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Abstract The NIMA-related kinases represent a family of serine/threonine kinases implicated in cell cycle control. The founding member of this family, the NIMA kinase of Aspergillus nidulans, as well as the fission yeast homologue Fin1, contribute to multiple aspects of mitotic progression including the timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Mammals contain a large family of eleven NIMA-related kinases, named Nek1 to Nek11. Of these, there is now substantial evidence that Nek2, Nek6, Nek7 and Nek9 also regulate mitotic events. At least three of these kinases, as well as NIMA and Fin1, have been localized to the microtubule organizing centre of their respective species, namely the centrosome or spindle pole body. Here, they have important functions in microtubule organization and mitotic spindle assembly. Other Nek kinases have been proposed to play microtubule-dependent roles in non-dividing cells, most notably in regulating the axonemal microtubules of cilia and flagella. In this review, we discuss the evidence that NIMA-related kinases make a significant contribution to the orchestration of mitotic progression and thereby protect cells from chromosome instability. Furthermore, we highlight their potential as novel chemotherapeutic targets.

The EML4-ALK oncogene drives tumour progression in approximately 5% of cases of non-small-cell lung cancers. At least 15 EML4-ALK variants have been identified, which elicit differential responses to conventional ALK inhibitors. Unfortunately, most, if not all, patients eventually acquire resistance to these inhibitors and succumb to the disease, which warrants the need for alternative targets to be identified. The most aggressive variant, EML4-ALK variant 3 (V3), assembles into a complex on interphase microtubules together with the NEK9 and NEK7 kinases, which leads to the downstream phosphorylation of NEK7 substrates. Overall, this promotes an elongated cell morphology and an enhanced migratory phenotype, which likely contributes to the increased metastasis often seen in V3 patients. Here, using two separate approaches to displace V3 from microtubules and a variety of in vitro assays, we show that microtubule association of EML4-ALK V3 is required for both V3 phenotypes, as removal of the oncogenic fusion protein from microtubules led to the dissociation of the V3-NEK9-NEK7 complex and the reversal of both phenotypic changes. Overall, we propose that targeting the interaction between EML4-ALK V3 and microtubules might offer a novel therapeutic option, independent of ALK activity, for V3+ NSCLC patients with acquired resistance to ALK inhibitors.

Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) oncogenic fusion proteins are found in approximately 5% of non-small cell lung cancers. Different EML4-ALK fusion variants exist with variant 3 (V3) being associated with a significantly higher risk than other common variants, such as variant 1 (V1). Patients with V3 respond less well to targeted ALK inhibitors, have accelerated rates of metastasis, and have poorer overall survival. A pathway has been described downstream of EML4-ALK V3 that is independent of ALK catalytic activity but dependent on the NEK9 and NEK7 kinases. It has been proposed that assembly of an EML4-ALK V3-NEK9-NEK7 complex on microtubules leads to cells developing a mesenchymal-like morphology and exhibiting enhanced migration. However, downstream targets of this complex remain unknown. Here, we show that the microtubule-based kinesin, Eg5, is recruited to interphase microtubules in cells expressing EML4-ALK V3, whereas chemical inhibition of Eg5 reverses the mesenchymal morphology of cells. Furthermore, we show that depletion of NEK7 interferes with Eg5 recruitment to microtubules in cells expressing EML4-ALK V3 and cell length is reduced, but this is reversed by coexpression of a phosphomimetic mutant of Eg5, in a site, S1033, phosphorylated by NEK7. Intriguingly, we also found that expression of Eg5-S1033D led to cells expressing EML4-ALK V1 adopting a more mesenchymal-like morphology. Together, we propose that Eg5 acts as a substrate of NEK7 in cells expressing EML4-ALK V3 and Eg5 phosphorylation promotes the mesenchymal morphology typical of these cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Dominance of the left hemisphere for language processing is a prominent feature of brain organisation. Whereas structural models clarify the functional asymmetry due to direct access to local language circuits, dynamic models propose functional states of intrahemispheric activation and interhemispheric inhibition that are coupled with attentional processes. Real word settings often require modulations of lateralised neural processing and further express individual heterogeneity. In this research, we tested left- and right-handers, and used a behavioural paradigm with presentation of lateralised cue-target pairs to the same or opposite visual field. We observed that handedness distinctly affected word processing in the left hemisphere following contralateral cueing. Moreover, left-hemispheric dominance strengthened for right-handers vs abolished for left-handers, influencing behavioural efficiency. In combination with eye dominance recordings, these data suggest that attentional biases guided the processing strategies of both groups and in turn their achievements. Therefore, hand and eye dominance are both essential factors with a functional role in directing the communication of visual information between both hemispheres. Overall, the findings underline the importance of interacting hand-eye control systems in contributing to interhemispheric patterns in the context of language processing., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Admission to an Intensive Care Unit (ICU) in obstetrics is often used as a proxy for maternal near miss/severe maternal morbidity (MNM/SMM) events. Understanding incidence and management of pregnant or postpartum patients requiring critical care (CC) is thus important for continued improvement of maternity care. This study aims to describe provision of critical care in obstetrics in the Republic of Ireland., Material and Methods: The national clinical audit on critical care included 15 of 19 maternity units in Ireland (2014-2016). 960 pregnant or postpartum (within 42 days) individuals who required CC were included. Data were reported on all cases requiring level 2 or level 3 CC. We calculated basic descriptive statistics for diagnosis and process of care variables, and compared characteristics of women requiring level 2 care to those requiring level 3. Outcomes included diagnoses necessitating critical care; additional complications; level of care required; care process outcomes such as length of stay, consultation with non-obstetric specialties, location of maternal critical care, and neonatal care provision., Results: Overall, the rate of critical care in obstetrics for these hospitals was 1 in 131 live births; 900 of the 960 cases required level 2 care only. Hypertensive disorders contributed to the need for critical care for 1 in 242; hemorrhage, 1 in 422; and infections, 1 in 926. A substantial minority (15.7%) had more than one diagnosis, accounting for 40% of level 3 care. Serious complications were rare (eg, hysterectomy, 1 in 3846). Parity, hospital size, and identification as high-risk antenatally (<50% cases) were associated with requiring level 3 care. Critical care was provided in multiple locations, including ICUs, HDUs, and operating theatres. Only 23.8% of patients received CC in an ICU, suggesting ICU admission is not an ideal method for identifying severe maternal morbidity., Conclusions: We reported rates of critical care admission and primary diagnoses within the range of other published estimates, but huge variability exists in the literature, and within our data. ICU admission in and of itself iss not a reliable proxy for having received level 2 or 3 obstetric critical care in Ireland., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

EML4-ALK is an oncogenic fusion protein that accounts for approximately 5% of NSCLC cases. Targeted inhibitors of ALK are the standard of care treatment, often leading to a good initial response. Sadly, some patients do not respond well, and most will develop resistance over time, emphasizing the need for alternative treatments. This review discusses recent advances in our understanding of the mechanisms behind EML4-ALK-driven NSCLC progression and the opportunities they present for alternative treatment options to ALK inhibitor monotherapy. Targeting ALK-dependent signalling pathways can overcome resistance that has developed due to mutations in the ALK catalytic domain, as well as through activation of bypass mechanisms that utilise the same pathways. We also consider evidence for polytherapy approaches that combine targeted inhibition of these pathways with ALK inhibitors. Lastly, we review combination approaches that use targeted inhibitors of ALK together with chemotherapy, radiotherapy or immunotherapy. Throughout this article, we highlight the importance of alternative breakpoints in the EML4 gene that result in the generation of distinct EML4-ALK variants with different biological and pathological properties and consider monotherapy and polytherapy approaches that may be selective to particular variants.

EML4-ALK is an oncogenic fusion protein present in approximately 5% of non-small cell lung cancers (NSCLC). Alternative breakpoints in the gene encoding EML4 result in distinct variants that are linked to markedly different patient outcomes. Patients with EML4-ALK variant 3 (V3) respond poorly to ALK inhibitors and have lower survival rates compared with patients with other common variants, such as V1. Here, we use isogenic Beas-2B bronchial epithelial cell lines expressing EML4-ALK V1 or V3, as well as ALK-positive NSCLC patient cells that express V1 (H3122 cells) or V3 (H2228 cells), to show that EML4-ALK V3 but not V1 leads to hyperstabilized K-fibers in mitosis, as well as errors in chromosome congression and segregation. This is consistent with our observation that EML4-ALK V3 but not V1 localizes to spindle microtubules and that wild-type EML4 is a microtubule stabilizing protein. In addition, cells expressing EML4-ALK V3 exhibit loss of spindle assembly checkpoint control that is at least in part dependent on ALK catalytic activity. Finally, we demonstrate that cells expressing EML4-ALK V3 have increased sensitivity to microtubule poisons that interfere with mitotic spindle assembly, whereas combination treatment with paclitaxel and clinically approved ALK inhibitors leads to a synergistic response in terms of reduced survival of H2228 cells., Implications: This study suggests that combining the microtubule poison, paclitaxel, with targeted ALK inhibitors may provide an effective new treatment option for patients with NSCLC with tumors that express the EML4-ALK V3 oncogenic fusion., (©2022 The Authors; Published by the American Association for Cancer Research.)

Hemispheric lateralisation is a fundamental principle of functional brain organisation. We studied two core cognitive functions-language and visuospatial attention-that typically lateralise in opposite cerebral hemispheres. In this work, we tested both left- and right-handed participants on lexical decision-making as well as on symmetry detection by means of a visual half-field paradigm with various target-distractor combinations simultaneously presented to opposite visual fields. Laterality indexes were analysed using a behavioural metrics in single individuals as well as between individuals. We observed that lateralisation of language and visuospatial attention as well as their relationship generally followed a left-right profile, albeit with differences as a function of handedness and target-distractor combination. In particular, right-handed individuals tended towards a typical pattern whereas left-handed individuals demonstrated increased individual variation and atypical organisation. That the atypical variants varied as a function of target-distractor combination and thus interhemispheric communication underlines its dynamic role in characterising lateralisation properties. The data further revealed distinctive relationships between right-handedness and left-hemispheric dominance for language together with right-hemispheric dominance for visuospatial processing. Overall, these findings illustrate the role of broader mechanisms in supporting hemispheric lateralisation of cognition and behaviour, relying on common principles but controlled by internal and external factors., (© 2022. The Author(s).)

The human retinal pigment epithelial RPE-1 cell line immortalized with hTERT retains a stable karyotype with a modal chromosome number of 46 and has been widely used to study physiological events in human cell culture systems. To facilitate inducible knock-out or knock-in experiments in this cell line, we have modified the AAVS1 locus to harbour a DNA fragment encoding ERT2-Cre-ERT2 fusion protein under regulation of a Tet-On expression system. In the generated cell line, active Cre recombinase was induced by simple addition of doxycycline and tamoxifen to the culture medium. As proof of concept, we successfully introduced an oncogenic point mutation to the endogenous KRAS gene locus of this cell line. The cell line will serve as a powerful tool to conduct functional analyses of human genes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Mesothelioma is a universally lethal cancer lacking effective therapy. The spindle poison vinorelbine exhibits clinical activity in the relapsed setting, and in preclinical models requires BRCA1 to initiate apoptosis. However, the mechanisms underlying this regulation and the clinical implications have not been explored. Here, we show that BRCA1 silencing abrogated vinorelbine-induced cell-cycle arrest, recruitment of BUBR1 to kinetochores, and apoptosis. BRCA1 silencing led to codepletion of MAD2L1 at the mRNA and protein levels consistent with its status as a transcriptional target of BRCA1 Silencing of MAD2L1 phenocopied BRCA1 and was sufficient to confer resistance to vinorelbine. This was recapitulated in cell lines selected for resistance to vinorelbine, which acquired loss of both BRCA1 and MAD2L1 expression. Following ex vivo vinorelbine in 20 primary tumor explants, apoptotic response rate was 59% in BRCA1/MAD2L1 -positive explants compared with 0% in BRCA1/MAD2L1 -negative explants. In 48 patients, BRCA1 and/or MAD2L1 loss of expression was not prognostic; however, in a subset of patients treated with vinorelbine, survival was shorter for patients lacking BRCA1/MAD2L1 expression compared with double-positive patients (5.9 vs. 36.7 months, P = 0.03). Our data implicate BRCA1/MAD2L1 loss as a putative predictive marker of resistance to vinorelbine in mesothelioma and warrant prospective clinical evaluation., (©2020 American Association for Cancer Research.)

EML4-ALK is an oncogenic fusion present in ∼5% of non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants of EML4-ALK with different patient outcomes. Here, we show that, in cell models, EML4-ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. EML4-ALK V3 also recruits the NEK9 and NEK7 kinases to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but does not require ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4-ALK patients. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through NEK9 and NEK7, leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4-ALK lung cancer., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser 144 and Ser 146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Adaptive behaviour requires cognitive control for shielding current goals from distractors (stability) but at the same time for switching between alternative goals (flexibility). In this behavioural study, we examine the stability-flexibility balance in left- and right-handers during two types of decision-making, instructed (sensory cued) and voluntary (own choice), by means of distractor inhibition and hand/task switching. The data revealed that both groups showed opposite tendencies for instructed decision-making. Moreover, right-handers resisted distracting information more efficiently whereas left-handers showed superior switching abilities. When participants were involved in voluntary decision-making, no effects of handedness were noted, which suggests that free-choice processing alters the balance between stability and flexibility. These data illustrate that handedness is an index of individual variation during instructed decision-making, biasing the proficiency of cognitive control towards stability and flexibility of information processing. These biases can however be overruled by top-down strategies that dominate during voluntary decision-making. Overall, the research underlines the antagonistic functions of stability and flexibility in decision-making, and offers an approach for examining cognitive control and the role of internal and external factors in balancing the stability-flexibility trade-off., Competing Interests: The authors have declared that no competing interests exist.

Language and spatial processing are cognitive functions that are asymmetrically distributed across both cerebral hemispheres. In the present study, we compare left- and right-handers on word comprehension using a divided visual field paradigm and spatial attention using a landmark task. We investigate hemispheric asymmetries by assessing the participants' behavioral metrics; response accuracy, reaction time and their laterality index. The data showed that right-handers benefitted more from left-hemispheric lateralization for language comprehension and right-hemispheric lateralization for spatial attention than left-handers. Furthermore, left-handers demonstrated a more variable distribution across both hemispheres, supporting a less focal profile of functional brain organization. Taken together, the results underline that handedness distinctively modulates hemispheric processing and behavioral performance during verbal and nonverbal tasks. In particular, typical lateralization is most prevalent for right-handers whereas atypical lateralization is more evident for left-handers. These insights contribute to the understanding of individual variation of brain asymmetries and the mechanisms related to changes in cerebral dominance.

Hsp72 is a member of the 70-kDa heat shock family of molecular chaperones (Hsp70s) that comprise a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) connected by a linker that couples the exchange of adenosine diphosphate (ADP) for adenosine triphosphate (ATP) with the release of the protein substrate. Mitotic phosphorylation of Hsp72 by the kinase NEK6 at Thr 66 located in the NBD promotes the localization of Hsp72 to the mitotic spindle and is required for efficient spindle assembly and chromosome congression and segregation. We determined the crystal structure of the Hsp72 NBD containing a genetically encoded phosphoserine at position 66. This revealed structural changes that stabilized interactions between subdomains within the NBD. ATP binding to the NBD of unmodified Hsp72 resulted in the release of substrate from the SBD, but phosphorylated Hsp72 retained substrate in the presence of ATP. Mutations that prevented phosphorylation-dependent subdomain interactions restored the connection between ATP binding and substrate release. Thus, phosphorylation of Thr 66 is a reversible mechanism that decouples the allosteric connection between nucleotide binding and substrate release, providing further insight into the regulation of the Hsp70 family. We propose that phosphorylation of Hsp72 on Thr 66 by NEK6 during mitosis promotes its localization to the spindle by stabilizing its interactions with components of the mitotic spindle., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cancer cells frequently possess extra amplified centrosomes clustered into two poles whose pseudo-bipolar spindles exhibit reduced fidelity of chromosome segregation and promote genetic instability. Inhibition of centrosome clustering triggers multipolar spindle formation and mitotic catastrophe, offering an attractive therapeutic approach to selectively kill cells with amplified centrosomes. However, mechanisms of centrosome clustering remain poorly understood. Here, we identify a new pathway that acts through NIMA-related kinase 6 (Nek6) and Hsp72 to promote centrosome clustering. Nek6, as well as its upstream activators polo-like kinase 1 and Aurora-A, targeted Hsp72 to the poles of cells with amplified centrosomes. Unlike some centrosome declustering agents, blocking Hsp72 or Nek6 function did not induce formation of acentrosomal poles, meaning that multipolar spindles were observable only in cells with amplified centrosomes. Inhibition of Hsp72 in acute lymphoblastic leukemia cells resulted in increased multipolar spindle frequency that correlated with centrosome amplification, while loss of Hsp72 or Nek6 function in noncancer-derived cells disturbs neither spindle formation nor mitotic progression. Hence, the Nek6-Hsp72 module represents a novel actionable pathway for selective targeting of cancer cells with amplified centrosomes. Cancer Res; 77(18); 4785-96. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Time is a fundamental dimension of our behavior and enables us to guide our actions and to experience time such as predicting collisions or listening to music. In this study, we investigate the regulation and covariation of motor timing and time perception functions in left- and right-handers who are characterized by distinct brain processing mechanisms for cognitive-motor control. To this purpose, we use a combination of tasks that assess the timed responses during movements and the perception of time intervals. The results showed a positive association across left- and right-handers between movement-driven timing and perceived interval duration when adopting a preferred tempo, suggesting cross-domain coupling between both abilities when an intrinsic timescale is present. Handedness guided motor timing during externally-driven conditions that required cognitive intervention, which specifies the relevance of action expertise for the performance of timed-based motor activities. Overall, our results reveal that individual variation across domain-general and domain-specific levels of organization plays a steering role in how one predicts, perceives and experiences time, which accordingly impacts on cognition and behavior.

The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Mitotic spindle assembly requires the regulated activities of protein kinases such as Nek7 and Nek9. Nek7 is autoinhibited by the protrusion of Tyr97 into the active site and activated by the Nek9 non-catalytic C-terminal domain (CTD). CTD binding apparently releases autoinhibition because mutation of Tyr97 to phenylalanine increases Nek7 activity independently of Nek9. Here we find that self-association of the Nek9-CTD is needed for Nek7 activation. We map the minimal Nek7 binding region of Nek9 to residues 810-828. A crystal structure of Nek7(Y97F) bound to Nek9(810-828) reveals a binding site on the C-lobe of the Nek7 kinase domain. Nek7(Y97F) crystallizes as a back-to-back dimer between kinase domain N-lobes, in which the specific contacts within the interface are coupled to the conformation of residue 97. Hence, we propose that the Nek9-CTD activates Nek7 through promoting back-to-back dimerization that releases the autoinhibitory tyrosine residue, a mechanism conserved in unrelated kinase families.

The mitotic spindle is the apparatus upon which chromosomes are segregated during cell division. We have discovered new roles for two members of the NIMA-related kinase (NEK) family in different molecular processes of spindle assembly. Moreover, loss of these proteins leads to segregation errors that drive cancer progression.

Hsp70 proteins represent a family of chaperones that regulate cellular homeostasis and are required for cancer cell survival. However, their function and regulation in mitosis remain unknown. In this paper, we show that the major inducible cytoplasmic Hsp70 isoform, Hsp72, is required for assembly of a robust bipolar spindle capable of efficient chromosome congression. Mechanistically, Hsp72 associates with the K-fiber-stabilizing proteins, ch-TOG and TACC3, and promotes their interaction with each other and recruitment to spindle microtubules (MTs). Targeting of Hsp72 to the mitotic spindle is dependent on phosphorylation at Thr-66 within its nucleotide-binding domain by the Nek6 kinase. Phosphorylated Hsp72 concentrates on spindle poles and sites of MT-kinetochore attachment. A phosphomimetic Hsp72 mutant rescued defects in K-fiber assembly, ch-TOG/TACC3 recruitment and mitotic progression that also resulted from Nek6 depletion. We therefore propose that Nek6 facilitates association of Hsp72 with the mitotic spindle, where it promotes stable K-fiber assembly through recruitment of the ch-TOG-TACC3 complex., (© 2015 O’Regan et al.)

Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1-4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

This research aimed to explore the neural correlates of relational learning by recording high-density EEG during a behavioural task involving derivation levels of varying complexity. A total of 15 participants (5 male; age range 18-23 years; mean age=20.0 years) completed contextual cue training, relational learning, function training and a derivation task while 128-channel event-related potentials (ERPs) were recorded from the scalp (Background). Differences in response latencies were observed between the two derived (symmetry and equivalence) and directly trained relations, with longest latencies found for equivalence and shortest for the directly trained relations. This pattern failed to reach statistical significance. Importantly, ERPs revealed an early P3a positivity (from 230 to 350ms) over right posterior scalp sites. Significantly larger mean amplitudes were found at three channels (P6, E115 and E121) for the equivalence relations compared to the two other types (Results). We believe this may constitute a first demonstration of differences in brain electrophysiology in the transformation of stimulus functions through derived relations of hierarchical levels of complexity (Conclusions)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Histone deacetylases 1 and 2 (HDAC1/2) form the core catalytic components of corepressor complexes that modulate gene expression. In most cell types, deletion of both Hdac1 and Hdac2 is required to generate a discernible phenotype, suggesting their activity is largely redundant. We have therefore generated an ES cell line in which Hdac1 and Hdac2 can be inactivated simultaneously. Loss of HDAC1/2 resulted in a 60% reduction in total HDAC activity and a loss of cell viability. Cell death is dependent upon cell cycle progression, because differentiated, nonproliferating cells retain their viability. Furthermore, we observe increased mitotic defects, chromatin bridges, and micronuclei, suggesting HDAC1/2 are necessary for accurate chromosome segregation. Consistent with a critical role in the regulation of gene expression, microarray analysis of Hdac1/2-deleted cells reveals 1,708 differentially expressed genes. Significantly for the maintenance of stem cell self-renewal, we detected a reduction in the expression of the pluripotent transcription factors, Oct4, Nanog, Esrrb, and Rex1. HDAC1/2 activity is regulated through binding of an inositol tetraphosphate molecule (IP4) sandwiched between the HDAC and its cognate corepressor. This raises the important question of whether IP4 regulates the activity of the complex in cells. By rescuing the viability of double-knockout cells, we demonstrate for the first time (to our knowledge) that mutations that abolish IP4 binding reduce the activity of HDAC1/2 in vivo. Our data indicate that HDAC1/2 have essential and pleiotropic roles in cellular proliferation and regulate stem cell self-renewal by maintaining expression of key pluripotent transcription factors.

Proteins of the echinoderm microtubule-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule network. They contain a unique hydrophobic EML protein (HELP) motif and a variable number of WD40 repeats. Recurrent gene rearrangements in nonsmall cell lung cancer fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression of several fusion oncoprotein variants. We have determined a 2.6-Å crystal structure of the representative ∼70-kDa core of EML1, revealing an intimately associated pair of β-propellers, which we term a TAPE (tandem atypical propeller in EMLs) domain. One propeller is highly atypical, having a discontinuous subdomain unrelated to a WD40 motif in place of one of its blades. This unexpected feature shows how a propeller structure can be assembled from subdomains with distinct folds. The HELP motif is not an independent domain but forms part of the hydrophobic core that joins the two β-propellers. The TAPE domain binds α/β-tubulin via its conserved, concave surface, including part of the atypical blade. Mapping the characteristic breakpoints of each EML4-ALK variant onto our structure indicates that the EML4 TAPE domain is truncated in many variants in a manner likely to make the fusion protein structurally unstable. We found that the heat shock protein 90 (Hsp90) inhibitor ganetespib induced degradation of these variants whereas others lacking a partial TAPE domain were resistant in both overexpression models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins involve breakpoints in disordered regions of both partners.

Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer.

Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the alphaC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

Aims: To describe alprazolam poisoning and the relative toxicity of alprazolam compared with other benzodiazepines., Methods: A database of consecutive poisoning admissions to a regional toxicology service was searched to identify consecutive benzodiazepine deliberate self poisonings, which were coded as alprazolam, diazepam or other benzodiazepine. Major outcomes used were length of stay (LOS), intensive care (ICU) admission, coma (GCS < 9), flumazenil administration and requirement for mechanical ventilation. Prescription data were obtained for benzodiazepines for the study period., Results: There were 2063 single benzodiazepine overdose admissions: 131 alprazolam overdoses, 823 diazepam overdoses and 1109 other benzodiazepine overdoses. The median LOS for alprazolam overdoses was 19 h which was 1.27 (95% CI 1.04, 1.54) times longer compared with other benzodiazepines by multiple linear regression. For patients with alprazolam overdoses, 22% were admitted to ICU which was 2.06 (95% CI 1.27, 3.33) times more likely compared with other benzodiazepines after multivariate analysis adjusting for age, dose, gender, time to ingestion and co-ingested drugs. Flumazenil was administered to 14% of alprazolam patients and 16% were ventilated, which was significantly more than for other benzodiazepine overdoses (8% and 11%, respectively). Twelve percent of alprazolam overdoses had a GCS < 9 compared with 10% for other benzodiazepines. From benzodiazepine prescription data, total alprazolam prescriptions in Australia increased from 0.13 million in 1992 to 0.41 million in 2001. Eighty five percent of prescriptions were for panic disorder, anxiety, depression or mixed anxiety/depression., Conclusions: Alprazolam was significantly more toxic than other benzodiazepines. The increased prescription of alprazolam to groups with an increased risk of deliberate self poisoning is concerning and needs review.