A force-sensitive mutation reveals a non-canonical role for dynein in anaphase progression.

The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.

Recapturing Communicative Erasure: Black Women Farmers' Lived Experience, Political Voice and Cultural Knowledge as Critical Health Communication Praxis.

In this paper, we conduct a case study analysis of the National Women in Agriculture Association (NWIAA), an international, Black women-led farm assistance organization founded in 2008 and based in Oklahoma City, Oklahoma. Drawing on the Culture-Centered Approach (CCA) and grounded in interviews and observational fieldwork, we center the perspectives of NWIAA chapter leaders (n = 16) to examine how they describe motivations for farming, challenge power inequities, engage with intersectional barriers, and develop locally situated solutions across agricultural and community health contexts. The analysis argues that Black women farmer's historical lived experiences, political voice, and shared deep-rooted agricultural knowledge provide an innovative and emancipatory praxis for rethinking health communication intervention approaches that address food system-generated disparities. This study contributes important takeaways for health communication practitioners, policymakers, and advocates addressing food inequities. It extends the CCA as a first step in developing community-driven, context-specific food insecurity health communication interventions within marginalized communities across the United States and beyond.

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron.

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

Molecular mechanism of dynein-dynactin complex assembly by LIS1.

Cytoplasmic dynein is a microtubule motor vital for cellular organization and division. It functions as a ~4-megadalton complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognize diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as lissencephaly-1 (LIS1) protein (LIS1) remain unclear. In this study, we determined the cryo-electron microscopy structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. This structure reveals the molecular basis of interactions occurring during dynein activation. We show how JIP3 activates dynein despite its atypical architecture. Unexpectedly, LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Our data suggest that LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.

Dynein and dynactin move long-range but are delivered separately to the axon tip.

Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein, which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level, it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here, we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction, we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with the initiation of dynein transport. Intriguingly, in the anterograde direction, dynein/LIS1 moves faster than dynactin/NDEL1, consistent with transport on different cargos. Therefore, neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required.

The use of NexoBrid™ enzymatic debridement and coagulation abnormalities.

OBJECTIVES: Current evidence on how the use of bromelain-based enzymatic debridement techniques (NexoBrid™) affect patient coagulation is limited. A single patient case report [1] suggests that a patient with 15% total body surface area (TBSA) burn developed decreased coagulation activity following debridement with NexoBrid™ enzymatic debridement (ED). Regional Burns Centres in the United Kingdom may be reluctant to use ED, particularly in larger burns, citing concerns regarding coagulation. At our centre we have routinely used ED on deep partial thickness burns since 2017 including on patients with burns over 15% TBSA. This study aims to investigate whether there is a significant disruption in coagulation in patients undergoing ED with burns > 15% TBSA or admitted to intensive care compared to the standard of care (SOC) which is surgical debridement in theatre. METHODS: This single-centre retrospective study includes all patients with a burn treated with ED at Pinderfields General Hospital Regional Burns Centre intensive care unit (ITU) from 2017 to 2020. Patients were matched to those treated with SOC at the same centre by age, % TBSA burn and presence of inhalational injury. These parameters correlate with the Baux score [9]. Percentage of burn debrided was matched as closely as possible, with coagulation profiles and platelet count taken the day before, the day of and three days following surgery. RESULTS: Thirty-one patients were treated with ED in the intensive care unit between 2017 and 2020. Four patients were excluded due to insufficient records and one patient was anti-coagulated. Twenty-six patients were included and matched as described above. Average age of patients receiving ED was 44 years, the same in the matched group. Average TBSA burn is 35.5% (35.8% in matched group). No statistically significant difference in coagulation was seen between patients undergoing ED compared to SOC when considering prothrombin time (PT), activated partial thromboplastin time (aPTT) and platelet count. Both groups slightly breached the upper limit of normal on day 2 post ED and SOC. There was a slight breach of the lower limit of the average platelet count on day 2 post-ED which was neither statistically nor clinically significant. CONCLUSIONS: Large burns are associated with coagulation abnormalities, therefore isolating a single variable in this cohort is challenging. However, this study found no significant change following ED use when compared to SOC and therefore no convincing evidence that ED is associated with coagulation abnormalities. This study represents one of the largest focusing on coagulation abnormalities following the use of ED, as the current literature is limited. Our study suggests that concerns regarding coagulation abnormalities should not prevent patients with large, deep partial thickness burns or full thickness burns being treated with ED.

Facility-based approach for the management of acute ST segment elevation myocardial infarction with cardiogenic shock in a rural medical centre: the Durango model.

INTRODUCTION: Cardiogenic shock (CS) complicates 5%-15% of cases of acute myocardial infarction (AMI) with inpatient mortality greater than 40%. The implementation of standardised protocols may improve clinical outcomes in patients with AMI-CS. METHODS AND ANALYSIS: The Durango model is a prospective single-centre registry designed to enable early identification of patients with STEMI-CS to facilitate primary reperfusion therapy with a shock team management algorithm in a rural level II heart attack centre. This prospective registry includes all patients >18 years of age presenting with STEMI with or without CS beginning on 1 February 2023. The primary outcome measures are adherence to model-based documentation of SCAI shock Classification prehospital and in the ED with appropriate STEMI shock alert for AMI and stages C, D, E shock; use of mechanical circulatory support Pre-PCI and door to support time <90 min. ETHICS AND DISSEMINATION: This study was approved by the Institutional Review Board with a waiver of informed consent. The findings will be submitted for publication in a peer-review open access journal on completion of the study. CONCLUSIONS: The Durango model will demonstrate that the implementation of a STEMI shock team can be feasible in a rural medical centre through comprehensive education of a diverse group providers with different levels of experience, continuous model/device proficiency training and performance feedback.

Sowing Seeds of Sovereignty: A Qualitative Exploration of Chapter Leader Experiences Who Belong to an African, Caribbean, and Black Women's Agriculture Advocacy Collective.

African, Caribbean, and Black (ACB) populations are disproportionately impacted by food insecurity since the COVID-19 pandemic, with reports including limited access to healthy, affordable, and available food resulting in the exacerbation of existing health disparities and poorer living conditions and environments. Many interventions addressing food insecurity among ACB populations incorporate downstream models that emphasize consumer-level solutions (e.g., food donation or assistance programs). While useful for developing short-term solutions, these approaches do not meaningfully engage broader structures and root-causes-and, in many ways, reify existing inequities among these populations. Missing from mainstream dialogue is how food insecurity inequities at the individual level are often generated at higher structural levels and the unique assets of a targeted community or population to grow/distribute their own food and restore frayed transnational cultural foodways. This paper documents a case study of an international, Black women-led agriculture education collective headquartered in the South-Central region of the United States. Drawing on in-depth interviews with affiliate chapter leaders in the United States, US Virgin Islands, and Africa (n = 22), the research investigates how chapter leaders describe unique opportunities and barriers to farming and developing food insecurity solutions. Emergent themes from the data analysis were: (1) diasporically linked barriers, (a) infrastructural/political barriers and (b) patriarchal barriers; (2) mobilizing strategies; and (3) cultural and context-specific solutions. Findings from this study contribute important insights for food security development research centering community-based and grassroots organizing models addressing inequities in food production and health.

CryoET shows cofilactin filaments inside the microtubule lumen.

Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.

A force-sensitive mutation reveals a spindle assembly checkpoint-independent role for dynein in anaphase progression.

The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate in vivo studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors.

Novel Iron Chelator SK4 Drives Cytotoxicity through Inhibiting Mitochondrial Metabolism in Ovarian and Triple Negative Breast Cancer Cell Lines.

Anti-cancer therapy by iron chelation has been shown to inhibit many cellular processes including DNA replication, mitochondrial metabolism and oncogenic signalling pathways (e.g., EGFR). Iron chelator SK4 represents a double pronged approach towards treating cancer. SK4 enters through LAT1, a commonly overexpressed amino acid transporter in tumours, thus targeting iron addiction and LAT1 overexpression. The aim of this study was to characterise the mode of action of SK4 through proteomics, metabolomics, lipidomics and seahorse real-time analysis in ovarian cell line SKOV3 and triple negative breast cancer cell line MDA MB 231. Pathway enrichment of proteomics data showed an overrepresentation of metabolism related pathways. Metabolic change after SK4 exposure have been confirmed in investigations of changes in basal and maximal mitochondrial respiration using seahorse real-time analysis of mitochondrial metabolism. Metabolomics also showed an increase in AMP and glucose-1-phosphate. Interestingly, our lipidomics data show a decrease in phospholipid synthesis in the SKOV3 cells which is in contrast with previous data which showed an upregulation of ceramide driven apoptosis. In summary, our data highlight impairment of energy metabolism as a mechanism of action underlying SK4 apoptosis, but also suggest a potential role of ceramide induction in the phenotypic outcome of the cell model.

Digital Storytelling as a Tool to Increase Colorectal Cancer Screening Intention in a Latinx Church Community.

Although colorectal cancer screening (CRCS) rates have improved for all racial groups due to wider availability of screening, Latinx continue to have lower screening rates and are more likely to be diagnosed with later stages of colorectal cancer compared to non-Latinx whites. More culturally tailored educational interventions are needed to reach this population. This study introduced a digital storytelling (DST) intervention in a church community setting and explored its potential to influence CRCS intention and perception among Latinx and the acceptability of the DST intervention. Participants (n=20) between the ages of 50 and 75 who were not up-to-date with CRCS were recruited to view digital stories developed by fellow church members with previous CRCS experience. They completed surveys assessing their intention to complete CRCS before and after the viewing and were asked to participate in focus groups to understand, qualitatively, how the digital stories influenced their perceptions and intentions related to CRCS. Analysis of participant narratives revealed three overarching themes related to their perceptions and intentions of CRCS after the DST intervention: (1) the duality of the faith-health connection and fatalism, (2) willingness to consider other screening methods, and (3) the push-pull of individual barriers and interpersonal facilitators. Participants felt the DST intervention humanized the CRCS process and that it would be acceptable and well received in other church settings. The introduction of a community-based DST intervention within a church setting is a novel strategy with the potential to influence members of the Latinx church population to complete CRCS.

Dietary interventions improve diabetic kidney disease, but not peripheral neuropathy, in a db/db mouse model of type 2 diabetes.

Patients with type 2 diabetes often develop the microvascular complications of diabetic kidney disease (DKD) and diabetic peripheral neuropathy (DPN), which decrease quality of life and increase mortality. Unfortunately, treatment options for DKD and DPN are limited. Lifestyle interventions, such as changes to diet, have been proposed as non-pharmacological treatment options for preventing or improving DKD and DPN. However, there are no reported studies simultaneously evaluating the therapeutic efficacy of varying dietary interventions in a type 2 diabetes mouse model of both DKD and DPN. Therefore, we compared the efficacy of a 12-week regimen of three dietary interventions, low carbohydrate, caloric restriction, and alternate day fasting, for preventing complications in a db/db type 2 diabetes mouse model by performing metabolic, DKD, and DPN phenotyping. All three dietary interventions promoted weight loss, ameliorated glycemic status, and improved DKD, but did not impact percent fat mass and DPN. Multiple regression analysis identified a negative correlation between fat mass and motor nerve conduction velocity. Collectively, our data indicate that these three dietary interventions improved weight and glycemic status and alleviated DKD but not DPN. Moreover, diets that decrease fat mass may be a promising non-pharmacological approach to improve DPN in type 2 diabetes given the negative correlation between fat mass and motor nerve conduction velocity.

CryoET shows cofilactin filaments inside the microtubule lumen.

Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes their accumulation has not been conclusively established. Here, we used cryogenic electron tomography (cryoET) of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) with the small-molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, cofilin was activated in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNAi knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.

Mapping the Influence: A Nexus Analysis Approach to Addressing Food Access.

One innovative approach toward addressing community-level food access is nexus analysis. This framework suggests that social actions do not happen outside of context, but rather are embedded within unique political, cultural and economic histories. In this paper, I conduct a case study analysis of the South Memphis Farmers Market (SMFM), a community-based, resident-led farmers market located in South Memphis, TN. Drawing on an understanding that neighborhood-level institutions such as farmers markets serve as an intermediary space to examine the nexus of macro-level (e.g., how resources are drawn within a community) and micro-level processes (e.g., how residents determine what food to buy and who to buy it from), this paper traces the success of the SMFM as an illustration of positive local response to the question of "good food" access. Through its history, location and connection to its predominantly Black patrons, the SMFM was able to effectively address structural and cultural barriers as a means of improving food access. Findings from the study may offer insights to the theorization of culture and space in community-based health campaigns.

An Examination of Culturally Relevant Health Messages in African-American Churches.

This quantitative study examined the presence of culturally relevant health messages for African-Americans based on a preexisting dataset from 21 African-American churches in South Carolina (USA). Content analysis served as the primary methodological approach to code printed media messages based on their cultural relevance among African-Americans (Cohen's kappa = .74). Within the dataset (n = 2166), 477 (22%) items were identified as culturally relevant. A low prevalence of culturally relevant messages was found across the three message topics, two media types, and one media source. Due to the limited presence of culturally relevant messages, researchers should collaborate with African-American churches to design health promotion messages.

Novel iron chelator SK4 demonstrates cytotoxicity in a range of tumour derived cell lines.

Iron is an essential micronutrient due to its involvement in many cellular processes including DNA replication and OXPHOS. Tumors overexpress iron metabolism linked proteins which allow for iron accumulation driving high levels of proliferation. Our group has designed novel iron chelator SK4 which targets cancer's "iron addiction." SK4 comprises of two key moieties: an iron chelation moiety responsible for cytotoxicity and an amino acid moiety which allows entry through amino acid transporter LAT1. We selected LAT1 as a route of entry as it is commonly overexpressed in malignant tumors. SK4 has previously demonstrated promising results in an in vitro model for melanoma. We hypothesized SK4 would be effective against a range of tumor types. We have screened a panel of tumor-derived cell lines from different origins including breast, prostate, ovarian and cervical cancer for SK4 sensitivity and we have found a range of differential sensitivities varying from 111.3 to >500 µM. We validated the iron chelation moiety as responsible for inducing cytotoxicity through control compounds; each lacking a key moiety. Following the screen, we conducted a series of assays to elucidate the mechanism of action behind SK4 cytotoxicity. SK4 was shown to induce apoptosis in triple negative breast cancer cell line MDA MB 231 but not ovarian cancer cell line SKOV3 suggesting SK4 may induce different modes of cell death in each cell line. As MDA MB 231 cells harbor a mutation in p53, we conclude SK4 is capable of inducing apoptosis in a p53-independent manner. SK4 upregulated NDRG1 expression in MDA MB 231 and SKOV3 cells. Interestingly, knockdown of NDRG1 antagonized SK4 in MDA MB 231 cells but not SKOV3 cells suggesting SK4's mechanism of action may be mediated through NDRG1 in MDA MB 231 cells. In conclusion, we have shown tagging iron chelators with an amino acid moiety to allow entry through the LAT1 transporter represents a double pronged approach to cancer therapy, targeting "iron addiction" and amino acid metabolism dysregulation.

Conformational transitions of the Spindly adaptor underlie its interaction with Dynein and Dynactin.

Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end-directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein-Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein-Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein-Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.

SARS-CoV-2 Delta and Omicron variants evade population antibody response by mutations in a single spike epitope.

Population antibody response is thought to be important in selection of virus variants. We report that SARS-CoV-2 infection elicits a population immune response that is mediated by a lineage of VH1-69 germline antibodies. A representative antibody R1-32 from this lineage was isolated. By cryo-EM, we show that it targets a semi-cryptic epitope in the spike receptor-binding domain. Binding to this non-ACE2 competing epitope results in spike destruction, thereby inhibiting virus entry. On the basis of epitope location, neutralization mechanism and analysis of antibody binding to spike variants, we propose that recurrent substitutions at 452 and 490 are associated with immune evasion of the identified population antibody response. These substitutions, including L452R (present in the Delta variant), disrupt interactions mediated by the VH1-69-specific hydrophobic HCDR2 to impair antibody-antigen association, enabling variants to escape. The first Omicron variants were sensitive to antibody R1-32 but subvariants that harbour L452R quickly emerged and spread. Our results provide insights into how SARS-CoV-2 variants emerge and evade host immune responses.