Leak-proof probe for accurate detection of Neisseria gonorrhoeae by recombinase polymerase amplification-mediated lateral flow strip.

Neisseria gonorrhoeae is the only pathogen contributing to gonorrhea, a common infectious disease. Clinically, approximately 50-80% of female and 40% of male patients are asymptomatic, and these carriers are the key to gonorrhea transmission. The rapid detection of N. gonorrhoeae recessive infection is vital to curb the spread of gonorrhea. Therefore, the development of a specific, sensitive, rapid, and convenient method for the diagnosis of N. gonorrhoeae is a priority. In this study, we identified the highly conserved fitA gene of N. gonorrhoeae as a detection target through bioinformatics analysis. Then, we constructed a convenient, economical, and effective biosensor to detect N. gonorrhoeae without false-positive results based on recombinase polymerase amplification-mediated lateral flow strip by leak-proof probe. The biosensor has high sensitivity, is capable of detecting N. gonorrhoeae at concentrations as low as 102 copies/µL within 28 min, and has high specificity, which allows N. gonorrhoeae to be differentiated from other genito-urinary bacteria and fungi. Finally, this biosensor has been successfully applied to the detection of N. gonorrhoeae in clinical samples, and the results have been consistent with those determined using qRT-PCR.

Differential SW16.1 allelic effects and genetic backgrounds contributed to increased seed weight after soybean domestication.

Although seed weight has increased following domestication from wild soybean (Glycine soja) to cultivated soybean (Glycine max), the genetic basis underlying this change is unclear. Using mapping populations derived from chromosome segment substitution lines of wild soybean, we identified SW16.1 as the causative gene underlying a major quantitative trait locus controlling seed weight. SW16.1 encodes a nucleus-localized LIM domain-containing protein. Importantly, the GsSW16.1 allele from wild soybean accession N24852 had a negative effect on seed weight, whereas the GmSW16.1 allele from cultivar NN1138-2 had a positive effect. Gene expression network analysis, reverse-transcription quantitative polymerase chain reaction, and promoter-luciferase reporter transient expression assays suggested that SW16.1 regulates the transcription of MT4, a positive regulator of seed weight. The natural variations in SW16.1 and other known seed weight genes were analyzed in soybean germplasm. The SW16.1 polymorphism was associated with seed weight in 247 soybean accessions, showing much higher frequency of positive-effect alleles in cultivated soybean than in wild soybean. Interestingly, gene allele matrix analysis of the known seed weight genes revealed that G. max has lost 38.5% of the G. soja alleles and that most of the lost alleles had negative effects on seed weight. Our results suggest that eliminating negative alleles from G. soja led to a higher frequency of positive alleles and changed genetic backgrounds in G. max, which contributed to larger seeds in cultivated soybean after domestication from wild soybean. Our findings provide new insights regarding soybean domestication and should assist current soybean breeding programs.

Identifying Wild Versus Cultivated Gene-Alleles Conferring Seed Coat Color and Days to Flowering in Soybean.

Annual wild soybean (G. soja) is the ancestor of the cultivated soybean (G. max). To reveal the genetic changes from soja to max, an improved wild soybean chromosome segment substitution line (CSSL) population, SojaCSSLP5, composed of 177 CSSLs with 182 SSR markers (SSR-map), was developed based on SojaCSSLP1 generated from NN1138-2(max)×N24852(soja). The SojaCSSLP5 was genotyped further through whole-genome resequencing, resulting in a physical map with 1366 SNPLDBs (SNP linkage-disequilibrium blocks), which are composed of more markers/segments, shorter marker length and more recombination breakpoints than the SSR-map and caused 721 new wild substituted segments. Using the SNPLDB-map, two loci co-segregating with seed-coat color (SCC) and six loci for days to flowering (DTF) with 88.02% phenotypic contribution were identified. Integrated with parental RNA-seq and DNA-resequencing, two SCC and six DTF candidate genes, including three previously cloned (G, E2 and GmPRR3B) and five newly detected ones, were predicted and verified at nucleotide mutant level, and then demonstrated with the consistency between gene-alleles and their phenotypes in SojaCSSLP5. In total, six of the eight genes were identified with the parental allele-pairs coincided to those in 303 germplasm accessions, then were further demonstrated by the consistency between gene-alleles and germplasm phenotypes. Accordingly, the CSSL population integrated with parental DNA and RNA sequencing data was demonstrated to be an efficient platform in identifying candidate wild vs. cultivated gene-alleles.

Comprehensive Identification of Drought Tolerance QTL-Allele and Candidate Gene Systems in Chinese Cultivated Soybean Population.

Drought is one of the most important factors affecting plant growth and productivity. The previous results on drought tolerance (DT) genetic system in soybean indicated a complex of genes not only few ones were involved in the trait. This study is featured with a relatively thorough identification of QTL-allele/candidate-gene system using an efficient restricted two-stage multi-locus multi-allele genome-wide association study, on two comprehensive DT indicators, membership index values of relative plant weight (MPW) and height (MPH), instead of a single biological characteristic, in a large sample (564 accessions) of the Chinese cultivated soybean population (CCSP). Based on 24,694 multi-allele markers, 75 and 64 QTL with 261 and 207 alleles (2-12/locus) were detected for MPW and MPH, explaining 54.7% and 47.1% of phenotypic variance, respectively. The detected QTL-alleles were organized into a QTL-allele matrix for each indicator, indicating DT is a super-trait conferred by two (even more) QTL-allele systems of sub-traits. Each CCSP matrix was separated into landrace (LR) and released cultivar (RC) sub-matrices, which showed significant differentiation in QTL-allele constitutions, with 58 LR alleles excluded and 16 new ones emerged in RC. Using the matrices, optimal crosses with great DT transgressive recombinants were predicted. From the detected QTL, 177 candidate genes were annotated and validated with quantitative Real-time PCR, and grouped into nine categories, with ABA and stress responders as the major parts. The key point of the above results is the establishment of relatively full QTL-allele matrices composed of numerous gene functions jointly conferring DT, therefore, demonstrates the complexity of DT genetic system and potential of CCSP in DT breeding.

Prevalence and Antifungal Susceptibility of Pathogenic Yeasts in China: A 10-Year Retrospective Study in a Teaching Hospital.

To determine the dynamic changes of pathogenic yeast prevalence and antifungal susceptibility patterns in tertiary hospitals in China, we analyzed 527 yeast isolates preserved in the Research Center for Medical Mycology at Peking University, Beijing, China, between Jan 2010 and Dec 2019 and correctly identified 19 yeast species by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and ribosomal DNA sequencing. Antifungal susceptibility testing was performed following a Sensititre YeastOne colorimetric microdilution panel with nine clinically available antifungals. The Clinical and Laboratory Standards Institute (CLSI)-approved standard M27-A3 (S4) and newly revised clinical breakpoints or species-specific and method-specific epidemiological cutoff values were used for the interpretation of susceptibility test data. In this study, although Candida albicans was the predominant single species, non-C. albicans species constituted >50% of isolates in 6 out of 10 years, and more rare species were present in the recent 5 years. The non-C. albicans species identified most frequently were Candida parapsilosis sensu stricto, Candida tropicalis, and Candida glabrata. The prevalence of fluconazole and voriconazole resistance in the C. parapsilosis sensu stricto population was <3%, but C. tropicalis exhibited decreased susceptibility to fluconazole (42, 57.5%) and voriconazole (31, 42.5%), and 22 (30.1%) C. tropicalis isolates exhibited wild-type minimum inhibitory concentrations (MICs) to posaconazole. Furthermore, fluconazole and voriconazole cross-resistance prevalence in C. tropicalis was 19 (26.1%). The overall prevalence of fluconazole resistance in the C. glabrata population was 14 (26.9%), and prevalence of isolates exhibiting voriconazole non-wild-type MICs was 33 (63.5%). High-level echinocandin resistance was mainly observed in C. glabrata, and the prevalence rates of isolate resistance to anidulafungin, micafungin, and caspofungin were 5 (9.6%), 5 (9.6%), and 4 (7.7%), respectively. Moreover, one C. glabrata isolate showed multidrug resistant to azoles, echinocandins, and flucytosine. Overall, the 10-year surveillance study showed the increasing prevalence of non-C. albicans species over time; the emergence of azole resistance in C. tropicalis and multidrug resistance in C. glabrata over the years reinforced the need for epidemiological surveillance and monitoring.

Preliminary study on the effect of brazilin on biofilms of Staphylococcus aureus.

Biofilms significantly enhance antibiotic resistance by inhibiting penetration of antibiotics and are shielded from the immune system via the formation of an extracellular polymeric matrix. Innovative and novel approaches are required for the inhibition of biofilm formation and treatment of biofilm-associated infectious diseases. In the current study, a biofilm model of Staphylococcus aureus was established in vitro to explore inhibitory effects of brazilin (BN) on biofilm formation and to evaluate damaging effects of BN in the presence and absence of vancomycin (VCM) on the biofilm. Antibiofilm-infection mechanisms of BN were observed. In these experiments, the clinical strain of S. aureus C-4-4 was isolated for biofilm formation. Crystal violet staining and fluorescence microscopy revealed that BN inhibited biofilm formation in vitro and the best effect was observed with two times the minimum inhibitory concentration of BN following 48 h incubation. Additionally, the results demonstrated that BN in combination with VCM enhanced the damage to biofilms, whereas VCM alone did not. The results of the reverse transcription-quantitative polymerase chain reaction analyses demonstrated that BN downregulated gene expression of intercellular adhesion (ica)A and upregulated icaR and the quorum-sensing (QS) system regulator accessory gene regulator A. In summary, BN inhibited S. aureus biofilm formation and destroyed biofilms, while simultaneously increasing permeability to VCM. BN was able to reduce production of the extracellular polymeric matrix and inhibited the QS system. These results support the use of BN as a novel drug and treatment strategy for S. aureus biofilm-associated infections.

A novel gene delivery system targeting Tie2 for cancer gene therapy.

UNLABELLED: The aim of this study was to explore a novel gene vector for targeting gene therapy. MATERIALS AND METHODS: We conjugated a peptide ligand (named GA3) for endothelial TEK tyrosine kinase (Tie2) with polyethylenimine (PEI) to construct a GA3-PEI complex and used the vector to transfer reporter and therapeutic gene in vitro and in vivo respectively. RESULTS: The results demonstrated the vehicle was able to transfer reporter genes specifically into lung cancer SPC-A1 cells and SPC-A1 xenografts highly expressing Tie2 and epithelial cells of bronchus, but not in heart, liver, spleen, kidney, lung alveolar and vascular tissues. In the gene therapy study, tumor growth was significantly inhibited in SPC-A1 xenograft-bearing mice treated with GA3-PEI/p53 complexes compared with control groups (p<0.05). CONCLUSION: Our results indicated that GA3-PEI is an efficient gene delivery system targeting Tie2.

Identification and characterization of a novel peptide ligand of Tie2 for targeting gene therapy.

Tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2) has been considered as a rational target for gene therapy in solid tumors. In order to identify a novel peptide ligand of Tie2 for targeted gene therapy, we screened a phage display peptide library and identified a candidate peptide ligand NSLSNASEFRAPY (designated GA5). Binding assays and Scatchard analysis revealed that GA5 could specifically bind to Tie2 with a dissociation constant of 2.1x10(-8)M. In addition, we showed that GA5 was internalized into tumor cells highly expressing Tie2. In the biodistribution assay, (125)I-GA5 was mainly accumulated in SPC-A1 xenograft tumors that express Tie2. In gene delivery studies, GA5-conjugated polyethylenimine vector could achieve greater transgene transduction than non-targeted vectors both in vitro and in vivo. Tumor growth inhibition was observed in SPC-A1 xenograft-bearing mice that received eight intratumoral injections of GA5-polyethylenimine/p53 complexes in 3 weeks. The difference in tumor volume between the experiment and control groups was significant (P<0.05). Our results showed that GA5 is a potentially efficient targeting element for cancer gene or molecular therapy.

Kinetics and subcellular localization of 5-ALA-induced PpIX in DHL cells via two-photon excitation fluorescence microscopy.

Two-photon excitation fluorescence (TPEF) microscopy was used to measure the 5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence in follicular lymphoma DHL cells. Kinetics of 5-ALA-induced PpIX accumulation in DHL cells under various 5-ALA concentrations was studied. We found that during the course of continuous incubation with 5-ALA, the relationship between the DHL cell fluorescence signal and the incubation time showed a biphasic variation. Initially the PpIX signal increased with the incubation time and reached the maximal value at about 3 h, and then it decreased with time during the subsequent incubation period. By labeling the 5-ALA incubated DHL cells with different organelle-specific fluorescence probes: Rhodamine 123 (for mitochondria), DioC6(3) (for endoplasmic reticulum) and LysoTracker Green (for lysosomes) respectively, we found that 5-ALA-induced PpIX was primarily localized in endoplasmic reticulum and mitochondria; its concentration in the lysosome was much lower. The results suggested that 5-ALA could potentially be an effective photosensitizer in photodynamic purging of DHL cells. Two-photon excitation fluorescence microscope is a useful tool for studying 5-ALA-induced PpIX subcellular localization.

Large-scale cDNA transfection screening for genes related to cancer development and progression.

A large-scale assay was performed by transfecting 29,910 individual cDNA clones derived from human placenta, fetus, and normal liver tissues into human hepatoma cells and 22,926 cDNA clones into mouse NIH 3T3 cells. Based on the results of colony formation in hepatoma cells and foci formation in NIH 3T3 cells, 3,806 cDNA species (8,237 clones) were found to possess the ability of either stimulating or inhibiting cell growth. Among them, 2,836 (6,958 clones) were known genes, 372 (384 clones) were previously unrecognized genes, and 598 (895 clones) were unigenes of uncharacterized structure and function. A comprehensive analysis of the genes and the potential mechanisms for their involvement in the regulation of cell growth is provided. The genes were classified into four categories: I, genes related to the basic cellular mechanism for growth and survival; II, genes related to the cellular microenvironment; III, genes related to host-cell systemic regulation; and IV, genes of miscellaneous function. The extensive growth-regulatory activity of genes with such highly diversified functions suggests that cancer may be related to multiple levels of cellular and systemic controls. The present assay provides a direct genomewide functional screening method. It offers a better understanding of the basic machinery of oncogenesis, including previously undescribed systemic regulatory mechanisms, and also provides a tool for gene discovery with potential clinical applications.

A novel small peptide as a targeting ligand for receptor tyrosine kinase Tie2.

Tie2 is an endothelium-specific receptor tyrosine kinase known to play an important role in tumor angiogenesis. We sought to identify a small peptide ligand against Tie2 for developing a delivery targeting agent. We used hydrophobic analysis and comparative sequence/structure analysis to select a minimal peptide based on angiopoietin-2 amino acid sequence. The resulting peptide named GA3(WTIIQRREDGSVDFQRTWKEYK) was synthesized and labeled with iodine-125 at the C-terminal tyrosine residue to characterize its binding capability. In in vitro binding assays, GA3 can not only specifically bind to SMMC7721-Tie2 but also compete with angiopoietin-2 in binding. Via mouse tail vein injection, 125I-labeled GA3 was found to favorably accumulate in SPC-A1 xenograft tumor tissues which positively express Tie2. These results demonstrated that GA3 may be useful as a drug or gene delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy.