A cell-permeable Ub-Dha probe for profiling E1-E2-E3 enzymes in live cells.

Activity-based ubiquitin probes (Ub-ABPs) have recently been developed as effective tools for studying the capabilities of E1-E2-E3 enzymes, but most of them can only be used in cell lysates. Here, we report the first cell-penetrating Ub-Dha probes based on thiazolidine-protected cysteines, which enable successful delivery into cells confirmed by a fluorophore at the N-terminus of Ub and live-cell fluorescence microscopy. A total of 18 E1-E2-E3 enzymes in live cells were labelled and enriched in combination with label-free quantification (LFQ) mass spectrometry. This work provided a new cell-penetrating Ub tool for studying the activity and function of Ub-related enzymes.

Gene association analysis to determine the causal relationship between immune cells and juvenile idiopathic arthritis.

BACKGROUND: Juvenile idiopathic arthritis (JIA) is a type of chronic childhood arthritis with complex pathogenesis. Immunological studies have shown that JIA is an acquired self-inflammatory disease, involving a variety of immune cells, and it is also affected by genetic and environmental susceptibility. However, the precise causative relationship between the phenotype of immune cells and JIA remains unclear to date. The objective of our study is to approach this inquiry from a genetic perspective, employing a method of genetic association analysis to ascertain the causal relationship between immune phenotypes and the onset of JIA. METHODS: In this study, a two-sample Mendelian randomization (MR) analysis was used to select single nucleotide polymorphisms (SNPs) significantly associated with immune cells as instrumental variables to analyze the bidirectional causal relationship between 731 immune cells and JIA. There were four types of immune features (median fluorescence intensity (MFI), relative cellular (RC), absolute cellular (AC), and morphological parameters (MP)). Finally, the heterogeneity and horizontal reproducibility of the results were verified by sensitivity analysis, which ensured more robust results. RESULTS: We found that CD3 on CM CD8br was causally associated with JIA at the level of 0.05 significant difference (95% CI = 0.630 ~ 0.847, P = 3.33 × 10-5, PFDR = 0.024). At the significance level of 0.20, two immunophenotypes were causally associated with JIA, namely: HLA DR on CD14+ CD16- monocyte (95% CI = 0.633 ~ 0.884, P = 6.83 × 10-4, PFDR = 0.16) and HLA DR on CD14+ monocyte (95% CI = 0.627 ~ 0.882, P = 6.9 × 10-4, PFDR = 0.16). CONCLUSION: Our study assessed the causal effect of immune cells on JIA from a genetic perspective. These findings emphasize the complex and important role of immune cells in the pathogenesis of JIA and lay a foundation for further study of the pathogenesis of JIA.

A van der Waals p-n heterostructure of GaSe/SnS2: a high thermoelectric figure of merit and strong anisotropy.

In recent years, van der Waals heterostructures (vdWHs) with controllable and peculiar properties have attracted extensive attention in the fields of electronics, optoelectronics, spintronics and electrochemistry. However, vdWHs with good thermoelectric performance are few due to the complex coupling of thermoelectric coefficients. Here, we employ density functional theory and Boltzmann's transport equation to explore the thermoelectric properties of the p-n vdWH of GaSe/SnS2, which has been experimentally observed to exhibit high performance as an optoelectronic device. We reveal that GaSe/SnS2 possesses strong anisotropy in terms of electronic transport resulting from the anisotropic carrier relaxation time. The longer carrier relaxation time in the y-direction for n-type induces a high power factor of 0.084 W m-1 K-2 at 300 K, while it is only 0.0087 W m-1 K-2) in the x-direction. The strong coupling of low-mid frequency phonon branches and the relatively weak Sn-S bond-induced anharmonicity hinder the phonon transport, which results in the lattice thermal conductivity of GaSe/SnS2 (14.61 and 15.43 W m-1 K-1 along the x- and y-directions at 300 K) being much smaller than the average value of GaSe and SnS2 (43.44 W m-1 K-1 at 300 K). The optimal thermoelectric figure of merit at 700 K for GaSe/SnS2 reaches 2.99, which is significantly higher than those of the constituents of GaSe (0.58) and SnS2 (1.04). The present work highlights the potential thermoelectric applications and the understanding of the thermoelectric transport mechanism for the recently synthesized p-n vdWH of GaSe/SnS2 with a high thermoelectric figure of merit and strong anisotropy.

Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells.

Dynamic monitoring of intracellular ubiquitin (Ub) conjugates is instrumental to understanding the Ub regulatory machinery. Although many biochemical approaches have been developed to characterize protein ubiquitination, chemical tools capable of temporal resolution probing of ubiquitination events remain to be developed. Here, we report the development of the first cell-permeable and stimuli-responsive Ub probe and its application for the temporal resolution profiling of ubiquitinated substrates in live cells. The probe carrying the photolabile group N-(2-nitrobenzyl)-Gly (Nbg) on the amide bond between Ub Gly75 and Gly76 is readily prepared through chemical synthesis and can be delivered to live cells by conjugation via a disulfide bond with the cyclic cell-penetrating peptide cR10D (i.e., 4-((4-(dimethylamino)phenyl)-azo)-benzoic acid-modified cyclic deca-arginine). Both in vitro and in vivo experiments showed that Ub-modifying enzymes (E1, E2s, and E3s) could not install the Ub probe onto substrate proteins prior to removal of the nitrobenzyl group, which was easily accomplished via photoirradiation. The utility and practicality of this probe were exemplified by the time-resolved biochemical and proteomic investigation of ubiquitination events in live cells during a H2O2-mediated oxidative stress response. This work shows a conceptually new family of chemical Ub tools for the time-resolved studies on dynamic protein ubiquitination in different biological processes and highlights the utility of modern chemical protein synthesis in obtaining custom-designed tools for biological studies.

How AI can learn from the law: putting humans in the loop only on appeal.

While the literature on putting a "human in the loop" in artificial intelligence (AI) and machine learning (ML) has grown significantly, limited attention has been paid to how human expertise ought to be combined with AI/ML judgments. This design question arises because of the ubiquity and quantity of algorithmic decisions being made today in the face of widespread public reluctance to forgo human expert judgment. To resolve this conflict, we propose that human expert judges be included via appeals processes for review of algorithmic decisions. Thus, the human intervenes only in a limited number of cases and only after an initial AI/ML judgment has been made. Based on an analogy with appellate processes in judiciary decision-making, we argue that this is, in many respects, a more efficient way to divide the labor between a human and a machine. Human reviewers can add more nuanced clinical, moral, or legal reasoning, and they can consider case-specific information that is not easily quantified and, as such, not available to the AI/ML at an initial stage. In doing so, the human can serve as a crucial error correction check on the AI/ML, while retaining much of the efficiency of AI/ML's use in the decision-making process. In this paper, we develop these widely applicable arguments while focusing primarily on examples from the use of AI/ML in medicine, including organ allocation, fertility care, and hospital readmission.

CISH impairs lysosomal function in activated T cells resulting in mitochondrial DNA release and inflammaging.

Chronic systemic inflammation is one of the hallmarks of the aging immune system. Here we show that activated T cells from older adults contribute to inflammaging by releasing mitochondrial DNA (mtDNA) into their environment due to an increased expression of the cytokine-inducible SH2-containing protein (CISH). CISH targets ATP6V1A, an essential component of the proton pump V-ATPase, for proteasomal degradation, thereby impairing lysosomal function. Impaired lysosomal activity caused intracellular accumulation of multivesicular bodies and amphisomes and the export of their cargos, including mtDNA. CISH silencing in T cells from older adults restored lysosomal activity and prevented amphisomal release. In antigen-specific responses in vivo, CISH-deficient CD4+ T cells released less mtDNA and induced fewer inflammatory cytokines. Attenuating CISH expression may present a promising strategy to reduce inflammation in an immune response of older individuals.

T cell receptor repertoires associated with control and disease progression following Mycobacterium tuberculosis infection.

Antigen-specific, MHC-restricted αß T cells are necessary for protective immunity against Mycobacterium tuberculosis, but the ability to broadly study these responses has been limited. In the present study, we used single-cell and bulk T cell receptor (TCR) sequencing and the GLIPH2 algorithm to analyze M. tuberculosis-specific sequences in two longitudinal cohorts, comprising 166 individuals with M. tuberculosis infection who progressed to either tuberculosis (n = 48) or controlled infection (n = 118). We found 24 T cell groups with similar TCR-ß sequences, predicted by GLIPH2 to have common TCR specificities, which were associated with control of infection (n = 17), and others that were associated with progression to disease (n = 7). Using a genome-wide M. tuberculosis antigen screen, we identified peptides targeted by T cell similarity groups enriched either in controllers or in progressors. We propose that antigens recognized by T cell similarity groups associated with control of infection can be considered as high-priority targets for future vaccine development.

Shear stress enhances anoikis resistance of cancer cells through ROS and NO suppressed degeneration of Caveolin-1.

Circulating tumor cells (CTCs) acquire enhanced anti-anoikis abilities after experiencing flow shear stress in the circulatory system. Our previous study demonstrated that low shear stress (LSS) promotes anoikis resistance of human breast carcinoma cells via caveolin-1 (Cav-1)-dependent extrinsic and intrinsic apoptotic pathways. However, the underlying mechanism how LSS enhanced Cav-1 expression in suspended cancer cells remains unclear. Herein, we found that LSS induced redox signaling was involved in the regulation of Cav-1 level and anoikis resistance in suspension cultured cancer cells. Exposure of human breast carcinoma MDA-MB-231 cells to LSS (2 dyn/cm2) markedly induced ROS and •NO generation, which promoted the cell viability and reduced the cancer cell apoptosis. Furthermore, ROS and •NO scavenging inhibited the upregulation of Cav-1 by interfering ubiquitination, and suppressed the anoikis resistance of suspended tumor cells. These findings provide new insight into the mechanism by which LSS-stimulated ROS and •NO generation increases Cav-1 stabilization in suspended cancer cells through inhibition of ubiquitination and proteasomal degradation, which could be a potential target for therapy of metastatic tumors.

A strain-induced considerable decrease of lattice thermal conductivity in 2D KAgSe with Coulomb interaction.

Based on first-principles calculations in combination with the Boltzmann transport theory, we investigate the effects of onsite Coulomb interaction and strain on the lattice thermal conductivity of the KAgSe monolayer, a recently discovered 2D thermoelectric system with a low lattice thermal conductivity when the onsite Coulomb interaction was not considered (X. Zhang, C. Liu, Y. Tao, Y. Li, Y. Guo, Y. Chen, X. C. Zeng and J. Wang, Adv. Funct. Mater., 2020, 30, 2001200). Our calculations reveal that the onsite Coulomb interaction leads to an increase in the lattice thermal conductivity from 1.22 to 1.82 W m-1 K-1 at room temperature due to the increased phonon group velocity and relaxation time. However, with onsite Coulomb interaction, small 3% biaxial tensile strain can give rise to a 75% considerable decrease in the lattice thermal conductivity at room temperature, from 1.82 to 0.45 W m-1 K-1, which is also much lower than the lattice thermal conductivity of 1.22 W m-1 K-1 without onsite Coulomb interaction and strain. The strain induced decrease of phonon group velocity and enhancement of lattice anharmonicity (large Grüneisen parameter and phase space volume) are responsible for the reduced lattice thermal conductivity. The present work highlights that the onsite Coulomb interaction is indispensable when determining the lattice thermal conductivity of 2D KAgSe, and small tensile strain can greatly decrease the lattice thermal conductivity.

A High-Throughput Strategy for T-Cell Receptor Cloning and Expression.

Expression of T-cell receptor (TCR) genes is a critical step for TCR characterization and epitope identification. The recent interest in using specific TCRs for cancer immunotherapy has further increased the demand for practical and robust methods to rapidly clone and express TCRs. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TCR transgene into different expression systems. In this protocol, we first constructed all the human TRAV and TRBV genes into individual plasmid. To clone any TCR, we only need to ligate a short CDR3 fragment to its corresponding V gene plasmid using Golden Gate cloning. This strategy significantly improves the efficiency of individual TCR cloning and mutagenesis, providing a flexible high-throughput method for TCR analysis and TCR-mediated therapeutics.

Effective photodegradation of rhodamine B and levofloxacin over CQDs modified BiOCl and BiOBr composite: Mechanism and toxicity assessment.

In this contribution, carbon quantum dots (CQDs) modified 3D-flower like BiOX (X = Cl, Br, I) photocatalyst were successfully prepared via a facile mechanical compounding method. The crystal structure, surface composition, morphologies, optical properties and photocatalytic activities were investigated in detail. The photocatalytic activity of the as-obtained photocatalyst were evaluated by degradation of rhodamine B (RhB) and Levofloxacin (LEV) under near IR-UV-vis light irradiation, the CQDs/BiOX composite displayed enhanced photocatalytic activity as compared with individual BiOX materials. The CQDs/BiOX composite had the outstanding light harvesting and electron transfer ability because of the ordered ultrathin nanosheet structure of the BiOX, the formation of metal Bi under photoinduction, and the synergistic effects between CQDs and pure BiOX. Antibacterial activity and effects on Rye seeds growth of the LEV degradation intermediate were also researched. Reactive-species-trapping experiments exhibited that h+ and O2- were the active reactive species during photodegradation process. This work provided an effective and simple strategy for designing QDs modified Bi-rich oxyhalides in organic pollutant containing wastewater treatment.

LncRNA LUCAT1 as a Plasma Biomarker for Assessing Disease Activity in Adult Patients with Crohn's Disease.

AIM: To explore the expression of long noncoding RNA (LncRNA) LUCAT1 in adult patients with Crohn's disease (CD) and evaluate the relationship between LncRNA LUCAT1 and the disease activity in Chinese patients with CD. METHODS: Patients with CD and healthy participants (≥18 years old) were enrolled in this study between January 2018 and December 2019. The expression of LncRNA LUCAT1 in plasma samples was evaluated by quantitative reverse transcription-polymerase chain reaction. Basic characteristics of patients with CD were collected, including gender, age, clinical stage, disease behavior, disease location, C-reactive protein (CRP), platelet (PLT), erythrocyte sedimentation rate (ESR), fecal calprotectin (FC), Crohn's disease activity index (CDAI) score, and simplified Crohn's disease endoscopic score (SES-CD). RESULTS: In total, 168 patients with CD and 65 healthy participants (≥18 years old) were enrolled in this study. Among them, ninety patients with clinically active CD, seventy-eight patients with CD in clinical remission, forty-eight patients with endoscopically active CD, thirty patients with endoscopically inactive CD, and sixty-five healthy participants. LncRNA LUCAT1 was increased in plasma of patients with CD compared with the control group. The plasma LncRNA LUCAT1 level of patients with CD both in the clinical and endoscopic active phase was higher than that of both the clinical and endoscopic remission phase. The plasma level of LncRNA LUCAT1 in patients with CD was positively correlated with ESR, CRP, FC, CDAI, and SES-CD. There was no significant correlation between the level of LUCAT1 and platelets. The plasma LncRNA LUCAT1 level in patients with CD had significant differences between severe active patients and mild/moderate active patients. CONCLUSION: The plasma LncRNA LUCAT1 is positively associated with the disease activity in patients with CD, and it may act as a noninvasive biomarker to identify the degree of disease activity.

Activation of mTORC1 at late endosomes misdirects T cell fate decision in older individuals.

The nutrient-sensing mammalian target of rapamycin (mTOR) is integral to cell fate decisions after T cell activation. Sustained mTORC1 activity favors the generation of terminally differentiated effector T cells instead of follicular helper and memory T cells. This is particularly pertinent for T cell responses of older adults who have sustained mTORC1 activation despite dysfunctional lysosomes. Here, we show that lysosome-deficient T cells rely on late endosomes rather than lysosomes as an mTORC1 activation platform, where mTORC1 is activated by sensing cytosolic amino acids. T cells from older adults have an increased expression of the plasma membrane leucine transporter SLC7A5 to provide a cytosolic amino acid source. Hence, SLC7A5 and VPS39 deficiency (a member of the HOPS complex promoting early to late endosome conversion) substantially reduced mTORC1 activities in T cells from older but not young individuals. Late endosomal mTORC1 is independent of the negative-feedback loop involving mTORC1-induced inactivation of the transcription factor TFEB that controls expression of lysosomal genes. The resulting sustained mTORC1 activation impaired lysosome function and prevented lysosomal degradation of PD-1 in CD4+ T cells from older adults, thereby inhibiting their proliferative responses. VPS39 silencing of human T cells improved their expansion to pertussis and to SARS-CoV-2 peptides in vitro. Furthermore, adoptive transfer of CD4+ Vps39-deficient LCMV-specific SMARTA cells improved germinal center responses, CD8+ memory T cell generation, and recall responses to infection. Thus, curtailing late endosomal mTORC1 activity is a promising strategy to enhance T cell immunity.

Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects.

Several existing technologies enable short genomic alterations including generating indels and short nucleotide variants, however, engineering more significant genomic changes is more challenging due to reduced efficiency and precision. Here, we developed RecT Editor via Designer-Cas9-Initiated Targeting (REDIT), which leverages phage single-stranded DNA-annealing proteins (SSAP) RecT for mammalian genome engineering. Relative to Cas9-mediated homology-directed repair (HDR), REDIT yielded up to a 5-fold increase of efficiency to insert kilobase-scale exogenous sequences at defined genomic regions. We validated our REDIT approach using different formats and lengths of knock-in templates. We further demonstrated that REDIT tools using Cas9 nickase have efficient gene-editing activities and reduced off-target errors, measured using a combination of targeted sequencing, genome-wide indel, and insertion mapping assays. Our experiments inhibiting repair enzyme activities suggested that REDIT has the potential to overcome limitations of endogenous DNA repair steps. Finally, our REDIT method is applicable across cell types including human stem cells, and is generalizable to different Cas9 enzymes.

Internet False News Information Feature Extraction and Screening Based on 5G Internet of Things Combined with Passive RFID.

In this paper, the authenticity of news information on the 5G Internet of Things (IoT) is studied, and a network false news information screening platform is designed and optimized by IoT combined with passive RFID. The electronic license chain based on data sovereignty is established, in which, combined with the identity identification and strong correlation ability based on the electronic license chain, a cross-industry, cross-business, and cross-field behavior record base database is formed; then, a digital library is constructed based on this base library; finally, through data sharing and management, a false news information feature extraction and screening platform is formed for the orderly management and reasonable dispatch of government resources and reducing various risks. The main functional modules implemented by the platform are the acquisition of news data and comment data, the retrieval and analysis of news data, the false detection of online news, and the visualization of false news data. However, there is still much public who are not aware or do not understand that news truth is this dynamic form. Therefore, this paper aims to inform the public that news truth in news context is a dynamic process by 5G Internet of Things combined with passive RFID. The public understands the circumstances where news truth may be dynamic truth to avoid being misled by false news.

CRISPR-Cas12a System With Synergistic Phage Recombination Proteins for Multiplex Precision Editing in Human Cells.

The development of CRISPR-based gene-editing technologies has brought an unprecedented revolution in the field of genome engineering. Cas12a, a member of the Class 2 Type V CRISPR-associated endonuclease family distinct from Cas9, has been repurposed and developed into versatile gene-editing tools with distinct PAM recognition sites and multiplexed gene targeting capability. However, with current CRISPR/Cas12a technologies, it remains a challenge to perform efficient and precise genome editing of long sequences in mammalian cells. To address this limitation, we utilized phage recombination enzymes and developed an efficient CRISPR/Cas12a tool for multiplexed precision editing in mammalian cells. Through protein engineering, we were able to recruit phage recombination proteins to Cas12a to enhance its homology-directed repair efficiencies. Our phage-recombination-assisted Cas12a system achieved up to 3-fold improvements for kilobase-scale knock-ins in human cells without compromising the specificity of the enzyme. The performance of this system compares favorably against Cas9 references, the commonly used enzyme for gene-editing tasks, with improved specificity. Additionally, we demonstrated multi-target editing with similar improved activities thanks to the RNA-processing activity of the Cas12a system. This compact, multi-target editing tool has the potential to assist in understanding multi-gene interactions. In particular, it paves the way for a gene therapy method for human diseases that complements existing tools and is suitable for polygenic disorders and diseases requiring long-sequence corrections.

Identification of infectious spleen and kidney necrosis virus (ISKNV)-encoded microRNAs.

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression by complementary binding to target mRNAs. Virus-encoded miRNAs play important roles in virus life cycle and virus-host interactions. Viruses from the Megalocytivirus genus, family Iridoviridae, infect a wide range of fishes, bringing great challenges to aquaculture. Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus. In this study, using Illumina sequencing coupled with miRNA precursor prediction and stem-loop real-time PCR, 14 putative ISKNV-encoded miRNAs were preliminarily identified from ISKNV-infected mandarin fish MFF-1 cells. To initially study their functions, inhibitors of the 14 viral miRNAs were synthesized and transfected into MFF-1 cells, which were further infected with ISKNV. The results showed that these viral miRNAs could affect the virus titers in the supernatant of ISKNV-infected cells and the expression of major capsid protein (MCP). Moreover, we observed that inhibition of several ISKNV miRNAs had different effects on MCP expression and on titer of released virus, suggesting complex roles of viral miRNAs in ISKNV infection. The current study may provide a fundamental information for further identification and functional studies on miRNAs encoded by Megalocytivirus.

Shear stress stimulates integrin ß1 trafficking and increases directional migration of cancer cells via promoting deacetylation of microtubules.

In egress routes of malignancy, cancer cells are constantly subjected to shear stress imposed by blood/lymph flow. Increasing evidence points toward the regulatory roles of shear stress in tumor cell adhesion and motility. Although it is known that integrin endocytic trafficking governs focal adhesion (FA) turnover and cell migration, the effect and biological consequences of low shear stress (LSS) on integrin trafficking remain unclear. Here, we identified the critical role of integrin ß1 trafficking and caveolin-1 (Cav-1) mediated endocytosis in LSS-induced cell directional migration. LSS altered the distribution of integrin ß1 in MDA-MB-231 cells and significantly promoted its internalization and recycling, which in turn facilitated FA turnover and directional cell migration. Furthermore, LSS induced cytoskeleton remodeling, which was required for internalization of integrin ß1. LSS down-regulated the acetylation level of microtubules (MTs) via activating ROCK/HDAC6 pathway, resulting in elevation of MTs dynamics, Cav-1 motility, and Cav-1-dependent integrin ß1 recycling. We also showed that high HDAC6 expression was a ROCK-dependent prognostic factor, which was correlated with poor outcomes in breast cancer patients. Taken together, these results defined a novel mechanism by which LSS enhanced integrin ß1 trafficking via actin cytoskeleton remodeling and ROCK/HDAC6 mediated deacetylation of MTs, thereby promoting FAs turnover and directional cell migration.

Matrix stiffness modulates ILK-mediated YAP activation to control the drug resistance of breast cancer cells.

One of the hallmarks of cancer progression is strong drug resistance during clinical treatments. The tumor microenvironment is closely associated with multidrug resistance, the optimization of tumor microenvironments may have a strong therapeutic effect. In this study, we configured polyacrylamide hydrogels of varying stiffness [low (10 kPa), intermediate (38 kPa) and high (57 kPa)] to simulate tissue physical matrix stiffness across different stages of breast cancer. After treatment with doxorubicin, cell survival rates on intermediate stiffness substrate are significantly higher. We find that high expression of ILK and YAP reduces the survival rates of breast cancer patients. Drug resistance is closely associated with the inactivation of the hippo pathway protein Merlin/MST/LATS and the activation of YAP. These results not only highlight the understanding of drug resistance mechanisms but also serve as a new basis for developing breast cancer treatment delivery systems.