Development of Allosteric Small Molecule APOBEC3B Inhibitors from In Silico Screening.

APOBEC3B cytosine deaminase contributes to the mutational burdens of tumors, resulting in tumor progression and therapy resistance. Small molecule APOBEC3B inhibitors have potential to slow or mitigate these detrimental outcomes. Through molecular dynamics (MD) simulations and computational solvent mapping analysis, we identified a novel putative allosteric pocket on the C-terminal domain of APOBEC3B (A3Bctd), and virtually screened the ChemBridge Diversity Set (N~110,000) against both the active and potential allosteric sites. Selected high-scoring compounds were subsequently purchased, characterized for purity and composition, and tested in biochemical assays, which yielded 13 hit compounds. Orthogonal NMR assays verified binding to the target protein. Initial selectivity studies suggest these compounds preferentially target A3Bctd over related deaminase APOBEC3A (A3A), and MD simulations indicate this selectivity may be due to the steric repulsion from H56 that is unique to A3A. Taken together, our studies represent the first virtual screening effort against A3Bctd that has yielded candidate inhibitors suitable for further development.

Mutational signature-based identification of DNA repair deficient gastroesophageal adenocarcinomas for therapeutic targeting.

Homologous recombination (HR) and nucleotide excision repair (NER) are the two most frequently disabled DNA repair pathways in cancer. HR-deficient breast, ovarian, pancreatic and prostate cancers respond well to platinum chemotherapy and PARP inhibitors. However, the frequency of HR deficiency in gastric and esophageal adenocarcinoma (GEA) still lacks diagnostic and functional validation. Using whole exome and genome sequencing data, we found that a significant subset of GEA, but very few colorectal adenocarcinomas, show evidence of HR deficiency by mutational signature analysis (HRD score). High HRD gastric cancer cell lines demonstrated functional HR deficiency by RAD51 foci assay and increased sensitivity to platinum chemotherapy and PARP inhibitors. Of clinical relevance, analysis of three different GEA patient cohorts demonstrated that platinum treated HR deficient cancers had better outcomes. A gastric cancer cell line with strong sensitivity to cisplatin showed HR proficiency but exhibited NER deficiency by two photoproduct repair assays. Single-cell RNA-sequencing revealed that, in addition to inducing apoptosis, cisplatin treatment triggered ferroptosis in a NER-deficient gastric cancer, validated by intracellular GSH assay. Overall, our study provides preclinical evidence that a subset of GEAs harbor genomic features of HR and NER deficiency and may therefore benefit from platinum chemotherapy and PARP inhibitors.

Revealing the Impacts of Chemical Complexity on Submicrometer Sea Spray Aerosol Morphology.

Sea spray aerosol (SSA) ejected through bursting bubbles at the ocean surface is a complex mixture of salts and organic species. Submicrometer SSA particles have long atmospheric lifetimes and play a critical role in the climate system. Composition impacts their ability to form marine clouds, yet their cloud-forming potential is difficult to study due to their small size. Here, we use large-scale molecular dynamics (MD) simulations as a "computational microscope" to provide never-before-seen views of 40 nm model aerosol particles and their molecular morphologies. We investigate how increasing chemical complexity impacts the distribution of organic material throughout individual particles for a range of organic constituents with varying chemical properties. Our simulations show that common organic marine surfactants readily partition between both the surface and interior of the aerosol, indicating that nascent SSA may be more heterogeneous than traditional morphological models suggest. We support our computational observations of SSA surface heterogeneity with Brewster angle microscopy on model interfaces. These observations indicate that increased chemical complexity in submicrometer SSA leads to a reduced surface coverage by marine organics, which may facilitate water uptake in the atmosphere. Our work thus establishes large-scale MD simulations as a novel technique for interrogating aerosols at the single-particle level.

Deep Sequencing and Molecular Characterisation of BK Virus and JC Virus WHO International Reference Materials for Clinical Diagnostic Use.

BACKGROUND: Reactivation of JC and BK polyomaviruses during immunosuppression can lead to adverse clinical outcomes. In renal transplant recipients, BKV-associated nephropathy can result in graft loss, while in patients with autoimmune disorders, prolonged immunomodulatory drug use can cause rare onset of progressive multifocal leukoencephalopathy due to JCV reactivation. In such patients, accurate BK and JC viral load determinations by molecular technologies are important for diagnosis and clinical management; however, comparability across centres requires effective standardisation of diagnostic molecular detection systems. In October 2015, the WHO Expert Committee for Biological Standardisation (ECBS) established the 1st WHO International Standards (ISs) for use as primary-order calibrants for BKV and JCV nucleic acid detection. Two multi-centre collaborative studies confirmed their utility in harmonising agreement across the wide range of BKV and JCV assays, respectively. Previous Illumina-based deep sequence analysis of these standards, however, identified deletions in different regions, including the large T-antigen coding region. Hence, further detailed characterization was warranted. METHODS: Comprehensive sequence characterisation of each preparation using short- and long-read next-generation sequencing technologies was performed with additional corroborative independent digital PCR (dPCR) determinations. Potential error rates associated with long-read sequencing were minimised by applying rolling circle amplification (RCA) protocols for viral DNA (circular dsDNA), generating a full validation of sequence identity and composition and delineating the integrity of full-length BK and JC genomes. RESULTS: The analysed genomes displayed subpopulations frequently characterised by complex gene re-arrangements, duplications and deletions. CONCLUSIONS: Despite the recognition of such polymorphisms using high-resolution sequencing methodologies, the ability of these reference materials to act to enhance assay harmonisation did not appear significantly impacted, based on data generated by the 2015 WHO collaborative studies, but highlights cautionary aspects of IS generation and commutability for clinical molecular diagnostic application.

Bacterial and Chemical Evidence of Coastal Water Pollution from the Tijuana River in Sea Spray Aerosol.

Roughly half of the human population lives near the coast, and coastal water pollution (CWP) is widespread. Coastal waters along Tijuana, Mexico, and Imperial Beach (IB), USA, are frequently polluted by millions of gallons of untreated sewage and stormwater runoff. Entering coastal waters causes over 100 million global annual illnesses, but CWP has the potential to reach many more people on land via transfer in sea spray aerosol (SSA). Using 16S rRNA gene amplicon sequencing, we found sewage-associated bacteria in the polluted Tijuana River flowing into coastal waters and returning to land in marine aerosol. Tentative chemical identification from non-targeted tandem mass spectrometry identified anthropogenic compounds as chemical indicators of aerosolized CWP, but they were ubiquitous and present at highest concentrations in continental aerosol. Bacteria were better tracers of airborne CWP, and 40 tracer bacteria comprised up to 76% of the bacteria community in IB air. These findings confirm that CWP transfers in SSA and exposes many people along the coast. Climate change may exacerbate CWP with more extreme storms, and our findings call for minimizing CWP and investigating the health effects of airborne exposure.

Aberrant cell state plasticity mediated by developmental reprogramming precedes colorectal cancer initiation.

Cell state plasticity is carefully regulated in adult epithelia to prevent cancer. The aberrant expansion of the normally restricted capability for cell state plasticity in neoplasia is poorly defined. Using genetically engineered and carcinogen-induced mouse models of intestinal neoplasia, we observed that impaired differentiation is a conserved event preceding cancer development. Single-cell RNA sequencing (scRNA-seq) of premalignant lesions from mouse models and a patient with hereditary polyposis revealed that cancer initiates by adopting an aberrant transcriptional state characterized by regenerative activity, marked by Ly6a (Sca-1), and reactivation of fetal intestinal genes, including Tacstd2 (Trop2). Genetic inactivation of Sox9 prevented adenoma formation, obstructed the emergence of regenerative and fetal programs, and restored multilineage differentiation by scRNA-seq. Expanded chromatin accessibility at regeneration and fetal genes upon Apc inactivation was reduced by concomitant Sox9 suppression. These studies indicate that aberrant cell state plasticity mediated by unabated regenerative activity and developmental reprogramming precedes cancer development.

#COVIDisAirborne: AI-enabled multiscale computational microscopy of delta SARS-CoV-2 in a respiratory aerosol.

We seek to completely revise current models of airborne transmission of respiratory viruses by providing never-before-seen atomic-level views of the SARS-CoV-2 virus within a respiratory aerosol. Our work dramatically extends the capabilities of multiscale computational microscopy to address the significant gaps that exist in current experimental methods, which are limited in their ability to interrogate aerosols at the atomic/molecular level and thus obscure our understanding of airborne transmission. We demonstrate how our integrated data-driven platform provides a new way of exploring the composition, structure, and dynamics of aerosols and aerosolized viruses, while driving simulation method development along several important axes. We present a series of initial scientific discoveries for the SARS-CoV-2 Delta variant, noting that the full scientific impact of this work has yet to be realized.

Anthropogenic and Biogenic Contributions to the Organic Composition of Coastal Submicron Sea Spray Aerosol.

The organic composition of coastal sea spray aerosol is important for both atmospheric chemistry and public health but remains poorly characterized. Coastal waters contain an organic material derived from both anthropogenic processes, such as wastewater discharge, and biological processes, including biological blooms. Here, we probe the chemical composition of the organic fraction of sea spray aerosol over the course of the 2019 SeaSCAPE mesocosm experiment, in which a phytoplankton bloom was facilitated in natural coastal water from La Jolla, California. We apply untargeted two-dimensional gas chromatography to characterize submicron nascent sea spray aerosol samples, reporting ∼750 unique organic species traced over a 19 day phytoplankton bloom experiment. Categorization and quantitative compositional analysis reveal three major findings. First, anthropogenic species made up 30% of total submicron nascent sea spray aerosol organic mass under the pre-bloom condition. Second, biological activity drove large changes within the aerosolized carbon pool, decreasing the anthropogenic mass fraction by 89% and increasing the biogenic and biologically transformed fraction by a factor of 5.6. Third, biogenic marine organics are underrepresented in mass spectral databases in comparison to marine organic pollutants, with more than twice as much biogenic aerosol mass attributable to unlisted compounds.

Isotopic Insights into Organic Composition Differences between Supermicron and Submicron Sea Spray Aerosol.

To elucidate the seawater biological and physicochemical factors driving differences in organic composition between supermicron and submicron sea spray aerosol (SSAsuper and SSAsub), carbon isotopic composition (δ13C) measurements were performed on size-segregated, nascent SSA collected during a phytoplankton bloom mesocosm experiment. The δ13C measurements indicate that SSAsuper contains a mixture of particulate and dissolved organic material in the bulk seawater. After phytoplankton growth, a greater amount of freshly produced carbon was observed in SSAsuper with the proportional contribution being modulated by bacterial activity, emphasizing the importance of the microbial loop in controlling the organic composition of SSAsuper. Conversely, SSAsub exhibited no apparent relationship with biological activity but tracked closely with surface tension measurements probing the topmost ∼0.2-1.5 µm of the sea surface microlayer. This probing depth is similar to a bubble's film thickness at the ocean surface, suggesting that SSAsub organic composition may be influenced by the presence of surfactants at the air-sea interface that are transferred into SSAsub by bubble bursting. Our findings illustrate the substantial impact of seawater dynamics on the pronounced organic compositional differences between SSAsuper and SSAsub and demonstrate that these two SSA populations should be considered separately when assessing their contribution to marine aerosols and climate.

The Sea Spray Chemistry and Particle Evolution study (SeaSCAPE): overview and experimental methods.

Marine aerosols strongly influence climate through their interactions with solar radiation and clouds. However, significant questions remain regarding the influences of biological activity and seawater chemistry on the flux, chemical composition, and climate-relevant properties of marine aerosols and gases. Wave channels, a traditional tool of physical oceanography, have been adapted for large-scale ocean-atmosphere mesocosm experiments in the laboratory. These experiments enable the study of aerosols under controlled conditions which isolate the marine system from atmospheric anthropogenic and terrestrial influences. Here, we present an overview of the 2019 Sea Spray Chemistry and Particle Evolution (SeaSCAPE) study, which was conducted in an 11 800 L wave channel which was modified to facilitate atmospheric measurements. The SeaSCAPE campaign sought to determine the influence of biological activity in seawater on the production of primary sea spray aerosols, volatile organic compounds (VOCs), and secondary marine aerosols. Notably, the SeaSCAPE experiment also focused on understanding how photooxidative aging processes transform the composition of marine aerosols. In addition to a broad range of aerosol, gas, and seawater measurements, we present key results which highlight the experimental capabilities during the campaign, including the phytoplankton bloom dynamics, VOC production, and the effects of photochemical aging on aerosol production, morphology, and chemical composition. Additionally, we discuss the modifications made to the wave channel to improve aerosol production and reduce background contamination, as well as subsequent characterization experiments. The SeaSCAPE experiment provides unique insight into the connections between marine biology, atmospheric chemistry, and climate-relevant aerosol properties, and demonstrates how an ocean-atmosphere-interaction facility can be used to isolate and study reactions in the marine atmosphere in the laboratory under more controlled conditions.

#COVIDisAirborne: AI-Enabled Multiscale Computational Microscopy of Delta SARS-CoV-2 in a Respiratory Aerosol.

We seek to completely revise current models of airborne transmission of respiratory viruses by providing never-before-seen atomic-level views of the SARS-CoV-2 virus within a respiratory aerosol. Our work dramatically extends the capabilities of multiscale computational microscopy to address the significant gaps that exist in current experimental methods, which are limited in their ability to interrogate aerosols at the atomic/molecular level and thus ob-scure our understanding of airborne transmission. We demonstrate how our integrated data-driven platform provides a new way of exploring the composition, structure, and dynamics of aerosols and aerosolized viruses, while driving simulation method development along several important axes. We present a series of initial scientific discoveries for the SARS-CoV-2 Delta variant, noting that the full scientific impact of this work has yet to be realized. ACM REFERENCE FORMAT: Abigail Dommer 1† , Lorenzo Casalino 1† , Fiona Kearns 1† , Mia Rosenfeld 1 , Nicholas Wauer 1 , Surl-Hee Ahn 1 , John Russo, 2 Sofia Oliveira 3 , Clare Morris 1 , AnthonyBogetti 4 , AndaTrifan 5,6 , Alexander Brace 5,7 , TerraSztain 1,8 , Austin Clyde 5,7 , Heng Ma 5 , Chakra Chennubhotla 4 , Hyungro Lee 9 , Matteo Turilli 9 , Syma Khalid 10 , Teresa Tamayo-Mendoza 11 , Matthew Welborn 11 , Anders Christensen 11 , Daniel G. A. Smith 11 , Zhuoran Qiao 12 , Sai Krishna Sirumalla 11 , Michael O'Connor 11 , Frederick Manby 11 , Anima Anandkumar 12,13 , David Hardy 6 , James Phillips 6 , Abraham Stern 13 , Josh Romero 13 , David Clark 13 , Mitchell Dorrell 14 , Tom Maiden 14 , Lei Huang 15 , John McCalpin 15 , Christo- pherWoods 3 , Alan Gray 13 , MattWilliams 3 , Bryan Barker 16 , HarindaRajapaksha 16 , Richard Pitts 16 , Tom Gibbs 13 , John Stone 6 , Daniel Zuckerman 2 *, Adrian Mulholland 3 *, Thomas MillerIII 11,12 *, ShantenuJha 9 *, Arvind Ramanathan 5 *, Lillian Chong 4 *, Rommie Amaro 1 *. 2021. #COVIDisAirborne: AI-Enabled Multiscale Computational Microscopy ofDeltaSARS-CoV-2 in a Respiratory Aerosol. In Supercomputing '21: International Conference for High Perfor-mance Computing, Networking, Storage, and Analysis . ACM, New York, NY, USA, 14 pages. https://doi.org/finalDOI.

Moving beyond solutionism: Re-imagining placements through an activity systems lens.

INTRODUCTION: Clinical placements are central to the process of preparing future health professionals for practice. Health care environments are increasingly complex and demanding with clinical placements often being perceived as a burden on busy health professionals giving rise to a service-education tension. This tension creates a situation ripe for simplistic solutionist approaches. For example, characterising the problem of clinical placements as students negatively impacting on service productivity results in a reductionist solution such as universities compensating health services for student education. Challenges faced by placement seekers and placement providers are multifaceted and complex requiring a more sophisticated understanding and response to the challenges of involving students in the workplace to prepare them for the future workforce. RE-CONCEPTUALISATION: We argue that the health and education systems have become de-coupled. Learning and working are seen as distinct activities that are at odds with one another. Re-imagining the purposes and practices of clinical placements for the mutual benefit of patients, health services and students may fruitfully address this disconnect. WORKED EXAMPLE: We present a worked example using the conceptual and analytical tools of cultural-historical activity theory to articulate what we have learnt about this health-education disconnect. Our worked example draws on research involving a series of clinical education case studies within acute care contexts. CONCLUSION: Through the lens of cultural-historical activity theory, we highlight that solutionist approaches are entrenched in a de-coupling of health from education where the shared object of preparing the future workforce is fragmented. Successful re-coupling requires a partnership that is founded on a shared commitment to preparing the future workforce; recognises that learning and practice are inseparable; and understands that both activity systems are fluid and that collaboration to stay focused on the shared object of preparing the future workforce is complex, challenging and ongoing work.

Expansive learning in medical education: Putting Change Laboratory to work.

This paper explores the purposeful use of conceptual and methodological tools provided by Cultural Historical Activity Theory (CHAT) to transform learning cultures and practices within and across diverse clinical learning environments. We describe how Change Laboratory methodology helped clinicians and others who support student, intern, and resident education to make changes collaboratively. A case study in undergraduate medical education shows how this created new forms of medical student placement and a postgraduate study shows how it addressed supervisors' undermining behaviour towards Obstetrics and Gynaecology residents. This empirical work illustrates ways of modifying the classical Change Laboratory process to fit local contexts, resources, and needs. We conclude with lessons learned and future directions for practitioner-researchers who wish to broaden the range of methodological tools they use to transform clinical learning environments.

Complete Genome Sequence of Original Material Used To Derive the WHO International Standard for Human Polyomavirus BK DNA.

The complete genomic sequence was determined for the original biological material used to derive the WHO international standard for BK polyomavirus (BKV) DNA. The entire coding sequence and noncoding regions were assigned BKV subtype 1, subgroup 1b-1. This information will aid development and evaluation of human BKV DNA amplification assays.

Biofilm management using monofilament fibre debridement technology: outcomes and clinician and patient satisfaction.

OBJECTIVE: Best practice in wound bed preparation and biofilm-based wound management includes debridement to create a clean wound bed and to assist in minimising the redevelopment of biofilm. Biofilm that is not removed inhibits healing and redevelops if not prevented from doing so with topical antimicrobial agents. Monofilament fibre debriding technology (MFDT) is used for effective and rapid mechanical debridement of loose material, slough and biofilm. The objective of this evaluation was to determine the clinical effect and consequential levels of health professional and patient satisfaction with the results of a biofilm pathway that included MFDT to achieve debridement. METHODS: This non-comparative, open label evaluation was conducted in static and non-static wounds that required debridement. MFDT was used to debride in a two-week evaluation of a biofilm pathway. Wounds were debrided three times in week one and twice in week two. Each debridement was followed by treatment with an antimicrobial dressing. Other care included secondary dressings and compression delivered according to local practice, guidelines and formularies. After the clinical evaluation, health professionals were invited to complete an online survey of the clinical outcomes and their satisfaction with the biofilm pathway. RESULTS: There were 706 health professionals who provided answers to the survey questions. Wound types evaluated were leg ulcers (67.4%), pressure ulcers (10%), dehisced surgical wounds (1.7%), diabetic foot ulcers (7.4%) and other wounds (13.4%). Of the wounds, 9% were reported as non-static despite the eligibility criteria. Not all wounds followed the pathway. The most frequently-used antimicrobial was silver. Non-antimicrobial products used included all-in-one dressings, other secondary dressings and compression. There was a change in 77% of wounds overall after two weeks. Change was reported almost equally for both static and non-static wounds. Health professionals who did or did not follow the pathway were 'completely satisfied' or 'satisfied' with the overall clinical outcome 96% and 95%, respectively. Of the patients, 77% were 'completely satisfied' or 'satisfied' with healing after following the pathway, as reported by the treating health professional. CONCLUSION: The biofilm pathway that includes MFDT appears effective. Wounds managed on the pathway were debrided effectively and healing progressed to the satisfaction of both health professionals and patients.

Rapid debridement with monofilament fibre debridement technology: clinical outcomes and practitioner satisfaction.

OBJECTIVE: To determine the clinical effect and consequential levels of health professionals and patient satisfaction with the results of debridement episodes of wounds with visible slough and/or scaly skin using monofilament fibre debridement technology. METHODS: This was a non-comparative, open label evaluation conducted in static/non-healing acute and chronic wounds with visible slough and/or scaly skin that required debridement. Monofilament fibre debridement technology was applied in 1-2 sequential treatment episodes during normal clinical practice which followed local practice, guidelines or formularies. Following the clinical phase of the evaluation, health professionals were invited to complete an online survey of the clinical outcomes and their satisfaction with them. RESULTS: Survey questions were answered by 1129 health professionals. Wounds managed using the monofilament fibre debridement technology during this evaluation included leg ulcers (63%), pressure ulcers (10%), dehisced surgical wounds (3%), diabetic foot ulcers (8%) and other wounds (13%). 'Other' wound types included acute dirty wounds, burns, cellulitis, psoriasis, diabetic amputation wounds, dry flaky skin, moisture wounds, trauma, varicose eczema. Of the wounds, 12% were reported as non-static. There was visible change in the wound and/or skin after first use of the monofilament fibre debridement technology in a high proportion of all wound types, and a further increase in the proportion of wounds with visible change after the second use. The visible difference was significant for both static and non-static wounds. User and patient satisfaction with all clinical outcomes were high, whether or not the user and patient had previous experience of monofilament fibre debridement technology. CONCLUSION: Monofilament fibre debridement technology provides rapid, visible and effective debridement of slough and scaly skin after one application and further visible improvement after two applications in static and non-static wounds. Health professionals and patients report high levels of satisfaction with outcomes following application of the monofilament fibre debridement technology.

Utility of reference materials for Zika Virus nucleic acid testing.

The emergence of Zika virus (ZIKV) in the Americas has resulted in increased nucleic acid amplification testing (NAT) of clinical samples and blood donations. New molecular diagnostic assays have been developed resulting in a corollary requirement for ZIKV reference material. To address this we have produced and calibrated two African lineage ZIKV reference materials: a highly concentrated secondary standard (NIBSC: 16/110) and a lower concentration external quality control (QC) reagent (NIBSC: 16/124) and compared their performance in three ZIKV NAT assays in relation with the First International Standard (IS) for Zika Virus NAT assays (PEI: 11468/16). In summary the African lineage ZIKV reference materials were detected by all three assays. The ZIKV lineage did not affect the performance of the secondary standard. The external QC reagent (16/124) was detected by all three assays highlighting its suitability for use as a low positive control to monitor assay performance on a regular basis. The relative potency of 16/110 to the IS was 5.49E+06IU/mL (95% CI: 1.46E+06-2.06E+07) and 16/124 to 16/110 was 8.36E+03 (95% CI: 7.83E+03-8.92E+03). The global availability of African lineage ZIKV reference materials will facilitate standardization of ZIKV molecular diagnostic assays between and within laboratories whilst preserving the IS.

The development and establishment of the 1st WHO BKV International Standard for nucleic acid based techniques.

Immunocompromised patients are at significant risk from BKV reactivation, causing allograft dysfunction or loss. Patient management relies on viral DNA monitoring, using a viral load cut-off to reduce immunosuppression. However, consistency between viral load detection assays cannot be achieved without an effective means of standardisation. We have worked with the WHO's Expert Committee on Biological Standardisation to develop suitable reference materials and undertake an international collaborative study to establish the 1st WHO International Standard for BKV detection assays. We report on the evaluation of two lyophilised candidate cell culture derived, whole virus preparations, undertaken by 33 expert laboratories. By employing the principles of biological standardisation, we show improved agreement across laboratories, demonstrating the suitability of either candidate for use as a primary order calibrant. Candidate 14/212 was established by the WHO ECBS with an assigned potency of 7.2 log10 International Units/mL intended for the calibration of BKV secondary reference materials.