Interactions between Dental MSCs and Biomimetic Composite Scaffold during Bone Remodeling Followed by In Vivo Real-Time Bioimaging.

Oral-maxillofacial tumor removal can generate critical bone defects and major problems for patients, causing dysfunctionalities and affecting oral competencies such as mastication, swallowing, and breathing. The association of novel biomaterials and cell therapies in tissue engineering strategies could offer new strategies to promote osteomucosa healing. This study focused on the development of a bioengineered construct loaded with human dental follicle cells (MSCs). To increase the bioconstruct integration to the surrounding tissue, a novel and comprehensive approach was designed combining an injectable biomimetic hydrogel and dental stem cells (hDFMSCs) expressing luminescence/fluorescence for semi-quantitative tissue imaging in live animals. This in vivo model with human MSCs was based on an intramembranous bone regeneration process (IMO). Biologically, the biocomposite based on collagen/nanohydroxyapatite filled with cell-loaded osteopontin-fibrin hydrogel (Coll/nanoHA OPN-Fb) exhibited a high cellular proliferation rate, increased bone extracellular matrix deposition (osteopontin) and high ALP activity, indicating an early osteogenic differentiation. Thus, the presence of human OPN enhanced hDFMSC adhesion, migration, and spatial distribution within the 3D matrix. The developed 3D bioconstruct provided the necessary pro-regenerative effect to modulate the biological response, precisely fitting the bone defect with fine-tuned adjustment to the surrounding original structure and promoting oral osteomucosa tissue regeneration. We were also able to track the cells in vivo and evaluate their behavior (migration, proliferation, and differentiation), providing a glimpse into bone regeneration and helping in the optimization of patient-specific therapies.

Stathmin recruits tubulin to Listeria monocytogenes-induced actin comets and promotes bacterial dissemination.

The tubulin cytoskeleton is one of the main components of the cytoarchitecture and is involved in several cellular functions. Here, we examine the interplay between Listeria monocytogenes (Lm) and the tubulin cytoskeleton upon cellular infection. We show that non-polymeric tubulin is present throughout Lm actin comet tails and, to a less extent, in actin clouds. Moreover, we demonstrate that stathmin, a regulator of microtubule dynamics, is also found in these Lm-associated actin structures and is required for tubulin recruitment. Depletion of host stathmin results in longer comets containing less F-actin, which may be correlated with higher levels of inactive cofilin in the comet, thus suggesting a defect on local F-actin dynamics. In addition, intracellular bacterial speed is significantly reduced in stathmin-depleted cells, revealing the importance of stathmin/tubulin in intracellular Lm motility. In agreement, the area of infection foci and the total bacterial loads are also significantly reduced in stathmin-depleted cells. Collectively, our results demonstrate that stathmin promotes efficient cellular infection, possibly through tubulin recruitment and control of actin dynamics at Lm-polymerized actin structures.

An open computational toolbox to analyze multi- and single-unit sympathetic nerve activity in microneurography.

Microelectrode recordings from human peripheral and cranial nerves provide a means to study both afferent and efferent axonal signals at different levels of detail, from multi- to single-unit activity. Their analysis can lead to advancements both in diagnostic and in the understanding of the genesis of neural disorders. However, most of the existing computational toolboxes for the analysis of microneurographic recordings are limited in scope or not open-source. Additionally, conventional burst-based metrics are not suited to analyze pathological conditions and are highly sensitive to distance of the microelectrode tip from the active axons. To address these challenges, we developed an open-source toolbox that offers advanced analysis capabilities for studying neuronal reflexes and physiological responses to peripheral nerve activity. Our toolbox leverages the observation of temporal sequences of action potentials within inherently cyclic signals, introducing innovative methods and indices to enhance analysis accuracy. Importantly, we have designed our computational toolbox to be accessible to novices in biomedical signal processing. This may include researchers and professionals in healthcare domains, such as clinical medicine, life sciences, and related fields. By prioritizing user-friendliness, our software application serves as a valuable resource for the scientific community, allowing to extract advanced metrics of neural activity in short time and evaluate their impact on other physiological variables in a consistent and standardized manner, with the final aim to widen the use of microneurography among researchers and clinicians.

The Role of Spastin in Axon Biology.

Neurons are highly polarized cells with elaborate shapes that allow them to perform their function. In neurons, microtubule organization-length, density, and dynamics-are essential for the establishment of polarity, growth, and transport. A mounting body of evidence shows that modulation of the microtubule cytoskeleton by microtubule-associated proteins fine tunes key aspects of neuronal cell biology. In this respect, microtubule severing enzymes-spastin, katanin and fidgetin-a group of microtubule-associated proteins that bind to and generate internal breaks in the microtubule lattice, are emerging as key modulators of the microtubule cytoskeleton in different model systems. In this review, we provide an integrative view on the latest research demonstrating the key role of spastin in neurons, specifically in the context of axonal cell biology. We focus on the function of spastin in the regulation of microtubule organization, and axonal transport, that underlie its importance in the intricate control of axon growth, branching and regeneration.

Listeria monocytogenes Interferes with Host Cell Mitosis through Its Virulence Factors InlC and ActA.

Listeria monocytogenes is among the best-characterized intracellular pathogens. Its virulence factors, and the way they interfere with host cells to hijack host functions and promote the establishment and dissemination of the infection, have been the focus of multiple studies over the last 30 years. During cellular infection, L. monocytogenes was shown to induce host DNA damage and delay the host cell cycle to its own benefit. However, whether the cell cycle stage would interfere with the capacity of Listeria to infect human cultured cell lines was never assessed. We found here that L. monocytogenes preferentially infects cultured cells in G2/M phases. Inside G2/M cells, the bacteria lead to an increase in the overall mitosis duration by delaying the mitotic exit. We showed that L. monocytogenes infection causes a sustained activation of the spindle assembly checkpoint, which we correlated with the increase in the percentage of misaligned chromosomes detected in infected cells. Moreover, we demonstrated that chromosome misalignment in Listeria-infected cells required the function of two Listeria virulence factors, ActA and InlC. Our findings show the pleiotropic role of Listeria virulence factors and their cooperative action in successfully establishing the cellular infection.

Profilin 1 delivery tunes cytoskeletal dynamics toward CNS axon regeneration.

After trauma, regeneration of adult CNS axons is abortive, causing devastating neurologic deficits. Despite progress in rehabilitative care, there is no effective treatment that stimulates axonal growth following injury. Using models with different regenerative capacities, followed by gain- and loss-of-function analysis, we identified profilin 1 (Pfn1) as a coordinator of actin and microtubules (MTs), powering axonal growth and regeneration. In growth cones, Pfn1 increased actin retrograde flow, MT growth speed, and invasion of filopodia by MTs, orchestrating cytoskeletal dynamics toward axonal growth. In vitro, active Pfn1 promoted MT growth in a formin-dependent manner, whereas localization of MTs to growth cone filopodia was facilitated by direct MT binding and interaction with formins. In vivo, Pfn1 ablation limited regeneration of growth-competent axons after sciatic nerve and spinal cord injury. Adeno-associated viral (AAV) delivery of constitutively active Pfn1 to rodents promoted axonal regeneration, neuromuscular junction maturation, and functional recovery of injured sciatic nerves, and increased the ability of regenerating axons to penetrate the inhibitory spinal cord glial scar. Thus, we identify Pfn1 as an important regulator of axonal regeneration and suggest that AAV-mediated delivery of constitutively active Pfn1, together with the identification of modulators of Pfn1 activity, should be considered to treat the injured nervous system.

Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System.

Mesenchymal stromal cells (MSC) hold great promise for tissue engineering and cell-based therapies due to their multilineage differentiation potential and intrinsic immunomodulatory and trophic activities. Over the past years, increasing evidence has proposed extracellular vesicles (EVs) as mediators of many of the MSC-associated therapeutic features. EVs have emerged as mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. EVs are derived from cell membranes, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. In this work, EVs were produced by MSC isolated from different human tissue sources [bone marrow (BM), adipose tissue (AT), and umbilical cord matrix (UCM)]. A serum-/xeno-free microcarrier-based culture system was implemented in a Vertical-WheelTM bioreactor (VWBR), employing a human platelet lysate culture supplement (UltraGROTM-PURE), toward the scalable production of MSC-derived EVs (MSC-EVs). The morphology and structure of the manufactured EVs were assessed by atomic force microscopy, while EV protein markers were successfully identified in EVs by Western blot, and EV surface charge was maintained relatively constant (between -15.5 ± 1.6 mV and -19.4 ± 1.4 mV), as determined by zeta potential measurements. When compared to traditional culture systems under static conditions (T-flasks), the VWBR system allowed the production of EVs at higher concentration (i.e., EV concentration in the conditioned medium) (5.7-fold increase overall) and productivity (i.e., amount of EVs generated per cell) (3-fold increase overall). BM, AT and UCM MSC cultured in the VWBR system yielded an average of 2.8 ± 0.1 × 1011, 3.1 ± 1.3 × 1011, and 4.1 ± 1.7 × 1011 EV particles (n = 3), respectively, in a 60 mL final volume. This bioreactor system also allowed to obtain a more robust MSC-EV production, regarding their purity, compared to static culture. Overall, we demonstrate that this scalable culture system can robustly manufacture EVs from MSC derived from different tissue sources, toward the development of novel therapeutic products.

The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction.

Neurons have a membrane periodic skeleton (MPS) composed of actin rings interconnected by spectrin. Here, combining chemical and genetic gain- and loss-of-function assays, we show that in rat hippocampal neurons the MPS is an actomyosin network that controls axonal expansion and contraction. Using super-resolution microscopy, we analyzed the localization of axonal non-muscle myosin II (NMII). We show that active NMII light chains are colocalized with actin rings and organized in a circular periodic manner throughout the axon shaft. In contrast, NMII heavy chains are mostly positioned along the longitudinal axonal axis, being able to crosslink adjacent rings. NMII filaments can play contractile or scaffolding roles determined by their position relative to actin rings and activation state. We also show that MPS destabilization through NMII inactivation affects axonal electrophysiology, increasing action potential conduction velocity. In summary, our findings open new perspectives on axon diameter regulation, with important implications in neuronal biology.

Aspirin and blood pressure: Effects when used alone or in combination with antihypertensive drugs.

Arterial hypertension is a major risk factor for cardiovascular and renal events. Lowering blood pressure is thus an important strategy for reducing morbidity and mortality. Since low-dose aspirin is a cornerstone in the prevention of adverse cardiovascular outcomes, combined treatment with aspirin and antihypertensive drugs is very common. However, the impact of aspirin therapy on blood pressure control remains a subject of intense debate. Recent data suggest that the cardioprotective action of aspirin extends beyond its well-known antithrombotic effect. Aspirin has been shown to trigger the synthesis of specialized pro-resolving lipid mediators from arachidonic acid and omega-3 fatty acids. These novel anti-inflammatory and pro-resolving mediators actively stimulate the resolution of inflammation and tissue regeneration. Additionally, they may contribute to other protective effects on redox status and vascular reactivity that have also been attributed to aspirin. Of note, aspirin has been shown to improve vasodilation through cyclooxygenase-independent mechanisms. On the other hand, higher aspirin doses have been reported to exert a negative impact on blood pressure due to inhibition of cyclooxygenase-2 activity, which reduces renal blood flow, glomerular filtration rate and sodium and water excretion. This review aims to provide an overview of the effects of aspirin on blood pressure and the underlying mechanisms, focusing on the interaction between aspirin and antihypertensive drugs. Studies in both experimental and human hypertension are presented.

Computed tomography enterography or magnetic resonance enterography in Crohn's disease - which to choose? / Enterografia por tomografia computadorizada ou por ressonância magnética na doença de Crohn - qual escolher?

ABSTRACT Rationale and objectives: Evaluation of Crohn's disease by computed tomography enterography, magnetic resonance imaging enterography and colonoscopy is essential for disease monitoring. The aim of this study is to evaluate this exams acuity. Materials and methods: Patients with histological diagnosis of Crohn's disease who underwent computed tomography enterography, magnetic resonance imaging enterography and colonoscopy in the period of January 1st, 2009 and July 31st, 2016 and the realization of these exams did not exceed a time interval of 6 months was included. Sensitivity, specificity, positive and negative predictive values (PPV, NPV), Cohen's kappa (K), agreement and disagreement were calculated. Results: Comparing computed tomography enterography and magnetic resonance imaging enterography with colonoscopy, there was an agreement of 85.7% and a disagreement of 14.3% in Crohn's disease overall detection, for both exams. Computed tomography enterography and colonoscopy showed greater agreement in abscesses and lumen reduction detection (C = 95.2%) and magnetic resonance imaging enterography and colonoscopy in abscesses detection (C = 92.9%). Comparing magnetic resonance imaging enterography and computed tomography enterography, greater agreement was observed in detection of lumen reduction/dilatation (C = 96%). K showed considerable agreement in detection of mesenteric lymph nodes, fistulas, mural inflammation and thickening. The sensitivity, specificity, PPV and NPV were respectively set at 94.12% (95% CI 71.31-99.85), 50% (95% CI 6.76-93.24), 88.89% (95% CI 65.29-98.62) and 66.67% (95% CI 9.43-99.16) for CTE and 90.62% (95% CI 80.70-96.48), 33.33% (95% CI 4.33-77.72), 93.55% (95% CI 84.30-98.21) and 25% (95% CI 3.19-65.09) for MRIE. Conclusions: Although computed tomography enterography presents better sensitivity and specificity than magnetic resonance imaging enterography, both present high agreement values in detection of characteristic Crohn's disease findings, therefore the selection of the best test to monitor Crohn's disease should take into account aspects such as age, tolerability, disease phenotype and resources availability.

RESUMO Fundamentação e objetivos: A avaliação da doença de Crohn por enterografia por tomografia computadorizada, enterografia por ressonância magnética e colonoscopia é essencial para o monitoramento da doença. Este estudo teve como objetivo avaliar a acuidade desses exames. Materiais e métodos: O estudo incluiu pacientes com diagnóstico histológico de doença de Crohn submetidos à enterografia por tomografia computadorizada, enterografia por ressonância magnética e colonoscopia no período entre 1° de janeiro de 2009 e 31 de julho de 2016; os exames foram realizados em um intervalo de máximo de seis meses. Calculou-se a sensibilidade, especificidade, valores preditivos positivo e negativo (VPP, VPN), Kappa (K) de Cohen, concordância e discordância. Resultados: Ao comparar enterografia por tomografia computadorizada e enterografia por ressonância magnética com colonoscopia, observou-se uma concordância de 85,7% e discordância de 14,3% na detecção global da doença de Crohn para ambos os exames. A enterografia por tomografia computadorizada e a colonoscopia mostraram maior concordância nos abscessos e na detecção da redução da luz (C = 95,2%) e enterografia por ressonância magnética e colonoscopia, na detecção de abscessos (C = 92,9%). Ao comparar a enterografia por ressonância magnética e a enterografia por tomografia computadorizada, observou-se maior concordância na detecção da redução/dilatação do lúmen (C = 96%). Os valores de K mostraram concordância considerável na detecção de linfonodos mesentéricos, fístulas, inflamação e espessamento mural. A sensibilidade, especificidade, VPP e VPN foram, respectivamente, 94,12% (IC 95%: 71,31 ± 99,85), 50% (IC 95%: 6,76 ± 93,24), 88,89% (IC 95%: 65,29 ± 98,62) e 66,67% (IC 95%: 9,43 ± 99,16) para ETC e 90,62% (IC 95%: 80,70 ± 96,48), 33,33% (IC 95%: 4,33 ± 77,72), 93,55% (IC 95%: 84,30 ± 98,21) e 25% (IC 95%: 3,19 ± 65,09) para enterografia por ressonância magnética. Conclusões: Embora a enterografia por tomografia computadorizada apresente melhor sensibilidade e especificidade que a enterografia por ressonância magnética, ambas apresentam altos valores de concordância na detecção dos achados característicos da doença de Crohn. Assim, a seleção do teste mais adequado para monitorar a doença de Crohn deve levar em consideração aspectos como idade, tolerabilidade, fenótipo da doença e disponibilidade de recursos.

Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection.

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.

CagA associates with c-Met, E-cadherin, and p120-catenin in a multiproteic complex that suppresses Helicobacter pylori-induced cell-invasive phenotype.

BACKGROUND: Helicobacter pylori induces an invasive phenotype in gastric epithelial cells through a mechanism that requires the type IV secretion system and the phosphorylation of c-Met. The E-cadherin-catenin complex is a major component of the adherens junctions and functions as an invasion suppressor. We investigated whether E-cadherin has a role in H. pylori-induced, c-Met phosphorylation-dependent cell-invasive phenotype. METHODS: AGS cells that lack E-cadherin and that are invasive to H. pylori stimulation were transduced with E-cadherin and infected with H. pylori. NCI-N87 cells, which endogenously express E-cadherin, were also used for infection experiments. RESULTS: E-cadherin was sufficient to suppress not only H. pylori-mediated cell-invasive phenotype but also c-Met and p120-catenin tyrosine phosphorylation. H. pylori infection led to increased interactions between E-cadherin and p120-catenin, c-Met and E-cadherin, and c-Met and p120-catenin. Using in vitro infection assays, we showed that H. pylori CagA interacts with E-cadherin, p120-catenin, and c-Met. Finally, using small interfering RNA, we showed that interactions between CagA and E-cadherin and between CagA and p120-catenin were established through c-Met. CONCLUSIONS: We suggest that H. pylori alters the E-cadherin-catenin complex, leading to formation of a multiproteic complex composed of CagA, c-Met, E-cadherin, and p120-catenin. This complex abrogates c-Met and p120-catenin tyrosine phosphorylation and suppresses the cell-invasive phenotype induced by H. pylori.