Predictive Performance of Cardiovascular Risk Scores in Cancer Survivors From the UK Biobank.

Background: Cardiovascular preventive strategies are guided by risk scores with unknown validity in cancer cohorts. Objectives: This study aimed to evaluate the predictive performance of 7 established cardiovascular risk scores in cancer survivors from the UK Biobank. Methods: The predictive performance of QRISK3, Systematic Coronary Risk Evaluation 2 (SCORE2)/Systematic Coronary Risk Evaluation for Older Persons (SCORE-OP), Framingham Risk Score, Pooled Cohort equations to Prevent Heart Failure (PCP-HF), CHARGE-AF, QStroke, and CHA2DS2-VASc was calculated in participants with and without a history of cancer. Participants were propensity matched on age, sex, deprivation, health behaviors, family history, and metabolic conditions. Analyses were stratified into any cancer, breast, lung, prostate, brain/central nervous system, hematologic malignancies, Hodgkin lymphoma, and non-Hodgkin lymphoma. Incident cardiovascular events were tracked through health record linkage over 10 years of follow-up. The area under the receiver operating curve, balanced accuracy, and sensitivity were reported. Results: The analysis included 31,534 cancer survivors and 126,136 covariate-matched controls. Risk score distributions were near identical in cases and controls. Participants with any cancer had a significantly higher incidence of all cardiovascular outcomes than matched controls. Performance metrics were significantly worse for all risk scores in cancer cases than in matched controls. The most notable differences were among participants with a history of hematologic malignancies who had significantly higher outcome rates and poorer risk score performance than their matched controls. The performance of risk scores for predicting stroke in participants with brain/central nervous system cancer was very poor, with predictive accuracy more than 30% lower than noncancer controls. Conclusions: Existing cardiovascular risk scores have significantly worse predictive accuracy in cancer survivors compared with noncancer comparators, leading to an underestimation of risk in this cohort.

Sward type alters enteric methane emissions, nitrogen output and the relative abundance of the rumen microbial ecosystem in sheep.

Observed improvements in animal and sward performance, coupled with a desire for more sustainable pasture-based feeding systems has triggered a surge in the implementation of more botanically diverse pastures. However, thus far, there has been limited research investigating the effects of botanically diverse sward types on enteric methane (CH4) or nitrogen (N) excretion, alongside the ruminal microbiota and fermentation profile, in sheep. Hence, this study investigates the effect of sward type on CH4 production and N excretion, in addition to assessing the rumen microbiome, volatile fatty acid (VFA) proportions, and ammonia nitrogen (NH3-N) concentration in sheep. A 5×5 Latin square design experiment was implemented to investigate five dietary treatments; perennial ryegrass (Lolium perenne L.;PRG) only or PRG plus white clover (Trifolium repens L.;PRG+WC), red clover (Trifolium pratense L.;PRG+RC), chicory (Chicorium intybus L.;PRG+Chic) or plantain (Plantago lanceolata L.;PRG+Plan). Diets were mixed at a ratio of 75% PRG and 25% of the respective companion forage and 100% PRG for the PRG treatment, on a dry matter basis. Twenty castrated male sheep were housed in metabolism crates across five feeding periods. Methane measurements were acquired utilizing portable accumulation chambers. Rumen fluid was harvested using a transoesophageal sampling device. Microbial rumen DNA was extracted and subjected to 16S rRNA amplicon sequencing and fermentation analysis. Data were analysed using PROC MIXED in SAS. Results show that animals consuming PRG+WC ranked lower for CH4 production (g/d) than sheep offered PRG, PRG+Chic or PRG+Plan (P<0.01) while the addition of any companion forage ranked CH4 yield (g/kg DMI) lower (P<0.001) than PRG. There was a moderate positive correlation between DMI and CH4 (g/d; r=0.51). Ruminal NH3-N was lowest in animals consuming the PRG diet (P<0.01). There was a greater abundance of Methanobrevibacter and reduced abundance of Methanosphaera (P<0.001) in sheep offered PRG, compared with any binary sward. On average, herb diets (PRG+Chic or PRG+Plan) reduced urinary nitrogen concentration of sheep by 34% in comparison to legume diets (PRG+WC or PRG+RC) and 13% relative to the PRG diet (P<0.001). Sheep offered PRG+Chic had a greater dietary nitrogen use efficiency than PRG+RC (P<0.05). This study demonstrates the potential for sward type to influence rumen function and the microbial community, along with CH4 and N output from sheep.

Repetitive Transcranial Magnetic Stimulation Modulates Brain Connectivity in Children with Self-limited Epilepsy with Centrotemporal Spikes.

Objective: Interictal epileptiform discharges (IEDs) alter brain connectivity in children with epilepsy; this connectivity change may be a mechanism by which epilepsy induces cognitive deficits. Here, we test whether repetitive transcranial magnetic stimulation (rTMS), a non-invasive neuromodulation technique, modulates connectivity and reduces IEDs in children with epilepsy. Methods: Nineteen children with self-limited epilepsy with centrotemporal spikes (SeLECTS) participated in a cross-over study comparing the impact of active vs. sham rTMS on IEDs and brain connectivity. SeLECTS is an epilepsy syndrome affecting the motor cortex, and prior studies show that motor cortices become pathologically hyper-connected to frontal and temporal language cortices. Using a crossover design, we compared the effect of single doses of active versus sham motor cortex rTMS. Connectivity, which was quantified by the weighted phase lag index (wPLI), was measured before and after rTMS using single pulses of TMS combined with EEG (spTMS-EEG). Analyses focused on six regions: bilateral motor cortices and bilateral inferior frontal and superior temporal regions. IEDs were counted in the five minutes before and after rTMS. Results: Active, but not sham, rTMS significantly and globally decreased wPLI connectivity between multiple regions, with the greatest reductions seen in the superior temporal region connections in the stimulated hemisphere. Additionally, there was a trend suggesting that rTMS decreases IED frequency. Interpretation: These findings underscore the potential of low-frequency rTMS to target pathologic hyperconnectivity and reduce IEDs in children with SeLECTS and potentially other pediatric epilepsy syndromes, offering a promising avenue for therapeutic intervention.

Transplanted fibroblasts take the pressure.

Weight-bearing skin cells show promising therapeutic potential.

KECORT Study: An International e-Delphi Study on the Treatment of KEloids Using Intralesional CORTicosteroids in Clinical Practice.

BACKGROUND: Intralesional corticosteroid administration (ICA) is a first-line keloid treatment. However, it faces significant variability in current clinical and scientific practice, which hinders comparability of treatment results. OBJECTIVES: The aim of the study was to reach consensus on different aspects of ICA using hypodermic needles in keloids among an international group of dermatologists and plastic surgeons specialized in keloid treatment to provide consensus-based clinical treatment recommendations for all physicians treating keloids. METHODS: The keloid expert panel of 12 dermatologists and 11 plastic surgeons rated 30 statements. Two online e-Delphi rounds were held, both with a response rate of 100%. Fifteen (65%) keloid experts participated in the final consensus meetings. Consensus was defined as ≥ 75% of the participants choosing agree or strongly agree on a 7-point Likert scale. RESULTS: Consensus was reached on treatment goals, indication for ICA, triamcinolone acetonide (TAC) 40 mg/mL as the preferred corticosteroid administered at a maximum of 80 mg per month and at intervals of 4 weeks, minimizing pain during ICA, the use of 1 mL syringes and 25 or 27 Gauge needles, blanching as endpoint of successful infiltration, caution of not injecting subcutaneously, and the option of making multiple passes in very firm keloids prior to infiltration. Consensus could not be reached on TAC dosing, methods of prior local anesthesia, and location of injection. CONCLUSIONS: This e-Delphi study provides important clinical treatment recommendations on essential aspects of ICA in keloids. By implementing these recommendations, uniformity of ICA in keloid treatment will increase and better treatment results may be achieved.

A diffusion tensor imaging white matter atlas of the domestic canine brain.

There is increasing reliance on magnetic resonance imaging (MRI) techniques in both research and clinical settings. However, few standardized methods exist to permit comparative studies of brain pathology and function. To help facilitate these studies, we have created a detailed, MRI-based white matter atlas of the canine brain using diffusion tensor imaging. This technique, which relies on the movement properties of water, permits the creation of a three-dimensional diffusivity map of white matter brain regions that can be used to predict major axonal tracts. To generate an atlas of white matter tracts, thirty neurologically and clinically normal dogs underwent MRI imaging under anesthesia. High-resolution, three-dimensional T1-weighted sequences were collected and averaged to create a population average template. Diffusion-weighted imaging sequences were collected and used to generate diffusivity maps, which were then registered to the T1-weighted template. Using these diffusivity maps, individual white matter tracts-including association, projection, commissural, brainstem, olfactory, and cerebellar tracts-were identified with reference to previous canine brain atlas sources. To enable the use of this atlas, we created downloadable overlay files for each white matter tract identified using manual segmentation software. In addition, using diffusion tensor imaging tractography, we created tract files to delineate major projection pathways. This comprehensive white matter atlas serves as a standard reference to aid in the interpretation of quantitative changes in brain structure and function in clinical and research settings.

Multi-omics analysis reveals regime shifts in the gastrointestinal ecosystem in chickens following anticoccidial vaccination and Eimeria tenella challenge.

Coccidiosis, caused by Eimeria parasites, significantly impacts poultry farm economics and animal welfare. Beyond its direct impact on health, Eimeria infection disrupts enteric microbial populations leading to dysbiosis and increases vulnerability to secondary diseases such as necrotic enteritis, caused by Clostridium perfringens. The impact of Eimeria infection or anticoccidial vaccination on host gastrointestinal phenotypes and enteric microbiota remains understudied. In this study, the metabolomic profiles and microbiota composition of chicken caecal tissue and contents were evaluated concurrently during a controlled experimental vaccination and challenge trial. Cobb500 broilers were vaccinated with a Saccharomyces cerevisiae-vectored anticoccidial vaccine and challenged with 15,000 Eimeria tenella oocysts. Assessment of caecal pathology and quantification of parasite load revealed correlations with alterations to caecal microbiota and caecal metabolome linked to infection and vaccination status. Infection heightened microbiota richness with increases in potentially pathogenic species, while vaccination elevated beneficial Bifidobacterium. Using a multi-omics factor analysis, data on caecal microbiota and metabolome were integrated and distinct profiles for healthy, infected, and recovering chickens were identified. Healthy and recovering chickens exhibited higher vitamin B metabolism linked to short-chain fatty acid-producing bacteria, whereas essential amino acid and cell membrane lipid metabolisms were prominent in infected and vaccinated chickens. Notably, vaccinated chickens showed distinct metabolites related to the enrichment of sphingolipids, important components of nerve cells and cell membranes. Our integrated multi-omics model revealed latent biomarkers indicative of vaccination and infection status, offering potential tools for diagnosing infection, monitoring vaccination efficacy, and guiding the development of novel treatments or controls.IMPORTANCEAdvances in anticoccidial vaccines have garnered significant attention in poultry health management. However, the intricacies of vaccine-induced alterations in the chicken gut microbiome and its subsequent impact on host metabolism remain inadequately explored. This study delves into the metabolic and microbiotic shifts in chickens post-vaccination, employing a multi-omics integration analysis. Our findings highlight a notable synergy between the microbiome composition and host-microbe interacted metabolic pathways in vaccinated chickens, differentiating them from infected or non-vaccinated cohorts. These insights pave the way for more targeted and efficient approaches in poultry disease control, enhancing both the efficacy of vaccines and the overall health of poultry populations.

A comprehensive audit of difficult airway trolleys in selected Victorian hospitals.

This study aimed to assess the availability, design, and contents of difficult airway trolleys in hospitals in Victoria, Australia. A survey audit was conducted with a 92.3% reply rate, and the responses from 22 major Victorian hospitals were analysed. The results showed that difficult airway trolleys were available in 100% of operating theatres, emergency departments and intensive care units, and the rate of standardisation was high. Compliance with recommended design features and resources was on average 68.3%. There was no significant difference in the compliance rate of major tertiary centres compared with other hospitals. The carriage of non-essential items was reduced compared with earlier audits. However, there was heterogeneity in the brands of supraglottic airway devices, videolaryngoscopes and cognitive aids used. The study highlights the need for ongoing improvement to the organization and content of difficult airway trolleys, and for further discussion regarding the safety of equipment variation across institutions.

Gene identification for ocular congenital cranial motor neuron disorders using human sequencing, zebrafish screening, and protein binding microarrays.

Purpose: To functionally evaluate novel human sequence-derived candidate genes and variants for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). Methods: Through exome and genome sequencing of a genetically unsolved human oCCDD cohort, we previously identified variants in 80 strong candidate genes. Here, we further prioritized a subset of these (43 human genes, 57 zebrafish genes) using a G0 CRISPR/Cas9-based knockout assay in zebrafish and generated F2 germline mutants for seventeen. We tested the functionality of variants of uncertain significance in known and novel candidate transcription factor-encoding genes through protein binding microarrays. Results: We first demonstrated the feasibility of the G0 screen by targeting known oCCDD genes phox2a and mafba . 70-90% of gene-targeted G0 zebrafish embryos recapitulated germline homozygous null-equivalent phenotypes. Using this approach, we then identified three novel candidate oCCDD genes ( SEMA3F , OLIG2, and FRMD4B ) with putative contributions to human and zebrafish cranial motor development. In addition, protein binding microarrays demonstrated reduced or abolished DNA binding of human variants of uncertain significance in known and novel sequence-derived transcription factors PHOX2A (p.(Trp137Cys)), MAFB (p.(Glu223Lys)), and OLIG2 (p.(Arg156Leu)). Conclusions: This study nominates three strong novel candidate oCCDD genes ( SEMA3F , OLIG2, and FRMD4B ) and supports the functionality and putative pathogenicity of transcription factor candidate variants PHOX2A p.(Trp137Cys), MAFB p.(Glu223Lys), and OLIG2 p.(Arg156Leu). Our findings support that G0 loss-of-function screening in zebrafish can be coupled with human sequence analysis and protein binding microarrays to aid in prioritizing oCCDD candidate genes/variants.

Projected Healthcare System Cost Burden of Metabolic Dysfunction-Associated Steatotic Liver Disease in Canada.

Background and Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of chronic liver disease worldwide. The current and projected cost of treating individuals with MASLD in Canada remains unknown. Our objective was to calculate the projected liver-specific and total health-care costs for people living with MASLD in Canada from 2020 to 2050. Methods: The health-care usage of a cohort of patients diagnosed with MASLD in Calgary, Alberta was calculated using administrative data. Liver-specific encounters were identified and the average costs per year per patient were calculated. Projected costs were calculated by multiplying the average cost per patient within each health state by the projected prevalence of each health state. Results: There were 6358 patients in the cohort. The annual average liver-specific cost per patient was $7.02 for F0/F1, $35.30 for F2, $60.46 for F3, and $72.55 for F4. The projected Canada-wide liver-specific cost was $85.5 million in 2020 and was expected to increase by $51 million by 2050. The average annual total health-care cost per patient was $397.90 for F0/F1, $781.53 for F2, $2881.84 for F3, and $1598.82 for F4. Thus, the projected Canada-wide total health-care cost was $3.76 billion in 2020 and was expected to increase by almost $2 billion by 2050. Conclusion: These estimates underscore the need for a MASLD framework that focuses on both prevention and innovative care models to change the predicted trajectory of health-care costs.

Forensic alcohol calculations in individuals under the age of 18.

Alcohol calculations are carried out in many forensic case types. On occasion, individuals under the age of 18 are involved, and alcohol calculations may be beneficial. To date, there are no studies that have determined the best way to estimate total body water (TBW) or alcohol elimination rates in juveniles for alcohol calculations. We utilized a data set of 207 females and 218 males, aged 3-18 years, from a variety of ethnic backgrounds, where TBW had been empirically measured, to evaluate the best anthropometric equation to use to estimate TBW in juveniles. For males aged 3-15 years and females aged 2-13 years, the equation of Wells et al. was the most accurate and precise (RMSE of ≤10.4% in males and ≤9.9% in females). For males aged 16+ years and females aged 14+ years, the equation of Watson et al. was more appropriate (RMSE ≤11.5% and ≤12.4%, respectively). Based on published studies where the alcohol elimination rate was determined in 43 juveniles (aged 10-17 years) who were hospitalized due to alcohol consumption, a mean alcohol elimination rate of 16 mg/100 mL/h should be used. The recommended range being 9-25 mg/100 mL (5th-95th percentile). This study provides evidence that there are valid anthropometric equations to determine TBW and alcohol elimination rates that can be used for alcohol calculations in juveniles between the ages of 10 and 17.

Virus-Based Separation of Rare Earth Elements.

The utilization of biomaterials for the separation of rare earth elements (REEs) has attracted considerable interest due to their inherent advantages, including diverse molecular structures for selective binding and the use of eco-friendly materials for sustainable systems. We present a pioneering methodology for developing a safe virus to selectively bind REEs and facilitate their release through pH modulation. We engineered the major coat protein of M13 bacteriophage (phage) to incorporate a lanthanide-binding peptide. The engineered lanthanide-binding phage (LBPh), presenting ∼3300 copies of the peptide, serves as an effective biological template for REE separation. Our findings demonstrate the LBPh's preferential binding for heavy REEs over light REEs. Moreover, the LBPh exhibits remarkable robustness with excellent recyclability and stability across multiple cycles of separations. This study underscores the potential of genetically integrating virus templates with selective binding motifs for REE separation, offering a promising avenue for environmentally friendly and energy-efficient separation processes.

A neuronal circuit driven by GLP-1 in the olfactory bulb regulates insulin secretion.

Glucagon-like peptide 1 (GLP-1) stimulates insulin secretion and holds significant pharmacological potential. Nevertheless, the regulation of energy homeostasis by centrally-produced GLP-1 remains partially understood. Preproglucagon cells, known to release GLP-1, are found in the olfactory bulb (OB). We show that activating GLP-1 receptors (GLP-1R) in the OB stimulates insulin secretion in response to oral glucose in lean and diet-induced obese male mice. This is associated with reduced noradrenaline content in the pancreas and blocked by an α2-adrenergic receptor agonist, implicating functional involvement of the sympathetic nervous system (SNS). Inhibiting GABAA receptors in the paraventricular nucleus of the hypothalamus (PVN), the control centre of the SNS, abolishes the enhancing effect on insulin secretion induced by OB GLP-1R. Therefore, OB GLP-1-dependent regulation of insulin secretion relies on a relay within the PVN. This study provides evidence that OB GLP-1 signalling engages a top-down neural mechanism to control insulin secretion via the SNS.

Esophageal Bronchus-the Hidden Link. A Case Report.

An esophageal bronchus is a subtype of congenital bronchopulmonary foregut malformations in which a lobar bronchus arises directly from the esophagus, creating a communication between the esophagus and lung tissue. Early diagnosis is crucial to prevent worsening pulmonary sequelae but is challenging due to the rarity of the anomaly and nonspecific respiratory symptoms. We present a child whose esophageal bronchus was identified incidentally during preanesthetic assessment for craniosynostosis repair and discuss the role an anesthesiologist can play in identifying and managing this diagnosis.

Abscopal Effects, Clastogenic Effects and Bystander Effects: 70 Years of Non-Targeted Effects of Radiation.

In vitro and in vivo observations accumulated over several decades have firmly shown that the biological effects of ionizing radiation can spread from irradiated cells/tissues to non-targeted cells/tissues. Redox-modulated intercellular communication mechanisms that include a role for secreted factors and gap junctions, can mediate these non-targeted effects. Clearly, the expression of such effects and their transmission to progeny cells has implications for issues related to radiation protection. Their elucidation is also relevant towards enhancing the efficacy of cancer radiotherapy and reducing its impact on the development of normal tissue toxicities. In addition, the study of non-targeted effects is pertinent to our basic understanding of intercellular communications under conditions of oxidative stress. This review will trace the history of non-targeted effects of radiation starting with early reports of abscopal effects which described radiation induced effects in tissues distant from the site of radiation exposure. A related effect involved the production of clastogenic factors in plasma following irradiation which can induce chromosome damage in unirradiated cells. Despite these early reports suggesting non-targeted effects of radiation, the classical paradigm that a direct deposition of energy in the nucleus was required still dominated. This paradigm was challenged by papers describing radiation induced bystander effects. This review will cover mechanisms of radiation-induced bystander effects and the potential impacts on radiation protection and radiation therapy.

Neutral evolution of snoRNA Host Gene long non-coding RNA affects cell fate control.

A fundamental challenge in molecular biology is to understand how evolving genomes can acquire new functions. Actively transcribed, non-coding parts of the genome provide a potential platform for the development of new functional sequences, but their biological and evolutionary roles remain largely unexplored. Here, we show that a set of neutrally evolving long non-coding RNAs (lncRNAs) whose introns encode small nucleolar RNAs (snoRNA Host Genes, SNHGs) are highly expressed in skin and dysregulated in inflammatory conditions. Using SNHG7 and human epidermal keratinocytes as a model, we describe a mechanism by which these lncRNAs can increase self-renewal and inhibit differentiation. The activity of SNHG7 lncRNA has been recently acquired in the primate lineage and depends on a short sequence required for microRNA binding. Taken together, our results highlight the importance of understanding the role of fast-evolving transcripts in normal and diseased epithelia, and show how poorly conserved, actively transcribed non-coding sequences can participate in the evolution of genomic functionality.

Effectiveness of dairy products to protect against cognitive decline in later life: a narrative review.

As the world's population ages the prevalence of age-related health concerns is increasing, including neurodegeneration disorders such as mild cognitive impairment, vascular dementia and Alzheimer's disease. Diet is a key modifiable risk factor for the development of neurodegeneration, likely due to gut-brain axis interactions related to neuroinflammation. Analyses of dietary patterns identified dairy as being part of a cognitively healthy diet; however, its contribution to cognitive outcomes is difficult to discern. This narrative review evaluates the literature to determine whether there is sufficient evidence that the consumption of dairy products helps to maintain cognitive function in later life. A search using the terms (dairy OR milk OR cheese OR yogurt OR yogurt) AND ("mild cognitive impairment" OR dementia OR "Alzheimer's disease") identified 796 articles. After screening and sorting, 23 observational studies and 6 intervention studies were identified. The results of the observational studies implied that the relationship between total dairy consumption and cognitive outcomes is inverse U-shaped, with moderate consumption (1-2 servings per day) being the most beneficial. The analysis of the intake of different types of dairy products indicated that fermented products, particularly cheese, were most likely responsible for the observed benefits. The experimental studies all used dairy-derived peptides produced during fermentation as the dietary intervention, and the results indicated that these could be an effective treatment for early-stage cognitive impairment. Further experimental studies with whole dairy products, particularly fermented dairy, are needed to determine whether the regular consumption of these foods should be recommended to maximize the likelihood of healthy cognitive aging.

Understanding symptom contribution to sex inequality in bladder and renal cancer stage at diagnosis.

Background: Understanding sex-specific factors contributing to advanced-stage diagnosis can guide interventions to reduce sex inequality in patients with urological cancers. Method: We used linked primary care and cancer registry data to examine associations between symptoms and advanced-stage in 1151 bladder cancer and 440 renal cancer patients diagnosed between January 2012 and December 2015 in England. We performed logistic regression, adjusting for sex, age, deprivation and routes to diagnosis, including interaction terms between symptoms and sex and symptoms and age. Results: Female sex (OR vs. men 1.89 [1.28-2.79]; p = 0.001) and patients presenting with urinary tract infections (OR 2.22 [1.34-3.69]) and abdominal symptoms (OR 2.19 [1.30-3.70]) were associated with increased odds of advanced-stage bladder cancer (vs. haematuria, p = 0.016 for both). Women with haematuria and men with abdominal symptoms (compared with the opposite sex with the same presenting symptom) were more likely to have advanced-stage bladder cancer. Neither sex nor symptom associations were observed for renal cancer. Conclusion: Non-haematuria symptoms are associated with higher risk of advanced-stage bladder cancer. Greater risk of advanced-stage bladder cancer in women may reflect biological differences in haematuria onset and sex differences during diagnostic process. Identifying higher risk women with haematuria may reduce sex inequalities in bladder cancer outcomes.

Comparative study of Eimeria tenella development in different cell culture systems.

Cell culture systems have long been recognised as great resources to mitigate the use of animals in research, offering effective solutions for replacement or reduction with benefits commonly including lower costs, shorter duration and improved reproducibility. The use of in vitro culture methods has been extensively explored for many apicomplexan parasites, supporting significant research advances, but studies with Eimeria are often limited since they still depend on the animal host. In this study we have used 2.5D and 3D culture systems for the first time to evaluate the growth of Eimeria tenella parasites using a panel of cell lines (MDBK, HD11, COLO-680N and HCC4006). Results were compared to growth in 2D monolayers following established protocols. Observations using the fluorescent transgenic strain Et-dYFP showed invasion and development of parasites inside cells suspended in a collagen matrix (2.5D or 3D), supporting the development of asexual stages with the release of first-generation merozoites. Similar findings were observed when Scaffold-free 3D cell spheroids of HD11 cells were infected with sporozoites. No subsequent developmental stages were identified while evaluating these cell lines and further work will be required to improve in vitro culture systems to a point where reduction and replacement of animal use becomes routine.