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In this work, we study the first passage statistics of amino acid primary sequences, that is the probability of observing an amino acid for the first time at a certain number of residues away from a fixed amino acid. By using this rich mathematical framework, we are able to capture the background distribution for an organism, and infer lengths at which the first passage has a probability that differs from what is expected. While many features of an organism's genome are due to natural selection, others are related to amino acid chemistry and the environment in which an organism lives, constraining the randomness of genomes upon which selection can further act. We therefore use this approach to infer amino acid correlations, and then study how these correlations vary across a wide range of organisms under a wide range of optimal growth temperatures. We find a nearly universal exponential background distribution, consistent with the idea that most amino acids are globally uncorrelated from other amino acids in genomes. When we are able to extract significant correlations, these correlations are reliably dependent on optimal growth temperature, across phylogenetic boundaries. Some of the correlations we extract, such as the enhanced probability of finding, for the first time, a cysteine three residues away from a cysteine or glutamic acid two residues away from an arginine, likely relate to thermal stability. However, other correlations, likely appearing on alpha helical surfaces, have a less clear physiochemical interpretation and may relate to thermal stability or unusual metabolic properties of organisms that live in a high temperature environment.

Segregation of genetic material occurs when chromosomes move to opposite spindle poles during mitosis. This movement depends on K-fibers, specialized microtubule (MT) bundles attached to the chromosomes' kinetochores. A long-standing assumption is that continuous K-fibers connect every kinetochore to a spindle pole and the force for chromosome movement is produced at the kinetochore and coupled with MT depolymerization. However, we found that chromosomes still maintained their position at the spindle equator during metaphase and segregated properly during anaphase when one of their K-fibers was severed near the kinetochore with a laser microbeam. We also found that, in normal fully assembled spindles, K-fibers of some chromosomes did not extend to the spindle pole. These K-fibers connected to adjacent K-fibers and/or nonkinetochore MTs. Poleward movement of chromosomes with short K-fibers was uncoupled from MT depolymerization at the kinetochore. Instead, these chromosomes moved by dynein-mediated transport of the entire K-fiber/kinetochore assembly. Thus, at least two distinct parallel mechanisms drive chromosome segregation in mammalian cells.

Most sounds of interest consist of complex, time-dependent admixtures of tones of diverse frequencies and variable amplitudes. To detect and process these signals, the ear employs a highly nonlinear, adaptive, real-time spectral analyzer: the cochlea. Sound excites vibration of the eardrum and the three miniscule bones of the middle ear, the last of which acts as a piston to initiate oscillatory pressure changes within the liquid-filled chambers of the cochlea. The basilar membrane, an elastic band spiraling along the cochlea between two of these chambers, responds to these pressures by conducting a largely independent traveling wave for each frequency component of the input. Because the basilar membrane is graded in mass and stiffness along its length, however, each traveling wave grows in magnitude and decreases in wavelength until it peaks at a specific, frequency-dependent position: low frequencies propagate to the cochlear apex, whereas high frequencies culminate at the base. The oscillations of the basilar membrane deflect hair bundles, the mechanically sensitive organelles of the ear's sensory receptors, the hair cells. As mechanically sensitive ion channels open and close, each hair cell responds with an electrical signal that is chemically transmitted to an afferent nerve fiber and thence into the brain. In addition to transducing mechanical inputs, hair cells amplify them by two means. Channel gating endows a hair bundle with negative stiffness, an instability that interacts with the motor protein myosin-1c to produce a mechanical amplifier and oscillator. Acting through the piezoelectric membrane protein prestin, electrical responses also cause outer hair cells to elongate and shorten, thus pumping energy into the basilar membrane's movements. The two forms of motility constitute an active process that amplifies mechanical inputs, sharpens frequency discrimination, and confers a compressive nonlinearity on responsiveness. These features arise because the active process operates near a Hopf bifurcation, the generic properties of which explain several key features of hearing. Moreover, when the gain of the active process rises sufficiently in ultraquiet circumstances, the system traverses the bifurcation and even a normal ear actually emits sound. The remarkable properties of hearing thus stem from the propagation of traveling waves on a nonlinear and excitable medium.

Background: Alpha-hemolysin (Hla) is a major virulence factor in the pathogenesis of Staphylococcus aureus infection, being active against a wide range of host cells. Although hla is ubiquitous in S. aureus, its genetic diversity and variation in expression in different genetic backgrounds is not known. We evaluated nucleotide sequence variation and gene expression profiles of hla among representatives of hospital (HA) and community-associated (CA) S. aureus clones. Methods: 51 methicillin-resistant S. aureus and 22 methicillin-susceptible S. aureus were characterized by PFGE, spa typing, MLST and SCCmec typing. The internal regions of hla and the hla promoter were sequenced and gene expression was assessed by RT-PCR. Results: Alpha-hemolysin encoding- and promoter sequences were diverse, with 12 and 23 different alleles, respectively. Based on phylogenetic analysis, we suggest that hla may have evolved together with the S. aureus genetic background, except for ST22, ST121, ST59 and ST93. Conversely, the promoter region showed lack of co-evolution with the genetic backgrounds. Four non-synonymous amino acid changes were identified close to important regions of hla activity. Amino acid changes in the RNAIII binding site were not associated to hla expression. Although expression rates of hla were in general strain-specific, we observed CA clones showed significantly higher hla expression (p = 0.003) when compared with HA clones. Conclusion: We propose that the hla gene has evolved together with the genetic background. Overall, CA genetic backgrounds showed higher levels of hla expression than HA, and a high strain-to-strain variation of gene expression was detected in closely related strains.

In mammalian cells, tumor suppressor p53 plays critical roles in the regulation of glucose metabolism, including glycolysis and oxidative phosphorylation, but whether and how p53 also regulates gluconeogenesis is less clear. Here, we report that p53 efficiently down-regulates the expression of phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), which encode rate-limiting enzymes in gluconeogenesis. Cell-based assays demonstrate the p53-dependent nuclear exclusion of forkhead box protein O1 (FoxO1), a key transcription factor that mediates activation of PCK1 and G6PC, with consequent alleviation of FoxO1-dependent gluconeogenesis. Further mechanistic studies show that p53 directly activates expression of the NAD+-dependent histone deacetylase sirtuin 6 (SIRT6), whose interaction with FoxO1 leads to FoxO1 deacetylation and export to the cytoplasm. In support of these observations, p53-mediated FoxO1 nuclear exclusion, down-regulation of PCK1 and G6PC expression, and regulation of glucose levels were confirmed in C57BL/J6 mice and in liver-specific Sirt6 conditional knockout mice. Our results provide insights into mechanisms of metabolism-related p53 functions that may be relevant to tumor suppression.

Myxovirus resistance 2 (Mx2/MxB) has recently been uncovered as an effector of the anti-HIV-1 activity of type I interferons (IFNs) that inhibits HIV-1 at an early stage postinfection, after reverse transcription but prior to proviral integration into host DNA. The mechanistic details of Mx2 antiviral activity are not yet understood, but a few substitutions in the HIV-1 capsid have been shown to confer resistance to Mx2. Through a combination of in vitro evolution and unbiased mutagenesis, we further map the determinants of sensitivity to Mx2 and reveal that multiple capsid (CA) surfaces define sensitivity to Mx2. Intriguingly, we reveal an unanticipated sensitivity determinant within the C-terminal domain of capsid. We also report that Mx2s derived from multiple primate species share the capacity to potently inhibit HIV-1, whereas selected nonprimate orthologs have no such activity. Like TRIM5 alpha, another CA targeting antiretroviral protein, primate Mx2s exhibit species-dependent variation in antiviral specificity against at least one extant virus and multiple HIV-1 capsid mutants. Using a combination of chimeric Mx2 proteins and evolution-guided approaches, we reveal that a single residue close to the N terminus that has evolved under positive selection can determine antiviral specificity. Thus, the variable N-terminal region can define the spectrum of viruses inhibited by Mx2. IMPORTANCE Type I interferons (IFNs) inhibit the replication of most mammalian viruses. IFN stimulation upregulates hundreds of different IFN-stimulated genes (ISGs), but it is often unclear which ISGs are responsible for inhibition of a given virus. Recently, Mx2 was identified as an ISG that contributes to the inhibition of HIV-1 replication by type I IFN. Thus, Mx2 might inhibit HIV-1 replication in patients, and this inhibitory action might have therapeutic potential. The mechanistic details of how Mx2 inhibits HIV-1 are currently unclear, but the HIV-1 capsid protein is the likely viral target. Here, we determine the regions of capsid that specify sensitivity to Mx2. We demonstrate that Mx2 from multiple primates can inhibit HIV-1, whereas Mx2 from other mammals (dogs and sheep) cannot. We also show that primate variants of Mx2 differ in the spectrum of lentiviruses they inhibit and that a single residue in Mx2 can determine this antiviral specificity.

Background: DNA barcode differences within animal species are usually much less than differences among species, making it generally straightforward to match unknowns to a reference library. Here we aim to better understand the evolutionary mechanisms underlying this usual "barcode gap'' pattern. We employ avian barcode libraries to test a central prediction of neutral theory, namely, intraspecific variation equals 2 N mu, where N is population size and mu is mutations per site per generation. Birds are uniquely suited for this task: they have the best-known species limits, are well represented in barcode libraries, and, most critically, are the only large group with documented census population sizes. In addition, we ask if mitochondrial molecular clock measurements conform to neutral theory prediction of clock rate equals mu. Results: Intraspecific COI barcode variation was uniformly low regardless of census population size (n = 142 species in 15 families). Apparent outliers reflected lumping of reproductively isolated populations or hybrid lineages. Re-analysis of a published survey of cytochrome b variation in diverse birds (n = 93 species in 39 families) further confirmed uniformly low intraspecific variation. Hybridization/gene flow among species/populations was the main limitation to DNA barcode identification. Conclusions/Significance: To our knowledge, this is the first large study of animal mitochondrial diversity using actual census population sizes and the first to test outliers for population structure. Our finding of universally low intraspecific variation contradicts a central prediction of neutral theory and is not readily accounted for by commonly proposed ad hoc modifications. We argue that the weight of evidence-low intraspecific variation and the molecular clock-indicates neutral evolution plays a minor role in mitochondrial sequence evolution. As an alternate paradigm consistent with empirical data, we propose extreme purifying selection, including at synonymous sites, limits variation within species and continuous adaptive selection drives the molecular clock.

Themultidrug-resistant Streptococcus pneumoniae Taiwan (19F)-14, or PMEN14, clone was first observed with a 19F serotype, which is targeted by the heptavalent polysaccharide conjugate vaccine (PCV7). However, "vaccine escape" PMEN14 isolates with a 19A serotype became an increasingly important cause of disease post-PCV7. Whole genome sequencing was used to characterize the recent evolution of 173 pneumococci of, or related to, PMEN14. This suggested that PMEN14 is a single lineage that originated in the late 1980s in parallel with the acquisition of multiple resistances by close relatives. One of the four detected serotype switches to 19A generated representatives of the sequence type (ST) 320 isolates that have been highly successful post-PCV7. A second produced an ST236 19A genotype with reduced resistance to beta-lactams owing to alteration of pbp1a and pbp2x sequences through the same recombination that caused the change in serotype. A third, which generated a mosaic capsule biosynthesis locus, resulted in serotype 19A ST271 isolates. The rapid diversification through homologous recombination seen in the global collection was similarly observed in the absence of vaccination in a set of isolates from the Maela refugee camp in Thailand, a collection that also allowed variation to be observed within carriage through longitudinal sampling. This suggests that some pneumococcal genotypes generate a pool of standing variation that is sufficiently extensive to result in "soft" selective sweeps: The emergence of multiple mutants in parallel upon a change in selection pressure, such as vaccine introduction. The subsequent competition between these mutants makes this phenomenon difficult to detect without deep sampling of individual lineages.

BACKGROUND Dupilumab, a fully human monoclonal antibody that blocks interleukin-4 and interleukin-13, has shown efficacy in patients with asthma and elevated eosinophil levels. The blockade by dupilumab of these key drivers of type 2 helper T-cell (Th2)-mediated inflammation could help in the treatment of related diseases, including atopic dermatitis. METHODS We performed randomized, double-blind, placebo-controlled trials involving adults who had moderate-to-severe atopic dermatitis despite treatment with topical glucocorticoids and calcineurin inhibitors. Dupilumab was evaluated as monotherapy in two 4-week trials and in one 12-week trial and in combination with topical glucocorticoids in another 4-week study. End points included the Eczema Area and Severity Index (EASI) score, the investigator's global assessment score, pruritus, safety assessments, serum biomarker levels, and disease transcriptome. RESULTS In the 4-week monotherapy studies, dupilumab resulted in rapid and dose-dependent improvements in clinical indexes, biomarker levels, and the transcriptome. The results of the 12-week study of dupilumab monotherapy reproduced and extended the 4-week findings: 85% of patients in the dupilumab group, as compared with 35% of those in the placebo group, had a 50% reduction in the EASI score (EASI-50, with higher scores in the EASI indicating greater severity of eczema) (P<0.001); 40% of patients in the dupilumab group, as compared with 7% in the placebo group, had a score of 0 to 1 (indicating clearing or near-clearing of skin lesions) on the investigator's global assessment (P<0.001); and pruritus scores decreased (indicating a reduction in itch) by 55.7% in the dupilumab group versus 15.1% in the placebo group (P<0.001). In the combination study, 100% of the patients in the dupilumab group, as compared with 50% of those who received topical glucocorticoids with placebo injection, met the criterion for EASI-50 (P = 0.002), despite the fact that patients who received dupilumab plus glucocorticoids used less than half the amount of topical glucocorticoids used by those who received placebo plus the topical medication (P = 0.16). Adverse events, such as skin infection, occurred more frequently with placebo; nasopharyngitis and headache were the most frequent adverse events with dupilumab. CONCLUSIONS Patients treated with dupilumab had marked and rapid improvement in all the evaluated measures of atopic dermatitis disease activity. Side-effect profiles were not doselimiting.

Objective Hepatitis C virus (HCV) infection causes severe liver disease and affects more than 160 million individuals worldwide. People undergoing liver organ transplantation face universal re-infection of the graft. Therefore, affordable antiviral strategies targeting the early stages of infection are urgently needed to prevent the recurrence of HCV infection. The aim of the study was to determine the potency of turmeric curcumin as an HCV entry inhibitor. Design The antiviral activity of curcumin and its derivatives was evaluated using HCV pseudo-particles (HCVpp) and cell-culture-derived HCV (HCVcc) in hepatoma cell lines and primary human hepatocytes. The mechanism of action was dissected using R18-labelled virions and a membrane fluidity assay. Results Curcumin treatment had no effect on HCV RNA replication or viral assembly/release. However, co-incubation of HCV with curcumin potently inhibited entry of all major HCV genotypes. Similar antiviral activities were also exerted by other curcumin derivatives but not by tetrahydrocurcumin, suggesting the importance of alpha,beta-unsaturated ketone groups for the antiviral activity. Expression levels of known HCV receptors were unaltered, while pretreating the virus with the compound reduced viral infectivity without viral lysis. Membrane fluidity experiments indicated that curcumin affected the fluidity of the HCV envelope resulting in impairment of viral binding and fusion. Curcumin has also been found to inhibit cell-to-cell transmission and to be effective in combination with other antiviral agents. Conclusions Turmeric curcumin inhibits HCV entry independently of the genotype and in primary human hepatocytes by affecting membrane fluidity thereby impairing virus binding and fusion.