Biomedical subjects

Yinghua Chen

Publications and source records attributed to Yinghua Chen.

10 recordsLinked to original sources

PubMed2025

Sensitive and Visualized Detection of Hantavirus Using CRISPR/Cas12a Based on AutoCORDSv2 Design.

In recent years, detection technologies based on the CRISPR/Cas12a method have been extensively utilized in the fields of nucleic acid, enzyme, and macromolecule detection, thereby reinforcing their significant role in the detection landscape. Enhancing the simplicity of design, efficiency, and automation of the CRISPR/Cas12a detection system is essential for advancing its application in diagnostics. Recently, we developed an automated CRISPR/Cas12a design system named AutoCORDSv2. This system can process published genomic sequences of pathogenic bacteria in a high-throughput manner and automatically generate conserved and highly specific crRNA sequences, along with primer sequences for target amplification. This capability facilitates the specific and precise design of the CRISPR/Cas12a detection system. In this study, crRNAs targeting the Hantaan virus (HTNV) and Seoul virus (SEOV), as well as RT-PCR primers and RT-RPA primers, were designed using AutoCORDSv2. The experimental results demonstrated that the CRISPR/Cas12a system, automatically designed by AutoCORDSv2, was specific for the detection of both the HTNV and SEOV, with no cross-reactivity observed with other pathogens. The detection sensitivity reached 6 copies/μL (equivalent to 111 copies per amplification reaction), whether measured by a microplate reader or directly observed with the naked eye. The detection results for 50 samples were consistent with those obtained from commercial RT-qPCR kits, indicating high precision. Furthermore, the CRISPR/Cas12a system designed by AutoCORDSv2 can also be utilized for the development of a single-tube detection system with a sensitivity of 42 copies per reaction. This system combined with a 5-min extraction step and RT-RPA, further underscoring its potential for application.

CRISPR-Cas Systems↗

PubMed2006

Heme crevice disorder after sixth ligand displacement in the cytochrome c-551 family.

1H NMR and visible absorption spectroscopy were used to monitor sixth ligand methionine displacement reactions in four members of the ferricytochrome c-551 family from Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas stutzeri substrain ZoBell, and Nitrosomonas europae. Potassium cyanide displaces the methionine ligand with very modest changes in the visible spectra, but profound changes in the NMR spectra. The initial product formed kinetically, designated complex I, changes with time and/or heating to a more thermodynamically favored product termed complex II. Spectra indicate that both I and II are actually a family of closely related conformational isomers. Low temperature NMR spectra of complex II indicate that some of the isomers are in chemical exchange on the NMR time scale. High pH also displaces the methionine ligand in a manner similar to the well-known alkaline transition of mitochondrial cytochrome c. However, the reaction occurs at higher pH values and over a narrower pH range for the c-551 family, and the transition pH range is different for the different proteins studied. The final alkaline forms also show peak widths and a number of peaks indicative of multiple conformational isomers.

Bacterial Proteins↗

PubMed2006

Primary structure-based function characterization of BRCT domain replicates in BRCA1.

BRCA1 is a large protein that exhibits a multiplicity of functions in its apparent role in DNA repair. Certain mutations of BRCA1 are known to have exceptionally high penetrance with respect to familial breast and ovarian cancers. The structures of the N-terminus and C-terminus of the protein have been determined. The C-terminus unit consists of two alpha-beta-alpha domains designated BRCT. We predicated two homologous BRCT regions in the BRCA1 internal region, and subsequently produced and purified these protein domains. Both recombinant domains show significant self-association capabilities as well as a preferential tendency to interact with each other. These results suggest a possible regulatory mechanism for BRCA1 function. We have demonstrated p53-binding activity by an additional region, and confirmed previous results showing that two regions of BRCA1 protein bind p53 in vitro. Based on sequence analysis, we predict five p53-binding sites. Our comparison of binding by wild-type and mutant domains indicates the sequence specificity of BRCA1-p53 interaction.

Amino Acid Sequence↗

PubMed2006

Tob1 controls dorsal development of zebrafish embryos by antagonizing maternal beta-catenin transcriptional activity.

Maternal beta-catenin and Nodal signals are essential for the formation of the dorsal organizer, which, in turn, induces neural and other dorsal tissue development in vertebrate embryos. Tob (Transducer of ErbB2) proteins possess antiproliferative properties and are known to influence BMP signaling, but their relationship to other signaling pathways and to embryonic patterning in general was unclear. In this study, we demonstrate that zebrafish tob1a is required for correct dorsoventral patterning. Mechanistically, Tob1a inhibits beta-catenin transcriptional activity by physically associating with beta-catenin and preventing the formation of beta-catenin/LEF1 complexes. Although Tob1a can also inhibit the transcriptional activity of the Nodal effector Smad3, its role in limiting dorsal development is executed primarily by antagonizing the beta-catenin signal. We further demonstrate that Tob family members across species share similar biochemical properties and biological activities.

Animals↗

PubMed2006

CcpA causes repression of the phoPR promoter through a novel transcription start site, P(A6).

The Bacillus subtilis PhoPR two-component system is directly responsible for activation or repression of Pho regulon genes in response to phosphate deprivation. The response regulator, PhoP, and the histidine kinase, PhoR, are encoded in a single operon with a complex promoter region that contains five known transcription start sites, which respond to at least two regulatory proteins. We report here the identification of another direct regulator of phoPR transcription, carbon catabolite protein A, CcpA. This regulator functions in the presence of glucose or other readily metabolized carbon sources. The maximum derepression of phoPR expression in a ccpA mutant compared to a wild-type stain was observed under excess phosphate conditions with glucose either throughout growth in a high-phosphate defined medium or in a low-phosphate defined medium during exponential growth, a growth condition when phoPR transcription is low in a wild-type strain due to the absence of autoinduction. Either HPr or Crh were sufficient to cause CcpA dependent repression of the phoPR promoter in vivo. A ptsH1 (Hpr) crh double mutant completely relieves phoPR repression during phosphate starvation but not during phosphate replete growth. In vivo and in vitro studies showed that CcpA repressed phoPR transcription by binding directly to the cre consensus sequence present in the promoter. Primer extension and in vitro transcription studies revealed that the CcpA regulation of phoPR transcription was due to repression of P(A6), a previously unidentified promoter positioned immediately upstream of the cre box. Esigma(A) was sufficient for transcription of P(A6), which was repressed by CcpA in vitro. These studies showed direct repression by CcpA of a newly discovered Esigma(A)-responsive phoPR promoter that required either Hpr or Crh in vivo for direct binding to the putative consensus cre sequence located between P(A6) and the five downstream promoters characterized previously.

Bacillus subtilis↗

PubMed2005

Bacillus subtilis phosphorylated PhoP: direct activation of the E(sigma)A- and repression of the E(sigma)E-responsive phoB-PS+V promoters during pho response.

The phoB gene of Bacillus subtilis encodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the complete phoB promoter or individual phoB promoter fusions were used to assess expression from each promoter under both induction conditions. The phoB promoter responsible for expression during sporulation, phoB-P(S), was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter, phoB-P(V). E(sigma)E was necessary and sufficient for P(S) expression in vitro. P(S) expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of sigma(E) is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoP approximately P) repressed P(S) in vitro via direct binding to the promoter, the first example of an E(sigma)E-responsive promoter that is repressed by PhoP approximately P. Whereas either PhoP or PhoP approximately P in the presence of E(sigma)A was sufficient to stimulate transcription from the phoB-P(V) promoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoP approximately P were required for P(V) promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during P(i)-replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.

Bacillus subtilis↗

PubMed2004

Bacillus subtilis YdiH is a direct negative regulator of the cydABCD operon.

During aerobic respiration, Bacillus subtilis utilizes three terminal oxidases, cytochromes aa3, caa3, and bd. Cytochrome bd is encoded by the cydABCD operon. We report here the first identification of a regulator for the cydABCD operon, YdiH. While working with DeltaresDE mutant strains, we identified colonies which contained suppressor mutations (cmp) which bypassed the requirement for ResD for all phenotypes not associated with cytochrome aa3 or caa3. Mapping identified a class of Tn10 insertions which were close to the cmp locus (Tn10-2) and a second class (Tn10-1) which was inserted in cydD, a gene which appears to be essential to the cmp phenotype. Sequencing of the cmp loci from four independent DeltaresDE cmp isolates yielded four loss-of-function alleles of ydiH, a gene encoding a protein with homology to AT-rich DNA-binding proteins. Additionally, we determined that cytochrome bd was aberrantly expressed in the DeltaresDE cmp background. Together these data led to the hypothesis that YdiH serves as a negative regulator of cydABCD expression, a hypothesis supported by both gel-shift and DNase I footprinting analyses. YdiH protected the cydA promoter region at three 22-bp repeats located in the long 5' untranslated region (193 bp). Induction of the cydABCD operon in a DeltaresDE background showed that expression of the terminal oxidase bd was responsible for the bypass phenotype observed in a DeltaresDE cmp strain, indicating that cytochrome bd expression complemented the loss of cytochromes aa3 and caa3 in the DeltaresDE strain.

Bacillus subtilis↗

PubMed2004

Residues required for Bacillus subtilis PhoP DNA binding or RNA polymerase interaction: alanine scanning of PhoP effector domain transactivation loop and alpha helix 3.

Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP alpha loop and alpha helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoP(I206A), PhoP(R210A), PhoP(L209A), and PhoP(H208A)) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoP(H205A) and PhoP(V204A)) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoP(V202A) and PhoP(D203A), had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoP(V190A), PhoP(W191A), PhoP(Y193A), PhoP(F195A), PhoP(G197A,) PhoP(T199A), and PhoP(R200A)) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoP(W191A) and PhoP(R200A) could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short alpha helix 3, more similar to OmpR than to PhoB of Escherichia coli.

Alanine↗

PubMed2003

The crystal structure of the phosphorylation domain in PhoP reveals a functional tandem association mediated by an asymmetric interface.

PhoP from Bacillus subtilis belongs to the OmpR subfamily of response regulators. It regulates the transcription of several operons and participates in a signal transduction network that controls adaptation of the bacteria to phosphate deficiency. The receiver domains of two members of this subfamily, PhoB from Escherichia coli and DrrD from Thermotoga maritima, have been structurally characterized. These modules have similar overall folds but display remarkable differences in the conformation of the beta4-alpha4 and alpha4 regions. The crystal structure of the receiver domain of PhoP (PhoPN) described in this paper illustrates yet another geometry in this region. Another major issue of the structure determination is the dimeric state of the protein and the novel mode of association between receiver domains. The protein-protein interface is provided by two different surfaces from each protomer, and the tandem unit formed through this asymmetric interface leaves free interaction surfaces. This design is well suited for further association of PhoP dimers to form oligomeric structures. The interprotein interface buries 970 A(2) from solvent and mostly involves interactions between charged residues. As described in the accompanying paper, mutations of a single residue in one salt bridge shielded from solvent prevented dimerization of the unphosphorylated and phosphorylated response regulator and had drastic functional consequences. The three structurally documented members of the OmpR family (PhoB, DrrD, and PhoP) provide a framework to consider possible relationships between structural features and sequence signatures in critical regions of the receiver domains.

Amino Acid Sequence↗

PubMed2003

Residue R113 is essential for PhoP dimerization and function: a residue buried in the asymmetric PhoP dimer interface determined in the PhoPN three-dimensional crystal structure.

Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which P(i) is growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3' of PhoP-binding consensus repeats in PhoP-repressed promoters. The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoP(R113E), PhoP(R113A), or PhoP(D60K) resulted in a Pho-negative phenotype. In vitro analysis showed that PhoP(R113E) was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoP(R113E) approximately P was deficient in DNA binding, contributing to the PhoP(R113E) in vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.

Alkaline Phosphatase↗