Biomedical subjects
Publications and source records attributed to Ning Sun.
We report the draft genomes of two Enterococcus faecium blood isolates: one vancomycin-susceptible and one vancomycin-resistant. The assemblies comprised 2,991,512 bp (7 contigs) for the susceptible strain and 3,305,414 bp (7 contigs) for the resistant strain.
Deciphering the functionality of the noncoding genome, which includes important cis-regulatory elements (CREs) and transcribed noncoding RNA genes, remains technically challenging. Here, using massively parallel genetic screening, we systematically benchmark the performance of five representative loss-of-function perturbation tools, including single-guide RNA (gRNA) mediated SpCas9 cleavage or CRISPR interference, and paired gRNA (pgRNA) involved dual-SpCas9, Big Papi (paired SpCas9 and SaCas9) or dual-enAsCas12a fragment deletion methods, in decoding the roles of the noncoding genome. For targeting CREs such as enhancers, dual-SpCas9 outperforms other methods with superior efficiency in destroying functional genomic regions. For perturbing noncoding RNA genes, in addition to dual-SpCas9, other RNA-targeting methods such as RNA interference are recommended to discriminate transcript-dependent or -independent roles. A deep learning model, DeepDC, with an associated web server, is built to facilitate optimal dual-SpCas9 pgRNA design for efficiently deleting a genomic fragment. Together, our work provides practical guidance on selecting appropriate loss-of-function tools to resolve the functional complexity of the noncoding genome.
Hepatitis B virus (HBV) persistence is sustained by the viral covalently closed circular DNA (cccDNA) minichromosome, which remains a major barrier to curative antiviral therapies. The lack of reliable quantitative cccDNA detection methods and surrogate markers has hindered efforts to target cccDNA in antiviral high-throughput screening (HTS). Here, we established a novel inducible cccDNA-dependent nanoluciferase (NLuc) reporter cell line, designated HepBLE12, by inserting an in-frame 11-amino acid split-NLuc HiBiT tag into the precore (pC) coding region of an HBV transgene. The resulting 1.3-kDa HiBiT tag on pC serves as the detection module of the split NLuc system, generating quantitative luminescence upon high-affinity complementation with the cognate 18-kDa LgBiT subunit in cell lysates. Notably, the HiBiT assay enables direct detection of intracellular HiBiT-pC protein rather than secreted HBeAg, providing a reporter signal more closely linked to cccDNA activity. HepBLE12 cells exhibited inducible and robust viral DNA replication, and the cccDNA-dependent HiBiT signal was validated under diverse experimental conditions that modulate cccDNA formation or transcription. We further miniaturized the assay to a 384-well format and optimized key parameters following standard HTS assay development practices. The assay was successfully automated and demonstrated excellent performance in a multi-day variability study and a pilot screen, with signal-to-background (S/B) ≈ 9, coefficient of variance (CV) < 10%, and average Z-factor value of 0.74, exceeding canonical HTS quality benchmarks. Together, the HepBLE12 cell-based HTS platform provides a robust and practical tool for identifying inhibitors targeting HBV cccDNA.
Enterococci are common commensals of the human gut and important opportunistic pathogens, with Enterococcus faecium and Enterococcus faecalis being the most clinically prevalent species. A significant epidemiological shift has emerged with an increasing clinical burden of E. faecium. To compare genomic evolution of E. faecium and E. faecalis, we performed whole-genome sequencing on 93 E. faecium and 32 E. faecalis isolates causing bloodstream infections at a single hospital (2022-2024). Analysis of patient demographics revealed that E. faecium infections originated from fewer sources than E. faecalis, with a higher proportion deriving from intra-abdominal infections. Multilocus sequence typing identified ST78 and ST789 as the predominant sequence types for E. faecium, whereas ST16 and ST179 were most common for E. faecalis. E. faecium carried more antimicrobial resistance genes and putative virulence marker (PVM)-type virulence genes than E. faecalis, with vancomycin resistance predominantly mediated by vanHAX (33/93, 35.5%) and a single E. faecalis isolate also carrying vanHAX (1/32, 3.1%); the structurally incomplete vanHMX gene cluster was detected in 11 E. faecium isolates. Pan-genome analysis indicated a larger core genome in E. faecalis compared to E. faecium, consistent with greater plasmid replicon diversity in the latter. Intra-host comparisons showed that two E. faecalis pairs from the same patient were clonally related, with one isolate acquiring a vanHAX plasmid conferring vancomycin resistance. In contrast, E. faecium isolates exhibited marked genomic diversity even among clonally related pairs. These findings suggest that E. faecium possesses greater genomic plasticity and adaptive potential to the clinical environment.IMPORTANCEThis study provides a detailed comparison of clinical and genomic features between Enterococcus faecium and Enterococcus faecalis from the same hospital setting. We show that E. faecium isolates, mainly ST78/ST789, carry more antimicrobial resistance genes and a higher number of putative virulence marker (PVM) genes than E. faecalis, reflecting their hospital-adapted nature. E. faecium also exhibits a smaller core genome and greater diversity of plasmid replicon types, indicating higher genomic plasticity and capacity for horizontal gene transfer. By contrast, E. faecalis retains a larger core genome and a set of classical virulence factors, and its within-host isolates are clonally related. These distinct genomic profiles help to understand how the two species adapt to clinical environments and may inform more targeted infection control strategies and resistance surveillance.
T cell activation and tolerance are regulated by costimulatory molecules. Although PD-1 serves as a crucial negative regulator of T cells, the function of its ligands, PDL1 and PDL2, is still controversial. In this study, we created a PDL2-deficient mouse to characterize its function in T cell activation and tolerance. Antigen-presenting cells from PDL2-/- mice were found to be more potent in activation of T cells in vitro over the wild-type controls, which depended on PD-1. Upon immunization with chicken ovalbumin, PDL2-/- mice exhibited increased activation of CD4(+) and CD8(+) T cells in vivo when compared with WT animals. In addition, T cell tolerance to an oral antigen was abrogated by the lack of PDL2. Our results thus demonstrate that PDL2 negatively regulates T cells in immune responses and plays an essential role in immune tolerance.
Transcription regulation is a fundamental biological process, and extensive efforts have been made to dissect its mechanisms through direct biological experiments and regulation modeling based on physical-chemical principles and mathematical formulations. Despite these efforts, transcription regulation is yet not well understood because of its complexity and limitations in biological experiments. Recent advances in high throughput technologies have provided substantial amounts and diverse types of genomic data that reveal valuable information on transcription regulation, including DNA sequence data, protein-DNA binding data, microarray gene expression data, and others. In this article, we propose a Bayesian error analysis model to integrate protein-DNA binding data and gene expression data to reconstruct transcriptional regulatory networks. There are two unique aspects to this proposed model. First, transcription is modeled as a set of biochemical reactions, and a linear system model with clear biological interpretation is developed. Second, measurement errors in both protein-DNA binding data and gene expression data are explicitly considered in a Bayesian hierarchical model framework. Model parameters are inferred through Markov chain Monte Carlo. The usefulness of this approach is demonstrated through its application to infer transcriptional regulatory networks in the yeast cell cycle.
A sensitive high-performance liquid chromatographic method with fluorescence detection has been developed for determination of MCC-555 (5-[[6-(2-fluorbenzyl)-oxy-2-naphy] methyl]-2,4-thiazolidinedione) in beagle dog plasma. Sample preparation was done by protein precipitation with acetonitrile and a synthetic intermediate of MCC-555 (5-[[6-(2-fluorbenzyl)-oxy-2-naphy] methylene]-2,4-thiazolidinedione) was used as the internal standard (IS). The isocratic mobile phase consisted of acetonitrile-10 mmol/l sodium phosphate buffer (pH 4.5) (65:35, v/v) was delivered at a flow rate of 1 ml/min to a Kromasil C(18) reversed-phase column (250 mm x 4.6 mm, 5 microm). The compounds were detected by fluorescence detection, using an excitation wavelength of 232 nm, and emission wavelength of 352 nm. Calibration curves of MCC-555 were linear in the concentration range of 0.005-2.0 microg/ml. Intra- and inter-day precision ranged from 3.4 to 5.4% and 3.0 to 8.8%, respectively. No endogenous interferences were observed with either MCC-555 or IS. The assay is simple, economical, precise, and is directly applicable to pharmacokinetic studies in beagle dogs involving three dose administrations.
Cell survival is regulated by the balance between death and survival signals. Previous studies have shown that the N-methyl-d-aspartate receptors (NMDARs) are responsible for the glutamate-induced excitotoxicity in the postischemic brain. Meanwhile, nerve growth factor (NGF) is critically involved in cell survival and neuroprotective effects via the extracellular signal-related kinase (ERK) pathway or the phosphatidylinositol 3-kinase (PI3-K) pathway mediated by the high affinity NGF receptor, tropomyosin-related kinase A (TrkA). Clinically, electroacupuncture (EA) has been shown to produce beneficial effects on stroke patients. However, the detailed mechanisms mediating the beneficial effects of EA on stroke are still unknown. In the present study, we found that EA treatment reversed the high expression of NR1 subunit and up-regulated the level of TrkA in a rat model of middle cerebral artery occlusion. Using protein kinase inhibitors of specific intracellular signaling pathways, we found that the neuroprotective effects of EA appear to be mediated by stimulation of the PI3-K pathway, but not ERK pathway. These findings may provide important experimental evidence for the clinical application of EA treatment for stroke patients.
Two families of a new generation of ionic liquids, in which the chiral cations are directly derived from naturally occurring alpha-amino acids and alpha-amino acid ester salts, have been obtained via very simple preparations.
The effect of electroacupuncture (EA) on TRPM7 mRNA expression of focal cerebral ischemia in rats and further the role of EA in the relationship between TRPM7 and trkA pathway was investigated. Thirty SD rats were randomly divided into 5 groups : normal group, ischemia/reperfusion group, EA treated group (ischemic rats with EA treatment), TE infusion group (ischemic rats with EA treatment and TE buffer infusion), AS-ODN group (ischemic rats with EA treatment and antisense trkA oligonucleotide infusion). The stroke animal model was established by the modified method of middle cerebral artery occlusion. Antisense trkA oligonucleotide that blocked NGFs effects was injected into cerebroventricle before EA. The TRPM7 mRNA was detected by RT-PCR method. The results showed that there were low TRPM7 mRNA levels in cortex and hippocampus in normal group. Compared with normal group, TRPM7 mRNA expression was increased significantly in ischemia/reperfusion group (P<0.05). A significant reduction in the expression of TR-PM7 mRNA was found in EA treated group in contrast to ischemia/reperfusion group (P<0.05). The expression of TRPM7 mRNA in AS-ODN group was remarkably increased compared with EA treated group and TE infusion group (P<0.05). The results indicated that TRPM7 channels in the ischemic cortex and hippocampus in rats might play a key role in ischemic brain injury. EA could reverse the overexpression of TRPM7 in cerebral ischemia/reperfusion rats. And the inhibitory effect of EA on TRPM7 channels might be through trkA pathway.
To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthase isoforms (nNOS and iNOS), rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen-glucose deprivation (OGD) for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD (P < 0.05). The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase (LDH) efflux into the incubation solution. The results suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.
Arabidopsis and rice are the only two model plants whose finished phase genome sequence has been completed. Here we report the construction of an oligomer microarray based on the presently known and predicted gene models in the rice genome. This microarray was used to analyze the transcriptional activity of the gene models in representative rice organ types. Expression of 86% of the 41,754 known and predicted gene models was detected. A significant fraction of these expressed gene models are organized into chromosomal regions, about 100 kb in length, that exhibit a coexpression pattern. Compared with similar genome-wide surveys of the Arabidopsis transcriptome, our results indicate that similar proportions of the two genomes are expressed in their corresponding organ types. A large percentage of the rice gene models that lack significant Arabidopsis homologs are expressed. Furthermore, the expression patterns of rice and Arabidopsis best-matched homologous genes in distinct functional groups indicate dramatic differences in their degree of conservation between the two species. Thus, this initial comparative analysis reveals some basic similarities and differences between the Arabidopsis and rice transcriptomes.
The development of complex eukaryotic organisms can be viewed as the selective expression of distinct fractions of the genome in different organs or tissue types in response to developmental and environmental cues. Here, we generated a genome expression atlas of 18 organ or tissue types representing the life cycle of Arabidopsis (Arabidopsis thaliana). We showed that each organ or tissue type had a defining genome expression pattern and that the degree to which organs share expression profiles is highly correlated with the biological relationship of organ types. Further, distinct fractions of the genome exhibited expression changes in response to environmental light among the three seedling organs, despite the fact that they share the same photo-perception and transduction systems. A significant fraction of the genes in the Arabidopsis genome is organized into chromatin domains exhibiting coregulated expression patterns in response to developmental or environmental signals. The knowledge of organ-specific expression patterns and their response to the changing environment provides a foundation for dissecting the molecular processes underlying development.
Advances in genomic research have provided many types of large-scale data that contain rich information on various biological pathways. Intensive efforts have been made to qualitatively or quantitatively model biological pathways using these genomic data. Some general network properties, such as the scale-free property and network motifs, have been discussed and various network models have been applied to reconstruct pathways. However, there is a lack of systematic integration of prior knowledge and different genomic data in these analyses. In this review, we discuss pathway reconstruction under the consideration of the complexity embedded in the biological system, and the global and local properties of biological pathways. We review major methodologies, including clustering methods, scale-free networks models, Bayesian networks models, Boolean networks models, systems of differential equations, and data integration methods. We focus on the difficulty of each methodology in modeling biological pathways, and emphasize that different models capture different aspects of biological pathways or genomic data. The 'noisy' large-scale genomic data require the mathematical models and computational methods to be both robust and identifiable. In addition, we believe that ideal models should have the capability of incorporating various data types and these models need to be assessed through rigorous comparisons with empirical data.
OBJECTIVE: To study the clinicopathological, immunohistochemical and electron microscopic characteristics of pediatric rhabdomyosarcomas (RMS). METHODS: One hundred and forty-five cases of pediatric rhabdomyosarcomas were studied by routine histological, immunohistochemical and electron microscopic studies. RESULTS: There were 97 male and 48 female patients with ages ranging from 4 months to 13 years and a mean of 4.2 years. The follow-up period of 100 patients was from 1 year to 20 years with a mean of 5 years after diagnosis. All cases were subtyped into the following histological categories: embryonal RMS, botryoid RMS, spindle cell RMS, alveolar RMS and solid RMS. Histopathological subtypes, tumor site and tumor stage correlated significantly with the patients' 5 years survival. The best prognosis was observed in spindle cell and botryoid RMS. Embryonal RMS carried an intermediate prognosis. Patients with alveolar RMS and solid RMS had the worst prognosis. Tumors involving bladder, head and neck carried a favorable clinical outcome. Patients with tumors involving trunk extremities retroperitoneum and pelvis did poorly. Immunohistochemically, all cases were positive for Vimentin. The positive staining rates for desmin, SMA and myoglobin were 78%, 75% and 37%, respectively. All tumors were negative for NSE, CD99 and LCA. Electron microscopy study showed features of myofilament and sarcomere in 10 of 15 cases. CONCLUSIONS: RMS is the most common soft tissue sarcoma of childhood. Immunohistochemistry and electron microscopy are helpful in diagnosis and classification of RMS.
BACKGROUND: To date, many genomic and pathway-related tools and databases have been developed to analyze microarray data. In published web-based applications to date, however, complex pathways have been displayed with static image files that may not be up-to-date or are time-consuming to rebuild. In addition, gene expression analyses focus on individual probes and genes with little or no consideration of pathways. These approaches reveal little information about pathways that are key to a full understanding of the building blocks of biological systems. Therefore, there is a need to provide useful tools that can generate pathways without manually building images and allow gene expression data to be integrated and analyzed at pathway levels for such experimental organisms as Arabidopsis. RESULTS: We have developed PathMAPA, a web-based application written in Java that can be easily accessed over the Internet. An Oracle database is used to store, query, and manipulate the large amounts of data that are involved. PathMAPA allows its users to (i) upload and populate microarray data into a database; (ii) integrate gene expression with enzymes of the pathways; (iii) generate pathway diagrams without building image files manually; (iv) visualize gene expressions for each pathway at enzyme, locus, and probe levels; and (v) perform statistical tests at pathway, enzyme and gene levels. PathMAPA can be used to examine Arabidopsis thaliana gene expression patterns associated with metabolic pathways. CONCLUSION: PathMAPA provides two unique features for the gene expression analysis of Arabidopsis thaliana: (i) automatic generation of pathways associated with gene expression and (ii) statistical tests at pathway level. The first feature allows for the periodical updating of genomic data for pathways, while the second feature can provide insight into how treatments affect relevant pathways for the selected experiment(s).
A microarray based on PCR amplicons of 1864 confirmed and predicted Arabidopsis transcription factor genes was produced and used to profile the global expression pattern in seedlings, specifically their light regulation. We detected expression of 1371 and 1241 genes in white-light- and dark-grown 6-d-old seedlings, respectively. Together they account for 84% of the transcription factor genes examined. This array was further used to study the kinetics of transcription factor gene expression change of dark-grown seedlings in response to blue light and the role of specific photoreceptors in this blue-light regulation. The expression of about 20% of those transcription factor genes are responsive to blue-light exposure, with 249 and 115 genes up or down-regulated, respectively. A large portion of blue-light-responsive transcription factor genes exhibited very rapid expression changes in response to blue light, earlier than the bulk of blue-light-regulated genes. This result suggests the involvement of transcription cascades in blue-light control of genome expression. Comparative analysis of the expression profiles of wild type and various photoreceptor mutants demonstrated that during early seedling development cryptochromes are the major photoreceptors for blue-light control of transcription factor gene expression, whereas phytochrome A and phototropins play rather limited roles.
To explore the anti-apoptotic role of electroacupuncture (EA) and its molecular mechanisms after cerebral ischemia/reperfusion (IR) of rats, by using animal model of middle cerebral artery occlusion (MCAO), the changes of the cleavage of PARP were observed by Western blot and the mRNA of heat shock protein (Hsp) 70 and Hsp90 beta detected by competitive RT-PCR after cerebral IR and EA treatment. The results were as follows: (1) The cleavage of PARP was increased in ischemic hippocampus, and EA treatment could attenuate the level of the cleavage remarkably; (2) The mRNA expression of Hsp70 was increased in the ischemic cortex and hippocampus and was further increased after EA treatment; (3) The mRNA expression of Hsp90 beta was decreased in ischemic cortex and hippocampus and the decrease was relatively slight after EA treatment. The above results demonstrated EA treatment could protect neurons from apoptosis after cerebral IR. One of the molecular mechanisms was the promotion of the inducible expression of Hsp70 and the improvement of the inhibition of the expression of Hsp90.