The century-old idea that stripes make zebras cryptic to large carnivores has never been examined systematically. We evaluated this hypothesis by passing digital images of zebras through species-specific spatial and colour filters to simulate their appearance for the visual systems of zebras’ primary predators and zebras themselves. We also measured stripe widths and luminance contrast to estimate the maximum distances from which lions, spotted hyaenas, and zebras can resolve stripes. We found that beyond ca. 50 m (daylight) and 30 m (twilight) zebra stripes are difficult for the estimated visual systems of large carnivores to resolve, but not humans. On moonless nights, stripes are difficult for all species to resolve beyond ca. 9 m. In open treeless habitats where zebras spend most time, zebras are as clearly identified by the lion visual system as are similar-sized ungulates, suggesting that stripes cannot confer crypsis by disrupting the zebra’s outline. Stripes confer a minor advantage over solid pelage in masking body shape in woodlands, but the effect is stronger for humans than for predators. Zebras appear to be less able than humans to resolve stripes although they are better than their chief predators. In conclusion, compared to the uniform pelage of other sympatric herbivores it appears highly unlikely that stripes are a form of anti-predator camouflage. The functional significance of the unique high-contrast black and white stripes of zebra species is a subject of considerable debate. Historically, four major hypotheses have been offered to account for the function of zebra stripes: an antipredator defense operating either through crypsis, aposematism, or confusion of predators –3; a meaPanthera leo) and spotted hyaenas (Crocuta crocuta) (the chief predators of zebras), and of zebras themselves (Equus sp.) to resolve zebra stripes, relative to the ability of humans. To do so, we used spatial-vision-based digital image filtering techniques to compare images of zebras and other prey animals as they might be seen in high-, intermediate- or low-light conditions.Unfortunately, we still have little idea of how predators or even conspecific zebras view striped coats in natural situations. This problem is exacerbated by humans’ anomalously acute photopic spatial vision that far surpasses the ability of non-primate mammals to resolve detail, an ability that may have led us to overestimate the conspicuity of striped pelage for other species . In thisSpatial vision refers generally to the ability to resolve spatially defined features. The term commonly applies to the perception of stationary two-dimensional luminance patterns measured by resolution acuity or the contrast sensitivity function (CSF). By testing the level of contrast needed to resolve harmonic targets—typically sinusoidal gratings that vary in spatial frequency—the CSF provides a more comprehensive measure of spatial vision than does acuity, a measure of the finest high contrast detail  that can be resolved. Consequently, the CSF is more useful for predicting performance on real-world tasks –16. In tAs noted by Shapley , linear The possible anti-predator benefits of zebra striping founded on the visual systems of different observer species have not been evaluated (but see ). Here, If zebra stripes can be resolved by predators at a distance or under low lighting conditions or both, this would support a critical component of the argument that striping is a form of camouflage, albeit a rather unique one . StripinEquus burchellii), mountain (E. quagga) and Grevy’s zebras (E. grevyii) could be resolved by different predators, other zebras, and humans. We also measured the luminance of the black and white stripes of live captive zebras to quantify the mean Michelson luminance contrast [(Lmax—Lmin)/(Lmax+Lmin)], where L = luminance, in ambient conditions ranging from daylight to twilight.Zebra stripes, although of irregular width, can broadly be thought of as naturally occurring square wave gratings, the frequency  of which varies according to body region and viewing distance. A species’ acuity can be measured using gratings of high contrast, typically 0.5–0.8, of varying spatial frequency –23. UsinE. grevyii) and the immediate surrounding area  above, below, to the left and to the right of the animal was recorded by ADM using an LS-110 Minolta Spot Photometer with a one-degree acceptance angle on August 29th 2012 at the Calgary Zoo. Luminance under photopic conditions was recorded at 2:50 pm under full sun. Luminance under photopic (sun with light cloud) then mesopic (civil twilight) conditions was measured from 7:50 pm past sunset (8:27 pm) until ambient levels fell below the range of the meter at 8:43 pm (civic twilight end at 9:01 pm). Zebras were measured in both open (full sun) and shaded  conditions. In the former case, the stripe luminance was measured above and below the natural body shadow. Absolute luminance was higher in stripes exposed to full sun but luminance contrast was highly conserved across conditions , California Academy of Sciences , and UC Berkeley’s Museum of Vertebrate Zoology (2 E. burchellii) , topi (Connochaetes taurinus) and impala (Aepyceros melanopus); pelts of plains zebras, wildebeest (Alcelaphus buselaphus) and impala; and of two-dimensional life-size models of striped and “solid” zebras constructed of plywood and painted with glossy paint, were taken in the field in Katavi National Park, western Tanzania in August and September 2012 by TC. Following guidelines for using digital photography to study animal colouration [rd, on auto focus, and shot in RAW format. Objects  were photographed from 100–200 m away; precise distances were subsequently recorded using a rangefinder. Within 30 s after a photo was taken, TC went to the object’s location and erected a standard 24-square GretagMacbeth Colour Chart 1 m above ground. He then returned to the vehicle and photographed the colour checker card with the same zoom (70 mm) aperture and shutter speed settings. We used the PictoColor in Camera Plug-In for Photoshop to achieve consistent colour profiles across images. To overcome digital photography dynamic range limitations under dark conditions, the photographs were taken over a range from of light levels from high mesopic to high photopic. A complete dataset of all original and filtered images is available at the Harvard Dataverse, DOI: 10.7910/DVN/NZNAEH). Field research permission was granted by the Tanzania Wildlife Research Institute.Photographs of live adult and subadult plains zebras, waterbuck , where V is the minimum resolvable visual angle in degrees (acuity), S is the width of a stripe, and D is distance. In this study, species-specific acuity measures are extrapolated from similar species for which spatial vision has been measured behaviourally.Felis catus) and equids  [CSFs have been determined behaviourally under varying illumination conditions for humans and for other species, including carnivores  , 25, 26.aballus) , 27; (2)aballus) –30; (3) aballus) ; and (4)aballus) .2), mesopic (0.16 cd/m2) and scotopic (0.000016 cd/m2) conditions [lion = bcat + (VAlion—VAcat). The slope, which differed across luminance conditions, was held constant among species. This approach is a reasonable first-order approximation as lions and hyaenas have broadly similar retinal morphologies to cats, including topography and density of retinal cones [Acinonyx jubatus), as they have increased cone densities and other adaptations that increase acuity relative to domestic cats [Our specific approach for predators was as follows: we fitted cat CSFs recorded under the photopic  supporttic cats . HoweverWe followed a similar procedure to estimate the CSF of zebras. However, horses have considerably better acuity (cut-off ca. 23.3 cpd) than mammals of similar size . We theWe use a linear systems approach to image filtering, which is based on the premise that an image can be represented equivalently in both the spatial and frequency domains, and that a linear transformation  between them preserves the identity of the original image. Here, a Fourier transform refers to a convolution procedure in which the spectra of natural world scenes were modified by the digital weighting functions  representative of the spatial vision abilities of a lion, spotted hyaena, zebra or human in high, intermediate or low light conditions.Two-dimensional patterns can be represented by their Fourier spectrum—the linear sum of their component sine waves of specific spatial frequency, amplitude, phase and orientation. Since the CSF is assumed to represent the sensitivity of the visual system to these same components, it can be used as a “quasi-modulation transfer function” (MTF) through which an image’s components can be passed to arrive at a description of the source observer’s image pattern , 42. In In this study, the convolutions were performed by DK with a Vision and Aging Lab (VAL) custom CSF plug-in for ImageJ, a U.S. National Institutes of Health public domain Java-based image-processing program. The plug-in allows digital image spatial filtering with user-defined Butterworth, exponential or CSF filters. In this study, a Test Filter/Control Filter protocol was used wherein digital scenes were convolved using an observer-specific “test” photopic, mesopic or scotopic CSF filter in relation to a “gold standard” control filter–a young human adult photopic CSF . This apmacula lutea), it does allow for the specification of cone sensitivities. Therefore, it provides the best available emulation of species-specific colour vision, and serves our general purpose of generating a better final representation of a scene experienced through the eyes of dichromatic viewers.Given the primary intent of our study was to examine visibility of largely achromatic zebra stripes in three lighting conditions, one of them achromatic , to avoid potentially spurious exacerbation of luminance contrast differences we performed spatial filtering prior to the final colour adjustment of the images. Species-specific dichromatic colour vision filters were applied to the images by ADM to adjust for the effects of non-human species differences on spectral sensitivity and thus, for net luminance and chromatic contrast. We used a customizable Colour Vision Simulator software program , which aFinally, to estimate the impact of natural lighting changes on animal visibility, the mean bright-area luminance of each image was adjusted photometrically by DK to high, intermediate or low luminance within the photopic range using a Minolta LS-110 Spot Photometer. To maintain consistent luminance contrast while adjusting overall scene luminance, spot photometric measurements were carried out to find the mean contrast ratio of the highest on-screen luminance point on the animal relative to four immediately adjacent background locations  for the human observer images. The luminance of the brightest of the four points was then used as the reference for using the “brightness” control in Photoshop to find the highest  and lowest (“scotopic”) luminance levels at which the mean animal/background contrast was maintained . Due to natural variations in the images  the scotopic-photopic luminance range of the reference location varied across scenes. The luminance of the reference point for mesopic images was set at one-third of the range above the scotopic level, and thus, two-thirds of the range below the photopic level. Each image was adjusted separately, with the same brightness increment/decrement applied to all four observer species.tapetum lucidum, a reflective “eye-shine” layer immediately behind the retina. Prior studies report that domestic cats have six times greater sensitivity to light than humans [Non-primate mammals generally have better low-light sensitivity than humans due to the presence of a n humans . Additiord the width of the widest stripes (Grevy’s) to 1/5th the width of the widest stripes found in mountain zebras. Stripes may also be found on the legs of other congeners   .Under photopic (daylight) conditions, humans with the best acuity possible (20/10) should be able resolve the widest stripes of our main species of interest–a plains zebra–at distances up to 360 m. A human with good average  acuity would resolve stripes at half this distance (180m). The limit of a lion’s and spotted hyaena’s ability to resolve these same stripes under photopic (daylight) conditions is projected to be at distances of approximately 80 m and 48 m respectively, whereas other zebras might resolve conspecific stripes up to 140 m away. The maximum distance at which the widest stripes could be resolved is slightly extended for the mountain zebra (ranging from 439 m to 59 m–from best (human), to worst (hyaena))–and is much less for the Grevy’s zebra (195 m–26 m), which has the thinnest stripes ; Fig 1.The maximum distance of resolution decreases for areas of the body with thinner stripes. For example, the forelimbs of a plains zebra may appear to be uniform grey at distances of 82 m (humans), 18 m (lions), 11 m (hyaenas), and 32 m zebras; . These vProjected acuity declines with light level . Under tA separate approach to determining whether stripes aid in concealment is to compare images of striped and unstriped prey species modeled for a human versus a lion . If striTo add greater objectivity to our conclusions, CH applied a Sobel edge-detection algorithm to these images. This biologically-relevant analysis is well suited to detecting naturally-occurring features, including body contours and pelage patterns. We set a detection threshold—which determines the cut-off intensity of edges displayed relative to maximum intensity of the image—of 0.15. Adjustment of the threshold to 0.3 decreases the detail rendered but does not alter the result. This analysis confirmed that outlines of animals are readily distinguishable from the background scene . FinallyTo assess the conspicuity of zebra stripes amidst a background of trees, we compared photographs of live zebras and also life-sized models of a striped zebra with a hypothetical solid grey zebra in woodland. A solitary zebra appears less conspicuous in a forested setting than on the open plains, and a striped zebra model was less conspicuous than a solid grey model in woodland to all visual systems simulated . This suWe combine field data, digital images and psychophysical approaches to address the long-standing crypsis hypothesis for the function of zebra stripes by modeling their perception by chief predators, conspecifics and humans. Our analyses indicate that, under daylight conditions, humans can resolve zebra stripes at considerably greater distances than can large carnivores or zebras, but that all four observer types perform in a roughly equivalent fashion under scotopic conditions; . Under pStripe visibility decreases dramatically as light falls. At dusk, when hunting by carnivores normally begins, humans can resolve stripes from greater distances than other mammals: three times those of lions, five times further than spotted hyaenas, and 1.9 times more distant than zebras . Under sThese findings suggest strongly that stripes themselves are unlikely to be a form of crypsis at far distances because predators would not be able to discern the black stripes against a treed background, or see white stripes blending in with bright shafts of light between trees (background matching). Nor by the same reasoning could striping operate by disrupting the zebra’s body outline. At dusk or during bright moonlight  theSince the chief predators of zebras cannot resolve distant stripes, early arguments concerning zebras being cryptic as advanced by Galton and others, arguments that have persisted for more than a century, are contradicted by our analyses. Furthermore, comparisons between uniformly coloured species and zebras  show thaIn this study we were also able to estimate how zebras see stripes. Assuming that zebra spatial vision is similar to that of horses, zebras can likely see stripes from considerably greater distances than can their predators, although less well than humans. We therefore cannot reject the hypothesis that stripes may assist recognition of conspecifics or individuals, although stripes promoting species recognition seem improbable given the limited extent of allopatry in the three species of zebra. Field observations do not support the idea of stripes enhancing allogrooming, social bonding, individual recognition or being an indicator of phenotypic quality or health . Nor is In sum, our results do not lend support to stripes being a form of anti-predator crypsis, leading us to reject this longstanding hypothesis on the adaptive significance of zebra striping. Zebra stripes have also been hypothesized to reduce predation through aposematism , 54. OurS1 Dataset(CSV)Click here for additional data file.S2 Dataset(CSV)Click here for additional data file.S1 FigThe CSF for a domestic cat is illustrated for reference.(TIFF)Click here for additional data file.S2 FigImages are modeled under a 0.30 threshold (left panels) and 0.15 threshold (right panels) for a lion’s visual system.(TIFF)Click here for additional data file.S3 FigImages are modeled for a lion visual system under photopic conditions.(TIFF)Click here for additional data file.S1 TableValues represent how much closer to the target the species listed vertically would need to be to resolve the same level of spatial detail as the species listed horizontally.(DOCX)Click here for additional data file.


Malaria invasion of red blood cells involves multiple parasite-specific targets that are easily accessible to inhibitory compounds, making it an attractive target for antimalarial development. However, no current antimalarial agents act against host cell invasion.Plasmodium falciparum) and rodent (P. berghei) malarias. Multiple lines of evidence demonstrate that the action of azithromycin in inhibiting parasite invasion of red blood cells is independent of its inhibition of protein synthesis in the parasite apicoplast, opening up a new strategy to develop a single drug with multiple parasite targets. We identified derivatives of azithromycin and erythromycin that are better invasion inhibitors than parent compounds, offering promise for development of this novel antimalarial strategy.Here, we demonstrate that the clinically used macrolide antibiotic azithromycin, which is known to kill human malaria asexual blood-stage parasites by blocking protein synthesis in their apicoplast, is also a rapid inhibitor of red blood cell invasion in human  contains supplementary material, which is available to authorized users. Plasmodium falciparum. Current disease control measures rely on reducing exposure to the mosquito vector or treatment of clinical malaria cases with antimalarial drugs that target the disease causing blood-stage trophozoite; most of these act against the parasite’s food vacuole or interfere with pyrimidine synthesis of maturing trophozoites [Malaria is a mosquito-borne parasitic disease, which causes nearly 600,000 deaths every year, mainly in children under 5 years of age in sub-Saharan Africa . Most dehozoites –6. Of mahozoites –9. The phozoites . This deInvasion of blood-stage merozoites into host cells has been proposed as a potential target for antimalarial chemotherapy –14. InvaP. falciparum merozoite invasion, partly through lack of tractable invasion inhibition assays. The ability to assess the invasion inhibitory potential of compounds and their biological activity in vitro has been greatly enhanced with the development of a robust and efficient merozoite purification protocol and techniques to directly measure invasion inhibition [P. falciparum and P. berghei merozoite invasion in vitro. This represents the first time that a drug or drug class that has clinical applications against malaria has been demonstrated to inhibit parasite invasion of the red blood cell.Few compounds have been directly tested for ability to inhibit hibition , 18–20, hibition , 22, hephibition  and calchibition , 24) havhibition . In thisin vivo [In vitro experiments at clinically relevant concentrations show that these antibiotics have a ‘delayed death’ drug response. Treated parasites grow normally during the first lifecycle under treatment. Only during the second post-treatment cycle (after replication and reinvasion) are drug effects observed. Parasite death at this stage is thought to result from the inheritance of a defective apicoplast that is unable to synthesize isoprenoid precursors required for development [Antibiotics such as azithromycin and clindamycin (lincosamide antibiotic) have been proposed as potential partner drugs for artemisinin combination therapies owing to their extremely long half-life and good safety profile in vivo –29. Bothin vivo –32. In velopment –33. Thiselopment .50 of invasion inhibitory activity could be lowered through modification. These results provide the basis for a novel strategy of antimalarial drug development by advancing compounds that have dual mechanisms of action. Given the established safety and low cost of macrolides in widespread clinical use, development of macrolides as antimalarials with dual modalities against merozoite invasion and protein translation in the apicoplast is a promising strategy to counter the rapid emergence of drug resistance.In this study, we aimed to identify existing, and novel, compounds with invasion inhibitory activity that have the potential for development as effective antimalarials. We demonstrate that the antibiotic azithromycin can specifically inhibit merozoite invasion. Related macrolide antibiotics were also found to inhibit invasion, and the ICP. falciparum asexual blood-stage merozoite invasion of the host erythrocyte  or DMSO (38 μM), suggesting that choice of vehicle can impact azithromycin potency in vitro. Comparative screens of azithromycin potency over different treatment times demonstrated that the IC50 of merozoite invasion assays (1 hour exposure to drug) were surprisingly similar to the in cycle  invasion 10 μm; in cycle 6 μM; 1 cycle 7 μM, −(drug prepared in DMSO) invasion 38 μM (DMSO); in cycle 12 μM; 1 cycle assay 7 μM, Fig. in vitro.Application of the merozoite purification method of Boyle et al.  identifi ethanol 0 μM or D80mero DMSO 151 μM) and the invasion inhibitory control compound, heparin, was significantly more inhibitory to parasite growth than the same treatment of early ring stages at the time-point of 20 minutes post-invasion (T = 20 (exposed for 40 minutes); azithromycin P <0.001; heparin P <0.01). Additionally, pretreatment of erythrocytes, followed by washout of drug, had little or no inhibitory effect on invasion . This indicated that exposure of merozoites, but not very early ring stages or uninfected erythrocytes, was inhibitory to parasite growth.To confirm that azithromycin was inhibiting merozoite invasion directly in our assays and was not acting downstream of invasion during intracellular parasite growth, the effect of exposure of merozoites or early ring-stage parasites to the drug was examined; azithromycin was washed out of the cultures within 1 hour and parasitaemia assessed by flow cytometry 40 hours post-assay setup for all treatments (prior to the next round of parasite rupture and invasion of new host cells). Treatment of merozoites (10 minute incubation) and early ring stages for less than 1 hour (T = 0) with azithromycin (1 × IC80mero (380 μM (drug prepared in ethanol to limit nonspecific effects of vehicle)) prior to washing and addition of merozoites. There was minimal loss of invasion into erythrocytes pretreated with a 10 × IC80mero concentration of azithromycin for 1 hour relative to control , supporting the conclusion that it is the merozoite and its interaction with the erythrocyte, and not the erythrocyte itself, that is the target of inhibition.In order to confirm that the inhibitory activity was against purified merozoites and not the uninfected erythrocyte prior to merozoite invasion, erythrocytes were pretreated with azithromycin at a 10 × ICrol Fig. . In cont80mero), confirming that azithromycin is indeed inhibitory to merozoite invasion  and spiramycin (123 μM), while erythromycin (420 μM) was 42-fold less potent than azithromycin. Of interest was the very poor invasion inhibitory activity of dirithromycin (521 μM), which was 52-fold less potent than azithromycin, even though the IC50 values determined using 1 cycle growth assays were very similar  further confirmed specific inhibition of merozoite invasion .After identifying that azithromycin was inhibitory to merozoite invasion, we examined whether related drugs with a history of clinical use as antibiotics also had this property. Macrolides with a 14-membered macrolactone ring  and 16-membered ring (spiramycin) were tested using the same methods as azithromycin  than seen for azithromycin (10 μM). However, pre-treatment of erythrocytes with 2,972 μM of clindamycin (IC80mero) resulted in a 65 % reduction in merozoite invasion relative to untreated controls and there was evidence of erythrocyte lysis using these high levels of clindamycin  selected for reduced sensitivity to both azithromycin and erythromycin in 2 cycle (delayed death) assays. Sequencing of apicoplast ribosome genes revealed a G91D mutation in the rpl4 gene product, corresponding to the G112D mutation in Chlamydomonas reinhardtii, known to confer resistance to erythromycin [50120hr D10-AZRr, 9 μM; D10 parental, 0.16 μM; P <0.0001; Fig. 50mero , erythromycin  or clindamycin . Together, these data suggest that the invasion inhibitory activity of azithromycin is largely independent of apicoplast ribosomal protein synthesis, consistent with the current view that the apicoplast does not play a role in invasion.We tested further whether apicoplast ribosomal protein translation was the target of the inhibitory activity at the merozoite stage by comparison of the invasion inhibitory activity of azithromycin, erythromycin and clindamycin between the D10-PfPHG (azithromycin-sensitive) parasite used throughout this study and an azithromycin-resistant D10 derivative  that has a lower IC50 in 1 cycle growth inhibition assays [50 of merozoite invasion inhibition (13 μM) 32-fold compared to the parent compound  almost 3-fold compared to erythromycin A. The increased potency of Meg-erythromycin and erythromycin oxime compared to the parent drug indicates that various modifications to macrolides can lead to more potent invasion inhibitory activity.We tested a panel of macrolide analogues to determine whether the ICn assays  was test50 values lower than azithromycin in a 72 hour growth inhibition assay  had a substantially lower IC50 for merozoite invasion inhibition than azithromycin (38 μM (when prepared in DMSO); Fig. in vitro.Next we obtained a small panel of azithromycin analogues (in DMSO) from GlaxoSmithKline  –44 with 50 of the modified analogues was between 10- and 14-fold lower than that of azithromycin  and 90 hours  the IC80) reduced invasion to only 8 % of total contact events, reduced contacts leading to erythrocyte deformation to 27 % of contact events and increased the number of merozoites releasing without invading or deforming the erythrocyte to 65 % of all contact events, resulting in failure to invade for 92 % of all contact events. These results support the live filming observations for azithromycin in ethanol . As expected, at the same concentration the less potent parent azithromycin was less inhibitory to invasion (25 % contact events invaded) and as a result had fewer failed invasion events (75 %). Together, these findings establish a proof-of-concept that macrolides with more potent invasion inhibitory activity can be developed through chemical modification and that the dual activities of invasion inhibition and other inhibitory activities can be developed in single compounds and are not mutually exclusive.In order to assess whether the invasion inhibitory phenotype of a more potent analogue was similar to our observations for azithromycin in ethanol see Fig. , live finol Fig.  and agai50 for both in cycle  and 1 cycle  drug assays . Comparison of these compounds indicates that the cladinosyl group has a role in lowering the IC50 of invasion inhibition for azithromycin, but the presence of both the cladinosyl and desosamine groups is critical for azithromycin’s invasion inhibitory activity. While these data suggest that the desosamine group plays the critical role during invasion inhibition, technical limitations prevented the selective removal of the desosamine group.We were further able to remove both the cladinosyl and desosamine groups from azithromycin  [P. berghei sporozoites to enter liver cells in vitro using an established assay. GFP-expressing sporozoites were allowed to settle on and invade HepG2 cells. After free sporozoites were removed by washing, cells were fixed and then antibody-labeled without cell permeabilization, thereby preventing labeling of sporozoites that are inside host cells. We then compared the number of labeled cells  to the total number of cells  to determine invasion efficiency. There was a statistically significant, but modest, effect indicating that azithromycin may inhibit sporozoite invasion of hepatocytes at concentrations up to 100 μM azithromycin ; Fig. P. berghei sporozoites  ; the efftes Fig. .Toxoplasma gondii, which has orthologues of many Plasmodium invasion proteins and a similar invasion process [80 of azithromycin against P. falciparum merozoite , clearly indicating that azithromycin is a less potent inhibitor of T. gondii invasion in vitro. Importantly, there was no evidence of tachyzoite invasion inhibition in the presence of 500 μM erythromycin A, reflecting the results of the P. falciparum studies which showed that erythromycin A is a poor inhibitor of merozoite invasion compared to azithromycin. We next explored whether the azithromycin analogue 12e, which was found to be a more potent inhibitor of P. falciparum merozoite invasion than azithromycin (DMSO as vehicle), had improved tachyzoite invasion inhibitory activity compared to azithromycin. Analogue 12e (tested at 42 μM due to limited compound availability) was found to be a consistently more potent inhibitor of T. gondii tachyzoite invasion (48 % inhibition compared to non-inhibitory control) than much higher concentrations of azithromycin  to the low nanomolar range [In addition to its ‘delayed death’ effect on parasite growth, azithromycin has been reported to kill parasites within the first cycle of trations , 32. It trations . Given gar range –44. Yet Several lines of evidence indicate that azithromycin has a secondary mechanism and target(s) of action to inhibit invasion, in addition to binding to the 50S apicoplast ribosomal subunit as described for the ‘delayed death’ phenotype , 32. Cli50 values seen for both inhibitory phenotypes raises the intriguing possibility that this secondary mechanism of action could be targeted throughout the disease causing blood-stage of P. falciparum malaria.The near instantaneous inhibition of merozoite invasion caused by azithromycin also suggests that this drug can rapidly kill parasites through a secondary mechanism of action. Typical ‘delayed death’ inhibition by macrolide antibiotics results in the loss of apicoplast functionality , 32, witThe ability of azithromycin to rapidly inhibit merozoite invasion, in addition to the apicoplast-targeting ‘delayed death’ mechanism of action, is an exciting prospect for the development of macrolide-based drugs in combination therapies. This dual modality could see azithromycin assist in rapid clearance of parasites as well as offering longer-term treatment of the remaining low-level blood and liver-stage parasites through its activity against the apicoplast. Such a drug treatment strategy has the potential to increase drug efficacy, while reducing the chances for the development of resistance.in vitro merozoite invasion inhibitory activity of azithromycin required drug concentrations at the upper end of what is achieved clinically [in vivo fermentation process [50 by up to 5-fold. The azithromycin derivatives were tested only when solubilized in DMSO due to limitations in the amount of available material. Importantly, the use of DMSO as vehicle for solubilization reduced the potency of the parent compound azithromycin 4-fold relative to ethanol as vehicle. If this observation were to hold true for the derivatives, the invasion inhibitory IC50 of the compound 1j (5) [Although the inically , 32, we  process  produced process –44 lower50 of azithromycin (10 μM) and analogues  is comparable to that achieved for inhibitors that specifically target the function of essential merozoite proteins. Two recent proof-of-concept studies identified small molecule invasion inhibitory compounds targeting interactions between essential invasion ligands RON4-AMA1 (inhibitory range 30–6 μM) and MSP1-19 (replication inhibition 21.7 μM) [50 values in a very similar range to azithromycin and analogues. Another highly selective invasion inhibitor, the 3D7-AMA1-specific peptide inhibitor R1 [50 of 2.5 μM in assays using purified merozoites [The invasion inhibitory IC21.7 μM) , and repbitor R1 , has an rozoites . AzithroRemoval of azithromycin’s glycan groups provided insight into the structural requirements for invasion inhibitory activity. Removal of the cladinosyl sugar resulted in a 5-fold reduction in invasion inhibitory activity. Strikingly, removal of both sugars resulted in complete loss of invasion inhibitory activity, strongly suggesting that the desosamine sugar, rather than the cladinosyl, is critical for invasion inhibitory activity. Interestingly, the desosamine sugar is also considered the critical glycosylated group in binding of macrolides to microbial ribosomes  with cliPlasmodium merozoites  and T. gondii tachyzoites parasites suggests that the target of inhibition is shared amongst some apicomplexan invasive stages. The fact that the potency of azithromycin against P. falciparum (human) and P. berghei (rodent) malarias was very similar raises the possibility that targeting this pathway could be an effective strategy to treat other malaria species. The results of the T. gondii invasion assays suggest that host cell invasion of this more distantly related apicomplexan parasite can also be targeted by azithromycin, albeit at higher concentrations. Importantly, the analogue 12e had equal or better invasion inhibitory potency than azithromycin  when tested at a 6-fold lower concentration. This strongly suggests that analogues of azithromycin could be developed with superior inhibitory activity against tachyzoite invasion and confirms that the invasion inhibitory activity of azithromycin and analogues is shared across distantly related apicomplexan parasites.The fact that azithromycin inhibits host invasion by 2+ and MAPK through an as yet undefined mechanism . Intraciewed in ) so inteiewed in , 56. IntPlasmodium spp. merozoite invasion into erythrocytes in vitro. Invasion inhibition assays and live filming experiments show that azithromycin acts rapidly to stop merozoite entry very early during the invasion process. The mechanism of invasion inhibition appears to be independent of apicoplast ribosomal translation inhibition, which is the currently described mechanism of action of these drugs. Modification of both azithromycin and erythromycin A resulted in promising reductions in the invasion inhibitory IC50 to levels nearing those achieved clinically for the parent compounds, providing a proof-of-concept for the potential development of potent invasion inhibitors. Elucidation of the mechanism of action and subsequent improvements in compound potency could pave the way for development of macrolide antibiotics with dual modality as novel and potent antimalarials to complement frontline treatments for malaria. Furthermore, the use of drugs that inhibit merozoite invasion in combination with drugs that inhibit intra-erythrocytic parasite development may be an effective strategy to facilitate rapid parasite clearance to help improve clinical outcomes, reduce transmission, and minimize the development of resistance.The results of this study have identified for the first time that macrolide antibiotics, a diverse group of well-tolerated antibiotic drugs with antimalarial properties, inhibit + erythrocytes  according to established protocols [D10 parental and GFP fluorescent D10-PfPHG  parasiterotocols , 58. Synrotocols , 23, 57.rotocols , 18.P. falciparum growth and invasion inhibition is available in Fig. A diagram outlining the different drug inhibition assays used in this study to measure P. falciparum and P. berghei merozoites from E64-treated schizonts and merozoite invasion inhibition assays have been previously described [The protocols for filtration of viable escribed , 18, 45.P. berghei ANKA sporozoites dissected from infected Anopheles stephensi mosquitoes. Two-color invasion assays followed the protocol of Sinnis et al. [P. berghei constitutively expressing GFP [P. berghei merozoite surface proteins [.Liver-stage assays used s et al.  with thesing GFP  were addproteins  (500:1) proteins .Toxoplasma gondii invasion assays were performed using the Rh parasite strain according to existing protocols [rotocols  with the50 and IC80 concentrations were determined using GraphPad Prism  as previously described [90hr, 120hr and mero where appropriate.The ICescribed . The inh2, 5 % O2 and 5 % CO2), selected schizonts were observed until they ruptured and released their merozoites. Time-lapse videos of the invading merozoites were recorded with a high-resolution AxioCam MRm camera (Zeiss). ImageJ was used to perform image analysis and GraphPad Prism used to perform statistical analyses using a Chi-squared test.Live cell microscopy experiments were undertaken according to the method of Weiss et al. . BrieflyA summary of the chemical structures of the compounds used in this study is available in Fig. A tabular summary of data used to generate graphs and perform statistical tests presented in this manuscript Figs. ,e,f is a


Here, we discuss the potency and selectivity of PLK-1-targeted inhibitors and their molecular interactions with PLK-1 domains.Mitotic kinases are the key components of the cell cycle machinery and play vital roles in cell cycle progression. PLK-1 (Polo-like kinase-1) is a crucial mitotic protein kinase that plays an essential role in both the onset of G2/M transition and cytokinesis. The overexpression of PLK-1 is strongly correlated with a wide spectrum of human cancers and poor prognosis. The (si)RNA-mediated depletion of PLK-1 arrests tumor growth and triggers apoptosis in cancer cells without affecting normal cells. Therefore, PLK-1 has been selected as an attractive anticancer therapeutic drug target. Some small molecules have been discovered to target the catalytic and noncatalytic domains of PLK-1. These domains regulate the catalytic activation and subcellular localization of PLK-1. However, while PLK-1 inhibitors block tumor growth, they have been shown to cause severe adverse complications, such as toxicity, neutropenia, and bone marrow suppression during clinical trials, due to a lack of selectivity and specificity within the human kinome. To minimize these toxicities, inhibitors should be tested against all protein kinases  In this study, we will discuss the potential, selectivity, and specificity profiling of PLK-1 targeted inhibitors against binding sites in an attempt to provide more selective and potent antitumor therapeutic agents.Polo-like kinase-1 (PLK-1), a serine/threonine (Ser/Thr) protein kinase, is highly conserved from yeast to humans and is reported to play a role in the mitotic events of the fruit fly. Five PLK family members are known in humans: PLK-1, -2, -3, -4, and -5 , 7. Therin-vitro –22. Thesin-vitro –29. Thesin-vitro , 31. AltClassification(a) Depend upon the SourceNatural source: Aristolactam AIIIa, Scytonemin, Wortmannin,Synthetic source: ON01910.Na, BI 2536, BI 6727, GSK461364A, HMN-176, SBE13, ZK-thiazolidinone (TAL), Compound 36, Compound 15, Compound 38, NMS-P937, LFM- A13, RO3280, TAK-960,Peptides: MQSpTPL or MAGPMQSpTPLNGAKK, LLCSpTPNG, PLHSpT,RNAi: TKM-080301.(b) Binding Site Based ClassificationInhibitor Interaction with kinase domain:Non ATP-competitive type: ON01910.Na, Cyclapolin 1,ATP-competitive type: BI 2536, BI 6727, GSK-461364A, HMN-176, SBE13, ZK-thiazolidinone (TAL), Scytoemin, Wortmannin, Compound 36, Compound 15, Compound 38, NMS-P937, LFM-A13, RO3280, TAK-960,RNAi: TKM-080301,Others: DAP81, PHA680626.Inhibitor Interaction with PBD domain:Natural or semisynthetic: Poloxin, Poloxipan, Purpurogallin, Aristolochia AIIIa,Peptide: MQSpTPL or MAGPMQSpTPLNGAKK, LLCSpTPNG, PLHSpT.γ-phosphate group of ATP to substrates containing Ser/Thr/Tyr amino acid residues. Many studies have documented that the kinase domain is one of the most promising drug targets [The protein kinases catalyze the transfer of the  targets . During  targets .aa36-345 construct [Structure-guided drug design is an improved strategy to design more selective and potent drug like molecules. Various groups of researchers have determined the crystal structure of the kinase domain. The first kinase domain was crystallized by using ankyrin repeat proteins (DARPins) to address the active confirmation . Meanwhionstruct . The kin Calothrix sp., and Lyngbya aestuarii [50 of 2 ± 0.1 μM in a concentration-dependent manner in vitro and disrupts the activation of CDC25C [50 of 3-4 μM [Initially, scytonemin was identified as a PLK-1 inhibitor and was isolated from many strains of Cyanobacteria,estuarii . The strestuarii . PLK-1 aestuarii –6. Scytof CDC25C , 8. Anotf 3-4 μM . It was f 3-4 μM . Scytone50 ranging from 50 to 250 nM in-vivo and in-vitro [μM in an in vitro kinase assay and was unable to show RNAi-induced cellular phenotypic resemblance [ON018910.Na is a hydrophilic benzyl styryl sulphone analog that was first reported as a PLK-1 inhibitor that functions in an ATP-independent manner. ON01910.Na treated cells exhibited inhibition of the PI3K/AKT pathways and downregulation of cyclin D1 with activation of the apoptosis-related genes NOXA and BIM. Furthermore, another study demonstrated that ON01910.Na treated cells showed inhibition of phosphorylation of CDC25C, a downstream protein of cyclin B, and activated the caspase pathway by the downregulation of Bid, Bcl-xl and Mcl-1 in B-cell lymphoma , 37. A win-vitro . Howeveremblance . Furtheremblance . Moreoveemblance . ON01910 Penicillium funiculosum and Penicillium wortmannin and was initially characterized as a phosphatidylinositol-3-kinase (PI3K) inhibitor. However, a later study observed that wortmannin also inhibited PLK-1 with an IC50 of 24 nM [μM) [Wortmannin is a steroid metabolite of the fungiof 24 nM . Accordiof 24 nM . Further nM [μM) .50 value of 118 nM in vitro [ MDR1 by inhibiting the transcription factor Nuclear Transcription Factor Y subunit alpha (NF-Y) and inducing mitotic cell cycle arrest [HMN-176 is an active metabolite of the synthetic antitumor oral prodrug, HMN-214, and was developed by the Japan-based company Nippon Shinyaku Co. Ltd. HMN-176 did not show direct catalytic inhibition of PLK-1. A wide range of human cancers have been screened to validate the potential of HMN-176 as an anticancer chemotherapeutic agent with an ICin vitro . HMN-176e arrest . HMN-176μM. Another study has documented that NMS-P937 is well-tolerated with good potency and efficacy in preclinical xenograft tumor models. Moreover, the 2′-trifluoro-methoxy moiety of NMS-P937 determined the molecular selectivity for PLK-1 and the rest of the kinases, and the 2′-trifluoro-methoxy moiety fits in the ATP-binding pocketsformed by Arg57 and the hinge residues Leu132-Cys133-Arg134 [https://clinicaltrials.gov/ct2/show/record/NCT01014429).NMS-P937 is a derivative of pyrazolo quinazoline that was developed by Nerviano Medical Science, Milano, Italy. NMS-P937 is an oral available, selective ATP-competitive inhibitor . NMS-P933-Arg134 . NMS-P933-Arg134 . Further50 value of 50 nM. Moreover, GSK461364A was also screened against a panel of 260 protein kinases and exhibited an IC50 value of <1 μM against only 10 additional kinases [ in vitro inhibition of PLK-1 but also dose-dependent tumor growth inhibition in various established tumor xenografts. Moreover, GSK461364A also prevented brain metastases from breast cancer. This study suggests that PLK-1 may be a therapeutic target for the inhibition of metastases [GSK461364A is a selective thiophene amide derivative PLK-1 inhibitor that was designed by Glaxo Smith Kline . It is a kinases , 25. GSKtastases . GSK4613tastases . GSK461350 of 1 nM in vitro. Nonetheless, it also showed activity against other members of the PLK family (except PLK-4 and 5) at different concentrations, PLK-2 and PLK-3 with IC50 values of 3.5–4.0 nM and 9 nM, respectively. Moreover, BI 2536 showed 1000-fold better selectivity against a panel of tyrosine and serine/threonine kinases [ in vivo, and it reduced tumor size in several well-established xenograft models [BI 2536 is a dihydropteridinone derivative that was developed by Boehringer Ingelheim. BI 2536 is the most potent ATP-competitive type inhibitor of PLK-1, arresting a large number of cell lines in the G2/M phase with mitotic abnormalities, such as the monopolar spindle and aberrant accumulation of cyclin B1 , 31. BI  kinases . BI 2536t models , 32. In t models , 39. Thet models . These c in vitro. BI 6727-treated cells experienced G2/M phase arrest, along with a polo-like spindle resemblance phenotype and subsequently induced apoptosis [https://clinicaltrials.gov/ct2/show/study/NCT00824408). BI 6727 phase II clinical trials are also ongoing with platinum-resistant/refractory ovarian cancer (http://meetinglibrary.asco.org/content/112794-132). In addition, BI 6727 has been tested with Aurora kinase inhibitor BI 811283 against pediatric malignancy cell lines, including Ewing sarcomas, leukemias, medulloblastomas, neuroblastomas, and osteo- and rhabdomyosarcomas in vitro. In this study, it was observed that BI 6727 showed a GI50 ranging from 2.89 nM/L to 5.05 nM/L and also showed G2/M arrest at 24 hr, leading to apoptotic cell death at 48 hrs in an RMS-1 xenograft model of rhabdo blastoma in pediatrics tumors [BI 6727, which was also developed by Boehringer Ingelheim, is another PLK-1-selective ATP-competitive inhibitor in the dihydropteridinone series with improved properties. It exhibits inhibition activity against the targeted catalytic domain of PLK-1/2/3 at different half-inhibitory concentrations of 0.85, 5.0, and 56 nM, respectively. The inhibitory potential of BI 6727 has been screened against a wide range of tumor cell linespoptosis . BI 6727poptosis . Howeverpoptosis . Moreoves tumors . Drosophila cell lines HeLa and S2 cells, respectively. Moreover, Cyclapolin1 reduced the centrosome integrity and microtubule nucleation ability. Several cancer cell lines have been screened against cyclapolin1 to evaluate the potential for an IC50 of ~20 nM [Cyclapolin1 is a selective, non-ATP-competitive type inhibitor of PLK-1, developed by Cyclacel Ltd., Cambridge, UK. Cyclapolin is a benzthiazole-N-oxide derivative that is currently undergoing preclinical evaluation to improve its efficacy with minimum toxicity . Cyclapof ~20 nM .50 of 0.9 μM and shows the polo-like phenotype resemblance. Moreover, DAP-81 has also been proven to be an inhibitor by reducing the phosphorylation of CDC25C in a dose-dependent manner [DAP-81 is a diamino-pyrimidine derivative designed at Rockefeller University, New York. DAP81 destabilized the kinetochore-microtubule assembly, whereas other spindle tubules are stabilized, leading to the monopolar spindle phenotype . DAP-81 t manner . DAP-81 t manner .50 of 19 ± 12 nM in vitro [50 of 0.2–1.3 μM. TAL-treated cells exhibited mitotic G2/M arrest and showed monopolar-phenotypic resemblance, along with apoptosis. Moreover, TAL-mediated inhibition also participated in impairment of centrosome maturation and lead to the failure of cytokinesis [TAL is an ATP-competitive type inhibitor of PLK-1 designed by Bayer Schering Pharma AG, Berlin, Germany. TAL inhibits the activity of the catalytic domain of PLK-1 with an ICin vitro . A numbeokinesis . Preclin50 of 0.07 μM [50 of 0.53/0.007/1.67 μM in vitro, respectively [PHA-680626 is a pyrrolo-pyrazole derivative, and it inhibits PLK-1. Initially, it was developed as an Aurora-A inhibitor with an IC 0.07 μM . Later, ectively . PHA-68050 of 12–39 μM [http://www.tandfonline.com/doi/pdf/10.4161/cc.9.3.10721). SBE13 is currently undergoing a preclinical phase of development [SBE13 is a vanillin derivative, and it inhibits PLK-1 with an IC12–39 μM . SBE13 selopment .50 of 9.8 nM in vitro, whereas it inhibited the other PLK family members, PLK-2 and PLK-3 with an IC50 of 21 nM and 178 nM, respectively. Compound 36 is also currently undergoing preclinical development [Compound 36 belongs to the imidazopyridine class of inhibitors, and is selective for the inhibition of PLK-1. Compound 36 exhibited high selectivity against PLK-1 among a panel of 212 kinases . Compounelopment .50 of 32.5 μM, whereas inhibits PLK-3 with an IC50 of 61 μM in vitro. On the basis of molecular docking studies, it has been predicted that LFM-A13 binds to the catalytic site of the Plx1 kinase domain by using a GST-CDC25 substrate peptide interaction. LFM-A13-treated cells exhibited mitotic arrest and prevented bipolar spindle assembly formation in human cancer cell lines, including breast cancer and glioblastoma. Furthermore, LFM-A13 showed tolerable toxicity in mouse and rat tumor models, without hematological toxicity such as peripheral blood counts and bone marrow suppression [ in-vivo. Furthermore, the combined study of LFM-A13 with the antileukemic drug vincristine, exhibited inhibitory activity against PLK-1 without any sign of morbidity or mortality. The preclinical studies of LFM-A13 support the hypothesis that chemoresistance of relapsed B-cell precursor acute lymphoblastic leukemia can be cured by using LFM-A13 in combination with vincristine. LFM-A13 continues ongoing preclinical development [LFM-A13 is a leflunomide metabolite analog and a selective inhibitor of PLK-1 . It inhipression . Moreovepression . In addielopment .50 of 3 nM and EC50 of 6.0–82 nM against several cancer cell lines in vitro. Furthermore, RO3280 has also been screened against a wide panel of 318 kinases (wild type and mutant) to evaluate its selectivity and potency. The binding potential of RO3280 to PLK-1 is 500-fold tighter than any other protein kinase in the panels. Moreover, RO3280 also showed higher efficacy against a series of mouse xenograft models. Additionally, RO3280 exhibited robust antitumor activity at a dose 40 mg/kg/week and reduced 72% of tumor growth, whereas at higher dose of RO3280 reduced tumor growth completely in vivo [RO3280 is a derivative of the pyrimidodiazepines series and is recognized as a PLK-1 inhibitor . RO3280  in vivo . RO3280-50 of 0.051 μM [ in vitro. However, compound 15 also inhibited other members of the PLK family, namely, PLK-2/3, with an IC50 of 0.172 μM and 1.382 μM, respectively. Compound 15-treated HCT116 colorectal carcinoma cell lines displayed monopolar spindles phenotype resemblance and mitotic arrest, which induced apoptosis [This compound is a derivative of a novel 2-amino-isoxazolopyridine and inhibits PLK-1 activity with an IC50 of 0.042 μM and an EC50 of 3.64 μM in vitro. Compound 38-treated HCT116 colorectal cancer cell line exhibited G2/M cell cycle arrest and lead to apoptosis. In addition, Compound 38 showed 50-fold more inhibition selectivity to PLK-1 over PLK-2 and PLK-3 [This compound is a derivative of 2-amino-pyrazolopyridine and is identified as a selective ATP-competitive type inhibitor of PLK-1 . Compounnd PLK-3 .50 of 0.8 nM and the other PLK family members, namely, PLK-2 and PLK-3, with an IC50 of 16.9 nM and 50.2 nM in vitro, respectively [ in vitro. Furthermore, TAK-960 was also evaluated against a wide range of cancer cell lines with a status of mutation in TP53 and KRAS, including multidrug-resistant cell lines (MDR1), and exhibited an EC50 in the range of 8.4 to 46.9 nM. TAK-960-treated HT-29 colorectal cancer cells displayed a monopolar spindle phenotype, and the cells were arrested in the G2/M phase, subsequently lead to apoptosis. Furthermore, TAK-960 inhibited a wide range of tumor xenograft models by using a dose of 10 mg/kg once daily for 2 weeks, which was selected on the basis of experiments with HT-29 colorectal cancer xenografts. In addition, TAK is well-tolerated an oral dose of 7.5 mg/kg once daily for 9 days and showed significant growth inhibition for an MV4-11 human leukemia model in-vivo [https://clinicaltrials.gov/ct2/show/results/NCT01179399).TAK-960 is a novel, potent, orally bioavailable and selective ATP-competitive inhibitor of PLK-1 . TAK-960ectively . Moreovehttps://clinicaltrials.gov/ct2/show/NCT01262235, February 20, 2015).TKM-080301, which was developed by Tekmira Pharmaceuticals Corp., is a lipid nanoparticle- (LNP-) based formulation of a (si)RNA that targeting human PLK-1 mRNA and has shown not only potent antiproliferative activity and gene-specific silencing against many human cancer cell lines but also potent antitumor activity against several xenograft models of human cancers. TKM-080301 is a highly selective inhibitor for PLK-1 without affecting the remaining members of the PLK-1 family. Moreover, it also demonstrated potential antitumor activity in implanted xenografts intrahepatically and subcutaneously. TKM-080301-mediated silencing of PLK-1 mRNA also persisted for up to 7–10 days/single administration without any notable stimulation of the innate immune response. TKM-080301 is highly restricted to the liver and spleen because the toxicity profile of TKM-080301 was governed by the LNP distribution. In a phase I study, the estimated MTD of TKM-080301 is 0.75 mg/Kg against various types of human cancers with hepatic metastases and showed tolerated side effects, including fever, rigors pyrexia, chills, nausea, vomiting, and fatigue. Moreover, TKM-080301 was well-tolerated at the MTD in a phase I study, and phase II study is currently underway. TKM-080301 has been administered intravenously against specific neuroendocrine tumors (NET) and adrenocortical carcinoma (ACC) , and a single point mutation in the catalytic site of PLK-1 (S67V) results in dramatic resistance to the structurally diverse ATP-competitive inhibitors . The PBD50 of 3.2 ± 0.3 μM, 1.7 ± 0.2 μM and 3.0 ± 0.1 μM, respectively, along with the inhibition of other phosphoserine/phosphothreonine-binding domains, such as the forkhead-associated (FHA) domain of CHK-2, WW domain of peptidyl-prolyl cis/trans isomerase (PIN1) and phosphotyrosine-binding domains (SH2 domains of STAT1/3/5 and lymphocyte-specific protein tyrosine kinase) [Poloxipan is a pan-specific inhibitor of the PBD that was isolated through the screening of chemical libraries. Poloxipan inhibits the PBD of PLK-1/2/3 with an IC kinase) .50 of 4.8 ± 1.3 μM in vitro and in vivo [50 of 18.7 ± 1.8 μM and 53.9 ± 0.8 μM, respectively. Moreover, poloxin-treated cells showed similar phenotypic appearances, such as catalytic inhibition of PLK-1 [Poloxin is a synthetic derivative of a well-known PBD antineoplastic inhibitor named thymoquinone, which was identified using a fluorescence polarization-based high-throughput screening assay against a wide library of 20,000 small molecules. Poloxin showed a better specificity profile in comparison to its parent molecule. Poloxin inhibited the PBD with an IC in vivo . Furtherof PLK-1 . Furtherof PLK-1 . The recof PLK-1 .50 of ~0.3 μM in a GST-pull down assay using the PBD as the target for WEE1 in vivo and in vitro. Moreover, PPG also inhibits PLK-2 but not PLK-3 [-XL-BH3 and BAD inhibition in comparison to PLK-1. Moreover, PPG-treated cells exhibited prolonged progression of mitosis and delay its onset. PPG-treated cells did not show the monopolar spindle like appearance, but exhibited improper chromosome alignment [PPG is a natural benzo tropolone compound derived from nutgall and oak bark. PPG was identified after high-throughput screening of 2500 natural compound repositories at RIKEN Saitama, Japan. PPG inhibits the PBD with an ICot PLK-3 . Furtherlignment . In addilignment .50 of 47.5 μM in vitro. Moreover, Aristolactam AIIIa also inhibited PBD-dependent binding with an IC50 of 10 μM in an SBR assay [This compound is a derivative of the natural compound Aristolactam, which was isolated from a natural product library by random screening against the PBD of PLK-1 . AristolBR assay . Xenopus, and yeast. The crystal structure of the PBD-phosphopeptide complex shows that the phosphopeptide binds within a positively charged cleft located at the edge of PBD interface. This cleft is essential for PLK-1 to recognize the substrate and to regulate PLK-1 [ in vitro and in vivo. Therefore, these studies collectively provide robust evidence that PBD-phosphopeptide binding is essential for PLK-1 targeting to recognize the substrate and also regulate the PLK-1. The inhibition of PBD leads to cell cycle arrest, inducing the apoptosis [PLK-1 was identified as a cell cycle-regulating mitotic kinase because the inhibition of PLK-1 caused the polo-like structure of the spindle fiber in the nucleus. The PBD domain is identified as a chemotherapeutic target for cancer based on its implication in PBD-dependent substrate targeting and subcellular localization of PLK-1. PBD has an evolutionarily conserved phosphoserine/phosphothreonine motif in humans,te PLK-1 . A loss poptosis .50 of 5 μM in a dose-dependent manner in vitro. Furthermore, MQSpTPL phosphopeptide is also recognized by the pincer grip-like pockets of PB1 and PB2, which are formed by Lys540 and His538 residues. These residues directly interact with the phosphate group of the phosphopeptide [This inhibitory peptide was designed after a study of the phosphopeptide-PBD complex. MAGPMQSpTPLNGAKK (Poloboxtide) was the first designed phosphopeptide, and it was successfully used in various biochemical assays. Poloboxtide inhibited the enzymatic activity of PLK-1 with an ICopeptide , 74. Moropeptide .This phosphopeptide was designed on the basis of the crystal structure of PBD-phosphatase CDC25C complex. LLCSpTPNG is recognized by the Trp414 residue of PB1. Moreover, loss-of-function studies revealed that the mutation of Trp414-Phe abolished the molecular recognition and subcellular localization of PLK-1to the centrosome , 75.D of 0.445 mM. Moreover, the phosphatase-resistant pThr mimetic -2-amino-3-methyl-4-phosphonobutyric acid- (Pmab-) containing peptide PLHS-Pmab bound to the PBD without reducing the affinity and specificity of the original peptide [This optimal phosphopeptide was designed on the basis of the molecular interaction of PBD and PBD-binding protein-1(PBIP1) with a high level of binding affinity and specificity via the PBIP1-p-T78 motif . Further peptide . As a co peptide . in vivo and in vitro, they also inhibit other vital protein kinases due to their similarity in the highly conserved catalytic domain. These inhibitors, therefore, inhibit the catalytic activity of targeted protein along with nontargeted proteins. Thus, these inhibitors should be validated against the 518 protein kinases of the human kinome to minimize adverse effects and maximize the selectivity during treatment or clinical trials.The essentiality of PLK-1 for normal cell cycle progression and mitosis has raised lingering questions regarding the targeting value of PLK-1 in cancer chemotherapy , 81, 82.PLK-1 is the most promising mitotic kinase validated as a chemotherapeutic cancer target because PLK-1 is critically essential for cancer cell survival and not for normal cells. The overexpression of PLK-1 is actively implicated in the failure of mitotic checkpoint arrest and tumor progression. Several studies collectively have proven that the (si)RNA-mediated severe inhibition of PLK-1 reduced tumor size without affecting the viability of normal cells . Therefo


Here we report that the salt-inducible kinases , members of the AMPK-related kinase family, play a key role as gluconeogenic suppressors downstream of LKB1 in the liver. The selective SIK inhibitor HG-9-91-01 promotes dephosphorylation of transcriptional co-activators CRTC2/3 resulting in enhanced gluconeogenic gene expression and glucose production in hepatocytes, an effect that is abolished when an HG-9-91-01-insensitive mutant SIK is introduced or LKB1 is ablated. Although SIK2 was proposed as a key regulator of insulin-mediated suppression of gluconeogenesis, we provide genetic evidence that liver-specific ablation of SIK2 alone has no effect on gluconeogenesis and insulin does not modulate SIK2 phosphorylation or activity. Collectively, we demonstrate that the LKB1–SIK pathway functions as a key gluconeogenic gatekeeper in the liver.LKB1 is a master kinase that regulates metabolism and growth through adenosine monophosphate-activated protein kinase (AMPK) and 12 other closely related kinases. Liver-specific ablation of LKB1 causes increased glucose production in hepatocytes  The liver is an important regulator of glucose homeostasis. Here, the authors provide insight into the molecular signalling pathways controlling hepatic gluconeogenesis by showing that SIK protein kinases suppress gluconeogenesis, and that glucagon—but not insulin—regulates phosphorylation of SIK2. In the postprandial state, these processes are largely suppressed by insulin released from pancreatic β-cells and the concomitant decrease in glucagon in response to elevated blood glucose and glucose itself. Deregulation of one or more of these processes contributes to development of metabolic disorders such as type 2 diabetes. Therefore delineating the molecular control of glucose homoeostasis is fundamental to combating the rising tide of metabolic disease in modern populations.Blood glucose concentration is tightly controlled in healthy humans to prevent the toxic effects of prolonged hyperglycaemia (diabetic complications) and the acute debilitating effects of hypoglycemia (coma). Two vital components to this control are the suppression of hepatic glucose output following a meal, and the induction of hepatic glucose production during prolonged fasting. The latter involves increased breakdown of stored hepatic glycogen and G6pc gene) and cytosolic phosphoenolpyruvate carboxykinase . Two major signalling pathways that suppress gluconeogenic gene transcription are the insulin signalling pathway and also putatively the LKB1/adenosine monophosphate-activated protein kinase (AMPK) pathway. Substantial evidence supports a model in which insulin, via Akt-mediated phosphorylation and inhibition of forkhead box protein-O (FoxO), reduces the expression of G6pc and Pck1 , an AMP mimetic per se but indicate that AMPK is not required for suppression of hepatic glucose production in response to metformin or feeding. Interestingly, there is now evidence for mitochondrial inhibition (independent of AMPK) influencing the action of the cyclic AMP (cAMP)-dependent protein kinase ) signalling pathway in the regulation of hepatic gluconeogenesis9A key point of control of hepatic gluconeogenesis is transcriptional regulation of genes encoding rate-controlling enzymes, including the catalytic subunit of glucose-6-phosphatase  by regulating phosphorylation of the cAMP-response element-binding protein (CREB)-regulated transcription co-activator-2 (CRTC2) by SIK2 , we focused on the Thr484 site. Moreover, Ser379 is not conserved between human (MHSPNMRLL) and mouse (LHPPNVRLM) and was not further studied. The LC-MS-MS analysis coverage of HA–SIK2 was ~80% of the total sequence for all conditions studied (Recombinant haemagglutinin (HA)-tagged SIK2 was expressed in mouse primary hepatocytes using adenovirus and the cells were left untreated or treated with glucagon or insulin. HA–SIK2 protein was affinity-purified , in-gel  studied . To vali studied . The eff studied . In all  studied  and aden studied  was robu studied . Enhance studied . Similar studied  as well  studied . Althoug studied , we fail studied  of SIK2  studied .in vitro2-cAMP). SIK2 was phosphorylated on Ser358 in response to Bt2-cAMP in both genotypes, but not in cells treated with insulin (Previous work reported that SIK2 Ser358 phosphorylation (as measured using a phospho–Akt substrate antibody) was increased by insulin through the PI3K–Akt2-dependent pathway in mouse liver tissue and primary hepatocytes, and that this was associated with an increase in SIK2 catalytic activity measured  insulin . To furt insulin . As anti insulin , CRTC2 d insulin .in vitro kinase assays performed. As shown in To determine if glucagon-induced phosphorylation of SIK2 had any impact on its catalytic activity, endogenous SIK2 was immunoprecipitated from cells incubated with or without hormones, and in vitro after immunoprecipitation. Since we cannot rule out the possibility that glucagon-mediated phosphorylation of SIK2 may modulate its downstream targets in intact cells without altering intrinsic activity, we overexpressed single or multi-mutants (3A or 4A) in mouse primary hepatocytes and assessed phosphorylation of CRTC2/3 and histone deacetylase 4 . All the single mutants tested showed no effect on glucagon-mediated dephosphorylation. However interestingly, in SIK2 3A or 4A mutant-expressing cells glucagon-induced dephosphorylation of CRTC2/3 and HDAC4 was prevented  and liver-specific SIK2fl/flCre+/− (KO) mice are indistinguishable from SIK2+/+ (WT) mice, displaying normal growth and no overt phenotype. Immunoblot analysis showed that SIK2 protein expression  was comparable between the SIK2+/+ and SIK2fl/flCre−/− mice, while a profound liver-specific depletion of SIK2 protein was observed in SIK2fl/flCre+/− mice  in primary hepatocytes was not altered by genetic deletion of SIK2  and lipogenic  and hepatic PEPCK protein levels were similar between control and triple KO (TKO) mice (2-cAMP- or glucagon-induced glucose production were comparable between control and the triple AMPKα1/α2/SIK2-deficient hepatocytes (2-AMP or glucagon) conditions was comparable between the triple AMPKα1/α2/SIK2-deficient and control hepatocytes  expression was comparable between control and the TKO hepatocytes in unstimulated or stimulated (Bt2-AMP or glucagon) conditions  mice . Consistatocytes . CRTC2 gatocytes . In contatocytes  as previnditions .These results highlight that deletion of AMPK or SIK2, alone or in combination, are not sufficient to mimic the alteration in the regulation of gluconeogenesis , 3, 4, 54in vitro and in intact cells (macrophages)The previously described experiments suggest that SIK2 is dispensable for control of hepatic gluconeogenesis, perhaps because loss of SIK2 can be compensated by other SIK isoforms (but not by AMPK). To fully delineate the role of SIKs in hepatic gluconeogenesis, it would be necessary to ablate the activity of all SIK isoforms in the liver. We took advantage of recently identified pan-SIK inhibitors whose potency and specificity have been well-characterized We first performed a dose-response (0.1–4 μM) and time-course (4–12 h) study to determine the concentration of SIK inhibitor (HG-9-91-01) that is required for a robust and sufficient suppression of SIK activity in mouse primary hepatocytes. We confirmed that 4 μM is the saturating concentration required for promoting CRTC2 dephosphorylation and glucose production in mouse primary hepatocytes (data not shown). We assessed SIK activity by monitoring the phosphorylation state of CRTC2 as indicated by band-mobility shift and observed that HG-9-91-01 caused a dose-dependent increase in a faster migrating form of CRTC2 (dephosphorylation) with a reciprocal reduction in the slower migrating phosphorylated form . CRTC2 dIt has been reported that in unstimulated hepatocytes, CRTC2 is highly phosphorylated and mostly localized in the cytoplasm. Upon treatment with glucagon or stimuli that induce cellular cAMP elevation, CRTC2 is dephosphorylated and translocates into the nucleus11Pck1 and G6pc and that effect was similar in cells treated with 4 μM HG-9-91-01 or 0.1 μM glucagon  would not display an additive increase in glucose production compared with individual treatments. Contrary to our hypothesis, there was a significant additive increase in glucose production when the two agents were given to hepatocytes simultaneously , which wThe possible mechanism behind this additive effect was investigated by measuring the phosphorylation of glucagon-regulated signalling molecules proposed to be involved in hepatic gluconeogenesis. In line with the observations shown in previous studies, glucagon-induced phosphorylation of CREB, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (PFKFB1) and IP3R , which a1019LKB1-dependent kinases, including AMPK and SIKs20Catalytic activity of SIK requires LKB1-dependent T-loop phosphorylation and thus SIK activity in LKB1-deficient HeLa cells and mouse embryonic fibroblasts is profoundly reducedin vitro kinase assay, respectively. We observed that SIK2 protein expression was comparable between control and LKB1-deficient liver, while T-loop (Thr175) phosphorylation was undetectable in liver extracts derived from liver LKB1 KO mice  or GFP alone (control) and the cells were then left untreated, treated with HG-9-91-01 (1 or 4μM) or with glucagon (0.1 μM) . In untr2 mutant , demonstet al.et al.in vivo.Previous work by Dentin We believe that the identification of Ser358 as a potential Akt phosphorylation site in SIK2 in the previous workpSer/Thr, Xaa, any amino acid) and the phospho–PKA substrate-motif (Arg–Arg–Xaa–pSer/T) antibodies both detect multiple phosphorylation sites on SIK2 due to their overlapping epitopes. To eliminate glucagon-induced phosphorylation detected using these antibodies we had to simultaneously mutate three  or four (3A and Ser587) sites on SIK2  of the glucagon-induced phosphorylation sites had no effect on SIK2 activity and did not alter phosphorylation in the activation T-loop Thr175 site  significantly increases hepatic gluconeogenesis in primary hepatocytes. On the other hand, we also provided genetic evidence that ablation of SIK2 in the liver does not affect hormone-mediated changes in gluconeogenesis and we confirmed that there were no compensatory changes in the activity of other SIK isoforms (SIK1/3) in SIK2-deficient liver. These results suggest that SIK2 has no significant role in the control of hepatic gluconeogenesis, or alternatively, there is a degree of redundancy among the SIK isoforms that does not require upregulation of SIK protein. To test these hypotheses we performed short hairpin RNA interference -mediated knockdown of the SIK isoforms (singly and in combination) using adenovirus infection of SIK2-null mouse primary hepatocytes. However, we could achieve only ~80% knockdown of SIK1 or 3, which did not result in significant changes in CRTC2 phosphorylation and the amount of adenovirus (scramble control) required for dual (SIK1&3) knockdown itself affected glucose production . Therefore, this approach could not provide compelling evidence to conclude whether there is a dominant SIK isoform or whether SIK isoforms play a redundant role in the control of hepatic gluconeogenesis. As a next step, it would be interesting to generate and analyze liver-specific SIK1 or SIK3, and if viable, double (SIK1/3) or TKO (SIK1/2/3) mouse models.2-cAMP, but not the SIK inhibitor promoted glucose production in LKB1-deficient hepatocytes (2+ through phosphorylation of IP3R (Ser1756), which in turn activates calcineurin  leading to dephosphorylation and nuclear translocation of CRTCs, and a resultant activation of CREB-regulated gluconeogenic genes in hepatocytes2+-CaMKII-mediated FoxO1 nuclear localization101924A key finding of this study is that the cAMP–PKA pathway and SIKs appear to regulate CRTCs and gluconeogenesis at least partially through distinct mechanisms. This notion is based on the following: 1) Btatocytes , suggest BtatocytIn summary, we provide evidence that SIK isoforms significantly contribute to suppression of hepatic gluconeogenesis by regulating downstream transcription factors/co-activators and that glucagon (but not insulin) can regulate phosphorylation of SIK2 . However32P]-labelled ATP was from Perkin Elmer . Precision Plus protein markers and SsoFast EvaGreen Supermix were from Bio-Rad . Insulin-like growth factor 1 (IGF-1) was from Cell Signaling Technology . Protease inhibitor cocktail tablets were purchased from Roche . Anti-HA-agarose, anti-FLAG-agarose, collagenase , triiodothyronine, Bt2-cAMP and lactate were from Sigma-Aldrich . Infinity glucose assay kit was from Thermo Scientific . Insulin and glucagon were from Novo Nordisk . Cell culture media and reagents including Dulbecco’s modified eagle medium (DMEM) and M199 with glutamax were from Life Technologies . All other chemicals unless specified were obtained from Sigma-Aldrich.Forskolin, MK-2206 and PI-103 were from Tocris . Protein G-Sepharose, glutathione-Sepharose and enhanced chemiluminescence reagents were purchased from GE Healthcare . -labelled ATP. Control reactions either lacked purified kinase or contained pre-immune immunoglobulin G-coupled beads. Phosphorylation reactions were initiated by adding reaction mix to the protein kinase and incubating on a thermomixer at 30 °C for 20 min. These reactions were terminated by spotting 40 μl of mixture onto P81 phosphocellulose paper and immersing them in 75 mM orthophosphoric acid. P81 papers were washed three times for 10 min with 75 mM orthophosphoric acid and finally in acetone for 5 min. P81 papers were air-dried and the incorporation of 32P into peptide substrate was determined by Cherenkov counting. Results were expressed in mU mg−1 protein where 1 mU equals 1 pmol of phosphate incorporation into a substrate in 1 min at 30 °C (refs SIK isoforms were immunoprecipitated from liver tissue/hepatocyte extracts (0.5–2 mg). SIK activity assay was performed in a final volume of 50 μl containing 15 μl of protein kinase (immune purified/precipitated) and 35 μl of reaction mix. The reaction mix contained 200 μM of Sakamoto-tide peptide (ALNRTSSDSALHRRR), 50 mM HEPES, 1 mM EGTA, 10 mM magnesium acetate and 0.1 mM [γ °C refs , 39.Primary mouse hepatocytes were fixed on glass cover slips with 3.7% paraformaldehyde after the treatment indicated in figure legends. Cells were washed three times with PBS containing 5% (w/v) BSA followed by incubation with 0.1% NP-40 in PBS for 5 min. Subsequently, cells were blocked with PBS containing 5% (w/v) BSA for 20 min and incubated with anti-rabbit SIK2 antibody  or anti-rabbit CRTC2 antibody  at a dilution of 1:500 for 1 h at 37 °C. Excess antibodies were removed by washing the cells three times with PBS containing 5% (w/v) BSA. This was followed by a 30 min incubation with secondary antibody (Alexa Fluor 488). Subsequently the cells were further washed three times with PBS containing 5% (w/v) BSA before mounting the cover slips on a glass slide with mounting media containing 4′,6-diamidino-2-phenylindole. Cells were later analyzed with a Nikon Eclipse Ti fluorescent microscope and images were captured using Nikon NIS Elements BR 3.1 software.t-test or one-way or two-way analysis of variance using Tukey’s or Dunnet’s test for multiple comparisons where appropriate. Differences between groups were considered statistically significant at P<0.05.Data are expressed as mean±standard deviation or standard error. Statistical analysis was performed using Student’s K.S. and M.F. came up with the concept of the study. K.S., M.F., C.S. and K.P. designed the overall experiments. K.P. performed the experiments and analysis shown in Accession codes: The mass spectrometry data has been deposited to the ProteomeXchange ConsortiumHow to cite this article: Patel, K. et al. The LKB1-Salt-inducible kinase pathway functions as a key gluconeogenic suppressor in the liver. Nat. Commun. 5:4535 doi: 10.1038/ncomms5535 (2014).Supplementary Figures 1-12


Drosophila serves as a paradigm for gradient formation in textbooks. To explain the generation of the gradient, the ARTS model, which is based on the observation of a bcd mRNA gradient, proposes that the bcd mRNA, localized at the anterior pole at fertilization, migrates along microtubules (MTs) at the cortex to the posterior to form a bcd mRNA gradient which is translated to form a protein gradient. To fulfil the criteria of the ARTS model, an early cortical MT network is thus a prerequisite. We report hitherto undiscovered MT activities in the early embryo important for bcd mRNA transport: (i) an early and omnidirectional MT network exclusively at the anterior cortex of early nuclear cycle embryos showing activity during metaphase and anaphase only, (ii) long MTs up to 50 µm extending into the yolk at blastoderm stage to enable basal-apical transport. The cortical MT network is not anchored to the actin cytoskeleton. The posterior transport of the mRNA via the cortical MT network critically depends on maternally-expressed αTubulin67C and the minus-end motor Ncd. In either mutant, cortical transport of the bcd mRNA does not take place and the mRNA migrates along another yet undisclosed interior MT network, instead. Our data strongly corroborate the ARTS model and explain the occurrence of the bcd mRNA gradient.The Bicoid (Bcd) protein gradient in  Drosophila blastoderm egg Synthesis, Diffusion and Degradation) model proposes that the gradient arises through translation of an anteriorly-localized bcd mRNA source, followed by diffusion of Bcd throughout the embryo, and uniform degradation. In contrast, the ARTS (Active RNA Transport and Synthesis) model proposes that the bcd mRNA is actively transported along the cortex in a posterior direction from its anterior pole to form an mRNA gradient which serves as template for the synthesis of Bcd.The Bicoid (Bcd) protein is a paradigm for morphogen gradient formation taught in textbooks and studied for more than two decades. The hallmark of the Bcd morphogen is its spectacular gradient along the anterior-posterior axis of the early a priori that the SDD model is the correct one. Furthermore, the model still lacks a direct proof of the existence of long-range Bcd diffusion, e. g. by tracking single Bcd molecules during the early nuclear cycles. Equally important to note: the ARTS model does not argue against a high diffusion coefficient of Bcd, it is only largely irrelevant for the model.Recently, the SDD model encountered some severe difficulties: the diffusion coefficient of Bcd was found to be two orders of magnitude too low to establish a steady-state Bcd gradient by the blastoderm stage bcd mRNA gradient, which is the hallmark of the ARTS model, was first described in 1986 bcd mRNA distribution during early Drosophila embryogenesis bcd mRNA gradient forms from a bcd mRNA source that at fertilization is strictly localized to the anterior pole of the embryo.The existence of a bcd mRNA during oogenesis, using in vivo imaging of the movement of the bcd mRNA bcd mRNA by the nurse cells into the oocyte starts at stage 10b, accompanied by an active cytoplasmic movement within the oocyte  there is an extensive MT network exclusively in the anterior half, (ii) this MT network is only formed at the cortex, and (iii) Taxol induces artefacts giving the impression that the egg contains a dense and ubiquitous network of cortical MTs. Further, we show that bcd mRNA gradient in DrosophilaLucilia sericata, the gradient appears very similar  mutation) αTub67C is required for the formation of long MTs bcd mRNA transport, embryos do not show any cortical transport, rather the mRNA formed a long streak in the interior of the embryo in parallel to the A-P axis  locus encoding an unconventional Kinesin which transports cargos to the minus-end αTub67Cncd allele, 1ncd which again showed a large bcd mRNA streak  a permeabilization and fixation protocol permitted to overcome the limitations of rapid fixation of MT structures in the Drosophila egg, and 2) an antibody with an excellent signal-to-noise ratio that detected tyrosinated Tubulin.The ARTS model bcd mRNA molecules from the yolk to the apical side of the nuclei at early nc 14 Initially, visibility of the cortical network was limited to a short 2 minute-window which let us conclude that it is a highly dynamic and short-lived MT network, consistent with the reported specificity of the mab against tyrosinated Tubulin 1,2. Mature tubulin shows this terminal tyrosine cleaved off, often referred to as Glu-Tubulin, using an enzyme, Tubulin tyrosine carboxypeptidase (TTCP). If necessary, tyrosination of the de-tyrosinated end using an enzyme, Tubulin tyrosine ligase (TTL) could convert the COOH-end back to its original status , most likely because there is no ‘conventional’ microtubule organizing center (MTOC) at the cortex. Notably, the same minus-end markers usually detected the MTOCs associated with the internal nuclei without difficulty. Conversely, antibodies against plus-end markers do not stain well following our improved fixation method. We were also unable to stain the MT threads using Tubulin-GFP-constructs (unpublished), most likely due to the fact that the signal from their fine structures is difficult to detect within the autofluorescence of the yolk. The only protein that co-stains the cortical MT network is Chromosome bows (Chb), formerly called Mast/Orbit/CLASP bcd mRNA transport is concerned, the velocity of Staufen-bcd-RNP complexes was measured in the oocyte, resulting in values of 0.36 to 2.15 µm/sec. bcd mRNA within minutes from the anterior to the posterior pole. As was discussed for the ARTS model oskar mRNA particle transport As far as the velocity of the αTub67C bcd mRNA bcd mRNA, thus making the claim of a basal degradation of the mRNA without apical transport inconsistent. How do we reconcile these opposing results? We surmise that the discrepancy between our result demonstrating the presence of an apical mRNA and their result showing absence was caused by the fact that many embryos in the Little et al. analysis revealed poor preservation of the periplasm which appeared largely rubbed off, best exemplified in embryo E and F of their Fig. 5, respectively. Consequently, little or no fluorescent signal could emerge from the missing tissue. As far as the discrepancy of a long versus a short mRNA gradient is concerned, we surmise (i) a similar cause owing to the rubbed periplasm, and (ii) possibly a general loss of mRNA of the poorly-preserved embryos. Indeed, the long mRNA gradient was most conspicuous from the apical mRNA species bcd mRNA increases with posterior migration such that the posterior-most particles harbour the longest poly(A) tail. As a consequence, only few mRNA molecules would be required to efficiently translate the Bcd protein in more posterior regions, molecules that current in situ hybridization protocols are unable to detect. Interestingly, Wispy, a poly(A) polymerase was recently shown to be part of the bcd ribonucleoprotein particle bcd mRNA. Like this, the poly(A) tail length of the bcd mRNA does not only vary with time bcd mRNA gradient. Further, we provide evidence of a long-range transport of bcd mRNA particles up to 40% EL bcd mRNA gradient forms, is answered and confirms the earlier postulate of a MT-mediated transport To take up the question that was asked in the introduction: we demonstrate that the molecular structures which the ARTS model predicted exist and described two proteins that play critical roles for the transport machinery that form the rX21kavar, referred to as nullkavar, the only known null allele of αTub67C is described in Df(3L)55, to generate embryos without functional αTub67C activity kavar alleles were described in 1, αTub67C2αTub67C and 3αTub67C alleles were described in Dncd, 1ncd and 3 ncdDαTub67C double mutants in 3, ncdD/ncdDαTub67C embryos were obtained by crossing 3 ncdDαTub67C/+females to D/ncdDncd males, as described by 05884ncd allele and the over-expression P(EPgy2)EY03397ncd allele were obtained from Bloomington. The maternal GAL4-driver line V32 was obtained from the Perrimon lab.In order to visualize the cortical network, we modified a recent permeabilization protocol without heptane After dechorionation, embryos were subjected to 10 µg/ml Taxol for 2 minutes, or to a 50 µg/ml Colcemide/20 µg/ml Colchicine mixture for 10 minutes in permeabilization buffer 1,2 against tyrosinated tubulin (Millipore) was used at 1∶2000. Anti-αTub67C rabbit polyclonal antibodies were made against a peptide from amino acids (aa) 35–61 of αTub67C, affinity-purified and used at 1∶400. Anti-αTub84B+D guinea pig polyclonal antibodies were made against the last 15 aa including the tyrosine residue of the common COOH-termini of αTub84B and αTub84D, respectively, and used as crude serum at 1∶300. DM1A was used as a FITC-conjugate (Sigma) at 1∶50 sequentially to mab YL1,2 in 1,2, we preferentially used 2nd antibodies coupled to 594 nm fluorochromes to obtain an optimal signal-to-noise ratio. All pictures were recorded on a Zeiss LSM 710.Mab YL0–2 h extracts were separated on 12% PAGE and probed with Tubulin-specific mab’s and a polyclonal antiserum using standard methods, as described in in situ hybridization was used according to Fluorescent For colour conversion and interpretation of signal intensities in Figure S1bcd mRNA gradients in Diptera. (A) a Drosophila embryo at fertilization hybridised with a bcd probe and alkaline phosphatase to reveal the strict accumulation of the mRNA at the anterior pole. (B) a Drosophila nc 14 embryo hybridised with a bcd probe and alkaline phosphatase showing an extended gradient. (C) a Lucilia sericata nc 14 embryo hybridised with a bcd probe and alkaline phosphatase showing an extended gradient. (D) a Lucilia sericata nc 14 embryo hybridised with a bcd probe using fluorescence. Methods and colour conversion as in (TIF)Click here for additional data file.Figure S2Mislocalization of the bcd mRNA in αTub67C and motor protein mutants. (A) a nc 1 1/αTub67C1αTub67C embryo, stained for the bcd mRNA (green) along with DAPI (blue) to reveal a streak of the mRNA. (B) a nc 5 2/αTub67C2αTub67 embryo, stained for the bcd mRNA (green), along with DAPI (blue). (C) 3-D reconstruction of the tip of a nc 5 3/αTub67C3αTub67Cembryo, stained with mab YL1,2 (red) and DAPI (blue) to reveal a dense MT network and aster-like MT bundles without nuclei (asteriks). The positions of the nuclei are indicated with yellow arrows, one normal metaphase nucleus is indicated with a white arrow. A movie of this 3-D construction is available as 21GKavar/+ embryo, stained for the bcd mRNA (green), along with DAPI (blue). (E) a nc 1 18CKavar/+ embryo, stained for the bcd mRNA (green), along with DAPI (blue). (F) a nc 3 05884ncd/05884ncd embryo, stained for the bcd mRNA (green) along with DAPI (blue). (G) wild-type stage 10 oocyte, stained for the bcd mRNA (green), along with DAPI (blue). (H) nullkavar/Df(3L)55 stage 10 oocyte, stained for the bcd mRNA (green), along with DAPI (blue). The anterior localization is largely normal.(TIF)Click here for additional data file.Figure S3Specificity of Tubulin antibodies. Western analysis of 0–2 h embryonic extracts Click here for additional data file.Video S1An exclusive anterior MT network. Video of the 3D-reconstruction of the confocal stack used in (ZIP)Click here for additional data file.Video S2Independence of the early MT network from the actin sheet in a nc 2 embryo. Video of the 3D-reconstruction of the confocal stack used in (ZIP)Click here for additional data file.Video S3Independence of the early MT network from the actin sheet in a nc 6 embryo. Video of the 3D-reconstruction of the confocal stack used in (ZIP)Click here for additional data file.Video S4Asters and intensive MT network activity at the cortex of 3αTub67C embryos. Video of the 3D-reconstruction of the confocal stack used in (ZIP)Click here for additional data file.


In a subsequent antiviral assay, the cytopathic effect of EV71 was significantly and dose-dependently relieved by the administration of luteoloside . Using a plaque reduction assay, we administered luteoloside at various time points and found that the compound reduced EV71 viability in RD cells rather than increasing defensive mobilization or viral absorption. Moreover, biochemical studies focused on VP1  mRNA transcript and protein levels also revealed the inhibitory effects of luteoloside on the EV71 viral yield. Finally, we performed inhibition assays using luteoloside to evaluate its effect on recombinant 3C protease activity. Our results demonstrated that luteoloside blocked 3C protease enzymatic activity in a dose-dependent manner (IC50 = 0.36 mM) that was similar to the effect of rutin, which is a well-known C3 protease inhibitor. Collectively, the results from this study indicate that luteoloside can block 3C protease activity and subsequently inhibit EV71 production in vitro.Luteoloside is a member of the flavonoids family that exhibits several bioactivities including anti-microbial and anti-cancer activities. However, the antiviral activity of luteoloside against enterovirus 71 (EV71) and the potential mechanism(s) responsible for this effect remain unknown. In this study, the antiviral potency of luteoloside against EV71 and its inhibitory effects on 3C protease activity were evaluated. First, we investigated the cytotoxicity of luteoloside against rhabdomyosarcoma (RD) cells, which was the cell line selected for an  Luteoloside is a member of the flavonoids family and was extracted from several traditional Chinese herbs –4 and otPicornaviridae family and is the causative agent of hand, foot, and mouth disease (HFMD) in infants and young children. Without timely or effective treatments, EV71 induces severe nervous and respiratory system complications that lead to poor prognoses and high fatality rates  and H2O2 and monitored at A405. To determine the inhibitory effects, 2-fold dilutions of luteoloside were added to the 3C protease analysis system, and the 3C protease inhibition was defined as 100%×(AHRP+drug+3C protease-AHRP++3C protease)/(AHRP+drug-AHRP+3C protease). The dose that elicited an inhibition of 50% (IC50) was also calculated [The activity of the 3C protease was analysed according the method detailed in a previous study with slight modifications . Brieflylculated . Rutin . The asterisks indicate significance at the P<0.05 level.All data are expressed as the means ± the SEs. The statistical significances of the differences in the mean values were assessed with P<0.05). However, at ≥3 mM, luteoloside was significantly toxic to the RD cells (P<0.01) with a CC50 of approximately 2.3 mM. Based on these results, we identified the nontoxic concentration range of luteoloside for RD cells.The molecular structure of luteoloside is illustrated in RD cells , P<0.01 P<0.05). However, the other concentrations of luteoloside elicit only slight mitigations of the CPE induced by the virus . In contrast, when the compound was administered at 12–24 hpi, we observed limited inhibitory effects against EV71. Thus, the inhibitory effects of luteoloside against EV71 were only elicited following administration within the first 12 hours after infection, which suggests that luteoloside inhibits EV71 yield during the early stage of infection.To investigate the antiviral effects of luteoloside, the compound was added to EV71-infected RD cells at various time points (0–24 hpi). As illustrated in P<0.05). The addition of luteoloside prior to or simultaneously with viral inoculation , which is a well-known 3C protease inhibitor [50 of luteoloside was 0.36 mM. Based on the results of our HRP-based enzymatic assay, luteoloside appears to inhibit the 3C protease in a dose-dependent manner in vitro.To identify the potential antiviral mechanism(s) and target(s) of luteoloside, the effects of the compound (0–0.5 mM) on 3C protease activity were investigated. For this enzymatic assay, we used an HRP type VI-A containing a 3C protease cleavage site (Gln-Gly) as the 3C protease substrate. As illustrated in nhibitor . All of Lonicera japonica is a well-known traditional Chinese herb that has been widely used in HFMD treatments [in vitro.eatments  due to ieatments . Luteoloeatments ,2. Howevin vitro [RD cells have previously been demonstrated to be sensitive to EV71 infection , and EV7in vitro . CollectVP1 mRNA and protein syntheses. The VP1 protein contains the majority of the EV71 epitopes and is used to detect EV71 infections [Time course analysis indicated that the addition of luteoloside at 0–9 hpi significantly inhibited the EV71-induced CPE, which contrasts with the minimally inhibitory effect observed following the addition of luteoloside at 12–24 hpi . Previoufections ,47. In t50 = 1.19 mM) of rutin on RD cells are illustrated. Rutin inhibits EV71 replication by blocking 3C protease activity (IC50≈0.4 mM) [50 of luteoloside was 0.36 mM, which is approximately equal to the EC50 calculated based on the in vitro RD cell assay; these findings further support the antiviral effects of luteoloside. Several antiviral compounds have been reported to act as 3C protease inhibitors and antagonists [The 3C protease plays a major role in the EV71 life cycle; it cleaves the precursor polyprotein into structural and functional proteins  and acti≈0.4 mM) . The resagonists . The antagonists . The abiIn summary, this study indicated that luteoloside has antiviral effects against EV71 that are mediated by blocking 3C protease activity, and these effects subsequently inhibited EV71 production. These results suggest that luteoloside may be a moderately potent agent that could be utilized for the treatment of EV71 infection.S1 Filen = 3). Asterisk meant the data differed from the blank (0 mM) significantly at P<0.05 level according to t-test.Click here for additional data file.