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BACKGROUND Vitrification of human blastocysts is being used increasingly to cryopreserve supernumerary embryos following IVF. In this study, we investigate the effects of aseptic vitrification on the cytoskeleton and development of human blastocysts, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy. METHODS A total of 55 fresh blastocysts and 55 day 5 dimethylsulphoxide/ethylene glycol vitrified blastocysts, which were allowed to remain in culture for 24 h post-warming, were rapidly fixed in ice cold methanol, and immunostained with an a-tubulin antibody to visualize microtubules in combination with antibodies against acetylated tubulin (to visualize spindles, poles and mid bodies), gamma tubulin (to identify spindle poles) and 4(6-diamidino-2-phenylindole) to visualize DNA. RESULTS In total, 213 spindles were analysed in the control (fresh) group of which 183/213 (85.9%) were normal, 20/213 (9.4%) were abnormally shaped, 9/213 (4.2%) were multipolar and 1/213 (0.5%) was monopolar. A total of 175 spindles were analysed in the vitrified group, of which 120/175 (68.6%) were normal, 39/175 (22.3%) were abnormally shaped, 10/175 (5.7%) were multipolar and 6/175 (3.4%) were monopolar. The incidence of multipolar spindles was similar in the two groups, but the level of abnormally shaped spindles, often associated with chromosome lagging, or congression failure, was significantly higher in the vitrified group compared with the fresh group (P< 0.05). CONCLUSIONS The high survival rate following thawing and the large proportion of normal spindle/chromosome configurations suggests that vitrification at the blastocyst stage on Day 5 does not adversely affect the development of human embryos and the ability of spindles to form and continue normal cell divisions. However, there was a significantly higher incidence of abnormal spindles in the vitrified group compared with the fresh group, notably of spindles with a focused and an unfocused pole as well as chromosome bridging and disorganized middle spindle fibres at telophase. Further investigation is warranted to elucidate the mitotic stages that are more vulnerable to damage during vitrification, the fate of the abnormal spindles and any potential effects that may be reflected on the chromosomal constitution of the developing blastocysts. [ABSTRACT FROM AUTHOR]

A new [ONO] tridentate phenolate ligand (H2LI) containing an aliphatic alcohol as a side arm has been synthesized, deprotonated and attached to lanthanide(II) and (III) ions, which are employed as catalysts for ring-opening polymerization of cyclic esters. In contrast to many other mono-phenolate lanthanide compounds, these have been found to be inert to polymer incorporation during the polymerization reactions. Three new divalent ytterbium compounds have been synthesized in high yield containing ancillary ligands; two via a transamination reaction between [Yb(N(SiMe3)2)2(THF)2] and one equivalent of the phenols, HOC6H2-(2,4-tBu)-6-CH2N(Me)CH2CH2OH (H2LI) or HOC6H2-(2,4-tBu)- 6-CH2N(Me)CH2CH2N(Me)CH2-6-(2,4-tBu)-C6H2OH (H2LII) in hexanes to yield [Yb(LI)]2 (1) and [Yb(LII)]2 (2), respectively. The third divalent ytterbium compound [Yb(L2)] (3) was prepared by treatment of [Yb(N(SiMe3)2)2(THF)2] with two equivalents of a related monoanionic ancillary phenol, HOC6H2-(2,4-tBu)-6-CH2N(Me)CH2CH2NMe2 (HL) in hexanes. Additionally, the oxidation chemistry of these divalent systems was explored where compound 1 was treated with silver triflate and phenol to form corresponding heteroleptic trivalent ytterbium phenolate complexes [Yb(LI)(O3SCF3)(THF)] (4) and [Yb(LI)(OPh)] (5), respectively. Finally, three new heteroleptic trivalent lanthanide silylamido compounds were synthesized via a ligand exchange transamination reaction between the homoleptic trivalent [Ln(N(SiMe3)2)3] compound and one equivalent of the new dianionic ligand (H2LI) in THF {[La(LI)(N(SiMe3)2)(THF)2] (6); [Sm(LI)(N(SiMe3)2)(THF)] (7); [Yb(LI)(N(SiMe3)2)(THF)] (8)}. These lanthanide(II) and (III) compounds were assessed as catalyst precursors towards the ring-opening polymerization of both L-lactide and e-caprolactone. End-group analyses and detailed kinetics studies {rate law: -d[LA]/dt = k[LA]1[catalyst]1} of the most efficacious lanthanum compound (6) further corroborated the substitutionally inert characteristics of the new stationary ancillary [ONO] tridentate dianionic ligand. [ABSTRACT FROM AUTHOR]

A new [ONO] tridentate phenolate ligand (H2LI) containing an aliphatic alcohol as a side arm has been synthesized, deprotonated and attached to lanthanide(ii) and (iii) ions, which are employed as catalysts for ring-opening polymerization of cyclic esters. In contrast to many other mono-phenolate lanthanide compounds, these have been found to be inert to polymer incorporation during the polymerization reactions. Three new divalent ytterbium compounds have been synthesized in high yield containing ancillary ligands; two viaa transamination reaction between [Yb(N(SiMe3)2)2(THF)2] and one equivalent of the phenols, HOC6H2-(2,4-tBu)-6-CH2N(Me)CH2CH2OH (H2LI) or HOC6H2-(2,4-tBu)-6-CH2N(Me)CH2CH2N(Me)CH2-6-(2,4-tBu)-C6H2OH (H2LII) in hexanes to yield [Yb(LI)]2(1) and [Yb(LII)]2(2), respectively. The third divalent ytterbium compound [Yb(L2)] (3) was prepared by treatment of [Yb(N(SiMe3)2)2(THF)2] with two equivalents of a related monoanionic ancillary phenol, HOC6H2-(2,4-tBu)-6-CH2N(Me)CH2CH2NMe2(HL) in hexanes. Additionally, the oxidation chemistry of these divalent systems was explored where compound 1was treated with silver triflate and phenol to form corresponding heteroleptic trivalent ytterbium phenolate complexes [Yb(LI)(O3SCF3)(THF)] (4) and [Yb(LI)(OPh)] (5), respectively. Finally, three new heteroleptic trivalent lanthanide silylamido compounds were synthesized viaa ligand exchange transamination reaction between the homoleptic trivalent [Ln(N(SiMe3)2)3] compound and one equivalent of the new dianionic ligand (H2LI) in THF {[La(LI)(N(SiMe3)2)(THF)2] (6); [Sm(LI)(N(SiMe3)2)(THF)] (7); [Yb(LI)(N(SiMe3)2)(THF)] (8)}. These lanthanide(ii) and (iii) compounds were assessed as catalyst precursors towards the ring-opening polymerization of both l-lactide and -caprolactone. End-group analyses and detailed kinetics studies {rate law: −d[LA]/dt= k[LA]1[catalyst]1} of the most efficacious lanthanum compound (6) further corroborated the substitutionally inert characteristics of the new stationary ancillary [ONO] tridentate dianionic ligand. [ABSTRACT FROM AUTHOR]

In humans and most mammals, with the exception of some rodents notably the mouse, the centrosome, which is the organizing centre of the spindle, is uniparentally (paternally) inherited. The sperm centrosome is transmitted to the egg at fertilization, forming an aster comprising radially arrayed microtubules that brings the male and female pronuclei into close apposition and organizes the first mitotic spindle in the zygote. Each centrosome contains a pair of centrioles that are oriented perpendicular to one another, surrounded by dense fibrillar pericentriolar material, within which functional and regulatory molecules are embedded. The centrosome is a complex organelle with a crucial role to play in human fertility and the ability of human embryos to undergo normal development. Centrosomal defects can lead to failure in fertilization, and cause embryonic arrest through the formation of abnormal spindles and the accumulation of chromosomally abnormal ceils that derive from them. This paper is a brief review of the role of the centrosome and the implications of its dysfunction in early embryogenesis, with particular reference to the human embryo. [ABSTRACT FROM AUTHOR]

Five new heteroleptic lanthanide(iii) phenolate compounds have been synthesised in high yield, four via a transamination reaction between Ln(N(SiMe3)2)3 and two equivalents of the phenol, HOC6H4(2,4-But)CH2N(Me)CH2CH2NMe2 (LH) in thf {L2LnN(SiMe3)2 where Ln = La (1); Nd (2); Sm (3); Yb (4)}. The fifth compound, [L2La][BPh4] 5 was formed by conversion of 1 by treatment with one equivalent of [Et3NH][BPh4] in toluene. Compound 3 was subjected to a single-crystal X-ray analysis and revealed a five-coordinate, distorted trigonal bipyramidal samarium(iii) metal centre where each phenolate ligand is bidentate coordinating through the phenolate oxygen and nitrogen yielding six-membered chelate rings. Compound 1 exhibited fluxional behaviour in C4D8O solution which was temperature dependent. All five compounds were assessed as catalyst precursors towards the ring-opening polymerisation of both l-lactide and ɛ-caprolactone. These polymerisation studies revealed that catalysts containing larger lanthanide metals were more efficacious than those with smaller lanthanide metals. Furthermore, replacement of the [N(SiMe3)2] initiating group in 1 with [BPh4] in 5 reduced catalytic activity by this compound. Detailed kinetics analysis of the ring-opening polymerisation of l-lactide by compound 1, the most efficacious catalyst precursor analysed in this study, revealed the following rate law: −d[LA]/dt = k[LA]2[1]1 which is second order in lactide and first order in catalyst. End-group analysis by ESI mass spectrometry revealed the presence of phenolate end-groups and lactide cycles, the latter formed by intra-molecular, intrachain transesterification. [ABSTRACT FROM AUTHOR]

Two ytterbium(ii) bis(phenolate) complexes, [LRYb] where R = NMe21 and OMe 2, have been synthesized and characterized, with 1 being structurally defined to be a dimeric species with an unsymmetrical coordination of the bis(phenolate) ligand which is preserved in solution. Both 1 and 2 have been oxidized by a variety of oxidants (AgX, ROH) to form heteroleptic ytterbium(iii) bis(phenolate) complexes: [(LNMe2)YbPF6] (3), [(LNMe2)YbOSO2CF3(thf)] (4), [(LNMe2)YbOBut] (5), [(LNMe2)YbOPh] (6), [(LOMe)YbOPh] (7). Compound 4 has been structurally characterized as having a quasi-octahedral environment around ytterbium, with significant inter species hydrogen bonding between CHx and triflate fluorine atoms. Ligand exchange between Yb(N(SiMe3)2)3(thf)2 and H2LR yielded [(LNMe2)YbN(SiMe3)2] (8) and [(LOMe)YbN(SiMe3)2] (9), while metathesis from YbI2(thf)2 and K2LOMe reproducibly afforded the surprising oxidized product [(LOMe)2YbK(dme)2] (10), which was structurally characterized as having a distorted octahedral environment around the ytterbium(iii) centre. Compounds 1–9 were used to polymerize ɛ-caprolactone at room temperature in toluene, with only compounds 1, 2, 8 and 9 exhibiting significant catalytic activities. The polycaprolactone formed in these reactions was generally of high molecular weight and polydispersities <1.90 in all but one case. [ABSTRACT FROM AUTHOR]

Use of a non-contact infrared laser (IRL) or acid Tyrode's for zona drilling before embryo biopsy was compared by assessing blastomere viability using various fluorescent markers or culture of the single biopsied blastomere, and, by cytoskeletal and molecular cytogenetic analysis of the biopsied embryos following culture to the blastocyst stage. There was no significant difference in the proportion of biopsied embryos that showed no damage in both the biopsied blastomere and in the remaining embryo (acid Tyrode's: 75% versus IRL: 68%), or in the proportion of single biopsied blastomeres that divided in culture (P > 0.05). However, single biopsied blastomeres from laser drilled embryos showed a greater tendency to form miniblastocysts. The proportion of laser or acid Tyrode's biopsied embryos that reached the blastocyst stage by day 6 was similar, although evident earlier (day 5) in the laser biopsied embryos. Spindle abnormalities at the blastocyst stage included tripolar and tetrapolar spindles, but their incidence was not significantly different from controls. In addition, no significant difference was observed in the incidence of chromosomal abnormalities and mosaicism between the two groups. It is concluded that using an IRL at a safe working distance does not cause adverse immediate or longer term effects on the development of human biopsied embryos, although damage can occur if drilling within this distance is unavoidable. Acid Tyrode's drilling can also cause damage, and tended to retard blastocyst development. [ABSTRACT FROM AUTHOR]

BACKGROUND: Despite recent technical improvements, many human preimplantation embryos fail to develop to the blastocyst stage or implant after transfer to the uterus. A possible cause for this developmental arrest is the high incidence of nuclear and postzygotic chromosomal abnormalities observed during cleavage, including chaotic chromosome complements, suggestive of defects in mitotic chromosomal segregation. The underlying mechanisms are largely unknown, but similarities with chromosome instability in human cancers led to the proposal that cell cycle checkpoints may not operate at these early stages. METHODS: To investigate this and to examine whether spindle abnormalities contribute to chromosome malsegregation, we have used fluorescence and confocal laser scanning microscopy, following immunolabelling with antibodies specific for a-tubulin, ?-tubulin, or acetylated tubulin, combined with a DNA fluorochrome to visualize nuclei, spindle and chromosome configurations in normal and arrested human embryos, from cleavage to blastocyst stages. RESULTS: In addition to frequent interphase nuclear abnormalities, we identify for the first time various spindle abnormalities including abnormal shape and chromosome loss and multipolar spindles at cleavage and blastocyst stages. CONCLUSIONS: We propose that a major pathway leading to postzygotic chromosomal abnormalities is the formation of binucleate blastomeres with two centrosomes which result either in a bipolar spindle and division to two tetraploid blastomeres, or in a multipolar spindle, chromosome malsegregation and chromosomal chaos. [ABSTRACT FROM AUTHOR]

Kinesin-1 is a molecular transporter that trafficks along microtubules. There is some evidence that kinesin-1 targets specific cellular sites, but it is unclear how this spatial regulation is achieved. To investigate this process, we used a combination of in vivo imaging of kinesin heavy-chain Kif5c (an isoform of kinesin-1) fused to GFP, in vitro analyses and mathematical modelling. GFP-Kif5c fluorescent puncta localised to a subset of microtubules in live cells. These puncta moved at speeds of up to 1 µm second¹ and exchanged into cortically labelled clusters at microtubule ends. This behaviour depended on the presence of a functional motor domain, because a rigor-mutant GFP-Kif5c bound to microtubules but did not move along them. Further analysis indicated that the microtubule subset decorated by GFP-Kif5c was highly stable and primarily composed of detyrosinated tubulin. In vitro motility assays showed that the motor domain of Kif5c moved detyrosinated microtubules at significantly lower velocities than tyrosinated (unmodified) microtubules. Mathematical modelling predicted that a small increase in detyrosination would bias kinesin-1 occupancy towards detyrosinated microtubules. These data suggest that kinesin-1 preferentially binds to and trafficks on detyrosinated microtubules in vivo, providing a potential basis for the spatial targeting of kinesin-1-based cargo transport. [ABSTRACT FROM AUTHOR]

Background Bluetongue virus (BTV) is an arbovirus that is responsible for 'bluetongue', an economically important disease of livestock. Although BTV is well characterised at the protein level, less is known regarding its interaction with host cells. During studies of virus inclusion body formation we observed what appeared to be a large proportion of cells in mitosis. Although the modulation of the cell cycle is well established for many viruses, this was a novel observation for BTV. We therefore undertook a study to reveal in more depth the impact of BTV upon cell division. Methods We used a confocal microscopy approach to investigate the localisation of BTV proteins in a cellular context with their respective position relative to cellular proteins. In addition, to quantitatively assess the frequency of aberrant mitosis induction by the viral non-structural protein (NS) 2 we utilised live cell imaging to monitor HeLa-mCherry tubulin cells transfected with a plasmid expressing NS2. Results Our data showed that these 'aberrant mitoses' can be induced in multiple cell types and by different strains of BTV. Further study confirmed multiplication of the centrosomes, each resulting in a separate mitotic spindle during mitosis. Interestingly, the BTV NS1 protein was strongly localised to the centrosomal regions. In a separate, yet related observation, the BTV NS2 protein was co-localised with the condensed chromosomes to a region suggestive of the kinetochore. Live cell imaging revealed that expression of an EGFP-NS2 fusion protein in HeLa-mCherry tubulin cells also results in mitotic defects. Conclusions We hypothesise that NS2 is a microtubule cargo protein that may inadvertently disrupt the interaction of microtubule tips with the kinetochores during mitosis. Furthermore, the BTV NS1 protein was distinctly localised to a region encompassing the centrosome and may therefore be, at least in part, responsible for the disruption of the centrosome as observed in BTV infected mammalian cells. [ABSTRACT FROM AUTHOR]

Treatment of an array of bis(phenol)s H2Lx {Lx = [(-OC6H2(2,4-R)(6-CH2))2 NCH2CH2X], where X = CH2NMe2, NMe2, NEt2, OMe and R = But, Pet where Pet = C(CH3)2Et} with [Ln(N(SiMe3)2)2(THF)2] (Ln = Yb, Sm) in a 1:1 molar ratio in hexanes afforded a multitude of new divalent lanthanide bis(phenolate) complexes: [L2cYb]2 (X = NEt2, R = But) 1, [L2bYb]2 (X = CH2NMe2, R = But) 2, [L3aYb]2 (X = NMe2, R = Pet) 3, [L3bYb]2 (X = OMe, R = Pet) 4, [L2cSm] (X = NEt2, R = But) 5, [L3cYb] (X = NEt2, R = Pet) 6, [L3cSm] (X = NEt2, R = Pet) 7. X-Ray crystallographic analyses of compounds 1 and 3 reveal dimeric, centrosymmetric structures with 5-coordinate ytterbium centers arising from bridging and terminal phenolate groups. A selection of divalent compounds (1, 3, 4, 5, 6 and 7) were tested as catalyst precursors in the polymerization of ϵ-caprolactone and/or L-lactide which resulted in high molecular weight polymers with PDIs of 1.11-2.81 and 1.13-1.56 for ϵ-caprolactone and L-lactide respectively. All polymerization studies were performed in either toluene or THF and at room temperature (ϵ-caprolactone) or 70 °C (L-lactide) at a variety of catalyst:monomer ratios (1:100-1:300). Kinetics analyses of the polymerization of L-lactide by compounds 1 and 5 indicated pseudo-first order response with respect to L-lactide. [ABSTRACT FROM AUTHOR]

Treatment of an array of bis(phenol)s H2Lx{Lx= [(−OC6H2(2,4-R)(6-CH2))2NCH2CH2X], where X = CH2NMe2, NMe2, NEt2, OMe and R = But, Petwhere Pet= C(CH3)2Et} with [Ln(N(SiMe3)2)2(THF)2] (Ln = Yb, Sm) in a 1:1 molar ratio in hexanes afforded a multitude of new divalent lanthanide bis(phenolate) complexes: [L2cYb]2(X = NEt2, R = But) 1, [L2bYb]2(X = CH2NMe2, R = But) 2, [L3aYb]2(X = NMe2, R = Pet) 3, [L3bYb]2(X = OMe, R = Pet) 4, [L2cSm] (X = NEt2, R = But) 5, [L3cYb] (X = NEt2, R = Pet) 6, [L3cSm] (X = NEt2, R = Pet) 7. X-Ray crystallographic analyses of compounds 1and 3reveal dimeric, centrosymmetric structures with 5-coordinate ytterbium centers arising from bridging and terminal phenolate groups. A selection of divalent compounds (1, 3, 4, 5, 6and 7) were tested as catalyst precursors in the polymerization of -caprolactone and/or L-lactide which resulted in high molecular weight polymers with PDIs of 1.11–2.81 and 1.13–1.56 for -caprolactone and L-lactide respectively. All polymerization studies were performed in either toluene or THF and at room temperature (-caprolactone) or 70 °C (L-lactide) at a variety of catalyst:monomer ratios (1:100–1:300). Kinetics analyses of the polymerization of L-lactide by compounds 1and 5indicated pseudo-first order response with respect to L-lactide. [ABSTRACT FROM AUTHOR]

Highlights • Undifferentiated/differentiated SH-SY5Y cells provide distinct model systems. • Parkin specifically sensitises to mitochondrial uncoupling. • Kinases differentially regulate c-Jun activity depending on stress levels. • Parkin exists in a feedback loop with c-Jun and its upstream kinases. Abstract Parkinson's disease (PD) is defined by the progressive loss of dopaminergic neurons. Mitochondrial dysfunction and oxidative stress are associated with PD although it is not fully understood how neurons respond to these stresses. How adaptive and apoptotic neuronal stress response pathways are regulated and the thresholds at which they are activated remains ambiguous. Utilising SH-SY5Y neuroblastoma cells, we show that MAPK/AP-1 pathways are critical in regulating the response to mitochondrial uncoupling. Here we found the AP-1 transcription factor c-Jun can act in either a pro- or anti-apoptotic manner, depending on the level of stress. JNK-mediated cell death in differentiated cells only occurred once a threshold of stress was surpassed. We also identified a novel feedback loop between Parkin activity and the c-Jun response, suggesting defective mitophagy may initiate MAPK/c-Jun-mediated neuronal loss observed in PD. Our data supports the hypothesis that blocking cell death pathways upstream of c-Jun as a therapeutic target in PD may not be appropriate due to crossover of the pro- and anti-apoptotic responses. Boosting adaptive responses or targeting specific aspects of the neuronal death response may therefore represent more viable therapeutic strategies. [ABSTRACT FROM AUTHOR]

Aims The aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples. Background Telomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear. MCPH1 (also known as BRIT1 , BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. Methods qPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. Results The wild type α+/β+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/β− hTERT , a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue ( p  = 0.05) and primary cultures ( p  = 0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/β+ hTERT ( p  = 0.04), and a strong positive association between MCPH1/BRIT1 and both α−/β+ hTERT and α−/β− hTERT (both p  = 0.02). A positive association was observed between BRCA1 and α−/β+ hTERT and α−/β− hTERT expression ( p  = 0.003 and p  = 0.04, respectively). Conclusions These findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/β+). [ABSTRACT FROM AUTHOR]

Tubulin alpha 8 (Tuba8) is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG) syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to investigate the interaction and co-localization of novel interacting proteins with the Leber congenital amaurosis (LCA) associated protein aryl hydrocarbon receptor interacting protein-like 1 (AIPL1). Methods: The CytoTrapXR yeast two-hybrid system was used to screen a bovine retinal cDNA library. A novel interaction between AIPL1 and members of the family of EB proteins was confirmed by directed yeast two-hybrid analysis and co-immunoprecipitation assays. The localization of AIPL1 and the EB proteins in cultured cells and in retinal cryosections was examined by immunofluorescence microscopy and cryo-immunogold electron microscopy. Results: Yeast two-hybrid (Y2H) analysis identified the interaction between AIPL1 and the EB proteins, EB1 and EB3. EB1 and EB3 were specifically co-immunoprecipitated with AIPL1 from SK-N-SH neuroblastoma cells. In directed 1:1 Y2H analysis, the interaction of EB1 with AIPL1 harbouring the LCA-causing mutations A197P, C239R and W278X was severely compromised. Immunofluorescent confocal microscopy revealed that AIPL1 did not co-localize with endogenous EB1 at the tips of microtubules, endogenous EB1 at the microtubule organising centre following disruption of the microtubule network, or with endogenous β-tubulin. Moreover, AIPL1 did not localize to primary cilia in ARPE-19 cells, whereas EB1 co-localized with the centrosomal marker pericentrin at the base of primary cilia. However, both AIPL1 and the EB proteins, EB1 and EB3, co-localized with centrin-3 in the connecting cilium of photoreceptor cells. Cryo-immunogold electron microscopy confirmed the co-localization of AIPL1 and EB1 in the connecting cilia in human retinal photoreceptors. Conclusions: AIPL1 and the EB proteins, EB1 and EB3, localize at the connecting cilia of retinal photoreceptor cells, but do not co-localize in the cellular microtubule network or in primary cilia in non-retinal cells. These findings suggest that AIPL1 function in these cells is not related to the role of EB proteins in microtubule dynamics or primary ciliogenesis, but that their association may be related to a specific role in the specialized cilia apparatus of retinal photoreceptors. [ABSTRACT FROM AUTHOR]

Mutations in the MCPH1 (Microcephalin) and ASPM (abnormal spindle-like microcephaly associated) genes cause primary microcephaly. Both are centrosomal associated proteins involved in mitosis. Microcephalin plays an important role in DNA damage response and ASPM is required for correct division of proliferative neuro-epithelial cells of the developing brain. Reduced MCPH1 mRNA expression and ASPM mRNA over-expression have been implicated in the development of human carcinomas. Epithelial ovarian cancer (EOC) is characterised by highly aneuploid tumours. Previously we have reported low Microcephalin and high ASPM protein levels and associations with clinico-pathological parameters in malignant cells from ascitic fluids. To confirm these previous findings on a larger scale Microcephalin and ASPM expression levels and localisations were evaluated by immunohistochemistry in two cohorts; a training set of 25 samples and a validation set of 322 EOC tissue samples. Results were correlated to the associated histopathological data. In normal ovarian tissues the Microcephalin nuclear staining pattern was consistently strong. In the cancer tissues, we identified low nuclear Microcephalin expression in high grade and advanced stage tumours (p<0.0001 and p = 0.0438 respectively). ASPM had moderate to high nuclear and low to moderate cytoplasmic expression in normal tissue. Cytoplasmic ASPM expression decreased with tumour grade and stage in the serous subtype of EOC (p = 0.023 and p = 0.011 respectively). Cytoplasmic ASPM increased with tumour stage in the endometrioid subtype (p = 0.023). Increasing tumour invasiveness (T3) and lymph node involvement (N1) also correlated with a decrease in cytoplasmic ASPM in EOC (p = 0.02 and p = 0.04 respectively). We have validated previous findings of deregulated expression of Microcephalin and ASPM in EOC by confirming associations for low nuclear Microcephalin levels and high cytoplasmic ASPM levels in a larger scale tumour tissue study. Microcephalin and ASPM may prove useful biomarkers in EOC. [ABSTRACT FROM AUTHOR]

Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays. [ABSTRACT FROM AUTHOR]

Abstract: A library of flavonol analogues was synthesised and evaluated as potential anticancer agents against a human prostate cancer cell line, 22rν1. Compounds 3, 8 and 11 (IC50 2.6, 3.3 and 4.0 μM respectively) showed potent cancer cell growth inhibition, comparable to the lead compound 3′,4′,5′-trimethoxyflavonol (1) (IC50 3.1 μM) and superior to the naturally occurring flavonols quercetin (16) and fisetin (22) (both >15 μM). Results indicate that the 3′,4′,5′- arrangement of either hydroxy (OH) or methoxy (OMe) residues is important for growth arrest of these cells and that the OMe analogues may be superior to their OH counterparts. Compounds 1, 3, 8 and 11 warrant further investigation as potential agents for the management of prostate cancer. [Copyright &y& Elsevier]

Highly aneuploid tumours are common in epithelial ovarian cancers (EOC). We investigated whether NuMA expression was associated with this phenomenon. NuMA protein levels in normal and tumour tissues, ovarian cell lines and primary cultures of malignant cells derived from ovarian ascitic fluids were analysed by Affymetrix microarray analysis, immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF), with results correlated to associated clinical data. Aneuploidy status in primary cultures was determined by FACS analysis. Affymetrix microarray data indicated that NuMA was overexpressed in tumour tissue, primary cultures and cell lines compared to normal ovarian tissue. IHC revealed low to weak NuMA expression in normal tissues. Expression was upregulated in tumours, with a significant association with disease stage in mucinous EOC subtypes (p = 0.009), lymph node involvement (p = 0.03) and patient age (p = 0.04). Additional discontinuous data analysis revealed that high NuMA levels in tumours decreased with grade (p = 0.02) but increased with disease stage (p = 0.04) in serous EOC. NuMA expression decreased in late disease stage 4 endometrioid EOCs. High NuMA levels decreased with increased tumour invasion in all subtypes (p = 0.03). IF of primary cultures revealed that high NuMA levels at mitotic spindle poles were significantly associated with a decreased proportion of cells in cytokinesis (p = 0.05), increased binucleation (p = 0.021) and multinucleation (p = 0.007), and aneuploidy (p = 0.008). NuMA is highly expressed in EOC tumours and high NuMA levels correlate with increases in mitotic defects and aneuploidy in primary cultures. [ABSTRACT FROM AUTHOR]

This experiment was conducted to test the predictions of two behavioral-economic approaches to quantifying relative reinforcer efficacy. The normalized demand analysis suggests that characteristics of averaged normalized demand curves may be used to predict progressive-ratio breakpoints and peak responding. By contrast, the demand analysis holds that traditional measures of relative reinforcer efficacy (breakpoint, peak response rate, and choice) correspond to specific characteristics of non-normalized demand curves. The accuracy of these predictions was evaluated in rats' responding for food or water: two reinforcers known to function as complements. Consistent with the first approach, predicted peak normalized response output values obtained under single-schedule conditions ordinally predicted progressive-ratio breakpoints and peak response rates obtained in a separate condition. Combining the minimum-needs hypothesis with the normalized demand analysis helped to interpret prior findings, but was less useful in predicting choice between food and water--two strongly complementary reinforcers. Predictions of the demand analysis had mixed success. Peak response outputs predicted from the non-normalized water demand curves were significantly correlated with obtained peak responding for water in a separate condition, but none of the remaining three predicted correlations was statistically significant. The demand analysis fared better in predicting choice--relative consumption of food and water under single schedules of reinforcement predicted preference under concurrent schedules significantly better than chance. [ABSTRACT FROM AUTHOR]

This experiment was conducted to test predictions of two behavioral-economic approaches to quantifying relative reinforcer efficacy. According to the first of these approaches, characteristics of averaged normalized demand curves may be used to predict progressive-ratio breakpoints and peak responding. The second approach, the demand analysis, rejects the concept of reinforcer efficacy, arguing instead that traditional measures of relative reinforcer efficacy (breakpoint, peak response rate, and choice) correspond to specific characteristics of non-normalized demand curves. The accuracy of these predictions was evaluated in rats' responding for food or fat: two reinforcers known to function as partial substitutes. Consistent with the first approach, predicted peak normalized response output values (Omax) obtained under single-schedule conditions ordinally predicted progressive-ratio breakpoints and peak responding. Predictions of the demand analysis had mixed success. Pmax and Omax were significantly correlated with PR breakpoints and peak responding (respectively) when fat, but not when food, was the reinforcer. Relative consumption of food and fat under single schedules of reinforcement did not predict preference better than chance. The normalized demand analysis is supplemented with the economic concept of diminishing marginal utility, to predict preference shifts across the range of food and fat prices examined. [ABSTRACT FROM AUTHOR]

Most information about the roles of the adenomatous polyposis coli protein (APC) and its binding partner EB1 in mitotic cells has come from siRNA studies. These suggest functions in chromosomal segregation and spindle positioning whose loss might contribute to tumourigenesis in cancers initiated by APC mutation. However, siRNA-based approaches have drawbacks associated with the time taken to achieve significant expression knockdown and the pleiotropic effects of EB1 and APC gene knockdown. Here we describe the effects of microinjecting APC- or EB1- specific monoclonal antibodies and a dominant-negative EB1 protein fragment into mammalian mitotic cells. The phenotypes observed were consistent with the roles proposed for EB1 and APC in chromosomal segregation in previous work. However, EB1 antibody injection also revealed two novel mitotic phenotypes, anaphase-specific cortical blebbing and asymmetric spindle pole movement. The daughters of microinjected cells displayed inequalities in microtubule content, with the greatest differences seen in the products of mitoses that showed the severest asymmetry in spindle pole movement. Daughters that inherited the least mobile pole contained the fewest microtubules, consistent with a role for EB1 in processes that promote equality of astral microtubule function at both poles in a spindle. We propose that these novel phenotypes represent APC-independent roles for EB1 in spindle pole function and the regulation of cortical contractility in the later stages of mitosis. Our work confirms that EB1 and APC have important mitotic roles, the loss of which could contribute to CIN in colorectal tumour cells. [ABSTRACT FROM AUTHOR]

Background: Mutations in the Abnormal Spindle Microcephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation. ASPM encodes a mitotic spindle pole associated protein. It is suggested that the MCPH phenotype arises from proliferation defects in neural progenitor cells (NPC). Results: We show that ASPM is a microtubule minus end-associated protein that is recruited in a microtubuledependent manner to the pericentriolar matrix (PCM) at the spindle poles during mitosis. ASPM siRNA reduces ASPM protein at the spindle poles in cultured U2OS cells and severely perturbs a number of aspects of mitosis, including the orientation of the mitotic spindle, the main determinant of developmental asymmetrical cell division. The majority of ASPM depleted mitotic cells fail to complete cytokinesis. In MCPH patient fibroblasts we show that a pathogenic ASPM splice site mutation results in the expression of a novel variant protein lacking a tripeptide motif, a minimal alteration that correlates with a dramatic decrease in ASPM spindle pole localisation. Moreover, expression of dominant-negative ASPM C-terminal fragments cause severe spindle assembly defects and cytokinesis failure in cultured cells. Conclusions: These observations indicate that ASPM participates in spindle organisation, spindle positioning and cytokinesis in all dividing cells and that the extreme C-terminus of the protein is required for ASPM localisation and function. Our data supports the hypothesis that the MCPH phenotype caused by ASPM mutation is a consequence of mitotic aberrations during neurogenesis. We propose the effects of ASPM mutation are tolerated in somatic cells but have profound consequences for the symmetrical division of NPCs, due to the unusual morphology of these cells. This antagonises the early expansion of the progenitor pool that underpins cortical neurogenesis, causing the MCPH phenotype. [ABSTRACT FROM AUTHOR]

Background: The trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actinassociated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented. Results: Using live imaging of transfected COS-7 cells we observed intercellular junctionassociated pools of GFP-APC in addition to previously documented microtubule-associated GFPAPC and a variety of minor localisations. Although both microtubule and junction-associated populations could co-exist within individual cells, they differed in their subcellular location, dynamic behaviour and sensitivity to cytoskeletal poisons. GFP-APC deletion mutant analysis indicated that a protein truncated immediately after the APC armadillo repeat domain retained the ability to localise to adhesive membranes in transfected cells. Supporting this, we also observed junctional APC immunostaining in cultures of human colorectal cancer cell line that express truncated forms of APC. Conclusion: Our data indicate that APC can be found in two spatially separate populations at the cell periphery and these populations can co-exist in the same cell. The first localisation is highly dynamic and associated with microtubules near free edges and in cell vertices, while the second is comparatively static and is closely associated with actin at sites of cell-cell contact. Our imaging confirms that human GFP-APC possesses many of the localisations and behaviours previously seen by live imaging of Xenopus GFP-APC. However, we report the novel finding that GFP-APC puncta can remain associated with the ends of shrinking microtubules. Deletion analysis indicated that the N-terminal region of the APC protein mediated its junctional localisation, consistent with our observation that truncated APC proteins in colon cancer cell lines are still capable of localising to the cell cortex. This may have implications for the development of colorectal cancer. [ABSTRACT FROM AUTHOR]

Background: EB1 is a microtubule tip-associated protein that interacts with the APC tumour suppressor protein and the p150glued subunit of dynactin. We previously reported that an EB1 deletion mutant that retains both of these interactions but does not directly associate with microtubules (EB1-ΔN2-GFP) spontaneously formed perinuclear aggregates when expressed in COS-7 cells. Results: In the present study live imaging indicated that EB1-ΔN2-GFP aggregates underwent dynamic microtubule-dependent changes in morphology and appeared to be internally cohesive. EB1-ΔN2-GFP aggregates were phase-dense structures that displayed microtubule-dependent accumulation around the centrosome, were immunoreactive for both the 20s subunit of the proteasome and ubiquitin, and induced the collapse of the vimentin cytoskeleton. Fractionation studies revealed that a proportion of EB1-ΔN2-GFP was detergent-insoluble and ubiquitylated, indicating that EB1-ΔN2-GFP aggregates are aggresomes. Immunostaining also revealed that APC and p150glued were present in EB1-ΔN2-GFP aggregates, whereas EB3 was not. Furthermore, evidence for p150glued degradation was found in the insoluble fraction of EB1-ΔN2-GFP transfected cultures. Conclusion: Our data indicate that aggresomes can be internally cohesive and may not represent a simple "aggregate of aggregates" assembled around the centrosome. Our observations also indicate that a partially misfolded protein may retain the ability to interact with its normal physiological ligands, leading to their co-assembly into aggresomes. This supports the idea that the trapping and degradation of co-aggregated proteins might contribute to human pathologies characterised by aggresome formation. [ABSTRACT FROM AUTHOR]

Autosomal recessive primary microcephaly is a potential model in which to research genes involved in human brain growth. We show that two forms of the disorder result from homozygous mutations in the genes CDK5RAP2 and CENPJ. We found neuroepithelial expression of the genes during prenatal neurogenesis and protein localization to the spindle poles of mitotic cells, suggesting that a centrosomal mechanism controls neuron number in the developing mammalian brain. [ABSTRACT FROM AUTHOR]