More Like this

1. Cardiac physiology and metabolic gene expression during late organogenesis among F. heteroclitus embryo families from crosses between pollution-sensitive and -resistant parents

   https://doi.org/10.1186/s12862-022-01959-1  

   Bozinovic, Goran ; Feng, Zuying ; Shea, Damian ; Oleksiak, Marjorie F. ( December 2022 , BMC Ecology and Evolution)

   Abstract Background The teleost fish Fundulus heteroclitus inhabit estuaries heavily polluted with persistent and bioaccumulative chemicals. While embryos of parents from polluted sites are remarkably resistant to toxic sediment and develop normally, embryos of parents from relatively clean estuaries, when treated with polluted sediment extracts, are developmentally delayed, displaying deformities characteristic of pollution-induced embryotoxicity. To gain insight into parental effects on sensitive and resistant phenotypes during late organogenesis, we established sensitive, resistant, and crossed embryo families using five female and five male parents from relatively clean and predominantly PAH-polluted estuaries each, measured heart rates, and quantified individual embryo expression of 179 metabolic genes. Results Pollution-induced embryotoxicity manifested as morphological deformities, significant developmental delays, and altered cardiac physiology was evident among sensitive embryos resulting from crosses between females and males from relatively clean estuaries. Significantly different heart rates among several geographically unrelated populations of sensitive, resistant, and crossed embryo families during late organogenesis and pre-hatching suggest site-specific adaptive cardiac physiology phenotypes relative to pollution exposure. Metabolic gene expression patterns (32 genes, 17.9%, at p < 0.05; 11 genes, 6.1%, at p < 0.01) among the embryo families indicate maternal pollutant deposition in the eggs and parental effects on gene expression and metabolic alterations. Conclusion Heartmore »rate differences among sensitive, resistant, and crossed embryos is a reliable phenotype for further explorations of adaptive mechanisms. While metabolic gene expression patterns among embryo families are suggestive of parental effects on several differentially expressed genes, a definitive adaptive signature and metabolic cost of resistant phenotypes is unclear and shows unexpected sensitive-resistant crossed embryo expression profiles. Our study highlights physiological and metabolic gene expression differences during a critical embryonic stage among pollution sensitive, resistant, and crossed embryo families, which may contribute to underlying resistance mechanisms observed in natural F. heteroclitus populations living in heavily contaminated estuaries.« less
2. Evolutionary Physiology and Genomics in the Highly Adaptable Killifish (Fundulus heteroc

   https://doi.org/10.1002/cphy.c190004  

   Crawford, D. L. Schulte ( April 2020 , Comprehensive physiology)

   By investigating evolutionary adaptations that change physiological functions, we can enhance our understanding of how organisms work, the importance of physiological traits, and the genes that influence these traits. This approach of investigating the evolution of physiological adaptation has been used with the teleost fish Fundulus heteroclitus and has produced insights into (i) how protein polymorphisms enhance swimming and development; (ii) the role of equilibrium enzymes in modulating metabolic flux; (iii) how variation in DNA sequences and mRNA expression patterns mitigate changes in temperature, pollution, and salinity; and (iv) the importance of nuclear-mitochondrial genome interactions for energy metabolism. Fundulus heteroclitus provides so many examples of adaptive evolution because their local population sizes are large, they have significant standing genetic variation, and they experience large ranges of environmental conditions that enhance the likelihood that adaptive evolution will occur. Thus, F. heteroclitus research takes advantage of evolutionary changes associated with exposure to diverse environments, both across the North American Atlantic coast and within local habitats, to contrast neutral versus adaptive divergence. Based on evolutionary analyses contrasting neutral and adaptive evolution in F. heteroclitus populations, we conclude that adaptive evolution can occur readily and rapidly, at least in part because it depends onmore »large amounts of standing genetic variation among many genes that can alter physiological traits. These observations of polygenic adaptation enhance our understanding of how evolution and physiological adaptation progresses, thus informing both biological and medical scientists about genotype-phenotype relationships« less
3. Combined noninvasive metabolic and spindle imaging as potential tools for embryo and oocyte assessment

   https://doi.org/10.1093/humrep/dez210  

   Sanchez, Tim ; Venturas, Marta ; Aghvami, S. Ali ; Yang, Xingbo ; Fraden, Seth ; Sakkas, Denny ; Needleman, Daniel J. ( December 2019 , Human Reproduction)

   Is the combined use of fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging and second harmonic generation (SHG) spindle imaging a feasible and safe approach for noninvasive embryo assessment?

   Metabolic imaging can sensitively detect meaningful metabolic changes in embryos, SHG produces high-quality images of spindles and the methods do not significantly impair embryo viability.

   Proper metabolism is essential for embryo viability. Metabolic imaging is a well-tested method for measuring metabolism of cells and tissues, but it is unclear if it is sensitive enough and safe enough for use in embryo assessment.

   This study consisted of time-course experiments and control versus treatment experiments. We monitored the metabolism of 25 mouse oocytes with a noninvasive metabolic imaging system while exposing them to oxamate (cytoplasmic lactate dehydrogenase inhibitor) and rotenone (mitochondrial oxidative phosphorylation inhibitor) in series. Mouse embryos (n = 39) were measured every 2 h from the one-cell stage to blastocyst in order to characterize metabolic changes occurring during pre-implantation development. To assess the safety of FLIM illumination, n = 144 illuminated embryos were implanted into n = 12 mice, and n = 108 nonilluminated embryos were implanted into n = 9 mice.

   Experiments were performed in mouse embryos and oocytes. Samples weremore »monitored with noninvasive, FLIM-based metabolic imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. Between NADH cytoplasm, NADH mitochondria and FAD mitochondria, a single metabolic measurement produces up to 12 quantitative parameters for characterizing the metabolic state of an embryo. For safety experiments, live birth rates and pup weights (mean ± SEM) were used as endpoints. For all test conditions, the level of significance was set at P < 0.05.

   Measured FLIM parameters were highly sensitive to metabolic changes due to both metabolic perturbations and embryo development. For oocytes, metabolic parameter values were compared before and after exposure to oxamate and rotenone. The metabolic measurements provided a basis for complete separation of the data sets. For embryos, metabolic parameter values were compared between the first division and morula stages, morula and blastocyst and first division and blastocyst. The metabolic measurements again completely separated the data sets. Exposure of embryos to excessive illumination dosages (24 measurements) had no significant effect on live birth rate (5.1 ± 0.94 pups/mouse for illuminated group; 5.7 ± 1.74 pups/mouse for control group) or pup weights (1.88 ± 0.10 g for illuminated group; 1.89 ± 0.11 g for control group).

   The study was performed using a mouse model, so conclusions concerning sensitivity and safety may not generalize to human embryos. A limitation of the live birth data is also that although cages were routinely monitored, we could not preclude that some runt pups may have been eaten.

   Promising proof-of-concept results demonstrate that FLIM with SHG provide detailed biological information that may be valuable for the assessment of embryo and oocyte quality. Live birth experiments support the method’s safety, arguing for further studies of the clinical utility of these techniques.

   Supported by the Blavatnik Biomedical Accelerator Grant at Harvard University and by the Harvard Catalyst/The Harvard Clinical and Translational Science Center (National Institutes of Health Award UL1 TR001102), by NSF grants DMR-0820484 and PFI-TT-1827309 and by NIH grant R01HD092550-01. T.S. was supported by a National Science Foundation Postdoctoral Research Fellowship in Biology grant (1308878). S.F. and S.A. were supported by NSF MRSEC DMR-1420382. Becker and Hickl GmbH sponsored the research with the loaning of equipment for FLIM. T.S. and D.N. are cofounders and shareholders of LuminOva, Inc., and co-hold patents (US20150346100A1 and US20170039415A1) for metabolic imaging methods. D.S. is on the scientific advisory board for Cooper Surgical and has stock options with LuminOva, Inc.

   « less
4. Measurement of oxygen consumption rates of human renal proximal tubule cells in an array of organ-on-chip devices to monitor drug-induced metabolic shifts

   https://doi.org/10.1038/s41378-022-00442-7  

   Kann, Samuel H. ; Shaughnessey, Erin M. ; Coppeta, Jonathan R. ; Azizgolshani, Hesham ; Isenberg, Brett C. ; Vedula, Else M. ; Zhang, Xin ; Charest, Joseph L. ( September 2022 , Microsystems & Nanoengineering)

   Measurement of cell metabolism in moderate-throughput to high-throughput organ-on-chip (OOC) systems would expand the range of data collected for studying drug effects or disease in physiologically relevant tissue models. However, current measurement approaches rely on fluorescent imaging or colorimetric assays that are focused on endpoints, require labels or added substrates, and lack real-time data. Here, we integrated optical-based oxygen sensors in a high-throughput OOC platform and developed an approach for monitoring cell metabolic activity in an array of membrane bilayer devices. Each membrane bilayer device supported a culture of human renal proximal tubule epithelial cells on a porous membrane suspended between two microchannels and exposed to controlled, unidirectional perfusion and physiologically relevant shear stress for several days. For the first time, we measured changes in oxygen in a membrane bilayer format and used a finite element analysis model to estimate cell oxygen consumption rates (OCRs), allowing comparison with OCRs from other cell culture systems. Finally, we demonstrated label-free detection of metabolic shifts in human renal proximal tubule cells following exposure to FCCP, a drug known for increasing cell oxygen consumption, as well as oligomycin and antimycin A, drugs known for decreasing cell oxygen consumption. The capability to measure cellmore »OCRs and detect metabolic shifts in an array of membrane bilayer devices contained within an industry standard microtiter plate format will be valuable for analyzing flow-responsive and physiologically complex tissues during drug development and disease research.

   « less
5. Biosensor for branched-chain amino acid metabolism in yeast and applications in isobutanol and isopentanol production

   https://doi.org/10.1038/s41467-021-27852-x  

   Zhang, Yanfei ; Cortez, Jeremy D. ; Hammer, Sarah K. ; Carrasco-López, César ; García Echauri, Sergio Á. ; Wiggins, Jessica B. ; Wang, Wei ; Avalos, José L. ( January 2022 , Nature Communications)

   Branched-chain amino acid (BCAA) metabolism fulfills numerous physiological roles and can be harnessed to produce valuable chemicals. However, the lack of eukaryotic biosensors specific for BCAA-derived products has limited the ability to develop high-throughput screens for strain engineering and metabolic studies. Here, we harness the transcriptional regulator Leu3p fromSaccharomyces cerevisiaeto develop a genetically encoded biosensor for BCAA metabolism. In one configuration, we use the biosensor to monitor yeast production of isobutanol, an alcohol derived from valine degradation. Small modifications allow us to redeploy Leu3p in another biosensor configuration that monitors production of the leucine-derived alcohol, isopentanol. These biosensor configurations are effective at isolating high-producing strains and identifying enzymes with enhanced activity from screens for branched-chain higher alcohol (BCHA) biosynthesis in mitochondria as well as cytosol. Furthermore, this biosensor has the potential to assist in metabolic studies involving BCAA pathways, and offers a blueprint to develop biosensors for other products derived from BCAA metabolism.