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A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell 1 . For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP4 2 , 3 . Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an ‘upright’ to a ‘reversed’ conformation. The reversed structure projects a previously buried ‘foot’ domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses. Electron cryomicroscopy and cryotomography studies reveal that rotaviruses attach to a target cell through the outer-layer protein VP4, which—following cleavage—rearranges to enable perforation of the membrane and delivery of the viral genome into the host cell.

Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex--for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼ 700 Å inner capsid particle (\"double-layered particle\", DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼ 10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.

Metal coordination is a key structural and functional component of a large fraction of proteins. Given this dual role we considered the possibility that metal coordination may have played a templating role in the early evolution of protein folds and complexes. We describe here a rational design approach, Metal Templated Interface Redesign (MeTIR), that mimics the time course of a hypothetical evolutionary pathway for the formation of stable protein assemblies through an initial metal coordination event. Using a folded monomeric protein, cytochrome cb 562 , as a building block we show that its non-self-associating surface can be made self-associating through a minimal number of mutations that enable Zn coordination. The protein interfaces in the resulting Zn-directed, D 2 -symmetrical tetramer are subsequently redesigned, yielding unique protein architectures that self-assemble in the presence or absence of metals. Aside from its evolutionary implications, MeTIR provides a route to engineer de novo protein interfaces and metal coordination environments that can be tuned through the extensive noncovalent bonding interactions in these interfaces.

Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex--for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ~700 Å inner capsid particle (\"double-layered particle\", DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ~10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.

Rotaviruses, like other non-enveloped, double-strand RNA (dsRNA) viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the \"double-layer particle\" or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each fivefold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual fivefold positions. We have analyzed intact virions (\"triple-layer particles\" or TLPs), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg2+, and S-adenosyl methionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, dsRNA genome.

The de novo design of protein-protein interactions (PPIs) has proven to be an immense challenge due to the difficulty in controlling and predicting the weak forces that govern them. In order to circumvent this challenge, we set out to induce new PPIs between monomeric proteins through the coordination of metal ions. In our strategy, the strength of the metal bonding interactions should be sufficient to drive the formation of PPIs without initial consideration of the weak non-covalent interactions along the newly formed interfaces. Additionally, the distinct geometric preferences of metal ions should only allow for a limited number of organizations of proteins around a given metal center, thus permitting a degree of foresight into the oligomeric architectures that will be formed. As a model system, we chose a stable, four-helix bundle protein, cytochrome cb562, onto which two i and i+4 bis-histidine \"clamp\" coordination motifs were installed. The resulting metal-binding construct, MBPC-1, is shown to be capable of forming discrete oligomeric species whose supramolecular architectures depend on the stereochemical preferences of the added metal. Further studies to exert control over the morphology of a Zn-mediated tetrameric assembly of MBPC-1 (Zn4:MBPC-14) highlighted the fact that, although we initially ignored secondary, non-covalent interactions along our newly induced interfaces, these interactions do play a role in determining the overall structure of the complex. This idea led us to computationally \"evolve\" our protein by re-designing residues along interfaces of the Zn4:MBPC-14 tetramer to include more favorable hydrophobic packing interactions. The resulting constructs were shown to not only form a significantly stabilized tetramer, but also to have the ability to form oligomers in the absence of metals. Further stabilization of this tetrameric architecture by way of chemical crosslinking has allowed for the formation of a potentially metal-selective complex that can serve as a stable platform onto which functionalized metal centers, and in turn enzymatic activity, may be engineered in the future.

Aim: Tropical forests account for a quarter of the global carbon storage and a third of the terrestrial productivity. Few studies have teased apart the relative importance of environmental factors and forest attributes for ecosystem functioning, especially for the tropics. This study aims to relate aboveground biomass (AGB) and biomass dynamics (i.e., net biomass productivity and its underlying demographic drivers: biomass recruitment, growth and mortality) to forest attributes (tree diversity, community-mean traits and stand basal area) and environmental conditions (water availability, soil fertility and disturbance). Location: Neotropics. Methods: We used data from 26 sites, 201 1-ha plots and >92,000 trees distributed across the Neotropics. We quantified for each site water availability and soil total exchangeable bases and for each plot three key community-weighted mean functional traits that are important for biomass stocks and productivity. We used structural equation models to test the hypothesis that all drivers have independent, positive effects on biomass stocks and dynamics. Results: Of the relationships analysed, vegetation attributes were more frequently associated significantly with biomass stocks and dynamics than environmental conditions (in 67 vs. 33% of the relationships). High climatic water availability increased biomass growth and stocks, light disturbance increased biomass growth, and soil bases had no effect. Rarefied tree species richness had consistent positive relationships with biomass stocks and dynamics, probably because of niche complementarity, but was not related to net biomass productivity. Community-mean traits were good predictors of biomass stocks and dynamics. Main conclusions: Water availability has a strong positive effect on biomass stocks and growth, and a future predicted increase in (atmospheric) drought might, therefore, potentially reduce carbon storage. Forest attributes, including species diversity and community-weighted mean traits, have independent and important relationships with AGB stocks, dynamics and ecosystem functioning, not only in relatively simple temperate systems, but also in structurally complex hyper-diverse tropical forests.

Anthracycline–taxane chemotherapy for early-stage breast cancer substantially improves survival compared with no chemotherapy. However, concerns about short-term and long-term side-effects of anthracyclines have led to increased use of taxane chemotherapy without anthracycline, which could compromise efficacy. We aimed to better characterise the benefits and risks of including anthracycline, and the comparative benefits of different anthracycline–taxane regimens. We did an individual patient-level meta-analysis of randomised trials comparing taxane regimens with versus without anthracycline, and updated our previous meta-analysis of anthracycline regimens with versus without taxane, as well as analysing 44 trials in six related comparisons. We searched databases, including MEDLINE, Embase, the Cochrane Library, and meeting abstracts to identify trials assessing anthracycline and taxane chemotherapy. Adjuvant or neoadjuvant trials were eligible if they began before Jan 1, 2012. Primary outcomes were breast cancer recurrence and cause-specific mortality. Log-rank analyses yielded first-event rate ratios (RRs) and CIs. 28 trials of taxane regimens with or without anthracycline were identified, of which 23 were deemed eligible, and 15 provided data on 18 103 women. Across all 15 trials that provided individual data, recurrence rates were 14% lower on average (RR 0·86, 95% CI 0·79–0·93; p=0·0004) with taxane regimens including anthracycline than those without. Non-breast cancer deaths were not increased but there was one additional acute myeloid leukaemia case per 700 women treated. The clearest reductions in recurrence were found when anthracycline was added concurrently to docetaxel plus cyclophosphamide versus the same dose of docetaxel plus cyclophosphamide (10-year recurrence risk 12·3% vs 21·0%; risk difference 8·7%, 95% CI 4·5–12·9; RR 0·58, 0·47–0·73; p<0·0001). 10-year breast cancer mortality in this group was reduced by 4·2% (0·4–8·1; p=0·0034). No significant reduction in recurrence risk was found for sequential schedules of taxane plus anthracycline when compared with docetaxel plus cyclophosphamide (RR 0·94, 0·83–1·06; p=0·30). For the analysis of anthracycline regimens with versus without taxane, 35 trials (n=52 976) provided individual patient data. Larger recurrence reductions were seen from adding taxane to anthracycline regimens when the cumulative dose of anthracycline was the same in each group (RR 0·87, 0·82–0·93; p<0·0001; n=11 167) than in trials with two-fold higher cumulative doses of non-taxane (mostly anthracycline) in the control group than in the taxane group (RR 0·96, 0·90–1·03; p=0·27; n=14 620). Direct comparisons between anthracycline and taxane regimens showed that a higher cumulative dose and more dose-intense schedules were more efficacious. The proportional reductions in recurrence for taxane plus anthracycline were similar in oestrogen receptor-positive and oestrogen receptor-negative disease, and did not differ by age, nodal status, or tumour size or grade. Anthracycline plus taxane regimens are most efficacious at reducing breast cancer recurrence and death. Regimens with higher cumulative doses of anthracycline plus taxane provide the greatest benefits, challenging the current trend in clinical practice and guidelines towards non-anthracycline chemotherapy, particularly shorter regimens, such as four cycles of docetaxel–cyclophosphamide. By bringing together data from almost all relevant trials, this meta-analysis provides a reliable evidence base to inform individual treatment decisions, clinical guidelines, and the design of future clinical trials. Cancer Research UK, UK Medical Research Council.

Incidence rates of colorectal cancer vary geographically and have changed over time 1 . Notably, in the past two decades, the incidence of early-onset colorectal cancer, which affects individuals below 50 years of age, has doubled in many countries 2 , 3 , 4 – 5 . The reasons for this increase are unknown. Here we investigate whether mutational processes contribute to geographic and age-related differences by examining 981 colorectal cancer genomes from 11 countries. No major differences were found in microsatellite-unstable cancers, but variations in mutation burden and signatures were observed in the 802 microsatellite-stable cases. Multiple signatures, most with unknown aetiologies, exhibited varying prevalence in Argentina, Brazil, Colombia, Russia and Thailand, indicating geographically diverse levels of mutagenic exposure. Signatures SBS88 and ID18, caused by the bacteria-produced mutagen colibactin 6 , 7 , had higher mutation loads in countries with higher colorectal cancer incidence rates. SBS88 and ID18 were also enriched in early-onset colorectal cancers, being 3.3 times more common in individuals who were diagnosed before 40 years of age than in those over 70 years of age, and were imprinted early during colorectal cancer development. Colibactin exposure was further linked to APC driver mutations, with ID18 being responsible for about 25% of APC driver indels in colibactin-positive cases. This study reveals geographic and age-related variations in colorectal cancer mutational processes, and suggests that mutagenic exposure to colibactin-producing bacteria in early life may contribute to the increasing incidence of early-onset colorectal cancer. Analysis of genomic data from 981 colorectal cancers from participants in 11 countries reveals variations in mutational signatures of microsatellite-stable cancers that are dependent on geographical origin and age at which the cancer was diagnosed.

In the randomized, phase 3 KEYNOTE-119 study, overall survival (OS) was not significantly improved with pembrolizumab 200 mg Q3W versus investigator’s choice of chemotherapy in participants with previously treated metastatic TNBC. In this exploratory analysis, we evaluated associations of tumor-infiltrating lymphocytes (TILs), T-cell‒inflamed gene expression profile (Tcell inf GEP), BRCA1 / BRCA2 mutation (BRCAm) status, homologous recombination deficiency (HRD) status, and tumor mutational burden (TMB) with clinical outcomes. TIL level was associated with improved objective response rate (ORR), progression-free survival (PFS), and OS with pembrolizumab but not with chemotherapy or after adjusting for Tcell inf GEP. Associations were also identified between Tcell inf GEP and improved ORR, PFS, and OS with pembrolizumab. Participants with TMB ≥ 10 mut/Mb showed a trend toward increased benefit with pembrolizumab versus chemotherapy. No association was seen between BRCAm/HRD status and treatment response. These findings suggest a positive association between TILs, Tcell inf GEP, and TMB with clinical outcomes in patients with metastatic TNBC receiving pembrolizumab. ClinicalTrials.gov Identifier: NCT02555657 (date of registration: September 18, 2015).