Screening HCV genotype-specific epitope peptides based on conserved sequence analysis and B cell epitope prediction in HCV E2 region

The high mutation rate of the hepatitis C virus (HCV) genome increases the genotype diversity and renders the detection of the virus more difficult. Therefore, prediction and assessment of highly conserved and strongly antigenic epitope polypeptide sequences have become a focus of current research. The E2 region is the target binding region of neutralizing antibodies. HCV genomics, especially the high mutation rate of E2 region sequence, makes its genotyping more and more diverse, and the detection of HCV and genotype is becoming more and more strict. In this study, four HCV B cell epitope polypeptides were constructed based on assessment of conserved sequences in the HCV E2 region and prediction of B cell epitopes, including sequences specific to genotype 1A (DC-13: 434-DTGWLAGLFYYHK-446), genotype 1B (HC-13: 434-HTGFLAALFYAKS-446), genotype 4D (NC-13: 434-NTGFLASLFYTHK-446), and a consensus sequence (FC-9: 447-FNSSGCPER-455). Epitope polypeptides combined with serum from 29 HCV-infected or 25 non-HCV-infected individuals were assayed by enzyme-linked immunosorbent assay (ELISA), and differences were analyzed by T/T’ test methods in SPSS v20.0 software. Binding levels of genotype 1A, 4D, and consensus epitope polypeptides with sera of HCV-infected patients were higher than those of non-infected individuals. Moreover, binding of genotype 1B epitope polypeptides with serum of HCV 1B-infected patients was higher than that of HCV 2A-infected patients. While the screening results of HCV genotype-specific epitope polypeptides were preliminary, these findings indicated that we successfully established an HCV and genotype serological ELISA detection method. Such an approach would facilitate the discovery of epitope polypeptides which may become new antigen candidates in peptide vaccine development for the prevention of HCV infection.     

Compensatory force measurement and multimodal force feedback for remote-controlled vascular interventional robot

Minimally invasive vascular interventional surgery is widely used and remote-controlled vascular interventional surgery robots (RVIRs) are being developed to reduce the occupational risk of the intervening physician in minimally invasive vascular interventional surgeries. Skilled surgeon performs surgeries mainly depending on the detection of collisions. Inaccurate force feedback will be difficult for surgeons to perform surgeries or even results in medical accidents. In addition, the surgeon cannot quickly and easily distinguish whether the proximal force exceeds the safety threshold of blood vessels or not, and thus it results in damage to the blood vessels. In this paper, we present a novel method comprising compensatory force measurement and multimodal force feedback (MFF). Calibration experiments and performance evaluation experiments were carried out. Experimental results demonstrated that the proposed method can measure the proximal force of catheter/guidewire accurately and assist surgeons to distinguish the change of proximal force more easily. This novel method is suitable for use in actual surgical operations.     

Pathogenic gene screening in 91 Chinese patients with short stature of unknown etiology with a targeted next-generation sequencing panel

Background

Dwarfism is a common severe growth disorder, but the etiology is unclear in the majority of cases. Recombinant human growth hormone may be a treatment option, but it has limited efficacy. The currently known laboratory assays do not meet the precision requirements for clinical diagnosis. Here, we have constructed a targeted next-generation sequencing (NGS) panel of selected genes that are suspected to be associated with dwarfism for genetic screening.

Methods

Genetic screening of 91 children with short stature of unknown etiology was performed with the help of the NGS panel. All the coding regions and exon-intron boundaries of 166 genes were included in the panel. To clarify the pathogenicity of these mutations, their clinical data were reviewed and analyzed.

Results

The assay identified p.A72G, p.I282V, and p.P491S variants of the PTPN11 gene and a p.I437T variant of the SOS1 gene in 4 cases with Noonan syndrome. A frameshift mutation (p.D2407fs) of the ACAN gene was identified in a case of idiopathic short stature with moderately advanced bone age. A p.R904C variant of the COL2A1 gene was found in a patient, who was accordingly diagnosed with Stickler syndrome. Severe short stature without limb deformity was associated with a p.G11A variant of HOXD13. In addition, we evaluated evidence that a p.D401N variant of the COMP gene may cause multiple epiphyseal dysplasia.

Conclusions

Our findings suggest that syndromes, particularly Noonan syndrome, may be overlooked due to atypical clinical features. This gene panel has been verified to be effective for the rapid screening of genetic etiologies associated with short stature and for guiding precision medicine-based clinical management.

     

Lentiviral Vectors and Adeno‐Associated Virus Vectors: Useful Tools for Gene Transfer in Pain Research

Pain, especially chronic pain, has always been a heated point in both basic and clinical researches since it puts heavy burdens on both individuals and the whole society. A better understanding of the role of biological molecules and various ionic channels involved in pain can shed light on the mechanism under pain and advocate the development of pain management. Using viral vectors to transfer specific genes at targeted sites is a promising method for both research and clinical applications. Lentiviral vectors and adeno‐associated virus (AAV) vectors which allow stable and long‐term expression of transgene in non‐dividing cells are widely applied in pain research. In this review, we thoroughly outline the structure, category, advantages and disadvantages and the delivery methods of lentiviral and AAV vectors. The methods through which lentiviral and AAV vectors are delivered to targeted sites are closely related with the sites, level and period of transgene expression. Focus is placed on the various delivery methods applied to deliver vectors to spinal cord and dorsal root ganglion both of which play important roles in primary nociception. Our goal is to provide insight into the features of these two viral vectors and which administration approach can be chosen for different pain researches. Anat Rec, 301:825–836, 2018. © 2017 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

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.