A transformer architecture for retention time prediction in liquid chromatography mass spectrometry-based proteomics

Accurate retention time (RT) prediction is important for spectral library-based analysis in data-independent acquisition mass spectrometry-based proteomics. The deep learning approach has demonstrated superior performance over traditional machine learning methods for this purpose. The transformer architecture is a recent development in deep learning that delivers state-of-the-art performance in many fields such as natural language processing, computer vision, and biology. We assess the performance of the transformer architecture for RT prediction using datasets from five deep learning models Prosit, DeepDIA, AutoRT, DeepPhospho, and AlphaPeptDeep. The experimental results on holdout datasets and independent datasets exhibit state-of-the-art performance of the transformer architecture. The software and evaluation datasets are publicly available for future development in the field.     

Application of an Escherichia coli triple reporter strain for at-line monitoring of single-cell physiology during L-phenylalanine production

Biotechnological production processes are sustainable approaches for the production of biobased components such as amino acids for food and feed industry. Scale-up from ideal lab-scale bioreactors to large-scale processes is often accompanied by loss in productivity. This may be related to population heterogeneities of cells originating from isogenic cultures that arise due to dynamic non-ideal conditions in the bioreactor. To better understand this phenomenon, deeper insights into single-cell physiologies in bioprocesses are mandatory before scale-up. Here, a triple reporter strain (3RP) was developed by chromosomally integrating the fluorescent proteins mEmerald, CyOFP1, and mTagBFP2 into the L-phenylalanine producing Escherichia coli strain FUS4 (pF81_kan) to allow monitoring of growth, oxygen availability, and general stress response of the single cells. Functionality of the 3RP was confirmed in well-mixed lab-scale fed-batch processes with glycerol as carbon source in comparison to the strain without fluorescent proteins, leading to no difference in process performance. Fluorescence levels could successfully reflect the course of related process state variables, revealed population heterogeneities during the transition between different process phases and potentially subpopulations that exhibit superior process performance. Furthermore, indications were found for noise in gene expression as regulation strategy against environmental perturbation.     

Crystal structure and conformational analysis of ampullosporin A.

Ampullosporin A is a 15-mer peptaibol type polypeptide that induces pigment formation by the fungus Phoma destructiva, forms voltage-dependent ion channels in membranes and exhibits hypothermic effects in mice. The structure of ampullosporin A has been determined by x-ray crystallography. This is the first three-dimensional (3D) structure of the peptaibol subfamily SF6. From the N-terminus to residue 13 the molecule adopts an approximate right-handed alpha-helical geometry, whereas a less regular structure pattern with beta-turn characteristics is found in the C-terminus. Even though ampullosporin A does not contain a single proline or hydroxyproline it is significantly bent. It belongs to both the shortest and the most strongly bent peptaibol 3D structures. The straight structure part encompasses residues Ac-Trp(1)-Aib(10) and is thus less extended than the alpha-helical subunit. The 3D structure of ampullosporin A is discussed in relation to other experimentally determined peptaibol structures and in the context of its channel-forming properties. As a part of this comparison a novel bending analysis based on a 3D curvilinear axis describing the global structural characteristics has been proposed and applied to all 3D peptaibol structures. A sampling of 2500 conformations using different molecular dynamics protocols yields, for the complete ampullosporin A structure, an alpha-helix as the preferred conformation in vacuo with almost no bend. This indicates that solvent or crystal effects may be important for the experimentally observed peptide backbone bending characteristics of ampullosporin A.     

Tagging and mapping the thermo-sensitive genic male-sterile gene in rice (Oryza sativa L.) with molecular markers

The thermo-sensititve genic male-sterile (TGMS) gene in rice can alter fertility in response to temperature and is useful in the two-line system of hybrid rice production. However, little is known about the TGMS gene at the molecular level. The objective of this study was to identify molecular markers tightly linked with the TGMS gene and to map the gene onto a specific rice chromosome. Bulked segregant analysis of an F₂ population from 5460s (a TGMS mutant line) x Hong Wan 52 was used to identify RAPD markers linked to the rice TGMS gene. Four hundred RAPD primers were screened for polymorphisms between the parents and between two bulks representing fertile and sterile plants; of these, 4 primers produced polymorphic products. Most of the polymorphic fragments contained repetitive sequences. Only one singlecopy sequence fragment was found, a 1.2-kb fragment amplified by primer OPB-19 and subsequently named TGMS1.2. TGMS1.2 was mapped on chromosome 8 with a RIL population and confirmed by remapping with a DHL population. Segregation analysis using TGMS1.2 as a probe indicated that TGMS1.2 both consegregated and was lined with the TGMS gene in this population. It is located about 6.7 cM from the TGMS gene. As TGMS1.2 is linked to the TGMS gene, the TGMS gene must be located on chromosome 8.This research was supported by the Rockefeller Foundation and China National High-Tech Research and Development Program. The first author is a Rockefeller Career Fellow at Texas Tech University     

Production of isoleucine by overexpression of ilvA in a Corynebacterium lactofermentum threonine producer

Overproduction of isoleucine, an essential amino acid, was achieved by amplification of the gene encoding threonine dehydratase, the first enzyme in the threonine to isoleucine pathway, in a Corynebacterium lactofermentum threonine producer. Threonine overproduction was previously achieved with C. lactofermentum ATCC 21799, a lysine-hyperproducing strain, by introduction of plasmid pGC42 containing the Corynebacterium hom ^dr and thrB genes (encoding homoserine dehydrogenase and homoserine kinase respectively) under separate promoters. The pGC42 derivative, pGC77, also contains ilvA, which encodes threonine dehydratase. In a shake-flask fermentation, strain 21799(pGC77) produced 15 g/l isoleucine, along with small amounts of lysine and glycine. A molar carbon balance indicates that most of the carbon previously converted to threonine, lysine, glycine and isoleucine was incorporated into isoleucine by the new strain. Thus, in our system, simple overexpression of wild-type ilvA sufficed to overcome the effects of feedback inhibition of threonine dehydratase by the end-product, isoleucine.     

Morphometrics of Infective Juveniles of Steinernema spp. and Heterorhabditis bacteriophora (Nemata: Rhabditida)

Selected morphometrics of Heterorhabditis bacteriophora and seven species of Steinernema from in vivo culture were compared in relation to time of harvest. In addition, five Steinernema species were reared in vitro and their morphometrics were compared with those from in vivo culture. With in vivo culture, there was generally a negative linear relationship between body length of infective juveniles (IJ) and time of harvest. The distance from the anterior end to the excretory pore (EP) and the tail length (T) of IJ also varied with time of harvest. The E percentage (= EP/T x 100) was the least variable. Body lengths of IJ reared in vitro were much less than those of IJ reared in vivo. The study suggests that IJ harvested from in vivo culture within 1 week of emergence from cadavers are best for species identification. Infective juveniles from in vitro culture should not be used for species identification.     

Regulation,purification and partial characterization of glutamine synthetase fromStreptomyces aureofaciens

The activity of glutamine synthetase (GS) fromStreptomyces aureofaciens was regulated by the availability of the nitrogen source. Rich nitrogen sources repressed GS synthesis and increased GS adenylylation. The enzyme was purified 270-fold to virtual homogeneity with 37% recovery. The molar mass of the native enzyme and its subunits was determined to be 620 and 55 kDa, respectively, indicating that GS is composed of 12 identical subunits. The enzyme has a hexagonal-bilayered structure as observed by electron microscopy. The isoelectric point of the purified GS was at pH 4.2. The enzyme was stable for 1 h at 50°C but lost activity rapidly when incubated at 65 and 70°C. Mg²⁺ supported relative synthetic activity of 100 and 72%, respectively, with the corresponding pH optima of 7.3 and 7.0. Mn²⁺ ions activated transferase activity at a pH optimum of 7.0. The temperature optimum for all GS activities was 50°C. Intermediates of the citric acid cycle exerted insignificant effects on the synthetic activities. There was no SH-group essential for the GS activity.     

Caenorhabditis Elegans Mutants Resistant to Inhibitors of Acetylcholinesterase

We characterized 18 genes from Caenorhabditis elegans that, when mutated, confer recessive resistance to inhibitors of acetylcholinesterase. These include previously described genes as well as newly identified genes; they encode essential as well as nonessential functions. In the absence of acetylcholinesterase inhibitors, the different mutants display a wide range of behavioral deficits, from mild uncoordination to almost complete paralysis. Measurements of acetylcholine levels in these mutants suggest that some of the genes are involved in presynaptic functions.     

Identification of Trans-Acting Genes Necessary for Centromere Function in Drosophila Melanogaster Using Centromere-Defective Minichromosomes

Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere, but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centromere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function.