Modified Unit-Mediated Strand Displacement Reactions for Direct Detection of Single Nucleotide Variants in Active Double-Stranded DNA.

Accurate identification of single nucleotide variants (SNVs) in key driver genes holds a significant value for disease diagnosis and treatment. Fluorescent probes exhibit tremendous potential in specific, high-resolution, and rapid detection of SNVs. However, additional steps are required in most post-PCR assays to convert double-stranded DNA (dsDNA) products into single-stranded DNA (ssDNA), enabling them to possess hybridization activity to trigger subsequent reactions. This process not only prolongs the complexity of the experiment but also introduces the risk of losing target information. In this study, we proposed two strategies for enriching active double-stranded DNA, involving PCR based on obstructive groups and cleavable units. Building upon this, we explored the impact of modified units on the strand displacement reaction (SDR) and assessed their discriminatory efficacy for mutations. The results showed that detection of low variant allele frequencies (VAF) as low as 0.1% can be achieved. The proposed strategy allowed orthogonal identification of 45 clinical colorectal cancer tissue samples with 100% specificity, and the results were generally consistent with sequencing results. Compared to existing methods for enriching active targets, our approach offers a more diverse set of enrichment strategies, characterized by the advantage of being simple and fast and preserving original information to the maximum extent. The objective of this study is to offer an effective solution for the swift and facile acquisition of active double-stranded DNA. We anticipate that our work will facilitate the practical applications of SDR based on dsDNA.

Bronchial oncocytic carcinoma in an adult: a case report and literature review.

BACKGROUND: Lung salivary-type tumors originating from bronchial submucosal glands are rare, only four types of salivary gland-type tumors are listed in 2015 WHO classification of lung tumors. Here, we report a rare case of oncocytic carcinoma (OC) in the right main bronchus. CASE PRESENTATION: A 34-year-old man presented to our hospital with a two-month history of recurrent hemoptysis and with one month of inspiratory dyspnea. Pulmonary function tests showed mild restrictive ventilatory dysfunction and severe diffusion dysfunction. Furthermore, the flow volume loop showed a variable extra-thoracic obstruction. Computed tomography (CT) of the chest revealed that a polypiform nodule of 13 mm in diameter was at the proximal right main bronchus. Testing for purified protein derivative was positive (category 2). The nodule was resected under bronchoscopy. The bronchial aspirate was negative for mycobacterium tuberculosis and tumor cells. The biopsy sample showed a solid and acinar predominant pattern with abundant eosinophilic cytoplasm. The bronchial mucosa was destroyed and replaced by tumor cells. The loose edematous stromal reaction could be seen in a local area. Immunohistochemically, tumor cells were positive for CK, EMA, Vimentin, CD117, CK7, S100, Mammaglobin and SOX10. Only scattered tumor cells were stained by basal cell markers, including CK5/6, P40 and P63. Electron microscopy revealed numerous swelling mitochondria with lacking mitochondrial cristae in tumor cells. Fluorescence in situ hybridization (FISH) testing for MAML2 and ETV6 rearrangement were negative. Next-generation sequencing analysis of 520 genes in the tissue biopsy specimen showed no somatic mutation. The diagnosis of OC was made. Subsequently, the patient underwent a right upper lobectomy with sleeve resection of the main bronchus and lymph dissection. No recurrent evidence was seen during two years of chest CT follow-up. CONCLUSIONS: To our knowledge, this is the first case of primary OC in the bronchus. This patient has no recurrence during two years of follow-up, indicating that primary OC in the bronchus has the same favorable prognosis as in salivary glands. Moreover, complete excision and thorough sampling to know the invasive growth pattern is important to reach the correct diagnosis.

Cardio-oncology in China.

OPINION STATEMENT: Cardio-oncology is going under rapid development in various areas across an increasing number of provinces in China. However there are still a myriad of challenges that need to be overcome in order to ensure its gradual and consistent expansion. The Cardio-Oncology Knowledge Transfer Model (KTM) forms the basis to allow exponential development of effective cardio-oncology services. This would ensure the implementation of precision-based practice while dynamically evolving cardio-oncology to integrate both Western and Chinese medical practices to become an official clinical sub-speciality in its own right in China, for the ultimate benefit of the patient.

Asymmetric Hairpins DNA Encapsulated Silver Nanoclusters for In Situ Fluorescence Imaging of Fusion Gene Isoforms in Bone Marrow.

Rapid and accurate imaging of the BCR/ABL fusion gene isoforms (e.g., e13a2, e14a2 and co-expression type) of chronic myeloid leukemia (CML) is of vital importance to first-line drug selection, but there is no assay that meets clinical needs (e.g., clinical kits > 18 h without isoforms information). Herein, an in situ imaging platform is developed for the rapid and accurate detection of CML fusion gene isoforms using asymmetric sequence-enhanced hairpins DNA encapsulated silver nanoclusters (ADHA) and catalyzed hairpin assembly (CHA). The specific detection of e13a2 and e14a2 fusion gene isoforms with detection limits of 19.2 am (11.558 copies µL-1 ) and 32.56 am (19.601 copies µL-1 ) in one-pot is achieved. The feasibility of the developed assay for real-world applications are demonstrated by one-step fluorescence imaging (40 min) of e13a2, e14a2 and co-expression type in bone marrow quantitatively (International Standard: 15.66%-168.878%) and further validated by cDNA-sequencing. This work suggests that the developed imaging platform holds great potential for rapid identification of the fusion gene isoforms and isoform related treatment monitoring.

Comprehensive analysis of NGS and ARMS-PCR for detecting EGFR mutations based on 4467 cases of NSCLC patients.

BACKGROUND: By comparing the detection rate and type of targeted gene mutations in non-small cell lung cancer (NSCLC) between amplification refractory mutation system PCR (ARMS-PCR) and next-generation sequencing (NGS), the characteristics and application advantages of non-small cell lung cancer detection are explained, providing a basis for clinicians to effectively select the corresponding detection methods. METHODS AND MATERIALS: The cases of targeted genes for lung cancer were selected from the First Affiliated Hospital of Chongqing Medical University from January 2016 to October 2020. A sample of 4467 cases was selected, and they were diagnosed with NSCLC by Pathological biopsy. Sample sources include surgical resection, bronchoscope biopsy, metastatic biopsy, blood, sputum, cytology of pleural effusion. Among them, 3665 cases were detected by ARMS-PCR technique, and 802 cases were detected by NGS technology. The detection rate and type of ARMS-PCR and NGS techniques for EGFR gene mutations (including exon 18, exon 19, exon 20, exon 21 and so on) in different NSCLC samples were compared, respectively. RESULTS: The total mutation rate of EGFR gene detected by ARMS-PCR was 47.6% while 42.4% detected by NGS which indicated that there was a significant difference between the two methods in detecting total mutation of EGFR gene (P < 0.001). In different exons, the EGFR mutation rate detected by two methods is various. The mutation rate of exon 19 by ARMS-PCR detection was evidently higher than that of NGS detection, while the mutation rate of exons 20 and 21 by ARMS-PCR detection were statistically significantly lower than that of NGS detection. Moreover, the multiple mutation rate detected by NGS was 16.3% which was much higher than the 2.7% detected by ARMS-PCR with statistically different. CONCLUSION: It showed that NGS could direct the drug use for the resistant patients. However, some rare loci could be detected by NGS but the importance and directed meaning are still unknown and the number of rare mutations is rare too. Further research on new biomarkers and technique is still needed for early diagnosis, directing drug use and assessing the therapy prognosis.

A Prospective Study of Etiological Agents Among Febrile Patients in Sierra Leone.

INTRODUCTION: Sierra Leone has one of the highest burdens of febrile illnesses in the world. As the incidence of malaria diminishes, a better understanding of the spectrum of etiological agents was important for accurate diagnosis and empirical treatment of febrile illness. METHODS: Blood, nasopharyngeal, and fecal specimens were collected from febrile patients for serological, molecular detection, and microbiologic culture to identify potential pathogens. RESULTS: For this prospective study, 142 febrile patients were enrolled. The prevalence of malaria was higher in children aged 5-15 years old (P = 0.185) and adults (P = 0.018). Acute respiratory infection (ARI) presented more commonly in the under 5 years old group (P = 0.009). For diarrhea, all children groups (P = 0.024) were predominant. A total of 22.5% of the febrile patients had malaria infection, 19.7% had typhoid infection, and 2.8% were coinfected with malaria and typhoid. ARI was the most common causes of fever, accounting for 31.7% of patients, influenza A virus, Mycoplasma pneumoniae, and five other respiratory pathogens were found. Diarrhea accounted for 16.2%, and seven kinds of diarrhea bacteria were isolated. Hepatitis B accounted for 8.5%, including five cases of spontaneous bacterial peritonitis, and ascites smear staining were both Gram-negative bacteria. Tuberculous encephalitis, parasitic diseases (ascaris and filariasis), and skin infection caused by Staphylococcus aureus accounted for 0.7%, 2.1%, and 0.7%, respectively. CONCLUSIONS: Evidence of a wide spectrum of febrile etiological agents other than malaria was identified. The spread of malaria rapid diagnostic tests (RDTs) out of hospital and establishment of a national standard for Widal test will reduce the misdiagnosis of febrile diseases. Antibiotics against Gram-negative bacteria are helpful for empirical treatment.

Sierra Leone has one of the highest burdens of febrile illnesses in the world. Evidence of a wide spectrum of febrile pathogens other than malaria has been proven in this study. We considered that the etiology of febrile patients was closely related to local geography, heredity, immune features, economic industry, living habits, air pollution, medical and health conditions, and this was fully analyzed and discussed. The screening process used in this study can further simplify and identify the etiological agents of fever in more than 70% of the study population. This laid the foundation for the establishment of a more simplified and efficient diagnosis and treatment process in the local area. We also found the characteristics of age distribution of different febrile diseases. Children were an important susceptible population to fever. This study indicated the importance of reliable diagnostic tests for febrile pathogens and provided the necessary information for RDT requirements. The spread of malaria RDTs out of hospital and establishment of a national standard for Widal test will reduce the misdiagnosis of febrile diseases. For empirical treatment, antimalarial treatment was still targeted at falciparum malaria in Sierra Leone. Antibiotics against Gram-negative bacteria contributed to the empirical treatment of febrile diseases. For patients with acute respiratory tract infection, Gram-positive coccal antibiotics could be candidates for treatment. In addition, systematic and professional treatment of liver diseases should be promoted to reduce infection complications.

Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell Lines.

Gliomas are the most common type of primary brain tumor, yet the prognosis for glioma patients remains poor. Mutations in the promoter region of the telomerase reverse transcriptase gene (TERTp) are associated with diagnosis and poor prognosis in gliomas. Here, we developed a precise and rapid Sanger sequencing assay to screen or TERTp mutations. We established the Sanger sequencing approach for the detection of TERTp mutations based on human glioma cell lines U251 and assessed the analytical validation by determining the accuracy, sensitivity, precision, and specificity. In our study, we verified the accuracy of Sanger sequencing by the real-time polymerase chain reaction method. Our data showed that TERTp mutations were detected at an analytical sensitivity of 10% per mutant. The precision and specificity validation also showed the desired results. In total, 147 glioma patients were investigated for TERTp mutations, and of each patient, clinical data and molecular characteristics were analyzed. We found that anaplastic oligodendroglioma had the highest frequency of TERTp mutations (66.7%). No differences in TERTp mutation frequency were observed between frozen tissue specimens and formalin-fixed and paraffin-embedded tissue. TERTp mutations were associated with older patients (≥45 years), whereas isocitrate dehydrogenase (IDH) mutations were inclined to a younger age (<45 years), frontal location, and pathologic stage II-III patients. IDH mutations were significantly associated with O6-methylguanine-DNA methyltransferase (MGMT) methylation (P = 0.003) and lower Ki-67 protein expression (P = 0.011). Moreover, MGMT methylation was enriched in IDH-mutant/TERTp-mutant gliomas, and Ki-67 protein expression was the highest in the IDH-wild type/TERTp-mutant group. Taken together, the findings of this study indicate the establishment of a rapid, precise, and practical Sanger sequencing technique for TERTp mutations in gliomas that may show promising results in clinical applications.

Highly Sensitive Detection of Low-Abundance BRAF V600E Mutation in Fine-Needle Aspiration Samples by Zip Recombinase Polymerase Amplification.

Papillary thyroid carcinoma (PTC) is the most common thyroid cancer with high incidence in endocrine tumors, which emphasizes the significance of accurate diagnostics. Still, the commonly used cytological method (fine-needle aspiration (FNA) cytology) and molecular diagnostic methods (such as PCR and sequencing) are limited in terms of diagnostic time, sensitivity, and user-friendliness. In this study, we introduce a novel Zip recombinase polymerase amplification (Z-RPA) strategy to efficiently detect rare mutant alleles in PTC fine-needle aspiration samples, which is sensitive, fast, and simple to manipulate. Using Zip nucleic acid (ZNA) probes to clamp the mutation region, the phi 29 polymerase could selectively displace mismatched ZNA probes and start amplification, while leaving complementary ZNA probes untouched and blocking amplification according to genotype. We demonstrated the good sensitivity and specificity of this strategy with optimized conditions and design, which enabled detection of BRAF V600E mutation in a total 4 ng of genomic DNA within 40 min (≈1 copy). Robust behavior in clinical specimen analysis was also demonstrated. The Z-RPA strategy provides a pragmatic approach to rapidly, sensitively, and easily detect BRAF V600E mutation in clinical fine-needle aspiration samples, which is a promising method for early cancer diagnosis and treatment guideline.

Effect of rebiopsy methods and clinical features on T790M mutation after first-line EGFR-TKI treatment failure.

Aim: This study investigated the association between clinical data and T790M mutation in rebiopsy after EGFR tyrosine kinase inhibitors (EGFR-TKIs) failure, and explored the prognosis of T790M-positive patients. Methods: Patients with non-small-cell lung cancer undergoing rebiopsy after first-generation TKI failure were reviewed. Results & conclusion: Patients with brain metastases, negative TP53, initial 19del and longer initial PFS had higher positive rate of T790M. The median progression-free survival (PFS) of T790M-positive patients with cytology and tissue rebiopsy were longer than patients with liquid rebiopsy. The median PFS of T790M-positive patients rebiopsied by ordinary bronchoscope and endobronchial ultrasound-guided transbronchial lung biopsy with a guided sheath (EBUS-GS-TBLB) were longer than that of the patients rebiopsied by EBUS transbronchial needle aspiration (TBNA).

Lay abstract A specific genetic mutation, T790M, is the main cause of drug resistance in patients with lung cancer receiving a type of drug called EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib. We explored the factors that influence the prognosis of T790M-positive patients and how this mutation causes resistance. We found a number of biomarkers that are linked to higher expression of T790M, including brain metastases and longer initial treatment period of EGFR-TKI. The median length of time before tumors started progressing in T790M-positive patients who underwent cytology and tissue rebiopsy was longer than patients who received a blood biopsy. The length of time before progression in patients undergoing different rebiopsy methods (such as ordinary bronchoscope and endobronchial ultrasound bronchoscope) was different.

Comprehensive evaluation of risk factors for lymph node metastasis in patients with papillary thyroid carcinoma.

With the increasing incidence of papillary thyroid cancer (PTC), it is important to risk-stratify patients who may have a more aggressive tumor biology. The present study aimed to evaluate the risk factors for lymph node metastasis (LNM) in patients with PTC, which may provide a significant reference for clinical diagnosis and treatment. In total, 1,045 patients with PTC [313 with PT microcarcinoma (PTMC) and 732 with non-PTMC] between August 2016 and August 2019 were investigated. The B-type Raf kinase (BRAF) V600E mutation was tested in all samples. The clinical data (sex, age, tumor location, sample type and pathological features) were retrospectively analyzed. Logistic regression analysis was performed to evaluate independent risk factors for LNM. A total of 181/313 (57.8%) PTMC cases and 145/732 (19.8%) non-PTMC cases had a BRAF V600E mutation. In the PTMC cases, significant differences in sex and sample type were identified (BRAF V600E mutation vs. wild-type). In the non-PTMC cases, significant differences in sex and age were identified (BRAF V600E mutation vs. wild-type). Female sex and tumor diameter ≤1 cm were significant independent predictors of LNM in PTC. In PTMC, female sex was a significant independent predictor of LNM. A bilateral tumor was an independent protective factor for LNM in PTC, PTMC and non-PTMC. The BRAF V600E mutation rate of ultrasound-guided fine-needle aspiration cytology was higher compared with FFPE in PTMC (P=0.018). In contrast to previous studies, the results of the present study suggested that being female and having a tumor of diameter ≤1 cm were risk factors for LNM, and that the BRAF wild-type of PTMC may be more aggressive than other types. Notably, the position of the tumor in the bilateral thyroid was also an independent protective factor for LNM. Therefore, ultrasound-guided fine-needle aspiration should be recommended for gene analysis (BRAF V600E) in PTMC. In addition, clinicians should consider an individualized treatment according to gene mutations, sex, age, tumor size and the location of the tumor, in order to achieve an improved therapeutic efficacy.

Application of large-scale targeted sequencing to distinguish multiple lung primary tumors from intrapulmonary metastases.

The effective differentiation between multiple primary lung tumors (MPs) and intrapulmonary metastases (IMs) in patients is imperative to discover the exact disease stage and to select the most appropriate treatment. In this study, the authors was to evaluate the efficacy and validity of large-scale targeted sequencing (LSTS) as a supplement to estimate whether multifocal lung cancers (MLCs) are primary or metastatic. Targeted sequencing of 520 cancer-related oncogenes was performed on 36 distinct tumors from 16 patients with MPs. Pairing analysis was performed to evaluate the somatic mutation pattern of MLCs in each patient. A total of 25 tumor pairs from 16 patients were sequenced, 88% (n = 22) of which were classified as MPs by LSTS, consistent with clinical diagnosis. One tumor pair from a patient with lymph node metastases had highly consistent somatic mutation profiles, thus predicted as a primary-metastatic pair. In addition, some matched mutations were observed in the remaining two paired ground-glass nodules (GGNs) and classified as high-probability IMs by LSTS. Our study revealed that LSTS can potentially facilitate the distinction of MPs from IMs. In addition, our results provide new genomic evidence of the presence of cancer invasion in GGNs, even pure GGNs.

Tuning the specificity of DNA probes using bulge-loops for low-abundance SNV detection.

Tuning the free energy difference between a molecular probe and the target has been regarded as a feasible way to realize selective mutant recognition. But due to limited extent of variation on the probing sequences, it remains a challenge to moderately leverage the thermodynamic kinetics simply by changing the base composition of probes. Herein we propose the modulation of discrimination capability for single nucleotide variations (SNVs) detection by insertion of bulge-loop into duplex DNA probes. Based on controllable tuning of free energy change (ΔG) before and after strand exchange with either mutated or wild-type DNAs, much higher specificity than conventional linear probes is obtained. As-proposed bulge-loop probes allows excellent discrimination of SNVs in high guanine and cytosine (GC) rich regions, and reaches a detection limit of 0.02% abundance with down to 2 femtomolar target gene. The probes also demonstrate excellent consistence with droplet digital PCR (ddPCR) in identifying low abundant L858R mutant in lung tissue samples that are not resolved by either a commercial PCR kit or Sanger sequencing. Our work not only provides insight into the rational design of strand exchange probes for point-of-care diagnosis but also advance the construction of customizable cascade reactions in dynamic DNA nanotechnology more broadly.

One-step discrimination of BCR/ABLp210 transcript isoforms directly from RNA extraction with fusion-triggered rolling circle amplification.

BCR/ABLp210 fusion gene, the characteristic biomarker of chronic myelogenous leukemia (CML), contains two different transcription isoforms, e13a2 and e14a2, which lead to differences in the pathological features and response to targeted drug. At present, there is short of simple and fast technology to distinguish these two transcript isoforms. In this paper, RNA fusion-triggered rolling circle amplification (RF-RCA) strategy was developed to distinguish e13a2 and e14a2 transcripts directly from RNA extraction in one step. The simultaneous binding of dumbbell template and corresponding primer with target fused RNA can induce their proximal hybridization and trigger the RCA to produce lots of tandem repeat G-quadruplexes sequences for real time fluorescence readout with the interaction of Thioflavin T and G-quadruplex. The proposed strategy can detect as low as 0.1 aM target and discriminate e13a2 (0.01%) and e14a2 (0.1%) transcript isoforms directly from complex genomic RNA extraction, proving high sensitivity and specificity. Furthermore, the RF-RNA was successfully applied to analyze BCR/ABLp210 isoforms from clinical samples for accurately molecular subtyping and monitoring the response of imatinib treatment. The developed RF-RCA strategy presented an ultrasensitive, accurate and pragmatic toolbox to simple and rapid discriminate BCR/ABLp210 fusion isoforms for promoting clinical research and personalized treatment of CML.

Engineered zinc-finger transcription factors inhibit the replication and transcription of HBV in vitro and in vivo.

In the present study, an artificial zinc-finger transcription factor eukaryotic expression vector specifically recognizing and binding to the hepatitis B virus (HBV) enhancer (Enh) was constructed, which inhibited the replication and expression of HBV DNA. The HBV EnhI­specific pcDNA3.1­artificial transcription factor (ATF) vector was successfully constructed, and then transformed or injected into HepG2.2.15 cells and HBV transgenic mice, respectively. The results demonstrated that the HBV EnhI (1,070­1,234 bp)­specific ATF significantly inhibited the replication and transcription of HBV DNA in vivo and in vitro. The HBV EnhI­specific ATF may be a meritorious component of progressive combination therapies for eliminating HBV DNA in infected patients. A radical cure for chronic HBV infection may become feasible by using this bioengineering technology.

Structural and functional characterization of Escherichia coli toxin-antitoxin complex DinJ-YafQ.

Toxin YafQ functions as a ribonuclease in the dinJ-yafQ toxin-antitoxin system of Escherichia coli. Antitoxin DinJ neutralizes YafQ-mediated toxicity by forming a stable protein complex. Here, crystal structures of the (DinJ)2-(YafQ)2 complex and the isolated YafQ toxin have been determined. The structure of the heterotetrameric complex (DinJ)2-(YafQ)2 revealed that the N-terminal region of DinJ folds into a ribbon-helix-helix motif and dimerizes for DNA recognition, and the C-terminal portion of each DinJ exclusively wraps around a YafQ molecule. Upon incorporation into the heterotetrameric complex, a conformational change of YafQ in close proximity to the catalytic site of the typical microbial ribonuclease fold was observed and validated. Mutagenesis experiments revealed that a DinJ mutant restored YafQ RNase activity in a tetramer complex in vitro but not in vivo. An electrophoretic mobility shift assay showed that one of the palindromic sequences present in the upstream intergenic region of DinJ served as a binding sequences for both the DinJ-YafQ complex and the antitoxin DinJ alone. Based on structure-guided and site-directed mutagenesis of DinJ-YafQ, we showed that two pairs of amino acids in DinJ were important for DNA binding; the R8A and K16A substitutions and the S31A and R35A substitutions in DinJ abolished the DNA binding ability of the DinJ-YafQ complex.

The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein-protein interfaces.

Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow-brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein-protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners.

Structural basis of the novel S. pneumoniae virulence factor, GHIP, a glycosyl hydrolase 25 participating in host-cell invasion.

Pathogenic bacteria produce a wide variety of virulence factors that are considered to be potential antibiotic targets. In this study, we report the crystal structure of a novel S. pneumoniae virulence factor, GHIP, which is a streptococcus-specific glycosyl hydrolase. This novel structure exhibits an α/ß-barrel fold that slightly differs from other characterized hydrolases. The GHIP active site, located at the negatively charged groove in the barrel, is very similar to the active site in known peptidoglycan hydrolases. Functionally, GHIP exhibited weak enzymatic activity to hydrolyze the PNP-(GlcNAc)5 peptidoglycan by the general acid/base catalytic mechanism. Animal experiments demonstrated a marked attenuation of S. pneumoniae-mediated virulence in mice infected by ΔGHIP-deficient strains, suggesting that GHIP functions as a novel S. pneumoniae virulence factor. Furthermore, GHIP participates in allowing S. pneumoniae to colonize the nasopharynx and invade host epithelial cells. Taken together, these findings suggest that GHIP can potentially serve as an antibiotic target to effectively treat streptococcus-mediated infection.

[Prokaryotic expression, purification and identification of recombinant staphylokinase-HC fusion protein].

AIM: To construct the prokaryotic expression plasmid pEGX-6P-1-SAK-HC, express it in E.coli, and identify its biological activity. METHODS: The fusion gene (SAK-HC) was obtained by overlap-extension PCR and then inserted into prokaryotic soluble pEGX-6P-1 vector with GST tag to construct expression plasmid (pEGX-6P-1-SAK-HC). GST-SAK-HC was expressed by E.coli B834 (DE3) under the induction of IPTG and purified by Glutathion-Sepharose 4B (GST) affinity chromatography and negative-ion exchange column (DEAE) chromatography. PreScission protease was used to remove the GST tag. The purity of the fusion protein was analyzed by SDS-PAGE and the fibrinolytic activity of SAK-HC in vitro was characterized by soluble fibrin plate method. RESULTS: PCR, sequencing and restriction enzyme digestion analysis demonstrated that the recombinant plasmid was constructed successfully. The fusion protein was expressed in E.coli B834 (DE3), M(r); being 36 000 as shown by SDS-PAGE. After purified by GST affinity and DEAE chromatography, SAK-HC fusion protein of high purity was obtained from the cell supernantants. In vitro experiments showed that the fibrinolytic activity of the recombinant SAK-HC was about 9.4×10();4 IU/mg. CONCLUSION: The SAK-HC fusion protein we obtained was successfully expressed in E.coli and exhibited a fibrinolytic activity as high as the urokinase standard, which offers a base for the identification of immunogenicity of the fusion protein.

Cloning, purification, crystallization and preliminary X-ray studies of human α1-microglobulin.

α(1)-Microglobulin (α(1)m) is one of the phylogenetically most widespread lipocalins and is distributed in various organs and tissues, including liver, heart, eye, kidney, brain, lung, pancreas and skeletal muscle. α(1)m has been found to exert multifarious functions, including interacting with IgA, albumin and prothrombin, binding strongly to haem and exhibiting reductase activity. Nevertheless, little structural information is available regarding these functions of α(1)m. Since determination of three-dimensional structure is a powerful means of functional characterization, X-ray crystallography was used to accomplish this task. Here, the expression, purification, crystallization and preliminary crystallographic analysis of human α(1)m are reported. The crystal belonged to space group P4(3), with unit-cell parameters a = b = 36.45, c = 112.68 Å, and diffracted to a resolution of 2.0 Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a V(M) value of 1.63 Å(3) Da(-1).

[Predictive study of HBsAg in different stages of neonatal venous blood on failure of blocking HBV mother to infant transmission].

OBJECTIVE: In this study, we discuss the predictive value of different content of HBsAg in different stages of neotal venous blood on failure of blocking mother to infant transmission of HBV. METHODS: 150 infants born of chronically HBV infected mothers who were positive of both HBsAg and HBeAg and who also had a HBV DNA virus load above 10(5) copies/ml were enrolled. These infants were given hepatitis B virus immune globin (HBIG) 200 IU immediately after birth and were given hepatitis B vaccine 10 or 20 microg at brith, 1 month and 6 months after birth. HBV serological index of these infants were test at birth, 1 month and 7 months after birth respectively. Different content of HBsAg in different stages of neonatal venus blood were analyzed to predict the failure of blocking mother to infant transmission of HBV. RESULTS: 11 infants failed in blocking of HBV mother to infant transmission. The positive rate of HBsAg at birth, 1 month and 7 months after birth were 41.26%, 10.49% and 7.69% respectively, and were 97.90%, 65.73% and 13.29% of HBeAg. The positive predictive value of HBsAg > or = 0.05 and HBsAg > or = 1 IU/ml at birth were 18.64% and 70% respectively, and were 73.33% and 100% one month after birth. CONCLUSIONS: Infants with HBsAg > or = 1 IU/ml at birth should be suspicious of failure on blocking HBV mother-to-infant transmission and it should be more credible if the infant has HBsAg > or = 1 IU/ml one month after birth. How to improve the blocking rate of neonates who were positive of HBsAg at birth and one month after birth should be the focus of our future research.