The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology.

Harnessing the process of natural selection to obtain and understand new microbial phenotypes has become increasingly possible due to advances in culturing techniques, DNA sequencing, bioinformatics, and genetic engineering. Accordingly, Adaptive Laboratory Evolution (ALE) experiments represent a powerful approach both to investigate the evolutionary forces influencing strain phenotypes, performance, and stability, and to acquire production strains that contain beneficial mutations. In this review, we summarize and categorize the applications of ALE to various aspects of microbial physiology pertinent to industrial bioproduction by collecting case studies that highlight the multitude of ways in which evolution can facilitate the strain construction process. Further, we discuss principles that inform experimental design, complementary approaches such as computational modeling that help maximize utility, and the future of ALE as an efficient strain design and build tool driven by growing adoption and improvements in automation.

The small membrane protein MgrB regulates PhoQ bifunctionality to control PhoP target gene expression dynamics.

In Escherichia coli and other γ-proteobacteria, the PhoQ-PhoP two-component signaling system responds to low extracellular Mg++ and cationic antimicrobial peptides. On transition to inducing conditions, the expression of PhoP-dependent genes increases rapidly, but then decays to a new, intermediate steady-state level, a phenomenon often referred to as partial adaptation. The molecular basis for this partial adaptation has been unclear. Here, using time-lapse fluorescence microscopy to examine PhoP-dependent gene expression in individual E. coli cells we show that partial adaptation arises through a negative feedback loop involving the small protein MgrB. When E. coli cells are shifted to low Mg++ , PhoQ engages in multiple rounds of autophosphorylation and phosphotransfer to PhoP, which, in turn, drives the expression of mgrB. MgrB then feeds back to inhibit the kinase activity of PhoQ. PhoQ is bifunctional such that, when not active as a kinase, it can stimulate the dephosphorylation of PhoP. Thus, MgrB drives the inactivation of PhoP and the observed adaptation in PhoP-dependent gene expression. Our results clarify the source of feedback inhibition in the E. coli PhoQ-PhoP system and reveal how exogenous factors, such as MgrB, can combine with a canonical two-component signaling pathway to produce complex temporal dynamics in target gene expression.

Real-world Evidence of Diagnostic Testing and Treatment Patterns in US Patients With Breast Cancer With Implications for Treatment Biomarkers From RNA Sequencing Data.

OBJECTIVE/BACKGROUND: We performed a retrospective analysis of longitudinal real-world data (RWD) from patients with breast cancer to replicate results from clinical studies and demonstrate the feasibility of generating real-world evidence. We also assessed the value of transcriptome profiling as a complementary tool for determining molecular subtypes. METHODS: De-identified, longitudinal data were analyzed after abstraction from records of patients with breast cancer in the United States (US) structured and stored in the Tempus database. Demographics, clinical characteristics, molecular subtype, treatment history, and survival outcomes were assessed according to strict qualitative criteria. RNA sequencing and clinical data were used to predict molecular subtypes and signaling pathway enrichment. RESULTS: The clinical abstraction cohort (n = 4000) mirrored the demographics and clinical characteristics of patients with breast cancer in the US, indicating feasibility for RWE generation. Among patients who were human epidermal growth factor receptor 2-positive (HER2+), 74.2% received anti-HER2 therapy, with ∼70% starting within 3 months of a positive test result. Most non-treated patients were early stage. In this RWD set, 31.7% of patients with HER2+ immunohistochemistry (IHC) had discordant fluorescence in situ hybridization results recorded. Among patients with multiple HER2 IHC results at diagnosis, 18.6% exhibited intra-test discordance. Through development of a whole-transcriptome model to predict IHC receptor status in the molecular sequenced cohort (n = 400), molecular subtypes were resolved for all patients (n = 36) with equivocal HER2 statuses from abstracted test results. Receptor-related signaling pathways were differentially enriched between clinical molecular subtypes. CONCLUSIONS: RWD in the Tempus database mirrors the overall population of patients with breast cancer in the US. These results suggest that real-time, RWD analyses are feasible in a large, highly heterogeneous database. Furthermore, molecular data may aid deficiencies and discrepancies observed from breast cancer RWD.

Genetic Determinants Enabling Medium-Dependent Adaptation to Nafcillin in Methicillin-Resistant Staphylococcus aureus.

Antimicrobial susceptibility testing standards driving clinical decision-making have centered around the use of cation-adjusted Mueller-Hinton broth (CA-MHB) as the medium with the notion of supporting bacterial growth, without consideration of recapitulating the in vivo environment. However, it is increasingly recognized that various medium conditions have tremendous influence on antimicrobial activity, which in turn may have major implications on the ability of in vitro susceptibility assays to predict antibiotic activity in vivo. To elucidate differential growth optimization and antibiotic resistance mechanisms, adaptive laboratory evolution was performed in the presence or absence of the antibiotic nafcillin with methicillin-resistant Staphylococcus aureus (MRSA) TCH1516 in either (i) CA-MHB, a traditional bacteriological nutritionally rich medium, or (ii) Roswell Park Memorial Institute (RPMI), a medium more reflective of the in vivo host environment. Medium adaptation analysis showed an increase in growth rate in RPMI, but not CA-MHB, with mutations in apt, adenine phosphoribosyltransferase, and the manganese transporter subunit, mntA, occurring reproducibly in parallel replicate evolutions. The medium-adapted strains showed no virulence attenuation. Continuous exposure of medium-adapted strains to increasing concentrations of nafcillin led to medium-specific evolutionary strategies. Key reproducibly occurring mutations were specific for nafcillin adaptation in each medium type and did not confer resistance in the other medium environment. Only the vraRST operon, a regulator of membrane- and cell wall-related genes, showed mutations in both CA-MHB- and RPMI-evolved strains. Collectively, these results demonstrate the medium-specific genetic adaptive responses of MRSA and establish adaptive laboratory evolution as a platform to study clinically relevant resistance mechanisms.IMPORTANCE The ability of pathogens such as Staphylococcus aureus to evolve resistance to antibiotics used in the treatment of infections has been an important concern in the last decades. Resistant acquisition usually translates into treatment failure and puts patients at risk of unfavorable outcomes. Furthermore, the laboratory testing of antibiotic resistance does not account for the different environment the bacteria experiences within the human body, leading to results that do not translate into the clinic. In this study, we forced methicillin-resistant S. aureus to develop nafcillin resistance in two different environments, a laboratory environment and a physiologically more relevant environment. This allowed us to identify genetic changes that led to nafcillin resistance under both conditions. We concluded that not only does the environment dictate the evolutionary strategy of S. aureus to nafcillin but also that the evolutionary strategy is specific to that given environment.

Exposure of caged mussels to metals in a primary-treated municipal wastewater plume.

The biological availability of metals in municipal wastewater effluents is strongly influenced by the physical and chemical conditions of both the effluent and the receiving water. Aquatic organisms are exposed to both dissolved and particulate (food ingestion) forms of these metals. In the present study, the distribution of metals in specific tissues was used to distinguish between exposure routes (i.e. dissolved vs. particulate phase) and to examine metal bioavailability in mussels exposed to municipal effluents. Caged Elliptio complanata mussels were deployed at sites located between 1.5 km upstream and 12 km downstream of a major effluent outfall in the St. Lawrence River. Metals in surface water samples were fractionated by filtration techniques to determine their dissolved, truly-dissolved (<10 kDa), total-particulate and acid-reactive-particulate forms. At the end of the exposure period (90 days), pooled mussel soft tissues (digestive gland, gills, gonad, foot and mantle) were analyzed for several metals. The results showed that gills and digestive gland were generally the most important target tissues for metal bioaccumulation, while gill/digestive gland metal ratios suggest that both exposure routes should be considered for mussels exposed to municipal effluents. We also found that Ag and Cd in the dispersion plume nearest the outfall, in contrast to other metals such as Cu and Zn, are more closely associated with colloids and were generally less bioavailable than at the reference site in the St. Lawrence River.

Contaminant exposures at the 4H shell mounds in the Santa Barbara Channel.

Remobilization, bioavailability, and potential toxicity of chemical contaminants were evaluated at the 4H shell mounds - the site of abandoned offshore oil and gas production platforms in the Santa Barbara Channel region of the Southern California Bight. Evaluations used a weight-of-evidence approach based on results from bulk phase chemical analyses and laboratory toxicity testing of shell mound cores, in situ field bioassays using caged mussels, and surficial sediment chemistry. Shell mound cores contained elevated concentrations of metals associated with drilling wastes (e.g., Ba, Cr, Pb, and Zn), as well as monocyclic and polycyclic aromatic hydrocarbons (PAHs). The highest concentrations along with pockets of free oil were associated with the middle "cuttings" stratum. Sediments composited from all core strata caused significant acute toxicity and bioaccumulation of Ba and PAHs in test organisms during laboratory exposures. In contrast, caged mussels placed at each of the shell mounds for a period of 57-58 days had greater than 90% survival, and there were no significant differences in survival of mussels placed at the shell mounds and corresponding reference sites. While all mussel samples exhibited increases in shell length, whole animal weight, and tissue lipid content, in some cases growth metrics for the shell mound mussels were significantly higher than those for the reference sites. Concentrations of metals, PAHs, and polychlorinated biphenyls (PCBs) in tissues of the shell mound mussels were not significantly different from those at reference sites. The presence of labile aromatic hydrocarbons in shell mound cores and absence of significant contaminant accumulation of tissues of caged mussels indicated that chemical contaminants are not being remobilized from the 4H shell mounds. Surficial bottom sediments near the shell mounds contained elevated Ba concentrations that probably were associated with drilling wastes. However, concentrations did not exhibit clear spatial gradients with distance from the shell mounds. Despite a number of storm events during the mussel exposures, maximum currents were 34 cm s(-1) and unlikely to erode materials from the shell mounds. Thus, Ba distributions in bottom sediments probably were due to episodic disturbance such as platform removal or trawling rather than ongoing erosion and dispersion of shell mound solids by near-bottom currents. These results suggest that, in the absence of physical disturbances, contaminants are expected to remain sequestered in the shell mounds.

Temporal and evolutionary dynamics of two-component signaling pathways.

Bacteria sense and respond to numerous environmental signals through two-component signaling pathways. Typically, a given stimulus will activate a sensor histidine kinase to autophosphorylate and then phosphotransfer to a cognate response regulator, which can mount an appropriate response. Although these signaling pathways often appear to be simple switches, they can also orchestrate surprisingly sophisticated and complex responses. These temporal dynamics arise from several key regulatory features, including the bifunctionality of histidine kinases as well as positive and negative feedback loops. Two-component signaling pathways are also dynamic on evolutionary time-scales, expanding dramatically in many species through gene duplication and divergence. Here, we review recent work probing the temporal and evolutionary dynamics of two-component signaling systems.

Properties of a Method for Performing Adaptive, Multilevel QM Simulations of Complex Chemical Reactions in the Gas-Phase.

The properties of a new method of performing molecular dynamic simulations of complex chemical processes are presented. The method is formulated to give a time-dependent, multilevel representation of the total potential that is derived from spatially resolved quantum mechanical regions. An illustrative simulation is performed on a 110 atom system to demonstrate the continuity and energy conserving properties of the method. The effect of a discontinuous total potential upon the kinetic energy of the system is examined. The discontinuities in the magnitude of atomic force vectors due to changing the electronic structure during the simulation are examined as well as the effect that these discontinuities have upon the atomic kinetic energies. The method, while not conserving total energy, does yield canonical (NVT) simulations. The time reversibility property of the simulation with an extremely discontinuous total potential is discussed. The computational scaling associated with the formation of the spatially resolved, time-dependent groups is also investigated.

Review of aquatic in situ approaches for stressor and effect diagnosis.

Field-based (in situ) approaches are used increasingly for measuring biological effects and for stressor diagnoses in aquatic systems because these assessment tools provide realistic exposure environments that are rarely replicated in laboratory toxicity tests. Providing realistic exposure scenarios is important because environmental conditions can alter toxicity through complex exposure dynamics (e.g., multiple stressor interactions). In this critical review, we explore the information provided by aquatic in situ exposure and monitoring methods when compared with more traditional approaches and discuss the associated strengths and limitations of these techniques. In situ approaches can, under some circumstances, provide more valuable information to a decision maker than information from surveys of resident biota, laboratory toxicity tests, or chemical analyses alone. A decision tree is provided to assist decision makers in determining when in situ approaches can add value.

Molecular dynamics of complex gas-phase reactive systems by time-dependent groups.

A novel way of assembling the total potential for performing molecular dynamical studies of complex gas-phase reactive chemical systems is introduced. The method breaks the calculation of the total potential and gradients of the potential into time-dependent groups that are governed by spatial cutoffs. These groups evolve during the course of the simulation and their number may increase or diminish as the dynamics of the system determine. In an effort to extend the simulation time of these complex reactive processes and to use high levels of theory when necessary, multiple levels of theory may be used over the groups for the calculation of both the intragroup and intergroup interactions. Representative simulations are performed to illustrate the method and a computationally facile method of obtaining the groups of a simulation are also discussed.

CD14 tobacco gene-environment interaction modifies asthma severity and immunoglobulin E levels in Latinos with asthma.

BACKGROUND: A recent family-based genomewide screen revealed linkage between the 5q31 region and the diagnosis of asthma, but only in those exposed to environmental tobacco smoke (ETS). Among the candidate genes in this region is CD14. METHODS: To determine whether polymorphisms in the CD14 gene are related to this gene-by-environment interaction in Latinos, we used both family-based and cross-sectional cohort analysis to test for interactions between CD14 genotypes/haplotypes, exposure to ETS, and asthma-related phenotypes in 659 Mexican and Puerto Rican families. RESULTS: We identified 21 single nucleotide polymorphisms (SNPs) in the CD14 gene by sequencing 72 Puerto Ricans, Mexicans, and African Americans with asthma. Three SNPs, -810, -159, and +1437, were further genotyped in families with asthma. Among all subjects with asthma exposed to ETS, without regard to ethnicity, CD14 +1437 genotypes were associated with asthma severity. SNP +1437 GG or GC genotypes were significantly associated with lower baseline FEV1 using both family-based (p = 0.0009) and cross-sectional cohort (p = 0.03) analyses. Subjects with asthma with the GG or GC genotypes who were exposed to ETS had mean baseline FEV1 (% predicted) values 8.6% lower than subjects not exposed to ETS (p = 0.03). As previously observed in whites, we found an interaction between plasma IgE levels, SNP -159 genotypes, and ETS exposure (p = 0.0002). The lowest IgE levels were in those subjects with the TT genotype and who were exposed to ETS regardless of ethnicity. CONCLUSIONS: Our data suggest a gene-by-environment interaction between CD14 genotypes and ETS, which affects pulmonary function and IgE levels among Latinos with asthma.

Pharmacogenetic differences in response to albuterol between Puerto Ricans and Mexicans with asthma.

BACKGROUND: In the United States, Puerto Ricans and Mexicans have the highest and lowest asthma prevalence, morbidity, and mortality, respectively. Ethnic-specific differences in the response to drug treatment may contribute to differences in disease outcomes. Genetic variants at the beta(2)-adrenergic receptor (beta(2)AR) may modify asthma severity and albuterol responsiveness. We tested the association of beta(2)AR genotypes with asthma severity and bronchodilator response to albuterol in Puerto Ricans and Mexicans with asthma. METHODS: We used both family-based and cross-sectional tests of association with 8 beta(2)AR single nucleotide polymorphisms in 684 Puerto Rican and Mexican families. Regression analyses were used to determine the interaction between genotype, asthma severity, and bronchodilator drug responsiveness. RESULTS: Among Puerto Ricans with asthma, the arginine (Arg) 16 allele was associated with greater bronchodilator response using both family-based and cross-sectional tests (p = 0.00001-0.01). We found a strong interaction of baseline FEV(1) with the Arg16Glycine (Gly) polymorphism in predicting bronchodilator response. Among Puerto Ricans with asthma with baseline FEV(1) < 80% of predicted, but not in those with FEV(1) > 80%, there was a very strong association between the Arg16 genotype and greater bronchodilator responsiveness. No association was observed between Arg16Gly genotypes and drug responsiveness among Mexicans with asthma. CONCLUSIONS: Ethnic-specific pharmacogenetic differences exist between Arg16Gly genotypes, asthma severity, and bronchodilator response in Puerto Ricans and Mexicans with asthma. These findings underscore the need for additional research on racial/ethnic differences in asthma morbidity and drug responsiveness.

Global potential energy surfaces for the Al+(1S) + H2 system.

Global, three-dimensional multireference ab initio potential energy surfaces have been calculated for the AlH2+ system for the two lowest energy singlet states and the lowest energy triplet state. These surfaces were calculated using the multireference configuration interaction level of theory with a large basis set. The accuracy of the surfaces were checked against available experimental data and previous theoretical investigations. The areas of surface crossings between the ground state singlet surface and the lowest energy triplet surface and the first excited singlet surface have been thoroughly investigated in all three dimensions and found to give rise to two regions of surface crossings--an "early" crossing (reduced H2 distance) and a "late" crossing (enlarged H2 distance). It is anticipated that both of these crossings will be important in modeling the dynamics of the system. Each of the global potential energy surfaces were fit by interpolation methodology to obtain analytic representations of the surfaces. A representative classical simulation on the ground state singlet surface was performed and discussed.

Lower bronchodilator responsiveness in Puerto Rican than in Mexican subjects with asthma.

In the United States, Puerto Ricans and Mexicans have the highest and lowest asthma prevalence, morbidity, and mortality, respectively. To determine whether ethnicity-specific differences in therapeutic response, clinical response, and/or genetic factors contribute to differences in asthma outcomes, we compared asthma-related clinical characteristics among 684 Mexican and Puerto Rican individuals with asthma recruited from San Francisco, New York City, Puerto Rico, and Mexico City. Puerto Ricans with asthma had reduced lung function, greater morbidity, and longer asthma duration than did Mexicans with asthma. Bronchodilator responsiveness, measured as percentage change from baseline FEV1, was significantly lower among Puerto Ricans with asthma than among Mexicans with asthma. Puerto Ricans with asthma had on average 7.3% (95% confidence interval [CI], 4.6 to 9.9; p < 0.001) lower bronchodilator reversibility in FEV1, higher risk of an emergency department visit in the previous year (odds ratio, 2.63; 95% CI, 1.6 to 4.3; p < 0.001), and of previous hospitalization for asthma (odds ratio, 1.94; 95% CI, 1.2 to 3.2; p = 0.009) than Mexicans. Subgroup analysis corroborated that Puerto Ricans with asthma had more severe disease than did Mexicans on the basis of lung function measurements, responsiveness to beta2-adrenergic agonists, and health care use. We conclude that Puerto Ricans with asthma respond less to albuterol than do Mexicans with asthma. These findings underscore the need for additional research on racial/ethnic differences in asthma morbidity and response to therapy.