TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages.

Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.

Expanding the "Magic Triangle" of Reinforced Rubber Using a Supramolecular Filler Strategy.

A strategy for optimizing the rolling resistance, wet skid and cut resistance of reinforced rubber simultaneously using a supramolecular filler is demonstrated. A ß-alanine trimer-grafted Styrene Butadiene Rubber (A3-SBR) pristine polymer was designed and mechanically mixed with commercially available styrene butadiene rubber to help the dispersion of a ß-alanine trimer (A3) supramolecular filler in the rubber matrix. To increase the miscibility of A3-SBR with other rubber components during mechanical mixing, the pristine polymer was saturated with ethanol before mixing. The mixture was vulcanized using a conventional rubber processing method. The morphology of the assembles of the A3 supramolecular filler in the rubber matrix was studied by Differential Scanning Calorimetry (DSC) and Transmission Electron Microscopy (TEM). The Differential Scanning Calorimetry study showed that the melting temperature of ß-sheet crystals in the vulcanizates was around 179 °C and was broad. The melting temperature was similar to that of the pristine polymer, and the broad melting peak likely suggests that the size of the crystals is not uniform. The Transmission Electron Microscopy study revealed that after mixing the pristine polymer with SBR, some ß-sheet crystals were rod-like with several tens of nanometers and some ß-sheet crystals were particulate with low aspect ratios. Tensile testing with pre-cut specimens showed that the vulcanizate containing A3-SBR was more cut-resistant than the one that did not contain A3-SBR, especially at a large cut size. The rolling resistance and wet skid were predicted by dynamic mechanical analysis (DMA). DMA tests showed that the vulcanizates containing A3-SBR were significantly less hysteretic at 60 °C and more hysteretic at 0 °C based on loss factor. Overall, the "magic triangle" was expanded by optimizing the rolling resistance, wet-skid, and cut resistance simultaneously using a ß-alanine trimer supramolecular filler. The Payne effect also became less severe after introducing the ß-alanine trimer supramolecular filler into the system.

Porcine Epidemic Diarrhea Virus and Its nsp14 Suppress ER Stress Induced GRP78.

Porcine epidemic diarrhea virus (PEDV), a member of the α-coronavirus genus, can cause vomiting, diarrhea, and dehydration in piglets. Neonatal piglets infected with PEDV have a mortality rate as high as 100%. PEDV has caused substantial economic losses to the pork industry. Endoplasmic reticulum (ER) stress, which can alleviate the accumulation of unfolded or misfolded proteins in ER, involves in coronavirus infection. Previous studies have indicated that ER stress could inhibit the replication of human coronaviruses, and some human coronaviruses in turn could suppress ER stress-related factors. In this study, we demonstrated that PEDV could interact with ER stress. We determined that ER stress could potently inhibit the replication of GⅠ, GⅡ-a, and GⅡ-b PEDV strains. Moreover, we found that these PEDV strains can dampen the expression of the 78 kDa glucose-regulated protein (GRP78), an ER stress marker, while GRP78 overexpression showed antiviral activity against PEDV. Among different PEDV proteins, PEDV non-structural protein 14 (nsp14) was revealed to play an essential role in the inhibition of GRP78 by PEDV, and its guanine-N7-methyltransferase domain is necessary for this role. Further studies show that both PEDV and its nsp14 negatively regulated host translation, which could account for their inhibitory effects against GRP78. In addition, we found that PEDV nsp14 could inhibit the activity of GRP78 promotor, helping suppress GRP78 transcription. Our results reveal that PEDV possesses the potential to antagonize ER stress, and suggest that ER stress and PEDV nsp14 could be the targets for developing anti-PEDV drugs.

TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells.

Glaesserella parasuis is a common bacterium in the porcine upper respiratory tract that causes severe Glasser's disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis. TurboID is an enzyme that mediates the biotinylation of endogenous proteins that can fuse with proteins of interest to label protein interactors and local proteomes. To reveal the host proteins that interact with outer membrane protein P2 (OmpP2) by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage iPAM cells, 0.1 and 2.58 mg/mL His-tagged TurboID-OmpP2 and TurboID recombinant proteins were expressed and purified. By mass spectrometry, we identified 948 and 758 iPAM cell proteins that interacted with His-TurboID-OmpP2 and His-TurboID, respectively. After removal of background proteins through comparison with the TurboID-treated group, 240 unique interacting proteins were identified in the TurboID-OmpP2-treated group. Ultimately, only four membrane proteins were identified, CAV1, ARF6, PPP2R1A, and AP2M1, from these 240 host proteins. Our data indicated that CAV1, ARF6, and PPP2R1A could interact with OmpP2 of G. parasuis, as confirmed by coimmunoprecipitation assay. Finally, we found that CAV1, ARF6, and PPP2R1A were involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells by using overexpression and RNA interference assays. This study provides first-hand information regarding the interaction of the iPAM cell proteomes with G. parasuis OmpP2 protein by using the TurboID proximity labeling system and identifies three novel host membrane proteins involved in the recognition and phagocytosis of G. parasuis by iPAM cells. These results provide new insight for a better understanding of Glasser's disease pathogenesis. IMPORTANCE G. parasuis can cause serious Glasser's disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis in pigs. It can cause high morbidity and mortality in swine herds and major economic losses to the global pig industry. Understanding the mechanism of interactions between alveolar macrophages and pathogenic G. parasuis is essential for developing effective vaccines and targeted drugs against G. parasuis. To reveal the host proteins interacting with OmpP2 by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage (iPAM) cells, we identified 240 unique proteins from iPAM cells that could interact with G. parasuis OmpP2. Among them, only four membrane proteins, CAV1, ARF6, PPP2R1A, and AP2M1, were identified, and further study showed that CAV1, ARF6, and PPP2R1A are involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells. This study provides new insight into proteomic interactions between hosts and pathogenic microorganisms.

Transcriptome analysis of heat resistance regulated by quorum sensing system in Glaesserella parasuis.

The ability of bacteria to resist heat shock allows them to adapt to different environments. In addition, heat shock resistance is known for their virulence. Our previous study showed that the AI-2/luxS quorum sensing system affects the growth characteristics, biofilm formation, and virulence of Glaesserella parasuis. The resistance of quorum sensing system deficient G. parasuis to heat shock was obviously weaker than that of wild type strain. However, the regulatory mechanism of this phenotype remains unclear. To illustrate the regulatory mechanism by which the quorum sensing system provides resistance to heat shock, the transcriptomes of wild type (GPS2), ΔluxS, and luxS complemented (C-luxS) strains were analyzed. Four hundred forty-four differentially expressed genes were identified in quorum sensing system deficient G. parasuis, which participated in multiple regulatory pathways. Furthermore, we found that G. parasuis regulates the expression of rseA, rpoE, rseB, degS, clpP, and htrA genes to resist heat shock via the quorum sensing system. We further confirmed that rseA and rpoE genes exerted an opposite regulatory effect on heat shock resistance. In conclusion, the findings of this study provide a novel insight into how the quorum sensing system affects the transcriptome of G. parasuis and regulates its heat shock resistance property.

Association of Plasma Epstein-Barr Virus DNA With Outcomes for Patients With Recurrent or Metastatic Nasopharyngeal Carcinoma Receiving Anti-Programmed Cell Death 1 Immunotherapy.

IMPORTANCE: Anti-programmed cell death 1 (anti-PD-1) immunotherapy features a durable response and improved survival in a small subset of patients with recurrent or metastatic nasopharyngeal carcinoma (RM-NPC). The association between plasma Epstein-Barr virus (EBV) DNA titer dynamics and efficacy of anti-PD-1 monotherapy has been reported, while its value in predicting long-term outcomes and monitoring disease progression is unclear for patients with RM-NPC who are receiving anti-PD-1 monotherapy. OBJECTIVE: To evaluate the role of plasma EBV DNA titers in prognosis prediction and surveillance of disease progression for patients with RM-NPC who are receiving anti-PD-1 monotherapy. DESIGN, SETTING, AND PARTICIPANTS: Patients with RM-NPC from the POLARIS-02 prospective clinical trial, the largest cohort to receive anti-PD-1 monotherapy, were included in this study. From December 22, 2016, to February 19, 2019, 17 participating centers in China screened 279 patients with RM-NPC; 190 patients were enrolled and followed up until February 19, 2020. Plasma EBV DNA was detected before treatment and every 4 weeks until disease progression. MAIN OUTCOMES AND MEASURES: Plasma EBV DNA as a predictor for progression-free survival (PFS), overall survival (OS), durable clinical benefit (defined as PFS of ≥6 months), and disease progression. RESULTS: Of 179 patients with RM-NPC receiving anti-PD-1 therapy, 148 (82.7%) were men, and the median age was 46 years (range, 22-71 years). A higher baseline EBV DNA titer was associated with shorter median OS (hazard ratio, 1.88; 95% CI, 1.22-2.89; P = .004). Patients with a ratio of the EBV DNA titer at week 4 to that at baseline (W4 to baseline ratio) greater than 0.5 had shorter median OS (hazard ratio, 2.18; 95% CI, 1.30-3.65; P < .001) than those with a W4 to baseline ratio of 0.5 or less. Patients with higher baseline EBV DNA titers had a lower durable clinical benefit rate than those with lower baseline EBV DNA titers (19 of 97 [19.6%] vs 27 of 71 [38.0%]; P = .01). Similarly, patients with a W4 to baseline ratio greater than 0.5 had a lower durable clinical benefit rate than those with a W4 to baseline ratio of 0.5 or less (9 of 86 [10.5%] vs 32 of 54 [59.3%]; P < .001). In addition, a significant EBV DNA titer increase was present at a median of 2.6 months (IQR, 0.9-4.5 months) prior to radiographic progression. CONCLUSIONS AND RELEVANCE: This study of plasma EBV DNA in patients with RM-NPC who are receiving anti-PD-1 monotherapy suggests that plasma EBV DNA could be a useful biomarker for outcomes and monitoring disease progression.

Density-Dependent Emission Colors from a Conformation-Switching Chromophore in Polyurethanes.

Achieving full-color emission from a single chromophore is not only highly desirable from practical considerations, but also greatly challenging for fundamental research. Herein, we demonstrated the density-dependent emission colors from a single boron-containing chromophore, from which multi-color fluorescent polyurethanes were prepared as well. Originating from its switchable molecular conformations, the emission color of the chromophore was found to be governed by the packing density and strongly influenced by hydrogen bonding interactions. The chromophore was incorporated into polyurethanes to achieve full-color emitting materials; the emission color was only dependent on the chromophore density and could be tuned via synthetic approach by controlling the compositions. The emission colors could also be modulated by physical approaches, including by swelling/deswelling process, compression under high pressure, and even blending the fluorescent polyurethane with non-emitting ones.

Deletion of the crp gene affects the virulence and the activation of the NF-κB and MAPK signaling pathways in PK-15 and iPAM cells derived from G. parasuis serovar 5.

Glaesserella parasuis can cause serious systemic disease (Glasser's disease) that is characterized by fibrinous polyserositis, polyarthritis and meningitis. cAMP receptor protein (CRP) is among the well studied global regulator proteins which could modulate the virulence of many pathogenic bacteria. Our previous study showed that the crp gene was involved in the regulation of growth rate, biofilm formation, stress tolerance, serum resistance, and iron utilization in G. parasuis. However, whether the crp gene could regulate the virulence of G. parasuis has not been analyzed previously. In this study, it was observed that the crp gene in G. parasuis serovar 5 (HPS5) was involved in regulating the adhesion and invasion abilities on iPAM cells, and the mRNA expression of various virulence-related factors. It also possessed the ability to induce the mRNA expression of pro-inflammatory cytokines (IL-1α, IL-1ß, IL-6, IL-8 and TNF-α), promoted the activation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in porcine kidney epithelial (PK-15) and immortalized swine pulmonary alveolar macrophage (iPAM) cells, and contributed to the pathogenicity and organs colonization in mice. As compared with the wild type, both the expression of virulence-related factors in the crp mutant strain and its ability to induce the mRNA expression of pro-inflammatory cytokines, as well as the expression of phospho-p65 and phospho-p38 in PK-15 and iPAM cells was reduced significantly. Furthermore, it also found that the virulence of crp mutant was significantly reduced as compared with the wild type. However, the abilities of adherence and invasion on iPAM cell of Δcrp strain was noted to be significantly enhanced as compared with the wild type. These results suggested that the crp gene deletion could effectively attenuate the virulence of G. parasuis, and crp gene may act as an important potential target for the formulation of a novel vaccine against G. parasuis.

Generation of a RGS18 gene knockout cell line from a human embryonic stem cell line by CRISPR/Cas9.

RGS18 is a member of the RGS (Regulators of G-protein signaling) protein family, involved in megakaryopoiesis, megakaryocyte differentiation and chemotaxis. Here, we created a RGS18 knockout cell line from a human embryonic stem cell line by CRISPR/Cas9 mediated gene targeting, to further understand roles of RGS18 in these processes. The cell line maintains stem cell morphology and normal karyotype, and retains expression of pluripotent marker genes and differentiation potential in vivo. The RGS18-/- cell line will facilitate investigation of the role of RGS18 during multiple cellular processes in human pluripotent stem cell modeled hematopoiesis.

Ultrasmall T1-T2 Magnetic Resonance Multimodal Imaging Nanoprobes for the Detection of ß-amyloid Aggregates in Alzheimer's Disease Mice.

Alzheimer's disease (AD) is an irreversible brain disorder and imposes a severe burden upon patients and the public health system. Most research efforts have focused on the search for effective therapeutic drugs, but it is time to pursue efficient early diagnosis based on the reasonable assumption that AD may be easier to prevent than reverse. Recent studies have shown that there are several probes for detecting amyloid-ß (Aß) plaques, one of the neuropathological hallmarks found in AD brain. However, it is still a great challenge for nonradioactive, sensitive detection and location of Aß plaques by brain imaging with high spatial resolution. Herein, phenothiazine derivative (PZD)-conjugated sub-5 nm ultrasmall ferrite nanoprobes (UFNPs@PEG/PZD) are designed and prepared for efficient T1-T2 magnetic resonance multimodal imaging of Aß plaques. UFNPs@PEG/PZD not only possess high binding affinity to Aß plaques but also exhibit excellent properties of r1 and r2 relaxivities. This study thus provides a promising ultrasmall nanoplatform as an Aß-targeting multimodal imaging probe for the application of early diagnosis of AD.

Label-free electrochemical impedance spectroscopy biosensor for direct detection of cancer cells based on the interaction between carbohydrate and lectin.

A novel label-free electrochemical impedance spectroscopy (EIS) biosensor for direct cancer cell detection based on the interaction between carbohydrate and lectin has been developed with good sensitivity and selectivity. In the present work, concanavalin A (Con A), a mannose specific lectin, was immobilized on a gold disk electrode to fabricate the Con A sensor. This sensor was incubated with the cancer cell sample, and the binding of cancer cells with Con A resulted in a change of charge transfer resistance (Rct). EIS measurement was employed to measure the impedance change which reveals the concentration of cancer cells. This method has been successfully applied in human liver cancer cell Bel-7404 for direct and sensitive detection with a detection limit of 234cells/mL. This method could be extended to carry out multi-component diagnosis applications, thus providing enormous potential for applications of cancer monitoring and therapy.