The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data

Background  

The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues.     

False occurrences of functional motifs in protein sequences highlight evolutionary constraints

Background

False occurrences of functional motifs in protein sequences can be considered as random events due solely to the sequence composition of a proteome. Here we use a numerical approach to investigate the random appearance of functional motifs with the aim of addressing biological questions such as: How are organisms protected from undesirable occurrences of motifs otherwise selected for their functionality? Has the random appearance of functional motifs in protein sequences been affected during evolution?

Results

Here we analyse the occurrence of functional motifs in random sequences and compare it to that observed in biological proteomes; the behaviour of random motifs is also studied. Most motifs exhibit a number of false positives significantly similar to the number of times they appear in randomized proteomes (=expected number of false positives). Interestingly, about 3% of the analysed motifs show a different kind of behaviour and appear in biological proteomes less than they do in random sequences. In some of these cases, a mechanism of evolutionary negative selection is apparent; this helps to prevent unwanted functionalities which could interfere with cellular mechanisms.

Conclusion

Our thorough statistical and biological analysis showed that there are several mechanisms and evolutionary constraints both of which affect the appearance of functional motifs in protein sequences.

     

Improved chemical synthesis and demonstration of the relaxin receptor binding affinity and biological activity of mouse relaxin

The primary stored and circulating form of relaxin in humans, human gene-2 (H2) relaxin, has potent antifibrotic properties with rapidly occurring efficacy. However, when administered to experimental models of fibrosis, H2 relaxin can only be applied over short-term (2-4 week) periods, due to rodents mounting an antibody response to the exogenous human relaxin, resulting in delayed clearance and, hence, increased and variable circulating levels. To overcome this problem, the current study investigated the therapeutic potential of mouse relaxin over long-term exposure in vivo. Mouse relaxin is unique among the known relaxins in that it possesses an extra residue within the C-terminal region of its A-chain. To enable a detailed assessment of its receptor interaction and biological properties, it was chemically synthesized in good overall yield by the separate preparation of each of its A- and B-chains followed by regioselective formation of each of the intramolecular and two intermolecular disulfide bonds. Murine relaxin was shown to bind with high affinity to the human, mouse, and rat RXFP1 (primary relaxin) receptor but with a slightly lower affinity to that of H2 relaxin. When administered to relaxin-deficient mice (which undergo an age-dependent progression of organ fibrosis) over a 4 month treatment period, mouse relaxin was able to significantly inhibit the progression of collagen accumulation in several organs including the lung, kidney, testis, and skin (all p < 0.05 vs untreated group), consistent with the actions of H2 relaxin. These combined data demonstrate that mouse relaxin can effectively inhibit collagen deposition and accumulation (fibrosis) over long-term treatment periods.     

Sampling related individuals within ponds biases estimates of population structure in a pond‐breeding amphibian

Effective conservation and management of pond‐breeding amphibians depends on the accurate estimation of population structure, demographic parameters, and the influence of landscape features on breeding‐site connectivity. Population‐level studies of pond‐breeding amphibians typically sample larval life stages because they are easily captured and can be sampled nondestructively. These studies often identify high levels of relatedness between individuals from the same pond, which can be exacerbated by sampling the larval stage. Yet, the effect of these related individuals on population genetic studies using genomic data is not yet fully understood. Here, we assess the effect of within‐pond relatedness on population and landscape genetic analyses by focusing on the barred tiger salamanders (Ambystoma mavortium) from the Nebraska Sandhills. Utilizing genome‐wide SNPs generated using a double‐digest RADseq approach, we conducted standard population and landscape genetic analyses using datasets with and without siblings. We found that reduced sample sizes influenced parameter estimates more than the inclusion of siblings, but that within‐pond relatedness led to the inference of spurious population structure when analyses depended on allele frequencies. Our landscape genetic analyses also supported different models across datasets depending on the spatial resolution analyzed. We recommend that future studies not only test for relatedness among larval samples but also remove siblings before conducting population or landscape genetic analyses. We also recommend alternative sampling strategies to reduce sampling siblings before sequencing takes place. Biases introduced by unknowingly including siblings can have significant implications for population and landscape genetic analyses, and in turn, for species conservation strategies and outcomes.     

Autophagic and apoptotic response to stress signals in mammalian cells

Autophagy is a highly conserved catabolic programme for degrading proteins and organelles. This process has been shown to act as a pro-survival or pro-death mechanism in different physiological and pathological conditions. Several stress stimuli can induce autophagy, such as nutrient deprivation or critical steps in development of lower and higher eukaryotes. Apoptosis is an orchestrated form of cell death in which cells are actively involved in their own demise. Again, stress is a positive regulator of apoptosis and, in particular, of its apoptosome-mediated mitochondrial pathway. Besides discussing the individual roles played by the key molecules involved in autophagy in mammals in response to stress signals, we discuss here the interrelations between autophagy and apoptosis under these conditions.     

Biaryl isoxazolinone antibacterial agents

In an era of increasing resistance to classical antibacterial agents, the synthetic oxazolidinone series of antibiotics has attracted much interest. Zyvoxtrade mark was the first oxazolidinone to be approved for clinical use against infections caused by multi-drug resistant Gram-positive bacteria. In the course of studies directed toward the discovery of novel antibacterial agents, a new series of synthetic phenyl-isoxazolinone agents that displayed potent activity against Gram-positive bacterial strains was recently discovered at Bristol-Myers Squibb. Extensive investigation of various substitutions on the phenyl ring was then undertaken. We report here, the synthesis and antibacterial activity of a series of biaryl isoxazolinone compounds.     

Rat CNS neurons are not yet programmed to shorten their chromatin repeat length at the end of fetal neurogenesis

Neurons from rat fetal cerebral hemispheres were grown in a synthetic medium (Maat medium), as previously described, for different periods of time. The repeat length of their chromatin was determined by micrococcal nuclease digestion and compared with that of neurons isolated from postnatal rat brain of corresponding ages. In contrast to the in vivo situation, we found that neurons, dissociated at the 16th gestational day and cultured in vitro, did not undergo the shortening of their chromatin repeat, thus indicating that, at the end of their mitotic cycles, they are not yet programmed to this event.