Computational modeling of light processing in the habenula and dorsal raphe based on laser ablation of functionally-defined cells.

BACKGROUND: The habenula is a major regulator of serotonergic neurons in the dorsal raphe, and thus of brain state. The functional connectivity between these regions is incompletely characterized. Here, we use the ability of changes in irradiance to trigger reproducible changes in activity in the habenula and dorsal raphe of zebrafish larvae, combined with two-photon laser ablation of specific neurons, to establish causal relationships. RESULTS: Neurons in the habenula can show an excitatory response to the onset or offset of light, while neurons in the anterior dorsal raphe display an inhibitory response to light, as assessed by calcium imaging. The raphe response changed in a complex way following ablations in the dorsal habenula (dHb) and ventral habenula (vHb). After ablation of the ON cells in the vHb (V-ON), the raphe displayed no response to light. After ablation of the OFF cells in the vHb (V-OFF), the raphe displayed an excitatory response to darkness. After ablation of the ON cells in the dHb (D-ON), the raphe displayed an excitatory response to light. We sought to develop in silico models that could recapitulate the response of raphe neurons as a function of the ON and OFF cells of the habenula. Early attempts at mechanistic modeling using ordinary differential equation (ODE) failed to capture observed raphe responses accurately. However, a simple two-layer fully connected neural network (NN) model was successful at recapitulating the diversity of observed phenotypes with root-mean-squared error values ranging from 0.012 to 0.043. The NN model also estimated the raphe response to ablation of D-off cells, which can be verified via future experiments. CONCLUSION: Lesioning specific cells in different regions of habenula led to qualitatively different responses to light in the dorsal raphe. A simple neural network is capable of mimicking experimental observations. This work illustrates the ability of computational modeling to integrate complex observations into a simple compact formalism for generating testable hypotheses, and for guiding the design of biological experiments.

Computational modeling suggests binding-induced expansion of Epsin disordered regions upon association with AP2.

Intrinsically disordered regions (IDRs) are prevalent in the eukaryotic proteome. Common functional roles of IDRs include forming flexible linkers or undergoing allosteric folding-upon-binding. Recent studies have suggested an additional functional role for IDRs: generating steric pressure on the plasma membrane during endocytosis, via molecular crowding. However, in order to accomplish useful functions, such crowding needs to be regulated in space (e.g., endocytic hotspots) and time (e.g., during vesicle formation). In this work, we explore binding-induced regulation of IDR steric volume. We simulate the IDRs of two proteins from Clathrin-mediated endocytosis (CME) to see if their conformational spaces are regulated via binding-induced expansion. Using Monte-Carlo computational modeling of excluded volumes, we generate large conformational ensembles (3 million) for the IDRs of Epsin and Eps15 and dock the conformers to the alpha subunit of Adaptor Protein 2 (AP2α), their CME binding partner. Our results show that as more molecules of AP2α are bound, the Epsin-derived ensemble shows a significant increase in global dimensions, measured as the radius of Gyration (RG) and the end-to-end distance (EED). Unlike Epsin, Eps15-derived conformers that permit AP2α binding at one motif were found to be more likely to accommodate binding of AP2α at other motifs, suggesting a tendency toward co-accessibility of binding motifs. Co-accessibility was not observed for any pair of binding motifs in Epsin. Thus, we speculate that the disordered regions of Epsin and Eps15 perform different roles during CME, with accessibility in Eps15 allowing it to act as a recruiter of AP2α molecules, while binding-induced expansion of the Epsin disordered region could impose steric pressure and remodel the plasma membrane during vesicle formation.

Complications associated with paediatric airway management during the COVID-19 pandemic: an international, multicentre, observational study.

Respiratory adverse events in adults with COVID-19 undergoing general anaesthesia can be life-threatening. However, there remains a knowledge gap about respiratory adverse events in children with COVID-19. We created an international observational registry to collect airway management outcomes in children with COVID-19 who were having a general anaesthetic. We hypothesised that children with confirmed or suspected COVID-19 would experience more hypoxaemia and complications than those without. Between 3 April 2020 and 1 November 2020, 78 international centres participated. In phase 1, centres collected outcomes on all children (age ≤ 18 y) having a general anaesthetic for 2 consecutive weeks. In phase 2, centres recorded outcomes for children with test-confirmed or suspected COVID-19 (based on symptoms) having a general anaesthetic. We did not study children whose tracheas were already intubated. The primary outcome was the incidence of hypoxaemia during airway management. Secondary outcomes included: incidence of other complications; and first-pass success rate for tracheal intubation. In total, 7896 children were analysed (7567 COVID-19 negative and 329 confirmed or presumed COVID-19 positive). The incidence of hypoxaemia during airway management was greater in children who were COVID-19 positive (24 out of 329 (7%) vs. 214 out of 7567 (3%); OR 2.70 (95%CI 1.70-4.10)). Children who had symptoms of COVID-19 had a higher incidence of hypoxaemia compared with those who were asymptomatic (9 out of 51 (19%) vs. 14 out of 258 (5%), respectively; OR 3.7 (95%CI 1.5-9.1)). Children with confirmed or presumed COVID-19 have an increased risk of hypoxaemia during airway management in conjunction with general anaesthesia.

Transcompp: understanding phenotypic plasticity by estimating Markov transition rates for cell state transitions.

MOTIVATION: Gradual population-level changes in tissues can be driven by stochastic plasticity, meaning rare stochastic transitions of single-cell phenotype. Quantifying the rates of these stochastic transitions requires time-intensive experiments, and analysis is generally confounded by simultaneous bidirectional transitions and asymmetric proliferation kinetics. To quantify cellular plasticity, we developed Transcompp (Transition Rate ANalysis of Single Cells to Observe and Measure Phenotypic Plasticity), a Markov modeling algorithm that uses optimization and resampling to compute best-fit rates and statistical intervals for stochastic cell-state transitions. RESULTS: We applied Transcompp to time-series datasets in which purified subpopulations of stem-like or non-stem cancer cells were exposed to various cell culture environments, and allowed to re-equilibrate spontaneously over time. Results revealed that commonly used cell culture reagents hydrocortisone and cholera toxin shifted the cell population equilibrium toward stem-like or non-stem states, respectively, in the basal-like breast cancer cell line MCF10CA1a. In addition, applying Transcompp to patient-derived cells showed that transition rates computed from short-term experiments could predict long-term trajectories and equilibrium convergence of the cultured cell population. AVAILABILITY AND IMPLEMENTATION: Freely available for download at http://github.com/nsuhasj/Transcompp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Structural and white matter changes associated with duration of Braille education in early and late blind children.

In early (EB) and late blind (LB) children, vision deprivation produces cross-modal plasticity in the visual cortex. The progression of structural- and tract-based spatial statistics changes in the visual cortex in EB and LB, as well as their impact on global cognition, have yet to be investigated. The purpose of this study was to determine the cortical thickness (CT), gyrification index (GI), and white matter (WM) integrity in EB and LB children, as well as their association to the duration of blindness and education. Structural and diffusion tensor imaging data were acquired in a 3T magnetic resonance imaging in EB and LB children (n = 40 each) and 30 sighted controls (SCs) and processed using CAT12 toolbox and FSL software. Two sample t-test was used for group analyses with P < 0.05 (false discovery rate-corrected). Increased CT in visual, sensory-motor, and auditory areas, and GI in bilateral visual cortex was observed in EB children. In LB children, the right visual cortex, anterior-cingulate, sensorimotor, and auditory areas showed increased GI. Structural- and tract-based spatial statistics changes were observed in anterior visual pathway, thalamo-cortical, and corticospinal tracts, and were correlated with education onset and global cognition in EB children. Reduced impairment in WM, increased CT and GI and its correlation with global cognitive functions in visually impaired children suggests cross-modal plasticity due to adaptive compensatory mechanism (as compared to SCs). Reduced CT and increased FA in thalamo-cortical areas in EB suggest synaptic pruning and alteration in WM integrity. In the visual cortical pathway, higher education and the development of blindness modify the morphology of brain areas and influence the probabilistic tractography in EB rather than LB.

Frontal lobe metabolic alterations characterizing Parkinson's disease cognitive impairment.

BACKGROUND AND PURPOSE: Diagnosis of Parkinson's disease (PD) cognitive impairment at early stages is challenging compared to the stage of PD dementia where functional impairment is apparent and easily diagnosed. Hence, to evaluate potential early stage cognitive biomarkers, we assessed frontal lobe metabolic alterations using in vivo multi-voxel proton magnetic resonance spectroscopic imaging (1H-MRSI). METHOD: Frontal metabolism was studied in patients with PD with normal cognition (PD-CN) (n = 26), with cognitive impairment (PD-CI) (n = 27), and healthy controls (HC) (n = 30) using a single slice (two-dimensional) 1H-MRSI at 3 T. The acquired spectra were post-processed distinctly for voxels corresponding to the bilateral middle/superior frontal gray matter (GM) and frontal white matter (WM) regions (delineated employing neuromorphometrics atlas) using the LC-Model software. RESULT: Significant (post hoc p < 0.016) reduction in the concentration of N-acetyl aspartate (NAA) in the middle and superior frontal GMs and total choline (tCho) and total creatine (tCr) in the frontal WM was observed in PD-CI compared to PD-CN and HC, while that in HC and PD-CN groups were comparable. The NAA and tCr/tCho metabolite concentrations showed significant (p < 0.05) positive correlations with cognitive test scores in the frontal GM and WM, respectively. The receiver operating curve (ROC) analysis revealed significant (p < 0.05) "area under curve" for NAA/tNAA in the frontal GM and tCho in the frontal WM. CONCLUSION: The frontal metabolic profile is altered in cognitively impaired PD compared with cognitively normal PD. Neuronal function loss (NAA), altered energy metabolism (Cr), and cholinergic (Cho) neural transmission are implicated in PD cognitive pathology. Frontal neuro-metabolism may promisingly serve as PD cognitive biomarker.

Cortical thickness and gyrification index measuring cognition in Parkinson's disease.

OBJECTIVE: Cortical dynamics is driven by cortico-cortical connectivity and it characterizes cortical morphological features. These brain surface features complement volumetric changes and may offer improved understanding of disease pathophysiology. Hence, present study aims to investigate surface features; cortical thickness (CT) and gyrification index (GI) in Parkinson's disease (PD) patients of normal cognition (PD-CN), cognitively impaired patients with PD (PD-CI) in comparison with cognitively normal healthy controls (HC) to better elucidate cognition linked features in PD. METHOD: Anatomical MRI (3DT1) was carried out in 30 HC (56.53 ± 8.42 years), 30 PD-CN (58.8 ± 6.07 years), and 30 PD-CI (60.3 ± 6.43 years) subjects. Whole brain ROI based parcellation using Desikan-Killiany (DK-40) atlas followed by regional CT and GI differentiation [with 'age' and 'total intracranial volume' (TIV) correction], multiple linear regression (with 'age', 'TIV', and 'education' correction) with clinical variables, ROC analysis, and CT-GI correlation across the groups was used for data analysis. RESULTS: Widespread cortical thinning with regional GI reduction was evident in PD-CI with respect to other two groups (HC and PD-CN), and with absence of such alterations in PD-CN compared to HC. Frontal, parietal, and temporal CT/GI significantly correlated with cognition and presented classification abilities for cognitive state in PD. Mean regional CT and GI were found negatively correlated across groups with heterogeneous regions. CONCLUSION: Fronto-parietal and temporal regions suffer cognition associated cortical thinning and GI reduction. CT may serve better discriminator properties and may be more consistent than GI in studying cognition in PD. Heterogeneous surface dynamics across the groups may signify neuro-developmental alterations in PD.

Quantitative metabolomics of saliva using proton NMR spectroscopy in patients with Parkinson's disease and healthy controls.

INTRODUCTION: Parkinson's disease (PD) is a multisystem disorder of unknown etiology, highlights a broad array of symptoms and pathological features influencing organs throughout the body. The metabolic profile of saliva in patients with PD may be influenced by malabsorption in the gastroenteric tract, neurodegeneration, and mitochondrial dysfunction. In the present study, we apply a powerful NMR metabolomics approach for biomarker identification in PD using saliva, a non-invasive bio-fluid. METHODS: Metabolic profiling of saliva were studied in patients with PD (n = 76) and healthy controls (HC, n = 37) were analyzed and differentiated PD from HC. A total of 40 metabolites including aromatic amino acids, short-chain fatty acids, branched chain amino acids, taurine, and N-acetylglutamate were identified. Spectral binned data and concentration of metabolites were estimated for analysis. RESULTS: Increased concentration of phenylalanine, tyrosine, histidine, glycine, acetoacetate, taurine, TMAO, GABA, N-acetylglutamate, acetoin, acetate, alanine, fucose, propionate, isoleucine, and valine were observed in PD as compared to HC. Further, subgroup analysis among early PD, advanced PD, and HC groups, revealed increased metabolite concentration in early PD group as compared to advanced PD and HC group. DISCUSSION: Analysis revealed potential biomarkers and their involvement in amino acid metabolism, energy metabolism, neurotransmitters metabolism, and microflora system. Patients with early PD exhibited higher metabolite concentration as compared to advanced PD group which might be associated with dopaminergic treatment. CONCLUSION: The results of our data indicate that patients with PD might be characterized by metabolic imbalances like gut microflora system, energy metabolites, and neurotransmitters which may contribute to the PD pathogenesis.

Wnt proteins synergize to activate ß-catenin signaling.

Wnt ligands are involved in diverse signaling pathways that are active during development, maintenance of tissue homeostasis and in various disease states. While signaling regulated by individual Wnts has been extensively studied, Wnts are rarely expressed alone, and the consequences of Wnt gene co-expression are not well understood. Here, we studied the effect of co-expression of Wnts on the ß-catenin signaling pathway. While some Wnts are deemed 'non-canonical' due to their limited ability to activate ß-catenin when expressed alone, unexpectedly, we find that multiple Wnt combinations can synergistically activate ß-catenin signaling in multiple cell types. WNT1- and WNT7B-mediated synergistic Wnt signaling requires FZD5, FZD8 and LRP6, as well as the WNT7B co-receptors GPR124 (also known as ADGRA2) and RECK. Unexpectedly, this synergistic signaling occurs downstream of ß-catenin stabilization, and is correlated with increased lysine acetylation of ß-catenin. Wnt synergy provides a general mechanism to confer increased combinatorial control over this important regulatory pathway.

Effect of Doxorubicin on Squamous Cell Carcinoma of Skin: Assessment by MRI Relaxometry at 4.7T.

Squamous cell carcinoma (SCC) of skin has no standard treatment regimen, resulting in recurrences/metastasis. Although, doxorubicin (Dox), an anthracycline antibiotic has demonstrated some degree of efficacy. Molecular imaging can help in assessment of treatment response and prognosis of SCCs. MRI data showed that spin-spin relaxation (T2) time was longer (138 ± 2 msec) in Dox treated Test-II and there is no significant difference in spin-lattice relaxation (T1) time with respective controls. These findings further corroborated with the histology, proliferation index, apoptotic index, and HMGA1 protein expression. Thus, MRI may be a useful tool for monitoring treatment response noninvasively for skin tumor prognosis.

A qualitative analysis of students' perceptions of videos to support learning in a psychomotor skills course.

INTRODUCTION: This qualitative study analysed students' experiences and perceptions about different types of videos in a psychomotor skills course, relating to clinical knowledge and skills and how they benefited learners in and out of the classroom. METHODS: Subjects were recruited from 4th and 5th year dental undergraduate programme. The recruitment was terminated after 24 students participated since data saturation was reached. A semi-structured interview was conducted with each participating student, digitally recorded and transcribed verbatim. A thematic analysis was performed using an inductive approach to identify key themes. RESULTS: Four key domains were identified from thematic analysis: functionality and access; learning and understanding; clinical skills preparation and performance and supplementary learning resources. Students perceived videos to be an important and vital learning tool relating to its on-demand access, control of functionality and use for particular needs that is not possible from textbooks or lectures. Video learning materials allows students to clarify knowledge, improve cognitive thinking and enhance revision of theoretical concepts and clinical skills. Two types of video were regarded as particularly useful as students reported being actively and cognitively engaged in the learning content and broadened their clinical experience. In addition, students reported mental preparation of clinical psychomotor skills using their own mental visualisation which enabled them to prepare for the clinical care of their patients. CONCLUSION: This qualitative research supports the benefits of learning from videos in relation to convenience of access and functionality of the videos. In addition, it shows novel aspects to learning from videos that are more cognitively engaging and in mental preparation of clinical skills prior to patient care.

Randomized equivalence trial of the King Vision aBlade videolaryngoscope with the Miller direct laryngoscope for routine tracheal intubation in children <2 yr of age.

BACKGROUND.: We conducted a randomized equivalence trial to compare direct laryngoscopy using a Miller blade (DL) with the King Vision videolaryngoscope (KVL) for routine tracheal intubation. We hypothesized that tracheal intubation times with DL would be equivalent to the KVL in children <2 yr of age. METHODS.: Two hundred children were randomly assigned to tracheal intubation using DL or KVL. The primary outcome was the median difference in the total time for successful tracheal intubation. Secondary outcomes assessed were tracheal intubation attempts, time to best glottic view, time for tracheal tube entry, percentage of glottic opening score, airway manoeuvres needed, and complications. RESULTS.: The median difference between the groups was 5.7 s, with an upper 95% confidence interval of 7.5 s, which was less than our defined equivalence time difference of 10 s. There were no differences in the number of tracheal intubation attempts and the time to best glottic view [DL median 5.3 (4.1-7.6) s vs KVL 5.0 (4.0-6.3) s; P =0.19]. The percentage of glottic opening score was better when using the KVL [median 100 (100-100) vs DL median 100 (90-100); P <0.0001]. Use of DL was associated with greater need for airway manoeuvres during tracheal intubation (33 vs 7%; P <0.001). Complications did not differ between devices. CONCLUSIONS.: In children <2 yr of age, the KVL was associated with equivalent times for routine tracheal intubation when compared with the Miller blade. CLINICAL TRIAL REGISTRATION: NCT02590237.

A randomised comparison of the Ambu® AuraGain™ and the LMA® supreme in infants and children.

We conducted a randomised trial in 100 children in order to compare the clinical performance of the Ambu(®) AuraGain(™) and the LMA(®) Supreme(*) for airway maintenance during mechanical ventilation. The primary outcomes were initial and 10-min airway leak pressures. Ease, time and success rates for device and gastric tube insertion, fibreoptic grades of view, airway quality during anaesthetic maintenance, and complications were also assessed. There were no differences in the initial and ten min airway leak pressures between the Ambu AuraGain and LMA Supreme, median (IQR [range]) initial: 19 (16-22 [10-34]) vs 18 (14-24 [8-40]) cmH2 O, p = 0.4; and ten min: 22 (18-26 [11-40]) vs 20 (16-26 [12-40]) cmH2 O, p = 0.08, respectively. Ease, time and success rates for device placement, gastric tube insertion and complications were also not significantly different. Children receiving the LMA Supreme required more airway manouevers (7 vs 1 patient, p = 0.06) to maintain a patent airway. Our results suggest that the Ambu AuraGain may be a useful alternative to the LMA Supreme, as demonstrated by comparable overall clinical performance in children.

Randomized comparison of experts and trainees with nasal and oral fibreoptic intubation in children less than 2 yr of age.

BACKGROUND: We hypothesized that the time to successful fibreoptic tracheal intubation through the nasal route would be faster than the oral route for both experts and trainees in children <2 yr of age. METHODS: One hundred children, 24 months and under in age, were randomized to an operator (expert or trainee), and route (nasal or oral) for fibreoptic tracheal intubation. Three separate times were then measured: (i) time to first glottic view, (ii) time to carinal view, and (iii) total time to successful tracheal intubation. The number of attempts made, manoeuvres needed to obtain an adequate laryngeal view, and manoeuvres for tracheal tube passage were also recorded. RESULTS: Time to successful tracheal intubation was significantly faster for experts than trainees. There was no difference in the time to tracheal intubation between the nasal and oral routes for experts. In trainees, intubation times were shorter for the nasal route-median (inter-quartile range) time (s) to carinal view was 35 (27-63) for the nasal route vs 59 (38-94) for the oral route (P=0.03), and the median time to successful tracheal intubation were 62 (49-122) vs 117 (61-224), P=0.05, for the nasal and oral routes, respectively. For trainees, the oral route required a greater number of airway manoeuvres for adequate laryngeal views and passage of the tracheal tube compared with the nasal route. CONCLUSIONS: For clinicians with less experience in using paediatric bronchoscopes, fibreoptic tracheal intubation through the nasal route may be a more straightforward process than the oral route in children <2 yr of age. CLINICAL TRIAL REGISTRATION: NCT02029300 (www.clinicaltrials.gov).

Elective use of supraglottic airway devices for primary airway management in children with difficult airways.

BACKGROUND: Supraglottic airways (SGAs) have an established role in airway management of difficult airways in both adults and children. However, there are limited data regarding the use of SGAs for primary airway management in children. The aim of this study is to assess the success rates and adverse events related to the use of SGAs for primary airway management during anaesthesia in children with difficult airways. METHODS: A retrospective analysis of SGA use for primary airway management in the difficult airway population in a single centre over a 4-yr period was performed. Difficult airway was defined as either a history of difficult direct laryngoscopy (a documented Cormack and Lehane Grade 3 or greater and the need for an alternate device to direct laryngoscopy for successful tracheal intubation), a history of difficult mask ventilation, or both. The difficult airway condition, patient characteristic data, type and length of procedure, type and size of SGA placed, number of attempts for successful device placement, success/failure associated with the device during anaesthetic maintenance, and complications were recorded. RESULTS: A total of 77,272 children received general anaesthesia in a free-standing paediatric institution. Four hundred and fifty-nine patients were reported to have a difficult airway. Of those, 109 received general anaesthesia and an SGA for primary management, meeting the inclusion criteria for this study during a 4-yr period. An SGA was successfully used in 96% of these patients. In four patients, an alternative airway was needed. CONCLUSIONS: SGAs can be effectively utilized for airway maintenance in the paediatric difficult airway population.

A randomised comparison of free-handed vs air-Q assisted fibreoptic-guided tracheal intubation in children < 2 years of age.

We prospectively compared free-handed and air-Q™ assisted fibreoptic-guided tracheal intubation in children < 2 years of age. Eighty healthy children were enrolled and randomly assigned to a technique (free-handed or air-Q assisted) and operator (trainee or attending). Time, number of attempts and manoeuvres required were assessed. There was no difference in median (IQR [range]) time to successful tracheal intubation between the free-handed (52.2 (34.8-67.7 [19.7-108.0]) s), and the air-Q assisted (60.3 (45.5-75.1 [28.1-129.0]) s; p = 0.13) groups, or the number of attempts needed. The air-Q assisted group required fewer manoeuvres to optimise the laryngeal view (median (IQR [range]) 0 (0-1 [0-2])) than the free-handed group (1 (1-1 [0-3]); p < 0.001). In conclusion, fibreoptic-guided tracheal intubation times were similar with and without the use of the air-Q, but supraglottic airway devices may be a consideration for their other practical advantages.

A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells.

The manufacturing of autologous chimaeric antigen receptor (CAR) T cells largely relies either on fed-batch and manual processes that often lack environmental monitoring and control or on bioreactors that cannot be easily scaled out to meet patient demands. Here we show that human primary T cells can be activated, transduced and expanded to high densities in a 2 ml automated closed-system microfluidic bioreactor to produce viable anti-CD19 CAR T cells (specifically, more than 60 million CAR T cells from donor cells derived from patients with lymphoma and more than 200 million CAR T cells from healthy donors). The in vitro secretion of cytokines, the short-term cytotoxic activity and the long-term persistence and proliferation of the cell products, as well as their in vivo anti-leukaemic activity, were comparable to those of T cells produced in a gas-permeable well. The manufacturing-process intensification enabled by the miniaturized perfusable bioreactor may facilitate the analysis of the growth and metabolic states of CAR T cells during ex vivo culture, the high-throughput optimization of cell-manufacturing processes and the scale out of cell-therapy manufacturing.

Use of the air-Q intubating laryngeal airway for rapid-sequence intubation in infants with severe airway obstruction: a case series.

We describe a four-step method for fibreoptic-guided, rapid-sequence tracheal intubation through the air-Q intubating laryngeal airway in infants with severe airway obstruction. Our step-wise process provides an organised and controlled approach to safely securing the airway.