The earth is warming at an alarming rate, especially in the Arctic, where a marked decline in sea ice cover may have far-ranging consequences for endemic species. Little auks, endemic Arctic seabirds, are key bioindicators as they forage in the marginal ice zone and feed preferentially on lipid-rich Arctic copepods and ice-associated amphipods sensitive to the consequences of global warming. We tested how little auks cope with an ice-free foraging environment during the breeding season. To this end, we took advantage of natural variation in sea ice concentration along the east coast of Greenland. We compared foraging and diving behaviour, chick diet and growth and adult body condition between two years, in the presence versus nearby absence of sea ice in the vicinity of their breeding site. Moreover, we sampled zooplankton at sea when sea ice was absent to evaluate prey location and little auk dietary preferences. Little auks foraged in the same areas both years, irrespective of sea ice presence/concentration, and targeted the shelf break and the continental shelf. We confirmed that breeding little auks showed a clear preference for larger copepod species to feed their chick, but caught smaller copepods and nearly no ice-associated amphipod when sea ice was absent. Nevertheless, these dietary changes had no impact on chick growth and adult body condition. Our findings demonstrate the importance of bathymetry for profitable little auk foraging, whatever the sea-ice conditions. Our investigations, along with recent studies, also confirm more flexibility than previously predicted for this key species in a warming Arctic. Odobenus rosmarus), polar bears (Ursus maritimus) or ice-associated amphipods [The release of anthropogenic greenhouse gases into the atmosphere leads to climate warming on a worldwide scale . Consequmphipods ,4. Modifmphipods . These bmphipods –8. are endemic to the Arctic and the most abundant seabird in the North Atlantic Arctic, with an estimated population of 40–80 million individuals [Little auks ividuals . Recent ividuals –13. Beyoividuals –18, a beividuals . Moreoveividuals . Such orividuals , and litividuals ,12. Yet ividuals . In partividuals ,23. Aggrividuals –26. In tIn this study, we tested the hypothesis that little auk foraging behaviour during the breeding season is affected both by the presence/absence of sea ice and bathymetry. To this end, we looked at the effects of these two factors on (1) little auk foraging location and diving activity, and (2) zooplankton species composition of chick diet. We also investigated their impact on (3) chick growth and adult body condition.To test this hypothesis, we studied little auks from the breeding colony of Ukaleqarteq (Kap Höegh), located in East Greenland where foraging conditions are influenced by the East Greenland current carrying large volumes of Arctic sea ice southwards. Importantly, there is a strong inter-annual variability in this sea ice drift, allowing us to compare little auk foraging behaviour in the presence/nearly absence of sea ice within their foraging range . To this end, we used a multidisciplinary approach, combining satellite remote-sensing of sea ice concentration, land-based studies of little auk foraging behaviour and reproductive performance, and at-sea observations of little auk distribution and sampling of their zooplankton prey.The R software version 3.0.2 was used for numerical and statistical analyses . QGis 2 was usedFieldwork took place in Ukaleqarteq , measured (head-bill and wing length to the nearest millimeter), fitted with a metal ring for individual identification and equipped with a data logger . Additional breeding birds  were captured and measured only following the same procedure to access their body condition. All birds were released within less than 10 minutes of capture. The breeding status of adult birds was ascertained via the presence of a full gular pouch , of a brood patch, or through the presence of a chick at the nest. When birds had a full gular pouch, chick diet was collected following Harding et al [® tape . Devices were either removed upon recapture after 3–10 days, or fell off during the complete moult which immediately follows the breeding season in little auks [GPS-recorders or Temperature-Depth-Recorders (TDR) were attached dorsally (GPS) or ventrally (TDR) to feathers with Tesatle auks . Two GPSSV Argelvor between August, 16–18 2014, towards the end of the chick-rearing period. No sea ice was encountered during the whole survey. Two transects were performed, one at the latitude of the colony and the other 0.29° further North, from the coast to 135 km offshore. Along each transect, 10 plankton samples were collected using a WP2 net . Vertical net hauls were performed from 50 m depth to the surface with a manual winch at constant speed . Data from the Global Sea Ice Concentration reprocessing dataset, with a grid resolution of 12.5km, were used for the period 1978–2014 (http://www.natice.noaa.gov/products/daily_products.html) and for each map, we presented the daily ice extent on the day for which we had more tracks recorded.Daily sea ice concentrations were downloaded from the Eumetsat OSI SAF website . For ea-1  and a UTTDR data were analysed using MultiTrace . Dive duration, maximum dive depth and the number of foraging dives per 24h were measured. To compare depth data from different TDR types, we recalibrated them in a pressure chamber. We found that DST micro-TD underestimated real depth and the following correction was therefore applied on depth data before analyses: Depth = 1.0473 * Depth(Star Oddi) + 0.4498 (1).Zooplankton sampled at sea and in gular pouches was identified under a stereo microscope, to the lowest-possible taxonomic level using –35. Cala3) of the 3 Calanus species over the continental shelf, the shelf break and the open ocean.To compare species composition between the different at-sea sampling locations, the Bray-Curtis distance was calculated and a classification tree was constructed using Ward’s method in the R package ‘Vegan’ ,37. We ai−Ei, which is the difference between the relative abundance of prey i in the gular pouch GPi, and in the environment Ei [i and Ei which were assumed to have a normal distribution of observed means and standard deviations.The linear food selection index (LFSI) was calculated as LFSI = GPnment Ei . This inWe compared chick growth during the linear growth period  did not differ between years  were the main prey type in both years (85% in 2012 and 90% in 2014), but proportion of the three Calanus species present in the diet differed between years. The largest species C. hyperboreus was more abundant in 2012 (t(38) = 2.43, p = 0.02), whereas the smaller C. glacialis and C. finmarchicus were more abundant in 2014  = -5.28, p < 0.001 and t(38) = -2.75, p < 0.001 respectively). Further, ice-associated amphipods represented nearly 10% of the diet in 2012 and were virtually absent in 2014. In particular, the amphipod Apherusa glacialis represented up to 96% of single food loads in 2012, and only a few single specimens were found in 2014. The linear food selection index  were also avoided by parent little auks to feed their chick  in the East Bering Sea [Information provided by GPSs and at-sea observations show that little auks foraged preferentially on the shelf break and on the continental shelf Figs  and S1. nean Sea , black p Zealand , or blaclf slope . In addiring Sea , and plaring Sea ,49. Concring Sea . This diC. hyperboreus and C. glacialis , for instance, resting at the sea surface was estimated to be 50% more costly, energetically, than resting on sea ice [Further, sea ice is also important for little auks and other Arctic seabirds as a platform to rest during foraging trips , and its sea ice  and the  sea ice . This co sea ice . In our Birds equipped with GPSs showed significantly longer trip durations when compared with literature data for the same colony . MoreovCalanus finmarchicus [C. finmarchicus are smaller and contain less lipids than the two Arctic copepods C. glacialis and C. hyperboreus. Moreover, higher temperatures are expected to favour smaller zooplankton, both smaller species and smaller individuals within a species [C. finmarchicus in their environment  changes in the behaviour of their predators. For instance, enhanced polar bear predation on seabirds has been demonstrated in recent years , see alsarchicus . C. finm species ,73. Such species . While as study, ), the fos study, .Comparative studies conducted across the Greenland Sea do indicate that little auks have for now the capacity to buffer the consequences of current ocean warming, through marked foraging plasticity ,76. FurtS1 Fig-1) and black dots to travelling (instant speed >10 km.h-1). Sea ice extent data were downloaded from the U.S. National Ice Center (http://www.natice.noaa.gov/products/daily_products.html). White: pack ice with an ice concentration >80%. Light blue: marginal ice zone (MIZ) with an ice concentration <80%. In the marginal ice zone, sea ice concentration decreased between pack ice and open water. Only 1 out of 4 trips were complete, thus we did not include these tracks in our analyses. For the complete track, the maximum distance to the colony was 84.3 km and the trip duration was 22.4h. Projection: GR96/ UTM zone 27N.Red dots correspond to foraging or resting (speed <10 km.h(PDF)Click here for additional data file.S2 FigChicks were weighed every second day. Chick growth was compared between years during the linear growth period (delimited by dotted lines) using a linear mixed effect model with mass as an explanatory variable, chick age and year as fixed factors and chick as a random factor. Model selection process retained model with chick age as fixed factor and chick as random effect. Year factor was rejected meaning that there is no difference of chick growth between years.(PDF)Click here for additional data file.S3 Fig1,183 = 0.064, p = 0.8).There was no difference in body condition between both years (F(PDF)Click here for additional data file.S1 TableModel selection of all combination of factors, a chick random effect is included. K: number of parameters. ΔAIC is the difference of AIC between a given model and the model with the lowest AIC. Best model is number 1 with the smallest AIC and less parameters than model 2.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases. These results indicate that methylene blue or its derivatives could (1) have an additional effect in AD by inhibiting caspases, (2) be used as a drug to prevent caspase activation in other degenerative conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of effector Casp3.Since methylene blue inhibits the activity of Tau protein through its pro-oxidant activity on cysteines, here, we tested if it can also inhibit the cysteinyl caspase proteases. Our results showed that methylene blue and its derivatives efficiently inhibited active caspases in vitro assay. Caspase activities were sufficiently retained with 10 μM DTT  was detected with a neoepitope antiserum whereas full length uncleaved tubulin (upper tubulin band) and Tub∆Casp6 (lower protein band) were detected with a full-length tubulin antibody by western blot analyses (50 of 10.6 μM (r2 = 0.87), 0.5 μM (r2 = 0.89), and 34.4 μM (r2 = 0.90) with methylene blue, azure A, and azure B, respectively  plot (inset) shows that the data intersect the y axis at the same point, indicating a competitive mode of inhibition. Fitting the data to the competitive inhibition model, and using nonlinear regression analysis, we determined the Ki values to be 50.5 μM ± 2.3 μM for methylene blue, 0.42 μM ± 0.02 for azure A, and 7.89 μM ± 0.2 μM for azure B, respectively. These results suggest that methylene blue, azure A, and azure B prevent binding of substrate in a competitive manner.To determine the mechanism of methylene blue-mediated Casp6 inhibition, inhibitor kinetic studies were conducted with non-interfering concentrations of phenothiazine in the fluorogenic assay. Increasing concentrations of methylene blue , azure AHuman colon carcinoma cells (HCT116) and human primary neurons were first observed by phase contrast microscopy after treatment with increasing concentrations of methylene blue and its derivatives . At 100 To test the ability of phenothiazines to inhibit Casp6 in live cells, HCT116 cells, transfected with a self-activating form of Casp6 (Casp6p20p10) to induce high levels of Casp6 activityin vitro /galactosamine (GALN), a well-known model to assess drugs against liver Casp3 activityTo determine if methylene blue has the ability to inhibit caspases in vitro  by both methylene blue and azure B . LC/MS/MOur study provides the identification of a post-translational regulatory modification of the caspase catalytic cysteine and adds a new group of cysteinyl enzymes that are regulated by sulfenation. Oxidation-dependent inhibition of caspases, mainly Casp3, has been demonstrated previously, but the chemical modification was not identified383942in vivo. Similarly, inhibition of Casp1 and Casp6 by methylene blue could have beneficial effects by reducing inflammatory processes and axonal degeneration, but inhibition of Casp3 could alter neuronal function or promote cancer cell survival and be detrimental to tissue homeostasis. It is therefore important to monitor patients who are chronically treated with phenothiazines for deregulated inflammatory responses and cancers, especially in individuals at risk for these conditions.The inhibition of Casp1, Casp3, and Casp6 could have a major influence on inflammation, apoptosis and neurodegeneration, respectively. Casp1 is the main producer of interleukin-1-beta, which can mediate several neuroinflammatory processes452650 against Tau aggregation ranges from 2 μM 50 of methylene blue and azure B against Casp6 cleavage of tubulin are 10.6 μM and 34.4 μM, respectively, whereas azure A is 0.5 μM. Therefore, the abundant levels of active Casp6 observed in AD brainsBeneficial effects observed in AD clinical trials will have to consider the possibility that phenothiazines may benefit cognitive decline in AD not only by disaggregating Tau but by inhibiting Casp6-mediated axonal degeneration. The IC50 in the nM or pM range, phenothiazines are weak inhibitorsCompared to other caspase inhibitors, which have ICin vivo to fully understand how phenothiazines benefit age-dependent cognitive deficits and AD. Autopsied brains from AD individuals treated with phenothiazines will be invaluable to assess the effect of methylene blue on neurofibrillary tangles and Casp6 activity and obtain more selective therapies against AD.Methylene blue and its derivatives have other pleiotropic effects that could benefit AD (reviewed by51522425262728These results imply that methylene blue or its derivatives could (1) have an additional positive effect against AD by inhibiting caspases, (2) be used as a drug to prevent caspase activation in other degenerative conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases. In conclusion, this study identifies an important mechanism of action of methylene blue against activity of caspases that could impact tissue homeostasis in pathological and age-dependent conditions.The pET23b (+) recombinant human proCasp6-His*tag was a kind gift from Dr Guy Salvesen  and our laboratory cloned the human Casp6p20p10 in pCep4β vectorE. Coli BL21 (DE3)pLysS pET23b (+) recombinant pro-Casp6-His*tag was purified from in vitro fluorogenic assays with the Ac-Val-Glu-Ile-Asp-7-Amino-4-trifluoromethyl-couramin substrate  in Stennicke’s buffer Casp6 activity was assessed by 50 for methylene blue, azure A, and azure B was determined by performing an in vitro tubulin cleavage assay because concentrations of phenothiazines above 100 μM interfered with both fluorescence . The Ki values and type of inhibition was subsequently estimated from the x-axis  and y-axis (Vmax) intercepts of plots versus inhibitor concentrations. Linear and non-linear regression analyses were performed using the Prism version 5.0 software package.Phenothiazines were added to 10 nM RCasp6 and enzyme activity was measured on nine different Ac-VEID-AFC concentrations (2.5 μM to 200 μM). To estimate K6 cells/mL were serum-deprived for 2 hours in the presence of phenothiazines or an equivalent volume of PBS. Proteins were harvested in cell lysis buffer .Human colon carcinoma (HCT116) cells, cultured as recommended , were transfected with 1 μg pCep4βCasp6p20p10 and 8 μg polyethyleneimine , and grown 24 hrs before treatment with PBS and phenothiazines for 2 hours. Human primary neurons were cultured from fetal brain cortical areas obtained under ethical guidelines and approved by the McGill University’s institutional review board as previously described5 cells/well and human neurons, plated at a density of 6 × 106 cells/well on poly-L-lysine were treated with PBS or phenothiazines for 2 hours. The media was replaced before acquiring images with the Nikon Eclipse Ti microscope and the NIS-Elements (Version 3.10) software. MTT assays: Cells were plated in 96-well plates at 1 × 104 or 1 × 105 cells per well for 24 hours before treatments with phenothiazines or PBS. After 2 hours, fresh media containing 0.5 μg/ml MTT -2,5-diphenyltetrazolium bromide) (Sigma-Aldrich) was added and the cells were incubated for 4 hours at 37 °C in 5% CO2. The media was removed, formazan crystals were dissolved in DMSO, and the absorbance was measured at 560 nm and 670 nm using the BioTek Synergy H4 plate reader.HCT116 cells, plated at a density of 1 × 10The caspase-like activity of the proteasome was measured using 10 μM Ac-Nle-Pro-Nle-Asp-AMC  as described for caspase activity assays except for the use of 10 mM DTT and a 7-amino-4-methylcoumarin  standard curves. Mouse liver protein extracts were treated with 0.1 μM of epoxomicin (Enzo Lifesciences), bortezomib, 1 μM of Q-VD-OPh, or equal volumes of DMSO for 20 mins on ice before measuring proteasome or caspase activities.The 10630  and GN60622  neoepitope antibodies against the p20 subunit of active Casp6rd day, after gavage, a mixture of 100 mg/kg of LPS (Sigma) and 700 mg/kg of D-(+)-galactosamine (GALN) (Sigma) or PBS was injected i.p.2 5.5 hours after the i.p. injection. Left lobe liver sections (50–150 mg) were frozen rapidly and ground glass homogenized with 10x volume of iced CLB supplemented with fresh protease inhibitors . The homogenates were centrifuged at 1000 × g at 4 °C and the supernatant analysed for caspase activity in a blind fashion.All animal procedures followed the Canadian Council on Animal Care guidelines and were approved by the McGill Animal care committees. Male C57BL6/J mice  were gavaged with 3 mg/kg dose of methylene blue or vehicle (0.5% methylcellulose (Sigma)) for 3 consecutive days. On the 3RCasp6 (1 mg/ml) in 50 mM Tris, pH 8.0, 100 mM NaCl, 1 mM DTT was incubated for 15 min at 37 °C, divided equally in three tubes, and 2 mM methylene blue, 2 mM azure B or buffer were added to each tube and incubated for 45 min at 37 °C. The samples were snap-frozen in dry ice and ethanol, and stored at −80 °C until analysis at the IRIC Mass Spectrometry core facility . Briefly, 10 μg RCasp6 was digested with 1 μg of trypsin for 8 h at 37 °C, desalted with Ziptips, loaded on a homemade C18 pre-column (0.3 mm i.d. × 5 mm) connected directly to the switching valve and separated on a homemade reversed-phase column (150 μm i.d. × 150 mm) with a 56-min gradient from 10–60% acetonitrile (0.2% FA) and a 600 nl/min flow rate on a NanoLC-2D system (Eksigent) connected to an LTQ-Orbitrap Elite (Thermo Fisher Scientific). Each MS spectrum acquired with a 60,000 resolution was followed by 12 MS/MS spectra, where the 12 most abundant multiply charged ions were selected for MS/MS sequencing. Tandem MS experiments were performed using collision-induced dissociation in the linear ion trap. Peaks were identified using Mascot version 2.4 (Matrix science) and peptide sequence were blasted against the Human Uniprot database . Tolerance was set at 15 ppm for precursor and 0.5 Da for fragment ions during data processing. The occurrence of cysteine oxidations was considered.The number of independent experiments and statistical analyses conducted with ANOVA and post hoc Bonferroni or Dunnett’s tests were indicated in the figure legends.How to cite this article: Pakavathkumar, P. et al. Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine. Sci. Rep.5, 13730; doi: 10.1038/srep13730 (2015).


AbstractLimoniidae and Pediciidae from Croatia, with new distribution records for species in 12 different genera. Four genera and 18 species are newly recorded for Croatia. Until now, including this data, 87 Limoniidae and eight Pediciidae have been recorded from Croatia. In this paper we confirm presence of Ormosia (Oreophila) bergrothi  and we give the first records of Dicranota (Paradicranota) pavida  and Molophilus (Molophilus) repentinus Starý, 1971 from Balkan Peninsula.New records are provided for  Pediciidae or hairy-eyed crane flies comprise slender tipuloid like flies. Species belonging to subfamily Pediciinae have carnivorous larvae and are connected with the wet environments  occulta , Dicranota (Paradicranota) pavida ). Limoniidae or short-palped crane flies are a paraphyletic group (Antocha). From Western Palaearctic region 77 Pediciidae and 735 Limoniidae species are reported  tristis  is reported for the first time in Croatia. At the present a total of four Pediciidae and 73 Limoniidae are reported from Croatia , was fixed in the sediment of the stream, and contained 2% formaldehyde; six traps were placed at each location on various types of microhabitats. At majority of sites traps were placed for one year (two sites at Krka River) or two years (majority of sites in Plitvice Lakes), but at three sites at Plitvice Lakes  they were placed for seven years, for additional details see Limoniidae and Pediciidae specimens were placed in 80% ethanol. The literature used for identification is as follows: PageBreakDiptera Collection of the Faculty of Biology and Geology, Cluj-Napoca, Romania. GPS coordinates and altitudes for the localities where specimens were trapped and/or collected are given in Table Additional sampling using a sweep net took place between March 2011 and June 2014 at various sites in Croatia. All The following format is used for the records given here: name of the site, followed by the sampling date , and the number of sampled specimens. New species for Croatia are listed with * before the name of the species. All the sites are listed in Table PageBreakDicranota (Dicranota) bimaculata * New records. Spring of Bijela rijeka, Plitvice Lakes, 5.x.2010, P4, 1♀; same site, 29.v.2012, P3, 1♀Comments. Widespread in Europe except the Mediterranean region. In the Balkan Peninsula it has been recorded from Bulgaria.Dicranota (Paradicranota) pavida * New records. Upper reach of Bijele rijeka, Plitvice Lakes, 5.x.2010, P4, 1♂, 1♀; spring of Crna rijeka, Plitvice Lakes, 29.v.2011, 1♂Comments. Mostly Central and Western European distributed species. This is the first record for Balkan Peninsula.Pedicia  occulta * New records. Spring of Bijela rijeka, Plitvice Lakes, 28.iv.2007, P2, 1♀; same site and date, P3, 1♂; same site and date, P6, 1♀; same site, 29.v.2007, P3, 1♂, 2♀; same site, 28.vi.2007, P2, 1♀; same site and date, P5, 1♂; same site, 25.vii.2007, P2, 1♂; same site, 30.viii.2007, P2, 1♂; same site and date, P3, 1♂; same site, 2.x.2007, P3, 1♂; same site, 29.x.2007, P2, 3♂; same site and date, P3, 2♂, 3♀; same site and date, P5, 1♀; same site, 29.xi.2007, P2, 1♀; same site and date, P3, 1♀; same site, 26.xii.2007, P3, 2♂; same site, 2.iv.2008, P2, 1♂, 1♀; same site and date, P6, 1♂; same site, 23.iv.2008, P2, 1♂, 1♀; same site and date, P3, 1♂; same site and date, P4, 1♀; same site and date, P6, 1♂; same site, 1.vi.2008, P2, 1♀; same site and date, P4, 2♀; same site, 30.vi.2008, P3, 1♂; same site, 22.vii.2008, P4, 1♀; same site, 27.viii.2008, P4, 1♂; same site and date, P6, 1♀; same site, 26.xi.2008, P2, 1♂; same site and date, P3, 1♀; same site, 3.iii.2009, P2, 1♂; same site, 1.iv.2009, P2, 2♂, 2♀; same site and date, P4, 1♂; same site and date, P6, 1♀; same site, 2.v.2009, P2, 2♂; same site and date, P6, 1♀; same site, 30.x.2009, P2, 1♂, 1♀; same site, 8.iv.2010, P2, 1♀; same site and date, P3, 1♂; same site, 4.v.2010, P6, 1♀; same site, 4.vi.2010, P4, 1♂; same site, 25.vii.2010, P2, 1♂, 1♀; same site, 26.viii.2010, P4, 2♂; same site, 2.ix.2010, P3, 1♀; same site and date, P4, 1♀; same site and date, P6, 1♀; same site, 10.xii.2010, P3, 1♂, 1♀; same site, 25.ii.2011, P6, 1♂, 1♀; same site, 26.iv.2011, P4, 2♀; same site and date, P6, 1♂, 1♀; same site, 29.v.2011, P4, 1♂, 2♀; same site and date, P6, 1♂; same site, 27.vi.2011, P2, 1♂; same site, 2.viii.2011, P6, 1♂, 1♀; same site, 6.ix.2011, P3, PageBreak1♀; same site and date, P4, 2♀; same site, 4.viii.2012, P3, 1♂; same site, 4.ix.2012, P3, 1♂; same site and date, P4, 2♂, 1♀; same site, 6.xi.2012, P4, 1♂; same site and date, P5, 1♂, 1♀; same site and date, P6, 1♂; same site, 11.iii.2013, P2, 2♂; same site, 2.iv.2013, P3, 1♀; same site and date, P4, 1♀; same site, 3.v.2013, P4, 1♂; same site, 7.viii.2013, P4, 1♀; same site and date, P5, 1♂; same site, 29.x.2013, P3, 1♀; upper reach of Bijele rijeka, Plitvice Lakes, 30.iii.2007, P2, 1♀; same site and date, P4, 2♂; same site, 28.iv.2007, P2, 1♀; same site and date, P3, 2♂; same site and date, P4, 2♂; same site and date, P5, 2♀; same site, 29.v.2007, P2, 2♂; same site and date, P3, 1♀; same site and date, P4, 1♀; same site and date, P5, 1♂; same site and date, P6, 1♀; same site, 28.vi.2007, P2, 1♂, 2♀; same site and date, P3, 1♂; same site and date, P4, 1♂; same site, 25.vii.2007, P3, 2♀; same site and date, P4, 6♂, 1♀; same site, 30.viii.2007, P1, 1♀; same site and date, P2, 3♂; same site and date, P3, 1♂; same site and date, P4, 1♂; same site, 2.x.2007, P1, 2♂, 1♀; same site and date, P2, 2♀; same site and date, P3, 1♂, 1♀; same site and date, P4, 1♀; same site and date, P6, 1♂; same site, 29.x.2007, P1, 2♀; same site and date, P5, 1♀; same site, 29.xi.2007, P1, 1♀; same site and date, P2, 1♂, 3♀; same site, 25.ii.2008, P1, 1♂; same site, 2.iv.2008, P2, 1♂; same site and date, P3, 4♂; same site and date, P4, 2♂, 3♀; same site, 23.iv.2008, P2, 1♂, 1♀; same site and date, P4, 3♂; same site and date, P5, 2♀; same site, 1.vi.2008, P5, 1♀; same site, 30.vi.2008, P2, 2♂; same site and date, P3, 1♂, 1♀; same site and date, P4, 1♂; same site, 22.vii.2008, P3, 1♂; same site, 27.8.2008, P1, 1♀; same site and date, P2, 1♂; same site and date, P3, 1♂; same site and date, P5, 1♀; same site, 29.xi.2008, P6, 1♀; same site and date, 27.x.2008, P2, 2♀; same site and date, P3, 1♂; same site and date, P4, 1♂; same site and date, P6, 1♀; same site and date, 26.xi.2008, P2, 1♀; same site and date, P4, 1♀; same site, 3.iii.2009, P5, 1♂; same site, 1.iv.2009, P3, 1♀; same site and date, P5, 1♂; same site, 2.v.2009, P4, 1♂, 1♀; same site and date, P5, 1♂, 1♀; same site, 29.v.2009, P1, 2♀; same site and date, P2, 1♂; same site and date, P3, 1♂; same site, 26.vi.2009, P4, 1♀; same site and date, P5, 1♀; same site, 24.vii.2009, P4, 1♀; same site and date, P5, 1♀; same site, 27.viii.2009, P3, 1♂; same site and date, P4, 1♂; same site, 30.x.2009, P2, 3♂, 2♀; same site, 8.iv.2010, P6, 1♂; same site, 4.v.2010, P2, 1♂, 2♀; same site and date, P4, 1♀; same site, 1.vii.2010, P1, 1♂; same site and date, P4, 1♀; same site and date, P6, 1♂; same site, 25.vii.2010, P1, 2♂; same site and date, P2, 1♂; same site and date, P4, 6♀; same site and date, P6, 1♂; same site, 26.viii.2010, P2, 2♂; same site and date, P4, 2♂, 3♀; same site and date, P6, 1♂; same site, 5.x.2010, P2, 2♂, 1♀; same site and date, P3, 1♂, 1♀; same site and date, P4, 1♂; same site, 2.xi.2010, P1, 4♀; same site and date, P3, 1♂; same site and date, P4, 1♂, 3♀; same site and date, P5, 1♀; same site, 10.xii.2010, P1, 1♂; spring of Crna rijeka, Plitvice Lakes, 29.v.2007, P3, 1♂; same site, 30.viii.2007, P5, 1♀; Crna rijeka by the bridge, 28.vi.2007, P3, 1♂;Comments. Widespread in Europe, including the Balkan Peninsula. It is frequently collected in springs and headwater brooks . In the Balkan Peninsula it is recorded from Albania, Bosnia and Herzegovina, Bulgaria, Greece, Macedonia, Montenegro, Serbia and Slovenia.PageBreakTricyphona (Tricyphona) immaculata * New records. Spring of Bijela rijeka, Plitvice Lakes, 28.x.2011, P6, 1♂; upper reach of Bijele rijeka, Plitvice Lakes, 29.v.2007, P4, 2♀; spring of Crna rijeka, Plitvice Lakes, 30.iv.2007, P1, 5♀; same site, 29.v.2007, P1, 5♀; tufa barrier Labudovac, Plitvice Lakes, 28.x.2011, P1, 1♂Comments. Widespread species. In the Balkan Peninsula it is recorded from Bosnia and Herzegovina, Montenegro, Macedonia, Serbia and Slovenia.Ellipteroides (Ellipteroides) lateralis * New record. Tufa barrier Kozjak-Milanovac, Plitvice Lakes, 1.vii.2012, P4, 1♂Comments. The species is frequently collected in calcareous springs, fens and around the tufa sediment (sediment . It is wGonomyia (Gonomyia) tenella New records. Spring of Bijela rijeka, Plitvice Lakes, 29.x.2007, P5, 1♀; Korana Village, Plitvice Lakes, 29.vi.2007, P2, 1♀; same site and date, P6, 1♀; same site, 26.vii.2007, P1, 6♀; same site and date, P2, 1♂; same site and date, P3, 1♂; same site and date, P6, 3♂, 12♀; same site, 1.xi.2007, P1, 1♀; same site and date, P6, 3♀Comments. A semi-aquatic species which can be found along flowing and standing waters (g waters . In the Molophilus (Molophilus) brevihamatus Bangerter, 1947* New record. Spring of Krka River, 7.vii.2011, 1♂Comments. A characteristic species along mountainous waters in Carpathians, Alps and Balkan high mountains . Probably the microclimate of the spring provides right condition for the species in lower altitude. In the Balkan Peninsula it is recorded from Bulgaria and Slovenia.Molophilus (Molophilus) bifidus Goetghebuer, 1920* New record. Spring of Bijela rijeka, Plitvice Lakes, 4.9.2012, P1, 1♂PageBreakComments. This species is associated with springs and head waters (d waters . PreviouMolophilus (Molophilus) obscurus New records. Spring Krčić, 29.iv.2011, 2♂; Roški Slap, Krka River, 13.x.2011, 1♂Comments. Common species, with wide distribution in Europe, including Balkan Peninsula.Molophilus (Molophilus) repentinus Starý, 1971* New record. Korana Village, Plitvice Lakes, 30.v.2007, P2, 1♂, 1♀Comments. Mountains species with sporadic distribution in Alps and Carpathians and surrounding mountains area. Its presence in the low altitude (390 m) is explainable by the presence of mountainous microhabitats around fast flowing Korana River. First record for Balkan Peninsula.Ormosia (Oreophila) bergrothi * New record. Spring of Crna rijeka, Plitvice Lakes, 25.vii.2007, P1, 3♀; same site, 30.viii.2007, P1, 1♀Comments. This species has a sporadic distribution, with erroneous remark on type- locality. Rhabdomastix (Rhabdomastix) edwardsi Tjeder, 1967* New records. Korana Village, Plitvice Lakes, 29.vi.2007, P2, 1♂; same site, 29.ix.2008, P6, 1♀Comments. Riverine species associated with sandy sediments mostly in mountainous areas New record. Upper reach of Bijele rijeka, Plitvice Lakes, 25.vii.2007, P4, 1♀Comments. Common species associated with sandy sediment New records. Upper reach of Bijele rijeka, Plitvice Lakes, 2.x.2007, P5, 1♀; Crna rijeka by the bridge, Plitvice Lakes, 31.viii.2007, P2, 1♀Comments. Common species with wide European distribution.Eloeophilamiliaria * New record. Brzaja, before N. Zvečeva, Papuk Mountain, 14.vi.2012, 1♀Comments. Less common species than Eloeophilaapicata and Eloeophilamaculata. Reported from Albania, Bulgaria and Serbia.Epiphragma (Epiphragma) ocellare New records. Spring of Kupa River, 10.vi.2009, 1♀; spring of Crna rijeka, Plitvice Lakes, 29.v.2011, 1♂Comments. Very common species with wide distribution.Hexatoma (Eriocera) chirothecata New records. Tufa barrier Novakovića Brod, Plitvice Lakes, 25.vii.2007, P4, 1♂; Korana Village, Plitvice Lakes, 26.vii.2007, P4, 1♀Comments. Mostly distributed in southeastern Europe. Reported from majority of Balkan countries.PageBreakParadelphomyia (Oxyrhiza) senilis * New records. Upper reach of Bijele rijeka, Plitvice Lakes, 2.x.2007, P2, 1♂; same site and date, P4, 1♀; spring of Crna rijeka, Plitvice Lakes, 28.vi.2007, P1, 2♀Comments. A wide distributed species in Palearctic region, frequently collected around different flowing waters (g waters . In the Antocha (Antocha) vitripennis New record. Tufa barrier Kozjak-Milanovac, Plitvice Lakes, 27.vi.2013, P4, 1♂; tufa barrier Novakovića Brod, Plitvice Lakes, 1.vi.2008, P2, 1♂; Korana Village, Plitvice Lakes, 30.v.2007, P6, 1♂; same site and date, 29.vi.2007, P1, 1♂; same site and date, 30.vi.2008, P6, 1♂; Radmanove mlinice, Cetina River, 19.vii.2005, 1♂, 5♀.Comments. A very common species.Dicranomyia (Dicranomyia) chorea New records. Spring of Bijela rijeka, Plitvice Lakes, 1.vii.2010, P6, 1♂; same site, 2.viii.2011, P6, 1♂; upper reach of Bijele rijeka, Plitvice Lakes, 27.viii.2009, P3, 2♀; 1.vii.2010, P3, 1♂; same site, 25.vii.2010, P3, 1♂; same site, 26.viii.2010, P1, 1♂, 1♀; same site and date, P3, 4♂, 2♀; same site and date, P4, 2♂; same site, 5.x.2010, P1, 1♀; same site and date, P2, 1♂; tufa barrier Kozjak-Milanovac, Plitvice Lakes, 29.x.2013, P1, 1♂; stream Plitvica, Plitvice Lakes, 30.viii.2007, P3, 1♂; Korana village, Plitvice Lakes, 29.vi.2007, P1, 1♂; Roški Slap, Krka River, 28.iv.2014, P4, 1♀;Comments. A very common species.Dicranomyia (Dicranomyia) didyma New records. Spring of Bijela rijeka, Plitvice Lakes, 30.viii.2007, P5, 15♂, 9♀; same site and date, P6, 6♂, 5♀; same site, 2.x.2007, P2, 1♂, 2♀; same site and date, P5, 4♂, 2♀; same site, 29.x.2007, P5, 1♂; same site, 1.vi.2008, P5, 1♀; same site, 22.vii.2008, P6, 4♂; same site, 27.viii.2008, P6, 1♀; same site, 29.ix.2008, P5, 1♀; same site and date, P6, 2♂; same site, 1.x.2009, P5, 1♂; same site and date, P6, 3♂, 3♀; same site, 30.x.2009, P5, 1♂, 2♀; same site, 8.xii.2009, P6, 1♂, 1♀; same site, 1.vii.2010, P6, 1♀; same site, 26.viii.2010, P5, 4♂, 8♀; same site and date, P6, 1♂; same site, 5.x.2010, P2, 1♂, 1♀; same site and date, P5, 4♂, 3♀; same site and date, P6, 2♂, 2♀; same site, 29.v.2011, P6, 7♂, 6♀; same site, 2.viii.2011, P6, 2♂, 1♀; same site, 6.ix.2011, P6, 12♂, 10♀; same PageBreaksite, 28.ix.2011, P6, 2♀; same site, 3.ix.2013, P6, 7♂, 5♀; upper reach of Bijele rijeka, Plitvice Lakes, 24.vii.2009, P1, 1♂; same site and date, P3, 1♂; same site, 27.viii.2009, P1, 5♂, 1♀; same site and date, P2, 1♀; same site, 1.x.2009, P1, 8♂, 2♀; same site and date, P2, 2♀; same site and date, P3, 3♂, 4♀; same site, 30.x.2009, P2, 1♂; same site and date, P3, 1♀; same site, 1.vii.2010, P2, 4♂, 4♀; same site and date, P3, 1♂; same site, 25.vii.2010, P2, 4♂, 5♀; same site, 26.viii.2010, P1, 3♂, 3♀; same site and date, P3, 1♂, 3♀; same site and date, P4, 2♂; same site, 5.x.2010, P1, 1♂, 2♀; same site and date, P2, 2♂; same site and date, P3, 14♂; same site and date, P4, 1♂, 3♀; same site, 2.xi.2010, P3, 3♀; same site and date, P4, 1♂, 8♀; same site, 10.xii.2010, P4, 1♀; tufa barrier Kozjak-Milanovac, Plitvice Lakes, 1.vi.2008, P4, 1♂; same site, 27.viii.2008, P5, 1♀; same site, 1.vii.2010, P4, 1♂, 4♀; stream Plitvica, Plitvice Lakes, 25.v.2007, 1♂; same site, 27.vii.2007, P2, 1♂; Roški Slap, Krka River, 28.iv.2011, 3♂, 2♀; same site, 25.v.2011, 5♂, 3♀; same site, 30.viii.2011, 1♀; same site, 13.x.2011, 1 ♂; same site, 6.xi.2013, P4, 1♂, 1♀; same site, 7.ii.2014, P4, 5♂, 1♀; same site, 5.iii.2014, P4, 1♀; same site, 2.iv.2014, P3, 1♂; same site and date, P4, 1♂, 4♀; same site, 28.iv.2014, P4, 2♂, 3♀; same site, 2.vi.2014, P3, 1♂, 2♀; same site and date, P4, 3♂, 2♀; same site, 26.vi.2014, P3, 2♂, 1♀; same site and date, P4, 5♂, 1♀; same site, 26.vii.2014, P4, 1♂, 1♀; same site and date, P6, 2♂; Skradinski Buk, Krka River, 2.vi.2014, P3, 3♂, 2♀; same site, 26.vi.2014, P4, 1♀Comments. A very common and abundant species.Dicranomyia (Dicranomyia) goritiensis New records. Roški Slap, Krka River, 28.iv.2011, 2♂; same site, 25.v.2011, 2♂; same site, 2.vi.2014, P4, 1♀; same site, 26.vii.2014, P2, 1♂; same site and date, P4, 1♂, 2♀; same site, 2.ix.2014, P4, 1♀; same site, 2.x.2014, P1, 2♀; same site, 27.x.2014, P2, 2♀; same site and date, P4, 2♂; spring of Krka River, 25.v.2011, 1♂; spring of Krupa River, 26.v.2011, 1♂Comments. Sporadic distributed species, which is associated with moss and/or algae covered stones, mostly around waterfalls and rocky coast. In the Balkan Peninsula it is recorded from Croatia and Greece.Dicranomyia (Dicranomyia) imbecilla Lackschewitz, 1941* New records. Spring of Bijela rijeka, Plitvice Lakes, 29.v.2011, P2, 1♀; same site, 27.vi.2013, P1, 1♀; tufa barrier Kozjak-Milanovac, Plitvice Lakes, 27.vi.2013, P5, 1♂; spring Krčić, 29.iv.2011, 1♂; same site, 26.v.2011, 3♂, 4♀Comments. The species is known from calcareous seepages depositing tufaceous substrate. The species known from Bulgaria, Czech Republic, Great Britain, Germany, Slovakia and Switzerland (Stary and Stubbs 2015).PageBreakDicranomyia (Dicranomyia) lucida de Meijere, 1918* New record. Roški Slap, Krka River, 2.x.2014, P1, 1♀Comments. Relatively common species in woodlands around rivers. In the Balkan Peninsula it is reported from Bulgaria, Macedonia and Greece.Dicranomyia (Dicranomyia) mitis  complexNew records. Brzaja after N. Zvečevo, Papuk Mountain, 13.vi.2012, 1♂; upper reach of Bijele rijeka, Plitvice Lakes, 29.v.2009, P1, 1♂;; spring of Krka River, 7.vii.2011, 1♀Comments. A very common and widespread species.Dicranomyia (Dicranomyia) modesta New record. Roški Slap, Krka River, 25.v.2011, 2♂Comments. A common species which inhabits various habitats, but mostly found in forests.Ellipteraomissa Schiner, 1863* New record. Dubočanka stream, Papuk Mountain, 18.ix.2012, 1♀Comments. Mountainous species. In the Balkan Peninsula it is reported from Bosnia and Herzegovina, Bulgaria, Montenegro, Serbia and Slovenia.Limoniahercegovinae New records. Upper reach of Bijele rijeka, Plitvice Lakes, 28.iv.2007, P4, 1♂; spring of Crna rijeka, Plitvice Lakes, 30.iv.2007, P1, 1♂, 1♀; spring Jankovac, Papuk Mountain, 13.vi.2012, 1♀Comments. A widely distributed species, but mostly in Balkan Peninsula.Limoniamacrostigma New record. Brzaja, after N. Zvečeva, Papuk Mountain, 13.vi.2012, 1♂Comments. Widely distributed and common species.PageBreakLimoniaphragmitidis New record. Brzaja, after N. Zvečeva, Papuk Mountain, 14.vi.2012, 2♀Comments. Widely distributed and common species.Lipsothrixnobilis Loew, 1873* New record. Crna rijeka by the bridge, Plitvice Lakes, 28.vi.2007, P3, 1♂; tufa barrier Labudovac, Plitvice Lakes, 31.v.2007, P2, 2♂Comments. Less common species associated with woodland streams and springs.Lipsothrixremota * New records. Roški Slap, Krka River, 28.iv.2011, 1♂; same site, 25.v.2011, 1♂, 1♀Comments. Less common species associated with woodland streams and springs.Orimarga (Orimarga) virgo New record. Spring of Krka River, 7.vii.2011, 2♀Comments. Species reported from the different types of calcareous rocky habitats, which are covered with moss and/or algae. In the Balkan Peninsula it is reported from Croatia, Greece and Slovenia.Lipsothrix Loew, Paradelphomyia Alexander, Rhabdomastix Skuse and Tricyphona Zetterstedt) and 18 species new for Croatia. Including our results, eight Pediciidae and 87 Limoniidae have now been recorded from Croatia.In this paper we report for the first time four genera  and 106 of Limoniidae (L), while Hungary has 12 species of Pediciidae and 132 of Limoniidae, however a similar number of species was reported from Bosnia and Herzegovina (6 P and 69 L), Serbia (8 P and 68 L) and Montenegro (9 P and 59 L). It is evident from these numbers that much remains to be discovered about the Tipuloidea fauna of Croatia and entire Balkan Peninsula.PageBreak


Drosophila importin β in lateral neurons abolishes behavioral rhythms in flies. Collectively, these data show that KPNB1 is required for timely nuclear import of PER/CRY in the negative feedback regulation of the circadian clock.Regulated nuclear translocation of the PER/CRY repressor complex is critical for negative feedback regulation of the circadian clock of mammals. However, the precise molecular mechanism is not fully understood. Here, we report that KPNB1, an importin β component of the ncRNA repressor of nuclear factor of activated T cells (NRON) ribonucleoprotein complex, mediates nuclear translocation and repressor function of the PER/CRY complex. RNAi depletion of KPNB1 traps the PER/CRY complex in the cytoplasm by blocking nuclear entry of PER proteins in human cells. KPNB1 interacts mainly with PER proteins and directs PER/CRY nuclear transport in a circadian fashion. Interestingly, KPNB1 regulates the PER/CRY nuclear entry and repressor function, independently of importin α, its classical partner. Moreover, inducible inhibition of the conserved DOI:http://dx.doi.org/10.7554/eLife.08647.001 Most organisms have an internal clock—known as the circadian clock—that regulates many aspects of their biology and behavior in roughly 24-hr long cycles. In animals, the core of the circadian clock is made of two ‘activator’ proteins and two ‘repressor’ proteins that inhibit the activators so that the levels of all four proteins in cells fluctuate over the cycle.The activator proteins switch on the genes that encode the repressor proteins. This increases the production of the repressor proteins in an area of the cell called the cytoplasm. The repressor proteins then bind to each other and then move into the nucleus of the cell to inactivate the activator proteins. However, it was not clear how the repressor proteins move into the nucleus.Lee et al. used a technique called ‘RNA interference’ to study the circadian clock in human cells and fruit flies. The experiments show that a protein called importin β enables the repressor proteins to move into the nucleus. Importin β directly interacted with only one of the repressor proteins . Previous studies have shown that importin β is able to interact with another protein called importin α, but Lee et al.'s results show that this interaction is not important for importin β's role in the movement of the repressor proteins.Blocking importin β activity resulted in the loss of circadian rhythms in both human cells and fruit flies, which suggests that importin β performs the same role in many different animals. The circadian clock is disrupted in many cancers, so Lee et al.'s findings may also help to lead us to new treatments to fight these diseases.DOI:http://dx.doi.org/10.7554/eLife.08647.002 Nearly all organisms have circadian clocks, internal timing systems that anticipate and adapt physiology and behavior to daily changes in the light–dark cycle. In eukaryotes, the clock is an interlocked transcriptional–translational feedback loop, which drives rhythmic expression of both core clock genes that make up the oscillator and clock-output genes that drive biological rhythms. These include rhythms in the sleep wake cycle, body temperature, hormone secretion, and energy metabolism .PER1/PER2/PER3 (fly Per) and CRY1/CRY2 (fly Cry) genes. Once translated, PERs and CRYs (Per and Tim in flies) accumulate in a cytoplasmic complex and then translocate into the nucleus to repress the CLOCK-BMAL1 heterodimer, thereby repressing their own transcription  or PAR-domain protein-1 (dPDP1) make up this ancillary loop and stabilize the Drosophila clock by driving rhythmic expression of Clk  and BMAL1 (fly Cyc). This heterodimer binds E-box elements in the promoters of two families of transcriptional repressors, the cription . This prn of Clk . Furthern of Clk .KPNB1 is a nuclear import receptor that mediates docking of nuclear localization signal (NLS)-containing cargo bound to importin α to the nuclear envelope, thereby facilitating nuclear entry of the target protein . KPNB1 cNRON) (lncRNA repressor of the NFAT) RNA-protein-scaffold complex containing several kinases, CKIε, GSK3β, and DYRK2 that regulate NFAT phosphorylation and nuclear translocation in human and Drosophila cells  , GSK3β (sgg), and DYRK1a (mnb), are clock components in both flies and mammals. Moreover, NFAT nuclear translocation has been suggested to be mediated by KPNB1  analysis of cells expressing several flag-tagged clock proteins, knockdown of KPNB1 completely blocked , partly blocked (CRY1), or didn't significantly block  nuclearPer1, Dbp, Nr1d1, Nr1d2) targeted by the clock repressors in their nuclear accumulation phase  promoter revealed significantly less recruitment of both PER1 and PER2 to the CLOCK/BMAL1 responsive element in the KPNB1-depleted U2 OS cells  were upregulated as a result of KPNB1 knockdown  or importin α5 (KPNA1) did not affect the circadian rhythmicity  in dexamethasone (Dex)-synchronized cells with altered nuclear accumulation of PER and CRY proteins Ket, ketel, in the fly clock by RNAi followed by locomotor activity monitoring . We crossed ketel RNAi transgenic flies with pdf-GS and then drove ketel dsRNAs expression with a drug (RU486) during adulthood  and humans , across 600 million years of evolution. Indeed, the kinases that regulate PER/CRY translocation to the nucleus are also conserved . Because KPNB1 was required for rhythmicity in human cells, we investigated the role of the nitoring . We usedosophila . dicer we effect . Behavioe effect . With a e effect . To excldulthood . After 6activity . 81% of activity . These rpdf-GS > ketel RNAi flies at ZT1, when PER is nuclear in wild type flies. PDF is expressed in the large and small ventral lateral neurons (LNvs), so we focused on PER expression in these cells. In ketel knockdown flies, PER levels were low and primarily cytoplasmic in large LNvs, while PER staining was intensely nuclear in control flies  repressor complex is a critical and conserved step in maintaining rhythmicity in metazoans. However, the functional carrier molecules responsible for nuclear localization of the feedback regulators have yet to be determined. Our study provides the first evidence that KPNB1 plays a key role as a nuclear transporter of the PER/CRY complex in negative feedback repression in both mammalian and fly clocks .10.7554/Drosophila clock studies have shown that phosphorylation and nuclear translocation of PERs and CRYs are closely coupled  or were required for cellular rhythms (CSE1L) when silenced , data no. Thus, t2+ dynamics in circadian clock regulation. For example, periodic Ca2+ influx in the cytosol, resulting from circadian variation in membrane potential or by the release of Ca(2+) from ryanodine-sensitive stores in SCN neurons, is a critical process for circadian pacemaker function , modest (CRY1), or no effect (CRY2) on single tagged PER and CRY proteins, while it had strong effects on all combinations of PER/CRY complexes . Similare rhythm . These de rhythm .Recently, importin β emerged as a potential molecular target for cancer prevention . So far,2. The cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen) and DNA plasmids using Fugene HD reagents . The combined transfection of DNA plasmids with siRNA into the cells were performed using Lipofectamine 2000 (Invitrogen).U2 OS or HEK293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 1% L-Glutamine, and 1% penicillin-streptomycin  at 37°C under 5% CONRON in cells and generation of its transgenic cell lines, full-length genomic DNA fragments of NRON from U2 OS cells was obtained by PCR with primers containing the flanking restriction sites  and inserted into pcDNA 3.1 mammalian expression vector (Invitrogen). Plasmids encoding several flag-tagged clock proteins  were generated as described previously . For generation of plasmids encoding mutant KPNB1, DNA fragments of the KPNB1 gene with an importin-α binding domain deletion encoding a truncated protein (1–771) were sub-cloned into pCMV Sport6 expression vector (Invitrogen) using NotI and SalI restriction enzymes.For overexpression of eviously . For plaeviously . For lucHEK293T cells transfected with plasmids encoding proteins as indicated in the figures were assayed for luciferase reporter activity using DualGlo luciferin reagent (Promega) 24 hr post-transfection according to the manufacturer's protocol.Anti-PER2 used for immunoblot analysis of immunoprecipitated samples from mouse liver extracts were kindly provided by Dr Cheng Chi Lee's lab . Anti-KPNB1 (A300-482A) for immunoblotting analysis was obtained from Bethyl Labratories , and Anti-KPNB1 (ab2811) for immunoprecipiation with mouse liver extracts or immunostaining of U2 OS cells was purchased from Abcam . For ChIP or immunostaining experiments, Anti-PER1  and Anti-PER2  were used. Anti-Flag , Anti-GFP , Anti-PER1 , Anti-PER2 , Anti-CRY1 , Anti-CRY2 , were used for immunopreciptation, immunoblotting, and immunostaining experiments as indicated in figures. Normal mouse IgG (NI03) was purchased from Calbiochem. Anti-Tubulin , anti-TBP , anti-Lamin . and anti-GAPDH  were used as loading control antibodies for cytoplasmic, nuclear, and total protein extracts respectively.KPNB1. Cells were collected 48 hr post-transfection by briefly trypsinizing and washing twice in ice cold PBS. Nuclear–cytoplasmic fractionation of the cells was performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit  according to the manufacturer's protocol. The same kit protocol was used for preparation of cytoplasmic and nuclear extracts from mouse liver tissues collected at 4 hr interval for 24 hr in constant darkness.U2 OS cells were co-transfected with each of the following DNA plasmids encoding Venus (V)-tagged PER1, PER2, CRY1, or CRY2  and siRNA against 3VO4, and protease inhibitor cocktail ). Protein-G coated magnetic beads  were pre-incubated with 3 µg of anti-GFP  antibody at 4°C for 6 hr. The antibody conjugated beads were incubated with equal amounts of total protein at 4°C overnight. The final immune complexes were analyzed by immunoblot assay using antibodies as described. For tissue immunoprecipitation, liver tissue was homogenized in 1 vol of RIPA buffer 1 . Homogenates were cleared by dilution with 2 vol of RIPA buffer 2 (RIPA buffer 1 without NaCl). Further procedures were as described above. Immunoblot analyses were performed on 7.5% sodium dodecyl sulfatepolyacrylamide gels and transferred to polyvinylidene difluoride membranes . Target proteins were detected with antibodies as indicated. The immune complexes were visualized with an ECL detection kit .At 48 hr post-transfection of plasmids encoding PER2-Venus into U2 OS cells, the cell lysates were harvested in radioimmunoprecipitation assay (RIPA) buffer  were as follows: Forward primer: 5′-TCTCCCTCTCTCCTCCCTTCC-3′, Reverse primer: 5′-GCCTGATTGGCTAGTGGTCTT-3′, Probe: GTGTGTGACACAGCCCTGACC.For ChIP analysis, cell lysates were collected from U2 OS cells and the following ‘Materials and methods’ were performed as described previously . BrieflyAt 24 hr post-transfection of expression vectors encoding Venus-tagged clock proteins  or BiFC plasmids expressing  as indicated, the cells were fixed with 4% paraformaldehyde in PBS and visualized using GFP filter set in fluorescence microscopy. For IF analysis, U2 OS cells was incubated with various antibodies as indicated by secondary antibodies conjugated to Alexa Fluor 488 and/or 568 (Invitrogen). Cells were visualized using FITC/TRITC/DAPI filter set in fluorescence microscopy.Per2 or pBmal1 dLuc reporter U2 OS cells after synchronization with 1 µM Dex (Sigma) were monitored using a LumiCycle luminometer  as previously described . qPCR reactions were conducted using iTaq PCR mastermix  in combination with gene expression assays (Applied Biosystems) on a 7800HT Taqman machine (Applied Biosystems). 3–5 days old flies were collected at indicated ZT on the fourth day of LD entrainment. Fly heads of each genotype were opened and brains were immediately fixed with 4% paraformaldehyde (in 1× PBS) for 15–20 min, followed by dissection in 1× PBST (0.3% Triton X-100 in PBS) at room temperature. After 30min-wash with 1× PBST at room temperature, brains were blocked with 5% normal donkey serum (NSD) for 20 min, and then incubated overnight at 4°C with primary antibody: rat anti-PER  and mouse anti-PDF . After 30-min wash in 1× PBST at room temperature, brains were incubated with secondary antibodies  for 2 hr in NDS at room temperature, followed by extensive 30 min-wash. Samples were imaged using Leica TCS SP5 confocal microscope. 8–10 brains were examined at each time point.ketel RNAi in PDF+ cells and tim-expressing clock neurons were performed using the UAS/GAL4 system. We tested two independent UAS-ketel RNAi lines  and isogenic w1118 (iso31) strain was used as a wild-type control. Male flies of the UAS-ketel RNAi lines or iso31 (control) were crossed to female flies from Gal4 drivers. Male progenies of the crosses were entrained to a 12 hr:12 hr LD cycle at 25°C at least for 3 days. Individual flies were loaded in the glass locomotor tubes containing 5% sucrose and 2% agar. Locomotor activity was measured for 7–15 days in DD using the Drosophila Activity Monitoring System as previously described (Targeted expressions of escribed . Power o review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  in these proteins have been reported. The dynamics of this is important since it ensures the correct timing of negative feedback repression of clock genes. Here, the authors show that KPNB1 depletion results in primarily cytoplasmic localization of PER in human cells and Drosophila. Timely subcellular localization of PER2, CRY1, and KPNB1 coincides well and KPNB1 mainly interacts with PER1/2. Thus, the authors concluded that KPNB1 controls nuclear translocation of PER/CRY complex via direct interaction with PER. Intriguingly, KPNB1 mediated nuclear translocation of PER/CRY complex is not dependent on KPNA2 (importin α1) which is a well-known partner of importin β proteins and identified as major carrier protein for PER/TIM repressor complex in flies.The study by Lee et al. describes the importance for KPNB1, a member of the importin β family, for nuclear localization of clock proteins, in particular PERIOD proteins in both human cells as well as in Essential revisions:1) Clarify the role of KPNB1 knockdown on clock gene expression .2) Is KPNB1 really required for CIRCADIAN feedback repression ?3) Better define (and discuss) the roles of KPNA family members .Reviewer #1:Drosophila. Timed nuclear accumulation of members of the negative limb within the circadian clockwork has long been known to be critical for a functional circadian clock. Circadian rhythms of nuclear appearance of PERs, CRYs as well as TIM (in flys) as well as functional nuclear localization and nuclear export sequences (NLS and NES) in these proteins have been reported. The dynamics of this is important since it ensures the correct timing of negative feedback repression of clock genes.The study by Lee et al. describes the importance for KPNB1 (importin β) for nuclear localization of clock proteins, in particular PERIOD proteins in both human cells as well as in Drosophila: again, PER is shifted towards more cytosolic localization upon depletion of ketel, the Drosophila ortholog of KPNB1. Probably as a consequence, flies with KPNB1 depletion in pdf cells or all clock cells are largely arrhythmic.Here, the authors show that KPNB1 (but not KPNA2 – importin α) depletion in human cells results in primarily cytoplasmic localization of PER proteins  both for overexpressed as well as for endogenous proteins (a nice and complete set of experiments). In addition, PER proteins directly bind to KPNB1 (are the NLS required?). Also very convincing and carefully conducted are the results they found in Less convincing are the experiments aiming to demonstrate effects on the circadian clockwork in mammalian cells (see below).Overall, however, the manuscript is of high quality and should eventually be published, if the following points can be convincingly addressed:BMAL1 pre-mRNA (1) The effects of KPNB1 knockdown on clock gene expression show conflicting results: either upregulation of PER1 pre-mRNA and downregulation of pre-mRNA  or the opre-mRNA . In addiPer2-reporter show a low and the Bmal1-reporter a high magnitude, if KPNB1 knockdown is supposed to lead to less inhibition of E-boxes?2) In addition, the effects on circadian rhythmicity should be better characterized: are there period effects? Why does the BMAL1 as an antiphasic control.3) 4) Reviewer #2:In this study, the authors explored the molecular mechanism for nuclear translocation of circadian repressor complex, PER/CRY. They identified KPNB1, a member of the importin β family, plays major role for nuclear translocation of PER1/2, PER/CRY complex and hence feedback repression. Timely subcellular localization of PER2, CRY1, and KPNB1 coincides well and KPNB1 mainly interacts with PER1/2. Thus, the authors concluded that KPNB1 controls nuclear translocation of PER/CRY complex via direct interaction with PER. Intriguingly, KPNB1 mediated nuclear translocation of PER/CRY complex is not dependent on KPNA2 (importin α1) which is a well-known partner of importin β proteins and identified as major carrier protein for PER/TIM repressor complex in flies. Overall, the experiments are well executed and this finding will enhance our understanding of detailed biochemical mechanisms underlying timely controlled nuclear translocation of circadian repressor complex.Although the results demonstrating KPNB1 is the major carrier protein for PERs are very solid, the conclusion about KPNB1 being the major carrier protein for PER/CRY complex in mammals and PER/TIM complex in flies needs to be further qualified (see below).Drosophila TIM binds importin α1, and acts as an adapter to transport PER to the nucleus). 1) As cited in the text, ketel (fly homolog of KPNB1) in clock cells made flies arrhythmic via inhibiting nuclear translocation of PER, hence claiming KPNB1 mediates nuclear entry of PER. In another paper lead by some of the authors in this study, they demonstrated that importin α1 protein is critical for nuclear translocation of fly circadian repressor complex PER/TIM, through direct interaction between TIM and importin α1 . There, Jang AR et al. proposed the model in which TIM bound to importin α1 acts as a carrier for PER translocation together with importin β. Thus, this reviewer is wondering whether nuclear translocation of TIM is also inhibited in ketel mutant, which will be a slightly different case to the mammals. Or, very little amounts of PER translocate to the nucleus in a TIM-independent manner through ketel. The authors showed only the PER patterns in ketel knockdown flies. By showing TIM patterns in ketel knockdown flies, it will be clarified that PER translocate into the nucleus as a PER/TIM/importin α1/importin β complex or via two pathways one is TIM/importin α1 dependent and the other is ketel dependent as in mammals.2) The authors showed that knockdown of 3) In the Discussion, the authors state: “Similarly, knockdown of KPNB1 resulted in cytoplasmic retention of endogenous PER1, PER2, CRY1, and CRY2 substantially .” HoweveReviewer #3:Drosophila Importin β also blocks the nuclear translocation of PER proteins and results in the severe impairment of circadian locomotor activity rhythms in flies. By gathering these findings, the authors conclude that KPNB1 is the essential factor to elicit the cyclic nuclear translocation of PER/CRY in the negative-feedback regulation of the circadian clock.In the manuscript entitled “KPNB1 mediates PER/CRY nuclear translocation and circadian clock function” by Lee et al., the authors investigate the role of KPNB1 (Importin β subunit) for nuclear translocation of PER proteins and its functional implication for circadian clock oscillation in mammalian cells and flies. The authors clearly show that KPNB1 critically regulates the nuclear translocation of PER proteins which are largely independent from CRY proteins in U2OS cells. Interestingly, a knock-down of KPNB1 blocked the nuclear translocation of the PER/CRY complex, suggesting that PER proteins played a dominant role in the nuclear translocation of the PER/CRY complex in mammalian cells. In addition, the authors exhibit that the loss of KPNA2 does not block the nuclear translocation of PER proteins and circadian molecular rhythms in mammalian cells. Moreover, the authors show that a decrease in In general, the study includes novel findings, especially for the regulation mechanism of subcellular PER/CRY dynamics, which is essential for the circadian clock. However, the reviewer requires the authors discuss the following points before publication.Per2:dluc circadian rhythms . In addition, it has recently been reported that KPNA2 accelerates cytoplasmic localization of proteins in some cases . Through the accumulated findings, KPNA2 has been thought to be a unique protein among the KPNAs, and does not represent the function of other KPNAs in somatic cells. Thus, the reviewer feels curious as to why the authors performed knock-down study against KPNA2 instead of KPNA1 or any other KPNAs. The authors might have taken into account a recently reported study which mentioned KPNA2 as a factor to obstruct circadian clock cycling in mouse embryonic stem (ES) cells . Umemura et al. showed the high level expression of KPNA2 and cytoplasmic retention of PER protein in undifferentiated ES cells. In addition, overexpression of KPNA2 did not facilitate nuclear translocation, rather accelerated cytoplasmic retention of PER proteins. Strikingly, the authors here clearly showed that knock-down of KPNA2 in U2OS cells resulted in the exclusive nuclear localization of PER2 and increased amplitude of  rhythms . These r 1) Clarify the role of KPNB1 knockdown on clock gene expression .2) Is KPNB1 really required for CIRCADIAN feedback repression ?3) Better define (and discuss) the roles of KPNA family members .Reviewer #1:1) The effects of KPNB1 knockdown on clock gene expression show conflicting results: either upregulation of PER1 pre-mRNA and downregulation of BMAL1 pre-mRNA  or the opposite . In addition, the effects inare very subtle  error bars are SEM with n=3). What about endogenous clock gene levels upon overexpression of KPNB1? Maybe this helps to solve the issue.We agree and did the experiment you suggested. In parallel, we tested both KPNB1 knockdown and overexpression on endogenous clock components regulated primarily by the E-box loop. This showed that KPNB1 knockdown led to significant up-regulation of most of the E-box-controlled genes, PER1, CRY1, DBP, REV-ERBβ. Conversely, these genes are repressed by KPBN1 over-expression , legend.2) In addition, the effects on circadian rhythmicity should be better characterized: are there period effects? Why does the Per2-reporter show a low and the Bmal1-reporter a high magnitude, if KPNB1 knockdown is supposed to lead to less inhibition of E-boxes?Per2 and Bmal1 reporters get longer and dampen more quickly, as representatively shown in the detrended graph : controls do oscillate with a 16-18 hour period (in conflict withand in conflict with knockdown time series). This is odd and could be normalization artefact - at least the authors should show BMAL1 as an antiphasic control.BMAL1. Levels of Per1 go up, while levels of Bmal1 are unchanged, consistent with Bmal1 is also antiphase to Per1 and Per2.Per your suggestion, we re-did this experiment in the revised 4): The authors claim that KPNB1 is responsible for circadian accumulation and feedback repression, hence they only observe correlations. To unambiguously show that KPNB1 is required for circadian feedback repression, they should analyze circadian protein accumulation  in wild-type and KPNB1 depleted cells together with analysis of clock gene pre-mRNA over time.We agree and did a time course of control and KPNB1 knockdown cells for both cytoplasmic and nuclear fractions of clock proteins and pre-mRNAs levels. As shown in Reviewer #2:1) As cited in the text,demonstrated that importin α family members  control nuclear translocation of CRY2 but not importin β. The data from Sakakida et al., is consistent with this study showing that depletion of KPNB1 produces minimal effects on CRY2 translocation and partial effects on CRY1 translocation . Based on these two studies, this reviewer thinks that Importin α1 might control translocation of mainly CRY2 and some of CRY1. This is somewhat similar to the case of flies, where translocation of TIM  is controlled by importin α1 .luciferase results indicated that CRY1/CRY2 represses even better than PER1/PER2. Then, confusion comes from the data showing KPNA1 deficiency did not produce any defects in cellular rhythms. Thus, this reviewer is wondering how CRY2 localized in KPNA1 knockdown cells. By performing this experiment we might be able to clarify the reason why KPNA1 depletion did not produce any effects on cellular rhythms. Is that due to either  KPNA1 does not playing any role in translocation of clock proteins (mainly CRY2) on the contrary to previous report and different from the fly case (or there might be cell-type specific issue?) or  nuclear translocation defects of CRY2 not producing any effects on feedback repression suggesting that main repressor of feedback circuitry is PER not CRY?Thank you for your comments and suggestions. The nomenclature of the KPNA1/2 genes is confusing. In both human and mouse KPNA genes, KPNA1 is importin α5 (IPOA5), while KPNA2 is Importin α1 (IPOA1).Per2::luc as well as PER2/CRY2 localization and found no significant changes  knockdown effect on subcellular localization of endogenous or ectopically expressed CRY2 localization and found no significant effects in our nuclear cytoplasmic fractionation and immunofluorescence analysis , legend. changes  legend. 2) The authors showed that knockdown of ketel (fly homolog of KPNB1) in clock cells made flies arrhythmic via inhibiting nuclear translocation of PER, hence claiming KPNB1 mediates nuclear entry of PER. In another paper lead by some of the authors in this study, they demonstrated that importin α1 protein is critical for nuclear translocation of fly circadian repressor complex PER/TIM, through direct interaction between TIM and importin α1 . There, Jang AR et al. proposed the model in which TIM bound to importin α1 acts as a carrier for PER translocation together with importin β. Thus, this reviewer is wondering whether nuclear translocation of TIM is also inhibited in ketel mutant, which will be a slightly different case to the mammals. Or, very little amounts of PER translocate to the nucleus in a TIM-independent manner through ketel. The authors showed only the PER patterns in ketel knockdown flies. By showing TIM patterns in ketel knockdown flies, it will be clarified that PER translocate into the nucleus as a PER/TIM/importin α1/importin β complex or via two pathways one is TIM/importinαα1 dependent and the other is ketel dependent as in mammals.ketel mutants. For one thing, a second ketel-dependent (and TIM-independent mechanism) would result in PER being partly nuclear in ketel mutants. Also, PER would be partly nuclear in tim mutants. Neither one is the case. While PER is unstable in the cytoplasm without TIM, it is stable in the nucleus, so nuclear PER would have been visible in tim mutants. Thus we believe that Importin alpha typically requires an importin beta, and PER likely translocates into the nucleus as a PER/TIM/importin α1/importin β complex in flies.We think it is very unlikely that TIM would be nuclear in 3) In the Discussion, the authors state: “Similarly, knockdown of KPNB1 resulted in cytoplasmic retention of endogenous PER1, PER2, CRY1, and CRY2 substantially .” However, I do not see any substantial effects on CRY2 protein. Rather, this data clearly indicated KPNB1 did not play any roles in nuclear translocation of CRY2 protein. Thus, this statement needs to be rewritten.We agree that KPNB1 does not affect nuclear translocation of CRY2 protein when ectopically expressed. In fact, if we looked at endogenous CRY2 localization carefully upon KPNB1 knockdown as shown in Reviewer #3:[…] Different from KPNA1, expression level of KPNA2 is usually very low in normal somatic cells. On the other hand, KPNA2 highly expressed in early embryo or pluripotent stem cells as well as in some cancer cells. For example, the expression level of the Kpna2 transcript is extremely low in adult mice liver . In addition, it has recently been reported that KPNA2 accelerates cytoplasmic localization of proteins in some cases . Through the accumulated findings, KPNA2 has been thought to be a unique protein among the KPNAs, and does not represent the function of other KPNAs in somatic cells. Thus, the reviewer feels curious as to why the authors performed knock-down study against KPNA2 instead of KPNA1 or any other KPNAs. The authors might have taken into account a recently reported study which mentioned KPNA2 as a factor to obstruct circadian clock cycling in mouse embryonic stem (ES) cells . Umemura et al. showed the high level expression of KPNA2 and cytoplasmic retention of PER protein in undifferentiated ES cells. In addition, overexpression of KPNA2 did not facilitate nuclear translocation, rather accelerated cytoplasmic retention of PER proteins. Strikingly, the authors here clearly showed that knock-down of KPNA2 in U2OS cells resulted in the exclusive nuclear localization of PER2 and increased amplitude of Per2:dluc circadian rhythms . These results are all well compatible with the results mentioned in the paper by Umemura et al. Therefore, the reviewer recommends that the authors discuss this point.In several previous reports (1996∼2001), KPNA2 (importin α1) was the predominant form interacting with KPNB1 in both human and mouse species :1810-7; Kutay et al., Cell. 1997, 90(6):1061-71; Cingolani et al., Nature. 1999, 399(6733):221-9p; Köhler et al., Mol Cell Biol. 1999, 19(11):7782-91; Catimel et al., J Biol Chem. 2001. 276(36):34189-98). Thus we chose KPNA2 (importin α1) for our representative knockdown study.Per2::luc  knockdown effect on subcellular localization of endogenous or ectopically expressed PER2 or CRY2 localization and found no significant effects in fluorescence analysis , legend.er2::luc , legend Regarding the effect of KPNA2 overexpression on PER2 localization , intriguingly, we found that KPNA2 overexpression substantially reduced cells expressing PER2-Venus protein in our analysis , legend.Our results in U2 OS cells are different from Umemura et al.’s results in stem cells, probably due to the different cell types and conditions analyzed. We added some discussion in the text to reflect this point (Discussion section).


Tyrosine kinase inhibitors (TKIs) are anticancer drugs that may be co-administered with other drugs. The aims of this study are to investigate the inhibitory effects of TKIs on UDP-glucuronosyltransferase (UGT) activities, and to quantitatively evaluate their potential to cause drug-drug interactions (DDIs). Inhibition kinetic profiles of a panel of UGT enzymes  by four TKIs  were characterized by using hepatic microsomes and recombinant proteins. Lapatinib exhibited potent competitive inhibition against UGT1A1 activity with a Ki of 0.5 μM. Imatinib was found to exhibit broad inhibition on several UGTs, particularly potent competitive inhibition against UGT2B17 with a Ki of 0.4 μM. The TKIs also exerted intermediate inhibition against several UGTs . Results from modeling for the quantitative prediction of DDI risk indicated that the coadministration of lapatinib or imatinib at clinical doses could result in a significant increase in AUC of drugs primarily cleared by UGT1A1 or 2B17. Lapatinib and imatinib may cause clinically significant DDIs when co-administered UGT1A1 or 2B17 substrates. Tyrosine-kinase inhibitors (TKIs) are anticancer drugs. Tyrosine kinases phosphorylate the tyrosine residues of proteins involved in the activation of signal transduction cascades that play key roles in biological processes including growth, differentiation and apoptosis in cancer cells4UDP-glucuronosyltransferases (UGT), a class of phase II enzymes, catalyze the conjugation of glucuronic acid to endogenous substances and exogenous compounds. UGT-catalyzed glucuronidation reactions account for approximately 35% of drugs eliminated by phase II enzymes (or one-seventh of the drugs prescribed in the United States in 2002)10in vitro and in vivo studies indicate that TKIs may alter the hepatic elimination of co-administered drugs by inhibiting their metabolism. For example, nilotinib and erlotinib inhibit UGT1A1 activity, and gefitinib inhibits UGT1A1, 1A7, 1A9 and 2B7 activities121314Previous In this study, four commonly used TKIs−axitinib, imatinib, lapatinib and vandetanib −were eva50 values of TKIs were further estimated. The summary of IC50 values is shown in As a preliminary study, we first examined whether TKIs inhibit different UGTs. To this end, axitinib, imatinib, lapatinib, or vandetanib (or vehicle control) was incubated with a UGT substrate (4-methylumbelliferone (4-MU) for all UGTs except for UGT1A4; trifluoperazine (TFP) was used for UGT1A4) and one of recombinant UGT enzymes . Then, the extent of glucuronide metabolite production was examined. The results showed that at 100 μM concentration, TKIs inhibited the activity of UGT isoforms to varying extent . For UGTmax) is in the sub-micromolar rangemax values of the remaining TKIs tested in this study are at the micromolar level at typical oral doses. For TKIs that exhibit IC50 values lower or similar to the reported plasma Cmax , their UGT inhibitory kinetics profiles were further characterized.Except axitinib whose maximum plasma concentration   . The estapatinib  was non-apatinib .i/AUC). To this end, the hepatic or systemic concentrations of each TKI were first estimated using previously reported clinical pharmacokinetic data  were predicted to be significant. For example, UGT2B17 inhibition by imatinib was predicted to increase the AUC of 4-MU by 140% when the fraction of the UGT substrate metabolized by the inhibited enzyme (fm) of a victim drug was 90%, which was much greater than the cut-off value for potential clinical significance fm is 1, indicating a potential DDI risk with clinical significance. On the other hand, axitinib could not cause the increase in AUC of UGT1A4, 1A7, or 1A9 substrates when the fm is 1, and the AUCi/AUC ratios were close to 1. UGT1A9 inhibition by Vandetanib, UGT1A1 inhibition by imatinib, and UGT1A7 inhibition by lapatinib were predicted to increase the AUC of substrate by about 2%, 3%, and 1%, indicating minor clinical impacts of the TKIs on the pharmacokinetics of other UGT substrates.The risk of DDIs due to the inhibition of UGTs by TKIs was evaluated by predicting the ratio of area under the plasma drug concentration versus time curve (AUC) of a victim drug in the presence and absence of the TKI  and pyridine class (sorafenib) have been shown to inhibit UGTs121315122450 values greater than about 75–100 μM because sufficiently high levels are not clinically achieved50 and Ki values of TKIs were further estimated. Most notable finding in our study was UGT inhibition by imatinib and lapatinib. Our study has revealed that imatinib is a broad inhibitor of several UGTs. Imatinib is a potent inhibitor of UGT2B17; an intermediate inhibitor of UGT1A1; and a weak inhibitor of UGT1A7, 1A8, 1A9, 1A10, 2B7, and 2B15. The human UGT2B enzymes are involved in the metabolism of steroid hormones as well as bile acids, acidic steroids, and fatty acids2829Important inhibition is uncommon for compounds with ICin vitro evidence that lapatinib is a potent inhibitor of UGT1A1. UGT1A1 is broadly expressed in human organs including liver, intestines, and kidney3132in vivo systems can be underestimated when the prediction is based solely on in vitro parametersOur data offer Different from above two TKIs, vandetanib was found as an intermediate inhibitor of UGT1A9, which is also involved in the glucuronidation of a number of drugsin vitro data to in vivo drug interactions should be taken with caution. Various factors may affect the accuracy of prediction, including enzyme resources, incubation systems, protein binding, active transporters, the selection of probe substrates, and the accumulation of products in the reaction medium so on. In addition, UGTs in the gastrointestinal tract may contribute significantly to the first-pass metabolism of orally administered drugs that undergo glucuronidation. The concentrations of substrates in the intestine may be different from the concentrations used here to predict the AUC ratio. Therefore, further systemic studies are needed to clarify the in vivo effects.It is noteworthy that the extrapolation from In summary, the present findings highlight potential DDIs involving specific TKIs and UGT substrates, namely by inhibition of UGT1A1 and 2B17 by lapatinib and imatinib, respectively. Our study provides a basis for design of clinical studies for investigation of DDIs involving TKIs.Axitinib, imatinib, lapatinib, vandetanib, SN-38 and SN-38 glucuronide (SN-38G) were purchased from Toronto Research Chemicals, Inc . Tris-HCl, 4-methylumbelliferone (4-MU), 4-methylumbelliferone-β-D-glucuronide (4-MUG), alamethicin and uridine 5-diphosphoglucuronic acid trisodium salt (UDPGA) were purchased from Sigma-Aldrich . Trifluoperazine (TFP) was purchased from Enzo Life Science . All other reagents were of high-performance liquid chromatography (HPLC) grade or the highest grade commercially available. A panel of recombinant human UGT supersomes  expressed in baculovirusinfected insect cells was purchased from BD Gentest . Pooled human liver microsomes (HLMs) (n = 50 donors) were purchased from Invitrogen .2, 100 mM Tris-HCl buffer (pH 7.4), and 4-MU in the absence or presence of a TKI (100 μM). Incubations with 4-MU were performed at the final concentration corresponding to the apparent Km or S50 value reported for each isoform 1222The nonselective substrate of UGTs, 4-MU, was used as a probe substrate for all UGT isoforms except for UGT1A4. A typical incubation mixture with a total volume of 100 μl contained recombinant UGT isoform , 10 mM UDPGA, 5 mM MgCl4-MUG concentrations were determined by using an Agilent 1200 HPLC interfaced with Applied Biosystems Qtrap 3200 equipped with an electrospray ion source. Chromatographic separation was carried out with a Waters XTerra MS C18 column . The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B) and was delivered at 250 μl/min. The gradient was initiated at 85% mobile phase A to 90% mobile phase B over 1 min, held constant for 1.5 min, and then restored to the initial composition until 10 min. The MRM parameters for 4-MUG and phenytoin as the internal standard were 353.05/177.20 and 253.30/182.20 in positive ion mode.Signals for TFPG were detected by using Applied Biosystems Qtrap 4000 with an electrospray ion source interfaced with a Shimazu Prominence Modular HPLC. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The column was first equilibrated at 25% mobile phase B for 1.5 min at a 200 μl/min flow rate. The elution was then ramped linearly to 90% mobile phase B over 1.5 min, maintained for 5 min, and followed by a return to initial conditions. TFPG was detected using MRM pair of 584/408 in positive ionization modeSN-38 glucuronidation activity was determined with a slight modification of a previously published methodThe concentration of SN-38G was determined by using an Agilent 1200 HPLC interfaced with Applied Biosystems Qtrap 5500. The MRM setting for SN-38G was 569.2/393.2 in the positive mode. The same internal standard was used for quantification. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The column was first equilibrated at 10% mobile phase B for 1 min at 250 μl/min. The elution was then ramped linearly to 90% mobile phase B over 2 min, maintained for 2 min, and followed by a return to initial conditions.50) value for each TKI was estimated by using GraphPad Prism 5 software .The same incubation condition as for the preliminary inhibition study was used for each UGT isoform, except that the concentration of each inhibitor was ranged from 0 to 200 μM. The relative formation rate of 4-MUG, TFPG or SN-38G in the presence vs. absence of a TKI was estimated, and the half maximal inhibitory concentration (ICKm to 4 × Km) was incubated with recombinant UGT1A1, 1A3, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15 or 2B17 at a final protein concentration as described in the UGT glucuronidation inhibition assay in the presence or absence of TKI at four different concentrations (ranging from IC50/4 to 4 × IC50). The incubation time for each UGT isoform was the same as that used to determine IC50. SN-38  was incubated with UGT1A1 (0.1 mg/ml) or HLMs (0.4 mg/ml for) in the presence of lapatinib . Ki values were calculated using nonlinear regression according to the models for competitive inhibition, noncompetitive inhibition, or mixed inhibition. Global nonlinear regression analysis in the kinetics module of GraphPad Prism 5 software was used to simultaneously fit all data to the equations for competitive inhibition, non-competitive inhibition, and uncompetitive inhibition. The type of inhibition was determined based on Lineweaver-Burk plot and the best fitting of data to the equations. Goodness of fit to kinetic and inhibition models was assessed from the F statistic, r2 values, parameter standard error estimates and 95% confidence intervals. Kinetic constants are reported as the mean value ± standard error of the parameter estimate. Lineweaver-Burk plot and Dixon plot were also used as graphical representation of the type of inhibition and Ki.4-MU at four concentrations (ranging from 0.5 × av), the maximum systemic plasma concentration ([I]max), the maximum unbound systemic plasma concentration , the maximum hepatic input concentration ([I]in) and the maximum unbound hepatic input concentration  of TKIs after repeated oral administration were estimated based on the previously reported equations3738394041424344The average systemic plasma concentration ([I]i/AUC) of UGT substrates in the presence and absence of the inhibitor. The ratio was calculated based on the eq. 1 for drugs orally administeredThe magnitude of the inhibition of TKIs was estimated as the ratio of the area under the curve  was used for [I] in this study. Of note, clinically observed maximum unbound systemic plasma or blood concentration  was not used because the Cmax,u is known to be much lower than the [I]in,umax,u) was used to represent [I]. The prediction of AUC ratio is for any potential UGT substrates with undetermined fm, a range of fm between 0.1 and 1 was included for calculating AUCi/AUC ratio.In view of the general assumption that only unbound drug is available for interaction with the enzyme active site, and the aim of DDI research is to prevent the highest potential risk, the maximum unbound hepatic input concentration .


In the investigation of food allergy syndromes, serum assays detecting IgE antibodies (Ab) to food allergens use whole extracts on the solid phase, which contain specific allergen components in varying amounts. The interpretation of results assumes that the causative allergen components are adequately represented in the extract and that the presence and level of specific IgE titer to the whole extract of the relevant food(s) are diagnostic of the syndrome. IgE assays for beef or pork can underestimate the IgE Ab to a minor component of mammalian meat, such as galactose-α-1,3-galactose . The inciting food allergens in Eosinophilic Esophagitis (EoE) remain unclear, although IgE Ab to milk, wheat, soy, and peanut are frequently present in low titer.IgE Ab to relevant allergens and their components were measured in the sera of adults and children with peanut allergy, delayed anaphylaxis to mammalian meat, or esophageal biopsy-diagnosed EoE. Assay methods included: ImmunoCAP (CAP) using whole and component extracts, CAP assays on serial dilutions (1:2 to 1:8) of sera, and ImmunoCAP ISAC .Analysis with ISAC correlated well with results using component-specific CAP assays for both peanut allergens  and inhalant allergens (EoE). By contrast, no positive results for food allergens were found by ISAC in EoE sera that were positive for milk, wheat, or soy by CAP. Dilution assays showed no change  in either peanut allergy sera or EoE sera positive for aeroallergens (dust mite and cat); in contrast, calculated titers up to six times the undiluted value were noted for foods in EoE sera  and in mammalian meat allergy sera (beef and pork). CAP assays for 5 milk components revealed positivity to minor components in 50% of EoE sera. CAP assays for α-gal revealed positivity in 100% of patients presenting with delayed anaphylaxis to mammalian meat.Differences in the results of the dilution assays demonstrate that assaying undiluted sera can significantly underestimate IgE Ab levels if the IgE Ab are directed against a quantitatively minor component of the extract on the solid phase. These results strongly suggest that the IgE Ab to milk and other foods in EoE sera are directed against a minor component that has not yet been identified.


Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain . The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein–protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation,  Carica papaya  of  targets . P. falcdrolysis .2+), by an auto catalytic process with requirement of a significant drop in pH and by other enzymes as in trans-activation , cysteine proteases are also synthesized as inactive precursors (or zymogens). Cysteine protease zymogens contain a prodomain that block access of substrate to the active site . Besidestivation . This reFigure 1). Nucleophilic cysteine residue attacks to the carbon of the reactive peptide bond, producing the first tetrahedral thioester intermediate in the reaction with release of an amine or amino terminus fragment of the substrate . The prodomain of the cathepsin L subfamily  contain a prodomain of about 100 residues, with two conserved motifs: a highly conserved ERFNIN and GNFD motifs. An ERFNIN motif is lacking in cathepsin B, C, O, and X. Cathepsin B has a characteristic feature with ‘occluding loop’ which provide carboxydipeptidase activity .The mature enzymes or active enzymes of the family share a well-known fold of papain-like which consists of two domains: N-terminal helical domain and a C-terminal β-sheet domain. Catalytic Cys, His, and Asn are contributed by these two domains. These residues are located in the substrate binding cleft  lying at their interface  has shown inability to undergo auto-activation at its optimum pH (5.5) .The cathepsins B and L are well characterized papain-like enzymes, synthesized as zymogens . The pronterface . These rpH (5.5) . A conseIN motif . Due to Although there are differences but both the classes of enzymes have conserved mode of inhibition and share some common features such as interactions between the loop and the conserved hydrophobic residues in the prodomain and a conserved Gly residue near to the catalytic Cys residue. A conserved Gly in the hydrophobic pocket facilitate the C-terminus of the prodomain to push deep into the catalytic cleft and inhibit the catalytic site to perform catalysis. The prodomains exhibit a limited selectivity of inhibition against their related enzymes . First etrans-activation (65→Arg21 and Gln70→Arg31) within the prodomain .Auto-catalysis is a common mode of activation of cysteine proteases itself from their zymogens under the influence of pH change (acidic pH). The pH has great importance in auto catalytic mode of activation. The pH change triggers the disruption of important interactions between the prodomain and the mature domain, thereby make accessible the cleavage site within the prodomain loop to the active site. An N-terminal prodomain get cleaved off from the whole enzyme by active site and enzyme get activated (Table 1). Endopeptidases such as the cathepsins B, H, L, S, and K can be activated by autoactivation, whereas the true exopeptidases, such as the cathepsins C and X, needed endopeptidases, such as the cathepsins L and S   . Mutatiohepsin K .in vivo processing of cathepsins  (The activation of cysteine proteases are modulated by the glycosaminoglycans (GAGs) such as sulfated GAGs, heparin, heparan sulfate, and chondroitin sulfates A, B, C, E, and other negatively charged polysaccharides, e.g., dextran sulfate. Various studies have suggested the involvement of GAGs in the thepsins . Similarigure 5) . Furtherigure 5) . Howeverigure 5) . Besidesigure 5) . Most ofigure 5) .P. falciparum, synthesize as zymogens and get activated by auto activation in acidic environment (pH 5–5.5). 185–Glu221, Glu210–Lys403) in falcipain-2, and one salt bridge (Arg202–Glu238) in falcipain-3, and hydrophobic interactions present both in falcipain-2 (Phe214–Trp449Trp 453), and falcipain-3 (Phe231–Trp457Trp 461) also play important roles in the activation of these enzymes . Mutants were unable to undergo autocatalysis, although, activation can further be achieved by trans-activation  demonstrated that auto activation can occur within wide pH range 4.5–7.3 (F. hepatica (FhproCL1Gly25) cannot undergo auto-catalytic processing  expresses papain-like cysteine proteases including cathepsins  and an aspartic protease (cathepsin D) in the parasite gut, function in an acidic pH environment  or some other proteases that cleave within the residues lying at the junction of prodomain and mature domain such as pepsin, Cathepsin D, and legumain/asparaginyl endopeptidase    residues (F. hepatica cathepsin L (FhproCL1) enzyme is able to trans-process (FhproCL1) zymogens at specific cleavage site Leu12–Ser11↓His10motif and thus increase rate of activation exponentially. Mutational study has shown that alteration of the motif to a Pro12–Ser11↓His10 prevents or slows down FhproCL1 activation  lacks the conserved asparagine residues found in other homologues and could not be trans-processed by Opisthorchis viverrini asparaginyl endopeptidase. However, recent studies shown O. viverrini cathepsin B (Ov-CB-1) is capable of trans-activating cathepsin F (Ov-CF-1) at a specific site between the prodomain and the mature enzyme (igure 7) . Cathepsigure 7) . Howeverigure 7) . Falciparesidues . Pre-acttivation . A studyistosome . Furthere enzyme .in vitro from zymogen to mature enzyme by reducing the pH of solution  is a unique in terms of activation, as the zymogen does not auto catalytically activate at low pH . CrystalCathepsin-D, an aspartic protease also synthesize as inactive enzyme. The activation of cathepsin-D, an intermediate mechanism had been proposed, i.e., a partial auto-activation followed by a final processing by other enzymes. Studies have shown that the processing of endocytosed procathepsin-D is also independent of its catalytic function and requires cysteine proteases for its activation .trans-activation). Auto-activation is most economical mode of conversion since no involvement of other enzymes. However, trans-activation by other enzyme is required to enhance the conversion rate in critical conditions. For example, cathepsins F, a liver fluke (O. viverrini) enzyme, secreted as an inactive zymogen that auto-catalytically processes and activated to a mature enzyme at pH 4.5, but studies shows that it can also be activated via trans-processing by cathepsins B1 at pH 5.5, where it is unable to activate auto-catalytically. Both enzymes (cathepsin B1 and F1) work together to degrade the host tissue and contributing to develop liver-fluke-associated chalangiocarcinoma . Presently, targeting PPIs is an attractive and promising area of research. A special interest in cysteine proteases as targets derives from the recognition that they are critical to the life cycle or pathogenicity of many parasites. This functional diversity is derived from their unique nucleophilicity, adaptability to different substrates, stability in different biological environments, and their regulations. Parasite derived cysteine proteases play key roles in hemoglobin hydrolysis, breakdown of RBC proteins, immunoevasion, enzyme activation, virulence, tissue, and cellular invasion as well as excystment, hatching, and molting . PreviouIn this review, we have discussed targeting the cysteine proteases before activation, which may prevent their involvement in various diseases. The possible ways are:1. Targeting promature domain interactions which are important for the activation.2. Targeting the residues involved in pH sensing and activation.Pro-mature domain interactions can be categorized as PPIs. Earlier targeting PPIs were not in trend due to technological hurdles. Studies have demonstrated that small molecules can disrupt the large and complex protein interactions by interacting with interface residues, known as hot-spots . In a st69 of prodomain act as pH sensor that regulates the compartment-specific cleavages of the prodomain. Although, structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His69 does not contribute directly to the prodomain–enzyme interface. But, rather, triggers movement of a loop region in the prodomain that modulates access to the cleavage site and thus, allows the tight pH regulation of furin activation (The pH has great importance in activation of zymogens of proteases in specific compartment . It is vtivation . Like seSV and KP conceived and designed the review. SV, KP, and RD wrote the review paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.