Microbial population dynamics decouple growth response from environmental nutrient concentration.

How the growth rate of a microbial population responds to the environmental availability of chemical nutrients and other resources is a fundamental question in microbiology. Models of this response, such as the widely used Monod model, are generally characterized by a maximum growth rate and a half-saturation concentration of the resource. What values should we expect for these half-saturation concentrations, and how should they depend on the environmental concentration of the resource? We survey growth response data across a wide range of organisms and resources. We find that the half-saturation concentrations vary across orders of magnitude, even for the same organism and resource. To explain this variation, we develop an evolutionary model to show that demographic fluctuations (genetic drift) can constrain the adaptation of half-saturation concentrations. We find that this effect fundamentally differs depending on the type of population dynamics: Populations undergoing periodic bottlenecks of fixed size will adapt their half-saturation concentrations in proportion to the environmental resource concentrations, but populations undergoing periodic dilutions of fixed size will evolve half-saturation concentrations that are largely decoupled from the environmental concentrations. Our model not only provides testable predictions for laboratory evolution experiments, but it also reveals how an evolved half-saturation concentration may not reflect the organism's environment. In particular, this explains how organisms in resource-rich environments can still evolve fast growth at low resource concentrations. Altogether, our results demonstrate the critical role of population dynamics in shaping fundamental ecological traits.

Extended Multimodal Flat Detector CT Imaging in Acute Ischemic Stroke: A Pilot Study.

By using Flat detector computed tomography (FD-CT), a one-stop-shop approach in the diagnostic workup of acute ischemic stroke (AIS) might be achieved. Although information on upstream vessels is warranted, dedicated FD-CT protocols which include the imaging of the cervical vasculature are still lacking. We aimed to prospectively evaluate the implementation of a new multimodal FD-CT protocol including cervical vessel imaging in AIS patients. In total, 16 patients were included in this study. Eight patients with AIS due to large vessel occlusion (LVO) prospectively received a fully multimodal FD-CT imaging, including non-enhanced flat detector computed tomography (NE-FDCT), dynamic perfusion flat detector computed tomography (FD-CTP) and flat detector computed tomography angiography (FD-CTA) including cervical imaging. For comparison of time metrics and image quality, eight AIS patients, which received multimodal CT imaging, were included retrospectively. Although image quality of NE-FDCT and FD-CTA was rated slightly lower than NE-CT and CTA, all FD-CT datasets were of diagnostic quality. Intracerebral hemorrhage exclusion and LVO detection was reliably possible. Median door-to-image time was comparable for the FD-CT group and the control group (CT:30 min, IQR27-58; FD-CT:44.5 min, IQR31-55, p = 0.491). Door-to-groin-puncture time (CT:79.5 min, IQR65-90; FD-CT:59.5 min, IQR51-67; p = 0.016) and image-to-groin-puncture time (CT:44 min, IQR30-50; FD-CT:14 min, IQR12-18; p < 0.001) were significantly shorter, when patients were directly transferred to the angiosuite, where FD-CT took place. Our study indicates that using a new fully multimodal FD-CT approach including imaging of cervical vessels for first-line imaging in AIS patients is feasible and comparable to multimodal CT imaging with substantial potential to streamline the stroke workflow.

High-speed flat-detector computed tomography for temporal bone imaging and postoperative control of cochlear implants.

PURPOSE: Flat-detector computed tomography (FD-CT) is the standard for cochlear implant (CI) imaging. FD-CT systems differ in technical characteristics. Our aim was an evaluation of two different FD-CT generations with different protocols and hardware regarding image quality, radiation dose, and scan time. METHODS: Two temporal bone specimens (- / + CI = TB0/TB1) were scanned using three different scanners: two FD-CT systems with different scanning protocols (standard FD-CT: 20 s 70 kV, 20 s 109 kV; high-speed FD-CT [HS-FD-CT]: 7 s 109 kV, 9 s 109 kV, 14 s 72 kV) and MS-CT (5 s 120 kV). Acquired datasets were evaluated in consensus reading regarding qualitative and quantitative parameters: addressing CI- and cochlea-specific parameters, cochlea delineation, lamina spiralis ossea visibility, distinction of single CI electrodes, determination of intracochlear implant position, stapes delineation, and mastoidal septation were assessed. Addressing protocol-specific parameters, radiation dose (dose-length-product/DLP), and scan time were assessed. RESULTS: Two HS-FD-CT protocols (14 s/9 s) provide higher or equivalent diagnostic information regarding CI- and cochlea-specific parameters compared to both standard FD-CT protocols. The fastest HS-FD-CT protocol (7 s)-providing inferior diagnostic information compared to all other FD-CT protocols-still exceeds MS-CT. The highest DLP was recorded for the 14 s HS-FD-CT protocol (TB1 = 956 mGycm); the lowest DLPs were recorded for the 7 s HS-FD-CT protocol (TB0 = 188 mGycm) and for MS-CT (TB0 = 138 mGycm), respectively. HS-FD-CT allows a significant reduction of scan time compared to standard FD-CT. CONCLUSION: High-speed FD-CT improves visualization of temporal bone anatomy and postoperative assessment of CIs by combining excellent image quality, fast scan time, and reasonable radiation exposure.

Evolutionary regain of lost gene circuit function.

Evolutionary reversibility-the ability to regain a lost function-is an important problem both in evolutionary and synthetic biology, where repairing natural or synthetic systems broken by evolutionary processes may be valuable. Here, we use a synthetic positive-feedback (PF) gene circuit integrated into haploid Saccharomyces cerevisiae cells to test if the population can restore lost PF function. In previous evolution experiments, mutations in a gene eliminated the fitness costs of PF activation. Since PF activation also provides drug resistance, exposing such compromised or broken mutants to both drug and inducer should create selection pressure to regain drug resistance and possibly PF function. Indeed, evolving 7 PF mutant strains in the presence of drug revealed 3 adaptation scenarios through genomic, PF-external mutations that elevate PF basal expression, possibly by affecting transcription, translation, degradation, and other fundamental cellular processes. Nonfunctional mutants gained drug resistance without ever developing high expression, while quasifunctional and dysfunctional PF mutants developed high expression nongenetically, which then diminished, although more slowly for dysfunctional mutants where revertant clones arose. These results highlight how intracellular context, such as the growth rate, can affect regulatory network dynamics and evolutionary dynamics, which has important consequences for understanding the evolution of drug resistance and developing future synthetic biology applications.

Natural Family Planning and Marital Chastity: The Effects of Periodic Abstinence on Marital Relationships.

Marital chastity is the practice of periodic abstinence with use of natural family planning (NFP). The purpose of this study was to determine the influence of the most common methods of contraception (female sterilization, oral contraceptive pills, and condoms) and NFP on divorce/separation and cohabitation rates among reproductive age women. The study involved an extensive review of the literature on the effects of practice of NFP on marital dynamics and a statistical analysis of 2,550 ever-married women in the (2015-2017) National Survey of Family Growth data set. Importance of religion and frequency of church attendance were included in the analysis. With ever-use of NFP, 14 percent were divorced or separated, and 27 percent to 39 percent were divorced or separated with ever-use of oral contraceptive pills. Stepwise logistic regression indicated that ever-use of contraception was associated with increased odds of divorce or separation (odds ratio [OR] = 2.05; confidence interval [CI]: 1.96-2.49) and cohabitation (2.95, CI: 2.20-3.95). Ever-use of NFP yielded 58 percent lower odds for divorce or separation. Frequent church attendance was associated with lower odds of divorce or separation and cohabitation. Although there are lower odds of divorce among NFP users, the reason might be due to their religiosity. SUMMARY: This study showed that ever-use of natural family planning (NFP) among ever-married women was associated with 58 percent lower odds of divorce than among women who never-used NFP. Ever-use of contraceptive methods was associated with two times the odds of divorce and four times for cohabitation compared to those women who never-used those methods. Use of periodic abstinence with NFP is the practice of marital chastity and is thought to strengthen the marital relationship.

Hormonally Active Contraceptives, Part II: Sociological, Environmental, and Economic Impact.

To investigate the sociological, environmental, and economic impact of hormonally active contraceptives, a series of comprehensive literature surveys were employed. Sociological effects are discussed including abortion, exploitation of women, a weakening of marriage, and an increase in divorce with deleterious effects on children such as child poverty, poorer health, lower educational achievement, suicide risks, drug and alcohol abuse, criminality, and incarceration, among others. The environmental impact is discussed briefly and includes the feminization and trans-gendering of male fish downstream from the effluent of city wastewater treatment plants with declining fish populations. The potential economic impact of most of these side effects is estimated based on epidemiologic data and published estimates of costs of caring for the diseases which are linked to the use of hormonally active contraceptives. Hormonally active contraceptives appear to have a deleterious impact on multiple aspects of women's health as well as negative economic and environmental impacts. These risks can be avoided through the use of nonhormonal methods and need to be more clearly conveyed to the public. SUMMARY: Hormonal contraceptives have wide-ranging effects.  The potential economic impact of the medical side effects is estimated. Sociological effects are discussed including abortion, exploitation of women, a weakening of marriage and an increase in divorce with negative effects on children such as child poverty, poorer health, lower educational achievement, suicide risks, drug and alcohol abuse, criminality and incarceration among others. The environmental impact includes hormonal effects on fish with declining fish populations. Women seeking birth control have a right to know about how to avoid these risks by using effective hormone-free methods like Fertility Awareness Methods.

Hormonally Active Contraceptives Part I: Risks Acknowledged and Unacknowledged.

Hormonal contraceptives have been on the market for over fifty years and, while their formulations have changed, the basic mechanism of action has remained the same. During this time, numerous studies have been performed documenting side effects, some of which appear over time, some within weeks or months, but all can have a serious impact on health and quality of life. An effort was made to perform a series of comprehensive literature surveys to better understand immediate and long-term side effects of these agents. The results of this literature review uncovered a number of potential side effects, some of which are acknowledged and many of which are not noted in the prescribing information for these agents. Among the unacknowledged side effects are: an increased risk of HIV transmission for depot medroxyprogesterone acetate (DMPA), and for combination contraceptives breast cancer, cervical cancer, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, depression, mood disorders and suicides (especially among women twenty-five years of age and younger, in the first six months of use), multiple sclerosis, interstitial cystitis, female sexual dysfunction, osteoporotic bone fractures (especially for progesterone-only contraceptives), and fatty weight gain. Misleading prescribing information regarding cardiovascular and thrombotic risks are also noted. Women seeking birth control have a right to be informed and educated about risk avoidance through the use of effective nonhormonal methods like fertility awareness methods. In one case-that of DMPA-the increased risk of HIV acquisition has been conclusively demonstrated to be both real and unique to this drug. Considering the availability of numerous alternatives, there is no justification for the continued marketing of DMPA to the public. SUMMARY: We reviewed the effect of hormonal contraceptives on women's health. A number of potential side effects were noted including increased risks of breast cancer, cervical cancer, inflammatory bowel  disease, lupus, multiple sclerosis, cystitis, bone fractures, depression, mood disorders and suicides,  fatty weight gain, and female sexual dysfunction.  With the long-acting injectable contraceptives there is an increased risk of getting HIV.  Misleading prescribing information regarding the risks of heart attacks, strokes and blood clotting problems were also noted. Women seeking birth control have a right to know about how to avoid these risks by using effective hormone-free Fertility Awareness Methods.

A Comparison of User Behaviors for a Fertility-Tracking App: Does Training in an NFP Method Improve Persistence and Use?

This study explores differences in the use of CycleProGo™ (CPG), a fertility-tracking app developed by Couple to Couple League (CCL), between those exposed to it as a part of natural family planning (NFP) instruction versus those who find it on their own. An anonymous data set of 17,543 CPG accounts opened between April 2013 and June 2016 was used for analysis. Nonmember users opened the most accounts (58 percent, n = 10,134), CCL members represented 38 percent (n = 6,758) of new accounts, and 207 CCL teachers (4 percent) were using CPG for personal charting. Significantly more nonmember accounts had zero days of use after the initial opening compared to CCL member accounts (61 percent vs. 23 percent, respectively, χ2 = 2,405.9, p < .001). Conversely, significantly more CCL member accounts were used for ninety days or longer than nonmember accounts (47 percent vs. 13 percent, respectively, χ2 = 2,404.2, p < .001). CCL students-those who began using the app as part of a formal NFP teaching curriculum-were more likely to use the app for > six cycles compared to nonmembers. In accounts with at least one complete cycle, CCL students were the most diligent at daily recording (95 percent of cycle days with observation recorded) followed by CCL members (88 percent) and nonmembers (76 percent). CCL teachers had the lowest frequency of cycle days with a recorded observation (73 percent). Within each cohort, accounts with > six recorded cycles had a lower proportion of cycle days with an observation recorded, likely reflecting increasing knowledge of their personal fertility patterns. Long-term users who had no known formal training in NFP still had the lowest proportion cycle days with a fertility observation. We conclude formal NFP instruction increases the probability of long-term app use, and regardless of training, long-term users will likely record observations on about 70 percent of cycle days. SUMMARY: "CycleProGo™ users with NFP training were more persistent and diligent about daily data input than those without training."

Trade-offs between microbial growth phases lead to frequency-dependent and non-transitive selection.

Mutations in a microbial population can increase the frequency of a genotype not only by increasing its exponential growth rate, but also by decreasing its lag time or adjusting the yield (resource efficiency). The contribution of multiple life-history traits to selection is a critical question for evolutionary biology as we seek to predict the evolutionary fates of mutations. Here we use a model of microbial growth to show that there are two distinct components of selection corresponding to the growth and lag phases, while the yield modulates their relative importance. The model predicts rich population dynamics when there are trade-offs between phases: multiple strains can coexist or exhibit bistability due to frequency-dependent selection, and strains can engage in rock-paper-scissors interactions due to non-transitive selection. We characterize the environmental conditions and patterns of traits necessary to realize these phenomena, which we show to be readily accessible to experiments. Our results provide a theoretical framework for analysing high-throughput measurements of microbial growth traits, especially interpreting the pleiotropy and correlations between traits across mutants. This work also highlights the need for more comprehensive measurements of selection in simple microbial systems, where the concept of an ordinary fitness landscape breaks down.

Prototype metal artefact reduction algorithm in flat panel computed tomography - evaluation in patients undergoing transarterial hepatic radioembolisation.

OBJECTIVE: To investigate the effect of an on-site prototype metal artefact reduction (MAR) algorithm in cone-beam CT-catheter-arteriography (CBCT-CA) in patients undergoing transarterial radioembolisation (RE) of hepatic masses. METHODS AND MATERIALS: Ethical board approved retrospective study of 29 patients (mean 63.7±13.7 years, 11 female), including 16 patients with arterial metallic coils, undergoing CBCT-CA (8s scan, 200 degrees rotation, 397 projections). Image reconstructions with and without prototype MAR algorithm were evaluated quantitatively (streak-artefact attenuation changes) and qualitatively (visibility of hepatic parenchyma and vessels) in near- (<1cm) and far-field (>3cm) of artefact sources (metallic coils and catheters). Quantitative and qualitative measurements of uncorrected and MAR corrected images and different artefact sources were compared RESULTS: Quantitative evaluation showed significant reduction of near- and far-field streak-artefacts with MAR for both artefact sources (p<0.001), while remaining stable for unaffected organs (all p>0.05). Inhomogeneities of attenuation values were significantly higher for metallic coils compared to catheters (p<0.001) and decreased significantly for both after MAR (p<0.001). Qualitative image scores were significantly improved after MAR (all p<0.003) with by trend higher artefact degrees for metallic coils compared to catheters. CONCLUSION: In patients undergoing CBCT-CA for transarterial RE, prototype MAR algorithm improves image quality in proximity of metallic coil and catheter artefacts. KEY POINTS: • Metal objects cause artefacts in cone-beam computed tomography (CBCT) imaging. • These artefacts can be corrected by metal artefact reduction (MAR) algorithms. • Corrected images show significantly better visibility of nearby hepatic vessels and tissue. • Better visibility may facilitate image interpretation, save time and radiation exposure.

Protein folding and binding can emerge as evolutionary spandrels through structural coupling.

Binding interactions between proteins and other molecules mediate numerous cellular processes, including metabolism, signaling, and gene regulation. These interactions often evolve in response to changes in the protein's chemical or physical environment (such as the addition of an antibiotic). Several recent studies have shown the importance of folding stability in constraining protein evolution. Here we investigate how structural coupling between folding and binding--the fact that most proteins can only bind their targets when folded--gives rise to an evolutionary coupling between the traits of folding stability and binding strength. Using a biophysical and evolutionary model, we show how these protein traits can emerge as evolutionary "spandrels" even if they do not confer an intrinsic fitness advantage. In particular, proteins can evolve strong binding interactions that have no functional role but merely serve to stabilize the protein if its misfolding is deleterious. Furthermore, such proteins may have divergent fates, evolving to bind or not bind their targets depending on random mutational events. These observations may explain the abundance of apparently nonfunctional interactions among proteins observed in high-throughput assays. In contrast, for proteins with both functional binding and deleterious misfolding, evolution may be highly predictable at the level of biophysical traits: adaptive paths are tightly constrained to first gain extra folding stability and then partially lose it as the new binding function is developed. These findings have important consequences for our understanding of how natural and engineered proteins evolve under selective pressure.

Protein Homeostasis Imposes a Barrier on Functional Integration of Horizontally Transferred Genes in Bacteria.

Horizontal gene transfer (HGT) plays a central role in bacterial evolution, yet the molecular and cellular constraints on functional integration of the foreign genes are poorly understood. Here we performed inter-species replacement of the chromosomal folA gene, encoding an essential metabolic enzyme dihydrofolate reductase (DHFR), with orthologs from 35 other mesophilic bacteria. The orthologous inter-species replacements caused a marked drop (in the range 10-90%) in bacterial growth rate despite the fact that most orthologous DHFRs are as stable as E.coli DHFR at 37°C and are more catalytically active than E. coli DHFR. Although phylogenetic distance between E. coli and orthologous DHFRs as well as their individual molecular properties correlate poorly with growth rates, the product of the intracellular DHFR abundance and catalytic activity (kcat/KM), correlates strongly with growth rates, indicating that the drop in DHFR abundance constitutes the major fitness barrier to HGT. Serial propagation of the orthologous strains for ~600 generations dramatically improved growth rates by largely alleviating the fitness barriers. Whole genome sequencing and global proteome quantification revealed that the evolved strains with the largest fitness improvements have accumulated mutations that inactivated the ATP-dependent Lon protease, causing an increase in the intracellular DHFR abundance. In one case DHFR abundance increased further due to mutations accumulated in folA promoter, but only after the lon inactivating mutations were fixed in the population. Thus, by apparently distinguishing between self and non-self proteins, protein homeostasis imposes an immediate and global barrier to the functional integration of foreign genes by decreasing the intracellular abundance of their products. Once this barrier is alleviated, more fine-tuned evolution occurs to adjust the function/expression of the transferred proteins to the constraints imposed by the intracellular environment of the host organism.

The State of the Science of Natural Family Planning Fifty Years after Humane Vitae: A Report from NFP Scientists' Meeting Held at the US Conference of Catholic Bishops, April 4, 2018.

A one-day meeting of physicians, professional nurses, and scientists actively involved in Natural Family Planning (NFP) research was held to review the state of the science of NFP and consider future priorities. The meeting had four objectives: (i) determine the gaps in research evidence for secure methods of NFP among women of all reproductive categories, (ii) determine the gaps in the research and development of new technology for providing NFP services, (iii) determine the gaps in the research that determine the benefits and challenges with use of NFP among married couples, and (iv) provide prioritized ideas for future research needs from the analysis of evidence gaps from objectives above. This article summarizes the discussion and conclusions drawn from topics reviewed. While much has been accomplished in the fifty years since Humane vitae, there are still many gaps to address. Five areas for future research in NFP were identified as high priority: (1) well-designed method effectiveness studies among various reproductive categories including important subpopulations (postpartum, perimenopause, posthormonal contraceptive), normally cycling women (especially US women), and comparative studies between NFP methods; (2) validation studies to establish the benefit of charting fertility signs (both currently known and potential new indicators) as a screening tool for women's health issues; (3) ongoing independent evaluation of fertility monitoring apps to provide users perspective on the relative merits of each and to identify those most worthy of further effectiveness testing; (4) studies evaluating the impact of new technologies on NFP adoption, use, and persistence; and (5) creation of a shared database across various NFP methods to collaborate on shared research interests, longitudinal studies, and so on. This summarizes a meeting to review the scientific and medical progress related to natural family planning made in the 50 years since Humane Vitae and to define priorities for future work. Areas reviewed included the evidence for avoiding pregnancy in normally cycling, postpartum, and perimenopausal women, the impact of new technology, including fertility charting apps, on NFP, and the impact on relationships and personal well-being from use of NFP. Five priority focus areas for future research were also identified.

Competitive Assays of Label-Free DNA Hybridization with Single-Molecule Fluorescence Imaging Detection.

Single-molecule imaging of fluorescently labeled biomolecules is a powerful technique for measuring association interactions; however, care must be taken to ensure that the fluorescent labels do not influence the system being probed. Label-free techniques are needed to understand biomolecule interactions free from the influence of an attached label, but these techniques often lack sensitivity and specificity. To solve these challenges, we have developed a competitive assay that uses single-molecule detection to track the population of unlabeled target single-stranded DNA (ssDNA) hybridized with probe DNA immobilized at a glass interface by detecting individual duplexes with a fluorescently labeled "tracer" ssDNA. By labeling a small fraction (<0.2%) of target molecules, the "tracer" DNA tracks the available probe DNA sites without significant competition with the unlabeled target population. Single-molecule fluorescence imaging is a good read-out scheme for competitive assays, as it is sufficiently sensitive to detect tracer DNA on substrates with relatively low densities of probe DNA, ∼10(-3) of a monolayer, so that steric interactions do not hinder DNA hybridization. Competitive assays are used to measure the association constant of complementary strand DNA hybridization of 9- and 10-base pair targets, where the tracer assay predicts the same association constant as a traditional displacement competitive assay. This methodology was used to compare the Ka of hybridization for identical DNA strands differing only by the presence of a fluorescent label tethered to the 5' end of the solution-phase target. The addition of the fluorescent label significantly stabilizes the DNA duplex by 3.6 kJmol(-1), adding more stability than an additional adenine-thymine base-pairing interaction, 2.7 kJmol(-1). This competitive tracer assay could be used to screen a number of labeled and unlabeled target DNA strands to measure the impact of fluorescent labeling on duplex stability. This single-molecule competitive hybridization scheme could be easily adapted into a sensitive assay, where competition between tracer and target oligonucleotides for probe sites could be used to measure concentrations of unlabeled DNA or RNA.

Single-Molecule Fluorescence Imaging of Interfacial DNA Hybridization Kinetics at Selective Capture Surfaces.

Accurate knowledge of the kinetics of complementary oligonucleotide hybridization is integral to the design and understanding of DNA-based biosensors. In this work, single-molecule fluorescence imaging is applied to measuring rates of hybridization between fluorescently labeled target ssDNA and unlabeled probe ssDNA immobilized on glass surfaces. In the absence of probe site labeling, the capture surface must be highly selective to avoid the influence of nonspecific adsorption on the interpretation of single-molecule imaging results. This is accomplished by increasing the probe molecule site densities by a factor of ∼100 compared to optically resolvable sites so that nonspecific interactions compete with a much greater number of capture sites and by immobilizing sulfonate groups to passivate the surface between probe strands. The resulting substrates exhibit very low nonspecific adsorption, and the selectivity for binding a complementary target sequence exceeds that of a scrambled sequence by nearly 3 orders of magnitude. The population of immobilized DNA probe sites is quantified by counting individual DNA duplexes at low target concentrations, and those results are used to calibrate fluorescence intensities on the same sample at much higher target concentrations to measure a full binding isotherm. Dissociation rates are determined from interfacial residence times of individual DNA duplexes. Equilibrium and rate constants of hybridization, K(a) = 38 (±1) µM(-1), k(on) = 1.64 (±0.06) × 10(6) M(-1) s(-1), and k(off) = 4.3 (±0.1) × 10(-2) s(-1), were found not to change with surface density of immobilized probe DNA, indicating that hybridization events at neighboring probe sites are independent. To test the influence of probe-strand immobilization on hybridization, the kinetics of the probe target reaction at the surface were compared with the same reaction in free solution, and the equilibrium constants and dissociation and association rates were found to be nearly equivalent. The selectivity of these capture surfaces should facilitate sensitive investigations of DNA hybridization at the limit of counting molecules. Because the immobilized probe DNA on these surfaces is unlabeled, photobleaching of a probe label is not an issue, allowing capture substrates to be used for long periods of time or even reused in multiple experiments.

Stress-response balance drives the evolution of a network module and its host genome.

Stress response genes and their regulators form networks that underlie drug resistance. These networks often have an inherent tradeoff: their expression is costly in the absence of stress, but beneficial in stress. They can quickly emerge in the genomes of infectious microbes and cancer cells, protecting them from treatment. Yet, the evolution of stress resistance networks is not well understood. Here, we use a two-component synthetic gene circuit integrated into the budding yeast genome to model experimentally the adaptation of a stress response module and its host genome in three different scenarios. In agreement with computational predictions, we find that: (i) intra-module mutations target and eliminate the module if it confers only cost without any benefit to the cell; (ii) intra- and extra-module mutations jointly activate the module if it is potentially beneficial and confers no cost; and (iii) a few specific mutations repeatedly fine-tune the module's noisy response if it has excessive costs and/or insufficient benefits. Overall, these findings reveal how the timing and mechanisms of stress response network evolution depend on the environment.

Scaling properties of evolutionary paths in a biophysical model of protein adaptation.

The enormous size and complexity of genotypic sequence space frequently requires consideration of coarse-grained sequences in empirical models. We develop scaling relations to quantify the effect of this coarse-graining on properties of fitness landscapes and evolutionary paths. We first consider evolution on a simple Mount Fuji fitness landscape, focusing on how the length and predictability of evolutionary paths scale with the coarse-grained sequence length and alphabet. We obtain simple scaling relations for both the weak- and strong-selection limits, with a non-trivial crossover regime at intermediate selection strengths. We apply these results to evolution on a biophysical fitness landscape that describes how proteins evolve new binding interactions while maintaining their folding stability. We combine the scaling relations with numerical calculations for coarse-grained protein sequences to obtain quantitative properties of the model for realistic binding interfaces and a full amino acid alphabet.

Biophysical fitness landscapes for transcription factor binding sites.

Phenotypic states and evolutionary trajectories available to cell populations are ultimately dictated by complex interactions among DNA, RNA, proteins, and other molecular species. Here we study how evolution of gene regulation in a single-cell eukaryote S. cerevisiae is affected by interactions between transcription factors (TFs) and their cognate DNA sites. Our study is informed by a comprehensive collection of genomic binding sites and high-throughput in vitro measurements of TF-DNA binding interactions. Using an evolutionary model for monomorphic populations evolving on a fitness landscape, we infer fitness as a function of TF-DNA binding to show that the shape of the inferred fitness functions is in broad agreement with a simple functional form inspired by a thermodynamic model of two-state TF-DNA binding. However, the effective parameters of the model are not always consistent with physical values, indicating selection pressures beyond the biophysical constraints imposed by TF-DNA interactions. We find little statistical support for the fitness landscape in which each position in the binding site evolves independently, indicating that epistasis is common in the evolution of gene regulation. Finally, by correlating TF-DNA binding energies with biological properties of the sites or the genes they regulate, we are able to rule out several scenarios of site-specific selection, under which binding sites of the same TF would experience different selection pressures depending on their position in the genome. These findings support the existence of universal fitness landscapes which shape evolution of all sites for a given TF, and whose properties are determined in part by the physics of protein-DNA interactions.

Path statistics, memory, and coarse-graining of continuous-time random walks on networks.

Continuous-time random walks (CTRWs) on discrete state spaces, ranging from regular lattices to complex networks, are ubiquitous across physics, chemistry, and biology. Models with coarse-grained states (for example, those employed in studies of molecular kinetics) or spatial disorder can give rise to memory and non-exponential distributions of waiting times and first-passage statistics. However, existing methods for analyzing CTRWs on complex energy landscapes do not address these effects. Here we use statistical mechanics of the nonequilibrium path ensemble to characterize first-passage CTRWs on networks with arbitrary connectivity, energy landscape, and waiting time distributions. Our approach can be applied to calculating higher moments (beyond the mean) of path length, time, and action, as well as statistics of any conservative or non-conservative force along a path. For homogeneous networks, we derive exact relations between length and time moments, quantifying the validity of approximating a continuous-time process with its discrete-time projection. For more general models, we obtain recursion relations, reminiscent of transfer matrix and exact enumeration techniques, to efficiently calculate path statistics numerically. We have implemented our algorithm in PathMAN (Path Matrix Algorithm for Networks), a Python script that users can apply to their model of choice. We demonstrate the algorithm on a few representative examples which underscore the importance of non-exponential distributions, memory, and coarse-graining in CTRWs.

Flat-panel Detector Perfusion Imaging and Conventional Multidetector Perfusion Imaging in Patients with Acute Ischemic Stroke : A Comparative Study.

PURPOSE: Flat-panel detector computed tomography (FDCT) is increasingly used in (neuro)interventional angiography suites. This study aimed to compare FDCT perfusion (FDCTP) with conventional multidetector computed tomography perfusion (MDCTP) in patients with acute ischemic stroke. METHODS: In this study, 19 patients with large vessel occlusion in the anterior circulation who had undergone mechanical thrombectomy, baseline MDCTP and pre-interventional FDCTP were included. Hypoperfused tissue volumes were manually segmented on time to maximum (Tmax) and time to peak (TTP) maps based on the maximum visible extent. Absolute and relative thresholds were applied to the maximum visible extent on Tmax and relative cerebral blood flow (rCBF) maps to delineate penumbra volumes and volumes with a high likelihood of irreversible infarcted tissue ("core"). Standard comparative metrics were used to evaluate the performance of FDCTP. RESULTS: Strong correlations and robust agreement were found between manually segmented volumes on MDCTP and FDCTP Tmax maps (r = 0.85, 95% CI 0.65-0.94, p < 0.001; ICC = 0.85, 95% CI 0.69-0.94) and TTP maps (r = 0.91, 95% CI 0.78-0.97, p < 0.001; ICC = 0.90, 95% CI 0.78-0.96); however, direct quantitative comparisons using thresholding showed lower correlations and weaker agreement (MDCTP versus FDCTP Tmax 6 s: r = 0.35, 95% CI -0.13-0.69, p = 0.15; ICC = 0.32, 95% CI 0.07-0.75). Normalization techniques improved results for Tmax maps (r = 0.78, 95% CI 0.50-0.91, p < 0.001; ICC = 0.77, 95% CI 0.55-0.91). Bland-Altman analyses indicated a slight systematic underestimation of FDCTP Tmax maximum visible extent volumes and slight overestimation of FDCTP TTP maximum visible extent volumes compared to MDCTP. CONCLUSION: FDCTP and MDCTP provide qualitatively comparable volumetric results on Tmax and TTP maps; however, direct quantitative measurements of infarct core and hypoperfused tissue volumes showed lower correlations and agreement.