Targeting NAT10 inhibits osteosarcoma progression via ATF4/ASNS-mediated asparagine biosynthesis.

Despite advances in treatment, the prognosis of patients with osteosarcoma remains unsatisfactory, and searching for potential targets is imperative. Here, we identify N4-acetylcytidine (ac4C) acetyltransferase 10 (NAT10) as a candidate therapeutic target in osteosarcoma through functional screening. NAT10 overexpression is correlated with a poor prognosis, and NAT10 knockout inhibits osteosarcoma progression. Mechanistically, NAT10 enhances mRNA stability of activating transcription factor 4 (ATF4) through ac4C modification. ATF4 induces the transcription of asparagine synthetase (ASNS), which catalyzes asparagine (Asn) biosynthesis, facilitating osteosarcoma progression. Utilizing virtual screening, we identify paliperidone and AG-401 as potential NAT10 inhibitors, and both inhibitors are found to bind to NAT10 proteins. Inhibiting NAT10 suppresses osteosarcoma progression in vivo. Combined treatment using paliperidone and AG-401 produces synergistic inhibition for osteosarcoma in patient-derived xenograft (PDX) models. Our findings demonstrate that NAT10 facilitates osteosarcoma progression through the ATF4/ASNS/Asn axis, and pharmacological inhibition of NAT10 may be a feasible therapeutic approach for osteosarcoma.

Ac-HSP20 regulates autophagy and promotes the encystation of Acanthamoeba castellanii by inhibiting the PI3K/AKT/mTOR signaling pathway.

BACKGROUND: The encystation of Acanthamoeba castellanii has important ecological and medical significance. Blocking encystation is the key to preventing transmission and curing infections caused by A. castellanii. The formation of autophagosomes is one of the most important changes that occur during the encystation of Acanthamoeba. Our previous studies have shown that the heat shock protein 20 of A. castellanii (Ac-HSP20) is involved in its encystation. This study aimed to determine the role and mechanism of Ac-HSP20 in regulating autophagy involved in the encystation of A. castellanii. METHODS: Immunofluorescence assay, western blotting and transmission electron microscopy were used to analyze the dynamic changes in autophagy during the initiation and continuation of encystation. The knockdown of Ac-HSP20 was performed to clarify its regulation of encystation and autophagy and to elucidate the molecular mechanism by which Ac-HSP20 participates in autophagy to promote cyst maturation. RESULTS: The encystation rates and autophagosomes were significantly decreased by treatment with the autophagy inhibitor 3-MA. The autophagy marker LC3B and autophagic lysosomes increased with the induced duration of encystation and reached the maximum at 48 h. The encystation rate, LC3B expression and autophagosomes decreased when Ac-HSP20 was knocked down by siRNA transfection. In addition, the expression levels of Ac-HSP20 and LC3B increased and the expressions of p-AKT and p-mTOR decreased after 48 h of encystation without knockdown. However, the expressions of p-AKT and p-mTOR increased while the expression of LC3B decreased under the knockdown of Ac-HSP20. Furthermore, the protein expression of LC3B increased when the PI3K/AKT/mTOR signaling pathway was inhibited but decreased when the pathway was activated. CONCLUSIONS: The results demonstrated that autophagy is positively correlated with the encystation of A. castellanii, and Ac-HSP20 regulates autophagy to maintain the homeostasis of A. castellanii by inhibiting the PI3K /AKT /mTOR signaling pathway, thus promoting the maturation and stability of encystation.

RNA modification-related genes illuminate prognostic signature and mechanism in esophageal squamous cell carcinoma.

Emerging studies have demonstrated the link between RNA modifications and various cancers, while the predictive value and functional mechanisms of RNA modification-related genes (RMGs) in esophageal squamous cell carcinoma (ESCC) remain unclear. Here we established a prognostic signature for ESCC based on five RMGs. The analysis of ESCC clinical samples further verified the prognostic power of the prognostic signature. Moreover, we found that the knockdown of NSUN6 promotes ESCC progression in vitro and in vivo, whereas the overexpression of NSUN6 inhibits the malignant phenotype of ESCC cells. Mechanically, NSUN6 mediated tRNA m5C modifications selectively enhance the translation efficiency of CDH1 mRNA in a codon dependent manner. Rescue assays revealed that E-cadherin is an essential downstream target that mediates NSUN6's function in the regulation of ESCC progression. These findings offer additional insights into the link between ESCC and RMGs, as well as provide potential strategies for ESCC management and therapy.

Ceramide metabolism-related prognostic signature and immunosuppressive function of ST3GAL1 in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor with elevated disability and mortality rates in children and adolescents and the therapeutic effect for osteosarcoma has remained stagnant in the past 30 years. Emerging evidence has shown ceramide metabolism plays a vital role in tumor progression, but its mechanisms in osteosarcoma progression remain unknown. Through consensus clustering and LASSO regression analysis based on the osteosarcoma cohorts from TARGET database, we constructed a ceramide metabolism-related prognostic signature including ten genes for osteosarcoma, with ST3GAL1 exhibiting the highest hazard ratio. Biological signatures analysis demonstrated that ceramide metabolism was associated with immune-related pathways, immune cell infiltration and the expression of immune checkpoint genes. Single-cell profiling revealed that ceramide metabolism was enriched in myeloid, osteoblast and mesenchymal cells. The interaction between TAMs and CD8+ T cells played an essential role in osteosarcoma. ST3GAL1 regulated the SPP1-CD44 interaction between TAMs and CD8+ T cells and IL-10 secretion in TAMs through α2,3 sialic acid receptors, which inhibited CD8+ T cell function. IHC analysis showed that ST3GAL1 expression correlated with the prognosis of osteosarcoma patients. Co-culture assay revealed that upregulation of ST3GAL1 in tumor cells regulated the differentiation of TAMs and cytokine secretion. Collectively, our findings demonstrated that ceramide metabolism was associated with clinical outcome in osteosarcoma. ST3GAL1 facilitated tumor progression through regulating tumor immune microenvironment, providing a feasible therapeutic approach for patients with osteosarcoma.

First report of environmental bla PAC-1-carrying Aeromonas enteropelogenes.

The bla PAC-1 has been reported in Central Asia and Europe countries like Afghanistan and France in Aeromonas caviae and Pseudomonas aeruginosa strains from animals and patients, respectively. However, there is no record of bla PAC-1-carrying strain from the natural environment, and bla PAC-1-carrying Aeromonas has not been reported in the Asia Pacific. Here, we report the first known enviromental bla PAC-1-carrying Aeromonas enteropelogenes in the world from reservoir water in Singapore. We have performed a comprehensive genetic environment alignment and comparison of bla PAC-1 between our strain and other strains from different countries and sources and found the bla PAC-1 located on a highly conserved gene cluster. We suggest that environmental Aeromonas strains may act as a hidden reservoir involved in the circulating of bla PAC-1. The finding of conserved bla PAC-1 cluster also suggested the existence of multiple transmission pathways of bla PAC-1 in the Asia-Pacific region, involving multiple sources and different species.

NAT10-mediated ac4C tRNA modification promotes EGFR mRNA translation and gefitinib resistance in cancer.

Aberrant RNA modifications are frequently associated with cancers, while the underlying mechanisms and clinical significance remain poorly understood. Here, we find that the ac4C RNA acetyltransferase NAT10 is significantly upregulated in esophageal cancers (ESCAs) and associated with poor ESCA prognosis. In addition, using ESCA cell lines and mouse models, we confirm the critical functions of NAT10 in promoting ESCA tumorigenesis and progression in vitro and in vivo. Mechanistically, NAT10 depletion reduces the abundance of ac4C-modified tRNAs and decreases the translation efficiencies of mRNAs enriched for ac4C-modified tRNA-decoded codons. We further identify EGFR as a key downstream target that facilitates NAT10's oncogenic functions. In terms of clinical significance, we demonstrate that NAT10 depletion and gefitinib treatment synergistically inhibit ESCA progression in vitro and in vivo. Our data indicate the mechanisms underlying ESCA progression at the layer of mRNA translation control and provide molecular insights for the development of effective cancer therapeutic strategies.

METTL1/WDR4-mediated tRNA m7G modification and mRNA translation control promote oncogenesis and doxorubicin resistance.

Osteosarcoma is the most common bone tumor that leads to high mortality in adolescents and children. The tRNA N7-methylguanosine methyltransferase METTL1 is located in chromosome 12q14.1, a region that is frequently amplified in osteosarcoma patients, while its functions and underlying mechanisms in regulation of osteosarcoma remain unknown. Herein we show that METTL1 and WDR4 are overexpressed in osteosarcoma and associated with poor patient prognosis. Knockdown of METTL1 or WDR4 causes decreased tRNA m7G modification level and impairs osteosarcoma progression in vitro and in vivo. Conversely, METTL1/WDR4 overexpression promotes osteosarcoma proliferation, migration and invasion capacities. tRNA methylation and mRNA translation profiling indicate that METTL1/WDR4 modified tRNAs enhance translation of mRNAs with more m7G tRNA-decoded codons, including extracellular matrix (ECM) remodeling effectors, which facilitates osteosarcoma progression and chemoresistance to doxorubicin. Our study demonstrates METTL1/WDR4 mediated tRNA m7G modification plays crucial oncogenic functions to enhance osteosarcoma progression and chemoresistance to doxorubicin via alteration of oncogenic mRNA translation, suggesting METTL1 inhibition combined with chemotherapy is a promising strategy for treatment of osteosarcoma patients.

mRNA alternative polyadenylation (APA) in regulation of gene expression and diseases.

The mRNA polyadenylation plays essential function in regulation of mRNA metabolism. Mis-regulations of mRNA polyadenylation are frequently linked with aberrant gene expression and disease progression. Under the action of polyadenylate polymerase, poly(A) tail is synthesized after the polyadenylation signal (PAS) sites on the mRNAs. Alternative polyadenylation (APA) often occurs in mRNAs with multiple poly(A) sites, producing different 3' ends for transcript variants, and therefore plays important functions in gene expression regulation. In this review, we first summarize the classical process of mRNA 3'-terminal formation and discuss the length control mechanisms of poly(A) in nucleus and cytoplasm. Then we review the research progress on alternative polyadenylation regulation and the APA site selection mechanism. Finally, we summarize the functional roles of APA in the regulation of gene expression and diseases including cancers.

N6-methyladenosine (m6A) in 18S rRNA promotes fatty acid metabolism and oncogenic transformation.

Aberrant RNA modifications lead to dysregulated gene expression and cancer progression. Ribosomal RNA (rRNA) accounts for more than 80% of a cell's total RNA, but the functions and molecular mechanisms underlying rRNA modifications in cancers are poorly understood. Here, we show that the 18S rRNA N6-methyladenosine (m6A) methyltransferase complex METTL5-TRMT112 is upregulated in various cancer types and correlated with poor prognosis. In addition, we demonstrate the critical functions of METTL5 in promoting hepatocellular carcinoma (HCC) tumorigenesis in vitro and in mouse models. Mechanistically, depletion of METTL5-mediated 18S rRNA m6A modification results in impaired 80S ribosome assembly and decreased translation of mRNAs involved in fatty acid metabolism. We further reveal that ACSL4 mediates the function of METTL5 on fatty acid metabolism and HCC progression, and targeting ACSL4 and METTL5 synergistically inhibits HCC tumorigenesis in vivo. Our study uncovers mechanistic insights underlying mRNA translation control and HCC tumorigenesis through lipid metabolism remodeling and provides a molecular basis for the development of therapeutic strategies for HCC treatment.

N7-methylguanosine tRNA modification promotes esophageal squamous cell carcinoma tumorigenesis via the RPTOR/ULK1/autophagy axis.

Mis-regulated RNA modifications promote the processing and translation of oncogenic mRNAs to facilitate cancer progression, while the molecular mechanisms remain unclear. Here we reveal that tRNA m7G methyltransferase complex proteins METTL1 and WDR4 are significantly up-regulated in esophageal squamous cell carcinoma (ESCC) tissues and associated with poor ESCC prognosis. In addition, METTL1 and WDR4 promote ESCC progression via the tRNA m7G methyltransferase activity in vitro and in vivo. Mechanistically, METTL1 or WDR4 knockdown leads to decreased expression of m7G-modified tRNAs and reduces the translation of a subset of oncogenic transcripts enriched in RPTOR/ULK1/autophagy pathway. Furthermore, ESCC models using Mettl1 conditional knockout and knockin mice uncover the essential function of METTL1 in promoting ESCC tumorigenesis in vivo. Our study demonstrates the important oncogenic function of mis-regulated tRNA m7G modification in ESCC, and suggest that targeting METTL1 and its downstream signaling axis could be a promising therapeutic target for ESCC treatment.

Loss of m6A Methyltransferase METTL5 Promotes Cardiac Hypertrophy Through Epitranscriptomic Control of SUZ12 Expression.

Enhancement of protein synthesis from mRNA translation is one of the key steps supporting cardiomyocyte hypertrophy during cardiac remodeling. The methyltransferase-like5 (METTL5), which catalyzes m6A modification of 18S rRNA at position A1832, has been shown to regulate the efficiency of mRNA translation during the differentiation of ES cells and the growth of cancer cells. It remains unknown whether and how METTL5 regulates cardiac hypertrophy. In this study, we have generated a mouse model, METTL5-cKO, with cardiac-specific depletion of METTL5 in vivo. Loss function of METTL5 promotes pressure overload-induced cardiomyocyte hypertrophy and adverse remodeling. The regulatory function of METTL5 in hypertrophic growth of cardiomyocytes was further confirmed with both gain- and loss-of-function approaches in primary cardiomyocytes. Mechanically, METTL5 can modulate the mRNA translation of SUZ12, a core component of PRC2 complex, and further regulate the transcriptomic shift during cardiac hypertrophy. Altogether, our study may uncover an important translational regulator of cardiac hypertrophy through m6A modification.

METTL3-mediated m6A mRNA modification promotes esophageal cancer initiation and progression via Notch signaling pathway.

Esophageal cancer is a lethal malignancy with a high mortality rate, while the molecular mechanisms underlying esophageal cancer pathogenesis are still poorly understood. Here, we found that the N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) is significantly upregulated in esophageal squamous cell carcinoma (ESCC) and associated with poor patient prognosis. Depletion of METTL3 results in decreased ESCC growth and progression in vitro and in vivo. We further established ESCC initiation and progression models using Mettl3 conditional knockout mouse and revealed that 3METTL3-mediated m6A modification promotes ESCC initiation and progression in vivo. Moreover, using METTL3 overexpression ESCC cell model and Mettl3 conditional knockin mouse model, we demonstrated the critical function of METTL3 in promoting ESCC tumorigenesis in vitro and in vivo. Mechanistically, METTL3-catalyzed m6A modification promotes NOTCH1 expression and the activation of the Notch signaling pathway. Forced activation of Notch signaling pathway successfully rescues the growth, migration, and invasion capacities of METTL3-depleted ESCC cells. Our data uncovered important mechanistical insights underlying ESCC tumorigenesis and provided molecular basis for the development of novel strategies for ESCC diagnosis and treatment.

One-Step Synthesis of Water-Soluble CdS Quantum Dots for Silver-Ion Detection.

To realize fast synthesis of cadmium sulfide (CdS) quantum dots with a low-toxic material, a one-step synthesis method is investigated and conducted. Potato extract is used as a stabilizer and modifier, by which aqueous CdS quantum dots can be prepared at a lower temperature with a shorter time. Through systematic characterization and analysis, a green and fast synthesis mechanism is demonstrated in detail. And the nanoscale CdS quantum dots are uniform in size and dispersity. With low cost and high sensitivity, the prepared CdS quantum dots show promising application in silver-ion detection. This method shows great significance for an environmentally friendly and facile synthesis of CdS quantum dots.

Effect of High-Dispersible Graphene on the Strength and Durability of Cement Mortars.

Graphene's outstanding properties make it a potential material for reinforced cementitious composites. However, its shortcomings, such as easy agglomeration and poor dispersion, severely restrict its application in cementitious materials. In this paper, a highly dispersible graphene (TiO2-RGO) with better dispersibility compared with graphene oxide (GO) is obtained through improvement of the graphene preparation method. In this study, both GO and TiO2-RGO can improve the pore size distribution of cement mortars. According to the results of the mercury intrusion porosity (MIP) test, the porosity of cement mortar mixed with GO and TiO2-RGO was reduced by 26% and 40%, respectively, relative to ordinary cement mortar specimens. However, the TiO2-RGO cement mortars showed better pore size distribution and porosity than GO cement mortars. Comparative tests on the strength and durability of ordinary cement mortars, GO cement mortars, and TiO2-RGO cement mortars were conducted, and it was found that with the same amount of TiO2-RGO and GO, the TiO2-RGO cement mortars have nearly twice the strength of GO cement mortars. In addition, it has far higher durability, such as impermeability and chloride ion penetration resistance, than GO cement mortars. These results indicate that TiO2-RGO prepared by titanium dioxide (TiO2) intercalation can better improve the strength and durability performance of cement mortars compared to GO.

Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Jurong Lake, Singapore with Whole-Genome-Sequencing.

Background: The fast-spreading of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli (ESBL-producing E. coli) and ESBL genes has become a big challenge to public health. The risk of spreading ESBL genes and pathogens in the environment and community has raised public health concern. The characterizing and whole-genome sequencing studies of ESBL-producing bacteria from reservoir water in Singapore is still limited. Materials and methods: The reservoir water sample was taken from two randomly selected sampling points of the Chinese Garden (Jurong river reservoir), which is a popular reservoir park in Singapore. The bacteria of the water sample were collected with 0.45 µm filter membranes and enriched before processing with ESBL-producing E. coli screening. The collected ESBL positive isolates were further characterized by both phenotypic tests including disc diffusion and microdilution Minimum Inhibitory Concentration (MIC) test, and also genotypic test as whole-genome sequencing analysis. Besides, to investigate the transferability of the resistance gene, a conjugation test was performed with the J53 E. coli strain as the gene receptor. Result: Nine ESBL-producing E. coli isolates were collected and confirmed as ESBL-producing with both phenotypic and genotypic tests. A potential pathogen as ST131 clade A isolate was identified, and all isolates were determined to harbor a blaCTX-M gene. Among them, strain J1E4 was resistant to polymyxin E and confirmed to harboring a conjugatable mcr-1 gene. Further genetic environment analysis has reflected a conversed gene cluster formed by insert sequence (IS), blaCTX-M-15, and WbuC family cupin-fold metalloprotein, which may potentially jump from the plasmids to the chromosome. Conclusion: The first time we reported the whole genome sequencing (WGS) data of ESBL-producing E. coli including potential pathogen (ST131) present in reservoir water in Singapore. The ESBL-producing E. coli from reservoir water also carrying conjugatable colistin resistance genes which may become a risk to human health.

Prevalence and genomic analysis of ESBL-producing Escherichia coli in retail raw meats in Singapore.

OBJECTIVES: To determine the prevalence and genetic characteristics of ESBL-producing Escherichia coli in retail raw meats from Singapore markets. METHODS: A total of 634 raw meat (chicken, pork and beef) samples were collected from markets in Singapore during June 2017-October 2018. The samples were enriched overnight and then incubated on Brilliance™ ESBL Agar. Presumptive ESBL isolates were confirmed using the double-disc synergy test. Confirmed ESBL-producing E. coli were sent for WGS and bioinformatic analysis was performed. RESULTS: The prevalence of ESBL-producing E. coli in chicken, pork and beef meats was 51.2% (109/213), 26.9% (58/216) and 7.3% (15/205), respectively. A total of 225 ESBL-producing E. coli were isolated from 184 samples. ß-Lactam resistance genes were detected in all isolates. After ß-lactam resistance genes, the most common antimicrobial resistance genes detected were aminoglycoside resistance genes (92.4%). One hundred and seventy-two (76.4%), 102 (45.3%) and 52 (23.1%) isolates carried blaCTX-M genes, blaTEM genes and blaSHV genes, respectively. blaCTX-M-55 (57/225, 25.3%) and blaCTX-M-65 (40/225, 17.8%) were the most frequent ESBL genes. Colistin resistance genes (including mcr-1, mcr-3 and mcr-5) were found in 15.6% of all isolates. CONCLUSIONS: This study indicates that ESBL-producing E. coli are widely found in retail raw meats, especially chicken, in Singapore. Occurrence of MDR (resistance to at least three classes of antimicrobial) and colistin resistance genes in retail raw meat suggests potential food safety and public health risks.

Occurrence and Antimicrobial Resistance Traits of Escherichia coli from Wild Birds and Rodents in Singapore.

Antimicrobial resistance (AMR) in Escherichia coli (E. coli) poses a public health concern worldwide. Wild birds and rodents, due to their mobility, are potential vehicles for transmission of AMR bacteria to humans. Ninety-six wild birds' faecal samples and 135 rodents' droppings samples were collected and analysed in 2017. Forty-six E. coli isolates from wild birds and rodents were subjected to AMR phenotypic and genotypic characterisation. The proportion of E. coli isolates resistant to at least one of the antimicrobials tested from wild birds (80.8%) was significantly higher than that of isolates from rodents (40.0%). The proportion of E. coli isolates resistant to each antimicrobial class for wild birds was 3.8% to 73.1% and that for rodents was 5.0% to 35.0%. Six out of 26 E. coli isolates from wild birds (23.1%) and two out of 20 (10.0%) isolates from rodents were multi-drug resistant (MDR) strains. These MDR E. coli isolates were detected with various antimicrobial resistance genes such as blaTEM-1B and qnrS1 and could be considered as part of the environmental resistome. Findings in this study suggested that wild birds and rodents could play a role in disseminating antimicrobial resistant E. coli, and this underscores the necessity of environment management and close monitoring on AMR bacteria in wild birds and rodents to prevent spreading of resistant organisms to other wildlife animals and humans.

Porous nickel doped titanium dioxide nanoparticles with improved visible light photocatalytic activity.

A green hydrothermal synthesis route to prepare a porous nickel doped titanium dioxide (Ni-TiO2) nanostructured photocatalyst has been developed in this research. The results show that Ni doping can greatly increase the visible light photocatalytic performance of TiO2 through the introduction of impurity bands in the band gap of TiO2. After 5 cycles of reuse, Ni-TiO2 nanoparticles still show stable photocatalytic activity for MB degradation. The Ni-TiO2 nanoparticles developed in the present study are expected to have great potential applications in wastewater treatment due to the advantages of strong visible light photocatalytic performance, a simple synthetic process and high cycle utilization performance.

Extended-spectrum ß-lactamase-producing Proteus mirabilis with multidrug resistance isolated from raw chicken in Singapore: Genotypic and phenotypic analysis.

OBJECTIVES: Proteus mirabilis is ubiquitous in soil and water. It is an important catheter-associated urinary tract pathogen and has reportedly been associated with antimicrobial-resistant infections. This study reports the draft genome of a multidrug-resistant P. mirabilis isolated from raw retail chicken meat in Singapore. METHODS: The P. mirabilis strain was isolated on BrillianceTM ESBL Agar and was screened for antimicrobial susceptibility against 29 antimicrobial agents using a MicroScan® Neg MIC Panel Type 44. The double-disk synergy test (DDST) was used for confirmation of extended-spectrum ß-lactamase (ESBL) production. Genomic DNA from the pure culture isolate was extracted and was sent for sequencing based on Illumina HiSeq 2500 technology. Further bioinformatics analysis was performed using online tools available at the Center for Genomic Epidemiology. RESULTS: Species identification of the isolate was performed by KmerFinder. Antimicrobial susceptibility testing of the isolate showed multidrug resistance to broad-spectrum ß-lactams, fluoroquinolones and aminoglycosides, among others. ESBL production was confirmed by the DDST. A total of 29 antimicrobial resistance genes were detected by ResFinder. CONCLUSION: To the best of our knowledge, this is the first report of the whole-genome sequence of a multidrug-resistant P. mirabilis producing an ESBL from raw chicken meat in Singapore. This indicates that raw meat in Singapore can be a reservoir for drug-resistant pathogens.