A technical review of canonical correlation analysis for neuroscience applications

Collecting comprehensive data sets of the same subject has become a standard in neuroscience research and uncovering multivariate relationships among collected data sets have gained significant attentions in recent years. Canonical correlation analysis (CCA) is one of the powerful multivariate tools to jointly investigate relationships among multiple data sets, which can uncover disease or environmental effects in various modalities simultaneously and characterize changes during development, aging, and disease progressions comprehensively. In the past 10 years, despite an increasing number of studies have utilized CCA in multivariate analysis, simple conventional CCA dominates these applications. Multiple CCA‐variant techniques have been proposed to improve the model performance; however, the complicated multivariate formulations and not well‐known capabilities have delayed their wide applications. Therefore, in this study, a comprehensive review of CCA and its variant techniques is provided. Detailed technical formulation with analytical and numerical solutions, current applications in neuroscience research, and advantages and limitations of each CCA‐related technique are discussed. Finally, a general guideline in how to select the most appropriate CCA‐related technique based on the properties of available data sets and particularly targeted neuroscience questions is provided.     

β-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR

Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly β-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified β-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of β-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a β-helical architecture, in which 18 β-strand segments are arranged in six consecutive windings of a β-helix.Similar to eukaryotes, bacteria use a number of different cytoskeletal elements to ensure the proper temporal and spatial organization of their cellular machinery. Various studies proved the existence of bacterial homologs of eukaryotic cytoskeleton proteins including tubulin homologs such as FtsZ (1), actin homologs such as MreB (2), and intermediate filament (IF)-like proteins (3), which together have important roles in cell division, morphogenesis, polarity determination, and DNA segregation (4–7). In addition, several groups of polymer-forming proteins that are limited to the bacterial domain have been described (6).A recent addition to these bacteria-specific cytoskeletal proteins are the so-called bactofilins (8), a class of proteins that is widespread among most bacterial lineages and involved in a variety of different cellular processes. In the prosthecate α-proteobacterium Caulobacter crescentus, for instance, the two bactofilin paralogues BacA and BacB (Fig. 1A) assemble into membrane-associated polymeric sheets that are specifically localized to the cell pole carrying the stalk (8), a thin protrusion of the cell body involved in cell attachment and nutrient acquisition (9). These assemblies serve as spatial landmarks mediating the polar localization of a cell wall biosynthetic enzyme, PbpC, involved in stalk biogenesis (8) and organization (10). The δ-proteobacterial species Myxococcus xanthus, by contrast, possesses four bactofilin paralogues (BacMNOP) with, at least partly, distinct functions. BacM was shown to form cable- or rod-like structures that are critical for proper cell shape (8, 11). BacP, on the other hand, assembles into short filamentous structures that emanate from the cell poles, recruiting and thus controlling a small GTPase involved in type IV pili-dependent motility (12). As another well-characterized example, the ε-proteobacterium Helicobacter pylori, a human pathogen notorious for causing peptic ulcers, was shown to depend on a bactofilin homolog (CcmA) for maintaining its characteristic helical cell shape, a feature required for cells to efficiently colonize the gastric mucus (13). Moreover, in the γ-proteobacterium Proteus mirabilis, a homolog of the bactofilin CcmA has been implicated in cell shape and swarming motility (14).Open in a separate windowFig. 1.High-resolution ssNMR spectra of BacA filaments. (A) Selected bacterial bactofilins. The total number of amino acids is indicated in parentheses. (B) Amino acid sequence of C. crescentus BacA, together with a synthetic linker peptide (of 17 residues, in red) and a His₆-tag (in blue) attached at the C terminus. The DUF583 domain is highlighted in magenta, and prolines are shown in green. (C) Carbon–carbon 2D correlation spectrum of uniformly [¹³C, ¹⁵N]-labeled BacA. The carbon–carbon magnetization transfer is achieved by PDSD. A short PDSD mixing time of 20 ms was applied, optimal for intraresidue transfer. In the spectrum, a trace through Ile60 is shown to illustrate sensitivity and line width.Bactofilins are usually small proteins (∼20 kDa) that are composed of a central conserved domain of unknown function (DUF583) and flanking N- and C-terminal regions of variable length and sequence. A characteristic of bactofilins is their ability to polymerize spontaneously in the absence of nucleotides or other cofactors (8). Native BacM protofilaments have been isolated from M. xanthus whole-cell lysates by sucrose density centrifugation (11). Moreover, polymers of C. crescentus BacA and BacB were obtained after heterologous expression in Escherichia coli, a species that lacks chromosomally encoded bactofilin homologs (8). Similarly, polymerization was observed for heterologously produced M. xanthus BacN, BacO, and BacP (8, 12). In all cases, the filamentous structures formed were biochemically inert and resistant to nonphysiological salt concentrations and pH values. This behavior is reminiscent of IFs, although there is no evolutionary relationship between these two groups of cytoskeletal elements (6). In particular, bactofilins lack predicted coiled-coil regions, which are a key feature of IF proteins.Up to now, the molecular structure of bactofilins and the mechanism(s) underlying their assembly have remained unknown. This is in large part due to the spontaneous formation, inertness, and insolubility of bactofilin polymers, which makes them difficult substrates for crystallography studies as well as conventional liquid-state NMR methods. We therefore resorted to the use of solid-state NMR (ssNMR) spectroscopy, a technique that has recently been adapted to obtain high-resolution structural information on insoluble and noncrystalline protein assemblies, including functional oligomeric assemblies (15–17), disease-related amyloid fibrils (18–21), and membrane proteins in a lipid bilayer environment (22–26). In this study, we have applied state-of-the-art magic-angle spinning ssNMR spectroscopy in combination with a range of other biophysical methods to filaments of the C. crescentus bactofilin BacA (161 residues). We show that the DUF583 domain serves as a polymerization module that forms the rigid core of BacA filaments, whereas the terminal regions of the protein remain flexible. The core domain folds exclusively into β-sheets, but with an arrangement different from that typically found in amyloids. On the basis of the diffraction pattern of 2D crystalline assemblies observed by transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM) measurements that yield a value for the mass per length (MPL), and the β-strand segments identified by ssNMR, we propose a β-helical arrangement of the BacA subunit. Finally, we performed a mutational analysis of conserved residues in the β-strand segments, which suggests that the folding and/or polymerization of bactofilins are mediated by an extensive and redundant network of hydrophobic interactions. These findings provide for the first time, to our knowledge, insight into the atomic structure of a bacteria-specific cytoskeletal filament and highlight ssNMR as a powerful technique for the analysis of cytoskeletal elements that are unamenable to standard structural biological approaches.     

Systemic Arterial Endothelial Function in Children and Young Adults with Idiopathic Pulmonary Arterial Hypertension: Is there a Relation to Pulmonary Endothelium-Dependent Relaxation?

Pulmonary arterial endothelial function is known to be affected in patients with idiopathic pulmonary arterial hypertension (IPAH). Current reports also detected peripheral systemic arterial dysfunction in IPAH patients. The purpose of this study was to assess whether there is a relation between pulmonary arterial and systemic arterial endothelial function. Pulmonary arterial endothelium-dependent relaxation was assessed by changes in pulmonary blood flow in response to acetylcholine which were determined using intravascular Doppler flow measurements. Pulmonary flow reserve (PFR) was calculated as the ratio of pulmonary blood flow velocity in response to acetylcholine relative to baseline values. Systemic arterial endothelial function was assessed by the vascular response to reactive hyperemia, and was recorded non-invasively by peripheral arterial finger tonometry under standardized conditions. Thirteen children and young adults [mean age 16.7 (±5.6) years] with IPAH and 13 age-/gender-matched controls were included in the study. Digital reactive hyperemic index (RHI) of the IPAH patients was 1.54 (±0.69), and of the controls was 1.67 (±0.66) [p = 0.64]. The mean baseline flow velocity in the segmental pulmonary artery of all patients was 18.5 (±5.5) cm/s, increasing to 27.4 (±12.3) cm/s (p = 0.003) during acetylcholine infusion. The calculated mean PFR was 1.48 (±0.4). There was no significant correlation between the PFR and RHI (r = 0.19; p = 0.54). According to our results, systemic arterial endothelial function assessed by peripheral arterial finger tonometry was not significantly impaired in children and young adults with IPAH compared with age-/gender-matched controls. There was no correlation between systemic arterial and pulmonary arterial endothelial function, suggesting that different mechanisms may contribute to their pathogenesis and progression.