External Stresses Affect Gonococcal Type 4 Pilus Dynamics

Bacterial type 4 pili (T4P) are extracellular polymers that serve both as adhesins and molecular motors. Functionally, they are involved in adhesion, colony formation, twitching motility, and horizontal gene transfer. T4P of the human pathogen Neisseria gonorrhoeae have been shown to enhance survivability under treatment with antibiotics or hydrogen peroxide. However, little is known about the effect of external stresses on T4P production and motor properties. Here, we address this question by directly visualizing gonococcal T4P dynamics. We show that in the absence of stress gonococci produce T4P at a remarkably high rate of similar to 200 T4P min(-1). T4P retraction succeeds elongation without detectable time delay. Treatment with azithromycin or ceftriaxone reduces the T4P production rate. RNA sequencing results suggest that reduced piliation is caused by combined downregulation of the complexes required for T4P extrusion from the cell envelope and cellular energy depletion. Various other stresses including inhibitors of cell wall synthesis and DNA replication, as well as hydrogen peroxide and lactic acid, inhibit T4P production. Moreover, hydrogen peroxide and acidic pH strongly affect pilus length and motor function. In summary, we show that gonococcal T4P are highly dynamic and diverse external stresses reduce piliation despite the protective effect of T4P against some of these stresses

Global, in situ analysis of the structural proteome in individuals with Parkinson's disease to identify a new class of biomarker

Parkinson’s disease (PD) is a prevalent neurodegenerative disease for which robust biomarkers are needed. Because protein structure reflects function, we tested whether global, in situ analysis of protein structural changes provides insight into PD pathophysiology and could inform a new concept of structural disease biomarkers. Using limited proteolysis–mass spectrometry (LiP–MS), we identified 76 structurally altered proteins in cerebrospinal fluid (CSF) of individuals with PD relative to healthy donors. These proteins were enriched in processes misregulated in PD, and some proteins also showed structural changes in PD brain samples. CSF protein structural information outperformed abundance information in discriminating between healthy participants and those with PD and improved the discriminatory performance of CSF measures of the hallmark PD protein α-synuclein. We also present the first analysis of inter-individual variability of a structural proteome in healthy individuals, identifying biophysical features of variable protein regions. Although independent validation is needed, our data suggest that global analyses of the human structural proteome will guide the development of novel structural biomarkers of disease and enable hypothesis generation about underlying disease processes

Global, in situ analysis of the structural proteome in individuals with Parkinson's disease to identify a new class of biomarker

Parkinson's disease (PD) is a prevalent neurodegenerative disease for which robust biomarkers are needed. Because protein structure reflects function, we tested whether global, in situ analysis of protein structural changes provides insight into PD pathophysiology and could inform a new concept of structural disease biomarkers. Using limited proteolysis-mass spectrometry (LiP-MS), we identified 76 structurally altered proteins in cerebrospinal fluid (CSF) of individuals with PD relative to healthy donors. These proteins were enriched in processes misregulated in PD, and some proteins also showed structural changes in PD brain samples. CSF protein structural information outperformed abundance information in discriminating between healthy participants and those with PD and improved the discriminatory performance of CSF measures of the hallmark PD protein alpha-synuclein. We also present the first analysis of inter-individual variability of a structural proteome in healthy individuals, identifying biophysical features of variable protein regions. Although independent validation is needed, our data suggest that global analyses of the human structural proteome will guide the development of novel structural biomarkers of disease and enable hypothesis generation about underlying disease processes.ISSN:1545-9993ISSN:1545-998

Genetic effects on molecular network states explain complex traits

The complexity of many cellular and organismal traits results from the integration of genetic and environmental factors via molecular networks. Network structure and effect propagation are best understood at the level of functional modules, but so far, no concept has been established to include the global network state. Here, we show when and how genetic perturbations lead to molecular changes that are confined to small parts of a network versus when they lead to modulation of network states. Integrating multi-omics profiling of genetically heterogeneous budding and fission yeast strains with an array of cellular traits identified a central state transition of the yeast molecular network that is related to PKA and TOR (PT) signaling. Genetic variants affecting this PT state globally shifted the molecular network along a single-dimensional axis, thereby modulating processes including energy and amino acid metabolism, transcription, translation, cell cycle control, and cellular stress response. We propose that genetic effects can propagate through large parts of molecular networks because of the functional requirement to centrally coordinate the activity of fundamental cellular processes.ISSN:1744-429

Plane-by-plane inscription of grating structures in optical fibers

Plane-by-plane fabrication of fiber Bragg gratings in optical fibers using short-pulse femtosecond IR laser is proposed and demonstrated. By incorporating a cylindrical lens in the fabrication setup, a plane of index modification can be directly inscribed in fiber core by a single laser pulse through the proposed method. This plane-by-plane method simplifies the grating inscription process and allows for the fabrication of complicated grating structures.Peer reviewed: YesNRC publication: Ye

The impact of genomic variation on protein phosphorylation states and regulatory networks

Genomic variation impacts on cellular networks by affecting the abundance (e.g., protein levels) and the functional states (e.g., protein phosphorylation) of their components. Previous work has focused on the former, while in this context, the functional states of proteins have largely remained neglected. Here, we generated high-quality transcriptome, proteome, and phosphoproteome data for a panel of 112 genomically well-defined yeast strains. Genetic effects on transcripts were generally transmitted to the protein layer, but specific gene groups, such as ribosomal proteins, showed diverging effects on protein levels compared with RNA levels. Phosphorylation states proved crucial to unravel genetic effects on signaling networks. Correspondingly, genetic variants that cause phosphorylation changes were mostly different from those causing abundance changes in the respective proteins. Underscoring their relevance for cell physiology, phosphorylation traits were more strongly correlated with cell physiological traits such as chemical compound resistance or cell morphology, compared with transcript or protein abundance. This study demonstrates how molecular networks mediate the effects of genomic variants to cellular traits and highlights the particular importance of protein phosphorylation.ISSN:1744-429

Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ

Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology

Effective high-throughput RT-qPCR screening for SARS-CoV-2 infections in children

Systematic SARS-CoV-2 testing is a valuable tool for infection control and surveillance. However, broad application of high sensitive RT-qPCR testing in children is often hampered due to unpleasant sample collection, limited RT-qPCR capacities and high costs. Here, we developed a high-throughput approach ('Lolli-Method') for SARS-CoV-2 detection in children, combining non-invasive sample collection with an RT-qPCR-pool testing strategy. SARS-CoV-2 infections were diagnosed with sensitivities of 100% and 93.9% when viral loads were >10(6) copies/ml and >10(3) copies/ml in corresponding Naso-/Oropharyngeal-swabs, respectively. For effective application of the Lolli-Method in schools and daycare facilities, SEIR-modeling indicated a preferred frequency of two tests per week. The developed test strategy was implemented in 3,700 schools and 698 daycare facilities in Germany, screening over 800,000 individuals twice per week. In a period of 3 months, 6,364 pool-RT-qPCRs tested positive (0.64%), ranging from 0.05% to 2.61% per week. Notably, infections correlated with local SARS-CoV-2 incidences and with a school social deprivation index. Moreover, in comparison with the alpha variant, statistical modeling revealed a 36.8% increase for multiple (>= 2 children) infections per class following infections with the delta variant. We conclude that the Lolli-Method is a powerful tool for SARS-CoV-2 surveillance and can support infection control in schools and daycare facilities. Dewald et al. combine a non-invasive sampling approach (Lolli-Test) with an RT qPCR-pool testing strategy to screen for SARS-CoV-2 infections in children and use the method for surveillance and infection control in > 4000 school and daycare settings