Designer membraneless organelles sequester native factors for control of cell behavior.

Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron-size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to 90% of targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.

Effect of vitamin D source and amount on vitamin D status and response to endotoxin challenge.

The objectives were to test the effects of dietary vitamin D3 [cholecalciferol (CHOL)] compared with 25-hydroxyvitamin D3 [calcidiol (CAL)] on vitamin D status and response to an endotoxin challenge. Forty-five Holstein bull calves (5 ± 2 d of age) were blocked into weekly cohorts, fed a basal diet that provided 0.25 µg/kg body weight (BW) CHOL, and assigned randomly to 1 of 5 treatments: control [(CON) no additional vitamin D], 1.5 µg/kg BW CHOL (CHOL1.5), 3 µg/kg BW CHOL (CHOL3), 1.5 µg/kg BW CAL (CAL1.5), or 3 µg/kg BW CAL (CAL3). Calves were fed milk replacer until weaning at 56 d of age and had ad libitum access to water and starter grain throughout the experiment. Treatments were added daily to the diet of milk replacer until weaning and starter grain after weaning. Measures of growth, dry matter intake, and serum concentrations of vitamin D, Ca, Mg, and P were collected from 0 to 91 d of the experiment. At 91 d of the experiment, calves received an intravenous injection of 0.1 µg/kg BW lipopolysaccharide (LPS). Clinical and physiological responses were measured from 0 to 72 h relative to LPS injection. Data were analyzed with mixed models that included fixed effects of treatment and time, and random effect of block. Orthogonal contrasts evaluated the effects of (1) source (CAL vs. CHOL), (2) dose (1.5 vs. 3.0 µg/kg BW), (3) interaction between source and dose, and (4) supplementation (CON vs. all other treatments) of vitamin D. From 21 to 91 d of the experiment, mean BW of supplemented calves was less compared with CON calves, but the effect was predominantly a result of the CHOL calves, which tended to weigh less than the CAL calves. Supplementing vitamin D increased concentrations of 25-hydroxyvitamin D in serum compared with CON, but the increment from increasing the dose from 1.5 to 3.0 µg/kg BW was greater for CAL compared with CHOL (CON = 18.9, CHOL = 24.7 and 29.6, CAL = 35.6 and 65.7 ± 3.2 ng/mL, respectively). Feeding CAL also increased serum Ca and P compared with CHOL. An interaction between source and dose of treatment was observed for rectal temperature and derivatives of reactive metabolites after LPS challenge because calves receiving CHOL3 and CAL1.5 had lower rectal temperatures and plasma derivatives of reactive metabolites compared with calves receiving CHOL1.5 and CAL3. Supplementing vitamin D increased plasma P concentrations post-LPS challenge compared with CON, but plasma concentrations of Ca, Mg, fatty acids, glucose, ß-hydroxybutyrate, haptoglobin, tumor necrosis factor-α, and antioxidant potential did not differ among treatments post-LPS challenge. Last, supplementing vitamin D increased granulocytes as a percentage of blood leukocytes post-LPS challenge compared with CON. Supplementing CAL as a source of vitamin D to dairy calves was more effective at increasing serum 25-hydroxyvitamin D, Ca, and P concentrations compared with feeding CHOL. Supplemental source and dose of vitamin D also influenced responses to the LPS challenge.

The Current Landscape of Research Advocacy and Education for Patients with Colorectal Cancer.

OPINION STATEMENT: Research advocacy is an evolving concept and should be tailored for the colorectal cancer research community. Research advocacy training and evaluation must be designed for the patient community with their insight included at each step of engagement, training, and implementation. Patient advocates bring a great deal of expertise to the research review process, but it is important to ensure that their insight is appropriately placed, and they bring an appropriate orientation to the research process as the most informed patient. This can be accomplished in part by providing advocates with the proper training, employing universal core competencies, and applying principles of adult learning. Additionally, the research community, advocacy organizations, and industry partners must understand the need to diversify the voices that are being leveraged to guide research, recognizing the importance of adequate mental health tools and compensation commensurate with their experience. As a community, it is necessary that we create and implement training programs, as well as evaluate and measure their impact to continually improve and tailor the delivery of this specific education. Research advocacy has become a necessity to the field, and when implemented effectively, research advocates can have a significant impact on the delivery of health care research, improving health outcomes for all those affected by colorectal cancer.

Priorities of Unmet Needs for Those Affected by Colorectal Cancer: Considerations From a Series of Nominal Group Technique Sessions.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer among men and women in the United States. Patients and survivors experience a range of challenges, including anxiety, financial issues, long-term adverse effects, and more. The intent of this project was to assess the needs of the CRC community directly from survivors and their caregivers and to lay a foundation for ongoing support. METHODS: Twelve nominal group technique sessions were facilitated. Participants were randomized and presented with the following questions: "What information do you wish you had at the time of diagnosis?" and "What information do you need now as a survivor?" After the nominal group technique process, each statement's score was divided by the number of people in the session, providing the average to identify the top-ranked statements. Themes and subthemes were applied to statements. Results were compared between coders. RESULTS: There was a total of 79 participants, 49 of whom self-identified as a patient with or survivor of cancer. Patient/survivor demographics were as follows: stage IV disease (n=20), stage III disease (n=22), stage II disease (n=5), stage I disease (n=2), caregiver/family member (n=30), male (n=16), female (n=63), White (n=50), Native Hawaiian/Pacific Islander (n=1), Hispanic/Latino (n=13), Black/African American (n=11), Asian (n=1), and more than one race/ethnicity (n=3). The most frequent themes among responses to the first question were communication and coordination with care team and access to CRC resources. The most frequent themes among responses to the second question were psychosocial support and family/caregiver support. Frequent themes among responses across both questions were understanding treatment options and adverse effects. CONCLUSIONS: These findings highlight gaps in support for individuals affected by CRC, and lay a foundation for ongoing assistance. Future studies exploring differences based on disease stage, race/ethnicity, age, gender identity, geographic location, and tumor location are needed to further tailor support for those experiencing CRC. Themes identified in this project require a multidisciplinary approach to ensure that the unmet needs of survivors are addressed.

Evaluation of solar photovoltaic systems to shade cows in a pasture-based dairy herd.

The combined use of solar photovoltaics and agriculture may provide farmers with an alternative source of income and reduce heat stress in dairy cows. The objective of this study was to determine the effects on grazing cattle under shade from a solar photovoltaic system. The study was conducted at the University of Minnesota West Central Research and Outreach Center in Morris, Minnesota on a grazing dairy. Twenty-four crossbred cows were randomly assigned to 2 treatment groups (shade or no shade) from June to September in 2019. The replicated (n = 4) treatment groups of 6 cows each were provided shade from a 30-kW photovoltaic system. Two groups of cows had access to shade in paddocks, and 2 groups of cows had no shade in paddocks. All cows were located in the same pasture during summer. Behavior observations and milk production were evaluated for cows during 4 periods of summer. Boluses and an eartag sensor monitored internal body temperature, activity, and rumination on all cows, respectively. Independent variables were the fixed effects of breed, treatment group, coat color, period, and parity, and random effects were replicate group, date, and cow. No differences in fly prevalence, milk production, fat and protein production, or drinking bouts were observed between the treatment groups. Shade cows had more ear flicks (11.4 ear flicks/30 s) than no-shade cows (8.6 ear flicks/30 s) and had dirtier bellies and lower legs (2.2 and 3.2, respectively) than no-shade cows (1.9 and 2.9, respectively). During afternoon hours, shade cows had lower respiration rates (66.4 breaths/min) than no-shade cows (78.3 breaths/min). From 1200 to 1800 h and 1800 to 0000 h, shade cows had lower body temperature (39.0 and 39.2°C, respectively) than no-shade cows (39.3 and 39.4°C, respectively). Furthermore, between milking times (0800 and 1600 h), the shade cows had lower body temperature (38.9°C) than no-shade cows (39.1°C). Agrivoltaics incorporated into pasture dairy systems may reduce the intensity of heats stress in dairy cows and increase well-being of cows and the efficiency of land use.

Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

Role of the Δ Resonance in the Population of a Four-Nucleon State in the

The ^{54}Fe nucleus was populated from a ^{56}Fe beam impinging on a Be target with an energy of E/A=500 MeV. The internal decay via γ-ray emission of the 10^{+} metastable state was observed. As the structure of this isomeric state has to involve at least four unpaired nucleons, it cannot be populated in a simple two-neutron removal reaction from the ^{56}Fe ground state. The isomeric state was produced in the low-momentum (-energy) tail of the parallel momentum (energy) distribution of ^{54}Fe, suggesting that it was populated via the decay of the Δ^{0} resonance into a proton. This process allows the population of four-nucleon states, such as the observed isomer. Therefore, it is concluded that the observation of this 10^{+} metastable state in ^{54}Fe is a consequence of the quark structure of the nucleons.

Assessing susceptibility for polycyclic aromatic hydrocarbon toxicity in an in vitro 3D respiratory model for asthma.

There is increased emphasis on understanding cumulative risk from the combined effects of chemical and non-chemical stressors as it relates to public health. Recent animal studies have identified pulmonary inflammation as a possible modifier and risk factor for chemical toxicity in the lung after exposure to inhaled pollutants; however, little is known about specific interactions and potential mechanisms of action. In this study, primary human bronchial epithelial cells (HBEC) cultured in 3D at the air-liquid interface (ALI) are utilized as a physiologically relevant model to evaluate the effects of inflammation on toxicity of polycyclic aromatic hydrocarbons (PAHs), a class of contaminants generated from incomplete combustion of fossil fuels. Normal HBEC were differentiated in the presence of IL-13 for 14 days to induce a profibrotic phenotype similar to asthma. Fully differentiated normal and IL-13 phenotype HBEC were treated with benzo[a]pyrene (BAP; 1-40 µg/mL) or 1% DMSO/PBS vehicle at the ALI for 48 h. Cells were evaluated for cytotoxicity, barrier integrity, and transcriptional biomarkers of chemical metabolism and inflammation by quantitative PCR. Cells with the IL-13 phenotype treated with BAP result in significantly (p < 0.05) decreased barrier integrity, less than 50% compared to normal cells. The effect of BAP in the IL-13 phenotype was more apparent when evaluating transcriptional biomarkers of barrier integrity in addition to markers of mucus production, goblet cell hyperplasia, type 2 asthmatic inflammation and chemical metabolism, which all resulted in dose-dependent changes (p < 0.05) in the presence of BAP. Additionally, RNA sequencing data showed that the HBEC with the IL-13 phenotype may have increased potential for uncontrolled proliferation and decreased capacity for immune response after BAP exposure compared to normal phenotype HBEC. These data are the first to evaluate the role of combined environmental factors associated with inflammation from pre-existing disease and PAH exposure on pulmonary toxicity in a physiologically relevant human in vitro model.

Genome sequence of Soos: a siphovirus of the CP cluster infecting Gordonia rubripertincta  .

Novel actinobacteriophage Soos was isolated and purified from Southern Indiana soil using host Gordonia rubripertincta NRRL B-16540. Sequencing revealed a 57,509 bp circularly permuted genome encoding 87 predicted protein-coding genes. Soos is only the third phage in cluster CP, along with phages Clawz and Sting.

Effects of cooled floor pads combined with chilled drinking water on behavior and performance of lactating sows under heat stress.

This study was conducted to evaluate whether cooled floor pads combined with chilled drinking water could alleviate negative impacts of heat stress on lactating sows. Thirty sows (Landrace × Yorkshire, Parity = 1 to 6) were housed in individual farrowing stalls in two rooms with temperatures being controlled at 29.4°C (0700-1900 hours) and 23.9°C (1900-0700 hours). Sows in one room (Cool), but not in the other room (Control) were provided cooled floor pads (21-22°C) and chilled drinking water (13-15°C). Behavior of sows (15 sows/treatment) was video recorded during farrowing, and days 1, 3, 7, 14, and 21 after farrowing. Videos were viewed continuously to register the birth time of each piglet, from which total farrowing duration and birth intervals were calculated. The number of drinking bouts and the duration of each drinking bout were registered for each sow through viewing videos continuously for 2 h (1530-1730 hours) each video-recording day. Postures (lying laterally, lying ventrally, sitting, and standing) were recorded by scanning video recordings at 5-min intervals for 24 h each video-recording day, and time budget for each posture was calculated. Rectal temperature and respiration rate were measured for all sows the day before and after farrowing, and then once weekly. Sow and litter performance was recorded. Data were analyzed using the Glimmix procedure of SAS. The cooling treatment did not affect sow behavior or litter performance. Sows in the Cool room had lower rectal temperature (P = 0.03) and lower respiration rate (P < 0.001), consumed more feed (P = 0.03), tended to have reduced weight loss (P = 0.07), and backfat loss (P = 0.07) during lactation than sows in the Control room. As lactation progressed, sows increased drinking frequency (P < 0.001) and time spent lying ventrally (P < 0.0001), standing (P < 0.001), and sitting (P < 0.0001), and decreased time spent lying laterally (P < 0.0001) in both Cool and Control rooms. While cooled floor pads combined with chilled drinking water did not affect sow behavior, they did alleviate heat stress partially, as indicated by decreased rectal temperature, respiration rate, weight, and backfat loss, and increased feed intake in lactating sows.

Phylogenetic diversity of 200+ isolates of the ectomycorrhizal fungus Cenococcum geophilum associated with Populus trichocarpa soils in the Pacific Northwest, USA and comparison to globally distributed representatives.

The ectomycorrhizal fungal symbiont Cenococcum geophilum is of high interest as it is globally distributed, associates with many plant species, and has resistance to multiple environmental stressors. C. geophilum is only known from asexual states but is often considered a cryptic species complex, since extreme phylogenetic divergence is often observed within nearly morphologically identical strains. Alternatively, C. geophilum may represent a highly diverse single species, which would suggest cryptic but frequent recombination. Here we describe a new isolate collection of 229 C. geophilum isolates from soils under Populus trichocarpa at 123 collection sites spanning a ~283 mile north-south transect in Western Washington and Oregon, USA (PNW). To further understanding of the phylogenetic relationships within C. geophilum, we performed maximum likelihood and Bayesian phylogenetic analyses to assess divergence within the PNW isolate collection, as well as a global phylogenetic analysis of 789 isolates with publicly available data from the United States, Japan, and European countries. Phylogenetic analyses of the PNW isolates revealed three distinct phylogenetic groups, with 15 clades that strongly resolved at >80% bootstrap support based on a GAPDH phylogeny and one clade segregating strongly in two principle component analyses. The abundance and representation of PNW isolate clades varied greatly across the North-South range, including a monophyletic group of isolates that spanned nearly the entire gradient at ~250 miles. A direct comparison between the GAPDH and ITS rRNA gene region phylogenies, combined with additional analyses revealed stark incongruence between the ITS and GAPDH gene regions, consistent with intra-species recombination between PNW isolates. In the global isolate collection phylogeny, 34 clades were strongly resolved using Maximum Likelihood and Bayesian approaches (at >80% MLBS and >0.90 BPP respectively), with some clades having intra- and intercontinental distributions. Together these data are highly suggestive of divergence within multiple cryptic species, however additional analyses such as higher resolution genotype-by-sequencing approaches are needed to distinguish potential species boundaries and the mode and tempo of recombination patterns.

Isolation of oligodendrocyte-like cells from human umbilical cord blood.

BACKGROUND: As human umbilical cord blood (UCB) is known to be a rich source of progenitor cells, the prospect of isolating a subset of these cells that could differentiate into cells of non-hematopoietic lineages suggests a therapeutic use for patients with inherited lysosomal and peroxisomal storage diseases currently treated with UCB transplantation. METHODS: Oligodendrocyte-like cells were isolated from UCB by density-gradient centrifugation and expanded using selective media. We then characterized this population of cells using standard immunohistochemical staining methods for neural cell proteins and polymerase chain reaction (PCR) to detect RNA sequences for myelin basic protein (MBP). We also developed a functional assay demonstrating myelination of neurons in vitro. RESULTS: Cells with oligodendrocyte-like morphology were reproducibly cultured ex vivo from fresh human UCB. Cells stained positively for multiple oligodendria cell markers (O1, MBP and CNPase) via immunohistochemical staining and flow cytometry. PCR confirmed the presence of MBP and CNPase mRNA. A further in vitro functional assay demonstrated the myelination of mature neuronal cells from the brain of a myelin-deficient murine model co-cultured with the oligodendrocyte-like cells. DISCUSSION: After human UCB transplant, donor-derived cells have been noted to migrate to the brain over time. Although is not known whether these cells solely deliver enzyme replacement or a subset engrafts and differentiates into mature neural cells, the clinical improvements noted in these patients suggest a potential role for targeted cellular therapy. Oligodendrocyte-like cells isolated ex vivo and expanded from human UCB could provide a potential cellular therapy for patients with demyelinating or dismyelinating diseases.

Controllable protein phase separation and modular recruitment to form responsive membraneless organelles.

Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles. Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. Here we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. First, we demonstrate enzymatically triggered droplet assembly and disassembly, whereby miscibility and RGG domain valency are tuned by protease activity. Second, we control droplet composition by selectively recruiting cargo molecules via protein interaction motifs. We then demonstrate protease-triggered controlled release of cargo. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells. This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells.

Specific affinity between fibronectin and the epidermolysis bullosa acquisita antigen.

Autoantibodies in the skin and sera of patients with epidermolysis bullosa acquisita bind to a large matrix molecule within the lamina densa region of skin basement membrane. At the site of these immune complexes, the epidermis separates from the dermis, which creates a subepidermal blister just below the lamina densa. The target molecule for the autoantibodies is in close apposition to fibronectin, a major extracellular matrix molecule that is abundant in the upper dermis of skin. In this report, we show specific affinity between fibronectin and the 290,000-D chain of the epidermolysis bullosa acquisita antigen, and that this affinity is mediated by the gelatin/collagen-binding domain of fibronectin (Mr = 60,000). Since blistering in epidermolysis bullosa acquisita often occurs in the absence of clinical and histological inflammation, a direct interruption in the fibronectin-epidermolysis bullosa acquisita antigen bond may be involved in the pathogenesis of epidermal-dermal disadherence that occurs in this bullous disease.

Epidermolysis bullosa acquisita antigen is the globular carboxyl terminus of type VII procollagen.

Epidermolysis bullosa acquisita (EBA) is a severe, chronic blistering disease of the skin. EBA patients have circulating and tissue-bound autoantibodies to a large (Mr = 290,000) macromolecule that is localized within the basement membrane zone between the epidermis and dermis of skin, the site of blister formation. The "EBA antigen" is known to be distinct from laminin, heparan sulfate proteoglycan, fibronectin, the bullous pemphigoid antigen, elastin, and collagen types I, II, III, IV, and V. Sera from patients with EBA, two monoclonal antibodies to the EBA antigen, and a monoclonal antibody to the carboxyl terminus of type VII procollagen identically label human amnion and skin by immunofluorescent and immunoelectron microscopy. Western immunoblots of the EBA antigen extracted from skin and of type VII procollagen labeled with the above sera and antibodies are identical. None of the sera or antibodies labels Western blots of pepsinized type VII collagen which is missing the globular amino and carboxyl terminal domains. These data show that the EBA antigen is the carboxyl terminus of type VII procollagen.

Annotating eukaryote genomes.

The Genome Annotation Assessment Project tested current methods of gene identification, including a critical assessment of the accuracy of different methods. Two new databases have provided new resources for gene annotation: these are the InterPro database of protein domains and motifs, and the Gene Ontology database for terms that describe the molecular functions and biological roles of gene products. Efforts in genome annotation are most often based upon advances in computer systems that are specifically designed to deal with the tremendous amounts of data being generated by current sequencing projects. These efforts in analysis are being linked to new ways of visualizing computationally annotated genomes.

Niobium direct detectors for fast and sensitive terahertz spectroscopy.

We report the performance of a niobium hot-electron bolometer designed for laboratory terahertz spectroscopy. The antenna-coupled detector can operate above 4.2 K and has fast (subnanosecond) response. Detailed microwave measurements of performance over a wide range of operating conditions were correlated with quantitative terahertz measurements. The maximum responsivity is 4 x 10(4) VW with a noise equivalent power at the detector of 2 x 10(-14) W/Hz(12), approaching the intrinsic thermal fluctuation limit for the device. This detector enables a variety of novel laboratory spectroscopy measurements.

Tumor progression in vivo: increased soybean agglutinin lectin binding, N-acetylgalactosamine-specific lectin expression, and liver metastasis potential.

Tumors which grew out from threshold s.c. inocula of L5178Y-F9 and SL2-5 murine T-cell lymphomas in syngeneic DBA/2 mice exhibited a unified natural defense-resistant phenotype including an increased tumorigenicity and correlating reductions in susceptibility to natural antibodies, natural killer cells, and activated macrophages in vitro. The metastatic potential and cell surface saccharide expression of these cells were determined to assess the impact of growth from a small tumor focus in vivo on subsequent metastatic ability and to determine whether there was any association with changes in cell surface carbohydrates, which have been implicated now for many years in tumor development. A significantly increased liver-colonizing ability was observed following i.v. injection. The most consistent change in cell surface saccharide expression detected in studies using five lectins was an increase in N-acetyl-D-galactosamine (D-GalNAc)-specific soybean agglutinin (SBA) binding. The log of experimental liver metastasis, SBA binding, and the percentage of hepatocyte rosetting of the parental and in vivo-selected cells exhibited significant direct correlations. While inhibition of rosetting with in vivo-selected lines by D-GalNAc and galactose was consistent with the involvement of the D-galactose/D-GalNAc-specific hepatocyte receptor, preincubation of the tumor cells but not hepatocytes with D-GalNAc inhibited hepatocyte rosetting and D-GalNAc inhibited homotypic tumor cell binding. These data suggest a role for a saccharide-specific, lectin-like receptor on tumor cells in both interactions and therefore in the increased experimental liver metastasis. Furthermore, the increased expression of D-GalNAc-inhibitable SBA binding sites on the in vivo-selected variants should increase the homotypic binding by the D-GalNAc-specific lectin-like receptors on the tumor cells providing a rationale for the direct relationship observed between increased SBA binding and i.v. metastatic potential.

Perceptions of Threats to Physical Safety, Sexual Autonomy, Values, and of Discrimination Drive LGB Prejudices Toward Heterosexuals.

Many studies have investigated heterosexuals' prejudices toward nonheterosexuals, yet LGB's prejudices toward heterosexuals remain largely unexplored. Therefore, we sought to determine the threats and opportunities (i.e., affordances) LGB perceive heterosexuals to pose and whether those affordances explain their sexual prejudices toward heterosexuals. Study 1 analyzed LGB's reasons for liking and disliking heterosexuals, which determined whether the threats predicted to be salient for LGB mirrored the affordances they generated. Study 2 measured these perceived affordances and examined the extent to which they drove LGB's prejudices toward heterosexuals. Generally, perceptions of discrimination and unreciprocated sexual interest threats drove anger, physical safety and sexual autonomy threats drove fear, and values threats drove moral disgust toward heterosexuals, although results varied slightly by perceiver and target groups. Goals to alleviate the tensions between heterosexuals and LGB require an understanding of the dynamics between these groups. This research provides preliminary insights into understanding those dynamics.

An exploration of the sequence of a 2.9-Mb region of the genome of Drosophila melanogaster: the Adh region.

A contiguous sequence of nearly 3 Mb from the genome of Drosophila melanogaster has been sequenced from a series of overlapping P1 and BAC clones. This region covers 69 chromosome polytene bands on chromosome arm 2L, including the genetically well-characterized "Adh region." A computational analysis of the sequence predicts 218 protein-coding genes, 11 tRNAs, and 17 transposable element sequences. At least 38 of the protein-coding genes are arranged in clusters of from 2 to 6 closely related genes, suggesting extensive tandem duplication. The gene density is one protein-coding gene every 13 kb; the transposable element density is one element every 171 kb. Of 73 genes in this region identified by genetic analysis, 49 have been located on the sequence; P-element insertions have been mapped to 43 genes. Ninety-five (44%) of the known and predicted genes match a Drosophila EST, and 144 (66%) have clear similarities to proteins in other organisms. Genes known to have mutant phenotypes are more likely to be represented in cDNA libraries, and far more likely to have products similar to proteins of other organisms, than are genes with no known mutant phenotype. Over 650 chromosome aberration breakpoints map to this chromosome region, and their nonrandom distribution on the genetic map reflects variation in gene spacing on the DNA. This is the first large-scale analysis of the genome of D. melanogaster at the sequence level. In addition to the direct results obtained, this analysis has allowed us to develop and test methods that will be needed to interpret the complete sequence of the genome of this species. Before beginning a Hunt, it is wise to ask someone what you are looking for before you begin looking for it. Milne 1926