Fasting Animals Research Articles

Decompensated MASH-Cirrhosis Model by Acute and Toxic Effects of Phenobarbital.

Metabolic dysfunction-associated Steatohepatitis (MASH), is a prominent cause for liver cirrhosis. MASH-cirrhosis is responsible for liver complications and there is no specific treatment. To develop new therapeutic approaches, animal models are needed. The aim of this study was to develop a fast animal model of MASH-cirrhosis in rats reflecting the human disease. Carbon tetrachloride (CCl4) injections in combination with a high-fat Western diet (WD) were used to induce MASH-cirrhosis. To accelerate liver injury, animals received phenobarbital (PB) in their drinking water using two different regimens. Rats developed advanced MASH-cirrhosis characterized by portal hypertension, blood biochemistry, hepatic ballooning, steatosis, inflammation and fibrosis. Importantly, rats receiving low-dose PB for the long term (LT) showed ascites after 6 weeks, whereas rats with high-dose short-term (ST) PB developed ascites after 8 weeks. ST- and LT-treated rats showed increased portal pressure (PP) and decreased mean arterial pressure (MAP). Of note, hepatocyte ballooning was only observed in the LT group. The LT administration of low-dose PB with CCl4 intoxication and WD represents a fast and reproducible rat model mimicking decompensated MASH-cirrhosis in humans. Thus, CCl4 + WD with LT low-dose phenobarbital treatment might be the preferred rat animal model for drug development in MASH-cirrhosis.

EVALUATION OF ANTI-DIABETIC ACTIVITY OF METHANOLIC EXTRACT FROM THE FRUIT PEEL OF ATALANTIA MONOPHYLLA (LINN.) IN ALLOXAN INDUCED DIABETIC MICE

Objective: Atlantia monophylla is an indigenous medicinal plant, with a folk reputation as hypoglycemic agent throughout India. Considerable research has shown that plants rich in flavonoids content with anti-oxidant potential are known to be bioactive for the management of diabetes mellitus (DM). In the present investigation, hypoglycemic activity of aqueous fruit peel extract of A. monophylla was evaluated using alloxan-induced hyperglycemic mice. Methods: Aqueous extract of the fruit peel of A. monophylla (AEAM) was subjected to preliminary phytochemical screening and acute oral toxicity study as per Organization for Economic Cooperation and Development guidelines 425. AEAM was evaluated for hypoglycemic activity, Alloxan induced DM in mice, oral glucose tolerance test in fasted mice. In vitro α- amylase inhibitory in albino mice method was employed for evaluation of hypoglycemic activity. Results: All of the mice in each group had their blood glucose levels measured both before and after the treatments on fasting animals. The extract was given at doses of 200 mg/kg and 400 mg/kg body weight, or 100 mg/kg body weight of the conventional antidiabetic medication Metformin. A glucometer was used to measure the blood glucose level. After 1 h, 2 h, and 4 h, the extract-treated diabetic mice had a substantial (p≤0.05) drop in blood glucose levels. The hypoglycemic action was similar to that of the medication Metformin used as a benchmark. Conclusion: The results of this study indicate that AEAM has strong hypoglycemic properties. One such method might include the activation of insulin receptors and subsequent release of insulin once β cells are stimulated. The hypoglycemic effect of the plant could be achieved through increasing insulin release from the pancreas.

Predictive musculoskeletal simulations reveal the mechanistic link between speed, posture and energetics among extant mammals

An unusual pattern among the scaling laws in nature is that the fastest animals are neither the largest, nor the smallest, but rather intermediately sized. Because of the enormous diversity in animal shape, the mechanisms underlying this have long been difficult to determine. To address this, we challenge predictive human musculoskeletal simulations, scaled in mass from the size of a mouse (0.1 kg) to the size of an elephant (2000 kg), to move as fast as possible. Our models replicate patterns observed across extant animals including: (i) an intermediate optimal body mass for speed; (ii) a reduction in the cost of transport with increasing size; and (iii) crouched postures at smaller body masses and upright postures at larger body masses. Finally, we use our models to determine the mechanical limitations of speed with size, showing larger animals may be limited by their ability to produce muscular force while smaller animals are likely limited by their ability to produce larger ground reaction forces. Despite their bipedal gait, our models replicate patterns observed across quadrupedal animals, suggesting these biological phenomena likely represent general rules and are not the result of phylogenetic or other ecological factors that typically hinder comparative studies.

Pre-housing fasting and diet physical form impact broiler chicks’ gastrointestinal development along the first week of housing

This study aimed to evaluate body and gastrointestinal development in broilers chicks subjected or not to pre-housing fasting and different diet physical forms during the first week of housing. Nine hundred chicks were distributed into a completely randomized design in a 3×3 factorial arrangement: 0, 24 and 48 hours(h) of fasting; mashed (MM), crushed (CC) and micropelletized (MP) physical forms. For each fasting period, 300 animals were housed in protection circles with 100 chicks each. On days 1, 3, 5 and 7 of feeding, six animals were randomly selected, previously weighed and euthanized to obtain organs biometrical and histological data. Chicks fasted for 24 h and 48 h were lighter compared to the 0 h group and the 24-h fasting x MP interaction had a positive impact on body weight at d7 (P<0.05). Although fasted chicks presented lower body weight, small intestine, liver and gizzard were heavier, and presented greater yolk sac consumption (YSC; P<0.05). There were no alterations in the duodenal mucosa (DM) in pre-housing fasting animals (P>0.05). Despite the diet physical form and pre-housing fasting interactions for villus height (VH) and absorptive area (AA) on d1, only diet physical form affected DM, where greater VH and AA on d7 were observed in CC and MP groups (P<0.05). Animals fed MP after 48 h fasting presented smaller VH and AA compared to chicks fed the same diet after 24 h and were similar to the 0 h group (P<0.05). On d1, YSC was similar among groups of the same diet, but for those fed with MP(P<0.05). The 48 h-fasted group presented heavier intestines relative weights (IRW) at d1(P<0.05). Furthermore, the 0 h group fed with CC presented higher IRW, whereas MP showed the lowest IRW over the first week (P<0.05). The DM development (kinetics) showed an increase in VH and AA overtime in all groups, which was more pronounced in 48 h-fasting chicks throughout the first week, showing the highest increase in VH when fed with MP (P<0.05). VH:CD ratio was similar along the first week in MP groups, regardless of the fasting period but more pronounced in the 24 h group fed MM and CC (P<0.05). Therefore, 24 h fasting associated to MP diet may be the most efficient management practice. Collectively, these findings suggest pre-housing fasting is strongly associated with diet physical form at the first week of housing and should be considered to achieve greater performance in broilers.

Voronoi diagrams and Delaunay triangulation for modelling animal territorial behaviour.

We explore the use of movable automata in numerical modelling of male competition for territory. We used territorial dragonflies as our biological inspiration for the model, assuming two types of competing males: (a) faster and larger males that adopt a face-off strategy and repulse other males; (b) slower and smaller males that adopt a non-aggressive strategy. The faster and larger males have higher noise intensity, leading to faster motion and longer conservation of motion direction. The velocity distributions resemble the Maxwell distributions of velocity, expected in Brownian dynamics, with two probable velocities and distribution widths for the two animal subpopulations. The fast animals' trajectories move between visually fixed density folds of the slower animal subpopulation. A correlation is found between individual velocity and individual area distribution, with smaller animals concentrated in a region of small velocities and areas. Attraction between animals results in a modification of the system behaviour, with larger animals spending more time being surrounded by smaller animals and being slowed down by their interaction with the surroundings. Overall, the study provides insights into the dynamics of animal competition for territory and the impact of attraction between animals.

Considering preferences, speed and the animation of multiple symbols in developing P300 brain-computer interface for children

Purpose Prior research has begun establishing the efficacy of animation in brain-computer interfaces access to augmentative and alternative communication (BCI-AAC). However, the use of animation in P300-BCI-AAC for children is in the early stages and largely limited to single item highlighting of extended durations. In pursuit of practical application, the present study aims to evaluate children’s event-related potential (ERP) characteristics and user experience during a task involving an animated P300-BCI-AAC system. Materials and methods The system utilizes multi-item zoom animations to access a 28-pictorial symbols. Participants completed a fast (100 ms) and slow (200 ms) zoom animation highlighting conditions wherein four pictorial symbols were highlighted concurrently. Results The proposed display appears feasible, eliciting all targeted ERPs. However, ERP amplitudes may be reduced in comparison to single-item animation highlighting, possibly due to distraction. Ratings of mental effort were significantly higher for the 100 ms condition, though differences in the frontal P200/P300 ERP did not achieve significance. Most participants identified a preference for the 100 ms condition, though age may impact preference. Conclusions Overall, findings support the preliminary feasibility of a proposed 28-item interface that utilises group zoom animation highlighting of pictorial symbols. Further research is needed evaluating ERP characteristics and outcomes from online (real-time) use of animation-based P300-BCI-AAC for children with severe speech and physical impairments across multiple training sessions.

Dynamic similarity and the peculiar allometry of maximum running speed

Animal performance fundamentally influences behaviour, ecology, and evolution. It typically varies monotonously with size. A notable exception is maximum running speed; the fastest animals are of intermediate size. Here we show that this peculiar allometry results from the competition between two musculoskeletal constraints: the kinetic energy capacity, which dominates in small animals, and the work capacity, which reigns supreme in large animals. The ratio of both capacities defines the physiological similarity index Γ, a dimensionless number akin to the Reynolds number in fluid mechanics. The scaling of Γ indicates a transition from a dominance of muscle forces to a dominance of inertial forces as animals grow in size; its magnitude defines conditions of “dynamic similarity“ that enable comparison and estimates of locomotor performance across extant and extinct animals; and the physical parameters that define it highlight opportunities for adaptations in musculoskeletal “design” that depart from the eternal null hypothesis of geometric similarity. The physiological similarity index challenges the Froude number as prevailing dynamic similarity condition, reveals that the differential growth of muscle and weight forces central to classic scaling theory is of secondary importance for the majority of terrestrial animals, and suggests avenues for comparative analyses of locomotor systems.

Fast and on-site animal species identification in processed meat via centrifugal microfluidics and isothermal amplification.

We present a novel centrifugal microfluidic approach to rapidly identify animal species in meat products. The workflow requires a centrifugal cartridge for DNA extraction and for preparation of a recombinant polymerase amplification (RPA) reaction, a programmable centrifuge for processing the cartridge and an isothermal reader to perform the RPA. Liquid reagents are pre-stored on the cartridge and the meat sample can be added directly without any pre-treatment. With this system, we are able to identify six different animal species in a single run within one hour. In pork salami containing horse, turkey, sheep, chicken and beef meat, it was possible to identify species levels as low as 0.01%. In beef salami and cooked pork sausages 0.1% of foreign meat could be detected. This novel workflow enables rapid and sensitive species identification in processed meat at the point of need.

Intestinal and Renal Adaptations to Changes of Dietary Phosphate Concentrations in Rat.

We have studied the role of the intestine, kidney, and several hormones when adapting to changes in dietary P concentration. Normal and parathyroidectomized (PTX) rats were fed pH-matched diets containing 0.1%, 0.6%, and 1.2% P concentrations. 32Pi uptake was determined in the jejunum and kidney cortex brush border membrane vesicles. Several hormone and ion concentrations were determined in the blood and urine of rats. Both jejunum and kidney cortex Pi transport was regulated with 5 d of chronic feeding of P diets in normal rats. Acute adaptation was determined by switching foods on day 6, which was only clearly observed in the kidney cortex of normal rats, with more statistical variability in the jejunum. However, no paradoxical increase of Pi uptake in the jejunum was reproduced after the acute switch to the 1.2% P diet. Pi uptake in the jejunum was parathyroid hormone (PTH)-independent, but in the kidney, the chronic adaptation was reduced, and no acute dietary adaptations were observed. The NaPi2a protein was more abundant in the PTX than the sham kidneys, but contrary to the modest or absent changes in Pi uptake adaptation, the transporter was similarly regulated by dietary P, as in the sham rats. PTH and fibroblast growth factor 23 (FGF23) were the only hormones regulated by all diet changes, even in fasting animals, which exhibited regulated Pi transport despite similar phosphatemia. Evidence of Pi appetite effects was also observed. In brief, our results show new characteristics of Pi adaptations, including a lack of correlation between Pi transport, NaPi2a expression, and PTH/FGF23 concentrations.

Open field activity is linked to, but is not affected by, the rate of recovery from reward downshift in female Wistar rats

Frustration is an aversive emotion triggered by unexpected reward downshifts. Using the consummatory successive negative contrast (cSNC) task, a 32-to-2% sucrose downshift was shown to initially suppress consummatory behavior. Such suppression was followed by behavioral recovery over subsequent sessions. Individual differences often emerge in the rate of recovery after the initial consummatory suppression. These experiments were designed to determine whether a stable trait of sensation/novelty seeking (SNS) is related to such individual differences in recovery from reward downshift. In Experiment 1, open field (OF) activity in the central area served as a measure of SNS. A week later, animals received training in the cSNC task involving ten 5-min sessions of access to 32% sucrose followed by four sessions of access to 2% sucrose. Higher OF activity predicted greater consummatory suppression after downshift, but a steeper recovery rate across downshifted sessions. Controls not exposed to the OF showed cSNC, but downshifted animals performed at equivalent levels whether they had OF exposure or not. In Experiment 2, after a 32-to-2% sucrose downshift, fast vs. slow recovery animals displayed similar levels of central activity in the OF. In Experiment 3, animals exhibited similar levels of central activity whether after a 32-to-2% or an 8-to-2% sucrose downshift. In both experiments, activity levels were similar whether immediately after session 12 (onset of recovery) or after session 15 (fully recovered). These results suggest that individual variations in recovery from reward downshift are correlated with levels of SNS as a stable trait.

Not so fast: Paradoxically increased variability in the glucose tolerance test due to food withdrawal in continuous glucose-monitored mice

ObjectiveThis study was performed to determine the effect of fasting on reproducibility of the glucose tolerance test. Due to individual variation in animal feeding behaviors, fasting animals prior to metabolic and behavioral experiments is widely held to reduce inter-subject variation in glucose and metabolic parameters of preclinical rodent models. Reducing variability is especially important for studies where initial metabolite levels can influence the magnitude of experimental interventions, but fasting also imposes stress that may distort the variables of interest. One such intervention is the glucose tolerance test (GTT) which measures the maximum response and recovery following a bolus of exogenous glucose. We sought to investigate how fasting affects the response of individual mice to a GTT. MethodsUsing simultaneous continuous glucose monitoring (CGM) and indirect calorimetry, we quantified blood glucose, physical activity, body temperature, metabolic rates, and food consumption levels on a minute-to-minute basis in adult male mice for 4 weeks. We tested the effects of a 4-h or 18-h fast on the GTT to examine the effect of food withdrawal in light or dark photoperiods. Studies were also performed with 4-h fasting in additional mice without implanted CGM probes. ResultsContrary to our expectations, a 4-h fast during the light photoperiod promotes a paradoxical increase in inter-animal variation in metabolic rate, physical activity, body temperature, glycemia, and glucose tolerance. This hyperglycemic and hyper-metabolic phenotype promotes increased corticosterone levels and is consistent with a behavioral stress response to food deprivation, even in well-fed mice. We find that mice undergoing an 18-h fast entered torpor, a hibernation-like state. In addition to low body temperature and metabolic rate, torpor is also associated with glucose levels 56 mg/dl lower than those seen in mice with ad libitum access to food. Moreover, the time spent in torpor affects the response to a GTT. ConclusionOur results suggest fasting mice before glucose tolerance testing, and perhaps other experiments, can have the opposite of the intended effect where fasting can increase, rather than decrease, experimental variability.

Reduced Fasting Duration in Cynomolgus Monkeys Enhances Animal Welfare During Toxicology Studies.

During toxicology studies, fasting animals prior to clinical pathology blood collection is believed to reduce variability in some clinical chemistry analytes. However, fasting adds stress to animals that are already stressed from the administration of potentially toxic doses of the test article. The purpose of this study was to assess the impacts of different fasting durations on cynomolgus monkeys' welfare during toxicology studies. To this end, we assessed the cynomolgus monkeys traditional and ancillary clinical pathology endpoints at different fasting times. We showed that most clinical pathology endpoints were largely comparable between different fasting times suggesting that cynomolgus monkeys could be fasted for as little as 4 hours for toxicology studies, as longer fasting times (up to 20 hours) resulted in stress, dehydration, and significant decreases in blood glucose- changes that impacts animal welfare. Shorter fasting times were associated with higher triglycerides variability among individual animals. Therefore, we propose that shorter fasting time (i.e., 4 hours) should be adequate for most toxicology studies except when: (1) parameters that could be affected by non-fasting conditions are important for safety and pharmacodynamic assessments (i.e., glucose and lipids) and (2) fasting would be needed for the bioavailability of an orally administered test article.

The effect of single and repeated doses of rivastigmine on gastric myoelectric activity in experimental pigs.

Rivastigmine is a pseudo-irreversible cholinesterase inhibitor used for therapy of Alzheimer's disease and non-Alzheimer dementia syndromes. In humans, rivastigmine can cause significant gastrointestinal side effects that can limit its clinical use. The aim of this study was to assess the impact of rivastigmine on gastric motor function by means of electrogastrography (EGG) in experimental pigs. Six experimental adult female pigs (Sus scrofa f. domestica, hybrids of Czech White and Landrace breeds; 3-month-old; mean weight 30.7 ± 1.2 kg) were enrolled into the study twice and created two experimental groups. In group A, a single intragastric dose of 6 mg rivastigmine hydrogen tartate was administered in the morning to fasting pigs before EGG recording. In group B, rivastigmine was administered to overnight fasting animals in a dietary bolus in the morning for 7 days (6 mg per day). On day 8, an intragastric dose of 12 mg rivastigmine was given in the morning to fasting pigs before EGG. EGG recording was accomplished by means of an EGG standalone system. Recordings from both groups were evaluated in dominant frequency and EGG power (areas of amplitudes). In total, 1,980 one-minute EGG intervals were evaluated. In group A, basal EGG power (median 1290.5; interquartile range 736.5-2330 μV2) was significantly higher in comparison with the power of intervals T6 (882; 577-1375; p = 0.001) and T10 (992.5; 385-2859; p = 0.032). In group B, the dominant frequency increased significantly from basal values (1.97 ± 1.57 cycles per minute) to intervals T9 (3.26 ± 2.16; p < 0.001) and T10 (2.14 ± 1.16; p = 0.012), respectively. In group B, basal EGG power (median 1030.5; interquartile range 549-5093) was significantly higher in comparison with the power of intervals T7 (692.5; 434-1476; p = 0.002) and T8 (799; 435-1463 μV2; p = 0.004). Both single as well as repeated intragastric administration of rivastigmine hydrogen tartrate caused a significant decrease of EGG power (areas of amplitudes) in experimental pigs. EGG power may serve as an indirect indicator of gastric motor competence. These findings might provide a possible explanation of rivastigmine-associated dyspepsia in humans.

On dissecting the wakes of flapping wings

Flapping is an energy-demanding mode of fast animal locomotion that requires physiology, wing kinematics, and unsteady mechanics to work in unison. The trailing flow or wake is a signature of flapping mechanics, which makes it a popular candidate for visualization and analysis. While there are multiple ways to analyze wakes, we need a general framework to ensure utility of the analysis in bio-inspired flapping flight. In this article, we develop a theoretical framework to analyze the transport phenomena of wakes. To illustrate, we apply the theory to a two-dimensional modeled hovering flight comprising engineered rigid flapping plates, which vary in aspect ratio, wingbeat frequency, and the use of winglet. The results potentially explain certain observations in natural flight.

Drosophila melanogaster Systemic Infection Model to Study Altered Virulence during Polymicrobial Infection by Aeromonas.

Polymicrobial infections are complex infections associated with worse outcomes compared to monomicrobial infections. We need simple, fast, and cost-effective animal models to assess their still poorly known pathogenesis. We developed a Drosophila melanogaster polymicrobial infection model for opportunistic pathogens and assessed its capacity to discriminate the effects of bacterial mixtures taken from cases of human polymicrobial infections by Aeromonas strains. A systemic infection was obtained by needle pricking the dorsal thorax of the flies, and the fly survival was monitored over time. Different lineages of the flies were infected by a single strain or paired strains (strain ratio 1:1). Individual strains killed more than 80% of the flies in 20 h. The course of infection could be altered with a microbial mix. The model could distinguish between the diverse effects (synergistic, antagonistic, and no difference) that resulted in a milder, more severe, or similar infection, depending on the paired strain considered. We then investigated the determinants of the effects. The effects were maintained in deficient fly lineages for the main signaling pathways (Toll deficient and IMD deficient), which suggests an active microbe/microbe/host interaction. These results indicate that the D. melanogaster systemic infection model is consistent with the study of polymicrobial infection.

An Accurate and Fast Animal Species Detection System for Embedded Devices

Encounters between humans and wildlife often lead to injuries, especially in remote wilderness regions, and highways. Therefore, animal detection is a vital safety and wildlife conservation component that can mitigate the negative impacts of these encounters. Deep learning techniques have achieved the best results compared to other object detection techniques; however, they require many computations and parameters. A lightweight animal species detection model based on YOLOv2 was proposed. It was designed as a proof of concept of and as a first step to build a real-time mitigation system with embedded devices. Multi-level features merging is employed by adding a new pass-through layer to improve the feature extraction ability and accuracy of YOLOv2. Moreover, the two repeated 3 × 3 convolutional layers in the seventh block of the YOLOv2 architecture are removed to reduce computational complexity, and thus increase detection speed without reducing accuracy. Animal species detection methods based on regular Convolutional Neural Networks (CNNs) have been widely applied; however, these methods are difficult to adapt to geometric variations of animals in images. Thus, a modified YOLOv2 with the addition of deformable convolutional layers (DCLs) was proposed to resolve this issue. Our experimental results show that the proposed model outperforms the original YOLOv2 by 5.0% in accuracy and 12.0% in speed. Furthermore, our analysis shows that the modified YOLOv2 model is more suitable for deployment than YOLOv3 and YOLOv4 on embedded devices.

Anti-hyperglycemic potential and chemical constituents of Aristolochia triangularis Cham. leaves − A medicinal species native to Brazilian forests

Ethnopharmacological relevanceAristolochia triangularis Cham. has been used in Brazilian traditional medicine for various therapeutic purposes, including as a leaf-based infusion for diabetes management. Aim of the study: This study was designed to chemically characterize an infusion of in natura A. triangularis leaves and evaluate the in vivo anti-hyperglycemic properties of this infusion. Materials and methods: Chemical composition was examined using liquid–liquid extraction procedure, chromatographic methods, NMR, and LC−MS/MS. The in vivo anti-hyperglycemic activity of the freeze-dried infusion of A. triangularis leaves (Inf-L-At) was assessed using oral glucose tolerance test (OGTT). Initially, normoglycemic male rats were pre-treated with orally administered Inf-L-At at doses of 62.5, 125, and 250 mg/kg for two consecutive days. On the day of the OGTT, fasting animals received a glucose load (4 g/kg) 30 min after treatment with Inf-L-At, and the blood glucose levels were verified at 15, 30, 60, and 180 min. Intestinal maltase, lactase, and sucrase activities and muscle and liver glycogen contents were also assessed after the OGTT. Results: Inf-L-At extract led to glycemic reduction with no dose-response at 15, 30, and 60 min comparable to that of the antidiabetic drug glibenclamide and was accompanied by an increase in hepatic and muscle glycogen contents. Additionally, there was a significant statistically decrease in the in vitro activity of disaccharidases. Maltase and sucrase activities were inhibited at all doses, whereas lactase activity was inhibited only at 62.5 and 250 mg/kg. In total, 75 compounds were found in the infusion, including seven new ones, (7S*,8S*,7ꞌS*,8ꞌR*)-4,4ꞌ-dihydroxy-3,3ꞌ-dimethoxy-7,9ꞌ-epoxylignan-7ꞌ-ol; 4ꞌ-hydroxy-3ꞌ-methoxy-3,4-methylenedioxy-7,9ꞌ-epoxylignan-9,7ꞌ-diol; triangularisines A, B, and C; N-ethyl-N-methyl-affineine; and N-methyl pachyconfine, and one previously not described as a natural product, epi-secoisolariciresinol monomethyl ether. Conclusion: The results demonstrated the anti-hyperglycemic activity of the infusion from A. triangularis leaves and showed that it is a rich source of lignoids, alkaloids, and glycosylated flavonoids, which are known to exhibit antidiabetic effects and other biological properties that can be beneficial for patients with chronic hyperglycemia, thus certifying the popular use of this herbal drink.

CNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals

NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively.

Combined proton radiography and irradiation for high-precision preclinical studies in small animals.

Proton therapy has become a popular treatment modality in the field of radiooncology due to higher spatial dose conformity compared to conventional radiotherapy, which holds the potential to spare normal tissue. Nevertheless, unresolved research questions, such as the much debated relative biological effectiveness (RBE) of protons, call for preclinical research, especially regarding in vivo studies. To mimic clinical workflows, high-precision small animal irradiation setups with image-guidance are needed. A preclinical experimental setup for small animal brain irradiation using proton radiographies was established to perform planning, repositioning, and irradiation of mice. The image quality of proton radiographies was optimized regarding the resolution, contrast-to-noise ratio (CNR), and minimal dose deposition in the animal. Subsequently, proof-of-concept histological analysis was conducted by staining for DNA double-strand breaks that were then correlated to the delivered dose. The developed setup and workflow allow precise brain irradiation with a lateral target positioning accuracy of<0.26mm for in vivo experiments at minimal imaging dose of<23mGy per mouse. The custom-made software for image registration enables the fast and precise animal positioning at the beam with low observer-variability. DNA damage staining validated the successful positioning and irradiation of the mouse hippocampus. Proton radiography enables fast and effective high-precision lateral alignment of proton beam and target volume in mouse irradiation experiments with limited dose exposure. In the future, this will enable irradiation of larger animal cohorts as well as fractionated proton irradiation.

Acute binge alcohol alters whole body metabolism and the time-dependent expression of skeletal muscle-specific metabolic markers for multiple days in mice.

Alcohol is a myotoxin that disrupts skeletal muscle function and metabolism, but specific metabolic alternations following a binge and the time course of recovery remain undefined. The purpose of this work was to determine the metabolic response to binge alcohol, the role of corticosterone in this response, and whether nutrient availability mediates the response. Female mice received saline (control) or alcohol (EtOH) (5 g/kg) via intraperitoneal injection at the start of the dark cycle. Whole body metabolism was assessed for 5 days. In a separate cohort, gastrocnemius muscles and liver were collected every 4 h for 48 h following intoxication. Metyrapone was administered before alcohol and gastrocnemius was collected 4 h later. Lastly, alcohol-treated mice were compared with fed or fasted controls. Alcohol disrupted whole body metabolism for multiple days. Alcohol altered the expression of genes and proteins in the gastrocnemius related to the promotion of fat oxidation (Pparα, Pparδ/β, AMPK, and Cd36) and protein breakdown (Murf1, Klf15, Bcat2). Changes to select metabolic genes in the liver did not parallel those in skeletal muscle. An alcohol-induced increase in circulating corticosterone was responsible for the initial change in protein breakdown factors but not the induction of FoxO1, Cebpβ, Pparα, and FoxO3. Alcohol led to a similar, but distinct metabolic response when compared with fasting animals. Overall, these data show that an acute alcohol binge rapidly disrupts macronutrient metabolism including sustained disruption to the metabolic gene signature of skeletal muscle in a manner similar to fasting at some time points.NEW & NOTEWORTHY Herein, we demonstrate that acute alcohol intoxication immediately alters whole body metabolism coinciding with rapid changes in the skeletal muscle macronutrient gene signature for at least 48 h postbinge and that this response diverges from hepatic effects and those of a fasted animal.