A universal molecular control for DNA, mRNA and protein expression.

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

Structural Factors Contributing to Compassion Fatigue, Burnout, and Secondary Traumatic Stress Among Hospital-Based Healthcare Professionals During the COVID-19 Pandemic.

High levels of burnout among healthcare providers (HCPs) have been a widely documented phenomenon, which have been exacerbated during the COVID-19 pandemic. In the United States, qualitative studies that are inclusive of HCPs in diverse professional roles have been limited. Therefore, we utilized a qualitative-quantitative design to examine professional quality of life in terms of compassion fatigue, burnout, and secondary traumatic stress among hospital-based HCPs, including social workers, hospitalists, residents, and palliative care team members during COVID-19. HCPs (n = 26) participated in virtual semi-structured focus groups or individual interviews and online surveys (n = 30) including the Professional Quality of Life (ProQOL) Scale. While ProQOL scores indicated low levels of compassion fatigue, burnout, and secondary traumatic stress, thematic analysis of our qualitative data included rich descriptions of compassion fatigue, burnout, and secondary traumatic stress. Safety concerns and value misalignment characterized structural stressors perceived to contribute to HCP compassion fatigue, burnout, and secondary traumatic stress. The discrepancy between our qualitative and quantitative findings may be indication that modifications to current screenings are warranted. These findings also suggest a need to identify and implement structural and policy changes that increase HCPs' physical and emotional safety and promote better alignment of institutional interests with HCP values.

Bringing Generalists to Global Health: a Missed Opportunity and Call to Action.

The credo of the generalist physician has always been the promotion of health for all, in every aspect: not just multiple vulnerable organ systems, but multiple social, cultural, and political factors that contribute to poor health and exacerbate health inequity. In recent years, the field of global health has also adopted this same mission: working across both national and clinical specialty borders to improve health for all and end health disparities worldwide. Yet within the Society for General Internal Medicine, and among American generalists, engagement in global health, both within and outside the USA, remains uncommon. We see this gap as an opportunity, because in fact generalists in America already have the skills and experience that global health badly needs. SGIM could promote generalists to global health's vanguard, with three core steps. First, we generalists must continue to integrate health for the vulnerable into our domestic work, generating care models applicable in low-resource settings around the globe. Conversely, we must also engage with and implement international ideas and solutions for universal access to primary care for vulnerable patients in the USA. And lastly, we must build platforms to connect ourselves with colleagues worldwide to exchange these learnings.

mRNA vaccine quality analysis using RNA sequencing.

The success of mRNA vaccines has been realised, in part, by advances in manufacturing that enabled billions of doses to be produced at sufficient quality and safety. However, mRNA vaccines must be rigorously analysed to measure their integrity and detect contaminants that reduce their effectiveness and induce side-effects. Currently, mRNA vaccines and therapies are analysed using a range of time-consuming and costly methods. Here we describe a streamlined method to analyse mRNA vaccines and therapies using long-read nanopore sequencing. Compared to other industry-standard techniques, VAX-seq can comprehensively measure key mRNA vaccine quality attributes, including sequence, length, integrity, and purity. We also show how direct RNA sequencing can analyse mRNA chemistry, including the detection of nucleoside modifications. To support this approach, we provide supporting software to automatically report on mRNA and plasmid template quality and integrity. Given these advantages, we anticipate that RNA sequencing methods, such as VAX-seq, will become central to the development and manufacture of mRNA drugs.

Community engagement to inform development of strategies to improve referral for hypertension: perspectives of patients, providers and local community members in western Kenya.

BACKGROUND: Hypertension is the leading cause of death and disability. Clinical care for patients with hypertension in Kenya leverages referral networks to provide basic and specialized healthcare services. However, referrals are characterized by non-adherence and delays in completion. An integrated health information technology (HIT) and peer-based support strategy to improve adherence to referrals and blood pressure control was proposed. A formative assessment gathered perspectives on barriers to referral completion and garnered thoughts on the proposed intervention. METHODS: We conducted a qualitative study in Kitale, Webuye, Kocholya, Turbo, Mosoriot and Burnt Forest areas of Western Kenya. We utilized the PRECEDE-PROCEED framework to understand the behavioral, environmental and ecological factors that would influence uptake and success of our intervention. We conducted four mabaraza (customary heterogenous community assemblies), eighteen key informant interviews, and twelve focus group discussions among clinicians, patients and community members. The data obtained was audio recorded alongside field note taking. Audio recordings were transcribed and translated for onward coding and thematic analysis using NVivo 12. RESULTS: Specific supply-side and demand-side barriers influenced completion of referral for hypertension. Key demand-side barriers included lack of money for care and inadequate referral knowledge. On the supply-side, long distance to health facilities, low availability of services, unaffordable services, and poor referral management were reported. All participants felt that the proposed strategies could improve delivery of care and expressed much enthusiasm for them. Participants appreciated benefits of the peer component, saying it would motivate positive patient behavior, and provide health education, psychosocial support, and assistance in navigating care. The HIT component was seen as reducing paper work, easing communication between providers, and facilitating tracking of patient information. Participants also shared concerns that could influence implementation of the two strategies including consent, confidentiality, and reduction in patient-provider interaction. CONCLUSIONS: Appreciation of local realities and patients' experiences is critical to development and implementation of sustainable strategies to improve effectiveness of hypertension referral networks. Incorporating concerns from patients, health care workers, and local leaders facilitates adaptation of interventions to respond to real needs. This approach is ethical and also allows research teams to harness benefits of participatory community-involved research. TRIAL REGISTRATION: Clinicaltrials.gov, NCT03543787, Registered June 1, 2018. https://clinicaltrials.gov/ct2/show/NCT03543787.

Identifying barriers and facilitators along the hepatitis C care cascade to inform human-centered design of contextualized treatment protocols for vulnerable populations in Austin, Texas: a qualitative study.

BACKGROUND: Hepatitis C virus (HCV) is a leading cause of liver-related mortality and morbidity. Despite effective direct acting antivirals and a simplified treatment algorithm, limited access to HCV treatment in vulnerable populations, including people experiencing homelessness (PEH) and people who inject drugs (PWID), hinders global elimination. Adapting the evidence-based, simplified HCV treatment algorithm to the organizational and contextual realities of non-traditional clinic settings serving vulnerable populations can help overcome specific barriers to HCV care. The first phase of the Erase Hep C study aimed to identify barriers and facilitators specific to these vulnerable populations to design the site-specific, simplified treatment protocols. METHODS: Forty-two semi-structured qualitative interviews, guided by the Practical, Robust Implementation and Sustainability Model (PRISM) framework, were conducted with clinic staff, community-based organizations providing screening and linkage to care, and patients diagnosed with HCV, to identify contextual barriers and facilitators to treatment at a local community health center's Health Care for the Homeless program in Austin, Texas. Audio-recorded interviews were systematically analyzed using thematic analysis informed by the PRISM framework and design thinking, to anchor barriers and facilitators along the HCV care cascade. Findings were fed into human-centered design workshops to co-design, with clinic staff, site-specific, simplified HCV treatment protocols. RESULTS: The specific needs of PEH and PWID patient populations informed barriers and facilitators of HCV care. Barriers included tracking patients who miss critical appointments or labs, medication access and adherence, and patient HCV knowledge. Clinical teams leveraged existing facilitators and incorporated solutions to barriers into clinic workflows to improve care coordination and medication access. Actionable solutions included augmenting existing staff roles, employing HCV care navigation throughout the cascade, and standardizing medication adherence counseling. CONCLUSIONS: Clinic staff identified HCV care facilitators to leverage, and designed actionable solutions to address barriers, to incorporate into site-specific treatment protocols to improve patient HCV outcomes. Methods used to incorporate staff and patient experiential knowledge into the design of contextualized treatment protocols in non-traditional clinic settings could serve as a model for future implementation research. The next phase of the study is protocol implementation and patient enrollment into a single-arm trial to achieve HCV cure.

Development, implementation, and feasibility of site-specific hepatitis C virus treatment workflows for treating vulnerable, high-risk populations: protocol of the Erase Hep C study - a prospective single-arm intervention trial.

BACKGROUND: Hepatitis C virus (HCV) is the leading indication for liver transplantation and liver-related mortality. The development of direct-acting antivirals (DAA) and a simplified treatment algorithm with a > 97% cure rate should make global elimination of HCV an achievable goal. Yet, vulnerable populations with high rates of HCV still have limited access to treatment. By designing locally contextualized site-specific HCV treatment workflows, we aim to cure HCV in vulnerable, high-risk populations, including people experiencing homelessness (PEH) and people who inject drugs (PWID), in Austin, TX, USA. METHODS: Our implementation science study will utilize a qualitative and design thinking approach to characterize patient and systemic barriers and facilitators to HCV treatment in vulnerable, high-risk populations seeking care across seven diverse primary care clinics serving PEHs and PWIDs. Qualitative interviews guided by the Practical, Robust Implementation and Sustainability Model (PRISM) framework will identify barriers and facilitators by leveraging knowledge and experience from both clinic staff and patients. Data synthesized using thematic analysis and design thinking will feed into workshops with clinic stakeholders for idea generation to design site-specific HCV treatment workflows. Providers will be trained on the use of a simplified HCV treatment algorithm with DAAs and clinic staff on the new site-specific HCV treatment workflows. These workflows will be implemented by the seven diverse primary care clinics serving vulnerable, high-risk populations. Implementation and clinical outcomes will be measured using data collected through interviews with staff as well as through medical chart review. DISCUSSION: Our study provides a model of how to contextualize and implement site-specific HCV treatment workflows targeting vulnerable, high-risk populations in other geographic locations. This model can be adopted for future implementation research programs aiming to develop and implement site-specific treatment workflows for vulnerable, high-risk populations and in primary care clinical settings for other disease states beyond just HCV. TRIAL REGISTRATION: Registered on ClinicalTrials.gov on July, 14, 2022. Identifier: NCT05460130 .

Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing.

Library adaptors are short oligonucleotides that are attached to RNA and DNA samples in preparation for next-generation sequencing (NGS). Adaptors can also include additional functional elements, such as sample indexes and unique molecular identifiers, to improve library analysis. Here, we describe Control Library Adaptors, termed CAPTORs, that measure the accuracy and reliability of NGS. CAPTORs can be integrated within the library preparation of RNA and DNA samples, and their encoded information is retrieved during sequencing. We show how CAPTORs can measure the accuracy of nanopore sequencing, evaluate the quantitative performance of metagenomic and RNA sequencing, and improve normalisation between samples. CAPTORs can also be customised for clinical diagnoses, correcting systematic sequencing errors and improving the diagnosis of pathogenic BRCA1/2 variants in breast cancer. CAPTORs are a simple and effective method to increase the accuracy and reliability of NGS, enabling comparisons between samples, reagents and laboratories, and supporting the use of nanopore sequencing for clinical diagnosis.

Bioengineered textiles with peptide binders that capture SARS-CoV-2 viral particles.

The use of personal protective equipment (PPE), face masks and ventilation are key strategies to control the transmission of respiratory viruses. However, most PPE provides physical protection that only partially prevents the transmission of viral particles. Here, we develop textiles with integrated peptide binders that capture viral particles. We fuse peptides capable of binding the receptor domain of the spike protein on the SARS-CoV-2 capsid to the cellulose-binding domain from the Trichoderma reesei cellobiohydrolase II protein. The hybrid peptides can be attached to the cellulose fibres in cotton and capture SARS-CoV-2 viral particles with high affinity. The resulting bioengineered cotton captures 114,000 infective virus particles per cm2 and reduces onwards SARS-CoV-2 infection of cells by 500-fold. The hybrid peptides could be easily modified to capture and control the spread of other infectious pathogens or for attachment to different materials. We anticipate the use of bioengineered protective textiles in PPE, facemasks, ventilation, and furnishings will provide additional protection to the airborne or fomite transmission of viruses.

Collective skyrmion motion under the influence of an additional interfacial spin-transfer torque.

Here we study the effect of an additional interfacial spin-transfer torque, as well as the well-established spin-orbit torque and bulk spin-transfer torque, on skyrmion collections-group of skyrmions dense enough that they are not isolated from one another-in ultrathin heavy metal/ferromagnetic multilayers, by comparing modelling with experimental results. Using a skyrmion collection with a range of skyrmion diameters and landscape disorder, we study the dependence of the skyrmion Hall angle on diameter and velocity, as well as the velocity as a function of diameter. We show that inclusion of the interfacial spin-transfer torque results in reduced skyrmion Hall angles, with values close to experimental results. We also show that for skyrmion collections the velocity is approximately independent of diameter, in marked contrast to the motion of isolated skyrmions, as the group of skyrmions move together at an average group velocity. Moreover, the calculated skyrmion velocities are comparable to those obtained in experiments when the interfacial spin-transfer torque is included. Our results thus show the significance of the interfacial spin-transfer torque in ultrathin magnetic multilayers, which helps to explain the low skyrmion Hall angles and velocities observed in experiment. We conclude that the interfacial spin-transfer torque should be considered in numerical modelling for reproduction of experimental results.

Network characteristics of a referral system for patients with hypertension in Western Kenya: results from the Strengthening Referral Networks for Management of Hypertension Across the Health System (STRENGTHS) study.

BACKGROUND: Health system approaches to improve hypertension control require an effective referral network. A national referral strategy exists in Kenya; however, a number of barriers to referral completion persist. This paper is a baseline assessment of a hypertension referral network for a cluster-randomized trial to improve hypertension control and reduce cardiovascular disease risk. METHODS: We used sociometric network analysis to understand the relationships between providers within a network of nine geographic clusters in western Kenya, including primary, secondary, and tertiary care facilities. We conducted a survey which asked providers to nominate individuals and facilities to which they refer patients with controlled and uncontrolled hypertension. Degree centrality measures were used to identify providers in prominent positions, while mixed-effect regression models were used to determine provider characteristics related to the likelihood of receiving referrals. We calculated core-periphery correlation scores (CP) for each cluster (ideal CP score = 1.0). RESULTS: We surveyed 152 providers (physicians, nurses, medical officers, and clinical officers), range 10-36 per cluster. Median number of hypertensive patients seen per month was 40 (range 1-600). While 97% of providers reported referring patients up to a more specialized health facility, only 55% reported referring down to lower level facilities. Individuals were more likely to receive a referral if they had higher level of training, worked at a higher level facility, were male, or had more job experience. CP scores for provider networks range from 0.335 to 0.693, while the CP scores for the facility networks range from 0.707 to 0.949. CONCLUSIONS: This analysis highlights several points of weakness in this referral network including cluster variability, poor provider linkages, and the lack of down referrals. Facility networks were stronger than provider networks. These shortcomings represent opportunities to focus interventions to improve referral networks for hypertension. TRIAL REGISTRATION: Trial Registered on ClinicalTrials.gov NCT03543787 , June 1, 2018.

The potential of long noncoding RNA therapies.

The human genome expresses vast numbers of long noncoding RNAs (lncRNA) that fulfil diverse roles in gene regulation, cell biology, development, and human disease. These roles are often mediated by sequence motifs and secondary structures bound by proteins and can regulate epigenetic, transcriptional, and translational pathways. These functional domains can be further optimised and engineered into RNA devices that are widely used in synthetic biology. We propose that natural lncRNA structures can be explored and exploited for the rational design and assembly of synthetic RNA therapies. This potential has been enabled by advances in the stability, immunogenicity, manufacture, and delivery of other RNA-based therapies, from which we can anticipate the pharmacological properties of lncRNA therapies that have not yet otherwise entered clinical trials.

Using synthetic chromosome controls to evaluate the sequencing of difficult regions within the human genome.

BACKGROUND: Next-generation sequencing (NGS) can identify mutations in the human genome that cause disease and has been widely adopted in clinical diagnosis. However, the human genome contains many polymorphic, low-complexity, and repetitive regions that are difficult to sequence and analyze. Despite their difficulty, these regions include many clinically important sequences that can inform the treatment of human diseases and improve the diagnostic yield of NGS. RESULTS: To evaluate the accuracy by which these difficult regions are analyzed with NGS, we built an in silico decoy chromosome, along with corresponding synthetic DNA reference controls, that encode difficult and clinically important human genome regions, including repeats, microsatellites, HLA genes, and immune receptors. These controls provide a known ground-truth reference against which to measure the performance of diverse sequencing technologies, reagents, and bioinformatic tools. Using this approach, we provide a comprehensive evaluation of short- and long-read sequencing instruments, library preparation methods, and software tools and identify the errors and systematic bias that confound our resolution of these remaining difficult regions. CONCLUSIONS: This study provides an analytical validation of diagnosis using NGS in difficult regions of the human genome and highlights the challenges that remain to resolve these difficult regions.

Term Newborn Care Recommendations Provided in a Kenyan Postnatal Ward: A Rapid, Focused Ethnographic Assessment.

BACKGROUND: Neonatal mortality (death within 0-28 d of life) in Kenya is high despite strong evidence that newborn care recommendations save lives. In public healthcare facilities, nurses counsel caregivers on term newborn care, but knowledge about the content and quality of nurses' recommendations is limited. PURPOSE: To describe the term newborn care recommendations provided at a tertiary-level, public referral hospital in Western Kenya, how they were provided, and related content taught at a university nursing school. METHODS: A rapid, focused ethnographic assessment, guided by the culture care theory, using stratified purposive sampling yielded 240 hours of participant observation, 24 interviews, 34 relevant documents, and 268 pages of field notes. Data were organized using NVivo software and key findings identified using applied thematic analysis. RESULTS: Themes reflect recommendations for exclusive breastfeeding, warmth, cord care, follow-up examinations, and immunizations, which were provided orally in Kiswahili and some on a written English discharge summary. Select danger sign recommendations were also provided orally, if needed. Some recommendations conflicted with other providers' guidance. More recommendations for maternal care were provided than for newborn care. IMPLICATIONS FOR PRACTICE: There is need for improved consistency in content and provision of recommendations before discharge. Findings should be used to inform teaching, clinical, and administrative processes to address practice competency and improve nursing care quality. IMPLICATIONS FOR RESEARCH: Larger studies are needed to determine whether evidence-based recommendations are provided consistently across facilities and other populations, such as community-born and premature newborns, who also experience high rates of neonatal mortality in Kenya.

Emerging technologies and their impact on regulatory science.

There is an evolution and increasing need for the utilization of emerging cellular, molecular and in silico technologies and novel approaches for safety assessment of food, drugs, and personal care products. Convergence of these emerging technologies is also enabling rapid advances and approaches that may impact regulatory decisions and approvals. Although the development of emerging technologies may allow rapid advances in regulatory decision making, there is concern that these new technologies have not been thoroughly evaluated to determine if they are ready for regulatory application, singularly or in combinations. The magnitude of these combined technical advances may outpace the ability to assess fit for purpose and to allow routine application of these new methods for regulatory purposes. There is a need to develop strategies to evaluate the new technologies to determine which ones are ready for regulatory use. The opportunity to apply these potentially faster, more accurate, and cost-effective approaches remains an important goal to facilitate their incorporation into regulatory use. However, without a clear strategy to evaluate emerging technologies rapidly and appropriately, the value of these efforts may go unrecognized or may take longer. It is important for the regulatory science field to keep up with the research in these technically advanced areas and to understand the science behind these new approaches. The regulatory field must understand the critical quality attributes of these novel approaches and learn from each other's experience so that workforces can be trained to prepare for emerging global regulatory challenges. Moreover, it is essential that the regulatory community must work with the technology developers to harness collective capabilities towards developing a strategy for evaluation of these new and novel assessment tools.

The splicing effect of variants at branchpoint elements in cancer genes.

PURPOSE: Branchpoint elements are required for intron removal, and variants at these elements can result in aberrant splicing. We aimed to assess the value of branchpoint annotations generated from recent large-scale studies to select branchpoint-abrogating variants, using hereditary cancer genes as model. METHODS: We identified branchpoint elements in 119 genes associated with hereditary cancer from 3 genome-wide experimentally-inferred and 2 predicted branchpoint data sets. We then identified variants that occur within branchpoint elements from public databases. We compared conservation, unique variant observations, and population frequencies at different nucleotides within branchpoint motifs. Finally, selected minigene assays were performed to assess the splicing effect of variants at branchpoint elements within mismatch repair genes. RESULTS: There was poor overlap between predicted and experimentally-inferred branchpoints. Our analysis of cancer genes suggested that variants at -2 nucleotide, -1 nucleotide, and branchpoint positions in experimentally-inferred canonical motifs are more likely to be clinically relevant. Minigene assay data showed the -2 nucleotide to be more important to branchpoint motif integrity but also showed fluidity in branchpoint usage. CONCLUSION: Data from cancer gene analysis suggest that there are few high-risk alleles that severely impact function via branchpoint abrogation. Results of this study inform a general scheme to prioritize branchpoint motif variants for further study.