NGN2-based neuronal programming of hiPSCs in an automated microfluidic platform.

The generation of induced pluripotent stem cells (iPSCs) via somatic cell reprogramming allowed to have an unlimited in vitro source of patient-specific cells. This achievement has introduced a new revolutionary way to create human in vitro models and to study human diseases starting from patient's own cells, especially important for inaccessible tissues like the brain. Recently, lab-on-a-chip technology has opened new reliable alternatives to conventional in vitro models able to replicate key aspects of human physiology, thanks to the intrinsic high surface-area-to-volume ratio, which allows fine control of the cellular microenvironment. The development of automated microfluidic platforms allowed the implementation of this technology to perform high-throughput, standardized and parallelized assays, suitable for drug screenings and developing new therapeutic approaches in a cost-effective way. However, the major challenges in the broad application of automated lab-on-a-chip in biological research are the lack of production robustness and ease of use of the devices. Here, we present an automated microfluidic platform able to host the rapid conversion of human iPSCs (hiPSCs) into neurons via viral-mediated overexpression of Neurogenin 2 (NGN2) in a user-friendly manner. The design of the platform, built with multilayer soft-lithography techniques, shows easiness in the fabrication and assembly thanks to the simple geometry and experimental reproducibility at the same time. All operations are managed automatically, from the cell seeding, medium change, doxycycline-mediated neuronal induction, selection of the genetically engineered cells, and analysis of the output of differentiation, including immunofluorescence assay. Our results show a high-throughput, efficient and homogenous conversion of hiPSCs into neurons in 10 days, characterized by the expression of the mature neuronal marker MAP2 and calcium signaling. The neurons-on-chip model here described represents a fully automated loop system able to address the challenges in the field of neurological diseases modelling in vitro and improve current preclinical models.

Specific mesoderm subset derived from human pluripotent stem cells ameliorates microvascular pathology in type 2 diabetic mice.

Human induced pluripotent stem cells (hiPSCs) were differentiated into a specific mesoderm subset characterized by KDR+CD56+APLNR+ (KNA+) expression. KNA+ cells had high clonal proliferative potential and specification into endothelial colony-forming cell (ECFCs) phenotype. KNA+ cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient mice and when injected into the vitreous of type 2 diabetic mice (db/db mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA+ cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA+ cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of db/db mice injected with either control or diabetic donor-derived KNA+ cells showed correction of aberrant signaling in db/db retinas toward normal healthy retina. These data provide "proof of principle" that KNA+ cells restore perfusion and correct vascular dysfunction in db/db mice.

Fasting and fasting-mimicking treatment activate SIRT1/LXRα and alleviate diabetes-induced systemic and microvascular dysfunction.

AIMS/HYPOTHESIS: Homo sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting-mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage. METHODS: The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as liver X receptor alpha [LXRα]) acetylation was measured via western blot analysis. RESULTS: IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression. CONCLUSIONS/INTERPRETATION: Taken together, activation of SIRT1 signalling by IF or through pharmacological activation represents an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimens and prevention of microvascular and bone marrow dysfunction in diabetes.

Electrospun Nanofibrous Architectures of Thrombin-Loaded Poly(ethylene oxide) for Faster in Vivo Wound Clotting.

Wound healing materials to prevent blood loss are crucial during emergency medical treatment because uncontrolled bleeding can lead to patient death. Herein, bioabsorbable fibrous architectures of thrombin-loaded poly(ethylene oxide)-PEO/thrombin-are conceptualized and accomplished via electrospinning for faster wound clotting. Membranes with average fiber diameters ranging from 188 to 264 nm are achieved, where the active thrombin is entrapped within the nanofibers. The results of in vitro and in vivo wound healing activity tests revealed that when the nanofibers with thrombin-loaded capacity are in contact with the wound, the presence of water in the skin or blood catalyzes the degradation of the membranes, thus releasing thrombin. Thrombin then accelerates the wound clotting process. In contrast to other hemostatic materials, PEO/thrombin nanofibers do not require mechanical removal after application, and the viscoelastic nature of such biomaterials enables their conformation to a variety of wound topographies. Remarkably, PEO/thrombin membranes are promising functional materials and their use is a powerful strategy for hemostatic treatment, ranging from simple first aid and sealing to a wound to small surgical procedures.

Selective LXR agonist DMHCA corrects retinal and bone marrow dysfunction in type 2 diabetes.

In diabetic dyslipidemia, cholesterol accumulates in the plasma membrane, decreasing fluidity and thereby suppressing the ability of cells to transduce ligand-activated signaling pathways. Liver X receptors (LXRs) make up the main cellular mechanism by which intracellular cholesterol is regulated and play important roles in inflammation and disease pathogenesis. N, N-dimethyl-3ß-hydroxy-cholenamide (DMHCA), a selective LXR agonist, specifically activates the cholesterol efflux arm of the LXR pathway without stimulating triglyceride synthesis. In this study, we use a multisystem approach to understand the effects and molecular mechanisms of DMHCA treatment in type 2 diabetic (db/db) mice and human circulating angiogenic cells (CACs), which are hematopoietic progenitor cells with vascular reparative capacity. We found that DMHCA is sufficient to correct retinal and BM dysfunction in diabetes, thereby restoring retinal structure, function, and cholesterol homeostasis; rejuvenating membrane fluidity in CACs; hampering systemic inflammation; and correcting BM pathology. Using single-cell RNA sequencing on lineage-sca1+c-Kit+ (LSK) hematopoietic stem cells (HSCs) from untreated and DMHCA-treated diabetic mice, we provide potentially novel insights into hematopoiesis and reveal DMHCA's mechanism of action in correcting diabetic HSCs by reducing myeloidosis and increasing CACs and erythrocyte progenitors. Taken together, these findings demonstrate the beneficial effects of DMHCA treatment on diabetes-induced retinal and BM pathology.

Lecithochirium monticellii digenetic trematode parasites of Trichiurus lepturus (Actinopterygii) from the state of Rio de Janeiro, Brazil, with notes on its taxonomy.

Trichiurus lepturus (Actinopterygii, Perciformes) is a commercially and economically important fish. A total of 60 specimens of this cutlassfish were collected of the coast the municipalities of Niterói and Cabo Frio, state of Rio de Janeiro, Brazil. The fish were measured, necropsied, filleted and had their organs investigated for digenetic trematodes. Taxonomic identification was based on morphological and morphometric characters. The specimens of T. lepturus were parasitized with adult specimens of Lecithochirium monticellii. Parasite indices of prevalence, intensity, mean intensity, abundance, mean abundance, range of infection, and site of infection of parasitic species were evaluated. Notes on the taxonomy of the parasite were also included. This is the first report of L. monticellii parasitizing T. lepturus in Brazil.

Effect of high-intensity ultrasound on the nutritional profile and volatile compounds of a prebiotic soursop whey beverage.

This study evaluated the nutritional profile and volatile compounds present in a novel prebiotic (inulin) soursop whey beverage, due to the effects of high-intensity ultrasound (HIUS). The prebiotic soursop whey beverage was produced and processed by non-thermal high-intensity ultrasound varying the power (0, 200, 400 and 600 W) and by high-temperature short time (72 °C for 15 s) thermal treatment. Total acidity, pH, ascorbic acid content, total phenolics compounds content, antioxidant activity, hypertensive activity, fatty acid profile, volatile organic compounds, macro and micro minerals, as well as the heavy metals in these products, were analyzed. Overall, the HIUS technology induced some positive changes in the nutritional profile of the soursop whey beverage including beneficial effects, e.g., increase of phenolic content, improvement of the antioxidant and anti-hypertensive activity and reduction of undesired minerals. Although some negative changes, such as degradation of the ascorbic acid, decrease of some minerals and production of certain volatile compounds were found, the beneficial effects were prominent, thus, opening new opportunities to develop healthy functional beverages.

Administration of endothelial progenitor cells accelerates the resolution of arterial thrombus in mice.

BACKGROUND: Endothelial progenitor cells (EPCs) are circulating progenitor cells that can play an essential role in vascular remodelling. In this work, we compared the role of two EPCs cultivated with different mediums in the resolution of the arterial thrombus induced by FeCl3 lesion and in vessel re-endothelization in the mouse carotid artery. METHODS: Mice mononuclear cells were differentiated into EPCs using Dulbecco's Modified Eagle's Medium (DMEM) and vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and IGF (Insulin Growth Factor) called EPCs--M1) or with EGM2(endothelial growth medium) (media supplemented with growth factors from Lonza called (EPCs-M2) for 30days and characterized using flow cytometry. The animals received three EPC injections post-lesion, and we analyzed thrombosis time, vessel re-endothelization, metalloproteinases activities, eNOS (endothelial Nitric oxide synthase) presence and SDF-1(Stromal Derived Factor- 1) levels in circulation. RESULTS: EPC-M1 presented a more immature progenitor profile than EPC-M2 cells. The injection of EPC-M1 prolonged the thrombosis time, and the treatment with the different EPCs increased eNOS expression and MMP2 (Metalloproteinase 2) activity and decreased SDF-1 in plasma. Only EPC-M1 treatment increased both MMP2 and MMP9 and reduced thrombus after 7days. Also, both EPCs decreased platelet aggregation in vitro. CONCLUSIONS: EPCs-M1 were more efficient in all of the analyzed assays. EPCsM2 may be a more mature EPC, proliferating less and promoting a less significant matrix remodelling. EPCs can promote vascular remodelling by inhibiting thrombosis and stimulating vascular wall remodelling and the treatment with a more immature progenitor may be more efficient in this process.

Hyperbaric oxygen affects endothelial progenitor cells proliferation in vitro.

Hyperbaric oxygen is a clinical treatment that contributes to wound healing by increasing fibroblasts proliferation, collagen synthesis, and production of growth factors, inducing angiogenesis and inhibiting antimicrobial activity. It also has been shown that hyperbaric oxygen treatment (HBO), through the activation of nitric oxide synthase promotes an increase in the nitric oxide levels that may improve endothelial progenitor cells (EPC) mobilization from bone marrow to the peripheral blood and stimulates the vessel healing process. However, cellular mechanisms involved in cell proliferation and activation of EPC after HBO treatment remain unknown. Therefore, the present work aimed to analyze the effect of HBO on the proliferation of pre-treated bone marrow-derived EPC with TNF-alpha. Also, we investigated the expression of ICAM and eNOS by immunochemistry, the production of reactive species of oxygen and performed an in vitro wound healing. Although 1h of HBO treatment did not alter the rate of in vitro wound closure or cell proliferation, it increased eNOS expression and decreased ICAM expression and reactive oxygen species production in cells pre-treated with TNF-alpha. These results indicate that HBO can decrease the inflammatory response in endothelial cells mediated by TNF-alpha, and thus, promote vascular recovery after injury.

Height Prediction From Ulna Length of Critically Ill Patients.

BACKGROUND: Ulna length (UL) has been used in mathematical formulas to predict the body height of healthy and sick individuals. However, the evaluation of its use with patients admitted to intensive care units (ICU) is scarce. The objective of this study was to develop a mathematical equation to estimate critically ill patients' height using the UL measure and to evaluate its agreement with measured standing height. METHODS: This cross-sectional study was performed at the ICU of a tertiary hospital in Brazil. A total of 100 patients aged ≥18 years who had their body height measured before ICU admission were enrolled. The equation was developed through multiple linear regression, and its agreement was assessed through paired Student's t test and Bland-Altman plot. RESULTS: The following formula was obtained: height in cm = 153.492 - (7.97 × sex [sex: male = 1, female = 2]) + (0.974 × UL [in cm]). The difference between means of measured height (MH) and height estimated from UL was not significant (166.26 ± 8.75 cm and 166.30 ± 5.29 cm, respectively, P = .96), and a significant correlation (r = 0.624, P < .001) was detected. In the Bland-Altman analysis, UL was in agreement with MH; however, there was a significant bias (P < .001) suggesting that it may be disproportional and dependent on the average's height value. CONCLUSION: The mathematical equation for height estimation using UL developed in this study matched the MH of critically ill patients. However, we suggest more studies for its validation.

Tumor-Derived Exosomes Induce the Formation of Neutrophil Extracellular Traps: Implications For The Establishment of Cancer-Associated Thrombosis.

Cancer patients are at an increased risk of developing thromboembolic complications. Several mechanisms have been proposed to explain cancer-associated thrombosis including the release of tumor-derived extracellular vesicles and the activation of host vascular cells. It was proposed that neutrophil extracellular traps (NETs) contribute to the prothrombotic phenotype in cancer. In this study, we evaluated the possible cooperation between tumor-derived exosomes and NETs in cancer-associated thrombosis. Female BALB/c mice were orthotopically injected with 4T1 breast cancer cells. The tumor-bearing animals exhibited increased levels of plasma DNA and myeloperoxidase in addition to significantly increased numbers of circulating neutrophils. Mice were subjected to either Rose Bengal/laser-induced venous thrombosis or ferric chloride-induced arterial thrombosis models. The tumor-bearing mice exhibited accelerated thrombus formation in both models compared to tumor-free animals. Treatment with recombinant human DNase 1 reversed the prothrombotic phenotype of tumor-bearing mice in both models. Remarkably, 4T1-derived exosomes induced NET formation in neutrophils from mice treated with granulocyte colony-stimulating factor (G-CSF). In addition, tumor-derived exosomes interacted with NETs under static conditions. Accordingly, the intravenous administration of 4T1-derived exosomes into G-CSF-treated mice significantly accelerated venous thrombosis in vivo. Taken together, our observations suggest that tumor-derived exosomes and neutrophils may act cooperatively in the establishment of cancer-associated thrombosis.

Combined dermatan sulfate and endothelial progenitor cell treatment: action on the initial inflammatory response after arterial injury in C57BL/6 mice.

BACKGROUND AIMS: Dermatan sulfate (DS), an anticoagulant and antithrombotic glycosaminoglycan, also has anti-inflammatory activity. In this study, we investigated the effect of DS treatment in the presence or absence of bone marrow mononuclear cells (MNCs) or endothelial progenitor cells (EPCs) in the vascular response to carotid artery lesion in C57BL6 mice. METHODS: Thrombus formation, the expression of adhesion molecules and factors involved in vascular remodeling, inflammation or vascular tone were analyzed by histologic examination, Western blotting and enzyme-linked immunoassay 1 and 3 days after vascular injury. RESULTS: DS injections prevented thrombus formation and decreased P-selectin expression after 3 days of the injury. DS treatment also increased plasma SDF-1 levels but failed to rescue endothelial nitric oxide synthase (eNOS) expression, which is responsible for vascular tone. Treatment with MNCs alone failed to prevent thrombus formation 1 day after injury and increased intercellular adhesion molecule-1 expression, likely because of the inflammatory nature of these cells. Treatment with EPCs with DS was the most efficient among all therapies studied. Dual administration of EPCs and DS promoted an increase in the expression of adhesion molecules and, at the same time, induced a higher expression of eNOS at the injury site. Furthermore, it stimulated an elevated number of EPCs to migrate and adhere to the vascular wall. DISCUSSION: Simultaneous treatment with EPCs and DS increased the expression of adhesion molecules, prevented thrombosis, rescued the expression of eNOS and increased migration of EPCs to the site of injury, thereby affecting thrombus remodeling and inflammation and can be involved in vessel hemostasis.

Pentylenetetrazol-induced seizures are associated with Na⁺,K⁺-ATPase activity decrease and alpha subunit phosphorylation state in the mice cerebral cortex.

The present study aimed to investigate whether Na(+),K(+)-ATPase activity and phosphorylation state of the catalytic α subunit are altered by pentylenetetrazol (PTZ)-induced seizures. PTZ (30, 45 or 60 g/kg, i.p.) was administered to adult male Swiss mice, and Na(+),K(+)-ATPase activity and phosphorylation state were measured in the cerebral cortex 15 min after PTZ administration. Na(+),K(+)-ATPase activity significantly decreased after PTZ-induced seizures (60 mg/kg). Immunoreactivity of phosphorylated Ser943 at α subunit was increased after PTZ-induced seizures. A significant positive correlation between Na(+),K(+)-ATPase activity and latency to myoclonic jerks and generalized seizures was found. Conversely, a strong negative correlation between Ser943 phosphorylation and latency to generalized seizures was detected. Given the role of Na(+),K(+)-ATPase as a major regulator of brain excitability, Ser943 at Na(+),K(+)-ATPase α subunit may represent a potentially valuable new target for drug development for seizure disorders.

Clinical pharmacology and vascular risk.

Pharmacological treatment and several drugs of abuse have been associated with ischemic heart disease (IHD) and cerebrovascular diseases (CVD). However, there is a paucity of data on the independent risk of vascular disease (VD) associated with pharmacological treatment and no controlled trials demonstrating a reduction in risk with abstinence. Information about IHD and CVD-related drug abuse is mainly limited to epidemiological studies focused on urban populations. The potential link between some pharmacological treatments (estrogen, some oncologic drugs and some atypical antipsychotics) and cerebrovascular adverse events was analyzed, but disagreement about an association persists. Drugs of abuse, including cocaine, amphetamines and heroin, have been associated with an increased vascular risk. These drugs can cause abrupt changes in blood pressure, vasculitic-type changes, lead to embolization caused by infective endocarditis, and hemostatic and hematologic abnormalities that can result in increased blood viscosity and platelet aggregation. Long-term treatment strategies based on medication, psychological support, and outreach programs play an important role in treatment of drug dependency. In these last years public interest in risk factors for VD has been constantly increasing and the successful identification and management of pharmacological treatment and drug abuse can be challenging. One of the major public health issues for the future will be to focus more on new vascular risk factor recognition and management. The objective of this chapter is to review the relevance of IHD and CVD associated with various pharmacological treatments and drug abuse with focusing on ischemic disease. This chapter reports the clinical evidence of this association and analyzes the experimental role of new drugs as a growing risk factor of VD with the hypothetical new association. In conclusion, in this chapter great attention is paid to evaluating the scientific and real evidence of cerebrovascular effect and drug use and abuse so as to identify a new groups of "modifiable" risk factors.

Cervical artery dissection: emerging risk factors.

Cervical artery dissection (CAD) represents an increasingly recognized cause of stroke and the most common cause of ischemic stroke in young adults. Many factors have been identified in association with CAD such as primary disease of arterial wall (fibrodysplasia) and other non-specific diseases related to CAD like Ehlers Danlos-syndrome IV, Marfan's syndrome, vessel tortuosity. Moreover, an underlying arteriopathy which could be in part genetically determined, has been suspected. The rule of emerging risk factors for CAD such as recent respiratory tract infection, migraine and hyperhomocysteinemia are still a matter of research. Other known risks factors for CAD are major head/neck trauma like chiropractic maneuver, coughing or hyperextension injury associated to car. We examined emerging risks factors for CAD detected in the last years, as CAD pathogenesis is still not completely understood and needs further investigations.

[Castleman's disease in childhood: a case report]. / Malattia di castleman in età infantile: case report.

We report a case of Castleman's disease in 3-month old infant with cervical swelling, treated with surgical excision. This case is interesting because of the rarity of this disease in childhood, of the cervical localization and of the complexity of long- term follow-up.

[Proposal for a protocol for the staging of incontinentia pigmenti in pediatric age]. / Proposta di un protocollo per la stadiazione della Incontinentia Pigmenti in età pediatrica.

Incontinentia Pigmenti (IP) is an X-linked dominant disorder of skin with neurologic and ophthalmologic involvement. IP predominantly affects females because the mutations are usually lethal in males in utero. IP is characterized by abnormalities of neuroectodermal tissues. IP is caused by mutations in a gene called NEMO, which is required to activate the NF-kB pathway. We present a diagnostic protocol for IP and a meta-analysis of the clinical spectrum of IP in 82 patients cited by MEDLINE in the European literature from 2000 to 2006.

[Incontinentia pigmenti: case report]. / Incontinentia pigmenti: descrizione di un caso clinico.

IP is an uncommon X-linked dominant disorder (incidence: 1/40.000 newborn). It is caused by mutations in NEMO. It is characterized by cutaneous lesions and dental, ocular, neurologic, nails, hair disorders. The ocular and neurologic sequelae represent the major morbidity in IP. We present a case-report with classical cutaneous features diagnostic for IP. The clinical, ophtalomologic and neurologic examinations revealed no other pathological manifestations.

Isolated monoparesis following stroke.

BACKGROUND: Some investigators have stated that monoparesis is almost never the result of a lacunar infarct or cerebral haemorrhage. OBJECTIVE: To describe the topography and aetiology in a consecutive population where first ever stroke was manifested by isolated monoparesis. METHODS: Patients with motor paresis of only one limb were included consecutively in the study. A neuroradiologist determined stroke location, while a neurologist reviewed the clinical records to assign stroke subtype. Both physicians worked blind to each other's findings. RESULTS: 51 of 2003 patients (2.5%) had isolated monoparesis, and of these 39 (76.5%) were ischaemic strokes and 12 (23.5%) were haemorrhagic. Cardioembolism was the cause of stroke in 15.7%, atherosclerosis in 9.8%, and small artery disease in 39.2%. Most of the haemorrhages were in the thalamic-capsular region (5/12). Most of the ischaemic lesions were in the deep territory of the middle cerebral artery, the corona radiate, or the centrum semiovale (20/39); 16 of 39 were in the cortical territories or the watershed region. CONCLUSIONS: Isolated monoparesis is a rare symptom in stroke patients and is often caused by small artery disease or a small haemorrhage.