The thalamic covariance network is associated with cognitive deficits in patients with cerebral small vascular disease.

OBJECTIVE: Abnormalities in the gray matter structure of cerebral small vessel disease (CSVD) have been observed throughout the brain. However, whether cortico-cortical connections exist between regions of gray matter atrophy in patients with CSVD has not been fully elucidated. This question was tested by comparing the gray matter covariance networks in CSVD patients with and without cognitive impairment (CI). METHODS: We performed multivariate modeling of the gray matter volume measurements of 61 patients with CI (CSVD-CI), 85 patients without CI (CSVD-NC), and 108 healthy controls using source-based morphological analysis (SBM) to obtain gray matter structural covariance networks at the population level. Then, correlations between structural covariance networks and cognitive functions were analyzed in CSVD patients. Finally, a support vector machine (SVM) classifier was used with the gray matter covariance network as a classification feature to identify CI among the CSVD population. RESULTS: The results of the analysis of all the subjects showed that compared with healthy controls, the expression of the thalamic covariance network, cerebellum covariance network, and calcarine cortex covariance network was reduced in patients with CSVD. Moreover, CSVD-CI patients showed a significant reduction in the expression of the thalamic covariance network, encompassing the thalamus and the parahippocampal gyrus, relative to CSVD-NC patients, which persisted after excluding CSVD patients with thalamic lacunes. In patients with CSVD, cognitive functions were positively correlated with measures of the thalamic covariance network. More than 80% of CSVD patients with CI were correctly identified by the SVM classifier. INTERPRETATION: Our findings provide new evidence to explain the distribution state of gray matter reduction in CSVD patients, and the thalamic covariance network is the core region for early gray matter reduction during the development of CSVD disease, which is related to cognitive deficits. Reduced expression of thalamic covariance networks may provide a neuroimaging biomarker for the early identification of cognitive impairment in CSVD patients.

Origin of the multi-phasic quenching dynamics in the BLUF domains across the species.

Blue light using flavin (BLUF) photoreceptors respond to light via one of nature's smallest photo-switching domains. Upon photo-activation, the flavin cofactor in the BLUF domain exhibits multi-phasic dynamics, quenched by a proton-coupled electron transfer reaction involving the conserved Tyr and Gln. The dynamic behavior varies drastically across different species, the origin of which remains controversial. Here, we incorporate site-specific fluorinated Trp into three BLUF proteins, i.e., AppA, OaPAC and SyPixD, and characterize the percentages for the Wout, WinNHin and WinNHout conformations using 19F nuclear magnetic resonance spectroscopy. Using femtosecond spectroscopy, we identify that one key WinNHin conformation can introduce a branching one-step proton transfer in AppA and a two-step proton transfer in OaPAC and SyPixD. Correlating the flavin quenching dynamics with the active-site structural heterogeneity, we conclude that the quenching rate is determined by the percentage of WinNHin, which encodes a Tyr-Gln configuration that is not conducive to proton transfer.

Dissecting the Ultrafast Stepwise Bidirectional Proton Relay in a Blue-Light Photoreceptor.

Proton relays through H-bond networks are essential in realizing the functionality of protein machines such as in photosynthesis and photoreceptors. It has been challenging to dissect the rates and energetics of individual proton-transfer steps during the proton relay. Here, we have designed a proton rocking blue light using a flavin (BLUF) domain with the flavin mononucleotide (FMN)-glutamic acid (E)-tryptophan (W) triad and have resolved the four individual proton-transfer steps kinetically using ultrafast spectroscopy. We have found that after the photo-induced charge separation forming FMN·-/E-COOH/WH·+, the proton first rapidly jumps from the bridging E-COOH to FMN- (τfPT2 = 3.8 ps; KIE = 1.0), followed by a second proton transfer from WH·+ to E-COO- (τfPT1 = 336 ps; KIE = 2.6) which immediately rocks back to W· (τrPT1 = 85 ps; KIE = 6.7), followed by a proton return from FMNH· to E-COO- (τrPT2 = 34 ps; KIE = 3.3) with the final charge recombination between FMN·- and WH·+ to close the reaction cycle. Our results revisited the Grotthuss mechanism on the ultrafast timescale using the BLUF domain as a paradigm protein.

The nature of proton-coupled electron transfer in a blue light using flavin domain.

Proton-coupled electron transfer (PCET) is key to the activation of the blue light using flavin (BLUF) domain photoreceptors. Here, to elucidate the photocycle of the central FMN-Gln-Tyr motif in the BLUF domain of OaPAC, we eliminated the intrinsic interfering W90 in the mutant design. We integrated the stretched exponential function into the target analysis to account for the dynamic heterogeneity arising from the active-site solvation relaxation and the flexible H-bonding network as shown in the molecular dynamics simulation results, facilitating a simplified expression of the kinetics model. We find that, in both the functional wild-type (WT) and the nonfunctional Q48E and Q48A, forward PCET happens in the range of 105 ps to 344 ps, with a kinetic isotope effect (KIE) measured to be ∼1.8 to 2.4, suggesting that the nature of the forward PCET is concerted. Remarkably, only WT proceeds with an ultrafast reverse PCET process (31 ps, KIE = 4.0), characterized by an inverted kinetics of the intermediate FMNH˙. Our results reveal that the reverse PCET is driven by proton transfer via an intervening imidic Gln.

Prognostic nomogram for percutaneous balloon compression in the treatment of trigeminal neuralgia.

Because of its convenience and safety, percutaneous balloon compression (PBC) has become a more popular remedy for trigeminal neuralgia (TN) recently. The objective of this study was to establish a nomogram that can be used to preoperatively prognosticate the likelihood of pain-free based on preoperative disease characteristics. Clinical data were collected from those TN cases who had undergone PBC during the period of 2015 and 2020 in Qingdao Municipal Hospital. We excluded the cases caused by space-occupying lesion or had undergone MVD, percutaneous glycerol rhizotomy (PGR), and glycerol rhizotomy (GR). A nomogram was established based on the results of multivariable logistic analysis. A receiver operating characteristic curve (ROC) analysis was applied to evaluate the reliability of models. The plotted decision curves were also used to assess the net benefit of nomogram-assisted decisions. Internal validation was performed using the ROC by bootstrap sampling. Finally, 16 cases and 69 cases were included into the ineffective and effective groups respectively. In the crude, adjust I and adjust II models, response to carbamazepine positively, the grade II or III compression severity score, and classical TN type were all considered to be significant predictors of pain relief (BNI grades I-III) at 3 months' follow-up. The AUC, accuracy, specificity, and sensitivity of the nomogram system were 0.83, 0.85, 0.75, and 0.87 respectively for predicting patient outcomes. The decision curves showed good performance for the nomogram system in terms of clinical application, while more research with validation in multiple, external independent patient populations is needed.

A Nomogram for Predicting Acute Respiratory Failure After Cervical Traumatic Spinal Cord Injury Based on Admission Clinical Findings.

OBJECTIVES: Acute respiratory failure (ARF) is a common medical complication in patients with cervical traumatic spinal cord injury (TSCI). To identify independent predictors for ARF onset in patients who underwent cervical TSCI without premorbid respiratory diseases and to apply appropriate medical supports based on accurate prediction, a nomogram relating admission clinical information was developed for predicting ARF during acute care period. METHODS: We retrospectively reviewed clinical profiles of patients who suffered cervical TSCI and were emergently admitted to Qingdao Municipal Hospital from 2014 to 2020 as the training cohort. Univariate analysis was performed using admission clinical variables to estimate associated factors and a nomogram for predicting ARF occurrence was generated based on the independent predictors from multivariate logistic regression analysis. This nomogram was assessed by concordance index for discrimination and calibration curve with internal-validated bootstrap strategy. Receiver operating characteristic curve was conducted to compare the predictive accuracy between the nomogram and the traditional gold standard, which combines neuroimaging and neurological measurements by using area under the receiver operating characteristic curve (AUC). An additional 56-patient cohort from another medical center was retrospectively reviewed as the test cohort for external validation of the nomogram. RESULTS: 162 patients were eligible for this study and were included in the training cohort, among which 25 individuals developed ARF and were recorded to endure more complications. Despite the aggressive treatments and prolonged intensive care unit cares, 14 patients insulted with ARF died. Injury level, American Spinal Injury Association Impairment Scale (AIS) grade, admission hemoglobin (Hb), platelet to lymphocyte ratio, and neutrophil percentage to albumin ratio (NPAR) were independently associated with ARF onset. The concordance index of the nomogram incorporating these predictors was 0.933 in the training cohort and 0.955 in the test cohort, although both calibrations were good. The AUC of the nomogram was equal to concordance index, which presented better predictive accuracy compared with previous measurements using neuroimaging and AIS grade (AUC 0.933 versus 0.821, Delong's test p < 0.001). Similar significant results were also found in the test cohort (AUC 0.955 versus 0.765, Delong's test p = 0.034). In addition, this nomogram was translated to a Web-based calculator that could generate individual probability for ARF in a visualized form. CONCLUSIONS: The nomogram incorporating the injury level, AIS grade, admission Hb, platelet to lymphocyte ratio, and NPAR is a promising model to predict ARF in patients with cervical TSCI who are absent from previous respiratory dysfunction. This nomogram can be offered to clinicians to stratify patients, strengthen evidence-based decision-making, and apply appropriate individualized treatment in the field of acute clinical care.

Direct Observation of Ultrafast Proton Rocking in the BLUF Domain.

We present direct observation of ultrafast proton rocking in the central motif of a BLUF domain protein scaffold. The mutant design has taken consideration of modulating the proton-coupled electron transfer (PCET) driving forces by replacing Tyr in the original motif with Trp, in order to remove the interference of a competing electron transfer pathway. Using femtosecond pump-probe spectroscopy and detailed kinetics analysis, we resolved an electron-transfer-coupled Grotthuss-type forward and reverse proton rocking along the FMN-Gln-Trp proton relay chain. The rates of forward and reverse proton transfer are determined to be very close, namely 51 ps vs. 52 ps. The kinetic isotope effect (KIE) constants associated with the forward and reverse proton transfer are 3.9 and 5.3, respectively. The observation of ultrafast proton rocking is not only a crucial step towards revealing the nature of proton relay in the BLUF domain, but also provides a new paradigm of proton transfer in proteins for theoretical investigations.

Generation of gradient photonic moiré lattice fields.

We designed and generated gradient photonic moiré lattice fields comprising three varying periodic moiré wavefields. Because of the common twisted angles between periodic triangular and hexagonal moiré wavefields, gradient patterns can be easily obtained through coherent superposition of hexagonal-triangular-hexagonal photonic moiré lattice fields. In addition, two specific twisted angles of Δα|C=3 and Δα|C=5 are proposed, which not only guarantee the periodicity of moiré fields but also provide an additional degree of freedom to control the structural arrangement of the gradient photonic moiré lattice fields. Further study reveals the non-diffracting character of the gradient photonic moiré lattice field generated using the holographic method. This study proposes an easy way to generate and control the structures of gradient moiré lattice fields that can be used to fabricate photonic lattices in optical storage media for light modulation.

Tuning Magnetic Soliton Phase via Dimensional Confinement in Exfoliated 2D Cr1/3NbS2 Thin Flakes.

Thin flakes of Cr1/3NbS2 are fabricated successfully via microexfoliation techniques. Temperature-dependent and field-dependent magnetizations of thin flakes with various thicknesses are investigated. When the thickness of the flake is around several hundred nanometers, the softening and eventual disappearance of the bulk soliton peak is accompanied by the appearance of other magnetic peaks at lower magnetic fields. The emergence and annihilation of the soliton peaks are explained and simulated theoretically by the change in spin spiral number inside the soliton lattice due to dimensional confinement. Compared to the conventional magnetic states in nanoscale materials, the stability and thickness tunability of quantified spin spirals make Cr1/3NbS2 a potential candidate for spintronics nanodevices beyond Moore's law.

Dimensionality Effects in FeGe2 Nanowires: Enhanced Anisotropic Magnetization and Anomalous Electrical Transport.

We report the synthesis of single-crystal iron germanium nanowires via chemical vapor deposition without the assistance of any catalysts. The assembly of single-crystal FeGe2 nanowires with tetragonal C16 crystal structure shows anisotropic magnetic behavior along the radial direction or the growth axial direction, with both antiferromagnetic and ferromagnetic orders. Single FeGe2 nanowire devices were fabricated using e-beam lithography. Electronic transport measurement in these devices show two resistivity anomalies near 250 K and 200 K which are likely signatures of the two spin density wave states in FeGe2.

Ferromagnetism and nonmetallic transport of thin-film α-FeSi(2): a stabilized metastable material.

A metastable phase α-FeSi_{2} was epitaxially stabilized on a silicon substrate using pulsed laser deposition. Nonmetallic and ferromagnetic behaviors are tailored on α-FeSi_{2} (111) thin films, while the bulk material of α-FeSi_{2} is metallic and nonmagnetic. The transport property of the films renders two different conducting states with a strong crossover at 50 K, which is accompanied by the onset of a ferromagnetic transition as well as a substantial magnetoresistance. These experimental results are discussed in terms of the unusual electronic structure of α-FeSi_{2} obtained within density functional calculations and Boltzmann transport calculations with and without strain. Our finding sheds light on achieving ferromagnetic semiconductors through both their structure and doping tailoring, and provides an example of a tailored material with rich functionalities for both basic research and practical applications.

Quantitative proteomic dissection of a native 14-3-3ε interacting protein complex associated with hepatocellular carcinoma.

The 14-3-3 proteins regulate diverse biological processes that are implicated in cancer development, and seven 14-3-3 isoforms were identified with isoform-specific roles in different human tumors. In our previous work, we dissected the interactome of 14-3-3ε formed during the DNA damage response in a hepatocellular carcinoma (HCC) cell using an AACT/SILAC-based quantitative proteomic approach. In this study, we used a similar proteomic approach to profile/identify the 14-3-3ε interactome formed in native HCC cells. Functional categorization and data-dependent network analysis of the native HCC-specific 14-3-3ε interactome revealed that 14-3-3ε is involved in the regulation of multiple biological processes (BPs)/pathways, including cell cycle control, apoptosis, signal transduction, transport, cell adhesion, carbohydrate metabolism, and nucleic acid metabolism. Biological validation further supports that 14-3-3ε, via association with multiple BP/pathway-specific proteins, coordinates the regulation of proliferation, survival, and metastasis of HCC. The findings in this study, together with those of our previous study, provide an extensive profile of the 14-3-3ε interaction network in HCC cells, which should be valuable for understanding the pathology of HCC and HCC therapy.

14-3-3ε boosts bleomycin-induced DNA damage response by inhibiting the drug-resistant activity of MVP.

Major vault protein (MVP) is the predominant constituent of the vault particle, the largest known ribonuclear protein complex. Although emerging evidence have been establishing the links between MVP (vault) and multidrug resistance (MDR), little is known regarding exactly how the MDR activity of MVP is modulated during cellular response to drug-induced DNA damage (DDR). Bleomycin (BLM), an anticancer drug, induces DNA double-stranded breaks (DSBs) and consequently triggers the cellular DDR. Due to its physiological implications in hepatocellular carcinoma (HCC) and cell fate decision, 14-3-3ε was chosen as the pathway-specific bait protein to identify the critical target(s) responsible for HCC MDR. By using an LC-MS/MS-based proteomic approach, MVP was first identified in the BLM-induced 14-3-3ε interactome formed in HCC cells. Biological characterization revealed that MVP possesses specific activity to promote the resistance to the BLM-induced DDR. On the other hand, 14-3-3ε enhances BLM-induced DDR by interacting with MVP. Mechanistic investigation further revealed that 14-3-3ε, in a phosphorylation-dependent manner, binds to the phosphorylated sites at both Thr52 and Ser864 of the monomer of MVP. Consequently, the phosphorylation-dependent binding between 14-3-3ε and MVP inhibits the drug-resistant activity of MVP for an enhanced DDR to BLM treatment. Our findings provide an insight into the mechanism underlying how the BLM-induced interaction between 14-3-3ε and MVP modulates MDR, implicating novel strategy to overcome the chemotherapeutic resistance through interfering specific protein-protein interactions.

PPE38 of Mycobacterium marinum triggers the cross-talk of multiple pathways involved in the host response, as revealed by subcellular quantitative proteomics.

The PE/PPE family of proteins which are in high abundance in pathogenic species such as Mycobacterium tuberculosis and M. marinum , play the critical role in generating antigenic variation and evasion of host immune responses. However, little is known about their functional roles in mycobacterial pathogenesis. Previously, we found that PPE38 is associated with the virulence of mycobacteria, presumably by modulating the host immune response. To clarify the link between PPE38 and host response, we employed a subcellular, amino acid-coded mass tagging (AACT)/SILAC-based quantitative proteomic approach to determine the proteome changes during host response to M. marinum PPE38. As a result, 291 or 290 proteins were found respectively to be up- or down-regulated in the nucleus. Meanwhile, 576 upregulated and 272 downregulated proteins were respectively detected in the cytosol. The data of quantitative proteomic changes and concurrent biological validations revealed that M. marinum PPE38 could trigger extensive inflammatory responses in macrophages, probably through interacting with toll-like receptor 2 (TLR2). We also found that PPE38 may arrest MHC-1 processing and presentation in infected macrophages. Using bioinformatics tools to analyze global changes in the host proteome, we obtained a PPE38-respondor network involved in various transcriptional factors (TFs) and TF-associated proteins. The results of our systems investigation now indicate that there is cross-talk involving a broad range of diverse biological pathways/processes that coordinate the host response to M. marinum PPE38.

14-3-3ε mediates the cell fate decision-making pathways in response of hepatocellular carcinoma to Bleomycin-induced DNA damage.

BACKGROUND: Lack of understanding of the response of hepatocellular carcinoma (HCC) to anticancer drugs causes the high mortality of HCC patients. Bleomycin (BLM) that induces DNA damage is clinically used for cancer therapy, while the mechanism underlying BLM-induced DNA damage response (DDR) in HCC cells remains ambiguous. Given that 14-3-3 proteins are broadly involved in regulation of diverse biological processes (BPs)/pathways, we investigate how a 14-3-3 isoform coordinates particular BPs/pathways in BLM-induced DDR in HCC. METHODOLOGY/PRINCIPAL FINDINGS: Using dual-tagging quantitative proteomic approach, we dissected the 14-3-3ε interactome formed during BLM-induced DDR, which revealed that 14-3-3ε via its associations with multiple pathway-specific proteins coordinates multiple pathways including chromosome remodeling, DNA/RNA binding/processing, DNA repair, protein ubiquitination/degradation, cell cycle arrest, signal transduction and apoptosis. Further, "zoom-in" investigation of the 14-3-3ε interacting network indicated that the BLM-induced interaction between 14-3-3ε and a MAP kinase TAK1 plays a critical role in determining cell propensity of apoptosis. Functional characterization of this interaction further revealed that BLM triggers site-specific phosphorylations in the kinase domain of TAK1. These BLM-induced changes of phosphorylations directly correlate to the strength of the TAK1 binding to 14-3-3ε, which govern the phosphorylation-dependent TAK1 activation. The enhanced 14-3-3ε-TAK1 association then inhibits the anti-apoptotic activity of TAK1, which ultimately promotes BLM-induced apoptosis in HCC cells. In a data-dependent manner, we then derived a mechanistic model where 14-3-3ε plays the pivotal role in integrating diverse biological pathways for cellular DDR to BLM in HCC. CONCLUSIONS: Our data demonstrated on a systems view that 14-3-3ε coordinates multiple biological pathways involved in BLM-induced DDR in HCC cells. Specifically, 14-3-3ε associates with TAK1 in a phosphorylation-dependent manner to determine the cell fate of BLM-treated HCC cells. Not only individual proteins but also those critical links in the network could be the potential targets for BLM-mediated therapeutic intervention of HCC.

14-3-3 epsilon dynamically interacts with key components of mitogen-activated protein kinase signal module for selective modulation of the TNF-alpha-induced time course-dependent NF-kappaB activity.

Inflammation is tightly regulated by nuclear factor-kappa B (NF-kappaB), and if left unchecked excessive NF-kappaB activation for cytokine overproduction can lead to various pathogenic consequences including carcinogenesis. A proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), can be used to explore possible mechanisms whereby unknown functional pathways modulate the NF-kappaB activity for regulating TNF-alpha-induced inflammation. Given the multifunctional nature of 14-3-3 family proteins and the recent finding of their presence in the TNF-alpha/NF-kappaB pathway network, we used a dual-tagging quantitative proteomic method to first profile the TNF-alpha-inducible interacting partners of 14-3-3 epsilon, the least characterized 14-3-3 isomer in the family. For the first time, we found that TNF-alpha stimulation enhances the interactions between 14-3-3 epsilon and some key components in the mitogen-activated protein kinase (MAPK) signal module which is located at the immediate upstream of NF-kappaB, including transforming growth factor-beta activated kinase-1 (TAK1) and its interacting protein, protein phosphatase 2C beta (PPM1B). By using confocal laser scanning, we observed the TNF-alpha-induced colocalizations among 14-3-3 epsilon, TAK1, and protein phosphatase 2C beta (PPM1B), and these interactions were also TNF-alpha-inducible in different cell types. Further, we found that during the full course of the cellular response to TNF-alpha, the interactions between 14-3-3 epsilon and these two proteins were dynamic and were closely correlated with the time course-dependent changes in NF-kappaB activity, suggesting that these 14-3-3 epsilon interactions are the critical points of convergence for TNF-alpha signaling for modulating NF-kappaB activity. We then postulated a mechanistic view describing how 14-3-3 epsilon coordinates its dynamic interactions with TAK1 and PPM1B for differentially modulating TNF-alpha-induced changes in NF-kappaB activity. By using bioinformatics tools, we constructed the network involving most of the 14-3-3 epsilon interacting proteins identified in our proteomic study. We revealed that 14-3-3 epsilon coordinates the cross talks between the MAPK signal module and other molecular pathways/biological processes primarily including protein metabolism and synthesis, DNA repair, and cell cycle regulation where pharmacological targets for therapeutic intervention could be systematically located.

Subcellular quantitative proteomics reveals multiple pathway cross-talk that coordinates specific signaling and transcriptional regulation for the early host response to LPS.

Toll-like receptor 4 (TLR4) specifically recognizes lipopolysaccharide (LPS) to initiate signal transduction events that modulate host inflammatory responses. Although increasing numbers of genes have been characterized individually for their involvement in TLR4 signaling, the LPS-induced TLR4-mediated signaling pathway and connected networks are incompletely delineated. Given that most components of signaling pathways are activated at an early phase of the LPS-induced response, we have employed a subcellular, SILAC-based quantitative proteomics approach to identify proteins in LPS-stimulated macrophages showing either cytosolic- or nuclear-specific changes in abundance. Subcellular fractionation not only reduces the spectral complexity for identifying maximum numbers of proteins but also enriches for low-abundance proteins within the compartment in which they function. Following 10 min LPS stimulation, the abundances of 508 proteins were found elevated in the cytosol, while the elevated levels of 678 proteins together with the decreased abundances of another 80 proteins were quantified in nuclei. Coincident with observations that many key proteins involved in signal relays in the MAPK and NF-kappaB cascades were found simultaneously regulated in the cytosol, various transcriptional factors (TFs) such as IRFs were found activated in the nuclei. We also extended links between these intracellular pathways and various biological processes by identifying multiple pathway modules. For the first time, our combined data sets from quantitative proteomics and bioinformatics analyses provide a direct, system-wide insight into how cross-talk between upstream signaling pathways modulates the activities of particular TFs for regulating sets of pro-inflammatory genes.

Biotin tagging coupled with amino acid-coded mass tagging for efficient and precise screening of interaction proteome in mammalian cells.

In mammalian cells, when tandem affinity purification approach is employed, the existence of untagged endogenous target protein and repetitive washing steps together result in overall low yield of purified/stable complexes and the loss of weakly and transiently interacting partners of biological significance. To avoid the trade-offs involving in methodological sensitivity, precision, and throughput, here we introduce an integrated method, biotin tagging coupled with amino acid-coded mass tagging, for highly sensitive and accurate screening of mammalian protein-protein interactions. Without the need of establishing a stable cell line, using a short peptide tag which could be specifically biotinylated in vivo, the biotin-tagged target/bait protein was then isolated along with its associates efficiently by streptavidin magnetic microbeads in a single step. In a pulled-down complex amino acid-coded mass tagging serves as "in-spectra" quantitative markers to distinguish those bait-specific interactors from non-specific background proteins under stringent criteria. Applying this biotin tagging coupled with amino acid-coded mass tagging approach, we first biotin-tagged in vivo a multi-functional protein family member, 14-3-3epsilon, which was expressed at close to endogenous level. Starting with approximately 20 millions of 293T cells which were significantly less than what needed for a tandem affinity purification run, 266 specific interactors of 14-3-3epsilon were identified in high confidence.