Supraglottic airway devices: a powerful strategy in airway management.

The escalating airway management demands of cancer patients have prompted us to continually curate airway devices, with supraglottic airway devices (SADs) playing a significant role in this regard. SADs serve as instrumental tools for maintaining an open upper airway. Since the inception of the earliest SADs in the early 1980s, an array of advanced and enhanced second-generation devices have been employed in clinical settings. These upgraded SADs integrate specific features designed to enhance positive-pressure ventilation and mitigate the risk of aspiration. Nowadays, they are extensively used in general anesthesia procedures and play a critical role in difficult airway management, pre-hospital care, and emergency medicine. In certain situations, SADs may be deemed a superior alternative to endotracheal tube (ETT) and can be employed in a broader spectrum of surgical and non-surgical cases. This review provides an overview of the current evidence, a summary of classifications, relevant application scenarios, and areas for improvement in the development or clinical application of future SADs.

Discovery of toxoflavin, a potent IRE1α inhibitor acting through structure-dependent oxidative inhibition.

Inositol-requiring enzyme 1α (IRE1α) is the most conserved endoplasmic reticulum (ER) stress sensor with two catalytic domains, kinase and RNase, in its cytosolic portion. IRE1α inhibitors have been used to improve existing clinical treatments against various cancers. In this study we identified toxoflavin (TXF) as a new-type potent small molecule IRE1α inhibitor. We used luciferase reporter systems to screen compounds that inhibited the IRE1α-XBP1s signaling pathway. As a result, TXF was found to be the most potent IRE1α RNase inhibitor with an IC50 value of 0.226 µM. Its inhibitory potencies on IRE1α kinase and RNase were confirmed in a series of cellular and in vitro biochemical assays. Kinetic analysis showed that TXF caused time- and reducing reagent-dependent irreversible inhibition on IRE1α, implying that ROS might participate in the inhibition process. ROS scavengers decreased the inhibition of IRE1α by TXF, confirming that ROS mediated the inhibition process. Mass spectrometry analysis revealed that the thiol groups of four conserved cysteine residues (CYS-605, CYS-630, CYS-715 and CYS-951) in IRE1α were oxidized to sulfonic groups by ROS. In molecular docking experiments we affirmed the binding of TXF with IRE1α, and predicted its binding site, suggesting that the structure of TXF itself participates in the inhibition of IRE1α. Interestingly, CYS-951 was just near the docked site. In addition, the RNase IC50 and ROS production in vitro induced by TXF and its derivatives were negative correlated (r = -0.872). In conclusion, this study discovers a new type of IRE1α inhibitor that targets a predicted new alternative site located in the junction between RNase domain and kinase domain, and oxidizes conserved cysteine residues of IRE1α active sites to inhibit IRE1α. TXF could be used as a small molecule tool to study IRE1α's role in ER stress.

Molecular Deceleration Regulates Toxicant Release to Prevent Cell Damage in Pseudomonas putida S16 (DSM 28022).

The underlying molecular mechanisms of flavin-dependent amine oxidases remain relatively poorly understood, even though many of these enzymes have been reported. The nicotine oxidoreductase NicA2 is a crucial enzyme for the first step of nicotine degradation in Pseudomonas putida S16 (DSM 28022). Here, we present the crystal structure of a ternary complex comprising NicA2 residues 21 to 482, flavin adenine dinucleotide (FAD), and nicotine at 2.25 Å resolution. Unlike other, related structures, NicA2 does not have an associated diacyl glycerophospholipid, wraps its substrate more tightly, and has an intriguing exit passage in which nine bulky amino acid residues occlude the release of its toxic product, pseudooxynicotine (PN). The replacement of these bulky residues by amino acids with small side chains effectively increases the catalytic turnover rate of NicA2. Our results indicate that the passage in wild-type NicA2 effectively controls the rate of PN release and thus prevents its rapid intracellular accumulation. It gives ample time for PN to be converted to less-harmful substances by downstream enzymes such as pseudooxynicotine amine oxidase (Pnao) before its accumulation causes cell damage or even death. The temporal metabolic regulation mode revealed in this study may shed light on the production of cytotoxic compounds.IMPORTANCE Flavin-dependent amine oxidases have received extensive attention because of their importance in drug metabolism, Parkinson's disease, and neurotransmitter catabolism. However, the underlying molecular mechanisms remain relatively poorly understood. Here, combining the crystal structure of NicA2 (an enzyme in the first step of the bacterial nicotine degradation pathway in Pseudomonas putida S16 (DSM 28022)), biochemical analysis, and mutant construction, we found an intriguing exit passage in which bulky amino acid residues occlude the release of the toxic product of NicA2, in contrast to other, related structures. The selective product exportation register for NicA2 has proven to be beneficial to cell growth. Those seeking to produce cytotoxic compounds could greatly benefit from the use of such an export register mechanism.

Multiple Roles for Two Efflux Pumps in the Polycyclic Aromatic Hydrocarbon-Degrading Pseudomonas putida Strain B6-2 (DSM 28064).

Microbial bioremediation is a promising approach for the removal of polycyclic aromatic hydrocarbon (PAH) contaminants. Many degraders of PAHs possess efflux pump genes in their genomes; however, their specific roles in the degradation of PAHs have not been clearly elucidated. In this study, two efflux pumps, TtgABC and SrpABC, were systematically investigated to determine their functions in a PAH-degrading Pseudomonas putida strain B6-2 (DSM 28064). The disruption of genes ttgABC or srpABC resulted in a defect in organic solvent tolerance. TtgABC was found to contribute to antibiotic resistance; SrpABC only contributed to antibiotic resistance under an artificial overproduced condition. Moreover, a mutant strain without srpABC did not maintain its activity in long-term biphenyl (BP) degradation, which correlated with the loss of cell viability. The expression of SrpABC was significantly upregulated in the course of BP degradation. BP, 2-hydroxybiphenyl, 3-hydroxybiphenyl, and 2,3-dihydroxybiphenyl (2,3-DHBP) were revealed to be the inducers of srpABC 2,3-DHBP was verified to be a substrate of pump SrpABC; SrpABC can enhance the tolerance to 2,3-DHBP by pumping it out. The mutant strain B6-2ΔsrpS prolonged BP degradation with the increase of srpABC expression. These results suggest that the pump SrpABC of strain B6-2 plays a positive role in BP biodegradation by pumping out metabolized toxic substances such as 2,3-DHBP. This study provides insights into the versatile physiological functions of the widely distributed efflux pumps in the biodegradation of PAHs.IMPORTANCE Polycyclic aromatic hydrocarbons (PAHs) are notorious for their recalcitrance to degradation in the environment. A high frequency of the occurrence of the efflux pump genes was observed in the genomes of effective PAH degraders; however, their specific roles in the degradation of PAHs are still obscure. The significance of our study is in the identification of the function and mechanism of the efflux pump SrpABC of Pseudomonas putida strain B6-2 (DSM 28064) in the biphenyl degradation process. SrpABC is crucial for releasing the toxicity caused by intermediates that are unavoidably produced in PAH degradation, which enables an understanding of how cells maintain the intracellular balance of materials. The findings from this study provide a new perspective on PAH recalcitrance and shed light on enhancing PAH degradation by genetic engineering.

Structural basis for the transcriptional repressor NicR2 in nicotine degradation from Pseudomonas.

Nicotine is an environmental toxicant in tobacco wastes, imposing severe hazards for the health of human and other mammalians. NicR2, a TetR-like repressor from Pseudomonas putida S16, plays a critical role in regulating nicotine degradation. Here, we determined the crystal structures of NicR2 and its complex with the inducer 6-hydroxy-3-succinoyl-pyridine (HSP). The N-terminal domain of NicR2 contains a conserved helix-turn-helix (HTH) DNA-binding motif, while the C-terminal domain contains a cleft for its selective recognition for HSP. Residues R91, Y114 and Q118 of NicR2 form hydrogen bonds with HSP, their indispensable roles in NicR2's recognition with HSP were confirmed by structure-based mutagenesis combined with isothermal titration calorimetry analysis. Based on sequence alignment and structure comparison, Tyr67, Tyr68 and Lys72 of HTH motif were corroborated to take the major responsibility for DNA-binding using site-directed mutants. The 30-residue N-terminal extension of NicR2, especially residues 21-30 in the TFR arm, is required for the association with the operator DNA. Finally, we proposed that either NicR2 or the DNA would undergo a conformational change upon their association. Altogether, our structural and biochemical investigations unravel how NicR2 selectively recognizes HSP and DNA, and provide new insights into the TetR family of repressors.

Disruption of the unfolded protein response (UPR) by lead compound selectively suppresses cancer cell growth.

Identifying chemotherapy candidates with high selectivity against cancer cells is a major challenge in cancer treatment. Tumor microenvironments cause chronic endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) as an adaptive response. Here, one novel small-molecule compound, 17#, was discovered as a potent pan-UPR inhibitor. It exhibited good selection for growth inhibition when cancer cells were cultured in 2-deoxy-D-glucose (2DG), mimicking an in vitro glucose-deprived status. Additionally, 17# alone could mildly suppress the growth of HeLa tumor xenografts, and a synergistic anti-cancer effect was observed when 17# was combined with 2DG. A mechanistic study showed that 17#-induced selective anti-cancer effects were highly dependent on UPR inhibition, and overexpressing GRP78 or XBP1s reversed the 17#-induced growth inhibition and cell cycle arrest, partially by delaying the downregulation of the cell cycle regulator cyclin B1. Furthermore, 17# improved the sensitivity of anti-cancer drugs such as doxorubicin or etoposide. Our study presents evidence that disrupting the UPR has selective therapeutic potential and may enhance drug sensitivity.

Azoxystrobin, a mitochondrial complex III Qo site inhibitor, exerts beneficial metabolic effects in vivo and in vitro.

BACKGROUND: Several anti-diabetes drugs exert beneficial effects against metabolic syndrome by inhibiting mitochondrial function. Although much progress has been made toward understanding the role of mitochondrial function inhibitors in treating metabolic diseases, the potential effects of these inhibitors on mitochondrial respiratory chain complex III remain unclear. METHODS: We investigated the metabolic effects of azoxystrobin (AZOX), a Qo inhibitor of complex III, in a high-fat diet-fed mouse model with insulin resistance in order to elucidate the mechanism by which AZOX improves glucose and lipid metabolism at the metabolic cellular level. RESULTS: Acute administration of AZOX in mice increased the respiratory exchange ratio. Chronic treatment with AZOX reduced body weight and significantly improved glucose tolerance and insulin sensitivity in high-fat diet-fed mice. AZOX treatment resulted in decreased triacylglycerol accumulation and down-regulated the expression of genes involved in liver lipogenesis. AZOX increased glucose uptake in L6 myotubes and 3T3-L1 adipocytes and inhibited de novo lipogenesis in HepG2 cells. The findings indicate that AZOX-mediated alterations to lipid and glucose metabolism may depend on AMP-activated protein kinase (AMPK) signaling. CONCLUSIONS: AZOX, a Qo inhibitor of mitochondrial respiratory complex III, exerts whole-body beneficial effects on the regulation of glucose and lipid homeostasis in high-fat diet-fed mice. GENERAL SIGNIFICANCE: These findings provide evidence that a Qo inhibitor of mitochondrial respiratory complex III could represent a novel approach for the treatment of obesity.

Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas.

Microorganisms such as Pseudomonas putida play important roles in the mineralization of organic wastes and toxic compounds. To comprehensively and accurately elucidate key processes of nicotine degradation in Pseudomonas putida, we measured differential protein abundance levels with MS-based spectral counting in P. putida S16 grown on nicotine or glycerol, a non-repressive carbon source. In silico analyses highlighted significant clustering of proteins involved in a functional pathway in nicotine degradation. The transcriptional regulation of differentially expressed genes was analyzed by using quantitative reverse transcription-PCR. We observed the following key results: (i) The proteomes, containing 1,292 observed proteins, provide a detailed view of enzymes involved in nicotine metabolism. These proteins could be assigned to the functional groups of transport, detoxification, and amino acid metabolism. There were significant differences in the cytosolic protein patterns of cells growing in a nicotine medium and those in a glycerol medium. (ii) The key step in the conversion of 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine was catalyzed by a multi-enzyme reaction consisting of a molybdopeterin binding oxidase (spmA), molybdopterin dehydrogenase (spmB), and a (2Fe-2S)-binding ferredoxin (spmC) with molybdenum molybdopterin cytosine dinucleotide as a cofactor. (iii) The gene of a novel nicotine oxidoreductase (nicA2) was cloned, and the recombinant protein was characterized. The proteins and functional pathway identified in the current study represent attractive targets for degradation of environmental toxic compounds.