A GRAPHICAL APPROACH TO DESIGN AND OPTIMIZATION OF MOS AMPLIFIER

The component values and transistor dimensions of a single stage amplifier are the only designable parameters which can be adjusted to achieve optimum performance. The resulting parameters can be set to form an objective function and constraint equations to graphically shape a feasible region which is a polytope. The required optimum value of design parameters is then derived by navigating over each of the corner and internal points of the polytope shaped. Accordingly, the amplifier design problem is conveyed as an optimization problem termed Non Linear Programming (NLP) enlisting decipherable global optimization methods. The present technique yields an automatic analysis of single stage amplifiers inferred from the specification values. In this paper, the proposed technique is first used to express the design problem of a particular amplifier circuit as NLP and then applied to a varied amplifier designs. Comparison of result with existing work implies a superior outcome with respect to achievement of required small signal gain (Av) and unity gain frequency (UGF). The optimal trade-off curves related to performance metrics such as Av, power and UGF are derived in order to observe the corresponding dependencies

Downstream Improvement for Recombinant Adeno-Associated Viruses (rAAV) Produced in iCELLis Nano 4 m2 Adherent Bioreactor

In the clarification of recombinant adeno-associated virus cell culture (rAAV), unwanted cellular material is separated from the vector to increase its purity and enable further downstream processing. In industrial settings, primary clarification with depth filters is used to remove larger particles, such as cells and cellular debris originating from cell lysis and benzonase treatment. To reduce the challenge on the downstream process a clarification step that can eliminate these contaminants with minimal rAAV loss is highly desirable for robust GMP manufacture. We are working towards improving the clarification and downstream steps for rAAV5, 9 and other rAAV subtypes. We produced rAAV on the iCELLis Nano 4 m2 adherent bioreactor with 8 L media. At the point of harvest, the cells were lysed and were treated with benzonase. ‘The upstream feed was assessed for turbidity and subsequently processed and concentrated by depth filtration and Tangential Flow Filtration (TFF) and the reduction in turbidity determined

Protecting mammalian hair cells from aminoglycoside-toxicity: assessing phenoxybenzamine’s potential

Aminoglycosides (AGs) are widely used antibiotics because of their low cost and high efficacy against gram-negative bacterial infection. However, AGs are ototoxic,causing the death of sensory hair cells in the inner ear. Strategies aimed at developing or discovering agents that protect against aminoglycoside ototoxicity have focused on inhibiting apoptosis or more recently, on preventing antibiotic uptake by the hair cells. Recent screens for ototoprotective compounds using the larval zebrafish lateral line identified phenoxybenzamine as a potential protectant for aminoglycoside induced hair cell death. Here we used live imaging of FM1-43 uptake as a proxy for aminoglycoside entry, combined with hair-cell death assays to evaluate whether phenoxybenzamine can protect mammalian cochlear hair cells from the deleterious effects of the aminoglycoside antibiotic neomycin. We show that phenoxybenzamine can block FM1-43 entry into mammalian hair cells in a reversible and dose-dependent manner, but pre-incubation is required for maximal inhibition of entry. We observed differential effects of phenoxybenzamine on FM1-43 uptake in the two different types of cochlear hair cell in mammals, the outer hair cells (OHCs) and inner hair cells (IHCs).The requirement for pre-incubation and reversibility suggests an intracellular rather than an extracellular site of action for phenoxybenzamine. We also tested the efficacy of phenoxybenzamine as an otoprotective agent. In mouse cochlear explants the hair cell death resulting from 24 h exposure to neomycin was steeply dose-dependent, with 50% cell death occurring at ~230 uM for both IHC and OHC. We used 250 uM neomycin in subsequent hair-cell death assays. At 100 uM with 1 h pre-incubation, phenoxybenzamine conferred significant protection to both IHCs and OHCs, however at higher concentrations phenoxybenzamine itself showed clear signs of ototoxicity and an additive toxic effect when combined with neomycin. These data do not support the use of phenoxybenzamine as a therapeutic agent in mammalian inner ear. Our findings do share parallels with the observations from the zebrafish lateral line model but they also highlight the necessity for validation in the mammalian system and the potential for differential effects on sensory hair cells from different species, in different systems and even between cells in the same organ

New insights into purinergic receptor signaling in neuronal differentiation, neuroprotection, and brain disorders

Ionotropic P2X and metabotropic P2Y purinergic receptors are expressed in the central nervous system and participate in the synaptic process particularly associated with acetylcholine, GABA, and glutamate neurotransmission. As a result of activation, the P2 receptors promote the elevation of free intracellular calcium concentration as the main signaling pathway. Purinergic signaling is present in early stages of embryogenesis and is involved in processes of cell proliferation, migration, and differentiation. The use of new techniques such as knockout animals, in vitro models of neuronal differentiation, antisense oligonucleotides to induce downregulation of purinergic receptor gene expression, and the development of selective inhibitors for purinergic receptor subtypes contribute to the comprehension of the role of purinergic signaling during neurogenesis. In this review, we shall discuss the participation of purinergic receptors in developmental processes and in brain physiology, including neuron-glia interactions and pathophysiology

Analysis of P2X2 e P2X4 receptors during neuronal differentiation

Durante o desenvolvimento do sistema nervoso, as oscilações da concentração de cálcio intracelular livre resultam na proliferação celular, migração e diferenciação neuronal. Nesta tese foram investigadas a participação dos receptores ionotrópicos purinérgicos dos tipos P2X2 e P2X4 seletivos ao influxo de cálcio durante a diferenciação neuronal in vitro das células de carcinoma embrionário murino P19. Identificamos o padrão diferencial de expressão de receptores purinérgicos nas células indiferenciadas e neurônios P19. O receptor P2X4 é expresso durante toda a diferenciação neuronal e o receptor P2X2 é detectado na fase tardia da diferenciação em neurônios. Através de ensaios farmacológicos, foi possível identificar a participação dos receptores metabotropicos P2Y e do receptor P2X4 na formação dos corpos embriônicos, na proliferação celular e ou na determinação do fenótipo de progenitor neural. Durante a maturação neuronal os receptores P2X2 e P2Y1 participam da determinação do fenótipo neuronal glutamatérgico NMDA e os receptores P2X2 e P2Y2 no fenótipo neuronal colinérgico. A ausência de inibidores específicos e seletivos aos receptores purinérgicos levou-nos a empregar a técnica SELEX (Systematic Evolution of Ligands by EXponential enrichment) a fim de identificar inibidores seletivos aos receptores P2X2 e P2X4. A técnica envolve a utilização da biblioteca combinatória randômica de RNA 2\'- F pirimidina modificadas resistentes a nucleases. Após 9 ciclos de seleção in vitro de SELEX (ciclo 9-P2X4), as sequências selecionadas mostraram-se seletivas a ligação somente ao receptor P2X4 e não aos receptores P2X2 ou P2X7 através de ensaios de ligação radioligante-receptor. Por patch clamping na configuração whole cell recording identificou-se que além de seletividade ao receptor, que a aplicação do RNA ciclo 9- P2X4 promoveu inibição da corrente ativada pelo ATP somente nos receptores P2X4 e não em P2X2 em celulas 1321N1 astrocitoma transfectadas. A incubação do RNA ciclo 9-P2X4 na concentração de 200 nM com as células no estágio indiferenciado inibiu a formação dos corpos embriônicos. Já utilização de 25 nM, resultou em mudanças morfológicas nas células diferenciadas. Estes dados corroboram com os dados farmacológicos que identificaram a participação do receptor P2X4 na diferenciação precoce. Após 11 ciclos P2X2 de seleção, identificou-se sequências com especificidade de ligação aos receptores P2X2. Aptâmeros, moleculas de RNA com sequência identificada e com alta afinidade ao alvo da seleção, foram isolados de ambas as bibliotecas, ciclo 9 P2X4 e ciclo 11 P2X2. A co-aplicação destes aptâmeros e ATP em ensaios de whole-cell recording resultou na inibição de 30 a 80% da corrente ativada pelo ATP nos receptores P2X2 ou P2X4. Estes testes em células PC12 de rato, que expressa os receptores endógenos, resultou em inibição da corrente ativada pelo ATP de modo semelhante. Além de termos desenvolvido aptâmeros como ferramentas para elucidar as funções dos receptores P2X2 e P2X4 durante o desenvolvimento, diferenciação, em processos fisiológicos e patológicos, estas moléculas resistentes a nucleases são as primeiras identificadas capazes de reconhecer, discernir e inibir dois subtipos de receptores purinérgicos sendo promissores para utilização terapêutica.During the development of the nervous system, oscillations of intracellular calcium concentrations activate programs of gene expression resulting in proliferation, migration and neuronal differentiation of embryonic cells. In this thesis, the participation of ionotropic P2X2 and P2X4 receptor subtypes, whose receptor channels are highly permeable for calcium influx in the cells, was studied during the process of neuronal differentiation. We have identified differential gene expression of purinergic receptors in undifferentiated and neuronal-differentiated P19 cells. P2X4 receptor expression was present along neuronal differentiation of P19 cells, whereas P2X2 receptor expression was only detected when P19 cells became neurons. Based on purinergic receptor pharmacology we have determined the participation of P2X4 receptors in addition to metabotropic P2Y2 receptors in the formation of embryonic bodies as prerequisites for phenotype determination of P19 neural progenitor cells. Final neuronal maturation of P19 cells in the presence or absence of agonists or antagonists of purinergic receptors implicated the involvement of P2X2, P2Y1, and P2Y2 in the determination of the final neuronal phenotype, such as expression of NMDA-glutamate and cholinergic receptors. In order to further evaluate the functions of these P2X receptors and due to the absence of specific inhibitors for these receptor subtypes, we have used the SELEX technique (Systematic Evolution of Ligands by EXponential enrichment) to select for specific inhibitors for P2X2 and P2X4 receptors. The 2\' -F-pyrimidine modified, nuclease- resistant combinatorial SELEX RNA pool enriched with inhibitors of P2X4 receptors following nine cycles of in vitro selection (cycle 9-P2X4) specifically interacted with P2X4 receptors and not with P2X2 or P2X7 receptors as verified in radioligand-receptor binding studies. Moreover, whole-cell recording measurements using astrocytoma cells expressing recombinant rat P2X2 or P2X4 receptors showed inhibition of P2X4 but not of P2X2 receptors by the selected RNA molecules. RNA molecules selected in vitro in 11 reiterative SELEX cycles using the P2X2 receptor as target specifically bound to membrane extracts containing recombinant P2X2 receptors. From both selected RNA libraries (against P2X4 and P2X2 receptors) aptamers, as RNA molecules with identified sequences and high-affinity binding, were identified by cloning and DNA sequencing. The presence of these aptamers in whole-cell recording experiments resulted in 30-80% inhibition of ATP-induced receptor activity and did not provoke any inhibitory effects on P2X receptors which had not been used as selection target. The activity of the aptamers selected using recombinant receptors as targets in inhibiting wild-type P2X4 or P2X2 receptors was verified in whole-cell recording experiments with PC12 cells which endogenously express both receptor subtypes. In addition of having developed aptamers as tools to elucidate P2X2 and P2X4 receptor functions during neuronal differentiation, these nuclease-resistant aptamers are suitable for in vivo use and may turn into therapeutics in the inhibition of purinergic receptor participation in pathophysiological conditions

Multiphoton NAD(P)H FLIM reveals metabolic changes in individual cell types of the intact cochlea upon sensorineural hearing loss

Article Open Access Published: 11 December 2019 Multiphoton NAD(P)H FLIM reveals metabolic changes in individual cell types of the intact cochlea upon sensorineural hearing loss Paromita Majumder, Thomas S. Blacker, Lisa S. Nolan, Michael R. Duchen & Jonathan E. Gale Scientific Reports volume 9, Article number: 18907 (2019) Cite this article Article metrics 298 Accesses 9 Altmetric Metricsdetails Abstract An increasing volume of data suggests that changes in cellular metabolism have a major impact on the health of tissues and organs, including in the auditory system where metabolic alterations are implicated in both age-related and noise-induced hearing loss. However, the difficulty of access and the complex cyto-architecture of the organ of Corti has made interrogating the individual metabolic states of the diverse cell types present a major challenge. Multiphoton fluorescence lifetime imaging microscopy (FLIM) allows label-free measurements of the biochemical status of the intrinsically fluorescent metabolic cofactors NADH and NADPH with subcellular spatial resolution. However, the interpretation of NAD(P)H FLIM measurements in terms of the metabolic state of the sample are not completely understood. We have used this technique to explore changes in metabolism associated with hearing onset and with acquired (age-related and noise-induced) hearing loss. We show that these conditions are associated with altered NAD(P)H fluorescence lifetimes, use a simple cell model to confirm an inverse relationship between τbound and oxidative stress, and propose such changes as a potential index of oxidative stress applicable to all mammalian cell types

DNA and RNA aptamers: From tools for basic research towards therapeutic applications

The systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules are selected by their ability to bind their targets with high affinity and specificity, comparable to those of antibodies. Nucleic acids with high affinity for their targets have been selected against a wide variety of compounds, from small molecules, such as ATP, to membrane proteins and even whole organisms. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands, also known as aptamers, for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. the number of aptamers generated as inhibitors of various target proteins has increased following automatization of the SELEX process. Their diagnostic and therapeutic efficacy can be enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Several aptamers are currently being tested in preclinical and clinical trials, and aptamers are in the process of becoming a new class of therapeutic agents. Recently, the anti-VEGF aptamer pegaptanib received FDA approval for treatment of human ocular vascular disease.Univ São Paulo, Inst Quim, Dept Bioquim, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Neurociencias, BR-0402362 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Neurociencias, BR-0402362 São Paulo, BrazilWeb of Scienc

Density of CD3+ and CD8+ cells in gingivo-buccal oral squamous cell carcinoma is associated with lymph node metastases and survival.

The tumor immune microenvironment is emerging as a critical player in predicting cancer prognosis and response to therapies. However, the prognostic value of tumor-infiltrating immune cells in Gingivo-Buccal Oral Squamous Cell Carcinoma (GBOSCC) and their association with tumor size or lymph node metastases status require further elucidation. To study the relationship of tumor-infiltrating immune cells with tumor size (T stage) and lymph node metastases (N stages), we analyzed the density of tumor-infiltrating immune cells in archived, whole tumor resections from 94 patients. We characterized these sections by immune-histochemistry using 12 markers and enumerated tumor-infiltrating immune cells at the invasive margins (IM) and centers of tumors (CT). We observed that a higher density of CD3+ cells in the IM and CT was associated with smaller tumor size (T1-T2 stage). Fewer CD3+ cells was associated with larger tumor size (T3-T4 stage). High infiltration of CD3+and CD8+ cells in IM and CT as well as high CD4+ cell infiltrates in the IM was significantly associated with the absence of lymph node metastases. High infiltrates of CD3+ and CD8+ cells in CT was associated with significantly improved survival. Our results illustrate that the densities and spatial distribution of CD3+ and CD8+ cell infiltrates in primary GBOSCC tumors is predictive of disease progression and survival. Based on our findings, we recommend incorporating immune cell quantification in the TNM classification and routine histopathology reporting of GBOSCC. Immune cell quantification in CT and IM may help predict the efficacy of future therapies