Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

Microanatomy of the human tunnel of Corti structures and cochlear partition-tonotopic variations and transcellular signaling.

Auditory sensitivity and frequency resolution depend on the optimal transfer of sound-induced vibrations from the basilar membrane (BM) to the inner hair cells (IHCs), the principal auditory receptors. There remains a paucity of information on how this is accomplished along the frequency range in the human cochlea. Most of the current knowledge is derived either from animal experiments or human tissue processed after death, offering limited structural preservation and optical resolution. In our study, we analyzed the cytoarchitecture of the human cochlear partition at different frequency locations using high-resolution microscopy of uniquely preserved normal human tissue. The results may have clinical implications and increase our understanding of how frequency-dependent acoustic vibrations are carried to human IHCs. A 1-micron-thick plastic-embedded section (mid-modiolar) from a normal human cochlea uniquely preserved at lateral skull base surgery was analyzed using light and transmission electron microscopy (LM, TEM). Frequency locations were estimated using synchrotron radiation phase-contrast imaging (SR-PCI). Archival human tissue prepared for scanning electron microscopy (SEM) and super-resolution structured illumination microscopy (SR-SIM) were also used and compared in this study. Microscopy demonstrated great variations in the dimension and architecture of the human cochlear partition along the frequency range. Pillar cell geometry was closely regulated and depended on the reticular lamina slope and tympanic lip angle. A type II collagen-expressing lamina extended medially from the tympanic lip under the inner sulcus, here named "accessory basilar membrane." It was linked to the tympanic lip and inner pillar foot, and it may contribute to the overall compliance of the cochlear partition. Based on the findings, we speculate on the remarkable microanatomic inflections and geometric relationships which relay different sound-induced vibrations to the IHCs, including their relevance for the evolution of human speech reception and electric stimulation with auditory implants. The inner pillar transcellular microtubule/actin system's role of directly converting vibration energy to the IHC cuticular plate and ciliary bundle is highlighted.

Multivalent design of the monoclonal SynO2 antibody improves binding strength to soluble α-Synuclein aggregates.

Soluble aggregates are reported to be the most neurotoxic species of α-Synuclein (αSyn) in Parkinson's disease (PD) and hence are a promising target for diagnosis and treatment of PD. However, the predominantly intracellular location of αSyn limits its accessibility, especially for antibody-based molecules and prompts the need for exceptionally strong soluble αSyn aggregate binders to enhance their sensitivity and efficacy for targeting the extracellular αSyn pool. In this study, we have created the multivalent antibodies TetraSynO2 and HexaSynO2, derived from the αSyn oligomer-specific antibody SynO2, to increase avidity binding to soluble αSyn aggregate species through more binding sites in close proximity. The multivalency was achieved through recombinant fusion of single-chain variable fragments of SynO2 to the antibodies' original N-termini. Our ELISA results indicated a 20-fold increased binding strength of the multivalent formats to αSyn aggregates, while binding to αSyn monomers and unspecific binding to amyloid ß protofibrils remained low. Kinetic analysis using LigandTracer revealed that only 80% of SynO2 bound bivalently to soluble αSyn aggregates, whereas the proportion of TetraSynO2 and HexaSynO2 binding bi- or multivalently to soluble αSyn aggregates was increased to ~ 95% and 100%, respectively. The overall improved binding strength of TetraSynO2 and HexaSynO2 implies great potential for immunotherapeutic and diagnostic applications with targets of limited accessibility, like extracellular αSyn aggregates. The ability of the multivalent antibodies to bind a wider range of αSyn aggregate species, which are not targetable by conventional bivalent antibodies, thus could allow for an earlier and more effective intervention in the progression of PD.

Signatures in in vitro infection of NSC-34 mouse neurons and their cell nucleus with Rickettsia helvetica.

BACKGROUND: Rickettsia helvetica, a spotted fever rickettsia, is transmitted to humans via ticks in Europe, North Africa, and Asia. The central nervous system is a crucial target for rickettsial diseases, which has been reported for 12 of the 31 species, of which R. helvetica is one. This study aimed, in an experimental model, to identify characteristics of R. helvetica infection in a mouse neuronal cell line, NSC-34. RESULTS: NSC-34, a fusion cell line of mouse motor spinal cord neurons and neuroblastoma cells, was used as a model. Propagation of R. helvetica in neurons was confirmed. Short actin tails were shown at the polar end of the bacteria, which makes it likely that they can move intracellularly, and even spread between cells. Another protein, Sca4, which with the cell adhesion protein vinculin enables the passage of the cell membrane, was expressed during infection. No significant increase in TNFα levels was seen in the infected neurons, which is of interest because TNFα protects the host cell from infection-induced apoptotic death which is crucial for host cell survival. The bacteria were also shown to invade and grow in the cell nucleus of the neuron. CONCLUSIONS: The findings suggest that a R. helvetica infection may be harmful to NSC-34 neurons under these in vitro conditions, but the full effects of the infection on the cell need to be studied further, also on human neurons, to also understand the possible significance of this infection in relation to pathogenetic mechanisms.

The ultrastructure of a stria vascularis in the auditory organ of the cuban crocodile (Crocodylus rhombifer).

Background: An endocochlear potential (EP) exists in the mammalian cochlea generated by the stria vascularis and an associated fibrocyte network. It plays an essential role for sensory cell function and hearing sensitivity. In non-mammalian ectothermic animals the endocochlear potential is low and its origin somewhat unclear. In this study, we explored the crocodilian auditory organ and describe the fine structure of a stria vascularis epithelium that has not been verified in birds. Material and Methods: Three Cuban crocodiles (Crocodylus rhombifer) were analyzed with light and transmission electron microscopy. The ears were fixed in glutaraldehyde The temporal bones were drilled out and decalcified. The ears were dehydrated, and embedded and was followed by semi-thin and thin sectioning. Results: The fine structure of the crocodile auditory organ including the papilla basilaris and endolymph system was outlined. The upper roof of the endolymph compartment was specialized into a Reissner membrane and tegmentum vasculosum. At the lateral limbus an organized, multilayered, vascularized epithelium or stria vascularis was identified. Discussion: Electron microscopy demonstrates that the auditory organ in Crocodylus rhombifer, unlike in birds, contains a stria vascularis epithelium separate from the tegmentum vasculosum. It is believed to secrete endolymph and to generate a low grade endocochlear potential. It may regulate endolymph composition and optimize hearing sensitivity alongside the tegmentum vasculosum. It could represent a parallel evolution essential for the adaptation of crocodiles to their diverse habitats.

Regeneration in the Auditory Organ in Cuban and African Dwarf Crocodiles (Crocodylus rhombifer and Osteolaemus tetraspis) Can We Learn From the Crocodile How to Restore Our Hearing?

Background: In several non-mammalian species, auditory receptors undergo cell renewal after damage. This has raised hope of finding new options to treat human sensorineural deafness. Uncertainty remains as to the triggering mechanisms and whether hair cells are regenerated even under normal conditions. In the present investigation, we explored the auditory organ in the crocodile to validate possible ongoing natural hair cell regeneration. Materials and Methods: Two male Cuban crocodiles (Crocodylus rhombifer) and an adult male African Dwarf crocodile (Osteolaemus tetraspis) were analyzed using transmission electron microscopy and immunohistochemistry using confocal microscopy. The crocodile ears were fixed in formaldehyde and glutaraldehyde and underwent micro-computed tomography (micro-CT) and 3D reconstruction. The temporal bones were drilled out and decalcified. Results: The crocodile papilla basilaris contained tall (inner) and short (outer) hair cells surrounded by a mosaic of tightly connected supporting cells coupled with gap junctions. Afferent neurons with and without ribbon synapses innervated both hair cell types. Supporting cells occasionally showed signs of trans-differentiation into hair cells. They expressed the MAFA and SOX2 transcription factors. Supporting cells contained organelles that may transfer genetic information between cells, including the efferent nerve fibers during the regeneration process. The tectorial membrane showed signs of being replenished and its architecture being sculpted by extracellular exosome-like proteolysis. Discussion: Crocodilians seem to produce new hair cells during their life span from a range of supporting cells. Imposing efferent nerve fibers may play a role in regeneration and re-innervation of the auditory receptors, possibly triggered by apoptotic signals from wasted hair cells. Intercellular signaling may be accomplished by elaborate gap junction and organelle systems, including neural emperipolesis. Crocodilians seem to restore and sculpt their tectorial membranes throughout their lives.

The link between gliomas infiltration and white matter architecture investigated with electron microscopy and diffusion tensor imaging.

Diffuse low-grade gliomas (DLGG) display different preferential locations in eloquent and secondary associative brain areas. The reason for this tendency is still unknown. We hypothesized that the intrinsic architecture and water diffusion properties of the white matter bundles in these regions may facilitate gliomas infiltration. Magnetic resonance imaging of sixty-seven diffuse low-grade gliomas patients were normalized to/and segmented in MNI space to create three probabilistic infiltration weighted gradient maps according to the molecular status of each tumor group (IDH mutated, IDH wild-type and IDH mutated/1p19q co-deleted). Diffusion tensor imaging (DTI)- based parameters were derived for five major white matter bundles, displaying regional differences in the grade of infiltration, averaged over 20 healthy individuals acquired from the Human connectome project (HCP) database. Transmission electron microscopy (TEM) was used to analyze fiber density, fiber diameter and g-ratio in 100 human white matter regions, sampled from cadaver specimens, reflecting areas with different gliomas infiltration in each white matter bundle. Histological results and DTI-based parameters were compared in anatomical regions of high- and low grade of infiltration (HIF and LIF) respectively. We detected differences in the white matter infiltration of five major white matter bundles in three groups. Astrocytomas IDHm infiltrated left fronto-temporal subcortical areas. Astrocytomas IDHwt were detected in the posterior-temporal and temporo-parietal regions bilaterally. Oligodendrogliomas IDHm/1p19q infiltrated anterior subcortical regions of the frontal lobes bilaterally. Regional differences within the same white matter bundles were detected by both TEM- and DTI analysis linked to different topographical variables. Our multimodal analysis showed that HIF regions, common to all the groups, displayed a smaller fiber diameter, lower FA and higher RD compared with LIF regions. Our results suggest that the both morphological features and diffusion parameters of the white matter may be different in regions linked to the preferential location of DLGG.