Graft-derived neurons and bystander effects are maintained for six months after human iPSC-derived NESC transplantation in mice's cerebella.

Machado-Joseph disease (MJD) is a neurodegenerative disorder characterized by widespread neuronal death affecting the cerebellum. Cell therapy can trigger neuronal replacement and neuroprotection through bystander effects providing a therapeutic option for neurodegenerative diseases. Here, human control (CNT) and MJD iPSC-derived neuroepithelial stem cells (NESC) were established and tested for their therapeutic potential. Cells' neuroectodermal phenotype was demonstrated. Brain organoids obtained from the Control NESC showed higher mRNA levels of genes related to stem cells' bystander effects, such as BDNF, NEUROD1, and NOTCH1, as compared with organoids produced from MJD NESC, suggesting that Control NESC have a higher therapeutic potential. Graft-derived glia and neurons, such as cells positive for markers of cerebellar neurons, were detected six months after NESC transplantation in mice cerebella. The graft-derived neurons established excitatory and inhibitory synapses in the host cerebella, although CNT neurons exhibited higher excitatory synapse numbers compared with MJD neurons. Cell grafts, mainly CNT NESC, sustained the bystander effects through modulation of inflammatory interleukins (IL1B and IL10), neurotrophic factors (NGF), and neurogenesis-related proteins (Msi1 and NeuroD1), for six months in the mice cerebella. Altogether this study demonstrates the long-lasting therapeutic potential of human iPSC-derived NESC in the cerebellum.

TERTmonitor-qPCR Detection of TERTp Mutations in Glioma.

Telomerase promoter (TERTp) mutations are frequently observed in various types of tumours and commonly characterised by two specific hotspots located at positions -124 and -146 upstream of the start codon. They enhance TERTp activity, resulting in increased TERT expression. In central nervous system (CNS) tumours, they are integrated as biomarkers, aiding in the diagnosis and with a role in prognosis, where, in some settings, they are associated with aggressive behaviour. In this study, we evaluated the performance of TERTmonitor for TERTp genotyping in a series of 185 gliomas in comparison to the traditional method, Sanger sequencing. Against the gold-standard Sanger method, TERTmonitor performed with a 97.8% accuracy. Inaccuracy was mainly due to the over-detection of variants in negative cases (by Sanger) and the presence of variants that can modify the chemistry of the probe detection. The distribution of the mutations was comparable to other series, with the -124 being the most represented (38.92% for Sanger and TERTmonitor) and more prevalent in the higher-grade tumours, gliosarcoma (50.00%) and glioblastoma (52.6%). The non-matched cases are debatable, as we may be dealing with the reduced sensitivity of Sanger in detecting rare alleles, which strengthens the use of the TERTmonitor. With this study, we present a reliable and rapid potential tool for TERTp genotyping in gliomas.

Combining inflammatory miRNA molecules as diagnostic biomarkers for depression: a clinical study.

Background: Inflammation has been implicated in core features of depression pathophysiology and treatment resistance. Therefore, new challenges in the discovery of inflammatory mediators implicated in depression have emerged. MicroRNAs (miRNAs) have been found aberrantly expressed in several pathologies, increasing their potential as biomarkers and therapeutical targets. In this study, the aim was to assess the changes and biomarker potential of inflammation-related miRNAs in depression patients. Methods: Depression diagnosis was performed according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5). 40 healthy controls and 32 depression patients were included in the study. The levels of inflammatory cytokines were measured in plasma, and expression levels of cytokines and inflammation-related miRNAs were evaluated in peripheral blood mononuclear cells (PBMCs). Results: Depression patients were found to have a pro-inflammatory profile in plasma, with significantly higher levels of TNF-α and CCL2 compared with controls. In PBMCs of depression patients, TNF-α and IL-6 expression levels were significantly up and downregulated, respectively. Moreover, miR-342 levels were found upregulated, while miR-146a and miR-155 were significantly downregulated. miR-342 expression levels were positively correlated with TNF-α. Importantly, when analyzed as a diagnostic panel, receiver operating characteristics (ROC) analysis of miR-342, miR-146a, miR-155 in combination, showed to be highly specific and sensitive in distinguishing between depression patients and healthy controls. Conclusion: In summary, these findings suggest that inflammation-related miRNAs are aberrantly expressed in depression patients. Moreover, we show evidences on the potential of the combination of dysregulated miRNAs as a powerful diagnostic tool for depression.

A challenging case of sexsomnia in an adolescent female presenting with depressive-like symptoms.

Sleep-related sexualized behaviors occur in the parasomnia known as sexsomnia, recognized as a variant of confusional arousals in the International Classification of Sleep Disorders, third edition. These instinctive behaviors of a sexual nature emerge from deep non-rapid eye movement sleep, and patients often present with distinguishing features within this sleep disorder category. There are often adverse psychosocial consequences and not uncommonly medicolegal implications. While associations to psychiatric consequences from the sexsomnia have been demonstrated and efforts to further typify this condition have been made, sexsomnia remains incompletely characterized in the more than 200 published cases to date, with male predominance. We now present the first reported case of an adolescent female with sexsomnia that was triggered by the onset of Crohn's disease and its treatment with azathioprine and with interpersonal consequences leading to an initial psychiatric consultation on account of depressive symptoms. These symptoms were deemed to be secondary to the sexsomnia. In addition to describing unusual and clinically relevant features in this case of sexsomnia, this original case provides insights into triggers, predisposing factors, perpetuating factors, and therapeutic considerations that are important for raising awareness in sleep clinicians, primary care providers, and mental health professionals. CITATION: Brás J, Schenck CH, Andrade R, et al. A challenging case of sexsomnia in an adolescent female presenting with depressive-like symptoms. J Clin Sleep Med. 2023;19(10):1845-1847.

Establishment and characterization of human pluripotent stem cells-derived brain organoids to model cerebellar diseases.

The establishment of robust human brain organoids to model cerebellar diseases is essential to study new therapeutic strategies for cerebellum-associated disorders. Machado-Joseph disease (MJD) is a cerebellar hereditary neurodegenerative disease, without therapeutic options able to prevent the disease progression. In the present work, control and MJD induced-pluripotent stem cells were used to establish human brain organoids. These organoids were characterized regarding brain development, cell type composition, and MJD-associated neuropathology markers, to evaluate their value for cerebellar diseases modeling. Our data indicate that the organoids recapitulated, to some extent, aspects of brain development, such as astroglia emerging after neurons and the presence of ventricular-like zones surrounded by glia and neurons that are found only in primate brains. Moreover, the brain organoids presented markers of neural progenitors proliferation, neuronal differentiation, inhibitory and excitatory synapses, and firing neurons. The established brain organoids also exhibited markers of cerebellar neurons progenitors and mature cerebellar neurons. Finally, MJD brain organoids showed higher ventricular-like zone numbers, an indication of lower maturation, and an increased number of ataxin-3-positive aggregates, compared with control organoids. Altogether, our data indicate that the established organoids recapitulate important characteristics of human brain development and exhibit cerebellar features, constituting a resourceful tool for testing therapeutic approaches for cerebellar diseases.

Stress-induced depressive-like behavior in male rats is associated with microglial activation and inflammation dysregulation in the hippocampus in adulthood.

Neuroinflammation is increasingly recognized as playing a critical role in depression. Early-life stress exposure and constitutive differences in glucocorticoid responsiveness to stressors are two key risk factors for depression, but their impacts on the inflammatory status of the brain is still uncertain. Moreover, there is a need to identify specific molecules involved in these processes with the potential to be used as alternative therapeutic targets in inflammation-related depression. Here, we studied how peripubertal stress (PPS) combined with differential corticosterone (CORT)-stress responsiveness (CSR) influences depressive-like behaviors and brain inflammatory markers in male rats in adulthood, and how these alterations relate to microglia activation and miR-342 expression. We found that high-CORT stress-responsive (H-CSR) male rats that underwent PPS exhibited increased anhedonia and passive coping responses in adulthood. Also, animals exposed to PPS showed increased hippocampal TNF-α expression, which positively correlated with passive coping responses. In addition, PPS caused long-term effects on hippocampal microglia, particularly in H-CSR rats, with increased hippocampal IBA-1 expression and morphological alterations compatible with a higher degree of activation. H-CSR animals also showed upregulation of hippocampal miR-342, a mediator of TNF-α-driven microglial activation, and its expression was positively correlated with TNF-α expression, microglial activation and passive coping responses. Our findings indicate that individuals with constitutive H-CSR are particularly sensitive to developing protracted depression-like behaviors following PPS exposure. In addition, they show neuro-immunological alterations in adulthood, such as increased hippocampal TNF-α expression, microglial activation and miR-342 expression. Our work highlights miR-342 as a potential therapeutic target in inflammation-related depression.

Measurement of Water Level in Urban Streams under Bad Weather Conditions.

Flood control and water resources management require monitoring the water level in rivers and streams. Water level measurement techniques increasingly consider image processing procedures. Most of the systems use a staff gauge to support the waterline detection. However, these techniques can fail when applied to urban stream channels due to water undulation, debris on the water surface, and traces of rain captured by the camera, and other adverse effects on images can be quite dramatic on the results. The importance of considering these effects is that they are usually associated with the variation in the water level with the occurrence of rain. The technique proposed in this work uses a larger detection zone to minimize the effects that tend to obstruct the waterline. The developed system uses an infrared camera to operate during the day and night. Images acquired in different weather conditions helped to evaluate the proposed technique. The water level measurement accuracy was about 1.8 cm for images taken during the day and 2.8 cm for images taken at night. During short periods of heavy rain, the accuracy was 2.6 cm for the daytime and 3.4 cm for the nighttime. Infrared lighting can improve detection accuracy at night. The developed technique provides good accuracy under different weather conditions by combining information from various detection positions to deal with waterline detection issues.

Fibrinogen and magnesium combination biomaterials modulate macrophage phenotype, NF-kB signaling and crosstalk with mesenchymal stem/stromal cells.

Macrophage behavior upon biomaterial implantation conditions the inflammatory response and subsequent tissue repair. The hypothesis behind this work was that fibrinogen (Fg) and magnesium (Mg) biomaterials, used in combination (FgMg) could act synergistically to modulate macrophage activation, promoting a pro-regenerative phenotype. Materials were characterized by scanning electron microscopy, Fg and Mg degradation products were quantified by atomic absorption spectroscopy and ELISA. Whole blood immune cells and primary human monocyte-derived macrophages were exposed to the biomaterials extracts in unstimulated (M0) or pro-inflammatory LPS or LPS-IFNγ (M1) conditions. Macrophage phenotype was evaluated by flow cytometry, cytokines secreted by whole blood cells and macrophages were measured by ELISA, and signaling pathways were probed by Western blotting. The secretomes of macrophages preconditioned with biomaterials extracts were incubated with human mesenchymal stem/stromal cells (MSC) and their effect on osteogenic differentiation was evaluated via Alkaline Phosphatase (ALP) activity and alizarin red staining. Scaffolds of Fg, alone or in the FgMg combination, presented similar 3D porous architectures. Extracts from FgMg materials reduced LPS-induced TNF-α secretion by innate immune cells, and macrophage M1 polarization upon LPS-IFNγ stimulation, resulting in lower cell surface CD86 expression, lower NFκB p65 phosphorylation and reduced TNF-α secretion. Moreover, while biomaterial extracts per se did not enhance MSC osteogenic differentiation, macrophage secretome, particularly from cells exposed to FgMg extracts, increased MSC ALP activity and alizarin red staining, compared with extracts alone. These findings suggest that the combination of Fg and Mg synergistically influences macrophage pro-inflammatory activation and crosstalk with MSC. STATEMENT OF SIGNIFICANCE: Modulating macrophage phenotype by degradable and bioactive biomaterials is an increasingly explored strategy to promote tissue repair/regeneration. Fibrinogen (Fg) and magnesium (Mg)-based materials have been explored in this context. Previous work from our group showed that monocytes interact with fibrinogen adsorbed onto chitosan surfaces through TLR4 and that fibrinogen scaffolds promote in vivo bone regeneration. Also, magnesium ions have been reported to modulate macrophage pro-inflammatory M1 stimulation and to promote bone repair. Here we report, for the first time, the combination of Fg and Mg materials, hypothesizing that it could act synergistically on macrophages, directing them towards a pro-regenerative phenotype. As a first step towards proving/disproving our hypothesis we used extracts obtained from Fg, Mg and FgMg multilayer constructs. We observed that FgMg extracts led to a reduction in the polarization of macrophages towards a pro-inflammatory phenotype. Also, the secretome of macrophages exposed to extracts of the combination material promoted the expression of osteogenic markers by MSCs.

TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity.

Growing evidences suggest that sustained neuroinflammation, caused by microglia overactivation, is implicated in the development and aggravation of several neurological and psychiatric disorders. In some pathological conditions, microglia produce increased levels of cytotoxic and inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), which can reactivate microglia in a positive feedback mechanism. However, specific molecular mediators that can be effectively targeted to control TNF-α-mediated microglia overactivation, are yet to be uncovered. In this context, we aim to identify novel TNF-α-mediated micro(mi)RNAs and to dissect their roles in microglia activation, as well as to explore their impact on the cellular communication with neurons. A miRNA microarray, followed by RT-qPCR validation, was performed on TNF-α-stimulated primary rat microglia. Gain- and loss-of-function in vitro assays and proteomic analysis were used to dissect the role of miR-342 in microglia activation. Co-cultures of microglia with hippocampal neurons, using a microfluidic system, were performed to understand the impact on neurotoxicity. Stimulation of primary rat microglia with TNF-α led to an upregulation of Nos2, Tnf, and Il1b mRNAs. In addition, ph-NF-kB p65 levels were also increased. miRNA microarray analysis followed by RT-qPCR validation revealed that TNF-α stimulation induced the upregulation of miR-342. Interestingly, miR-342 overexpression in N9 microglia was sufficient to activate the NF-kB pathway by inhibiting BAG-1, leading to increased secretion of TNF-α and IL-1ß. Conversely, miR-342 inhibition led to a strong decrease in the levels of these cytokines after TNF-α activation. In fact, both TNF-α-stimulated and miR-342-overexpressing microglia drastically affected neuron viability. Remarkably, increased levels of nitrites were detected in the supernatants of these co-cultures. Globally, our findings show that miR-342 is a crucial mediator of TNF-α-mediated microglia activation and a potential target to tackle microglia-driven neuroinflammation.

miR-99a in bone homeostasis: Regulating osteogenic lineage commitment and osteoclast differentiation.

BACKGROUND: The tight coupling between osteoblasts and osteoclasts is essential to maintain bone homeostasis. Deregulation of this process leads to loss and deterioration of the bone tissue causing diseases, such as osteoporosis. MicroRNAs are able to control bone-related mechanisms and have been explored as therapeutic tools. In this study, we investigated the potential of miR-99a-5p to modulate osteogenic differentiation, osteoclastogenesis, and the osteoblasts-osteoclasts crosstalk. METHODS: To achieve this goal, human primary Mesenchymal Stem/Stromal Cells (MSC) were differentiated into osteoblasts and adipocytes, and miR-99a-5p expression was evaluated by RT-qPCR. Knockdown and overexpression experiments were conducted to modulate miR-99a-5p expression in MC3T3 cells. Cell proliferation and cell death/apoptosis were evaluated by resazurin assay and flow cytometry, respectively. Proteomic analysis was used to identify the miR-99a-5p regulatory network, and ELISA to evaluate OPG levels in the cell culture supernatant. Conditioned media from MC3T3-transfected cells was used to culture RAW 264.7 cells and the effect on osteoclast differentiation was assessed. Human primary monocytes were isolated to induce osteoclastogenesis and evaluate miR-99a-5p expression. Finally, levels of miR-99a-5p were modulated in RAW 264.7 cells to understand the impact on osteoclastogenesis. RESULTS: The results show that miR-99a-5p is significantly downregulated during the early stages of human primary MSCs osteogenic differentiation and during MC3T3 osteogenic differentiation. On the other hand, miR-99a-5p levels are increased during the initial stages of adipogenic differentiation. Inhibition of miR-99a-5p in MC3T3 pre-osteoblastic cells promoted osteogenic differentiation, whereas its overexpression suppressed the levels of osteogenic specific genes (Runx2 and Alpl), as well as mineralization, with no effect on proliferation or apoptosis. Proteomic analysis of miR-99a-5p-transfected cells showed that numerous proteins known to be involved in cell differentiation were altered, including osteogenic differentiation markers and extracellular matrix proteins. While inhibition of miR-99a-5p increased the Tnfrsf11b (OPG encoding gene)/Tnfsf11 (RANKL encoding gene) mRNA expression ratio, in addition to increasing OPG secretion, miR-99a-5p overexpression resulted in the opposite effect. The cell culture supernatant of miR-99a-5p-inhibited MC3T3 cells impaired the osteoclastogenic potential of RAW 264.7 cells by decreasing the number of multinucleated cells and reducing the expression of osteoclastogenic markers. Interestingly, miR-99a-5p expression is increased during osteoclasts differentiation, both in human primary monocytes and RAW 264.7. These results show that miR-99a-5p per se is a positive regulator of osteoclastogenic differentiation. CONCLUSIONS: Globally, our findings show that miR-99a-5p inhibition promotes the commitment into osteogenic differentiation, impairs osteoclastogenic differentiation, and control bone cells communication. Ultimately, it supports miR-99a-5p as a target candidate for future miRNA-based therapies for bone diseases associated with bone remodeling deregulation.

SLMP53-2 Restores Wild-Type-Like Function to Mutant p53 through Hsp70: Promising Activity in Hepatocellular Carcinoma.

Half of human cancers harbor TP53 mutations that render p53 inactive as a tumor suppressor. In these cancers, reactivation of mutant p53 (mutp53) through restoration of wild-type-like function constitutes a valuable anticancer therapeutic strategy. In order to search for mutp53 reactivators, a small library of tryptophanol-derived oxazoloisoindolinones was synthesized and the potential of these compounds as mutp53 reactivators and anticancer agents was investigated in human tumor cells and xenograft mouse models. By analysis of their anti-proliferative effect on a panel of p53-null NCI-H1299 tumor cells ectopically expressing highly prevalent mutp53, the compound SLMP53-2 was selected based on its potential reactivation of multiple structural mutp53. In mutp53-Y220C-expressing hepatocellular carcinoma (HCC) cells, SLMP53-2-induced growth inhibition was mediated by cell cycle arrest, apoptosis, and endoplasmic reticulum stress response. In these cells, SLMP53-2 restored wild-type-like conformation and DNA-binding ability of mutp53-Y220C by enhancing its interaction with the heat shock protein 70 (Hsp70), leading to the reestablishment of p53 transcriptional activity. Additionally, SLMP53-2 displayed synergistic effect with sorafenib, the only approved therapy for advanced HCC. Notably, it exhibited potent antitumor activity in human HCC xenograft mouse models with a favorable toxicological profile. Collectively, SLMP53-2 is a new mutp53-targeting agent with promising antitumor activity, particularly against HCC.

Cataract quantification using swept-source optical coherence tomography.

PURPOSE: To develop and evaluate a cataract quantification method using a swept-source optical coherence tomography (SS-OCT) device (IOLMaster 700). SETTING: Hanusch Hospital, Vienna, Austria. DESIGN: Prospective multicenter case series. METHODS: This study included patients with cataract in at least 1 eye. Two independent examiners performed Lens Opacities Classification System II (LOCS II) grading at the slitlamp independently. Corrected distance visual acuity, contrast sensitivity, and SS-OCT measurements were also performed. In addition, the phacoemulsification energy and time were recorded. To develop an objective SS-OCT-based cataract quantification system, all SS-OCT scans were segmented and the local pixel intensity unit of the lens was analyzed using Matlab's grayscale counting. The pixel intensity unit of the lens nucleus (OCT-based cataract quantification system score) was equated to the clinician's subjective nuclear opalescence grading. RESULTS: The study evaluated 186 eyes (113 patients). The correlation between the independent examiners' LOCS grading was good (0.91) (P < .01). The correlation between the LOCS grading and OCT-based cataract quantification system score was 0.86 for examiner 1 and 0.76 for examiner 2 (both P < .01). In 24 patients, phacoemulsification time, power, and energy; visual acuity; and contrast sensitivity were available and included in the study. The OCT-based cataract quantification system scores correlated significantly with phacoemulsification time (0.71) and energy (0.64) (both P < .01). CONCLUSIONS: Cataract density could be evaluated using an SS-OCT device, and the OCT-based cataract quantification system values correlated with the conventional LOCS II classification. Swept-source OCT measurements allowed quantification and documentation cataract density.

miR-195 inhibits macrophages pro-inflammatory profile and impacts the crosstalk with smooth muscle cells.

Macrophages are a main component of atherosclerotic plaques. Recent studies suggest that pro-inflammatory M1 macrophages are pro-atherogenic while M2 macrophages promote plaque stability. Moreover, toll-like receptor signalling pathways are implicated in atherosclerotic plaque formation, evolution and regression. We propose microRNAs as key regulators of these processes. In this context, our goal is to promote inflammation resolution using miR-195 to reduce M1-like macrophage polarization and to evaluate the molecular mechanisms underlying such effect, as well as to explore the functional consequences for smooth muscle cell recruitment. Human primary macrophages were differentiated from peripheral blood monocytes and stimulated with LPS or IL-10 to promote M1 or M2c polarization, respectively. miR-195 levels were upregulated in M2c macrophages compared with M1 macrophages. In THP-1 macrophages stimulated with LPS and IFN-γ, results show that TLR2 levels were reduced by miR-195 overexpression compared with scrambled control. In addition, phosphorylated forms of p54 JNK, p46 JNK and p38 MAPK were decreased by miR-195 in macrophages following M1 stimulation. Moreover, miR-195 significantly decreased levels of IL-1ß, IL-6 and TNF-α pro-inflammatory cytokines in the supernatants of M1-stimulated macrophage cultures. At the functional level, results from smooth muscle cell recruitment and migration models showed that miR-195 impairs the capacity of M1 macrophages to promote smooth muscle cells migration. In conclusion, miR-195 is involved in macrophage polarization and inhibits TLR2 inflammatory pathway mediators. Moreover, miR-195 impairs the effect of macrophages on smooth muscle cells recruitment capacity and migration profile. Thus, miR-195 might be used as a new potential tool to promote inflammation resolution in cardiovascular research.