UV-Vis Spectra-Activated Droplet Sorting for Label-Free Chemical Identification and Collection of Drople

We introduce the UV-vis spectra-activated droplet sorter (UVADS) for high-throughput label-free chemical identification and enzyme screening. In contrast to previous absorbance-based droplet sorters that relied on single-wavelength absorbance in the visible range, our platform collects full UV-vis spectra from 200 to 1050 nm at up to 2100 spectra per second. Our custom-built open-source software application, "SpectraSorter," enables real-time data processing, analysis, visualization, and selection of droplets for sorting with any set of UV-vis spectral features. An optimized UV-vis detection region extended the absorbance path length for droplets and allowed for the direct protein quantification down to 10 μM of bovine serum albumin at 280 nm. UV-vis spectral data can distinguish a variety of different chemicals or spurious events (such as air bubbles) that are inaccessible at a single wavelength. The platform is used to measure ergothionase enzyme activity from monoclonal microcolonies isolated in droplets. In a label-free manner, we directly measure the ergothioneine substrate to thiourocanic acid product conversion while tracking the microcolony formation. UVADS represents an important new tool for high-throughput label-free in-droplet chemical analysi

Differential impact of dietary branched chain and aromatic amino acids on chronic kidney disease progression in rats

The metabolism of dietary proteins generates waste products that are excreted by the kidney, in particular nitrogen-containing urea, uric acid, ammonia, creatinine, and other metabolites such as phosphates, sulfates, and protons. Kidney adaptation includes an increase in renal plasma flow (RPF) and glomerular filtration rate (GFR) and represents a burden for diseased kidneys increasing the progression rate of CKD. The present study aimed at identifying potential differences between amino acid (AA) groups constituting dietary proteins regarding their impact on RPF, GFR, and CKD progression. We utilized the well-established 5/6 nephrectomy (5/6 Nx) CKD model in rats and submitted the animals for 5 weeks to either the control diet (18% casein protein) or to diets containing 8% casein supplemented with 10% of a mix of free amino acids, representing all or only a subset of the amino acids contained in casein. Whereas the RPF and GFR measured in free moving animals remained stable during the course of the diet in rats receiving the control mix, these parameters decreased in animals receiving the branched chain amino acid (BCAA) supplementation and increased in the ones receiving the aromatic amino acids (AAAs). In animals receiving essential amino acids (EAAs) containing both BCAAs and AAAs, there was only a small increase in RPF. The kidneys of the 5/6 Nx rats receiving the BCAA diet showed the strongest increase in smooth muscle actin and collagen mRNA expression as a result of higher level of inflammation and fibrosis. These animals receiving BCAAs also showed an increase in plasma free fatty acids pointing to a problem at the level of energy metabolism. In contrast, the animals under AAA diet showed an activation of AMPK and STAT3. Taken together, our results demonstrate that subsets of EAAs contained in dietary proteins, specifically BCAAs and AAAs, exert contrasting effects on kidney functional parameters and CKD progression

Kidney mass reduction leads to L‐arginine metabolism‐dependent blood pressure increase in mice

Background Uninephrectomy (UNX) is performed for various reasons, including kidney cancer or donation. Kidneys being the main site of L‐arginine production in the body, we tested whether UNX mediated kidney mass reduction impacts L‐arginine metabolism and thereby nitric oxide production and blood pressure regulation in mice. Methods and Results In a first series of experiments, we observed a significant increase in arterial blood pressure 8 days post‐UNX in female and not in male mice. Further experimental series were performed in female mice, and the blood pressure increase was confirmed by telemetry. L‐citrulline, that is used in the kidney to produce L‐arginine, was elevated post‐UNX as was also asymmetric dimethylarginine, an inhibitor of nitric oxide synthase that competes with L‐arginine and is a marker for renal failure. Interestingly, the UNX‐induced blood pressure increase was prevented by supplementation of the diet with 5% of the L‐arginine precursor, L‐citrulline. Because L‐arginine is metabolized in the kidney and other peripheral tissues by arginase‐2, we tested whether the lack of this metabolic pathway also compensates for decreased L‐arginine production in the kidney and/or for local nitric oxide synthase inhibition and consecutive blood pressure increase. Indeed, upon uninephrectomy, arginase‐2 knockout mice (Arg‐2−/−) neither displayed an increase in asymmetric dimethylarginine and L‐citrulline plasma levels nor a significant increase in blood pressure. Conclusions UNX leads to a small increase in blood pressure that is prevented by L‐ citrulline supplementation or arginase deficiency, 2 measures that appear to compensate for the impact of kidney mass reduction on L‐arginine metabolism

Protocol for a systematic review of good surgical practice guidelines for experimental rodent surgery

OBJECTIVE: Surgery is an integral part of many experimental studies. Aseptic and minimal invasive surgical technique and optimal perioperative and post-operative care are prerequisites to achieve surgical success and best possible animal welfare outcomes. Good surgical practice cannot only improve the animal's postoperative recovery, but also study outcome and validity. There seems to be a lack of implementation of good surgical practice during rodent surgery. The aim of this systematic review is to identify, critically evaluate and compare the currently recommended standards and underlying guidelines for rodent surgery-and finally to compile a comprehensive guideline of good surgical practice for rodent surgery. SEARCH STRATEGY: PubMed, Embase and Web of Science were searched to identify guidelines published in peer-reviewed journals. To identify grey literature and unpublished guidelines, we will perform a Google search for published guidelines and search laboratory animal sciences books for relevant book chapters. Additionally, we will conduct a survey among animal researchers enquiring about the guidelines they use. SCREENING AND STUDY SELECTION: For publications retrieved by the systematic search, unique references are screened by two reviewers, first for eligibility based on title and abstract and subsequently for final inclusion based on full text. Eligibility of books is based on title and content, final inclusion based on chapter full text. Guidelines are either retrieved by Google searches or a survey. Google searches will be conducted by at least four of the authors. Thereafter, guidelines will be screened by two of the authors. DATA EXTRACTION AND SYNTHESIS: We will extract data from publications, book chapters and guidelines. Based on the extracted data, we will perform a descriptive synthesis of the bibliographical details, guideline development and endorsement, and the prevalence of individual recommendations, including subgroup analysis of the guidance per continent or country and differences between peer-reviewed versus non-peer-reviewed guidance

Error-prone protein synthesis recapitulates early symptoms of Alzheimer disease in aging mice

Age-related neurodegenerative diseases (NDDs) are associated with the aggregation and propagation of specific pathogenic protein species (e.g., Aβ, α-synuclein). However, whether disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation, with its frequent outcome of random protein misfolding, is sufficient to recapitulate many early features of NDDs, including perturbed Ca2+ signaling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in behavioral changes reminiscent of early Alzheimer disease (AD), such as learning and memory deficits, maladaptive emotional responses, epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation, accompanied by hippocampal NPY expression and cerebral glucose hypometabolism. Collectively, our findings suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs, providing an alternative framework for understanding the initiation of AD. Keywords: Alzheimer; CP: Neuroscience; error-prone translation; neurodegenerative diseases; pathogenesis; protein misfolding; synaptic homeostasi

UV-Vis Spectra-Activated Droplet Sorting for Label-Free Chemical Identification and Collection of Droplets

We introduce the UV-vis spectra-activated droplet sorter (UVADS) for high-throughput label-free chemical identification and enzyme screening. In contrast to previous absorbancebased droplet sorters that relied on single-wavelength absorbance in the visible range, our platform collects full UV-vis spectra from 200 to 1050 nm at up to 2100 spectra per second. Our custombuilt open-source software application, "SpectraSorter," enables real-time data processing, analysis, visualization, and selection of droplets for sorting with any set of UV-vis spectral features. An optimized UV-vis detection region extended the absorbance path length for droplets and allowed for the direct protein quantification down to 10 mu M of bovine serum albumin at 280 nm. UV-vis spectral data can distinguish a variety of different chemicals or spurious events (such as air bubbles) that are inaccessible at a single wavelength. The platform is used to measure ergothionase enzyme activity from monoclonal microcolonies isolated in droplets. In a label-free manner, we directly measure the ergothioneine substrate to thiourocanic acid product conversion while tracking the microcolony formation. UVADS represents an important new tool for high-throughput label-free in-droplet chemical analysis.ISSN:1520-6882ISSN:0003-270

Insights of posture dependent pressure characteristics in five rats

Current shunt treatments of hydrocephalus, a condition characterized by excessive accumulation of cerebrospinal fluid (CSF) and intracranial pressure (ICP) fluctuations, suffer malfunctions caused by changes in patient’s posture. Research toward a quantitative model describing posture dependent dynamics of CSF related pressures such as ICP and blood pressure (BP) shall provide rele vant information that can lead to a better understanding of CSF dynamics and thus, improved treatment outcomes. In this pilot study, ICP and femoral blood pressure (FBP) were measured concurrently in anaesthetized as well as awake and freely moving rats using radio telemetry. It was shown that despite the inherent challenges of limited space for sensor implants and rapid movements leading to strong artefacts, influences on CSF related pressure fluctuations due to posture changes can be observed in individual rats.ISSN:2364-550

First insights of posture related pressure dynamics in awake and freely moving rats

Introduction: To improve outcomes of current shunt treatments for hydrocephalus, a better understanding of cerebrospinal fluid (CSF) physiology is needed. Because malfunctions arise from posture changes, measurements of intracranial pressure (ICP) fluctuations and their relation to blood pressure during these changes shall provide valuable insights. Methods: ICP and femoral blood pressure (FBP) of five healthy rats were continuously measured in a chronic trial via radio telemetry implants and sampled at 1 kHz. While being awake and moving freely in an observation box, the rats were monitored with a camera system at 30 fps. Means and correlation coefficients of ICP and FBP during ten natural rear ups per rat were analyzed with t-tests. Results: Rear ups lasted on average 2.13 s. During these, FBP assessed as mean±SD (106.5 ± 17.4 mmHg) and ICP (1.4 ± 3.8 mmHg) were on average lower than FBP (118.9 ± 11.9 mmHg) and ICP (1.6 ± 4.0 mmHg) before rear ups. Changes of FBP were significant (p < 0.05) in all rats, whereas changes in ICP were significant (p < 0.05) in only two rats. In one of these two rats, correlation coefficients were significant (p < 0.01). ICP and FBP during these rear ups were on average moderately positively correlated (r = 0.24). Conclusion: Concurrent measurements of CSF related pressures in rats are inherently challenging due to the limited space for sensor implants and rapid movements leading to strong artefacts. However, statistically significant CSF dynamics due to posture changes could be observed using high resolution pressure and video recordings.ISSN:2045-811

Elevated FGF23 and disordered renal mineral handling with reduced bone mineralization in chronically erythropoietin over-expressing transgenic mice

Fibroblast Growth Factor 23 (FGF23) is a phosphaturic factor causing increased renal phosphate excretion as well as suppression of 1,25 (OH)$_{2}$-vitamin D$_{3.}$ Highly elevated FGF23 can promote development of rickets and osteomalacia. We and others previously reported that acute application of erythropoietin (EPO) stimulates FGF23 production. Considering that EPO is clinically used as chronic treatment against anemia, we used here the Tg6 mouse model that constitutively overexpresses human EPO in an oxygen-independent manner, to examine the consequences of long-term EPO therapy on mineral and bone metabolism. Six to eight weeks old female Tg6 mice showed elevated intact and C-terminal fragment of FGF23 but normal plasma levels of PTH, calcitriol, calcium and phosphate. Renal function showed moderate alterations with higher urea and creatinine clearance and mild albuminuria. Renal phosphate excretion was normal whereas mild hypercalciuria was found. Renal expression of the key proteins TRPV5 and calbindin D28k involved in active calcium reabsorption was reduced in Tg6 mice. Plasma levels of the bone turnover marker osteocalcin were comparable between groups. However, urinary excretion of deoxypyridinoline (DPD) was lower in Tg6 mice. MicroCT analysis showed reduced total, cortical, and trabecular bone mineral density in femora from Tg6 mice. Our data reveal that chronic elevation of EPO is associated with high FGF23 levels and disturbed mineral homeostasis resulting in reduced bone mineral density. These observations imply the need to study the impact of therapeutically applied EPO on bone mineralization in patients, especially those suffering from chronic kidney disease