Magnetosomes eliminate intracellular reactive oxygen species in Magnetospirillum gryphiswaldense MSR-1.

Magnetotactic bacteria synthesize magnetic particles called magnetosomes that cause them to orient to their external magnetic fields. However, the physiological significance and other possible functions of these magnetosomes have not been explored in detail. In this study, we have investigated the biological functions of magnetosomes with respect to their ability to scavenge reactive oxygen species (ROS) in Magnetospirillum gryphiswaldense MSR-1. To assess the changes in ROS levels under different conditions, cells were cultured under aerobic or micro-aerobic conditions in medium containing high and low amounts of iron. To ensure that the observed results were not due to nonspecific interactions, reactions were carried out using a mutant deficient in synthesizing magnetite (mamO-deficient mutant), its complementary strain or the wild-type MSR-1. We observed that the levels of intercellular ROS under micro-aerobic conditions with high-iron medium were much higher when the non-synthetic Fe(3) O(4) crystals mutant Mu21-415 was employed for the assay, compared with the wild-type or complementary strain, or when conditions were aerobic with low-iron medium. These results indicated that magnetosomes function in the scavenging of intracellular ROS. Furthermore, we have demonstrated that the magnetosomes exhibit peroxidase-like properties, by using the earlier reported in vitro horseradish peroxidase assay for artificial magnetic nanoparticles. In addition to possessing peroxidase-like activity, the magnetosomes also exhibited a more enzymatic kinetic response, suggesting that proteins on the membranes of the magnetosomes likely contribute to the enzymatic activity. This is the first study to demonstrate that magnetosomes play an important role in decreasing or eliminating ROS.

High-sensitivity detection of fruit tree viruses using bacterial magnetic particles.

Prunus necrotic ring spot virus (PNRSV) and grapevine fanleaf virus (GFLV) were detected by fluoroimmunoassay using bacterial magnetic particles (BMPs), and a double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). For the fluoroimmunoassay, fluorescein isothiocyanate labeled anti-PNRSV antibody or anti-GFLV antibody was conjugated onto BMPs of Magnetospirillum gryphiswaldense MSR-1. With this method, a very low minimum antigen concentration (1 x 10(6) dilution of the original sample concentration) could be detected. Using DAS-ELISA, the minimum antigen detection concentration was the original sample concentration. Thus, comparing these two methods, a BMP-based method could increase the sensitivity up to six orders of magnitude (10(6)) higher than an ELISA-based method of detection PNRSV and GFLV.

A new technology for reducing anastomotic fistula in the neck after esophageal cancer surgery.

BACKGROUND: To explore the risk factors and prevention methods of cervical mechanical anastomotic fistula and stenosis after the radical resection of esophageal cancer. METHODS: From March 2018 to November 2018, 128 patients undergoing mechanical anastomosis of esophageal cancer were selected from the Department of Thoracic Surgery of The First Affiliated Hospital of Zhengzhou University. All the enrolled patients were operated on using the Mckeown method, and a retrospective study was conducted. Data for preoperative and postoperative test indices, intraoperative embedding materials, postoperative complications, and preoperative and postoperative treatment were collected, and the relationship between various factors and the incidence of cervical anastomotic fistula and stenosis was analysed. Univariate analysis was conducted using t tests or Fisher's exact probability method, and multivariate analysis was conducted using logistic regression models. RESULTS: All 128 patients successfully underwent surgery without dying. The enrolled patients were evaluated using the Stooler classification, with 28 patients having grade 0, 41 patients having grade 1, 34 patients having grade 2, 21 patients having grade 3, and 4 patients having grade 4 stenosis. Patients with stenosis of grade 3 or above had obvious choking sensation, which could only be relieved by balloon dilation. Symptoms in all patients with stenosis were relieved by balloon dilation. There were no significant differences between the two groups regarding embedding materials, preoperative choking history, history of alcohol consumption, history of hypertension, history of coronary heart disease, history of diabetes, postoperative calcium concentration, average albumin concentration, average platelet concentration, body mass index, anastomotic fistula, preoperative chemotherapy, postoperative chemotherapy, or postoperative cough (P>0.05). There were significant differences in postoperative reflux (χ2=11.338, P<0.05) and scar constitution (χ2=12.497, P<0.05). The effects of embedding materials in patients with anastomotic fistula were significantly different (χ2=4.372, P<0.05). CONCLUSIONS: Postoperative reflux and scar constitution may be risk factors for postoperative anastomotic stenosis after resection of esophageal cancer. There was almost no difference in the effects on esophageal anastomotic stenosis between embedding materials and the omentum majus, but Neoveil® may have certain advantages in preventing cervical anastomotic fistula, and thus may have certain clinical application value.

Isolation of a flagellar operon in Azospirillum brasilense and functional analysis of FlbD.

A 10 kb fragment containing fliF, fliH, fliN, motA, flbD, flhA, flhF and fleN genes was cloned from the genomic DNA of Azospirillum brasilense Yu62. These eight genes appear to be structurally organized as an operon. FlbD, encoded by flbD, has a HTH DNA binding domain and shows homology to sigma(54)-dependent transcriptional activators such as NtrC, NifA and DctD. An in-frame deletion of flbD in A. brasilense abolishes biosynthesis of lateral flagella and swarming ability when grown on semi-solid surfaces. An intact copy of flbD on a plasmid complemented the DeltaflbD mutant by restoring lateral flagellation and swarming ability. Transcriptional analysis demonstrated that FlbD is involved in the genetic regulation of flagella biosynthesis and acts as both an activator and a repressor of flagellum gene expression in A. brasilense. DNA binding assays indicated direct interaction between FlbD and the promoter regions of laf1, fliF and flgB genes. We propose that A. brasilense has a genetic regulation profile for flagella biosynthesis similar to that observed in Caulobacter crescentus.

Computed tomography guided microcoil localization for pulmonary small nodules and ground-glass opacity prior to thoracoscopic resection.

BACKGROUND: To evaluate the feasibility, safety and efficacy of computed tomography (CT)-guided microcoil localization for small pulmonary lesions prior to thoracoscopic resection. METHODS: We retrospectively reviewed the medical data of patients with pulmonary solid nodules and ground-glass opacity (GGO) who underwent CT-guided microcoil localization prior to thoracoscopic surgery. The microcoil was deployed with the proximal end of the microcoil coiling beyond the parietal pleura while the distal part anchoring in the lung parenchyma. After marking with microcoil, the pulmonary lesions were removed by thoracoscopic surgery. RESULTS: CT-guided microcoil placements were successful in all 98 lesions, including 14 solid nodules, 11 part-solid GGO, and 73 pure GGO. The mean distance from the lesions to the pleura surface was 11.1±6.6 mm. Eighty-four microcoils (85.7%) were successfully placed with the tails coiled beyond the parietal pleura. Seventeen patients (17.3%) had mild complications after the procedure of localization. Thirteen patients with asymptomatic pneumothorax, only one patient required further thoracentesis, four patients with pulmonary hematoma. Removal of the pulmonary lesions was successful in all patients. Sixty-six lesions (67.3%) were localized through the proximal end of the microcoil beyond the visceral pleura by visual inspection, 29 lesions were localized by palpation of the microcoil or the nodule, and 3 lesions had dislocation of the microcoil, resulting in a success rate of 96.9% for intraoperative localization. CONCLUSIONS: CT-guided microcoil localization prior to thoracoscopic resection is a feasible, safe, and effective method for localization of pulmonary small nodules and GGO.

Characterization of Two GAS1 Genes and Their Effects on Expression and Secretion of Heterologous Protein Xylanase B in Kluyveromyces lactis.

ß-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative ß-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of ß-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The ß-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion- and transport-related genes. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.

Rapid determination of L-glutamine using engineered Escherichia coli overexpressing glutamine synthetase.

A genetically engineered Escherichia coli was developed as the source of enzyme for rapidly quantifying glutamine. E. coli BL21 (DE3) cells overexpressing a glutamine synthetase from Bacillus subtilis were prepared as tube aliquots and used in a small volume of nontoxic mixture. The current method was compared to high performance liquid chromatography analysis, Sigma kit (GLN-1) and Mecke method. The method is applicable to a wide range of glutamine concentrations (0.05-2.5 mM) and correlates well to the detection results obtained from high performance liquid chromatography (Pearson correlation is 0.978 at the 0.01 level). Moreover, the whole assay procedure takes less than 15 min and uses nontoxic reagents, so it can be applied to monitor glutamine production and utilization conveniently.

High-yield growth and magnetosome formation by Magnetospirillum gryphiswaldense MSR-1 in an oxygen-controlled fermentor supplied solely with air.

Magnetotactic bacteria are difficult to grow under defined conditions in culture, which has presented a major obstacle to commercial application of magnetosomes. We studied the relationships among the cell growth, magnetosome formation, dissolved oxygen concentration (DO), and the ability to supply oxygen to the cells. Mass culture of Magnetospirillum gryphiswaldense MSR-1 for the production of magnetosomes was established in a 42-L fermentor under the following conditions: (1) sterile air was the sole gas supplied in the fermentor, and DO could be regulated at any level below 10% saturation by cascading the stir rate to DO, (2) to resolve the paradoxical situation that the cell growth requires higher DO whereas magnetosome formation requires low DO below the detectable range of regular oxygen electrode, DO was controlled to optimal level using the change of cell growth rate, rather than reading from the highly sensitive oxygen electrode, as the signal for determining appropriate DO, and (3) timing and rate of supplying the substrates were determined by measuring cell density and Na-lactate concentration. Under these conditions, cell density (OD565) of strain MSR-1 reached 7.24 after 60-h culture in a 42-L fermentor, and cell yield (dry weight) was 2.17 g/L, the highest yield so far being reported. The yield of magnetosomes (dry weight) was 41.7 mg/L and 16.7 mg/L/day, which were 2.8 and 2.7 times higher than the previously reported yields.

Functional analysis of the GAF domain of NifA in Azospirillum brasilense: effects of Tyr-->Phe mutations on NifA and its interaction with GlnB.

Regulation of NifA activity in Azospirillum brasilense depends on GlnB (a PII protein), and it was previously reported that the target of GlnB activity is the N-terminal domain of NifA. Furthermore, mutation of the Tyr residue at position 18 in the N-terminal domain resulted in a NifA protein that did not require GlnB for activity under nitrogen fixation conditions. We report here that a NifA double mutant in which the Tyr residues at positions 18 and 53 of NifA N-were simultaneously replaced by Phe (NifA-Y1853F) displays high nitrogenase activity, which is still regulatable by ammonia, but not by GlnB. The yeast two-hybrid technique was used to investigate whether GlnB can physically interact with wild-type and mutant NifA proteins. GlnB was found to interact directly with the N-terminal GAF domain of wild-type NifA, but not with its central or C-terminal domain. GlnB could still bind to the single NifA mutants Y18F and Y53F. In contrast, no interaction was detected between GlnB and the double mutant NifA-Y18/53F or between GlnB and NifA-Y43.