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Summary In contrast to most DNA viruses, poxviruses replicate their genomes in the cytoplasm without host involvement. We find that vaccinia virus induces cytoplasmic activation of ATR early during infection, before genome uncoating, which is unexpected because ATR plays a fundamental nuclear role in maintaining host genome integrity. ATR, RPA, INTS7, and Chk1 are recruited to cytoplasmic DNA viral factories, suggesting canonical ATR pathway activation. Consistent with this, pharmacological and RNAi-mediated inhibition of canonical ATR signaling suppresses genome replication. RPA and the sliding clamp PCNA interact with the viral polymerase E9 and are required for DNA replication. Moreover, the ATR activator TOPBP1 promotes genome replication and associates with the viral replisome component H5. Our study suggests that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm., Graphical Abstract, Highlights • Vaccinia activates cytoplasmic ATR early during infection and before genome uncoating • Canonical ATR pathway activation promotes viral genome replication • RPA is recruited to the viral genome • PCNA, RPA, and TOPBP1 associate with the viral polymerase to promote DNA replication, Postigo et al. suggest that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm.

Intercellular heterogeneity, exacerbated by chromosomal instability (CIN), fosters tumor heterogeneity and drug resistance. However, extreme CIN correlates with improved cancer outcome, suggesting that karyotypic diversity required to adapt to selection pressures might be balanced in tumors against the risk of excessive instability. Here, we used a functional genomics screen, genome editing, and pharmacologic approaches to identify CIN-survival factors in diploid cells. We find partial anaphase-promoting complex/cyclosome (APC/C) dysfunction lengthens mitosis, suppresses pharmacologically induced chromosome segregation errors, and reduces naturally occurring lagging chromosomes in cancer cell lines or following tetraploidization. APC/C impairment caused adaptation to MPS1 inhibitors, revealing a likely resistance mechanism to therapies targeting the spindle assembly checkpoint. Finally, CRISPR-mediated introduction of cancer somatic mutations in the APC/C subunit cancer driver gene CDC27 reduces chromosome segregation errors, whereas reversal of an APC/C subunit nonsense mutation increases CIN. Subtle variations in mitotic duration, determined by APC/C activity, influence the extent of CIN, allowing cancer cells to dynamically optimize fitness during tumor evolution. Significance: We report a mechanism whereby cancers balance the evolutionary advantages associated with CIN against the fitness costs caused by excessive genome instability, providing insight into the consequence of CDC27 APC/C subunit driver mutations in cancer. Lengthening of mitosis through APC/C modulation may be a common mechanism of resistance to cancer therapeutics that increase chromosome segregation errors. Cancer Discov; 7(2); 218–33. ©2017 AACR. See related commentary by Burkard and Weaver, p. 134. This article is highlighted in the In This Issue feature, p. 115

Genome-wide CRISPR screening is a powerful tool to identify genes required under selective conditions. However, the inherent scale of genome-wide libraries can limit their application in experimental settings where cell numbers are restricted, such as in vivo infections or single cell analysis. The use of small scale CRISPR libraries targeting gene subsets circumvents this problem. Here we develop a method for rapid generation of custom guide RNA (gRNA) libraries using arrayed single-stranded oligonucleotides for reproducible pooled cloning of CRISPR/Cas9 libraries. We use this system to generate mutant pools of different sizes in the protozoan parasite Toxoplasma gondi and describe optimised analysis methods for small scale libraries. An in vivo genetic screen in the murine host identifies novel and known virulence factors and we confirm results using cloned knock-out parasites. Our study also reveals a potential trans-rescue of individual knock-out parasites in pools of mutants compared to homogenous knock-out lines of the key virulence factor MYR1., Targeted CRISPR libraries expand the use of genetic screens across experimental conditions. Here, the authors develop a method for generating and analysing small scale custom CRISPR libraries and use it in the human and livestock pathogen Toxoplasma gondii to identify virulence factors in mice.

The majority of the heredity of melanoma remains unexplained, however inherited copy number changes have not yet been systematically studied. The genetic environment is highly relevant to treatment stratification, and new gene discovery is therefore desirable. Using an unbiased whole genome screening approach for copy number we identify here a novel melanoma predisposing factor, familial duplications of genePPP2R3B, encoding a regulatory unit of critical phosphatase PP2A. Significant correlation between expression ofPPP2R3Bin tumour tissue and survival in a large melanoma cohort was confirmed, and associated with a non-immunological expression profile. Mechanistically, construction and extensive characterization of a stable, inducible cellular model forPPP2R3Boverexpression revealed induction of pigment cell switching towards proliferation and away from migration. Importantly, this was independent of the known microphthalmia-associated transcription factor(MITF)-controlled pigment cell phenotype switch, and was instead driven by uncharacterised geneC21orf91. Bioinformatic studies point toC21orf91as a novel target ofMITF,and therefore a potential hub in the control of phenotype switching in melanoma. This study identifies novel germline copy number variants inPPP2R3Bpredisposing to melanocytic neoplasia, and uncovers a new potential therapeutic targetC21orf91in the control of pigment cell proliferation.

BACKGROUND: Intraputamenal glial cell line-derived neurotrophic factor (GDNF), administered every 4 weeks to patients with moderately advanced Parkinson's disease, did not show significant clinical improvements against placebo at 40 weeks, although it significantly increased [18F]DOPA uptake throughout the entire putamen.OBJECTIVE: This open-label extension study explored the effects of continued (prior GDNF patients) or new (prior placebo patients) exposure to GDNF for another 40 weeks.METHODS: Using the infusion protocol of the parent study, all patients received GDNF without disclosing prior treatment allocations (GDNF or placebo). The primary outcome was the percentage change from baseline to Week 80 in the OFF state Unified Parkinson's Disease Rating Scale (UPDRS) motor score.RESULTS: All 41 parent study participants were enrolled. The primary outcome decreased by 26.7±20.7% in patients on GDNF for 80 weeks (GDNF/GDNF; N = 21) and 27.6±23.6% in patients on placebo for 40 weeks followed by GDNF for 40 weeks (placebo/GDNF, N = 20; least squares mean difference: 0.4%, 95% CI: -13.9, 14.6, p = 0.96). Secondary endpoints did not show significant differences between the groups at Week 80 either. Prespecified comparisons between GDNF/GDNF at Week 80 and placebo/GDNF at Week 40 showed significant differences for mean OFF state UPDRS motor (-9.6±6.7 vs. -3.8±4.2 points, p = 0.0108) and activities of daily living score (-6.9±5.5 vs. -1.0±3.7 points, p = 0.0003). No treatment-emergent safety concerns were identified.CONCLUSIONS: The aggregate study results, from the parent and open-label extension suggest that future testing with GDNF will likely require an 80- rather than a 40-week randomized treatment period and/or a higher dose.

Trials of GDNF in Parkinson’s disease have yielded inconsistent results. In a randomised controlled trial, Whone et al. administer GDNF using a paradigm designed to optimize delivery to the putamen. [18F]DOPA PET reveals putamen-wide uptake, but GDNF does not differ from placebo in its effects on motor function., We investigated the effects of glial cell line-derived neurotrophic factor (GDNF) in Parkinson’s disease, using intermittent intraputamenal convection-enhanced delivery via a skull-mounted transcutaneous port as a novel administration paradigm to potentially afford putamen-wide therapeutic delivery. This was a single-centre, randomized, double-blind, placebo-controlled trial. Patients were 35–75 years old, had motor symptoms for 5 or more years, and presented with moderate disease severity in the OFF state [Hoehn and Yahr stage 2–3 and Unified Parkinson’s Disease Rating Scale motor score (part III) (UPDRS-III) between 25 and 45] and motor fluctuations. Drug delivery devices were implanted and putamenal volume coverage was required to exceed a predefined threshold at a test infusion prior to randomization. Six pilot stage patients (randomization 2:1) and 35 primary stage patients (randomization 1:1) received bilateral intraputamenal infusions of GDNF (120 µg per putamen) or placebo every 4 weeks for 40 weeks. Efficacy analyses were based on the intention-to-treat principle and included all patients randomized. The primary outcome was the percentage change from baseline to Week 40 in the OFF state (UPDRS-III). The primary analysis was limited to primary stage patients, while further analyses included all patients from both study stages. The mean OFF state UPDRS motor score decreased by 17.3 ± 17.6% in the active group and 11.8 ± 15.8% in the placebo group (least squares mean difference: −4.9%, 95% CI: −16.9, 7.1, P = 0.41). Secondary endpoints did not show significant differences between the groups either. A post hoc analysis found nine (43%) patients in the active group but no placebo patients with a large clinically important motor improvement (≥10 points) in the OFF state (P = 0.0008). 18F-DOPA PET imaging demonstrated a significantly increased uptake throughout the putamen only in the active group, ranging from 25% (left anterior putamen; P = 0.0009) to 100% (both posterior putamina; P < 0.0001). GDNF appeared to be well tolerated and safe, and no drug-related serious adverse events were reported. The study did not meet its primary endpoint. 18F-DOPA imaging, however, suggested that intermittent convection-enhanced delivery of GDNF produced a putamen-wide tissue engagement effect, overcoming prior delivery limitations. Potential reasons for not proving clinical benefit at 40 weeks are discussed.

For image-based infection biology, accurate unbiased quantification of host–pathogen interactions is essential, yet often performed manually or using limited enumeration employing simple image analysis algorithms based on image segmentation. Host protein recruitment to pathogens is often refractory to accurate automated assessment due to its heterogeneous nature. An intuitive intelligent image analysis program to assess host protein recruitment within general cellular pathogen defense is lacking. We present HRMAn (Host Response to Microbe Analysis), an open-source image analysis platform based on machine learning algorithms and deep learning. We show that HRMAn has the capacity to learn phenotypes from the data, without relying on researcher-based assumptions. Using Toxoplasma gondii and Salmonella enterica Typhimurium we demonstrate HRMAn’s capacity to recognize, classify and quantify pathogen killing, replication and cellular defense responses. HRMAn thus presents the only intelligent solution operating at human capacity suitable for both single image and high content image analysis.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Summary On-target, cell-active chemical probes are of fundamental importance in chemical and cell biology, whereas poorly characterized probes often lead to invalid conclusions. Human N-myristoyltransferase (NMT) has attracted increasing interest as target in cancer and infectious diseases. Here we report an in-depth comparison of five compounds widely applied as human NMT inhibitors, using a combination of quantitative whole-proteome N-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis, and cell-cycle assays. We find that N-myristoylation is unaffected by 2-hydroxymyristic acid (100 μM), D-NMAPPD (30 μM), or Tris-DBA palladium (10 μM), with the latter compounds causing cytotoxicity through mechanisms unrelated to NMT. In contrast, drug-like inhibitors IMP-366 (DDD85646) and IMP-1088 delivered complete and specific inhibition of N-myristoylation in a range of cell lines at 1 μM and 100 nM, respectively. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies that used off-target compounds., Graphical Abstract, Highlights • IMP-1088 selectively blocks N-myristoylation in cells at low nanomolar concentration • Three widely used human NMT probes are invalidated by 6 orthogonal assay modalities • 2-Hydroxymyristate is inactive up to 100 μM; D-NMAPPD is non-specifically cytotoxic • Tris-DBA palladium does not inhibit NMT in cells, and is cytotoxic by precipitation, Specific inhibitors of protein N-myristoylation are powerful probes for exploring the role of this post-translational modification in cells and have therapeutic potential. Kallemeijn et al. identify three flawed but commonly applied human N-myristoyltransferase inhibitors, and advocate two classes of high-affinity inhibitors as probes with a well-validated on-target cellular mode of action.

Autophagy is a highly regulated process, and its deregulation can contribute to various diseases, including cancer, immune diseases, and neurodegenerative disorders. Here we describe the design, protocol, and analysis of an imaging-based high-throughput screen with an endogenous autophagy readout. The screen uses a genome-wide siRNA library to identify autophagy regulators in mammalian cells.

Autophagy is a highly regulated process, and its deregulation can contribute to various diseases, including cancer, immune diseases, and neurodegenerative disorders. Here we describe the design, protocol, and analysis of an imaging-based high-throughput screen with an endogenous autophagy readout. The screen uses a genome-wide siRNA library to identify autophagy regulators in mammalian cells.

This study examines the relationship between internal barriers, professional development, and computer integration outcomes among a sample of fourth- and fifth-grade teachers in an urban, low-income school district in the Southeastern United States. Specifically, we examine the impact of teachers’ computer attitudes, computer anxiety, and computer training on the quality of computer integration in their classrooms. Using data from the Integrating Computing Across the Curriculum project, we utilize a mixed-methods approach to explore these relationships. Our results indicate that teacher attitudes and participation in an intensive computer-based training have a positive effect on computer integration practices. Findings from this study support providing teachers with more computer-based training which aims to improve the quality of classroom integration. This may lead to improvements in teacher attitudes toward computing and an increase in levels of computer integration in the elementary school classroom.

BACKGROUND Measurement scales that quickly and rigorously evaluate the effectiveness of filler treatment in hands are important tools in clinical practice. The Merz Hand Grading Scale (MHGS) is used to grade the appearance of the dorsal hand. The MHGS has been validated for photographic and live assessment of the hands. OBJECTIVE To evaluate the sensitivity of the 5-point MHGS to detect clinically meaningful and aesthetically pleasing changes in hand appearance after treatment with a calcium hydroxylapatite (CaHA)-based dermal filler. METHODS The controlled 4-week study randomized 30 subjects (60 hands) 2:1 to a Treatment group (treatment at enrollment) or a Control group (treatment at end of study). Effectiveness was evaluated with live MHGS ratings and photographic assessments with the Global Aesthetic Improvement Scale (GAIS). RESULTS At Week 4, all Treatment group subjects (20/20) achieved a ≥1-point improvement on the MHGS compared with 0/10 (0%) of the Control group (p < .0001). Subjects and treating physicians rated 92.5% (37/40) and 100% (40/40), respectively, of hands as at least "improved," using the GAIS. CONCLUSION The MHGS is an appropriate and validated tool that clinicians can use to counsel patients and evaluate clinically meaningful and aesthetically pleasing changes after hand treatment with CaHA.

Summary During early development, extrinsic triggers prompt pluripotent cells to begin the process of differentiation. When and how human embryonic stem cells (hESCs) irreversibly commit to differentiation is a fundamental yet unanswered question. By combining single-cell imaging, genomic approaches, and mathematical modeling, we find that hESCs commit to exiting pluripotency unexpectedly early. We show that bone morphogenetic protein 4 (BMP4), an important differentiation trigger, induces a subset of early genes to mirror the sustained, bistable dynamics of upstream signaling. Induction of one of these genes, GATA3, drives differentiation in the absence of BMP4. Conversely, GATA3 knockout delays differentiation and prevents fast commitment to differentiation. We show that positive feedback at the level of the GATA3-BMP4 axis induces fast, irreversible commitment to differentiation. We propose that early commitment may be a feature of BMP-driven fate choices and that interlinked feedback is the molecular basis for an irreversible transition from pluripotency to differentiation., Graphical Abstract, Highlights • Irreversible commitment to BMP4-driven hESC differentiation is fast • SMAD activation is sustained, bistable, and irreversible due to positive feedback • GATA3 mirrors SMAD dynamics and mediates fast commitment to differentiation • GATA3 is an early commitment gene, Gunne-Braden et al. show that GATA3 is an early commitment gene that mediates fast commitment to BMP4-driven differentiation in human embryonic stem cells (hESCs). Interlinked feedback regulation at the level of the GATA3-BMP4 axis allows for an irreversible exit from pluripotency and an early commitment to differentiation.

For image-based infection biology, accurate unbiased quantification of host-pathogen interactions is essential, yet often performed manually or using limited enumeration employing simple image analysis algorithms based on image segmentation. Host protein recruitment to pathogens is often refractory to accurate automated assessment due to its heterogeneous nature. An intuitive intelligent image analysis program to assess host protein recruitment within general cellular pathogen defense is lacking. We present HRMAn (Host Response to Microbe Analysis), an open-source image analysis platform based on machine learning algorithms and deep learning. We show that HRMAn has the capability to learn phenotypes from the data, without relying on researcher-based assumptions. Using Toxoplasma gondii and Salmonella typhimurium we demonstrate HRMAn’s capacity to recognize, classify and quantify pathogen killing, replication and cellular defense responses.

Magnetic resonance imaging (MRI) of glioblastoma multiforme (GBM) with molecular imaging agents would allow for the specific localization of brain tumors. Prior studies using T 1-weighted MR imaging demonstrated that the SBK2-Tris-(Gd-DOTA)3 molecular imaging agent labeled heterotopic xenograft models of brain tumors more intensely than non-specific contrast agents using conventional T 1-weighted imaging techniques. In this study, we used a dynamic quantitative T 1 mapping strategy to more objectively compare intra-tumoral retention of the SBK2-Tris-(Gd-DOTA)3 agent over time in comparison to non-targeted control agents. Our results demonstrate that the targeted SBK2-Tris-(Gd-DOTA)3 agent, a scrambled-Tris-(Gd-DOTA)3 control agent, and the non-specific clinical contrast agent Optimark(™) all enhanced flank tumors of human glioma cells with similar maximal changes on T 1 mapping. However, the retention of the agents differs. The non-specific agents show significant recovery within 20 min by an increase in T 1 while the specific agent SBK2-Tris-(Gd-DOTA)3 is retained in the tumors and shows little recovery over 60 min. The retention effect is demonstrated by percent change in T 1 values and slope calculations as well as by calculations of gadolinium concentration in tumor compared to muscle. Quantitative T 1 mapping demonstrates the superior binding and retention in tumors of the SBK2-Tris-(Gd-DOTA)3 agent over time compared to the non-specific contrast agent currently in clinical use.

Metabolic reprogramming in cancer enhances macromolecule biosynthesis and supports cell survival. Oncogenic drivers affect metabolism by altering distinct metabolic processes and render cancer cells sensitive to perturbations of the metabolic network. This study aimed to identify selective metabolic dependencies in breast cancer by investigating 17 breast cancer cells lines representative of the genetic diversity of the disease. Using a functional screen, we demonstrate here that monocarboxylate transporter 4 (MCT4) is an important regulator of breast cancer cell survival. MCT4 supports pH maintenance, lactate secretion and non-oxidative glucose metabolism in breast cancer cells. Moreover, MCT4 depletion caused an increased dependence of cancer cells on mitochondrial respiration and glutamine metabolism. MCT4 depletion reduced the ability of breast cancer cells to grow in a three-dimensional (3D) matrix or as multilayered spheroids. Moreover, MCT4 expression is regulated by the PI3K-Akt signalling pathway and highly expressed in HER2-positive breast cancers. These results suggest that MCT4 is a potential therapeutic target in defined breast cancer subtypes and reveal novel avenues for combination treatment.

WISp39 associates with Hsp90, Coronin 1B, and Slingshot phosphatase to regulate Cofilin activation and Arp2/3 complex localization at the leading edge of migrating cells., We previously identified Waf1 Cip1 stabilizing protein 39 (WISp39) as a binding partner for heat shock protein 90 (Hsp90). We now report that WISp39 has an essential function in the control of directed cell migration, which requires WISp39 interaction with Hsp90. WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge. WISp39 binds Coronin 1B, known to regulate the Arp2/3 complex and Cofilin at the leading edge. WISp39 preferentially interacts with phosphorylated Coronin 1B, allowing it to complex with Slingshot phosphatase (SSH) to dephosphorylate and activate Cofilin. WISp39 also regulates Arp2/3 complex localization at the leading edge. WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant. We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge.

Summary A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment., Graphical Abstract, Highlights • ACSS2 expression positively correlates with tumor stage and patient survival • Hypoxia and low lipid availability synergistically stimulate ACSS2 expression • Acetate is a major source of carbon for lipid synthesis during metabolic stress • ACSS2 is required for growth of tumor xenografts harboring ACSS2 copy-number gains, Schug et al. show that ACSS2 expression is increased in cancer cells under metabolic stress, and it is critical for cancer cells to use acetate as a nutritional source for lipid biomass production under this condition. Importantly, the ACSS2 expression level correlates with breast cancer progression.

Summary: Signal transduction pathways stimulated by secreted growth factors are tightly regulated at multiple levels between the cell surface and the nucleus. The trafficking of cell surface receptors is emerging as a key step for regulating appropriate cellular responses, with perturbations in this process contributing to human diseases, including cancer. For receptors recognizing ligands of the transforming growth factor β (TGF-β) family, little is known about how trafficking is regulated or how this shapes signaling dynamics. Here, using whole genome small interfering RNA (siRNA) screens, we have identified the ESCRT (endosomal sorting complex required for transport) machinery as a crucial determinant of signal duration. Downregulation of ESCRT components increases the outputs of TGF-β signaling and sensitizes cells to low doses of ligand in their microenvironment. This sensitization drives an epithelial-to-mesenchymal transition (EMT) in response to low doses of ligand, and we demonstrate a link between downregulation of the ESCRT machinery and cancer survival. : Miller et al. demonstrate, using whole genome siRNA screening, that TGF-β receptors are targeted for degradation by the ESCRT machinery. Inhibiting ESCRT components upregulates long-term TGF-β signaling and enhances functional outputs of the pathway to sensitize cells to low levels of ligand in the micro-environment. Keywords: epithelial-to-mesenchymal transition, ESCRT machinery, receptor trafficking, signaling dynamics, SMAD2, TGF-β

SummaryThe transcriptional regulator YAP1 is critical for the pathological activation of fibroblasts. In normal fibroblasts YAP1 is predominantly located in the cytoplasm, while in activated cancer-associated fibroblasts it exhibits nuclear localization and promotes the expression of many genes required for pro-tumorigenic functions. Here, we investigate the dynamics of YAP1 shuttling in normal and activated fibroblasts, using EYFP-YAP1, quantitative photo-bleaching methods, and mathematical modeling. We find that both 14-3-3 and TEAD binding modulate YAP1 shuttling, but neither affects nuclear import. Instead, we find that YAP1 serine phosphorylation is required for nuclear export. Furthermore, YAP1 nuclear accumulation in activated fibroblasts results from Src and actomyosin-dependent suppression of phosphorylated YAP1 export. Finally, we show that nuclear constrained YAP1, upon XPO1 depletion, remains sensitive to blockade of actomyosin function. Together, these data place nuclear export at the center of YAP1 regulation and indicate that the cytoskeleton can regulate YAP1 within the nucleus.

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability., Recent findings suggest that forces acting on the cell nucleus can cause DNA damage, but the mechanisms are unclear. Here Takaki et al. report that actomyosin is a determinant of nuclear shape and that unrestrained contractility elicits nuclear envelope rupture and genome instability in cancer cells.

Summary The transcriptional regulator YAP1 is critical for the pathological activation of fibroblasts. In normal fibroblasts, YAP1 is located in the cytoplasm, while in activated cancer-associated fibroblasts, it is nuclear and promotes the expression of genes required for pro-tumorigenic functions. Here, we investigate the dynamics of YAP1 shuttling in normal and activated fibroblasts, using EYFP-YAP1, quantitative photobleaching methods, and mathematical modeling. Imaging of migrating fibroblasts reveals the tight temporal coupling of cell shape change and altered YAP1 localization. Both 14-3-3 and TEAD binding modulate YAP1 shuttling, but neither affects nuclear import. Instead, we find that YAP1 nuclear accumulation in activated fibroblasts results from Src and actomyosin-dependent suppression of phosphorylated YAP1 export. Finally, we show that nuclear-constrained YAP1, upon XPO1 depletion, remains sensitive to blockade of actomyosin function. Together, these data place nuclear export at the center of YAP1 regulation and indicate that the cytoskeleton can regulate YAP1 within the nucleus., Graphical Abstract, Highlights • Photobleaching coupled with mathematical modeling identifies YAP1 dynamics • Regulation of nuclear export is key determinant of YAP1 localization • Serine phosphorylation is required for YAP1 nuclear export through XPO1 • Nuclear YAP1 remains sensitive to actin and Src-family kinase regulation, Ege et al. use quantitative analysis of photobleaching experiments to reveal that nuclear export is a convergent point of YAP1 regulation by actomyosin, Src-family kinases, and LATS1/2. Further, actomyosin and Src-family kinases additionally regulate YAP1 transcription competence via tyrosine phosphorylation.

Summary The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage., Graphical Abstract, Highlights • UV elicits elongation slowdown and restricts transcription to the 5′ end of genes • UV induces a switch from long to short alternative last exon (ALE) transcript isoforms • ASCC3 short and long ALE isoforms have antagonistic functions in the UV response • The UV-induced ASCC3 short isoform functions as a long non-coding RNA, UV damage generates a functional non-coding RNA through alternative pre-mRNA processing of a damage response factor transcript, identifying a pathway for repurposing protein coding genes under selective conditions.

Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation during cell division. Using functional genomic screening, we identify a set of 26 pre-mRNA splicing factors that are required for sister chromatid cohesion in human cells. Loss of spliceosome subunits increases the dissociation rate of cohesin from chromatin and abrogates cohesion after DNA replication, ultimately causing mitotic catastrophe. Depletion of splicing factors causes defective processing of the pre-mRNA encoding sororin, a factor required for the stable association of cohesin with chromatin, and an associated reduction of sororin protein level. Expression of an intronless version of sororin and depletion of the cohesin release protein WAPL suppress the cohesion defect in cells lacking splicing factors. We propose that spliceosome components contribute to sister chromatid cohesion and mitotic chromosome segregation through splicing of sororin pre-mRNA. Our results highlight the loss of cohesion as an early cellular consequence of compromised splicing. This may have clinical implications because SF3B1, a splicing factor that we identify to be essential for cohesion, is recurrently mutated in chronic lymphocytic leukaemia.

An important purpose of integrating computer use into everyday classroom instruction is to help students approach technology as a learning tool. Effective classroom integration is dependent not only on access to computers but also teachers' implementation of computing into learning. Successful implementation, in turn, depends largely on teachers' beliefs about classroom computing. The purpose of this study is to examine the effects of a teacher-focused technology intervention on students' attitudes toward and use of computers as learning tools. Teachers' attitudes, anxiety, and classroom computer use are explored as mediators of this relationship. Data were collected during a technology intervention in fourth and fifth grade classrooms in an urban public school district. Results suggest that the technology intervention itself had a positive effect on students' attitudes toward and use of computers for educational purposes. There was no evidence, however, that teachers' use or attitudes had any mediating effect on this relationship. These results suggest that it is possible to increase students' attitudes toward computer use through intense interventions aimed at their teachers. Future research should further investigate the mechanisms through which this relationship exits.

Summary Background Type 1 diabetes results from autoimmune targeting of the pancreatic β cells, likely mediated by effector memory T (Tem) cells. CD2, a T cell surface protein highly expressed on Tem cells, is targeted by the fusion protein alefacept, depleting Tem cells and central memory T (Tcm) cells. We postulated that alefacept would arrest autoimmunity and preserve residual β cells in patients newly diagnosed with type 1 diabetes. Methods The T1DAL study is a phase 2, double-blind, placebo-controlled trial in patients with type 1 diabetes, aged 12–35 years who, within 100 days of diagnosis, were enrolled at 14 US sites. Patients were randomly assigned (2:1) to receive alefacept (two 12-week courses of 15 mg intramuscularly per week, separated by a 12-week pause) or a placebo. Randomisation was stratified by site, and was computer-generated with permuted blocks of three patients per block. All participants and site personnel were masked to treatment assignment. The primary endpoint was the change from baseline in mean 2 h C-peptide area under the curve (AUC) at 12 months. Secondary endpoints at 12 months were the change from baseline in the 4 h C-peptide AUC, insulin use, major hypoglycaemic events, and HbA 1c concentrations. This trial is registered with ClinicalTrials.gov, number NCT00965458. Findings Of 73 patients assessed for eligibility, 33 were randomly assigned to receive alefacept and 16 to receive placebo. The mean 2 h C-peptide AUC at 12 months increased by 0·015 nmol/L (95% CI −0·080 to 0·110) in the alefacept group and decreased by 0·115 nmol/L (–0·278 to 0·047) in the placebo group, and the difference between groups was not significant (p=0·065). However, key secondary endpoints were met: the mean 4 h C-peptide AUC was significantly higher (mean increase of 0·015 nmol/L [95% CI −0·076 to 0·106] vs decrease of −0·156 nmol/L [–0·305 to −0·006]; p=0·019), and daily insulin use (0·48 units per kg per day for placebo vs 0·36 units per kg per day for alefacept; p=0·02) and the rate of hypoglycaemic events (mean of 10·9 events per person per year for alefacept vs 17·3 events for placebo; p 1c concentrations at week 52 were not different between treatment groups (p=0·75). So far, no serious adverse events were reported and all patients had at least one adverse event. In the alefacept group, 29 (88%) participants had an adverse event related to study drug versus 15 (94%) participants in the placebo group. In the alefacept group, 14 (42%) participants had grade 3 or 4 adverse events compared with nine (56%) participants in the placebo group; no deaths occurred. Interpretation Although the primary outcome was not met, at 12 months, alefacept preserved the 4 h C-peptide AUC, lowered insulin use, and reduced hypoglycaemic events, suggesting efficacy. Safety and tolerability were similar in the alefacept and placebo groups. Alefacept could be useful to preserve β-cell function in patients with new-onset type 1 diabetes. Funding US National Institutes of Health and the Juvenile Diabetes Research Foundation.

The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z' factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

The specification of tissue size during development involves the coordinated action of many signalling pathways responding to organ-intrinsic signals, such as morphogen gradients, and systemic cues, such as nutrient status. The conserved Hippo (Hpo) pathway, which promotes both cell cycle exit and apoptosis, is a major determinant of size control.The pathway core is a kinase cassette, comprising the kinases Hpo and Warts (Wts) and the scaffold proteins Salvador (Sav) and Mats, which inactivates the pro-growth transcriptional co-activator Yorkie (Yki). We performed a split TEV-based genome-wide RNAi screen for modulators of Hpo signalling. We characterise the Drosophila salt-inducible kinases (Sik2 and Sik3) as negative regulators of Hpo signalling. Activated Sik kinases increase Yki target expression and promote tissue overgrowth through phosphorylation of Sav at Ser413. Since Sik kinases have been implicated in nutrient sensing, this suggests a link between the Hippo pathway and systemic growth control.

Smad4 in partnership with R-Smads (receptor-regulated Smads) activates TGF-β (transforming growth factor-β)-dependent signalling pathways essential for early mouse development. Smad4 null embryos die shortly after implantation due to severe defects in cell proliferation and visceral endoderm differentiation. In the basal state, Smad4 undergoes continuous shuttling between the cytoplasm and the nucleus due to the combined activities of an N-terminal NLS (nuclear localization signal) and an NES (nuclear export signal) located in its linker region. Cell culture experiments suggest that Smad4 nucleocytoplasmic shuttling plays an important role in TGF-β signalling. In the present study we have investigated the role of Smad4 shuttling in vivo using gene targeting to engineer two independent mutations designed to eliminate Smad4 nuclear export. As predicted this results in increased levels of Smad4 in the nucleus of homozygous ES cells (embryonic stem cells) and primary keratinocytes, in the presence or absence of ligand. Neither mutation affects Smad4 expression levels nor its ability to mediate transcriptional activation in homozygous cell lines. Remarkably mouse mutants lacking the Smad4 NES develop normally. Smad4 NES mutants carrying one copy of a Smad4 null allele also fail to display developmental defects. The present study clearly demonstrates that Smad4 nucleocytoplasmic shuttling is not required for embryonic development or tissue homoeostasis in normal, healthy adult mice. Abbreviations: BMP, bone morphogenetic proteins; CRM1, chromosomal region maintenance 1; d.p.c., days post-coitum; ES cell, embryonic stem cell; FBS, foetal bovine serum; GDF, growth and differentiation factor; LMB, leptomycin B; MEF, murine embryonic fibroblast; MH domain, Mad homology domain; NES, nuclear export signal; NLS, nuclear localization signal; RPA, ribonuclease protection assay; R-Smad, receptor-regulated Smad; SnoN, Ski-related novel protein N; TGF-β, transforming growth factor-β

Human lysyl aminoacyl tRNA synthetase (hLysRS) is integral to a variety of different functions ranging from protein biosynthesis, initiation of a proinflammatory response as well as signal transduction. Another important, non-canonical function of hLysRS is that it chaperones tRNA(Lys,3), the HIV-1 reverse transcription primer molecule into new HIV-1 particles. Since the N-terminal domain of hLysRS has been shown to be essential for such primer uptake, NMR studies of this domain are being conducted to obtain a better understanding of how hLysRS interacts with the primer tRNA. In order to study the RNA binding behavior of this domain, we are studying its complex with a fragment of the cognate tRNA corresponding to the tRNA anticodon loop. We report herein the backbone and side chain NMR resonance assignments of uniformly (15)N-, (13)C-labeled hLysRS N-terminal domain alone, as well as complexed to RNA.

SummaryThe exocyst complex plays a critical role in targeting and tethering vesicles to specific sites of the plasma membrane. These events are crucial for polarized delivery of membrane components to the cell surface, which is critical for cell motility and division. Though Rho GTPases are involved in regulating actin dynamics and membrane trafficking, their role in exocyst-mediated vesicle targeting is not very clear. Herein, we present evidence that depletion of GEF-H1, a guanine nucleotide exchange factor for Rho proteins, affects vesicle trafficking. Interestingly, we found that GEF-H1 directly binds to exocyst component Sec5 in a Ral GTPase-dependent manner. This interaction promotes RhoA activation, which then regulates exocyst assembly/localization and exocytosis. Taken together, our work defines a mechanism for RhoA activation in response to RalA-Sec5 signaling and involvement of GEF-H1/RhoA pathway in the regulation of vesicle trafficking.

We examine poverty's effect in two ways. First, we study the relationship between poverty and capacity for innovation in the U.S. states; second, we study the combined effects of poverty and innovation capacity on U.S. state economic output and employment. Because many of the relationships among poverty, innovation capacity and economic performance are simultaneous, we employ the Arellano Bond Difference GMM estimator to estimate various models using panel data (1980–1999). The findings reveal a negative indirect effect of socio-economic need (poverty) on human and U.S. state and local financial innovation capacity, though there is no empirical link between poverty and federal financial capacity. We find no statistically significant evidence of the contemporaneous effect of poverty on state economic performance, holding innovation capacity constant. This suggests that poverty primarily affects state economic performance indirectly through reduction of innovation capacity. Overall, our findings suggest that U.S. officials ought to be concerned about the role poverty plays in diminishing their state economies' capacity to innovate.

Oncogenic mutations in RAS genes are very common in human cancer, resulting in cells with well-characterized selective advantages, but also less well-understood vulnerabilities. We have carried out a large-scale loss-of-function screen to identify genes that are required by KRAS-transformed colon cancer cells, but not by derivatives lacking this oncogene. Top-scoring genes were then tested in a larger panel of KRAS mutant and wild-type cancer cells. Cancer cells expressing oncogenic KRAS were found to be highly dependent on the transcription factor GATA2 and the DNA replication initiation regulator CDC6. Extending this analysis using a collection of drugs with known targets, we found that cancer cells with mutant KRAS showed selective addiction to proteasome function, as well as synthetic lethality with topoisomerase inhibition. Combination targeting of these functions caused improved killing of KRAS mutant cells relative to wild-type cells. These observations suggest novel targets and new ways of combining existing therapies for optimal effect in RAS mutant cancers, which are traditionally seen as being highly refractory to therapy.

Alterations in metabolic activity contribute to the proliferation and survival of cancer cells. We investigated the effect of siRNA-mediated gene silencing of 222 metabolic enzymes, transporters, and regulators on the survival of 3 metastatic prostate cancer cell lines and a nonmalignant prostate epithelial cell line. This approach revealed significant complexity in the metabolic requirements of prostate cancer cells and identified several genes selectively required for their survival. Among these genes was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), an isoform of phosphofructokinase 2 (PFK2). We show that PFKFB4 is required to balance glycolytic activity and antioxidant production to maintain cellular redox balance in prostate cancer cells. Depletion of PFKFB4 inhibited tumor growth in a xenograft model, indicating that it is required under physiologic nutrient levels. PFKFB4 mRNA expression was also found to be greater in metastatic prostate cancer compared with primary tumors. Taken together, these results indicate that PFKFB4 is a potential target for the development of antineoplastic agents. Significance: Cancer cells undergo several changes in their metabolism that promote growth and survival. Using an unbiased functional screen, we found that the glycolytic enzyme PFKFB4 is essential for prostate cancer cell survival by maintaining the balance between the use of glucose for energy generation and the synthesis of antioxidants. Targeting PFKFB4 may therefore present new therapeutic opportunities. Cancer Discov; 2(4); 328–43. ©2012 AACR. Read the Commentary on this article by Dang, p. 304 This article is highlighted in the In This Issue feature, 288

Autophagy is a catabolic process by which cytoplasmic components are sequestered and transported by autophagosomes to lysosomes for degradation, enabling recycling of these components and providing cells with amino acids during starvation. It is a highly regulated process and its deregulation contributes to multiple diseases. Despite its importance in cell homeostasis, autophagy is not fully understood. To find new proteins that modulate starvation-induced autophagy, we performed a genome-wide siRNA screen in a stable human cell line expressing GFP–LC3, the marker-protein for autophagosomes. Using stringent validation criteria, our screen identified nine novel autophagy regulators. Among the hits required for autophagosome formation are SCOC (short coiled-coil protein), a Golgi protein, which interacts with fasciculation and elongation protein zeta 1 (FEZ1), an ULK1-binding protein. SCOC forms a starvation-sensitive trimeric complex with UVRAG (UV radiation resistance associated gene) and FEZ1 and may regulate ULK1 and Beclin 1 complex activities. A second candidate WAC is required for starvation-induced autophagy but also acts as a potential negative regulator of the ubiquitin-proteasome system. The identification of these novel regulatory proteins with diverse functions in autophagy contributes towards a fuller understanding of autophagosome formation.