Correlation of SARS-CoV-2 in Wastewater and Individual Testing Results in a Jail, Atlanta, Georgia, USA.

Institution-level wastewater-based surveillance was implemented during the COVID-19 pandemic, including in carceral facilities. We examined the relationship between COVID-19 diagnostic test results of residents in a jail in Atlanta, Georgia, USA (average population ≈2,700), and quantitative reverse transcription PCR signal for SARS-CoV-2 in weekly wastewater samples collected during October 2021‒May 2022. The jail offered residents rapid antigen testing at entry and periodic mass screenings by reverse transcription PCR of self-collected nasal swab specimens. We aggregated individual test data, calculated the Spearman correlation coefficient, and performed logistic regression to examine the relationship between strength of SARS-CoV-2 PCR signal (cycle threshold value) in wastewater and percentage of jail population that tested positive for COVID-19. Of 13,745 nasal specimens collected, 3.9% were COVID-positive (range 0%-29.5% per week). We observed a strong inverse correlation between diagnostic test positivity and cycle threshold value (r = -0.67; p<0.01). Wastewater-based surveillance represents an effective strategy for jailwide surveillance of COVID-19.

Rapid Lewis Acid Screening and Reaction Optimization Using 3D-Printed Catalyst-Impregnated Stirrer Devices in the Synthesis of Heterocycles.

We describe the development of Lewis acid (LA) catalyst-impregnated 3D-printed stirrer devices and demonstrate their ability to facilitate the rapid screening of reaction conditions to synthesize heterocycles. The stereolithography 3D-printed stirrer devices were designed to fit round-bottomed flasks and Radleys carousel tubes using our recently reported solvent-resistant resin, and using CFD modeling studies and experimental data, we demonstrated that the device design leads to rapid mixing and rapid throughput over the device surface. Using a range of LA 3D-printed stirrers, the reaction between a diamine and an aldehyde was optimized for the catalyst and solvent, and we demonstrated that use of the 3D-printed catalyst-embedded devices led to higher yields and reduced reaction times. A library of benzimidazole and benzothiazole compounds were synthesized, and the use of devices led to efficient formation of the product as well as low levels of the catalyst in the resultant crude mixture. The use of these devices makes the process of setting up multiple reactions simpler by avoiding weighing out of catalysts, and the devices, once used, can be simply removed from the reaction, making the process of compound library synthesis more facile.

Life Cycle Assessment of Urine Diversion and Conversion to Fertilizer Products at the City Scale.

Urine diversion has been proposed as an approach for producing renewable fertilizers and reducing nutrient loads to wastewater treatment plants. Life cycle assessment was used to compare environmental impacts of the operations phase of urine diversion and fertilizer processing systems [via (1) a urine concentration alternative and (2) a struvite precipitation and ion exchange alternative] at a city scale to conventional systems. Scenarios in Vermont, Michigan, and Virginia were modeled, along with additional sensitivity analyses to understand the importance of key parameters, such as the electricity grid and wastewater treatment method. Both urine diversion technologies had better environmental performance than the conventional system and led to reductions of 29-47% in greenhouse gas emissions, 26-41% in energy consumption, approximately half the freshwater use, and 25-64% in eutrophication potential, while acidification potential ranged between a 24% decrease to a 90% increase. In some situations, wastewater treatment chemical requirements were eliminated. The environmental performance improvement was usually dependent on offsetting the production of synthetic fertilizers. This study suggests that urine diversion could be applied broadly as a strategy for both improving wastewater management and decarbonization.

A comparative carbon footprint analysis of disposable and reusable vaginal specula.

BACKGROUND: Healthcare systems in the United States have increasingly turned toward the use of disposable medical equipment in an attempt to save time, lower costs, and reduce the transmission of infections. However, the use of disposable instruments is associated with increased solid waste production and may have negative impacts on the environment, such as increased greenhouse gas emissions. OBJECTIVE: The purpose of this study was to inform this discussion; we applied life cycle assessment methods to evaluate the carbon footprints of 3 vaginal specula: a single-use acrylic model and 2 reusable stainless steel models. STUDY DESIGN: The functional unit of the study was defined as the completion of 20 gynecologic examinations by either type of speculum. The greenhouse gas emissions (eg, carbon dioxide, methane, nitrous oxide) across all life cycle stages, which includes material production and manufacturing, transportation, use and reprocessing, and end-of-life, were analyzed with the use of SimaPro life cycle assessment software and converted into carbon dioxide equivalents. RESULTS: The reusable stainless steel grade 304 speculum was found to have a lesser carbon footprint over multiple model scenarios (different reprocessing techniques, autoclave loading/efficiency, and number of uses) than either the reusable stainless steel grade 316 or the disposable acrylic specula. The material production and manufacturing phase contributed most heavily to the total life cycle carbon footprint of the acrylic speculum, whereas the use and reprocessing phase contributed most to the carbon footprints of both stainless steel specula. CONCLUSION: The use of disposable vaginal specula is associated with increased greenhouse gas equivalents compared with reusable alternatives with no significant difference in clinical utility. These findings can be used to inform decision-making by healthcare systems, because they weigh a wide range of considerations in making final purchase decisions; similar analytic methods can and should be applied to other components of health systems' waste streams.

Novel epidithiodiketopiperazines as anti-viral zinc ejectors of the Feline Immunodeficiency Virus (FIV) nucleocapsid protein as a model for HIV infection.

Focused libraries of multi-substituted epidithiodiketopiperazines (ETP) were prepared and evaluated for efficacy of inhibiting the nucleocapsid protein function of the Feline Immunodeficiency Virus (FIV) as a model for HIV. This activity was compared and contrasted to observed toxicity utilising an in-vitro cell culture approach. This resulted in the identification of several promising lead compounds with nanomolar potency in cells with low toxicity and a favorable therapeutic index.

Physiological signature of a novel potentiator of AMPA receptor signalling.

We have synthesized a novel small molecule based on the pyrrolidinone-containing core structure of clausenamide, which is a candidate anti-dementia drug. The synthetic route yielded multi-gram quantities of an isomeric racemate mixture in a short number of steps. When tested in hippocampal slices from young adult rats the compound enhanced AMPA receptor-mediated signalling at mossy fibre synapses, and potentiated inward currents evoked by local application of l-glutamate onto CA3 pyramidal neurons. It facilitated the induction of mossy fibre LTP, but the magnitude of potentiation was smaller than that observed in untreated slices. The racemic mixture was separated and it was shown that only the (-) enantiomer was active. Toxicity analysis indicated that cell lines tolerated the compound at concentrations well above those enhancing synaptic transmission. Our results unveil a small molecule whose physiological signature resembles that of a potent nootropic drug.

Short Total Synthesis of (±)-γ-Lycorane by a Sequential Intramolecular Acylal Cyclisation (IAC) and Intramolecular Heck Addition Reaction.

An intramolecular acylal cyclisation (IAC) approach to the synthesis of a range of bicyclic heterocycles is reported. As an example of the utility of the IAC reaction, the methodology was applied in a protecting-group-free five-step total synthesis of (±)-γ-lycorane, incorporating a new intramolecular Heck addition reaction to generate the pentacyclic core structure of the natural product in good yield.

Intramolecular Acylal Cyclisation (IAC) as an Efficient Synthetic Strategy towards the Total Synthesis of Erythrina Alkaloid Derivatives.

Compounds that comprise the erythrina alkaloid class of natural products are based on a tetracyclic spiroamine framework and exhibit a range of biological activities on the central nervous system. Herein, we report a new and efficient total synthesis of this multiple-ring system based on an intramolecular acylal cyclisation (IAC) approach. Using this methodology, the tetracyclic core was rapidly assembled over a two-step domino process catalysed by a Lewis acid. The effect of heteroatoms, substituents and ring size on the IAC has also been investigated, and the broad application of this procedure is demonstrated by the synthesis of a library of derivatives in good yields with excellent regioselectivity.

Novel fused tetrathiocines as antivirals that target the nucleocapsid zinc finger containing protein of the feline immunodeficiency virus (FIV) as a model of HIV infection.

A novel series of fused tetrathiocines were prepared for evaluation of activity against the nucleocapsid protein of the feline immunodeficiency virus (FIV) in an in vitro cell culture approach. The results demonstrated that the compounds display potent nanomolar activity and low toxicity against this key model of HIV infection.

Evaluation of the antiviral efficacy of bis[1,2]dithiolo[1,4]thiazines and bis[1,2]dithiolopyrrole derivatives against the nucelocapsid protein of the Feline Immunodeficiency Virus (FIV) as a model for HIV infection.

A diverse library of bis[1,2]dithiolo[1,4]thiazines and bis[1,2]dithiolopyrrole derivatives were prepared for evaluation of activity against the nucleocapsid protein of the Feline Immunodeficiency Virus (FIV) as a model for HIV, using an in vitro cell culture approach, yielding nanomolar active compounds with low toxicity.

Synthesis of CHF2-Containing Heterocycles through Oxy-difluoromethylation Using Low-Cost 3D Printed PhotoFlow Reactors.

We report here a highly straightforward access to a variety of CHF2-containing heterocycles, including lactones, tetrahydrofurans, tetrahydropyrans, benzolactones, phthalanes, and pyrrolidines, through a visible light-mediated intramolecular oxy-difluoromethylation under continuous flow. The method, which relies on the use of readily available starting materials, low-cost 3D printed photoflow reactors, and difluoromethyltriphenylphosphonium bromide used here as a CHF2 radical precursor, is practical and scalable and provides the desired products in moderate to excellent yields and excellent regio- and stereoselectivities.

The influence of positional isomerism on G-quadruplex binding and anti-proliferative activity of tetra-substituted naphthalene diimide compounds.

The synthesis together with biophysical and biological evaluation of a series of tetra-substituted naphthalene diimide (ND) compounds, are presented. These compounds are positional isomers of a recently-described series of quadruplex-binding ND derivatives, in which the two N-methyl-piperidine-alkyl side-chains have now been interchanged with the positions of side-chains bearing a range of end-groups. Molecular dynamics simulations of a pair of positional isomers are in accord with the quadruplex stabilization and biological data for these compounds. Analysis of structure-activity data indicates that for compounds where the side-chains are not of equivalent length then the positional isomers described here tend to have improved cell proliferation potency and in some instances, superior quadruplex stabilization ability.

Rapid plugged flow synthesis of nucleoside analogues via Suzuki-Miyaura coupling and heck Alkenylation of 5-Iodo-2'-deoxyuridine (or cytidine).

Nucleosides modification via conventional cross-coupling has been performed using different catalytic systems and found to take place via long reaction times. However, since the pandemic, nucleoside-based antivirals and vaccines have received widespread attention and the requirement for rapid modification and synthesis of these moieties has become a major objective for researchers. To address this challenge, we describe the development of a rapid flow-based cross-coupling synthesis protocol for a variety of C5-pyrimidine substituted nucleosides. The protocol allows for facile access to multiple nucleoside analogues in very good yields in a few minutes compared to conventional batch chemistry. To highlight the utility of our approach, the synthesis of an anti-HSV drug, BVDU was also achieved in an efficient manner using our new protocol. Supplementary Information: The online version contains supplementary material available at 10.1007/s41981-023-00265-1.

Comparison of Nanotrap® Microbiome A Particles, membrane filtration, and skim milk workflows for SARS-CoV-2 concentration in wastewater.

Introduction: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA monitoring in wastewater has become an important tool for Coronavirus Disease 2019 (COVID-19) surveillance. Grab (quantitative) and passive samples (qualitative) are two distinct wastewater sampling methods. Although many viral concentration methods such as the usage of membrane filtration and skim milk are reported, these methods generally require large volumes of wastewater, expensive lab equipment, and laborious processes. Methods: The objectives of this study were to compare two workflows (Nanotrap® Microbiome A Particles coupled with MagMax kit and membrane filtration workflows coupled with RNeasy kit) for SARS-CoV-2 recovery in grab samples and two workflows (Nanotrap® Microbiome A Particles and skim milk workflows coupled with MagMax kit) for SARS-CoV-2 recovery in Moore swab samples. The Nanotrap particle workflow was initially evaluated with and without the addition of the enhancement reagent 1 (ER1) in 10 mL wastewater. RT-qPCR targeting the nucleocapsid protein was used for detecting SARS-CoV-2 RNA. Results: Adding ER1 to wastewater prior to viral concentration significantly improved viral concentration results (P < 0.0001) in 10 mL grab and swab samples processed by automated or manual Nanotrap workflows. SARS-CoV-2 concentrations in 10 mL grab and Moore swab samples with ER1 processed by the automated workflow as a whole showed significantly higher (P < 0.001) results than 150 mL grab samples using the membrane filtration workflow and 250 mL swab samples using the skim milk workflow, respectively. Spiking known genome copies (GC) of inactivated SARS-CoV-2 into 10 mL wastewater indicated that the limit of detection of the automated Nanotrap workflow was ~11.5 GC/mL using the RT-qPCR and 115 GC/mL using the digital PCR methods. Discussion: These results suggest that Nanotrap workflows could substitute the traditional membrane filtration and skim milk workflows for viral concentration without compromising the assay sensitivity. The manual workflow can be used in resource-limited areas, and the automated workflow is appropriate for large-scale COVID-19 wastewater-based surveillance.

When case reporting becomes untenable: Can sewer networks tell us where COVID-19 transmission occurs?

Monitoring SARS-CoV-2 in wastewater is a valuable approach to track COVID-19 transmission. Designing wastewater surveillance (WWS) with representative sampling sites and quantifiable results requires knowledge of the sewerage system and virus fate and transport. We developed a multi-level WWS system to track COVID-19 in Atlanta using an adaptive nested sampling strategy. From March 2021 to April 2022, 868 wastewater samples were collected from influent lines to wastewater treatment facilities and upstream community manholes. Variations in SARS-CoV-2 concentrations in influent line samples preceded similar variations in numbers of reported COVID-19 cases in the corresponding catchment areas. Community sites under nested sampling represented mutually-exclusive catchment areas. Community sites with high SARS-CoV-2 detection rates in wastewater covered high COVID-19 incidence areas, and adaptive sampling enabled identification and tracing of COVID-19 hotspots. This study demonstrates how a well-designed WWS provides actionable information including early warning of surges in cases and identification of disease hotspots.

Development and analysis of a novel loading technique for FDM 3D printed systems: Microwave-assisted impregnation of gastro-retentive PVA capsular devices.

In this paper, we describe a modular post-printing loading protocol for a 3D printed gastro-retentive drug delivery system. Fused Deposition Modelling (FDM) 3D printing was exploited for the rapid prototyping of a modular floating system (caps-in-cap). Optimized models were produced as blank PVA scaffolds, and a morphological analysis of the FDM printed models was conducted to develop a straightforward protocol for drug-loading. The 3D printed gastro-retentive systems were then subjected to microwave irradiation in oversaturated solutions of anhydrous caffeine for drug loading, and research focused on an analysis of the impact of microwave irradiation on the chemical and physical properties of the polymer and the drug. The drug-loading efficiency, thermal and chemical characteristics of components, the stability of the drug and the morphology of processed printouts are characterised and described. Parameters of this unexplored microwave-assisted post-printing loading technique were evaluated and adequately set up, and the process resulted in the preservation of the polymeric matrix and enhancement of drug loading. Hence, microwave impregnation confirmed its potential in superseding the traditional pre- and post-printing loading methods, such as soaking techniques, being faster and more efficient and providing a new paradigm approach to personalised drug delivery.

Epidithiodiketopiperazines block the interaction between hypoxia-inducible factor-1alpha (HIF-1alpha) and p300 by a zinc ejection mechanism.

The hypoxic response in humans is regulated by the hypoxia-inducible transcription factor system; inhibition of hypoxia-inducible factor (HIF) activity has potential for the treatment of cancer. Chetomin, a member of the epidithiodiketopiperazine (ETP) family of natural products, inhibits the interaction between HIF-alpha and the transcriptional coactivator p300. Structure-activity studies employing both natural and synthetic ETP derivatives reveal that only the structurally unique ETP core is required and sufficient to block the interaction of HIF-1alpha and p300. In support of both cell-based and animal work showing that the cytotoxic effect of ETPs is reduced by the addition of Zn(2+) through an unknown mechanism, our mechanistic studies reveal that ETPs react with p300, causing zinc ion ejection. Cell studies with both natural and synthetic ETPs demonstrated a decrease in vascular endothelial growth factor and antiproliferative effects that were abrogated by zinc supplementation. The results have implications for the design of selective ETPs and for the interaction of ETPs with other zinc ion-binding protein targets involved in gene expression.

Identification and characterisation of 2-aminopyridine inhibitors of checkpoint kinase 2.

5-(Hetero)aryl-3-(4-carboxamidophenyl)-2-aminopyridine inhibitors of CHK2 were identified from high throughput screening of a kinase-focussed compound library. Rapid exploration of the hits through straightforward chemistry established structure-activity relationships and a proposed ATP-competitive binding mode which was verified by X-ray crystallography of several analogues bound to CHK2. Variation of the 5-(hetero)aryl substituent identified bicyclic dioxolane and dioxane groups which improved the affinity and the selectivity of the compounds for CHK2 versus CHK1. The 3-(4-carboxamidophenyl) substituent could be successfully replaced by acyclic omega-aminoalkylamides, which made additional polar interactions within the binding site and led to more potent inhibitors of CHK2. Compounds from this series showed activity in cell-based mechanistic assays for inhibition of CHK2.

Selective Modulation of α5 GABAA Receptors Exacerbates Aberrant Inhibition at Key Hippocampal Neuronal Circuits in APP Mouse Model of Alzheimer's Disease.

Selective negative allosteric modulators (NAMs), targeting α5 subunit-containing GABAA receptors (GABAARs) as potential therapeutic targets for disorders associated with cognitive deficits, including Alzheimer's disease (AD), continually fail clinical trials. We investigated whether this was due to the change in the expression of α5 GABAARs, consequently altering synaptic function during AD pathogenesis. Using medicinal chemistry and computational modeling, we developed aqueous soluble hybrids of 6,6-dimethyl-3-(2-hydroxyethyl) thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophene-4(5H)-one, that demonstrated selective binding and high negative allosteric modulation, specifically for the α5 GABAAR subtypes in constructed HEK293 stable cell-lines. Using a knock-in mouse model of AD (APP NL-F/NL-F), which expresses a mutant form of human amyloid-ß (Aß), we performed immunofluorescence studies combined with electrophysiological whole-cell recordings to investigate the effects of our key molecule, α5-SOP002 in the hippocampal CA1 region. In aged APP NL-F/NL-F mice, selective preservation of α5 GABAARs was observed in, calretinin- (CR), cholecystokinin- (CCK), somatostatin- (SST) expressing interneurons, and pyramidal cells. Previously, we reported that CR dis-inhibitory interneurons, specialized in regulating other interneurons displayed abnormally high levels of synaptic inhibition in the APP NL-F/NL-F mouse model, here we show that this excessive inhibition was "normalized" to control values with bath-applied α5-SOP002 (1 µM). However, α5-SOP002, further impaired inhibition onto CCK and pyramidal cells that were already largely compromised by exhibiting a deficit of inhibition in the AD model. In summary, using a multi-disciplinary approach, we show that exposure to α5 GABAAR NAMs may further compromise aberrant synapses in AD. We, therefore, suggest that the α5 GABAAR is not a suitable therapeutic target for the treatment of AD or other cognitive deficits due to the widespread neuronal-networks that use α5 GABAARs.

A Preliminary Investigation of Additive Manufacture to Fabricate Human Nail Plate Surrogates for Pharmaceutical Testing.

In vitro permeation studies using nail clippings or nail plates are commonly used in the development of transungual formulations. However, there are ethical, safety and cost issues associated with sourcing such tissues. Herein, we describe a preliminary approach is described for the design and manufacture of a human nail model surrogate based on 3D printing. To evaluate these 3D printed constructs, nails were mounted in conventional glass Franz cells and a commercial antifungal lacquer formulation containing ciclopirox olamine was applied daily to the surrogate printed surfaces for a period of 14 days. On days 8 and 14, the surfaces of the 3D printed nails were washed with ethanol to remove excess formulation. Confocal Raman spectroscopy (CRS) was used to profile the drug in the 3D printed nail. At the end of the Franz cell studies, no drug was observed in the receptor phase. CRS studies confirmed penetration of the active into the model nails with reproducible depth profiles. Our ongoing work is focused on synthesising commercial and non-commercial printable resins that can replicate the physical and chemical characteristics of the human nail. This will allow further evaluation of actives for ungual therapy and advance the development of the surrogate nail tissue model.