A specific inhibitor of lactate dehydrogenase overcame the resistance toward gemcitabine in hypoxic mesothelioma cells,and modulated the expression of the human equilibrative transporter-1

ABSTRACT

Malignant pleural mesothelioma (MPM) is a very hypoxic malignancy, and hypoxia has been associated with resistance towards gemcitabine. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis. Therefore we investigated the combination of a new LDH-A inhibitor (NHI-1) with gemcitabine in MPM cell lines. Under hypoxia (O₂ tension of 1%) the cell growth inhibitory effects of gemcitabine, were reduced, as demonstrated by a 5- to 10-fold increase in IC₅₀s. However, the simultaneous addition of NHI-1 was synergistic (combination index < 1). Flow cytometry demonstrated that hypoxia caused a G1 arrest, whereas the combination of NHI-1 significantly increased gemcitabine-induced cell death. Finally, the mRNA expression levels of the human equilibrative transporter-1 (hENT1) were significantly down-regulated under hypoxia, but treatment with NHI-1 was associated with a recovery of hENT1 expression. In conclusion, our data show that hypoxia increased MPM resistance to gemcitabine. However, cell death induction and modulation of the key transporter in gemcitabine uptake may contribute to the synergistic interaction of gemcitabine with the LDH-A inhibitor NHI-1 and support further studies for the rational development of this combination.     

Comprehensive manipulation of glycosylation profiles across development scales

The extent and pattern of glycosylation on therapeutic antibodies can influence their circulatory half-life, engagement of effector functions, and immunogenicity, with direct consequences to efficacy and patient safety. Hence, controlling glycosylation patterns is central to any drug development program, yet poses a formidable challenge to the bio-manufacturing industry. Process changes, which can affect glycosylation patterns, range from manufacturing at different scales or sites, to switching production process mode, all the way to using alternative host cell lines. In the emerging space of biosimilars development, often times all of these aspects apply. Gaining a deep understanding of the direction and extent to which glycosylation quality attributes can be modulated is key for efficient fine-tuning of glycan profiles in a stage appropriate manner, but establishment of such platform knowledge is time consuming and resource intensive. Here we report an inexpensive and highly adaptable screening system for comprehensive modulation of glycans on antibodies expressed in CHO cells. We characterize 10 media additives in univariable studies and in combination, using a design of experiments approach to map the design space for tuning glycosylation profile attributes. We introduce a robust workflow that does not require automation, yet enables rapid process optimization. We demonstrate scalability across deep wells, shake flasks, AMBR-15 cell culture system, and 2 L single-use bioreactors. Further, we show that it is broadly applicable to different molecules and host cell lineages. This universal approach permits fine-tuned modulation of glycan product quality, reduces development costs, and enables agile implementation of process changes throughout the product lifecycle.     

Protection by herpes simplex virus glycoprotein D against Fas-mediated apoptosis: role of nuclear factor kappaB

Signals involved in protection against apoptosis by herpes simplex virus 1 (HSV-1) were investigated. Using U937 monocytoid cells as an experimental model, we have demonstrated that HSV-1 rendered these cells resistant to Fas-induced apoptosis promptly after infection. UV-inactivated virus as well as the envelope glycoprotein D (gD) of HSV-1, by itself, exerted a protective effect on Fas-induced apoptosis. NF-kappaB was activated by gD, and protection against Fas-mediated apoptosis by gD was abolished in cells stably transfected with a dominant negative mutant I-kappaBalpha, indicating that NF-kappaB activation plays a role in the antiapoptotic activity of gD in our experimental model. Moreover, NF-kappaB-dependent protection against Fas-mediated apoptosis was associated with decreased levels of caspase-8 activity and with the up-regulation of intracellular antiapoptotic proteins.     

Ocean Acidification and Increased Temperature Have Both Positive and Negative Effects on Early Ontogenetic Traits of a Rocky Shore Keystone Predator Species

The combined effect of ocean acidification and warming is expected to have significant effects on several traits of marine organisms. The gastropod Concholepas concholepas is a rocky shore keystone predator characteristic of the south-eastern Pacific coast of South America and an important natural resource exploited by small-scale artisanal fishermen along the coast of Chile and Peru. In this study, we used small juveniles of C. concholepas collected from the rocky intertidal habitats of southern Chile (39°S) to evaluate under laboratory conditions the potential consequences of projected near-future levels of ocean acidification and warming for important early ontogenetic traits. The individuals were exposed long-term (5.8 months) to contrasting pCO₂ (ca. 500 and 1400 μatm) and temperature (15 and 19°C) levels. After this period we compared body growth traits, dislodgement resistance, predator-escape response, self-righting and metabolic rates. With respect to these traits there was no evidence of a synergistic interaction between pCO₂ and temperature. Shell growth was negatively affected by high pCO₂ levels only at 15°C. High pCO₂ levels also had a negative effect on the predator-escape response. Conversely, dislodgement resistance and self-righting were positively affected by high pCO₂ levels at both temperatures. High tenacity and fast self-righting would reduce predation risk in nature and might compensate for the negative effects of high pCO₂ levels on other important defensive traits such as shell size and escape behaviour. We conclude that climate change might produce in C. concholepas positive and negative effects in physiology and behaviour. In fact, some of the behavioural responses might be a consequence of physiological effects, such as changes in chemosensory capacity (e.g. predator-escape response) or secretion of adhesive mucous (e.g. dislodgement resistance). Moreover, we conclude that positive behavioural responses may assist in the adaptation to negative physiological impacts, and that this may also be the case for other benthic organisms.     

The effect of ADF/cofilin and profilin on the dynamics of monomeric actin

The main goal of the work was to uncover the dynamical changes in actin induced by the binding of cofilin and profilin. The change in the structure and flexibility of the small domain and its function in the thermodynamic stability of the actin monomer were examined with fluorescence spectroscopy and differential scanning calorimetry (DSC). The structure around the C-terminus of actin is slightly affected by the presence of cofilin and profilin. Temperature dependent fluorescence resonance energy transfer measurements indicated that both actin binding proteins decreased the flexibility of the protein matrix between the subdomains 1 and 2. Time resolved anisotropy decay measurements supported the idea that cofilin and profilin changed similarly the dynamics around the fluorescently labeled Cys-374 and Lys-61 residues in subdomains 1 and 2, respectively. DSC experiments indicated that the thermodynamic stability of actin increased by cofilin and decreased in the presence of profilin. Based on the information obtained it is possible to conclude that while the small domain of actin acts uniformly in the presence of cofilin and profilin the overall stability of actin changes differently in the presence of the studied actin binding proteins. The results support the idea that the small domain of actin behaves as a rigid unit during the opening and closing of the nucleotide binding pocket in the presence of profilin and cofilin as well. The structural arrangement of the nucleotide binding cleft mainly influences the global stability of actin while the dynamics of the different segments can change autonomously.     

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.     

Ciliate–adenovirus interactions in experimental co-cultures of Euplotes octocarinatus and in wastewater environment

The aim of the present work was to study the dynamics of the interactions between human adenovirus and ciliates under both experimental and field conditions. Experimental co-cultures of the ciliated protozoan Euplotes octocarinatus and human adenovirus (HAdV) type 2 were established and virus internalization was investigated using nested PCR and direct immunofluorescence (IF). In addition, to study protozoa-virus interactions in the field, wild ciliates were isolated from active sludges of a wastewater treatment plant and analyzed for the presence of adenovirus using direct IF. In vitro experiments revealed HAdV type 2 inside Euplotes cells after 15 min of contact and its persistence until at least 35 days post infection. In addition, our results showed the adsorption of adenovirus on the surface of wild ciliates. We conclude that HAdV is taken up by ciliates, however more studies are necessary in order to better investigate the mechanisms, the infectivity of internalized virus and the protective effects of internalization against disinfection.     

Density dependence and environmental factors affect population stability of an agricultural pest and its specialist parasitoid

Host–parasitoid dynamics are intrinsically unstable unless the risk of parasitism is sufficiently heterogeneous among hosts. Spatial aggregation of parasitoids can contribute to this heterogeneity, stabilising host–parasitoid population dynamics and thereby reducing pest outbreaks. We examined the spatial distribution of mango gall fly (Procontarinia matteiana, Kiefer and Cecconi), a non-native pest of South African mango orchards, which is controlled by a single parasitoid (Chrysonotomyia pulcherrima, Kerrich). We assessed whether spatial aggregation of parasitoids is associated with proximity to natural vegetation and/or to host density-dependent and host density-independent factors at three spatial scales. We found evidence for higher parasitism rates near natural vegetation at the field scale, and inverse host-density dependent and density-independent parasitoid aggregation at both the leaf scale and field scale. Therefore, we conclude that natural vegetation plays a role in promoting stabilising aggregation of parasitoids, possibly through provision of non-host resources (nectar, pollen), in this system.     

Autophagy Inhibition Favors Survival of Rubrospinal Neurons After Spinal Cord Hemisection

Spinal cord injuries (SCIs) are devastating conditions of the central nervous system (CNS) for which there are no restorative therapies. Neuronal death at the primary lesion site and in remote regions that are functionally connected to it is one of the major contributors to neurological deficits following SCI.Disruption of autophagic flux induces neuronal death in many CNS injuries, but its mechanism and relationship with remote cell death after SCI are unknown. We examined the function and effects of the modulation of autophagy on the fate of axotomized rubrospinal neurons in a rat model of spinal cord dorsal hemisection (SCH) at the cervical level. Following SCH, we observed an accumulation of LC3-positive autophagosomes (APs) in the axotomized neurons 1 and 5 days after injury. Furthermore, this accumulation was not attributed to greater initiation of autophagy but was caused by a decrease in AP clearance, as demonstrated by the build-up of p62, a widely used marker of the induction of autophagy. In axotomized rubrospinal neurons, the disruption of autophagic flux correlated strongly with remote neuronal death and worse functional recovery. Inhibition of AP biogenesis by 3-methyladenine (3-MA) significantly attenuated remote degeneration and improved spontaneous functional recovery, consistent with the detrimental effects of autophagy in remote damage after SCH. Collectively, our results demonstrate that autophagic flux is blocked in axotomized neurons on SCI and that the inhibition of AP formation improves their survival. Thus, autophagy is a promising target for the development of therapeutic interventions in the treatment of SCIs.     

Structural and biochemical insights into 7β‐hydroxysteroid dehydrogenase stereoselectivity

Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo‐ and regio‐selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7β‐hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active‐site accessibility, the bases of the specificity for NADP⁺, and the general architecture of the steroid binding site. Comparison with 7α‐hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C‐terminal extension reshapes the substrate site of the β‐selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859–865. © 2016 Wiley Periodicals, Inc.