An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders.

BACKGROUND: Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. METHODS: Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. RESULTS: The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls. CONCLUSIONS: We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities.

Cold seep formation from salt diapir-controlled deep biosphere oases.

Deep sea cold seeps are sites where hydrogen sulfide, methane, and other hydrocarbon-rich fluids vent from the ocean floor. They are an important component of Earth's carbon cycle in which subsurface hydrocarbons form the energy source for highly diverse benthic micro- and macro-fauna in what is otherwise vast and spartan sea scape. Passive continental margin cold seeps are typically attributed to the migration of hydrocarbons generated from deeply buried source rocks. Many of these seeps occur over salt tectonic provinces, where the movement of salt generates complex fault systems that can enable fluid migration or create seals and traps associated with reservoir formation. The elevated advective heat transport of the salt also produces a chimney effect directly over these structures. Here, we provide geophysical and geochemical evidence that the salt chimney effect in conjunction with diapiric faulting drives a subsurface groundwater circulation system that brings dissolved inorganic carbon, nutrient-rich deep basinal fluids, and potentially overlying seawater onto the crests of deeply buried salt diapirs. The mobilized fluids fuel methanogenic archaea locally enhancing the deep biosphere. The resulting elevated biogenic methane production, alongside the upward heat-driven fluid transport, represents a previously unrecognized mechanism of cold seep formation and regulation.

Characterizing enteric neurons in dopamine transporter (DAT)-Cre reporter mice reveals dopaminergic subtypes with dual-transmitter content.

The enteric nervous system (ENS) comprises a complex network of neurons whereby a subset appears to be dopaminergic although the characteristics, roles, and implications in disease are less understood. Most investigations relating to enteric dopamine (DA) neurons rely on immunoreactivity to tyrosine hydroxylase (TH)-the rate-limiting enzyme in the production of DA. However, TH immunoreactivity is likely to provide an incomplete picture. This study herein provides a comprehensive characterization of DA neurons in the gut using a reporter mouse line, expressing a fluorescent protein (tdTomato) under control of the DA transporter (DAT) promoter. Our findings confirm a unique localization of DA neurons in the gut and unveil the discrete subtypes of DA neurons in this organ, which we characterized using both immunofluorescence and single-cell transcriptomics, as well as validated using in situ hybridization. We observed distinct subtypes of DAT-tdTomato neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, while others were positive for a slew of canonical DAergic markers (TH, VMAT2 and GIRK2). Interestingly, we uncovered a seemingly novel population of DA neurons unique to the ENS which was ChAT/DAT-tdTomato-immunoreactive and expressed Grp, Calcb, and Sst. Given the clear heterogeneity of DAergic gut neurons, further investigation is warranted to define their functional signatures and decipher their implication in disease.

MerTK is a mediator of alpha-synuclein fibril uptake by human microglia.

Mer tyrosine kinase (MerTK) is a receptor tyrosine kinase that mediates non-inflammatory, homeostatic phagocytosis of diverse types of cellular debris. Highly expressed on the surface of microglial cells, MerTK is of importance in brain development, homeostasis, plasticity and disease. Yet, involvement of this receptor in the clearance of protein aggregates that accumulate with ageing and in neurodegenerative diseases has yet to be defined. The current study explored the function of MerTK in the microglial uptake of alpha-synuclein fibrils which play a causative role in the pathobiology of synucleinopathies. Using human primary and induced pluripotent stem cell-derived microglia, the MerTK-dependence of alpha-synuclein fibril internalization was investigated in vitro. Relevance of this pathway in synucleinopathies was assessed through burden analysis of MERTK variants and analysis of MerTK expression in patient-derived cells and tissues. Pharmacological inhibition of MerTK and siRNA-mediated MERTK knockdown both caused a decreased rate of alpha-synuclein fibril internalization by human microglia. Consistent with the non-inflammatory nature of MerTK-mediated phagocytosis, alpha-synuclein fibril internalization was not observed to induce secretion of pro-inflammatory cytokines such as IL-6 or TNF, and downmodulated IL-1ß secretion from microglia. Burden analysis in two independent patient cohorts revealed a significant association between rare functionally deleterious MERTK variants and Parkinson's disease in one of the cohorts (P = 0.002). Despite a small upregulation in MERTK mRNA expression in nigral microglia from Parkinson's disease/Lewy body dementia patients compared to those from non-neurological control donors in a single-nuclei RNA-sequencing dataset (P = 5.08 × 10-21), no significant upregulation in MerTK protein expression was observed in human cortex and substantia nigra lysates from Lewy body dementia patients compared to controls. Taken together, our findings define a novel role for MerTK in mediating the uptake of alpha-synuclein fibrils by human microglia, with possible involvement in limiting alpha-synuclein spread in synucleinopathies such as Parkinson's disease. Upregulation of this pathway in synucleinopathies could have therapeutic values in enhancing alpha-synuclein fibril clearance in the brain.

Comparative analysis of methods to reduce activation signature gene expression in PBMCs.

Preserving the in vivo cell transcriptome is essential for accurate profiling, yet factors during cell isolation including time ex vivo and temperature induce artifactual gene expression, particularly in stress-responsive immune cells. In this study, we investigated two methods to mitigate ex vivo activation signature gene (ASG) expression in peripheral blood mononuclear cells (PBMCs): transcription and translation inhibitors (TTis) and cold temperatures during isolation. Comparative analysis of PBMCs isolated with TTis revealed reduced ASG expression. However, TTi treatment impaired responsiveness to LPS stimulation in subsequent in vitro experiments. In contrast, cold isolation methods also prevented ASG expression; up to a point where the addition of TTis during cold isolation offered minimal additional advantage. These findings highlight the importance of considering the advantages and drawbacks of different isolation methods to ensure accurate interpretation of PBMC transcriptomic profiles.

Multi-year seabed environmental baseline in deep-sea offshore oil prospective areas established using microbial biodiversity.

Microorganisms are the ocean's first responders to marine pollution events, yet baseline studies rarely focus on microbial communities. Temporal and spatial microbial biodiversity baselines were established using bacterial 16S rRNA gene amplicon sequencing of seafloor sediments in a deep-water oil prospective area along the Scotian Slope off Canada's east coast sampled during 2015-2018. Bacterial diversity was generally similar in space and time, with members of the family Woeseiaceae detected consistently in >1 % relative abundance, similar to seabed sediments in other parts of the world. Anomalous biodiversity results at one site featured lower Woeseiaceae as well as higher levels of bacterial groups specifically associated with cold seeps such as Aminicenantes. This was unexpected given that site selection was based on sediment geochemistry not revealing any petroleum hydrocarbons in these locations. This finding highlights the sensitivity and specificity of microbial DNA sequencing in environmental monitoring. Microbiome assessments like this one represent an important strategy for incorporating microbial biodiversity as a new and useful metric for establishing robust environmental baselines that are necessary for understanding ecosystem responses to marine pollution.

SituSeq: an offline protocol for rapid and remote Nanopore 16S rRNA amplicon sequence analysis.

Microbiome analysis through 16S rRNA gene sequencing is a crucial tool for understanding the microbial ecology of any habitat or ecosystem. However, workflows require large equipment, stable internet, and extensive computing power such that most of the work is performed far away from sample collection in both space and time. Performing amplicon sequencing and analysis at sample collection would have positive implications in many instances including remote fieldwork and point-of-care medical diagnoses. Here we present SituSeq, an offline and portable workflow for the sequencing and analysis of 16S rRNA gene amplicons using Nanopore sequencing and a standard laptop computer. SituSeq was validated by comparing Nanopore 16S rRNA gene amplicons, Illumina 16S rRNA gene amplicons, and Illumina metagenomes, sequenced using the same environmental DNA. Comparisons revealed consistent community composition, ecological trends, and sequence identity across platforms. Correlation between the abundance of taxa in each taxonomic level in Illumina and Nanopore data sets was high (Pearson's r > 0.9), and over 70% of Illumina 16S rRNA gene sequences matched a Nanopore sequence with greater than 97% sequence identity. On board a research vessel on the open ocean, SituSeq was used to analyze amplicon sequences from deep sea sediments less than 2 h after sequencing, and 8 h after sample collection. The rapidly available results informed decisions about subsequent sampling in near real-time while the offshore expedition was still underway. SituSeq is a portable and user-friendly workflow that helps to bring the power of microbial genomics and diagnostics to many more researchers and situations.

Geological processes mediate a microbial dispersal loop in the deep biosphere.

The deep biosphere is the largest microbial habitat on Earth and features abundant bacterial endospores. Whereas dormancy and survival at theoretical energy minima are hallmarks of microbial physiology in the subsurface, ecological processes such as dispersal and selection in the deep biosphere remain poorly understood. We investigated the biogeography of dispersing bacteria in the deep sea where upward hydrocarbon seepage was confirmed by acoustic imagery and geochemistry. Thermophilic endospores in the permanently cold seabed correlated with underlying seep conduits reveal geofluid-facilitated cell migration pathways originating in deep petroleum-bearing sediments. Endospore genomes highlight adaptations to life in anoxic petroleum systems and bear close resemblance to oil reservoir microbiomes globally. Upon transport out of the subsurface, viable thermophilic endospores reenter the geosphere by sediment burial, enabling germination and environmental selection at depth where new petroleum systems establish. This microbial dispersal loop circulates living biomass in and out of the deep biosphere.

MicroRNA-210 regulates the metabolic and inflammatory status of primary human astrocytes.

BACKGROUND: Astrocytes are the most numerous glial cell type with important roles in maintaining homeostasis and responding to diseases in the brain. Astrocyte function is subject to modulation by microRNAs (miRs), which are short nucleotide strands that regulate protein expression in a post-transcriptional manner. Understanding the miR expression profile of astrocytes in disease settings provides insight into the cellular stresses present in the microenvironment and may uncover pathways of therapeutic interest. METHODS: Laser-capture microdissection was used to isolate human astrocytes surrounding stroke lesions and those from neurological control tissue. Astrocytic miR expression profiles were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Primary human fetal astrocytes were cultured under in vitro stress conditions and transfection of a miR mimic was used to better understand how altered levels of miR-210 affect astrocyte function. The astrocytic response to stress was studied using qPCR, enzyme-linked immunosorbent assays (ELISAs), measurement of released lactate, and Seahorse. RESULTS: Here, we measured miR expression levels in astrocytes around human ischemic stroke lesions and observed differential expression of miR-210 in chronic stroke astrocytes compared to astrocytes from neurological control tissue. We also identified increased expression of miR-210 in mouse white matter tissue around middle cerebral artery occlusion (MCAO) brain lesions. We aimed to understand the role of miR-210 in primary human fetal astrocytes by developing an in vitro assay of hypoxic, metabolic, and inflammatory stresses. A combination of hypoxic and inflammatory stresses was observed to upregulate miR-210 expression. Transfection with miR-210-mimic (210M) increased glycolysis, enhanced lactate export, and promoted an anti-inflammatory transcriptional and translational signature in astrocytes. Additionally, 210M transfection resulted in decreased expression of complement 3 (C3) and semaphorin 5b (Sema5b). CONCLUSIONS: We conclude that miR-210 expression in human astrocytes is modulated in response to ischemic stroke disease and under in vitro stress conditions, supporting a role for miR-210 in the astrocytic response to disease conditions. Further, the anti-inflammatory and pro-glycolytic impact of miR-210 on astrocytes makes it a potential candidate for further research as a neuroprotective agent.

Single Cell Transcriptomics of Ependymal Cells Across Age, Region and Species Reveals Cilia-Related and Metal Ion Regulatory Roles as Major Conserved Ependymal Cell Functions.

Ependymal cells are ciliated-epithelial glial cells that develop from radial glia along the surface of the ventricles of the brain and the spinal canal. They play a critical role in cerebrospinal fluid (CSF) homeostasis, brain metabolism, and the clearance of waste from the brain. These cells have been implicated in disease across the lifespan including developmental disorders, cancer, and neurodegenerative disease. Despite this, ependymal cells remain largely understudied. Using single-cell RNA sequencing data extracted from publicly available datasets, we make key findings regarding the remarkable conservation of ependymal cell gene signatures across age, region, and species. Through this unbiased analysis, we have discovered that one of the most overrepresented ependymal cell functions that we observed relates to a critically understudied role in metal ion homeostasis. Our analysis also revealed distinct subtypes and states of ependymal cells across regions and ages of the nervous system. For example, neonatal ependymal cells maintained a gene signature consistent with developmental processes such as determination of left/right symmetry; while adult ventricular ependymal cells, not spinal canal ependymal cells, appeared to express genes involved in regulating cellular transport and inflammation. Together, these findings highlight underappreciated functions of ependymal cells, which will be important to investigate in order to better understand these cells in health and disease.

Thermogenic hydrocarbon biodegradation by diverse depth-stratified microbial populations at a Scotian Basin cold seep.

At marine cold seeps, gaseous and liquid hydrocarbons migrate from deep subsurface origins to the sediment-water interface. Cold seep sediments are known to host taxonomically diverse microorganisms, but little is known about their metabolic potential and depth distribution in relation to hydrocarbon and electron acceptor availability. Here we combined geophysical, geochemical, metagenomic and metabolomic measurements to profile microbial activities at a newly discovered cold seep in the deep sea. Metagenomic profiling revealed compositional and functional differentiation between near-surface sediments and deeper subsurface layers. In both sulfate-rich and sulfate-depleted depths, various archaeal and bacterial community members are actively oxidizing thermogenic hydrocarbons anaerobically. Depth distributions of hydrocarbon-oxidizing archaea revealed that they are not necessarily associated with sulfate reduction, which is especially surprising for anaerobic ethane and butane oxidizers. Overall, these findings link subseafloor microbiomes to various biochemical mechanisms for the anaerobic degradation of deeply-sourced thermogenic hydrocarbons.

Associative and sensorimotor learning for parenting involves mirror neurons under the influence of oxytocin.

Mirror neuron-based associative learning may be understood according to associative learning theories, in addition to sensorimotor learning theories. This is important for a comprehensive understanding of the role of mirror neurons and related hormone modulators, such as oxytocin, in complex social interactions such as among parent-infant dyads and in examples of mirror neuron function that involve abnormal motor systems such as depression.

Modeling the solid-liquid phase transition in saturated triglycerides.

We investigated theoretically two competing published scenarios for the melting transition of the triglyceride trilaurin (TL): those of (1) Corkery et al. [Langmuir 23, 7241 (2007)], in which the average state of each TL molecule in the liquid phase is a discotic "Y" conformer whose three chains are dynamically twisted, with an average angle of approximately 120 degrees between them, and those of (2) Cebula et al. [J. Am. Oil Chem. Soc. 69, 130 (1992)], in which the liquid-state conformation of the TL molecule in the liquid phase is a nematic h*-conformer whose three chains are in a modified "chair" conformation. We developed two competing models for the two scenarios, in which TL molecules are in a nematic compact-chair (or "h") conformation, with extended, possibly all-trans, chains at low-temperatures, and in either a Y conformation or an h* conformation in the liquid state at temperatures higher than the phase-transition temperature, T*=319 K. We defined an h-Y model as a realization of the proposal of Corkery et al. [Langmuir 23, 7241 (2007)], and explored its predictions by mapping it onto an Ising model in a temperature-dependent field, performing a mean-field approximation, and calculating the transition enthalpy DeltaH. We found that the most plausible realization of the h-Y model, as applied to the solid-liquid phase transition in TL, and likely to all saturated triglycerides, gave a value of DeltaH in reasonable agreement with the experiment. We then defined an alternative h-h* model as a realization of the proposal of Cebula et al. [J. Am. Oil Chem. Soc. 69, 130 (1992)], in which the liquid phase exhibits an average symmetry breaking similar to an h conformation, but with twisted chains, to see whether it could describe the TL phase transition. The h-h* model gave a value of DeltaH that was too small by a factor of approximately 3-4. We also predicted the temperature dependence of the 1132 cm(-1) Raman band for both models, and performed measurements of the ratios of three TL Raman bands in the temperature range of -20 degrees C < or = T < or = 90 degrees C. The experimental results were in accord with the predictions of the h-Y model and support the proposal of Corkery et al. [Langmuir 23, 7241 (2007)] that the liquid state is made up of molecules that are each, on average, in a Y conformation. Finally, we carried out computer simulations of minimal-model TLs in the liquid phase, and concluded that although the individual TL molecules are, on average, Y conformers, long-range discotic order is unlikely to exist.

Binary weighted averaging of an ensemble of coherently collected image frames.

Recent interest in the collection of remote laser radar imagery has motivated novel systems that process temporally contiguous frames of collected imagery to produce an average image that reduces laser speckle, increases image SNR, decreases the deleterious effects of atmospheric distortion, and enhances image detail. This research seeks an algorithm based on Bayesian estimation theory to select those frames from an ensemble that increases spatial resolution compared to simple unweighted averaging of all frames. The resulting binary weighted motion-compensated frame average is compared to the unweighted average using simulated and experimental data collected from a fielded laser vision system. Image resolution is significantly enhanced as quantified by the estimation of the atmospheric seeing parameter through which the average image was formed.

Monitoring exercise-induced changes in glycemic control in type 2 diabetes.

PURPOSE: The present study determined the efficacy of the Continuous Glucose Monitoring System (CGMS) during moderate exercise and monitored the changes in whole-day glucose profiles using the CGMS in individuals with and without type 2 diabetes. METHODS: Six, obese, diet-treated individuals with and four age-matched individuals without type 2 diabetes were monitored using the CGMS for 3 d. Subjects cycled at 90% of a predetermined lactate threshold for 1 h at approximately 09:00 h on day 2, during which venous blood was sampled at 10-min intervals and immediately analyzed for glucose concentrations. RESULTS: Venous blood glucose and CGMS values declined during exercise in the diabetes (P < 0.001) but not the control group (P = 0.085). The CGMS overestimated blood glucose in the control (P = 0.003) and the diabetes (P = 0.045) groups during exercise. The number of data points outside of the 95% confidence intervals was <5% in both groups, showing that there is a statistically acceptable level of agreement between venous blood glucose and CGMS values during exercise. Moderate exercise improved whole-day average glucose concentrations (P = 0.007) and whole-day area under the glucose curve (P = 0.016) values (AUCglu), and the time spent within +/-10% of fasting venous glucose (FVG) increased in the diabetes group (P = 0.021). No such effects were seen in the control group. CONCLUSION: Using continuous glucose monitoring we were able to demonstrate that a period of moderate exercise improved whole-day glycemic control in obese individuals with type 2 diabetes. The CGMS should only be used as an adjunct and not as an alternative to frequent blood glucose sampling when examining the changes in glucose values during exercise in individuals with and without type 2 diabetes.

Precooling leg muscle improves intermittent sprint exercise performance in hot, humid conditions.

We used three techniques of precooling to test the hypothesis that heat strain would be alleviated, muscle temperature (Tmu) would be reduced, and as a result there would be delayed decrements in peak power output (PPO) during exercise in hot, humid conditions. Twelve male team-sport players completed four cycling intermittent sprint protocols (CISP). Each CISP consisted of twenty 2-min periods, each including 10 s of passive rest, 5 s of maximal sprint against a resistance of 7.5% body mass, and 105 s of active recovery. The CISP, preceded by 20 min of no cooling (Control), precooling via an ice vest (Vest), cold water immersion (Water), and ice packs covering the upper legs (Packs), was performed in hot, humid conditions (mean +/- SE; 33.7 +/- 0.3 degrees C, 51.6 +/- 2.2% relative humidity) in a randomized order. The rate of heat strain increase during the CISP was faster in Control than Water and Packs (P < 0.01), but it was similar to Vest. Packs and Water blunted the rise of Tmu until minute 16 and for the duration of the CISP (40 min), respectively (P < 0.01). Reductions in PPO occurred from minute 32 onward in Control, and an increase in PPO by approximately 4% due to Packs was observed (main effect; P < 0.05). The method of precooling determined the extent to which heat strain was reduced during intermittent sprint cycling, with leg precooling offering the greater ergogenic effect on PPO than either upper body or whole body cooling.

Lactate--a signal coordinating cell and systemic function.

Since its first documented observation in exhausted animal muscle in the early 19th century, the role of lactate (lactic acid) has fascinated muscle physiologists and biochemists. Initial interpretation was that lactate appeared as a waste product and was responsible in some way for exhaustion during exercise. Recent evidence, and new lines of investigation, now place lactate as an active metabolite, capable of moving between cells, tissues and organs, where it may be oxidised as a fuel or reconverted to form pyruvate or glucose. The questions now to be asked concern the effects of lactate at the systemic and cellular level on metabolic processes. Does lactate act as a metabolic signal to specific tissues, becoming a metabolite pseudo-hormone? Does lactate have a role in whole-body coordination of sympathetic/parasympathetic nerve system control? And, finally, does lactate play a role in maintaining muscle excitability during intense muscle contraction? The concept of lactate acting as a signalling compound is a relatively new hypothesis stemming from a combination of comparative, cell and whole-organism investigations. It has been clearly demonstrated that lactate is capable of entering cells via the monocarboxylate transporter (MCT) protein shuttle system and that conversion of lactate to and from pyruvate is governed by specific lactate dehydrogenase isoforms, thereby forming a highly adaptable metabolic intermediate system. This review is structured in three sections, the first covering pertinent topics in lactate's history that led to the model of lactate as a waste product. The second section will discuss the potential of lactate as a signalling compound, and the third section will identify ways in which such a hypothesis might be investigated. In examining the history of lactate research, it appears that periods have occurred when advances in scientific techniques allowed investigation of this metabolite to expand. Similar to developments made first in the 1920s and then in the 1980s, contemporary advances in stable isotope, gene microarray and RNA interference technologies may allow the next stage of understanding of the role of this compound, so that, finally, the fundamental questions of lactate's role in whole-body and localised muscle function may be answered.