Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype,induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

Background

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.

Methods

EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.

Results

Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.

Conclusions

Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

     

Reactive extraction of penicillin G in a pilot plant Karr-column II. Fermentation broths

Summary Penicillin G was extracted from mycelfree fermentation broths by means of the carrier (Amberlite LA-2) in n-butylacetate at pH 5 in a 7.6 m high pilot plant Karr-column with degrees of extraction E=98–99% and penicillin enrichments up to 3. The reextraction was carried out with phosphate buffer at pH-values above 7.5 with degree of extractions E=86–88% and penicillin enrichments up to 3. The penicillin and carrier losses were negligible. The influence of the process variables on the extraction degree was investigated. The penicillin extraction of the model medium and the fermentation broths were compared. Recommendations are given for the optimal penicillin recovery with reactive extraction.Symbols a specific interfacial area with regard to the volume of the continuous phase - cA concentration of carrier - cAHP,O concentration of complex in feed - cP,cP,O concentration of penicillin acid anion in theaqueous phase, in the feed - d ₃₂ Sauter droplet diameter - E degree of extraction - f stroke frequency - V _aq throughput of the aqueous phase - V ₀ throughput of the organic phase - Z dimensionsless longitudinal coordinate of the column with regard to its active length (4m) - holdup of the organic phase     

Three major mesoplanktonic communities resolved by in situ imaging in the upper 500 m of the global ocean

Aim

The distribution of mesoplankton communities has been poorly studied at global scale, especially from in situ instruments. This study aims to (1) describe the global distribution of mesoplankton communities in relation to their environment and (2) assess the ability of various environmental-based ocean regionalizations to explain the distribution of these communities.

Location

Global ocean, 0–500 m depth.

Time Period

2008–2019.

Major Taxa Studied

Twenty-eight groups of large mesoplanktonic and macroplanktonic organisms, covering Metazoa, Rhizaria and Cyanobacteria.

Methods

From a global data set of 2500 vertical profiles making use of the Underwater Vision Profiler 5 (UVP5), an in situ imaging instrument, we studied the global distribution of large (>600 μm) mesoplanktonic organisms. Among the 6.8 million imaged objects, 330,000 were large zooplanktonic organisms and phytoplankton colonies, the rest consisting of marine snow particles. Multivariate ordination (PCA) and clustering were used to describe patterns in community composition, while comparison with existing regionalizations was performed with regression methods (RDA).

Results

Within the observed size range, epipelagic plankton communities were Trichodesmium-enriched in the intertropical Atlantic, Copepoda-enriched at high latitudes and in upwelling areas, and Rhizaria-enriched in oligotrophic areas. In the mesopelagic layer, Copepoda-enriched communities were also found at high latitudes and in the Atlantic Ocean, while Rhizaria-enriched communities prevailed in the Peruvian upwelling system and a few mixed communities were found elsewhere. The comparison between the distribution of these communities and a set of existing regionalizations of the ocean suggested that the structure of plankton communities described above is mostly driven by basin-level environmental conditions.

Main Conclusions

In both layers, three types of plankton communities emerged and seemed to be mostly driven by regional environmental conditions. This work sheds light on the role not only of metazoans, but also of unexpected large protists and cyanobacteria in structuring large mesoplankton communities.     

Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice

Bacteriophages have been known to be present in the gut for many years, but studies of relationships between these viruses and their hosts in the intestine are still in their infancy. We isolated three bacteriophages specific for an enteroaggregative O104:H4 Escherichia coli (EAEC) strain responsible for diarrhoeal diseases in humans. We studied the replication of these bacteriophages in vitro and in vivo in a mouse model of gut colonization. Each bacteriophage was able to replicate in vitro in both aerobic and anaerobic conditions. Each bacteriophage individually reduced biofilms formed on plastic pegs and a cocktail of the three bacteriophages was found to be more efficient. The cocktail was also able to infect bacterial aggregates formed on the surface of epithelial cells. In the mouse intestine, bacteriophages replicated for at least 3 weeks, provided the host was present, with no change in host levels in the faeces. This model of stable and continuous viral replication provides opportunities for studying the long-term coevolution of virulent bacteriophages with their hosts within a mammalian polymicrobial ecosystem.     

Proteomic Strategy for Identifying Mollusc Shell Proteins Using Mild Chemical Degradation and Trypsin Digestion of Insoluble Organic Shell Matrix: A Pilot Study on Haliotis tuberculata

A successful strategy for the identification of shell proteins is based on proteomic analyses where soluble and insoluble fractions isolated from organic shell matrix are digested with trypsin with the aim of generating peptides, which are used to identify novel shell proteins contained in databases. However, using trypsin as a sole degradative agent is limited by the enzyme's cleavage specificity and is dependent upon the occurrence of lysine and arginine in the shell protein sequence. To bypass this limitation, we investigated the ability of trifluoroacetic acid (TFA), a low-specificity chemical degradative agent, to generate clusters of analyzable peptides from organic shell matrix, suitable for database annotation. Acetic acid-insoluble fractions from Haliotis tuberculata shell were processed by trypsin followed by TFA digestion. The hydrolysates were used to annotate an expressed sequence tag library constructed from the mantle tissue of Haliotis asinina, a tropical abalone species. The characterization of sequences with repeat motifs featured in some of the shell matrix proteins benefited from TFA-induced serial cutting, which can result in peptide ladder series. Using the degradative specificities of TFA and trypsin, we were able to identify five novel shell proteins. This pilot study indicates that a mild chemical digestion of organic shell matrix combined with trypsin generates peptides suitable for proteomic analysis for better characterization of mollusc shell matrix proteins.     

Structural insights into the allosteric effects of 4EBP1 on the eukaryotic translation initiation factor eIF4E

The eukaryotic translation initiation factor eIF4E plays key roles in cap-dependent translation and mRNA export. These functions rely on binding the 7-methyl-guanosine moiety (5'cap) on the 5'-end of all mRNAs. eIF4E is regulated by proteins such as eIF4G and eIF4E binding proteins (4EBPs) that bind the dorsal surface of eIF4E, distal to the cap binding site, and modulate cap binding activity. Both proteins increase the affinity of eIF4E for 5'cap. Our understanding of the allosteric effects and structural underpinnings of 4EBP1 or eIF4G binding can be advanced by obtaining structural data on cap-free eIF4E bound to one of these proteins. Here, we report the crystal structure of apo-eIF4E and cap-free eIF4E in complex with a 4EBP1 peptide. We also monitored 4EBP1 binding to cap-free eIF4E in solution using NMR. Together, these studies suggest that 4EBP1 transforms eIF4E into a cap-receptive state. NMR methods were also used to compare the allosteric routes activated by 4EBP1, eIF4G, and the arenavirus Z protein, a negative regulator of cap binding. We observed chemical shift perturbation at the dorsal binding site leading to alterations in the core of the protein, which were ultimately communicated to the unoccupied cap binding site of eIF4E. There were notable similarities between the routes taken by 4EBP1 and eIF4G and differences from the negative regulator Z. Thus, binding of 4EBP1 or eIF4G allosterically drives alterations throughout the protein that increase the affinity of eIF4E for the 5'cap.     

Determinants of sustained viral suppression in HIV-infected patients with self-reported poor adherence to antiretroviral therapy

Background

Good adherence to antiretroviral therapy (ART) is critical for successful HIV treatment. However, some patients remain virologically suppressed despite suboptimal adherence. We hypothesized that this could result from host genetic factors influencing drug levels.

Methods

Eligible individuals were Caucasians treated with efavirenz (EFV) and/or boosted lopinavir (LPV/r) with self-reported poor adherence, defined as missing doses of ART at least weekly for more than 6 months. Participants were genotyped for single nucleotide polymorphisms (SNPs) in candidate genes previously reported to decrease EFV (rs3745274, rs35303484, rs35979566 in CYP2B6) and LPV/r clearance (rs4149056 in SLCO1B1, rs6945984 in CYP3A, rs717620 in ABCC2). Viral suppression was defined as having HIV-1 RNA <400 copies/ml throughout the study period.

Results

From January 2003 until May 2009, 37 individuals on EFV (28 suppressed and 9 not suppressed) and 69 on LPV/r (38 suppressed and 31 not suppressed) were eligible. The poor adherence period was a median of 32 weeks with 18.9% of EFV and 20.3% of LPV/r patients reporting missed doses on a daily basis. The tested SNPs were not determinant for viral suppression. Reporting missing >1 dose/week was associated with a lower probability of viral suppression compared to missing 1 dose/week (EFV: odds ratio (OR) 0.11, 95% confidence interval (CI): 0.01–0.99; LPV/r: OR 0.29, 95% CI: 0.09–0.94). In both groups, the probability of remaining suppressed increased with the duration of continuous suppression prior to the poor adherence period (EFV: OR 3.40, 95% CI: 0.62–18.75; LPV/r: OR 5.65, 95% CI: 1.82–17.56).

Conclusions

The investigated genetic variants did not play a significant role in the sustained viral suppression of individuals with suboptimal adherence. Risk of failure decreased with longer duration of viral suppression in this population.     

Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. Identifying novel regulators of mitochondrial bioenergetics will broaden our understanding of regulatory checkpoints that coordinate complex metabolic pathways. We previously showed that Nur77, an orphan nuclear receptor of the NR4A family, regulates the expression of genes linked to glucose utilization. Here we demonstrate that expression of Nur77 in skeletal muscle also enhances mitochondrial function. We generated MCK-Nur77 transgenic mice that express wild-type Nur77 specifically in skeletal muscle. Nur77-overexpressing muscle had increased abundance of oxidative muscle fibers and mitochondrial DNA content. Transgenic muscle also exhibited enhanced oxidative metabolism, suggestive of increased mitochondrial activity. Metabolomic analysis confirmed that Nur77 transgenic muscle favored fatty acid oxidation over glucose oxidation, mimicking the metabolic profile of fasting. Nur77 expression also improved the intrinsic respiratory capacity of isolated mitochondria, likely due to the increased abundance of complex I of the electron transport chain. These changes in mitochondrial metabolism translated to improved muscle contractile function ex vivo and improved cold tolerance in vivo. Our studies outline a novel role for Nur77 in the regulation of oxidative metabolism and mitochondrial activity in skeletal muscle.     

Development of sensory innervation in chick skin: comparison of nerve fibre and chondroitin sulphate distributions in vivo and in vitro

In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using themonoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.