Chemical dissection of the cell cycle: probes for cell biology and anti-cancer drug development.

Cancer cell proliferation relies on the ability of cancer cells to grow, transition through the cell cycle, and divide. To identify novel chemical probes for dissecting the mechanisms governing cell cycle progression and cell division, and for developing new anti-cancer therapeutics, we developed and performed a novel cancer cell-based high-throughput chemical screen for cell cycle modulators. This approach identified novel G1, S, G2, and M-phase specific inhibitors with drug-like properties and diverse chemotypes likely targeting a broad array of processes. We further characterized the M-phase inhibitors and highlight the most potent M-phase inhibitor MI-181, which targets tubulin, inhibits tubulin polymerization, activates the spindle assembly checkpoint, arrests cells in mitosis, and triggers a fast apoptotic cell death. Importantly, MI-181 has broad anti-cancer activity, especially against BRAF(V600E) melanomas

Structure of the dynein-2 complex and its assembly with intraflagellar transport trains

Dynein-2 assembles with polymeric intraflagellar transport (IFT) trains to form a transport machinery crucial for cilia biogenesis and signaling. Here we recombinantly expressed the ~1.4 MDa human dynein-2 complex and solved its cryo-EM structure to near-atomic resolution. The two identical copies of the dynein-2 heavy chain are contorted into different conformations by a WDR60-WDR34 heterodimer and a block of two RB and six LC8 light chains. One heavy chain is steered into a zig-zag, which matches the periodicity of the anterograde IFT-B train. Contacts between adjacent dyneins along the train indicate a cooperative mode of assembly. Removal of the WDR60-WDR34-light chain subcomplex renders dynein-2 monomeric and relieves auto-inhibition of its motility. Our results converge on a model in which an unusual stoichiometry of non-motor subunits control dynein-2 assembly, asymmetry, and activity, giving mechanistic insight into dynein-2’s interaction with IFT trains and the origin of diverse functions in the dynein family

TRIM E3 Ubiquitin Ligases in Rare Genetic Disorders

The TRIM family comprises proteins characterized by the presence of the tripartite motif composed of a RING domain, one or two B-box domains and a coiled-coil region. The TRIM shared domain structure underscores a common biochemical function as E3 ligase within the ubiquitination cascade. The TRIM proteins represent one of the largest E3 ligase families counting in human more than 70 members. These proteins are implicated in a plethora of cellular processes such as apoptosis, cell cycle regulation, muscular physiology, and innate immune response. Consistently, their alteration results in several pathological conditions emphasizing their medical relevance. Here, the genetic and pathogenetic mechanisms of rare disorders directly caused by mutations in TRIM genes will be reviewed. These diseases fall into different pathological areas, from malformation birth defects due to developmental abnormalities, to neurological disorders and progressive teenage neuromuscular disorders. In many instances, TRIM E3 ligases act on several substrates thus exerting pleiotropic activities: the need of unraveling disease-specific TRIM pathways for a precise targeting therapy avoiding dramatic side effects will be discussed

10-year stroke prevention after successful carotidendarterectomy for asymptomatic stenosis (ACST-1):a multicentre randomised trial

Backgroun: If carotid artery narrowing remains asymptomatic (ie, has caused no recent stroke or other neurological symptoms), successful carotid endarterectomy (CEA) reduces stroke incidence for some years. We assessed the longterm effects of successful CEA. Methods Between 1993 and 2003, 3120 asymptomatic patients from 126 centres in 30 countries were allocated equally, by blinded minimised randomisation, to immediate CEA (median delay 1 month, IQR 0·3–2·5) or to indefi nite deferral of any carotid procedure, and were followed up until death or for a median among survivors of 9 years (IQR 6–11). The primary outcomes were perioperative mortality and morbidity (death or stroke within 30 days) and non-perioperative stroke. Kaplan-Meier percentages and logrank p values are from intention-to-treat analyses. This study is registered, number ISRCTN26156392. Findings 1560 patients were allocated immediate CEA versus 1560 allocated deferral of any carotid procedure. The proportions operated on while still asymptomatic were 89·7% versus 4·8% at 1 year (and 92·1% vs 16·5% at 5 years). Perioperative risk of stroke or death within 30 days was 3·0% (95% CI 2·4–3·9; 26 non-disabling strokes plus 34 disabling or fatal perioperative events in 1979 CEAs). Excluding perioperative events and non-stroke mortality, stroke risks (immediate vs deferred CEA) were 4·1% versus 10·0% at 5 years (gain 5·9%, 95% CI 4·0–7·8) and 10·8% versus 16·9% at 10 years (gain 6·1%, 2·7–9·4); ratio of stroke incidence rates 0·54, 95% CI 0·43–0·68, p<0·0001. 62 versus 104 had a disabling or fatal stroke, and 37 versus 84 others had a non-disabling stroke. Combining perioperative events and strokes, net risks were 6·9% versus 10·9% at 5 years (gain 4·1%, 2·0–6·2) and 13·4% versus 17·9% at 10 years (gain 4·6%, 1·2–7·9). Medication was similar in both groups; throughout the study, most were on antithrombotic and antihypertensive therapy. Net benefi ts were signifi cant both for those on lipid-lowering therapy and for those not, and both for men and for women up to 75 years of age at entry (although not for older patients). Interpretation Successful CEA for asymptomatic patients younger than 75 years of age reduces 10-year stroke risks. Half this reduction is in disabling or fatal strokes. Net benefit in future patients will depend on their risks from unoperated carotid lesions (which will be reduced by medication), on future surgical risks (which might differ from those in trials), and on whether life expectancy exceeds 10 years