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Plants grown under field conditions experience fluctuating light. Understanding the natural genetic variations for a similarly dynamic photosynthetic response among untapped germplasm resources, as well as the underlying mechanisms, may offer breeding strategies to improve production using molecular approaches. Here, we measured gas exchange under fluctuating light, along with stomatal density and size, in eight wild tomato species and two tomato cultivars. The photosynthetic induction response showed significant diversity, with some wild species having faster induction rates than the two cultivars. Species with faster photosynthetic induction rates had higher daily integrated photosynthesis, but lower average water use efficiency because of high stomatal conductance under natural fluctuating light. The variation in photosynthetic induction was closely associated with the speed of stomatal responses, highlighting its critical role in maximizing photosynthesis under fluctuating light conditions. Moreover, stomatal size was negatively correlated with stomatal density within a species, and plants with smaller stomata at a higher density had a quicker photosynthetic response than those with larger stomata at lower density. Our findings show that the response of stomatal conductance plays a pivotal role in photosynthetic induction, with smaller stomata at higher density proving advantageous for photosynthesis under fluctuating light in tomato species. The interspecific variation in the rate of stomatal responses could offer an untapped resource for optimizing dynamic photosynthetic responses under field conditions., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Genetically engineered mouse models only capture a small fraction of the genetic lesions that drive human cancer. Current CRISPR-Cas9 models can expand this fraction but are limited by their reliance on error-prone DNA repair. Here we develop a system for in vivo prime editing by encoding a Cre-inducible prime editor in the mouse germline. This model allows rapid, precise engineering of a wide range of mutations in cell lines and organoids derived from primary tissues, including a clinically relevant Kras mutation associated with drug resistance and Trp53 hotspot mutations commonly observed in pancreatic cancer. With this system, we demonstrate somatic prime editing in vivo using lipid nanoparticles, and we model lung and pancreatic cancer through viral delivery of prime editing guide RNAs or orthotopic transplantation of prime-edited organoids. We believe that this approach will accelerate functional studies of cancer-associated mutations and complex genetic combinations that are challenging to construct with traditional models., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Local adaptation of populations results from an interplay between their environment and genetics. If functional trait variation influences plant performance, populations can adapt to their local environment. However, populations may also respond plastically to environmental challenges, altering phenotype without shifting allele frequencies. The level of local adaptation in crop landraces and their capacity for plasticity in response to environmental change may predict their continued utility to farmers facing climate change. Yet we understand little about how physiological traits potentially underlying local adaptation of cultivars influence fitness. Farmers in Mexico-the crop center of origin for maize-manage and rely upon a high diversity of landraces. We studied maize grown in Chiapas, Mexico, where strong elevational gradients cover a relatively small geographic area. We reciprocally transplanted 12 populations sourced from three elevational zones (600, 1550 and 2150 m) back into those elevations for two years using a modified split-split plot design to model effects of environment, genetics, and their interaction. We studied physiological and growth traits, including photosynthetic rate, stomatal conductance, stomatal density, relative growth rate (RGR), and seed production. Maize fitness showed indications of local adaptation with highland and midland types performing poorly at warmer lowland locations, though patterns depended on the year. Several physiological traits, including stomatal conductance, were affected by G x E interactions, some of which indicated non-adaptive plastic responses with potential fitness implications. We discerned a significant positive relationship between fitness and relative growth rate. Growth rates in highland landraces were outperformed by midland and lowland landraces grown in high temperature, lowland garden. Lowland landrace stomatal conductance was diminished compared to that of highland landraces in the cooler highland garden. Thus, both adaptive and non-adaptive physiological responses of maize landraces in southern Mexico may have implications for fitness, as well as responses to climate change., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pace et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Domestication is an ongoing well-described process. However, while many have studied the changes domestication causes in plant genetics, few have explored its impact on the portion of the geographic landscape in which the plants exist. Therefore, the goal of this study was to understand how the process of domestication changed the geographic space suitable for chile pepper ( Capsicum annuum ) in its center of origin (domestication). C. annuum is a major crop species globally whose center of domestication, Mexico, has been well-studied. It provides a unique opportunity to explore the degree to which ranges of different domestication classes diverged and how these ranges might be altered by climate change. To this end, we created ecological niche models for four domestication classes (wild, semiwild, landrace, modern cultivar) based on present climate and future climate scenarios for 2050, 2070, and 2090. Considering present environment, we found substantial overlap in the geographic niches of all the domestication classes. Yet, environmental and geographic aspects of the current ranges did vary among classes. Wild and commercial varieties could grow in desert conditions, while landraces could not. With projections into the future, habitat was lost asymmetrically, with wild, semiwild, and landraces at greater risk of territorial declines than modern cultivars. Further, we identified areas where future suitability overlap between landraces and wilds is expected to be lost. While range expansion is widely associated with domestication, we found little support of a constant niche expansion (either in environmental or geographical space) throughout the domestication gradient in chile peppers in Mexico. Instead, particular domestication transitions resulted in loss, followed by capturing or recapturing environmental or geographic space. The differences in environmental characterization among domestication gradient classes and their future potential range shifts increase the need for conservation efforts to preserve landraces and semiwild genotypes., Competing Interests: Authors declare that they have no conflict of interest, including commercial or intellectual property interest in wild, local, or any germplasm., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Background: Peppers, bell and chile, are a culturally and economically important worldwide. Domesticated Capsicum spp. are distributed globally and represent a complex of valuable genetic resources., Objectives: Explore population structure and diversity in a collection of 467 peppers representing eight species, spanning the spectrum from highly domesticated to wild using 22,916 SNP markers distributed across the twelve chromosomes of pepper., Results: These species contained varied levels of genetic diversity, which also varied across chromosomes; the species also differ in the size of genetic bottlenecks they have experienced. We found that levels of diversity negatively correlate to levels of domestication, with the more diverse being the least domesticated., (© 2023. The Author(s).)

Crop wild relatives have been used as a source of genetic diversity for over one hundred years. The wild tomato relative Solanum galapagense accession LA1141 demonstrates the ability to tolerate deficit irrigation, making it a potential resource for crop improvement. Accessing traits from LA1141 through introgression may improve the response of cultivated tomatoes grown in water-limited environments. Canopy temperature is a proxy for physiological traits which are challenging to measure efficiently and may be related to water deficit tolerance. We optimized phenotypic evaluation based on variance partitioning and further show that objective phenotyping methods coupled with genomic prediction lead to gain under selection for water deficit tolerance. The objectives of this work were to improve phenotyping workflows for measuring canopy temperature, mapping quantitative trait loci (QTLs) from LA1141 that contribute to water deficit tolerance and comparing selection strategies. The phenotypic variance attributed to genetic causes for canopy temperature was higher when estimated from thermal images relative to estimates based on an infrared thermometer. Composite interval mapping using BC 2 S 3 families, genotyped with single nucleotide polymorphisms, suggested that accession LA1141 contributed alleles that lower canopy temperature and increase plant turgor under water deficit. QTLs for lower canopy temperature were mapped to chromosomes 1 and 6 and explained between 6.6 and 9.5% of the total phenotypic variance. QTLs for higher leaf turgor were detected on chromosomes 5 and 7 and explained between 6.8 and 9.1% of the variance. We advanced tolerant BC 2 S 3 families to the BC 2 S 5 generation using selection indices based on phenotypic values and genomic estimated breeding values (GEBVs). Phenotypic, genomic, and combined selection strategies demonstrated gain under selection and improved performance compared to randomly advanced BC 2 S 5 progenies. Leaf turgor, canopy temperature, stomatal conductance, and vapor pressure deficit (VPD) were evaluated and compared in BC 2 S 5 progenies grown under deficit irrigation. Progenies co-selected for phenotypic values and GEBVs wilted less, had significantly lower canopy temperature, higher stomatal conductance, and lower VPD than randomly advanced lines. The fruit size of water deficit tolerant selections was small compared to the recurrent parent. However, lines with acceptable yield, canopy width, and quality parameters were recovered. These results suggest that we can create selection indices to improve water deficit tolerance in a recurrent parent background, and additional crossing and evaluation are warranted., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fenstemaker, Cho, McCoy, Mercer and Francis.)

Premise: Capsicum annuum (Solanaceae) was originally domesticated in Mexico, where wild (C. annuum var. glabriusculum) and cultivated (C. annuum var. annuum) chile pepper populations (>60 landraces) are common, and wild-resembling individuals (hereafter semiwild) grow spontaneously in anthropogenic environments. Here we analyze the role of elevation and domestication gradients in shaping the genetic diversity in C. annuum from the state of Oaxaca, Mexico., Methods: We collected samples of 341 individuals from 28 populations, corresponding to wild, semiwild (C. annuum var. glabriusculum) and cultivated C. annuum, and closely related species Capsicum frutescens and C. chinense. From the genetic variation of 10 simple sequence repeat (SSR) loci, we assessed the population genetic structure, inbreeding, and gene flow through variance distribution analyses, genetic clustering, and connectivity estimations., Results: Genetic diversity (H E ) did not differ across domestication levels. However, inbreeding coefficients were higher in semiwild and cultivated chiles than in wild populations. We found evidence for gene flow between wild populations and cultivated landraces along the coast. Genetic structure analysis revealed strong differentiation between most highland and lowland landraces., Conclusions: Gene flow between wild and domesticated populations may be mediated by backyards and smallholder farms, while mating systems may facilitate gene flow between landraces and semiwild populations. Domestication and elevation may overlap in their influence on genetic differentiation. Lowland Gui'ña dani clustered with highland landraces perhaps due to the social history of the Zapotec peoples. In situ conservation may play an important role in preserving semiwild populations and private alleles found in landraces., (© 2022 Botanical Society of America.)

Global climate change is having a significant effect on agriculture by causing greater precipitation variability and an increased risk of drought. To mitigate these effects, it is important to identify specific traits, adaptations, and germplasm that improve tolerance to soil water deficit. Local varieties, known as landraces, have undergone generations of farmer-mediated selection and can serve as sources of variation, specifically for tolerance to abiotic stress. Landraces can possess local adaptations, where accessions adapted to a particular environment will outperform others grown under the same conditions. We explore adaptations to water deficit in chile pepper landraces from across an environmental gradient in Mexico, a center of crop domestication and diversity, as well in improved varieties bred for the US. In the present study, we evaluated 25 US and Mexico accessions in a greenhouse experiment under well-watered and water deficit conditions and measured morphological, physiological, and agronomic traits. Accession and irrigation regime influenced plant biomass and height, while branching, CO2 assimilation, and fruit weight were all influenced by an interaction between accession and irrigation. A priori group contrasts revealed possible adaptations to water deficit for branching, CO2 assimilation, and plant height associated with geographic origin, domestication level, and pepper species. Additionally, within the Mexican landraces, the number of primary branches had a strong relationship with precipitation from the environment of origin. This work provides insight into chile pepper response to water deficit and adaptation to drought and identifies possibly tolerant germplasm., Competing Interests: The authors have declared that no competing interests exist.

The GATA4 transcription factor acts as a master regulator of development of multiple tissues. GATA4 also acts in a distinct capacity to control a stress-inducible pro-inflammatory secretory program that is associated with senescence, a potent tumor suppression mechanism, but also operates in non-senescent contexts such as tumorigenesis. This secretory pathway is composed of chemokines, cytokines, growth factors, and proteases. Since GATA4 is deleted or epigenetically silenced in cancer, here we examine the role of GATA4 in tumorigenesis in mouse models through both loss-of-function and overexpression experiments. We find that GATA4 promotes non-cell autonomous tumor suppression in multiple model systems. Mechanistically, we show that Gata4-dependent tumor suppression requires cytotoxic CD8 T cells and partially requires the secreted chemokine CCL2. Analysis of transcriptome data in human tumors reveals reduced lymphocyte infiltration in GATA4-deficient tumors, consistent with our murine data. Notably, activation of the GATA4-dependent secretory program combined with an anti-PD-1 antibody robustly abrogates tumor growth in vivo., (© 2022. The Author(s).)

Crop diversity underpins the productivity, resilience and adaptive capacity of agriculture. Loss of this diversity, termed crop genetic erosion, is therefore concerning. While alarms regarding evident declines in crop diversity have been raised for over a century, the magnitude, trajectory, drivers and significance of these losses remain insufficiently understood. We outline the various definitions, measurements, scales and sources of information on crop genetic erosion. We then provide a synthesis of evidence regarding changes in the diversity of traditional crop landraces on farms, modern crop cultivars in agriculture, crop wild relatives in their natural habitats and crop genetic resources held in conservation repositories. This evidence indicates that marked losses, but also maintenance and increases in diversity, have occurred in all these contexts, the extent depending on species, taxonomic and geographic scale, and region, as well as analytical approach. We discuss steps needed to further advance knowledge around the agricultural and societal significance, as well as conservation implications, of crop genetic erosion. Finally, we propose actions to mitigate, stem and reverse further losses of crop diversity., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8 + T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation., Competing Interests: Declaration of interests T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific, and a co-Founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. T.J. is also President of Break Through Cancer. His laboratory currently receives funding from Johnson & Johnson and The Lustgarten Foundation; funds from the Lustgarten Foundation supported the research described in this manuscript. A.R. is a founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and until August 31, 2020, was an SAB member of Syros Pharmaceuticals, Neogene Therapeutics, Asimov and Thermo Fisher Scientific. From August 1, 2020, A.R. is an employee of Genentech, a member of the Roche Group. A.R. and Regev lab members' work was conducted at the Broad Institute, unrelated to these later affiliations. T.D.H., P.D., and J.W.R. are employees and stockholders at NanoString Technologies, Inc. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Responses to drought within a single species may vary based on plant developmental stage, drought severity, and the avoidance or tolerance mechanisms employed. Early drought stress can restrict emergence and seedling growth. Thus, in areas where water availability is limited, rapid germination leading to early plant establishment may be beneficial. Alternatively, germination without sufficient water to support the seedling may lead to early senescence, so reduced germination under low moisture conditions may be adaptive at the level of the population. We studied the germination response to osmotic stress of diverse chile pepper germplasm collected in southern Mexico from varied ecozones, cultivation systems, and of named landraces. Drought stress was simulated using polyethylene glycol solutions. Overall, survival time analysis revealed delayed germination at the 20% concentration of PEG across all ecozones. The effect was most pronounced in the genotypes from hotter, drier ecozones. Additionally, accessions from wetter and cooler ecozones had the fastest rate of germination. Moreover, accessions of the landraces Costeño Rojo and Tusta germinated more slowly and incompletely if sourced from a drier ecozone than a wetter one, indicating that slower, reduced germination under drought stress may be an adaptive avoidance mechanism. Significant differences were also observed between named landraces, with more domesticated types from intensive cultivation systems nearly always germinating faster than small-fruited backyard- or wild-types, perhaps due to the fact that the smaller-fruited accessions may have undergone less selection. Thus, we conclude that there is evidence of local adaptation to both ecozone of origin and source cultivation system in germination characteristics of diverse chile peppers., Competing Interests: The authors have declared that no competing interests exist.

Approximately 20-30% of human lung adenocarcinomas (LUAD) harbor loss-of-function (LOF) mutations in Kelch-like ECH Associated-Protein 1 ( KEAP1 ), which lead to hyperactivation of the nuclear factor, erythroid 2-like 2 (NRF2) antioxidant pathway and correlate with poor prognosis 1-3 . We previously showed that Keap1 mutation accelerates KRAS-driven LUAD and produces a marked dependency on glutaminolysis 4 . To extend the investigation of genetic dependencies in the context of Keap1 mutation, we performed a druggable genome CRISPR-Cas9 screen in Keap1 -mutant cells. This analysis uncovered a profound Keap1 mutant-specific dependency on solute carrier family 33 member 1 ( Slc33a1 ), an endomembrane-associated protein with roles in autophagy regulation 5 , as well as a series of functionally-related genes implicated in the unfolded protein response. Targeted genetic and biochemical experiments using mouse and human Keap1 -mutant tumor lines, as well as preclinical genetically-engineered mouse models (GEMMs) of LUAD, validate Slc33a1 as a robust Keap1 -mutant-specific dependency. Furthermore, unbiased genome-wide CRISPR screening identified additional genes related to Slc33a1 dependency. Overall, our study provides a strong rationale for stratification of patients harboring KEAP1 -mutant or NRF2-hyperactivated tumors as likely responders to targeted SLC33A1 inhibition and underscores the value of integrating functional genetic approaches with GEMMs to identify and validate genotype-specific therapeutic targets., Competing Interests: Declaration of Interest: T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific. He is also a co-Founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript. T.J. laboratory currently also receives funding from the Johnson & Johnson Lung Cancer Initiative, but this funding did not support the research described in this manuscript. This work was supported by the Howard Hughes Medical Institute and Calico Life Sciences LLC.

Globally, farmers cultivate and maintain crop landraces (i.e., traditional varieties). Landraces contain unique diversity shaped in part by natural and human-mediated selection and are an indispensable resource for farmers. Since environmental conditions change with elevation, crop landraces grown along elevational gradients have provided ideal locations to explore patterns of local adaptation. To further probe traits underlying this differentiation, transcriptome signatures can help provide a foundation for understanding the ways in which functional genetic diversity may be shaped by environment. In this study, we returned to an elevational gradient in Chiapas, Mexico, to assess transcriptional differentiation of genes underlying UV-B protection in locally adapted maize landraces from multiple elevations. We collected and planted landraces from three elevational zones (lowland, approximately 600 m; midland, approximately 1,550 m; highland approximately 2,100 m) in a common garden at 1,531 m. Using RNA-seq data derived from leaf tissue, we performed differential expression analysis between maize from these distinct elevations. Highland and lowland landraces displayed differential expression in phenylpropanoid and flavonoid biosynthesis genes involved in the production of UV-B protectants and did so at a rate greater than expected based on observed background transcriptional differentiation across the genome. These findings provide evidence for the differentiation of suites of genes involved in complex ecologically relevant pathways. Thus, while neutral evolutionary processes may have played a role in the observed patterns of differentiation, UV-B may have also acted as a selective pressure to differentiate maize landraces in the region. Studies of the distribution of functional crop genetic diversity across variable landscapes can aid us in understanding the response of diversity to abiotic/biotic change and, ultimately, may facilitate its conservation and utilization., Competing Interests: None declared., (© 2020 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer that remains among the most lethal of solid tumor malignancies. Recent genomic sequencing studies have identified many recurrently mutated genes in human SCLC tumors. However, the functional roles of most of these genes remain to be validated. Here, we have adapted the CRISPR-Cas9 system to a well-established murine model of SCLC to rapidly model loss-of-function mutations in candidate genes identified from SCLC sequencing studies. We show that loss of the gene p107 significantly accelerates tumor progression. Notably, compared with loss of the closely related gene p130 , loss of p107 results in fewer but larger tumors as well as earlier metastatic spread. In addition, we observe differences in proliferation and apoptosis as well as altered distribution of initiated tumors in the lung, resulting from loss of p107 or p130 Collectively, these data demonstrate the feasibility of using the CRISPR-Cas9 system to model loss of candidate tumor suppressor genes in SCLC, and we anticipate that this approach will facilitate efforts to investigate mechanisms driving tumor progression in this deadly disease., Competing Interests: Competing interest statement: T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific. He is also a co-founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this paper. Dr. Jacks’s laboratory currently also receives funding from the Johnson & Johnson Lung Cancer Initiative and Calico, but this funding did not support the research described in this paper., (Copyright © 2020 the Author(s). Published by PNAS.)

Small cell lung cancer (SCLC) is an aggressive lung cancer subtype with extremely poor prognosis. No targetable genetic driver events have been identified, and the treatment landscape for this disease has remained nearly unchanged for over 30 years. Here, we have taken a CRISPR-based screening approach to identify genetic vulnerabilities in SCLC that may serve as potential therapeutic targets. We used a single-guide RNA (sgRNA) library targeting ~5000 genes deemed to encode "druggable" proteins to perform loss-of-function genetic screens in a panel of cell lines derived from autochthonous genetically engineered mouse models (GEMMs) of SCLC, lung adenocarcinoma (LUAD), and pancreatic ductal adenocarcinoma (PDAC). Cross-cancer analyses allowed us to identify SCLC-selective vulnerabilities. In particular, we observed enhanced sensitivity of SCLC cells toward disruption of the pyrimidine biosynthesis pathway. Pharmacological inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in this pathway, reduced the viability of SCLC cells in vitro and strongly suppressed SCLC tumor growth in human patient-derived xenograft (PDX) models and in an autochthonous mouse model. These results indicate that DHODH inhibition may be an approach to treat SCLC., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Hybridization is a common phenomenon in plants and can lead to the introgression of alleles from one population into another, generate new hybrid lineages, or cause species extinction. The environmental conditions and the genetic background of the participating populations may influence these outcomes since they can affect the fitness of hybrids, thereby increasing or decreasing the chances of introgression. Thus, it is important to understand the context-dependent prospects for introgression of alleles into diverse populations and under multiple ecological environments. Crop-wild hybridization presents an opportunity to explore these dynamics in agroecosystems. To this end, we used diverse wild and hybrid sunflowers from across the northern United States as a basis for evaluating variation in morphological traits and assessing context-dependent selection. These crop-wild hybrids and their wild counterparts were grown under agricultural conditions in the field with and without wheat competition. Interactions between origin and cross type affected expression of early functional traits, while interactions between competition and cross type acted on reproductive traits. A smattering of early and reproductive traits was affected by interactions between cross type and competition that varied by origin (i.e., 3-way interactions). Seven functional traits, especially number of branches and tertiary head diameter, underwent net and direct directional selection, while six out of these seven traits appear to also be experiencing nonlinear selection dynamics. In general, wild-like traits were favored under both sets of conditions, while, under wheat competition, some crop-like traits related to fast growth and primary head diameter became important. These data reaffirm the hypothesis that stressful conditions establish a scenario more suitable for crop introgression and clarify that nonlinear selection dynamics may play a role in this process., Competing Interests: None declared.

Background: Landrace farmers are the keepers of crops locally adapted to the environments where they are cultivated. Patterns of diversity across the genome can provide signals of past evolution in the face of abiotic and biotic change. Understanding this rich genetic resource is imperative especially since diversity can provide agricultural security as climate continues to shift., Results: Here we employ RNA sequencing (RNA-seq) to understand the role that conditions that vary across a landscape may have played in shaping genetic diversity in the maize landraces of Chiapas, Mexico. We collected landraces from three distinct elevational zones and planted them in a midland common garden. Early season leaf tissue was collected for RNA-seq and we performed weighted gene co-expression network analysis (WGCNA). We then used association analysis between landrace co-expression module expression values and environmental parameters of landrace origin to elucidate genes and gene networks potentially shaped by environmental factors along our study gradient. Elevation of landrace origin affected the transcriptome profiles. Two co-expression modules were highly correlated with temperature parameters of landrace origin and queries into their 'hub' genes suggested that temperature may have led to differentiation among landraces in hormone biosynthesis/signaling and abiotic and biotic stress responses. We identified several 'hub' transcription factors and kinases as candidates for the regulation of these responses., Conclusions: These findings indicate that natural selection may influence the transcriptomes of crop landraces along an elevational gradient in a major diversity center, and provide a foundation for exploring the genetic basis of local adaptation. While we cannot rule out the role of neutral evolutionary forces in the patterns we have identified, combining whole transcriptome sequencing technologies, established bioinformatics techniques, and common garden experimentation can powerfully elucidate structure of adaptive diversity across a varied landscape. Ultimately, gaining such understanding can facilitate the conservation and strategic utilization of crop genetic diversity in a time of climate change.

Since plant mating choices are flexible and responsive to the environment, rates of spontaneous hybridization may vary across ecological clines. Developing a robust and predictive framework for rates of plant gene flow requires assessing the role of environmental sensitivity on plant reproductive traits, relative abundance, and pollen vectors. Therefore, across a soil moisture gradient, we quantified pollinator movement, life-history trait variation, and unidirectional hybridization rates from crop (Raphanus sativus) to wild (Raphanus raphanistrum) radish populations. Both radish species were grown together in relatively dry (no rain), relatively wet (double rain), or control soil moisture conditions in Ohio, USA. We measured wild and crop radish life-history, phenology and pollinator visitation patterns. To quantify hybridization rates from crop-to-wild species, we used a simply inherited morphological marker to detect F1 hybrid progeny. Although crop-to-wild hybridization did not respond to watering treatments, the abundance of hybrid offspring was higher in fruits produced late in the period of phenological overlap, when both species had roughly equal numbers of open flowers. Therefore, the timing of fruit production and its relationship to flowering overlap may be more important to hybrid zone formation in Raphanus spp. than soil moisture or pollen vector movements., Competing Interests: The authors have declared that no competing interests exist.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Hybridization produces strong evolutionary forces. In hybrid zones, selection can differentially occur on traits and selection intensities may differ among hybrid generations. Understanding these dynamics in crop-wild hybrid zones can clarify crop-like traits likely to introgress into wild populations and the particular hybrid generations through which introgression proceeds. In a field experiment with four crop-wild hybrid Helianthus annuus (sunflower) cross types, we measured growth and life history traits and performed phenotypic selection analysis on early season traits to ascertain the likelihood, and routes, of crop allele introgression into wild sunflower populations. All cross types overwintered, emerged in the spring, and survived until flowering, indicating no early life history barriers to crop allele introgression. While selection indirectly favored earlier seedling emergence and taller early season seedlings, direct selection only favored greater early season leaf length. Further, there was cross type variation in the intensity of selection operating on leaf length. Thus, introgression of multiple early season crop-like traits, due to direct selection for greater early season leaf length, should not be impeded by any cross type and may proceed at different rates among generations. In sum, alleles underlying early season sunflower crop-like traits are likely to introgress into wild sunflower populations.

Domestication has resulted in selection upon seed traits found in wild populations, yet crop-wild hybrids retain some aspects of both parental phenotypes. Seed fates of germination, dormancy, and mortality can influence the success of crop allele introgression in crop-wild hybrid zones, especially if crop alleles or crop-imparted seed coverings result in out-of-season germination. We performed a seed burial experiment using crop, wild, and diverse hybrid sunflower (Helianthus annuus) cross types to test how a cross type's maternal parent and nuclear genetic composition might affect its fate under field conditions. We observed higher maladaptive fall germination in the crop- and F1- produced seeds than wild-produced seeds and, due to an interaction with percent crop alleles, fall germination was higher for cross types with more crop-like nuclear genetics. By spring, crop-produced cross types had the highest overwintering mortality, primarily due to higher fall germination. Early spring germination was identical across maternal types, but germination continued for F1-produced seeds. In conclusion, the more wild-like the maternal parent or the less proportion of the cross type's genome contributed by the crop, the greater likelihood a seed will remain ungerminated than die. Wild-like dormancy may facilitate introgression through future recruitment from the soil seed bank.

Understanding the likelihood and extent of introgression of novel alleles in hybrid zones requires comparison of lifetime fitness of parents and hybrid progeny. However, fitness differences among cross types can vary depending on biotic conditions, thereby influencing introgression patterns. Based on past work, we predicted that increased competition would enhance introgression between cultivated and wild sunflower (Helianthus annuus) by reducing fitness advantages of wild plants. To test this prediction, we established a factorial field experiment in Kansas, USA where we monitored the fitness of four cross types (Wild, F1, F2, and BCw hybrids) under different levels of interspecific and intraspecific competition. Intraspecific manipulations consisted both of density of competitors and of frequency of crop-wild hybrids. We recorded emergence of overwintered seeds, survival to reproduction, and numbers of seeds produced per reproductive plant. We also calculated two compound fitness measures: seeds produced per emerged seedling and seeds produced per planted seed. Cross type and intraspecific competition affected emergence and survival to reproduction, respectively. Further, cross type interacted with competitive treatments to influence all other fitness traits. More intense competition treatments, especially related to density of intraspecific competitors, repeatedly reduced the fitness advantage of wild plants when considering seeds produced per reproductive plant and per emerged seedling, and F2 plants often became indistinguishable from the wilds. Wild fitness remained superior when seedling emergence was also considered as part of fitness, but the fitness of F2 hybrids relative to wild plants more than quadrupled with the addition of interspecific competitors and high densities of intraspecific competitors. Meanwhile, contrary to prediction, lower hybrid frequency reduced wild fitness advantage. These results emphasize the importance of taking a full life cycle perspective. Additionally, due to effects of exogenous selection, a given hybrid generation may be especially well-suited to hastening introgression under particular environmental conditions.

• Premise of the study: The fitness of an offspring may depend on its nuclear genetic composition (via both parental genotypes) as well as on genetic maternal effects (via only the maternal parent). Understanding the relative importance of these two genetic factors is particularly important for research on crop-wild hybridization, since traits with important genetic maternal effects (e.g., seed size) often differ among crops and their relatives. We hypothesized that the effects of these genetic factors on fitness components would change across the life cycle of hybrids.• Methods: We followed seed, plant size, and reproductive traits in field experiments with wild and four crop-wild hybrids of sunflower (Helianthus annuus), which differed in nuclear genetic composition and maternal parent (wild or F1 hybrid).• Key results: We identified strong genetic maternal effects for early life cycle characteristics, with seeds produced on an F1 mother having premature germination, negligible seed dormancy, and greater seedling size. Increased percentages of crop alleles also increased premature germination and reduced dormancy in seeds produced on a wild mother. For mature plants, nuclear genetic composition dominated: greater percentages of crop alleles reduced height, branching, and fecundity.• Conclusions: Particular backcrosses between hybrids and wilds may differentially facilitate movement of crop alleles into wild populations due to their specific features. For example, backcross seeds produced on wild mothers can persist in the seed bank, illustrating the importance of genetic maternal effects, whereas backcross individuals with either wild or F1 mothers have high fecundity, resulting from their wild-like nuclear genetic composition., (© 2014 Botanical Society of America, Inc.)

Premise: Variation in seedling emergence timing is considered adaptive over the long term in wild populations, but early emergence can result in a fitness advantage. To explore the adaptive significance of seedling emergence timing, it should be studied under realistic conditions and in the context of other traits that influence fitness., Methods: In a common garden, we monitored maternal families from seed to flowering (including over winter) with intra- and interspecific competition. We assessed the effects of emergence timing and plant size on survival to anthesis in different genetic backgrounds and under varying competition., Key Results: We found genetic variation for emergence (probability and timing), size, and survival to anthesis. We also found negative selection, both phenotypic and genetic, on emergence time, such that early emergers (day 8) had almost twice as great a predicted probability of surviving as later emergers (day 28). Size had strong positive effects on survival and, furthermore, the beneficial effects of early emergence may be mediated through size. Maternal family and competitive environment can also affect selection on emergence timing., Conclusions: Our results indicate that early emergence is related to greater survival in wild sunflower, although there may be little direct selection on this trait; rather, its importance may be mediated by its effects on highly adaptive traits associated with size. Also, the effects of early emergence may vary across genetic backgrounds and competitive conditions, facilitating the maintenance of variation for this trait across a diverse landscape.

Landraces cultivated in centers of crop diversity result from past and contemporary patterns of natural and farmer-mediated evolutionary forces. Successful in situ conservation of crop genetic resources depends on continuity of these evolutionary processes. Climate change is projected to affect agricultural production, yet analyses of impacts on in situ conservation of crop genetic diversity and farmers who conserve it have been absent. How will crop landraces respond to alterations in climate? We review the roles that phenotypic plasticity, evolution, and gene flow might play in sustaining production, although we might expect erosion of genetic diversity if landrace populations or entire races lose productivity. For example, highland maize landraces in southern Mexico do not express the plasticity necessary to sustain productivity under climate change, but may evolve in response to altered conditions. The outcome for any given crop in a given region will depend on the distribution of genetic variation that affects fitness and patterns of climate change. Understanding patterns of neutral and adaptive diversity from the population to the landscape scale is essential to clarify how landraces conserved in situ will continue to evolve and how to minimize genetic erosion of this essential natural resource.

Chemotherapy resistance is a major obstacle in cancer treatment, yet the mechanisms of response to specific therapies have been largely unexplored in vivo. Employing genetic, genomic, and imaging approaches, we examined the dynamics of response to a mainstay chemotherapeutic, cisplatin, in multiple mouse models of human non-small-cell lung cancer (NSCLC). We show that lung tumors initially respond to cisplatin by sensing DNA damage, undergoing cell cycle arrest, and inducing apoptosis-leading to a significant reduction in tumor burden. Importantly, we demonstrate that this response does not depend on the tumor suppressor p53 or its transcriptional target, p21. Prolonged cisplatin treatment promotes the emergence of resistant tumors with enhanced repair capacity that are cross-resistant to platinum analogs, exhibit advanced histopathology, and possess an increased frequency of genomic alterations. Cisplatin-resistant tumors express elevated levels of multiple DNA damage repair and cell cycle arrest-related genes, including p53-inducible protein with a death domain (Pidd). We demonstrate a novel role for PIDD as a regulator of chemotherapy response in human lung tumor cells.

Acute exposure to ionizing radiation can cause lethal damage to the gastrointestinal (GI) tract, a condition called the GI syndrome. Whether the target cells affected by radiation to cause the GI syndrome are derived from the epithelium or endothelium and whether the target cells die by apoptosis or other mechanisms are controversial issues. Studying mouse models, we found that selective deletion of the proapoptotic genes Bak1 and Bax from the GI epithelium or from endothelial cells did not protect mice from developing the GI syndrome after sub-total-body gamma irradiation. In contrast, selective deletion of p53 from the GI epithelium, but not from endothelial cells, sensitized irradiated mice to the GI syndrome. Transgenic mice overexpressing p53 in all tissues were protected from the GI syndrome after irradiation. These results suggest that the GI syndrome is caused by the death of GI epithelial cells and that these epithelial cells die by a mechanism that is regulated by p53 but independent of apoptosis.

Arsenic sorption to hydrous ferric oxide (HFO) is an effective treatment method for removing dissolved arsenic from fresh drinking water sources. However, detailed information is limited regarding arsenic removal from solutions of high ionic strength such as brackish groundwater, seawater, or high-pressure membrane process residuals. Bench-scale treatment experiments were conducted exploring arsenic removal from simple solutions with ionic strengths ranging from 0.008 to 1.5 M by addition of ferric chloride followed by solid/liquid separation (microfiltration or ultrafiltration). Arsenic removal from these solutions during in situ iron precipitation was approximately 90% at Fe:As molar ratios of 10 to 15 and > 95% for Fe:As molar ratios greater than 20. Arsenic removal at iron doses of 10(-6) to 10(-4) mol-Fe/L improved when pH was lowered from 8 to less than 6.5 at ionic strength 0.2 M; this improvement was not as significant at ionic strength 0.7 M. Arsenic removal diminished when alkalinity was increased from 400 to 1,400 mg/L as calcium carbonate; however, arsenic removal at the higher alkalinity improved when pH was lowered from approximately 8 to less than 7. Arsenic removal with preformed HFO solids and subsequent microfiltration was significantly less than that observed with in situ HFO precipitation. Increased removal by in situ precipitation compared to that of preformed solids is explained by an increased number of adsorption sites due to uptake during iron oxy-hydroxide polymerization as well as an increase in surface area resulting in diminished surface charge effects. Model simulations of arsenic uptake by in situ precipitation adequately captured these effect by changing the model parameters used to model arsenic uptake by preformed HFO, specificallythe total number of surface sites and surface area.

Oncogenic K-ras is one of the most common genetic alterations in human lung adenocarcinomas. In addition, inactivation of clusters of tumor suppressor genes is required to bring about classical characteristics of cancer including angiogenesis as a prelude to invasion and metastasis. Transforming growth factor-beta (TGF-beta) 1 is a tumor suppressor gene that is implicated in lung cancer progression. Although in vitro studies have shown that TGF-beta1 and Ras pathways cooperate during tumorigenesis, the biology of interaction of TGF-beta1 and Ras has not been studied in in vivo tumorigenesis. We hypothesized that inactivation of TGF-beta1 in addition to oncogeneic activation of K-ras would lead to early initiation and faster progression to lung adenocarcinoma and invasion and metastasis. Heterozygous (HT) TGF-beta1 mice were mated with latent activatable (LA) mutated K-ras mice to generate TGF-beta1(+/+), K-ras LA (wild-type (WT)/LA) and TGF-beta1(+/-), K-ras LA (HT/LA) mice. Both HT/LA and WT/LA mice developed spontaneous lung tumors, but HT/LA mice progressed to adenocarcinomas significantly earlier compared with WT/LA mice. In addition, WT/LA adenocarcinomas had significantly higher angiogenic activity compared with HT/LA adenocarcinomas. Thus, while oncogenic K-ras mutation and insensitivity to the growth regulatory effects of TGF-beta1 is essential for initiation and progression of mouse lung tumors to adenocarcinoma, a full gene dosage of TGF-beta1 is required for tumor-induced angiogenesis and invasive potential. This study identifies a number of genes not previously associated with lung cancer that are involved in tumor induction and progression. In addition, we provide evidence that progression to invasive angiogenic lesions requires TGF-beta1 responsiveness in addition to Ras mutation.

After a decade of transgenic crop production, the dynamics of gene introgression into wild relatives remain unclear. Taking an ecological genetics approach to investigating fitness in crop-wild hybrid zones, we uncovered both conditions and characteristics that may promote introgression. We compared diverse crop-wild hybrid genotypes relative to wild Helianthus annuus under one benign and three stressful agricultural environments. Whereas relative fitness of crop-wild hybrids averaged 0.25 under benign conditions, with herbicide application or competition it reached 0.45 and was more variable. In some instances, hybrid fitness matched wild fitness (approximately 1). Thus, wild populations under agronomic stress may be more susceptible to introgression. Although 'domestication' traits are typically considered unlikely to persist in wild populations, we found some (e.g. rapid growth and early flowering) that may enhance hybrid fitness, especially in stressful environments. Rigorous assessment of how particular genotypes, phenotypes, and environments affect introgression will improve risk assessment for transgenic crops.

Gene flow between crop fields and wild populations often results in hybrids with reduced fitness compared to their wild counterparts due to characteristics imparted by the crop genome. But the specifics of the evolutionary outcome of crop-wild gene flow may depend on context, varying due to local environmental conditions and genetic variation within and among wild populations and among crop lines. To evaluate context-dependence of fitness of F1 hybrids, sunflower crop lines were crossed with nine wild populations from across the northern United States. These crop-wild hybrids and their wild counterparts were grown under agricultural conditions in the field with and without wheat competition. Hybrids were far less fecund than wild plants, yet more likely to survive to reproduce. There was considerable variability among wild populations for fecundity and the specific crop line used to generate the crop-wild hybrid significantly affected fecundity. The fitness deficit suffered by crop-wild hybrids varied by population, as did the rankings of the crop-wild hybrids from three different crop lines. Wheat competition decreased fecundity and survival considerably and hampered seed production of wild plants more than that of hybrids. Genotype x environment interactions indicated that the response of fitness to competition differed by population. Consequently, the fitness of hybrids relative to wild plants varied considerably among wild populations and was not consistent across environments. Notably, relative fitness of hybrids was greater under competitive conditions. This research is the first study of its kind to demonstrate that the consequences of crop-wild gene flow are context dependent and contingent on the genetics of the specific wild populations and the local biotic and abiotic conditions.

Gene flow from crop fields to wild populations produces hybrids that often differ from their wild counterparts in growth form, phenology, and life history characteristics. Germination and dormancy dynamics have a strong influence on population persistence, competitive dynamics, and ultimately, plant fitness. They may also play a role in modifying crop gene introgression, which has been of primary interest since the release of transgenic crops. We investigated how seed germination and dormancy were affected by sunflower crop wild hybridization in both laboratory and field experiments. Hybridization increased seed germination and decreased dormancy. Of the nine wild populations we assayed, most of their hybrids had higher germination than the wilds of the same population. However, absolute germination levels varied by population and testing environment. Hybrids produced by three different crop lines differed in germination, and their germination rankings shifted across populations. Increased germination in hybrids could accelerate crop gene introgression, provided that hybrids germinate in an appropriate period. Differences in relative germination of wild and hybrid seed indicated that the effect of germination on introgression will likely vary by population due, in part, to initial levels of dormancy in the population. Therefore, the implications of gene flow from crops with novel characteristics or from transgenic crops will also vary by population.

Activating mutations in the ras oncogene are not considered sufficient to induce abnormal cellular proliferation in the absence of cooperating oncogenes. We demonstrate that the conditional expression of an endogenous K-ras(G12D) allele in murine embryonic fibroblasts causes enhanced proliferation and partial transformation in the absence of further genetic abnormalities. Interestingly, K-ras(G12D)-expressing fibroblasts demonstrate attenuation and altered regulation of canonical Ras effector signaling pathways. Widespread expression of endogenous K-ras(G12D) is not tolerated during embryonic development, and directed expression in the lung and GI tract induces preneoplastic epithelial hyperplasias. Our results suggest that endogenous oncogenic ras is sufficient to initiate transformation by stimulating proliferation, while further genetic lesions may be necessary for progression to frank malignancy.

Epidermal progenitor cells (EpPCs) were long thought to be unipotent, giving rise only to other keratinocytes but recent studies question this assumption. Here, we investigated whether mouse EpPCs can adopt other antigenic and functional phenotypes. To test this, we injected freshly isolated and cultured EpPCs and transient amplifying cells into diabetic and non-diabetic mouse ischemic hindlimb and followed the cells' fate and the recovery of the ischemic limb blood flow over time. Both freshly isolated and cultured EpPCs and transient amplifying cells were incorporated into the vasculature of the ischemic limb 2 and 5 weeks post-injection, and some expressed endothelial cell but not keratinocyte antigens. Additionally, in the non-diabetic animals, first transient amplifying cells and then EpPCs accelerated the restoration of the blood flow. By contrast, in diabetic animals, only injected EpPCs or unsorted epidermal cells accelerated the restoration of the blood flow. These data indicate that epidermal cells can adopt non-skin phenotypes and functions, and that this apparent pluripotency is not lost by differentiation of EpPCs into transient amplifying cells. They also suggest that epidermal cell therapy might be of therapeutic value in the treatment of diabetic ischemia. Finally, because epidermal cells are readily accessible and expandable, they appear to be ideally suited for use as a non-viral gene delivery therapy.

Mice heterozygous for the retinoblastoma (Rb) tumor suppressor gene develop pituitary and thyroid tumors with high penetrance. We demonstrate here that loss of the ARF tumor suppressor strongly accelerates intermediate lobe pituitary tumorigenesis in Rb heterozygous mice. These effects in the pituitary are greater than those conferred by p53 loss in that Rb+-;ARF-- mice display significantly more early atypical lesions than Rb+-; p53-- mice. Also, Rb+-;ARF-- compound mutants do not develop many of the novel tumors or precancerous lesions seen in Rb+-;p53-- compound mutants. Although complete loss of ARF expression is not obligatory for pituitary tumorigenesis in Rb+- mice, alterations of the ARF locus are observed in tumors from Rb+-;ARF+- mice, consistent with a selective advantage of ARF inactivation in this context. We conclude that inactivation of ARF acts more broadly than that of p53 in connecting abrogation of the Rb pathway to tumorigenesis.

The bacterial cell division protein FtsW has been suggested to perform two functions: stabilize the FtsZ cytokinetic ring, and facilitate septal peptidoglycan synthesis by the transpeptidase FtsI (penicillin-binding protein 3). We show here that depleting Escherichia coli cells of FtsW had little effect on the abundance of FtsZ rings but abrogated recruitment of FtsI to potential division sites. Analysis of FtsW localization confirmed and extended these results; septal localization of FtsW required FtsZ, FtsA, FtsQ, and FtsL but not FtsI. Thus, FtsW is a late recruit to the division site and is essential for subsequent recruitment of its cognate transpeptidase FtsI but not for stabilization of FtsZ rings. We suggest that a primary function of FtsW homologues--which are found in almost all bacteria and appear to work in conjunction with dedicated transpeptidases involved in division, elongation, or sporulation--is to recruit their cognate transpeptidases to the correct subcellular location.

Mycobacterium marinum causes long-term subclinical granulomatous infection in immunocompetent leopard frogs (Rana pipiens). These granulomas, organized collections of activated macrophages, share many morphological features with persistent human tuberculous infection. We examined organs of frogs with chronic M. marinum infection using transmission electron microscopy in conjunction with immunohistochemistry and acid phosphatase cytochemistry to better define the bacterium-host interplay during persistent infection. Bacteria were always found within macrophage phagosomes. These phagosomes were often fused to lysosomes, in sharp contrast to those formed during in vitro infection of J774 macrophage-like cells by M. marinum. The infected macrophages in frog granulomas showed various levels of activation, as evidenced by morphological changes, including epithelioid transformation, recent phagocytic events, phagolysosomal fusion, and disintegration of bacteria. Our results demonstrate that even long-term granulomas are dynamic environments with regard to the level of host cell activation and bacterial turnover and suggest a continuum between constantly replicating bacteria and phagocytic killing that maintains relatively constant bacterial numbers despite an established immune response. Infection with a mutant bacterial strain with a reduced capacity for intracellular replication shifted the balance, leading to a greatly reduced bacterial burden and inflammatory foci that differed from typical granulomas.

Two-dimensional crystals of cholera toxin bound to receptors in a lipid membrane give diffraction extending to 15 A resolution. Three-dimensional structure determination reveals a ring of five B subunits on the membrane surface, with one-third of the A subunit occupying the center of the ring. The remaining mass of the A subunit appears to penetrate the hydrophobic interior of the membrane. Cleavage of a disulfide bond in the A subunit, which activates the toxin, causes a major conformational change, with the A subunit mostly exiting from the B ring.

The development of synapses in the visual cortex (VC) and superior colliculus (SC) of the rabbit has been examined with the electron microscope. In both areas, the number of synapses reaches adult levels by 20--25 days of postnatal age, but the development in the visual cortex is delayed in comparison to that in the superior colliculus. When S synapses (spheroidal vesicles, asymmetric thickening) are compared with F synapses (flattened vesicles, symmetric thickening), even greater differences are seen. In both the VC and SC, S synapses develop earlier than F synapses, though there is considerable overlap. Of interest is that fact that synapses in the visual cortex seem to overshoot their adult levels late in development, suggesting that an excess of synapses may be formed in this system. Multiple synapses, probably of retinal origin, increase in the first 3 weeks of synaptic development in the SC, but never are present in significant proportions in the VC. Synapse formation most often is characterized by formation of a junction and a postsynaptic thickening, followed by acquisition of synaptic vesicles. After 15 days, there is only a small number of such "non-vesicle synapses" in either the SC or VC.