Dual regulations of cell cycle regulator DPa by auxin in Arabidopsis root distal stem cell maintenance.

The plant hormone auxin plays a key role to maintain root stem cell identity which is essential for root development. However, the molecular mechanism by which auxin regulates root distal stem cell (DSC) identity is not well understood. In this study, we revealed that the cell cycle factor DPa is a vital regulator in the maintenance of root DSC identity through multiple auxin signaling cascades. On the one hand, auxin positively regulates the transcription of DPa via AUXIN RESPONSE FACTOR 7 and ARF19. On the other hand, auxin enhances the protein stability of DPa through MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3)/MPK6-mediated phosphorylation. Consistently, mutation of the identified three threonine residues (Thr10, Thr25, and Thr227) of DPa to nonphosphorylated form alanine (DPa3A) highly decreased the phosphorylation level of DPa, which decreased its protein stability and affected the maintenance of root DSC identity. Taken together, this study provides insight into the molecular mechanism of how auxin regulates root distal stem cell identity through the dual regulations of DPa at both transcriptional and posttranslational levels.

A PilZ domain protein interacts with the transcriptional regulator HinK to regulate type VI secretion system in Pseudomonas aeruginosa.

Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZR5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression.

Development of novel lysosome-related signatures and their potential target drugs based on bulk RNA-seq and scRNA-seq for diabetic foot ulcers.

BACKGROUND: Diabetic foot ulcers (DFU) is the most serious complication of diabetes mellitus, which has become a global health problem due to its high morbidity and disability rates and the poor efficacy of conventional treatments. Thus, it is urgent to identify novel molecular targets to improve the prognosis and reduce disability rate in DFU patients. RESULTS: In the present study, bulk RNA-seq and scRNA-seq associated with DFU were downloaded from the GEO database. We identified 1393 DFU-related DEGs by differential analysis and WGCNA analysis together, and GO/KEGG analysis showed that these genes were associated with lysosomal and immune/inflammatory responses. Immediately thereafter, we identified CLU, RABGEF1 and ENPEP as DLGs for DFU using three machine learning algorithms (Randomforest, SVM-RFE and LASSO) and validated their diagnostic performance in a validation cohort independent of this study. Subsequently, we constructed a novel artificial neural network model for molecular diagnosis of DFU based on DLGs, and the diagnostic performance in the training and validation cohorts was sound. In single-cell sequencing, the heterogeneous expression of DLGs also provided favorable evidence for them to be potential diagnostic targets. In addition, the results of immune infiltration analysis showed that the abundance of mainstream immune cells, including B/T cells, was down-regulated in DFUs and significantly correlated with the expression of DLGs. Finally, we found latamoxef, parthenolide, meclofenoxate, and lomustine to be promising anti-DFU drugs by targeting DLGs. CONCLUSIONS: CLU, RABGEF1 and ENPEP can be used as novel lysosomal molecular signatures of DFU, and by targeting them, latamoxef, parthenolide, meclofenoxate and lomustine were identified as promising anti-DFU drugs. The present study provides new perspectives for the diagnosis and treatment of DFU and for improving the prognosis of DFU patients.

Correction: PRH1 mediates ARF7-LBD dependent auxin signaling to regulate lateral root development in Arabidopsis thaliana.

[This corrects the article DOI: 10.1371/journal.pgen.1008044.].

Synergetic collision and space separation in microfluidic chip for efficient affinity-discriminated molecular selection.

Efficient molecular selection is a prerequisite for generating molecular tools used in diagnosis, pathology, vaccinology, and therapeutics. Selection efficiency is thermodynamically highly dependent on the dissociation equilibrium that can be reached in a single round. Extreme shifting of equilibrium towards dissociation favors the retention of high-affinity ligands over those with lower affinity, thus improving the selection efficiency. We propose to synergize dual effects by deterministic lateral-displacement microfluidics, including the collision-based force effect and the two-dimensional (2D) separation-based concentration effect, to greatly shift the equilibrium. Compared with previous approaches, this system can remove more low- or moderate-affinity ligands and maintain most high-affinity ligands, thereby improving affinity discrimination in selection. This strategy is demonstrated on phage display in both experiment and simulation, and two peptides against tumor markers ephrin type-A receptor 2 (EphA2) and CD71 were obtained with high affinity and specificity within a single round of selection, which offers a promising direction for discovery of robust binding ligands for a wide range of biomedical applications.

Hydrophobization of Ribonucleic Acids for Facile Systemic Delivery and Multifaceted Cancer Immunotherapy.

Ribonucleic acids (RNAs) enable disease-related gene inhibition, expression, and editing and represent promising therapeutics in various diseases. The efficacy of RNA relies heavily on the presence of a secure and effective delivery system. Herein, we found that RNA could be hydrophobized by cationic lipid and ionizable lipid and conveniently coassemble with amphiphilic polymer to achieve micelle-like nanoparticles (MNP). The results of the study indicate that MNP exhibits a high level of efficiency in delivering RNA. Besides, the MNP encapsulating siRNA that targets CD47 and PD-L1 remarkably blocked these immune checkpoints in a melanoma tumor model and elicited a robust immune response. Moreover, the MNP encapsulating the mRNA of OVA achieved antigen translation and presentation, leading to an effective antitumor immunoprophylaxis outcome against OVA-expressing melanoma model. Our findings suggest that RNA hydrophobization could serve as a viable approach for delivering RNA, thereby facilitating the exploration of RNA therapy in disease treatment.

Metabolic and Molecular Insights Into Nicotiana benthamiana Trichome Exudates: An Ammunition Depot for Plant Resistance Against Insect Pests.

Nicotiana benthamiana, a widely acknowledged laboratory model plant for molecular studies, exhibits lethality to certain insect pests and can serve as a dead-end trap plant for pest control in the field. However, the underlying mechanism of N. benthamiana's resistance against insects remains unknown. Here, we elucidate that the lethal effect of N. benthamiana on the whitefly Bemisia tabaci arises from the toxic glandular trichome exudates. By comparing the metabolite profiles of trichome exudates, we found that 51 metabolites, including five O-acyl sugars (O-AS) with medium-chain acyl moieties, were highly accumulated in N. benthamiana. Silencing of two O-AS biosynthesis genes, branched-chain keto acid dehydrogenase (BCKD) and Isopropyl malate synthase-C (IPMS-C), significantly reduced the O-AS levels in N. benthamiana and its resistance against whiteflies. Additionally, we demonstrated that the higher expression levels of BCKD and IPMS-C in the trichomes of N. benthamiana contribute to O-AS synthesis and consequently enhance whitefly resistance. Furthermore, overexpression of NbBCKD and NbIPMS-C genes in the cultivated tobacco Nicotiana tabacum enhanced its resistance to whiteflies. Our study revealed the metabolic and molecular mechanisms underlying the lethal effect of N. benthamiana on whiteflies and presents a promising avenue for improving whitefly resistance.

Minimal residual disease monitoring in childhood Philadelphia chromosome-positive acute lymphoblastic leukemia: prognostic significance and correlation between multiparameter flow cytometry and real-time quantitative polymerase chain reaction

Not available.

Effects of sand substrate removal on the intestinal antioxidant and metabolism in Marsupenaeus japonicus.

M. japonicus is an important specie for factory farming, and factory farming requires an environment with sand at the bottom of the pond. However, the physiological responses as well as survival in the process of factory farming without laying sand are currently unknown. In the present study, we explored the effect of sand substrate removal on the intestinal histomorphology, antioxidant enzyme activity, and metabolic profile of M. japonicus. Our results indicate a gradual increase in the mortality rate of kuruma shrimp in ponds lacking sand substrate. The intestinal mucosa exhibited necrosis and the presence of vacuoles, with their number gradually increasing over time. The intestinal villi showed significant erosion, accompanied by a decrease in intestinal superoxide dismutase (SOD) activity and catalase (CAT) activity, and consistent with an upregulation in the expression of apoptosis-related genes such as caspase-3, indicating an adaptive response to the adverse environmental conditions. Additionally, the metabolomic analysis revealed that most significantly differential metabolites were linked to amino acid and lipid metabolism. These findings enhance our understanding of the sand substrate removal on the intestinal health of kuruma shrimp, which provides a basis for the factory farming.

Effective dose of propofol combined with intravenous esketamine for smooth flexible laryngeal mask airway insertion in two distinct age groups of preschool children.

BACKGROUND: There is limited research on the combined use of propofol and esketamine for anesthesia induction during flexible laryngeal mask airway (FLMA) in pediatric patients, and the effective dosage of propofol for FLMA smooth insertion remains unclear. We explored the effective dose of propofol combined with intravenous esketamine for the smooth insertion of FLMA in two distinct age groups of preschool children. METHODS: This is a prospective, observer-blind, interventional clinical study. Based on age, preschool children scheduled for elective surgery were divided into group A (aged 1-3 years) and group B (aged 3-6 years). Anesthesia induction was started with intravenous administration of esketamine (1.0 mg.kg- 1) followed by propofol administration. The FLMA was inserted 2 min after propofol administration at the target dose. The initial dose of propofol in group A and group B was 3.0 mg.kg- 1 and 2.5 mg.kg- 1, respectively. The target dose of propofol was determined with Dixon's up-and-down method, and the dosing interval of propofol was 0.5 mg.kg- 1. If there was smooth insertion of FLMA in the previous patient, the target dose of propofol for the next patient was reduced by 0.5 mg.kg- 1; otherwise, it was increased by 0.5 mg.kg- 1. The median 50% effective dose (ED50) for propofol was estimated using Dixon's up-and-down method and Probit analysis, while the 95% effective dose (ED95) was estimated through Probit analysis. Vital signs and adverse events during induction were recorded. RESULTS: Each group included 24 pediatric patients. Using Dixon's up-and-down method, the ED50 of propofol combined with esketamine for smooth insertion of FLMA in group A was 2.67 mg.kg- 1 (95%CI: 1.63-3.72), which was higher than that in group B (2.10 mg. kg- 1, 95%CI: 1.36-2.84) (p = 0.04). Using Probit analysis, the ED50 of propofol was calculated as 2.44 (95% CI: 1.02-3.15) mg.kg- 1 in group A and 1.93 (95% CI: 1.39-2.32) mg.kg- 1 in group B. The ED95 of propofol was 3.72 (95%CI: 3.07-15.18) mg.kg- 1 in group A and 2.74 (95%CI: 2.34-5.54) mg.kg- 1 in group B. In Group B, one pediatric patient experienced laryngospasm. CONCLUSION: The effective dose of propofol when combined with intravenous esketamine for smooth insertion of FLMA in children aged 1-3 years is 2.67 mg.kg- 1, which is higher than that in children aged 3-6 years (2.10 mg. kg- 1). TRIAL REGISTRATION: Chinese Clinical Trial Registry Center (Registration Number: ChiCTR2100044317; Registration Date: 2021/03/16).

Thermally activated persulfate (TAP)-enhanced tris(2-chloroethyl) phosphate removal in real-world waters based on a response-surface approach as well as toxicological evaluation on its degradation products.

As a typical organophosphorus flame retardant, tris(2-chloroethyl) phosphate (TCEP) is refractory in aqueous environment. The application of TAP is a promising method for removing pollutants. Herein, the removal of TCEP using TAP was rigorously investigated, and the effects of some key variables were optimized by the one-factor-at-a-time approach. To further evaluate the interactions among variables, the response surface methodology (RSM) based on central composite design was employed. Under optimized conditions (pH 5, [PS]0: [TCEP]0 = 500:1), the maximum removal efficiency (RE) of TCEP reached up to 90.6%. In real-world waters, the RE of TCEP spanned the range of 56%- 65% in river water, pond water, lake water and sanitary sewage. The low-concentration Cl- (0.1 mM) promoted TCEP degradation, but the contrary case occurred when the high-concentration Cl-, NO3-, CO32-, HCO3-, HPO42-, H2PO4-, NH4+ and humic acid were present owing to their prominently quenching effects on SO4•-. Both EPR and scavenger experiments revealed that the main radicals in the TAP system were SO4•- and •OH, in which SO4•- played the most crucial role in TCEP degradation. GC-MS/MS analysis disclosed that two degradation products appeared, sourcing from the replacement, oxidation, hydroxylation and water-molecule elimination reactions. The other two products were inferred from the comprehensive literature. As for acute toxicity to fish, daphnid and green algae, product A displayed the slightly higher toxicity, whereas other three products exhibited the declining toxicity as compared to their parent molecule. These findings offer a theoretical/practical reference for high-efficiency removal of TCEP and its ecotoxicological risk evaluation.

Synthesizing biomaterials in living organisms.

Living organisms fabricate biomacromolecules such as DNA, RNA, and proteins by the self-assembly process. The research on the mechanism of biomacromolecule formation also inspires the exploration of in vivo synthesized biomaterials. By elaborate design, artificial building blocks or precursors can self-assemble or polymerize into functional biomaterials within living organisms. In recent decades, these so-called in vivo synthesized biomaterials have achieved extensive applications in cell-fate manipulation, disease theranostics, bioanalysis, cellular surface engineering, and tissue regeneration. In this review, we classify strategies for in vivo synthesis into non-covalent, covalent, and genetic types. The development of these approaches is based on the chemical principles of supramolecular chemistry and synthetic chemistry, biological cues such as enzymes and microenvironments, and the means of synthetic biology. By summarizing the design principles in detail, some insights into the challenges and opportunities in this field are provided to  enlighten further research.

Carbon Dots: A Versatile Platform for Cu2+ Detection, Anti-Counterfeiting, and Bioimaging.

Carbon dots (CDs) have garnered extensive interest in basic physical chemistry as well as in biomedical applications due to their low cost, good biocompatibility, and great aqueous solubility. However, the synthesis of multi-functional carbon dots has always been a challenge for researchers. Here, we synthesized novel CDs with a high quantum yield of 28.2% through the straightforward hydrothermal method using Diaminomaleonitrile and Boc-D-2, 3-diaminopropionic acid. The size, chemical functional group, and photophysical properties of the CDs were characterized by TEM, FTIR, XPS, UV, and fluorescence. It was demonstrated in this study that the prepared CDs have a high quantum yield, excellent photostability, and low cytotoxicity. Regarding the highly water-soluble property of CDs, they were proven to possess selective and sensitive behavior against Cu2+ ions (linear range = 0-9 µM and limit of detection = 1.34 µM). Moreover, the CDs were utilized in fluorescent ink in anti-counterfeiting measures. Because of their low cytotoxicity and good biocompatibility, the CDs were also successfully utilized in cell imaging. Therefore, the as-prepared CDs have great potential in fluorescence sensing, anti-counterfeiting, and bioimaging.

MYB2 and MYB108 regulate lateral root development by interacting with LBD29 in Arabidopsis thaliana.

AUXIN RESPONSE FACTOR 7 (ARF7)-mediated auxin signaling plays a key role in lateral root (LR) development by regulating downstream LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor genes, including LBD16, LBD18, and LBD29. LBD proteins are believed to regulate the transcription of downstream genes as homodimers or heterodimers. However, whether LBD29 forms dimers with other proteins to regulate LR development remains unknown. Here, we determined that the Arabidopsis thaliana (L.) Heynh. MYB transcription factors MYB2 and MYB108 interact with LBD29 and regulate auxin-induced LR development. Both MYB2 and MYB108 were induced by auxin in an ARF7-dependent manner. Disruption of MYB2 by fusion with an SRDX domain severely affected auxin-induced LR formation and the ability of LBD29 to induce LR development. By contrast, overexpression of MYB2 or MYB108 resulted in greater LR numbers, except in the lbd29 mutant background. These findings underscore the interdependence and importance of MYB2, MYB108, and LBD29 in regulating LR development. In addition, MYB2-LBD29 and MYB108-LBD29 complexes promoted the expression of CUTICLE DESTRUCTING FACTOR 1 (CDEF1), a member of the GDSL (Gly-Asp-Ser-Leu) lipase/esterase family involved in LR development. In summary, this study identified MYB2-LBD29 and MYB108-LBD29 regulatory modules that act downstream of ARF7 and intricately control auxin-mediated LR development.

The Effect of Different Doses of Ropivacaine for Caudal Block in School-aged Children Undergoing Shang Ring Circumcision.

PURPOSE: The present study aimed to evaluate the effects of three different doses of ropivacaine in Shang Ring circumcision in school-aged children. DESIGN: This is a prospective, randomized, controlled study. METHODS: A total of 148 American Society of Anesthesiologists I to II children were enrolled and randomly assigned into the R0.2%, R0.25%, and R0.3% groups. These groups received 0.2%, 0.25%, and 0.3% of ropivacaine (0.5 mL/kg) for caudal block, respectively. The perioperative data on anesthesia quality (including adequate analgesia rate, analgesic duration, lower extremity numbness duration, and postoperative first urination time), and adverse events were collected. Hemodynamic variables were also measured perioperatively. FINDINGS: The adequate analgesia rate of caudal block in the R0.2% group (75.5%) was significantly lower than that in the R0.25% (94.0%) and R0.3% groups (98.0%) (P = .001). The analgesic duration of the R0.2% and R0.25% groups was significantly less than that of the R0.3% group (P < .001). The duration of lower extremity numbness in R0.2% group was significantly shorter than that in R0.25% (P < .05) and R0.3% groups (P < .01), and there was no significant difference between the R0.25% and R0.3% groups. The first urination time of R0.2% was significantly shorter than the R0.3% group (P < .05). There was no significant difference between the R0.2% and R0.25% or the R0.25% and R0.3% groups. No significant difference was found in adverse effects among groups (P > .05). CONCLUSIONS: Caudal block with 0.3% ropivacaine can provide more satisfactory intraoperative analgesia quality for school-aged children receiving Shang Ring circumcision, without increasing the risk of adverse effects.

Dynamic changes in intestinal microbiota and metabolite composition of pre-weaned beef calves.

Gut microbes and their metabolites are essential for maintaining host health and production. The intestinal microflora of pre-weaned calves gradually tends to mature with growth and development and has high plasticity, but few studies have explored the dynamic changes of intestinal microbiota and metabolites in pre-weaned beef calves. In this study, we tracked the dynamics of faecal microbiota in 13 new-born calves by 16S rRNA gene sequencing and analysed changes in faecal amino acid levels using metabolomics. Calves were divided into the relatively high average daily gain group (HA) and the relatively low average daily gain group (LA) for comparison. The results demonstrated that the alpha diversity of the faecal microbiota increased with calf growth and development. The abundance of Porphyromonadaceae bacterium DJF B175 increased in the HA group, while that of Lactobacillus reuteri decreased. The results of the LEfSe analysis showed that the microbiota of faeces of HA calves at eight weeks of age was enriched with P. bacterium DJF B175, while Escherichia coli and L. reuteri were enriched in the microbiota of faeces of LA calves. Besides, the total amino acid concentration decreased significantly in the eighth week compared with that in the first week (P < 0.05). Overall, even under the same management conditions, microorganisms and their metabolites interact to play different dynamic regulatory roles. Our results provide new insights into changes in the gut microbiota and metabolites of pre-weaned calves.

MPK3/6-induced degradation of ARR1/10/12 promotes salt tolerance in Arabidopsis.

Cytokinins are phytohormones that regulate plant development, growth, and responses to stress. In particular, cytokinin has been reported to negatively regulate plant adaptation to high salinity; however, the molecular mechanisms that counteract cytokinin signaling and enable salt tolerance are not fully understood. Here, we provide evidence that salt stress induces the degradation of the cytokinin signaling components Arabidopsis (Arabidopisis thaliana) response regulator 1 (ARR1), ARR10 and ARR12. Furthermore, the stress-activated mitogen-activated protein kinase 3 (MPK3) and MPK6 interact with and phosphorylate ARR1/10/12 to promote their degradation in response to salt stress. As expected, salt tolerance is decreased in the mpk3/6 double mutant, but enhanced upon ectopic MPK3/MPK6 activation in an MKK5DD line. Importantly, salt hypersensitivity phenotypes of the mpk3/6 line were significantly alleviated by mutation of ARR1/12. The above results indicate that MPK3/6 enhance salt tolerance in part via their negative regulation of ARR1/10/12 protein stability. Thus, our work reveals a new molecular mechanism underlying salt-induced stress adaptation and the inhibition of plant growth, via enhanced degradation of cytokinin signaling components.

Ambient Air Pollution and Atherosclerosis: A Potential Mediating Role of Sphingolipids.

BACKGROUND: The pathophysiological mechanisms of air pollution-induced atherosclerosis are incompletely understood. Sphingolipids serve as biological intermediates during atherosclerosis development by facilitating production of proatherogenic apoB (apolipoprotein B)-containing lipoproteins. We explored whether sphingolipids mediate the proatherogenic effects of air pollution. METHODS: This was a prospective panel study of 110 participants (mean age 56.5 years) followed from 2013 to 2015 in Beijing, China. Targeted lipidomic analyses were used to quantify 24 sphingolipids in 579 plasma samples. The mass concentrations of ambient particulate matter ≤2.5 µm in diameter (PM2.5) were continuously monitored by a fixed station. We evaluated the associations between sphingolipid levels and average PM2.5 concentrations 1-30 days before clinic visits using linear mixed-effects models and explored whether sphingolipids mediate PM2.5-associated changes in the levels of proatherogenic apoB-containing lipoproteins (LDL-C [low-density lipoprotein cholesterol] and non-HDL-C [nonhigh-density lipoprotein cholesterol]) using mediation analyses. RESULTS: We observed significant increases in the levels of non-HDL-C and fourteen sphingolipids associated with PM2.5 exposure, from short- (14 days) to medium-term (30 days) exposure time windows. The associations exhibited near-monotonic increases and peaked in 30-day time window. Increased levels of the sphingolipids, namely, sphinganine, ceramide C24:0, sphingomyelins C16:0/C18:0/C18:1/C20:0/C22:0/C24:0, and hexosylceramides C16:0/C18:0/C20:0/C22:0/C24:0/C24:1 significantly mediated 32%, 58%, 35% to 93%, and 23% to 86%, respectively, of the positive association between 14-day PM2.5 average and the non-HDL-C level, but not the LDL-C level. Similar mediation effects (19%-91%) of the sphingolipids were also observed in 30-day time window. CONCLUSIONS: Our results suggest that sphingolipids may mediate the proatherogenic effects of short- and medium-term PM2.5 exposure.

SKP2-Promoted Ubiquitination of FOXO3 Promotes the Development of Asthma.

Asthma is a respiratory disease with a dramatically increasing incidence globally. The present study explored the roles of S-phase kinase-associated protein 2 (SKP2) and forkhead box O3 (FOXO3) in asthma and their involvement in the Krüppel-like factor 15-lipoprotein receptor-related protein 5 (KLF15-LRP5) axis. SKP2 expression in patients with asthma and OVA-induced asthmatic Sprague Dawley rats was detected by reverse transcription quantitative PCR and Western blot assays. Alterations in SKP2 and LRP5 expression were evaluated in OVA-induced asthmatic rats, followed by measurement of inflammatory cytokines using ELISA and airway resistance using a methacholine challenge test. We applied TGF-ß1 to establish the airway smooth muscle cell (ASMC) proliferation model of asthma. The FOXO3 ubiquitination and changes in cell biological behaviors were detected using immunoprecipitation, MTT, and Annexin V/propidium iodide assays. Flow cytometry was adopted to detect cell cycle, and ELISA was used to measure the concentrations of IL-4, IL-5, IL-13, and IgE in rat bronchoalveolar lavage fluid. SKP2 was highly expressed and FOXO3 was poorly expressed in patients with asthma and in OVA-induced asthmatic rats. SKP2 silencing decreased IL-4, IL-5, IL-13, and IgE expression in rat bronchoalveolar lavage fluid, whereas SKP2 enhanced FOXO3 ubiquitination to upregulate KLF15, which bound to the LRP5 promoter in TGF-ß1-induced ASMCs and increased LRP5 expression. SKP2 enhanced airway hyperresponsiveness and inflammation in the OVA-induced rat model and augmented TGF-ß1-induced ASMC proliferation by inhibiting the FOXO3/KLF15/LRP5 axis. Additionally, overexpressed SKP2 resulted in reduced numbers of ASMCs in the G1 phase but increased numbers in the G2/M phase. Collectively, we show that SKP2 promotes FOXO3 ubiquitination to suppress the KLF15-LRP5 axis, thereby exacerbating asthma.

Development and validation of a simple and rapid HILIC-MS/MS method for the quantification of low-abundant lysoglycerophospholipids in human plasma.

Lysoglycerophospholipids (Lyso-GPLs) are an essential class of signaling lipids with potential roles in human diseases, such as cancer, central nervous system diseases, and atherosclerosis. Current methods for the quantification of Lyso-GPLs involve complex sample pretreatment, long analysis times, and insufficient validation, which hinder the research of Lyso-GPLs in human studies, especially for Lyso-GPLs with low abundance in human plasma such as lysophosphatidic acid (LPA), lysophosphatidylinositol (LPI), lysophosphatidylglycerol (LPG), lysophosphatidylserine (LysoPS), lyso-platelet-activating factor (LysoPAF), and cyclic phosphatidic acid (cPA). Herein, we report the development and validation of a simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of Lyso-GPLs with low abundance in plasma. Protein precipitation using MeOH for Lyso-GPL extraction, quick separation (within 18 min) based on hydrophilic interaction liquid chromatography (HILIC), and sensitive MS detection under dynamic multiple reaction monitoring (dMRM) mode enabled efficient quantification of 22 Lyso-GPLs including 2 cPA, 4 LPG, 11 LPA, 2 LysoPS, and 3 LysoPAF in 50 µL of human plasma. The present method showed good linearity (goodness of fit, 0.99823-0.99995), sensitivity (lower limit of quantification, 0.03-14.06 ng/mL), accuracy (73-117%), precision (coefficient of variation ≤ 28%), carryover (≤ 17%), recovery (80-110%), and stability (83-123%). We applied the method in an epidemiological study and report concentrations of 18 Lyso-GPLs in 567 human plasma samples comparable to those of previous studies. Significant negative associations of LysoPAF C18, LysoPAF C18:1, and LysoPAF C16 with homeostatic model assessment for insulin resistance (HOMA-IR) level were observed; this indicates possible roles of LysoPAF in glucose homeostasis. The application of the present method will improve understanding of the roles of circulating low-abundant Lyso-GPLs in health and diseases.