Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.     

Inhibition of hydrogen sulfide production by gene silencing attenuates inflammatory activity of LPS-activated RAW264.7 cells

Hydrogen sulfide is an inflammatory mediator and is produced by the activity of the enzyme cystathionine γ-lyase (CSE) in macrophages. Previously, pharmacological inhibition of CSE has been reported to have conflicting results, and this may be due to the lack of specificity of the pharmacological agents. Therefore, this study used a very specific approach of small interfering RNA (siRNA) to inhibit the production of the CSE in an in vitro setting. We found that the activation of macrophages by lipopolysaccharide (LPS) resulted in higher levels of CSE mRNA and protein as well as the increased production of proinflammatory cytokines and nitric oxide (NO). We successfully used siRNA to specifically reduce the levels of CSE mRNA and protein in activated macrophages. Furthermore, the levels of proinflammatory cytokines in LPS-activated macrophages were significantly lower in siRNA-transfected cells compared to those in untransfected controls. However, the production levels of NO by the transfected cells were higher, suggesting that CSE activity has an inhibitory effect on NO production. These findings suggest that the CSE enzyme has a crucial role in the activation of macrophages, and its activity has an inhibitory effect on NO production by these cells.     

Understanding and modeling retention of mammalian cells in fluidized bed centrifuges

Within the last decade, fully disposable centrifuge technologies, fluidized‐bed centrifuges (FBC), have been introduced to the biologics industry. The FBC has found a niche in cell therapy where it is used to collect, concentrate, and then wash mammalian cell product while continuously discarding centrate. The goal of this research was to determine optimum FBC conditions for recovery of live cells, and to develop a mathematical model that can assist with process scaleup. Cell losses can occur during bed formation via flow channels within the bed. Experimental results with the kSep400 centrifuge indicate that, for a given volume processed: the bed height (a bed compactness indicator) is affected by RPM and flowrate, and dead cells are selectively removed during operation. To explain these results, two modeling approaches were used: (i) equating the centrifugal and inertial forces on the cells (i.e., a force balance model or FBM) and (ii) a two‐phase computational fluid dynamics (CFD) model to predict liquid flow patterns and cell retention in the bowl. Both models predicted bed height vs. time reasonably well, though the CFD model proved more accurate. The flow patterns predicted by CFD indicate a Coriolis‐driven flow that enhances uniformity of cells in the bed and may lead to cell losses in the outflow over time. The CFD‐predicted loss of viable cells and selective removal of the dead cells generally agreed with experimental trends, but did over‐predict dead cell loss by up to 3‐fold for some of the conditions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1520–1530, 2016     

Cystathionine-γ-lyase gene silencing with siRNA in monocytes/macrophages attenuates inflammation in cecal ligation and puncture-induced sepsis in the mouse

Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H₂S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture (CLP)-induced sepsis is characterized by increased levels of myeloperoxidase (MPO) activity, morphological changes in liver and pro-inflammatory cytokines and chemokines in the liver and lung. SiRNA treatment attenuated inflammation in the liver and lungs of mice following CLP-induced sepsis. Liver MPO activity increased in CLP-induced sepsis and treatment with siRNA significantly reduced this. Similarly, lung MPO activity increased following induction of sepsis with CLP while siRNA treatment significantly reduced MPO activity. Liver and lung cytokine and chemokine levels in CLP-induced sepsis reduced following treatment with siRNA. These findings show a crucial pro-inflammatory role for H₂S synthesized by CSE in macrophages in sepsis and suggest CSE gene silencing with siRNA as a potential therapeutic approach for this condition.     

An Ultra‐Mechanosensitive Visco‐Poroelastic Polymer Ion Pump for Continuous Self‐Powering Kinematic Triboelectric Nanogenerators

A mechanosensitive, visco‐poroelastic polymer ion pump that can rapidly establish a dense electrical double layer via mechanical pressure, thereby significantly enhancing output performance of an ionic triboelectric nanogenerator (iTENG), is described. A working mechanism of an iTENG using a highly mechanosensitive, visco‐poroelastic ion pump is suggested and the optimal characteristics of the polymer ion pump are reported by investigating optical, mechanical, electrical, and electrochemical properties. Surprisingly, the pressure sensitivity of the iTENG reaches 23.3 V kPa^?1, which is tens of times the record value. To achieve controlled high‐frequency pulses from an iTENG, kinematic systems using a gear train and a cam are integrated with a single grounded iTENG, which produces a maximum of 600 V and 22 mA (≈2.2 W cm^?2) at an input frequency of 1.67 Hz; after power transforming, those values are converted to 1.42 V and 225 mA. A capacitor of 1 mF can be fully charged to 2 V in only 60 s, making it possible to continuously operate a wireless‐communicating self‐powered humidity sensor. Also, due to the high transparency and deformability of the polymer ion pump, a self‐powered transparent tactile sensor is successfully assembled using a 5 × 5 iTENG array.     

Prophylactic Potential of Synbiotic (Lactobacillus casei and Inulin) in Malnourished Murine Giardiasis: an Immunological and Ultrastructural Study

Probiotics and Antimicrobial Proteins - Giardiasis is a re-emerging infectious disease with outbreaks reported globally specially in children and malnourished individuals leading to malabsorption,...     

The potential of biobanked liquid based cytology samples for cervical cancer screening using Raman spectroscopy

Patient samples are unique and often irreplaceable. This allows biobanks to be a valuable source of material. The aim of this study was to assess the ability of Raman spectroscopy to screen for histologically confirmed cases of Cervical Intraepithelial neoplasia (CIN) using biobanked liquid based cytology (LBC) samples. Two temperatures for long term storage were assessed; 80°C and ?25°C. The utility of Raman spectroscopy for the detection of CIN was compared for fresh LBC samples and biobanked LBC samples. Two groups of samples were used for the study with one group associated with disease (CIN 3) and the other associated with no disease (cytology negative). The data indicates that samples stored at ?80°C are not suitable for assessment by Raman spectroscopy due to a lack of cellular material and the presence of cellular debris. However, the technology can be applied to fresh LBC samples and those stored at ?25°C and is, moreover, effective in the discrimination of negative samples from those where CIN 3 has been confirmed. Pooled fresh and biobanked samples are also amenable to the technology and achieve a similar sensitivity and specificity for CIN 3. This study demonstrates that cervical cytology samples stored within biobanks at temperatures that preclude cell lysis can act as a useful resource for Raman spectroscopy and will facilitate research and translational studies in this area.      

Breast MRI in nonpalpable breast lesions: a randomized trial with diagnostic and therapeutic outcome – MONET – study

Background

In orthodontic treatment, anchorage control is a fundamental aspect. Usually conventional mechanism for orthodontic anchorage control can be either extraoral or intraoral that is headgear or intermaxillary elastics. Their use are combined with various side effects such as tipping of occlusal plane or undesirable movements of teeth. Especially in cases, where key-teeth are missing, conventional anchorage defined as tooth-borne anchorage will meet limitations. Therefore, the use of endosseous implants for anchorage purposes are increasingly used to achieve positional stability and maximum anchorage.

Methods/Design

The intended study is designed as a prospective, multicenter randomized controlled trial (RCT), comparing and contrasting the effect of early loading of palatal implant therapy versus implant loading after 12 weeks post implantation using the new ortho-implant type II anchor system device (Orthosystem Straumann, Basel, Switzerland). 124 participants, mainly adult males or females, whose diagnoses require temporary stationary implant-based anchorage treatment will be randomized 1:1 to one of two treatment groups: group 1 will receive a loading of implant standard therapy after a healing period of 12 week (gold standard), whereas group 2 will receive an early loading of orthodontic implants within 1 week after implant insertion. Participants will be at least followed for 12 months after implant placement. The primary endpoint is to investigate the behavior of early loaded palatal implants in order to find out if shorter healing periods might be justified to accelerate active orthodontic treatment. Secondary outcomes will focus e.g. on achievement of orthodontic treatment goals and quantity of direct implant-bone interface of removed bone specimens. As tertiary objective, a histologic and microtomography evaluation of all retrieved implants will be performed to obtain data on the performance of the SLA surface in human bone evaluation of all retrieved implants. Additionally, resonance frequency analysis (RFA, Osstell? mentor) will be used at different times for clinically monitoring the implant stability and for histological comparison in order to measure the reliability of the resonance frequency measuring device.

Trial registration

Current Controlled Trials ISRCTN97142521.