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Lan, R.

Publications and source records attributed to Lan, R..

25 records · Page 2Linked to original sources

Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton

Increased intracellular iron spurs mitochondrial biogenesis and respiration to satisfy high-energy demand during osteoclast differentiation and bone-resorbing activities. Transferrin receptor 1 (TFR1) mediates cellular iron uptake through endocytosis of iron-loaded transferrin and its expression increases during osteoclast differentiation. Nonetheless, the precise functions of TFR1 and TFR1-mediated iron uptake in osteoclast biology and skeletal homeostasis remain incompletely understood. To investigate the role of TFR1 in osteoclast lineage cells, we conditionally deleted Tfr1 gene in myeloid precursors or mature osteoclasts by crossing Tfr1- floxed mice with LysM-Cre and Ctsk-Cre mice, respectively. Skeletal phenotyping by {micro}CT and histology unveiled that loss of Tfr1 in osteoclast progenitor cells resulted in a three-fold increase in trabecular bone mass in the long bones of 10-week old female but not male mice. Although high trabecular bone volume in long bones was seen in both male and female mice with deletion of Tfr1 in mature osteoclasts, this phenotype was more pronounced in female knockout mice. Mechanistically, disruption of Tfr1 expression attenuated mitochondrial metabolism and cytoskeletal organization in mature osteoclasts, leading to decreased bone resorption with no impact on osteoclastogenesis. These results indicate that Tfr1-mediated iron uptake is specifically required for osteoclast function and is indispensable for bone remodeling.

cell biology↗

Comparative genomics of Chinese and international isolates of Escherichia albertii: population structure and evolution of virulence and antimicrobial resistance

Escherichia albertii is a newly recognized species in the genus Escherichia that causes diarrhea. The population structure, genetic diversity and genomic features has not been fully examined. Here, 169 E. albertii isolates from different sources and regions in China were sequenced and combined with 312 publicly available genomes for phylogenetic and genomic analyses. The E. albertii population was divided into 2 clades and 8 lineages, with lineage 3 (L3), L5 and L8 more common in China. Clinical isolates were observed in all clades/lineages. Virulence genes were found to be distributed differently among lineages: subtypes of the intimin encoding gene eae and the cytolethal distending toxin (Cdt) gene cdtB were lineage associated, the second type three secretion system (ETT2) island was truncated in L3 and L6. Seven new eae subtypes and 1 new cdtB subtype (cdtB-VI) were found. Alarmingly, 85.9% of the Chinese E. albertii isolates were predicted to be multidrug resistant (MDR) with 35.9% harboured genes capable of conferring resistance to 10 to 14 different drug classes. By in silico multi-locus sequence typing, majority of the MDR isolates belonged to 4 STs (ST4638, ST4479, ST4633 and ST4488). Thirty-four intact plasmids carrying MDR and virulence genes, and 130 intact prophages were identified from 17 complete E. albertii genomes. Ten plasmid replicon types were found to be significantly associated with MDR. The 130 intact prophages were clustered into 5 groups, with group 5 prophages harbouring more virulence genes. Our findings provided fundamental insights into the population structure, virulence variation and MDR of E. albertii. Impact statementE. albertii is newly recognized foodborne pathogen causing diarrhea. Elucidation of its genomic features is important for surveillance and control of E. albertii infections. In this work, 169 E. albertii genomes from difference sources and regions in China were collected and sequenced, which contributed to the currently limited genomic data pool of E. albertii. In combination with 312 publicly available genomes from 14 additional countries, the population structure of E. albertii was defined. The presence and subtypes of virulence genes in different lineages were significantly different, indicating potential pathogenicity variation. Additionally, the presence of multidrug resistance (MDR) genes was alarmingly high in the Chinese dominated lineages. MDR associated STs and plasmid subtypes were identified, which could be used as sentinels for MDR surveillance. Moreover, the subtypes of plasmids and prophages were distributed differently across lineages, and were found to contribute to the acquisition of virulence and MDR genes of E. albertii. Altogether, this work reveals the diversity of E. albertii and characterized its genomic features in unprecedented detail. Data SummaryAll newly sequenced data in this work were deposited in National Center for Biotechnology Information (NCBI) under the BioProject of PRJNA693666, including 6 complete genomes and raw reads of 164 E. albertii isolates.

genomics↗

Cluster-specific gene marker enhance Shigella and Enteroinvasive Escherichia coli in silico serotyping

Shigella and enteroinvasive Escherichia coli (EIEC) cause human bacillary dysentery with similar invasion mechanisms and share similar physiological, biochemical and genetic characteristics. The ability to differentiate Shigella and EIEC from each other is important for clinical diagnostic and epidemiologic investigations. The existing genetic signatures may not discriminate between Shigella and EIEC. However, phylogenetically, Shigella and EIEC strains are composed of multiple clusters and are different forms of E. coli. In this study, we identified 10 Shigella clusters, 7 EIEC clusters and 53 sporadic types of EIEC by examining over 17,000 publicly available Shigella/EIEC genomes. We compared Shigella and EIEC accessory genomes to identify the cluster-specific gene markers or marker sets for the 17 clusters and 53 sporadic types. The gene markers showed 99.63% accuracy and more than 97.02% specificity. In addition, we developed a freely available in silico serotyping pipeline named Shigella EIEC Cluster Enhanced Serotype Finder (ShigEiFinder) by incorporating the cluster-specific gene markers and established Shigella/EIEC serotype specific O antigen genes and modification genes into typing. ShigEiFinder can process either paired end Illumina sequencing reads or assembled genomes and almost perfectly differentiated Shigella from EIEC with 99.70% and 99.81% cluster assignment accuracy for the assembled genomes and mapped reads respectively. ShigEiFinder was able to serotype over 59 Shigella serotypes and 22 EIEC serotypes and provided a high specificity with 99.40% for assembled genomes and 99.38% for mapped reads for serotyping. The cluster markers and our new serotyping tool, ShigEiFinder (https://github.com/LanLab/ShigEiFinder), will be useful for epidemiologic and diagnostic investigations. Impact statementThe differentiation of Shigella strains from enteroinvasive E. coli (EIEC) is important for clinical diagnosis and public health epidemiologic investigations. The similarities between Shigella and EIEC strains make this differentiation very difficult as both share common ancestries within E. coli. However, Shigella and EIEC are phylogenetically separated into multiple clusters, making high resolution separation using cluster specific genomic markers possible. In this study, we identified 17 Shigella or EIEC clusters including five that were newly identified through examination of over 17,000 publicly available Shigella and EIEC genomes. We further identified an individual or a set of cluster-specific gene markers for each cluster using comparative genomic analysis. These markers can then be used to classify isolates into clusters and were used to develop an in silico pipeline, ShigEiFinder (https://github.com/LanLab/ShigEiFinder) for accurate differentiation, cluster typing and serotyping of Shigella and EIEC from Illumina sequencing reads or assembled genomes. This study will have broad application from understanding the evolution of Shigella/EIEC to diagnosis and epidemiology. Data summarySequencing data have been deposited at the National Center for Biotechnology Information under BioProject number PRJNA692536. RepositoriesRaw sequence data are available from NCBI under the BioProject number PRJNA692536.

microbiology↗

Elucidation of global and local epidemiology of Salmonella Enteritidis through multilevel genome typing

Salmonella enterica serovar Enteritidis is a major cause of foodborne Salmonella infections and outbreaks in humans. Effective surveillance and timely outbreak detection are essential for public health control. Multilevel genome typing (MGT) with multiple levels of resolution has been previously demonstrated as a promising tool for this purpose. In this study, we developed MGT with nine levels for S. Enteritidis and characterised the genomic epidemiology of S. Enteritidis in detail. We examined 26,670 publicly available S. Enteritidis genome sequences from isolates spanning 101 years from 86 countries to reveal their spatial and temporal distributions. Using the lower resolution MGT levels, globally prevalent and regionally restricted sequence types (STs) were identified; avian associated MGT4-STs were found that were common in human cases in the USA were identified; temporal trends were observed in the UK with MGT5-STs from 2014 to 2018, revealing both long lived endemic STs and the rapid expansion of new STs. Using MGT3 to MGT6, we identified MDR associated STs at various MGT levels, which improves precision of detection and global tracking of MDR clones. We also found that the majority of the global S. Enteritidis population fell within two predominant lineages, which had significantly different propensity of causing large scale outbreaks. An online open MGT database has been established for unified international surveillance of S. Enteritidis. We demonstrated that MGT provides a flexible and high-resolution genome typing tool for S. Enteritidis surveillance and outbreak detection. Impact statementSalmonella enterica serovar Enteritidis is a common foodborne pathogen that can cause large outbreaks. Surveillance and high-resolution typing are essential for outbreak prevention and control. Genome sequencing offers unprecedented power for these purposes and a standardised method or platform for the interpretation, comparison and communication of genomic typing data is highly desirable. In this work, we developed a genomic typing scheme called Multilevel Genome Typing (MGT) for S. Enteritidis. We analysed 26,670 publicly available genomes of S. Enteritidis using MGT. We characterised the geographic and temporal distribution of S. Enteritidis MGT types as well as their association with multidrug resistance (MDR) and virulence genes. A publicly available MGT database for S. Enteritidis was established, which has the potential facilitate the unified global public health surveillance for this pathogen. Data SummaryO_LIThe MGT database for S. Enteritidis is available at https://mgtdb.unsw.edu.au/enteritidis/. C_LIO_LIAll accession numbers of the public available genomes were available in the MGT database and Data Set S1, Tab 1. And there were no newly sequenced data in this study. C_LIO_LISupplementary material: Supplementary Fig. S1 to S7, supplementary methods and supporting results about the evaluation of potential repeat sequencing bias. C_LIO_LIData Set S1: Supporting tables of the main results. C_LIO_LIData Set S2. Supporting tables of the repeat sequencing bias evaluation by removing the potential repeat sequencing isolates. Note outbreak isolates may also be removed. C_LI

genomics↗

Nucleus accumbens melanin-concentrating hormone signaling promotes feeding in a sex-specific manner

Melanin-concentrating hormone (MCH) is an orexigenic neuropeptide produced in the lateral hypothalamus and zona incerta that increases food intake. The neuronal pathways and behavioral mechanisms mediating the orexigenic effects of MCH are poorly understood, as is the extent to which MCH-mediated feeding outcomes are sex-dependent. Here we investigate the hypothesis that MCH-producing neurons act in the nucleus accumbens shell (ACBsh) to promote feeding behavior and motivation for palatable food in a sex-dependent manner. We utilized ACBsh MCH receptor (MCH1R)-directed pharmacology as well as a dual virus chemogenetic approach to selectively activate MCH neurons that project to the ACBsh. Results reveal that both ACBsh MCH1R activation and activating ACBsh-projecting MCH neurons increase consumption of standard chow and palatable sucrose in male rats without affecting motivated operant responding for sucrose, general activity levels, or anxiety-like behavior. In contrast, food intake was not affected in female rats by either ACBsh MCH1R activation or ACBsh-projecting MCH neuron activation. To determine a mechanism for this sexual dimorphism, we investigated whether the orexigenic effect of ACBsh MCH1R activation is reduced by endogenous estradiol signaling. In ovariectomized female rats on a cyclic regimen of either estradiol (EB) or oil vehicle, ACBsh MCH1R activation increased feeding only in oil-treated rats, suggesting that EB attenuates the ability of ACBsh MCH signaling to promote food intake. Collective results show that that MCH ACBsh signaling promotes feeding in an estrogen- and sex-dependent manner, thus identifying novel neurobiological mechanisms through which MCH and female sex hormones interact to influence food intake.

neuroscience↗

Integrin β3, a RACK1 interacting protein, is required for porcine reproductive and respiratory syndrome virus infection and NF-κB activation in Marc-145 cells

Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen of porcine reproductive and respiratory syndrome (PRRS), which is one of the most economically harmful diseases in modern pig production worldwide. Receptor of activated protein C kinase 1 (RACK1) was previously shown to be indispensable for the PRRSV replication and NF-{kappa}B activation in Marc-145 cells. Here we identified a membrane protein, integrin {beta}3 (ITGB3), as a RACK1-interacting protein. PRRSV infection in Marc-145 cells upregulated the ITGB3 expression. Abrogation of ITGB3 by siRNA knockdown or antibody blocking inhibited PRRSV infection and NF-{kappa}B activation, while on the other hand, overexpression of ITGB3 enhanced PRRSV infection and NF-{kappa}B activation. Furthermore, inhibition of ITGB3 alleviated the cytopathic effects and reduced the TCID50 titer in Marc-145 cells. We also showed that RACK1 and ITGB3 were NF-{kappa}B target genes during PRRSV infection, and that they regulate each other. Our data indicate that ITGB3, presumably as a co-receptor, plays an imperative role for PRRSV infection and NF-{kappa}B activation in Marc-145 cells. PRRSV infection activates a positive feedback loop involving the activation of NF-{kappa}B and upregulation of ITGB3 and RACK1 in Marc-145 cells. The findings would advance our elaborated understanding of the molecular host-pathogen interaction mechanisms underlying PRRSV infection in swine and suggest ITGB3 and NF-{kappa}B signaling pathway as potential therapeutic targets for PRRS control. IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the pathogens in pig production worldwide. Several cell surface receptors, such as heparan sulphate, sialoadhesin, vimentin and CD163, were identified to be involved in PRRSV infection in porcine alveolar macrophages (PAMs). We identified a cell surface protein, integrin {beta}3 (ITGB3), as an interacting protein with receptor of activated protein C kinase 1 (RACK1) from Marc-145 cells. ITGB3 interacts with RACK1 and facilitates PRRSV infection and NF-{kappa}B activation in Marc-145 cells, presumably as a co-receptor of CD136 or vimentin. Both ITGB3 and RACK1 were NF-{kappa}B target genes, and they regulate each other. The activation of NF-{kappa}B and the transcription of its downstream genes are beneficial for PRRSV infection/replication. The novel findings would advance our elaborated understanding of the molecular host-pathogen interaction mechanisms underlying PRRSV infection in swine and suggest ITGB3-RACK1-NF-{kappa}B axis as a potential therapeutic target for PRRS control.

microbiology↗

Beyond a ribosomal RNA methyltransferase, the wider role of MraW in DNA methylation, motility and colonization in Escherichia coli O157:H7

MraW (RsmH) is an AdoMet-dependent 16S rRNA methyltransferase conserved in bacteria and plays a role in the fine-tuning of the ribosomal decoding center. It was recently found to contribute to the virulence of Staphylococcus aureus in host animals. In this study, we examined the function of MraW in Escherichia coli O157:H7 and found that deletion of mraW led to decreased motility and flagellar production. Whole-genome bisulfite sequencing showed genome wide decrease of methylation of 336 genes and 219 promoters in the mraW mutant. The methylation level of 4 flagellar gene sequences were further confirmed by bisulfite PCR sequencing. Quantitative reverse transcription PCR results indicated the transcription of these genes was also affected. MraW was observed to directly bind to the four flagellar gene sequences by electrophoretic mobility shift assay (EMSA). A common motif in differentially methylated regions of promoters and coding regions of the 4 flagellar genes was identified. Reduced methylation was correlated with altered expression of 21 of the 24 genes tested. DNA methylation activity of MraW was confirmed by DNA methyltransferase (DNMT) activity assay in vitro. The mraW mutant colonized poorer than wild type in mice. we further found that the expression of mraZ in the mraW mutant was increased confirming the antagonistic effect of mraW on mraZ. In conclusion, mraW was found to be a DNA methylase and has a wide-ranging effect on E. coli O157:H7 including motility and virulence in vivo via genome wide methylation and mraZ antagonism.\n\nIMPORTANCEMraW is a well-studied 16S rRNA methyltransferase and was recently found have an impact on bacterial virulence. Here we demonstrated its new function as a DNA methylase and effect on motility, colonization in mice, DNA methylation in genome wide and contribution to virulence. Its direct binding of differentially methylated flagellar-encoding DNA sequences was observed, indicating a correlation between DNA methylation and regulation of flagellar genes. In addition, the expression of mraZ which function as an antagonist of mraW was increased in the mraW mutant. mraW plays an important role in gene regulation likely through DNA methylation. Clearly it plays a role in virulence in E. coli O157:H7. It also opens a new research field for virulence study in bacteria.

microbiology↗