AbstractSpinolyprops Pic, 1917  are described from Thailand and China . The species characters within the genus are discussed, photographs of all seven Oriental species are added, a species key for the Oriental species is compiled, and a map with the distributional patterns is provided.Two new species of the genus  Spinolyprops Pic, 1917  was based on the type species Spinolyprops rufithorax Pic, 1917 from Zanzibar . All known species have fully developed wings and thus possess a high ability for dispersal. Specimens are usually collected by sifting litter and similar substrates, and are also attracted by light.The Natural History Museum, London (Max Barclay)BMNHCollection Dr. Roland Grimm, NeuenbürgCRGTHungarian Natural History Museum, Budapest (Dr. Ottó Merkl)HNHMMuséum d’Histoire Naturelle, Genève (Dr. Giulio Cuccodoro)MHNGMuseum für Naturkunde, Berlin (Dr. Manfred Uhlig)MNBMuseo di Storia Naturale, Firenze (Dr. Luca Bartolozzi)MSNFNaturhistorisches Museum, Basel (Dr. Michel Brancucci †)NHMBNational Museum , Prague (Dr. Jiří Hájek)NMPCStaatliches Museum für Naturkunde, Karlsruhe (Dr. Alexander Riedel)SMNKStaatliches Museum für Naturkunde, StuttgartSMNSSpinolyprops pakistanicus), absent in all other species. Specific shape of the apicale of aedeagus, considering a certain variability , leg. A. Schulz & K. Vock, SMNS. – Paratypes: N Thailand, Chiang Mai Prov., Doi Inthanon, 1800 m, 14.V.2006, leg. R. Grimm, 4 ex. CRGT, 1 ex. SMNS. – NW Thailand, Doi Pui, 1600–1685 m, 7.–9.V.2004, leg. R. Grimm, 4 ex. CRGT. – NW Thailand, Doi Pui, 1600–1685 m, 22.–23.V.2006, leg. R. Grimm, 1 ex. SMNS. – China, Yunnan, 22 km NE Dali, NE bank of Er Hai Lake, 2010 m, 12.VI.2007, leg. M. Schülke, 1 ex. MNB, 1 ex. SMNS. – China, S Yunnan, Mengyang NR, 500 m, 12.IX.1994, leg. S. Kurbatov, 2 ex. HNHM. – China, NE Guangxi, 15 km N Longsheng, 1000 m, 15.–22.VI.1995, leg. S. Kurbatov, 1 ex. HNHM.Spinolyprops cribricollis sp. n. shares with Spinolyprops lateralis the rough dorsal punctation of pronotum and elytra, the shape of the pronotum with lateral parts broadly separated from disc and bent up, the frons between eyes smaller than dorsal eye diameter, and the antennomeres 8–10 longer than broad. Both can be separated mainly by the anterior margin of the pronotum with feeble (Spinolyprops cribricollis sp. n.) or deep excavation , and by different shape of the aedeagus , and the elytra are slightly more rounded. Spinolyprops maculatus has a similar shape of the pronotum with feebly excavated anterior margin, but the aedeagus has the apicale of the aedeagus different triangular with straight sides. Spinolyprops trautneri has also a different aedeagus with broad pentagonal apicale  Chinese specimens from Yunnan and Guangxi to the same species. Shape and punctation of the pronotum, elytral colour and shape of aedeagus are not distinctly different from the specimens from Thailand. The type locality lies in a lowland habitat (Island Ko Chang), and the paratypes from Thailand were collected in higher altitudes (Doi Pui and Doi Inthanon). Obviously, this species has a wide ecological range.The name refers to the rough punctation of the pronotum.Kaszab, 1965http://species-id.net/wiki/Spinolyprops_himalayicusIndia, Darjeeling , Peshok, 710 ft., 19.IX.1959, leg. F. Schmid, holotype HNHM.New material. Nepal, Gorkha Distr., Arughat Bazar, 600 m, 26.VII.1983, leg. J. Martens & W. Schawaller, 1 ex. SMNS. – Nepal, Surkhet Distr., Bheri Khola Bridge, 500 m, 24.–25.V.1998, leg. W. Schawaller, 1 ex. SMNS. – Nepal, Chitwan NP, Rampur, V.2005, leg. D. Ahrens, 1 ex. SMNS. – N India, Darjeeling, Sukna, 180 m, 21.–23.V.1980, leg. G. Topál, 1 ex. HNHM. – N India, Uttar Pradesh, Mussorie, 1300 m, 10.VII.1989, leg. A. Riedel, 1 ex. SMNS. – NE India, Meghalaya, 1 km E Tura, 500–600 m, 13.–18.V.2002, leg. M. Trýzna & P. Benda, 1 ex. SMNS. – W India, Maharasthra State, 70 km S Pune, Wai, 3.–6.X.2005, leg. F. & L. Kantner, 1 ex. SMNS. – W India, Maharasthra State, 40 km W Pune, Mulshi, 7.–11.X.2005, leg. F. & L. Kantner, 1 ex. SMNS. – S India, Anamalai Hills, Cinchona, 3500 ft., 1959, no collector labelled, 9 ex. NHMB . – Bhutan, Samchi, 300 m, 7.–11.V.1972, Basel Expedition, 2 ex. HNHM. – Andaman Islands, Havelock Island, village no. 7, 22.IV.–14.V.1998, leg. K. & S. Majer, 2 ex. NHMB, 1 ex. SMNS. – N Thailand, Chiang Mai, Doi Pui, 1500 m, 19.XII.1988, leg. K. Geigenmüller & J. Trautner, 1 ex. SMNS. – N Thailand, Chiang Dao, 9.I.1989, leg. K. Geigenmüller & J. Trautner, 1 ex. SMNS. – N Thailand, Chiang Dao, 70 km N ChiangPageBreak Mai, 26.–28.IV.2003, leg. O. Šafránek, 2 ex. SMNS. – N Thailand, Chiang Mai Prov., Ban San Pakia, 1700 m, 25.IV.–7.V.1996, leg. S. Bilý, 7 ex. NHMB, 1 ex. SMNS. – N Thailand, Nan, 22.–24.V.1999, leg. R. Grimm, 16 ex. CRGT, 1 ex. SMNS. – N Thailand, Nan, 2.–4.V.2003, leg. R. Grimm, 2 ex. CRGT. – NW Thailand, Doi Pui, 1600–1685 m, 15.–16.IV.2004, leg. R. Grimm, 1 ex. CRGT. – N Thailand, Chiang Mai, Doi Pui, 1600 m, 15.–16.IV.2004, leg. W. Schawaller, 2 ex. SMNS. – NW Thailand, Chiang Dao, 700–800 m, 4.V.2004, leg. R. Grimm, 3 ex. CRGT. – NW Thailand, Mae Hong Son, 5.V.2004, leg. R. Grimm, 2 ex. CRGT. – NW Thailand, 5 km E Pai, 700 m, 19.IV.2004, leg. W. Schawaller, 1 ex. SMNS. – NW Thailand, Soppong, 700 m, 23.IV.2004, leg. W. Schawaller, 9 ex. SMNS. – NW Thailand, Soppong (Pangmapa), 17.–18.V.2006, leg. R. Grimm, 5 ex. CRGT. – Thailand, Chumphon Prov., Pha To, 27.III.–14.IV.1996, leg. K. Majer, 3 ex. NHMB. – Thailand, Thanon Thong Chai, Palong, 750 m, 26.–28.V.1991, leg. V. Kubán, 3 ex. NHMB. – Thailand, Prachin Buri Prov., Sakaerat Ecology Research Institute, 4.VI.2001, leg. E. Horváth & G. Sziráki, 5 ex. HNHM. – Burma, N Shan State, Namhsan, 1500–1900 m, 18.–28.II.1996, leg. S. Kasantsev, 1 ex. NHMB. – C Laos, Khammouan Prov., Ban Khoun Ngeun, 200 m, 19.–31.V.2001, leg. L. Dembický, 5 ex. SMNS. – C Laos, Khammouan Prov., Ban Khoun Ngeun, 17.V.–6.VI.2007, leg. M. Štrba, 1 ex. SMNS. – CE Laos, Boli Kham Xai Prov., 8 km NE Ban Nape, 600 m, 1.–18.V.2001, leg. L. Dembický, 6 ex. SMNS. – Laos, Champassak Prov., Bolavens Plateau, 3 km SE Ban Lak, 1070 m, 9.V.2010, leg. J. Hájek, 1 ex. NMPC. – Laos, Champassak Prov., Ban Nong Luang, 12 km S Paksong, 800 m, 6.IV.1998, leg. O. Merkl & G. Csorba, 1 ex. HNHM. – Laos, Phongsaly Prov., Phongsaly, 1500 m, 28.V.–20.VI.2003, leg. M. Brancucci, 2 ex. NHMB. – Laos, Phongsaly Prov., Phongsaly, 1500 m, 6.–17.V.2004, leg. M. Brancucci, 1 ex. SMNS. – Vietnam, Daklak Prov., Buon Ma Thuot, Dak Linn, 500 m, 28.–29.IV.1986, leg. S. Golovatch & L. Medvedev, 7 ex. SMNS. – Vietnam, Bac Kan Prov., Ba Be NP, 350 m, 3.–8.VI.2011, leg. L. Bartolozzi et al., 3 ex. MSNF. – Java, Batavia (now Jakarta), III.1921, no further data, 1 ex. HNHM. – C Bali, Bedugul, Tamlingan, 1210 m, 6.XI.2007, leg. A. Riedel, 10 ex. SMNK, 4 ex. SMNS.India , Nepal, Bhutan, N Thailand, Vietnam ; AndamanPic, 1917http://species-id.net/wiki/Spinolyprops_lateralisSpinolyprops rufithoraxlateralis Pic, 1917 var. PageBreakSabah, Sapulut, Batu Pungull, 24.–26.VI.1998, leg. J. Kodada & F. Čiampor, 1 ex. SMNS. – Borneo, Sabah, Poring, 650 m, 15.V.2005, leg. R. Grimm, 1 ex. CRGT. – Borneo, Sarawak, Gunung Santubong, 10–200 m, 4.–8.IV.2009, leg. R. Grimm, 1 ex. CRGT. – Borneo, Sarawak, Gunung Santubong, 30–200 m, 30.XI.–5.XII.2010, leg. R. Grimm, 4 ex. CRGT. – Borneo, Sarawak, Gunung Gading NP, 100–300 m, 31.III.–4.IV.2009, leg. R. Grimm, 1 ex. CRGT. – Borneo, Sarawak, Gunung Gading NP, 50–200 m, 8.–10.XII.2010, leg. R. Grimm, 2 ex. CRGT. – NW Thailand, Mae Hong Son Prov., 32 km NNE Mae Hong Son, 5.V.2004, leg. R. Grimm, 2 ex. CRGT. – S Thailand, Khao Lak NP, Thone Chong Fa Waterfall, 100–300 m, 6.–15.I.1998, leg. A. Schulz & K. Vock, 3 ex. SMNS, 1 ex. MNB. – W Malaysia, Perak, 25 km NE Ipoh, Banjaran Titi Wangsa Mts., Mt. Korbu, 1200 m, 6.–12.V.2001, leg. P. Čechovský, 1 ex. SMNS.NE Sumatra, Tebing-Tinggi, 1 ex. NHMB (Frey collection). – E Sumatra, Lampung, Bawang, Pedada Bay, Gn. Tanggang, 660 m, 9.VIII.2006, leg. A. Riedel, 1 ex. SMNS. – Borneo, Brunei, Temburong Distr., ridge NE Kuala Belalong, 300 m, X.1992, leg. J. H. Martin, 1 ex. BMNH. – Borneo, Sabah, Crocker Range, Tenom, Kalang waterfall, 17.VI.1998, leg. J. Kodada & F. Čiampor, 5 ex. SMNS. – Borneo, Sumatra ; Borneo, Thailand, W Malaysia (new records).Kulzer, 1954http://species-id.net/wiki/Spinolyprops_maculatusSri Lanka, Colombo, III.1953, leg. G. Frey, holotype and 1 paratype NHMB (Frey collection), 2 paratypes HNHM.Sri Lanka, Uva, Diyaluma Falls, 400 m, 23.I.1970, leg. C. Besuchet, I. Löbl & R. Mussard, 3 ex. MHNG, 1 ex. SMNS. – Sri Lanka, Uva, Monaragala, 300 m, 13.II.1970, leg. C. Besuchet, I. Löbl & R. Mussard, 1 ex. HNHM. – Sri Lanka, Uva, S Wellawaya, 300 m, 25.I.1970, leg. C. Besuchet, I. Löbl & R. Mussard, 1ex. HNHM. – Sri Lanka, Periyapullumalai, 11.II.1970, leg. C. Besuchet, I. Löbl & R. Mussard, 1 ex. HNHM. – Sri Lanka, Kandy, 18.III.1973, leg. G. Zimmermann, 1 ex. SMNS. – S Burma (labelled as Tenasserim), no additional data, 1 ex. HNHM (det. Kaszab).Spinolyprops maculatus and shares with the specimens from Sri Lanka the elytral colour pattern apically with an arrow-shaped dark element .The specimen from Tenasserim was already published by  element . It is tSri Lanka , ? S Burma .Schawaller, 1996http://species-id.net/wiki/Spinolyprops_pakistanicusPakistan, Hazara, Malkandi, 1500 m, 3.VI.1983, leg. C. Besuchet & I. Löbl, 2 paratypes SMNS. – Pakistan, Swat, Madyan, 1400 m, 16.V.1983, leg. C. Besuchet & I. Löbl, 1 paratype SMNS.PageBreakNorthern Pakistan in Hazara and Swat.urn:lsid:zoobank.org:act:E51CF1A3-852E-4A33-8196-46C799657133http://species-id.net/wiki/Spinolyprops_thailandicusPageBreak4 ex. CRGT. – N Thailand, Chiang Mai, Doi Pui, 1600-1685 m, 7.–9.V.2004, leg. R. Grimm, 6 ex. CRGT. – N Thailand, Chiang Mai, Doi Pui, 1600-1685 m, 12.–13.V.2006, leg. R. Grimm, 7 ex. CRGT, 4 ex. SMNS. – N Thailand, Chiang Mai, Doi Pui, 1600-1685 m, 22.–23.V.2006, leg. R. Grimm, 6 ex. CRGT, 3 ex. HNHM.Holotype male: N Thailand, Chiang Mai, Doi Pui, 1600 m, 15.–16.IV.2004, leg. W. Schawaller, SMNS. – Paratypes: same data as holotype, 3 ex. SMNS. – N Thailand, Chiang Mai, Doi Pui, 1600-1685 m, 23.IV.–12.V.2003, leg. R. Grimm, Spinolyprops thailandicus sp. n. is characterized by the shape of the pronotum with deeply excavated anterior margin and with the lateral parts broadly separated from disc and bent up, in combination with rough dorsal punctation of pronotum and elytra and the frons between eyes smaller than dorsal eye diameter. The aedeagus of Spinolyprops thailandicus sp. n.PageBreak is similar as in Spinolyprops cribricollis sp. n..


Phil Biggin, PhD, is a group leader in the Department of Biochemistry, University of Oxford. He is interested in computational approaches to receptor dynamics and ligand-binding. His group uses a wide range of methodologies ranging from molecular dynamics simulations through to machine learning techniques to address fundamental problems in pharmacology. He is the Section Editor for Computational, in silico and modelling studies for BMC Pharmacology and Toxicology. In this interview we find out a little more about the key issues in this field of research.I became interested in chemistry and computers as a teenager which led to my decision to read Computer-aided Chemistry for my first degree. During that time I spent a fascinating industrial year at SmithKline Beecham (now GSK) developing software to aid the computational chemistry group. Although at that time I did not know I wanted to do a PhD, I did know that I wanted to continue investigating the way in which drugs work. I then undertook a PhD in the biophysics of ion channels, which helped fill in some of the biological gaps in my knowledge. The rest is history as they say.Computational chemistry and molecular modelling have been embedded within the drug-design process for many years now and indeed most pharmaceutical companies have some kind of computational chemistry group within their Research and Development programmes. From early quantitative structure activity relationship (QSAR) studies and visualization through to cheminformatics and molecular dynamics, the scope and power of these techniques is ever increasing and they are starting to yield important results that are not only interestingly academically, but can result in substantial financial savings for drug companies.One of the major challenges for computational chemists remains the prediction of affinity for small molecules with protein (and nucleic acid) targets. There have been many approaches to this ranging from thermodynamically rigorous free energy calculations through to machine learning methods. Although there have been examples of good success, the prediction of absolute affinities remains a major challenge for the field. If the prediction of affinity was not challenging enough, the next problem will be the prediction of efficacy. Just because a compound binds with high affinity does not necessarily mean it has high efficacy. In fact the problem may be even worse than that, because it is becoming apparent that some systems exhibit 'agonist bias' whereby the efficacy of the same agonist is not maintained across different pathways as discussed by Terry Kenakin in his review article on this topic . These aBoth of the problems above will become more tractable with physical models as the computational power available increases. In fact, it is already possible to explore timescales that are not only experimentally relevant but also able to address problems that are difficult if not impossible to address by experiment alone . We can Many of the challenges we face are still at the one protein-one drug level, but in order to predict pharmacology and toxicology in the future effectively, more consideration will have to be given to absorption, distribution, metabolism, excretion and toxicity (ADMET). Many adverse drug reactions are caused by unintended activity at off-targets. There has been some recent success in those types of predictions , and altThe best predictions will be those that account for all of the effects of a compound within the body and to that end aspects of systems biology will certainly have a key role to play. Indeed, physiologically based pharmacokinetic (PBPK) modelling is already used to guide the decision-making process within regulatory review . One migThe quality of future predictions will be underpinned by two main factors: 1) Knowledge or data and 2) computing power. As both of those are rising exponentially, one can look forward to more accurate computational pharmacology sooner rather than later.in silico and modelling studies.In keeping with the journal ethos to publish work deemed by peer review to be a coherent and sound addition to scientific knowledge I would be particularly interested in seeing papers on drug interactions with other macromolecular systems, especially those that are supported by experimental data and shed light on the underlying mechanism. Of course, that is a slightly personal view and we really would welcome submissions from across the whole field of Computational, BMC Pharmacology and Toxicology.Philip Biggin is a Section Editor on PB wrote and approved the final text.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2050-6511/13/2/prepub


Foxg1/−− null embryos and this expansion is more pronounced in the nasal retina where Foxg1 normally shows its highest expression levels. Results on expression of a reporter allele for Wnt/β-catenin signalling and of Lef1, a target of Wnt/β-catenin signalling, displayed significant upregulation of this pathway in Foxg1/−− nulls at embryonic days 12.5 and 14.5. Interestingly, this upregulation was observed specifically in the nasal retina, where normally very few Wnt-responsive cells are observed. These results indicate a suppressive role of Foxg1 on this signalling pathway. Our results reveal a new role of Foxg1 in limiting CM development in the nasal peripheral retina and add a new molecular player in the developmental network involved in CM specification.The ciliary margin (CM) develops in the peripheral retina and gives rise to the iris and the ciliary body. The Wnt/β-catenin signalling pathway has been implicated in ciliary margin development. Here, we tested the hypothesis that in the developing mouse retina Foxg1 is responsible for suppressing the Wnt/β-catenin pathway and restricting CM development. We showed that there is excess CM tissue in  •Foxg1 is expressed in a nasal-high to temporal-low gradient in developing retina.•Foxg1/−− mutant retina.Ciliary margin expansion is observed nasally in the •Foxg1/−− peripheral retina nasally.Wnt/β-catenin signalling is upregulated in the •A new role of Foxg1 in controlling ciliary margin development is proposed. During eye development the optic cup gives rise to the centrally located neural retina (NR), an outer located layer known as retinal pigment epithelium (RPE) and to the peripherally located ciliary margin (CM), which lies at the interface between the NR and the RPE. Although of neural origin, the CM generates two non-neural epithelial eye structures: the ciliary body (CB) proximally, which is responsible for the production of the aqueous humor and maintenance of the intraocular pressure, and the iris distally, whose role is to control the amount of light entering the eye and the circulation of the aqueous humor  have micropthalmic eyes with a large ventral coloboma  12.5 and E14.5. Interestingly, this upregulation is observed specifically in the nasal retina, where normally very few Wnt-responsive cells are observed. These results indicate a suppressive role of Foxg1 on this signalling pathway. Our results reveal a new role of Foxg1 in maintaining the normal size of the CM in the nasal peripheral retina and add a new molecular player in the developmental network involved in CM specification.Here, we have examined the hypothesis that in the developing mouse retina Foxg1 is responsible for suppressing the Wnt/β-catenin pathway and restricting the development of CM tissue. We show that CM tissue is expanded in Mice were maintained, bred and handled according to Home Office (UK) regulations.Foxg1+/cre mouse to generate compound Foxg1-cre heterozygous/BAT-gal mutants. For the GFP expression experiment, a Gsh2-cre strain  and genotyping .The following mouse lines were used for this study: a Foxg1-lacZ heterozygous strain  kept on e strain  was crosr allele . HeterozFoxg1+/+) and heterozygotes (Foxg1+/−) these embryos were both used in the control group.As no differences were detected between wild types  0.5.Embryos were collected and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. They were processed for paraffin or cryostat embedding according to previously described protocols .Immunohistochemistry and immunofluorescence were carried out as previously described . The folBmp4, Tbx5 and Vax2 (kindly provided by Dr R. Hindges); Foxg1 and Wnt2b (kindly provided Dr T. Theil); Msx1 (kindly provided by Dr L. Lettice); Lef1 (kindly provided by Dr O. Machon) and Sfrp2 (kindly provided by Dr A. Rattner).In situ hybridisation was performed as previously described . RiboproDAB and in situ images were taken with a Leica DFC480 camera connected to a Leica DMNB epifluorescence microscope. Fluorescence images were taken with a Leica DM5500B automated upright microscope connected to a DFC360FX camera. Confocal images were taken with a Zeiss LSM510 confocal system coupled to a Coherent Mira mode-locked Ti:Sapphire multi-photon laser.The labelling index (LI) was calculated as the ratio of BrdU-positive to total number of cells on E11.5 control and mutant sections. Pregnant females were injected with 50–70 mg/kg BrdU  and sacrificed 30 min later. Cell counts were performed within three 50 µm bins, placed along the peripheral retina and were the average of at least six control and four mutant 10 µm thick sections. A total of three eyes from two different specimens were used for each genotype.P values ≤0.01.A one way ANOVA was used to calculate statistical differences in the LI between bins of the same genotype. A student's t-test was used to calculate statistical differences between corresponding bins of controls and mutants. Differences were considered significant for Total numbers of β-gal-positive cells were counted along the entire retina on consecutive 10 μm thick sections 30 μm apart. A total of 6 eyes from 3 different specimens were used for each genotype.Numbers of nasal and temporal β-gal-positive cells were counted on consecutive 10 µm thick horizontal sections 30 µm apart. The most dorsal and ventral sections, where the nasal and temporal retina could not be defined with precision, were excluded from the counts. A total of 6 eyes from 3 different specimens for controls and 4 eyes from 2 different specimens for mutants were used.Lef1-immunopositive cells were the average from two consecutive 10 μm thick sections, 30 μm apart, counted in the nasal peripheral retina of 3 control and 3 mutant eyes.Foxg1/−− mutant total, nasal, and temporal β-gal-positive cells and Lef1-positive nasal cells. Differences were considered significant for P values ≤0.01.A non-parametric test (Mann-Whitney) was used to calculate statistical differences between control and Foxg1lacZ/+ heterozygous embryos  is located  and this was confirmed quantitatively by RT-qPCR (data not shown).We had previously described high expression of Foxg1 in the dorsal and nasal retina at embryonic day (E) 14.5, based on beta-galactosidase  expression in  embryos . Here, wat E12.5 . Our resgradient A–E. Also located H. In temCM cells I. The ovFoxg1/−− optic cup fails to close at the optic fissure leading to a large ventral coloboma and the retina elongates abnormally  of the differentiating layer of the central retina , in agreement with previous findings using other patterning molecules in these mutants  of the peripheral retinal region in controls and mutants. We performed this study at E11.5, when the morphological differences between Foxg1/−− and control retinas are not as severe as those observed at later developmental stages  was significantly different to the LI of bins 2 and 3 .Wnt2b and the canonical Wnt pathway have been implicated in the development of the CM in both chicks and mice . We testgnalling   or the broader CM area , with only very few detected nasally . As with the E12.5 sections this increase in mutants was attributed to an increase in nasally located CM cells. The majority of β-gal-positive cells for both groups was located in the most dorsal sections of the retina (not shown).A similar significant increase in β-gal-positive cells was also observed in E14.5 Foxg1/−− mutant retinas, we performed immunohistochemistry for Lef1, a downstream target of this pathway. Lef1 protein was detected at high levels in the nasal and temporal CM of controls Fig. 9).BATgal+;Foxg1/−− mutants, we observed an upregulation in the number of Wnt/β-catenin responsive cells nasally, indicating that loss of Foxg1 has a striking effect on the development of the nasal CM. How does Foxg1 control CM development nasally? Our in situ hybridisation results demonstrate high levels of Foxg1 expression in the nasal retina and CM and lower levels in the corresponding temporal structures of controls. Based on our experimental results we propose that, nasally, Foxg1 normally represses Wnt/β-catenin signalling, restricting CM expansion BATgal+;Fwnt8b, a direct target of foxg1 in the zebrafish telencephalon  of the downstream targets of the Wnt/β-catenin pathway expressed in the peripheral retina, such as nd Axin2 . To datend Axin2 , which bnd Axin2 , raises nd Axin2 , inhibitFoxg1/−− retinal phenotype and find an expansion of CM tissue, which is more pronounced in nasal retina where Foxg1 is normally expressed at high levels. Our Wnt/β-catenin signalling reporter mouse reveals that in controls this pathway is mainly active in the temporal CM, while in Foxg1/−− mutants it is over-activated nasally. This suggests that Foxg1 restricts CM expansion nasally, while a molecule other than Foxg1 plays an analogous role in the temporal CM. Our data are in agreement with a new role of Foxg1 in restricting the development of the CM nasally and establishing the boundary between the neurogenic and the non-neurogenic retina.In this report, we examine the The Wellcome Trust and MRC, UK.


To investigate the expression of cyclin-dependent kinase 8 (CDK8) and β-catenin in colon cancer and evaluate the role of CDK8 in the proliferation, apoptosis and cell cycle progression of colon cancer cells, especially in HCT116 cell line.Colon cancer cell line HCT116 was transfected with small interfering RNA (siRNA) targeting on CDK8. After CDK8-siRNA transfection, mRNA and protein expression levels of CDK8 and β-catenin were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot assay in HCT116 cells. Cell proliferation was measured by 3--2, 5-diphenyltetrazolium bromide Methylthiazolyl tetrazolium (MTT) assay, and cell cycle distribution and apoptosis were analyzed by flow cytometry analysis (FACS). CDK8 and β-catenin protein levels were also examined by real-time PCR and immunohistochemistry (IHC) in colon cancer tissues and adjacent normal tissues.P < 0.05). CDK8 specific siRNA transfection inhibited HCT116 cells' proliferation and facilitated their apoptosis significantly (P < 0.05). In addition, the proportion of HCT116 cells in the G0/G1 phase was remarkably increased after CDK8-siRNA transfection (P < 0.05). The expression levels of CDK8 and β-catenin in adjacent normal tissues were lower than in tumor tissues (P < 0.05). Moreover, the expression of CDK8 was correlated with the expression of β-catenin in both tumor and adjacent normal tissues (P < 0.05).After CDK8 specific siRNA transfection, mRNA and protein expression levels of CDK8 and β-catenin in HCT116 cells were noticeably decreased (CDK8 and β-catenin were expressed in colon cancer at a high frequency. CDK8 specific siRNA transfection down-regulated the expression of CDK8 in colon cancer cells, which was also associated with a decrease in the expression of β-catenin Moreover, CDK8 specific siRNA inhibited the proliferation of colon cancer cells, promoted their apoptosis and arrested these cells in the G0/G1 phase. Interference of CDK8 might be an effective strategy through β-catenin regulation of colon cancer. Colon cancer is a result of an evolving process characterized by alterations of multiple genes and dysregulated cell signal transduction pathways. It has been well known that mutations of key genes in the Wnt/β-catenin signaling pathway play an important role in the occurrence and development of colon cancer ,2. UnderCyclin-dependent kinase (CDK) 8 is located in chromosome 13q12.13 and is a member of the CDK family ,6. CDK iRabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA). Lipofectin2000 was provided by Invitrogen (USA). RT-PCR kits were purchased from Fermentas (USA). Annexin V apoptosis kit  and siRNA-CDK8  were used in the present study.5/well and maintained in RPMI1640  supplemented with 10% fetal bovine serum (FBS). All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.The human colon cancer cell line, HCT116 cell line was purchased from Shanghai Cell Biology Institutes, Chinese Academy of Sciences . HCT116 cell line was seeded in 6-well plate at a density of 1.5 × 105) were divided into three groups: (a) siRNA-CDK8 group, (b) scrambled siRNA group, and (c) non-siRNA control group. One hour before transfection, the medium was replaced with 1.5 ml of serum free Opti-MEM. Then, the following reagents were mixed as follows: (a) 4 μl of Lipofectin2000 were added to 250 μl of Opti-MEM, and the mixture was kept at room temperature for 5 min; (b) 4 μl of siRNA stock solution were added to 250 μl of Opti-MEM (siRNA dilution); and (c) the above two mixtures were then mixed and kept at room temperature for 20 min (siRNA-Lipofectin2000 mixture). After shaking, this siRNA-Lipofectin2000 mixture was then added to a 6-well plate (1.5 ml of Opti-MEM in each well). Six hours later, the medium was replaced with complete medium. Our previous study confirmed that we obtained the maximal transfection efficacy when the ratio of Lipofectin2000 to siRNA was 4 μl:4 μl.CDK8 siRNA sequence 5'-AUAUAAUAGUGACUUCACCAUUCCCTT-3' (S) 5'-GGGAAUGGUGAAGUVAVUAUUAUAUTT-3' (AS) and scrambled siRNA sequence 5'-UUCUCCGAACGUGUCACGUTT-3' (S) 5'-ACGUGACACGUUCGGAGAATT-3' (AS) were designed and synthesized by Genepharma . HCT116 cells -2, 5-diphenyltetrazolium bromide Methylthiazolyl tetrazolium (MTT) solution (5 mg/ml) at 37°C for 4 h and subsequently made soluble in 150 μl of DMSO. Absorbance (A) was measured at 490 nm with an automated plate reader. Each sample was triplicated and the experiment was repeated three times. Cell growth curves were calculated as mean values of each group.Cells were trypsinized and centrifuged at 1500 rpm/min for 5 min at 48 h after transfection. Cells were harvested and washed with Phosphate Buffered Saline (PBS) twice. Reagents for apoptosis detection were added, and then cells were incubated in dark for 30 min and subjected to flow cytometry analysis (FACS). Additionally, cells were collected, washed with PBS, fixed with 75% ethanol at-20°C overnight, and centrifuged at 1500 rpm/min for 5 min. Then, ethanol was removed and cells were washed with PBS twice. Propidium iodide (PI) and 500 μl of RNAse were added, and then cells were incubated in dark at 4°C for 60 min. Lastly, cells were subjected to cell cycle analysis by FACS.The mRNA expression of CDK8 and β-catenin in HCT116 cells after CDK8-siRNA transfection were quantified by RT-PCR. Total RNA was extracted from cells with Trizol and subjected to reverse transcription into cDNA. CDK8 and β-catenin were amplified from the cDNA by RT-PCR. The PCR conditions consisted of 5 min at 94°C one cycle, 30 s at 94°C, 40 s at 55°C, 45 s at 72°C, and 7 min at 72°C 40 cycles. The primer sequences were as follows: 5'-TCACCTTTGAAGCCTTTAGC-3' (forward) and 5'-CTGATGTAGGAAGTGGGTCT-3' (reverse) for CDK8; 5'-TGCCAAGTGGGTGGTATAGAG-3' (forward) and 5'-TGGGATGGTGGGTGTAAGAG-3' (reverse) for β-catenin; 5'CTGGGACGACATGGAGAAAA3' (forward) and 5'AAGGAAGGCTGGAAGAGTGC3' (reverse) for β-actin.-ΔΔCT method was applied to analyze the relative changes in gene expression.The mRNA expression of CDK8 and β-catenin in colon cancer samples (n = 12) were quantified by real-time PCR. Informed consent was obtained from all the patients, and research protocols were approved by Independent Ethics Committee (IEC) of our hospital. A total of 200 mg of tumor or adjacent normal tissues (at least 2 cm distant from the tumor site) were homogenized in liquid nitrogen. Total RNA was extracted and reverse transcribed into cDNA, which was then used for amplification of CDK8 and β-catenin. The real time PCR conditions consisted of 1 cycle at 94°C for 10 min followed by 40 cycles at 94°C for 30 s, at 55°C for 30 s, and at 72°C for 30 s. GAPDH was employed as an internal standard. The primer sequences were as follows: 5'-GAGCGGGTCGAGGACCTGTTTGAAT-3' (forward) and 5'-ACATGCCGACATAGAGATCCCAGTTCCTTC-3' (reverse) for CDK8; 5'-TGCCAAGTGGGTGGTATAGAG-3' (forward) and 5'-TGGGATGGTGGGTGTAAGAG-3' (reverse) for β-catenin; 5'AGGGGCCATCCACAGTCTTC3' (forward) and 5' AGAAGGCTGGGGCTCATTTG 3 (reverse) for GAPDH. The 2 As described previously , followiThe protein expression of CDK8 and β-catenin in 47 tumor tissues and adjacent normal tissues were detected by IHC. Samples were fixed in 10% neutral formaldehyde, embedded in paraffin, and sliced. Briefly, the paraffin-embedded tissues were serially cut into 4 μm sections, dewaxed, and rehydrated. Sections were then blocked with peroxide and non-immune animal serum and incubated sequentially with rat anti-human CDK8 and β-catenin (1:1000), and biotin-labeled goat anti-rabbit IgG (1:1000). Finally, the sections were stained with DBA, counterstained with hematoxylin, dehydrated, cleared in xylene, and fixed.Histological assessment was performed as described previously . ImmunosP values < 0.05 were considered to be statistically significant.All data were presented as means ± standard deviation (SD). A Student's t-test was used for comparisons between groups, and F test was applied for correlation analyses. Statistical analysis was performed with SPSS 13.0 statistic software package. Six hours after CDK8-siRNA transfection, the transfection efficiency was detected by FACS. Our previous study confirmed that the maximal transfection efficacy could be obtained when the ratio of Lipofectin 2000 to siRNA was 4 μL: 4 μL. Figure P < 0.01) . Similarly, there was no significant difference between the scrambled siRNA group and non-siRNA group.Following a 72 h CDK8-siRNA transfection of HCT116 cells, protein expression of CDK8 and β-catenin was determined by western blot assay. As shown in figure P < 0.05)    Figure . On the ) Figure .P < 0.05)  and adjacent normal tissues .To further confirm the expression of CDK8 and β-catenin in colon cancer, we detected the expression of CDK8 and β-catenin in fresh colon cancer tissues and adjacent normal tissues of the same patient. Real-time PCR was adopted to detect the mRNA levels. Results showed that mRNA expression levels of CDK8 and β-catenin in tumor tissues was significantly higher than in adjacent normal tissues  Figure . In addiP < 0.05). As predicted, the expression of CDK8 was also correlated with the expression of β-catenin in both tumor tissues  and adjacent normal tissues .Shown by IHC, the positive cells were expressed as brown particles distributed in the cytoplasm of tumor cells Figure . Of the Aberrant activation of the Wnt/β-catenin pathway has been shown to be associated with numerous human cancers ,2,16. Pret al [Ron Firestein et al  found thet al . Additioet al ,19. Furtet al  and p53'et al  and exeret al . Therefoet al [To investigate the effects of the activity of β-catenin on colon cancer through CDK8, CDK8 interference was constructed and transfected in colon cancer cells CT116 by the application of siRNA in our study. The alteration of the expression of β-catenin, proliferation, cell apoptosis and cell cycle distribution in HCT116 cells were determined. After silencing CDK8 by siRNA, our results showed that β-catenin expression was markedly lower at the mRNA and protein level, and it is considered that there was a significant association between CDK8 and β-catenin expression. Such results support the claim of Ron Firestein To confirm the expression and relationship of CDK8 and β-catenin based on colon cancer tissues, real-time PCR and IHC were performed in our study. As predicted, both CDK8 and β-catenin expression level were markedly higher in tumor compared to adjacent normal tissues. Furthermore, the expression of β-catenin showed positively related to CDK8 expression. Meanwhile, it is reported that the expression of β-catenin was still positive or high in some colon cancer cell lines that have negative expression of CDK8. It is suggested that there might be other factors for regulating the activity of β-catenin such as pancreatic adenocarcinoma up-regulated factor (PAUF)  and DeltBased on the high specificity of CDK8 to β-catenin, CDK8 may be used as an alternative target in the regulation of colon cancer. Given the number of CDK inhibitors are being applied in clinical practice ,26, futuIn this study, we demonstrated that CDK8 specific siRNA transfection down-regulated the expression of CDK8, which is expressed in a high fraction in colon cancer. We also found out that CDK8 specific siRNA inhibited the proliferation of colon cancer cells, promoted their apoptosis and arrested these cells in the G0/G1 phase. In addition, CDK8 inhibition may be associated with the down-regulation of β-catenin. Our results showed that CDK8 and β-catenin could be promising target in the regulation of colon cancer by the control of β-catenin through CDK8.The authors declare that they have no competing interests.SH, YY, and LW carried out literature research, experimental studies and data acquisition, participated in the study design, and drafted the manuscript. MY and YZ participated in the design of the study and performed the statistical analyses. XZ proposed the study, and participated in its design and coordination and helped to draft, and assisted writing the manuscript. All authors read and approved the final manuscript.


Forficula auricularia) if cues of maternal condition affect offspring behavior in terms of sibling cannibalism. We experimentally manipulated female condition by providing them with different amounts of food, kept nymph condition constant, allowed for nymph exposure to chemical maternal cues over extended time, quantified nymph survival  and extracted and analyzed the females’ cuticular hydrocarbons (CHC). Nymph survival was significantly affected by chemical cues of maternal condition, and this effect depended on the timing of breeding. Cues of poor maternal condition enhanced nymph survival in early broods, but reduced nymph survival in late broods, and vice versa for cues of good condition. Furthermore, female condition affected the quantitative composition of their CHC profile which in turn predicted nymph survival patterns. Thus, earwig offspring are sensitive to chemical cues of maternal condition and nymphs from early and late broods show opposite reactions to the same chemical cues. Together with former evidence on maternal sensitivities to condition-dependent nymph chemical cues, our study shows context-dependent reciprocal information exchange about condition between earwig mothers and their offspring, potentially mediated by cuticular hydrocarbons.The evolution of parent-offspring communication was mostly studied from the perspective of parents responding to begging signals conveying information about offspring condition. Parents should respond to begging because of the differential fitness returns obtained from their investment in offspring that differ in condition. For analogous reasons, offspring should adjust their behavior to cues/signals of parental condition: parents that differ in condition pay differential costs of care and, hence, should provide different amounts of food. In this study, we experimentally tested in the European earwig ( Parental care evolved due to its fitness benefits to offspring, and it often comes at a cost for parents Previous research has focused on offspring begging signals conveying information about offspring condition as signals of need or quality Parental condition is often related to the timing of breeding, for example because individuals in good condition are able to breed early Forficula auricularia) is an insect species with uniparental maternal care including egg- and offspring attendance and food provisioning F. auricularia an ideal model system to test the influence of maternal condition cues on offspring selfishness.The European earwig  were separated and transferred to a pair of Petri-dishes (10×2 cm) containing humid sand as substrate and plastic shelters, respectively. At this stage, the females were randomly assigned either to the high food (HF) or to the low food (LF) treatment. To obtain females in HF or in LF condition, while keeping nymph condition constant, we manipulated the degree of female food access . On day 40 after hatching, we counted the number of surviving nymphs, and we took again the weight of the female and of five randomly chosen nymphs .35 = 0.434, p = 0.667), clutch size , hatching success , female body weight at hatching , or nymph body weight at hatching .The sample size consisted of 37 replicates (Petri dish pairs), 18 females and their broods in the HF treatment and 19 females and their broods in the LF treatment. The experimental treatments were properly randomized as there were no significant differences between treatments in female egg-laying date . The female was then removed from the vial and the extract stored at −30°C. Chemical analysis was carried out using Gas-Chromatography/Mass-Spectrometry . For analysis, 2 µl extract were injected in the GC  in splitless mode (splitless time = 2 min.) and a constant inlet temperature of 250°C. The GC-MS system was equipped with a HP-5MS fused silica capillary column . The GC temperature program started with a temperature of 70°C (held for 2 min), then increased at 15°C/min to 232°C (held for 11 min), and then at 5°C/min to 300°C (held for 7 min). The column helium flow rate was 1 ml/min, ion detection started after a five minute solvent delay, and the MSD was set to a scan range of 40–550 m/z. For quantification of the CHC profiles, we integrated 31 peaks  from the chemical chromatogram using Chemstation software . For quantification, we divided the area of each peak by the area of the internal standard in the same chromatogram and multiplied this ratio by 5 ng to obtain an estimate of the quantity for each peak in ng. We provide peak identifications based on comparison with previous unpublished CHC identification from earwigs  and using fragmentation analysis After termination of the experiment on day 40, all females were individually frozen at −30°C for later CHC extraction. For extraction, each female was immersed for 10 minutes in 800 µl of the extraction solution which consisted of n-Heptane  and 2.5 ng/µl n-Octadecane as an internal standard , the number of nymphs alive as the dependent variable, the number of nymphs originally present at experimental set up as denominator, and the female condition treatment, hatching date and their interaction as fixed effects.y = x0.2 which yielded approximately normal distributions. The values y of each peak were then standardized to a mean = 0 and standard deviation = 1 (iz = (i-y-y)/yσ) [as recommended in 46]. Given the large number of peaks in the CHC data (k = 30 peaks) relative to sample size (n = 37), and in order to take into account tight correlations among individual peak quantities, we used a variable clustering approach as implemented in JMP®Pro 10.0.1 to reduce data dimensionality , and the treatment, hatching date and their interaction as fixed factors. To directly test for a quantitative relationship between the proportion of nymphs alive and maternal CHC we used a step-wise linear regression approach with hatching date dependent survival  as dependent variable and the cluster representatives as candidate explanatory variables. The model with the lowest value for the Bayesian Information Criterion (BIC) was chosen as the final model and confirmed using both forward and backward variable selection procedures. All statistical analyses were carried out using JMP®Pro 10.0.1 statistical software (SAS Institute Inc.) and all reported p-values are two-tailed.35 = −5.088, p<0.0001), but the female food treatment did not affect nymph weight gain (from day 1 to day 40) . Thus, our food manipulation successfully generated variation in female condition while keeping nymph condition unaffected.As intended, females from the HF treatment gained significantly more weight over the course of the experiment  than females from the LF treatment  and hatching date  were not significant. The interaction was due to a significantly higher proportion of nymphs alive in the LF treatment among early hatching broods  and the opposite, marginally non-significant, trend among late hatching broods   see .The statistical clustering of the 30 peaks resulted in six clusters of highly correlated peaks  or to an interaction between hatching date and treatment . Thus, the relative quantity of cluster 3 CHC compared to cluster 6 CHC was a cue for female condition and significantly predicted the hatching date dependent nymph survival pattern, but it was not in itself significantly related to hatching date.The quantity ratio of cluster 3 and cluster 6 CHC was affected by the maternal condition treatment (FUmbilia crassicornis) nymphs signal predator threat to their tending mother through vibrational signals Forficula auricularia that condition-dependent chemical cues/signals from the mother, as encoded in her CHC profile, predict offspring survival, and that the direction of this effect depended on the timing of breeding. The nymphs from both treatments had access to equal amounts of food throughout and did not differ in their weight, and cannibalism occurred in almost all cases of nymph death. Thus, the difference in survival between treatments was most likely due to variation in nymph siblicidal and cannibalistic drive, induced by cues of maternal condition.Parents may transmit information about their condition or environmental conditions through pre-birth maternal effects, through their behavioral interactions with offspring, the provisioning of resources Labidura riparia; F. auricularia), to which the nymphs are then exposed while in the burrow.The information transfer about female condition was not direct through a behavioral interaction from mothers to their offspring. We experimentally prevented any physical  contact between mothers and nymphs by keeping the mother and her nymphs in separate Petri dishes (and swapping them daily) to ensure that only chemical information about maternal condition, and not her behavior or the amount of maternal food provisioning, could mediate the observed effects on nymph siblicide and cannibalism. Thus, females must have released chemical cues in the form of non-volatile contact pheromones in the substrate, and the nymphs were exposed to these cues when subsequently placed in the same environment. Under natural conditions this indirect substrate-born signaling would occur in the breeding burrows during the period of maternal care. Female earwigs “mark” their breeding burrow with pheromone secretions . This is evidence for context-dependence of offspring responses to maternal cues/signals. If variation in hatching date has a genetic component, these results would show genotype × family environment interactions The effect of maternal chemical cues of condition on nymph siblicide and cannibalism depended on the timing of breeding. Among early broods, nymphs exposed to maternal cues of poor condition showed a significantly and about two-fold higher survival rate than nymphs exposed to maternal cues of high condition. Interestingly, the effect was in the opposite direction among late broods. This effect could be either due to a quantitative or qualitative difference in the condition-dependent chemical cues among early and late breeding females or a difference in the response to the same condition-dependent cues among nymphs from early and late broods. Although our data does not allow us to fully disentangle the two possibilities, our further analyses indicate that the latter is the more likely explanation. Variation between females in CHC profiles was quantitative in nature. Early and late breeding females, and females in poor and good condition, had qualitatively the same CHC profiles. The composition of the female CHC profile (in particular the quantity ratio between cluster 3 and cluster 6 CHC) varied quantitatively with female condition, but not with timing of breeding, and it explained the treatment effect on nymph survival. We cannot fully rule out that other cues not measured by CHC extraction and GC-MS analysis  may be the causal agents underlying this effect, but any such cue would have had to be correlated with maternal CHC. Thus, our results indicate that nymphs born early and nymphs born late had opposite responses to maternal substrate-born cues of condition, expressing behavioral reaction norms F. auricularia. But why should the benefits of cooperative versus competitive strategies vary with the timing of breeding? In earwigs, early broods are the first to emerge from their winter burrows and experience low densities, less cannibalism threat by other earwigs and more time for development before the next winter starts. The low density could imply that the costs of dispersing and self-foraging  that have immediate fitness consequences in terms of survival. Thus, there is selection on this cue, and it seems likely that variation in maternal CHC profiles may have evolved to some extent due to its signaling function. We do not know if the observed variation in CHC profiles carries strategic costs  or if it rather reflects a constraint of limited food intake , where adult CHC profiles display information about breeding status (breeding versus non-breeding), and to a lesser extent also about their sex and nutritional condition Lasius niger) it was shown that ant queen CHC convey information about queen reproductive potential, and inhibits worker ovarian development and aggression CHC are well known for their multitudes of functions in insect communication, especially their role as cues in insect (kin) recognition F. auricularia showing that earwig nymphs express condition-dependent CHC profiles that affect maternal behavior Taken together, our results on the effect of maternal condition-dependent cues on nymph siblicide and cannibalism reported here, and the former findings in


Carfilzomib, the next generation of proteasome inhibitor, may increase osteoblast-related markers in patients with multiple myeloma, but the molecular mechanism of its effect on mesenchymal stem cell differentiation to osteoblasts remains unknown. Herein, we demonstrated that carfilzomib significantly promoted mesenchymal stem cell differentiation into osteoblasts. In osteoprogenitor cells and primary mesenchymal stem cells from patients with myeloma, carfilzomib induced increases in alkaline phosphatase activity, matrix mineralization, and calcium deposition via Wnt-independent activation of β-catenin/TCF signaling. Using affinity pull-down assays with immunoblotting analysis and immunofluorescence, we found that carfilzomib induced stabilization of both free and active forms of β-catenin in a time- and dose-dependent manner that was not associated with β-catenin transcriptional regulation. Nuclear translocation of β-catenin protein was associated with TCF transcriptional activity that was independent of the effects of GSK3β-activation and of signaling induced by 19 Wnt ligands, 10 Frizzled receptors, and LRP5/6 co-receptors. Blocking activation of β-catenin/TCF signaling by dominant negative TCF1 or TCF4 attenuated carfilzomib-induced matrix mineralization. Thus, carfilzomib induced osteoblast differentiation via Wnt-independent activation of the β-catenin/TCF pathway. These results provide a novel molecular mechanism critical to understanding the anabolic role of carfilzomib on myeloma-induced bone disease. Multiple myeloma (MM) is characterized by malignant plasma cells infiltrating bone marrow, where they closely interact with mesenchymal stem/stromal cells (MSCs), osteoblasts (OBs), and osteoclasts, triggering osteolytic bone lesions. It has become clear that impaired formation of bone and function of OBs plays a role in development of MM bone lesions. A number of studies have reported fewer OBs and decreased bone formation in MM patients with higher plasma cell infiltration . Myelomaβ-catenin, a key element in Wnt signaling, plays important roles in regulating differentiation of OBs from MSCs  and is iCarfilzomib (CFZ) (formerly PR-171) has been investigated as a second-generation proteasome inhibitor and potential anti-MM agent . In clinClinical-grade CFZ  was dissolved in water (to 10 mM) and stored at -20°C. MG132 cell-permeable proteasome inhibitor was purchased from EMD Chemicals . Recombinant Wnt3a (rWnt3a) protein was purchased from R&D Systems .www.lonza.com/. Walkersville, MD). MSCs from bone marrow of patients with MM (designated as MMMSCs) were generated using methods previously described [The Institutional Review Board Committee of the University of Arkansas for Medical Sciences approved this study. Primary human MSCs used as normal control MSCs (NMSCs) were obtained from the Tulane Center for Preparation and Distribution of Adult Stem Cells , and two human MSC samples used as NMSCs were procured from Lonza , immortalized mouse embryonic MSCs; MG63 and Saos-2 (from ATCC), human OB-like cells; and HS5 and HS27A, human bone marrow stromal cells (as described in our previous study ). Cells Following treatment, cell lines and primary MSCs isolated from MM patients were isolated and subjected to immunoblotting analyses as previously described . Protein2; 0.5-mM CaCl2; 1-mM EDTA) and then homogenized by 30 strokes of a Dounce homogenizer. After removing the nuclear pellet, lysates were separated into cytosolic and membrane fractions by ultracentrifugation  for 60 minutes. The nuclear pellets were resuspended in hypotonic buffer containing 0.1% SDS. Proteins from each fraction were subjected to immunoblot analysis.Cytosolic and nuclear proteins were prepared by fractionating cell lysates as described , with miGST–E-cadherin binding assays were performed as previously described . The β-cALP activity was determined as described previously . BrieflyImmunofluorescence staining was used to identify the active form of β-catenin, as previously described . BrieflyMatrix mineralization was detected with von Kossa staining, using a slight modification of previously described methods . BrieflyTo determine TCF activity in response to CFZ treatment, luciferase reporter assays were performed as described previously .WNT family; 10 members of FZD family; DKK1; sFRP1, -2, -3, and -4; ALPL; BMP2; CTNNB/β-catenin1; OPG; and RANKL; reactions were performed as previously described [Total RNA was isolated  from cells treated with or without serial concentrations (2 to 20 nM) of CFZ. First-strand cDNA synthesis was performed as previously described . Quantitt-test as described previously [P values were less than 0.05, based on two-tailed test.Statistical significance of differences between experimental groups was determined with Student’s eviously . SignifiTo determine whether CFZ increases β-catenin protein levels in cell lines of OB progenitor cells and MSCs, we first used E-cadherin pull-down assays to separate the pool of free β-catenin from the pool of membrane-bound β-catenin; OBs and MSCs express abundant amounts of E-cadherin, which sequesters β-catenin in the membrane fraction  where itTo investigate whether these results obtained in cell lines were representative of those seen in primary samples of MSCs, MSCs isolated from bone marrow of two healthy donors (NMSCs) and from six patients with MM (MMMSCs) were cultured and incubated with CFZ. As with the cell lines, CFZ treatment of NMSCs resulted in increased levels of free β-catenin protein that were evident at 2 hours and peaked at 4 hours ; similarTo confirm the effects of CFZ on β-catenin in OBs and MSCs, we used a different proteasome inhibitor, MG132, to treat OBs, NMSCs, and MMMSCs. MG132 treatment led to stabilization of free β-catenin in MG63, HS5, and HS27A cell lines in a time-dependent manner . Levels We next determined whether CFZ treatment led to nuclear accumulation of β-catenin. CFZ induced an increase in levels of free β-catenin  and in tHaving demonstrated that CFZ significantly induced increases in both free and active β-catenin and induced β-catenin translocation into nuclei of OBs and MSCs, we next investigated whether increased nuclear β-catenin in OBs and MSCs affects TCF transcriptional activity. Using the TOPflash TCF reporter system , we founWnt/β-catenin signaling is required for MSC differentiation to OBs and for bone formation , so we iWe next used von Kossa staining to examine effects of CFZ on extracellular matrix mineralization (EMM), which indicates OB function during bone formation. In the presence of CFZ, mineralization was higher in C3H10T1/2 cells and in MMMSCs than in control cells not treated with CFZ . MicrogrTo confirm the effects of CFZ on MSC differentiation, cells were also stained with ARS to detect calcium deposition. We observed significant dose-dependent increases in ARS staining in C3H10T1/2 cells treated with serial concentrations of CFZ, with maximal effects observed at 10-nM CFZ ; similarTo investigate whether CFZ-stimulated activation of β-catenin/TCF was involved in regulating transcription of β-catenin, we analyzed the effects of CFZ on β-catenin mRNA levels. CFZ treatment did not result in changes in β-catenin gene expression in MG63 and Saos-2 cell lines  or in MMLRP5/6  in the differentiation medium for primary MSCs from two patients with MM. Administration of rDKK1 protein (200 ng/ml) did not significantly induce a decrease in ALP activity in the MSCs in the culture medium in the presence of CFZ , compareTCF1 and TCF4 were most abundant in OBs and MSCs [Given that CFZ activated β-catenin/TCF activity and induced MSC differentiation, we next investigated whether TCF signaling was required and/or sufficient for the action of CFZ. Because our previous studies demonstrated that both and MSCs , we asseWe next sought to determine whether CFZ-induced MSC differentiation was attenuated when TCF activity was blocked. In response to CFZ treatment, HS27A  and C3H1Suppression of the ability of MSCs to differentiate into OBs that occurs as a result of their interactions with myeloma cells is a fundamental part of the cellular basis for MM-triggered decreases in bone formation. Several in vitro and in vivo studies have shown that bortezomib, a first-generation proteasome inhibitor, is associated with inducing MSCs to differentiate into OBs ,30,31 anInitial studies demonstrated that CFZ enhanced activation of a β-catenin functional signaling pathway, as evidenced by accumulation of the transcriptionally active form of β-catenin and the uncomplexed, ubiquitinated form of β-catenin in the cytosol in mouse OB progenitor cells (C3H10T1/2 cell line), human OB-like cells (Saos-2 and MG63 cell line), NMSCs, and MMMSCs from six patients ; similarIt was previously reported that the β-catenin/TCF pathway is required for promoting MSC differentiation into OBs ,29, and Wnt ligands, LRP5/6, and the 10 members of the Frizzled/FZD receptors (data not shown). Second, CFZ did not synergize with Wnt3a to stimulate TCF activity. Third, blocking GSK3β activity  did not elicit a greater level of TCF transcriptional activity in the presence of CFZ in MG63 and Saos-2 cells (data not shown). Although CFZ decreased DKK1 mRNA levels, CFZ did not synergize with Wnt3a-induced TCF activity, indicating that its effects on stabilization of β-catenin are sufficient to overcome DKK1 inhibition of the canonical Wnt signaling in MSCs and OBs. Finally, CFZ did not affect β-catenin gene expression, implying that CFZ induces activation of β-catenin/TCF activity by blocking β-catenin degradation. CFZ activates Wnt-independent β-catenin/TCF signaling with a mechanism similar to that used by bortezomib [Although CFZ clearly stimulates activation of β-catenin/TCF signaling, it does so without regulating upstream targets of β-catenin, which are required for Wnt3a-initiated activation of the pathway. This conclusion is supported by several observations. First, CFZ did not influence expression of extracellular signaling components, including multiple rtezomib .DKK1 expression in MM cells [DKK1 expression in OBs and may contribute to osteoporosis induced by chronic exposure to this class of drug [DKK1 suppression. This observation is in agreement with previous studies revealing that bortezomib treatment results in decreased levels of DKK1 in serum of MM patients [DKK1 mRNA in MG63 cells but did not show statistically significant decrease in Saos-2 cells. CFZ suppressed growth of MG63 cells but not of Saos-2 cells, which do not express p53, which may account for the drug’s different effects on DKK1 expression in the two cell lines. It has been reported that DKK1 expression is dynamically regulated by altering cell cycle progression in human MSCs [DKK1 expression in these two cell lines. Because glucocorticoids, thalidomide, and lenalidomide are used to treat MM, combining CFZ with these compounds may be a potential benefit due to its ability to overcome the potential negative side-effects on bone that result from drug-induced activation of DKK1.The data presented here suggest that CFZ may be able to bypass DKK1’s suppressive effects on Wnt signaling by directly inhibiting the proteasome and leading to Wnt-independent β-catenin/TCF signaling in bone cells. We previously reported that thalidomide, lenalidomide, and dexamethasone—but not bortezomib—induce MM cells ,40. It i of drug . Interespatients -44. It sman MSCs . FurtherIn conclusion, these studies have revealed that CFZ-induced activation of β-catenin/TCF signaling contributes to promoting OB progenitors and MSCs to differentiate to OBs. CFZ activates β-catenin/TCF signaling via inhibition of proteasome-mediated degradation of β-catenin, which occurs independent of Wnt ligand, Frizzled/FZD receptors, and LRP5/6 co-receptor. These data provide mechanistic insights into CFZ’s bone-anabolic effects and a rationale for its use in treating Wnt/β-catenin–mediated suppression of MSC differentiation.Figure S1CFZ induced increases in active β-catenin protein in nuclei and cytoplasm of MSCs.HS27A (A), MG63 (B) and Saos-2 (C) cells were treated with indicated concentrations of CFZ for 12 hours. The active form of β-catenin in cells nuclei and cytoplasm was examined by immunofluorescence staining, using an antibody specific for active β-catenin and an FITC-labeled goat anti-mouse secondary antibody. Images were taken with an Axio Observer A1 fluorescence microscope with 10X objective lens  and SPOT camera .(TIF)Click here for additional data file.Figure S2CFZ induced increases in β-catenin protein in nuclei and cytoplasm of MSCs.HS5 (A), Saos-2 (B), MG63 (C), and human primary MSCs from bone marrow of one MM patient (D) were treated with indicated concentrations of CFZ for 12 hours and fixed as described in Methods. β-catenin protein in nuclei and cytoplasm of the cells was examined by immunofluorescence staining, using an antibody specific for β-catenin protein and a red-fluorescent-dye-labeled goat anti-mouse antibody. Nuclei were counterstained with DAPI. Images were taken and analyzed as described in (TIF)Click here for additional data file.Figure S3DKK1 did not block CFZ-induced increase in ALP activity in MSCs.Human primary MSCs isolated from bone marrow of two patients with MM  were cultured in medium alone (Cont.) or with 2-nM CFZ; DKK1; CFZ and DKK1; Wnt3a; Wnt3a and DKK1 (positive control indicating DKK1 function); or CFZ, DKK1, and Wnt3a. After 72 hours, cells were lysed and ALP activity determined as described in Methods. Data represent the mean ± SD (n=3) of three experiments. Statistical analyses to determine significance for each treatment group compared with control or compared among the treated groups was performed as described in Methods.(TIF)Click here for additional data file.Figure S4CFZ increased the free form of β-catenin in MSCs from patients with MM.MSCs from four patients with MM were treated with 20-nM CFZ for 6 hours, and proteins (500 µg) were isolated and subjected to GST-E-cadherin pull-down assays and immunoblotting analysis with anti-β-catenin antibody as described in Methods; tubulin was used as a loading control.(TIF)Click here for additional data file.


LOXL1) gene variants as alternative markers for EX.Previous studies have described a possible association between exfoliation syndrome (EX) and various ocular and systemic vascular disorders; however, the association between EX and branch retinal vein occlusion (BRVO) remains unclear. Because slit-lamp examination may overlook latent deposits of exfoliation materials, an ocular biopsy is usually needed for a precise diagnosis. We evaluated a possible association between EX and BRVO using lysyl oxidase-like 1  were determined for 78 consecutive Japanese patients with BRVO , and 158 patients with cataract without EX (CT) as controls.Allelic and genotypic frequencies of three The rs1048661 variant differed between the BRVO and CT groups in allelic and genotypic frequencies . Subgroup analysis, compared to the CT group, showed that BRVO EX+ had significantly different allelic and genotypic frequencies of rs1048661 , while BRVO EX- did not . The frequencies of rs3825942 and rs2165241 did not differ between the BRVO and CT groups.LOXL1 variants were used as disease markers for clinically undetectable EX. The results suggested that LOXL1 variants, well established markers for EX, are not likely genetic markers for BRVO in Japanese subjects.No association was found between BRVO and EX if  Retinal vein occlusions (RVOs), including central retinal vein occlusion (CRVO), an occlusion at the central trunk of the retinal vein, and branch retinal vein occlusion (BRVO), an occlusion at an arteriovenous crossing where the retinal artery and vein are bound by a common adventitial sheath, are important causes of ocular morbidity ,2. Althors2165241) and two exonic SNPs  in the first exon of the lysyl oxidase-like 1 (LOXL1) gene on chromosome 15q24.1 are highly associated with EX in Icelandic and Swedish populations, and that none of these SNPs was associated with primary open-angle glaucoma in the two populations [Exfoliation syndrome (EX), the most common identifiable cause of open-angle glaucoma worldwide, is an age-related, generalized disorder of the extracellular matrix characterized by the production and progressive accumulation of fibrillar extracellular material in many ocular tissues . A recenulations .LOXL1 polymorphism has been tested in several ocular [LOXL1 variants as a disease marker for EX.In addition to ocular tissues, production and progressive accumulation of exfoliation materials occur in skin and various visceral organs ,12. The l ocular  and systl ocular ,26 pathoLOXL1 variants and BRVO in a Japanese population to explore a possible association between EX and BRVO.In the current study, we tested the association between Unrelated Japanese subjects with BRVO (n=78) were consecutively recruited at the Shimane University Hospital and Iinan Hospital in Shimane, Japan. The BRVO group was divided into two subgroups based on the presence EX+, n=11) or absence  of clinically detectable ocular deposits of exfoliation material. The data set from patients with cataract without deposits of exfoliation material  reported in our previous study [ or absenThe current study adhered to the tenets of the Declaration of Helsinki. The institutional review boards of both hospitals reviewed and approved the research. All subjects provided written informed consent. All subjects underwent a dilated pupil examination of the anterior segments, ocular media, and fundus using a slit-lamp  and a funduscope . BRVO was diagnosed if the fundus examination revealed venous dilation and tortuosity with flame-shaped and dot-blot hemorrhages in a wedge-shaped region. Patients with CRVO and hemi-CRVO were excluded. Deposits of exfoliation material were identified if the slit-lamp examination revealed a typical pattern of exfoliation material on the anterior lens surface and/or pupillary margin.rs1048661 and rs3825942 (forward primer: 5′-AGG TGT ACA GCT TGC TCA ACT C-3′ and reverse primer: 5′-TAG TAC ACG AAA CCC TGG TCG T-3′) or only rs2165241 (forward primer: 5′-AGA ATG CAA GAC CTC AGC ATG AG-3′ and reverse primer: 5′-TAG TGG CCA GAG GTC TGC TAA G-3′). The sequence was determined based on the dideoxy terminator method using an ABI PRISM 3130xl Genetic Analyzer  according to the manufacturer’s protocol. We used SeqScape Software version 2.5 (Applied Biosystems) to analyze the sequence alignment.Genomic DNA was extracted from the peripheral white blood cells of each subject. A polymerase chain reaction was performed using primers designed to amplify the genomic region containing both Statistical analysis was performed using R version 2.6.2. Fisher’s exact test was used to compare the allele or genotype frequencies of each group with the controls.rs1048661, rs3825942, and rs2165241 within LOXL1 are shown in rs1048661 were higher in patients with BRVO . In subgroup analysis, compared to the CT group, the group with BRVO with exfoliation material deposits (EX+) had significantly different allelic and genotypic frequencies , while the group with BRVO without exfoliation material deposits (EX-) had no difference in allelic and genotypic frequencies . Compared to the CT group, the frequencies of the G allele of rs3825942 and the C allele of rs2165241 were higher in the BRVO EX+ groups with borderline significance , but the allelic and genotypic frequencies did not differ between any pairs of BRVO total or BRVO EX- and the CT group.The allelic and genotypic counts and frequencies of SNPs LOXL1 variants and BRVO. The prevalence of clinical EX increases with age, especially after age 60 [LOXL1 polymorphism has been tested in several pathologies including wet and dry age-related macular degeneration and polypoidal choroidal vasculopathy in a Japanese population [rs1048661 polymorphism and wet age-related macular degeneration in a Japanese population [LOXL1 polymorphism as an alternative marker of clinically undetectable EX other than invasive biopsy/histopathology.To the best of our knowledge, this is the first study to identify a possible association between r age 60 . Accordir age 60 ,28. A cor age 60 ; howeverpulation , Alzheimpulation , and carpulation . Fuse etpulation . These srs1048661 has been consistently suggested as the most significant indicator of EX/glaucoma in Icelandic, Swedish, and Japanese populations [rs1048661 between all subjects with BRVO and CT or BRVO EX+ and CT groups confirmed previous observations of the strong role of this SNP in EX. The results also suggested that using this SNP, we can detect a case–control association for EX even with such a small number of subjects (n=11) in a case group. In the same context, the other two SNPs, which showed only a borderline difference between BRVO EX+ and CT groups, may not have enough discriminatory power with this small number of subjects.Among the three SNPs reported , rs10486ulations . AccordiLOXL1 variants in BRVO. As a result, the significant difference observed in rs1048661 between the case and control groups was canceled in the analyses between the BRVO EX- and CT groups, suggesting that the percentage of the population at risk of EX is not significantly higher in the BRVO group. A retrospective chart review reported exfoliation material deposits in 6.0% of eyes with BRVO and 6.9% of eyes with CRVO [LOXL1 variants or EX in the development of BRVO in our Japanese subjects.Since both the BRVO EX- and CT groups, which were classified based on slit-lamp examination as not having EX, were identical except for the presence or absence of BRVO, comparison between these two groups should provide the most reliable information about the possible role of the ith CRVO , suggestith CRVO . By clinith CRVO . A retroith CRVO . In thisith CRVO ,20. DiffLOXL1 variants with BRVO. We did not find an association between BRVO and EX if the LOXL1 variants were used as disease markers for clinically undetectable EX.In summary, we tested the possible association of


There are widespread concerns about communication and support for patients and families, especially when they face clinical uncertainty, a situation most marked in intensive care units (ICUs). Therefore, we aimed to develop and evaluate an interventional tool to improve communication and palliative care, using the ICU as an example of where this is difficult.t-tests , the χ2 or Fisher’s exact test  and the Mann–Whitney U-test (for experience assessments) to compare the characteristics and experience for cases with and without PACE recorded.Our design was a phase I-II study following the Medical Research Council Guidance for the Development and Evaluation of Complex Interventions and the (Methods of Researching End-of-life Care (MORECare) statement. In two ICUs, with over 1900 admissions annually, phase I modeled a new intervention comprising implementation training and an assessment tool. We conducted a literature review, qualitative interviews, and focus groups with 40 staff and 13 family members. This resulted in the new tool, the Psychosocial Assessment and Communication Evaluation (PACE). Phase II evaluated the feasibility and effects of PACE, using observation, record audit, and surveys of staff and family members. Qualitative data were analyzed using the framework approach. The statistical tests used on quantitative data were U-test ranged from 616 to 1247, P-values ranged from 0.041 to 0.010) compared with those who did not.PACE provides individualized assessments of all patients entering the ICU. It is completed within 24 to 48 hours of admission, and covers five aspects , followed by recording of an ongoing communication evaluation. Implementation is supported by a training program with specialist palliative care. A post-implementation survey of 95 ICU staff found that 89% rated PACE assessment as very or generally useful. Of 213 family members, 165 (78%) responded to their survey, and two-thirds had PACE completed. Those for whom PACE was completed reported significantly higher satisfaction with symptom control, and the honesty and consistency of information from staff (Mann–Whitney PACE is a feasible interventional tool that has the potential to improve communication, information consistency, and family perceptions of symptom control. Communication and support for patients and their families are of central concern in healthcare, and yet too often are poorly addressed, especially at times of rapid changes in health status and in clinical uncertainty. The intensive care unit (ICU) is one setting in which patients’ circumstances can change rapidly. Although the central goal in the ICU is to preserve or extend life, the nature of illness or trauma means that more than one in five people admitted to an ICU die there . Many , and full hospital Research and Development approval.Development, implementation, and preliminary evaluation of the tool followed the Medical Research Council guidance for the development and evaluation complex interventions . Phase IThe study took place in two adult ICUs  in a 950-bed south London teaching hospital, serving a socially and culturally heterogeneous population. The two ICUs have 32 beds, and an annual intake of more than 1,900 admissions , associated with 250 to 300 deaths in the ICU. Most (70 the 80%) of these deaths follow life-sustaining treatment limitations. The ICU standardized mortality ratio  II) was 0.62 during the study, and these ICUs have a lower SMR than the median SMR within the UK casemix program . The ICUWe conducted qualitative interviews with families and staff, and observed their process of care and staff activity . Initial versions were tested in three 6-week pilot periods, and amended as necessary.Training to support PACE implementation  , validathttp://www.qsrinternational.com). Qualitative data were analyzed using the framework approach [All data were anonymized. All qualitative interviews (both phases) and focus groups were digitally audio-recorded, transcribed verbatim, and entered into NVivo9 , χ2 or Fisher’s exact test , and the Mann–Whitney U-test (for experience assessments) to compare the characteristics and experience of families for cases that had PACE completed with those that had not. When equality of variances was not assumed adjusted values are given . All tests were two-sided.Quantitative data were analyzed using simple descriptive statistics to describe PACE use and staff opinions. For the survey of family members we used Qualitative samples aimed to capture different groups in terms of age, gender, length of time in ICU, diagnosis, and social and cultural background, and thus capture the range of views. In the family members’ survey, most of the analysis was descriptive. However, if we assume 20% (a likely level based on previous research) of families have serious concerns, we needed 76 respondents to achieve a margin of error of 9% . Therefore, we estimated that 150 participants would provide reasonable descriptive accuracy and allow for subgroup analysis . Allowing for refusals, we aimed to approach 200 participants. For both the qualitative and quantitative data, the samples aimed to be sufficient for preliminary exploration, as we were not formally testing PACE but to exploring its potentially beneficial or adverse effects.We first interviewed 13 family members, representing 11 patients, and conducted observations on the care of these patients. Four the patients were women, age range 23 to 87 years) with diagnoses including infection, injury, and malignancy  Family details: key relationships; children; guardianship issues; vulnerable adultsb) Social details: financial concerns; religious/spiritual needs; language/cultural needs; transport/parking needs; other needsc) Patient preferences: previously expressed wishes; preferred place of care; presence of advance directive / statement or willd) Communication and information: patient / NOK awareness of situation; people to be given information; explanation of ICUe) Any other issues: which patient, family or staff feel is important.The next section of PACE gives space for a continuing log of any communication update.The final section of PACE provides a list of useful resources - as a prompt to seek additional support for patients and families. Additional file The PACE is logged by the admitting key worker, usually an ICU nurse. It is kept in the clinical records and can be updated by any member of the clinical team. The rationale for keeping PACE in the clinical record is to avoid miscommunication and improve continuity by holding psychosocial information in one place. The goal is that the assessment prompts changes in actions by staff in response to the assessment findings. The continuing log of communication enables information to flow between teams recording communication about new issues or changes in the patient or family circumstances.PACE requires staff to explain if an aspect is not explored (e.g. staff must give a reason if it is not appropriate to discuss preferences).In total, 43 nurses, 11 doctors and staff from palliative care, pharmacy, physiotherapy, occupational therapy, speech and language therapy, and chaplaincy departments attended the training during the launch week. The research process and feedback of the results led to much discussion about decision-making, communication, and end-of-life issues. In addition, there was increased input from the palliative care team on the units and psychosocial support by different providers, including chaplaincy and the palliative care team, as a consequence of the heightened awareness and identification of needs through the use of PACE.PACE was launched in the ICU after the three piloting phases. Further revisions were undertaken on two occasions, at 6 weeks and 4 months after the original launch, to address concerns and suggestions by staff. The PACE log was usually filed in the nursing notes. An audit of the completion of PACE logs of 81 consecutive patients found that half (41) were completed within 24 hours, and the remainder within 48 hours of admission to ICU. All aspects were completed for at least some patients, the most complete being family history, next of kin, and family understanding of the condition (both completed for over 90% of cases), and social history (70% of cases).Responses were received from 95/176 (54%) staff members, of whom 80% (76/95) had seen a PACE log. In the main; nurses  reported completing PACE rather than doctors . Just over half  reported completing PACE within 24 hours of admission all or most of the time (similar to the audit results), but 34% (27/80) had difficulty completing some sections, especially recording patient preferences. Most  reported having seen a completed PACE in patients’ notes. The majority  reported that PACE was very or generally useful, with only 3 respondents saying it was not useful, and 6 responses being neutral. The components of PACE most often judged to be useful were family details (65 respondents), social details (52 respondents), and communication/information (25 respondents).Response rate from the family members was 165/213(78%). Figure t = 1.95, P = 0.05) . Variation in cases between phase I and II made comparison difficult; however, the comments shed light on the quantitative results Table . One intet al. in a trauma unit of having an approach suitable for all patients irrespective of prognosis [PACE was developed and tested to help improve care in ICU, a setting in which uncertainty is common. PACE is a straightforward tool, building on the work of Mosenthal rognosis , but forrognosis . Resultset al. found that better documentation of pain does not in itself lead to better treatment [To embed palliative care principals and practice into ICUs, several countries and national and international associations have published recommendations, practice guides, and quality indicators ,52-54. Sreatment . Thus, wIn the ICU, it is difficult to accurately identify those who will die, and there is often a very short window to introduce palliative or end-of-life care and to support families. There is also a need to manage distressing and often complex symptoms, and at the same time, provide support and information to families at a time of clinical uncertainty and/or devastating news. A major problem is deciding on a trigger to use any pathway, something which has caused considerable debate in the UK with regard to the LCP ,64. For et al. [et al. [et al. [To underpin our results and to enable future evaluation, we developed a theoretical model of how PACE may be influencing the outcomes found in our study Figure . This buet al. , and Nel [et al. ,27 in de [et al.  in enhanOur study is limited by being based in two ICUs in a single hospital. However, PACE was used on a wide range of patients, with different ages, conditions, and cultural backgrounds. The findings from the surveys of staff and family members did not suggest that it was suitable for only some particular groups or circumstances. We found that PACE was used more often when patients were more critically ill, as indicated by the APACHE II score. This suggests that staff prioritized PACE for instances of greatest uncertainty. ICU wards have higher levels of staffing than general wards, and this may have supported the implementation of PACE. Therefore, wider testing of PACE in ICU units, and ideally general wards, is needed. Even if PACE is suitable only for the ICU, this represented over one-fifth of the deaths in our hospital, and so would affect a high number of patients and families.Our comparison of family members’ satisfaction when PACE was and was not used was restricted to the ICUs participating in this study. All ICU staff had been exposed to the palliative care training and awareness engendered by PACE implementation. This may have reduced the likelihood of finding a difference between groups, because the care of all patients (including those who did not have PACE completed) was influenced. Despite this limitation, our findings of a difference between groups suggests that PACE does improve care over and above that provided by general training, although we cannot exclude the possibility that some other factor reduced the quality of care for the ‘non-PACE’ patients, which also resulted in them not receiving PACE. For example, it may have been that those staff members choosing to complete PACE were also more skilled in communication and symptom management than those who did not complete PACE. The use of PACE increased during the project, so it might also have been that care generally improved during this time. To test PACE fully, a comparative trial is required, with before-and-after assessment of care experience, including units where PACE is not used. Finally, our study was limited by the high level of satisfaction found in the survey of family members’ experiences.PACE is an acceptable and feasible tool that has the potential to improve care of ICU patients and their families. Moreover, it appears to improve communication, information-sharing and family perceptions of symptom control. We did not find any harmful effects of the tool. PACE may help the better implementation of other tools or care bundles by establishing early an assessment of the patient and family circumstances beyond medical diagnosis and improving communication. It would need to be introduced using integrated working between specialist palliative care and ICU staff, with a shared training program, and now warrants evaluation and further development in comparative trials in the ICU. PACE may also be relevant for other hospital circumstances where there is a high level of clinical uncertainty.ICU: Intensive Care Unit; LCP: Liverpool Care Pathway for the care of the dying patient; MORECare: Methods of Researching End-of-life Care; PACE: Psychosocial Assessment and Communication Evaluation tool.All authors declare they have no competing interests; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; and no other relationships or activities that could appear to have influenced the submitted work.IJH, WP, JK, PH, SL, WB, and MM planned the study, wrote the protocol, won funding, oversaw the study, and contributed to all components. CS and CR were appointed to work on the study, reviewed the literature, collected research data, contributed to the design, and prepared preliminary reports for the investigators. WP, RB, PH, SL, OD, JN, and WB developed the PACE based on data from phase I. WP, RB, PH, SL, OD and JN led the implementation of PACE on ICU. WP and RB led the clinical palliative care input. PH, SL, and WB provided medical ICU leadership, and OD and JN nursing ICU leadership. SH analyzed the family members’ survey. CS, CR, MM, and JK analyzed the qualitative data. IJH, WP, JK, PH, SL, WB, MM, RB, OD, and JN formed the Project Advisory Group, contributed to planning the study, the PACE tool, its implementation, study design, and the interpretation of results; approved the final report; and made substantial critical revisions of the paper. IJH drafted this manuscript and revised it in response to critical revisions from all authors. IJH and CS are the co-guarantors of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/213/prepubPACE study procedures according to the MORECare Statement.Click here for fileDetailed description of phase I methods - Development and Modeling Leading to the Tool Development.Click here for filePACE: Psychosocial Assessment and Communication Evaluation.Click here for fileFamily survey responses: comparison of the patient and family characteristics where Psychosocial Assessment and Communication Evaluation (PACE) was and was not completed.Click here for file


In this study, we tested the ability of small molecule inhibitors of WNT/β-catenin signaling to block interleukin 1β (IL-1β)- and tumor necrosis factor α (TNFα)-induced cartilage degradation. Proinflammatory cytokines such as IL-1β and TNFα are potent inducers of cartilage degradation by upregulating matrix metalloproteinase (MMP) expression and activity. Because WNT/β-catenin signaling was found to be involved in IL-1β- and TNFα-induced upregulation of MMP activity, we hypothesized that inhibition of WNT/β-catenin signaling might block IL-1β- and TNFα-induced cartilage degradation. We tested the effect of small molecules that block the interaction between β-catenin and TCF/Lef transcription factors on IL-1β- and TNFα-induced cartilage degradation in mouse fetal metatarsals.ex vivo model for cytokine-induced cartilage degradation. Metatarsals were treated with IL-1β and TNFα in combination with the small molecules PKF115-584, PKF118-310 and CGP049090 at different concentrations and then harvested them for histological and gene expression analysis.We used mouse fetal metatarsals treated with IL-1β and TNFα as an We found that IL-1β- and TNFα-induced cartilage degradation in mouse fetal metatarsals was blocked by inhibiting WNT/β-catenin signaling using small molecule PKF115-584 and partially using CGP049090 dose-dependently. In addition, we found that PKF115-584 blocked IL-1β- and TNFα-induced MMP mRNA expression, but did not reverse the inhibitory effect of IL-1β on the expression of cartilage anabolic genes.In this study, we show that inhibition of WNT/β-catenin signaling by small molecules can effectively prevent IL-1β- and TNFα-induced cartilage degradation by blocking MMP expression and activity. Furthermore, we elucidate the involvement of WNT/β-catenin signaling in IL-1β- and TNFα-induced cartilage degradation. MMP1 (collagenase), MMP3 (stromelysin), MMP9 (gelatinase) and MMP13 (collagenase 3) mRNA has been found in chondrocytes in arthritic cartilage [MMP1 and MMP3 was also found in the synovial tissue of OA patients [Mmp13-deficient mice were resistant to cartilage damage in medial meniscus destabilization-induced cartilage degradation [In degenerative cartilage diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA), the balance between anabolic and catabolic processes is shifted toward breakdown of the extracellular cartilage matrix -3. Cartiartilage ,6. Increpatients . In agrepatients . The essradation .in vitro and in vivo [In vitro and in vivo studies have shown that proinflammatory cytokines such as IL-1β and TNFα are present in both OA and RA joint tissues and synovial fluid [Proinflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor α (TNFα) potently induce MMP expression and activity in cartilage, and these cytokines are associated with cartilage degradation  in vivo ,11,12. Tal fluid ,13. IL-1FRZB, an antagonist of canonical WNT signaling makes mice more susceptible to chemically induced articular cartilage degradation [Recently, the canonical WNT/β-catenin signaling pathway in the pathophysiology of cartilage degenerative disease has attracted much attention . The WNTradation . Furtherradation .Recently, a link between WNT/β-catenin signaling and IL-1β-induced cartilage degradation was found. Expression of WNT5a and WNT7a in articular chondrocytes was induced by IL-1β , and theBecause proinflammatory cytokine-induced cartilage degradation appears to involve WNT/β-catenin signaling and increased WNT/β-catenin signaling has been implicated in the initiation and progressive deterioration of cartilage degeneration, we hypothesized that small molecule inhibitors of the interaction between β-catenin and TCF4 and Lef1 could be used to prevent cytokine-induced cartilage degradation. The aim of this study was to assess the potential effects of small molecules that inhibit the WNT/β-catenin signaling pathway on the degeneration of cartilage. We have selected the small molecules PKF115-584, PKF118-310 and CGP049090, which block the binding of β-catenin to its transcription factor TCF4. PKF115-584 and CGP049090 also block the binding between β-catenin and transcription factor Lef1 -24. In a2 into 96-well plates  and cultured for 24 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 100 U of penicillin-streptomycin  prior to transfection with the TOPflash TCF/Lef luciferase reporter construct  and pRL-CMV control vector . Cells were stimulated with the GSK3β inhibitor BIO  to stimulate the WNT/β-catenin pathway in combination with the inhibitors 24 h after transfection. After 24 h of stimulation, luminescence was measured using the Dual-Glo Luciferase Assay System (Promega).HEK-293t cells were seeded at 7,500 cells/cmTo study the effect of small molecules on metabolic activity, the preosteoblast cell line KS483-4C3 was used . Twenty-4Cl for 10 min and incubated overnight at 4°C in NET GEL buffer . The next day cells were incubated with anti-β-catenin antibody  for 1 h at room temperature. Next, cells were incubated with anti-mouse fluorescein isothiocyanate-labeled secondary antibody  for 1 h at room temperature and mounted using VECTASHIELD mounting medium .Nuclear accumulation of β-catenin was detected by immunofluorescence staining. KS483-4C3 cells were seeded onto glass slides  and treated with LiCl, both with and without small molecule inhibitors. After 3 h, cells were washed in phosphate-buffered saline and fixed in 3.7% buffered formalin. Subsequently, cells were quenched in 50 mM NHMouse fetal metatarsals were isolated from FVB mouse embryos  at day 17.5 of gestation ,27. Afte2O (pH set to 1 using HCl) for 30 min and counterstained in 1% Nuclear Fast Red solution (Sigma-Aldrich) for 5 min. For immunohistochemical staining of collagen type II, sections were preincubated in 5 µg/ml proteinase K (Sigma-Aldrich) for 10 min, followed by 1 mg/ml hyaluronidase (Sigma-Aldrich) for 30 min, both at 37°C. Rabbit polyclonal collagen type II primary antibody  was diluted 1:1,000 and incubated overnight at 4°C. For visualization, the EnVision+ HRP kit  was used.Optical microscopy was performed at different time points, and the lengths of the metatarsals were measured along the sagittal axis of the bone using ImageJ image analysis software . For histological examination, metatarsals were fixed in 10% formalin and dehydrated in ethanol series before being embedded in paraffin. Five-micrometer sections were cut using a rotary microtome . Sections were stained for glycosaminoglycans using 0.5% Alcian Blue (Sigma-Aldrich) in HFive metatarsals were pooled and lysed in TRIzol reagent  for RNA isolation, using the NucleoSpin RNA II kit  according to the manufacturer's protocol. Subsequently, cDNA was synthesized using the iScript cDNA Synthesis Kit . Quantitative polymerase chain reaction was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories) in the MyiQ 2 Two-Color Real-Time PCR Detection System (Bio-Rad Laboratories). Gene expression was normalized using glyceraldehyde 3-phosphate dehydrogenase and expressed as fold change compared to controls. Primer sequences are listed in Table The results are expressed as mean values with 95% confidence intervals (CIs), and statistical significance was tested using analysis of variance with PASW Statistics 18 software .We first tested the efficacy and specificity of the small molecule compounds to inhibit canonical WNT/β-catenin signaling in HEK-293t cells transiently transfected with the TOPflash TCF/Lef luciferase reporter construct. The compounds were tested in the presence or absence of BIO, a potent activator of WNT/β-catenin signaling by blocking GSK3β . We founThe effect of small molecules on the metabolic activity of cells was tested in KS483-4C3 cells using an MTT assay . No signBecause β-catenin can effectively activate the WNT/β-catenin pathway only after nuclear translocation, the cellular localization of β-catenin was determined by immunofluorescence staining. Figure Metatarsals were cultured in medium containing IL-1β or TNFα or a combination of both at concentrations of 10 ng/ml. TNFα tended to blunt longitudinal bone growth, although this did not reach significance. In contrast, IL-1β alone induced bone and cartilage resorption, resulting in a significant reduction in bone length after 4 and 7 days of treatment. Cotreatment of IL-1β with TNFα caused even more abundant bone and cartilage resorption, resulting in a significant reduction in bone length Figures  and 2B. in vitro growth of the metatarsals was observed at any of the concentrations that were tested  No morphological changes were found in metatarsals treated with small molecules PKF115-584, PKF118-310 or CGP049090 at a concentration of 1.0 µM. A representative picture of two independent experiments is shown. Scale bar represents 500 µm. (B) No significant differences were found in the bone length when metatarsals were treated with small molecules. Data are means with 95% confidence intervals of three independent experiments.Click here for file