Generating and testing the efficacy of reagents for CRISPR/Cas9 homology directed repair-based manipulations in Tribolium.

CRISPR/Cas9 manipulations are possible in many insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of CRISPR-based (clustered regularly interspaced short palindromic repeats) genome-editing tools and methodologies are dependent upon direct experimentation. One useful technique is Cas9-dependent homologous recombination, which is a critical tool for studying gene function but also for developing pest related applications like gene drive. Here, we report our attempts to induce Cas9 homology directed repair (HDR) and subsequent gene drive in Tribolium castaneum (Herbst; Insecta: Coleoptera: Tenebrionidae). Utilizing constructs containing 1 or 2 target gRNAs in combination with Cas9 under 2 different promoters and corresponding homology arms, we found a high incidence of CRISPR/Cas9 induced mutations but no evidence of homologous recombination. Even though the generated constructs provide new resources for CRISPR/Cas9 modification of the Tribolium genome, our results suggest that additional modifications and increased sample sizes will be necessary to increase the potential and detection for HDR of the Tribolium genome.

Creating a Culturally Safe Online Data Collection Instrument to Measure Vaccine Confidence Among Indigenous Youth: Indigenous Consensus Method.

BACKGROUND:  Participating in surveys can shape the perception of participants related to the study topic. Administering a vaccine hesitancy questionnaire can have negative impacts on participants' vaccine confidence. This is particularly true for online and cross-cultural data collection because culturally safe health education to correct misinformation is typically not provided after the administration of an electronic survey. OBJECTIVE:  To create a culturally safe, online, COVID-19 vaccine confidence survey for Indigenous youth designed to collect authentic, culturally relevant data of their vaccine experiences, with a low risk of contributing to further vaccine confusion among participants. METHODS:  Using the Aboriginal Telehealth Knowledge Circle consensus method, a team of academics, health care providers, policy makers, and community partners reviewed COVID-19 vaccine hesitancy surveys used in public health research, analyzed potential risks, and created a framework for electronic Indigenous vaccine confidence surveys as well as survey items. RESULTS:  The framework for safer online survey items is based on 2 principles, a first do-no-harm approach and applying a strengths-based lens. Relevant survey domains identified in the process include sociodemographic information, participants' connection to their community, preferred sources for health information, vaccination uptake among family members and peers, as well as personal attitudes toward vaccines. A total of 44 survey items were developed, including 5 open-ended items to improve the authenticity of the data and the analysis of the experiences of Indigenous youth. CONCLUSIONS:  Using an Indigenous consensus method, we have developed an online COVID-19 vaccine confidence survey with culturally relevant domains and reduced the risk of amplifying misinformation and negative impacts on vaccine confidence among Indigenous participants. Our approach can be adapted to other online survey development in collaboration with Indigenous communities.

The investigation of the efficiency of basic life support education among high school students: Protocol, design and implementation of an interventional, prospective longitudinal, individually randomised, parallel 1:1 grouped trial.

Background: Basic life support (BLS) skills are crucial not only for healthcare workers but for all lay people as well. Timely recognition of out-of-hospital cardiac arrest (OHCA) and the initiation of BLS by bystanders before the arrival of healthcare personnel may improve survival. There are several methods of spreading BLS skills and improve BLS skill retention among lay people. One of these methods can be the education of adolescent school children. The introduction of mandatory BLS education in schools was very effective in some European countries to increase the rate of bystander BLS. Methods/design: The current study aims to investigate the efficacy of a BLS training and BLS curriculum among high school children in Hungary. Moreover, the investigators would like to optimise factors influencing skill retention in this first responder group and aim to compare two types of teaching methods: feedback given by the instructor or software-based feedback on the efficacy of chest compressions during the course. This study will be an interventional, assessor blinded, individually randomised parallel group trial recruiting 360 students. BLS skill retention will be assessed at the end of the course, two months after the training and six months after training. Discussion: The current study will increase our knowledge on the methods educating BLS among high school children. The results will help us to create an effective BLS curriculum at schools.Trial registration: ClinicalTrials.gov: NCT06016153. Prospectively registered on 08/2023.

Impacts of climate change on water quality, benthic mussels, and suspended mussel culture in a shallow, eutrophic estuary.

Climate change is a global problem that causes severe local changes to marine biota, ecosystem functioning, and ecosystem services. The Limfjorden is a shallow, eutrophic estuary influenced by episodic summer hypoxia with an important mussel fishery and suspended mussel culture industry. Three future climate change scenarios ranging from low greenhouse gas emissions (SSP1-2.6), to intermediate (SSP2-4.5) and very high emissions (SSP5-8.5) were combined with nutrient load reductions according to the National Water Plans to investigate potential impacts on natural benthic mussel populations and suspended mussel culture for the two periods 2051-2060 and 2090-2099, relative to a reference period from 2009 to 2018. The FlexSem model combined 3D hydrodynamics with a pelagic biogeochemical model, a sediment-benthos model, and a dynamic energy budget - farm scale model for mussel culture. Model results showed that the Limfjorden was sensitive to climate change impacts with the strongest responses of physics and water quality in the worst case SSP5-8.5 scenario with no nutrient reductions. In the two low emissions scenarios, expected improvements of bottom oxygen and Chlorophyll a concentrations due to reduced nutrient loads were counteracted by climate change impacts on water physics (warming, freshening, stronger stratification). Hence, higher nutrient reductions in the Water Plans would be needed to reach a good ecological status under the influence of climate change. Suspended mussel culture was intensified in all scenarios showing a high potential harvest, whereas the benthic mussels suffered from reduced food supply and hypoxia. Provided the environmental changes and trends in social demands, in the future, it is likely that suspended mussel cultivation will become the primary source of mussels for the industry. Model scenarios can be used to inform managers, mussel farmers, fishermen, and the local population on potential future changes in bivalve harvesting and ecosystem health, and to find solutions to mitigate climate change impacts.

The fluid dynamics of barnacle feeding.

Sessile barnacles feed by sweeping their basket-like cirral fan through the water, intercepting suspended prey. A primary component of the diet of adult barnacles is copepods that are sensitive to fluid disturbances and capable of escaping. How do barnacles manage to capture copepods despite the fluid disturbances they generate? We examined this question by describing the feeding current architecture of 1 cm sized Balanus crenatus using particle image velocimetry, and by studying the trajectories of captured copepods and the escapes of evading copepods. We found that barnacles produce a feeding current that arrives both from behind and the sides of the barnacle. The flow from the sides represents quiescent corridors of low fluid deformation and uninterrupted by the beating cirral fan. Potential prey arriving from behind are likely to encounter the cirral fan and, hence, capture here is highly unlikely. Accordingly, most captured copepods arrived through the quiet corridors, while most copepods arriving from behind managed to escape. Thus, it is the unique feeding flow architecture that allows feeding on evasive prey. We used the Landau-Squire jet as a simple model of the feeding current. For the Reynolds number of our experiments, the model reproduces the main features of the feeding current, including the lateral feeding corridors. Furthermore, the model suggests that smaller barnacle specimens, operating at lower Reynolds numbers, will produce a fore-aft symmetric feeding current without the lateral corridors. This suggests an ontogenetic diet shift from non-evasive prey to inclusion of evasive prey as the barnacle grows.

Directed evolution of enzymatic silicon-carbon bond cleavage in siloxanes.

Volatile methylsiloxanes (VMS) are man-made, nonbiodegradable chemicals produced at a megaton-per-year scale, which leads to concern over their potential for environmental persistence, long-range transport, and bioaccumulation. We used directed evolution to engineer a variant of bacterial cytochrome P450BM3 to break silicon-carbon bonds in linear and cyclic VMS. To accomplish silicon-carbon bond cleavage, the enzyme catalyzes two tandem oxidations of a siloxane methyl group, which is followed by putative [1,2]-Brook rearrangement and hydrolysis. Discovery of this so-called siloxane oxidase opens possibilities for the eventual biodegradation of VMS.

Individual differences in empathy in Danish university students: A cross-sectional study.

BACKGROUND: Empathy is widely recognized as a multi-dimensional construct, involving emotional and cognitive components. These may cause distinct experiences and behaviors that can be both beneficial and deleterious to individuals' well-being and mental health. AIM: We wished to examine the association between emotional and cognitive empathy of Danish university students as measured by the multidimensional Interpersonal Reactivity Index (IRI) and study major, sex, age, and parental status. Additionally, we aimed to gauge the validity of the Jefferson Scale of Empathy - Student version (JSE-S) as a measure of primarily cognitive empathy in the context of medical majors by comparing JSE-S scores with IRI cognitive scores. METHODS: In our national, cross-sectional study, conducted in October 2020, we used survey data from students in their first, third, and final study year. All students from University of Southern Denmark were invited to fill out IRI, and all medical students at Denmark's four medical educations were additionally invited to fill out the JSE-S. Associations were estimated by linear regression models. RESULTS: Of 14,072 invited, 2,595 students completed the questionnaire. Health majors scored statistically significantly higher on cognitive empathy than students from other study majors. The JSE-S correlated significantly with the cognitive empathy subscales of the IRI. Furthermore, the effects found in relation to sex, age-, and parental status were significant. CONCLUSION: Our study results show that large differences in empathy exist between university students and study majors. Overall, our results highlight (1) the relevance of investigating empathy as a multidimensional versus a global construct in young adult populations (including university students) and (2) the importance of focusing on differences in empathy across different student characteristics.

Spontaneous metastasis xenograft models link CD44 isoform 4 to angiogenesis, hypoxia, EMT and mitochondria-related pathways in colorectal cancer.

Hematogenous metastasis limits the survival of colorectal cancer (CRC) patients. Here, we illuminated the roles of CD44 isoforms in this process. Isoforms 3 and 4 were predominantly expressed in CRC patients. CD44 isoform 4 indicated poor outcome and correlated with epithelial-mesenchymal transition (EMT) and decreased oxidative phosphorylation (OxPhos) in patients; opposite associations were found for isoform 3. Pan-CD44 knockdown (kd) independently impaired primary tumor formation and abrogated distant metastasis in CRC xenografts. The xenograft tumors mainly expressed the clinically relevant CD44 isoforms 3 and 4. Both isoforms were enhanced in the paranecrotic, hypoxic tumor regions but were generally absent in lung metastases. Upon CD44 kd, tumor angiogenesis was increased in the paranecrotic areas, accompanied by reduced hypoxia-inducible factor-1α and CEACAM5 but increased E-cadherin expression. Mitochondrial genes and proteins were induced upon pan-CD44 kd, as were OxPhos genes. Hypoxia increased VEGF release from tumor spheres, particularly upon CD44 kd. Genes affected upon CD44 kd in xenografts specifically overlapped concordantly with genes correlating with CD44 isoform 4 (but not isoform 3) in patients, validating the clinical relevance of the used model and highlighting the metastasis-promoting role of CD44 isoform 4.

Culturally Safe Practices in the Co-creation of Medical Education Curriculum with Indigenous Animators: Outcomes From an Indigenous Learning Circle.

OBJECTIVES: To explore the experiences of Indigenous patient actors who co-created and enacted Indigenous patient scenarios in collaboration with medical school faculty. We critically examine the structures and systems in a medical school that mediate cultural safety for Indigenous patient actors. The Truth and Reconciliation Commission of Canada has called on medical schools and healthcare institutions to help address the intergenerational harms inflicted on Indigenous people by the Indian residential school (IRS) system. Institutions are striving to incorporate cultural competency, conflict resolution, human rights, and anti-racism education into their curricula. However, the structural inequities within undergraduate, postgraduate, and continuing medical education practices must be identified and challenged to ensure that medical education is authentic and culturally safe for those involved in the development and delivery of the Indigenous health curriculum. To explore potential structural inequities in the co-creation process of simulated cultural communication scenarios (SCCS), the Indigenous animators at Debajehmujig Storytellers and collaborating faculty and professional staff at the Northern Ontario School of Medicine University (NOSM U) examined cultural safety in their curriculum design and delivery process. METHODS: We utilized the qualitative Indigenous research methodology of the Learning Circle to deconstruct the co-creation process and to explore the experience of cultural safety from the Indigenous animators' perspective throughout the curriculum design and delivery process. RESULTS: A framework for culturally safe co-creation practices with Indigenous people, rooted within Indigenous teachings of the Medicine Wheel, emerged from the qualitative data. CONCLUSIONS: This framework has the potential to guide the practice of culturally safe co-creation of Indigenous patient simulations in medical education and healthcare workplace learning. While the Medicine Wheel teachings are held by specific Indigenous nations, we anticipate that the results and recommendations of this study will apply to Indigenous co-creators and academic medical educators internationally.

Can online and app-based interventions be used by people with diabetes to reduce diabetes distress? A protocol for a scoping review.

INTRODUCTION: Diabetes distress has been defined as "the negative emotional or affective experience resulting from the challenge of living with the demands of diabetes". Diabetes distress affects 20%-25% of individuals living with diabetes and can have negative effects on both diabetes regulation and quality of life. For people living with diabetes distress, innovative tools/interventions such as online or app-based interventions may potentially alleviate diabetes distress in a cost-effective way. The specific research questions of this scoping review are: (1) what are the effects of online or app-based interventions on diabetes distress for adults with type 1 or type 2 diabetes, and (2) what are the characteristics of these interventions (eg, type of intervention, duration, frequency, mode of delivery, underlying theories and working mechanisms)? METHODS AND ANALYSIS: A scoping review will be conducted, using the methodological framework of Arksey and O'Malley along with Levac et al. Eligible studies are: studies of adults ≥18 years old with type 1 or 2 diabetes using an online or app-based intervention and assessing diabetes distress as the primary or secondary outcome. Five databases (Medline, EMBASE, CINAHL, PsycINFO and Scopus) will be searched and is limited to articles written in English, Danish, Norwegian, Swedish or Dutch. Two reviewers will independently screen potentially eligible studies in Covidence, select studies, and together chart data, collate, summarise, and report the results. We will adhere to the Preferred reporting Items for Systematic Reviews and Meta-Analysis for Scoping Reviews (PRISMA-ScR). ETHICS AND DISSEMINATION: The scoping review has been exempt from full ethical review by the Regional Committees on Health Research Ethics for Southern Denmark (case number: S-20232000-88). The results of the review will be published in a peer-reviewed journal and presented at relevant conferences and workshops with relevant stakeholders.