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2.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 3122-3125, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018666

RESUMO

Previous works have shown that whitening improves the processed electromyogram (EMG) signal for use in end applications such as EMG to torque modelling. Traditional whitening methods fit each subject from calibration contractions, which is a hindrance to their widespread use. To eliminate this cumbersome calibration, a universal whitening filter was developed using the whitening filters from a pre-existing data set (64 subjects, 8 electrodes/subject). Since the shape of each subject-specific whitening filter was observed to be relatively consistent across subjects, the universal whitening filter was formed as their ensemble average. The processed EMG was then used to model surface EMG to torque about the elbow. Traditional and universal whitening provided the same EMG-torque benefit, each improving statistically over unwhitened processing by ~14% during dynamic contractions. We further studied the use of root difference of squares (RDS) post-processing to attenuate additive measurement noise in EMG channels. With and without whitening, RDS processing (vs. no RDS processing) better attenuated additive noise, reducing it from 2-4% (on average) of the processed EMG from a 50% contraction down to < 1%. The combined use of universal whitening filters and RDS processing should be a particular benefit in real-time applications such as prosthesis control.


Assuntos
Articulação do Cotovelo , Processamento de Sinais Assistido por Computador , Cotovelo , Eletromiografia , Humanos , Torque
3.
Materials (Basel) ; 13(18)2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32927682

RESUMO

To reduce the thermal-oxidative aging of asphalt and the release amount of harmful volatiles during the construction of asphalt pavement, a new composite anti-aging agent was developed. Since the volatiles were mainly released from saturates and aromatics during the thermal-oxidative aging of asphalt, expanded graphite (EG) was selected as a stabilizing agent to load magnesium hydroxide (MH) and calcium carbonate (CaCO3) nanoparticles for preparing the anti-aging agents of saturates and aromatics, respectively. Thermal stability and volatile constituents released from saturates and aromatics before and after the thermal-oxidative aging were characterized using the isothermal Thermogravimetry/Differential Scanning Calorimetry-Fourier Transform Infrared Spectrometer test (TG/DSC-FTIR test). Test results indicate that anti-aging agents of EG/MH and EG/CaCO3 effectively inhibit the volatilization of light components in asphalt and improve the thermal stability of saturates and aromatics. Then, the proportions of EG, MH, and CaCO3 added in the developed composite anti-aging agent of EG/MH/CaCO3 are 2:1:3 by weight. EG/MH/CaCO3 plays a synergetic effect on inhibiting the thermal-oxidative aging of asphalt, and reduces the release amount of harmful volatiles during the thermal-oxidative aging after EG/MH/CaCO3 is added into asphalt at the proposed content of 10 wt.%. EG plays a synergistic role with MH and CaCO3 nanoparticles to prevent the chain reactions, inhibiting the thermal-oxidative aging of asphalt.

4.
Immunity ; 53(2): 456-470.e6, 2020 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-32758419

RESUMO

Clinical evidence suggests that poor persistence of chimeric antigen receptor-T cells (CAR-T) in patients limits therapeutic efficacy. Here, we designed a CAR with recyclable capability to promote in vivo persistence and to sustain antitumor activity. We showed that the engagement of tumor antigens induced rapid ubiquitination of CARs, causing CAR downmodulation followed by lysosomal degradation. Blocking CAR ubiquitination by mutating all lysines in the CAR cytoplasmic domain (CARKR) markedly repressed CAR downmodulation by inhibiting lysosomal degradation while enhancing recycling of internalized CARs back to the cell surface. Upon encountering tumor antigens, CARKR-T cells ameliorated the loss of surface CARs, which promoted their long-term killing capacity. Moreover, CARKR-T cells containing 4-1BB signaling domains displayed elevated endosomal 4-1BB signaling that enhanced oxidative phosphorylation and promoted memory T cell differentiation, leading to superior persistence in vivo. Collectively, our study provides a straightforward strategy to optimize CAR-T antitumor efficacy by redirecting CAR trafficking.

5.
Cell ; 182(4): 855-871.e23, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32730808

RESUMO

A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3ε ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.

6.
Nat Commun ; 11(1): 2781, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493900

RESUMO

Mutations disrupting regulatory T (Treg) cell function can cause IPEX and IPEX-related disorders, but whether established disease can be reversed by correcting these mutations is unclear. Treg-specific deletion of the chromatin remodeling factor Brg1 impairs Treg cell activation and causes fatal autoimmunity in mice. Here, we show with a reversible knockout model that re-expression of Brg1, in conjunction with the severe endogenous proinflammatory environment, can convert defective Treg cells into powerful, super-activated Treg cells (SuperTreg cells) that can resolve advanced autoimmunity,  with  Brg1 re-expression in a minor fraction of Treg cells sufficient for the resolution in some cases. SuperTreg cells have enhanced trafficking and regulatory capabilities, but become deactivated as the inflammation subsides, thus avoiding excessive immune suppression. We propose a simple, robust yet safe gene-editing-based therapy for IPEX and IPEX-related disorders that exploits the defective Treg cells and the inflammatory environment pre-existing in the patients.


Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças do Sistema Imunitário/congênito , Linfócitos T Reguladores/imunologia , Alelos , Animais , Citocinas/metabolismo , DNA Helicases/deficiência , Diabetes Mellitus Tipo 1/imunologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Doenças do Sistema Imunitário/imunologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR3/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Linfócitos T Reguladores/efeitos dos fármacos , Tamoxifeno/farmacologia , Fatores de Transcrição/deficiência
7.
J Cell Biol ; 219(6)2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32437509

RESUMO

Blockade antibodies of the immunoinhibitory receptor PD-1 can stimulate the anti-tumor activity of T cells, but clinical benefit is limited to a fraction of patients. Evidence suggests that BTLA, a receptor structurally related to PD-1, may contribute to resistance to PD-1 targeted therapy, but how BTLA and PD-1 differ in their mechanisms is debated. Here, we compared the abilities of BTLA and PD-1 to recruit effector molecules and to regulate T cell signaling. While PD-1 selectively recruited SHP2 over the stronger phosphatase SHP1, BTLA preferentially recruited SHP1 to more efficiently suppress T cell signaling. Contrary to the dominant view that PD-1 and BTLA signal exclusively through SHP1/2, we found that in SHP1/2 double-deficient primary T cells, PD-1 and BTLA still potently inhibited cell proliferation and cytokine production, albeit more transiently than in wild type T cells. Thus, PD-1 and BTLA can suppress T cell signaling through a mechanism independent of both SHP1 and SHP2.

8.
Chemistry ; 26(36): 8017-8021, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32253796

RESUMO

Until now, all B≡B triple bonds have been achieved by adopting two ligands in the L→B≡B←L manner. Herein, we report an alternative route of designing the B≡B bonds based on the assumption that by acquiring two extra electrons, an element with the atomic number Z can have properties similar to those of the element with the atomic number Z+2. Specifically, we show that due to the electron donation from Al to B, the negatively charged B≡B kernel in the B2 Al3 - cluster mimics a triple N≡N bond. Comprehensive computational searches reveal that the global minimum structure of B2 Al3 - exhibits a direct B-B distance of 1.553 Å, and its calculated electron vertical detachment energies are in excellent agreement with the corresponding values of the experimental photoelectron spectrum. Chemical bonding analysis revealed one σ and two π bonds between the two B atoms, thus confirming a classical textbook B≡B triple bond, similar to that of N2 .

9.
Methods Mol Biol ; 2111: 59-70, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31933198

RESUMO

T-cell-based cancer immunotherapies have emerged as a promising approach for cancer treatment, highlighting the importance of understanding the regulation of T-cell function. However, the molecular mechanisms underlying T-cell activation are not fully understood. The CRISPR/Cas9 system can serve as a robust method to systematically study signaling pathways. In this chapter, we describe details of using the CRISPR screen to identify regulators in TCR signaling, from the sgRNA library construction to genomic DNA sequencing. We also add some notes to further help readers performing the CRISPR screen. This approach can be readily adapted to study the activation of other immune cells, including B cells and dendritic cells.

10.
IEEE Trans Neural Syst Rehabil Eng ; 27(12): 2328-2335, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31689197

RESUMO

Typical electromyogram (EMG) processors estimate EMG signal standard deviation (EMG σ ) via moving average root mean square (RMS) or mean absolute value (MAV) filters, whose outputs are used in force estimation, prosthesis/orthosis control, etc. In the inevitable presence of additive measurement noise, some processors subtract the noise standard deviation from EMG RMS (or MAV). Others compute a root difference of squares (RDS)-subtract the noise variance from the square of EMG RMS (or MAV), all followed by taking the square root. Herein, we model EMG as an amplitude-modulated random process in additive measurement noise. Assuming a Gaussian (or, separately, Laplacian) distribution, we derive analytically that the maximum likelihood estimate of EMG σ requires RDS processing. Whenever that subtraction would provide a negative-valued result, we show that EMG σ should be set to zero. Our theoretical models further show that during rest, approximately 50% of EMG σ estimates are non-zero. This result is problematic when EMG σ is used for real-time control, explaining the common use of additional thresholding. We tested our model results experimentally using biceps and triceps EMG from 64 subjects. Experimental results closely followed the Gaussian model. We conclude that EMG processors should use RDS processing and not noise standard deviation subtraction.


Assuntos
Algoritmos , Eletromiografia/estatística & dados numéricos , Eletromiografia/métodos , Músculos Isquiossurais/fisiologia , Humanos , Funções Verossimilhança , Modelos Teóricos , Contração Muscular , Distribuição Normal , Próteses e Implantes , Padrões de Referência , Processamento de Sinais Assistido por Computador
11.
Materials (Basel) ; 12(22)2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31717293

RESUMO

A styrene-butadiene-styrene (SBS) latex modifier can be used for asphalt modification due to the fact of its energy-saving, construction convenience, and economic advantages. The objective of this study was to investigate the influence of asphalt type and SBS latex dosage on the rheological properties, compatibility, and storage stability of asphalt through temperature and frequency sweep, steady-state flow, multiple stress creep and recovery (MSCR) tests, Cole-Cole plots and thermal storage tests. The results indicated that high SBS latex content is beneficial for improving anti-rutting, anti-fatigue, viscous flow resistance, and elastic recovery abilities of modified asphalt. The chemical composition of asphalt had a significant effect on the properties of the SBS latex-modified asphalt. High asphaltenes and low resins were favorable to enhancing anti-rutting and recovery properties but weakened the anti-fatigue, compatibility, and storage stability of modified asphalt. Furthermore, compared to SBS particle-modified asphalt, SBS latex-modified asphalt had greater rutting and fatigue resistance. However, SBS latex-modified asphalt had some disadvantages in compatibility and storage stability. Comprehensively considering the balance of viscoelastic properties, compatibility, and storage stability of SBS latex-modified asphalt, the mixing dosage of SBS latex modifier is recommended at 4.0 wt% which could feasibly replace SBS particle in asphalt modification.

12.
BMC Genomics ; 20(1): 107, 2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30727954

RESUMO

BACKGROUND: While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have thus far not investigated the influence of cellular heterogeneity on screen results. Instead most screens are analyzed by averaging the abundance of perturbed cells from a bulk population of cells. RESULTS: Here we developed multi-level barcoded sgRNA libraries to trace multiple clonal Cas9 cell lines exposed to the same environment. The first level of barcoding allows monitoring growth kinetics and treatment responses of multiplexed clonal cell lines under identical conditions while the second level enables in-sample replication and tracing of sub-clonal lineages of cells expressing the same sgRNA. CONCLUSION: Using our approach, we illustrate how heterogeneity in growth kinetics and treatment response of clonal cell lines impairs the results of pooled genetic screens.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Testes Genéticos/métodos , Tipagem Molecular/métodos , RNA Guia , Humanos , Células Jurkat
13.
Materials (Basel) ; 12(1)2019 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-30621178

RESUMO

Microwave heating is an encouraging heating technology for the maintenance, recycling, and deicing of asphalt pavement. To investigate the microwave heating properties of asphalt mixture, laboratory tests and numerical simulations were done and compared. Two types of Stone Mastic Asphalt (SMA) mixture samples (with basalt aggregates and steel slag aggregates) were heated using a microwave oven for different times. Numerical simulation models of microwave heating of asphalt mixture were developed with finite element software COMSOL Multiphysics. The main thermal and electromagnetic properties of asphalt mixture, served as the model input parameters, were measured through a series of laboratory tests. Both laboratory-measured and numerical simulated surface temperatures were recorded and analyzed. Results show that the replacement of basalt aggregates with steel slag aggregates can significantly increase the microwave heating efficiency of asphalt mixture. Numerical simulation results have a good correlation with laboratory test results. It is feasible to use the developed model coupling electromagnetic waves with heat transfer to simulate the microwave heating process of asphalt mixture.

14.
Nat Methods ; 15(9): 715-722, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30104635

RESUMO

The communication between cells and between cellular organelles is often controlled by the interaction of membrane proteins. Although many methods for the detection of protein-protein interactions (PPIs) exist, membrane PPIs remain difficult to detect. Here we developed a proximity-based tagging system, PUP-IT (pupylation-based interaction tagging), to identify membrane protein interactions. In this approach, a small protein tag, Pup, is applied to proteins that interact with a PafA-fused bait, enabling transient and weak interactions to be enriched and detected by mass spectrometry. Pup does not diffuse from the enzyme, which allows high-specificity labeling. We applied this approach to CD28, a critical costimulatory receptor for T lymphocyte activation, and identified known CD28 binding partners and multiple potential interacting proteins. In addition, we demonstrated that this method can identify the interaction between a cell surface receptor and its ligand.


Assuntos
Proteínas de Membrana/metabolismo , Mapas de Interação de Proteínas , Antígenos CD28/metabolismo , Humanos , Espectrometria de Massas , Ligação Proteica , Proteólise
15.
Angew Chem Int Ed Engl ; 57(43): 14060-14064, 2018 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-30129083

RESUMO

The discovery of homodinuclear multiple bonds composed of Group 13 elements represents one of the most challenging frontiers in modern chemistry. A classical triple bond such as N≡N and HC≡CH contains one σ bond and two π bonds constructed from the p orbitals perpendicular to the σ bond. However, the traditional textbook triple bond between two Al atoms has remained elusive. Here we report an Al≡Al triple bond in the designer Na3 Al2 - cluster predicted in silico, which was subsequently generated by pulsed arc discharge followed by mass spectrometry and photoelectron spectroscopy characterizations. Being effectively Al2- due to the electron donation from Na, the Al atoms in Na3 Al2 - undergo a double electronic transmutation into Group 15 elements, thus the Al2- ≡Al2- kernel mimics the P≡P and N≡N molecules. We anticipate this work will stimulate more endeavors in discovering materials using Al2- ≡Al2- as a building block in the gas phase and in the solid state.

16.
Proc Natl Acad Sci U S A ; 115(17): E4051-E4060, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29632189

RESUMO

Despite decades of research, mechanisms controlling T cell activation remain only partially understood, which hampers T cell-based immune cancer therapies. Here, we performed a genome-wide CRISPR screen to search for genes that regulate T cell activation. Our screen confirmed many of the known regulators in proximal T cell receptor signaling and, importantly, also uncovered a previously uncharacterized regulator, FAM49B (family with sequence similarity 49 member B). FAM49B deficiency led to hyperactivation of Jurkat T cells following T cell receptor stimulation, as indicated by enhancement of CD69 induction, PAK phosphorylation, and actin assembly. FAM49B directly interacted with the active form of the small GTPase Rac, and genetic disruption of the FAM49B-Rac interaction compromised FAM49B function. Thus, FAM49B inhibits T cell activation by repressing Rac activity and modulating cytoskeleton reorganization.


Assuntos
Ativação Linfocitária , Proteínas de Neoplasias/imunologia , Linfócitos T/imunologia , Actinas/genética , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Sistemas CRISPR-Cas , Citoesqueleto/genética , Citoesqueleto/imunologia , Estudo de Associação Genômica Ampla , Humanos , Células Jurkat , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Linfócitos T/citologia
17.
J Genet Genomics ; 44(9): 439-449, 2017 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-28967615

RESUMO

Reverse genetic screens are invaluable for uncovering gene functions, but are traditionally hampered by some technical limitations. Over the past few years, since the advent of the revolutionary CRISPR/Cas9 technology, its power in genome editing has been harnessed to overcome the traditional limitations in reverse genetic screens, with successes in various biological contexts. Here, we outline these CRISPR/Cas9-based screens, provide guidance on the design of effective screens and discuss the potential future directions of development of this field.


Assuntos
Sistemas CRISPR-Cas/genética , Testes Genéticos/métodos , Animais , Humanos
18.
Nat Struct Mol Biol ; 24(12): 1081-1092, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29058713

RESUMO

CD28 provides an essential costimulatory signal for T cell activation, and its function is critical in antitumor immunity. However, the molecular mechanism of CD28 transmembrane signaling remains elusive. Here we show that the conformation and signaling of CD28 are regulated by two counteractive charged factors, acidic phospholipids and Ca2+ ions. NMR spectroscopy analyses showed that acidic phospholipids can sequester CD28 signaling motifs within the membrane, thereby limiting CD28 basal signaling. T cell receptor (TCR) activation induced an increase in the local Ca2+ concentration around CD28, and Ca2+ directly disrupted CD28-lipid interaction, leading to opening and signaling of CD28. We observed that the TCR, Ca2+, and CD28 together form a dual-positive-feedback circuit that substantially amplifies T cell signaling and thus increases antigen sensitivity. This work unravels a new regulatory mechanism for CD28 signaling and thus contributes to the understanding of the dependence of costimulation signaling on TCR signaling and the high sensitivity of T cells.


Assuntos
Antígenos CD28/metabolismo , Cálcio/metabolismo , Ativação Linfocitária/imunologia , Fosfolipídeos/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Conformação Proteica , Receptores de Antígenos de Linfócitos T/imunologia
19.
Proc Natl Acad Sci U S A ; 114(29): E5891-E5899, 2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28659468

RESUMO

Antigen-triggered T-cell receptor (TCR) phosphorylation is the first signaling event in T cells to elicit adaptive immunity against invading pathogens and tumor cells. Despite its physiological importance, the underlying mechanism of TCR phosphorylation remains elusive. Here, we report a key mechanism regulating the initiation of TCR phosphorylation. The major TCR kinase Lck shows high selectivity on the four CD3 signaling proteins of TCR. CD3ε is the only CD3 chain that can efficiently interact with Lck, mainly through the ionic interactions between CD3ε basic residue-rich sequence (BRS) and acidic residues in the Unique domain of Lck. We applied a TCR reconstitution system to explicitly study the initiation of TCR phosphorylation. The ionic CD3ε-Lck interaction controls the phosphorylation level of the whole TCR upon antigen stimulation. CD3ε BRS is sequestered in the membrane, and antigen stimulation can unlock this motif. Dynamic opening of CD3ε BRS and its subsequent recruitment of Lck thus can serve as an important switch of the initiation of TCR phosphorylation.


Assuntos
Complexo CD3/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Sítios de Ligação , Complexo CD3/genética , Membrana Celular/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Fosforilação , Domínios Proteicos , Subunidades Proteicas , Transdução de Sinais , Especificidade por Substrato
20.
Anal Bioanal Chem ; 409(15): 3843-3851, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28374132

RESUMO

Elemental mass spectrometry offers quantitation and isotopic analysis without the need for compound-specific standards. We have recently introduced plasma assisted reaction chemical ionization (PARCI) as an efficient elemental ionization method for halogens. Here, we report a new ionization chemistry in PARCI for facile quantitation of elemental carbon in gas chromatography eluates. We demonstrate that in-plasma reactions of organic compounds followed by afterglow ionization lead to formation of polyatomic anions (CN-, OCN-, and CO3-), among which CN- offers the best analytical sensitivity with a detection limit of ~25 pg (21 pg/s) carbon on column. Using a mixture of pesticides with wide variations in structures and heteroatom content, we demonstrate that CN- ion response is quantitatively correlated with the carbon concentration over two orders of magnitude (r 2 = 0.985). We show that the novel GC-PARCI-MS method provides recoveries within 80-120% using a single standard for all analytes, highlighting the strength of elemental quantitation. Further, the ability of GC-PARCI-MS to identify 13C-tagged molecules without a priori knowledge of chemical formulas of analytes is demonstrated. Graphical abstract ᅟ.

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