Disturbed microcirculation and hyperaemic response in a murine model of systemic inflammation.

Systemic inflammation affects cognitive functions and increases the risk of dementia. This phenomenon is thought to be mediated in part by cytokines that promote neuronal survival, but the continuous exposure to which may lead to neurodegeneration. The effects of systemic inflammation on cerebral blood vessels, and their provision of adequate oxygen to support critical brain parenchymal cell functions, remains unclear. Here, we demonstrate that neurovascular coupling is profoundly disturbed in lipopolysaccharide (LPS) induced systemic inflammation in awake mice. In the 24 hours following LPS injection, the hyperaemic response of pial vessels to functional activation was attenuated and delayed. Concurrently, under steady-state conditions, the capillary network displayed a significant increase in the number of capillaries with blocked blood flow, as well as increased duration of 'capillary stalls'-a phenomenon previously reported in animal models of stroke and Alzheimer's disease pathology. We speculate that vascular changes and impaired oxygen availability may affect brain functions following acute systemic inflammation and contribute to the long-term risk of neurodegenerative changes associated with chronic, systemic inflammation.

Optimized High-Contrast Brightfield Microscopy Application for Noninvasive Proliferation Assays of Human Cell Cultures.

High-contrast brightfield (HCBF) microscopy has emerged as a strong tool for noninvasive counting of cells in culture. HCBF imaging delivers precise cell growth data and is completely label free rendering it an attractive alternative to common cell counting procedures that often adversely affect cell growth. With computational image analysis, HCBF achieves efficient high-throughput automated workflows, extremely relevant for drug and chemical screens in pharmaceutical, toxicological, and biomedical research. We demonstrate the applicability of HCBF microscopy to count three common cell types (HEK293, Huh7, and primary human dermal fibroblasts) with diverse morphology challenging the method. The three cell types required different analysis settings, and we identified two parameters of the computational image analysis, which after cell-specific optimization significantly improved the cell counting accuracy, namely the lower size limit and the intensity threshold. Three-dimensional (3D) imaging approaches, which have obtained great attention in recent years, were an interesting prospect to combine with HCBF microscopy. We optimized the analysis of two 3D outputs but found 3D HCBF imaging to be inferior to the optimized single-layer HCBF imaging for cell counting. HCBF cell counts were highly linearly correlated with (R2 > 0.99) and highly similar (<15% difference) to cell counts obtained through Hoechst staining, over a broad range of densities allowing at least this level of accuracy for two to three cell generations in Huh7 cells and fibroblasts. Counts of HEK293 cells correlated somewhat less. In conclusion, the HCBF cell counting method is excellently suited for cell proliferation assays and cytotoxicity assays.

Riboflavin Deficiency-Implications for General Human Health and Inborn Errors of Metabolism.

As an essential vitamin, the role of riboflavin in human diet and health is increasingly being highlighted. Insufficient dietary intake of riboflavin is often reported in nutritional surveys and population studies, even in non-developing countries with abundant sources of riboflavin-rich dietary products. A latent subclinical riboflavin deficiency can result in a significant clinical phenotype when combined with inborn genetic disturbances or environmental and physiological factors like infections, exercise, diet, aging and pregnancy. Riboflavin, and more importantly its derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), play a crucial role in essential cellular processes including mitochondrial energy metabolism, stress responses, vitamin and cofactor biogenesis, where they function as cofactors to ensure the catalytic activity and folding/stability of flavoenzymes. Numerous inborn errors of flavin metabolism and flavoenzyme function have been described, and supplementation with riboflavin has in many cases been shown to be lifesaving or to mitigate symptoms. This review discusses the environmental, physiological and genetic factors that affect cellular riboflavin status. We describe the crucial role of riboflavin for general human health, and the clear benefits of riboflavin treatment in patients with inborn errors of metabolism.

Deficiency of the mitochondrial sulfide regulator ETHE1 disturbs cell growth, glutathione level and causes proteome alterations outside mitochondria.

The mitochondrial enzyme ETHE1 is a persulfide dioxygenase essential for cellular sulfide detoxification, and its deficiency causes the severe and complex inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of well-described clinical symptoms of the disease, detailed cellular and molecular characterization is still ambiguous. Cellular redox regulation has been described to be influenced in ETHE1 deficient cells, and to clarify this further we applied image cytometry and detected decreased levels of reduced glutathione (GSH) in cultivated EE patient fibroblast cells. Cell growth initiation of the EE patient cells was impaired, whereas cell cycle regulation was not. Furthermore, Seahorse metabolic analyzes revealed decreased extracellular acidification, i. e. decreased lactate formation from glycolysis, in the EE patient cells. TMT-based large-scale proteomics was subsequently performed to broadly elucidate cellular consequences of the ETHE1 deficiency. More than 130 proteins were differentially regulated, of which the majority were non-mitochondrial. The proteomics data revealed a link between ETHE1-deficiency and down-regulation of several ribosomal proteins and LIM domain proteins important for cellular maintenance, and up-regulation of cell surface glycoproteins. Furthermore, several proteins of endoplasmic reticulum (ER) were perturbed including proteins influencing disulfide bond formation (e.g. protein disulfide isomerases and peroxiredoxin 4) and calcium-regulated proteins. The results indicate that decreased level of reduced GSH and alterations in proteins of ribosomes, ER and of cell adhesion lie behind the disrupted cell growth of the EE patient cells.