The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain’s microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB.

Introduction: There is much debate regarding the impact of COVID-induced lockdown on the standard of assessments, mainly since students were assessed at home via an online assessment platform. Regular orthodox lectures and assessments were carried out during the first term, while the strictly enforced South African COVID lockdown warranted that 2nd term lectures and assessments were based online. This created the fortunate control conditions to statistically compare orthodox face-to-face with online-home-based assessments.Methods: We compared the assessments of a cohort of second-year students studying physiology and anatomy during 2019 and 2020: Orthodox face-to-face teaching and assessments (2019) were compared to online teaching and their analogous online assessments (2020) during the COVID-19-induced lockdown.Results: Although class pass rates and marks for online assessments (2020) were significantly higher than for traditional assessments (2019), an essential finding of the study was that the Gaussian distribution of the marks across the class for both modes of assessment was statistically identical. This indicated that although students performed better with home-based online assessments, poor-performing students populated the lower spread of marks, modal students occupied the central distribution, while good students occupied the higher mark distribution of the curve.Discussion: The students were found to be resilient in adapting to things and learning, online presentations, and computer-based assessments. No gender-based difference or advantage to adjusting to newly introduced blended learning and concomitant changes in learning assessment strategies was found. The online-home-based assessments proved to incentivize prior learning and preparation for assessments by implementing strict time limits or assessments and randomizing the selections of questions and respective (MCQ) answer choices. We conclude that although home-based online assessments significantly improve the overall mark distribution, there was no distinction in the spread of the distribution of marks, which was indicative that the home-based online assessment process was able to provide an identical measure of course proficiency as in the orthodox sit-down assessment. Therefore, our statistical analysis of the performance of students under student assessment performances indicates that there is no rationale for thinking that the home-based online mode of assessment is equivalent to or better than the orthodox modes of assessment.

Cancer remains a major global concern, with metastasis responsible for most cancer-related deaths. Disseminated cancer cells become circulating tumour cells (CTCs) that travel until arresting at preferential vascular sites such as the blood-brain barrier (BBB), where they cross into the central nervous system (CNS). Given that the BBB robustly maintains CNS homeostasis, the mechanism whereby CTCs metastatically enter the brain is largely unknown. The triple-negative breast cancer (TNBC) (MDA-MB-231 cell line) cells were cultured under normoxic (21% O₂), physioxic (5% O₂), and hypoxic (1.5-2% O₂) conditions to generate conditioned media (CM). Diluted CM was used to treat brain endothelial cells (BECs: bEnd.5 cell line). Endothelial suppression of cell division was assessed using the trypan blue exclusion assay. Morphological changes and an abnormality index were evaluated using hematoxylin and eosin (H&E) staining. Transendothelial electrical resistance (TEER) measured the BEC monolayer integrity following CM exposure. Comparatively, all oxygen-derived CM suppressed BEC suppression of cellular division; however, hypoxic-derived CM was most severe, reducing cell division by 67.7% at 96 h. H&E staining similarly showed that hypoxic-derived CM induced an abnormality rate of 980 abnormalities per 1 000 cells. TEER measurements demonstrated that the hypoxic-derived CM overwhelmed BEC homeostatic mechanisms, significantly increasing monolayer permeability. A clear pattern of progressive dysfunction correlated with decreasing oxygen tension in MDA-MB-231 cell cultures. While the normoxic and physioxic CM had comparable effects, hypoxic-derived CM elicited exacerbated responses, with substantial impairment detectable as soon as 24 h. These findings suggest hypoxic tumour secretomes overwhelm the endothelial regulatory mechanisms, leading to BEC dysfunction and, ultimately, BBB compromise. Graphical Abstract The alternative text for this image may have been generated using AI. Illustration of breast cancer circulating tumour cells (CTCs) origin and metastasis to the blood-brain barrier (BBB). Illustrations from Server Medical Art (https://smart.servier.com), used under CC BY 4.0 license (https://creativecommons.org/licenses/by.4.0/).