Validated method for automated glioma diagnosis from GFAP immunohistological images: a complete pipeline

Background and objective: Glial fibrillar acid protein is a common marker for brain tumor because of its particular rearrangement during tumor development. It is commonly used in manually histological glioma detection and grading. An automatic pipeline for tumor diagnosis based on GFAP is proposed in the present manuscript for detecting and grading canine brain glioma in stages III and IV. Methods: The study was performed on canine brain tumor stages III and IV as well as healthy tissue immunohistochemically stained for gliofibrillar astroglial protein. Four stereological indexes were developed using the area of the image as reference unit: density of glioma protein, density of neuropil, density of astrocytes and the glioma nuclei number density. Images of the slides were subset for image analysis (n=1415) and indexed. The stereological indexes of each subset constituted an array of data describing the tumor phase of the subset. A 5% of these arrays were used as training set for decision tree classification with PCA. The other arrays were further classified in a supervised approach. ANOVA and PCA analysis were applied to the indexes. Results: The final pipeline is able to detect brain tumor and to grade it automatically. Added to it, the role the neuropil during tumor development has been quantified for the first time. While astroglial cells tend to disappear, glioma cells invade all the tumor area almost to a saturation in stage III before reducing the density in stage IV. The density of the neuropil is reduced during the tumour growth. Conclusions: The method validated ere allows the automated diagnosis and grading of glioma in dogs. This method opens the research of the role of the neuropil in tumor development.

Theoretical model of efficient phagocytosis driven by curved membrane proteins and active cytoskeleton forces

Phagocytosis is the process of engulfment and internalization of comparatively large particles by the cell, that plays a central role in the functioning of our immune system. We study the process of phagocytosis by considering a simplified coarse grained model of a three-dimensional vesicle, having uniform adhesion interaction with a rigid particle, in the presence of curved membrane proteins and active cytoskeletal forces. Complete engulfment is achieved when the bending energy cost of the vesicle is balanced by the gain in the adhesion energy. The presence of curved (convex) proteins reduces the bending energy cost by self-organizing with higher density at the highly curved leading edge of the engulfing membrane, which forms the circular rim of the phagocytic cup that wraps around the particle. This allows the engulfment to occur at much smaller adhesion strength. When the curved proteins exert outwards protrusive forces, representing actin polymerization, at the leading edge, we find that engulfment is achieved more quickly and at lower protein density. We consider spherical as well as non-spherical particles, and find that non-spherical particles are more difficult to engulf in comparison to the spherical particles of the same surface area. For non-spherical particles, the engulfment time crucially depends upon the initial orientation of the particles with respect to the vesicle. Our model offers a mechanism for the spontaneous self-organization of the actin cytoskeleton at the phagocytic cup, in good agreement with recent high-resolution experimental observations.

Maximum-likelihood model fitting for quantitative analysis of SMLM data

Quantitative analysis is an important part of any single-molecule localization microscopy (SMLM) data analysis workflow to extract biological insights from the coordinates of the single fluorophores, but current approaches are restricted to simple geometries or do not work on heterogenous structures. Here, we present LocMoFit (Localization Model Fit), an open-source framework to fit an arbitrary model directly to the localization coordinates in SMLM data. Using maximum likelihood estimation, this tool extracts the most likely parameters for a given model that best describe the data, and can select the most likely model from alternative models. We demonstrate the versatility of LocMoFit by measuring precise dimensions of the nuclear pore complex and microtubules. We also use LocMoFit to assemble static and dynamic multi-color protein density maps from thousands of snapshots. In case an underlying geometry cannot be postulated, LocMoFit can perform single-particle averaging of super-resolution structures without any assumption about geometry or symmetry. We provide extensive simulation and visualization routines to validate the robustness of LocMoFit and tutorials based on example data to enable any user to increase the information content they can extract from their SMLM data.

Formin Activity and mDia1 Contribute to Maintain Axon Initial Segment Composition and Structure

The axon initial segment (AIS) is essential for maintaining neuronal polarity, modulating protein transport into the axon, and action potential generation. These functions are supported by a distinctive actin and microtubule cytoskeleton that controls axonal trafficking and maintains a high density of voltage-gated ion channels linked by scaffold proteins to the AIS cytoskeleton. However, our knowledge of the mechanisms and proteins involved in AIS cytoskeleton regulation to maintain or modulate AIS structure is limited. In this context, formins play a significant role in the modulation of actin and microtubules. We show that pharmacological inhibition of formins modifies AIS actin and microtubule characteristics in cultured hippocampal neurons, reducing F-actin density and decreasing microtubule acetylation. Moreover, formin inhibition diminishes sodium channels, ankyrinG and βIV-spectrin AIS density, and AIS length, in cultured neurons and brain slices, accompanied by decreased neuronal excitability. We show that genetic downregulation of the mDia1 formin by interference RNAs also decreases AIS protein density and shortens AIS length. The ankyrinG decrease and AIS shortening observed in pharmacologically inhibited neurons and neuron-expressing mDia1 shRNAs were impaired by HDAC6 downregulation or EB1-GFP expression, known to increase microtubule acetylation or stability. However, actin stabilization only partially prevented AIS shortening without affecting AIS protein density loss. These results suggest that mDia1 maintain AIS composition and length contributing to the stability of AIS microtubules.

A feasibility study to test a novel approach to dietary weight loss with a focus on assisting informed decision making in food selection

Obesity is a significant contributor to the development of chronic diseases, some of which can be prevented or reversed by weight loss. However, dietary weight loss programs have shortcomings in success rate and magnitude or sustainability of weight loss. The objective of the Individualized Diet Improvement Program (iDip) was to test the feasibility of a novel approach that helps individuals self-select a sustainable diet for weight loss and maintenance instead of providing weight loss products or rigid diet instructions to follow. The iDip study consisted of 22 dietary improvement sessions over 12 months with six months of follow-up. Daily weights were collected, and a chart summarizing progress was provided weekly. Six 24-hour dietary records were collected, and dietary feedback was provided in the form of a protein-fiber plot, in which protein/energy and fiber/energy of foods were plotted two-dimensionally together with a target box specific to weight loss or maintenance. An exit survey was conducted at 12 months. Twelve (nine female, 46.3±3.1 years (mean±SE)) of the initial 14 participants (BMI>28 kg/m2) completed all sessions. Mean percent weight loss (n=12) at six and 12 months was -4.9%±1.1 (p=0.001) and -5.4%±1.7 (p=0.007), respectively. Weight loss varied among individuals at 12 months; top and bottom halves (n=6 each) achieved -9.7%±1.7 (p=0.0008) and -1.0%±1.4 weight loss, respectively. The 24-hour records showed a significant increase in protein density from baseline to final (4.1g/100kcal±0.3 vs. 5.7g/100kcal±0.5 (p=0.008)). Although mean fiber density showed no significant change from the first month (1.3g/100kcal±0.1), the top half had significantly higher fiber/energy intake than the bottom half group. The survey suggested that all participants valued the program and its individualized approach. In conclusion, half of the participants successfully lost >5% and maintained the lost weight for 12 months without strict diet instructions, showing the feasibility of the informed decision-making approach.

Principles of gene regulation quantitatively connect DNA to RNA and proteins in bacteria

Bacteria allocate their proteome to cellular functions differently in different growth conditions. It is largely unknown how such allocation arises from known mechanisms of gene regulation while constrained by limited translation capacity and fixed protein density. Here, we performed absolute transcriptomic and proteomic analysis for E. coli across many conditions, obtaining a plethora of results on promoters and mRNAs characteristics that clash with conventional expectations: the majority of mRNAs exhibit similar translational efficiencies, while the promoter strengths are vastly different across genes. These characteristics prescribe two principles of gene regulation guiding bacteria to attain the desired protein allocation under global constraints: Total transcriptional output is tightly coordinated with ribosomal activity, and the concentrations of individual proteins are largely set by transcription. These two principles lead to a quantitative formulation of Central Dogma which unravels the complex relationship between gene regulatory activities and mRNA/protein concentrations across conditions. The knowledge obtained will be invaluable for accurately inferring gene regulatory interactions from 'omics data, as well as for guiding the design of genetic circuits for synthetic biology applications in E. coli and other organisms.

Artemisinin-treatment in pre-symptomatic APP-PS1 mice increases gephyrin phosphorylation at Ser270: a modification regulating postsynaptic GABAAR density

Artemisinins, a group of plant-derived sesquiterpene lactones, are efficient antimalarial agents. They also share anti-inflammatory and anti-viral activities and were considered for treatment of neurodegenerative disorders like Alzheimer’s disease (AD). Additionally, artemisinins bind to gephyrin, the multifunctional scaffold of GABAergic synapses, and modulate inhibitory neurotransmission in vitro. We previously reported an increased expression of gephyrin and GABAA receptors in early pre-symptomatic stages of an AD mouse model (APP-PS1) and in parallel enhanced CDK5-dependent phosphorylation of gephyrin at S270. Here, we studied the effects of artemisinin on gephyrin in the brain of young APP-PS1 mice. We detected an additional increase of gephyrin protein level, elevated gephyrin phosphorylation at Ser270, and an increased amount of GABAAR-γ2 subunits after artemisinin-treatment. Interestingly, the CDK5 activator p35 was also upregulated. Moreover, we demonstrate decreased density of postsynaptic gephyrin and GABAAR-γ2 immunoreactivities in cultured hippocampal neurons expressing gephyrin with alanine mutations at two CDK5 phosphorylation sites. In addition, the activity-dependent modulation of synaptic protein density was abolished in neurons expressing gephyrin lacking one or both of these phosphorylation sites. Thus, our results reveal that artemisinin modulates expression as well as phosphorylation of gephyrin at sites that might have important impact on GABAergic synapses in AD.

Biomarker-Calibrated Macronutrient Intake and Chronic Disease Risk among Postmenopausal Women

ABSTRACT Background Knowledge about macronutrient intake and chronic disease risk has been limited by the absence of objective macronutrient measures. Recently, we proposed novel biomarkers for protein, protein density, carbohydrate, and carbohydrate density, using established biomarkers and serum and urine metabolomics profiles in a human feeding study. Objectives We aimed to use these biomarkers to develop calibration equations for macronutrient variables using dietary self-reports and personal characteristics and to study the association between biomarker-calibrated intake estimates and cardiovascular disease, cancer, and diabetes risk in Women's Health Initiative (WHI) cohorts. Methods Prospective disease association analyses are based on WHI cohorts of postmenopausal US women aged 50–79 y when enrolled at 40 US clinical centers (n = 81,954). We used biomarker intake values in a WHI nutritional biomarker study (n = 436) to develop calibration equations for each macronutrient variable, leading to calibrated macronutrient intake estimates throughout WHI cohorts. We then examined the association of these intakes with chronic disease incidence over a 20-y (median) follow-up period using HR regression methods. Results In analyses that included doubly labeled water–calibrated total energy, HRs for cardiovascular diseases and cancers were mostly unrelated to calibrated protein density. However, many were inversely related to carbohydrate density, with HRs (95% CIs) for a 20% increment in carbohydrate density of 0.81 (0.69, 0.95) and 0.83 (0.74, 0.93), respectively, for primary outcomes of coronary heart disease and breast cancer, as well as 0.74 (0.60, 0.91) and 0.87 (0.81, 0.93) for secondary outcomes of heart failure and total invasive cancer. Corresponding HRs (95% CIs) for type 2 diabetes incidence in relation to protein density and carbohydrate density were 1.17 (1.09, 1.75) and 0.73 (0.66, 0.80), respectively. Conclusions At specific energy intake, a diet high in carbohydrate density is associated with substantially reduced risk of major chronic diseases in a population of US postmenopausal women. This trial was registered at clinicaltrials.gov as NCT00000611.

Ecto-GPR37: a potential biomarker for Parkinson’s disease

Objective α-Synuclein has been studied as a potential biomarker for Parkinson’s disease (PD) with no concluding results. Accordingly, there is an urgent need to find out reliable specific biomarkers for PD. GPR37 is an orphan G protein-coupled receptor that toxically accumulates in autosomal recessive juvenile parkinsonism. Here, we investigated whether GPR37 is upregulated in sporadic PD, and thus a suitable potential biomarker for PD. Methods GPR37 protein density and mRNA expression in postmortem substantia nigra (SN) from PD patients were analysed by immunoblot and RT-qPCR, respectively. The presence of peptides from the N-terminus-cleaved domain of GPR37 (i.e. ecto-GPR37) in human cerebrospinal fluid (CSF) was determined by liquid chromatography-mass spectrometric analysis. An engineered in-house nanoluciferase-based immunoassay was used to quantify ecto-GPR37 in CSF samples from neurological control (NC) subjects, PD patients and Alzheimer’s disease (AD) patients. Results GPR37 protein density and mRNA expression were significantly augmented in sporadic PD. Increased amounts of ecto-GPR37 peptides in the CSF samples from PD patients were identified by mass spectrometry and quantified by the in-house ELISA method. However, the CSF total α-synuclein level in PD patients did not differ from that in NC subjects. Similarly, the cortical GPR37 mRNA expression and CSF ecto-GPR37 levels in AD patients were also unaltered. Conclusion GPR37 expression is increased in SN of sporadic PD patients. The ecto-GPR37 peptides are significantly increased in the CSF of PD patients, but not in AD patients. These results open perspectives and encourage further clinical studies to confirm the validity and utility of ecto-GPR37 as a potential PD biomarker.