Does the Formation of a Taylor Cone in a Pulsating Electrospray Directly Impact Mass Spectrometry Signals?

Electrospray ionization (ESI) remains the dominant technique in mass spectrometry (MS)-based analyses. Here, we investigated the relationship between a crucial aspect of ESI, the formation of the Taylor cone, and the MS ion current by utilizing a triple quadrupole (QqQ) mass spectrometer coupled with a streaming high-speed camera and a 3-ring electrode system. In one test, ion current over a 30-s plume gate (a ring electrode) opening was compared with the Taylor cone occurrence analyzed offline, with Spearman's correlation coefficients consistently near 0 despite parameter variations. In another test, real-time detection of Taylor cones was synchronized with QqQ-MS, selectively opening (de-energizing) the plume gate based on the Taylor cone status. This approach enabled matching the ion current with the Taylor cone occurrence. There was no apparent difference between the MS signals recorded in the presence and absence of a Taylor cone. Additionally, a Faraday plate was employed as a detector in offline experiments, revealing agreement between the frequency of liquid meniscus (Taylor cone) oscillation (∼1.92 kHz)-measured by high-speed imaging-and the frequency of spray current (∼1.93 kHz). We suggest that the lack of positive correlations in the MS experiments is due to intrinsic ion carryover during transit from the ion source to the detector and due to the insufficient data acquisition rate of the mass spectrometer, which erases short-term fluctuations of ion current.

Volatile Compounds Emitted by Plant Growth-Promoting Fungus Tolypocladium inflatum GT22 Alleviate Copper and Pathogen Stress.

Previous studies on the intricate interactions between plants and microorganisms have revealed that fungal volatile compounds (VCs) can affect plant growth and development. However, the precise mechanisms underlying these actions remain to be delineated. In this study, we discovered that VCs from the soilborne fungus Tolypocladium inflatum GT22 enhance the growth of Arabidopsis. Remarkably, priming Arabidopsis with GT22 VCs caused the plant to display an enhanced immune response and mitigated the detrimental effects of both pathogenic infections and copper stress. Transcriptomic analyses of Arabidopsis seedlings treated with GT22 VCs for 3, 24 and 48 h revealed that 90, 83 and 137 genes were differentially expressed, respectively. The responsive genes are known to be involved in growth, hormone regulation, defense mechanisms and signaling pathways. Furthermore, we observed the induction of genes related to innate immunity, hypoxia, salicylic acid biosynthesis and camalexin biosynthesis by GT22 VCs. Among the VCs emitted by GT22, exposure of Arabidopsis seedlings to limonene promoted plant growth and attenuated copper stress. Thus, limonene appears to be a key mediator of the interaction between GT22 and plants. Overall, our findings provide evidence that fungal VCs can promote plant growth and enhance both biotic and abiotic tolerance. As such, our study suggests that exposure of seedlings to T. inflatum GT22 VCs may be a means of improving crop productivity. This study describes a beneficial interaction between T. inflatun GT22 and Arabidopsis. Our investigation of microorganism function in terms of VC activities allowed us to overcome the limitations of traditional microbial application methods. The importance of this study lies in the discovery of T. inflatun GT22 as a beneficial microorganism. This soilborne fungus emits VCs with plant growth-promoting effects and the ability to alleviate both copper and pathogenic stress. Furthermore, our study offers a valuable approach to tracking the activities of fungal VC components via transcriptomic analysis and sheds light on the mechanisms through which VCs promote plant growth and induce resistance. This research significantly advances our knowledge of VC applications and provides an example for further investigations within this field.

Uncovering molecular mechanisms involved in microbial volatile compounds-induced stomatal closure in Arabidopsis thaliana.

Microbial volatile compounds (mVCs) may cause stomatal closure to limit pathogen invasion as part of plant innate immune response. However, the mechanisms of mVC-induced stomatal closure remain unclear. In this study, we co-cultured Enterobacter aerogenes with Arabidopsis (Arabidopsis thaliana) seedlings without direct contact to initiate stomatal closure. Experiments using the reactive oxygen species (ROS)-sensitive fluorescent dye, H2DCF-DA, showed that mVCs from E. aerogenes enhanced ROS production in guard cells of wild-type plants. The involvement of ROS in stomatal closure was then demonstrated in an ROS production mutant (rbohD). In addition, we identified two stages of signal transduction during E. aerogenes VC-induced stomatal closure by comparing the response of wild-type Arabidopsis with a panel of mutants. In the early stage (3 h exposure), E. aerogenes VCs induced stomatal closure in wild-type and receptor-like kinase THESEUS1 mutant (the1-1) but not in rbohD, plant hormone-related mutants (nced3, erf4, jar1-1), or MAPK kinase mutants (mkk1 and mkk3). However, in the late stage (24 h exposure), E. aerogenes VCs induced stomatal closure in wild-type and rbohD but not in nced3, erf4, jar1-1, the1-1, mkk1 or mkk3. Taken together, our results suggest that E. aerogenes mVC-induced plant immune responses modulate stomatal closure in Arabidopsis by a multi-phase mechanism.

Antifungal mechanism of volatile compounds emitted by Actinomycetota Paenarthrobacter ureafaciens from a disease-suppressive soil on Saccharomyces cerevisiae.

Increasing evidence suggests that in disease-suppressive soils, microbial volatile compounds (mVCs) released from bacteria may inhibit the growth of plant-pathogenic fungi. However, the antifungal activities and molecular responses of fungi to different mVCs remain largely undescribed. In this study, we first evaluated the responses of pathogenic fungi to treatment with mVCs from Paenarthrobacter ureafaciens. Then, we utilized the well-characterized fungal model organism Saccharomyces cerevisiae to study the potential mechanistic effects of the mVCs. Our data showed that exposure to P. ureafaciens mVCs leads to reduced growth of several pathogenic fungi, and in yeast cells, mVC exposure prompts the accumulation of reactive oxygen species. Further experiments with S. cerevisiae deletion mutants indicated that Slt2/Mpk1 and Hog1 MAPKs play major roles in the yeast response to P. ureafaciens mVCs. Transcriptomic analysis revealed that exposure to mVCs was associated with 1,030 differentially expressed genes (DEGs) in yeast. According to gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses, many of these DEGs are involved in mitochondrial dysfunction, cell integrity, mitophagy, cellular metabolism, and iron uptake. Genes encoding antimicrobial proteins were also significantly altered in the yeast after exposure to mVCs. These findings suggest that oxidative damage and mitochondrial dysfunction are major contributors to the fungal toxicity of mVCs. Furthermore, our data showed that cell wall, antioxidant, and antimicrobial defenses are induced in yeast exposed to mVCs. Thus, our findings expand upon previous research by delineating the transcriptional responses of the fungal model. IMPORTANCE Since the use of bacteria-emitted volatile compounds in phytopathogen control is of considerable interest, it is important to understand the molecular mechanisms by which fungi may adapt to microbial volatile compounds (mVCs). Paenarthrobacter ureafaciens is an isolated bacterium from disease-suppressive soil that belongs to the Actinomycetota phylum. P. ureafaciens mVCs showed a potent antifungal effect on phytopathogens, which may contribute to disease suppression in soil. However, our knowledge about the antifungal mechanism of mVCs is limited. This study has proven that mVCs are toxic to fungi due to oxidative stress and mitochondrial dysfunction. To deal with mVC toxicity, antioxidants and physical defenses are required. Furthermore, iron uptake and CAP proteins are required for antimicrobial defense, which is necessary for fungi to deal with the thread from mVCs. This study provides essential foundational knowledge regarding the molecular responses of fungi to inhibitory mVCs.

Are Most Micrometer Droplets (>10 µm) Wasted in Electrospray Ionization? An Insight from Real-Time High-Speed Imaging.

Electrospray ionization (ESI) is one of the main techniques used in mass spectrometry (MS) of nonvolatile compounds. ESI is a disordered process, in which a large number of polydisperse droplets are projected from a fluctuating Taylor cone and jet protruding ESI emitter. Here, we disclose a system for sectioning electrospray plumes to discrete packets with millisecond and submillisecond lifetime, which are introduced to the MS orifice, one at a time. A high-speed camera was triggered at 10,000 frames per second to capture consecutive images of the electrospray packets transmitted to the mass spectrometer. We further correlated the high-speed images of electrospray packets with MS signals of a test analyte (acetaminophen). Following computational treatment of the images, we determined the number of droplet observations (<300), average diameter of droplets (∼10-20 µm), and average volume of droplets (few tens of picoliters) in the individual electrospray packets. The result shows that most micrometer droplets (>10 µm) do not have any significant contribution to the MS signals. This finding is in agreement with the prior conjecture that most of the MS signals are mainly attributed to nanodroplets. Based on this finding, one can deduce that only a small number of the initial microdroplets effectively carry analyte molecules that undergo ionization. We discuss that, in future, one may propose a way to "recharge" the emitted initial micrometer droplets to increase the efficiency of conventional ESI setups.

Repair of a giant inguinoscrotal hernia with herniation of the ileum and sigmoid colon: A case report.

BACKGROUND: Giant inguinoscrotal hernias are huge inguinal hernias that extend below the midpoint of the inner thigh in the standing posture. Giant inguinoscrotal hernias are rare in developed countries because of their better medical resources and early treatment. However, they can develop in patients who refuse surgery or ignore their condition. Intervention is inevitable because strangulation and organ perforation can occur, leading to peritonitis and sepsis. Common surgical approaches include open abdominal and inguinal approaches or a combination of both. CASE SUMMARY: We present the case of a 73-year-old man who visited our emergency department with a huge mass in his left scrotum and septic complications. Abdominal computed tomography revealed a large left inguinoscrotal hernia that contained small bowel loops and the colon. Emergency surgical intervention was performed immediately because intestinal strangulation was highly suspected. The operative repair was performed using a combination of mini-exploratory laparotomy and the inguinal approach. The incarcerated organs, which included the ileum and sigmoid colon, had relatively good intestinal perfusion without perforation or ischemic changes. They were successfully reduced into the abdomen, and bowel resection was not necessary. A tension-free prosthetic mesh was used for the hernia repair. Two weeks after the initial surgery, and with adequate antimicrobial therapy, the patient recovered and was discharged from our hospital. No evidence of hernia relapse was noted during the outpatient follow-up examination 3 mo after surgery. CONCLUSION: Emergency surgery involving combined mini-exploratory laparotomy and the inguinal approach should be performed for serious incarcerated giant inguinoscrotal hernias.

The involvement of AtMKK1 and AtMKK3 in plant-deleterious microbial volatile compounds-induced defense responses.

KEY MESSAGE: Plant-deleterious microbial volatiles activate the transactivation of hypoxia, MAMPs and wound responsive genes in Arabidopsis thaliana. AtMKK1 and AtMKK3 are involved in the plant-deleterious microbial volatiles-induced defense responses. Microbial volatile compounds (mVCs) are a collection of volatile metabolites from microorganisms with biological effects on all living organisms. mVCs function as gaseous modulators of plant growth and plant health. In this study, the defense events induced by plant-deleterious mVCs were investigated. Enterobacter aerogenes VCs lead to growth inhibition and immune responses in Arabidopsis thaliana. E. aerogenes VCs negatively regulate auxin response and transport gene expression in the root tip, as evidenced by decreased expression of DR5::GFP, PIN3::PIN3-GFP and PIN4::PIN4-GFP. Data from transcriptional analysis suggests that E. aerogenes VCs trigger hypoxia response, innate immune responses and metabolic processes. In addition, the transcript levels of the genes involved in the synthetic pathways of antimicrobial metabolites camalexin and coumarin are increased after the E. aerogenes VCs exposure. Moreover, we demonstrate that MKK1 serves as a regulator of camalexin biosynthesis gene expression in response to E. aerogenes VCs, while MKK3 is the regulator of coumarin biosynthesis gene expression. Additionally, MKK1 and MKK3 mediate the E. aerogenes VCs-induced callose deposition. Collectively, these studies provide molecular insights into immune responses by plant-deleterious mVCs.

Microbial volatile compounds-induced cytotoxicity in the yeast Saccharomyces cerevisiae: The role of MAPK signaling and proteasome regulatory pathway.

Microbial volatile organic compounds (mVCs) are formed in the metabolism of microorganisms and widely distributed in nature and pose threats to human health. However, the air pollution by microorganisms is a situation which is poorly understood. In this study, the cytotoxicity of E. aerogenes VCs was evaluated in the model organism Saccharomyces cerevisiae. E. aerogenes VCs inhibited the survival of yeast and triggered the formation of intracellular reactive oxygen species (ROS). The hypersensitive of MAP kinase mpk1/slt2 and 19S regulatory assembly chaperone adc17 mutants to the E. aerogenes VCs indicated cell wall integrity (CWI) pathway together with stress-inducible proteasome assembly regulation are essentially involved in mVCs tolerance mechanism. Furthermore, exposure to the mVCs resulted in the transcriptional upregulation of the CWI pathway, the regulatory particle assembly chaperones, and genes involved in proteasome regulations. Our research suggested that the ROS/MAPK signaling and proteasome regulatory pathway play pivotal roles in the integration and fine-tuning of the mVCs stress response. This study provides a molecular framework for future study of the effects of mVCs on more complex organisms, such as humans.

Analysis of ambient temperature-responsive transcriptome in shoot apical meristem of heat-tolerant and heat-sensitive broccoli inbred lines during floral head formation.

BACKGROUND: Head formation of broccoli (Brassica oleracea var. italica) is greatly reduced under high temperature (22 °C and 27 °C). Broccoli inbred lines that are capable of producing heads at high temperatures in summer are varieties that are unique to Taiwan. However, knowledge of the early-activated pathways of broccoli head formation under high temperature is limited. RESULTS: We compared heat-tolerant (HT) and heat-sensitive (HS) transcriptome of broccoli under different temperatures. Weighted gene correlation network analysis (WGCNA) revealed that genes involved in calcium signaling pathways, mitogen-activated protein kinase (MAPK) cascades, leucine-rich repeat receptor-like kinases (LRR-RLKs), and genes coding for heat-shock proteins and reactive oxygen species homeostasis shared a similar expression pattern to BoFLC1, which was highly expressed at high temperature (27 °C). Of note, these genes were less expressed in HT than HS broccoli at 22 °C. Co-expression analysis identified a model for LRR-RLKs in survival-reproduction tradeoffs by modulating MAPK- versus phytohormones-signaling during head formation. The difference in head-forming ability in response to heat stress between HT and HS broccoli may result from their differential transcriptome profiles of LRR-RLK genes. High temperature induced JA- as well as suppressed auxin- and cytokinin-related pathways may facilitate a balancing act to ensure fitness at 27 °C. BoFLC1 was less expressed in HT than HS at 22 °C, whereas other FLC homologues were not. Promoter analysis of BoFLC1 showed fewer AT dinucleotide repeats in HT broccoli. These results provide insight into the early activation of stress- or development-related pathways during head formation in broccoli. The identification of the BoFLC1 DNA biomarker may facilitate breeding of HT broccoli. CONCLUSIONS: In this study, HT and HS broccoli genotypes were used to determine the effect of temperature on head formation by transcriptome profiling. On the basis of the expression pattern of high temperature-associated signaling genes, the HS transcriptome may be involved in stress defense instead of transition to the reproductive phase in response to heat stress. Transcriptome profiling of HT and HS broccoli helps in understanding the molecular mechanisms underlying head-forming capacity and in promoting functional marker-assisted breeding.

Integrating Early Transcriptomic Responses to Rhizotoxins in Rice (Oryza sativa. L.) Reveals Key Regulators and a Potential Early Biomarker of Cadmium Toxicity.

As sessile organisms, plants were constantly challenged with biotic and abiotic stresses. Transcriptional activation of stress-responsive genes is a crucial part of the plant adaptation to environmental changes. Here, early response of rice root to eight rhizotoxic stressors: arsenate, copper, cadmium, mercury, chromate, vanadate, ferulic acid and juglone, was analyzed using published microarray data. There were 539 general stress response (GSR) genes up-regulated under all eight treatments, including genes related to carbohydrate metabolism, phytohormone balance, and cell wall structure. Genes related to transcriptional coactivation showed higher Ka/Ks ratio compared to the other GSR genes. Network analysis discovered complicated interaction within GSR genes and the most connected signaling hubs were WRKY53, WRKY71, and MAPK5. Promoter analysis discovers enriched SCGCGCS cis-element in GSR genes. Moreover, GSR genes tend to be intronless and genes with shorter total intron length were induced in a higher level. Among genes uniquely up-regulated by a single stress, a phosphoenolpyruvate carboxylase kinase (PPCK) was identified as a candidate biomarker for detecting cadmium contamination. Our findings provide insights into the transcriptome dynamics of molecular response of rice to different rhizotoxic stress and also demonstrate potential use of comparative transcriptome analysis in identifying a novel potential early biomarker.

Microcomputed tomography analysis of particular autogenous bone graft in sinus augmentation at 5 months: differences on bone mineral density and 3D trabecular structure.

OBJECTIVES: This study investigated the effects of gender on the three-dimensional (3D) bone mineral density (BMD) and micromorphology of the trabeculae of matured autogenous bone grafts after sinus floor augmentation, and compared them to those of adjacent native bone. MATERIALS AND METHODS: Ten bone biopsy samples were removed from the implant placement areas of patients who had received second-stage sinus floor augmentation, and analyzed by microcomputed tomography. BMD phantoms with two calcium hydroxyapatite densities (0.25 and 0.75 g/cm(3)) were used to determine the BMD of the grafted and native bone samples. The 3D structural parameters of the trabeculae, including percentage of bone volume (bone volume/tissue volume, BV/TV), trabecular thickness (Tb.Th), trabecular number, trabecular separation, trabecular pattern factor (Tb.Pf), and structure model index, were analyzed between males and females and between grafted bone and native bone. RESULTS: No significant gender-specific differences in BMD and 3D trabecular structure of either native or grafted bone were found (P > 0.05). Compared to the adjacent native bone, the autogenous grafted bone exhibited lower BV/TV and Tb.Th as well as a higher Tb.Pf (P < 0.05). Additionally, there was a weak positive correlation between the Tb.Th values of grafted and native bone (R (2) = 0.58). CONCLUSIONS: In the maxillary sinus, autogenous grafted bone exhibited lower BV/TV, Tb.Th, and trabecular connectivity than the adjacent native bone. No significant gender-specific differences were found for either the BMD or 3D trabecular structure of grafted bone. CLINICAL RELEVANCE: After bone remodeling, autogenous grafted bone revealed different 3D trabecular structure as compared to native bone.