Nanomaterials in Medicine: Understanding Cellular Uptake, Localization, and Retention for Enhanced Disease Diagnosis and Therapy.

Nanomaterials (NMs) have emerged as promising tools for disease diagnosis and therapy due to their unique physicochemical properties. To maximize the effectiveness and design of NMs-based medical applications, it is essential to comprehend the complex mechanisms of cellular uptake, subcellular localization, and cellular retention. This review illuminates the various pathways that NMs take to get from the extracellular environment to certain intracellular compartments by investigating the various mechanisms that underlie their interaction with cells. The cellular uptake of NMs involves complex interactions with cell membranes, encompassing endocytosis, phagocytosis, and other active transport mechanisms. Unique uptake patterns across cell types highlight the necessity for customized NMs designs. After internalization, NMs move through a variety of intracellular routes that affect where they are located subcellularly. Understanding these pathways is pivotal for enhancing the targeted delivery of therapeutic agents and imaging probes. Furthermore, the cellular retention of NMs plays a critical role in sustained therapeutic efficacy and long-term imaging capabilities. Factors influencing cellular retention include nanoparticle size, surface chemistry, and the cellular microenvironment. Strategies for prolonging cellular retention are discussed, including surface modifications and encapsulation techniques. In conclusion, a comprehensive understanding of the mechanisms governing cellular uptake, subcellular localization, and cellular retention of NMs is essential for advancing their application in disease diagnosis and therapy. This review provides insights into the intricate interplay between NMs and biological systems, offering a foundation for the rational design of next-generation nanomedicines.

Comparisons of Forecasting for Survival Outcome for Head and Neck Squamous Cell Carcinoma by using Machine Learning Models based on Multi-omics.

Background: Machine learning methods showed excellent predictive ability in a wide range of fields. For the survival of head and neck squamous cell carcinoma (HNSC), its multi-omics influence is crucial. This study attempts to establish a variety of machine learning multi-omics models to predict the survival of HNSC and find the most suitable machine learning prediction method. Methods: The HNSC clinical data and multi-omics data were downloaded from the TCGA database. The important variables were screened by the LASSO algorithm. We used a total of 12 supervised machine learning models to predict the outcome of HNSC survival and compared the results. In vitro qPCR was performed to verify core genes predicted by the random forest algorithm. Results: For omics of HNSC, the results of the twelve models showed that the performance of multi-omics was better than each single-omic alone. Results were presented, which showed that the Bayesian network(BN) model (area under the curve [AUC] 0.8250, F1 score=0.7917) and random forest(RF) model (area under the curve [AUC] 0.8002,F1 score=0.7839) played good prediction performance in HNSC multi-omics data. The results of in vitro qPCR were consistent with the RF algorithm. Conclusion: Machine learning methods could better forecast the survival outcome of HNSC. Meanwhile, this study found that the BN model and the RF model were the most superior. Moreover, the forecast result of multi-omics was better than single-omic alone in HNSC.

Genomic analysis provides insights into the transmission and pathogenicity of Talaromyces marneffei.

Talaromyces marneffei (T. marneffei) is a medically important opportunistic dimorphic fungus that infects both humans and bamboo rats. However, the mechanisms of transmission and pathogenicity of T. marneffei are poorly understood. In our study, we combined Illumina and PacBio sequencing technologies to sequence and assemble a complete genome of T. marneffei. To elucidate the transmission route and source, we sequenced three additional T. marneffei isolates using Illumina sequencing technology. Variations among isolates were used to develop a multilocus sequence typing (MLST) system comprising five housekeeping genes that can be used to discriminate between isolates derived from different sources. Our analysis revealed that human and bamboo rat share identical genotypes in these five loci. Thus, we hypothesized that T. marneffei is transmitted to humans through inhalation of spores in the surrounding environment into the lungs and that the bamboo rat can serve as an important natural reservoir for pathogens. Furthermore, we also identified temperature-dependent polyketide synthases, non-ribosomal peptide synthetases and secreted proteins as putative pathogenicity-related factors. In addition, we identified antifungal drug targets that can be investigated in future studies to elucidate the mechanisms underlying drug resistance. In summary, our study presents the basic features of the T. marneffei genome and provides insights into the transmission and pathogenicity of T. marneffei, which warrant fundamental experimental research.

LncSSBP1 Functions as a Negative Regulator of IL-6 Through Interaction With hnRNPK in Bronchial Epithelial Cells Infected With Talaromyces marneffei.

Talaromyces marneffei (TM) is an important opportunistic pathogenic fungus capable of causing disseminated lethal infection. In our previous study, we identified host lncRNAs and mRNAs that are dysregulated in TM-infected bronchial epithelial cells. In this report, we verified that IL-6, a key factor in acute inflammatory response, is down-regulated in TM pathogenesis. To elucidate the mechanism of IL-6 regulation, we analyzed the coding/non-coding network, and identified lncSSBP1, a novel lncRNA that is up-regulated by TM. Our results demonstrate that overexpression of lncSSBP1 decreases IL-6 mRNA expression, whereas knockdown of lncSSBP1 enhances IL-6 mRNA expression. Though lncSSBP1 is primarily localized to the nucleus, bioinformatics analysis suggests that it is unlikely to function as competing endogenous RNA or to interact with IL-6 transcription factors. Instead, RNA pull down and RNA immunoprecipitation assays showed that lncSSBP1 binds specifically to heterogenous nuclear ribonucleoprotein K (hnRNPK), which is involved in IL-6 mRNA processing. Our findings suggest that lncSSBP1 may affect IL-6 mRNA expression during TM infection through interaction with hnRNPk in bronchial epithelial cells. Our results suggest a novel pathway by which TM may suppress the immune response to its advantage.

Lentivirus-mediated shRNA Targeting CNN2 Inhibits Hepatocarcinoma in Vitro and in Vivo.

Objective: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with a high rate of mortality. Our previous study shows the expression of calponin 2 (CNN2) is up-regulated in hepatocellular carcinoma tissues, especially in metastatic ones. To better understand the role of CNN2 in HCC, RNA interference (RNAi) was used to explore its role in tumor growth and metastasis. Methods: Lentivirus-mediated CNN2-shRNA was transfected into SK-hep-1 cells, and the efficacy of CNN2 expression, cell migration, invasion, proliferation and cell cycles were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB), Transwell assay, methyl thiazol tetrazolium assay and flow cytometry, respectively. SK-hep-1 cells transfected with Lentivirus-CNN2 shRNA were xenografted in Balb/C nude mice to explore the effect of CNN2-shRNA in tumor growth. Xenograft tumor tissues were examined for their histopathology, cell apoptosis, the expression of total protein and their corresponding phosphorylated protein of MEK1/2, ERK1/2, AKT, by hematoxylin and eosin stain (H & E staining), TUNEL assay, immunohistochemical technique, respectively. Results: Our research shows it is evident that CNN2 shRNA can effectively down-regulate the expressions of CNN2 mRNA and protein, inhibit cell proliferations, arrest cell cycles at the S phase and reduce cell migration and invasion. SK-hep-1 cells with CNN2 down-regulation have markedly attenuated tumor growth in nude mice. Xenograft tumor tissues have displayed typical tumor characteristics and no apoptosis is detected in shRNA group or in control group. No metastatic tumor was found in any group of nude mice. With CNN2 protein down-regulation, the protein of pMEK1/2 and pERK1/2 are effectively down-regulated, except pAKT, AKT, MEK1/2 and ERK1/2. Conclusions: CNN2 plays an important role in tumor growth and metastasis, possibly through MEK1/2-ERK1/2 signaling pathway. Our study illustrate that CNN2 might be a potential target in HCC molecular target therapy.

Microsatellite analysis of genetic diversity in the Tupaia belangeri yaoshanensis.

The Chinese tree shrew (Tupaia belangeri yaoshanensis) has long been proposed to serve as an animal model for studying human diseases. However, its overall genetic diversity and population structure remain largely unknown. In the present study, we investigated the genetic diversity of population microsatellite DNA in wild Tupaia belangeri yaoshanensis. Sixteen microsatellite loci were assessed in 76 wild Tupaia belangeri yaoshanensis. The target microsatellite DNA fragments were amplified from the peripheral blood DNA of the animals by polymerase chain reaction (PCR), and the PCR-amplified products were verified by DNA sequencing and used for the analysis of allele, effective allele, genetic heterozygosity, polymorphism and population structure. Our results showed that of the 16 microsatellite loci examined, 5 microsatellite loci were monomorphic and 11 microsatellite loci were polymorphic. We detected 61 alleles in the polymorphic loci and found 2-10 (with an average of 5.5455) alleles per locus. Our data also showed that the observed and expected heterozygosities ranged from 0.087 to 0.8947 and 0.1368 to 0.7892 with an average of 0.3968 and 0.4796, respectively. Taken together, the results revealed a considerably high heterozygosity and high genetic diversity at the molecular level in the population of wild Tupaia belangeri yaoshanensis. The identified markers from the present study may be useful for individual identification and parentage testing, as well as for the quantification of population heterogeneity in the Chinese tree shrew.

Acute Penicillium marneffei infection stimulates host M1/M2a macrophages polarization in BALB/C mice.

BACKGROUND: Penicillium marneffei (P. marneffei) is a thermally dimorphic fungus pathogen that causes fatal infection. Alveolar macrophages are innate immune cells that have critical roles in protection against pulmonary fungal pathogens and the macrophage polarization state has the potential to be a deciding factor in disease progression or resolution. The aim of this study was to investigate mouse alveolar macrophage polarization states during P. marneffei infection. RESULTS: We used enzyme-linked immunosorbent (ELISA) assays, quantitative real-time PCR (qRT-PCR), and Griess, arginase activity to evaluate the phenotypic markers of alveolar macrophages from BALB/C mice infected with P. marneffei. We then treated alveolar macrophages from infected mice with P. marneffei cytoplasmic yeast antigen (CYA) and investigated alveolar macrophage phenotypic markers in order to identify macrophage polarization in response to P. marneffei antigens. Our results showed: i) P. marneffei infection significantly enhanced the expression of classically activated macrophage (M1)-phenotypic markers (inducible nitric oxide synthase [iNOS] mRNA, nitric oxide [NO], interleukin-12 [IL-12], tumor necrosis factor-alpha [TNF-α]) and alternatively activated macrophage (M2a)-phenotypic markers (arginase1 [Arg1] mRNA, urea) during the second week post-infection. This significantly decreased during the fourth week post-infection. ii) During P. marneffei infection, CYA stimulation also significantly enhanced the expression of M1 and M2a-phenotypic markers, consistent with the results for P. marneffei infection and CYA stimulation preferentially induced M1 subtype. CONCLUSIONS: The data from the current study demonstrated that alveolar macrophage M1/M2a subtypes were present in host defense against acute P. marneffei infection and that CYA could mimic P. marneffei to induce a host immune response with enhanced M1 subtype. This could be useful for investigating the enhancement of host anti-P. marneffei immune responses and to provide novel ideas for prevention of P. marneffei-infection.

Expression profile of lncRNA in human bronchial epithelial cells response to Talaromyces marneffei infection: A microarray analysis.

Talaromyces marneffei is an important opportunistic pathogenic fungus capable of causing systemic lethal infection through inhalation of its conidia. However, little is known about the pathogenesis and interactions between Talaromyces marneffei and host. The aim of this study was to identify potential long noncoding RNAs (lncRNAs) and coding genes associated with interactions between airway epithelial cell and Talaromyces marneffei conidia. We carried out a microarray analysis to determine the expression profile of lncRNA and mRNA in human bronchial epithelial cell in response to Talaromyces marneffei infection. Compared to control group, we found that 370 and 149 lncRNAs were up and down regulated, respectively. Meanwhile, the expression level of 269 and 60 mRNAs was increased and decreased, respectively. To understand the potential role of the differentially expressed lncRNAs, we performed functional annotations of the corresponding coding genes using gene ontology and pathway analyses. Our results provide insights into the pathogenesis of early infection by Talaromyces marneffei.

Senescence marker protein 30 (SMP30) serves as a potential prognostic indicator in hepatocellular carcinoma.

Senescence marker protein 30 (SMP30) has been identified as a tumor-related molecule of hepatocellular carcinoma (HCC). Its clinical significance and underlying mechanisms in HCC tissues, however, remain largely unexplored. We have demonstrated a preferentially expressed SMP30 in normal liver using a tissue microarray. By employing real-time quantitative PCR, two tissue microarrays and Oncomine database analysis, we have also shown that the SMP30 in HCC tissues has significantly reduced when compared with that in paired adjacent non-tumor tissues (P = 0.0037). The reduced expression of SMP30 is very noticeably related to larger tumor size (P = 0.012), enhanced TNM (P = 0.009) and worse survival (P < 0.0001) in HCC patients. The analyses using Cox regression have indicated that the decreased SMP30 expression is an independent risk to the reduced overall survival rate of HCC patients (P = 0.001), and the down-regulation of SMP30 in HCC might be mediated by DNA methylation. Moreover, genes co-expressed with SMP30 may affect the prognosis through apoptotic process, biological adhesion and blood coagulation by PANTHER analyses. Our studies have indicated that the SMP30 may serve as a candidate of HCC clinical prognostic marker and a potential therapeutic target.

Transcriptome Sequencing of Gynostemma pentaphyllum to Identify Genes and Enzymes Involved in Triterpenoid Biosynthesis.

G. pentaphyllum (Gynostemma pentaphyllum), a creeping herbaceous perennial with many important medicinal properties, is widely distributed in Asia. Gypenosides (triterpenoid saponins), the main effective components of G. pentaphyllum, are well studied. FPS (farnesyl pyrophosphate synthase), SS (squalene synthase), and SE (squalene epoxidase) are the main enzymes involved in the synthesis of triterpenoid saponins. Considering the important medicinal functions of G. pentaphyllum, it is necessary to investigate the transcriptomic information of G. pentaphyllum to facilitate future studies of transcriptional regulation. After sequencing G. pentaphyllum, we obtained 50,654,708 unigenes. Next, we used RPKM (reads per kilobases per million reads) to calculate expression of the unigenes and we performed comparison of our data to that contained in five common databases to annotate different aspects of the unigenes. Finally, we noticed that FPS, SS, and SE showed differential expression of enzymes in DESeq. Leaves showed the highest expression of FPS, SS, and SE relative to the other two tissues. Our research provides transcriptomic information of G. pentaphyllum in its natural environment and we found consistency in unigene expression, enzymes expression (FPS, SS, and SE), and the distribution of gypenosides content in G. pentaphyllum. Our results will enable future related studies of G. pentaphyllum.

High expression and frequently humoral immune response of melanoma-associated antigen D4 in glioma.

MAGE-D4 is a novel member of MAGE super-family. It has preliminarily been demonstrated that MAGE-D4 mRNA is not expressed in majority of normal tissues except for brain and ovary in which only trace amount of MAGE-D4 mRNA can be detected, but predominantly expressed in glioma. MAGE-D4 protein expression and its immunogenicity in glioma have not been elucidated well. This study was designed to analyze MAGE-D4 expression both at mRNA and protein level, characteristic of humoral immune response, and their relationships with glioma patients' clinicopathological parameters. Recombinant MAGE-D4 protein and antiserum were generated. Quantitative RT-PCR analysis revealed that MAGE-D4 mRNA expression was overall up-regulated in 41 glioma specimens compared with that in 14 normal brain tissues. Immunohistochemistry analysis showed that 78% (21/27) glioma tissues expressed MAGE-D4 protein, which was predominantly located in the cytoplasm of tumor cells, but absent in any neuroglia cell of normal brain tissues. ELISA analysis demonstrated that humoral response against MAGE-D4 was detected in 17% (7/41) of glioma patients' sera but not in 77 healthy donors. No apparent correlation was observed between the expression and immunogenicity of MAGE-D4 with clinicopathological parameters of glioma. In summary, these results indicate that MAGE-D4 is highly expressed in glioma and can develop specifically humoral response in glioma patients, which supports that it may be a promising biomarker for glioma diagnosis and immunotherapy.

Glycine insertion at protease cleavage site of SNAP25 resists cleavage but enhances affinity for botulinum neurotoxin serotype A.

The light chain of botulinum neurotoxin A (BoNT/A-LC) is a Zn-dependent protease that specifically cleaves SNAP25 of the SNARE complex, thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions. The C-terminus of SNAP25 (residues 141-206) retains full activity for BoNT/A-LC-catalyzed cleavage at P1-P1' (Gln197-Arg198). Using the structure of a complex between the C-terminus of SNAP25 and BoNT/A-LC as a model to design SNAP25-derived pseudosubstrate inhibitors (SNAPIs) that prevent presentation of the scissile bond to the active site, we introduced multiple His residues to replace Ala-Asn-Gln-Arg (residues 195-198) at the substrate cleavage site, with the intent to identify possible side-chain interactions with the active site Zn. We also introduced multiple Gly residues between the P1-P1' residues to explore the spatial tolerance within the active-site cleft. Using a FRET substrate YsCsY, we compared a series of SNAPIs for inhibition of BoNT/A-LC. Among the SNAPIs tested, several known cleavage-resistant, single-point mutants of SNAP25 were poor inhibitors, with most of the mutants losing binding affinity. Replacement with His at the active site did not improve inhibition over wildtype substrate. In contrast, Gly-insertion mutants were not only resistant to cleavage, but also surprisingly showed enhanced affinity for BoNT/A-LC. Two of the Gly-insertion mutants exhibited 10-fold lower IC50 values than the wildtype 66-mer SNAP25 peptide. Our findings illustrate a scenario, where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed.

MoDUO1, a Duo1-like gene, is required for full virulence of the rice blast fungus Magnaporthe oryzae.

Duo1, a major component of the Dam1 complex which has been found in two species of yeast (the budding yeast Saccharomyces cerevisae and the fission yeast Schizosaccharomyces pombe), is involved in mitosis-related chromosome segregation, while its relevance to pathogenicity in filamentous fungi remains unclear. This report elucidated this very fact in the case of the rice blast fungus Magnaporthe oryzae. A gene designated MoDUO1 that encodes a Duo1-like homolog (MoDuo1) was discovered in the M. oryzae genome. Two types of MoDUO1 mutants were obtained using genetic approaches of Agrobacterium-mediated gene disruption and homologous recombination. Both disruption and deletion of MoDUO1 can exert profound effects on the formation pattern of conidiophores and conidial morphology, such as abnormal nucleic numbers in conidia and delayed extension of infectious hyphae. Intriguingly, plant infection assays demonstrated that inactivation of MoDUO1 significantly attenuates the virulence in its natural host rice leaves, and functional complementation can restore it. Subcellular localization assays showed that MoDuo1 is mainly distributed in the cytosol of fungal cells. Proteomics-based investigation revealed that the expression of four mitosis-related proteins is shut down in the MoDUO1 mutant, suggesting that MoDuo1 may have a function in mitosis. In light of the fact that Duo1 orthologs are widespread in plant and human fungal pathogens, our finding may represent a common mechanism underlying fungal virulence. To the best of our knowledge, this is the first example of linking a Duo1-like homolog to the pathogenesis of a pathogenic fungus, which might provide clues to additional studies on the role of Dam1 complex in M. oryzae and its interaction with rice.

CYP1, a hypovirus-regulated cyclophilin, is required for virulence in the chestnut blight fungus.

Cyclophilins are peptidyl-prolyl cis-trans isomerases that are highly conserved throughout eukaryotes and are the cellular target of the immunosuppressive drug cyclosporin A (CsA). We cloned cyp1, a cyclophilin A-encoding gene in the phytopathogenic fungus Cryphonectria parasitica, and showed that this gene was downregulated following infection by a virulence-attenuating hypovirus. The function of cyp1 was further investigated by construction of a cyp1 deletion mutant. Although the wild-type C. parasitica strain EP155 was sensitive to CsA, the Δcyp1 strain was highly tolerant to CsA, indicating that CYP1 was the target of CsA. Deletion of cyp1 resulted in reduced virulence when inoculated to chestnut stems. Transcriptional analysis revealed that deletion of cyp1 also reduced transcript levels for genes encoding key components of the heterotrimeric guanosine triphosphate-binding protein signalling pathway that are essential for sensing environmental cues and are involved in C. parasitica development and virulence.

Nm23-H1 suppresses hepatocarcinoma cell adhesion and migration on fibronectin by modulating glycosylation of integrin beta1.

BACKGROUND: Nm23 gene was isolated as a metastatic suppressor gene. The antimetastatic effect of Nm23 has been an enigma for more than 10 years. Little is known about its molecular mechanisms. In this study we overexpressed Nm23-H1 in H7721 cells and observed reduction of cell adhesion, migration and extension of actin stress fibers in cells stimulated by fibronectin (Fn). METHODS: pcDNA3/Nm23-H1 was introduced into H7721 cells, and expression of Nm23-H1 was monitored by RT-PCR and western blot. Cell adhesion, actin extension and wound-induced migration assays were done on dishes coated with fibronectin. Phosphorylation of focal adhesion kinase (FAK) and total amount of integrin alpha 5 and beta1 in Nm23-H1 transfected cells and control cells were measured by western blot. Flow cytometry was used to detect expression of surface alpha 5 and beta1 integrin. N-glycosylation inhibitor tunicamycin was used to deglycosylate the integrin beta1 subunit. RESULTS: Overexpression of nm23-H1 in H7721 cells reduced cell adhesion, migration and extension of actin stress fibers on dishes coated with Fn. Phosphorylation of FAK in Nm23-H1 transfected cells was also attenuated. Integrin alpha 5 and beta1 gene messages were unaltered in nm23-H1 overexpressed cells as detected by RT-PCR. However, while cell surface integrin alpha 5 was unchanged, surface expression of beta1 integrin was downregulated. Western blot also showed that the total amounts of integrin alpha 5 and beta1 were unaltered, but the level of mature integrin beta1 isoform was decreased significantly. Furthermore, partially glycosylated precursor beta1 was increased, which indicated that the impaired glycosylation of integrin beta1 precursor might contribute to the loss of cell surface integrin beta1 in nm23-H1 overexpressed cells. CONCLUSION: These results suggest that by modulating glycosylation of integrin beta1, nm23-H1 down-regulates integrin beta1 subunit on cell surface and mediates intracellular signaling and subsequent suppression of the invasive process, including cell adhesion and migration.

Effect of IPP5, a novel inhibitor of PP1, on apoptosis and the underlying mechanisms involved.

Genes encoding apoptosis-inducing proteins are postulated to be candidate tumour suppressors. The identification of such proteins may benefit the early diagnosis and therapy of tumours. In the present study, we characterized the function of a novel human BMSC (bone marrow stromal cell)-derived protein {IPP5 [inhibitor-5 of PP1 (protein phosphatase 1)]} by large-scale random sequencing of a human BMSC cDNA library. hIPP5 (human IPP5) cDNA encodes a protein of 116 amino acid residues, which shares high homology with human PPI-1 (inhibitor-1 of PP1). The effect of IPP5 on apoptosis and the underlying molecular mechanisms were investigated by overexpression of IPP5 in HeLa cells, a human cervical carcinoma cell line. Our results showed that overexpression of active mutant IPP5 inhibited anchorage-dependent growth and induced apoptosis in HeLa cells, which may be attributed to the up-regulation of p21(waf/cip1) (a 21 kDa cell-cycle regulatory protein), p53 and Bcl-2-antagonist/killer, and down-regulation of Bcl-2 and Bcl-X(L). We also showed that the expression of active mutant IPP5 in HeLa cells was further enhanced on TNF (tumour necrosis factor) treatment and overexpression of active mutant IPP5 sensitized HeLa cells to TNF-induced JNK (c-Jun N-terminal kinase) and p38 activation as well as TNF-mediated apoptosis. Thus overexpression of active mutant IPP5 may increase cell susceptibility to TNF-induced apoptosis by the activation of p38 and JNK pathways. In addition, IPP5 active mutant could interact with PP1alpha as demonstrated by the co-precipitation assay.

[Fingerprints identification of Gynostemma pentaphyllum by RAPD and cloning and analysis of its specific DNA fragment].

OBJECTIVE: To identify the resources of Gynostemma pentaphyllum and its spurious breed plant Cayratia japonica at level of DNA. METHODS: Two random primers ( WGS001, WGS004) screened were applied to do random amplification with genomic DNA extracted from Gynostemma pentaphyllum and Cayratia japonica which were collected from different habitats. After amplificated with WGS004, one characteristic fragment about 500 bp which was common to all Gynostemma pentaphyllum samples studied but not to Cayratia japonica was cloned and sequenced. Then these sequences obtained were analyzed for identity and compared by Blastn program in GenBank. RESULTS: There were obvious different bands amplified by above two primers in their fingerprints of genomic DNA. On the basis of these different bands of DNA fingerprints, they could distinguish Gynostemma pentaphyllum and Cayratia japonica obviously. Sequence alignment of seven cloned bands showed that their identities ranged from 45.7% - 94.5%. There was no similar genome sequences searched in GenBank. This indicated that these seven DNA fragments had not been reported before and they should be new sequences. CONCLUSION: RAPD technique can be used for the accurate identification of Gynostemma pentaphyllum and its counterfeit goods Cayratia japonica. Besides, these specific DNA sequences for Gynostemmna pentaphyllum in this study are useful for the further research on identification of species and assisted selection breeding in Gynostemma pentaphyllum.

Large-scale expressed sequence tag analysis for the chestnut blight fungus Cryphonectria parasitica.

Cryphonectria parasitica is the causal fungal agent responsible for the chestnut blight disease. We report the generation of 14,263 expressed sequence tags representing 6318 unisequences for the fungus. Functional annotation of these unisequences revealed different gene expression patterns for wild-type and hypovirus-infected cultures at the sporulation stage and allowed the reconstruction of key C. parasitica signal transduction pathways conserved in the Sorbidaryomycetes. A list of homologous genes implicated in pathogenicity, sporulation, and virus replication in other fungi were also identified. A large-scale gene comparison indicated that C. parasitica is most closely related to the plant pathogen Fusarium graminearum but more closely related to the non-pathogen Neurospora crassa than to the rice pathogen Magnaporthe grisea. This transcriptional information lays a new and solid ground for further investigation of both the biology of the fungus and interaction between a hypovirus and the host fungus.

Deletion of the cpku80 gene in the chestnut blight fungus, Cryphonectria parasitica, enhances gene disruption efficiency.

The chestnut blight fungus, Cryphonectria parasitica, and associated virulence-attenuating hypoviruses have emerged as an important model system for studying molecular mechanisms underlying fungal-plant pathogenic interactions. As more gene sequence information becomes available as a result of C. parasitica express sequence tags (ESTs) and ongoing whole genome sequencing projects, the development of an efficient gene disruption system has become an urgent need for functional genomics studies of this important forestry pathogen. Here, we report the cloning of the C. parasitica gene cpku80 that encodes a key component of the nonhomologous end joining DNA repair pathway and the construction of a corresponding deletion mutant strain. The cpku80 mutant was indistinguishable from the parental wild-type strain EP155 in colony morphology, ability to support hypovirus replication, conidiation and virulence. As predicted, the Deltacpku80 strain did exhibit an increased sensitivity to the mutagen methyl methanesulfonate. A test with three selected genes resulted in a gene disruption efficiency of about 80% for the Deltacpku80 strain, a significant increase over the 2-5% levels of homologous recombination generally observed for the wild-type strain EP155. This efficient homologous recombination system provides a powerful tool for large-scale analysis of gene functions in C. parasitica.

Genome sequence, full-length infectious cDNA clone, and mapping of viral double-stranded RNA accumulation determinant of hypovirus CHV1-EP721.

Cryphonectria parasitica strain EP721 is infected with a strain of hypovirus CHV1, CHV1-EP721, and exhibits typical hypovirulence-associated traits such as reduced pigmentation and reduced asexual sporulation. However, the accumulation of the viral double-stranded RNA (dsRNA) in this hypovirus-infected C. parasitica strain is atypically low. We now report the complete nucleotide sequence and construction of a full-length infectious cDNA clone for hypovirus CHV1-EP721. The genome sequence of CHV1-EP721 was determined to be 12,724 bp in length and to share extensive homology with two other hypovirus strains, CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at the nucleotide level and 99% and 92% identities at the amino acid level, respectively. CHV1-EP721 was successfully introduced into virus-free fungal host strain EP721(-v) by transfection with transcripts derived from a full-length viral cDNA. The transfected strain had a phenotype indistinguishable from that of EP721, and the accumulation of CHV1-EP721 dsRNA in the transfectant was lower than those transfected by CHV1-Euro7 and CHV1-EP713 transcripts. Through the construction of chimeric viruses by domain swapping using infectious cDNA clones of CHV1-EP721, CHV1-EP713, and CHV1-Euro7 hypoviruses, the determinant for the low level of viral dsRNA accumulation in CHV1-EP721 was mapped to the second of two CHV1-EP721 open reading frames (ORFs), ORF B. Further refined swapping of domains within ORF B identified a 2.5-kb coding region between p48 and the polymerase domain of CHV1-EP721 as being responsible for the low viral dsRNA accumulation. Evidence is also provided that low rates of hypovirus transmission through conidial spores correlates with low viral dsRNA accumulation.