Detection of Pathological Markers of Neurodegenerative Diseases following Microfluidic Direct Conversion of Patient Fibroblasts into Neurons

Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease are clinically diagnosed using neuropsychological and cognitive tests, expensive neuroimaging-based approaches (MRI and PET) and invasive and time-consuming lumbar puncture for cerebrospinal fluid (CSF) sample collection to detect biomarkers. Thus, a rapid, simple and cost-effective approach to more easily access fluids and tissues is in great need. Here, we exploit the chemical direct reprogramming of patient skin fibroblasts into neurons (chemically induced neurons, ciNs) as a novel strategy for the rapid detection of different pathological markers of neurodegenerative diseases. We found that FAD fibroblasts have a reduced efficiency of reprogramming, and converted ciNs show a less complex neuronal network. In addition, ciNs from patients show misfolded protein accumulation and mitochondria ultrastructural abnormalities, biomarkers commonly associated with neurodegeneration. Moreover, for the first time, we show that microfluidic technology, in combination with chemical reprogramming, enables on-chip examination of disease pathological processes and may have important applications in diagnosis. In conclusion, ciNs on microfluidic devices represent a small-scale, non-invasive and cost-effective high-throughput tool for protein misfolding disease diagnosis and may be useful for new biomarker discovery, disease mechanism studies and design of personalised therapies.     

Drosophila melanogaster Uncoupling Protein-4A (UCP4A) Catalyzes a Unidirectional Transport of Aspartate

Uncoupling proteins (UCPs) form a distinct subfamily of the mitochondrial carrier family (MCF) SLC25. Four UCPs, DmUCP4A-C and DmUCP5, have been identified in Drosophila melanogaster on the basis of their sequence homology with mammalian UCP4 and UCP5. In a Parkinson’s disease model, DmUCP4A showed a protective role against mitochondrial dysfunction, by increasing mitochondrial membrane potential and ATP synthesis. To date, DmUCP4A is still an orphan of a biochemical function, although its possible involvement in mitochondrial uncoupling has been ruled out. Here, we show that DmUCP4A expressed in bacteria and reconstituted in phospholipid vesicles catalyzes a unidirectional transport of aspartate, which is saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. Swelling experiments carried out in yeast mitochondria have demonstrated that the unidirectional transport of aspartate catalyzed by DmUCP4 is not proton-coupled. The biochemical function of DmUCP4A has been further confirmed in a yeast cell model, in which growth has required an efflux of aspartate from mitochondria. Notably, DmUCP4A is the first UCP4 homolog from any species to be biochemically characterized. In Drosophila melanogaster, DmUCP4A could be involved in the transport of aspartate from mitochondria to the cytosol, in which it could be used for protein and nucleotide synthesis, as well as in the biosynthesis of ß-alanine and N-acetylaspartate, which play key roles in signal transmission in the central nervous system.     

Differential Expression of Serum Extracellular Vesicle miRNAs in Multiple Sclerosis: Disease-Stage Specificity and Relevance to Pathophysiology

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Its first clinical presentation (clinically isolated syndrome, CIS) is often followed by the development of relapsing–remitting MS (RRMS). The periphery-to-CNS transmission of inflammatory molecules is a major pathophysiological pathway in MS. This could include signalling via extracellular vesicle (EV) microRNAs (miRNAs). In this study, we investigated the serum EV miRNome in CIS and RRMS patients and matched controls, with the aims to identify MS stage-specific differentially expressed miRNAs and investigate their biomarker potential and pathophysiological relevance. miRNA sequencing was conducted on serum EVs from CIS-remission, RRMS-relapse, and viral inflammatory CNS disorder patients, as well as from healthy and hospitalized controls. Differential expression analysis was conducted, followed by predictive power and target-pathway analysis. A moderate number of dysregulated serum EV miRNAs were identified in CIS-remission and RRMS-relapse patients, especially relative to healthy controls. Some of these miRNAs were also differentially expressed between the two MS stages and had biomarker potential for patient-control and CIS–RRMS separations. For the mRNA targets of the RRMS-relapse-specific EV miRNAs, biological processes inherent to MS pathophysiology were identified using in silico analysis. Study findings demonstrate that specific serum EV miRNAs have MS stage-specific biomarker potential and contribute to the identification of potential targets for novel, efficacious therapies.     

Biochemical and Cellular Characterization of New Radio-Resistant Cell Lines Reveals a Role of Natural Flavonoids to Bypass Senescence

Cancer is one of the main causes of death worldwide, and, among the most frequent cancer types, osteosarcoma accounts for 56% of bone neoplasms observed in children and colorectal cancer for 10.2% of tumors diagnosed in the adult population. A common and frequent hurdle in cancer treatment is the emergence of resistance to chemo- and radiotherapy whose biological causes are largely unknown. In the present work, human osteosarcoma (SAOS) and colorectal adenocarcinoma (HT29) cell lines were γ-irradiated at doses mimicking the sub-lethal irradiation in clinical settings to obtain two radio-resistant cellular sub-populations named SAOS400 and HT500, respectively. Since “therapy-induced senescence” (TIS) is often associated with tumor response to radiotherapy in cancer cells, we measured specific cellular and biochemical markers of senescence in SAOS400 and HT500 cells. In detail, both cell lines were characterized by a higher level of expression of cyclin-dependent kinase inhibitors p16^INK4 and p21^CIP1 and increased positivity to SAβ-gal (senescence-associated β-galactosidase) with respect to parental cells. Moreover, the intracellular levels of reactive oxygen species in the resistant cells were significantly lower compared to the parental counterparts. Subsequently, we demonstrated that senolytic agents were able to sensitize SAOS400 and HT500 to cell death induced by γ-irradiation. Employing two natural flavonoids, fisetin and quercetin, and a BH3-mimetic, ABT-263/navitoclax, we observed that their association with γ-irradiation significantly reduced the expression of p16^INK4, p21^CIP1 and synergistically (combination index < 1) increased cell death compared to radiation mono-alone treatments. The present results reinforce the potential role of senolytics as adjuvant agents in cancer therapy.     

Effects of P4 Antagonist RU486 on VEGF and Its Receptors’ Signaling during the In Vivo Transition from the Preovulatory to Periovulatory Phase of Ovarian Follicles

The development of an adequate blood vessel network is crucial for the accomplishment of ovarian follicle growth and ovulation, which is necessary to support the proliferative and endocrine functions of the follicular cells. Although the Vascular Endothelial Growth Factor (VEGF) through gonadotropins guides ovarian angiogenesis, the role exerted by the switch on of Progesterone (P₄) during the periovulatory phase remains to be clarified. The present research aimed to investigate in vivo VEGF-mediated mechanisms by inducing the development of periovulatory follicles using a pharmacologically validated synchronization treatment carried out in presence or absence of P₄ receptor antagonist RU486. Spatio-temporal expression profiles of VEGF, FLT1, and FLK1 receptors and the two major MAPK/ERKs and PI3K/AKT downstream pathways were analyzed on granulosa and on theca compartment. For the first time, the results demonstrated that in vivo administration of P₄ antagonist RU486 inhibits follicular VEGF receptors’ signaling mainly acting on the theca layer by downregulating the activation of ERKs and AKTs. Under the effect of RU486, periovulatory follicles’ microarchitecture did not move towards the periovulatory stage. The present evidence provides new insights on P₄ in vivo biological effects in driving vascular and tissue remodeling during the periovulatory phase.     

Direct Evidence of Macroscopic Quantum Effects at High Temperature

We present experiments on macroscopic quantum effects in Josephson junctions with different relevant junction parameters at a temperature well above the classical-quantum crossover temperature. This has been possible by extending the measurements on the escape rate out of the metastable state at higher sweeping frequency (dI/dt up to 100 A/sec.) in order to induce non-stationary conditions in the energy potential describing the junction dynamics. The non-stationary regime for the system allows a direct observation of energy level quantitation when the rate of change of the external energy, measured in terms of the level spacing, is fast with respect to the thermal transitions between levels.     

Women psychologists in academe: Mixed progress, unwarranted complacency.

This article reviews the characteristics of academic women in psychology. It also details what is known about women's participation in various academic roles and describes their successes in those roles. The article also discusses obstacles to women's success in psychology as a discipline and in academe more broadly. The article concludes with recommendations for the departments and institutions in which women work, as well as recommendations for APA and the field of psychology. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Enhanced injection in n++-poly/SiOx/SiO2/p-sub MOS capacitors for low-voltage nonvolatile memoryapplications: experiment

In this paper, n⁺⁺-poly/SiO_x/SiO₂/p-sub capacitors with enhanced electron injection under substrate accumulation are extensively studied. First, systematic investigation of the role of technology parameters in the PECVD deposition of the SiO_x films is presented. In particular, the effect of the silane dilution parameter on the device performance is investigated and the SiO_x film optimized in terms of reliability and electron injection enhancement. Then, investigation of the electrical behavior of n⁺⁺ -poly/SiO_x/SiO₂/p-sub MOS capacitors is presented. As a result, a picture of the space defect distribution in the SiO_x films is proposed. In SiO_x films, a relevant density of trapped charge adds to ionized impurities. In particular, the net charge is negative in the bulk of the dielectric, indicating that trapped electrons exceed all the other charge contributions. The space distribution of defects is strongly nonuniform and has the maximum in the vicinity of the SiO_x/SiO₂ interface. After dc current stress, the devices undergo electrical degradation, the dominant mechanism of degradation being the creation of interface hole traps. The trap generation model is based on the release of hydrogen and pairs generation in the SiO_x films. The time-scale of trap filling during the stress is tens of seconds, which suggests that the stress-induced traps are deep in the energy gap     

VLSI hardware architecture for complex fuzzy systems

This paper presents the design of a VLSI fuzzy processor, which is capable of dealing with complex fuzzy inference systems, i.e., fuzzy inferences that include rule chaining. The architecture of the processor is based on a computational model whose main features are: the capability to cope effectively with complex fuzzy inference systems; a detection phase of the rule with a positive degree of activation to reduce the number of rules to be processed per inference; parallel computation of the degree of activation of active rules; and representation of membership functions based on α-level sets. As the fuzzy inference can be divided into different processing phases, the processor is made up of a number of stages which are pipelined. In each stage several inference processing phases are performed parallelly. Its performance is in the order of 2 MFLIPS with 256 rules, eight inputs, two chained variables, and four outputs and 5.2 MFLIPS with 32 rules, three inputs, and one output with a clock frequency of 66 MHz     

Preimplantation embryotoxicity after mouse embryo exposition to reactive oxygen species.

Exposure of either gametes or embryos to conditions and/or factors that generate oxidative stress has been associated with impaired early embryogenesis. The effects of reactive oxygen species (ROS) on mouse preimplantation development, depending of the ROS-concentration and time of exposition, were studied. Two-cell embryos were incubated with 5, 10, 25 and 50 microM of hydrogen peroxide (H2O2) for 30 and 60 minutes of exposition and allowed to develop for 72 h to study the quality of development. The incubation with 50 microM H2O2 for 30 or 60 minutes, strongly inhibited the 2-cell embryo development as compared to the control (p < 0.001). Twenty-five microM H2O2 produced inhibition of blastocyst formation (p < 0.001) and 10 microM H2O2 significantly decreased the percentages of expanded and hatched blastocysts, which resulted morphologically altered (p < 0.05 and p < 0.01, respectively). The higher H2O2 concentrations were able to elicit necrotic morphology in the 2-cell arrested embryos, while 10 microM H2O2 induced moderate damage with the arrested embryos partially fragmented. In conclusion, important causes for defective preimplantation development and for early embryo losses may be due to oxidative stress because early mouse embryos exposed to ROS for short times arrested at the first cellular cycle (2-cell) and/or impaired embryo differentiation and morphogenesis, being these effects ROS-concentration-dependent.