An integrated multi-omics approach reveals polymethoxylated flavonoid biosynthesis in Citrus reticulata cv. Chachiensis.

Citrus reticulata cv. Chachiensis (CRC) is an important medicinal plant, its dried mature peels named "Guangchenpi", has been used as a traditional Chinese medicine to treat cough, indigestion, and lung diseases for several hundred years. However, the biosynthesis of the crucial natural products polymethoxylated flavonoids (PMFs) in CRC remains unclear. Here, we report a chromosome-scale genome assembly of CRC with the size of 314.96 Mb and a contig N50 of 16.22 Mb. Using multi-omics resources, we discover a putative caffeic acid O-methyltransferase (CcOMT1) that can transfer a methyl group to the 3-hydroxyl of natsudaidain to form 3,5,6,7,8,3',4'-heptamethoxyflavone (HPMF). Based on transient overexpression and virus-induced gene silencing experiments, we propose that CcOMT1 is a candidate enzyme in HPMF biosynthesis. In addition, a potential gene regulatory network associated with PMF biosynthesis is identified. This study provides insights into PMF biosynthesis and may assist future research on mining genes for the biosynthesis of plant-based medicines.

Isoprenoid emissions from Schima superba and Cunninghamia lanceolata: Their responses to elevated temperature by two warming facilities.

Isoprenoids (including isoprene (ISO) and monoterpenes (MTs)) are the majority of biogenic volatile organic compounds (BVOCs) which are important carbon-containing secondary metabolites biosynthesized by organisms, especially plant in terrestrial ecosystem. Results of the warming effects on isoprenoid emissions vary within species and warming facilities, and thus conclusions remain controversial. In this study, two typical subtropical tree species seedlings of Schima superba and Cunninghamia lanceolata were cultivated under three conditions, namely no warming (CK) and two warming facilities (with infrared radiators (IR) and heating wires (HW)) in open top chamber (OTC), and the isoprenoid emissions were measured with preconcentor-GC-MS system after warming for one, two and four months. The results showed that the isoprenoid emissions from S. superba and C. lanceolata exhibited uniformity in response to two warming facilities. IR and HW both stimulated isoprenoid emissions in two plants after one month of treatment, with increased ratios of 16.3 % and 72.5 % for S. superba, and 2.47 and 5.96 times for C. lanceolata. However, the emissions were suppressed after four months, with more pronounced effect for HW. The variation in isoprenoid emissions was primarily associated with the levels of Pn, Tr, monoterpene synthase (MTPS) activity. C. lanceolata predominantly released MTs (mainly α-pinene, α-terpene, γ-terpene, and limonene), with 39.7 % to 99.6 % of the total isoprenoid but ISO was only a very minor constituent. For S. superba, MTs constituted 24.7 % to 96.1 % of total isoprenoid. It is noteworthy that HW generated a greater disturbance to physiology activity in plants. Our study provided more comprehensive and more convincing support for integrating temperature-elevation experiments of different ecosystems and assessing response and adaptation of forest carbon cycle to global warming.

[Prognosis Analysis of Additional Surgical Treatment for High-Risk T1 Colorectal Cancer Patients After Endoscopic Resection]. / è¿½åŠ å¤–ç§‘æ‰‹æœ¯æ²»ç–—å¯¹é«˜é£Žé™©T1æœŸç»“ç›´è‚ ç™Œå† é•œåˆ‡é™¤æ‚£è€ çš„é¢„åŽå½±å“.

Objective: To analyze the effect of additional surgery on the survival and prognosis of high-risk T1 colorectal cancer patients who have undergone endoscopic resection. Methods: The clinical data of patients with high-risk T1 colorectal cancer were retrospectively collected. The patients were divided into the endoscopic resection (ER) plus additional surgical resection (SR) group, or the ER+SR group, and the ER group according to whether additional SR were performed after ER. Baseline data of the patients and information on the location, size, and postoperative pathology of the lesions were collected. Patient survival-related information was obtained through the medical record system and patient follow-up. The primary outcome indicators were the overall survival and the colorectal cancer-specific survival. Univariate Cox regression analysis was used to screen survival-related risk factors and hazard ratio (HR) was calculated. Multivariate Cox regression analysis was used to analyze the independent influencing factors. Results: The data of 109 patients with T1 high-risk colorectal cancer were collected, with 52 patients in the ER group and 57 patients in the ER+SR group. The mean age of patients in the ER group was higher than that in the ER+SR group (65.21 years old vs. 60.54 years old, P=0.035), and the median endoscopic measurement of the size of lesions in the ER group was slightly lower than that in the ER+SR group (2.00 cm vs. 2.50 cm, P=0.026). The median follow-up time was 30.00 months, with the maximum follow-up time being 119 months, in the ER+SR group and there were 4 patients deaths, including one colorectal cancer-related death. Whereas the median follow-up time in the ER group was 28.50 months, with the maximum follow-up time being 78.00 months, and there were 4 patient deaths, including one caused by colorectal cancer. The overall 5-year cumulative survival rates in the ER+SR group and the ER group were 94.44% and 81.65%, respectively, and the cancer-specific 5-year cumulative survival rates in the ER+SR group and the ER group were 97.18% and 98.06%, respectively. The Kaplan-Meier analysis showed no significant difference in the overall cumulative survival or cancer-specific cumulative survival between the ER+SR and the ER groups. Univariate Cox regression analysis showed that age and the number of reviews were the risk factors of overall survival (HR=1.16 and HR=0.27, respectively), with age identified as an independent risk factor of overall survival in the multivariate Cox regression analysis (HR=1.10, P=0.045). Conclusion: For T1 colorectal cancer patients with high risk factors after ER, factors such as patient age and their personal treatment decisions should not be overlooked. In clinical practice, additional caution should be exercised in decision-making concerning additional surgery.

Melatonin inhibits the stemness of head and neck squamous cell carcinoma by modulating HA synthesis via the FOSL1/HAS3 axis.

Hyaluronic acid (HA) is a glycosaminoglycan and the main component of the extracellular matrix (ECM), which has been reported to interact with its receptor CD44 to play critical roles in the self-renewal and maintenance of cancer stem cells (CSCs) of multiple malignancies. Melatonin is a neuroendocrine hormone with pleiotropic antitumor properties. However, whether melatonin could regulate HA accumulation in the ECM to modulate the stemness of head and neck squamous cell carcinoma (HNSCC) remains unknown. In this study, we found that melatonin suppressed CSC-related markers, such as CD44, of HNSCC cells and decreased the tumor-initiating frequency of CSCs in vivo. In addition, melatonin modulated HA synthesis of HNSCC cells by downregulating the expression of hyaluronan synthase 3 (HAS3). Further study showed that the Fos-like 1 (FOSL1)/HAS3 axis mediated the inhibitory effects of melatonin on HA accumulation and stemness of HNSCC in a receptor-independent manner. Taken together, melatonin modulated HA synthesis through the FOSL1/HAS3 axis to inhibit the stemness of HNSCC cells, which elucidates the effect of melatonin on the ECM and provides a novel perspective on melatonin in HNSCC treatment.

Advances and challenges of first-line immunotherapy for non-small cell lung cancer: A review.

The current use of immune checkpoint inhibitors (ICIs) for the treatment of lung cancer has dramatically changed the clinical strategy for metastatic non-small cell lung cancer (mNSCLC). As a result of great achievements in clinical trials, 6 programmed death-1 inhibitors (sintilimab, camrelizumab, tislelizumab, pembrolizumab, cemiplimab, and nivolumab), 2 programmed death-ligand 1 inhibitors (sugemalimab and atezolizumab), and 1 cytotoxic T lymphocyte-associated antigen-4 inhibitor (ipilimumab) have been approved as first-line treatment for mNSCLC by the US Food and Drug Administration. Recently, research on ICIs has shifted from a large number of second-line to first-line settings in clinical trials. Results from first-line trials have shown that almost all driver-negative mNSCLC are treated with ICIs and significantly prolong patient survival; however, the low response rate and adverse reactions to immunotherapy remain to be addressed. Here, we summarize the use of ICIs, including monotherapy and combination therapy, in the first-line treatment of mNSCLC in recent years and discuss the low response rate and adverse reactions of ICIs as well as the challenges and expectations for the first-line treatment of mNSCLC in the future.

Dysregulation of immunity by cigarette smoking promotes inflammation and cancer: A review.

Smoking is a serious global health issue. Cigarette smoking contains over 7000 different chemicals. The main harmful components include nicotine, acrolein, aromatic hydrocarbons and heavy metals, which play the key role for cigarette-induced inflammation and carcinogenesis. Growing evidences show that cigarette smoking and its components exert a remarkable impact on regulation of immunity and dysregulated immunity promotes inflammation and cancer. Therefore, this comprehensive and up-to-date review covers four interrelated topics, including cigarette smoking, inflammation, cancer and immune system. The known harmful chemicals from cigarette smoking were summarized. Importantly, we discussed in depth the impact of cigarette smoking on the formation of inflammatory or tumor microenvironment, primarily by affecting immune effector cells, such as macrophages, neutrophils, and T lymphocytes. Furthermore, the main molecular mechanisms by which cigarette smoking induces inflammation and cancer, including changes in epigenetics, DNA damage and others were further summarized. This article will contribute to a better understanding of the impact of cigarette smoking on inducing inflammation and cancer.

A simplified D3 -creatine dilution method for skeletal muscle mass determination with dynamic correction of creatinine and D3 -creatinine using ultra-performance liquid chromatography-tandem mass spectrometry.

This study developed a simple method for muscle mass determination based on D3 -creatine dilution by removing the matrix effects of ultra-performance liquid chromatography-tandem mass spectrometry analysis through mutual correction of creatinine and D3 -creatinine. Rats were administered an oral tracer dose of D3 -creatine at age 6 weeks. Creatinine and D3 -creatinine in urine were detected using ultra-performance liquid chromatography-tandem mass spectrometry after diluting 20 times to obtain D3 -creatinine enrichment factor (mole percent excess). The mole percent excess obtained from peak area could be used to calculate muscle mass using the improved formula. The limit of detection was 0.500 ng/mL for D3 -creatinine. Creatinine and D3 -creatinine could be mutually corrected because of the same matrix effect, and D3 -creatine spillage was negligible within 0.22%. Isotopic steady time was consistent with that obtained using conventional methods. Bland-Altman plots demonstrated the satisfying consistency between the proposed method and magnetic resonance imaging. This is a simple and rapid measuring method of muscle mass based on D3 -creatine dilution that requires no accurate quantification of creatinine and D3 -creatinine concentrations and no urine sample collection to obtain D3 -creatine spillage.

Tigecycline Resistance-Associated Mutations in the MepA Efflux Pump in Staphylococcus aureus.

Tigecycline is an important antibacterial drug for treating infection by clinical multidrug-resistant bacteria, and tigecycline-resistant Staphylococcus aureus (TRSA) has been increasingly reported in recent years. Notably, only rpsJ and mepA are associated with the tigecycline resistance of S. aureus. The mepA gene encodes MepA efflux pumps, and the overexpression of mepA has been confirmed to be directly related to tigecycline resistance. Although the mutations of MepA widely occur, the associations between TRSA and mutations of MepA are still unclear. In this study, we explored mutations in the mepA genes from various sources. Then, tigecycline resistance-associated mutations T29I, E287G, and T29I+E287G in MepA were identified, and their effects were evaluated through mutant deletion and complementation, tigecycline accumulation assay, and molecular docking experiments. Results showed that the MICs of tigecycline, gentamicin, and amikacin increased in special complementary transformants and recovered after the addition of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The tigecycline accumulation assay of the mepA-deleted mutant strain and its complementary transformants showed that T29I, E287G, and T29I+E287G mutations promoted tigecycline efflux, and molecular docking showed that mutations T29I, E287G, and T29I+E287G decreased the binding energy and contributed to ligand binding. Moreover, we inferred the evolutionary trajectory of S. aureus under the selective pressure of tigecycline in vitro. Overall, our study indicated that mutations in MepA play important roles in tigecycline resistance in S. aureus. IMPORTANCE Previous analysis has shown that overexpression of MepA is an exact mechanism involved in tigecycline resistance apart from the rpsJ mutation and is usually dependent on the mutant mepR. However, no research has evaluated the effects of diverse mutations discovered in TRSA in MepA. This study demonstrates that the mutations in MepA confer resistance to tigecycline without overexpression and provides genotypic references for identifying TRSA. Although tigecycline resistance-associated mutations in MepA identified in this study have not been observed in clinical isolates, the mechanism should be explored given that S. aureus strains are prevalent in the environment. Measures should be implemented to contain TRSA within the time window before tigecycline resistance-associated mutations in MepA are prevalent.

GWAS, MWAS and mGWAS provide insights into precision agriculture based on genotype-dependent microbial effects in foxtail millet.

Genetic and environmental factors collectively determine plant growth and yield. In the past 20 years, genome-wide association studies (GWAS) have been conducted on crops to decipher genetic loci that contribute to growth and yield, however, plant genotype appears to be insufficient to explain the trait variations. Here, we unravel the associations between genotypic, phenotypic, and rhizoplane microbiota variables of 827 foxtail millet cultivars by an integrated GWAS, microbiome-wide association studies (MWAS) and microbiome genome-wide association studies (mGWAS) method. We identify 257 rhizoplane microbial biomarkers associated with six key agronomic traits and validated the microbial-mediated growth effects on foxtail millet using marker strains isolated from the field. The rhizoplane microbiota composition is mainly driven by variations in plant genes related to immunity, metabolites, hormone signaling and nutrient uptake. Among these, the host immune gene FLS2 and transcription factor bHLH35 are widely associated with the microbial taxa of the rhizoplane. We further uncover a plant genotype-microbiota interaction network that contributes to phenotype plasticity. The microbial-mediated growth effects on foxtail millet are dependent on the host genotype, suggesting that precision microbiome management could be used to engineer high-yielding cultivars in agriculture systems.

A New High Selective and Sensitive Fluorescent Probe for Al3+ based on Photochromic Salicylaldehyde Hydrazyl Diarylethene.

A new diarylethene derivative 1O decorated with a salicylaldehyde hydrazine moiety was designed and synthesized successfully, and its structure was confirmed by NMR. Diarylethene 1O showed eminent photochromism and high selectivity and sensitivity for Al3+ with turn-on fluorescent performance. As the concentration of Al3+ in 1O solution increased, the color of solution remarkably changed from dark to bright green with 313-fold fluorescent emission intensity enhancement. The 1:1 combination stoichiometry between 1O and Al3+ was verified by Job's plot and MS analysis. The association constant between 1O and Al3+ was 3.9 × 102 mol-1 L, and the limit of detection toward Al3+ was 7.98 × 10-9 mol L-1. Meanwhile, the probe can be utilized in practical water and logic circuits.

P(v) intermediate-mediated E1cB elimination for the synthesis of glycals.

Glycals are highly versatile and useful building blocks in the chemistry of carbohydrate and natural products. However, the practical synthesis of glycals remains a long-standing and mostly unsolved problem in synthetic chemistry. Herein, we present an unprecedented approach to make a variety of glycals using phosphonium hydrolysis-induced, P(v) intermediate-mediated E1cB elimination. The method provides a highly efficient, practical and scalable strategy for the synthesis of glycals with good generality and excellent yields. Furthermore, the strategy was successfully applied to late-stage modification of complex drug-like molecules. Additionally, the corresponding 1-deuterium-glycals were produced easily by simple t BuONa/D2O-hydrolysis-elimination. Mechanistic investigations indicated that the oxaphosphorane intermediate-mediated E1cB mechanism is responsible for the elimination reaction.

[Determination of Haloacetic Acids, Disinfection Byproducts, in Tap Water with Reversed-Phase Ultra-Performance Liquid Chromatography-High Resolution Mass Spectrometry].

Objective: To establish a method for quantitative analysis of haloacetic acids (HAAs), disinfection byproducts, in tap water with reversed-phase ultra-performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry. Methods: Tap water samples were collected and 0.70 g/L ascorbic acid was added to eliminate residual chlorine. Then, the water samples were directly injected into the instrument for analysis after filtration. After separation on a pentafluorobenzene (PFP) column with an inner diameter of 1.0 mm at a higher linear velocity and a lower volume flow rate compared with those of a narrow-bore column, nine HAAs, namely, monochloroacetic acid (MCAA), monobromoacetic acid (MBAA), dichloroacetic acid (DCAA), bromochloroacetic acid (BCAA), dibromoacetic acid (DBAA), trichloroacetic acid (TCAA), bromodichloroacetic acid（BDCAA), chlorodibromoacetic acid (CDBAA) and tribromoacetic acid (TBAA), were examined by negative electrospray ionization and full MS/dd-MS 2 acquisition mode. In order to adjust for the matrix effect, matrix matching calibration curves were used to quantitate the nine HAAs. Results: Good linearity was obtained for each of the nine HAAs within their respective linear ranges. The detection limits and quantification limits of the method were 0.020-1.0 µg/L and 0.060-3.0 µg/L. The recoveries were 69.8%-119%. Conclusion: The proposed method showed strengths in separation speed and qualitative accuracy. It did not require for complicated pretreatment procedures and can meet the need of tap water sample analysis.

The microbial diversity in industrial effluents makes high-throughput sequencing-based source tracking of the effluents possible.

In order to explore the microbial diversity in industrial effluents, and on this basis, to verify the feasibility of tracking industrial effluents in sewer networks based on sequencing data, we collected 28 sewage samples from the industrial effluents relative to four factories in Shenzhen, China, and sequenced the 16S rRNA genes to profile the microbial compositions. We identified 5413 operational taxonomic units (OTUs) in total, and found that microbial compositions were highly diverse among samples from different locations in the sewer system, with only 107 OTUs shared by 90% of the samples. These shared OTUs were enriched in the phylum of Proteobacteria, the families of Comamonadaceae and Pseudomonadaceae, as well as the genus of Pseudomonas, with both degradation related and pathogenic bacteria. More importantly, we found differences in microbial composition among samples relevant to different factories, and identified microbial markers differentiating effluents from these factories, which can be used to track the sources of the effluents. This study improved our understanding of microbial diversity in industrial effluents, proved the feasibility of industrial effluent source tracking based on sequencing data, and provided an alternative technique solution for environmental surveillance and management.

An Updated Meta-analysis: Similar Clinical Efficacy of Anterior and Posterior Approaches in Peroral Endoscopic Myotomy (POEM) for Achalasia.

Introduction: Currently, there are few studies on the efficacy of peroral endoscopic myotomy (POEM) in the anterior or posterior approach; however, limited studies have shown contradictory findings. Thus, the goal was to obtain more quantitative and objective outcomes and further compare the clinical efficacy of these two approaches in this meta-analysis. Methods: A comprehensive search of PubMed, Embase, Cochrane Library, and Web of Science was conducted to find studies relevant to POEM. The retrieval time was from database inception to September 2021. Studies reporting the effects of POEM according to the anterior or posterior approach were included. STATA 16.0 was used to perform statistical analysis, mainly comparing the quantitative objective indicators (lower esophageal sphincter (LES) pressure and Eckardt scores, etc.) in anterior and posterior approaches by meta-analysis. Result: A total of 19 studies with 1261 patients were finally included. Except for shorter procedure time in the posterior approach, other factors (pooled difference of LES pressure, Eckardt scores, clinical success, length of total myotomy, hospital stays, gastroesophageal reflux (GERD), and adverse event) were compared, and all above confirmed that there is no difference between anterior and posterior approaches, and the safety of POEM is ensured. In addition, both anterior and posterior myotomy can improve LES pressure and Eckardt scores, and the difference in anterior and posterior myotomy was unconspicuous. Conclusion: The terms of the pooled difference in LES pressure, Eckardt scores, and other factors (clinical success, length of total myotomy, hospital stays, GERD, adverse events, and procedure time) seemed to be similar for the anterior and posterior approaches. However, the further prognosis after POEM via anterior and posterior approaches needs to be answered in the future.

[The Screening Approaches for 34 Common Drugs and Metabolites in Biological Samples by Liquid Chromatography Orbital Trap Mass Spectrometry].

Objective: To establish a high-performance liquid chromatography orbital trap mass spectrometry (HPLC-Obitrap MS) method for screening 34 common drugs and metabolites in biological samples. Methods: The target analytes in urine and blood samples were extracted with ethyl acetate, concentrated by nitrogen blowing and redissolved. The hair samples were washed with water and acetone, dried and cut into bits of about 1 mm, and then crushed in a freezing grinder. The analytes were extracted with methanol, and after filtration, the filtrate was used for instrumental analysis. Hypersil Gold PFP (2.1 mm×100 mm, 3 µm) column was used for chromatographic separation. Methanol and 5 mmol/L ammonium acetate solution were used as mobile phase with gradient elution at a flow rate of 400 µL/min. Mass spectrometry was done by electrospray positive and negative ion alternation mode. The data were collected using Full MS and Full MS/dd-MS2 mode. Xcalibur 4.0 software was used to control instruments and to collect data, and TraceFinder 3.3 was used for screening and identification. Results: The method's detection limits for 34 drugs and their metabolites in blood, urine and hair samples were 3.30-10700 ng/L, 4.43-5440 ng/L, 0.0350-4.21 µg/kg, respectively. The intra-day and inter-day precisions of the spiked samples at the levels of 5.0, 10, and 20 µg/L were 3.50%-6.00% and 4.18%-9.90%, respectively. A total of 1125 biological samples of urine, blood and hair were collected and screened. The results showed that 96.7% of the drug users were taking a single drug, while 3.3% were mixed drug users. The main types of drug of abuse were methamphetamine (75.8%), heroin (18.5%), ketamine (2.4%) and other drugs (3.3%), and 87.9% of the positive samples were from male users. Compared with the results of high-performance liquid chromatography triple quadrupole mass spectrometry, this method can be used to identify more types of drugs in one run and to conduct retrospective analysis. Conclusion: The method established in the study is simple and sensitive and is well suited for the screening of common drugs and metabolites in biological samples.

Melatonin inhibits EMT and PD-L1 expression through the ERK1/2/FOSL1 pathway and regulates anti-tumor immunity in HNSCC.

Melatonin is an endogenous hormone with various biological functions and possesses anti-tumor properties in multiple malignancies. Immune evasion is one of the most important hallmarks of head and neck squamous cell carcinoma (HNSCC) and is closely related to tumor progression. However, as an immune modulator under physiological conditions, the roles of melatonin in tumor immunity in HNSCC remains unclear. In this study, we found that the endogenous melatonin levels in patients with HNSCC were lower than those in patients with benign tumors in head and neck. Importantly, lower melatonin levels were related to lymph node metastasis among patients with HNSCC. Moreover, melatonin significantly suppressed programmed death-ligand 1 (PD-L1) expression and inhibited epithelial-mesenchymal transition (EMT) of HNSCC through the ERK1/2/FOSL1 pathway in vitro and in vivo. In SCC7/C3H syngeneic mouse models, anti-programmed death-1 (PD-1) antibody combined with melatonin significantly inhibited tumor growth and modulated anti-tumor immunity by increasing CD8+ T cell infiltration and decreasing the regulatory T cell (Treg) proportion in the tumor microenvironment. Taken together, melatonin inhibited EMT and downregulated PD-L1 expression in HNSCC through the ERK1/2/FOSL1 pathway and exerted synergistic effects with anti-PD-1 antibody in vivo, which could provide promising strategies for HNSCC treatment.

Genome of Hippophae rhamnoides provides insights into a conserved molecular mechanism in actinorhizal and rhizobial symbioses.

Sea buckthorn (Hippophae rhamnoides), a horticulturally multipurpose species in the family Elaeagnaceae, can build associations with Frankia actinomycetes to enable symbiotic nitrogen-fixing. Currently, no high-quality reference genome is available for an actinorhizal plant, which greatly hinders the study of actinorhizal symbiotic nodulation. Here, by combining short-read, long-read and Hi-C sequencing technologies, we generated a chromosome-level reference genome of H. rhamnoides (scaffold N50: 65 Mb, and genome size: 730 Mb) and predicted 30 812 protein-coding genes mainly on 12 pseudochromosomes. Hippophae rhamnoides was found to share a high proportion of symbiotic nodulation genes with Medicago truncatula, implying a shared molecular mechanism between actinorhizal and rhizobial symbioses. Phylogenetic analysis clustered the three paralogous NODULE INCEPTION (NIN) genes of H. rhamnoides with those of other nodulating species, forming the NIN group that most likely evolved from the ancestral NLP group. The genome of H. rhamnoides will help us to decipher the underlying genetic programming of actinorhizal symbiosis, and our high-quality genome and transcriptomic resources will make H. rhamnoides a new excellent model plant for actinorhizal symbiosis research.

HSP90/IKK-rich small extracellular vesicles activate pro-angiogenic melanoma-associated fibroblasts via the NF-κB/CXCL1 axis.

Hypoxia is a main feature of most solid tumors, but how melanoma cells under hypoxic conditions exploit tumor microenvironment (TME) to facilitate tumor progression remains poorly understood. In this study, we found that hypoxic melanoma-derived small extracellular vesicles (sEVs) could improve the proangiogenic capability of cancer-associated fibroblasts (CAFs). This improvement was due to the activation of the IKK/IκB/NF-κB signaling pathway and upregulation of CXCL1 expression and secretion in CAFs. By proteomic analysis, we verified that hypoxia could promote enrichment of chaperone HSP90 and client protein phosphorylated IKKα/ß (p-IKKα/ß) in melanoma-derived sEVs. Delivery of the HSP90/p-IKKα/ß complex by sEVs could activate the IKK/IκB/NF-κB/CXCL1 axis in CAFs and promote angiogenesis in vitro and in vivo. Taken together, these findings deepen the understanding of hypoxic response in melanoma progression and provide potential targets for melanoma treatment.

Structural Abnormalities of Spermatozoa in Triploid Gynogenetic Crucian Carp (Carassius auratus).

The spermatozoa of triploid gynogenetic crucian carp (Carassius auratus) (3nDTCC) possess a spermatogenesis process with a normal genetic background. However, the genetic materials of these spermatozoa do not completely inherit gynogenetic progeny in general. Understanding the intrinsic mechanism may be helpful for developing breeding strategies of gynogenetic fishes. In this study, the spermatozoa ultrastructure was systematically studied in diploid red crucian carp and 3nDTCC to demonstrate their cytological structural differences. In addition, the artificial breeding tests of 3nDTCC(♀) with different ploidy spermatozoa were performed to verify the contributions of genetic materials from 3nDTCC spermatozoa to the gynogenesis progeny. Furthermore, the mRNA expression of centriole-related genes (i.e., cep57, cetn1, rootletin, and nek2) involved in spermatozoa packaging was also determined by quantitative real-time PCR (qPCR) to illustrate the molecular expression characteristics of the spermatozoa packaging process in 3nDTCC. The results reveal the adaptive features of spermatozoa in 3nDTCC, including the loose midpiece structure, abnormal head structure, and abnormal expression of centriole-related genes, which may influence the motility of spermatozoa and make it not involved normally in the genetic composition of the gynogenesis offspring.

[Determination of halogenated carboxylic acids in drinking water by ion chromatography-orbitrap mass spectrometry method].

OBJECTIVE: To establish a method for determination of the common and emerging halogenated carboxylic acids(HCAs) in drinking water by ion chromatography(IC) and quadrupole-orbitrap(Q-Orbitrap) high resolution mass spectrometry(HRMS) combined the traditional quantitative detection with semi-target analysis. METHODS: Effects on the type of chromatographic column, the composition of mobile phase, the flow rate of acetonitrile added post column, the column temperature, and the injection volume were studied in detail for IC-HRMS method, also for HRMS conditions. Drinking water sample was directly injected into IC-HRMS for analysis after filtration. The chromatography separation was performed on an AS21 anion exchange chromatography column(2 mm×250 mm) with the gradient elution using 800 mmol/L methylamine-water as mobile phase, and acetonitrile was added after column. The detection was conducted on HRMS with the electrospray ionization negative mode. And the quantitative analysis of 8 haloacetic acids(HAAs) and semi-target screening of 19 HCAs were achieved by full MS/dd-MS~2 mode. RESULTS: Good linearity(r>0.996) was obtained for each of 8 HAAs for IC-HRMS. The method detection limits(MDLs) and method quantification limits(MQLs) were in the range of 0.50-2.5 µg/L and 1.7-8.3 µg/L, respectively. Intra-and inter-day relative standard deviations(RSDs) were in the range of 1.50%-11.0% and 4.58%-10.9%, respectively. The recoveries were in the range of 61.3%-117%(n=6). The proposed method was applied to analyze 39 drinking water samples, and dichloroacetic acid and trichloroacetic acid were detected and quantified, with concentrations ranging from 1.35 to 48.0 µg/L. Besides, five HCAs(difluoroacetic acid, trifluoroacetic acid, bromochloroacetic acid, monochloropropionic acid and dichloropropionic acid) were preliminary identified with semi-target screening method. CONCLUSION: The developed method was simple, rapid, no sample preparation except filtration and low reagent cosumption, which could meet the need of drinking water monitoring and achieve comprehensive screening of HCAs in drinking water. In addition, full MS/dd-MS~2 data acquisition mode could provide retrospective analysis of existing data by adding the emerging or interesting HCAs into the screening compound database.