Qualitative and quantitative rapid detection of VOCs differentially released by VAP-associated bacteria using PTR-MS and FGC-PTR-MS.

Ventilator-associated pneumonia (VAP) is a prevalent disease caused by microbial infection, resulting in significant morbidity and mortality within the intensive care unit (ICU). The rapid and accurate identification of pathogenic bacteria causing VAP can assist clinicians in formulating timely treatment plans. In this study, we attempted to differentiate bacterial species in VAP by utilizing the volatile organic compounds (VOCs) released by pathogens. We cultured 6 common bacteria in VAP in vitro, including Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Staphylococcus aureus, which covered most cases of VAP infection in clinic. After the VOCs released by bacteria were collected in sampling bags, they were quantitatively detected by a proton transfer reaction-mass spectrometry (PTR-MS), and the characteristic ions were qualitatively analyzed through a fast gas chromatography-proton transfer reaction-mass spectrometry (FGC-PTR-MS). After conducting principal component analysis (PCA) and analysis of similarities (ANOSIM), we discovered that the VOCs released by 6 bacteria exhibited differentiation following 3 h of quantitative cultivation in vitro. Additionally, we further investigated the variations in the types and concentrations of bacterial VOCs. The results showed that by utilizing the differences in types of VOCs, 6 bacteria could be classified into 5 sets, except for A. baumannii and E. cloacae which were indistinguishable. Furthermore, we observed significant variations in the concentration ratio of acetaldehyde and methyl mercaptan released by A. baumannii and E. cloacae. In conclusion, the VOCs released by bacteria could effectively differentiate the 6 pathogens commonly associated with VAP, which was expected to assist doctors in formulating treatment plans in time and improve the survival rate of patients.

Creating High-entropy Single Atoms on Transition Disulfides through Substrate-induced Redox Dynamics for Efficient Electrocatalytic Hydrogen Evolution.

The controllable anchoring of multiple metal single-atoms (SAs) into a single support exhibits scientific and technological opportunities, while marrying the concentration-complex multimetallic SAs and high-entropy SAs (HESAs) into one SAC system remains a substantial challenge. Here, we present a substrate-mediated SAs formation strategy to successfully fabricate a library of multimetallic SAs and HESAs on MoS2 and MoSe2 supports, which can precisely control the doping location of SAs. Specially, the contents of SAs can continuously increase until the accessible Mo atoms on TMDs carriers are completely replaced by SAs, thus allowing the of much higher metal contents. In-depth mechanistic study shows that the well-controlled synthesis of multimetallic SAs and HESAs is realized by controlling the reversible redox reaction occurred on the TMDs/TM ion interface. As a proof-of-concept application, a variety of SAs-TMDs were applied to hydrogen evolution reaction. The optimized HESAs-TMDs (Pt,Ru,Rh,Pd,Re-MoSe2) delivers a much higher activity and durability than state of-the-art Pt. Thus, our work will broaden the family of single-atom catalysts and provide a new guideline for the rational design of high-performance single-atom catalysts.

Evaluation of a New DC-Ion Funnel Drift Tube for Use in Proton Transfer Reaction Mass Spectrometry.

We have developed and characterized a novel drift tube called the direct current-ion funnel (DC-ion funnel) drift tube, consisting of 20 traditional ring electrodes and 5 new DC-focusing electrodes (DC-FEs) for use in proton transfer reaction mass spectrometry (PTR-MS). Ion trajectory simulations demonstrate the ion focusing effect of the DC-FE and DC-ion funnel drift tube. Further comparative experiments show that the PTR-MS with the novel DC-ion funnel drift tube has a higher sensitivity (3.8-7.3 times for the volatile organic compounds considered in this work) than the PTR-MS with a traditional drift tube. Different from conventional radiofrequency (rf) focusing methods, the DC-ion funnel drift tube can realize ion focusing with only a DC electric field and no additional rf power supply, which makes it especially suitable for instruments requiring miniaturization and low power consumption to improve detection sensitivity. In addition, the DC-ion funnel drift tube can easily be coupled to other types of mass spectrometers to increase their detection sensitivity.

Increased Sensitivity in Proton Transfer Reaction Mass Spectrometry by Using a Novel Focusing Quadrupole Ion Funnel.

Sensitivity enhancement in proton transfer reaction mass spectrometry (PTR-MS) is an important development direction. We developed a novel drift tube called a focusing quadrupole ion funnel (FQ-IF) for use in PTR-MS to improve the sensitivity. The FQ-IF consists of 20 layers of stainless steel electrodes, and each layer has 4 quarter rings. The first 6 layers have a constant inner hole diameter of 22 mm; the latter 14 layers taper the inner diameter down to 8 mm. The FQ-IF drift tube can also operate in the direct current (DC) mode (similar to a conventional drift tube) and ion funnel (IF) mode (similar to a conventional ion funnel drift tube) by changing the voltage loading method. The simulation results show that the transmission efficiency of the FQ-IF is significantly improved compared to that of the other two modes. Further experiments show that the product ions of limonene tend to convert into smaller m/z fragment ions at higher voltages for the DC and IF modes. However, unlike the DC and IF modes, the distribution of product ions is stable at higher voltages for the FQ-IF. In other words, a higher RF voltage for the FQ-IF will not increase the collision energy of ions. In addition, the improvements in sensitivity for the FQ-IF range from 13.8 to 87.9 times compared to the DC mode and from 1.7 to 4.8 times compared to the IF mode for the 12 test compounds. The improvements in the limit of detection (LOD) for the FQ-IF range from 2.7 to 35.7 times compared to the DC mode. The FQ-IF provides a valuable reference for improving the sensitivity of PTR-MS and other mass spectrometers.

Analysis of volatile organic compounds from deep airway in the lung through intubation sampling.

Exhaled volatile organic compounds (VOCs) have been widely applied for the study of disease biomarkers. Oral exhalation and nasal exhalation are two of the most common sampling methods. However, VOCs released from food residues and bacteria in the mouth or upper respiratory tract were also sampled and usually mistaken as that produced from body metabolism. In this study, exhalation from deep airway was first directly collected through intubation sampling and analyzed. The exhalation samples of 35 subjects were collected through a catheter, which was inserted into the trachea or bronchus through the mouth and upper respiratory tract. Then, the VOCs in these samples were detected by proton transfer reaction mass spectrometry (PTR-MS). In addition, fast gas chromatography proton transfer reaction mass spectrometry (FGC-PTR-MS) was used to further determine the VOCs with the same mass-to-charge ratios. The results showed that there was methanol, acetonitrile, ethanol, methyl mercaptan, acetone, isoprene, and phenol in the deep airway. Compared with that in oral exhalation, ethanol, methyl mercaptan, and phenol had lower concentrations. In detail, the median concentrations of ethanol, methyl mercaptan, and phenol were 7.3, 0.6, and 23.9 ppbv, while those in the oral exhalation were 80.0, 5.1, and 71.3 ppbv, respectively, which meant the three VOCs mainly originated from the food residues and bacteria in the mouth or upper respiratory tract, rather than body metabolism. The research results in our study can provide references for expiratory VOC research based on oral and nasal exhalation samplings, which are more feasible in clinical practice.

Cold Exposure Differentially Stimulates Angiogenesis in BAT and WAT of Mice: Implication in Adrenergic Activation.

BACKGROUND/AIMS: To characterize the temporal profile of cold-induced angiogenesis in brown and white adipose tissues of mice in vivo and the temporal changes of angiogenic factors in primary mice brown (BA) and white adipocytes (WA) treated with ß3-adrenoceptor agonist (CL316,243) in vitro. METHODS: 8-week old male C57BL/6J mice were individually housed in conventional cages under cold exposure (4°C) for 1, 2, 3, 4 and 5 days. Interscapular brown adipose tissue (iBAT), inguinal subcutaneous (sWAT) and epididymal white adipose tissues (eWAT) were harvested for immunohistochemical and gene expression analysis. In vitro, primary mice BA and WA treated with or without CL316,243 were harvested for gene expression and protein secretion analysis. RESULTS: A combination of morphological and genetic (Vegfa, Vegfr2, Hif-1α, Pai1 and Pedf) analyses demonstrated depot-specific angiogenesis in response to cold exposure. Upon CL316,243 treatment, angiogenic factors (Vegfa, Vegfr2, Hif-1α, Pai1 and Pedf) and secreted protein VEGFA were transiently increased in both BA and WA. CONCLUSION: Our results show that iBAT is highly responsive to cold-induced angiogenesis that is mainly supported by sWAT with a lesser extent by eWAT. Moreover, the angiogenesis is a transient process with the angiogenic factors may work in an autocrine/paracrine manner.

RNA-seq transcriptome analysis of extensor digitorum longus and soleus muscles in large white pigs.

Skeletal muscle fibers are mainly categorized into red and white fiber types, and the ratio of red/white fibers within muscle mass plays a crucial role in meat quality such as tenderness and flavor. To better understand the molecular difference between the two muscle fibers, this study takes advantage of RNA-seq to compare differences in the transcriptome between extensor digitorum longus (EDL; white fiber) and soleus (Sol; red fiber) muscles of large white pigs. In total, 89,658,562 and 46,723,568 raw reads from EDL and Sol were generated, respectively. Comparison between the two transcriptomes revealed 561 differentially expressed genes, with 408 displaying higher and 153 lower levels of expression in Sol. Quantitative real-time polymerase chain reaction validated the differential expression of nine genes. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis discovered several differentially enriched biological functions and processes of the two muscles. Moreover, transcriptome comparison between EDL and Sol identified many muscle-related genes (CSRP3, ACTN2, MYL1, and MYH6) and pathways related to myofiber formation, such as focal adhesion, tight junction formation, extracellular matrix (ECM)-receptor pathway, calcium signaling, and Wnt signaling. In addition, 58,362 and 58,359 single nucleotide polymorphisms were identified in EDL and Sol, respectively, and the sequence of 9069 genes was refined at the 5', 3' or both ends. Numerous novel transcripts and alternatively spliced RNAs were also identified. Our transcriptome analysis constitutes valuable sequence resource for uncovering important genes and pathways involved in muscle fiber type determination, and might help further our understanding of the molecular mechanisms in different types of muscle.

Cold-Induced Browning Dynamically Alters the Expression Profiles of Inflammatory Adipokines with Tissue Specificity in Mice.

Cold exposure or ß3-adrenoceptor agonist treatment induces the adipose tissues remodeling, relevant for beige adipogenesis within white adipose tissue (WAT). It remains unclear whether this process influences inflammatory adipokines expression in adipose tissues. We determine the temporal profile of cold or ß3-adrenoceptor agonist (CL316,243)-induced changes in the expression of inflammatory adipokines in adipose tissues in mice or primary mice adipocytes. Male C57BL/6J mice at eight weeks old were exposed to 4 °C for 1-5 days. Interscapular brown adipose tissue (iBAT), inguinal subcutaneous WAT (sWAT) and epididymal WAT (eWAT) were harvested for gene and protein expression analysis. In addition, cultured primary mice brown adipocyte (BA) and white adipocyte (WA) treated with or without CL316,243 were harvested for gene expression analysis. The inflammatory adipokines expressed significantly higher in WAT than BAT at baseline. They were rapidly changed in iBAT, while down-regulated in sWAT and up-regulated in eWAT during the cold acclimation. Upon CL316,243 treatment, detected inflammatory adipokines except Leptin were transiently increased in both BA and WA. Our in vivo and in vitro data demonstrate that the browning process alters the inflammatory adipokines expression in adipose tissues, which is acutely responded to in iBAT, dynamically decreased in sWAT whilst increased in eWAT for compensation.

MicroRNA-139-5p Suppresses 3T3-L1 Preadipocyte Differentiation Through Notch and IRS1/PI3K/Akt Insulin Signaling Pathways.

MicroRNAs (miRNAs) participate in the regulation of adipogenesis. Identification of the full repertoire of miRNAs expressed in adipose tissue is likely to significantly improve our understanding of adipose tissue growth and development. Here, miR-139-5p was identified as an inhibitor of 3T3-L1 adipocyte differentiation with significantly down-regulating the expression levels of adipogenic marker genes PPAR γ (P < 0.01), aP2 (P < 0.01) and FAS (P < 0.01). Importantly, flow cytometry and EdU incorporation assay indicated that this inhibition was partly due to the dysfunction of clonal expansion. Furthermore, we firstly demonstrated that miR-139-5p blocked adipogenesis via directly targeted the 3' untranslated regions (UTRs) of Notch1 and IRS1 mRNAs, a key member of Notch signaling and IRS1/PI3K/Akt insulin signaling, respectively. In addition, the overexpression of Notch1 or IRS1 partially restored the suppressive effects miR-139-5p on differentiation of 3T3-L1 cells. To our knowledge, this was the first report that miR-139-5p functioned negatively by targeting Notch1 and IRS1 during 3T3-L1 adipogenesis, regulating the transition from clonal expansion to terminal differentiation.

MicroRNA-139-5p regulates C2C12 cell myogenesis through blocking Wnt/ß-catenin signaling pathway.

MicroRNAs (miRNAs) are novel and potent regulators in myogenesis. However, the molecular mechanisms that many miRNAs regulate myoblast proliferation and differentiation which are largely unknown. Here, we found that miR-139-5p increased during C2C12 myoblast proliferation, while presenting an inverse trend during C2C12 myoblast differentiation. Flow cytometry and EdU incorporation assay showed that miR-139-5p slowed down the growth of C2C12 cells. Additional study demonstrated that ectopic introduction of miR-139-5p into C2C12 cells blocked myoblast differentiation. Importantly, we demonstrated for the first time that Wnt1, which is associated with the Wnt/ß-catenin signaling pathway, was a direct target of miR-139-5p. Moreover, we found that the expression level of Wnt1 was suppressed significantly (p < 0.01) by miR-139-5p, which triggered inhibition of Wnt/ß-catenin signaling through upregulation of glycogen synthase kinase 3 beta (GSK-3ß; p < 0.05) and downregulation of p-GSK-3ß (p < 0.01), ß-catenin (p < 0.05), and nuclear ß-catenin (p < 0.01). Taken together, these results suggest that miR-139-5p is an important negative regulator in myogenesis through blocking the Wnt1-mediated Wnt/ß-catenin signaling pathway.