Facile hermetic TEM grid preparation for molecular imaging of hydrated biological samples at room temperature.

Although structures of vitrified supramolecular complexes have been determined at near-atomic resolution, elucidating in situ molecular structure in living cells remains a challenge. Here, we report a straightforward liquid cell technique, originally developed for real-time visualization of dynamics at a liquid-gas interface using transmission electron microscopy, to image wet biological samples. Due to the scattering effects from the liquid phase, the micrographs display an amplitude contrast comparable to that observed in negatively stained samples. We succeed in resolving subunits within the protein complex GroEL imaged in a buffer solution at room temperature. Additionally, we capture various stages of virus cell entry, a process for which only sparse structural data exists due to their transient nature. To scrutinize the morphological details further, we used individual particle electron tomography for 3D reconstruction of each virus. These findings showcase this approach potential as an efficient, cost-effective complement to other microscopy technique in addressing biological questions at the molecular level.

Treg cell: Critical role of regulatory T-cells in depression.

Depression is a highly prevalent disorder of the central nervous system. The neuropsychiatric symptoms of clinical depression are persistent and include fatigue, anorexia, weight loss, altered sleep patterns, hyperalgesia, melancholia, anxiety, and impaired social behaviours. Mounting evidences suggest that neuroinflammation triggers dysregulated cellular immunity and increases susceptibility to psychiatric diseases. Neuroimmune responses have transformed the clinical approach to depression because of their roles in its pathophysiology and their therapeutic potential. In particular, activated regulatory T (Treg) cells play an increasingly evident role in the inflammatory immune response. In this review, we summarized the available data and discussed in depth the fundamental roles of Tregs in the pathogenesis of depression, as well as the clinical therapeutic potential of Tregs. We aimed to provide recent information regarding the potential of Tregs as immune-modulating biologics for the treatment and prevention of long-term neuropsychiatric symptoms of depression.

Biobased epoxidized natural rubber/sodium carboxymethyl cellulose composites with enhanced strength and healing ability.

Conventional vulcanized rubbers cause a non-negligible waste of resources due to the formation of 3D irreversible covalently cross-linked networks. The introduction of reversible covalent bonds, such as reversible disulfide bonds, into the rubber network, is an available solution to the above problem. However, the mechanical properties of rubber with only reversible disulfide bonds cannot meet most practical applications. In this paper, a strengthened bio-based epoxidized natural rubber (ENR) composite reinforced by sodium carboxymethyl cellulose (SCMC) was prepared. SCMC forms a mass of hydrogen bonds between its hydroxyl groups and the hydrophilic groups of ENR chain, which gives the ENR/2,2'-Dithiodibenzoic acid (DTSA)/SCMC composites an enhanced mechanical performance. With 20 phr SCMC, the tensile strength of the composite increases from 3.0 to 10.4 MPa, which is almost 3.5 times that of the ENR/DTSA composite without SCMC. Simultaneously, DTSA covalently cross-linked ENR with the introduction of reversible disulfide bonds, which enables the cross-linked network to rearrange its topology at low temperatures and thus endows the ENR/DTSA/SCMC composites with healing properties. The ENR/DTSA/SCMC-10 composite has a considerable healing efficiency of about 96 % after healing at 80 °C for 12 h.

Serum brain-derived neurotrophic factor as diagnosis clue for Alzheimer's disease: A cross-sectional observational study in the elderly.

Objective: Brain-derived neurotrophic factor (BDNF) has not been validated as a diagnostic marker for Alzheimer's disease (AD). To provide a different perspective, this study aimed to evaluate the relationship between serum levels of mature BDNF (mBDNF) and precursor BDNF (proBDNF) in AD and to investigate whether serum BDNF levels or the ratio of mBDNF levels to proBDNF levels (M/P) could be a valuable biomarker for determining the risk of AD in elderly individuals. Method: A total of 126 subjects who met the inclusion criteria were assigned to either the AD group (n = 62) or the healthy control group (HC, n = 64) in this cross-sectional observationl study. Serum levels of mBDNF and proBDNF were measured using enzyme immunoassay kits. We analyzed the Mini-Mental State Examination (MMSE) scores from the two groups and examined the associations between AD and BDNF metabolism. Results: The serum concentration of proBDNF was significantly higher in ADs (4140.937 pg/ml) than in HCs (2606.943 pg/ml; p < 0.01). The MMSE significantly correlated with proBDNF (p < 0.01, r = -0.686) and M/P (p < 0.01, r = 0.595) in all subjects. To determine the risk for AD, the area under the receiver operating characteristic curve was calculated, which was 0.896 (95% confidence interval 0.844-0.949) for proBDNF and 0.901 (95% 0.850-0.953) for proBDNF and M/P combined. Conclusion: We observed a correlation between low serum proBDNF levels and higher MMSE scores in AD. The most effective diagnostic strategy was the combination of proBDNF and M/P, whereas mBDNF levels performed poorly when we evaluated the predictive model.

The ubiquitin E3 ligase TRIM10 promotes STING aggregation and activation in the Golgi apparatus.

STING is an endoplasmic reticulum-resident protein regulating innate immunity. After binding with cyclic guanosine monophosphate-AMP (cGAMP), STING translocates from the endoplasmic reticulum (ER) to the Golgi apparatus to stimulate TBK1 and IRF3 activation, leading to expression of type I interferon. However, the exact mechanism concerning STING activation remains largely enigmatic. Here, we identify tripartite motif 10 (TRIM10) as a positive regulator of STING signaling. TRIM10-deficient macrophages exhibit reduced type I interferon production upon double-stranded DNA (dsDNA) or cGAMP stimulation and decreased resistance to herpes simplex virus 1 (HSV-1) infection. Additionally, TRIM10-deficient mice are more susceptible to HSV-1 infection and exhibit faster melanoma growth. Mechanistically, TRIM10 associates with STING and catalyzes K27- and K29-linked polyubiquitination of STING at K289 and K370, which promotes STING trafficking from the ER to the Golgi apparatus, formation of STING aggregates, and recruitment of TBK1 to STING, ultimately enhancing the STING-dependent type I interferon response. Our study defines TRIM10 as a critical activator in cGAS-STING-mediated antiviral and antitumor immunity.

Facile hermetic TEM grid preparation for molecular imaging of hydrated biological samples at room temperature.

Although structures of vitrified supramolecular complexes have been determined at near-atomic resolution, elucidating in situ molecular structure in living cells remains a major challenge. Here, we apply a novel but simple liquid-cell technique, developed previously for real-time imaging of the dynamics at a liquid-gas interface, to image wet biological samples. With extra scattering from the liquid phase, the transmission electron micrographs show amplitude contrast comparable to that in negatively stained samples. Single-molecule domains are resolved in the protein complex GroEL imaged in buffer solution at room temperature. Moreover, various stages of virus cell entry, which are transient events with very few structural information to date, are also captured. Morphological details are reconstructed using the technique of individual particle electron tomography. These results demonstrate that this approach can be a valuable yet cost-effective technique complementary to other microscopy techniques for addressing important biological questions at the molecular level.

Electron energy loss spectroscopy database synthesis and automation of core-loss edge recognition by deep-learning neural networks.

The ionization edges encoded in the electron energy loss spectroscopy (EELS) spectra enable advanced material analysis including composition analyses and elemental quantifications. The development of the parallel EELS instrument and fast, sensitive detectors have greatly improved the acquisition speed of EELS spectra. However, the traditional way of core-loss edge recognition is experience based and human labor dependent, which limits the processing speed. So far, the low signal-noise ratio and the low jump ratio of the core-loss edges on the raw EELS spectra have been challenging for the automation of edge recognition. In this work, a convolutional-bidirectional long short-term memory neural network (CNN-BiLSTM) is proposed to automate the detection and elemental identification of core-loss edges from raw spectra. An EELS spectral database is synthesized by using our forward model to assist in the training and validation of the neural network. To make the synthesized spectra resemble the real spectra, we collected a large library of experimentally acquired EELS core edges. In synthesize the training library, the edges are modeled by fitting the multi-Gaussian model to the real edges from experiments, and the noise and instrumental imperfectness are simulated and added. The well-trained CNN-BiLSTM network is tested against both the simulated spectra and real spectra collected from experiments. The high accuracy of the network, 94.9%, proves that, without complicated preprocessing of the raw spectra, the proposed CNN-BiLSTM network achieves the automation of core-loss edge recognition for EELS spectra with high accuracy.

Design of epoxidized natural rubber/poly(lipoic acid) elastomer with fast and efficient self-healing under a mild temperature.

Most of the dynamic covalent bonds (DCBs) for self-healing rubber must be activated at relatively high temperatures due to the requirement of high energy during the exchange of dynamic bonds, which may lead to unexpected degradation or excessive crosslinking of rubber. Herein, we designed and fabricated a highly stretchable, self-healable and reprocessable rubber by introducing dynamic disulfide bonds into the crosslink network of epoxidized natural rubber (ENR). Lipoic acid (LA) was firstly uniformly dispersed into ENR via a latex film formation technique, and then underwent a dynamic covalent ring-opening self-polymerization during hot pressing process, during which the carboxyl group of poly(LA) attacked the epoxy group of ENR to form ß-hydroxyl ester bond crosslinks. As a result, a revisable covalently crosslinked network without rigid steric hindrance groups was constructed, which exhibited a super self-healing efficiency of 99 % after self-healing at 80 °C for only 3 h. The elongation at break of the elastomer could reach 1115 % and the recovery rate of reprocessing was as high as 91 %.

Pharmacokinetics and Pharmacodynamics of Colistin Combined With Isopropoxy Benzene Guanidine Against mcr-1-Positive Salmonella in an Intestinal Infection Model.

Plasmid-borne colistin resistance mediated by mcr-1 is a growing problem, which poses a serious challenge to the clinical application of colistin for Gram-negative bacterial infections. Drug combination is one of the effective strategies to treat colistin-resistant bacteria. Here, we found a guanidine compound, namely, isopropoxy benzene guanidine (IBG), which boosted the efficacy of colistin against mcr-1-positive Salmonella. This study aimed to develop a pharmacokinetics/pharmacodynamics (PK/PD) model by combining colistin with IBG against mcr-1-positive Salmonella in an intestinal infection model. Antibiotic susceptibility testing, checkerboard assays and time-kill curves were used to investigate the antibacterial activity of the synergistic activity of the combination. PK studies of colistin in the intestine were determined through oral gavage of single dose of 2, 4, 8, and 16 mg/kg of body weight in broilers with intestinal infection. On the contrary, PD studies were conducted over 24 h based on a single dose ranging from 2 to 16 mg/kg. The inhibitory effect I max model was used for PK/PD modeling. The combination of colistin and IBG showed significant synergistic activity. The AUC0-24h /MIC index was used to evaluate the relationship between PK and PD, and the correlation was >0.9085. The AUC0-24h /MIC targets in combination required to achieve the bacteriostatic action, 3-log10 kill, and 4-log10 kill of bacterial counts were 47.55, 865.87, and 1894.39, respectively. These results can facilitate the evaluation of the use of IBG as a potential colistin adjuvant in the treatment of intestinal diseases in broilers caused by colistin-resistant Salmonella.

Single-molecule 3D imaging of HIV cellular entry by liquid-phase electron tomography.

Enveloped viruses, including human immunodeficiency virus (HIV) and SARS-CoV-2, target cells through membrane fusion process. The detailed understanding of the process is sought after for vaccine development but remains elusive due to current technique limitations for direct three-dimensional (3D) imaging of an individual virus during its viral entry. Recently, we developed a simple specimen preparation method for real-time imaging of metal dynamic liquid-vaper interface at nanometer resolution by transmission electron microscopy (TEM). Here, we extended this method to study biology sample through snapshot 3D structure of a single HIV (pseudo-typed with the envelope glycoprotein of vesicular stomatitis virus, VSV-G) at its intermediate stage of viral entry to HeLa cells in a liquid-phase environment. By individual-particle electron tomography (IPET), we found the viral surface release excess lipids with unbound viral spike proteins forming ~50-nm nanoparticles instead of merging cell membrane. Moreover, the spherical-shape shell formed by matrix proteins underneath the viral envelope does not disassemble into a cone shape right after fusion. The snapshot 3D imaging of a single virus provides us a direct structure-based understanding of the viral entry mechanism, which can be used to examine other viruses to support the development of vaccines combatting the current ongoing pandemic.

TRIM31 facilitates K27-linked polyubiquitination of SYK to regulate antifungal immunity.

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase, which plays an essential role in both innate and adaptive immunity. However, the key molecular mechanisms that regulate SYK activity are poorly understood. Here we identified the E3 ligase TRIM31 as a crucial regulator of SYK activation. We found that TRIM31 interacted with SYK and catalyzed K27-linked polyubiquitination at Lys375 and Lys517 of SYK. This K27-linked polyubiquitination of SYK promoted its plasma membrane translocation and binding with the C-type lectin receptors (CLRs), and also prevented the interaction with the phosphatase SHP-1. Therefore, deficiency of Trim31 in bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) dampened SYK-mediated signaling and inhibited the secretion of proinflammatory cytokines and chemokines against the fungal pathogen Candida albicans infection. Trim31-/- mice were also more sensitive to C. albicans systemic infection than Trim31+/+ mice and exhibited reduced Th1 and Th17 responses. Overall, our study uncovered the pivotal role of TRIM31-mediated K27-linked polyubiquitination on SYK activation and highlighted the significance of TRIM31 in anti-C. albicans immunity.

[Analysis of ACAT1 gene variants in a patient with ß-ketothiolase deficiency].

OBJECTIVE: To explore the genetic etiology of a child suspected for ß-ketothiolase deficiency by neonatal screening. METHODS: All coding exons and flanking sequences of the ACAT1 gene were subjected to targeted capture and high-throughput sequencing. Suspected variants were verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child was found to harbor compound heterozygous variants of the ACAT1 gene, namely c.121-3C>G and c.275G>A (p. Gly92Asp). The c.121-3C>G variant was also detected in his father and two sisters, while the c.275G>A (p. Gly92Asp) was a de novo variant. A c.334+ 172C>G (rs12226047) polymorphism was also detected in his mother and two sisters. Sanger sequencing has verified that the c.275G>A (p. Gly92Asp) and c.334+172C>G (rs12226047) variants are located on the same chromosome. Bioinformatics analysis suggested both c.121-3C>G and c.275G>A (p.G92D) variants to be damaging. Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.275G>A variant of the ACAT1 gene was predicted to be pathogenic (PS2+ PM2+ PM3+ PP3+PP4), the c.121-3C>G variant to be likely pathogenic (PM2+ PM3+ PP3+PP4). CONCLUSION: The c.121-3C>G and c.275G>A variants of the ACAT1 gene probably underlay the pathogenesis of the child. Above finding has enriched the variant spectrum of the ACAT1 gene.

Circ_0000396 inhibits rheumatoid arthritis synovial fibroblast growth and inflammatory response via miR-203/HBP1 axis.

BACKGROUND: Circ_0000396 was found to be down-regulated in the rheumatoid arthritis (RA) patients and had a high diagnostic value. However, the function and mechanisms underlying circ_0000396 in RA progression remain unclear. METHODS: The expression of circ_0000396, microRNA (miR)-203 and HMG-box transcription factor 1 (HBP1) was detected using qRT-PCR and western blot. The proliferative and apoptotic capabilities of rheumatoid arthritis synovial fibroblasts (RASFs) were measured by colony formation, CCK-8, flow cytometry and western blot assays, respectively. The levels of interleukins (IL)-6, IL-1ß, IL-8 and tumor necrosis factor-α (TNF-α) were detected using enzyme-linked immunosorbent assay (ELISA). The target correlations between miR-203 and circ_0000396 or HBP1 were validated using pull-down and dual-luciferase reporter assay. RESULTS: Circ_0000396 was decreased in RA synovial tissues and RASFs, and overexpression of circ_0000396 suppressed cell proliferation, induced cell apoptosis and reduced the release of inflammatory cytokine IL-6, IL-1ß, IL-8 and TNF-α in RASFs, while circ_0000396 deletion functioned oppositely. MiR-203 was confirmed to be a target of circ_0000396, and miR-203 reversed the protective effects of circ_0000396 on the dysfunction and inflammation of RASFs. HBP1 was a target of miR-203, and silencing miR-203 inhibited RASFs malignant changes by regulating HBP1. In addition, circ_0000396 could regulate HBP1 by sponging miR-203, and HBP1 decrease attenuated the effects of circ_0000396 on RASF growth and inflammation. CONCLUSION: Circ_0000396 inhibited the growth and inflammation in RASFs by regulating miR-203/HBP1 axis, providing a potential therapeutic target for RA.

OTUD5 promotes innate antiviral and antitumor immunity through deubiquitinating and stabilizing STING.

Stimulator of interferon genes (STING) is an adaptor protein that is critical for effective innate antiviral and antitumor immunity. The activity of STING is heavily regulated by protein ubiquitination, which is fine-tuned by both E3 ubiquitin ligases and deubiquitinases. Here, we report that the deubiquitinase OTUD5 interacts with STING, cleaves its K48-linked polyubiquitin chains, and promotes its stability. Consistently, knockout of OTUD5 resulted in faster turnover of STING and subsequently impaired type I IFN signaling following cytosolic DNA stimulation. More importantly, Lyz2-Cre Otud5fl/Y mice and CD11-Cre Otud5fl/Y mice showed more susceptibility to herpes simplex virus type 1 (HSV-1) infection and faster development of melanomas than their corresponding control littermates, indicating that OTUD5 is indispensable for STING-mediated antiviral and antitumor immunity. Our data suggest that OTUD5 is a novel checkpoint in the cGAS-STING cytosolic DNA sensing pathway.

Rhodojaponin II inhibits TNF-α-induced inflammatory cytokine secretion in MH7A human rheumatoid arthritis fibroblast-like synoviocytes.

Rhodojaponin II (R-II) has been shown to possess anti-inflammatory activity. Herein, we aimed to explore the effect of R-II on tumor necrosis factor-α (TNF-α)-induced inflammation in MH7A rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs). We found that R-II treatment at high concentration suppressed the viability of MH7A cells. R-II suppressed the levels of nitric oxide and prostaglandin E2, and inhibited messenger RNA expression and concentrations of interleukin-1ß (IL-1ß), IL-6 and matrix metalloproteinase-1 in TNF-α-stimulated RA-FLSs. Additionally, R-II repressed TNF-α-induced activation of the Akt, nuclear factor-κB (NF-κB), and toll-like receptor 4 (TLR4)/MyD88 pathways in MH7A cells. Inhibition of the Akt, NF-κB, and TLR4/MyD88 pathways by the corresponding inhibitors reinforced the inhibitory effect of R-II on TNF-α-induced inflammatory cytokine secretion in MH7A cells. R-II ameliorated the severity of collagen-induced arthritis in mice by inhibiting inflammation. In conclusion, R-II repressed TNF-α-induced inflammatory response in MH7A cells by inactivating the Akt, NF-κB, and TLR4/MyD88 pathways.

Protocatechuic acid inhibits proliferation, migration and inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a chronic joint inflammatory disease that is closely associated with dysregulation of fibroblast-like synoviocytes (FLSs). Protocatechuic acid (PCA), a phenolic compound of anthocyanins, has been proven to possess anti-inflammatory activity. However, the role of PCA in RA has not been investigated. In the present study, we aimed to explore the effects of PCA on the RA-FLSs. The results showed that PCA suppressed the proliferation, invasion, and migration of RA-FLSs in a dose-dependent manner. PCA treatment also inhibited the expressions of matrix metalloproteinase (MMP)-3 and MMP-13, as well as the secretion of inflammatory cytokines including TNF-α, IL-1ß, IL-6 in RA-FLSs. Moreover, cell apoptosis of RA-FLSs was significantly induced by PCA treatment. PCA was found to repress the activation of NF-κB signalling, which was evidenced by the decreased expression of p-p65 and increased expression of IκBα. Furthermore, PCA significantly decreased the phosphorylation levels of Akt and mTOR in RA-FLSs. In conclusion, the results indicated that PCA exhibited an inhibitory effect on RA-FLSs via inhibiting the NF-κB and Akt/mTOR signalling pathways. These findings supported the concept that PCA might be a therapeutic agent for RA treatment.

Cutting Edge: USP27X Deubiquitinates and Stabilizes the DNA Sensor cGAS to Regulate Cytosolic DNA-Mediated Signaling.

Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, catalyzes the formation of the second messenger 2'3'-cGAMP that binds to STING and triggers the type I IFN signaling. Activation of cGAS can be modulated by several protein posttranslational modifications, including ubiquitination. However, the cGAS activation regulated by protein deubiquitination remains poorly understood. In this study, we identified that deubiquitinase USP27X could interact with cGAS and cleave K48-linked polyubiquitination chains from cGAS, leading to cGAS stabilization. Consistently, knockout of Usp27x in mice macrophages resulted in an accelerated turnover of cGAS, decreased cGAMP production, phosphorylation of TBK1 and IRF3, and IFN-ß production. Furthermore, Usp27x knockout mice macrophages showed impaired innate antiviral responses against HSV type 1 infection. Our data suggest that USP27X is a novel regulator of the cGAS-STING cytosolic DNA sensing pathway.

Kinetics, thermodynamics, and physical characterization of corn stover (Zea mays) for solar biomass pyrolysis potential analysis.

Solar pyrolysis of agricultural waste has huge potential for sustainable production of fuel and chemical feedstock. In this paper, the kinetics, thermodynamics, and physical characterization of corn stover (CS) collected from Wyoming, USA was conducted with respect to solar pyrolysis. The kinetics and thermodynamics of the CS pyrolysis was analyzed in detail using the methods described by KAS (Kissinger-Akahira-Sunose) and FWO (Flynn-Wall-Ozawa), from which the activation energy, Gibbs energy, Arrhenius pre-exponential factor, enthalpy, and entropy were derived.14 other kinetics models based on reaction order, diffusion, nucleation, geometric contraction, power models were also examined, and models based on diffusion was found to be best suited. The CS was used for solar pyrolysis of biomass and the products were analyzed by mass spectroscopy, ICP-MS, GPC, micro-GC, and Elemental analyzer. The results show that CS is suitable for solar pyrolysis to produce chemicals and other fuels.