Precise control of the thyroid hormone (T3)-dependent transcriptional program is required by multiple cell systems, including muscle stem cells. Deciphering how this is achieved and how the T3 signal is controlled in stem cell niches is essentially unknown. We report that in response to proliferative stimuli such as acute skeletal muscle injury, type 3 deiodinase (D3), the thyroid hormone-inactivating enzyme, is induced in satellite cells where it reduces intracellular thyroid signaling. Satellite cell-specific genetic ablation of dio3 severely impairs skeletal muscle regeneration. This impairment is due to massive satellite cell apoptosis caused by exposure of activated satellite cells to the circulating TH. The execution of this proapoptotic program requires an intact FoxO3/MyoD axis, both genes positively regulated by intracellular TH. Thus, D3 is dynamically exploited in vivo to chronically attenuate TH signaling under basal conditions while also being available to acutely increase gene programs required for satellite cell lineage progression.

Neonatal β cells do not secrete glucose-responsive insulin and are considered immature. We previously showed the transcription factor MAFA is key for the functional maturation of β cells, but the physiological regulators of this process are unknown. Here we show that postnatal rat β cells express thyroid hormone (TH) receptor isoforms and deiodinases in an age-dependent pattern as glucose responsiveness develops. In vivo neonatal triiodothyronine supplementation and TH inhibition, respectively, accelerated and delayed metabolic development. In vitro exposure of immature islets to triiodothyronine enhanced the expression of Mafa, the secretion of glucose-responsive insulin, and the proportion of responsive cells, all of which are effects that were abolished in the presence of dominant-negative Mafa. Using chromatin immunoprecipitation and electrophoretic mobility shift assay, we show that TH has a direct receptor-ligand interaction with the Mafa promoter and, using a luciferase reporter, that this interaction was functional. Thus, TH can be considered a physiological regulator of functional maturation of β cells via its induction of Mafa.

Developing a safe and effective vaccine will be the principal way of controlling the COVID-19 pandemic. However, current COVID-19 vaccination trials are not adequately representing a diverse participant population in terms of age, ethnicity and comorbidities. Achieving the representative recruitment targets that are adequately powered to the study remains one of the greatest challenges in clinical trial management. To ensure accuracy and generalisability of the safety and efficacy conclusions generated by clinical trials, it is crucial to recruit patient cohorts as representative as possible of the future target population. Missing these targets can lead to reduced validity of the study results and can often slow down drug development leading to costly delays.

One of the main drivers within the field of bottom-up synthetic biology is to develop artificial chemical machines, perhaps even living systems, that have programmable functionality. Numerous toolkits exist to generate giant unilamellar vesicle-based artificial cells. However, methods able to quantitatively measure their molecular constituents upon formation is an underdeveloped area. We report an artificial cell quality control (AC/QC) protocol using a microfluidic-based single-molecule approach, enabling the absolute quantification of encapsulated biomolecules. While the measured average encapsulation efficiency was 11.4 ± 6.8%, the AC/QC method allowed us to determine encapsulation efficiencies per vesicle, which varied significantly from 2.4 to 41%. We show that it is possible to achieve a desired concentration of biomolecule within each vesicle by commensurate compensation of its concentration in the seed emulsion. However, the variability in encapsulation efficiency suggests caution is necessary when using such vesicles as simplified biological models or standards.

Serotonin (5-HT) is a well-known modulator of eating behavior. However, the molecular mechanisms linking its action to body weight balance have been only partially elucidated. Since platelets are a suitable peripheral model to study 5-HT transport, metabolism and release, we herein evaluated the expression of the platelet 5-HT re-uptake system (SERT) by [3H]-paroxetine binding assay. A cohort of 114 unrelated individuals (34 males, 80 females; age, mean ± SD: 38.57 ± 12.47 years) without major psychiatric disorders, was recruited following a naturalistic design regarding age or gender and classified accordingly to their body mass index (BMI). Subjects were divided into 5 groups: normal-weight (NW), overweight (OW) and grade I-III obese (OB) individuals. For gender analyses, data were transformed into [3H]-paroxetine density (Bmax)/BMI ratios to overcome both the disparity of women vs. men number and anthropometric differences between sexes.

Mammalian acyl-CoA thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoAs to form free fatty acids plus CoA, but their metabolic functions remain undefined. Thioesterase superfamily member 1 (Them1; synonyms Acot11, StarD14, and brown fat inducible thioesterase) is a long-chain fatty acyl-CoA thioesterase that is highly expressed in brown adipose tissue and is regulated by both ambient temperature and food consumption. Here we show that Them1(-/-) mice were resistant to diet-induced obesity despite greater food consumption. Them1(-/-) mice exhibited increased O(2) consumption and heat production, which were accompanied by increased rates of fatty acid oxidation in brown adipose tissue and up-regulation of genes that promote energy expenditure. Them1(-/-) mice were also protected against diet-induced inflammation in white adipose tissue, as well as hepatic steatosis, and demonstrated improved glucose homeostasis. The absence of Them1 expression in vivo and in cell culture led to markedly attenuated diet- or chemically induced endoplasmic reticulum stress responses, providing a mechanism by which Them1 deficiency protects against insulin resistance and lipid deposition. Taken together, these data suggest that Them1 functions to decrease energy consumption and conserve calories. In the setting of nutritional excess, the overproduction of free fatty acids by Them1 provokes insulin resistance that is associated with inflammation and endoplasmic reticulum stress.

Heterotrimeric G-proteins are ubiquitously expressed in several cancers, and they transduce signals from activated G-protein coupled receptors. These proteins have numerous biological functions, and they are becoming interesting target molecules in cancer therapy. Previously, we have shown that heterotrimeric G-protein subunit alphai2 (Gαi2) has an essential role in the migration and invasion of prostate cancer cells. Using a structure-based approach, we have synthesized optimized small molecule inhibitors that are able to prevent specifically the activation of the Gαi2 subunit, keeping the protein in its inactive GDP-bound state. We observed that two of the compounds (13 and 14) at 10 μΜ significantly inhibited the migratory behavior of the PC3 and DU145 prostate cancer cell lines. Additionally, compound 14 at 10 μΜ blocked the activation of Gαi2 in oxytocin-stimulated prostate cancer PC3 cells, and inhibited the migratory capability of DU145 cells overexpressing the constitutively active form of Gαi2, under basal and EGF-stimulated conditions. We also observed that the knockdown or inhibition of Gαi2 negatively regulated migration of renal and ovarian cancer cell lines. Our results suggest that small molecule inhibitors of Gαi2 have potential as leads for discovering novel anti-metastatic agents for attenuating the capability of cancer cells to spread and invade to distant sites.

Information from the retina is carried along the visual pathway with accuracy and spatial conservation as a result of topographically mapped axonal connections. The optic tectum in the midbrain is the primary region to which retinal ganglion cells project their axons in the chick. The two primary axes of the retina project independently onto the tectum using different sets of guidance cues to give rise to the retinotectal map. Specificity of the map is determined by attractive or repulsive interactions between molecular tags that are distributed in gradients in the retina and the tectum. Despite several studies, knowledge of the retinotectal guidance molecules is far from being complete. We screened for all molecules that are expressed differentially along the anterior-posterior and medial-lateral axes of the chick tectum using microarray based transcriptional profiling and identified several novel candidate retinotectal guidance molecules. Two such genes, encoding Wnt5a and Raldh2, the synthesizing enzymes for retinoic acid, were further analyzed for their function as putative regulators of retinotectal map formation. Wnt5a and retinoic acid were found to exhibit differential effects on the growth of axons from retinal explants derived from different quadrants of the retina. This screen also yielded a large number of genes expressed in a lamina-specific manner in the tectum, which may have other roles in tectal development. J. Comp. Neurol. 525:459-477, 2017. © 2016 Wiley Periodicals, Inc.

Hypophysiotropic thyrotropin-releasing hormone (TRH) neurons, the central regulators of the hypothalamic-pituitary-thyroid axis, are located in the hypothalamic paraventricular nucleus (PVN) in a partly overlapping distribution with non-hypophysiotropic TRH neurons. The distribution of hypophysiotropic TRH neurons in the rat PVN is well understood, but the localization of these neurons is unknown in mice. To determine the distribution and phenotype of hypophysiotropic TRH neurons in mice, double- and triple-labeling experiments were performed on sections of intact mice, and mice treated intravenously and intraperitoneally with the retrograde tracer Fluoro-Gold. TRH neurons were located in all parts of the PVN except the periventricular zone. Hypophysiotropic TRH neurons were observed only at the mid-level of the PVN, primarily in the compact part. In this part of the PVN, TRH neurons were intermingled with oxytocin and vasopressin neurons, but based on their size, the TRH neurons were parvocellular and did not contain magnocellular neuropeptides. Co-localization of TRH and cocaine- and amphetamine-regulated transcript (CART) were observed only in areas where hypophysiotropic TRH neurons were located. In accordance with the morphological observations, hypothyroidism increased TRH mRNA content of neurons only at the mid-level of the PVN. These data demonstrate that the distribution of hypophysiotropic TRH neurons in mice is vastly different from the pattern in rats, with a dominant occurrence of these neurosecretory cells in the compact part and adjacent regions at the mid-level of the PVN. Furthermore, our data demonstrate that the organization of the PVN is markedly different in mice and rats.

To determine the seroprevalence of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibody status amongst healthcare workers (HCWs) working through the first wave of the Coronavirus (COVID-19) pandemic in 2020. To examine the association of seroprevalence and self-reported COVID-19 symptoms with occupation, sex, and ethnicity; and how these factors were associated with physical and mental wellbeing.

Serial section electron microscopy (ssEM), a technique where volumes of tissue can be anatomically reconstructed by imaging consecutive tissue slices, has proven to be a powerful tool for the investigation of brain anatomy. Between the process of cutting the slices, or "sections," and imaging them, however, handling 10°-106 delicate sections remains a bottleneck in ssEM, especially for batches in the "mesoscale" regime, i.e., 102-103 sections. We present a tissue section handling device that transports and positions sections, accurately and repeatability, for automated, robotic section pick-up and placement onto an imaging substrate. The device interfaces with a conventional ultramicrotomy diamond knife, accomplishing in-line, exact-constraint trapping of sections with 100-μm repeatability. An associated mathematical model includes capillary-based and Stokes-based forces, accurately describing observed behavior and fundamentally extends the modeling of water-air interface forces. Using the device, we demonstrate and describe the limits of reliable handling of hundreds of slices onto a variety of electron and light microscopy substrates without significant defects (n = 8 datasets composed of 126 serial sections in an automated fashion with an average loss rate and throughput of 0.50% and 63 s/section, respectively. In total, this work represents an automated mesoscale serial sectioning system for scalable 3D-EM connectomics.

Serial section transmission electron microscopy (ssTEM) is the most promising tool for investigating the three-dimensional anatomy of the brain with nanometer resolution. Yet as the field progresses to larger volumes of brain tissue, new methods for high-yield, low-cost, and high-throughput serial sectioning are required. Here, we introduce LASSO (Loop-based Automated Serial Sectioning Operation), in which serial sections are processed in "batches." Batches are quantized groups of individual sections that, in LASSO, are cut with a diamond knife, picked up from an attached waterboat, and placed onto microfabricated TEM substrates using rapid, accurate, and repeatable robotic tools. Additionally, we introduce mathematical models for ssTEM with respect to yield, throughput, and cost to access ssTEM scalability. To validate the method experimentally, we processed 729 serial sections of human brain tissue (~40 nm x 1 mm x 1 mm). Section yield was 727/729 (99.7%). Sections were placed accurately and repeatably (x-direction: -20 ± 110 μm (1 s.d.), y-direction: 60 ± 150 μm (1 s.d.)) with a mean cycle time of 43 s ± 12 s (1 s.d.). High-magnification (2.5 nm/px) TEM imaging was conducted to measure the image quality. We report no significant distortion, information loss, or substrate-derived artifacts in the TEM images. Quantitatively, the edge spread function across vesicle edges and image contrast were comparable, suggesting that LASSO does not negatively affect image quality. In total, LASSO compares favorably with traditional serial sectioning methods with respect to throughput, yield, and cost for large-scale experiments, and represents a flexible, scalable, and accessible technology platform to enable the next generation of neuroanatomical studies.

The type 2 iodothyronine deiodinase (D2) converts the pro-hormone thyroxine into T3 within target tissues. D2 is essential for a full thermogenic response of brown adipose tissue (BAT), and mice with a disrupted Dio2 gene (D2KO) have an impaired response to cold. BAT is also activated by overfeeding.

A key challenge towards a successful COVID-19 vaccine uptake is vaccine hesitancy. We examine and provide novel insights on the key drivers and barriers towards COVID-19 vaccine uptake.

This study aims to determine differences in the on- and off-tumor microbiota between patients with right- and left-sided colorectal cancer. Microbiome profiling of tumor and tumor-adjacent biopsies from patients with right-sided (n = 17) and left-sided (n = 7) colorectal adenocarcinoma was performed using 16S ribosomal RNA sequencing. Off-tumor alpha and beta diversity were significantly different between right- and left-sided colorectal cancer patients. However, no differences in on-tumor diversity were observed between tumor locations. Comparing the off-tumor microbiota showed the right colon to be enriched with species of the Lachnoclostridium, Selenomonas, and Ruminococcus genera. Whereas the left colon is enriched with Epsilonbacteraeota phylum, Campylobacteria class, and Pasteurellales and Campylobacterales orders, in contrast, the on-tumor microbiota showed relatively fewer differences in bacterial taxonomy between tumor sites, with left tumors being enriched with Methylophilaceae and Vadin BE97 families and Alloprevotella, Intestinibacter, Romboutsia, and Ruminococcus 2 genera. Patients with left-sided colorectal cancer had large taxonomic differences between their paired on- and off-tumor microbiota, while patients with right-sided colorectal cancer showed relatively fewer taxonomic differences. Collectively, this suggests that the right and left colon show distinctive bacterial populations; however, the presence of a colonic tumor leads to a more consistent microbiota between locations.

The type 2 iodothyronine-deiodinase (D2) enzyme converts T4 to T3, and mice deficient in this enzyme [D2 knockout (D2KO) mice] have decreased T3 derived from T4 in skeletal muscle despite normal circulating T3 levels. Because slow skeletal muscle is particularly susceptible to changes in T3 levels, we expected D2 inactivation to result in more pronounced slow-muscle characteristics in the soleus muscle, mirroring hypothyroidism. However, ex vivo studies of D2KO soleus revealed higher rates of twitch contraction and relaxation and reduced resistance to fatigue. Immunostaining of D2KO soleus showed that these properties were associated with changes in muscle fiber type composition, including a marked increase in the number of fast, glycolytic type IIB fibers. D2KO soleus muscle fibers had a larger cross-sectional area, and this correlated with increased myonuclear accretion in myotubes formed from D2KO skeletal muscle precursor cells differentiated in vitro. Consistent with our functional findings, D2KO soleus gene expression was markedly different from that in hypothyroid wild-type (WT) mice. Comparison of gene expression between euthyroid WT and D2KO mice indicated that PGC-1α, a T3-dependent regulator of slow muscle fiber type, was decreased by ∼50% in D2KO soleus. Disruption of Dio2 in the C2C12 myoblast cell line led to a significant decrease in PGC-1α expression and a faster muscle phenotype upon differentiation. These results indicate that D2 loss leads to significant changes in soleus contractile function and fiber type composition that are inconsistent with local hypothyroidism and suggest that reduced levels of PCG-1α may contribute to the observed phenotypical changes.