Advancing brain barriers RNA sequencing: guidelines from experimental design to publication

Background: RNA sequencing (RNA-Seq) in its varied forms has become an indispensable tool for analyzing differential gene expression and thus characterization of specific tissues. Aiming to understand the brain barriers genetic signature, RNA seq has also been introduced in brain barriers research. This has led to availability of both, bulk and single-cell RNA-Seq datasets over the last few years. If appropriately performed, the RNA-Seq studies provide powerful datasets that allow for significant deepening of knowledge on the molecular mechanisms that establish the brain barriers. However, RNA-Seq studies comprise complex workflows that require to consider many options and variables before, during and after the proper sequencing process.Main body: In the current manuscript, we build on the interdisciplinary experience of the European PhD Training Network BtRAIN (https://www.btrain-2020.eu/) where bioinformaticians and brain barriers researchers collaborated to analyze and establish RNA-Seq datasets on vertebrate brain barriers. The obstacles BtRAIN has identified in this process have been integrated into the present manuscript. It provides guidelines along the entire workflow of brain barriers RNA-Seq studies starting from the overall experimental design to interpretation of results. Focusing on the vertebrate endothelial blood–brain barrier (BBB) and epithelial blood-cerebrospinal-fluid barrier (BCSFB) of the choroid plexus, we provide a step-by-step description of the workflow, highlighting the decisions to be made at each step of the workflow and explaining the strengths and weaknesses of individual choices made. Finally, we propose recommendations for accurate data interpretation and on the information to be included into a publication to ensure appropriate accessibility of the data and reproducibility of the observations by the scientific community.Conclusion: Next generation transcriptomic profiling of the brain barriers provides a novel resource for understanding the development, function and pathology of these barrier cells, which is essential for understanding CNS homeostasis and disease. Continuous advancement and sophistication of RNA-Seq will require interdisciplinary approaches between brain barrier researchers and bioinformaticians as successfully performed in BtRAIN. The present guidelines are built on the BtRAIN interdisciplinary experience and aim to facilitate collaboration of brain barriers researchers with bioinformaticians to advance RNA-Seq study design in the brain barriers community

Consensus statement on abusive head trauma in infants and young children

Abusive head trauma (AHT) is the leading cause of fatal head injuries in children younger than 2 years. A multidisciplinary team bases this diagnosis on history, physical examination, imaging and laboratory findings. Because the etiology of the injury is multifactorial (shaking, shaking and impact, impact, etc.) the current best and inclusive term is AHT. There is no controversy concerning the medical validity of the existence of AHT, with multiple components including subdural hematoma, intracranial and spinal changes, complex retinal hemorrhages, and rib and other fractures that are inconsistent with the provided mechanism of trauma. The workup must exclude medical diseases that can mimic AHT. However, the courtroom has become a forum for speculative theories that cannot be reconciled with generally accepted medical literature. There is no reliable medical evidence that the following processes are causative in the constellation of injuries of AHT: cerebral sinovenous thrombosis, hypoxic-ischemic injury, lumbar puncture or dysphagic choking/vomiting. There is no substantiation, at a time remote from birth, that an asymptomatic birth-related subdural hemorrhage can result in rebleeding and sudden collapse. Further, a diagnosis of AHT is a medical conclusion, not a legal determination of the intent of the perpetrator or a diagnosis of murder. We hope that this consensus document reduces confusion by recommending to judges and jurors the tools necessary to distinguish genuine evidence-based opinions of the relevant medical community from legal arguments or etiological speculations that are unwarranted by the clinical findings, medical evidence and evidence-based literature

A model of diurnal grazing patterns and herbage intake of a dairy cow, MINDY: Model description

Estimates of herbage intake and parallel measurements of ingestive and digestive behaviors of grazing ruminants pose considerable experimental and technical difficulties, owing to dynamic interactions between the plant, the rumen and the animal. As a consequence, advances in the area have been slow and costly. Model simulations that capture such interactions are critical for research and management decisions involving the grazing process. This work describes MINDY, a mathematical, mechanistic and dynamic simulation model of the diurnal grazing pattern of a dairy cow. MINDY is based on a cluster of three models: (1) Molly (Baldwin, 1995), a model of ruminant digestion and metabolism; (2) a model representing feed consumption as a function of diurnal fluctuations in the internal state of the animal; and (3) a sward structure, herbage quality and grazing behavior model. The objective of the work was to describe the diurnal grazing pattern, including ingestive actions and rumination behaviors, herbage intake, and nutrient supply to the animal in response to the animal's internal state and grazing environment. The model was coded in ACSL and simulations were conducted using ACSLXtreme. In addition to dietary nutrient composition required by Molly, MINDY requires sward surface height and mass, and grazing area offered to the cow. Key sub-model parameters were identified by sensitivity analyses and parameterized using two data sets from mid-lactation Friesian and late lactation Holstein dairy cows breeds under set stock conditions. The parameterized model predicted realistic estimates of ingestive behavior for different cow genotypes managed under set stocking and rotational grazing. It also predicted a realistic number of steps taken while eating and searching and sward defoliation dynamics as well as diurnal fluctuations of digestion and metabolism. Additional evaluations are required and further data may be needed to better define some parameters, but the model offers promise as an heuristic tool for feed intake and grazing process research and as an informative tool for grazing and cow management decisions

Representing interconversions among volatile fatty acids in the Molly cow model

The Molly cow model uses fixed stoichiometric coefficients for predicting volatile fatty acid (VFA) production from the fermented individual dietary nutrient fractions of forage and concentrate. We previously showed that predictions of VFA production had large errors and hypothesized that it was due to a lack of representation of carbon exchange among VFA. The objectives of the present study were to add VFA interconversion equations based on thermodynamics to the Molly cow model and evaluate the effect of these additions on model accuracy and precision of VFA predictions. Previously described thermodynamic equations were introduced to represent interconversions among VFA. The model was further modified to predict de novo acetate, propionate, and butyrate production coefficients based on forage-to-concentrate ratios rather than discrete, fixed sets of coefficients for forage-based, concentrate-based, and mixed diets. Both the original model and the modified one were reparameterized and evaluated against a common data set containing 8 studies reporting pH, VFA concentration, and VFA production rates using isotope dilution techniques and 62 studies reporting VFA concentrations and pH. Evaluations after parameter estimation revealed that predictions of VFA production rates were not improved, with root mean squared prediction errors (RMSPE) of 77, 60, and 51% for acetate, propionate, and butyrate, respectively, for the revised model versus 75, 63, and 55, respectively, for the original model. The RMSPE for predictions of VFA concentrations were reduced from 28, 46, and 40% to 22, 31, and 26% for acetate, propionate, and butyrate, respectively, simply by rederiving the VFA coefficients, but minimal further improvement was achieved with the addition of thermodynamically driven interconversion equations (RMSPE of 21, 32, and 27% for acetate, propionate, and butyrate, respectively). Thus, the results indicate that thermodynamically driven interchanges among VFA, as represented in this study, may not be a primary determinant for the accuracy of predictions of net production rates. Including the effect of pH on VFA absorption reduced the mean bias of propionate production and slope bias of acetate production, but not the overall RMSPE. The larger prediction errors for VFA production as compared with concentrations suggest the data quality may not be high, or that our representation of VFA production and absorption as well as ruminal digestion is inadequate. Additional data are required to discriminate among these hypotheses

Development of an improved representation of rumen digesta outflow in a mechanistic and dynamic model of a dairy cow, Molly

© 2015 Elsevier B.V. Accurate predictions of outflow of digesta from the rumen are critical for improving modeling of feed intake, rumen function and fermentation patterns of ruminants. The main objective of this work was to develop an improved representation of rumen digesta outflow in the Molly model. The work is primarily an integration of existing knowledge of rumen digestion responsible for variations in digesta outflows in ruminants, and describes the structure and function of the new development, assessing the new model in terms of ruminal outflow and fermentation in response to different feeding scenarios. The present development includes three changes to the model: (1) a medium-size particle pool was added to the rumen which was assumed to ferment and pass from the rumen; (2) particulate passage was made a function of particle size, particle concentrations in the rumen, and liquid passage rate; and (3) fermentation rate was made a function of particle surface area in the medium and small particle pools. Although prediction accuracy of digestive functions was not substantially improved by the change in model structure, the model now reproduces observed patterns of variation in rumen function as affected by the food intake and dietary particle size compared with those reported in the literature, which was not previously the case. It also reproduces more realistic trends in rumen fermentation patterns, digestion and methane yields. The concepts embedded in the new development capture underlying biological mechanisms driving the variation in digesta outflows from the rumen that were not captured before

Evaluation of morbidity, mortality and outcome following cervical spine injuries in elderly patients

We analysed the morbidity, mortality and outcome of cervical spine injuries in patients over the age of 65 years. This study was a retrospective review of 107 elderly patients admitted to our tertiary referral spinal injuries unit with cervical spine injuries between 1994 and 2002. The data was acquired by analysis of the national spinal unit database, hospital inpatient enquiry system, chart and radiographic review. Mean age was 74 years (range 66–93 years). The male to female ratio was 2.1:1 (M = 72, F = 35). The mean follow-up was 4.4 years (1–9 years) and mean in-hospital stay was 10 days (2–90 days). The mechanism of injury was a fall in 75 and road traffic accident in the remaining 32 patients. The level involved was atlanto-axial in 44 cases, sub-axial in 52 cases and the remaining 11 had no bony injury. Multilevel involvement occurred in 48 patients. C2 dominated the single level injury and most of them were type II odontoid fractures. Four patients had complete neurology, 27 had incomplete neurology, and the remaining 76 had no neurological deficit. Treatment included cervical orthosis in 67 cases, halo immobilization in 25, posterior stabilization in 12 patients and anterior cervical fusion in three patients. The overall complication rate was 18.6% with an associated in-hospital mortality of 11.2%. The complications included loss of reduction due to halo and Minerva loosening, non-union and delayed union among conservatively treated patients, pin site and wound infection, gastrointestinal bleeding and complication due to associated injuries. Among the 28.9% patients with neurological involvement, 37.7% had significant neurological recovery. Outcome was assessed using a cervical spine outcome questionnaire from Johns Hopkins School of Medicine. Sixty-seven patients (70%) completed the form, 20 patients (19%) were deceased at review and 8 patients (7%) were uncontactable. Functional disability was more marked in the patients with neurologically deficit at time of injury. Outcome of the injury was related to increasing age, co-morbidity and the severity of neurological deficit. Injuries of the cervical spine are not infrequent occurrence in the elderly and occur with relatively minor trauma. Neck pain in the elderly patients should be thoroughly evaluated to exclude C2 injuries. Most patients can be managed in an orthosis but unstable injuries require rigid external immobilization or surgical stabilization