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Highthroughput drug screening is an established approach to investigate tumor biology and identify therapeutic leads. Traditional platforms use two-dimensional cultures which do not accurately reflect the biology of human tumors. More clinically relevant model systems such as three-dimensional tumor organoids can be difficult to scale and screen. Manually seeded organoids coupled to destructive endpoint assays allow for the characterization of treatment response, but do not capture transitory changes and intra-sample heterogeneity underlying clinically observed resistance to therapy. We present a pipeline to generate bioprinted tumor organoids linked to label-free, time-resolved imaging via high-speed live cell interferometry (HSLCI) and machine learning-based quantitation of individual organoids. Bioprinting cells gives rise to 3D structures with unaltered tumor histology and gene expression profiles. HSLCI imaging in tandem with machine learning-based segmentation and classification tools enables accurate, label-free parallel mass measurements for thousands of organoids. We demonstrate that this strategy identifies organoids transiently or persistently sensitive or resistant to specific therapies, information that could be used to guide rapid therapy selection.

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Small multidrug resistance (SMR) transporters contribute to antibiotic resistance through proton-coupled efflux of toxic compounds. Previous biophysical studies of the E. coli SMR transporter EmrE suggest that it should also be able to perform proton/toxin symport or uniport, leading to toxin susceptibility rather than resistance in vivo. Here we show EmrE does confer susceptibility to several previously uncharacterized small-molecule substrates in E. coli, including harmane. In vitro electrophysiology assays demonstrate that harmane binding triggers uncoupled proton flux through EmrE. Assays in E. coli are consistent with EmrE-mediated dissipation of the transmembrane pH gradient as the mechanism underlying the in vivo phenotype of harmane susceptibility. Furthermore, checkerboard assays show this alternative EmrE transport mode can synergize with some existing antibiotics, such as kanamycin. These results demonstrate that it is possible to not just inhibit multidrug efflux, but to activate alternative transport modes detrimental to bacteria, suggesting a strategy to address antibiotic resistance.

Small multidrug resistance (SMR) transporters efflux toxic substrates from bacterial cells and were recently divided into two subfamilies: specific toxic metabolite transporters and promiscuous drug exporters. These drug exporters are thought to function similarly to EmrE, the model system for this subfamily of SMR transporters. Studies of EmrE homologs indicate that they are able to confer resistance to EmrE substrates inE. coliand in their native organisms. Recent work from our lab showed that functional EmrE can confer resistance or susceptibilityin vivodepending on the drug substrate. Here, we test whether this functional promiscuity of EmrE extends to SMR transporters from three additional human or animal pathogens: SAsmr fromStaphylococcus aureus, PAsmr fromPseudomonas aeruginosa, and FTsmr fromFrancisella tularensis. We find that these SMR homologs can confer either resistance or susceptibility to different toxic substrates inE. coli. This demonstrates that the ability of a single transporter to lead to opposite biological outcomes when transporting different substrates is a general property of the promiscuous multidrug transporters in the SMR family. It also suggests the potential for novel antibiotic development targeting these transporters with small molecules that trigger susceptibility. Such a strategy does not require that the target be the primary mode for antibiotic resistance because the goal is not simple inhibition of activity, but rather activation of an alternative transport function that is detrimental to bacteria.

EmrE, a small multidrug resistance (SMR) transporter from E. coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of 2 protons:1 cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14

EmrE, a small multidrug resistance (SMR) transporter from E. coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of 2 protons:1 cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14A and E14B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue while the other remains accessible to bind and transport a proton. Here we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Further, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.

Small multidrug resistance (SMR) transporters contribute to antibiotic resistance through proton-coupled efflux of toxic compounds. Previous biophysical studies of the E. coli SMR transporter EmrE suggest that it should also be able to perform proton/toxin symport or uniport, leading to toxin susceptibility rather than resistance in vivo. Here we show EmrE does confer susceptibility to several previously uncharacterized small-molecule substrates in E. coli, including harmane. In vitro electrophysiology assays demonstrate that harmane binding triggers uncoupled proton flux through EmrE. Assays in E. coli are consistent with EmrE-mediated dissipation of the transmembrane pH gradient as the mechanism underlying the in vivo phenotype of harmane susceptibility. Furthermore, checkerboard assays show this alternative EmrE transport mode can synergize with some existing antibiotics, such as kanamycin. These results demonstrate that it is possible to not just inhibit multidrug efflux, but to activate alternative transport modes detrimental to bacteria, suggesting a strategy to address antibiotic resistance.

Biodiversity monitoring plays an essential role in tracking changes in ecosystems, species distributions and abundances across the globe. Data collected through both structured and unstructured biodiversity recording can inform conservation measures designed to reduce, prevent, and reverse declines in valued biodiversity of many types. However, given that resources for biodiversity monitoring are limited, it is important that funding bodies prioritise investments relative to the requirements in any given region. We addressed this prioritisation requirement for a biodiverse Mediterranean island (Cyprus) using a three-stage process of expert-elicitation. This resulted in a structured list of twenty biodiversity monitoring needs; specifically, a hierarchy of three groups of these needs was created using a consensus approach. The most highly prioritised biodiversity monitoring needs were those related to the development of robust survey methodologies, and those ensuring that sufficiently skilled citizens are available to contribute. We discuss ways that the results of our expert-elicitation process could be used to support current and future biodiversity monitoring in Cyprus.

The homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance. We recently determined the structure of substrate-bound EmrE in phospholipid bilayers by measuring hundreds of protein-ligand HN–F distances for a fluorinated substrate, 4-fluoro-tetraphenylphosphonium (F4-TPP+), using solid-state NMR. This structure was solved at low pH where one of the two proton-binding Glu14 residues is protonated. Here, to understand how substrate transport depends on pH, we determine the structure of the EmrE-TPP complex at high pH, where both Glu14 residues are deprotonated. The high-pH complex exhibits an elongated and hydrated binding pocket in which the substrate is similarly exposed to the two sides of the membrane. In contrast, the low-pH complex asymmetrically exposes the substrate to one side of the membrane. These pH-dependent EmrE conformations provide detailed insights into the alternating-access model, and suggest that the high-pH conformation may facilitate proton binding in the presence of the substrate, thus accelerating the conformational change of EmrE to export the substrate.

We develop a method to apply a high-throughput quantitative phase imaging-based assay to functional biomass accumulation measurements of 3D tumor organoids with single-organoid resolution, and demonstrate its utility for drug screening and therapy selection.

The homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance. We recently determined the structure of substrate-bound EmrE in phospholipid bilayers by measuring hundreds of protein-ligand H

High-throughput drug screening is an established approach to investigate tumor biology and identify therapeutic leads. Traditional platforms for high-throughput screening use two-dimensional cultures of immortalized cell lines which do not accurately reflect the biology of human tumors. More clinically relevant model systems, such as three-dimensional tumor organoids, can be difficult to screen and scale. For example, manually seeded organoids coupled to destructive endpoint assays allow for the characterization of response to treatment, but do not capture the transitory changes and intra-sample heterogeneity underlying clinically observed resistance to therapy. We therefore developed a pipeline to generate bioprinted tumor organoids linked to label-free, real-time imaging via high-speed live cell interferometry (HSLCI) and machine learning-based quantitation of individual organoids. Bioprinting cells gives rise to 3D organoid structures that preserve tumor histology and gene expression. HSLCI imaging in tandem with machine learning-based image segmentation and organoid classification tools enables accurate, label-free parallel mass measurements for thousands of bioprinted organoids. We demonstrate that our method quantitatively identifies individual organoids as insensitive, transiently sensitive, or persistently sensitive to specific treatments. This opens new avenues for rapid, actionable therapeutic selection using automated tumor organoid screening.

As part of our continuing study of light variability in proto-planetary nebulae (PPNe), we present the results from a long-term study of nine southern hemisphere objects. We have monitored their light variations over a nine-year interval from 2010-2018. These were supplemented by data from the ASAS-SN and ASAS-3 surveys, leading to combined light curves from 2000 to 2020. Pulsation periods were found in seven of the objects, although the three shortest must be regarded as tentative. The periods range from 24 to 73 days. When compared with the results of previous studies of the light variations in PPNe, we find that they show the same trends of shorter period and smaller light variations with higher temperatures. Luminosities were calculated based on the spectral energy distributions, reddening, and Gaia distances, and these confirm the identification of all but one as post-AGB objects. Three of the stars possess long-period variations of 5 to 19 years. These are most likely due to the periodic obscuration of the star by a disk, suggesting the presence of a binary companion and a circumbinary disk., 27 pages, 6 tables, 8 figures. Accepted Sep 1, 2021 for publication in the Astronomical Journal: revised in attempt to correct pdf file

Biodiversity monitoring plays an essential role in tracking changes in ecosystems, species distributions and abundances across the globe. Data collected through both structured and unstructured biodiversity recording can inform conservation measures designed to reduce, prevent, and reverse declines in valued biodiversity of many types. However, given that resources for biodiversity monitoring are limited, it is important that funding bodies prioritise investments relative to the requirements in any given region. We addressed this prioritisation requirement for a biodiverse Mediterranean island (Cyprus) using a three-stage process of expert-elicitation. This resulted in a structured list of twenty biodiversity monitoring needs; specifically, a hierarchy of three groups of these needs was created using a consensus approach. The most highly prioritised biodiversity monitoring needs were those related to the development of robust survey methodologies, and those ensuring that sufficiently skilled citizens are available to contribute. We discuss ways that the results of our expert-elicitation process could be used to support current and future biodiversity monitoring in Cyprus.

We have carried out a new photometric V,Rc study of 12 protoplanetary nebulae, objects in the short-lived transition between the AGB and PN phases of stellar evolution. These had been the subjects of an earlier study, using data from 1994-2007, that found that all 12 varied periodically, with pulsation periods in the range of ~38 to ~150 days. They are all carbon-rich, with F-G spectral types. We combined our new (2008-2018) data with publicly-available ASAS-SN data and determined new periods for their variability. The older and newer period values were compared to investigate evidence of period change, for which there is theoretical support that it might be detectable in a decade or two in some cases. Such a detection is challenging since the light curves are complicated, with multiple periods, changing amplitudes, and evidence of shocks. Nevertheless, we found one, and possibly two, such cases, which are associated with the higher temperature stars in the sample (7250 and 8000 K). These results are most consistent with the evolution of stars at the lower end of the mass range of carbon stars, ~1.5-2 M(sun). Several of the stars show longer-term trends of increasing (six cases) or decreasing (one case) brightness, which we think most likely due to changes in the circumstellar dust opacity. There is one case of a possible ~1.8 yr period in addition to the shorter pulsation. This is interpreted as possible evidence of an orbiting companion., Comment: 26 pages, 9 figures. Accepted September 16, 2022 for publication in the ApJ

The efficient and non-derivatizing dissolution of natural biopolymers by ionic liquids (ILs) is poised to redefine the application of these ubiquitous and structurally robust materials on a grand industrial scale. In concert with this idea is Natural Fiber Welding (NFW), a technique which exploits the solvating properties of ionic liquids to partially dissociate the outer biopolymer layers in strands of fibrous thread, followed by solvent removal to collapse the structures in a reconfigured and entangled hydrogen bonding network. For cotton, this welding produces a layered motif of mesoporous cellulose atop a core of unmodified crystalline cellulose, creating a hierarchical structure which retains a semblance of the native biomaterial. Aggressive welding conditions increase the thickness and surface area of the mesoporous layer, though excessive welding leads to full dissolution of the crystalline core creating a tradeoff between an increase in surface area with loss of product integrity. In the present work, we seek to subvert aggressive welding conditions by supplementing dissolved cellulose as an additive in the welding solution. We will demonstrate how this extraneous cellulose can coat and weld to the underlying cotton substrate to increase the size of the amorphous shell around the crystalline core and, ultimately, bolster the mesoporous network and enhance the overall surface area. In addition, we will discuss how Raman-sensitive functionalization can be used to identify inclusion depth of the extraneous material and as a way to probe the potential for layer-by-layer deposition of functional cellulose during the NFW process.

A unique phenomenon, known locally as “town ball,” occurs every summer in Minnesota. Most individuals who support town ball teams are from rural communities. Despite small populations, teams often ...

As interest grows in using tumor organoids as models for drug discovery and precision medicine, platforms for the reliable functional testing and analysis of these organoids are needed. Existing functional approaches in precision medicine face difficulties in the creation, scaling, and analysis of physiological tumor organoid models. We present a method for the automated bioprinting of tumor organoids coupled with real-time biomass quantification using high-speed live cell interferometry (HSLCI). This workflow enhances consistency, preserves sample viability, and assesses response to treatment with single-organoid resolution. First, we describe the bioprinting protocol used to achieve the deposition of organoids in uniformly thin three-dimensional layers of Matrigel. Then we show that bioprinting preserves the histological and molecular characteristics of manually seeded organoids. In drug screening experiments, we demonstrate the simultaneous monitoring of thousands of individual organoids and show that HSLCI can rapidly identify drug sensitivity and resistance to treatment. We observe significant heterogeneity in the drug responses of single organoids and discuss the potential implications for use of this pipeline in the clinical setting. Citation Format: Peyton J. Tebon, Bowen Wang, Alexander L. Markowitz, Graeme Murray, Ardalan Davarifar, Huyen T. Nguyen, Nasrin Tavanaie, Thang L. Nguyen, Paul C. Boutros, Michael A. Teitell, Alice Soragni. High-speed live cell interferometry captures heterogeneity in bioprinted tumor organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3081.

Bone and soft tissue sarcomas span a spectrum of over one hundred histologic subtypes. Developing clinical trials and identifying effective therapies remains a challenge given the rarity of these tumors and scarcity of available tumor models. Patient-derived tumor organoids (PDTOs) are representative of the native physiology of tumors across an array of malignancies (Phan et al, 2019; Al Shihabi et al, 2021). For rare tumors such as bone and soft tissue sarcomas, PDTOs could constitute an important tool to better understand the biology of the disease and identify treatment options. Here, we present the pipeline we have established at UCLA to systematically procure tissue, develop and characterize individualized sarcoma PDTOs, and identify tumor sensitivities using high-throughput drug screening. We have collected tumor specimens from 114 patients diagnosed with sarcomas, representing more than 30 distinct subtypes of soft tissue and bone tumors. We have established suitable conditions to generate organoids from over 100 samples collected from needle biopsies, tumor resections, and metastasectomies, including soft tissue sarcomas (liposarcoma, undifferentiated pleomorphic sarcoma, leiomyosarcoma, etc.) and bone sarcomas (chondrosarcoma, osteosarcoma, Ewing sarcoma, etc.). We take advantage of our mini-ring technology to generate PDTOs in a format compatible with high-throughput drug screening (Phan et al, 2019; Nguyen et al, 2020). Our results highlight tumor-specific drug susceptibilities to chemotherapeutics, targeted agents, and combination therapies, generating results within a week from surgery. We will show how several of the organoid response patterns we identified transcend cancer subtype and patient characteristics. Our findings support the feasibility of rapidly generating individualized organoid models from bone and soft tissue sarcomas to facilitate biological interrogations and investigate response to treatment. Direct testing of PDTOs derived from clinical specimens can provide timely, actionable information for functional precision medicine approaches with the potential to improve patient outcomes. Citation Format: Ahmad Al Shihabi, Peyton J. Tebon, Jomjit Chantharasamee, Huyen T. Nguyen, Sara Sartini, Ardalan Davarifar, Nasrin Tavanaie, Scott D. Nelson, Noah Federman, Jane Yanagawa, Alice Soragni. A pipeline for functional precision medicine in bone and soft tissue sarcoma organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3078.

Small multidrug resistance (SMR) transporters efflux toxic substrates from bacterial cells. These transporters were recently divided into two subfamilies: the GdX-like and EmrE-like SMRs. The EmrE-like subfamily of SMRs is predicted to contain transporters that are highly promiscuous in both substrate specificity and mechanism based on extensive characterization of the founding member of this subfamily, EmrE. However, there is only limited functional analysis of other members of this family from pathogenic strains such as Staphylococcus aureus and Francisella tularensis. Here, we use a small compound screen to explore the substrate specificity and diversity of EmrE-subfamily SMRs from these two bacterial species and confirm that they are functionally more like EmrE than the GdX-like subfamily of toxic-metabolite transporters. The results of these experiments lay the foundation for understanding the complex substrate specificity profiles of SMR family transporters and assess the potential for targeting these transporters for future antibiotic development, either broadly or in a species-specific manner.

Significance We inferred the phylogeny and evolution of beetles using genomic data of an unprecedented scale. Moreover, we documented the diversification of plant-feeding (herbivorous) beetles, which account for nearly half of all beetle species and a similar proportion of herbivorous insects, following convergent horizontal transfers of bacterial and fungal genes enabling the digestion of lignocellulose in plant cell walls. Our findings clarify beetle phylogenetic relationships and reveal new insights into the evolution of specialized herbivory and why there are so many species of beetles. Furthermore, they underscore the intimacy and complexity of the evolutionary relationships between insects, plants, and microorganisms and show how analyses of large-scale genomic data are revealing the evolution and genomic basis of insect biodiversity., The order Coleoptera (beetles) is arguably the most speciose group of animals, but the evolutionary history of beetles, including the impacts of plant feeding (herbivory) on beetle diversification, remain poorly understood. We inferred the phylogeny of beetles using 4,818 genes for 146 species, estimated timing and rates of beetle diversification using 89 genes for 521 species representing all major lineages and traced the evolution of beetle genes enabling symbiont-independent digestion of lignocellulose using 154 genomes or transcriptomes. Phylogenomic analyses of these uniquely comprehensive datasets resolved previously controversial beetle relationships, dated the origin of Coleoptera to the Carboniferous, and supported the codiversification of beetles and angiosperms. Moreover, plant cell wall-degrading enzymes (PCWDEs) obtained from bacteria and fungi via horizontal gene transfers may have been key to the Mesozoic diversification of herbivorous beetles—remarkably, both major independent origins of specialized herbivory in beetles coincide with the first appearances of an arsenal of PCWDEs encoded in their genomes. Furthermore, corresponding (Jurassic) diversification rate increases suggest that these novel genes triggered adaptive radiations that resulted in nearly half of all living beetle species. We propose that PCWDEs enabled efficient digestion of plant tissues, including lignocellulose in cell walls, facilitating the evolution of uniquely specialized plant-feeding habits, such as leaf mining and stem and wood boring. Beetle diversity thus appears to have resulted from multiple factors, including low extinction rates over a long evolutionary history, codiversification with angiosperms, and adaptive radiations of specialized herbivorous beetles following convergent horizontal transfers of microbial genes encoding PCWDEs.

Several anti-cancer compounds, including chemotherapy and targeted agents such as ifosfamide, cyclophosphamide, and imatinib, are administered as prodrugs which are metabolized to become pharmacologically active. Prodrugs are particularly difficult to study in vitro or ex vivo as many are activated in the liver by Cytochrome P450 enzymes. Preclinical studies of these compounds are restricted to in vivo models, as cancer cells typically do not express the enzymes required to transform prodrugs into their active form. While animal models effectively recapitulate the complexity of systemic drug metabolism and distribution, in vivo studies are time-consuming, expensive, and unsuitable for high-throughput drug screening studies. In previous work, we have developed a platform for screening patient-derived tumor organoids that yields results within a week of surgery (Phan et al, Commun Biol 2, 78, 2019; Nguyen and Soragni, STAR Protoc 1, 2, 2020; Al Shihabi et al, bioRxiv, 2021). Tumor organoids are promising pre-clinical models as they closely recapitulate features of the parent tumor, including drug responses. Here, we expand the physiological relevance of tumor organoid screenings by including mini-livers to metabolize prodrugs of interest. To retain the ability to perform automated, high-throughput screenings, we engineered a miniature liver insert (MLI). The MLI is a removable system that facilitates the addition of liver organoids to specific wells of 96-well plates in which prodrugs are tested. The MLI is designed to integrate with our existing tumor organoid screening platform (Phan et al, Commun Biol 2, 78, 2019; Nguyen and Soragni, STAR Protoc 1, 2, 2020; Al Shihabi et al, bioRxiv, 2021), and allows for simultaneous monitoring of tumor organoids and hepatocytes with brightfield and fluorescent imaging. Hepatocytes seeded in the MLI are viable and maintain their expression of the key Cytochrome P450 enzymes responsible for the metabolism of many clinically used prodrugs. Finally, we demonstrate that incorporating the MLI into prodrug screening experiments sensitizes tumor organoids to treatment with chemotherapeutic and targeted agents. Due to the facile integration with existing screening protocols, the MLI is a simple and effective system for studying anti-cancer prodrugs ex vivo. The MLI is a novel tool that facilitates the co-culture of functional liver organoids with clinically relevant tumor organoid models for drug discovery studies and precision medicine applications. Citation Format: Peyton J Tebon, Alice Soragni. Engineered mini-livers for high-throughput tumor organoid screening of prodrugs [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA012.

Invasive alien species (IAS) are one of the major drivers of change that can negatively affect biodiversity, ecosystem functions and services and human health; islands are particularly vulnerable to biological invasions. Horizon scanning can lead to prioritisation of IAS to inform decision-making and action; its scale and scope can vary depending on the need. We focussed on IAS likely to arrive, establish and affect biodiversity and human health on the Mediterranean island of Cyprus. The scope of the horizon scanning was the entire island of Cyprus. We used a two-step consensus-building process in which experts reviewed and scored lists of alien species on their likelihood of arrival, establishment and potential to affect biodiversity, ecosystems and/or human health in the next 10 years. We reviewed 225 alien species, considered to be currently absent on Cyprus, across taxa and environments. We agreed upon 100 species that constituted very high, high or medium biodiversity risk, often arriving through multiple pathways of introduction. The remaining 125 species were ranked as low risk. The potential impacts on human health were documented for all 225 species; 82 species were considered to have a potentially negative impact on human health ranging from nuisance to disease transmission. The scope of the horizon scanning was the entire island of Cyprus, but the thematic groups also considered the relevance of the top 100 species to the Sovereign Base Areas of Cyprus, given their differing governance. This horizon scan provides the first systematic exercise to identify invasive alien species of potential concern to biodiversity and ecosystems but also human health within the Mediterranean region. The process and outcomes should provide other islands in the region and beyond with baseline data to improve IAS prioritisation and management. © 2019, The Author(s).

The spliceosome mediates precursor mRNA (pre-mRNA) splicing in eukaryotes, including the model organism Saccharomyces cerevisiae (yeast). Despite decades of study, no chemical inhibitors of yeast splicing in vivo are available. We have developed a system to efficiently inhibit splicing and block proliferation in living yeast cells using compounds that target the human spliceosome protein SF3B1. Potent inhibition is observed in yeast expressing a chimeric protein containing portions of human SF3B1. However, only a single point mutation in the yeast homolog of SF3B1 is needed for selective inhibition of splicing by pladienolide B (PB), herboxidiene (HB), or meayamycin (MAM) in liquid culture. Mutations which enable inhibition also improve splicing of branch sites containing mismatches between the intron and snRNA—suggesting a link between inhibitor sensitivity and usage of weak branch sites in humans. This approach provides powerful new tools for manipulating splicing in live yeast and studies of spliceosome inhibitors.

Proper transcription, the process of converting DNA to RNA, is crucial for the health and viability of an organism. This process is regulated by many proteins, such as co-transcriptional activators; one being the protein complex known as Spt-Ada-Gcn5-acetyltransferase, or SAGA. While much is known about the roles of SAGA in cell processes, how SAGA’s subunits promote functionality is still unknown. The focus of this study is to analyze the purpose of SAGA’s SF3B subunits. These subunits are also found in the spliceosome, the compound responsible for generating mature RNA. SAGA has no known functions relating to this process, so the reason the SF3B components are in SAGA is unclear. Spt7, another SAGA subunit, interacts with both SF3B subunits. In this study, a yeast two hybrid assay was performed where different Spt7 mutants were screened. This was done by transforming yeast with Spt7 mutants, analyzing the protein interactions and sequencing the mutants to determine their mutations. A key result of this study is in the determining that the two SF3B subunits interact with different regions of Spt7. Although the overall goal is to find an Spt7 mutant that does not interact with the SF3B components but still maintains interaction with other SAGA subunits, we now have a better idea of what type of Spt7 mutant is needed. This discovery will lay the foundation for future experiments where a mutated SAGA with no SF3B components will be expressed in Drosophila melanogaster and analyzed to determine the function of SF3B subunits in SAGA.

The interaction between splicing factors and the transcriptional machinery provides an intriguing link between the coupled processes of transcription and splicing. Here, we show that the two components of the SF3B complex, SF3B3 and SF3B5, that form part of the U2 small nuclear ribonucleoprotein particle (snRNP) are also subunits of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex in Drosophila melanogaster. Whereas SF3B3 had previously been identified as a human SAGA subunit, SF3B5 had not been identified as a component of SAGA in any species. We show that SF3B3 and SF3B5 bind to SAGA independent of RNA and interact with multiple SAGA subunits including Sgf29 and Spt7 in a yeast two-hybrid assay. Through analysis of sf3b5 mutant flies, we show that SF3B5 is necessary for proper development and cell viability but not for histone acetylation. Although SF3B5 does not appear to function in SAGA's histone-modifying activities, SF3B5 is still required for expression of a subset of SAGA-regulated genes independent of splicing. Thus, our data support an independent function of SF3B5 in SAGA's transcription coactivator activity that is separate from its role in splicing.

n order to support the prioritisation of invasive alien species for future risk assessments, a horizon scanning methodology for the Europe was developed and implemented. The outcome was a list of 95 species, including all taxa (except microorganisms) within marine, terrestrial and freshwater environments, considered as very high or high priority for risk assessment.

Proper gene regulation is vital to the health and development of an organism. Determining the relationship between splicing, transcription, and chromatin structure is vital for understanding gene regulation as a whole. There have been previous studies linking these elements pairwise; however, no evidence exists for a direct link between all three. Recent data shows that splicing components of the U2 small nuclear ribonucleic protein (snRNP) co-purify with Spt-Ada-Gcn5-acetyltransferase (SAGA), a highly conserved transcriptional co-activator and chromatin modifier. We hypothesize that SAGA binds with splicing components through a multi-protein binding surface with certain core components based on preliminary yeast two-hybrid data. Here, we examine the specific binding partners between SAGA and splicing components utilizing the yeast two-hybrid system in spt7Δ Saccharomyces cerevisiae as a validation for the preliminary yeast two-hybrid performed, producing recombinant proteins through sequence and ligation-independent cloning (SLIC) and Baculovirus transfections to obtain purified proteins, and co-immunoprecipitation (co-IP) to detect specific protein-protein interactions from recombinant proteins. Yeast two-hybrid results reveal that Spt7 is necessary for the transcription of reporter genes used in this assay. Therefore, this assay cannot validate previous results or detect false positives. Currently, recombinant proteins are being produced to perform co-IPs to test direct protein interactions. The results from these experiments will demonstrate the type of binding between SAGA subunits and splicing factors and provide direct evidence of a link between all three of the elements of gene regulation.

Fifty-five consecutive unselected patients were submitted for laparoscopic cholecystectomy, and the procedure completed laparoscopically in fifty cases. The outcome is presented with particular reference to the duration of surgery, postoperative pain and nausea, the length of hospital stay and the time taken to recover normal activities. This technique is shown to have major advantages over conventional gallbladder surgery for the majority of patients.

Previous empirical studies have interpreted results in regressions of output variables on export variables as providing support for an export promotion development strategy. Such an interpretation is questionable since these regressions provide no means of determining the direction of causality. This paper performs causality tests between exports and growth for 37 developing countries. The results cast considerable doubt on the validity of the export promotion hypothesis.

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An important debate has centered on the effects of inflation on economic growth. This paper employs Granger causality tests in a diverse array of countries in an attempt to discriminate between several competing hypotheses. A central question in this debate has been whether inflation contributes to or detracts from economic growth. Felix (1961), Seers (1962), Baer (1967), Georgescu-Roegen (1970), and Taylor (1979, 1983) have advanced a structuralist argument that inflation contributes favorably to real growth. Alternatively, Campos (1961), Harberger (1963), and Vogel (1974) have argued that the inefficiencies produced by inflation reduce real growth. The structuralist view that inflation has a positive effect on growth is based on the contention that inflation is a mechanism which induces forced savings (GeorgescuRoegen 1970; Taylor 1979). First, the government of a developing country, faced with an inadequate fiscal system, may resort to borrowing from the central bank as a way to finance expenditures. Thus inflationary finance may increase capital formation if the government uses its inflation-tax revenues to increase real investment. As long as private sector investment does not fall one-for-one, inflationary finance may contribute to real growth. Second, nominal wages may lag behind prices because of slowly adjusting expectations, sluggish wage bargains, or systematic governmental wage repression. If this is so, then inflation may increase growth in neoclassical fashion by shifting the income distribution in favor of higher saving capitalists and hence increasing savings and growth. From a more Keynesian perspective, inflation may increase growth by raising the rate of profit, thus increasing private investment. An alternative view is that inflation has a negative effect on growth (Mundell 1971; Taylor 1979). This position might be labeled the distortionary inflation view. Inflation may (with the help of government policies) create a variety of outputreducing inefficiencies (Baer 1967). First, inflation in a country with a fixed exchange rate will lead to a deteriorating trade balance and to speculative capital