Improved and expanded Q-system reagents for genetic manipulations.

The Q system is a repressible binary expression system for transgenic manipulations in living organisms. Through protein engineering and in vivo functional tests, we report here variants of the Q-system transcriptional activator, including QF2, for driving strong and ubiquitous expression in all Drosophila tissues. Our QF2, Gal4QF and LexAQF chimeric transcriptional activators substantially enrich the toolkit available for transgenic regulation in Drosophila melanogaster.

A platform to reproducibly evaluate human colon permeability and damage.

The intestinal epithelium comprises diverse cell types and executes many specialized functions as the primary interface between luminal contents and internal organs. A key function provided by the epithelium is maintenance of a barrier that protects the individual from pathogens, irritating luminal contents, and the microbiota. Disruption of this barrier can lead to inflammatory disease within the intestinal mucosa, and, in more severe cases, to sepsis. Animal models to study intestinal permeability are costly and not entirely predictive of human biology. Here we present a model of human colon barrier function that integrates primary human colon stem cells into Draper's PREDICT96 microfluidic organ-on-chip platform to yield a high-throughput system appropriate to predict damage and healing of the human colon epithelial barrier. We have demonstrated pharmacologically induced barrier damage measured by both a high throughput molecular permeability assay and transepithelial resistance. Using these assays, we developed an Inflammatory Bowel Disease-relevant model through cytokine induced damage that can support studies of disease mechanisms and putative therapeutics.

A High-Throughput, High-Containment Human Primary Epithelial Airway Organ-on-Chip Platform for SARS-CoV-2 Therapeutic Screening.

COVID-19 emerged as a worldwide pandemic in early 2020, and while the rapid development of safe and efficacious vaccines stands as an extraordinary achievement, the identification of effective therapeutics has been less successful. This process has been limited in part by a lack of human-relevant preclinical models compatible with therapeutic screening on the native virus, which requires a high-containment environment. Here, we report SARS-CoV-2 infection and robust viral replication in PREDICT96-ALI, a high-throughput, human primary cell-based organ-on-chip platform. We evaluate unique infection kinetic profiles across lung tissue from three human donors by immunofluorescence, RT-qPCR, and plaque assays over a 6-day infection period. Enabled by the 96 devices/plate throughput of PREDICT96-ALI, we also investigate the efficacy of Remdesivir and MPro61 in a proof-of-concept antiviral study. Both compounds exhibit an antiviral effect against SARS-CoV-2 in the platform. This demonstration of SARS-CoV-2 infection and antiviral dosing in a high-throughput organ-on-chip platform presents a critical capability for disease modeling and therapeutic screening applications in a human physiology-relevant in vitro system.

Base Editing of Somatic Cells Using CRISPR-Cas9 in Drosophila.

Cas9 and a guide RNA (gRNA) function to target specific genomic loci for generation of a double-stranded break. Catalytic dead versions of Cas9 (dCas9) no longer cause double-stranded breaks and instead can serve as molecular scaffolds to target additional enzymatic proteins to specific genomic loci. To generate mutations in selected genomic residues, dCas9 can be used for genomic base editing by fusing a cytidine deaminase (CD) to induce C > T (or G>A) mutations at targeted sites. In this study, we test base editing in Drosophila by expressing a transgenic Drosophila base editor (based on the mammalian BE2) that consists of a fusion protein of CD, dCas9, and uracil glycosylase inhibitor. We utilized transgenic lines expressing gRNAs along with pan-tissue expression of the Drosophila base editor (Actin5C-BE2) and found high rates of base editing at multiple targeted loci in the 20 bp target sequence. Highest rates of conversion of C > T were found in positions 3-9 of the gRNA-targeted site, with conversion reaching ∼100% of targeted DNA in somatic tissues. Surprisingly, the simultaneous use of two gRNAs targeting a genomic region spaced ∼50 bp apart led to mutations between the two gRNA targets, implicating a method to broaden the available sites accessible to targeting. These results indicate base editing is efficient in Drosophila, and could be used to induce point mutations at select loci.

Recurrent Urinary Tract Infection: A Mystery in Search of Better Model Systems.

Urinary tract infections (UTIs) are among the most common infectious diseases worldwide but are significantly understudied. Uropathogenic E. coli (UPEC) accounts for a significant proportion of UTI, but a large number of other species can infect the urinary tract, each of which will have unique host-pathogen interactions with the bladder environment. Given the substantial economic burden of UTI and its increasing antibiotic resistance, there is an urgent need to better understand UTI pathophysiology - especially its tendency to relapse and recur. Most models developed to date use murine infection; few human-relevant models exist. Of these, the majority of in vitro UTI models have utilized cells in static culture, but UTI needs to be studied in the context of the unique aspects of the bladder's biophysical environment (e.g., tissue architecture, urine, fluid flow, and stretch). In this review, we summarize the complexities of recurrent UTI, critically assess current infection models and discuss potential improvements. More advanced human cell-based in vitro models have the potential to enable a better understanding of the etiology of UTI disease and to provide a complementary platform alongside animals for drug screening and the search for better treatments.

Reduced Size Profile of Amniotic Membrane Particles Decreases Osteoarthritis Therapeutic Efficacy.

Osteoarthritis (OA) is a widespread disease that continues to lack approved and efficacious treatments that modify disease progression. Micronized dehydrated human amnion/chorion membrane (µ-dHACM) has been shown to be effective in reducing OA progression, but many of the engineering design parameters have not been explored. The objectives of this study were to characterize the particle size distributions of two µ-dHACM formulations and to investigate the influence of these distributions on the in vivo therapeutic efficacy of µ-dHACM. Male Lewis rats underwent medial meniscus transection (MMT) or sham surgery, and intra-articular injections of saline, µ-dHACM, or reduced particle size µ-dHACM (RPS µ-dHACM) were administered at 24 hours postsurgery (n = 9 per treatment group). After 3 weeks, the animals were euthanized, and left legs harvested for equilibrium partitioning of an ionic contrast agent microcomputed tomography and histological analysis. µ-dHACM and RPS µ-dHACM particles were fluorescently tagged and particle clearance was tracked in vivo for up to 42 days postsurgery. Protein elution from both formulations was quantified in vitro. Treatment with µ-HACM, but not RPS µ-dHACM, reduced lesion volume in the MMT model 3 weeks postsurgery. In contrast, RPS µ-dHACM increased cartilage surface roughness and osteophyte cartilage thickness and volume compared to saline treatment. There was no difference of in vivo fluorescently tagged particle clearance between the two µ-dHACM sizes. RPS µ-dHACM showed significantly greater protein elution in vitro over 21 days. Overall, delivery of RPS µ-dHACM did result in an increase of in vivo joint degeneration and in vitro protein elution compared to µ-dHACM, but did not result in differences in joint clearance in vivo. These results suggest that particle size and factor elution may be tailorable factors that are important to optimize for particulate amniotic membrane treatment to be an effective therapy for OA. Impact Statement Osteoarthritis (OA) is a widespread disease that continues to lack treatments that modify the progression of the disease. Micronized dehydrated human amnion/chorion membrane (µ-dHACM) has been shown to be effective in reducing OA progression, but many of the engineering design parameters have not been explored. This work investigates the effects of particle size profile of the µ-dHACM particles and lays out the methods used in these studies. The results of this work will guide engineers in designing µ-dHACM treatments specifically and disease-modifying OA therapeutics generally, and it demonstrates the utility of novel therapeutic evaluation methods such as contrast-enhanced microcomputed tomography.

Development of Pharmacist Independent Prescribing Clinics to Treat Opioid Analgesic Dependence in NHS Lanarkshire.

There has been an increase in opioid analgesic prescribing in general practice (GP). This is causing some concern around this contributing to dependency. NHS Lanarkshire have attempted to reduce the prescribing from GP surgeries through the development of specialised Pharmacist Independent Prescriber clinics being delivered from the practices. This article looks at the development of these services with pharmacist independent prescribers and the results from developing the services. The article aims to provide advice and recommendations for the development of other services and strategies to minimise the risks associated with Opioid Analgesic Dependence for patients.

Organization of olfactory centres in the malaria mosquito Anopheles gambiae.

Mosquitoes are vectors for multiple infectious human diseases and use a variety of sensory cues (olfactory, temperature, humidity and visual) to locate a human host. A comprehensive understanding of the circuitry underlying sensory signalling in the mosquito brain is lacking. Here we used the Q-system of binary gene expression to develop transgenic lines of Anopheles gambiae in which olfactory receptor neurons expressing the odorant receptor co-receptor (Orco) gene are labelled with GFP. These neurons project from the antennae and maxillary palps to the antennal lobe (AL) and from the labella on the proboscis to the suboesophageal zone (SEZ), suggesting integration of olfactory and gustatory signals occurs in this brain region. We present detailed anatomical maps of olfactory innervations in the AL and the SEZ, identifying glomeruli that may respond to human body odours or carbon dioxide. Our results pave the way for anatomical and functional neurogenetic studies of sensory processing in mosquitoes.

Plug-and-play genetic access to drosophila cell types using exchangeable exon cassettes.

Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here, we introduce a simple, versatile method for achieving cell-type-specific expression of transgenes that leverages the untapped potential of "coding introns" (i.e., introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted "plug-and-play" cassettes (called "Trojan exons") that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC (Minos-mediated integration cassette) transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system.

White vegetables: a forgotten source of nutrients: Purdue roundtable executive summary.

Purdue University convened a scientific roundtable, "White Vegetables: A Forgotten Source of Nutrients," in Chicago, IL, June 18-19, 2012, to bring together experts to address the contributions of white vegetables, including potatoes, as sources of key nutrients and other microconstituents within a dietary pattern supporting health and wellness. This paper summarizes the meeting and supplement papers, including discussion among participants. The group of researchers identified areas of ambiguity regarding classification of vegetables for research and dietary guidance, future research needs, and the imperative to draw on that research to enhance evidence-based dietary guidance about white vegetables, including potatoes. U.S. dietary guidance encourages consumption of a variety of fruits and vegetables, including at least 1 serving of a dark green and 1 orange vegetable daily. However, no such recommendation exists for white vegetables, such as potatoes, cauliflowers, turnips, onions, parsnips, mushrooms, corn, and kohlrabi. Vegetable subgrouping approaches need to be considered in the context of nutrients of concern and low fruits and vegetable consumption. This Roundtable and supplement provide a substantial body of evidence to demonstrate how the inclusion of white vegetables, such as potatoes, can increase shortfall nutrients, notably fiber, potassium, and magnesium, as well as help increase overall vegetable consumption among children, teens, and adults in the United States. In so doing, these increases can help consumers to effectively and economically meet the recommended 2010 Dietary Guidelines for Americans vegetable servings and improve nutrient intake for all age and sex categories. Although inclusion of many types of vegetables in the diet improves nutritional adequacy, a priority public health message is to increase vegetable consumption. Potatoes appear to be a pathway to increased vegetable consumption, thereby helping to meet the recommended 2010 Dietary Guidelines for Americans servings for vegetables provided the forms served limit the amount of added salt and fat. Potatoes, in all forms, when consumed in MyPlate serving sizes, can be part of health-promoting dietary patterns. More research is needed to determine the health contributions of white vegetables as a source of nutrients and bioactive constituents and their bioavailability beyond the isolated components.