Ontogeny of hemoglobin­oxygen binding and multiplicity in the obligate air-breathing fish Arapaima gigas.

The evolutionary and ontogenetic changes from water- to air-breathing result in major changes in the cardiorespiratory systems. However, the potential changes in hemoglobin's (Hb) oxygen binding properties during ontogenetic transitions to air-breathing remain poorly understood. Here we investigated Hb multiplicity and O2 binding in hemolysates and Hb components from juveniles and adults of the obligate air-breathing pirarucu (Arapaima gigas) that starts life as water-breathing hatchlings. Contrasting with previous electrophoresis studies that report one or two isoHbs in adults, isoelectric focusing (IEF) resolved the hemolysates from both stages into four major bands, which exhibited identical O2 binding properties (i.e. O2 affinities, cooperativity coefficients, and sensitivities to pH and the major organic phosphate effectors), also as compared to the cofactor-free hemolysates. Of note, the multiplicity pattern recurred upon reanalyses of the most-abundant fractions isolated from the juvenile and the adult stages, suggesting possible stabilization of different quaternary states with different isoelectric points during the purification procedure. The study demonstrates unchanged Hb-O2 binding properties during development, despite the pronounced differences in O2 availability between the two media, which harmonizes with findings based on a broader spectrum of interspecific comparisons. Taken together, these results disclose that obligate air-breathing in Arapaima is not contingent upon changes in Hb multiplicity and O2 binding characteristics.

Effect of NH2-terminal acetylation on the oxygenation properties of vertebrate haemoglobin.

In vertebrate haemoglobin (Hb), the NH2-terminal residues of the α- and ß-chain subunits are thought to play an important role in the allosteric binding of protons (Bohr effect), CO2 (as carbamino derivatives), chloride ions, and organic phosphates. Accordingly, acetylation of the α- and/or ß-chain NH2-termini may have significant effects on the oxygenation properties of Hb. Here we investigate the effect of NH2-terminal acetylation by using a newly developed expression plasmid system that enables us to compare recombinantly expressed Hbs that are structurally identical except for the presence or absence of NH2-terminal acetyl groups. Experiments with native and recombinant Hbs of representative vertebrates reveal that NH2-terminal acetylation does not impair the Bohr effect, nor does it significantly diminish responsiveness to allosteric cofactors, such as chloride ions or organic phosphates. These results suggest that observed variation in the oxygenation properties of vertebrate Hbs is principally explained by amino acid divergence in the constituent globin chains rather than post-translational modifications of the globin chain NH2-termini.

Molecular basis of hemoglobin adaptation in the high-flying bar-headed goose.

During the adaptive evolution of a particular trait, some selectively fixed mutations may be directly causative and others may be purely compensatory. The relative contribution of these two classes of mutation to adaptive phenotypic evolution depends on the form and prevalence of mutational pleiotropy. To investigate the nature of adaptive substitutions and their pleiotropic effects, we used a protein engineering approach to characterize the molecular basis of hemoglobin (Hb) adaptation in the high-flying bar-headed goose (Anser indicus), a hypoxia-tolerant species renowned for its trans-Himalayan migratory flights. To test the effects of observed substitutions on evolutionarily relevant genetic backgrounds, we synthesized all possible genotypic intermediates in the line of descent connecting the wildtype bar-headed goose genotype with the most recent common ancestor of bar-headed goose and its lowland relatives. Site-directed mutagenesis experiments revealed one major-effect mutation that significantly increased Hb-O2 affinity on all possible genetic backgrounds. Two other mutations exhibited smaller average effect sizes and less additivity across backgrounds. One of the latter mutations produced a concomitant increase in the autoxidation rate, a deleterious side-effect that was fully compensated by a second-site mutation at a spatially proximal residue. The experiments revealed three key insights: (i) subtle, localized structural changes can produce large functional effects; (ii) relative effect sizes of function-altering mutations may depend on the sequential order in which they occur; and (iii) compensation of deleterious pleiotropic effects may play an important role in the adaptive evolution of protein function.

Emergence of a Chimeric Globin Pseudogene and Increased Hemoglobin Oxygen Affinity Underlie the Evolution of Aquatic Specializations in Sirenia.

As limits on O2 availability during submergence impose severe constraints on aerobic respiration, the oxygen binding globin proteins of marine mammals are expected to have evolved under strong evolutionary pressures during their land-to-sea transition. Here, we address this question for the order Sirenia by retrieving, annotating, and performing detailed selection analyses on the globin repertoire of the extinct Steller's sea cow (Hydrodamalis gigas), dugong (Dugong dugon), and Florida manatee (Trichechus manatus latirostris) in relation to their closest living terrestrial relatives (elephants and hyraxes). These analyses indicate most loci experienced elevated nucleotide substitution rates during their transition to a fully aquatic lifestyle. While most of these genes evolved under neutrality or strong purifying selection, the rate of nonsynonymous/synonymous replacements increased in two genes (Hbz-T1 and Hba-T1) that encode the α-type chains of hemoglobin (Hb) during each stage of life. Notably, the relaxed evolution of Hba-T1 is temporally coupled with the emergence of a chimeric pseudogene (Hba-T2/Hbq-ps) that contributed to the tandemly linked Hba-T1 of stem sirenians via interparalog gene conversion. Functional tests on recombinant Hb proteins from extant and ancestral sirenians further revealed that the molecular remodeling of Hba-T1 coincided with increased Hb-O2 affinity in early sirenians. Available evidence suggests that this trait evolved to maximize O2 extraction from finite lung stores and suppress tissue O2 offloading, thereby facilitating the low metabolic intensities of extant sirenians. In contrast, the derived reduction in Hb-O2 affinity in (sub)Arctic Steller's sea cows is consistent with fueling increased thermogenesis by these once colossal marine herbivores.

Structure and function of crocodilian hemoglobins and allosteric regulation by chloride, ATP, and CO2.

Hemoglobins (Hbs) of crocodilians are reportedly characterized by unique mechanisms of allosteric regulatory control, but there are conflicting reports regarding the importance of different effectors, such as chloride ions, organic phosphates, and CO2. Progress in understanding the unusual properties of crocodilian Hbs has also been hindered by a dearth of structural information. Here, we present the first comparative analysis of blood properties and Hb structure and function in a phylogenetically diverse set of crocodilian species. We examine mechanisms of allosteric regulation in the Hbs of 13 crocodilian species belonging to the families Crocodylidae and Alligatoridae. We also report new amino acid sequences for the α- and ß-globins of these taxa, which, in combination with structural analyses, provide insights into molecular mechanisms of allosteric regulation. All crocodilian Hbs exhibited a remarkably strong sensitivity to CO2, which would permit effective O2 unloading to tissues in response to an increase in metabolism during intense activity and diving. Although the Hbs of all crocodilians exhibit similar intrinsic O2-affinities, there is considerable variation in sensitivity to Cl- ions and ATP, which appears to be at least partly attributable to variation in the extent of NH2-terminal acetylation. Whereas chloride appears to be a potent allosteric effector of all crocodile Hbs, ATP has a strong, chloride-independent effect on Hb-O2 affinity only in caimans. Modeling suggests that allosteric ATP binding has a somewhat different structural basis in crocodilian and mammalian Hbs.

Stability-Mediated Epistasis Restricts Accessible Mutational Pathways in the Functional Evolution of Avian Hemoglobin.

If the fitness effects of amino acid mutations are conditional on genetic background, then mutations can have different effects depending on the sequential order in which they occur during evolutionary transitions in protein function. A key question concerns the fraction of possible mutational pathways connecting alternative functional states that involve transient reductions in fitness. Here we examine the functional effects of multiple amino acid substitutions that contributed to an evolutionary transition in the oxygenation properties of avian hemoglobin (Hb). The set of causative changes included mutations at intradimer interfaces of the Hb tetramer. Replacements at such sites may be especially likely to have epistatic effects on Hb function since residues at intersubunit interfaces are enmeshed in networks of salt bridges and hydrogen bonds between like and unlike subunits; mutational reconfigurations of these atomic contacts can affect allosteric transitions in quaternary structure and the propensity for tetramer-dimer dissociation. We used ancestral protein resurrection in conjunction with a combinatorial protein engineering approach to synthesize genotypes representing the complete set of mutational intermediates in all possible forward pathways that connect functionally distinct ancestral and descendent genotypes. The experiments revealed that 1/2 of all possible forward pathways included mutational intermediates with aberrant functional properties because particular combinations of mutations promoted tetramer-dimer dissociation. The subset of mutational pathways with unstable intermediates may be selectively inaccessible, representing evolutionary roads not taken. The experimental results also demonstrate how epistasis for particular functional properties of proteins may be mediated indirectly by mutational effects on quaternary structural stability.

Hypoxia-induced changes in hemoglobins of Lake Victoria cichlids.

In a previous study, broods of the Lake Victoria cichlid Haplochromis ishmaeli raised under hypoxic or normoxic conditions showed striking differences in isohemoglobin (isoHb) pattern that were not observed in two other cichlids that do not belong to the Lake Victoria species flock. We therefore hypothesized that the adaptive mechanism seen in H. ishmaeli in response to hypoxia constitutes a trait that the Lake Victoria species flock inherited from ancestors that lived in hypoxic environments. We tested this hypothesis by designing split-brood experiments with three other representative species from the same species flock: the insectivorous Haplochromis thereuterion, the mollusk-shelling Platytaeniodus degeni and the zooplanktivorous Haplochromis piceatus, while keeping H. ishmaeli as a reference. Split broods were raised, under either normoxia or hypoxia. All hypoxia-raised (HR) individuals of each of the four species exhibited a distinctly different isoHb pattern compared with their normoxia-raised (NR) siblings. The hemoglobin of HR H. thereuterion showed higher O2 affinity compared with NR siblings particularly in the presence of ATP and GTP, indicating that blood of HR juveniles has significantly improved O2-binding affinity under hypoxic conditions. We also tested the capacity to acclimate at greater age in two species by reversing the O2 condition after 7 (H. thereuterion) and 4 (H. ishmaeli) months. After reacclimation for 1 and 2 months, respectively, we found incomplete reversal with intermediate isoHb patterns. As three of the four species do not encounter hypoxic conditions in their environment, this unique trait seems to be a relic inherited from predecessors that lived in hypoxic environments.

Allosteric mechanisms underlying the adaptive increase in hemoglobin-oxygen affinity of the bar-headed goose.

The high blood-O2 affinity of the bar-headed goose (Anser indicus) is an integral component of the biochemical and physiological adaptations that allow this hypoxia-tolerant species to undertake migratory flights over the Himalayas. The high blood-O2 affinity of this species was originally attributed to a single amino acid substitution of the major hemoglobin (Hb) isoform, HbA, which was thought to destabilize the low-affinity T state, thereby shifting the T-R allosteric equilibrium towards the high-affinity R state. Surprisingly, this mechanistic hypothesis has never been addressed using native proteins purified from blood. Here, we report a detailed analysis of O2 equilibria and kinetics of native major HbA and minor HbD isoforms from bar-headed goose and greylag goose (Anser anser), a strictly lowland species, to identify and characterize the mechanistic basis for the adaptive change in Hb function. We find that HbA and HbD of bar-headed goose have consistently higher O2 affinities than those of the greylag goose. The corresponding Hb isoforms of the two species are equally responsive to physiological allosteric cofactors and have similar Bohr effects. Thermodynamic analyses of O2 equilibrium curves according to the two-state Monod-Wyman-Changeaux model revealed higher R-state O2 affinities in the bar-headed goose Hbs, associated with lower O2 dissociation rates, compared with the greylag goose. Conversely, the T state was not destabilized and the T-R allosteric equilibrium was unaltered in bar-headed goose Hbs. The physiological implication of these results is that increased R-state affinity allows for enhanced O2 saturation in the lungs during hypoxia, but without impairing O2 delivery to tissues.

Contribution of a mutational hot spot to hemoglobin adaptation in high-altitude Andean house wrens.

A key question in evolutionary genetics is why certain mutations or certain types of mutation make disproportionate contributions to adaptive phenotypic evolution. In principle, the preferential fixation of particular mutations could stem directly from variation in the underlying rate of mutation to function-altering alleles. However, the influence of mutation bias on the genetic architecture of phenotypic evolution is difficult to evaluate because data on rates of mutation to function-altering alleles are seldom available. Here, we report the discovery that a single point mutation at a highly mutable site in the ß(A)-globin gene has contributed to an evolutionary change in hemoglobin (Hb) function in high-altitude Andean house wrens (Troglodytes aedon). Results of experiments on native Hb variants and engineered, recombinant Hb mutants demonstrate that a nonsynonymous mutation at a CpG dinucleotide in the ß(A)-globin gene is responsible for an evolved difference in Hb-O2 affinity between high- and low-altitude house wren populations. Moreover, patterns of genomic differentiation between high- and low-altitude populations suggest that altitudinal differentiation in allele frequencies at the causal amino acid polymorphism reflects a history of spatially varying selection. The experimental results highlight the influence of mutation rate on the genetic basis of phenotypic evolution by demonstrating that a large-effect allele at a highly mutable CpG site has promoted physiological differentiation in blood O2 transport capacity between house wren populations that are native to different elevations.

Convergent Evolution of Hemoglobin Function in High-Altitude Andean Waterfowl Involves Limited Parallelism at the Molecular Sequence Level.

A fundamental question in evolutionary genetics concerns the extent to which adaptive phenotypic convergence is attributable to convergent or parallel changes at the molecular sequence level. Here we report a comparative analysis of hemoglobin (Hb) function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergence in the oxygenation properties of Hb, and to assess the extent to which convergence in biochemical phenotype is attributable to repeated amino acid replacements. Functional experiments on native Hb variants and protein engineering experiments based on site-directed mutagenesis revealed the phenotypic effects of specific amino acid replacements that were responsible for convergent increases in Hb-O2 affinity in multiple high-altitude taxa. In six of the eight taxon pairs, high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique replacements, parallel replacements (involving identical-by-state variants with independent mutational origins in different lineages), and collateral replacements (involving shared, identical-by-descent variants derived via introgressive hybridization). In genome scans of nucleotide differentiation involving high- and low-altitude populations of three separate species, function-altering amino acid polymorphisms in the globin genes emerged as highly significant outliers, providing independent evidence for adaptive divergence in Hb function. The experimental results demonstrate that convergent changes in protein function can occur through multiple historical paths, and can involve multiple possible mutations. Most cases of convergence in Hb function did not involve parallel substitutions and most parallel substitutions did not affect Hb-O2 affinity, indicating that the repeatability of phenotypic evolution does not require parallelism at the molecular level.

Interval breast cancer characteristics before, during and after the transition from screen-film to full-field digital screening mammography.

BACKGROUND: To determine the proportion of "true" interval cancers and tumor characteristics of interval breast cancers prior to, during and after the transition from screen-film mammography screening (SFM) to full-field digital mammography screening (FFDM). METHODS: We included all women with interval cancers detected between January 2006 and January 2014. Breast imaging reports, biopsy results and breast surgery reports of all women recalled at screening mammography and of all women with interval breast cancers were collected. Two experienced screening radiologists reviewed the diagnostic mammograms, on which the interval cancers were diagnosed, as well as the prior screening mammograms and determined whether or not the interval cancer had been missed on the most recent screening mammogram. If not missed, the cancer was considered an occult ("true") interval cancer. RESULTS: A total of 442 interval cancers had been diagnosed, of which 144 at SFM with a prior SFM (SFM-SFM), 159 at FFDM with a prior SFM (FFDM-SFM) and 139 at FFDM with a prior FFDM (FFDM-FFDM). The transition from SFM to FFDM screening resulted in the diagnosis of more occult ("true") interval cancers at FFDM-SFM than at SFM-SFM (65.4% (104/159) versus 49.3% (71/144), P < 0.01), but this increase was no longer statistically significant in women who had been screened digitally for the second time (57.6% (80/139) at FFDM-FFDM versus 49.3% (71/144) at SFM-SFM). Tumor characteristics were comparable for the three interval cancer cohorts, except of a lower porportion (75.7 and 78.0% versus 67.2% af FFDM-FFDM, P < 0.05) of invasive ductal cancers at FFDM with prior FFDM. CONCLUSIONS: An increase in the proportion of occult interval cancers is observed during the transition from SFM to FFDM screening mammography. However, this increase seems temporary and is no longer detectable after the second round of digital screening. Tumor characteristics and type of surgery are comparable for interval cancers detected prior to, during and after the transition from SFM to FFDM screening mammography, except of a lower proportion of invasive ductal cancers after the transition.

O2 binding and CO2 sensitivity in haemoglobins of subterranean African mole rats.

Inhabiting deep and sealed subterranean burrows, mole rats exhibit a remarkable suite of specializations, including eusociality (living in colonies with single breeding queens), extraordinary longevity, cancer immunity and poikilothermy, and extreme tolerance of hypoxia and hypercapnia. With little information available on adjustments in haemoglobin (Hb) function that may mitigate the impact of exogenous and endogenous constraints on the uptake and internal transport of O2, we measured haematological characteristics, as well as Hb-O2 binding affinity and sensitivity to pH (Bohr effect), CO2, temperature and 2,3-diphosphoglycerate (DPG, the major allosteric modulator of Hb-O2 affinity in red blood cells) in four social and two solitary species of African mole rats (family Bathyergidae) originating from different biomes and soil types across Central and Southern Africa. We found no consistent patterns in haematocrit (Hct) and blood and red cell DPG and Hb concentrations or in intrinsic Hb-O2 affinity and its sensitivity to pH and DPG that correlate with burrowing, sociality and soil type. However, the results reveal low specific (pH independent) effects of CO2 on Hb-O2 affinity compared with humans that predictably safeguard pulmonary loading under hypoxic and hypercapnic burrow conditions. The O2 binding characteristics are discussed in relation to available information on the primary structure of Hbs from adult and developmental stages of mammals subjected to hypoxia and hypercapnia and the molecular mechanisms underlying functional variation in rodent Hbs.

Epistasis constrains mutational pathways of hemoglobin adaptation in high-altitude pikas.

A fundamental question in evolutionary genetics concerns the roles of mutational pleiotropy and epistasis in shaping trajectories of protein evolution. This question can be addressed most directly by using site-directed mutagenesis to explore the mutational landscape of protein function in experimentally defined regions of sequence space. Here, we evaluate how pleiotropic trade-offs and epistatic interactions influence the accessibility of alternative mutational pathways during the adaptive evolution of hemoglobin (Hb) function in high-altitude pikas (Mammalia: Lagomorpha). By combining ancestral protein resurrection with a combinatorial protein-engineering approach, we examined the functional effects of sequential mutational steps in all possible pathways that produced an increased Hb-O2 affinity. These experiments revealed that the effects of mutations on Hb-O2 affinity are highly dependent on the temporal order in which they occur: Each of three ß-chain substitutions produced a significant increase in Hb-O2 affinity on the ancestral genetic background, but two of these substitutions produced opposite effects when they occurred as later steps in the pathway. The experiments revealed pervasive epistasis for Hb-O2 affinity, but affinity-altering mutations produced no significant pleiotropic trade-offs. These results provide insights into the properties of adaptive substitutions in naturally evolved proteins and suggest that the accessibility of alternative mutational pathways may be more strongly constrained by sign epistasis for positively selected biochemical phenotypes than by antagonistic pleiotropy.

Intraspecific polymorphism, interspecific divergence, and the origins of function-altering mutations in deer mouse hemoglobin.

Major challenges for illuminating the genetic basis of phenotypic evolution are to identify causative mutations, to quantify their functional effects, to trace their origins as new or preexisting variants, and to assess the manner in which segregating variation is transduced into species differences. Here, we report an experimental analysis of genetic variation in hemoglobin (Hb) function within and among species of Peromyscus mice that are native to different elevations. A multilocus survey of sequence variation in the duplicated HBA and HBB genes in Peromyscus maniculatus revealed that function-altering amino acid variants are widely shared among geographically disparate populations from different elevations, and numerous amino acid polymorphisms are also shared with closely related species. Variation in Hb-O2 affinity within and among populations of P. maniculatus is attributable to numerous amino acid mutations that have individually small effects. One especially surprising feature of the Hb polymorphism in P. maniculatus is that an appreciable fraction of functional standing variation in the two transcriptionally active HBA paralogs is attributable to recurrent gene conversion from a tandemly linked HBA pseudogene. Moreover, transpecific polymorphism in the duplicated HBA genes is not solely attributable to incomplete lineage sorting or introgressive hybridization; instead, it is mainly attributable to recurrent interparalog gene conversion that has occurred independently in different species. Partly as a result of concerted evolution between tandemly duplicated globin genes, the same amino acid changes that contribute to variation in Hb function within P. maniculatus also contribute to divergence in Hb function among different species of Peromyscus. In the case of function-altering Hb mutations in Peromyscus, there is no qualitative or quantitative distinction between segregating variants within species and fixed differences between species.

Characteristics and prognosis of interval cancers after biennial screen-film or full-field digital screening mammography.

We determined the characteristics and prognosis of interval breast cancers (IC) at screen-film (SFM) and full-field digital (FFDM) screening mammography. The study population consisted of 417,746 consecutive screening mammograms (302,699 SFM screens and 115,047 FFDM screens), obtained between 2000 and 2011. During 2-year follow-up, we collected breast imaging reports, surgical reports, and pathology results. A total of 800 ICs had been diagnosed in the screened population, of which 288 detected in the first year (early ICs) and 512 in the second year (late ICs) after a negative screen. 31.3 % of early IC's and 19.1 % of late IC's, respectively, were visible in retrospect on the latest previous screens, but had been missed during screening (P < 0.001). Missed invasive ICs were larger (28.5 mm vs. 23.9 mm, P = 0.003) and showed a higher fraction of T3+cancers (16.9 vs. 8.5 %, P = 0.02) than true ICs (i.e., not visible at the latest screen). A higher portion of missed than true ICs underwent mastectomy (44.7 vs. 30.8 %, P = 0.002). We found no differences in mammographic and tumor characteristics for early ICs, detected either after SFM or FFDM. Late ICs following FFDM were more often true ICs than missed ICs (69.0 vs. 57.6 %, P = 0.03) and more often receptor triple negative (P = 0.02), compared to late ICs at SFM. Interval cancer subgroups showed comparable overall survival. Interval cancer subgroups show distinctive mammographic and tumor characteristics but a comparable overall survival.

Type and Extent of Surgery for Screen-Detected and Interval Cancers at Blinded Versus Nonblinded Double-Reading in a Population-Based Screening Mammography Program.

BACKGROUND: This study aimed to compare the type and extent of surgery in patients with screen-detected and interval cancers after blinded or nonblinded double-reading of screening mammograms. METHODS: The study investigated a consecutive series of screens double-read in either a blinded (n = 44,491) or nonblinded (n = 42,996) fashion between 2009 and 2011. During a 2 year follow-up period, the radiology reports, surgical correspondence, and pathology reports of all the screen-detected and interval cancers were collected. RESULTS: Screen-detected breast cancer was diagnosed for 325 women at blinded and 284 women at nonblinded double-reading. The majority of the women were treated by breast-conserving surgery (BCS) at both reading strategies (78.2 vs. 81.7 %; p = 0.51). Larger total resection volumes were observed at BCS for ductal carcinoma in situ (DCIS) treatment for patients after blinded double-reading (p = 0.005). The proportions of positive resection margins after BCS were comparable for patients with DCIS (p = 0.81) or invasive screen-detected cancers (p = 0.38) for the two reading strategies. A total of 158 interval cancers were diagnosed. The proportions of patients treated with BCS were comparable for the two reading strategies (p = 0.42). The total resection volume (p = 0.13) and the proportion of positive resection margins after BCS (p = 0.32) for invasive interval cancer were comparable for the two cohorts. The BCS rate was higher for women after nonblinded double-reading (p = 0.04). CONCLUSIONS: Blinded and nonblinded double-reading yielded comparable surgical treatments for women with screen-detected or interval breast cancer except for larger total resection volumes at BCS for screen-detected DCIS and a higher BCS rate for interval cancers at nonblinded double-reading.

Repeated elevational transitions in hemoglobin function during the evolution of Andean hummingbirds.

Animals that sustain high levels of aerobic activity under hypoxic conditions (e.g., birds that fly at high altitude) face the physiological challenge of jointly optimizing blood-O2 affinity for O2 loading in the pulmonary circulation and O2 unloading in the systemic circulation. At high altitude, this challenge is especially acute for small endotherms like hummingbirds that have exceedingly high mass-specific metabolic rates. Here we report an experimental analysis of hemoglobin (Hb) function in South American hummingbirds that revealed a positive correlation between Hb-O2 affinity and native elevation. Protein engineering experiments and ancestral-state reconstructions revealed that this correlation is attributable to derived increases in Hb-O2 affinity in highland lineages, as well as derived reductions in Hb-O2 affinity in lowland lineages. Site-directed mutagenesis experiments demonstrated that repeated evolutionary transitions in biochemical phenotype are mainly attributable to repeated amino acid replacements at two epistatically interacting sites that alter the allosteric regulation of Hb-O2 affinity. These results demonstrate that repeated changes in biochemical phenotype involve parallelism at the molecular level, and that mutations with indirect, second-order effects on Hb allostery play key roles in biochemical adaptation.

Screening outcome and surgical treatment during and after the transition from screen-film to digital screening mammography in the south of The Netherlands.

We determined screening outcome of subsequent screens during and after the transition from screen-film mammography (SFM) to full-field digital mammography (FFDM). A consecutive series of 102,863 subsequent (SFM screens with a prior SFM screen (SFM-SFM cohort), 91,941 FFDM screens with a prior SFM screen (FFDM-SFM cohort) and 90,407 FFDM screens with a prior FFDM screen (FFDM-FFDM cohort) were obtained between January 2006 and July 2013. The referral rate and cancer detection rate (CDR) per 1,000 screens were higher at FFDM-SFM than at SFM-SFM (2.7% vs. 1.2% (p < 0.001) and 7.0 vs. 4.9, p < 0.001), at the expense of a lower positive predictive value (PPV) of referral (25.8% vs. 39.6%, p < 0.001). These parameters were comparable for FFDM-SFM and FFDM-FFDM. Ductal carcinoma in situ (DCIS) and invasive cancer rates increased during transition and remained stable after transition. The rate of DCIS of intermediate grade increased during the transition from 0.2 per 1,000 screened women at SFM-SFM to 0.6 at FFDM-SFM (p < 0.001) and 0.5 at FFDM-FFDM (p = 0.001). Compared to SFM-SFM, a significantly higher rate of invasive cancers were stage T1a-b at FFDM-SFM (p < 0.001) and FFDM-FFDM (p < 0.001). Breast conserving surgery rates increased during transition (p < 0.001) and remained stable afterwards. The CDR and referral rate remained significantly higher at FFDM than at SFM, at the expense of a decreased PPV of referral. During transition, DCIS was more often of intermediate grade and invasive cancers were of smaller size.

Integrating evolutionary and functional tests of adaptive hypotheses: a case study of altitudinal differentiation in hemoglobin function in an Andean Sparrow, Zonotrichia capensis.

In air-breathing vertebrates, the physiologically optimal blood-O2 affinity is jointly determined by the prevailing partial pressure of atmospheric O2, the efficacy of pulmonary O2 transfer, and internal metabolic demands. Consequently, genetic variation in the oxygenation properties of hemoglobin (Hb) may be subject to spatially varying selection in species with broad elevational distributions. Here we report the results of a combined functional and evolutionary analysis of Hb polymorphism in the rufous-collared sparrow (Zonotrichia capensis), a species that is continuously distributed across a steep elevational gradient on the Pacific slope of the Peruvian Andes. We integrated a population genomic analysis that included all postnatally expressed Hb genes with functional studies of naturally occurring Hb variants, as well as recombinant Hb (rHb) mutants that were engineered through site-directed mutagenesis. We identified three clinally varying amino acid polymorphisms: Two in the α(A)-globin gene, which encodes the α-chain subunits of the major HbA isoform, and one in the α(D)-globin gene, which encodes the α-chain subunits of the minor HbD isoform. We then constructed and experimentally tested single- and double-mutant rHbs representing each of the alternative α(A)-globin genotypes that predominate at different elevations. Although the locus-specific patterns of altitudinal differentiation suggested a history of spatially varying selection acting on Hb polymorphism, the experimental tests demonstrated that the observed amino acid mutations have no discernible effect on respiratory properties of the HbA or HbD isoforms. These results highlight the importance of experimentally validating the hypothesized effects of genetic changes in protein function to avoid the pitfalls of adaptive storytelling.

Comparison of the diagnostic workup of women referred at non-blinded or blinded double reading in a population-based screening mammography programme in the south of the Netherlands.

BACKGROUND: To determine whether referred women experience differences in diagnostic workup at non-blinded or blinded double reading of screening mammograms. METHODS: We included a consecutive series of respectively 42.996 and 44.491 screens, double read either in a non-blinded or blinded manner between 2009 and 2011. This reading strategy was alternated on a monthly basis. RESULTS: The overall ultrasound-guided core needle biopsy (CNB) rate and stereotactic CNB (SCNB) rate per 1000 screens were higher at blinded than at non-blinded reading (7.5 vs 6.0, P=0.008 and 8.1 vs 6.6, P=0.009). Among women with benign workup, these rates were higher at blinded reading (2.6 vs 1.4, P<0.001 and 5.9 vs 4.7, P=0.013). The benign biopsy rates were higher at blinded double reading (P<0.001), whereas the positive predictive value of biopsy did not differ (P=0.103). CONCLUSIONS: Blinded double-reading results in higher overall CNB and SCNB rates than non-blinded double reading, as well as a higher benign biopsy rate.