Evaluation of pretreatment methods for filamentous fungal detection.

In order to obtain the best mass spectrometry identification results for using the most appropriate methods in clinical practice, we explore the optimal pretreatment methods for different species and morphologies of filamentous fungi. 98 fungal strains were treated with formic acid sandwich method, dispersion method, extraction method, and other methods using a medium element mass spectrometer (EXS3000) as a platform. Each strain had three targets, and the identification rates and confidence differences under different pre-treatment methods were compared to evaluate the identification effects of these methods. The mass spectrometry identification rates of 98 filamentous fungi obtained after pre-treatment with formic acid sandwich method, dispersion method, and extraction method were 85.71%, 82.65%, and 75.51%, respectively. The identification rate of the formic acid sandwich method was significantly higher than the other two methods (P < 0 005) has the best identification ability and the obtained confidence is also higher than the other two methods. The use of formic acid sandwich method for mass spectrometry identification of filamentous fungi can achieve ideal identification results, which is suitable for mass spectrometry identification of filamentous fungi in conventional laboratories.

Emergence of KPC-134, a KPC-2 variant associated with ceftazidime-avibactam resistance in a ST11 Klebsiella pneumoniae clinical strain.

The emergence of various new Klebsiella pneumoniae carbapenemase (KPC) variants leading to ceftazidime-avibactam treatment failure is a new challenge in current clinical anti-infection treatment. Here, we report a ceftazidime-avibactam-resistant K. pneumoniae 1072-2 clinical strain carrying a novel KPC variant, KPC-134, which differs from KPC-2 by both single mutation (D178A) and 8-amino acid insertions (asp-asp-asn-arg-ala-pro-asn-lys). The results of antimicrobial susceptibility testing showed that the isolate was resistant to meropenem (MIC = 4 mg/L), ceftazidime (MIC ≥ 32 mg/L), cefepime (MIC ≥128 mg/L), aztreonam (MIC ≥128 mg/L), and ceftazidime-avibactam (MIC ≥128 mg/L) but sensitive to imipenem (MIC = 0.5 mg/L), imepenem-relebactam (MIC = 0.5 mg/L), meropenem-vaborbactam (MIC = 2 mg/L), and aztreonam-avibactam (MIC = 4 mg/L). The plasmid containing blaKPC-134 was isolated from K. pneumoniae, and the blaKPC-134 gene was cloned into plasmid pHSG398 and transformed into an Escherichia coli DH5α to observe changes in antimicrobial resistance. The results indicated that the transformant was positive for blaKPC-134 and increased MICs of ceftazidime-avibactam, ceftazidime, cefepime, and aztreonam by 512-fold, 256-fold, 16-fold, and 4-fold, respectively, compared with the recipient. The results of third-generation sequencing showed that the blaKPC-134 gene was carried by a 133,789 bp IncFII-IncR plasmid, and many common resistance genes (including blaCTX-M-65, blaTEM-1B, blaSHV-12, rmtB, and catB4) along with the IS26, tnpR, ISkpn8, ISkpn6-like, and Tn1721 elements were identified. IMPORTANCE The emergence of various new KPC variants leading to ceftazidime-avibactam treatment failure is a new challenge for clinical anti-infection treatment. Here, we describe the characterization of a ceftazidime-avibactam-resistant blaKPC-134-positive Klebsiella pneumoniae clinical strain for the first time. K. pneumoniae bearing with KPC variant often mislead clinical anti-infection treatment because of their unique antimicrobial susceptibility profile and the tendency of conventional carbapenemase assays to give false negative results. Therefore, timely identification of KPC variants and effective anti-infective therapy are key to saving infected patients.

Direct common gram-negative bacterial identification from positive blood culture bottles by SELDI-TOF MS.

A protein database was constructed and validated with identification rate over 90% for the 4 most common Gram-negative bacteria on agar plates. By protein masses comparison, 120 bacteria of the 4 species from blood culture bottles were identified. The concordance was high (Kappa=0.906) between our method and conventional approach.

[Establishment of protein fingerprint database of Salmonella paratyphi A using SELDI-TOF-MS].

AIM: To establish a protein fingerprint database of Salmonella paratyphi A by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). METHODS: Thirty-six clinical bacterial isolates and 96 control bacteria isolates were collected and identified using 16S rDNA sequencing. Bacterial proteins were detected by SELDI-TOF-MS, and all protein fingerprints were analyzed by ProteinChip and Biomarker Wizard software. The analysis results were used to set up a classification tree model by means of BioMarker Patterns software. At the same time, the data were tested by a blinded validation. RESULTS: In the range of M(r); 3 000-20 000, we obtained 104 protein peaks, of which 90 were of statistical significance (P<0.01). A protein peak with mass-to-charge ratio(M/Z) 10 061.7 was chosen to establish the classification tree model of Salmonella paratyphi A, and the sensitivity and specificity of Salmonella paratyphi A diagnosis was 100% as shown by the blinded validation. CONCLUSION: The classification tree model of Salmonella paratyphi A can be not only established using SELDI-TOF-MS technology, but also used for the rapid identification of Salmonella paratyphi A.

Development of a method based on surface enhanced laser desorption and ionization time of flight mass spectrometry for rapid identification of Klebsiella pneumoniae.

A method based on surface enhanced laser desorption and ionization time of flight mass spectrometry (SELDI-TOF MS) was developed for the rapid identification of Klebsiella pneumoniae by directly applying bacterial colonies without further protein extraction. A total of 40 K. pneumoniae and 114 other related microorganisms isolated clinically were analyzed by SELDI-TOF MS. An identification model for K. pneumoniae was established by artificial neural networks (ANNs) with classification accuracy of 100%. The model was blindly tested with 43 K. pneumoniae and 53 control bacteria again. The results showed that the model was successful with accuracy of 96.9%, sensitivity of 100% and specificity of 943%. This strategy is potential for rapid identification of K. pneumoniae.

Mechanism of simvastatin-induced K562 cell apoptosis.

Statins are being widely used for the therapy and prevention of several types of tumors, including human chronic myelogenous leukemia, but the underlying molecular mechanisms still remain unknown. Therefore, inhibition of cell proliferation, apoptosis and involved molecules were investigated in K562 cells after incubation with simvastatin.The results showed that simvastatin diminished K562 cell proliferation and induced apoptosis. At the same time, the level of reactive oxygen species (ROS) and intracellular calcium concentration increased. Furthermore, nitric oxide (NO) content and inducible NO synthase (iNOS) mRNA expression were significantly higher in the simvastatin-treated group than in the corresponding control group. The elevated ROS level and intracellular calcium concentration, enhanced mRNA expression of iNOS and total NO content might be responsible for the apoptotic and anti-proliferative effects of simvastatin in K562 cells.

Rapid identification of Staphylococcus aureus by surface enhanced laser desorption and ionization time of flight mass spectrometry.

Staphylococcus aureus (S. aureus), a vital nosocomial pathogen, is responsible for several diseases. With the increasing isolation rate in clinical specimens, rapid identification of this bacterial species is required. But present identification via conventional methods is time-consuming and lacks accuracy. The purpose of the current study was to evaluate the use of surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) for rapid identification of S. aureus. A total of 120 clinical isolates of S. aureus and 153 non-S. aureus species were identified by conventional methods, and the species nature of all staphylococci was further confirmed by 16S rDNA sequencing. All strains observed were analyzed by SELDI-TOF MS. An identification model for S. aureus was developed and validated by an artificial neural network. The model based on 6 protein peaks exhibited a sensitivity of 98.4% and specificity of 98.6%. This strategy has the potential for rapid identification of S. aureus.

Preparation and identification of scFv and bsFv against transferrin receptor.

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.

In vitro and in vivo study of cell growth inhibition of simvastatin on chronic myelogenous leukemia cells.

BACKGROUND: Statins, a family of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase inhibitors, are being investigated for the therapy and prevention of cancers. Here we aimed to investigate the effects of simvastatin on chronic myelogenous leukemia (CML) cells in vitro and in vivo, and to elucidate the mechanisms. METHODS: Cell proliferation and cell cycle were measured after K562 cells were incubated with simvastatin, and differentially expressed genes were determined by oligonucleotide microarray. Changes of 2 genes obtained by oligonucleotide microarray were validated by real-time RT-PCR, and immunohistochemistry was performed to determine expression of proliferating cell nuclear antigen (PCNA). Finally, a xenograft tumor model was constructed to evaluate the effects of simvastatin in vivo. RESULTS: Simvastatin could inhibit K562 cell proliferation, and the inhibition rate was approximately 30% after treatment with 20 mumol/l simvastatin for 48 h. Cell cycle was arrested in G(1) phase, as shown by flow cytometry results. Fifteen downregulated, 9 upregulated cell cycle-related genes and decreased PCNA protein were observed in the presence of simvastatin. Furthermore, simvastatin exhibited impairment of xenograft tumor growth in nude mice and also blocked cell cycle in G(1) phase. CONCLUSION: Simvastatin can inhibit CML cell proliferation in vitro and in vivo, and its mechanisms might be involved in cell cycle regulation.

Preparation and Identification of scFv and bsFv against Transferrin Receptor / 华中科技大学学报（医学）（英德文版）

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complemenary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease Sill, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB 1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively.Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library.

AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.