Publications by authors named "Christine Strand"

14 Publications

  • Page 1 of 1

Neurodegeneration in juvenile Iberian pigs with diet-induced nonalcoholic fatty liver disease.

Am J Physiol Endocrinol Metab 2020 09 3;319(3):E592-E606. Epub 2020 Aug 3.

Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, California.

The objective of this study was to investigate whether juvenile Iberian pigs with diet-induced nonalcoholic fatty liver disease (NAFLD), cholestasis, and gut dysbiosis would develop histological and metabolic markers of neurodegeneration in the frontal cortex (FC) and whether supplementing probiotics would influence the response to the diet. Twenty-eight juvenile Iberian pigs were fed for 10 wk either a control (CON) or high-fructose high-fat (HFF) diet with or without a commercial probiotic mixture. Compared with CON, HFF-fed pigs had a decreased number of neurons and an increase in reactive astrocytes in FC tissue. There was also a decrease in one-carbon metabolites choline and betaine and a marked accumulation of bile acids, cholesteryl esters, and polyol pathway intermediates in FC of HFF-fed pigs, which were associated with markers of neurodegeneration and accentuated with the severity of NAFLD. Betaine depletion in FC tissue was negatively correlated with choline-derived phospholipids in colon content, whereas primary conjugated bile acids in FC were associated with cholestasis. Plasma kynurenine-to-tryptophan quotient, as a marker of indoleamine 2,3-dioxygenase activity, and intestinal dysbiosis were also correlated with neuronal loss and astrogliosis. Recognition memory test and FC levels of amyloid-β and phosphorylated Tau did not differ between diets, whereas probiotics increased amyloid-β and memory loss in HFF-fed pigs. In conclusion, our results show evidence of neurodegeneration in FC of juvenile Iberian pigs and establish a novel pediatric model to investigate the role of gut-liver-brain axis in diet-induced NAFLD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpendo.00120.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864229PMC
September 2020

Optimization of AsCas12a for combinatorial genetic screens in human cells.

Nat Biotechnol 2021 01 13;39(1):94-104. Epub 2020 Jul 13.

Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41587-020-0600-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854777PMC
January 2021

Genetic screens in isogenic mammalian cell lines without single cell cloning.

Nat Commun 2020 02 6;11(1):752. Epub 2020 Feb 6.

Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, MA, 02142, USA.

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs of mammalian cells, however, is labor-intensive, time-consuming, and, in some cell types, essentially impossible. Here, we present an approach to create isogenic pairs of cells that avoids single cell cloning, and screen these pairs with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We query the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, identifying both expected and uncharacterized buffering and synthetic lethal interactions. Additionally, we compare acute CRISPR-based knockout, single cell clones, and small-molecule inhibition. We observe that, while the approaches provide largely overlapping information, differences emerge, highlighting an important consideration when employing genetic screens to identify and characterize potential drug targets. We anticipate that this methodology will be broadly useful to comprehensively study gene function across many contexts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-14620-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005275PMC
February 2020

Natural variation in arsenic toxicity is explained by differences in branched chain amino acid metabolism.

Elife 2019 04 8;8. Epub 2019 Apr 8.

Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, United States.

We find that variation in the gene underlies natural differences in responses to the toxin arsenic. This gene encodes the E2 subunit of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, a core component of branched-chain amino acid (BCAA) metabolism. We causally linked a non-synonymous variant in the conserved lipoyl domain of DBT-1 to differential arsenic responses. Using targeted metabolomics and chemical supplementation, we demonstrate that differences in responses to arsenic are caused by variation in iso-branched chain fatty acids. Additionally, we show that levels of branched chain fatty acids in human cells are perturbed by arsenic treatment. This finding has broad implications for arsenic toxicity and for arsenic-focused chemotherapeutics across human populations. Our study implicates the BCKDH complex and BCAA metabolism in arsenic responses, demonstrating the power of natural genetic diversity to identify novel mechanisms by which environmental toxins affect organismal physiology.

Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.40260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453569PMC
April 2019

Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities.

Nat Commun 2018 12 21;9(1):5416. Epub 2018 Dec 21.

Broad Institute of Harvard and MIT, 75 Ames Street, Cambridge, MA, 02142, USA.

The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-07901-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303322PMC
December 2018

Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens.

PLoS One 2018 25;13(5):e0197547. Epub 2018 May 25.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.

Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197547PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969736PMC
December 2018

Food consumption increases cell proliferation in the python brain.

J Exp Biol 2018 04 6;221(Pt 7). Epub 2018 Apr 6.

Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA 93407-0401, USA

Pythons are model organisms for investigating physiological responses to food intake. While systemic growth in response to food consumption is well documented, what occurs in the brain is currently unexplored. In this study, male ball pythons () were used to test the hypothesis that food consumption stimulates cell proliferation in the brain. We used 5-bromo-12'-deoxyuridine (BrdU) as a cell-birth marker to quantify and compare cell proliferation in the brain of fasted snakes and those at 2 and 6 days after a meal. Throughout the telencephalon, cell proliferation was significantly increased in the 6 day group, with no difference between the 2 day group and controls. Systemic postprandial plasticity occurs quickly after a meal is ingested, during the period of active digestion; however, the brain displays a surge of cell proliferation after most digestion and absorption is complete.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jeb.173377DOI Listing
April 2018

Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.

Nat Biotechnol 2018 02 18;36(2):179-189. Epub 2017 Dec 18.

Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nbt.4048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800952PMC
February 2018

Natural variation in a single amino acid substitution underlies physiological responses to topoisomerase II poisons.

PLoS Genet 2017 Jul 12;13(7):e1006891. Epub 2017 Jul 12.

Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, Illinois, United States of America.

Many chemotherapeutic drugs are differentially effective from one patient to the next. Understanding the causes of this variability is a critical step towards the development of personalized treatments and improvements to existing medications. Here, we investigate sensitivity to a group of anti-neoplastic drugs that target topoisomerase II using the model organism Caenorhabditis elegans. We show that wild strains of C. elegans vary in their sensitivity to these drugs, and we use an unbiased genetic approach to demonstrate that this natural variation is explained by a methionine-to-glutamine substitution in topoisomerase II (TOP-2). The presence of a non-polar methionine at this residue increases hydrophobic interactions between TOP-2 and its poison etoposide, as compared to a polar glutamine. We hypothesize that this stabilizing interaction results in increased genomic instability in strains that contain a methionine residue. The residue affected by this substitution is conserved from yeast to humans and is one of the few differences between the two human topoisomerase II isoforms (methionine in hTOPIIα and glutamine in hTOPIIβ). We go on to show that this amino acid difference between the two human topoisomerase isoforms influences cytotoxicity of topoisomerase II poisons in human cell lines. These results explain why hTOPIIα and hTOPIIβ are differentially affected by various poisons and demonstrate the utility of C. elegans in understanding the genetics of drug responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1006891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529024PMC
July 2017

Experimentally altered navigational demands induce changes in the cortical forebrain of free-ranging northern pacific rattlesnakes (Crotalus o. oreganus).

Brain Behav Evol 2012 6;79(3):144-54. Epub 2012 Jan 6.

Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, Calif., USA.

The hippocampus of birds and mammals plays a crucial role in spatial memory and navigation. The hippocampus exhibits plasticity in adulthood in response to diverse environmental factors associated with spatial demands placed on an animal. The medial and dorsal cortices of the telencephalon of squamate reptiles have been implicated as functional homologues to the hippocampus. This study sought to experimentally manipulate the navigational demands placed on free-ranging northern Pacific rattlesnakes (Crotalus o. oreganus) to provide direct evidence of the relationship between spatial demands and neuroplasticity in the cortical telencephalon of the squamate brain. Adult male rattlesnakes were radio-tracked for 2 months, during which time 1 of 3 treatments was imposed weekly, namely 225-meter translocation in a random direction, 225-meter walk and release at that day's capture site (handling control) or undisturbed (control). Snakes were then sacrificed and the brains were removed and processed for histological analysis of cortical features. The activity range was larger in the translocated (Tr) group compared to the handled (Hd) and undisturbed control (Cn) groups when measured via 95% minimum convex polygon (MCP). At the 100% MCP level, Tr snakes had larger activity ranges than the Cn snakes only. The volume of the medial cortex (MC) was larger in the Tr group compared to the Cn group. The MC of Hd snakes was not significantly different from that of either of the other groups. No differences in dorsal cortex (DC) or lateral cortex volumes were detected among the groups. Numbers of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in the MC and DC 3 weeks after BrdU injection were not affected by treatment. This study establishes a causal relationship between navigational demands and greater MC volume in a free-ranging reptile.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000335034DOI Listing
August 2012

Human disturbance alters endocrine and immune responses in the Galapagos marine iguana (Amblyrhynchus cristatus).

Horm Behav 2010 Nov 11;58(5):792-9. Epub 2010 Aug 11.

Department of Biology, Utah State University, Logan, UT 84322, USA.

Anthropogenic disturbance is a relevant and widespread facilitator of environmental change and there is clear evidence that it impacts natural populations. While population-level responses to major anthropogenic changes have been well studied, individual physiological responses to mild disturbance can be equally critical to the long-term survival of a species, yet they remain largely unexamined. The current study investigated the impact of seemingly low-level anthropogenic disturbance (ecotourism) on stress responsiveness and specific fitness-related immune measures in different breeding stages of the marine iguana (Amblyrhynchus cristatus). Specifically, we found stress-induced elevations in plasma corticosterone among tourist-exposed populations relative to undisturbed populations. We also found changes in multiple immunological responses associated with stress-related effects of human disturbance, including bacterial killing ability, cutaneous wound healing, and hemolytic complement activity, and the responses varied according to reproductive state. By identifying health-related consequences of human disturbance, this study provides critical insight into the conservation of a well-known species that has a very distinct ecology. The study also broadens the foundation of knowledge needed to understand the global significance of various levels of human disturbance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yhbeh.2010.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2982938PMC
November 2010

Plasticity of the Rufous-winged Sparrow, Aimophila carpalis, song control regions during the monsoon-associated summer breeding period.

Horm Behav 2007 Sep 29;52(3):401-8. Epub 2007 Jun 29.

Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA 93407, USA.

In most temperate zone songbirds, exposure to increasing photoperiod in the spring stimulates the reproductive system and induces reproductive behaviors. Additionally, the brain regions that control singing (song control regions; SCRs) are larger during the breeding season, thus paralleling changes in the activity of the reproductive system. However, in some birds, environmental factors other than photoperiod initiate breeding. For example, free-living male Rufous-winged Sparrows develop their testes in March due to increasing photoperiod, but have relatively low plasma T until after they begin to breed, usually in July, during the monsoon period when day length is declining. We tested the hypothesis that SCRs grow and singing behavior increases after the monsoon rains begin. We captured adult male Rufous-winged Sparrows in July 2002, 7 days before and 20 days after the monsoon rains began, euthanized birds in the field, collected their brains, and measured SCR volumes from sections immunostained for the neuronal marker NeuN. In June and July 2006, we measured song rates in the field before and after the monsoon rains. SCR volumes were larger and singing behavior increased after the onset of the monsoon rains, coinciding with the initiation of breeding. Unlike in other species studied so far, SCR volumes grew as day length was decreasing. Comparative studies utilizing species that do not breed when day length is increasing may provide information on the relative contributions of various environmental factors to SCR neuroplasticity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yhbeh.2007.06.006DOI Listing
September 2007

Hormonal and environmental control of song control region growth and new neuron addition in adult male house finches, Carpodacus mexicanus.

Dev Neurobiol 2007 May;67(6):827-37

School of Life Sciences, Arizona State University, Tempe, Arizona 85287, USA.

In songbirds, testosterone (T) mediates seasonal changes in the sizes and neuroanatomical characteristics of brain regions that control singing (song control regions; SCRs). One model explaining the mechanisms of the growth of one SCR, the HVC, postulates that in the spring increasing photoperiod and circulating T concentrations enhance new neuron survival, thus increasing total neuron number. However, most research investigating the effects of T on new neuron survival has been done in autumn. The present study investigated the effects of photoperiod and T treatment on SCR growth and new neuron survival in the HVC in photosensitive adult male House Finches, Carpodacus mexicanus, under simulated spring-like conditions. Birds were castrated, given T-filled or empty Silastic capsules and maintained on short days (SD; 8L:16D) or long days (LD; 16L:8D). To mark new cells, birds received bromodeoxyuridine injections 11 days after experimental manipulations began and were sacrificed 28 days later. Testosterone treatment increased the sizes of two SCRs, the HVC and Robust nucleus of the arcopallium (RA). Exposure to LD did not affect HVC volume, but did increase RA volume. Testosterone treatment increased the total number of HVC neurons, but did not affect the number of new HVC neurons. Thus, T initiates SCR growth and increases neuron survival, but effects of T on new neuron incorporation may be limited in photosensitive birds under spring-like conditions. These results provide new insight into the effects of photoperiod and T treatment on vernal SCR growth and new neuron incorporation and support current models explaining this growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/dneu.20400DOI Listing
May 2007

Distribution of orexin/hypocretin immunoreactivity in the brain of a male songbird, the house finch, Carpodacus mexicanus.

J Chem Neuroanat 2006 Dec 30;32(2-4):81-9. Epub 2006 Jun 30.

Psychology Department and Institute for Neuroscience, 1 University Station, A8000, University of Texas, Austin, TX 78712, USA.

Previous research has shown orexin/hypocretin immunoreactive (orexin-ir) neurons in domesticated Galliformes. However, these findings may not be representative of other birds and these studies did not include a distribution of orexin-ir projections throughout the brain. The present study was carried out in a wild-caught passerine, the house finch, Carpodacus mexicanus, and includes a detailed description of orexin-ir neurons and their projections. Orexin A and B-ir neurons were located in a single population centered on the paraventricular nucleus of the hypothalamus extending into the lateral hypothalamic area, consistent with other studies in birds. Orexin A and B-ir fibers were similarly visible across the brain, with the highest density within the preoptic area, hypothalamus and thalamus. Orexin-ir projections extended from the paraventricular nucleus rostrally to the preoptic area, laterally towards the medial striatum, nidopallium, and dorsally along the lateral ventricle towards the mesopallium. Caudally, the highest densities of orexin-ir fibers were found along the third ventricle. The periaqueductal grey, substantia nigra pars compacta and the locus coeruleus also showed a high density of orexin-ir fibers. This study showed a detailed fiber distribution previously unreported in birds and showed that orexin-ir neurons were located in similar areas regardless of phylogeny or domestication in birds. The apparently conserved neural distribution of orexins suggests that these peptides play similar roles among birds. The widespread distribution of the projections in brain areas serving various roles indicates the potential involvement of these peptides in multiple behavioral and physiological functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jchemneu.2006.05.003DOI Listing
December 2006