Secretome of bone marrow mesenchymal stromal cells cultured in a dynamic system induces neuroprotection and modulates microglial responsiveness in an α-synuclein overexpression rat model.

BACKGROUND AIMS: Parkinson's disease (PD) is the second most common neurodegenerative disorder. The etiology of the disease remains largely unknown, but evidence have suggested that the overexpression and aggregation of alpha-synuclein (α-syn) play key roles in the pathogenesis and progression of PD. Mesenchymal stromal cells (MSCs) have been earning attention in this field, mainly due to their paracrine capacity. The bioactive molecules secreted by MSCs, i.e. their secretome, have been associated with enhanced neuronal survival as well as a strong modulatory capacity of the microenvironments where the disease develops. The selection of the appropriate animal model is crucial in studies of efficacy assessment. Given the involvement of α-syn in the pathogenesis of PD, the evidence generated from the use of animal models that develop a pathologic phenotype due to the action of this protein is extremely valuable. Therefore, in this work, we established an animal model based on the viral vector-mediated overexpression of A53T α-syn and studied the impact of the secretome of bone marrow mesenchymal stromal cells MSC(M) as a therapeutic strategy. METHODS: Adult male rats were subjected to α-syn over expression in the nigrostriatal pathway to model dopaminergic neurodegeneration. The impact of locally administered secretome treatment from MSC(M) was studied. Motor impairments were assessed throughout the study coupled with whole-region (striatum and substantia nigra) confocal microscopy evaluation of histopathological changes associated with dopaminergic neurodegeneration and glial cell reactivity. RESULTS: Ten weeks after lesion induction, the animals received secretome injections in the substantia nigra pars compacta (SNpc) and striatum (STR). The secretome used was produced from bone marrow mesenchymal stromal cells MSC(M) expanded in a spinner flask (SP) system. Nine weeks later, animals that received the viral vector containing the gene for A53T α-syn and treated with vehicle (Neurobasal-A medium) presented dopaminergic cell loss in the SNpc and denervation in the STR. The treatment with secretome significantly reduced the levels of α-syn in the SNpc and protected the dopaminergic neurons (DAn) within the SNpc and STR. CONCLUSIONS: Our results are aligned with previous studies in both α-syn Caenorhabditis elegans models, as well as 6-OHDA rodent model, revealing that secretome exerted a neuroprotective effect. Moreover, these effects were associated with a modulation of microglial reactivity supporting an immunomodulatory role for the factors contained within the secretome. This further supports the development of new studies exploring the effects and the mechanism of action of secretome from MSC(M) against α-syn-induced neurotoxicity.

Hemodialysis vascular access coordinator: Three-level model for access management.

Management of vascular access is a challenge for the dialysis team, particularly to keep the arteriovenous access working. The vascular access coordinator can positively contribute to increase the number of arteriovenous fistulas and reduce central venous catheters. In this article, we introduce a new approach to vascular access management centered on (the results of setting up) the role of vascular access coordinator. We described the three-level model (3Level_M) for vascular access management organized in three levels: vascular access nurse manager, vascular access coordinator, and vascular access consultant. We defined the instrumental skills and training required to be developed by each element and clarify the articulation between the model and all members of the dialysis team related to vascular access.

A Rare Presentation of Pica Disorder in Soap Ingestion Variant: Case Report and Review.

ABSTRACT: Pica indicates the persistent ingestion of inedible substances over a period of at least 1 month, being discordant to the individual's cognitive development level and not directly attributable to cultural or social normative practices. The literature suggests that the prevalence of pica varies significantly according to the social and clinical context. It can co-occur with psychiatric disorders, with its etiology being poorly understood and most likely multifactorial. In this article, we report the case of a patient in her 50s with a clinical setting compatible with soap ingestion variant of pica disorder. In addition to the variety of ingested substances, pica can be associated with heterogeneous behavior, mainly of the obsessive-compulsive spectrum. Despite recent research, this condition is still a target of much speculation. This is a very rare and challenging presentation of a soap ingestion variant of pica with very few similar cases described to date.

Perceived stress modulates the activity between the amygdala and the cortex.

The significant link between stress and psychiatric disorders has prompted research on stress's impact on the brain. Interestingly, previous studies on healthy subjects have demonstrated an association between perceived stress and amygdala volume, although the mechanisms by which perceived stress can affect brain function remain unknown. To better understand what this association entails at a functional level, herein, we explore the association of perceived stress, measured by the PSS10 questionnaire, with disseminated functional connectivity between brain areas. Using resting-state fMRI from 252 healthy subjects spanning a broad age range, we performed both a seed-based amygdala connectivity analysis (static connectivity, with spatial resolution but no temporal definition) and a whole-brain data-driven approach to detect altered patterns of phase interactions between brain areas (dynamic connectivity with spatiotemporal information). Results show that increased perceived stress is directly associated with increased amygdala connectivity with frontal cortical regions, which is driven by a reduced occurrence of an activity pattern where the signals in the amygdala and the hippocampus evolve in opposite directions with respect to the rest of the brain. Overall, these results not only reinforce the pathological effect of in-phase synchronicity between subcortical and cortical brain areas but also demonstrate the protective effect of counterbalanced (i.e., phase-shifted) activity between brain subsystems, which are otherwise missed with correlation-based functional connectivity analysis.

Tie me up, tie me down: inhibiting RNA polymerase.

Mechanistic understanding of antibiotic action can yield crucial insights that aid in the design of new antibiotics. In this issue, Mukhopadhyay et al. (2008) uncover the mechanism by which the antibiotic myxopyronin inhibits bacterial RNA polymerase, suggesting a new target region in RNA polymerase for inhibitor design.

Novel Benzo[a]phenoxazinium Chlorides Functionalized with Sulfonamide Groups as NIR Fluorescent Probes for Vacuole, Endoplasmic Reticulum, and Plasma Membrane Staining.

The demand for new fluorophores for different biological target imaging is increasing. Benzo[a]phenoxazine derivatives are fluorochromophores that show promising optical properties for bioimaging, namely fluorescent emission at the NIR of the visible region, where biological samples have minimal fluorescence emission. In this study, six new benzo[a]phenoxazinium chlorides possessing sulfonamide groups at 5-amino-positions were synthesized and their optical and biological properties were tested. Compared with previous probes evaluated using fluorescence microscopy, using different S. cerevisiae strains, these probes, with sulfonamide groups, stained the vacuole membrane and/or the perinuclear membrane of the endoplasmic reticulum with great specificity, with some fluorochromophores capable of even staining the plasma membrane. Thus, the addition of a sulfonamide group to the benzo[a]phenoxazinium core increases their specificity and attributes for the fluorescent labeling of cell applications and fractions, highlighting them as quite valid alternatives to commercially available dyes.

Association of amygdala size with stress perception: Findings of a transversal study across the lifespan.

Daily routines are getting increasingly stressful. Interestingly, associations between stress perception and amygdala volume, a brain region implicated in emotional behaviour, have been observed in both younger and older adults. Life stress, on the other hand, has become pervasive and is no longer restricted to a specific age group or life stage. As a result, it is vital to consider stress as a continuum across the lifespan. In this study, we investigated the relationship between perceived stress and amygdala size in 272 healthy participants with a broad age range. Participants were submitted to a structural magnetic resonance imaging (MRI) to extract amygdala volume, and the Perceived Stress Scale (PSS) scores were used as the independent variable in volumetric regressions. We found that perceived stress is positively associated with the right amygdala volume throughout life.

IL-15Rα membrane anchorage in either cis or trans is required for stabilization of IL-15 and optimal signaling.

Interleukin (IL)-15 plays an important role in the communication between immune cells. It delivers its signal through different modes involving three receptor chains: IL-15Rα, IL-2Rß and IL-2Rγc. The combination of the different chains result in the formation of IL-15Rα/IL-2Rß/γc trimeric or IL-2Rß/γc dimeric receptors. In this study, we have investigated the role of the IL-15Rα chain in stabilizing the cytokine in the IL-2Rß/γc dimeric receptor. By analyzing the key amino acid residues of IL-15 facing IL-2Rß, we provide evidence of differential interfaces in the presence or in the absence of membrane-anchored IL-15Rα. Moreover, we found that the anchorage of IL-15Rα to the cell surface regardless its mode of presentation - i.e. cis or trans - is crucial for complete signaling. These observations show how the cells can finely modulate the intensity of cytokine signaling through the quality and the level of expression of the receptor chains.

Mesenchymal stem cell secretome protects against alpha-synuclein-induced neurodegeneration in a Caenorhabditis elegans model of Parkinson's disease.

BACKGROUND AIMS: The capacity of the secretome from bone marrow-derived mesenchymal stem cells (BMSCs) to prevent dopaminergic neuron degeneration caused by overexpression of alpha-synuclein (α-syn) was explored using two Caenorhabditis elegans models of Parkinson's disease (PD). METHODS: First, a more predictive model of PD that overexpresses α-syn in dopamine neurons was subjected to chronic treatment with secretome. This strain displays progressive dopaminergic neurodegeneration that is age-dependent. Following chronic treatment with secretome, the number of intact dopaminergic neurons was determined. Following these initial experiments, a C. elegans strain that overexpresses α-syn in body wall muscle cells was used to determine the impact of hBMSC secretome on α-syn inclusions. Lastly, in silico analysis of the components that constitute the secretome was performed. RESULTS: The human BMSC (hBMSC) secretome induced a neuroprotective effect, leading to reduced dopaminergic neurodegeneration. Moreover, in animals submitted to chronic treatment with secretome, the number of α-syn inclusions was reduced, indicating that the secretome of MSCs was possibly contributing to the degradation of those structures. In silico analysis identified possible suppressors of α-syn proteotoxicity, including growth factors and players in the neuronal protein quality control mechanisms. CONCLUSIONS: The present findings indicate that hBMSC secretome has the potential to be used as a disease-modifying strategy in future PD regenerative medicine approaches.

Synthesis and applications of RNAs with position-selective labelling and mosaic composition.

Knowledge of the structure and dynamics of RNA molecules is critical to understanding their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be dramatically enhanced by methods that enable incorporation of modified or labelled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. Here we develop a hybrid solid-liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labelling. We demonstrate its use by successfully preparing various isotope- or fluorescently labelled versions of the 71-nucleotide aptamer domain of an adenine riboswitch for nuclear magnetic resonance spectroscopy or single-molecule Förster resonance energy transfer, respectively. Those RNAs include molecules that were selectively isotope-labelled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently labelled in and near kissing loops. These selectively labelled RNAs have the same fold as those transcribed using conventional methods, but they greatly simplify the interpretation of NMR spectra. The single-position isotope- and fluorescently labelled RNA samples reveal multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labelling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection, and disease diagnostics.

Hepatic osteodystrophy in cirrhosis due to alcohol-related liver disease.

INTRODUCTION: hepatic osteodystrophy, including osteoporosis, is an abnormal bone metabolism related with chronic liver diseases. Osteoporosis is associated with an increased risk of bone fractures, with a significant impact on morbidity, mortality and healthcare costs. Nevertheless, bone disorders tend to be undervalued in cirrhosis due to alcohol-related liver disease (ALD cirrhosis). This study aimed to assess the prevalence of hepatic osteodystrophy and osteoporosis in ALD cirrhosis. METHODS: a prospective observational study was performed that included patients with ALD cirrhosis, between September 2017 and December 2018. Bone mineral density was determined by dual energy X-ray absorptiometry at the lumbar spine and the femoral neck. Hepatic osteodystrophy was defined as a T-score below -1 SD and osteoporosis as a T-score below -2.5 SD. RESULTS: ninety-four patients were included; 24.5 % (n = 23) had prior fragility fractures and ten patients suffered new osteoporotic fractures during the study period. Hepatic osteodystrophy was diagnosed in 79.8 % (n = 75) and osteoporosis in 21.3 % (n = 20) of cases. Patients with hepatic osteodystrophy presented significantly worse Child-Turcotte-Pugh (p < 0.05) and Model for End-Stage Liver Disease (MELD-sodium) scores (p = 0.01). According to the multivariate analysis, lower body mass index (BMI) (OR = 0.787, 95 % CI: 0.688-0.901, p = 0.001) and vitamin D deficiency (OR = 6.798, 95 % CI: 1.775-26.038, p = 0.005) were significantly and independently associated with hepatic osteodystrophy. Patients with osteoporosis also had a lower BMI (p = 0.01). Female patients and those with prior fragility fractures were more likely to suffer from osteoporosis (p < 0.05). CONCLUSION: our study revealed a high prevalence of hepatic osteodystrophy and osteoporosis in patients with ALD cirrhosis (particularly in those with a lower BMI) and a concerning high rate of fragility fractures. Bone mineral density should be assessed in order to allow for an early diagnosis and the implementation of preventive measures.

Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution.

GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.

A rare case of diarrhea and ascites.

BACKGROUND: Eosinophilic enterocolitis is a rare condition included in the spectrum of the eosinophilic gastrointestinal disorders. Diagnosis is based on clinical presentation combined with an increase infiltration of eosinophils in the gastrointestinal tract, in the absence of other secondary causes of eosinophilic infiltration. CASE PRESENTATION: We report a case of a 22-year-old male with eosinophilic enterocolitis presenting with malabsorption syndrome (diarrhea, vomiting, weight loss), bowel wall thickening, and ascites. Secondary causes of intestinal eosinophilia were excluded, and diagnosis was established in a timely manner. Treatment plan included a 6-food elimination diet and corticosteroid therapy, with clinical remission after 2 weeks of therapy. The patient remains asymptomatic after 12 months of follow-up, with no relapse.

A dancer caught midstep: the structure of ATP-bound Hsp70.

Hsp70 ATP binding induces substrate release, but the transiency of this state has inhibited its characterization. In this issue, Kityk et al. determine the Hsp70(∗)ATP structure utilizing engineered disulfide bonds, providing insights into the workings of this essential molecular machine.

Enhanced microfracture using acellular scaffolds improves results after treatment of symptomatic focal grade III/IV knee cartilage lesions but current clinical evidence does not allow unequivocal recommendation.

PURPOSE: To systematically analyse post-operative outcomes following enhanced microfracture procedures in focal cartilage injuries of the knee. METHODS: Database searches were conducted in PubMed, EMBASE and Cochrane Library databases up to 30 November 2018, for clinical studies in humans that assessed surgical outcomes of enhanced microfracture procedures in focal cartilage injuries of the knee. The clinical, functional and imaging outcomes were assessed and summarized. The MINORS scale was used to assess the methodological quality of the studies included. RESULTS: Ten studies were included comprising a total of 331 patients (mean age of 37.0 ± 5.5 years, body mass 25.2 ± 1.7 kg m2, 56% male and 42% left knee), 278 femoral condyle chondral defects (147 medial, 35 lateral and 78 undefined) and 43 chondral defects distributed by the tibial plateau, patella and femoral trochlea. The chondral defects were mostly Outerbridge grade III or IV and the mean defect size was 3.2 ± 0.6 cm2. Studies consistently demonstrated significant improvement in the patient-reported outcome measures from baseline to final follow-up. Overall, imaging outcomes showed inconsistent results. Treatment-related adverse events were poorly reported. CONCLUSION: Enhanced microfracture techniques significantly result in improved patient-reported outcome measures over the MCID, but provide inconsistent imaging results. Current clinical evidence does not allow for unequivocal recommendation of enhanced microfracture to treat symptomatic focal grade III/IV knee cartilage lesions. LEVEL OF EVIDENCE: IV.

Impact of Aging on the 6-OHDA-Induced Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. The neurodegeneration leading to incapacitating motor abnormalities mainly occurs in the nigrostriatal pathway due to the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Several animal models have been developed not only to better understand the mechanisms underlying neurodegeneration but also to test the potential of emerging disease-modifying therapies. However, despite aging being the main risk factor for developing idiopathic PD, most of the studies do not use aged animals. Therefore, this study aimed at assessing the effect of aging in the unilateral 6-hydroxydopamine (6-OHDA)-induced animal model of PD. For this, female young adult and aged rats received a unilateral injection of 6-OHDA into the medial forebrain bundle. Subsequently, the impact of aging on 6-OHDA-induced effects on animal welfare, motor performance, and nigrostriatal integrity were assessed. The results showed that aging had a negative impact on animal welfare after surgery. Furthermore, 6-OHDA-induced impairments on skilled motor function were significantly higher in aged rats when compared with their younger counterparts. Nigrostriatal histological analysis further revealed an increased 6-OHDA-induced dopaminergic cell loss in the SNpc of aged animals when compared to young animals. Overall, our results demonstrate a higher susceptibility of aged animals to 6-OHDA toxic insult.

Di- and heptavalent nicotinic analogues to interfere with α7 nicotinic acetylcholine receptors.

In the field of nicotinic acetylcholine receptors (nAChRs), recognized as important therapeutic targets, much effort has been dedicated to the development of nicotinic analogues to agonize or antagonize distinct homo- and heteropentamers nAChR subtypes, selectively. In this work we developed di- and heptavalent nicotinic derivatives based on ethylene glycol (EG) and cyclodextrin cores, respectively. The compounds showed a concentration dependent inhibition of acetylcholine-induced currents on α7 nAChR expressed by Xenopus oocytes. Interesting features were observed with the divalent nicotinic derivatives, acting as antagonists with varied inhibitory concentrations (IC50) in function of the spacer arm length. The best divalent compounds showed a 16-fold lowered IC50 compared to the monovalent reference (12 vs 195 µM). Docking investigations provide guidelines to rationalize these experimental findings.

The Physics of Entropic Pulling: A Novel Model for the Hsp70 Motor Mechanism.

Hsp70s use ATP to generate forces that disassemble protein complexes and aggregates, and that translocate proteins into organelles. Entropic pulling has been proposed as a novel mechanism, distinct from the more familiar power-stroke and Brownian ratchet models, for how Hsp70s generate these forces. Experimental evidence supports entropic pulling, but this model may not be well understood among scientists studying these systems. In this review we address persistent misconceptions regarding the dynamics of proteins in solution that contribute to this lack of understanding, and we clarify the basic physics of entropic pulling with some simple analogies. We hope that increased understanding of the entropic pulling mechanism will inform future efforts to characterize how Hsp70s function as motors, and how they coordinate with their regulatory cochaperones in mechanochemical cycles that transduce the energy of ATP hydrolysis into physical changes in their protein substrates.

Mechanistic and Structural Insights on the IL-15 System through Molecular Dynamics Simulations.

Interleukin 15 (IL-15), a four-helix bundle cytokine, is involved in a plethora of different cellular functions and, particularly, plays a key role in the development and activation of immune responses. IL-15 forms receptor complexes by binding with IL-2Rß- and common γ(γc)-signaling subunits, which are shared with other members of the cytokines family (IL-2 for IL-2Rß- and all other γc- cytokines for γc). The specificity of IL-15 is brought by the non-signaling α-subunit, IL-15Rα. Here we present the results of molecular dynamics simulations carried out on four relevant forms of IL-15: its monomer, IL-15 interacting individually with IL-15Rα (IL-15/IL-15Rα), with IL-2Rß/γc subunits (IL-15/IL-2Rß/γc) or with its three receptors simultaneously (IL-15/IL-15Rα/IL-2Rß/γc). Through the analyses of the various trajectories, new insights on the structural features of the interfaces are highlighted, according to the considered form. The comparison of the results with the experimental data, available from X-ray crystallography, allows, in particular, the rationalization of the importance of IL-15 key residues (e.g. Asp8, Lys10, Glu64). Furthermore, the pivotal role of water molecules in the stabilization of the various protein-protein interfaces and their H-bonds networks are underlined for each of the considered complexes.