ASC-expressing pyroptotic extracellular vesicles alleviate sepsis by protecting B cells.

Pyroptosis is an inflammatory programmed cell death process characterized by membrane rupture. Interestingly, pyroptotic cells can generate plenty of nanosized vesicles. Non-inflammatory apoptotic cell death-derived apoptotic vesicles (apoVs) were systemically characterized and displayed multiple physiological functions and therapeutic potentials. However, the characteristics of pyroptotic cell-generated extracellular vesicles (EVs) are largely unknown. Here, we identified a group of pyroptotic EVs (pyroEVs) from in vitro cultured pyroptotic mesenchymal stem cells (MSCs), as well as from septic mouse blood. Compared with apoVs, pyroEVs express similar levels of annexin V, calreticulin, and common EV markers, but express a decreased level of apoptotic marker cleave caspase-3. PyroEVs, but not apoVs and exosomes, specifically express pyroptotic maker apoptosis-associated speck-like protein containing CARD (ASC). More importantly, MSC-derived pyroEVs protect B cells in the spleen and bone marrow to relieve inflammatory responses and enhance the survival rate of the septic mice. Mechanistically, pyroEV membrane-expressed ASC binds to B cells to repress cell death by repressing Toll-like receptor 4. This study uncovered the characteristics of pyroEVs and their therapeutic role in sepsis and B cell-mediated immune response.

Synonymous sites for accessibility around microRNA binding sites in bacterial spot and speck disease resistance genes of tomato.

The major causes of mass tomato infections in both covered and open ground are agents of bacterial spot and bacterial speck diseases. MicroRNAs (miRNAs) are 16-21 nucleotides in length, non-coding RNAs that inhibit translation and trigger mRNA degradation. MiRNAs play a significant part in plant resistance to abiotic and biotic stresses by mediating gene regulation via post-transcriptional RNA silencing. In this study, we analyzed a collection of bacterial resistance genes of tomato and their binding sites for tomato miRNAs and Pseudomonas syringe pv. tomato miRNAs. Our study found that two genes, bacterial spot disease resistance gene (Bs4) and bacterial speck disease resistance gene (Prf), have a 7mer-m8 perfect seed match with miRNAs. Bs4 was targeted by one tomato miRNA (sly-miR9470-3p) and three Pseudomonas syringe pv. tomato miRNAs (PSTJ4_3p_27246, PSTJ4_3p_27246, and PSTJ4_3p_27246). Again, Prf gene was found to be targeted by two tomato miRNAs namely, sly-miR9469-5p and sly-miR9474-3p. The accessibility of the miRNA-target site and its flanking regions and the relationship between relative synonymous codon usage and tRNAs were compared. Strong access to miRNA targeting regions and decreased rate of translations suggested that miRNAs might be efficient in binding to their particular targets. We also found the existence of rare codons, which suggests that it could enhance miRNA targeting even more. The codon usage pattern analysis of the two genes revealed that both were AT-rich (Bs4 = 63.2%; Prf = 60.8%). We found a low codon usage bias in both genes, suggesting that selective restriction might regulate them. The silencing property of miRNAs would allow researchers to discover the involvement of plant miRNAs in pathogen invasion. However, the efficient validation of direct targets of miRNAs is an urgent need that might be highly beneficial in enhancing plant resistance to multiple pathogenic diseases.

Isolation of Pseudomonas syringae pv. Tomato strains causing bacterial speck disease of tomato and marker-based monitoring for their virulence.

BACKGROUND: The bacterial speck disease of tomato caused by a bacterial pathogen Pseudomonas syringae pv. tomato is a most important disease causing severe crop losses. METHODS AND RESULTS: Present study was conducted to investigate and characterize the population diversity of P. syringae pv. tomato pathogen isolated from infected tomato plants from various regions of Egypt. Significant variation among the isolates was observed which demonstrated considerable virulence. All isolates were pathogenic and the CFU population recovered from inoculate tomato leaves by isolate Pst-2 was higher than other isolates. Genetic disparity among the isolates was investigated by PCR analysis by amplifying hrpZ gene using random amplified polymorphic DNA (RAPD), sequence-related amplified polymorphism (SRAP), and inter-simple sequence repeats (ISSR) markers. The amplified products for ITS1 were found to have 810 bp length whereas 536 bp length was observed for hrpZ gene using primer pairs (1406-f/23S-r) and (MM5-F, MM5-R) respectively. The restriction analysis of amplified regions "ITS" and hrpZ by using 5 and 4 endonucleases respectively demonstrated slight variation among the bacterial isolates. The results of RAPD, ISSR and SRAP showed higher polymorphism (60.52%) within the isolates which may assist for successful characterization by unique and specific markers based on geographical distribution, origin and virulence intensity. CONCLUSION: The results of present study suggested that the use of molecular approach may provide successful and valuable information to differentiate and classify P. syringae pv. tomato strains in future for the detection and confirmation of pathogenicity.

Quantum Neural Network Based Distinguisher on SPECK-32/64.

As IoT technology develops, many sensor devices are being used in our life. To protect such sensor data, lightweight block cipher techniques such as SPECK-32 are applied. However, attack techniques for these lightweight ciphers are also being studied. Block ciphers have differential characteristics, which are probabilistically predictable, so deep learning has been utilized to solve this problem. Since Gohr's work at Crypto2019, many studies on deep-learning-based distinguishers have been conducted. Currently, as quantum computers are developed, quantum neural network technology is developing. Quantum neural networks can also learn and make predictions on data, just like classical neural networks. However, current quantum computers are constrained by many factors (e.g., the scale and execution time of available quantum computers), making it difficult for quantum neural networks to outperform classical neural networks. Quantum computers have higher performance and computational speed than classical computers, but this cannot be achieved in the current quantum computing environment. Nevertheless, it is very important to find areas where quantum neural networks work for technology development in the future. In this paper, we propose the first quantum neural network based distinguisher for the block cipher SPECK-32 in an NISQ. Our quantum neural distinguisher successfully operated for up to 5 rounds even under constrained conditions. As a result of our experiment, the classical neural distinguisher achieved an accuracy of 0.93, but our quantum neural distinguisher achieved an accuracy of 0.53 due to limitations in data, time, and parameters. Due to the constrained environment, it cannot exceed the performance of classical neural networks, but it can operate as a distinguisher because it has obtained an accuracy of 0.51 or higher. In addition, we performed an in-depth analysis of the quantum neural network's various factors that affect the performance of the quantum neural distinguisher. As a result, it was confirmed that the embedding method, the number of the qubit, and quantum layers, etc., have an effect. It turns out that if a high-capacity network is needed, we have to properly tune properly to take into account the connectivity and complexity of the circuit, not just by adding quantum resources. In the future, if more quantum resources, data, and time become available, it is expected that an approach to achieve better performance can be designed by considering the various factors presented in this paper.

Deep-Learning-Based Cryptanalysis of Lightweight Block Ciphers Revisited.

With the development of artificial intelligence, deep-learning-based cryptanalysis has been actively studied. There are many cryptanalysis techniques. Among them, cryptanalysis was performed to recover the secret key used for cryptography encryption using known plaintext. In this paper, we propose a cryptanalysis method based on state-of-art deep learning technologies (e.g., residual connections and gated linear units) for lightweight block ciphers (e.g., S-DES, S-AES, and S-SPECK). The number of parameters required for training is significantly reduced by 93.16%, and the average of bit accuracy probability increased by about 5.3% compared with previous the-state-of-art work. In addition, cryptanalysis for S-AES and S-SPECK was possible with up to 12-bit and 6-bit keys, respectively. Through this experiment, we confirmed that the-state-of-art deep-learning-based key recovery techniques for modern cryptography algorithms with the full round and the full key are practically infeasible.

Side Channel Analysis of SPECK Based on Transfer Learning.

Although side-channel attacks based on deep learning are widely used in AES encryption algorithms, there is little research on lightweight algorithms. Lightweight algorithms have fewer nonlinear operations, so it is more difficult to attack successfully. Taking SPECK, a typical lightweight encryption algorithm, as an example, directly selecting the initial key as the label can only crack the first 16-bit key. In this regard, we evaluate the leakage of SPECK's operations (modular addition, XOR, shift), and finally select the result of XOR operation as the label, and successfully recover the last 48-bit key. Usually, the divide and conquer method often used in side-channel attacks not only needs to train multiple models, but also the different bytes of the key are regarded as unrelated individuals. Through the visualization method, we found that different key bytes overlap in the position of the complete electromagnetic leakage signal. That is, when SPECK generates a round key, there is a connection between different bytes of the key. In this regard, we propose a transfer learning method for different byte keys. This method can take advantage of the similarity of key bytes, improve the performance starting-point of the model, and reduce the convergence time of the model by 50%.

The zebrafish NLRP3 inflammasome has functional roles in ASC-dependent interleukin-1ß maturation and gasdermin E-mediated pyroptosis.

The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1ß (DrIL-1ß) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)-PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1ß secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC-/-) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis.

WRKY22 and WRKY25 transcription factors are positive regulators of defense responses in Nicotiana benthamiana.

KEY MESSAGE: NbWRKY22 and NbWRKY25 are required for full activation of bacteria-associated pattern- and effector-triggered immunity as well as for the response to other non-bacterial defense elicitors. Plants defend themselves against pathogens using a two-layered immune system. Pattern-triggered immunity (PTI) can be activated upon recognition of epitopes from flagellin including flg22. Pseudomonas syringae pv. tomato (Pst) delivers effector proteins into the plant cell to promote host susceptibility. However, some plants express resistance (R) proteins that recognize specific effectors leading to the activation of effector-triggered immunity (ETI). Resistant tomato lines such as Rio Grande-PtoR (RG-PtoR) recognize two Pst effectors, AvrPto and AvrPtoB, and activate ETI through the Pto/Prf protein complex. Using RNA-seq, we identified two tomato WRKY transcription factor genes, SlWRKY22 and SlWRKY25, whose expression is increased during Pst-induced ETI. Silencing of the WRKY25/22 orthologous genes in Nicotiana benthamiana led to a delay in programmed cell death normally associated with AvrPto recognition or several non-bacterial effector/R protein pairs. An increase in disease symptoms was observed in silenced plants infiltrated with Pseudomonas syringae pv. tabaci expressing AvrPto or HopQ1-1. Expression of both tomato WRKY genes is also induced upon treatment with flg22 and callose deposition and cell death suppression assays in WRKY25/22-silenced N. benthamiana plants supported their involvement in PTI. Our results reveal an important role for two WRKYs as positive regulators of plant immunity against bacterial and potentially non-bacterial pathogens.

Immune modulating effects of receptor interacting protein 2 (RIP2) in autoinflammation and immunity.

Receptor-interacting protein 2 (RIP2) is a kinase that is involved in downstream signaling of nuclear oligomerization domain (NOD)-like receptors NOD1 and 2 sensing bacterial peptidoglycans. RIP2-deficiency or targeting of RIP2 by pharmaceutical inhibitors partially ameliorates inflammatory diseases by reducing pro-inflammatory signaling in response to peptidoglycans. However, RIP2 is widely expressed and interacts with several other proteins suggesting additional functions outside the NOD-signaling pathway. In this review, we discuss the immunological functions of RIP2 and its possible role in autoinflammation and immunity.

First Report of bacterial speck caused by Pseudomonas syringae pv. tomato Race 1 affecting tomato in different Regions of Chile.

In Chile, tomato is one of the most widely cultivated vegetables, with around 5,000 ha for fresh market and 8,000 ha for processing industry. During recent years, symptoms of bacterial speck caused by Pseudomonas syringae pv. tomato, have been observed more frequently in tomato plants in different regions of Chile. This pathogen was first identified in Chile in 1987 (Latorre & Lolas, 1988) and the presence of an apparent new variant was reported in 2004 (Besoain et al. 2004). To characterize the pathogen that was affecting this crop, samples of diseased tomato plants were taken in three regions of Chile. The samples were collected in 2016 in Northern Chile in Lluta Valley from the Arica y Parinacota Region, and in Central Chile, in 2014 in Limache from Valparaíso Region and in 2015 in Pichidegua from O´Higgins Region. Affected tomato plants exhibited dark brown to black lesions surrounded by yellow halos in the leaves, and dark brown to black lesions in the stems, pedicels, and peduncles. Plants tissues were macerated, and the suspension was spread on King's B medium, resulting in fluorescent colonies visualized under 366 nm UV light. LOPAT tests results of three selected isolates from different Regions, were: levan production (+), oxidase reaction (-), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+) (Lelliot et al. 1966). Molecular identification was carried out by amplification and sequence analysis of housekeeping genes cts, encoding citrate synthase, gyrB, encoding DNA gyrase B, and rpoD, encoding sigma factor 70 (Hwang et al. 2005; Sarkar & Guttmann 2004) (GenBank Accessions No. OK001658-OK001666). BLAST analysis of cts and rpoD genes of the three isolates resulted in a match with a 100% identity (919 bp and 491 bp respectively) with Pseudomonas syringae pv. tomato strain B13-200 (GenBank: CP019871.1). BLAST analysis of gyrB gene of two isolates resulted in a match with a 100% identity (684 bp) and one isolate with 99.85% (683 bp) with Pseudomonas syringae pv. tomato strain B13-200. To identify the race 1, each strain was inoculated in five tomato plants cv. San Pedro, susceptible to both races of P. syringae pv. tomato, and cv. Rio Grande, resistant to race 0. The tomato plants were slightly wounded with a metal sponge and then sprayed with the bacterial suspension (108 CFU mL-1) of each isolate, including the reference strain DC3000 (race 0). Negative controls were sprayed with water. The plants inoculated with Chilean strains in both cv. San Pedro and cv. Rio Grande, showed symptoms of bacterial speck after 7 days. Plants inoculated with DC3000 strain showed symptoms only in cv. San Pedro, whereas control plants remained asymptomatic. Strains were re-isolated from symptomatic plants and identified by gene sequence analyses as Pseudomonas syryngae pv. tomato. This is the first report of Pseudomonas syryngae pv. tomato race 1 in Chile. Race 1 was previously reported in Canada (Lawton and MacNeill. 1986), in Italy (Buonaurio et al. 1996), in California (Arredondo and Davis 2000), in Portugal (Cruz et al. 2010), and in other states in the USA and countries in South America, Europe, Africa, and Australia, becoming the most commonly isolated race today (Cai et al 2011). These results will be the base for future studies of epidemiology, characterization, and virulence in order to explain the outbreak of this disease and the severity of symptoms observed.

Structure, Activation and Regulation of NLRP3 and AIM2 Inflammasomes.

The inflammasome is a three-component (sensor, adaptor, and effector) filamentous signaling platform that shields from multiple pathogenic infections by stimulating the proteolytical maturation of proinflammatory cytokines and pyroptotic cell death. The signaling process initiates with the detection of endogenous and/or external danger signals by specific sensors, followed by the nucleation and polymerization from sensor to downstream adaptor and then to the effector, caspase-1. Aberrant activation of inflammasomes promotes autoinflammatory diseases, cancer, neurodegeneration, and cardiometabolic disorders. Therefore, an equitable level of regulation is required to maintain the equilibrium between inflammasome activation and inhibition. Recent advancement in the structural and mechanistic understanding of inflammasome assembly potentiates the emergence of novel therapeutics against inflammasome-regulated diseases. In this review, we have comprehensively discussed the recent and updated insights into the structure of inflammasome components, their activation, interaction, mechanism of regulation, and finally, the formation of densely packed filamentous inflammasome complex that exists as micron-sized punctum in the cells and mediates the immune responses.

Sensory characteristics and volatile compounds of dry cured ham Speck are affected by pig dietary supplementation with antioxidant mixture.

BACKGROUND: Dry cured ham named Speck is a typical Italian product, with consolidated processing techniques and specific quality parameters. It has gained importance in the international market after obtaining the Protected Geographical Indication. Recent studies indicate that dietary plant extracts in pig may affect quality of processed meats but there are no data on their effect on Speck. Consequently, the effects of dietary supplementation with antioxidant mixture (AOX), containing vitamin E and polyphenols, in pigs on the nutritional characteristics, volatile compounds, sensory properties and consumers' preference of Speck were evaluated. RESULTS: The Speck weight loss during seasoning was lower in the AOX group, while products quality parameters were not affected by dietary treatments. Alcohol and terpenes concentrations were lower (P < 0.05) in the AOX samples than the control. Sensory profile revealed slight differences (P < 0.05) in Speck salty and sweet taste, whereas the consumer tests showed a higher (P < 0.05) preference, with mean score of seven, for Speck from pigs fed AOX. CONCLUSION: Our results provide new insights on the quality parameters of Speck. Dietary integration with antioxidant mixture decreased Speck weight losses during seasoning. Volatile compounds concentrations and sensory profile were slightly affected by dietary treatment. The consumer test revealed that dietary antioxidants, increased the overall preference for Speck, and can potentially increase its consumption. © 2020 Society of Chemical Industry.

Soluble Aß oligomers and protofibrils induce NLRP3 inflammasome activation in microglia.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder causing memory loss, language problems and behavioural disturbances. AD is associated with the accumulation of fibrillar amyloid-ß (Aß) and the formation of neurofibrillary tau tangles. Fibrillar Aß itself represents a danger-associated molecular pattern, which is recognized by specific microglial receptors. One of the key players is formation of the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome, whose activation has been demonstrated in AD patient brains and transgenic animal models of AD. Here, we investigated whether Aß oligomers or protofibrils that represent lower molecular aggregates prior to Aß deposition are able to activate the NLRP3 inflammasome and subsequent interleukin-1 beta (IL-1ß) release by microglia. In our study, we used Aß preparations of different sizes: small oligomers and protofibrils of which the structure was confirmed by atomic force microscopy. Primary microglial cells from C57BL/6 mice were treated with the respective Aß preparations and NLRP3 inflammasome activation, represented by caspase-1 cleavage, IL-1ß production, and apoptosis-associated speck-like protein containing a CARD speck formation was analysed. Both protofibrils and low molecular weight Aß aggregates induced a significant increase in IL-1ß release. Inflammasome activation was confirmed by apoptosis-associated speck-like protein containing a CARD speck formation and detection of active caspase-1. The NLRP3 inflammasome inhibitor MCC950 completely inhibited the Aß-induced immune response. Our results show that the NLRP3 inflammasome is activated not only by fibrillar Aß aggregates as reported before, but also by lower molecular weight Aß oligomers and protofibrils, highlighting the possibility that microglial activation by these Aß species may initiate innate immune responses in the central nervous system prior to the onset of Aß deposition. Cover Image for this issue: https://doi.org/10.1111/jnc.14773.

CRID3, a blocker of apoptosis associated speck like protein containing a card, ameliorates murine spinal cord injury by improving local immune microenvironment.

BACKGROUND: After spinal cord injury (SCI), destructive immune cell subsets are dominant in the local microenvironment, which are the important mechanism of injury. Studies have shown that inflammasomes play an important role in the inflammation following SCI, and apoptosis-associated speck-like protein containing a card (ASC) is the adaptor protein shared by inflammasomes. Therefore, we speculated that inhibiting ASC may improve the local microenvironment of injured spinal cord. Here, CRID3, a blocker of ASC oligomerization, was used to study its effect on the local microenvironment and the possible role in neuroprotection following SCI. METHODS: Murine SCI model was created using an Infinite Horizon impactor at T9 vertebral level with a force of 50 kdynes and CRID3 (50 mg/kg) was intraperitoneally injected following injury. ASC and its downstream molecules in inflammasome signaling pathway were measured by western blot. The immune cell subsets were detected by immunohistofluorescence (IHF) and flow cytometry (FCM). The spinal cord fibrosis area, neuron survival, myelin preservation, and functional recovery were assessed. RESULTS: Following SCI, CRID3 administration inhibited inflammasome-related ASC and caspase-1, IL-1ß, and IL-18 activation, which consequently suppressed M1 microglia, Th1 and Th1Th17 differentiation, and increased M2 microglia and Th2 differentiation. Accordingly, the improved histology and behavior have also been found. CONCLUSIONS: CRID3 may ameliorate murine SCI by inhibiting inflammasome activation, reducing proinflammatory factor production, restoring immune cell subset balance, and improving local immune microenvironment, and early administration may be a promising therapeutic strategy for SCI.

Imidazolium salts as alternative compounds to control diseases caused by plant pathogenic bacteria.

AIMS: To evaluate the inhibitory effect of five structurally different imidazolium salts on the in vitro growth of plant pathogenic bacteria that belong to divergent taxonomic genera as well as their ability to reduce the severity of common bacterial blight of common bean caused by Xanthomonas axonopodis pv. phaseoli and bacterial speck of tomato caused by Pseudomonas syringae pv. tomato. METHODS AND RESULTS: Growth inhibition of Xanthomonas, Pseudomonas, Erwinia, Pectobacterium and Dickeya strains by imidazolium salts was assessed in vitro by radial diffusion on agar medium and by ressazurin reduction in liquid medium. The reduction of common bacterial blight and bacterial speck symptoms and the area under de disease progress curves were determined by spraying two selected imidazolium salts on healthy plants 48 h prior to inoculation with virulent strains of the bacterial pathogens. All imidazolium salts inhibited the growth of all plant pathogenic bacteria when tested by radial diffusion on agar medium. The strength of inhibition differed among imidazolium salts when tested on the same bacterial strain and among bacterial strains when tested with the same imidazolium salt. In liquid medium, most imidazolium salts presented the same minimum inhibitory concentration (MIC) and minimum bactericidal concentration values (200 µmol l-1 ), the most notable exception of which was the MIC (at least 1000 µmol l-1 ) for the dicationic MImC10 MImBr2 . The imidazolium salts C16 MImBr and C16 MImCl caused significant reductions in the severity of common bacterial blight symptoms when compared with nontreated plants. CONCLUSION: Imidazolium salts inhibit the in vitro growth of plant pathogenic bacteria and reduce plant disease symptoms to levels comparable to an authorized commercial antibiotic product. SIGNIFICANCE AND IMPACT OF THE STUDY: New compounds exhibiting broad-spectrum antibacterial activity with potential use in agriculture were identified.

One Year Evaluation of Three Low-Cost PM2.5 Monitors.

The availability of low-cost monitors marketed for use in homes has increased rapidly over the past few years due to the advancement of sensing technologies, increased awareness of urban pollution, and the rise of citizen science. The user-friendly packages can make them appealing for use in research grade indoor exposure assessments, but a rigorous scientific evaluation has not been conducted for many monitors on the open market, which leads to uncertainty about the validity of the data. Furthermore, many previous sensor studies were conducted for a relatively short period of time, which may not capture the changes this type of instrument may exhibit over time (known as sensor aging). We evaluated three monitors (AirVisual Pro, Speck, and AirThinx) in an occupied, non-smoking residence over a 12-month period in order to assess the sensors, the built-in calibrations, and the need for additional data to achieve high accuracy for long deployments. Two units of each type of monitor were evaluated in order to assess the precision between units, and a personal DataRAM (pDR-1200) with a filter was placed in the home for about 20% of the sampling period (e.g., about a week each month) to evaluate the accuracy over time. The average PM2.5 mass concentration from the periods of colocation with the pDR were 5.31 µg/m3 for the gravimetric-corrected pDR (hereafter pDR-corrected), 5.11 and 5.03 µg/m3 for the AirVisual Pro units, 13.58 and 22.68 µg/m3 for the Speck units, and 7.56 and 7.57 µg/m3 for the AirThinx units. The AirVisual Pros exhibited the best accuracy compared to the filter at about 86%, which was slightly better than the nephelometric component of the pDR compared to the filter weight (84%). The accuracies of the Speck (-174 and -405%) and AirThinx (42 and 40%) monitors were much lower. When the 1-minute averaged PM2.5 mass concentrations were categorized by air quality index (AQI), the pDR-corrected matched the AirVisual Pro, Speck, and AirThinx bins about 97, 40, and 87% of the time, respectively. The Pearson correlation coefficients (R2) between the unit pairs and the pDR were 0.90/0.90, 0.50/0.27, and 0.92/0.93 for the AirVisual Pro, Speck, and AirThinx units, respectively. The R2 between units of the same type were 0.99, 0.17, and 1.00 for the AirVisual Pro, Speck, and AirThinx, respectively. All of the monitors could achieve better accuracy by adding filter corrections and post-processing to correct for known biases in addition to the manufacturer's correction routine. Monthly calibrations yielded the highest accuracies, but nearly as high of accuracies could be achieved with only one or two calibrations for the Air Visual Pro and the AirThinx for many applications. In general, this type of new low-cost monitor shows exciting potential for use in scientific research. However, only one of the three monitors exhibited high accuracy (within 20% of the true mass concentration) without any post processing or additional measurements, so an evaluation of each monitor is essential before the data can be used to confidently evaluate residential exposures.

Quantification of Viable Cells of Pseudomonas syringae pv. tomato in Tomato Seed Using Propidium Monoazide and a Real-Time PCR Assay.

Pseudomonas syringae pv. tomato is a seedborne pathogen that causes bacterial speck disease in tomato. P. syringae pv. tomato is typically detected in tomato seed using quantitative real-time PCR (qPCR) but the inability of qPCR to distinguish between viable and nonviable cells might lead to an overestimation of viable P. syringae pv. tomato cells. In the present study, a strategy involving a propidium monoazide (PMA) pretreatment followed by a qPCR (PMA-qPCR) assay was developed for quantifying viable P. syringae pv. tomato cells in contaminated tomato seed. PMA could selectively bind to the chromosomal DNA of dead bacterial cells and, therefore, block DNA amplification of qPCR. The primer pair Pst3F/Pst3R was designed based on gene hrpZ to specifically amplify and quantify P. syringae pv. tomato by qPCR. The PMA pretreatment protocol was optimized for selectively detecting viable P. syringae pv. tomato cells, and the optimal PMA concentration and light exposure time were 10 µmol liter-1 and 10 min, respectively. In the sensitivity test, the detection limit of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated tomato seed was 102 CFU ml-1 and 11.86 CFU g-1, respectively. For naturally contaminated tomato seed, viable P. syringae pv. tomato cells were quantified in 6 of the 19 samples, with infestation levels of approximately 102 to 104 CFU g-1. The results indicated that the PMA-qPCR assay is a suitable tool for quantifying viable P. syringae pv. tomato cells in tomato seed, which could be useful for avoiding the potential risks of primary inoculum sources from contaminated seed.

Hydrogen sulfide inhibits NLRP3 inflammasome activation and reduces cytokine production both in vitro and in a mouse model of inflammation.

A variety of stimuli, including monosodium urate (MSU) crystals, activate the NLRP3 inflammasome, and this activation involves several molecular mechanisms including xanthine oxidase (XO) up-regulation and mitochondrial dysfunction. Upon oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 becomes active and cleaves the proinflammatory cytokine IL-1ß into its active secreted form. Hydrogen sulfide (H2S), a gasotransmitter mainly produced by cystathionine γ-lyase (CSE) in macrophages, could modulate inflammation. Here, we sought to investigate the effects of exogenous and endogenous H2S on NLRP3 inflammasome activation in vitro and in vivo Primed bone marrow-derived macrophages (BMDM) isolated from wildtype (wt) or CSE-deficient mice and human macrophages (THP1 cells and primary macrophages), were stimulated with MSU crystals in the presence or absence of a H2S donor, sodium thiosulfate (STS) or GYY4137 (GYY). In murine and human macrophages in vitro, both STS and GYY inhibited MSU crystal-induced IL-1ß secretion in a dose-dependent manner. Moreover, the H2S donors inhibited MSU crystal-induced XO/caspase-1 activities, mitochondrial reactive oxygen species (ROS) generation, and ASC oligomerization. Accordingly, IL-1ß secretion and XO/caspase-1 activities were higher in CSE-deficient BMDMs than in wt BMDMs. For in vivo studies, we experimentally induced peritonitis by intraperitoneal injection of MSU crystals into mice. GYY pretreatment ameliorated inflammation, evidenced by decreased IL-6/monocyte chemoattractant protein-1 (MCP-1) released into peritoneal lavages. Taken together, our results suggest that both exogenous (via H2S donors) and endogenous (via CSE) H2S production may represent approaches for managing, for example, acute gout or other inflammation conditions.

Mai1 Protein Acts Between Host Recognition of Pathogen Effectors and Mitogen-Activated Protein Kinase Signaling.

The molecular mechanisms acting between host recognition of pathogen effectors by nucleotide-binding leucine-rich repeat receptor (NLR) proteins and mitogen-activated protein kinase (MAPK) signaling cascades are unknown. MAPKKKα (M3Kα) activates MAPK signaling leading to programmed cell death (PCD) associated with NLR-triggered immunity. We identified a tomato M3Kα-interacting protein, SlMai1, that has 80% amino acid identity with Arabidopsis brassinosteroid kinase 1 (AtBsk1). SlMai1 has a protein kinase domain and a C-terminal tetratricopeptide repeat domain that interacts with the kinase domain of M3Kα. Virus-induced gene silencing of Mai1 homologs in Nicotiana benthamiana increased susceptibility to Pseudomonas syringae and compromised PCD induced by four NLR proteins. PCD was restored by expression of a synthetic SlMai1 gene that resists silencing. Expression of AtBsk1 did not restore PCD in Mai1-silenced plants, suggesting SlMai1 is functionally divergent from AtBsk1. PCD caused by overexpression of M3Kα or MKK2 was unaffected by Mai1 silencing, suggesting Mai1 acts upstream of these proteins. Coexpression of Mai1 with M3Kα in leaves enhanced MAPK phosphorylation and accelerated PCD. These findings suggest Mai1 is a molecular link acting between host recognition of pathogens and MAPK signaling.

CASP1 variants influence subcellular caspase-1 localization, pyroptosome formation, pro-inflammatory cell death and macrophage deformability.

CASP1 variants result in reduced enzymatic activity of procaspase-1 and impaired IL-1ß release. Despite this, affected individuals can develop systemic autoinflammatory disease. These seemingly contradictory observations have only partially been explained by increased NF-κB activation through prolonged interaction of variant procaspase-1 with RIP2. To identify further disease underlying pathomechanisms, we established an in vitro model using shRNA-directed knock-down of procaspase-1 followed by viral transduction of human monocytes (THP-1) with plasmids encoding for wild-type procaspase-1, disease-associated CASP1 variants (p.L265S, p.R240Q) or a missense mutation in the active center of procaspase-1 (p.C285A). THP1-derived macrophages carrying CASP1 variants exhibited mutation-specific molecular alterations. We here provide in vitro evidence for abnormal pyroptosome formation (p.C285A, p.240Q, p.L265S), impaired nuclear (pro)caspase-1 localization (p.L265S), reduced pro-inflammatory cell death (p.C285A) and changes in macrophage deformability that may contribute to disease pathophysiology of patients with CASP1 variants. This offers previously unknown molecular pathomechanisms in patients with systemic autoinflammatory disease.