Xylosyltransferase Bump-and-hole Engineering to Chemically Manipulate Proteoglycans in Mammalian Cells.

Mammalian cells orchestrate signalling through interaction events on their surfaces. Proteoglycans are an intricate part of these interactions, carrying large glycosaminoglycan polysaccharides that recruit signalling molecules. Despite their importance in development, cancer and neurobiology, a relatively small number of proteoglycans have been identified. In addition to the complexity of glycan extension, biosynthetic redundancy in the first protein glycosylation step by two xylosyltransferase isoenzymes XT1 and XT2 complicates annotation of proteoglycans. Here, we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. By employing a tactic termed bump- and-hole engineering, we engineer the two isoenzymes XT1 and XT2 to specifically transfer a chemically modified xylose analogue to target proteins. The chemical modification contains a bioorthogonal tag, allowing the ability to visualise and profile target proteins modified by both transferases in mammalian cells. The versatility of our approach allows pinpointing glycosylation sites by tandem mass spectrometry, and exploiting the chemical handle to manufacture proteoglycans with defined GAG chains for cellular applications. Engineered XT enzymes permit a view into proteoglycan biology that is orthogonal to conventional techniques in biochemistry.

Chemokine Oligomers and the Impact of Fondaparinux Binding.

Heparin, a widely used clinical anticoagulant, is generally well-tolerated; however, approximately 1% of patients develop heparin-induced thrombocytopenia (HIT), a serious side effect. While efforts to understand the role of chemokines in HIT development are ongoing, certain aspects remain less studied, such as the stabilization of chemokine oligomers by heparin. Here, we conducted a combined ion mobility-native mass spectrometry study to investigate the stability of chemokine oligomers and their complexes with fondaparinux, a synthetic heparin analog. Collision-induced dissociation and unfolding experiments provided clarity on the specificity and relevance of chemokine oligomers and their fondaparinux complexes with varying stoichiometries, as well as the stabilizing effects of fondaparinux binding.

FAIR enough: Building an academic data ecosystem to make real-world data available for translational research.

The Stanford Population Health Sciences Data Ecosystem was created to facilitate the use of large datasets containing health records from hundreds of millions of individuals. This necessitated technical solutions optimized for an academic medical center to manage and share high-risk data at scale. Through collaboration with internal and external partners, we have built a Data Ecosystem to host, curate, and share data with hundreds of users in a secure and compliant manner. This platform has enabled us to host unique data assets and serve the needs of researchers across Stanford University, and the technology and approach were designed to be replicable and portable to other institutions. We have found, however, that though these technological advances are necessary, they are not sufficient. Challenges around making data Findable, Accessible, Interoperable, and Reusable remain. Our experience has demonstrated that there is a high demand for access to real-world data, and that if the appropriate tools and structures are in place, translational research can be advanced considerably. Together, technological solutions, management structures, and education to support researcher, data science, and community collaborations offer more impactful processes over the long-term for supporting translational research with real-world data.

Disproportionately High Rates of Burnout Among Disabled Caregivers During the COVID-19 Pandemic.

Background: Burnout is exhaustion caused by exposure to chronic stress. Prior to the COVID-19 pandemic, people with disabilities experienced high levels of burnout due to discrimination, barriers to accessing resources, and lack of accommodations. Caregivers have also experienced high levels of burnout during the COVID-19 pandemic. Background: While researchers have examined burnout among caregivers of disabled children, less research has focused on the experiences of disabled caregivers. We examined the association between caregiver disability and burnout during the pandemic. Methods: We distributed an online survey to caregivers of children enrolled in socially vulnerable elementary and middle schools in San Diego County, California between September and December, 2022. Our survey included demographic questions, questions about pandemic experiences, and a continuous burnout measure. We analyzed survey data to test our hypothesis that caregivers with a disability experienced higher levels of burnout than their non-disabled counterparts during the height of the COVID-19 pandemic. We used multivariable linear regression analysis adjusting for household income and caregiver education level. Results: Disabled caregivers self-reported higher levels of burnout than non-disabled caregivers (B = 0.72; p < 0.001) during the COVID-19 pandemic in bivariate and multivariable analyses. Caregivers with a higher household income (B = 0.04; p = 0.017) and more education (B = 0.13; p = 0.005) also reported higher levels of burnout. Conclusion: The COVID-19 pandemic exacerbated existing difficulties faced by disabled caregivers who often struggle to balance the demands of caregiving with their available resources. Targeted programs and policies are needed to support disabled caregivers during health emergencies that exacerbate existing inequities in access to resources.

Assessing the impact of institutional mistrust on parental endorsement for COVID-19 vaccination among school communities in San Diego County, California.

BACKGROUND: Institutional mistrust has weakened COVID-19 mitigation efforts. Assessing to what extent institutional mistrust impacts parental decision making is important in formulating structural efforts for improving future pandemic response. We hypothesized that institutional mistrust is associated with lower parental endorsement for COVID-19 vaccination. METHODS: We distributed an online survey among parents from schools in areas with high levels of social vulnerability relative to the rest of San Diego County. We defined vaccination endorsement as having a child aged 5 years or older who received at least one COVID-19 vaccine dose or being very likely to vaccinate their child aged 6 months-4 years when eligible. Institutional mistrust reflected the level of confidence in institutions using an aggregate score from 11 to 44. We built a multivariable logistic regression model with potential confounding variables. FINDINGS: Out of 290 parents in our sample, most were female (87.6%), reported their child as Hispanic/Latino (73.4%), and expressed vaccination endorsement (52.1%). For every one-point increase in mistrust score, there was an 8% reduction in the likelihood of participants endorsing vaccination for their child. Other statistically significant correlates that were positively associated with vaccination endorsement included parent vaccination status, child age, parent age, and Hispanic/Latino ethnicity. CONCLUSION: Our study further demonstrates how institutional mistrust hinders public response during health emergencies. Our findings also highlight the importance of building confidence in institutions and its downstream effects on pandemic preparedness and public health. One way that institutions can improve their relationship with constituents is through building genuine partnerships with trusted community figures.

Synovial Lipomatosis With Extra-articular Extension in the Arthritic Wrist: An Unexpected Diagnosis.

ABSTRACT: Synovial lipomatosis is a rare condition characterized by adipocyte proliferation within joint synovial tissue. It most commonly affects the knee and is typically intra-articular. Only 5 published case reports describe extra-articular synovial lipomatosis of the wrist. We present a case of a sexagenarian patient seen for his wrist arthropathy. His x-ray revealed pan-wrist arthritis and inflammatory soft tissue swelling. The patient was slated for a wrist fusion and Darrach procedure. Following the dorsal skin incision in the operating room, an unusual adipose mass was identified infiltrating all extensor compartments: midcarpal, radiocarpal, and distal radioulnar joints. The mass was excised and sent to pathology prior to proceeding with the slated surgery. Synovial lipomatosis was diagnosed postoperatively based on histopathology. Six weeks postoperatively, the wrist fusion had healed clinically and radiographically, and his pain had improved. There was no evidence of recurrence. Synovial lipomatosis is a rare entity that may imitate multiple other pathologies. It is possible that synovial lipomatosis may represent a secondary occurrence following degenerative articular disease or trauma in older patients. This is the first case report to date describing synovial lipomatosis of the wrist with extra-articular extension in the setting of pan-carpal wrist arthritis.

"They Don't See Us": Asian Students' Perceptions of Sexual Violence and Sexual Harassment on Three California Public University Campuses.

Sexual violence and sexual harassment (SVSH) are prevalent among college and university students; however, the experiences of ethnic minority students, especially Asians, are understudied. This study aimed to reduce this gap by exploring Asian students' perceptions of SVSH on three public university campuses in Southern California. We examined their perceptions about the campus environment related to SVSH, attitudes, and behaviors toward help seeking, and utilization of on-campus resources. A total of 23 in-depth interviews were conducted with Asian students enrolled at the three University of California campuses. Thematic coding was conducted to generate main themes and subthemes. Five main themes emerged: (a) SVSH is considered a "taboo" topic in Asian culture and family systems, and Asian student survivors are often reluctant to disclose incidents or seek support services. (b) Students did not feel their campus environments were tailored to understand or meet the sociocultural realities and needs of Asian student survivors. (c) Campus SVSH services and reporting processes were seen as non-transparent. (d) Peers were the major source of support and SVSH information, as opposed to official campus-based resources and training. (e) Survivors often conduct an internal cost-benefit analysis evaluating their decision about whether to report. This study highlights the lack of conversation surrounding SVSH in Asian families, and how the cultural stigma of sex and sexual violence prevented Asian students from receiving knowledge and resources about these topics in their families. Instead of relying on formal campus resources (e.g., Title IX and confidential advocacy services, mental health services), many students turn to their peers for support. Thus, facilitating peer support groups, training university students to support each other through SVSH incidents, and tailoring campus services to the diverse cultural backgrounds of students are key considerations to foster a safe campus environment and prevent SVSH.

Structure and function of Semaphorin-5A glycosaminoglycan interactions.

Integration of extracellular signals by neurons is pivotal for brain development, plasticity, and repair. Axon guidance relies on receptor-ligand interactions crosstalking with extracellular matrix components. Semaphorin-5A (Sema5A) is a bifunctional guidance cue exerting attractive and inhibitory effects on neuronal growth through the interaction with heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs), respectively. Sema5A harbors seven thrombospondin type-1 repeats (TSR1-7) important for GAG binding, however the underlying molecular basis and functions in vivo remain enigmatic. Here we dissect the structural basis for Sema5A:GAG specificity and demonstrate the functional significance of this interaction in vivo. Using x-ray crystallography, we reveal a dimeric fold variation for TSR4 that accommodates GAG interactions. TSR4 co-crystal structures identify binding residues validated by site-directed mutagenesis. In vitro and cell-based assays uncover specific GAG epitopes necessary for TSR association. We demonstrate that HS-GAG binding is preferred over CS-GAG and mediates Sema5A oligomerization. In vivo, Sema5A:GAG interactions are necessary for Sema5A function and regulate Plexin-A2 dependent dentate progenitor cell migration. Our study rationalizes Sema5A associated developmental and neurological disorders and provides mechanistic insights into how multifaceted guidance functions of a single transmembrane cue are regulated by proteoglycans.

Mechanistically Guided Materials Chemistry: Synthesis of Ternary Nitrides, CaZrN2 and CaHfN2.

Recent computational studies have predicted many new ternary nitrides, revealing synthetic opportunities in this underexplored phase space. However, synthesizing new ternary nitrides is difficult, in part because intermediate and product phases often have high cohesive energies that inhibit diffusion. Here, we report the synthesis of two new phases, calcium zirconium nitride (CaZrN2) and calcium hafnium nitride (CaHfN2), by solid state metathesis reactions between Ca3N2 and MCl4 (M = Zr, Hf). Although the reaction nominally proceeds to the target phases in a 1:1 ratio of the precursors via Ca3N2 + MCl4 → CaMN2 + 2 CaCl2, reactions prepared this way result in Ca-poor materials (CaxM2-xN2, x < 1). A small excess of Ca3N2 (ca. 20 mol %) is needed to yield stoichiometric CaMN2, as confirmed by high-resolution synchrotron powder X-ray diffraction. In situ synchrotron X-ray diffraction studies reveal that nominally stoichiometric reactions produce Zr3+ intermediates early in the reaction pathway, and the excess Ca3N2 is needed to reoxidize Zr3+ intermediates back to the Zr4+ oxidation state of CaZrN2. Analysis of computationally derived chemical potential diagrams rationalizes this synthetic approach and its contrast from the synthesis of MgZrN2. These findings additionally highlight the utility of in situ diffraction studies and computational thermochemistry to provide mechanistic guidance for synthesis.

Genomic surveillance reveals dynamic shifts in the connectivity of COVID-19 epidemics.

The maturation of genomic surveillance in the past decade has enabled tracking of the emergence and spread of epidemics at an unprecedented level. During the COVID-19 pandemic, for example, genomic data revealed that local epidemics varied considerably in the frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage importation and persistence, likely due to a combination of COVID-19 restrictions and changing connectivity. Here, we show that local COVID-19 epidemics are driven by regional transmission, including across international boundaries, but can become increasingly connected to distant locations following the relaxation of public health interventions. By integrating genomic, mobility, and epidemiological data, we find abundant transmission occurring between both adjacent and distant locations, supported by dynamic mobility patterns. We find that changing connectivity significantly influences local COVID-19 incidence. Our findings demonstrate a complex meaning of "local" when investigating connected epidemics and emphasize the importance of collaborative interventions for pandemic prevention and mitigation.

Glycoengineered keratinocyte library reveals essential functions of specific glycans for all stages of HSV-1 infection.

Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generate a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures have minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affects glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.

Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans.

Chemokine-driven leukocyte recruitment is a key component of the immune response and of various diseases. Therapeutically targeting the chemokine system in inflammatory disease has been unsuccessful, which has been attributed to redundancy. We investigated why chemokines instead have specific, specialized functions, as demonstrated by multiple studies. We analyzed the expression of genes encoding chemokines and their receptors across species, tissues, and diseases. This analysis revealed complex expression patterns such that genes encoding multiple chemokines that mediated recruitment of the same leukocyte type were expressed in the same context, such as the genes encoding the CXCR3 ligands CXCL9, CXCL10, and CXCL11. Through biophysical approaches, we showed that these chemokines differentially interacted with extracellular matrix glycosaminoglycans (ECM GAGs), which was enhanced by sulfation of specific GAGs. Last, in vivo approaches demonstrated that GAG binding was critical for the CXCL9-dependent recruitment of specific T cell subsets but not of others, irrespective of CXCR3 expression. Our data demonstrate that interactions with ECM GAGs regulated whether chemokines were presented on cell surfaces or remained more soluble, thereby affecting chemokine availability and ensuring specificity of chemokine action. Our findings provide a mechanistic understanding of chemokine-mediated immune cell recruitment and identify strategies to target specific chemokines during inflammatory disease.

A multi-taxon analysis of European Red Lists reveals major threats to biodiversity.

Biodiversity loss is a major global challenge and minimizing extinction rates is the goal of several multilateral environmental agreements. Policy decisions require comprehensive, spatially explicit information on species' distributions and threats. We present an analysis of the conservation status of 14,669 European terrestrial, freshwater and marine species (ca. 10% of the continental fauna and flora), including all vertebrates and selected groups of invertebrates and plants. Our results reveal that 19% of European species are threatened with extinction, with higher extinction risks for plants (27%) and invertebrates (24%) compared to vertebrates (18%). These numbers exceed recent IPBES (Intergovernmental Platform on Biodiversity and Ecosystem Services) assumptions of extinction risk. Changes in agricultural practices and associated habitat loss, overharvesting, pollution and development are major threats to biodiversity. Maintaining and restoring sustainable land and water use practices is crucial to minimize future biodiversity declines.

Examining the Nurse Manager Practice Environment Effects on Burnout: Results of a Cross-sectional Multisite Study.

OBJECTIVE: This study examined the relationships between the dimensions of the nurse manager (NM) practice environment (PE) and burnout. BACKGROUND: NMs are critical to the success of their unit(s). Understanding the degree to which their PE characteristics impact their level of burnout is important to NM retention. METHOD: A cross-sectional research design using a 71-item questionnaire was used to measure NM characteristics, hospital characteristics, NM PE, and burnout. There were 110 completed respondents across 22 hospitals in the United States. RESULTS: Moderate inverse relationships exist between the NM PE and 2 of the 3 (work and personal) dimensions of burnout. NM age and tenure also showed an inverse relationship with burnout. CONCLUSIONS: This study added evidence showing a statistically significant relationship between NM PE and the work and personal dimensions of burnout. These results also suggest the relationships NMs have with the patients on their unit(s) do not add to their level of burnout.

Direct observation of glycans bonded to proteins and lipids at the single-molecule level.

Proteins and lipids decorated with glycans are found throughout biological entities, playing roles in biological functions and dysfunctions. Current analytical strategies for these glycan-decorated biomolecules, termed glycoconjugates, rely on ensemble-averaged methods that do not provide a full view of positions and structures of glycans attached at individual sites in a given molecule, especially for glycoproteins. We show single-molecule analysis of glycoconjugates by direct imaging of individual glycoconjugate molecules using low-temperature scanning tunneling microscopy. Intact glycoconjugate ions from electrospray are soft-landed on a surface for their direct single-molecule imaging. The submolecular imaging resolution corroborated by quantum mechanical modeling unveils whole structures and attachment sites of glycans in glycopeptides, glycolipids, N-glycoproteins, and O-glycoproteins densely decorated with glycans.

Community-based participatory research to improve alumni transition from an intensive research training program for historically underrepresented undergraduates.

Various initiatives for undergraduates from historically underrepresented backgrounds attempt to address disparities in the completion of science, technology, engineering, mathematics (STEM) degrees and the pursuit of careers in scientific research. Intensive research training programs for historically underrepresented undergraduates may include multiple components, such as authentic research experiences, advising and mentoring, supplemental curriculum, and financial assistance. Following comprehensive support during program participation, the post-program transition may present a vulnerable period in students' career trajectories. This study used a community-based participatory research (CBPR) approach to investigate the experiences of students completing an intensive research training program to understand and develop recommendations for the post-program transition process. As a team of program alumni, academic researchers, and program staff, we developed, conducted, and analyzed semi-structured, open-ended interviews of recent program alumni and students approaching program completion (n=11; 55% female, 55% non-White). Applying thematic analysis at semantic and latent levels through a critical paradigm revealed the transition as a bittersweet experience, with feelings of pride and accomplishment mixed with sadness and anxiety. Findings also suggested the transition is described as a narrative influenced by preceding program experiences and adaptations. Financial concerns were prominent, and specific barriers and facilitators of successful transition included: aligned mentoring, negotiation of continued research employment, consideration of culture, planning for next steps, and engagement with the scholar community. Collaboratively, we developed recommendations for program improvements potentially relevant to similarly intensive STEM diversity programs. We also highlight the value of a CBPR approach that includes students equitably as co-researchers in program research and evaluation.

The relationship between underage initiation of selling sex and depression among female sex workers in Eswatini.

Background: Minors who sell sex are likely to have complex mental health needs that may persist into adulthood. This topic is understudied in sub-Saharan Africa. This study hypothesized that adult female sex workers in Eswatini who started selling sex as minors have a higher prevalence of depression than those who started as adults. We also examined correlates of depression and underage initiation of selling sex, including stigma and condom-related behaviors. Methods: From October-December 2014, women aged 18 or older who sold sex in the past 12 months in Eswatini were recruited through venue-based sampling. Participants completed a survey including the 9-item Patient Health Questionnaire (PHQ-9) and a question about the age at which they first sold sex for money. T-tests, χ2 tests and multivariable logistic regression were used to assess associations. Results: Overall, 43.1% of participants (332/770) had probable depression, and 16.6% (128/770) started selling sex as minors under the age of 18. Over half (55.5%, 71/128) of those who started selling sex as minors had depression. This was significantly higher than the 40.7% (261/642) prevalence of depression among participants who started selling sex as adults (p = 0.002). After adjusting for confounders, female sex workers who started selling sex as minors had higher odds of depression than those who started as adults (adjusted odds ratio [aOR] 1.70, 95% confidence interval 1.11-2.60). Conclusion: Results highlight the need for trauma-informed and adolescent-friendly mental health services in settings free of stigma toward female sex workers in Eswatini.

Determinants of parent-reported child mental health status in San Diego public schools during the height of the COVID-19 omicron outbreak: A serial cross-sectional study.

Despite extensive debate on the effects of COVID-19 mitigation measures in school settings, little evidence exists on trends in school children's mental health over the course of the COVID-19 pandemic. The objective of this paper was to identify factors affecting parent reports of school children's mental health during COVID-19 Omicron variant outbreak in a cohort of high-risk, socially vulnerable children attending public elementary schools. We analyzed four waves of cross-sectional, online-administered surveys completed by parents of children attending public elementary schools in San Diego between November 2021 and March of 2022. Children (n = 684) ranged in age from 2-17 years. We used multilevel linear mixed effects models to assess determinants of parent-reported child mental health status. The outcome was child mental health, as reported by the parent. Parents consistently rated their children's mental health as very good, though parents who experienced recent COVID-related challenges and who had older children reported lower levels of mental health in their children. Children's mental health was generally considered to be very good, as judged by their parents during a period of constant in-school masking and the Omicron variant outbreak. Structural support mechanisms aimed at mitigating COVID-related challenges for adults may offer benefit to children's mental health.