Understanding the Role of Transmembrane and Juxtamembrane Domains in Plexin and Neuropilin Signaling

Neuropilins (nrps) and plexins (plxns) are transmembrane (TM) proteins that form co-receptor complexes to guide neuronal, vascular, lymphatic, and bone development as well as cancer metastasis. While it is understood that nrp serves as the extracellular ligand-binding receptor and plxn as the signal-transducing portion of the complex, little is understood about the mechanism of activation of the signal transduction cascade beyond ligand binding. Understanding the mechanisms of plxn and nrp activation may provide insight necessary for rational design of novel cancer therapeutics.Co-receptor clustering is believed to induce activation. Previous studies suggest deletion of the plxn extracellular domain leads to a constitutively active plxn, but lack of membrane-anchorage of the cytosolic domain yields inactivity, implying a role for the plxn TM and juxtamembrane (JM) domains in clustering and subsequent activation. We demonstrate that a heptad repeat in the cytosolic JM domain modulates Danio rerio PlxnA3 homodimerization of the TM + JM domains in a bacterial membrane via the AraTM homodimer assay and of the TM + JM domains with a full extracellular domain intact via a bioluminescence resonance energy transfer (BRET2) assay. A specific mutation (M1281L) that enhances homodimerization in the BRET2 assay in the presence of a Nrp2a co-receptor and semaphorin-3F ligand also fails to rescue motor neuron patterning in PlxnA3-knockout zebrafish embryos, in contrast to the wild-type protein. We also demonstrate via these same techniques that a glycine-rich segment of the PlxnA3 TM domain modulates receptor homodimerization, competing with the dimerization motif of the JM domain. Specifically, mutations to small-x3-small motifs in the PlxnA3 TM domain enhance dimerization of the TM + JM domains in the AraTM assay. Mutations to both the TM and JM dimerization motifs demonstrate, in the context of the TM + JM system, the heptad repeat in the JM dominates TM + JM dimerization. Mutations to the small-x3-small TM dimerization motifs exhibit reduced functionality in the zebrafish embryo axonal guidance assay. Collectively, these results demonstrate that enhanced PlxnA3 dimerization does not correlate with enhanced function. The TM-driven dimerization serves to weaken the JM dimer, likely allowing switchability between co-receptors as well as active and inactive states.The nrp MAM domain is also believed to contribute to the observed clustering phenomenon with the intact, full-length plxn receptor. We show that cysteines in the Danio rerio Nrp2a MAM domain, in particular residue C711, modulate Nrp2 homodimerization, as determined via the BRET2 assay. Mutation of residue C711 also disrupts ligand binding. While zebrafish embryos injected with wild-type nrp2a RNA exhibit ectopic vascular branching, significantly fewer embryos injected with nrp2a RNA with the C711S mutation exhibit this overexpression phenotype.Collectively, this work provides insight into the dimerization mechanisms important for nrp and plxn activity. The structure-function correlations determined may assist in rational design of targeted therapeutics to alter nrp and plxn activity

Mechanisms of cohesin protection and removal during meiosis in Saccharomyces cerevisiae

Meiosis is a specialised form of cell division which results in the formation of haploid cells from a diploid progenitor, and thus meiosis halves the chromosome content of the parental cell. A tightly controlled sequence of chromosome segregation events is required to ensure that chromosome missegregation does not occur during meiosis. Chromosome missegregation causes aneuploidy, which in humans can result in the genetic disorder Down Syndrome, and is the leading cause of infertility and miscarriage. To avoid this, it is critical that in the first meiotic division homologous chromosomes are segregated, followed by sister chromatid segregation in meiosis II. Cohesin is a ring-shaped protein complex that holds that sister chromatids together during meiosis, and aids homologue pairing and recombination, as well as ensuring the correct timing of chromosome segregation. In meiosis I, cohesin must be cleaved by separase on the arms of the chromosomes, to allow homologue segregation, whilst at the centromere Sgo1-PP2A protects this pool of cohesin from cleavage and holds sister chromatids together. In meiosis II, the remaining centromeric cohesin is cleaved to allow segregation of sister chromatids. Therefore, correct regulation of Sgo1 and its interaction partners in meiosis is crucial to prevent aneuploidy. In this study I characterise the role of an Sgo1 binding partner, condensin, in meiotic chromosome segregation, and show that condensin is essential for faithful chromosome segregation in both meiosis I and II. Sgo1 is post-translationally modified in meiosis, and in this study Sgo1 phosphomutants were analysed, but were found to have no discernible effects on faithful chromosome segregation. However additional Sgo1 post-translational modifications were identified, leaving the regulation of Sgo1 by post-translational modification open to future study. Additional to the removal of cohesin by separase cleavage, there is a non-proteolytic pathway for cohesin removal. During mitotic prophase in higher eukaryotes, cohesin is destabilised from replicated sister chromatid arms through the action of Wapl. However, a subset of cohesin is protected from the destabilising effects of Wapl because acetylation of the Smc3 subunit of cohesin allows binding of sororin. Phosphorylation events prevent sororin association with chromosome arms, making cohesin susceptible to removal by Wapl. In contrast, at centromeres, shugoshin counteracts these phosphorylation events, thereby protecting centromeric cohesin from Wapl. During meiosis, Wapl is important in cohesin destabilisation in mouse, C. elegans and A. thaliana, and recent evidence suggests that this pathway is also active in budding yeast. However, it is unclear if the acetyltransferase, Eco1, and cohesin acetylation are important in the generation of cohesive cohesin in meiosis to allow faithful chromosome segregation. Additionally it is unclear if the destabilising activity of Wapl only contributes to cohesin removal on chromosome arms, or whether mechanisms are required to protect cohesin from destabilisation activity near centromeres. I aim to address these questions using budding yeast. Previous work showed that deletion of Wapl (Rad61) in meiosis prevents destabilisation of cohesin from the DNA prior to metaphase I. Consistently, I found Rad61 is regulated in meiosis, and has a role in promoting faithful chromosome segregation in tetranucleates. Additionally, Eco1 acetyltransferase is expressed during S phase of meiosis, and this expression coincides with Smc3 acetylation. In meiosis, disruption of Eco1 function and Smc3 acetylation has a detrimental impact on DNA segregation and cell viability. Further findings show that Sgo1 interacts with cohesin in budding yeast meiosis, and Sgo1 localisation to the chromatin is impacted in Eco1 mutants, but the precise interplay between Sgo1 and acetylated cohesin remains to be fully deduced

Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains

Cohesin organizes the genome by forming intra-chromosomal loops and inter-sister chromatid linkages. During gamete formation by meiosis, chromosomes are reshaped to support crossover recombination and two consecutive rounds of chromosome segregation. Here, we show that meiotic chromosomes are organized into functional domains by Eco1 acetyltransferase-dependent positioning of both chromatin loops and sister chromatid cohesion in budding yeast. Eco1 acetylates the Smc3 cohesin subunit in meiotic S phase to establish chromatin boundaries, independently of DNA replication. Boundary formation by Eco1 is critical for prophase exit and for the maintenance of cohesion until meiosis II, but is independent of the ability of Eco1 to antagonize the cohesin release factor, Wpl1. Conversely, prevention of cohesin release by Wpl1 is essential for centromeric cohesion, kinetochore mono-orientation, and co-segregation of sister chromatids in meiosis I. Our findings establish Eco1 as a key determinant of chromatin boundaries and cohesion positioning, revealing how local chromosome structuring directs genome transmission into gametes

A randomised controlled trial of three or one breathing technique training sessions for breathlessness in people with malignant lung disease.

BACKGROUND: About 90 % of patients with intra-thoracic malignancy experience breathlessness. Breathing training is helpful, but it is unknown whether repeated sessions are needed. The present study aims to test whether three sessions are better than one for breathlessness in this population. METHODS: This is a multi-centre randomised controlled non-blinded parallel arm trial. Participants were allocated to three sessions or single (1:2 ratio) using central computer-generated block randomisation by an independent Trials Unit and stratified for centre. The setting was respiratory, oncology or palliative care clinics at eight UK centres. Inclusion criteria were people with intrathoracic cancer and refractory breathlessness, expected prognosis ≥3 months, and no prior experience of breathing training. The trial intervention was a complex breathlessness intervention (breathing training, anxiety management, relaxation, pacing, and prioritisation) delivered over three hour-long sessions at weekly intervals, or during a single hour-long session. The main primary outcome was worst breathlessness over the previous 24 hours ('worst'), by numerical rating scale (0 = none; 10 = worst imaginable). Our primary analysis was area under the curve (AUC) 'worst' from baseline to 4 weeks. All analyses were by intention to treat. RESULTS: Between April 2011 and October 2013, 156 consenting participants were randomised (52 three; 104 single). Overall, the 'worst' score reduced from 6.81 (SD, 1.89) to 5.84 (2.39). Primary analysis [n = 124 (79 %)], showed no between-arm difference in the AUC: three sessions 22.86 (7.12) vs single session 22.58 (7.10); P value = 0.83); mean difference 0.2, 95 % CIs (-2.31 to 2.97). Complete case analysis showed a non-significant reduction in QALYs with three sessions (mean difference -0.006, 95 % CIs -0.018 to 0.006). Sensitivity analyses found similar results. The probability of the single session being cost-effective (threshold value of £20,000 per QALY) was over 80 %. CONCLUSIONS: There was no evidence that three sessions conferred additional benefits, including cost-effectiveness, over one. A single session of breathing training seems appropriate and minimises patient burden. TRIAL REGISTRATION: Registry: ISRCTN; TRIAL REGISTRATION NUMBER: ISRCTN49387307; http://www.isrctn.com/ISRCTN49387307 ; registration date: 25/01/2011

Spo13 prevents premature cohesin cleavage during meiosis [version 1; peer review: 2 approved, 1 approved with reservations]

Background: Meiosis produces gametes through two successive nuclear divisions, meiosis I and meiosis II. In contrast to mitosis and meiosis II, where sister chromatids are segregated, during meiosis I, homologous chromosomes are segregated. This requires the monopolar attachment of sister kinetochores and the loss of cohesion from chromosome arms, but not centromeres, during meiosis I. The establishment of both sister kinetochore mono-orientation and cohesin protection rely on the budding yeast meiosis I-specific Spo13 protein, the functional homolog of fission yeast Moa1 and mouse MEIKIN. Methods: Here we investigate the effects of loss of SPO13 on cohesion during meiosis I using a live-cell imaging approach. Results: Unlike wild type, cells lacking SPO13 fail to maintain the meiosis-specific cohesin subunit, Rec8, at centromeres and segregate sister chromatids to opposite poles during anaphase I. We show that the cohesin-destabilizing factor, Wpl1, is not primarily responsible for the loss of cohesion during meiosis I. Instead, premature loss of centromeric cohesin during anaphase I in spo13Δ cells relies on separase-dependent cohesin cleavage. Further, cohesin loss in spo13Δ anaphase I cells is blocked by forcibly tethering the regulatory subunit of protein phosphatase 2A, Rts1, to Rec8. Conclusions: Our findings indicate that separase-dependent cleavage of phosphorylated Rec8 causes premature cohesin loss in spo13Δ cells

LiGHT trial: 6-year results of primary selective laser trabeculoplasty versus eye drops for the treatment of glaucoma and ocular hypertension

PURPOSE: The LiGHT trial has shown selective laser trabeculoplasty (SLT) to be clinically and cost-effective as a primary treatment of open-angle glaucoma (OAG) and ocular hypertension (OHT) at 3 years. This paper reports health-related quality of life (HRQL) and clinical effectiveness of initial treatment with SLT compared to intra-ocular pressure (IOP) lowering eye drops, after 6 years of treatment. DESIGN: Prospective multicentre randomized controlled trial. PARTICIPANTS: Treatment-naïve eyes with OAG or OHT, initially treated with SLT or IOP-lowering drops. METHODS: Patients were randomly allocated to initial SLT or eye drops. Eye specific target IOP and monitoring intervals were based on international guidelines. After the initial 3 years of the trial, patients in the SLT arm were permitted a 3rd SLT if necessary; patients in the drops arm were allowed SLT as a treatment switch or escalation. Analysis was by intention to treat. This study is registered at controlled-trials.com (ISRCTN32038223). MAIN OUTCOME MEASURES: The primary outcome was HRQL at 6 years; secondary outcomes were clinical effectiveness and safety. RESULTS: Of the 692 patients completing 3 years in the LiGHT trial, 633 (91.5%) entered the extension and 524 patients completed 6 years in the trial (82.8% of those entering the extension phase, 73% of those initially randomised). At 6 years, there were no significant differences in HRQL for EQ-5D, GUI and GQL-15 (all p>0.05). The SLT arm had better GSS scores than the drops arm (83.6 (SD 18.1) vs 81.3 (SD 17.3), respectively). 69.8% of eyes in the SLT arm remained at or below target IOP without the need for medical or surgical treatment. More eyes in the drops arm exhibited disease progression (26.8% vs 19.6%, respectively, p=0.006). Trabeculectomy was required in 32 eyes in the drops arm compared to 13 eyes in the SLT arm (p<0.001); there were more cataract surgeries in the drops arm (95 compared to 57 eyes, p=0.03). There were no serious laser-related adverse events. CONCLUSIONS: SLT is a safe treatment for OAG and OHT, providing better long-term disease control than initial drop therapy, with reduced need for incisional glaucoma and cataract surgery over 6 years

Centromere localization and function of Mis18 requires Yippee-like domain-mediated oligomerization.

Mis18 is a key regulator responsible for the centromere localization of the CENP-A chaperone Scm3 in Schizosaccharomyces pombe and HJURP in humans, which establishes CENP-A chromatin that defines centromeres. The molecular and structural determinants of Mis18 centromere targeting remain elusive. Here, by combining structural, biochemical, and yeast genetic studies, we show that the oligomerization of S. pombe Mis18, mediated via its conserved N-terminal Yippee-like domain, is crucial for its centromere localization and function. The crystal structure of the N-terminal Yippee-like domain reveals a fold containing a cradle-shaped pocket that is implicated in protein/nucleic acid binding, which we show is required for Mis18 function. While the N-terminal Yippee-like domain forms a homodimer in vitro and in vivo, full-length Mis18, including the C-terminal α-helical domain, forms a homotetramer in vitro We also show that the Yippee-like domains of human Mis18α/Mis18β interact to form a heterodimer, implying a conserved structural theme for Mis18 regulation.Wellcome Trust Career Development Grant095822 Principal Research Fellowship095021065061 Wellcome Trust Centre Core Grant092076091020 Wellcome Trust‐University of Edinburgh Institutional Strategic Support Fund EpiGeneSys Network of ExcellenceHEALTH‐F4‐2010‐257082 EC FP7 Marie Curie International Incoming FellowshipPIIF‐GA‐2010‐275280 EMBO Long Term FellowshipALTF 1491‐2010 Wellcome Trus

The impact of cancer on subsequent chance of pregnancy: a population-based analysis

This study was funded by NHS Lothian Cancer and Leukaemia Endowments Fund. Part of this work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1 .STUDY QUESTION What is the impact of cancer in females aged ≤39 years on subsequent chance of pregnancy? SUMMARY ANSWER Cancer survivors achieved fewer pregnancies across all cancer types, and the chance of achieving a first pregnancy was also lower. WHAT IS KNOWN ALREADY The diagnosis and treatment of cancer in young females may be associated with reduced fertility but the true pregnancy deficit in a population is unknown. STUDY DESIGN, SIZE, DURATION We performed a retrospective cohort study relating first incident cancer diagnosed between 1981 and 2012 to subsequent pregnancy in all female patients in Scotland aged 39 years or less at cancer diagnosis (n = 23 201). Pregnancies were included up to end of 2014. Females from the exposed group not pregnant before cancer diagnosis (n = 10 271) were compared with general population controls matched for age, deprivation quintile and year of diagnosis. PARTICIPANTS/MATERIALS, SETTING, METHODS Scottish Cancer Registry records were linked to hospital discharge records to calculate standardized incidence ratios (SIR) for pregnancy, standardized for age and year of diagnosis. Linkage to death records was also performed. We also selected women from the exposed group who had not been pregnant prior to their cancer diagnosis who were compared with a matched control group from the general population. Additional analyses were performed for breast cancer, Hodgkin lymphoma, leukaemia, cervical cancer and brain/CNS cancers. MAIN RESULTS AND THE ROLE OF CHANCE Cancer survivors achieved fewer pregnancies: SIR 0.62 (95% CI: 0.60, 0.63). Reduced SIR was observed for all cancer types. The chance of achieving a first pregnancy was also lower, adjusted hazard ratio = 0.57 (95% CI: 0.53, 0.61) for women >5 years after diagnosis, with marked reductions in women with breast, cervical and brain/CNS tumours, and leukaemia. The effect was reduced with more recent treatment period overall and in cervical cancer, breast cancer and Hodgkin lymphoma, but was unchanged for leukaemia or brain/CNS cancers. The proportion of pregnancies that ended in termination was lower after a cancer diagnosis, and the proportion ending in live birth was higher (78.7 vs 75.6%, CI of difference: 1.1, 5.0). LIMITATIONS, REASONS FOR CAUTION Details of treatments received were not available, so the impact of specific treatment regimens on fertility could not be assessed. Limited duration of follow-up was available for women diagnosed in the most recent time period. WIDER IMPLICATIONS OF THE FINDINGS This analysis provides population-based quantification by cancer type of the effect of cancer and its treatment on subsequent pregnancy across the reproductive age range, and how this has changed in recent decades. The demonstration of a reduced chance of pregnancy across all cancer types and the changing impact in some but not other common cancers highlights the need for appropriate fertility counselling of all females of reproductive age at diagnosis. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by NHS Lothian Cancer and Leukaemia Endowments Fund. Part of this work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. RAA has participated in Advisory Boards and/or received speaker’s fees from Beckman Coulter, IBSA, Merck and Roche Diagnostics. He has received research support from Roche Diagnostics, Ansh labs and Ferring. The other authors have no conflicts to declare.Publisher PDFPeer reviewe