Effects of Site-Directed Mutagenesis of Cysteine on the Structure of Sip Proteins.

Bacillus thuringiensis, a gram-positive bacteria, has three insecticidal proteins: Vip (vegetative insecticidal protein), Cry (crystal), and Sip (secreted insecticidal protein). Of the three, Sip proteins have insecticidal activity against larvae of Coleoptera. However, the Sip1Aa protein has little solubility in the supernatant because of inclusion bodies. This makes it more difficult to study, and thus research on Sip proteins is limited, which hinders the study of their mechanistic functions and insecticidal mechanisms. This highlights the importance of further investigation of the Sip1Aa protein. Disulfide bonds play an important role in the stability and function of proteins. Here, we successfully constructed mutant proteins with high insecticidal activity. The tertiary structure of the Sip1Aa protein was analyzed with homologous modeling and bioinformatics to predict the conserved domain of the protein. Cysteine was used to replace amino acids via site-directed mutagenesis. We successfully constructed Sip149-251, Sip153-248, Sip158-243, and Sip178-314 mutant proteins with higher solubility than Sip1Aa. Sip153-248 and Sip158-243 were the most stable compared to Sip1Aa, followed by Sip149-251 and Sip178-314. The insecticidal activity of Sip153-248 (Sip158-243) was 2.76 (2.26) times higher than that of Sip1Aa. The insecticidal activity of Sip149-251 and Sip178-314 did not differ significantly from that of Sip1Aa. Basic structural properties, physicochemical properties, and the spatial structure of the mutation site of Sip1Aa and the mutant proteins were analyzed. These results provide a molecular basis for using Sip1Aa to control Coleopteran insects and contribute to the study of the Sip1Aa insecticidal mechanism.

Isolation and Identification of Pseudomonas sp. Strain DY-1 from Agricultural Soil and Its Degradation Effect on Prometryne.

Prometryne is a widely used herbicide in China to control annual grasses and broadleaf weeds. However, the stability of prometryne makes it difficult to be degraded, which poses a threat to human health. This study presents a bacterial strain isolated from soil samples with a prometryne application history, designated strain DY-1. Strain DY-1, identified as Pseudomonas sp., is capable of utilizing prometryne as a sole carbon source for growth and degrading 100% of prometryne within 48 h from an initial concentration of 50 mg L-1. To further optimize the degradation of prometryne, the prometryne concentration, temperature, pH, and salt concentration were examined. The optimal conditions for degradation of prometryne by strain DY-1 were an initial prometryne concentration of 50 mg L-1, 30 °C, pH 7-8, and NaCl concentration of 200 mg L-1. The same strain also degraded other s-triazine herbicides, including simetryne, ametryne, desmetryne, and metribuzin, under the same conditions. The biodegradation pathway of prometryne was established by isolating sulfoxide prometryne as the first metabolite and by the identification of sulfone prometryne and 2-hydroxy prometryne by liquid chromatography-mass spectrometry (LC-MS/MS). The results illustrated that strain DY-1 achieved the removal of prometryne by gradually oxidizing and hydrolyzing the methylthio groups. A bioremediation trial with contaminated soil and pot experiments showed that after treating the prometryne-contaminated soil with strain DY-1, the content of prometryne was significantly reduced (P < 0.05). This study provides an efficient bacterial strain and approach that could be potentially useful for detoxification and bioremediation of prometryne analogs.

Performance of the Xpert® MTB/RIF assay in the rapid diagnosis of tracheobronchial tuberculosis using bronchial washing fluid.

OBJECTIVE: To assess the diagnostic value of the Xpert® MTB/RIF (GeneXpert) assay for tracheobronchial tuberculosis (TBTB) using bronchial washing fluid (BWF). METHODS: This retrospective study enrolled patients suspected of having TBTB and patients with non-TB pulmonary disease as controls. BWF were used to undertake acid-fast bacillus (AFB) smears, the GeneXpert assay and the LÓ§wenstein-Jensen (LJ) culture method. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were compared among BWF AFB smears, BWF GeneXpert and the BWF LJ culture method. RESULTS: A total of 130 patients with TBTB and 102 patients with non-TB pulmonary disease were enrolled in the study. Sputum AFB smears were positive in 62 of 130 patients (47.7%) with TBTB. Using the clinical diagnosis of TBTB as the gold standard, the sensitivity, specificity, PPV and NPV of the three methods using BWF were as follows: 93.1%, 99.0%, 99.2% and 91.8% for BWF GeneXpert; 73.1%, 100.0%, 100.0% and 74.5% for BWF LJ cultures; 53.8%, 99.0%, 98.6% and 62.7% for BWF AFB smears. The diagnostic yield of BWF GeneXpert was significantly higher compared with BWF cultures for type III and IV TBTB. CONCLUSION: The Xpert® MTB/RIF assay using BWF exhibited higher sensitivity than bacteriological diagnostic methods and was particularly useful for the early diagnosis of smear-negative TBTB.

In silico Structure-Based Investigation of Key Residues of Insecticidal Activity of Sip1Aa Protein.

Colaphellus bowringi Baly mainly damages cruciferous vegetables, leading to huge economic losses. The secretory insecticidal protein (Sip) of Bacillus thuringiensis (Bt) has high insecticidal activity against C. bowringi Baly. The tertiary structure of Sip1Aa protein was analyzed by homologous modeling and other bioinformatics methods to predict the conserved domain of Sip1Aa protein. Acidic and basic amino acids in the conserved domain were selected, and alanine was used to replace these amino acids by site-directed mutation. The difference between the insecticidal activities of mutant protein and Sip1Aa protein was analyzed. The insecticidal activities of H99A, K109A, K128A, and E130A against C. bowringi Baly were significantly increased, among which that of K128A was the most obviously changed, and the LC50 value was decreased by about 10 times compared with that of Sip1Aa protein. The LC50 value of mutant E130A was 0.286 µg/mL, which was about six times less than that of Sip1Aa. K128 and E130 were both in the ß9-ß10 loop. The toxicity of D290A, H242A, and H303A to C. bowringi Baly was significantly reduced, and their LC50 value increased by about six, eight, and three times compared with that of Sip1Aa protein, respectively. This study showed that acidic and basic amino acid residues played a certain role in the toxicity of Sip1Aa protein, and the loss of side chains in key residues had a significant impact on the insecticidal activity of the protein. This study provides the theoretical basis for revealing the relationship between the structure and function of Sip1Aa protein and also provides a new method for the subsequent study of sip gene.

[Development of a simultaneous strain and temperature sensor with small-diameter FBG].

Manufacture of the small diameter FBG was designed. Cross sensitivity of temperature and strain at sensing point was solved. Based on coupled-mode theory, optical properties of the designed FBG were studied. The reflection and transmission spectra of the designed FBG in small diameter were studied A single mode optical fiber, whose cladding diameter is 80 microm, was manufactured to a fiber Bragg grating (phi80FBG). According to spectrum simulation, the grating length and period were chosen as the wavelength was 1528 nm. The connector of the small diameter FBG with demodulation was designed too. In applications, the FBG measures the total deformation including strain due to forces applied to the structures as well as thermal expansion. In order to overcome this inconvenience and to measure both parameters at the same time and location, a novel scheme for simultaneous strain and temperature sensor was presented. Since the uniform strength beam has same deformation at all points, a pair of phi80 FBG was attached on a uniform strength cantilever. One of the FBG was on the upper surface, with the other one on the below. Therefore, the strains at the monitoring points were equal in magnitude but of opposite sign. The strain and temperature in sensing point could be discriminated by matrix equation. The determination of the K is not null and thus matrix inversion is well conditioned, even the values for the K elements are close. Consequently, the cross sensitivity of the FBG with temperature and strain can be experimentally solved. Experiments were carried out to study the strain discriminability of small-diameter FBG sensors. The temperature and strain were calculated and the errors were, respectively, 5% and 6%.