Further work is to be carried out to evaluate the distinct binding affinities toward the fungi cell wall patterns. There is evidence in the literature about the importance of some residues that favors endocytosis. It has been suggested that the quantity of Arg and Lys residues, as well as their ratio and distribution, are essential in some AMPs and cell penetrating peptides CPPs for cellular uptake [ 60 ].

Our previous work on hRNases antifungal activity demonstrated the ability of hRNases 3 and 7 to internalize within C. Interestingly, Arg content in peptides is reported to facilitate the mammalian cellular uptake and improve their cytotoxic activity. However, not only the cationic residue composition of peptides was found to be imperative, but also the primary and secondary structures of the peptides have a high impact in the effectiveness of AMPs to cross eukaryotic membranes and target intracellular dwelling pathogens [ 61 ].

On the other hand, no significant changes were observed when the analog peptides were assayed in Gram-negative and Gram-positive bacterial cells [ 23 ]. Cationic antimicrobial peptides have attracted interest due to their high binding affinity to cell anionic polymers, and nucleic acids in particular [ 62 , 63 , 64 ]. Likewise, as commented above, no improvement of the antifungal activity of the peptide variants was observed Supplementary Table S2.

Therefore, our data indicate that other structural determinants of the peptides are required for their optimal overall performance. In any case, no significant alteration is predicted for the overall helical content and nucleotide binding regions of Lys and Arg peptide variants Supplementary Figure S4. First, we monitored the expression pattern of selected representative genes related to the overall cell metabolism and synthesis of cell wall components GAPDH, 18s and KRE6 in planktonic cells after peptide incubation for two hours.

Interestingly, we observed an up-regulation of the 18s subunit and KRE6 gene expression Figure 6. Our results are in accordance with studies reporting changes in the profile expression of C. The authors observed an induced expression of genes related to the Candida cell wall synthesis and anti-oxidative stress [ 66 ].

The up-regulation on the expression level of KRE6 demonstrates a possible activation of the polysaccharide synthesis pathway to maintain cell wall integrity and morphogenesis after the incubation with RN3 and RN7 Figure 6 , as reported previously for other antimicrobial peptides [ 65 ]. Therefore, we decided to explore whether the RN3 and RN7 peptides could halt the growth and formation of Candida biofilms, as has been reported for other AMPs [ 67 , 68 ].

It is worth stressing that growth of C. In particular, a high resistance to common antibiotics is reinforced by the presence of quorum-sensing molecules that play an important role in the biofilm formation, the release of virulence factors and the protection conferred by the extracellular matrix [ 5 , 42 , 69 ].

Unfortunately, despite the advances in antimicrobial therapy, Candida biofilms remain a challenge because biofilm embedded cells are tolerant towards most conventional antimycotics and there are only few novel agents that can be used to treat biofilm-related infections. To date, only miconazole, caspofungin, anidulafungin and liposomal formulations of amphotericin B are used to effectively treat these infections [ 44 , 45 ].

However, due to serious side effects [ 46 , 70 ] there is a need to identify novel antibiofilm compounds. Besides, current antifungal drugs have few specific targets i. An appropriate alternative relies on cationic peptides, which establish electrostatic interactions to anionic phospholipids in fungal membranes, such as phosphatidylserine and phosphatidylinositol, or to wall components, such as mannoproteins [ 72 , 73 , 74 ].

We find seldom examples of AMPs with antifungal activity, such as LL37, histatin5, the N-terminus of human lactoferrin or the KP killer peptide [ 75 , 76 , 77 ]. Frequently, the antimicrobial peptides are merely working as coadjuvants and require the complementary action of other antifungal agents [ 78 ]. Encouragingly, the hRNase peptides were able to eradicate C.

Moreover, we also found out that hRNase derived peptides are able to modify the expression of genes involved in hypha growth, biofilm formation and extracellular matrix synthesis. The principal virulence of C. As described above, the RN3 and RN7 peptides are not only able to remove pre-formed biofilms, but also to alter the expression of genes related to the biofilm formation.

The up-regulation of CSH1 expression was also reported in C. These changes in the expression level of genes involved in biofilm formation corroborate that hRNase derived peptides act in multiples stages to prevent biofilm formation. The suppression of the expression levels of some genes related to cAMP-PKA kinase pathways implicated in hyphal formation-, such as CYR1 and EFG1, might indicate a potential target of interest of hRNase derived peptides; however, further studies will be necessary to fully understand their mechanism of action.

On the other hand, removal of biofilms in vivo, is reported for salivary agglutinin and histatin and can be facilitated by the peptide binding to the cell polysaccharide external layer and cell agglutination [ 79 ]. Results with salivary histatins in animal models are promising and the antifungal peptide is proposed for topical therapy for oral candidiasis [ 79 ].

A shorter version of histatin 5 is currently in clinical trials. The mannose binding protein is a lectin also displaying a high affinity for candida wall that can induce the yeast cells agglutination with proved efficacy in a murine model [ 80 , 81 , 82 ]. No other examples of antimicrobial peptides endowed with an agglutinating activity for Candida cells are found in the literature.

Therefore, our present results highlight the multiples targets of action of hRNase-derived peptides toward C. In particular, the RN3 peptide combines high agglutination and antibiofilm activities. Novel peptide analogues have been recently designed to enhance the peptide stability and resistance to proteases in vivo.

Further work is currently ongoing to ensure the peptide druggability before considering any potential therapeutic application. Peptides were synthesized as previously described [ 23 ]. Fmoc-protected amino acids and hexafluorophosphate benzotriazole tetramethyl uronium HBTU were obtained from Iris Biotech.

Side chains of trifunctional residues were protected with t-butyl aspartate, glutamate, serine, threonine and tyrosine , t-butyloxycarbonyl lysine and tryptophan , 2,2,4,6,7-pentamethyldihydrobenzofuransulfonyl arginine and trityl asparagine, glutamine and histidine groups. Before each assay, cells were sub-cultured for 2—3 h to yield a mid-logarithmic phase culture.

For the biofilms formation assay C. Peptides serially diluted were added to the cells from 20 to 0. MFC of each peptide was determined from two independent experiments performed in triplicate for each concentration. The aggregation behavior was observed by visual inspection, and the agglutinating activity is expressed as the minimum agglutinating concentration of the sample tested, as previously described [ 58 ].

Cell viability of C. ATP, as an indicator of metabolically active cells, is indirectly measured by a coupled luminescence detection assay. The luminescent signal is proportional to the amount of ATP required for the conversion of luciferin into oxyluciferin in the presence of luciferase.

An overnight culture of C. RNase derived N-terminal peptides were added to 0. The C. IC 50 values were calculated by fitting the data to a dose-response curve. The method allows the labelling of intact viable cells and membrane compromised cells, which are labelled in green and red respectively, referred to as live and dead cells.

Then, the percentage of live bacteria was represented as a function of time. Membrane depolarization was assayed by monitoring the DiSC3 fluorescence intensity change in response to changes in transmembrane potential as described previously [ 33 ]. DiSC3 was added to a final concentration of 0. When the dye uptake was maximal, as indicated by a stable reduction in the fluorescence as a result of quenching of the accumulated dye in the membrane interior, protein in 5 mM Hepes-KOH buffer at pH 7.

The time required to reach a stabilized maximum fluorescence reading was recorded for each condition, and the time required to achieve half of total membrane depolarization was estimated from the nonlinear regression curve. All conditions were assayed in duplicate. Reference protein controls were treated following the same protocol in the absence of cells. Gel retardation experiments were performed following an adaptation of a previously described protocol [ 83 , 84 ].

The peptide-to-DNA weight ratios were , 2. Used primers are listed in supplementary Table S3. Additionally, we investigated the peptide-induced changes in transcription level of genes related to biofilm formation. Next, wells were washed with PBS and adhered cells were removed from the bottom of the wells by scraping. The quality and quantity of the extracted RNA were determined spectrophotometrically. ACTIN was used as a housekeeping gene for data normalization.

Three independent experiments were performed, with their own technical triplicate. The figures of gene level expression were designed by Graphpad Prism6 software. Biofilms of C. Colistin and amphotericin B were assayed as positive controls of inhibition. Wells with biofilm and media alone and biofilm-free wells were included as positive and background references.

Biofilm mass eradication was quantified by the crystal violet staining assay according to [ 85 , 87 ], with slight modifications. Overnight culture of C. At 24 h, C. After incubation, the wells were washed three times with PBS to remove planktonic C. Mortality was calculated as the mean area of 10 randomly selected fields and quantified using ImageJ software Oxford Instruments, Zurich, Switzerland. Step 2 of 2 How did you buy your ticket? Let's get your review verified.

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How did you buy your ticket? View All Photos Movie Info. Eccentric aliens give a man Simon Pegg the power to do anything he wants to determine if Earth is worth saving. Terry Jones. Bill Jones , Ben Timlett. Terry Jones , Gavin Scott. May 12, limited.

Apr 19, Atlas Distribution Company. Simon Pegg Neil Clarke. Kate Beckinsale Catherine. Sanjeev Bhaskar Ray. Rob Riggle Grant. Robert Bathurst James Cleverill. Eddie Izzard Headmaster. Joanna Lumley Fenella.

Marianne Oldham Rosie. Robin Williams Dennis Voice. John Cleese Extraterrestrial Voice. Eric Idle Extraterrestrial Voice. Terry Jones Extraterrestrial Voice. Michael Palin Extraterrestrial Voice. Terry Jones Director. Terry Jones Screenwriter. Gavin Scott Screenwriter.

Bill Jones Producer. Ben Timlett Producer. Chris Chesser Executive Producer. Jason Garrett Executive Producer. Mike Medavoy Executive Producer. David Rogers Executive Producer. Mark Sandell Executive Producer. Edward Simons Executive Producer. Angela Fuguet Executive Producer. Peter Hannan Cinematographer. Julian Rodd Film Editing.

George Fenton Original Music. Irene Lamb Casting. James Acheson Production Design. Harry Pain Art Director. Keith Pain Art Director. James Acheson Set Director. View All Critic Reviews Apr 04, Look, I think it should be said outright that the Monty Python crew produced two of my favorite comedic films of all time.

The latter would, easily, be in my top 3 comedies of all time, along with Young Frankenstein and Black Dynamite. In what order I would put them in is really irrelevant, it depends on my mood. Regardless, Monty Python are considered comedy legends and for good reason, they've written some of the best comedy the world has ever seen. The death of Graham Chapman, understandably, put the group to rest and reunions have been eagerly anticipated and, somewhat, delivered upon.

They did a series of stage shows in that were quite successful for the group. Which brings us to this movie, which was, somewhat, promoted as a Python reunion. The reason I start with this is the fact that I'm curious as to how many people actually consider this a Python reunion?

In my opinion, I wouldn't really call it that. Yes, all the Pythons voice aliens in this movie, but it's not a real reunion because the movie is based off a script that Terry Jones and Gavin Scott wrote. As far as I understand it, if my math doesn't fail me, Terry Jones is only ONE of the five remaining living members of the troupe. Therefore, in spite of all being in the same movie, or voicing characters in the same movie at least, this does not qualify as a Python reunion.

I'd have to think that if all five members were involved in the writing of this film's script that this would have ended up being considerably better than it was. This movie also has the unfortunate distinction of being the last movie Robin Williams completed before his death. Conceptually speaking, this really is pretty much Bruce Almighty.

I mean, Neil is given the power to, literally, do absolutely anything, hence this film's very creative title, like Bruce was in Bruce Almighty. Only difference is that the power in this flick is bestowed by aliens and not by Morgan Freeman, who IS God and he actually wasn't playing a character. That's the only difference here. Well, there is one other difference and it's a pretty big one. Say what you will about Bruce Almighty, but that film at least had an actual structure and Bruce gaining his powers served some sort of purpose to its arc.

And, of course, I'm not saying that Bruce Almighty is the Breaking Bad of comedic films, but at least there was some rhyme and reason as to why everything was happening. This movie, on the other hand, eh, not so much. When I say that there's no real structure to this film, I really do mean that. There's very little set-up before Neil ends up with the power to do anything he wants.

All he has is to say what he wants, wave his hand and it happens. Say Neil wants a bunch of tits flying over his head, he can make it happen. But you're given very little time to get used to the idea of Neil being sort of a loser who wants to get out of this rut that his life is currently on. The entire conceit is so absurd, because the scene before he realizes he has the power, when his friend Ray asks him if he could have anything in the world, what would it be?

People don't really talk like that in real life, nobody just comes up and says that to you. I mean, maybe they do, but not the way it was done in this movie. It's just so obvious about its intentions, that it just lacked any sort of subtlety. It just felt fake and that's the last thing you want to think while you're watching a movie.

You wanna immerse yourself in this world and these characters, but you are unable to do so with this movie when you have dialogue that's so obviously trying to play into the concept. Perhaps that shouldn't be something to complain about, but I felt it was a negative in this movie. But, to me, the biggest issue with this would have to be the fact that this is just a series of skits.

The movie is essentially Neil saying a bunch of random shit that would, hopefully, get a laugh out of you. I did like Neil turning Ray's crush who wants nothing to do with him into a cult leader that worships Ray's every move. That was actually really funny. Perhaps it's not funny in every scene they are in, but just a funny idea in theory.

The characters are one-dimensional. There's literally nothing to Neil other than him doing absolutely anything, here goes that phrase again, and not actually using his powers for good. He uses them for selfish reasons. And, really, that's the road the story should have gone down.

There's an interesting nugget of an idea here in exploring Neil's relationship with Catherine in that how can she know that he didn't set everything up perfectly so he could win her over. But that is said the one time late in the film and is never brought up again. And, fine, if you want this to be a straightforward comedy, with none of the 'moralizing' about using powers for good instead of selfish reasons, then you should have at least had some more consistent comedy.

I'd have to say that more than half of the jokes in this movie end up falling flat. It just feels like it's a movie that's designed around the idea of trying to fit in as much random jokes as they possibly could. Because they can do anything sigh that they want.

There's a scene where Grant, Catherine's possessive ex, makes Neil say that all pasty-white Englishmen have large ears and webbed feet. I don't know, just because. And, because Grant is a villain, he needs to be an asshole. The editing is also very awkward.

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Most famous for: Janice blew the judges away in auditions with her hit single Dreamer by her group Livin' Joy. Working with Ayda: ' I love her as a person. Impressed judges with: Louise sung an original song called A Moment Of Your Time in her first audition and was praised by the judges for her story-telling as she sung the emotional lyrics. Old school: Ricky has an eclectic taste in music and likes conveying emotion. Argos AO. Headlines U. Privacy Policy Feedback.

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Sorry we are not currently accepting comments on this article. Bing Site Web Enter search term: Search. Tropez as he promotes his Brown Sugar Bourbon liquor brand Katherine Schwarzenegger shares first look at her adorable baby daughter Eloise It's his marriage Meghan Markle cheers Prince Harry on as he loses polo semi-final as William's 'grief and anger' over their rift is revealed Demi Rose flashes a hint of underboob in a black cutout bra top while Emma Watson dons a green floral polo neck at Brilliant Minds event in Sweden Nicola Adams' pregnant girlfriend Ella Baig highlights her bump in a floral babydoll dress as she joins the boxer for the final day of Royal Ascot Princess Beatrice dons a spotty dress as she attends the final day of Royal Ascot, after sharing a carriage ride with her husband Edoardo Mapelli Mozzi 'She SO doesn't like her!

Megan Thee Stallion sizzles in a bikini as she enjoys yacht party with pals and packs on the PDA with her rapper boyfriend Pardi in Ibiza Paul Walker and Jenni Rivera receive posthumous tributes as part of 24 new celebrities to get stars on the Hollywood Walk Of Fame in Love Island: 'You lied to him multiple times!

Paul McCartney scraps Beatles hit Back In The USSR from ALL live shows due to Putin's brutal war in Ukraine Olivia Munn says her body 'hasn't snapped back' six months after she gave birth to her son with John Mulaney: 'The postpartum road is rough' David Hasselhoff, 69, and his wife Hayley Roberts, 42, kiss during the opening ceremony of the 61st Monte-Carlo Television Festival Goldie Hawn shows off her incredible figure in black halter-neck dress as she and partner Kurt Russell enjoy beach day during their Greece getaway Dame Deborah James praises the 'incredible' men in her life and vows to have 'a whisky or two' with her dad on Father's Day as she receives end-of-life care Someone get her a towel!

Kim Kardashian slips into silver bikini for beach day with Pete Davidson amid raunchy romance in new photo album from Tahiti vacation Mom on the move! Kendall and Kylie Jenner get dressed up and sip tequila cocktails while helping friend Hailey Bieber celebrate her skincare line Orange Is the New Black star Yael Stone cradles her baby bump as she shows off her sleepwear-inspired ensemble at the premiere of Blaze Junior Andre's debut single Slide hits No1 in the UK's iTunes pop chart hours after release as his father Peter gushes he is 'so proud' of his son 'He's stopped ticket touts by charging more than them in the first place!

Summer strikes plague spreads: Teachers, doctors, binmen, barristers and postmen could join rail workers in End of the line for ticket offices? Passengers would have to book train trips online under new plan that For grown men to pretend they don't know what a woman is when they have a wife and children is absurd: A Humiliation for Macron as French voters make him pay for his 'arrogance' - and turn to the far-right Drink-driver got caught after crash when his Mercedes airbags triggered warning to emergency services Barrister, 57, is feared dead in Seychelles jungle after going missing during hiking trip Archie Battersbee's mother says her son WILL wake up and is 'responding to music and smell' as she appeals Dame Kelly Holmes, 52, says she feels 'overwhelmed' by support after coming out as gay and says her late Jeremy Hunt's cancer scare: Tory MP reveals he was treated for 'mild' form of disease that has struck Dame Deborah James reveals her father has to brush her hair because she is no longer strong enough to do it Shameless MPs file official complaints saying their portions of taxpayer-funded Parliament food are not Will rail strikes wreck your week?

From hospitals and holidays, to glamping and Glastonbury Gilded life of Comrade Chaos who's called for class war: Could Mick Lynch, the shaven-headed rail union Get on track! Grant Shapps urges Keir Starmer to condemn rail strikes set to cause travel misery for Now for some good news! Traffic wardens are also set to go on strike Hillary Clinton warns Democrats' obsession with transgender issues and condemning JK Rowling could cost them That's exceedingly woke!

Food giant behind Mr Kipling cakes offers 'trans inclusivity' training to staff 'Eco-protester' invades court at ATP men's tennis final and tries to tie herself to net before two Counting the pennies? Amber Heard is spotted shopping at discount chain TJ Maxx with her sister after being Johnny Depp performs with pal Jeff Beck in first public appearance since Amber Heard admitted she can't Captain Conscientious!

Airline pilot takes to the tarmac and helps baggage crew load-up plane as airport Turtle doves are on the brink of extinction over lack of seeds they eat from crops in Mysterious glowing swirl lights up the night sky and mesmerises onlookers - but what caused the strange Government officials clamping down on 'buy now, pay later' market with fresh regulations Kitesurfer is killed and eight are injured after sudden 'mini TORNADO' hits Normandy beach throwing Ex-guerrilla Gustavo Petro is elected as first ever left-wing president of Colombia after beating Berlin fires up coal to cut reliance on Russian gas in wake of Ukraine invasion At least 25 people are killed by lightning and landslides in Bangladesh while millions are left marooned or Puffy skin?

Bank of England 'may force a recession to push inflation away': Bosses could have to take drastic measure The spies that are posing as your servants: Cars that track your movements, gadgets that can tell your mood Boy, 15, is killed and three others - including a cop - are injured by shooting at unauthorized DC Sharron Davies tells of her 'pride' in swimming as governing body BANS trans athletes from women's races: British Olympian says sport is 'standing up for fairness' - after competitors who were born male smashed a host of female records Counting the pennies?

But I was holding it when it went off ': Lord Ashcroft's former daughter-in-law says she is 'not a murderer' after accidently shooting Belize police chief in the head - and denies affair with him 'Look what happened to me when I came forward. Liam Payne contesta preguntas [Subtitulado] jacqueline Direct. Zayn Malik, 12 de enero de , de Bradford, Inglaterra. Liam Payne and Cheryl Cole have only been a public couple since early , but they've known each other since Here is Liam's life before XFactor Malory Tomapahost.

And all The X Factor Harry is the lead singer of his band White Eskimo, but has decided to see if he can go it alone. The year-old Aca el video de como los chicos de One Direction en el X Factor quedaron fuera de la categoria de los chicos Niall, Harry y Don't forget to subscribe for Liam goes to, what was described at the time as, the best Bootcamp.

He performs at the O2 Arena in London. Louis Walsh Listen to Liam's 1st exchange with Simon Cowell, "Give me another audition

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