Layered hydrogels accelerate iPSC-derived neuronal maturation and reveal migration defects caused by MeCP2 dysfunction

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.Neuronal migration and maturation is a key step in brain development. Defects in this process have been implicated in many disorders, including autism (1) and schizophrenia (2). Thoroughly understanding how neural progenitor cell (NPC) migration is affected in neurodevelopmental disorders requires a means of dissecting the process using cells with genetic alterations matching those in patients. Existing in vitro assays of migration generally involve measurement of cell movement across a scratch or gap or through a membrane toward a chemoattractant in 2D culture systems. Although widely used, such assays may not accurately reveal in vivo differences, as neuronal migration is tightly regulated by physical and chemical cues in the extracellular matrix (ECM) that NPCs encounter as they migrate.In vitro 3D culture systems offer a solution to these limitations (3–7). Compared with 2D culture, a 3D arrangement allows neuronal cells to interact with many more cells (4); this similarity to the in vivo setting has been shown to lengthen viability, enhance survival, and allow formation of longer neurites and more dense networks in primary neurons in uniform matrices or aggregate culture (8, 9). Indeed, 3D culture systems have been used to study nerve regeneration, neuronal and glial development (10–12), and amyloid-β and tau pathology (13). Thus, measuring neuronal migration through a soft 3D matrix would continue this trend toward using 3D systems to study neuronal development and pathology.We sought to develop a 3D assay to examine potential migration and neuronal maturation defects in Rett syndrome (RTT), a genetic neurodevelopmental disorder that affects 1 in 10,000 children in the United States and is caused by mutations in the X-linked methyl-CpG-binding protein-2 (MECP2) gene (14). Studies using induced pluripotent stem cells (iPSCs) from RTT patients in traditional 2D adherent culture have revealed reduced neurite outgrowth and synapse number, as well as altered calcium transients and spontaneous postsynaptic currents (1). However, 2D migration assays seemed unlikely to reveal inherent defects in this developmental process, which could be affected because MeCP2 regulates multiple developmental related genes (15). Migration of RTT iPSC-derived NPCs has not previously been studied.Using a previously unidentified 3D tissue culture system that allows creation of layered architectures, we studied differences in migration of MeCP2-mutant iPSC-derived versus control iPSC-derived NPCs. This approach revealed a defect in migration of MeCP2-mutant iPSC-derived NPCs induced by either astrocytes or neurons. Further, this 3D system accelerated maturation of neurons from human iPSC-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. With mature neurons derived from RTT patients and controls, we further confirmed defective neurite outgrowth and synaptogenesis in MeCP2-mutant neurons. Thus, this 3D system enables study of morphological features accessible in 2D system as well as previously unexamined phenotypes.     

Development and characterization of a new chitosan-based scaffold associated with gelatin,microparticulate dentin and genipin for endodontic regeneration

ObjectiveAn ideal scaffold for endodontic regeneration should allow the predictableness of the new tissue organization and limit the negative impact of residual bacteria. Therefore, composition and functionalization of the scaffold play an important role in tissue bioengineering. The objective of this study was to assess the morphological, physicochemical, biological and antimicrobial properties of a new solid chitosan-based scaffold associated with gelatin, microparticulate dentin and genipin.MethodsScaffolds based on chitosan (Ch); chitosan associated with gelatin and genipin (ChGG); and chitosan associated with gelatin, microparticulate dentin and genipin (ChGDG) were prepared by using the freeze-drying method. The morphology of the scaffolds was analyzed by scanning electron microscopy (SEM). The physicochemical properties were assessed for biodegradation, swelling and total released proteins. The biological aspects of the scaffolds were assessed using human cells from the apical papilla (hCAPs). Cell morphology and adhesion to the scaffolds were evaluated by SEM, cytotoxicity and cell proliferation by MTT reduction-assay. Cell differentiation in scaffolds was assessed by using alizarin red assay. The antimicrobial effect of the scaffolds was evaluated by using the bacterial culture method, and bacterial adhesion to the scaffolds was observed by SEM.ResultsAll the scaffolds presented porous structures. The ChCDG had more protein release, adhesion, proliferation and differentiation of hCAPs, and bacteriostatic effect on Enterococcus faecalis than Ch and ChGG (p < 0.05).SignificanceThe chitosan associated with gelatin, microparticulate dentin and genipin has morphological, physicochemical, biological and antibacterial characteristics suitable for their potential use as scaffold in regenerative endodontics.     

Severe Thrombotic Events Associated with Dengue Fever,Brazil

Dengue fever has been a major problem in hospital settings in Brazil for the past 15 years. The main concern has been the severe forms, i.e., dengue hemorrhagic fever and dengue shock syndrome. Hemorrhagic events of different degrees have also been a major concern. We report five cases of large vein thrombotic events associated with the acute phase of dengue fever, including a previously non-reported case of mesenteric vein thrombosis. Complications such as these could have been overlooked in the diagnosis of dengue fever, given that the major concern is the hemorrhagic event.Dengue fever frequently affects Brazil, where thousands of cases have been diagnosed annually and all four dengue virus serotypes (DENV-1 through DENV-4) have been reported, sometimes concurrently in the same region.^1,2 Hemorrhagic events of different degrees have often been described in dengue, but thrombotic events have not been extensively reported, despite the wide range of increased procoagulant activity during illness.^1,3–8 During a recent outbreak, several cases of thrombotic events affected large veins in dengue fever patients. We report cases of unusual and overlooked complication of dengue fever.From January 2011 through March 2011 during a local outbreak of dengue fever caused by DENV-1 and DENV-2 according to local health officials, five patients were given diagnoses by imaging techniques (pulmonary computed tomography angiography, cholangio-magnetic resonance imaging, and Doppler ultrasound of lower extremities) as having large vessel thrombosis. These patients were part of 92 serologically confirmed (by immunochromatographic strip test or IgM antigen-capture enzyme-linked immunosorbent assay) patients with dengue (60% women and 40% men, median age = 39 years, age range = 10–99 years) admitted to Monte Sinai Hospital (200 beds) in Juiz de Fora, (population = 500,000), Minas Gerais, Brazil. This study was approved by the Ethical Committee of Monte Sinai Hospital.Twenty-three patients (25%) were classified as having dengue shock syndrome or dengue hemorrhagic fever and 4 deaths were recorded (case-fatality rate = 4.3%). These thrombotic patients represented 5.4% of all dengue inpatients. All thrombotic events were identified within the first five days of illness, and all patients had symptoms compatible with the reported thrombotic event at hospital admission. No thrombotic events were identified among the remaining patients during hospitalization, and none of the patients them received drugs for thrombosis prophylaxis, which is contraindicated in dengue.The demographic and clinical characteristics of the five patients are summarized in Figure 1), which was associated with jaundice and severe sepsis by Escherichia coli demonstrated by blood culture. Known risk factors for thrombotic events such as smoking, use of oral contraceptives, and being overweight were absent in all patients and none had had any similar diagnosis or symptoms.Open in a separate windowFigure 1.T1-weighted cholangio-magnetic resonance image with contrast of dengue patient 5 showing the superior mesenteric vein (SMV) with a large thrombus (arrow) occupying the vein lumen. SMA = superior mesenteric artery; IVC = inferior vena cava; Ao = aorta.

Table 1

Demographic, clinical, and laboratory characteristics of five dengue fever patients with thrombotic events, Brazil^*

The effects of water-based strength exercise on quality of life in young women

Background

Exercise improves quality of life (QoL). However, little is known concerning the effects of different volumes of strength exercise on QoL. The aims of this study are to: (1) evaluate the effects of water-based strength exercise on QoL of healthy young women and (2) compare the effects of different volumes of water-based strength exercises on QoL of healthy young women.

Methods

Sixty-six participants were randomly allocated into four groups with different volumes of exercise. The participants performed water-based strength exercises for 20 weeks, two times a week, supervised by trained physiologists.

Results

A significant improvement was found in overall QoL (F = 5.96; p = 0.018) and in physical (F = 22.01; p < 0.001), psychological (F = 8.408; p = 0.006) and environment domains (F = 8.34; p = 0.006). In addition, a significant decrease of depressive symptoms was found (F = 22.32; p < 0.001). No difference was found between groups in any domain of QoL or depressive symptoms.

Conclusion

Water-based strength exercise improves specific domains of QoL and decreases depressive symptoms of young healthy women. Different volumes of exercise promote similar effects on QoL and depressive symptoms.

     

Effect of low-level laser therapy (808 nm) on markers of muscle damage: a randomized double-blind placebo-controlled trial

The aim of this randomized double-blind placebo-controlled study was to investigate the effect of low-level laser therapy (LLLT) on markers of muscle damage (creatine kinase (CK) and strength performance) in the biceps brachii. Twenty-two physically active men were randomized into two groups: placebo and laser. All volunteers were submitted to an exercise-induced muscle damage protocol for biceps brachii (biceps curl, 10 sets of 10 repetitions with load of 50 % of one-repetition maximum test (1RM)). Active LLLT (808 nm; 100 mW; 35.7 W/cm², 357.14 J/cm² per point, energy of 1 J per point applied for 10 s on four points of the biceps brachii belly of each arm) or placebo was applied between the sets of the biceps curl exercise. CK activity and maximum strength performance (1RM) were measured before, immediately after, 24, 48, and 72 h after the exercise-induced muscle damage protocol. There was an increase in CK activity after the muscle damage protocol in both groups; however, this increase was attenuated in the laser group compared to the placebo group at 72 h (placebo?=?841 vs. laser?=?357 %; p?<?0.05). Maximum strength performance was decreased immediately after the muscle damage protocol in both groups (p?<?0.05), but at 24, 48, and 72 h, and it returned to the baseline level in both groups. In conclusion, the LLLT attenuated CK activity 72 h after the muscle damage protocol but did not have a positive effect on the recovery of strength performance.     

The benefits of a high-intensity aquatic exercise program (HydrOS) for bone metabolism and bone mass of postmenopausal women

This study aimed to evaluate the 24-week effects of a high-intensity aquatic exercise program on bone remodeling markers and bone mass of postmenopausal women. In this randomized, controlled trial we studied 108 women (58.8 ± 6.4 years), randomized into Aquatic Exercise Group (AEG), n = 64, performing 24 weeks of aquatic exercises, and Control Group (CG), n = 44, sedentary. They had their fasting morning blood sample collected for the measures of intact parathyroid hormone (iPTH), procollagen type 1 amino-terminal propeptide (P1NP) and carboxy-terminal cross-linking telopeptide of type I collagen (CTx). Bone mass was measured by dual-energy X-ray absorptiometry before and after the intervention. Participants of both groups received a daily supplementation of 500 mg of elementary calcium and 1,000 IU of vitamin D (cholecalciferol). Results showed an augment in bone formation marker (P1NP) only in the AEG (15.8 %; p = 0.001), and although both groups experienced significant enhancements in bone resorption marker (CTx), this increase was less considerable in the AEG (15 % in the AEG and 29 % in the CG). IPTH was increased by 19 % in the CG (p = 0.003) at the end. The femoral trochanter BMD presented a 1.2 % reduction in the CG (p = 0.009), whereas in the AEG no change was observed (p = 0.069). The proposed aquatic exercise program was efficient in attenuating bone resorption raise and enhancing bone formation, which prevented the participants in the AEG from reducing the femoral trochanter BMD, as happened in the CG.     

Clinical hematological and biochemical parameters in Swiss,BALB/c,C57BL/6 and B6D2F1 Mus musculus

BackgroundAnimal models are widely used in scientific research in order to obtain information from a whole organism under a specific set of experimental conditions. Various lineages of mice have been used to investigate diseases and new therapeutic strategies, and, consequently, hematological and biochemical tests in these laboratory animals are essential to validate scientific studies. Our study seeks to establish reference values for hematological and biochemical parameters of four lineages of mice.MethodsWe evaluated the hematological and biochemical profiles of 20 males and 20 females from the lineages Swiss (heterogeneous), BALB/c and C57BL/6 (isogenic), and B6D2F1 (hybrid), totaling 160 mice. Analysis were standardized using the systems pocH‐100iV Diff™ for 19 hematological parameters and VITROS^® 350 for 12 biochemical parameters.ResultsResults are shown as means and standard deviation, grouped by lineage and genre. Comparing the values obtained in this study with the values from previous studies, some variations were detected, which could be explained by differences in methodologies or individual variability.ConclusionThus our study shows that knowledge and disclosure of the values of physiological parameters of laboratory animals is necessary, and emphasises the importance of considering variations influenced by gender, lineage and genotype in the choice of the best experimental model.     

Increase in Hepatitis A Cases Linked to Imported Strains to Rio de Janeiro,Brazil: A Cross-Sectional Study

This study aims to evaluate the epidemiological and molecular features associated with HAV transmission in adults in Rio de Janeiro during a period of increased registered cases of HAV (2017–2018). Socio-epidemiological data and serum samples from anti-HAV IgM+ individuals were obtained. HAV RNA was RT-PCR amplified and sequenced for further phylogenetic and phylogeographic analyses. From fifty-two HAV IgM+ individuals, most were men (78.85%; p = 0.024), aged 20–30 years old (84.61%; p < 0.001), resided in the Rio de Janeiro north zone (31/52; 59.62%; p = 0.001), and are men who have sex with men (MSM) (57.69%; p = 0.002). Sexual practices were more frequent (96%) than others risk factors (food-borne (44%), water-borne (42.31%), and parenteral (34.62%)). Individuals who traveled to endemic regions had a 7.19-fold (1.93–36.04; p < 0.01) increased risk of HAV. Phylogenetic analysis revealed four distinct clades of subgenotype IA, three of them comprised sequences from European/Asian MSM outbreaks and one from Brazilian endemic strains. Bayesian Inference showed that the imported strains were introduced to Brazil during large mass sportive events. Sexual orientation and sexual practices may play a role in acquiring HAV infection. Public policies targeting key populations must be implemented to prevent further dissemination of HAV and other STIs.