The bimolecular hydrogen-deuterium exchange behavior of protonated alkyl dipeptides in the gas phase

As part of an ongoing characterization of the intrinsic chemical properties of peptides, thermal hydrogen-deuterium exchange has been studied for a series of fast-atom-bombardment-generated protonated alkyldipeptides and related model compounds in the reaction with D2O, CH3OD, and ND3 in a Fourier transform ion cyclotron resonance mass spectrometer. Despite the very large basicity difference between the dipeptides and the D2O and CH3OD exchange reagents, efficient exchange of all active hydrogen atoms occurs. From the kinetic data it appears that exchange of the amino, amide, and hydroxyl hydrogens proceeds with different efficiencies, which implies that the proton in thermal protonated dipeptides is immobile. The selectivity of the exchange at the different basic sites is governed by the nature of both the dipeptide and the exchange reagent. The results indicate that reversible proton transfer in the reaction complexes, which effectuates the deuterium incorporation, is assisted by formation of multiple hydrogen bonds between the reagents. Exchange is considered to proceed via the intermediacy of different competing intermediate complexes, each of which specifically leads to deuterium incorporation at different basic sites. The relative stabilization of the competing intermediate complexes can be related to the relative efficiencies of deuterium incorporation at different basic sites in the dipeptide. For all protonated dipeptides studied, the exchange in the reaction with ND3 proceeds with unit efficiency, whereas all active hydrogen atoms are exchanged equally efficiently. Evidently specific multiple hydrogen bond formations are far less important in the reversible proton transfers with the relatively basic ammonia, which allows effective randomization of all active hydrogen atoms in the reaction complexes

Disulfide bond structure and domain organization of yeast beta(1,3)-glucanosyltransferases involved in cell wall biogenesis

The Gel/Gas/Phr family of fungal \u3b2(1,3)-glucanosyltransferases plays an important role in cell wall biogenesis by processing the main component \u3b2(1,3)-glucan. Two subfamilies are distinguished depending on the presence or absence of a C-terminal cysteine-rich domain, denoted "Cys-box." The N-terminal domain (NtD) contains the catalytic residues for transglycosidase activity and is separated from the Cys-box by a linker region. To obtain a better understanding of the structure and function of the Cys-box-containing subfamily, we identified the disulfide bonds in Gas2p from Saccharomyces cerevisiae by an improved mass spectrometric methodology. We mapped two separate intra-domain clusters of three and four disulfide bridges. One of the bonds in the first cluster connects a central Cys residue of the NtD with a single conserved Cys residue in the linker. Site-directed mutagenesis of the Cys residue in the linker resulted in an endoplasmic reticulum precursor that was not matured and underwent a gradual degradation. The relevant disulfide bond has a crucial role in folding as it may stabilize the NtD and facilitate its interaction with the C-terminal portion of a Gas protein. The four disulfide bonds in the Cys-box are arranged in a manner consistent with a partial structural resemblance with the plant X8 domain, an independent carbohydrate-binding module that possesses only three disulfide bonds. Deletion of the Cys-box in Gas2 or Gas1 proteins led to the formation of an NtD devoid of any enzymatic activity. The results suggest that the Cys-box is required for proper folding of the NtD and/or substrate binding

From Predicting Solar Activity to Forecasting Space Weather: Practical Examples of Research-to-Operations and Operations-to-Research

The successful transition of research to operations (R2O) and operations to research (O2R) requires, above all, interaction between the two communities. We explore the role that close interaction and ongoing communication played in the successful fielding of three separate developments: an observation platform, a numerical model, and a visualization and specification tool. Additionally, we will examine how these three pieces came together to revolutionize interplanetary coronal mass ejection (ICME) arrival forecasts. A discussion of the importance of education and training in ensuring a positive outcome from R2O activity follows. We describe efforts by the meteorological community to make research results more accessible to forecasters and the applicability of these efforts to the transfer of space-weather research.We end with a forecaster "wish list" for R2O transitions. Ongoing, two-way communication between the research and operations communities is the thread connecting it all.Comment: 18 pages, 3 figures, Solar Physics in pres

Comparison of illumina versus nanopore 16s rRNA gene sequencing of the human nasal microbiota

Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME (Oxford Nanopore Technologies—ONT, Oxford, UK) and an in-house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 (Oxford Nanopore Technologies—ONT, Oxford, UK) and R9.2 (Oxford Nanopore Technologies—ONT, Oxford, UK) were compared. In conclusion, the current study shows that the nanopore sequencing platform is comparable with the Illumina platform in detection bacterial genera of the nasal microbiota, but the nanopore platform does have problems in detecting bacteria within the genus Corynebacterium. Although advances are being made, thorough validation of the nanopore platform is still recommendable

Heterozygous missense variants of LMX1A lead to nonsyndromic hearing impairment and vestibular dysfunction

Unraveling the causes and pathomechanisms of progressive disorders is essential for the development of therapeutic strategies. Here, we identified heterozygous pathogenic missense variants of LMX1A in two families of Dutch origin with progressive nonsyndromic hearing impairment (HI), using whole exome sequencing. One variant, c.721G > C (p.Val241Leu), occurred de novo and is predicted to affect the homeodomain of LMX1A, which is essential for DNA binding. The second variant, c.290G > C (p.Cys97Ser), predicted to affect a zinc-binding residue of the second LIM domain that is involved in protein–protein interactions. Bi-allelic deleterious variants of Lmx1a are associated with a complex phenotype in mice, including deafness and vestibular defects, due to arrest of inner ear development. Although Lmx1a mouse mutants demonstrate neurological, skeletal, pigmentation and reproductive system abnormalities, no syndromic features were present in the participating subjects of either family. LMX1A has previously been suggested as a candidate gene for intellectual disability, but our data do not support this, as affected subjects displayed normal cognition. Large variability was observed in the age of onset (a)symmetry, severity and progression rate of HI. About half of the affected individuals displayed vestibular dysfunction and experienced symptoms thereof. The late-onset progressive phenotype and the absence of cochleovestibular malformations on computed tomography scans indicate that heterozygous defects of LMX1A do not result in severe developmental abnormalities in humans. We propose that a single LMX1A wild-type copy is sufficient for normal development but insufficient for maintenance of cochleovestibular function. Alternatively, minor cochleovestibular developmental abnormalities could eventually lead to the progressive phenotype seen in the families