Application of SWATH Mass Spectrometry and Machine Learning in the Diagnosis of Inflammatory Bowel Disease Based on the Stool Proteome.

Inflammatory bowel disease (IBD) flare-ups exhibit symptoms that are similar to other diseases and conditions, making diagnosis and treatment complicated. Currently, the gold standard for diagnosing and monitoring IBD is colonoscopy and biopsy, which are invasive and uncomfortable procedures, and the fecal calprotectin test, which is not sufficiently accurate. Therefore, it is necessary to develop an alternative method. In this study, our aim was to provide proof of concept for the application of Sequential Window Acquisition of All Theoretical Mass Spectra-Mass spectrometry (SWATH-MS) and machine learning to develop a non-invasive and accurate predictive model using the stool proteome to distinguish between active IBD patients and symptomatic non-IBD patients. Proteome profiles of 123 samples were obtained and data processing procedures were optimized to select an appropriate pipeline. The differentially abundant analysis identified 48 proteins. Utilizing correlation-based feature selection (Cfs), 7 proteins were selected for proceeding steps. To identify the most appropriate predictive machine learning model, five of the most popular methods, including support vector machines (SVMs), random forests, logistic regression, naive Bayes, and k-nearest neighbors (KNN), were assessed. The generated model was validated by implementing the algorithm on 45 prospective unseen datasets; the results showed a sensitivity of 96% and a specificity of 76%, indicating its performance. In conclusion, this study illustrates the effectiveness of utilizing the stool proteome obtained through SWATH-MS in accurately diagnosing active IBD via a machine learning model.

Proteomics Profiling of Stool Samples from Preterm Neonates with SWATH/DIA Mass Spectrometry for Predicting Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a life-threatening condition for premature infants in neonatal intensive care units. Finding indicators that can predict NEC development before symptoms appear would provide more time to apply targeted interventions. In this study, stools from 132 very-low-birth-weight (VLBW) infants were collected daily in the context of a multi-center prospective study aimed at investigating the potential of fecal biomarkers for NEC prediction using proteomics technology. Eight of the VLBW infants received a stage-3 NEC diagnosis. Stools collected from the NEC infants up to 10 days before their diagnosis were available for seven of them. Their samples were matched with those from seven pairs of non-NEC controls. The samples were processed for liquid chromatography-tandem mass spectrometry analysis using SWATH/DIA acquisition and cross-compatible proteomic software to perform label-free quantification. ROC curve and principal component analyses were used to explore discriminating information and to evaluate candidate protein markers. A series of 36 proteins showed the most efficient capacity with a signature that predicted all seven NEC infants at least a week in advance. Overall, our study demonstrates that multiplexed proteomic signature detection constitutes a promising approach for the early detection of NEC development in premature infants.

Active Yeast Telomerase Shares Subunits with Ribonucleoproteins RNase P and RNase MRP.

Telomerase is the ribonucleoprotein enzyme that replenishes telomeric DNA and maintains genome integrity. Minimally, telomerase activity requires a templating RNA and a catalytic protein. Additional proteins are required for activity on telomeres in vivo. Here, we report that the Pop1, Pop6, and Pop7 proteins, known components of RNase P and RNase MRP, bind to yeast telomerase RNA and are essential constituents of the telomerase holoenzyme. Pop1/Pop6/Pop7 binding is specific and involves an RNA domain highly similar to a protein-binding domain in the RNAs of RNase P/MRP. The results also show that Pop1/Pop6/Pop7 function to maintain the essential components Est1 and Est2 on the RNA in vivo. Consistently, addition of Pop1 allows for telomerase activity reconstitution with wild-type telomerase RNA in vitro. Thus, the same chaperoning module has allowed the evolution of functionally and, remarkably, structurally distinct RNPs, telomerase, and RNases P/MRP from unrelated progenitor RNAs.

Exposing secrets of telomere-telomerase encounters.

In order for telomeres to remain functional and stable, they must rendezvous with the enzyme telomerase in a productive manner. In human cells, this interaction is mediated by Cajal bodies as matchmaker, and now Zhong et al. reveal molecular determinants that establish good chemistry between the two partners.

Budding yeast telomerase RNA transcription termination is dictated by the Nrd1/Nab3 non-coding RNA termination pathway.

The RNA component of budding yeast telomerase (Tlc1) occurs in two forms, a non-polyadenylated form found in functional telomerase and a rare polyadenylated version with unknown function. Previous work suggested that the functional Tlc1 polyA- RNA is processed from the polyA+ form, but the mechanisms regulating its transcription termination and 3'-end formation remained unclear. Here we examined transcription termination of Tlc1 RNA in the sequences 3' of the TLC1 gene and relate it to telomere maintenance. Strikingly, disruption of all probable or cryptic polyadenylation signals near the 3'-end blocked the accumulation of the previously reported polyA+ RNA without affecting the level, function or specific 3' nucleotide of the mature polyA- form. A genetic approach analysing TLC1 3'-end sequences revealed that transcription terminates upstream of the polyadenylation sites. Furthermore, the results also demonstrate that the function of this Tlc1 terminator depends on the Nrd1/Nab3 transcription termination pathway. The data thus show that transcription termination of the budding yeast telomerase RNA occurs as that of snRNAs and Tlc1 functions in telomere maintenance are not strictly dependent on a polyadenylated precursor, even if the polyA+ form can serve as intermediate in a redundant termination/maturation pathway.

Abrupt telomere losses and reduced end-resection can explain accelerated senescence of Smc5/6 mutants lacking telomerase.

The highly conserved Structural Maintenance of Chromosome (SMC) proteins are crucial for the formation of three essential complexes involved in high fidelity chromosome transmission during cell division. Recently, the Smc5/6 complex has been reported to be important for telomere maintenance in yeast and also in cancerous human ALT cells, where it could function in a homologous recombination-based (HR) telomere maintenance pathway. Here, we investigate the possible roles of the budding yeast Smc5/6 complex in maintaining appropriate chromosome end-structures allowing cell survival in absence of telomerase. The results show that cells harbouring mutant alleles of genes encoding Smc5/6-complex proteins rapidly stop growing after telomerase loss. Furthermore, this telomerase-induced growth arrest is much more pronounced as compared to cultures with a functional Smc5/6-complex. Bulk telomere sequence loss is not increased in the mutant cells and the evidence suggests that Smc5/6 slows senescence through a partially HR-independent pathway. We propose that in yeast, the Smc5/6-complex is required for efficient and timely termination of DNA replication and repair at telomeres to avoid stochastic telomere loss during cell division. Consistent with this hypothesis, sequencing of telomeres from telomerase-positive smc5/6 mutant cells revealed a higher frequency of telomere breakage events. Finally, the results also show that on dysfunctional telomeres, the generation of 3'-single stranded DNA is impaired, suggesting that the complex may also participate in the formation of single-stranded overhangs which are thought to be the substrates for telomere repeat replenishment in the absence of telomerase.

RTAnalyzer: a web application for finding new retrotransposons and detecting L1 retrotransposition signatures.

Mobile genetic elements have significantly contributed to the shaping of mammalian genomes. The RTAnalyzer software tracks sequences of retrotransposed origin by scoring the signature results from an L1-mediated insertion within a genome. More specifically, a sequence of interest is searched for in genomic databases using BLAST. Each hit, along with additional 5' and 3' sequences of pre-defined lengths, is saved. RTAnalyzer searches for specific L1 retrotransposition signatures (i.e. target site duplication, endonuclease cleavage site and poly(A)), and then calculates an overall retrotransposition score. This score represents the likelihood of a given sequence originating from a retrotransposition event involving the L1 machinery. RTAnalyzer may be used under GNU public license, and is available at http://www.riboclub.org/rtanalyzer.

Retropseudogenes derived from the human Ro/SS-A autoantigen-associated hY RNAs.

We report the characterization in the human genome of 966 pseudogenes derived from the four human Y (hY) RNAs, components of the Ro/SS-A autoantigen. About 95% of the Y RNA pseudogenes are found in corresponding locations on the chimpanzee and human chromosomes. On the contrary, Y pseudogenes in mice are both infrequent and found in different genomic regions. In addition to this rodent/primate discrepancy, the conservation of hY pseudogenes relative to hY genes suggests that they occurred after rodent/primate divergence. Flanking regions of hY pseudogenes contain convincing evidence for involvement of the L1 retrotransposition machinery. Although Alu elements are found in close proximity to most hY pseudogenes, these are not chimeric retrogenes. Point mutations in hY RNA transcripts specifically affecting binding of Ro60 protein likely contributed to their selection for direct trans retrotransposition. This represents a novel requirement for the selection of specific RNAs for their genomic integration by the L1 retrotransposition machinery. Over 40% of the hY pseudogenes are found in intronic regions of protein-coding genes. Considering the functions of proteins known to bind subsets of hY RNAs, hY pseudogenes constitute a new class of L1-dependent non-autonomous retroelements, potentially involved in post-transcriptional regulation of gene expression.

Amino acid and divalent ion permeability of the pores formed by the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac in insect midgut brush border membrane vesicles.

The pores formed by Bacillus thuringiensis insecticidal toxins have been shown to allow the diffusion of a variety of monovalent cations and anions and neutral solutes. To further characterize their ion selectivity, membrane permeability induced by Cry1Aa and Cry1Ac to amino acids (Asp, Glu, Ser, Leu, His, Lys and Arg) and to divalent cations (Mg(2+), Ca(2+) and Ba(2+)) and anions (SO(4)(2-) and phosphate) was analyzed at pH 7.5 and 10.5 with midgut brush border membrane vesicles isolated from Manduca sexta and an osmotic swelling assay. Shifting pH from 7.5 to 10.5 increases the proportion of the more negatively charged species of amino acids and phosphate ions. All amino acids diffused well across the toxin-induced pores, but, except for aspartate and glutamate, amino acid permeability was lower at the higher pH. In the presence of either toxin, membrane permeability was higher for the chloride salts of divalent cations than for the potassium salts of divalent anions. These results clearly indicate that the pores are cation-selective.