Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.     

Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses

A systematic, comprehensive strategy that optimizes sample preparation and chromatography to minimize matrix effects in bioanalytical LC/MS/MS assays was developed. Comparisons were made among several sample preparation methods, including protein precipitation (PPT), liquid-liquid extraction (LLE), pure cation exchange solid-phase extraction (SPE), reversed-phase SPE and mixed-mode SPE. The influence of mobile phase pH and gradient duration on the selectivity and sensitivity for both matrix components and basic analytes was investigated. Matrix effects and overall sensitivity and resolution between UPLC technology and HPLC were compared. The amount of specific matrix components, or class of matrix components, was measured in the sample preparation extracts by LC/MS/MS with electrospray ionization (ESI) using both precursor ion scanning mode and multiple reaction monitoring (MRM). PPT is the least effective sample preparation technique, often resulting in significant matrix effects due to the presence of many residual matrix components. Reversed-phase and pure cation exchange SPE methods resulted in cleaner extracts and reduced matrix effects compared to PPT. The cleanest extracts, however, were produced with polymeric mixed-mode SPE (both reversed-phase and ion exchange retention mechanisms). These mixed-mode sorbents dramatically reduced the levels of residual matrix components from biological samples, leading to significant reduction in matrix effects. LLE also provided clean final extracts. However, analyte recovery, particularly for polar analytes, was very low. Mobile phase pH was manipulated to alter the retention of basic compounds relative to phospholipids, whose retention tends to be relatively independent of pH. In addition to the expected resolution, speed and sensitivity benefits of UPLC technology, a paired t-test demonstrated a statistically significant improvement with respect to matrix effects when this technology was chosen over traditional HPLC. The combination of polymeric mixed-mode SPE, the appropriate mobile phase pH and UPLC technology provides significant advantages for reducing matrix effects resulting from plasma matrix components and in improving the ruggedness and sensitivity of bioanalytical methods.     

Air pollution distribution patterns in the San Bernardino Mountains of southern California: a 40-year perspective

Since the mid-1950s, native pines in the San Bernardino Mountains (SBM) in southern California have shown symptoms of decline. Initial studies in 1963 showed that ozone (O3) generated in the upwind Los Angeles Basin was responsible for the injury and decline of sensitive trees. Ambient O3 decreased significantly by the mid-1990s, resulting in decreased O3 injury and improved tree growth. Increased growth of trees may also be attributed to elevated atmospheric nitrogen (N) deposition. Since most of the N deposition to mixed conifer forest stands in the SBM results from dry deposition of nitric acid vapor (HNO3) and ammonia (NH3), characterization of spatial and temporal distribution of these two pollutants has become essential. Although maximum daytime O3 concentrations over last 40 years have significantly decreased (approximately 3-fold), seasonal means have been reduced much less (approximately 1.5-fold), with 2-week long means occasionally exceeding 100 ppb in the western part of the range. In the same area, significantly elevated concentrations of HNO3 and NH3, up to 17.5 and 18.5 microg/m3 as 2-week averages, respectively, have been determined. Elevated levels of O3 and increased N deposition together with long-term drought predispose the SBM forests to massive bark beetle attacks making them susceptible to catastrophic fires.     

Comparative genomic analysis of Lactobacillus mucosae LM1 identifies potential niche-specific genes and pathways for gastrointestinal adaptation

Lactobacillus mucosae is currently of interest as putative probiotics due to their metabolic capabilities and ability to colonize host mucosal niches. L. mucosae LM1 has been studied in its functions in cell adhesion and pathogen inhibition, etc. It demonstrated unique abilities to use energy from carbohydrate and non-carbohydrate sources. Due to these functions, we report the first complete genome sequence of an L. mucosae strain, L. mucosae LM1. Analysis of the pan-genome in comparison with closely-related Lactobacillus species identified a complete glycogen metabolism pathway, as well as folate biosynthesis, complementing previous proteomic data on the LM1 strain. It also revealed common and unique niche-adaptation genes among the various L. mucosae strains. The aim of this study was to derive genomic information that would reveal the probable mechanisms underlying the probiotic effect of L. mucosae LM1, and provide a better understanding of the nature of L. mucosae sp.     

Phyllachora species infecting maize and other grass species in the Americas represents a complex of closely related species

The genus Phyllachora contains numerous obligate fungal parasites that produce raised, melanized structures called stromata on their plant hosts referred to as tar spot. Members of this genus are known to infect many grass species but generally do not cause significant damage or defoliation, with the exception of P. maydis which has emerged as an important pathogen of maize throughout the Americas, but the origin of this pathogen remains unknown. To date, species designations for Phyllachora have been based on host associations and morphology, and most species are assumed to be host specific. We assessed the sequence diversity of 186 single stroma isolates collected from 16 hosts representing 15 countries. Samples included both herbarium and contemporary strains that covered a temporal range from 1905 to 2019. These 186 isolates were grouped into five distinct species with strong bootstrap support. We found three closely related, but genetically distinct groups of Phyllachora are capable of infecting maize in the United States, we refer to these as the P. maydis species complex. Based on herbarium specimens, we hypothesize that these three groups in the P. maydis species complex originated from Central America, Mexico, and the Caribbean. Although two of these groups were only found on maize, the third and largest group contained contemporary strains found on maize and other grass hosts, as well as herbarium specimens from maize and other grasses that include 10 species of Phyllachora. The herbarium specimens were previously identified based on morphology and host association. This work represents the first attempt at molecular characterization of Phyllachora species infecting grass hosts and indicates some Phyllachora species can infect a broad range of host species and there may be significant synonymy in the Phyllachora genus.     

DEVELOPMENT,FUNCTION, AND THE QUANTITATIVE GENETICS OF WING MELANIN PATTERN IN PIERIS BUTTERFLIES

Do genetic correlations among phenotypic characters reflect developmental organization or functional coadaptation of the characters? We test these hypotheses for the wing melanin pattern of Pieris occidentalis butterflies, by comparing estimated genetic correlations among wing melanin characters with a priori predictions of the developmental organization and the functional (thermoregulatory) organization of melanin pattern. There were significant broad-sense heritabilities and significant genetic correlations for most melanin characters. Matrix correlation tests revealed significant agreement between the observed genetic correlations and both developmental and functional predictions in most cases; this occurred even when the overlap between developmental and functional predictions was eliminated. These results suggest that both developmental organization and functional coadaptation among melanin characters influence the genetic correlation structure of melanin pattern in this species. These results have two important implications for the evolution of melanin pattern in P. occidentalis and other butterflies: 1) most phenotypic variation in pattern may reflect variation among, rather than within, sets of developmentally homologous wing melanin characters; and 2) in a changing selective environment, genetic correlations may retard the disruption of functionally coupled melanin characters, thus affecting the evolutionary response to selection.     

A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK⁻ and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.