Alterations of the axon initial segment in multiple sclerosis grey matter.

Grey matter damage has been established as a key contributor to disability progression in multiple sclerosis. Aside from neuronal loss and axonal transections, which predominate in cortical demyelinated lesions, synaptic alterations have been detected in both demyelinated plaques and normal-appearing grey matter, resulting in functional neuronal damage. The axon initial segment is a key element of neuronal function, responsible for action potential initiation and maintenance of neuronal polarity. Despite several reports of profound axon initial segment alterations in different pathological models, among which experimental auto-immune encephalomyelitis, whether the axon initial segment is affected in multiple sclerosis is still unknown. Using immunohistochemistry, we analysed axon initial segments from control and multiple sclerosis tissue, focusing on layer 5/6 pyramidal neurons in the neocortex and Purkinje cells in the cerebellum and performed analysis on the parameters known to control neuronal excitability, i.e. axon initial segment length and position. We found that the axon initial segment length was increased only in pyramidal neurons of inactive demyelinated lesions, compared with normal appearing grey matter tissue. In contrast, in both cell types, the axon initial segment position was altered, with an increased soma-axon initial segment gap, in both active and inactive demyelinated lesions. In addition, using a computational model, we show that this increased gap between soma and axon initial segment might increase neuronal excitability. Taken together, these results show, for the first time, changes of axon initial segments in multiple sclerosis, in active as well as inactive grey matter lesions in both neocortex and cerebellum, which might alter neuronal function.

Patient-derived glioblastoma stem cells transfer mitochondria through tunneling nanotubes in tumor organoids.

Glioblastoma (GBM) is the most aggressive brain cancer and its relapse after surgery, chemo and radiotherapy appears to be led by GBM stem cells (GSCs). Also, tumor networking and intercellular communication play a major role in driving GBM therapy-resistance. Tunneling Nanotubes (TNTs), thin membranous open-ended channels connecting distant cells, have been observed in several types of cancer, where they emerge to drive a more malignant phenotype. Here, we investigated whether GBM cells are capable to intercommunicate by TNTs. Two GBM stem-like cells (GSLCs) were obtained from the external and infiltrative zone of one GBM from one patient. We show, for the first time, that both GSLCs, grown in classical 2D culture and in 3D-tumor organoids, formed functional TNTs which allowed mitochondria transfer. In the organoid model, recapitulative of several tumor's features, we observed the formation of a network between cells constituted of both Tumor Microtubes (TMs), previously observed in vivo, and TNTs. In addition, the two GSLCs exhibited different responses to irradiation in terms of TNT induction and mitochondria transfer, although the correlation with the disease progression and therapy-resistance needs to be further addressed. Thus, TNT-based communication is active in different GSLCs derived from the external tumoral areas associated to GBM relapse, and we propose that they participate together with TMs in tumor networking.

Morphological and Mechanical Characterization of DNA SAMs Combining Nanolithography with AFM and Optical Methods.

The morphological and mechanical properties of thiolated ssDNA films self-assembled at different ionic strength on flat gold surfaces have been investigated using Atomic Force Microscopy (AFM). AFM nanoshaving experiments, performed in hard tapping mode, allowed selectively removing molecules from micro-sized regions. To image the shaved areas, in addition to the soft contact mode, we explored the use of the Quantitative Imaging (QI) mode. QI is a less perturbative imaging mode that allows obtaining quantitative information on both sample topography and mechanical properties. AFM analysis showed that DNA SAMs assembled at high ionic strength are thicker and less deformable than films prepared at low ionic strength. In the case of thicker films, the difference between film and substrate Young's moduli could be assessed from the analysis of QI data. The AFM finding of thicker and denser films was confirmed by X-Ray Photoelectron Spectroscopy (XPS) and Spectroscopic Ellipsometry (SE) analysis. SE data allowed detecting the DNA UV absorption on dense monomolecular films. Moreover, feeding the SE analysis with the thickness data obtained by AFM, we could estimate the refractive index of dense DNA films.

Tunneling Nanotubes: The Fuel of Tumor Progression?

Tunneling nanotubes (TNTs) are thin membrane tubes connecting remote cells and allowing the transfer of cellular content. TNTs have been reported in several cancer in vitro, ex vivo, and in vivo models. Cancer cells exploit TNT-like connections to exchange material between themselves or with the tumoral microenvironment. Cells acquire new abilities (e.g., enhanced metabolic plasticity, migratory phenotypes, angiogenic ability, and therapy resistance) via these exchanges, contributing to cancer aggressiveness. Here, we review the morphological and functional features of TNT-like structures and their impact on cancer progression and resistance to therapies. Finally, we discuss the case of glioblastoma (GBM), in which a functional and resistant network between cancer cells in an in vivo model has been described for the first time.

Reproducibility warning: The curious case of polyethylene glycol 6000 and spheroid cell culture.

Reproducibility of results is essential for a well-designed and conducted experiment. Several reasons may originate failure in reproducing data, such as selective reporting, low statistical power, or poor analysis. In this study, we used PEG6000 samples from different distributors and tested their capability inducing spheroid formation upon surface coating. MALDI-MS, NMR, FTIR, and Triple SEC analysis of the different PEG60000s showed nearly identical physicochemical properties different, with only minor differences in mass and hydrodynamic radius, and AFM analysis showed no significant differences in the surface coatings obtained with the available PEG6000s. Despite these similarities, just one showed a highly reproducible formation of spheroids with different cell lines, such as HT-29, HeLa, Caco2, and PANC-1. Using the peculiar PEG6000 sample and a reference PEG6000 chosen amongst the others as control, we tested the effect of the cell/PEG interaction by incubating cells in the PEG solution prior to cell plating. These experiments indicate that the spheroid formation is due to direct interaction of the polymer with the cells rather than by interaction of cells with the coated surfaces. The experiments point out that for biological entities, such as cells or tissues, even very small differences in impurities or minimal variations in the starting product can have a very strong impact on the reproducibility of data.

How to Avoid and Manage Mental Nerve Injury in Transoral Thyroidectomy.

Transoral endoscopic thyroidectomy by vestibular approach (TOETVA) represents an innovative and scarless technique for thyroid surgery. The procedure is conducted via a three-port technique at the oral vestibule using a 10mm port for the 30° endoscope and two additional 5mm ports for the dissecting and coagulating instruments. Patients meeting the following criteria can be considered as candidates for TOETVA: (a) an ultrasonographically (US) estimated thyroid diameter =10cm; (b) US-estimated gland volume =45mL; (c) nodule size =50mm; (d) presence of a benign tumor, such as a thyroid cyst or a single- or multinodular goiter; (e) Bethesda 3 and/or 4 categories, and (f) papillary microcar-cinoma without the evidence of metastasis. Beyond the classic complications of thyroid surgery, namely cervical hematoma, recurrent laryngeal nerve injury and hypoparathyroidism, novel consequences can occur as mental nerve (MN) injury. In this paper, leading experts in the field report on their current clinical experience with the TOETVA approach for thyroid gland surgery, with emphasis given to tips and tricks to avoid and manage MN injury.

Functionalizing gold with single strand DNA: novel insight into optical properties via combined spectroscopic ellipsometry and nanolithography measurements.

We have studied the self-assembly of 22-base oligonucleotides bound by a short alkyl thiol linker (C6-ssDNA) on flat Au films. The self-assembled monolayer (SAM) was modified by addition of a spacer (mercaptohexanol, MCH). Molecular depositions were monitored in situ by spectroscopic ellipsometry (SE). SAMs were characterized in a liquid environment by coupling SE (difference spectra method) with Atomic Force Microscope (AFM) measurements. We exploited the biofilm thickness obtained by AFM nanolithography and imaging to solve the refractive index/thickness correlation in optical measurements on ultrathin molecular layers. The combined SE/AFM analysis provided reliable estimates of the thickness and the refractive index of the biofilm in the NIR region (650-1300 nm) and revealed new aspects of DNA molecular organization: exposure to MCH leads to an increase of both film thickness and refractive index, which points to a reorganization of C6-ssDNA film. We show that the contribution of the thiol/Au interface has to be included in the optical model to obtain a more reliable determination of the refractive index of the biofilm in a liquid. The careful, correlative characterization of the mixed C6-ssDNA/MCH SAM represents a key step towards the optimization of a robust detection scheme based on helix-helix hybridization.

Limitations of Continuous Neural Monitoring in Thyroid Surgery.

Continuous intraoperative neuromonitoring is currently the gold standard technique available to prevent recurrent laryngeal nerve injuries. It significantly reduces the complication rate compared with intermittent intraoperative neuromonitoring, and represents significant progress in thyroid surgery, particularly in cases of more difficult dissections. There are, however, some technological and interpretative limits related to the lack of standardization of continuous intraoperative neuromonitoring and the prolonged length of time employed in the surgical positioning of the probe, despite various proposed approaches to the vagal nerve. Nonetheless, this method can be considered a safe and modern approach to thyroid surgery that reduces post-surgical complications and provides useful information.

Postoperative Bleeding after Thyroid Surgery: Care Instructions.

Prospective studies on the incidence, etiology, and prognosis of well-characterized patients with bleeding after thyroid surgery are lacking. Bleeding after thyroid surgery cannot be predicted or prevented even if risk factors are known in every single procedure, which enhances the im-portance of the following issues: (a) meticulous hemostasis and surgical technique; (b) coopera-tion with the anesthesiologist, i.e., controlling the Valsalva maneuver, adequate blood pressure at the end of the operation as well as at extubation phase and (c) in case of bleeding, a prompt management to guarantee a better outcome. This requires an intensive postoperative clinical monitoring of patients, ideally, in a recovery room with trained staff for at least 4-6 h. Early recognition of postoperative bleeding with immediate intervention is the key to the management of this complication.