Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal-dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, joint hypermobility, and mild skin findings. A locus was mapped to an approximately 5.8 Mb region at 12p13.1 but no candidate gene was identified. In an international consortium we recruited 19 independent families comprising 107 individuals with pEDS to identify the locus, characterize the clinical details in those with defined genetic causes, and try to understand the physiological basis of the condition. In 17 of these families, we identified heterozygous missense or in-frame insertion/deletion mutations in C1R (15 families) or C1S (2 families), contiguous genes in the mapped locus that encode subunits C1r and C1s of the first component of the classical complement pathway. These two proteins form a heterotetramer that then combines with six C1q subunits. Pathogenic variants involve the subunit interfaces or inter-domain hinges of C1r and C1s and are associated with intracellular retention and mild endoplasmic reticulum enlargement. Clinical features of affected individuals in these families include rapidly progressing periodontitis with onset in the teens or childhood, a previously unrecognized lack of attached gingiva, pretibial hyperpigmentation, skin and vascular fragility, easy bruising, and variable musculoskeletal symptoms. Our findings open a connection between the inflammatory classical complement pathway and connective tissue homeostasis.

Three-dimensional (3D) fluorescence microscopy in general requires axial scanning to capture images of a sample at different planes. Here we demonstrate that a deep convolutional neural network can be trained to virtually refocus a 2D fluorescence image onto user-defined 3D surfaces within the sample volume. With this data-driven computational microscopy framework, we imaged the neuron activity of a Caenorhabditis elegans worm in 3D using a time-sequence of fluorescence images acquired at a single focal plane, digitally increasing the depth-of-field of the microscope by 20-fold without any axial scanning, additional hardware, or a trade-off of imaging resolution or speed. Furthermore, we demonstrate that this learning-based approach can correct for sample drift, tilt, and other image aberrations, all digitally performed after the acquisition of a single fluorescence image. This unique framework also cross-connects different imaging modalities to each other, enabling 3D refocusing of a single wide-field fluorescence image to match confocal microscopy images acquired at different sample planes. This deep learning-based 3D image refocusing method might be transformative for imaging and tracking of 3D biological samples, especially over extended periods of time, mitigating photo-toxicity, sample drift, aberration and defocusing related challenges associated with standard 3D fluorescence microscopy techniques. Text:Three-dimensional (3D) fluorescence microscopic imaging is essential for biomedical and physical sciences as well as engineering, covering various applications 1-7 . Despite its broad

Carbon dots were synthesized by a simple and green strategy for selective and sensitive Cu(2+) ion detection using both down and upconversion fluorescence. These fluorescent nanosensors show low cytotoxicity and are applied for intracellular sensing and imaging of Cu(2+) in biological systems.

Numerous studies on nanocarriers use fluorescent dye labeling to investigate their biodistribution or cellular trafficking. However, when the fluorescence dye is not grafted to the nanocarrier, the question of the stability of the labeling arises. How can it be validated that the fluorescence observed during an experiment corresponds to the nanocarriers, and not to the free dye released from the nanocarriers? Studying the integrity of the labeling is challenging. Therefore, an innovative approach to confirm the labeling stability was developed, based on the transfer of a fluorescent dye from its hosting nanocarrier to a lipophilic compartment. Lipid nanocapsules (LNC) and triglyceride oil were used as models. The protocol involved mixing of LNC suspension and oil, and then separation by centrifugation. The quality of the separation was controlled by light scattering, using the derived count rate tool. Dye transfer from loaded LNCs to the lipophilic compartment or from a lipophilic compartment containing dye to non-loaded LNC was investigated by varying the nature of the dye and the oil, the oil volume and the LNC dilution. Tensiometry was used to define the dye location in the nanocarrier. Results showed that when dyes such as Nile Red and Coumarin-6 are located in oily core, the transfer occurred in a partition-dependent manner. In contrast, when the dye was entrapped in the surfactant shell of LNCs such as lipophilic indocarbocyanines (i.e. DiO, DiI and DiD), no transfer was observed. Dye diffusion was also observed in cell culture, with Nile Red inside lipid bodies of HEI-OC1 cells, without uptake of LNCs. In contrast, DiO-loaded LNCs had to be internalized to observe fluorescence inside the cells, providing a further confirmation of the absence of transfer in this case, and the stability of fluorescence labeling of the LNCs.

Background:In order to improve therapy for HNSCC patients, novel methods to predict and combat local and/or distant tumour relapses are urgently needed. This study has been dedicated to the hypothesis that Rac1, a Rho GTPase, is implicated in HNSCC insensitivity to chemo-radiotherapy resulting in tumour recurrence development.Methods:Parental and radiation-resistant (IRR) HNSCC cells were used to support this hypothesis. All cells were investigated for their sensitivity to ionising radiation and cisplatin, Rac1 activity, its intracellular expression and subcellular localisation. Additionally, tumour tissues obtained from 60 HNSCC patients showing different therapy response were evaluated for intratumoral Rac1 expression.Results:Radiation-resistant IRR cells also revealed resistance to cisplatin accompanied by increased expression, activity and trend towards nuclear translocation of Rac1 protein. Chemical inhibition of Rac1 expression and activity resulted in significant improvement of HNSCC sensitivity to ionising radiation and cisplatin. Preclinical results were confirmed in clinical samples. Although Rac1 was poorly presented in normal mucosa, tumour tissues revealed increased Rac1 expression. The most pronounced Rac1 presence was observed in HNSCC patients with poor early or late responses to chemo-radiotherapy. Tissues taken at recurrence were characterised not only by enhanced Rac1 expression but also increased nuclear Rac1 content.Conclusions:Increased expression, activity and subcellular localisation of Rac1 could be associated with lower early response rate and higher risk of tumour recurrences in HNSCC patients and warrants further validation in larger independent studies. Inhibition of Rac1 activity can be useful in overcoming treatment resistance and could be proposed for HNSCC patients with primary or secondary chemo-radioresistance.

Objective: EMT (epithelial to mesenchymal transition) contributes to tumor progression and metastasis. We aimed to investigate the effects of EMT on CDDP resistance in HNSCC (head and neck squamous cell carcinoma)-cells.Methods: EMT was induced using conditioned medium from a tumor cell/fibroblast co-culture. HNSCC cells were alternatively treated with TGF-β1. The response to CDDP was evaluated with viability and clonogenic assays.Results Conclusions: Cell free medium from a co-culture was able to induce EMT in HNSCC cells. Co-culture treated HNSCC cells revealed increased viability and were less sensitive to CDDP treatment. TGF-β1 also induced a mesenchymal phenotype, but did not alter resistance to CDDP in HNSCC cells.

Sprouty (Spry) proteins are negative feedback inhibitors of receptor tyrosine kinase signaling. Downregulation of Spry2 has been demonstrated to promote elongative axon growth of cultured peripheral and central neurons. Here, we analyzed Spry2 global knockout mice with respect to axon outgrowth in vitro and peripheral axon regeneration in vivo. Neurons dissociated from adult Spry2 deficient sensory ganglia revealed stronger extracellular signal-regulated kinase activation and enhanced axon outgrowth. Prominent axon elongation was observed in heterozygous Spry2 1/2 neuron cultures, whereas homozygous Spry2 2/2 neurons predominantly exhibited a branching phenotype. Following sciatic nerve crush, Spry2 1/2 mice recovered faster in motor but not sensory testing paradigms (Spry2 2/2 mice did not tolerate anesthesia required for nerve surgery). We attribute the improvement in the rotarod test to higher numbers of myelinated fibers in the regenerating sciatic nerve, higher densities of motor endplates in hind limb muscles and increased levels of GAP-43 mRNA, a downstream target of extracellular regulated kinase signaling. Conversely, homozygous Spry2 2/2 mice revealed enhanced mechanosensory function (von Frey's test) that was accompanied by an increased innervation of the epidermis, elevated numbers of nonmyelinated axons and more IB4-positive neurons in dorsal root ganglia. The present results corroborate the functional significance of receptor tyrosine kinase signaling inhibitors for axon outgrowth during development and nerve regeneration and propose Spry2 as a novel potential target for pharmacological inhibition to accelerate long-distance axon regeneration in injured peripheral nerves.

Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of OSCC tumor cells. We hypothesized that Curcumin targets this dynamic mutual interaction between CAFs and tumor cells. Normal and 2 μM Curcumin-treated co-culture were performed for 4 days, followed by analysis of tumor cell invasivity, mRNA/protein expression of EMT-markers and mediators, activity measure of matrix metalloproteinase 9 (MMP-9), and western blot analysis of signal transduction in tumor cells and fibroblasts. In Curcumin-treated co-culture, in tumor cells, the levels of nuclear factor κB (NFκBα) and early response kinase (ERK)—decreased, in fibroblasts, integrin αv protein synthesis decreased compared to corresponding cells in normal co-culture. The signal modulatory changes induced by Curcumin caused decreased release of EMT-mediators in CAFs and reversal of EMT in tumor cells, which was associated with decreased invasion. These data confirm the palliative potential of Curcumin in clinical application.