Seeing Keratinocyte Proteins through the Looking Glass of Intrinsic Disorder

Epidermal keratinocyte proteins include many with an eccentric amino acid content (compositional bias), atypical ultrastructural fate (built-in protease sensitivity), or assembly visible at the light microscope level (cytoplasmic granules). However, when considered through the looking glass of intrinsic disorder (ID), these apparent oddities seem quite expected. Keratinocyte proteins with highly repetitive motifs are of low complexity but high adaptation, providing polymers (e.g., profilaggrin) for proteolysis into bioactive derivatives, or monomers (e.g., loricrin) repeatedly cross-linked to self and other proteins to shield underlying tissue. Keratohyalin granules developing from liquid–liquid phase separation (LLPS) show that unique biomolecular condensates (BMC) and proteinaceous membraneless organelles (PMLO) occur in these highly customized cells. We conducted bioinformatic and in silico assessments of representative keratinocyte differentiation-dependent proteins. This was conducted in the context of them having demonstrated potential ID with the prospect of that characteristic driving formation of distinctive keratinocyte structures. Intriguingly, while ID is characteristic of many of these proteins, it does not appear to guarantee LLPS, nor is it required for incorporation into certain keratinocyte protein condensates. Further examination of keratinocyte-specific proteins will provide variations in the theme of PMLO, possibly recognizing new BMC for advancements in understanding intrinsically disordered proteins as reflected by keratinocyte biology.

3D analysis of the synaptic organization in the Entorhinal cortex in Alzheimer’s disease

The entorhinal cortex (EC) is especially vulnerable in the early stages of Alzheimer’s disease (AD). In particular, cognitive deficits have been linked to alterations in the upper layers of EC. In the present report, we performed light microscopy analysis and 3D ultrastructural analyses of synapses in the EC using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) to examine possible alterations related to AD. We analyzed 5000 synaptic junctions that were 3D reconstructed, representing the largest 3D ultrastructural study of synapses in the EC of the human brain from cases with AD performed to date. Structural differences were found in the AD tissue at the light microscope level and at the ultrastructural level. These differences may play a role in the anatomical basis for the impairment of cognitive functions in AD.

Anatomical and Histological Study of Male Uropoetic Organs of Common Rabbit (Oryctolagus cuniculus)

Common rabbit or European rabbit (Oryctolagus cuniculus) is one of the mammals that belongs to the order Lagomorph. The study of uropoetic organs in adult male common rabbit included the anatomical and histological description at the light microscope level by using Hematoxyline and Eosine stain methods. The aims of this study was to determine the anatomical and histological featu res of male Oryctolagus cuniculus. Uropoetic organs of adult male common rabbit consists of four organs that is kidneys, ureters, bladder, and urethra. The result showed that Oryctolagus cuniculus have a bean-shaped kidney. The length of the right kidney is 3,4 cm and the width is 1,9 cm while the length of the left kidney is 3,2 cm and the width is 1,8 cm. The histological study showed that common rabbit kidne y consists of glomerulus, Bowman’s capsule, proximal tubules, distal tubules, and collecting ducts.

Enhanced Pain Sensitivity with Systemic Ultrastructural Changes of the Nervous Systems after Cobra Venom Injection is Reversed by Electroacupuncture Treatment

Background: Electroacupuncture (EA) has been proved to be effective in treating certain neuropathic pain conditions. The mechanisms of pain relief by EA are not fully understood. There have been sporadic reports of damage in the peripheral nervous system (PNS) and regions of the central nervous system (CNS) at the ultrastructural level following peripheral nerve injury. However, information about possible systemic changes in the PNS and CNS after nerve injury is scarce. Objectives: The goal of this study was to examine the ultrastructural changes of the nervous system induced by a local injection of cobra venom into the sciatic nerve and to compare the ultrastructural changes in rats with or without treatment with EA or pregabalin. Study Design: An experimental study. Setting: Department of Anesthesiology, Pain Medicine, and Critical Care Medicine, Aviation General Hospital of China Medical University. Methods: In this study, using an established model of sciatic neuralgia induced by local injection of cobra venom into the sciatic nerve, we examined ultrastructural changes of the PNS and CNS and how they respond to EA and pregabalin treatment. EA and pregabalin were given daily from postoperative day (POD) 14 to 36. Based on previous works, the frequency of EA stimulation of the ST36 and GB34 acupoints was held to 2/100 Hz variable. Pain sensitivity in the sciatic neuralgia rats with and without treatments was assessed using the von Frey test. Ultrastructural alterations were examined bilaterally in the prefrontal cortex, hippocampus, medulla oblongata; and the cervical, thoracic, and lumbar spinal cords on PODs 14, 40, and 60. Ultrastructural examinations were also carried out on the bilateral sciatic nerves and dorsal root ganglion (DRG) at the cervical, thoracic and lumbar levels. In rats treated with EA or pregabalin, the ultrastructure was examined on PODs 40 and 60. Results: Behavioral signs of pain and systemic ultrastructural changes including demyelination were observed at all levels of the PNS and CNS in rats with sciatic neuralgia. After intervention, the mechanical withdrawal thresholds of the EA group and pregabalin group were significantly higher than that of the cobra venom group (P < 0.05). Both EA and pregabalin treatments partially reversed increased cutaneous sensitivity to mechanical stimulation. However, only the EA treatment was able to repair the ultrastructural damages caused by cobra venom. Limitations: The results confirm that peripheral nerve injury led to the ultrastructural damage at different levels of the CNS as demonstrated with electron microscopy; however, we need to further verify this at both the molecular level and in light microscope level. Sciatic neuralgia induced by cobra venom is a chemical injury, and whether this exactly mimics a peripheral nerve mechanical injury is still unclear. Conclusions: Local cobra venom injection leads to systemic neurotoxicity. EA and pregabalin alleviate pain via different mechanisms. Key words: Sciatic neuralgia, cobra venom, demyelination, electroacupuncture, pregabalin, rat model

ANALYSIS OF STRUCTURAL PLASTICITY IN THE HONEY BEE BRAIN USING THE CAVALIERI ESTIMATOR OF VOLUME AND THE DISECTOR METHOD

The antennal lobe of the worker honey bee has been used as a model system to address the origins of structural plasticity in the central nervous system. A combination of light and electron microscopy was used to determine total synapse number within an easily identifiable sub-unit of the antennal lobe neuropil, the T4-2(1) glomerulus. The Cavalieri method was applied at the light microscope level to determine a reference volume (Vref) of this glomerulus. Using transmission electron microscopy, the physical disector was used to determine synaptic density (Nv) within the T4-2(1) glomerulus. An estimate of the total synapse number N(syn) was determined by; N(syn) = V(ref) Nv. Newly emerged, 4-day old,10-day old and forager-aged bees were analysed. Results showed that despite a significant increase in T4-2(1) volume with age, the total number of synapses in this glomerulus did not show a corresponding increase. Disturbingly, it is possible that huge variances within age groups, due to one or two outlying data points, could be masking the true trend of synapse number. This variance, the heterogeneous distribution of synapses within this glomerulus and the problems associated with reproducibility of synapse counts are discussed.

TEM identification of Pseudo-nitzschia species from Lisbon Bay (NE Atlantic)

Several species of the chain-forming marine diatom genus Pseudo-nitzschia produce domoic acid (DA), a neuroexcitant amino acid, responsible for a human amnesic syndrome after consumption of toxified shellfish (ASP). Since 1995, the Portuguese monitoring program has regularly detected the presence of DA in shellfish. So far only P. australis has been implicated, and references therein. Identification of the causative species of Pseudo-nitzchia in natural samples is hampered by difficulties in identification at the light microscope level. Recent studies have shown that ultrastructural details of the wall, observed by transmission electron microscopy, allow the identification of species in good agreement with the phylogenetic and biological species concept.

Increased Electrotonic Coupling in Spinal Motoneurons After Transient Botulinum Neurotoxin Paralysis in the Neonatal Rat

The effect of early postnatal blockade of neuromuscular transmission using botulinum neurotoxin (BoNT) type A on motoneuron gap junctional coupling was studied by means of intracellular recordings and biocytin labeling using the in vitro hemisected spinal cord preparation of neonatal rats. The somata of tibialis anterior (TA) motoneurons were retrogradely labeled at birth (P0) by intramuscular injection of fluorescent tracers. Two days later, BoNT was injected unilaterally into the TA muscle. The toxin blocked neuromuscular transmission for the period studied (P4–P7) as shown by tension recordings of the TA muscle. Retrograde horseradish peroxidase tracing in animals reared to adulthood demonstrated no significant cell death or changes in the soma size of BoNT-treated TA motoneurons. Intracellular recordings were carried out in prelabeled control and BoNT-treated TA motoneurons from P4 to P7. Graded stimulation of the ventral root at subthreshold intensities elicited short-latency depolarizing (SLD) potentials that consisted of several discrete components reflecting electrotonic coupling between two or more motoneurons. BoNT treatment produced a significant increase (67%) in the maximum amplitude of the SLD and in the number of SLD components as compared with control (3.1 ± 1.7 vs. 1.4 ± 0.7; means ± SD). The morphological correlates of electrotonic coupling were investigated at the light microscope level by studying the transfer of biocytin to other motoneurons and the putative sites of gap junctional interaction. The dye-coupled neurons clustered around the injected cell with close somato-somatic, dendro-somatic and -dendritic appositions that might represent the sites of electrotonic coupling. The size of the motoneuron cluster was, on average, 2.2 times larger after BoNT treatment. Our findings demonstrate that a short-lasting functional disconnection of motoneurons from their target muscle delays motoneuron maturation by halting the elimination of gap junctional coupling that normally occurs during early postnatal development.