Double-negative-2 B cells are the major synovial plasma cell precursor in rheumatoid arthritis.

B cells are key pathogenic drivers of chronic inflammation in rheumatoid arthritis (RA). There is limited understanding of the relationship between synovial B cell subsets and pathogenic antibody secreting cells (ASCs). This knowledge is crucial for the development of more targeted B-cell depleting therapies. While CD11c+ double-negative 2 (DN2) B cells have been suggested as an ASC precursor in lupus, to date there is no proven link between the two subsets in RA. We have used both single-cell gene expression and BCR sequencing to study synovial B cells from patients with established RA, in addition to flow cytometry of circulating B cells. To better understand the differentiation patterns within the diseased tissue, a combination of RNA-based trajectory inference and clonal lineage analysis of BCR relationships were used. Both forms of analysis indicated that DN2 B cells serve as a major precursors to synovial ASCs. This study advances our understanding of B cells in RA and reveals the origin of pathogenic ASCs in the RA synovium. Given the significant role of DN2 B cells as a progenitor to pathogenic B cells in RA, it is important to conduct additional research to investigate the origins of DN2 B cells in RA and explore their potential as therapeutic targets in place of the less specific pan-B cells depletion therapies currently in use.

AIRRSHIP: simulating human B cell receptor repertoire sequences.

SUMMARY: Adaptive Immune Receptor Repertoire Sequencing is a rapidly developing field that has advanced understanding of the role of the adaptive immune system in health and disease. Numerous tools have been developed to analyse the complex data produced by this technique but work to compare their accuracy and reliability has been limited. Thorough, systematic assessment of their performance is dependent on the ability to produce high quality simulated datasets with known ground truth. We have developed AIRRSHIP, a flexible and fast Python package that produces synthetic human B cell receptor sequences. AIRRSHIP uses a comprehensive set of reference data to replicate key mechanisms in the immunoglobulin recombination process, with a particular focus on junctional complexity. Repertoires generated by AIRRSHIP are highly similar to published data and all steps in the sequence generation process are recorded. These data can be used to not only determine the accuracy of repertoire analysis tools but can also, by tuning of the large number of user-controllable parameters, give insight into factors that contribute to inaccuracies in results. AVAILABILITY AND IMPLEMENTATION: AIRRSHIP is implemented in Python. It is available via https://github.com/Cowanlab/airrship and on PyPI at https://pypi.org/project/airrship/. Documentation can be found at https://airrship.readthedocs.io/.

Dietary and agricultural adaptations to drought among smallholder farmers in South Africa: A qualitative study.

Building resilience to environmental change is an integral part of long-term climate adaptation planning and local policy. There is an increased understanding of the impact of climate change on global crop production however, little focus has been given to local adaptation pathways and rural smallholder community responses, especially regarding food security. It is becoming increasingly evident that local level decision-making plays a vital role in reducing vulnerability to environmental change. This research aimed to qualitatively investigate coping and adaptive strategies adopted by smallholder farming households to respond to the impacts of drought in rural KwaZulu-Natal, South Africa. Focus group discussions (n = 7) consisting of 5-9 participants and individual interviews (n = 9) using pre-tested topic guides, involving a total of 57 adults were conducted in rural areas of drought-affected districts: Msinga, Richmond and Umbumbulu of KwaZulu-Natal, in July 2018. The data were analysed using thematic analysis in NVivo 12. Thematic analysis identified three principal themes: 1. Perceived effects of droughts on the local food system and diets; 2. Current coping strategies; and 3. Enablers for successful adaptation. All sites reported a change in food consumption habits, with the majority perceiving drought to be the main driver behind a shift from vegetable-based to starch-based diets and decreased animal source food consumption. Only short-term coping strategies were implemented across the study sites. However, knowledge of long-term adaptation strategies existed but was unattainable to most respondents. Recommendations of perceived context-specific long-term adaptation strategies that could be used at a local scale were communicated by the respondents. However, they would need external help to actualize them. A need exists to support smallholder communities' short-term response methods to drought to achieve more holistic resilience and successful adaptation. Short-term adaptation strategies, if implemented alone, often have significant trade-offs with longer-term adaptation and building resilience. This study highlights the need for targeted, contextualised policy solutions to improve smallholder productivity during drought through a strategic combination of both short- and longer-term adaptation measures, i.e. short-term adaptation should be guided by a long-term adaptation strategy. Proper planning, including the use of climate scenarios combined with information on nutritional status, is needed to develop context-specific and transformative adaptation strategies. These strategies should aim to strengthen resilience at a local level and should be included as policy recommendations.

Innovation for improved hand hygiene: Field testing the Autarky handwashing station in collaboration with informal settlement residents in Durban, South Africa.

Safe and accessible water services for hand hygiene are critical to human health and well-being. However, access to handwashing facilities is limited in cities in the Global South, where rapid urbanisation, service backlogs, lack of infrastructure and capacity, and water scarcity impact on the ability of local governments to provide them. Community participation and the co-production of knowledge in the development of innovative technologies, which are aligned with Water, Sanitation and Hygiene (WASH) principles, can lead to more sustainable and socially-acceptable hand hygiene systems. This paper presents the outcomes of the testing of the Autarky handwashing station, a technology that provides onsite treatment and recycling of handwashing water, in an informal settlement in Durban, South Africa. The transdisciplinary research approach adopted enabled the participation of multiple stakeholders with different knowledge systems in the framing, testing and evaluation of the system. The process of co-producing knowledge, as well as the outcomes of the testing, namely high levels of functionality and social acceptability of the technology, supported the WASH principles. The evaluation revealed that the Autarky handwashing station is a niche intervention that improved access to safe and appealing handwashing facilities in an informal settlement. Its novel design, socially desirable features, reliability and ability to save water increased its acceptance in the community. The testing of the system in a real-world context revealed the value of including communities in knowledge production processes for technology innovation. Further work is required to ensure that real-time monitoring of system function is feasible before such systems can be implemented at larger scale.

Socio-technical analysis of a sanitation innovation in a peri-urban household in Durban, South Africa.

The provision of water and sanitation for all that is safe, dignified, reliable, affordable and sustainable is a major global challenge. While centralized sewer-based sanitation systems remain the dominant approach to providing sanitation, the benefits of non-sewered onsite sanitation systems are increasingly being recognised. This paper presents the outcomes of the testing of the Blue Diversion Autarky Toilet (BDAT), a sanitation system providing hygiene and dignity without relying on water and wastewater infrastructure, in a peri-urban household in Durban, South Africa. The BDAT was used by a single household as their only form of sanitation during three months of technical and social testing. An analysis based on technical data in combination with interpretive, qualitative research methods revealed that the BDAT functioned well and achieved high levels of social acceptance in the test household. The flushing, cleanliness and odour-free nature of the sanitation technology, its functionality, the household's previous sanitation experience, and their experience with and understanding of water scarcity, were the main factors underpinning their positive response to this innovation in sanitation. The testing process resulted in broader developmental benefits for the household, including improved basic services due to the upgrading of the electrical and existing sanitation system, social learning, and improved relationships between household members and the local state. A transdisciplinary research process, which emerged through the assessment, enabled the integration of different forms of knowledge from multiple actors to address the complexity of problems related to the development of socially just sanitation. The benefit of engaging with societal actors in sanitation innovation and assessing its outcomes using both the technical and social sciences is evident in this paper.

A Conserved TCRß Signature Dominates a Highly Polyclonal T-Cell Expansion During the Acute Phase of a Murine Malaria Infection.

CD4+ αß T-cells are key mediators of the immune response to a first Plasmodium infection, undergoing extensive activation and splenic expansion during the acute phase of an infection. However, the clonality and clonal composition of this expansion has not previously been described. Using a comparative infection model, we sequenced the splenic CD4+ T-cell receptor repertoires generated over the time-course of a Plasmodium chabaudi infection. We show through repeat replicate experiments, single-cell RNA-seq, and analyses of independent RNA-seq data, that following a first infection - within a highly polyclonal expansion - T-effector repertoires are consistently dominated by TRBV3 gene usage. Clustering by sequence similarity, we find the same dominant clonal signature is expanded across replicates in the acute phase of an infection, revealing a conserved pathogen-specific T-cell response that is consistently a hallmark of a first infection, but not expanded upon re-challenge. Determining the host or parasite factors driving this conserved response may uncover novel immune targets for malaria therapeutic purposes.

Amplifying the Voice of the Patient in Clinical Research: Development of Toolkits for Use in Designing and Conducting Patient-Centered Clinical Studies.

Incorporating patient perspectives into clinical studies is recognized as important to the development of high-quality, safe, and effective fit-for-patient medicines. However, no widely accepted methodology to help design more patient-centered studies has been established systematically. TransCelerate Biopharma Inc., a non-profit organization promoting collaboration across biopharmaceutical companies, organized a Patient Experience (PE) Initiative to create tools to intentionally include the patient perspective into the design and implementation of clinical studies. The resulting tools include the Patient Protocol Engagement Toolkit (P-PET), to engage patients early in protocol development, and the Study Participant Feedback Questionnaire (SPFQ), to assess patient experiences during clinical studies. To develop these toolkits, TransCelerate conducted a literature review and identified aspects of clinical studies that patients find either valuable or burdensome, or that affect participation, adherence, and engagement in a clinical study. The concepts identified were refined through elicitation of feedback from patient advisors, clinical study site advisors, and subject matter experts from member companies (MCs) of TransCelerate. This feedback was considered in identifying gaps, defining scientific methodology to understand how to evaluate patients' needs, and developing and refining the P-PET and the SPFQ. As part of the development process, descriptions/drafts of the tools were shared with patients, clinical site advisory groups, MCs, and the US Food and Drug Administration, and then revised. MCs simulated use of the tools, and feedback was incorporated into the final versions of the P-PET and SPFQ prior to public release. The P-PET and SPFQ are available free on the TransCelerate website.

An Introduction to the Structure and Function of the Catalytic Core Enzyme of Escherichia coli RNA Polymerase.

RNA polymerase (RNAP) is the essential enzyme responsible for transcribing genetic information stored in DNA to RNA. Understanding the structure and function of RNAP is important for those who study basic principles in gene expression, such as the mechanism of transcription and its regulation, as well as translational sciences such as antibiotic development. With over a half-century of investigations, there is a wealth of information available on the structure and function of Escherichia coli RNAP. This review introduces the structural features of E. coli RNAP, organized by subunit, giving information on the function, location, and conservation of these features to early stage investigators who have just started their research of E. coli RNAP.

The calmodulin-binding site of sphingosine kinase and its role in agonist-dependent translocation of sphingosine kinase 1 to the plasma membrane.

Sphingosine kinases catalyze the formation of sphingosine 1-phosphate, a bioactive lipid involved in many aspects of cellular regulation, including the fundamental biological processes of cell growth and survival. A diverse range of cell agonists induce activation of human sphingosine kinase 1 (hSK1) and, commonly, its translocation to the plasma membrane. Although the activation of hSK1 in response to at least some agonists occurs directly via its phosphorylation at Ser225 by ERK1/2, many aspects governing the regulation of this phosphorylation and subsequent translocation remain unknown. Here, in an attempt to understand some of these processes, we have examined the known interaction of hSK1 with calmodulin (CaM). By using a combination of limited proteolysis, peptide interaction analysis, and site-directed mutagenesis, we have identified that the CaM-binding site of hSK1 resides in the region spanned by residues 191-206. Specifically, Phe197 and Leu198 are critically involved in the interaction because a version of hSK1 incorporating mutations of both Phe197 --> Ala and Leu198 --> Gln failed to bind CaM. We have also shown for the first time that human sphingosine kinase 2 (hSK2) binds CaM, and does so via a CaM binding region that is conserved with hSK1 because comparable mutations in hSK2 also ablate CaM binding to this protein. By using the CaM-binding-deficient version of hSK1, we have begun to elucidate the role of CaM in hSK1 regulation by demonstrating that disruption of the CaM-binding site ablates agonist-induced translocation of hSK1 from the cytoplasm to the plasma membrane, while having no effect on hSK1 phosphorylation and catalytic activation.

Elm1p is one of three upstream kinases for the Saccharomyces cerevisiae SNF1 complex.

BACKGROUND: The yeast SNF1 protein kinase and the mammalian AMP-activated protein kinase are highly conserved heterotrimeric complexes that are "metabolic master switches" involved in the switch from fermentative/anaerobic to oxidative metabolism. They are activated by cellular stresses that deplete cellular ATP, and SNF1 is essential in the response to glucose starvation. In both cases, activation requires phosphorylation at a conserved threonine residue within the activation loop of the kinase domain, but identifying the upstream kinase(s) responsible for this has been a challenging, unsolved problem. RESULTS: Using a library of strains that express 119 yeast protein kinases as GST fusions, we identified Elm1p as the sole kinase that could activate the kinase domain of AMP-activated protein kinase in vitro. Elm1p also activated the purified SNF1 complex, and this correlated with phosphorylation of Thr210 in the activation loop. Removal of the C-terminal domain increased the Elm1p kinase activity, indicating that it is auto-inhibitory. Expression of activated, truncated Elm1p from its own promoter gave a constitutive pseudohyphal growth phenotype that was rescued by deletion of SNF1, showing that Snf1p was acting downstream of Elm1p. Deletion of ELM1 does not give an snf- phenotype. However, Elm1p is closely related to Pak1p and Tos3p, and a pak1Delta tos3Delta elm1Delta triple mutant had an snf1- phenotype, i.e., it would not grow on raffinose and did not display hyperphosphorylation of the SNF1 target, Mig1p, in response to glucose starvation. CONCLUSIONS: Elm1p, Pak1p, and Tos3p are upstream kinases for the SNF1 complex that have partially redundant functions.