DRGKB: a knowledgebase of worldwide diagnosis-related groups' practices for comparison, evaluation and knowledge-guided application.

As a prospective payment method, diagnosis-related groups (DRGs)'s implementation has varying effects on different regions and adopt different case classification systems. Our goal is to build a structured public online knowledgebase describing the worldwide practice of DRGs, which includes systematic indicators for DRGs' performance assessment. Therefore, we manually collected the qualified literature from PUBMED and constructed DRGKB website. We divided the evaluation indicators into four categories, including (i) medical service quality; (ii) medical service efficiency; (iii) profitability and sustainability; (iv) case grouping ability. Then we carried out descriptive analysis and comprehensive scoring on outcome measurements performance, improvement strategy and specialty performance. At last, the DRGKB finally contains 297 entries. It was found that DRGs generally have a considerable impact on hospital operations, including average length of stay, medical quality and use of medical resources. At the same time, the current DRGs also have many deficiencies, including insufficient reimbursement rates and the ability to classify complex cases. We analyzed these underperforming parts by domain. In conclusion, this research innovatively constructed a knowledgebase to quantify the practice effects of DRGs, analyzed and visualized the development trends and area performance from a comprehensive perspective. This study provides a data-driven research paradigm for following DRGs-related work along with a proposed DRGs evolution model. Availability and implementation: DRGKB is freely available at http://www.sysbio.org.cn/drgkb/. Database URL: http://www.sysbio.org.cn/drgkb/.

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome.

Dedicated assembly factors orchestrate the stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here we report cryo-electron microscopy reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, as well as how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors and reveals conceptual principles underlying human proteasome biogenesis, thus providing an explanation for many previous biochemical and genetic observations.

Noncanonical assembly, neddylation and chimeric cullin-RING/RBR ubiquitylation by the 1.8 MDa CUL9 E3 ligase complex.

Ubiquitin ligation is typically executed by hallmark E3 catalytic domains. Two such domains, 'cullin-RING' and 'RBR', are individually found in several hundred human E3 ligases, and collaborate with E2 enzymes to catalyze ubiquitylation. However, the vertebrate-specific CUL9 complex with RBX1 (also called ROC1), of interest due to its tumor suppressive interaction with TP53, uniquely encompasses both cullin-RING and RBR domains. Here, cryo-EM, biochemistry and cellular assays elucidate a 1.8-MDa hexameric human CUL9-RBX1 assembly. Within one dimeric subcomplex, an E2-bound RBR domain is activated by neddylation of its own cullin domain and positioning from the adjacent CUL9-RBX1 in trans. Our data show CUL9 as unique among RBX1-bound cullins in dependence on the metazoan-specific UBE2F neddylation enzyme, while the RBR domain protects it from deneddylation. Substrates are recruited to various upstream domains, while ubiquitylation relies on both CUL9's neddylated cullin and RBR domains achieving self-assembled and chimeric cullin-RING/RBR E3 ligase activity.

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome.

Dedicated assembly factors orchestrate stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here, we report cryo-EM reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, and how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates, and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. The structural findings reported here explain many previous biochemical and genetic observations. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors, and reveals conceptual principles underlying human proteasome biogenesis.

Genome wide association and haplotype analyses for the crease depth trait in bread wheat (Triticum aestivum L.).

Wheat grain has a complex structure that includes a crease on one side, and tissues within the crease region play an important role in nutrient transportation during wheat grain development. However, the genetic architecture of the crease region is still unclear. In this study, 413 global wheat accessions were resequenced and a method was developed for evaluating the phenotypic data of crease depth (CD). The CD values exhibited continuous and considerable large variation in the population, and the broad-sense heritability was 84.09%. CD was found to be positively correlated with grain-related traits and negatively with quality-related traits. Analysis of differentiation of traits between landraces and cultivars revealed that grain-related traits and CD were simultaneously improved during breeding improvement. Moreover, 2,150.8-Mb genetic segments were identified to fall within the selective sweeps between the landraces and cultivars; they contained some known functional genes for quality- and grain-related traits. Genome-wide association study (GWAS) was performed using around 10 million SNPs generated by genome resequencing and 551 significant SNPs and 18 QTLs were detected significantly associated with CD. Combined with cluster analysis of gene expression, haplotype analysis, and annotated information of candidate genes, two promising genes TraesCS3D02G197700 and TraesCS5A02G292900 were identified to potentially regulate CD. To the best of our knowledge, this is the first study to provide the genetic basis of CD, and the genetic loci identified in this study may ultimately assist in wheat breeding programs.

Enhancing Detection of Control State for High-Speed Asynchronous SSVEP-BCIs Using Frequency-Specific Framework.

This study proposed a novel frequency-specific (FS) algorithm framework for enhancing control state detection using short data length toward high-performance asynchronous steady-state visual evoked potential (SSVEP)-based brain-computer interfaces (BCI). The FS framework sequentially incorporated task-related component analysis (TRCA)-based SSVEP identification and a classifier bank containing multiple FS control state detection classifiers. For an input EEG epoch, the FS framework first identified its potential SSVEP frequency using the TRCA-based method and then recognized its control state using one of the classifiers trained on the features specifically related to the identified frequency. A frequency-unified (FU) framework that conducted control state detection using a unified classifier trained on features related to all candidate frequencies was proposed to compare with the FS framework. Offline evaluation using data lengths within 1 s found that the FS framework achieved excellent performance and significantly outperformed the FU framework. 14-target FS and FU asynchronous systems were separately constructed by incorporating a simple dynamic stopping strategy and validated using a cue-guided selection task in an online experiment. Using averaged data length of 591.63±5.65 ms, the online FS system significantly outperformed the FU system and achieved an information transfer rate, true positive rate, false positive rate, and balanced accuracy of 124.95±12.35 bits/min, 93.16±4.4%, 5.21±5.85%, and 92.89±4.02%, respectively. The FS system was also of higher reliability by accepting more correctly identified SSVEP trials and rejecting more wrongly identified ones. These results suggest that the FS framework has great potential to enhance the control state detection for high-speed asynchronous SSVEP-BCIs.

A Strategy for Accessing Nanobody-Based Electrochemical Sensors for Analyte Detection in Complex Media.

Nanobodies are single variable domain antibodies isolated from camelids and are rapidly distinguishing themselves as ideal recognition elements in biosensors due to their comparative stability, ease of production and isolation, and high binding affinities. However, transducing analyte binding by nanobodies in real time is challenging, as most nanobodies do not directly produce an optical or electrical signal upon target recognition. Here, we report a general strategy to fabricate sensitive and selective electrochemical sensors incorporating nanobodies for detecting target analytes in heterogeneous media, such as cell lysate. Graphite felt can be covalently functionalized with recombinant HaloTag-modified nanobodies. Subsequent encapsulation with a thin layer of a hydrogel using a vapor deposition process affords encapsulated electrodes that directly display a decrease in current upon antigen binding, without added redox mediators. Differential pulse voltammetry affords clear and consistent decreases in electrode current across multiple electrode samples for specific antigen concentrations. The change in observed current vs increasing antigen concentration follows Langmuir binding characteristics, as expected. Importantly, selective and repeatable target binding in unpurified cell lysate is only demonstrated by the encapsulated electrode, with an antigen detection limit of ca. 30 pmol, whereas bare electrodes lacking encapsulation produce numerous false positive signals in control experiments.

Removal of Chromium (VI) by a Magnetic Nanoscale Zerovalent Iron-Assisted Chicken Manure-Derived Biochar: Adsorption Behavior and Synergetic Mechanism.

Using chicken manure as raw material to prepare activated carbon as a dispersant, a novel biochar-loaded nano-zerovalent iron composite (nZVI@CMBC) was developed and applied to remove hexavalent chromium, i.e., Cr(VI), in wastewater. The dispersion of nano-zerovalent iron (nZVI) particles on the surface of chicken manure-derived biochar (CMBC) successfully inhibited the aggregation of magnetic iron particles and effectively reduced the size of nZVI particles. The results demonstrated that under acidic conditions, the removal efficiency of Cr(VI) by the nZVI@CMBC composite could reach 124.12 mg g-1. The pseudosecond-order kinetic model had a good agreement with the adsorption kinetics of the nZVI@CMBC composite, implying that the adsorption of Cr(VI) is based on the multi-layer chemical adsorption. Therefore, this study provides a new clue and strategy for removing Cr(VI) in wastewater.

Ubiquitination drives COPI priming and Golgi SNARE localization.

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime coatomer protein I (COPI) assembly to ensure incorporation of these essential cargoes into vesicles, but the regulation of these events is poorly understood. Here, we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs in Saccharomyces cerevisiae. The ability of COPI to bind ubiquitin, but not the dilysine motif, through its N-terminal WD repeat domain of ß'-COP or through an unrelated ubiquitin-binding domain is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3, and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is proposed to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.

A cryptic K48 ubiquitin chain binding site on UCH37 is required for its role in proteasomal degradation.

Degradation by the 26 S proteasome is an intricately regulated process fine tuned by the precise nature of ubiquitin modifications attached to a protein substrate. By debranching ubiquitin chains composed of K48 linkages, the proteasome-associated ubiquitin C-terminal hydrolase UCHL5/UCH37 serves as a positive regulator of protein degradation. How UCH37 achieves specificity for K48 chains is unclear. Here, we use a combination of hydrogen-deuterium mass spectrometry, chemical crosslinking, small-angle X-ray scattering, nuclear magnetic resonance (NMR), molecular docking, and targeted mutagenesis to uncover a cryptic K48 ubiquitin (Ub) chain-specific binding site on the opposite face of UCH37 relative to the canonical S1 (cS1) ubiquitin-binding site. Biochemical assays demonstrate the K48 chain-specific binding site is required for chain debranching and proteasome-mediated degradation of proteins modified with branched chains. Using quantitative proteomics, translation shutoff experiments, and linkage-specific affinity tools, we then identify specific proteins whose degradation depends on the debranching activity of UCH37. Our findings suggest that UCH37 and potentially other DUBs could use more than one S1 site to perform different biochemical functions.

Immobilization of Nanobodies with Vapor-Deposited Polymer Encapsulation for Robust Biosensors.

To produce next-generation, shelf-stable biosensors for point-of-care diagnostics, a combination of rugged biomolecular recognition elements, efficient encapsulants, and innocuous deposition approaches is needed. Furthermore, to ensure that the sensitivity and specificity that are inherent to biological recognition elements are maintained in solid-state biosensing systems, site-specific immobilization chemistries must be invoked such that the function of the biomolecule remains unperturbed. In this work, we present a widely applicable strategy to develop robust solid-state biosensors using emergent nanobody (Nb) recognition elements coupled with a vapor-deposited polymer encapsulation layer. As compared to conventional immunoglobulin G antibodies, Nbs are smaller (12-15 kDa as opposed to ~150 kDa), have higher thermal stability and pH tolerance, boast greater ease of recombinant production, and are capable of binding antigens with high affinity and specificity. Photoinitiated chemical vapor deposition affords thin, protective polymer barrier layers over immobilized Nb arrays that allow for retention of Nb activity and specificity after both storage under ambient conditions and complete desiccation. Most importantly, we also demonstrate that vapor-deposited polymer encapsulation of Nb arrays enables specific detection of target proteins in complex heterogeneous samples, such as unpurified cell lysate, which is otherwise challenging to achieve with bare Nb arrays.

Real-Time and Label-Free Measurement of Deubiquitinase Activity with a MspA Nanopore.

Covalently attaching ubiquitin (Ub) to cellular proteins as a post-translational modification can result in altered function of modified proteins. Enzymes regulating Ub as a post-translational modification, such as ligases and deubiquitinases, are challenging to characterize in part due to the low throughput of in-vitro assays. Single-molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label-free, real-time fashion. Here we demonstrate the use of a MspA nanopore for discriminating and quantifying Ub proteins. We further applied the MspA pore to measure the Ub-chain disassembly activity of UCH37, a proteasome associated deubiquitinase. The implementation of this MspA system into nanopore arrays could enable high throughput characterizations of unknown deubiquitinases as well as drug screening against disease related enzymes.

Proteasome-Bound UCH37/UCHL5 Debranches Ubiquitin Chains to Promote Degradation.

The linkage, length, and architecture of ubiquitin (Ub) chains are all important variables in providing tight control over many biological paradigms. There are clear roles for branched architectures in regulating proteasome-mediated degradation, but the proteins that selectively recognize and process these atypical chains are unknown. Here, using synthetic and enzyme-derived ubiquitin chains along with intact mass spectrometry, we report that UCH37/UCHL5, a proteasome-associated deubiquitinase, cleaves K48 branched chains. The activity and selectivity toward branched chains is markedly enhanced by the proteasomal Ub receptor RPN13/ADRM1. Using reconstituted proteasome complexes, we find that chain debranching promotes degradation of substrates modified with branched chains under multi-turnover conditions. These results are further supported by proteome-wide pulse-chase experiments, which show that the loss of UCH37 activity impairs global protein turnover. Our work therefore defines UCH37 as a debranching deubiquitinase important for promoting proteasomal degradation.

The Effects of Transcranial Direct Current Stimulation (tDCS) on Working Memory Training in Healthy Young Adults.

Working memory (WM) is a fundamental cognitive ability to support complex thought, but it is limited in capacity. WM training has shown the potential benefit for those in need of a higher WM ability. Many studies have shown the potential of transcranial direct current stimulation (tDCS) to transiently enhance WM performance by delivering a low current to the brain cortex of interest, via electrodes on the scalp. tDCS has also been revealed as a promising intervention to augment WM training in a few studies. However, those few tDCS-paired WM training studies, focused more on the effect of tDCS on WM enhancement and its transferability after training and paid less attention to the variation of cognitive performance during the training procedure. The current study attempted to explore the effect of tDCS on the variation of performance, during WM training, in healthy young adults. All the participants received WM training with the load-adaptive verbal N-back task, for 5 days. During the training procedure, active/sham anodal high-definition tDCS (HD-tDCS) was used to stimulate the left dorsolateral prefrontal cortex (DLPFC). To examine the training effect, pre- and post-tests were performed, respectively, 1 day before and after the training sessions. At the beginning of each training session, stable-load WM tasks were performed, to examine the performance variation during training. Compared to the sham stimulation, higher learning rates of performance metrics during the training procedure were found when WM training was combined with active anodal HD-tDCS. The performance improvements (post-pre) of the active group, were also found to be higher than those of the sham group and were transferred to a similar untrained WM task. Further analysis revealed a negative relationship between the training improvements and the baseline performance. These findings show the potential that tDCS may be leveraged as an intervention to facilitate WM training, for those in need of a higher WM ability.

High-Definition Transcranial Direct Current Stimulation (HD-tDCS) enhances working memory training.

High-Definition transcranial direct current stimulation (HD-tDCS) is a noninvasive brain stimulation technique that can improve the performance of working memory (WM). However, the current researches have focused on the effects of stimulation, while ignored the process of stimulus and the neural mechanism. The targets of this study were to explore the effects of different stimulus categories (active or sham) applied on left dorsolateral prefrontal cortex (LDLPFC) on WM training, as well as the physiological changes in the brain after training. Behavioral and electroencephalography (EEG) results of 20 participants showed that HD-tDCS significantly enhanced training effects in the later training period. Furthermore, WM ability benefited from training combined with HD-tDCS, and active group found the time-dependent desynchronization (ERD) weakened in α and ß band, while sham group increased. The results supported the viewpoint that HD-tDCS can shorten the training time and alter neurons rhythm, it may be used as psychotherapy for the patients with brain injury.

A Cross-Subject SSVEP-BCI Based on Task Related Component Analysis.

SSVEP-BCIs have attracted extensive attention because of high information transfer rate. High-speed BCIs need to collect sufficient user's own data to train optimal subject-specific parameters. However, one of the challenges which limits the real-life application of BCIs is the time-consuming and tiring calibration process. This study developed two cross-subject frameworks. One of them uses data from all training subjects to train task-related component analysis based spatial filters (all-to-one, A2O), and the other uses data from each training subject to train task-related component analysis based spatial filters (one-to-one, O2O). Both of them do not need calibration process for a new user. The study further proposed O2O with threshold (O2O-Thr) to increase the reliability of recognition process. The proposed strategies can exploit information from existing subjects' SSVEP data and transfer it to new users. The performance of these methods was compared using an 8-class SSVEP dataset recorded from 10 subjects. O2O-Thr achieves average accuracy of 94.6% with data length of 1.5 seconds. The proposed methods have great potential for building subject-independent BCI that do not require any calibration data from new users, which make BCI more practical and user-friendly.

A two-step idle-state detection method for SSVEP BCI.

The role of detecting work/idle state in asynchronous Steady-state visual evoked potential (SSVEP) Brain-computer interface (BCI) or a self-paced SSVEP BCI has received increased attention in recent years. This study proposed a tree structure method which identifies the work/idle state based on the frequency recognition to detect work/idle state. Firstly, a frequency recognition estimated with task-related component analysis (TRCA). Then, the work/idle state is classified with step-wise linear discriminant analysis (SWLDA) using the data fusion of TRCA scores and power spectral density (PSD) as features. This method was evaluated by Electroencephalography (EEG) data from fourteen healthy participants with eight frequencies as work states and three idle state conditions. The averaged AUC of this method achieved 0.89 with data lengths of one second, which was significantly higher than that of the conventional power spectrum-based algorithm. The proposed method could identify the work/idle state fast and accurately, making the SSVEP BCI better suited for practical application.

An SSVEP-BCI in Augmented Reality.

Steady-State Visual Evoked Potentials (SSVEP) based Brain-Computer Interface (BCI) has achieved very high information transmission rate (ITR), but its portability and fundamental interactions with the surrounding environment were limited. The combination of Augmented Reality (AR) and BCI is expected to solve these problems. In this paper, we combined AR with the SSVEP-BCI to build a more portable and natural BCI system in Microsoft HoloLens. We designed the AR-BCI system and studied the influence of different algorithms on the system performance. The analysis of SSVEP signals collected in AR environment shows that the extended filter bank canonical correlation analysis was better than task-related component analysis. The average recognition accuracy and ITR obtained by using Electroencephalography (EEG) data of 1s, 1.5s, and 2s length were 87.7%,95.4%, 97.6% and 64.6 bit/min, 62.9 bit/min, 55.6 bit/min, respectively. Compared with the existing AR-BCI studies, the ITR has been greatly improved in this study.

A fluorescence polarization-based competition assay for measuring interactions between unlabeled ubiquitin chains and UCH37•RPN13.

Ubiquitin chains regulate distinct signaling events through cooperative interactions with effector proteins and deubiquitinases. Measuring the strength of these interactions is often challenging; either large amounts of material are required or one of the binding partners must be labeled for detection. We sought to develop a label-free method for measuring binding of ubiquitin chains to the proteasome-associated deubiquitinase UCH37 and its binding partner RPN13. The method we describe here is based on a fluorescence polarization competition (FPcomp) assay in which fluorescent monoubiquitin is competed off the UCH37•RPN13 complex by the addition of unlabeled ubiquitin chains. We show that the UCH37•RPN13 complex displays higher affinity toward chains with more than two ubiquitin subunits. Removing the ubiquitin-binding PRU domain of RPN13 does not change affinities. These results suggest UCH37•RPN13 acts to selectively recruit proteins modified with long chains (>2 subunits) to the proteasome for degradation. We also demonstrate that the FPcomp assay is suitable for high-throughput screening, which is important considering both UCH37 and RPN13 are potential targets for cancer therapy.

Optimize the field emission character of a vertical few-layer graphene sheet by manipulating the morphology.

Vertical few-layer graphene (FLG) sheets have been fabricated by using microwave-plasma-enhanced chemical vapour deposition. Their shape was manipulated through adjusting the growth time and hydrocarbon gas ratio. The growth mechanism during different growth stages is discussed. The field emission characteristics for different FLG shapes were tested and found to be strongly influenced by the tip shape, the height and the amorphous carbon content. The optimal shape of vertical FLG for field emission had fewer layers, sharp corners, large height and was free of amorphous carbon. The best field emission properties with the optimal shape were observed with a turn-on field of 1:8 V µm(-1) and maximum current density of 7 mA cm(-2).