Microglia experience dramatic molecular and functional changes when transferred from the central nervous system (CNS) to a cell culture environment. Investigators largely attribute these findings to the loss of CNS-specific microenvironmental cues that dictate the gene-regulatory networks specified by master regulator transcription factors such as V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB). MafB regulates macrophage differentiation and activation by activating or repressing target genes critical to these processes. Here, we show that basal MafB levels in the BV-2 microglial cell line depend on the availability of lipids in the cell culture environment. Depletion of lipids, either by serum deprivation or the use of lipid-depleted serum, reduced MafB protein levels in BV-2 cells. Using live imaging, we also observed the engulfment of apoptotic BV-2 cell debris by neighboring BV-2 cells, highlighting an additional potential source of lipids in the cell culture environment. This observation was supported by experiments showing reduced MafB protein levels in BV-2 cells cultured with various phagocytosis inhibitors (cytochalasin D, annexin V) and reduced BV-2 cell phagocytic activity with serum deprivation. In aggregate, our data suggest that serum exposure regulates the transcription factor MafB in BV-2 cells through direct and indirect mechanisms.
In 2015, meningeal lymphatic vessels (mLVs) were (re)discovered in mice and human dura specimens. Two years later, the first report was published showing that mLVs can be detected in humans in vivo by high-resolution 3 Tesla magnetic resonance imaging (MRI). In 2017 and 2018, two further studies reported the successful MRI-based detection of mLVs in vivo in humans. The aim of our study was to provide further evidence of the possibility to detect mLVs in vivo with MRI in humans. To this end, MR images already available from one subject (the first author) were analyzed. We detected mLVs in the coronal plane at the bilateral superior lateral corners of the superior sagittal sinus (SSS) as well as below the SSS, in agreement with the two other published reports about the in vivo detection of mLVS in humans with MRI. Our report is thus, to the best of our knowledge, the fourth published report about in vivo MR imaging of human mLVs.
Plankton provides an essential foundation for life on Earth, supplying most of the breathable oxygen, carbon sequestration, and larvae nutrition. Toxic chemicals introduced into the environment pose a potential danger to plankton and the ecosystem. Traditional plankton chemical toxicity assays measure the concentration required to cause death. Sublethal doses that affect plankton activities like foraging and escaping predators can have a cascading impact on ecosystems. We have developed a high-throughput device to measure the sublethal effect of chemicals on the plankton rate of movement. The device automatically creates a series of chemical dilutions, subjects each concentration to a group of plankton, and calculates the average group movement speed. We tested the device on groups of Stentor coeruleus (n=3 to 26, mean=10) with acetic acid dilutions (43 to 25,000 ppm) and measured a declining trend in average speed with increasing sublethal concentrations (170 to 502 ppm).
Plastid targeted proteins of diatoms and related algae can be predicted with high sensitivity and specificity using the ASAFind method published in 2015. ASAFind predictions rely on SignalP predictions of endoplasmic reticulum (ER) targeting signal peptides. Recently (in 2019), a new version of SignalP was released, SignalP 5.0. We tested the ability of SignalP 5.0 to recognize signal peptides of nucleus-encoded, plastid-targeted diatom pre-proteins, and to identify the signal peptide cleavage site. The results were compared to manual predictions of the characteristic cleavage site motif, and to previous versions of SignalP. SignalP 5.0 is less sensitive than the previous versions of SignalP in this specific task, and also in the detection of signal peptides of non-plastid proteins in diatoms. However, in combination with ASAFind, the resulting prediction performance for plastid proteins is high. In addition, we tested the multi-location prediction tool TargetP for its suitability to provide signal peptide information to ASAFind. The newest version, TargetP 2.0, had the highest prediction performances for diatom signal peptides and mitochondrial transit peptides compared to other versions of SignalP and TargetP, thus it provides a good basis for ASAFind predictions.
For many years, reports have been published describing the discovery and investigation of an additional vascular system in mammals. This primo vascular system (PVS) is distinct from the blood and lymph vascular system and consists of primo nodes (PNs) and primo vessels (PVs) as its main constituents. We investigated samples of the PVS from the intestine surface of rats and observed several instances of a red thread-like structure (RTLS) and a red oval or round structure (RORS) in PVs and PNs, respectively. We conclude that the RTLS and RORS are most likely due to erythrocytes, indicating the occurrence of extramedullary hematopoiesis inside the PVS of the intestine surface. To the best of our knowledge, this is the first report showing detailed microscopic images of an RTLS traversing four PNs and a single PV. Our report is intended to document our findings and also to motivate others to repeat and extend our study in order to investigate in detail the possible extramedullary hematopoiesis occurring inside the PVS.
Numerous enzymes have been demonstrated to be active in non-aqueous solutions, yet the utility of phosphatases under such conditions has been difficult to determine. Here, we demonstrate the ability to fluorescently detect naphthol AS‐MX in high percentages 1,4-dioxane with a fluorescence differential compared with naphthol AS‐MX phosphate. While intensities and maximum fluorescence wavelengths changed depending on solvent conditions, these results demonstrate this system’s potential for testing phosphatase activity in high amounts of dioxane.
Early embryo elongation involves coordinated cellular and tissue behaviors that are readily observable in the chick embryo vertebrate model system. Easily identifiable morphological landmarks are crucial to obtain reliable morphometric data, particularly when assessing tissue elongation over time. The posterior end of the primitive streak marks the caudal end of the chick embryonic tissue. However, the identification of its precise location is ambiguous, especially to the untrained eye. Herein, we assessed if the posterior limit of the area pellucida (pPL), which is readily recognizable due to the optical contrast with the area opaca, is a valid proxy for the caudal limit of the primitive streak. Measurements of total embryo length were performed in multiple images of chick embryos over time using both caudal landmarks. We found that the pPL offered greater precision and a higher degree of inter-user reproducibility, when compared to the end of the primitive streak. Importantly, our work uncovers a quantitative proportionality between embryo length measurements using the end of the primitive streak and the pPL as caudal landmarks. We have thus validated the pPL as a reliable morphological proxy for the end of the primitive streak in chick embryo elongation studies.
The complement protein C1q has lately emerged as an important molecule in the clearance of apoptotic cells. However, its role in the brain in physiological conditions is less explored. Using as a model the adult neurogenic cascade, where newborn cells undergo apoptosis and are phagocytosed by microglia, in the present study we have discovered three major findings using immunofluorescence and RTqPCR from FACSsorted microglia in fms-EGFP mice. First, we found that C1q is mainly produced by microglia in the hippocampus in physiological conditions. Second, we observed a relationship between phagocytic microglia and C1q, as suggested by the presence of C1q within the majority of the microglial phagocytic pouches. Finally, we discovered that the functions of C1q in microglia may go beyond phagocytosis, as C1q was also found in non-phagocytic microglia.
Many infectious diseases lack cheap, fast, and reliable serodiagnostic tools. Recently, glycans coupled to gold nanoparticles (GNPs) have shown potential to fill this void by utilizing them as a coating in ELISA experiments. In this study, we used GNPs functionalized either with galactofuranose (Galf), rhamnose (Rhap) or a mixture of these monosaccharides to detect the presence of IgM and IgG antibodies against these non-human carbohydrates in sera obtained from tuberculosis patients and control groups. Our findings confirm the wide abundance of anti-Rhap antibodies in humans and show for the first time the presence of anti-Galf antibodies in human sera. This study supports further investigation of using GNP-ELISA with microbe-specific carbohydrates for serodiagnosis of infectious diseases.
The branching of vertebrate neuronal processes, axons or dendrites, is predominantly binary, where a proximal neuronal process bifurcates into two distal progeny. Here, we illustrate counterexamples where neuronal processes branched into more than 2, up to 6, progeny branches, in one step. These were branch points of myelinated dendrites in the peripheral terminals of ALLn cranial nerve afferents innervating the Lorenziniantype ampullary electroreceptors on the rostrum of paddlefish, Polyodon spathula. We imaged afferent terminals fluorescently (in widefield stacks, with deconvolution) after immunolabling of afferent terminals for neuronal cytoplasmic neurofilament-H (NEFH), myelin markers (MBP, P0), or nodal ion channels (Nav1.x, Kv1.1), or after migration of DiI in dendrite membranes. Branched afferent terminals formed a laminar radial radiation beneath a receptive field, parallel to the skin surface, with two serial stages: (1) Starting at each afferent’s centric first branchpoint, each of a small group of 2–4 (most often 3) afferents branched radially into 2–4 (usually 3) generations of myelinated dendrites, whose internodes were covered by sheaths immunoreactive for antigenic markers of myelin (MBP+/P0+), ending distally at ~15 heminode presumed spike initiation zones. (2) From the latter, bundles of unmyelinated (MBP-/P0-) sensory neuron (NEFH+) processes projected distally to innervate electrosensory neuroepithelia of adjacent ampullary organs. The nonbinary branch points that we imaged were in stage-1, on myelinated dendrites. Branch points always coincided with composite systems of nodes, in which each progeny dendrite started at a narrowed nodal segment expressing voltage gated sodium ion channels at high density. These narrowed nodal segments of multiple progeny branched in parallel from a parent’s blunt distal end. Hence the branch point nodal complexes formed multisite excitable systems, likely strongly coupled due to proximity.
