The past as present in health promotion: the case for a 'public health humanities'.

Health promotion is conceived as a unifying concept for improving the health of populations. This means addressing the socio-cultural, economic and commercial causes of ill-health, which are necessarily informed by past policies and socio-cultural contexts. However, historical scholarship has rarely figured in health promotion practice or scholarship. This gap resides in the determinants of health, and notably in the analyses of tobacco control and skin cancer prevention, two long-running campaigns that have shaped modern health promotion in Australia. Both highlight a need for understanding the profound impact of history on the present and the value of learning from past successes and failures. Doing so requires integrating historical analyses into existing health promotion scholarship. To achieve this aim, we present a new 'public health humanities' methodology. This novel interdisciplinary framework is conceived as a spectrum in which historical studies integrate with existing health promotion disciplines to solve complex health problems. We draw on the many calls for more interdisciplinarity in health promotion and derive this methodology from proposals in the medical humanities and cognate fields that have wrestled with combining history and present-focused disciplines. Using tobacco control and skin cancer prevention as case studies, we demonstrate how public health humanities uses interdisciplinary teams and shared research questions to generate valuable new knowledge unavailable with traditional methods. Furthermore, we show how it creates evaluation criteria to consider the powerful impact of issues like colonialism on current inequities that hinder health promotion strategies, and from which lessons may be derived for the future.

Measuring the Change in Soft Palate Length and Shape Following Maxillary Advancement: A Cohort Study in Patients with Orofacial Clefts.

OBJECTIVE: To evaluate a method of measuring the change in palatal length and shape following maxillary advancement using synchronous lateral videofluoroscopy and voice recording in order to understand how movement of the maxilla may affect VPI risk in patients with cleft lip and/or palate (CL/P). DESIGN: Retrospective cohort study of children with cleft lip and/or palate. SETTING: Single center, tertiary children's hospital. PARTICIPANTS: Patients with cleft lip and/or palate who underwent maxillary advancement between 2016-21 inclusive. INTERVENTIONS: Maxillary advancement surgery, including those who underwent concurrent mandibular procedures. MAIN OUTCOME MEASURES: The length of the soft palate and the genu angle were measured throughout palatal dynamic range. Pre- and post-operative measurements were compared using a one sided T-test, with subgroup analysis for patients with clinical VPI. RESULTS: Ten patients were examined. The mean distance of maxillary advancement was 10.5 mm. The average increase in pre-genu soft palate length was 2.8 mm in the resting position and 2.9 mm in the closed position. The genu angle decreased in the closed position by 16.3 degrees. CONCLUSIONS: The soft palate showed limited ability to lengthen following maxillary advancement and this may explain the risk of VPI. There was partial compensation by the muscle sling of the palate as demonstrated by a more acute post-operative genu angle and this suggests one reason for the variability of VPI reported. Future research is required to investigate how length and shape changes measured using this method can predict VPI risk.

Practical extended use of antimicrobial silver (PExUS).

BACKGROUND: Antimicrobial silver has had a role in wound antisepsis throughout history and, with the rise in acquired antibiotic resistance, silver dressings are once again commonly used. Issues with silver dressings include the important environmental consideration of nanoparticle manufacture, and the significant financial cost of these products. One solution to these problems may be to adopt an opened-but-unused model of wound care whereby dressing materials are used in piecemeal fashion and excess stored in between dressing changes. Due to a lack of literature on the topic, this project was designed with the aim of testing the antimicrobial efficacy of available silver dressings during storage after opening. METHODS: Four commonly used silver dressings were tested for antimicrobial activity using a zone of inhibition assay against clinically important pathogens. The assay was performed on opening of dressings and repeated over 3 months in storage at 4, 25 or 37°C. Analysis was performed using repeated measures ANOVA. Swab cultures were taken at each simulated dressing change to detect microbial contamination of the dressings during storage. RESULTS: There was no effect of time or storage temperature on the zone of inhibition over the 12 week test period. No swabs taken returned culture consistent with microbial contamination of stored dressings. CONCLUSION: Opened silver dressings maintain antimicrobial activity for at least 12 weeks in storage and are resistant to contamination. An opened-but-unused model for wound care is likely to improve cost-effectiveness while preserving effectiveness and safety.

Antimicrobial silver dressings: a review of emerging issues for modern wound care.

Skin is an important barrier to pathogenic microorganisms and plays a critical role in a ctivation of innate immune responses. When the skin barrier is breached following wounding or burn injury, pathogens can invade and complicate healing with infection resulting in delayed healing and symptomatic scarring. Wound infection is a significant problem after burn injury and in patients with chronic wounds. Antimicrobial silver has had a significant role in wound antisepsis throughout history and, given the rise in community acquired antibiotic resistance, silver dressings are now commonly used to combat wound infection. The multi-modal mechanism of action, low potential for toxicity and formation of microbial resistance makes silver dressings suitable tools against a wide array of clinically important microbes. There are, however, a number of issues with silver dressings including a conflicting evidence base, the important environmental consideration of nanoparticle manufacture, and the significant cost of these products. One solution may be to adopt an 'opened-but-unused' means of wound care whereby bulk dressing materials are used piecemeal and stored in between dressing changes to increase the cost-effectiveness and reduced wastage. There is, however, little literature on this topic and so in vitro and clinical research must be performed to consider the efficacy of active ingredient dressings in wound care including silver dressings once opened and stored.

Generating Data Visualizations of Longitudinal Cohort Ambient Air Pollution Exposure: Report-Back Intervention Development in Participatory Action Research.

A civic engagement and data science design was used to develop a report-back intervention to address stakeholder concerns related to air emissions surrounding a coke oven factory near Buffalo, NY. This factory had historically emitted high levels of benzene pollution and ceased operation in October 2018 because of violations of the US Clean Air Act and US Resource Conservation and Recovery Act. Using publicly available air pollution and weather data, descriptive time series and wind-rose data visualizations were developed using open-source software as part of a two-page report-back brief. Data from two air toxics monitoring sites in this direction suggest that industrial sources were likely the major contributor to the benzene in the air at these locations prior to May 2018, after which traffic emissions became the likely major contributor. Wind-rose visualizations demonstrated that the wind typically blew toward the northeast, which was qualitatively consistent with locations of stakeholder concerns. With the factory closed, collective efforts subsequently shifted to address traffic emission air pollution sources, factory site cleanup, and ground and water pollution mitigation. Because this intervention utilized open-source software and publicly available data, it can serve as a blueprint for future data-driven nursing interventions and community-led environmental justice efforts.

Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors.

Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM (transmission electron cryomicroscopy), denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these approaches, we have made two major discoveries: (1) rAAV capsids have post-translational modifications (PTMs), including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms; and (2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data show that host cell protein impurities differ between platforms and can have their own PTMs, including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (p < 0.05-0.0001), in various mouse tissues in vivo (p < 0.03-0.0001), and in human liver in vivo (p < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity, as well as cost considerations.

Fast-Seq: A Simple Method for Rapid and Inexpensive Validation of Packaged Single-Stranded Adeno-Associated Viral Genomes in Academic Settings.

Adeno-associated viral (AAV) vectors have shown great promise in gene delivery as evidenced by recent FDA approvals. Despite efforts to optimize manufacturing for good manufacturing practice (GMP) productions, few academic laboratories have the resources to assess vector composition. One critical component of vector quality is packaged genome fidelity. Errors in viral genome replication and packaging can result in the incorporation of faulty genomes with mutations, truncations, or rearrangements, compromising vector potency. Thus, sequence validation of packaged genome composition is an important quality control (QC), even in academic settings. We developed Fast-Seq, an end-to-end method for extraction, purification, sequencing, and data analysis of packaged single-stranded AAV (ssAAV) genomes intended for non-GMP preclinical environments. We validated Fast-Seq on ssAAV vectors with three different genome compositions (CAG-GFP, CAG-tdTomato, EF1α-FLuc), three different genome sizes (2.9, 3.6, 4.4 kb), packaged in four different capsid serotypes (AAV1, AAV2, AAV5, and AAV8), and produced using the two most common production methods (Baculovirus-Sf9 and human HEK293), from both common commercial vendors and academic core facilities supplying academic laboratories. We achieved an average genome coverage of >1,400 × and an average inverted terminal repeat coverage of >280 × , despite the many differences in composition of each ssAAV sample. When compared with other ssAAV next-generation sequencing (NGS) methods for GMP settings, Fast-Seq has several unique advantages: Tn5 transposase-based fragmentation rather than sonication, 125 × less input DNA, simpler adapter ligation, compatibility with commonly available inexpensive sequencing instruments, and free open-source data analysis code in a preassembled customizable Docker container designed for novices. Fast-Seq can be completed in 18 h, is more cost-effective than other NGS methods, and is more accurate than Sanger sequencing, which is generally only applied at 1-2 × sequencing depth. Fast-Seq is a rapid, simple, and inexpensive methodology to validate packaged ssAAV genomes in academic settings.

Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation.

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Gas-Particle Partitioning of Vehicle Emitted Primary Organic Aerosol Measured in a Traffic Tunnel.

We measured the gas-particle partitioning of vehicle emitted primary organic aerosol (POA) in a traffic tunnel with three independent methods: artifact corrected bare-quartz filters, thermodenuder (TD) measurements, and thermal-desorption gas-chromatography mass-spectrometry (TD-GC-MS). Results from all methods consistently show that vehicle emitted POA measured in the traffic tunnel is semivolatile under a wide range of fleet compositions and ambient conditions. We compared the gas-particle partitioning of POA measured in both tunnel and dynamometer studies and found that volatility distributions measured in the traffic tunnel are similar to volatility distributions measured in the dynamometer studies, and predict similar gas-particle partitioning in the TD. These results suggest that the POA volatility distribution measured in the dynamometer studies can be applied to describe gas-particle partitioning of ambient POA emissions. The POA volatility distribution measured in the tunnel does not have significant diurnal or seasonal variations, which indicate that a single volatility distribution is adequate to describe the gas-particle partitioning of vehicle emitted POA in the urban environment.

Orbital occupancy and charge doping in iron-based superconductors.

The intrinsic Fe local magnetic moment and Fe orbital occupations of iron-based superconductors are unveiled through the local, real-space capability of aberration-corrected scanning transmission electron microscopy/electron energy loss spectroscopy (STEM/EELS). Although the ordering of Fe moments needs to be suppressed for superconductivity to arise, the local, fluctuating Fe magnetic moment is enhanced near optimal superconductivity.

Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis.

Cancer genome sequencing studies have identified numerous driver genes, but the relative timing of mutations in carcinogenesis remains unclear. The gradual progression from premalignant Barrett's esophagus to esophageal adenocarcinoma (EAC) provides an ideal model to study the ordering of somatic mutations. We identified recurrently mutated genes and assessed clonal structure using whole-genome sequencing and amplicon resequencing of 112 EACs. We next screened a cohort of 109 biopsies from 2 key transition points in the development of malignancy: benign metaplastic never-dysplastic Barrett's esophagus (NDBE; n=66) and high-grade dysplasia (HGD; n=43). Unexpectedly, the majority of recurrently mutated genes in EAC were also mutated in NDBE. Only TP53 and SMAD4 mutations occurred in a stage-specific manner, confined to HGD and EAC, respectively. Finally, we applied this knowledge to identify high-risk Barrett's esophagus in a new non-endoscopic test. In conclusion, mutations in EAC driver genes generally occur exceptionally early in disease development with profound implications for diagnostic and therapeutic strategies.

Clinical and pathological impact of VHL, PBRM1, BAP1, SETD2, KDM6A, and JARID1c in clear cell renal cell carcinoma.

VHL is mutated in the majority of patients with clear cell renal cell carcinoma (ccRCC), with conflicting clinical relevance. Recent studies have identified recurrent mutations in histone modifying and chromatin remodeling genes, including BAP1, PBRM1, SETD2, KDM6A, and JARID1c. Current evidence suggests that BAP1 mutations are associated with aggressive disease. The clinical significance of the remaining genes is unknown. In this study, targeted sequencing of VHL and JARID1c (entire genes) and coding regions of BAP1, PBRM1, SETD2, and KDM6A was performed on 132 ccRCCs and matched normal tissues. Associations between mutations and clinical and pathological outcomes were interrogated. Inactivation of VHL (coding mutation or promoter methylation) was seen in 75% of ccRCCs. Somatic noncoding VHL alterations were identified in 29% of ccRCCs and may be associated with improved overall survival. BAP1 (11%), PBRM1 (33%), SETD2 (16%), JARID1c (4%), and KDM6A (3%) mutations were identified. BAP1-mutated tumors were associated with metastatic disease at presentation (P = 0.023), advanced clinical stage (P = 0.042) and a trend towards shorter recurrence free survival (P = 0.059) when compared with tumors exclusively mutated for PBRM1. Our results support those of recent publications pointing towards a role for BAP1 and PBRM1 mutations in risk stratifying ccRCCs. Further investigation of noncoding alterations in VHL is warranted.

Gas-particle partitioning of primary organic aerosol emissions: (2) diesel vehicles.

Experiments were performed to investigate the gas-particle partitioning of primary organic aerosol (POA) emissions from two medium-duty (MDDV) and three heavy-duty (HDDV) diesel vehicles. Each test was conducted on a chassis dynamometer with the entire exhaust sampled into a constant volume sampler (CVS). The vehicles were operated over a range of driving cycles (transient, high-speed, creep/idle) on different ultralow sulfur diesel fuels with varying aromatic content. Four independent yet complementary approaches were used to investigate POA gas-particle partitioning: artifact correction of quartz filter samples, dilution from the CVS into a portable environmental chamber, heating in a thermodenuder, and thermal desorption/gas chromatography/mass spectrometry (TD-GC-MS) analysis of quartz filter samples. During tests of vehicles not equipped with diesel particulate filters (DPF), POA concentrations inside the CVS were a factor of 10 greater than ambient levels, which created large and systematic partitioning biases in the emissions data. For low-emitting DPF-equipped vehicles, as much as 90% of the POA collected on a quartz filter from the CVS were adsorbed vapors. Although the POA emission factors varied by more than an order of magnitude across the set of test vehicles, the measured gas-particle partitioning of all emissions can be predicted using a single volatility distribution derived from TD-GC-MS analysis of quartz filters. This distribution is designed to be applied directly to quartz filter data that are the basis for existing emissions inventories and chemical transport models that have implemented the volatility basis set approach.

A structural basis for the assembly and functions of a viral polymer that inactivates multiple tumor suppressors.

Evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction networks. The structural basis for the multiple and dominant functions of adenovirus oncoproteins has remained elusive. E4-ORF3 forms a nuclear polymer and simultaneously inactivates p53, PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressors. We identify oligomerization mutants and solve the crystal structure of E4-ORF3. E4-ORF3 forms a dimer with a central ß core, and its structure is unrelated to known polymers or oncogenes. E4-ORF3 dimer units coassemble through reciprocal and nonreciprocal exchanges of their C-terminal tails. This results in linear and branched oligomer chains that further assemble in variable arrangements to form a polymer network that partitions the nuclear volume. E4-ORF3 assembly creates avidity-driven interactions with PML and an emergent MRN binding interface. This reveals an elegant structural solution whereby a small protein forms a multivalent matrix that traps disparate tumor suppressors.

Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA.

Plasma of cancer patients contains cell-free tumor DNA that carries information on tumor mutations and tumor burden. Individual mutations have been probed using allele-specific assays, but sequencing of entire genes to detect cancer mutations in circulating DNA has not been demonstrated. We developed a method for tagged-amplicon deep sequencing (TAm-Seq) and screened 5995 genomic bases for low-frequency mutations. Using this method, we identified cancer mutations present in circulating DNA at allele frequencies as low as 2%, with sensitivity and specificity of >97%. We identified mutations throughout the tumor suppressor gene TP53 in circulating DNA from 46 plasma samples of advanced ovarian cancer patients. We demonstrated use of TAm-Seq to noninvasively identify the origin of metastatic relapse in a patient with multiple primary tumors. In another case, we identified in plasma an EGFR mutation not found in an initial ovarian biopsy. We further used TAm-Seq to monitor tumor dynamics, and tracked 10 concomitant mutations in plasma of a metastatic breast cancer patient over 16 months. This low-cost, high-throughput method could facilitate analysis of circulating DNA as a noninvasive "liquid biopsy" for personalized cancer genomics.

Macro-to-micro structural proteomics: native source proteins for high-throughput crystallization.

Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography.

The critical protein interactions and structures that elicit growth deregulation in cancer and viral replication.

One of the greatest challenges in biomedicine is to define the critical targets and network interactions that are subverted to elicit growth deregulation in human cells. Understanding and developing rational treatments for cancer requires a definition of the key molecular targets and how they interact to elicit the complex growth deregulation phenotype. Viral proteins provide discerning and powerful probes to understand both how cells work and how they can be manipulated using a minimal number of components. The small DNA viruses have evolved to target inherent weaknesses in cellular protein interaction networks to hijack the cellular DNA and protein replication machinery. In the battle to escape the inevitability of senescence and programmed cell death, cancers have converged on similar mechanisms, through the acquisition and selection of somatic mutations that drive unchecked cellular replication in tumors. Understanding the dynamic mechanisms through which a minimal number of viral proteins promote host cells to undergo unscheduled and pathological replication is a powerful strategy to identify critical targets that are also disrupted in cancer. Viruses can therefore be used as tools to probe the system-wide protein-protein interactions and structures that drive growth deregulation in human cells. Ultimately this can provide a path for developing system context-dependent therapeutics. This review will describe ongoing experimental approaches using viruses to study pathways deregulated in cancer, with a particular focus on viral cellular protein-protein interactions and structures.

A domain insertion in Escherichia coli GyrB adopts a novel fold that plays a critical role in gyrase function.

DNA topoisomerases manage chromosome supercoiling and organization in all forms of life. Gyrase, a prokaryotic heterotetrameric type IIA topo, introduces negative supercoils into DNA by an ATP-dependent strand passage mechanism. All gyrase orthologs rely on a homologous set of catalytic domains for function; however, these enzymes also can possess species-specific auxiliary regions. The gyrases of many gram-negative bacteria harbor a 170-amino acid insertion of unknown architecture and function in the metal- and DNA-binding TOPRIM domain of the GyrB subunit. We have determined the structure of the 212 kDa Escherichia coli gyrase DNA binding and cleavage core containing this insert to 3.1 Å resolution. We find that the insert adopts a novel, extended fold that braces the GyrB TOPRIM domain against the coiled-coil arms of its partner GyrA subunit. Structure-guided deletion of the insert greatly reduces the DNA binding, supercoiling and DNA-stimulated ATPase activities of gyrase. Mutation of a single amino acid at the contact point between the insert and GyrA more modestly impairs supercoiling and ATP turnover, and does not affect DNA binding. Our data indicate that the insert has two functions, acting as a steric buttress to pre-configure the primary DNA-binding site, and serving as a relay that may help coordinate communication between different functional domains.

Radio-frequency plasma transducer for use in harsh environments.

We describe a compact transducer used to generate and modulate low-intensity radio-frequency atmospheric pressure plasma (RF-APP) for high temperature gap measurement and generation of air-coupled ultrasound. The new transducer consists of a quarter-wave transmission line where the ground return path is a coaxial solenoid winding. The RF-APP is initiated at the open end of the transmission line and stabilized by passive negative feedback between the electrical impedance of the plasma and the energy stored in the solenoid. The electrical impedance of the plasma was measured at the lower-voltage source end of the transducer, eliminating the need to measure kilovolt-level voltages near the discharge. We describe the use of a 7 MHz RF-APP prototype as a harsh-environment clearance sensor to demonstrate the suitability of plasma discharges for a common nondestructive inspection application. Clearance measurements of 0-5 mm were performed on a rotating calibration target with a measurement precision of 0.1 mm and a 20 kHz sampling rate.