Fluorescent analogues of quinoline reveal amine ligand loss from cis and trans platinum(II) complexes in cancer cells

Analogues of cytotoxic cis and trans dichloridoplatinum(II) complexes with one ammonia and one aromatic amine (cis- and trans-[PtCl₂(aromatic amine)(NH₃)]) were synthesised in which the aromatic group was replaced by the fluorescent ligand 7-azaindole (1). Coordination resulted in almost complete quenching of the fluorescence and the ligand had a effect on the biological activities of the cis and trans isomers similar to that previously reported for aromatic amines as is exemplified by them having similar cytotoxicities (IC₅₀ 3.6(5) and 6.0(19) μM, respectively). Observation of fluorescence following treatment of the cis complex with cysteine, glutathione, or methionine suggests labilisation and subsequent loss of the putative non-leaving group ligands. No such effect was observed for the trans complex which does not experience trans labilisation. Two-photon excitation of cells that had been treated with the complexes gave rise to observable fluorescence, suggesting ligand displacement for both complexes. The fluorescence appears to be localised in the lysosomes or late endosomes. These complexes are excellent models of analogues of cytotoxic cis and trans complexes with aromatic amine ligands and can be used to study the metabolism of the non-leaving group positions.     

Essential roles of lipoyl domains in the activated function and control of pyruvate dehydrogenase kinases and phosphatase isoform 1.

Four pyruvate dehydrogenase kinase and two pyruvate dehydrogenase phosphatase isoforms function in adjusting the activation state of the pyruvate dehydrogenase complex (PDC) through determining the fraction of active (nonphosphorylated) pyruvate dehydrogenase component. Necessary adaptations of PDC activity with varying metabolic requirements in different tissues and cell types are met by the selective expression and pronounced variation in the inherent functional properties and effector sensitivities of these regulatory enzymes. This review emphasizes how the foremost changes in the kinase and phosphatase activities issue from the dynamic, effector-modified interactions of these regulatory enzymes with the flexibly held outer domains of the core-forming dihydrolipoyl acetyl transferase component.     

The elastin-binding protein of Staphylococcus aureus (EbpS) is expressed at the cell surface as an integral membrane protein and not as a cell wall-associated protein.

The elastin-binding proteins EbpS of Staphylococcus aureus strains Cowan and 8325-4 were predicted from sequence analysis to comprise 486 residues. Specific antibodies were raised against an N-terminal domain (residues 1-267) and a C-terminal domain (residues 343-486) expressed as recombinant proteins in Escherichia coli. Western blotting of lysates of wild-type 8325-4 and Newman and the corresponding ebpS mutants showed that EbpS migrated with an apparent molecular mass of 83 kDa. The protein was found exclusively in cytoplasmic membrane fractions purified from protoplasts or lysed cells, in contrast to the clumping factor ClfA, which was cell-wall-associated. EbpS was predicted to have three hydrophobic domains H1-(205-224), H2-(265-280), and H3-(315-342). A series of hybrid proteins was formed between EbpS at the N terminus and either alkaline phosphatase or beta-galactosidase at the C terminus (EbpS-PhoA, EbpS-LacZ). PhoA and LacZ were fused to EbpS between hydrophobic domains H1-H2 and H2-H3, and distal to H3. Expression of enzymatic activity in E. coli showed that EbpS is an integral membrane protein with two membrane-spanning domains H1 and H3. N-terminal residues 1-205 and C-terminal residues 343-486 were predicted to be exposed on the outer face of the cytoplasmic membrane. The ligand-binding domain of EbpS is known from previous studies to be present in the N terminus between residues 14-34 and probing whole cells with anti-EbpS1-267 antibodies indicated that this region is exposed on the surface of intact cells. This was also confirmed by the observation that wild-type S. aureus Newman cells bound labeled tropoelastin whereas the ebpS mutant bound 72% less. In contrast, the C terminus, which carries a putative LysM peptidoglycan-binding domain, is not exposed on the surface of intact cells and presumably remains buried within the peptidoglycan. Finally, expression of EbpS was correlated with the ability of cells to grow to a higher density in liquid culture, suggesting that EbpS may have a role in regulating cell growth.     

A Multi-Atlas Based Method for Automated Anatomical Rat Brain MRI Segmentation and Extraction of PET Activity

Introduction

Preclinical in vivo imaging requires precise and reproducible delineation of brain structures. Manual segmentation is time consuming and operator dependent. Automated segmentation as usually performed via single atlas registration fails to account for anatomo-physiological variability. We present, evaluate, and make available a multi-atlas approach for automatically segmenting rat brain MRI and extracting PET activies.

Methods

High-resolution 7T 2DT2 MR images of 12 Sprague-Dawley rat brains were manually segmented into 27-VOI label volumes using detailed protocols. Automated methods were developed with 7/12 atlas datasets, i.e. the MRIs and their associated label volumes. MRIs were registered to a common space, where an MRI template and a maximum probability atlas were created. Three automated methods were tested: 1/registering individual MRIs to the template, and using a single atlas (SA), 2/using the maximum probability atlas (MP), and 3/registering the MRIs from the multi-atlas dataset to an individual MRI, propagating the label volumes and fusing them in individual MRI space (propagation & fusion, PF). Evaluation was performed on the five remaining rats which additionally underwent [¹⁸F]FDG PET. Automated and manual segmentations were compared for morphometric performance (assessed by comparing volume bias and Dice overlap index) and functional performance (evaluated by comparing extracted PET measures).

Results

Only the SA method showed volume bias. Dice indices were significantly different between methods (PF>MP>SA). PET regional measures were more accurate with multi-atlas methods than with SA method.

Conclusions

Multi-atlas methods outperform SA for automated anatomical brain segmentation and PET measure’s extraction. They perform comparably to manual segmentation for FDG-PET quantification. Multi-atlas methods are suitable for rapid reproducible VOI analyses.     

Adaptive Evolution and Environmental Durability Jointly Structure Phylodynamic Patterns in Avian Influenza Viruses

Avian influenza viruses (AIVs) have been pivotal to the origination of human pandemic strains. Despite their scientific and public health significance, however, there remains much to be understood about the ecology and evolution of AIVs in wild birds, where major pools of genetic diversity are generated and maintained. Here, we present comparative phylodynamic analyses of human and AIVs in North America, demonstrating (i) significantly higher standing genetic diversity and (ii) phylogenetic trees with a weaker signature of immune escape in AIVs than in human viruses. To explain these differences, we performed statistical analyses to quantify the relative contribution of several potential explanations. We found that HA genetic diversity in avian viruses is determined by a combination of factors, predominantly subtype-specific differences in host immune selective pressure and the ecology of transmission (in particular, the durability of subtypes in aquatic environments). Extending this analysis using a computational model demonstrated that virus durability may lead to long-term, indirect chains of transmission that, when coupled with a short host lifespan, can generate and maintain the observed high levels of genetic diversity. Further evidence in support of this novel finding was found by demonstrating an association between subtype-specific environmental durability and predicted phylogenetic signatures: genetic diversity, variation in phylogenetic tree branch lengths, and tree height. The conclusion that environmental transmission plays an important role in the evolutionary biology of avian influenza viruses—a manifestation of the “storage effect”—highlights the potentially unpredictable impact of wildlife reservoirs for future human pandemics and the need for improved understanding of the natural ecology of these viruses.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

Migration patterns and navigation cues of Atlantic salmon post-smolts migrating from 12 rivers through the coastal zones around the Irish Sea

The freshwater phase of the first seaward migration of juvenile Atlantic salmon (Salmo salar) is relatively well understood when compared with our understanding of the marine phase of their migration. In 2021, 1008 wild and 60 ranched Atlantic salmon smolts were tagged with acoustic transmitters in 12 rivers in England, Scotland, Northern Ireland and Ireland. Large marine receiver arrays were deployed in the Irish Sea at two locations: at the transition of the Irish Sea into the North Atlantic between Ireland and Scotland, and between southern Scotland and Northern Ireland, to examine the early phase of the marine migration of Atlantic salmon smolts. After leaving their natal rivers' post-smolt migration through the Irish Sea was rapid with minimum speeds ranging from 14.03 to 38.56 km.day⁻¹ for Atlantic salmon smolts that entered the Irish Sea directly from their natal river, to 9.69–39.94 km.day⁻¹ for Atlantic salmon smolts that entered the Irish Sea directly from their natal estuary. Population minimum migration success through the study area was strongly correlated with the distance of travel, populations further away from the point of entry to the open North Atlantic exhibited lower migration success. Post-smolts from different populations experienced different water temperatures on entering the North Atlantic. This was largely driven by the timing of their migration and may have significant consequences for feeding and ultimately survivorship. The influence of water currents on post-smolt movement was investigated using data from previously constructed numerical hydrodynamic models. Modeled water current data in the northern Irish Sea showed that post-smolts had a strong preference for migrating when the current direction was at around 283° (west-north-west) but did not migrate when exposed to strong currents in other directions. This is the most favorable direction for onward passage from the Irish Sea to the continental shelf edge current, a known accumulation point for migrating post-smolts. These results strongly indicate that post-smolts migrating through the coastal marine environment are: (1) not simply migrating by current following (2) engage in active directional swimming (3) have an intrinsic sense of their migration direction and (4) can use cues other than water current direction to orientate during this part of their migration.     

Homer 1b regulates the trafficking of group I metabotropic glutamate receptors.

The molecular basis for glutamate receptor trafficking to the plasma membrane is not understood. In the present study, we demonstrate that Homer 1b (H1b), a constitutively expressed splice form of the immediate early gene product Homer (now termed Homer 1a) regulates the trafficking and surface expression of group I metabotropic glutamate receptors. H1b inhibits surface expression of the metabotropic glutamate receptor mGluR5 in heterologous cells, causing mGluR5 to be retained in the endoplasmic reticulum (ER). In contrast, mGluR5 alone or mGluR5 coexpressed with Homer 1a successfully travels through the secretory pathway to the plasma membrane. In addition, point mutations that disrupt mGluR5 binding to H1b eliminate ER retention of mGluR5, demonstrating that H1b affects metabotropic receptor localization via a direct protein-protein interaction. Electron microscopic analysis reveals that the group I metabotropic receptor mGluR1alpha is significantly enriched in the ER of Purkinje cells, suggesting that a similar mechanism may exist in vivo. Because H1b is found in dendritic spines of neurons, local retention of metabotropic receptors within dendritic ER provides a potential mechanism for regulating synapse-specific expression of group I metabotropic glutamate receptors.