Bt-Modified Transgenic Rice May Shift the Composition and Diversity of Rhizosphere Microbiota.

Plants significantly shape root-associated microbiota, making rhizosphere microbes useful environmental indicator organisms for safety assessment. Here, we report the pyrosequencing of the bacterial 16S ribosomal RNA in rhizosphere soil samples collected from transgenic cry1Ab/cry1Ac Bt rice Huahui No. 1 (GM crop) and its parental counterpart, Minghui63. We identified a total of 2579 quantifiable bacterial operational taxonomic units (OTUs). Many treatment-enriched microbial OTUs were identified, including 14 NonGM-enriched OTUs and 10 GM-enriched OTUs. OTUs belonging to the phyla Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes, Nitrospirae, Chlorobi and GN04 were identified as statistically different in abundance between GM and the other two treatments. Compared with the different impacts of different rice varieties on microbiota, the impact of rice planting on microbiota is more obvious. Furthermore, Huahui No. 1 transgenic Bt rice had a greater impact on the rhizosphere bacterial communities than Minghui63. Early developmental stages of the transgenic Bt rice had a significant impact on many Bacillaceae communities. Soil chemical properties were not significantly altered by the presence of transgenic Bt rice. The peak concentration level of Bt protein products was detected during the seedling stage of transgenic Bt rice, which may be an intriguing factor for bacterial diversity variations. Based on these findings, we conclude that transgenic Bt rice has a significant impact on root-associated bacteria. This information may be leveraged in future environmental safety assessments of transgenic Bt rice varieties.

Multicenter comparative study of three "non-destructive" methods of detecting the cleanliness of the da Vinci surgical robotic instrument.

BACKGROUND: The robotic instrument of the da Vinci surgical system determines the accuracy of robotic-assisted surgery, However, the most effective cleaning method of robotic equipment is a challenge for healthcare professionals. This study compared three da Vinci robot-assisted surgery manipulators to detect the effect of "non-destructive" testing of the cleaning effect by two different methods. METHODS: The post-surgical cleaning of the da Vinci robotic instruments in the Sun Yat-sen University Cancer Center, The First Affiliated Hospital of Guangzhou Medical University and the Shenzhen Second People's Hospital was performed using two different processes from January 2019 to January 2020: manual joint automatic ultrasonic cleaning and automatic mechanical cleaning. The efficacy of visual estimation, the residual protein assay (quantitative) and adenosine triphosphate (ATP) biological biofluorescence detection of the cleanliness of the mechanical instrument's work (distal working end) surface and the shaft's inner chamber was compared. If the cleaning effect of any position on the surface or inner cavity of the manipulator did not qualify, the entire robotic instrument was judged as disqualified. RESULTS: A total of 198 cases of da Vinci robotic instrument postoperative cleanliness data were collected. The qualifying rates of automatic ultrasonic cleaning were 96.97% by visual estimation, 93.94% by residual protein assay and 60.61% by ATP biological fluorescence detection. The respective rates for automatic mechanical cleaning were 100% by visual estimation, 90.91% by residual protein assay and 66.67% by ATP biological fluorescence detection. CONCLUSIONS: The cleaning of the da Vinci robotic instrument detected by "non-destructive" residual protein assay or ATP biological fluorescence detection is more accurate than visual estimation.

A new isolation device for shortening gene flow distance in small-scale transgenic maize breeding.

The transmission of pollen is the main cause of maize gene flow. Under the compulsory labeling system for genetically modified (GM) products in China, isolation measures are crucial. At present, there is no effective isolation device for preventing and controlling the short-range flow of GM maize pollen. The purposes of the present experiments were to overcome the deficiencies of existing technology and to demonstrate a new isolation device for decreasing the gene flow distance of GM maize. The isolation device we invented was shown to be more robust than traditional isolation methods, and it can be disassembled and repeatedly reused. The most important point was that the frequency of gene flow could be greatly reduced using this device. When the distance from the isolation device was more than 1 m, the gene flow rate could be decreased to less than 1%, and when the distance from the isolation device was more than 10 m, the gene flow rate could be reduced to less than 0.1%. When the isolation device was adopted to isolate GM maize in conjunction with bagging the tassels of GM maize at the pollination stage, the gene flow could be controlled to less than 0.1% when the distance from the isolation device was more than 1 m. This device was, however, only applicable for small plots and can shorten the isolation distance of GM maize planting and improve the purity of seeds, all while meeting the needs of close isolation breeding. The use of this device represents a feasible method for risk prevention and control of GM crops.

Comparative Proteomics of Phytase-transgenic Maize Seeds Indicates Environmental Influence is More Important than that of Gene Insertion.

Proteomic differences were compared between phytase-transgenic (PT) maize seeds and nontransgenic (NT) maize seeds through two-dimensional electrophoresis (2-DE) with mass spectrometry (MS). When maize was grown under field conditions, 30 differentially accumulated proteins (DAPs) were successfully identified in PT seeds (PT/NT). Clusters of Orthologous Groups (COG) functional classification of these proteins showed that the largest group was associated with posttranslational modifications. To investigate the effects of environmental factors, we further compared the seed protein profiles of the same maize planted in a greenhouse or under field conditions. There were 76 DAPs between the greenhouse- and field-grown NT maize seeds and 77 DAPs between the greenhouse- and field-grown PT maize seeds However, under the same planting conditions, there were only 43 DAPs (planted in the greenhouse) or 37 DAPs (planted in the field) between PT and NT maize seeds. The results revealed that DAPs caused by environmental factors were more common than those caused by the insertion of exogenous genes, indicating that the environment has much more important effects on the seed protein profiles. Our maize seed proteomics results also indicated that the occurrence of unintended effects is not specific to genetically modified crops (GMCs); instead, such effects often occur in traditionally bred plants. Our data may be beneficial for biosafety assessments of GMCs at the protein profile level in the future.

An improved protein extraction method applied to cotton leaves is compatible with 2-DE and LC-MS.

BACKGROUND: Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used in plant proteomics research. However, these two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome. RESULTS: Here, we evaluated the efficiency of three different protein extraction methods for 2-DE and LC-MS/MS analyses of total proteins obtained from cotton leaves. The protein yield of the borax/PVPP/phenol (BPP) method (0.14%) was significantly lower than the yields of the trichloroacetic acid/acetone (TCA) precipitation method (1.42%) and optimized TCA combined with BPP (TCA-B) method (0.47%). The BPP method was failed to get a clear 2-DE electrophoretogram. Fifty pairs of protein spots were randomly selected from the 2-DE gels of TCA- and TCA-B-extracted proteins for identification by MALDI TOF/TOF, and the results of 42 pairs were consistent. High-throughput proteomic analysis showed that 6339, 9282 and 9697 unique proteins were identified from the total cotton leaf proteins extracted by the TCA, BPP and TCA-B methods, respectively. Gene Ontology (GO) analysis revealed that the proteins specifically identified by TCA method were primarily distributed in the plasma membrane, while BPP and TCA-B methods specific proteins distributed in the cytosol, indicating the sub-cellular preference of different protein extraction methods. Further, ATP-dependent zinc metalloprotease FTSH 8 could be observed in the 2-DE gels of TCA and TCA-B methods, and could only be detected in the LC-MS/MS results of the BPP and TCA-B methods, showing that TCA-B method might be the optimized choice for both 2-DE and LC-MS/MS. CONCLUSION: Our data provided an improved TCA-B method for protein extraction that is compatible with 2-DE and LC-MS/MS for cotton leaves and similar plant tissues which is rich in polysaccharides and polyphenols.

Comparative physiological and proteomic analyses of the chloroplasts in halophyte Sesuvium portulacastrum under differential salt conditions.

Sesuvium portulacastrum, an important mangrove-associated true halophyte belongs to the family Aizoaceae, has excellent salt tolerance. Chloroplasts are the most sensitive organelles involved in the response to salinity. However, the regulation mechanism of chloroplasts of S. portulacastrum under salinity stress has not been reported. In this study, morphological and physiological analyses of leaves and comparative proteomics of chloroplasts isolated from the leaves of S. portulacastrum under different NaCl treatments were performed. Our results showed that the thickness of the palisade tissue, the leaf area, the maximum photochemical efficiency of photosystem II, and the electron transport rate increased remarkably after the plants were subjected to differential saline environments, indicating that salinity can increase photosynthetic efficiency and improve the growth of S. portulacastrum. Subsequently, 55 differentially expressed protein species (DEPs) from the chloroplasts of S. portulacastrum under differential salt conditions were positively identified by mass spectrometry. These DEPs were involved in multiple metabolic pathways, such as photosynthesis, carbon metabolism, ATP synthesis and the cell structure. Among these DEPs, the abundance of most proteins was induced by salt stress. Based on a combination of the morphological and physiological data, as well as the chloroplast proteome results, we speculated that S. portulacastrum can maintain photosynthetic efficiency and growth by maintaining the stability of the photosystem II complex, promoting the photochemical reaction rate, enhancing carbon fixation, developing plastoglobules, and preserving the biomembrane system of chloroplasts under salt stress.

Proteomic analysis of phytase transgenic and non-transgenic maize seeds.

Proteomics has become a powerful technique for investigating unintended effects in genetically modified crops. In this study, we performed a comparative proteomics of the seeds of phytase-transgenic (PT) and non-transgenic (NT) maize using 2-DE and iTRAQ techniques. A total of 148 differentially expressed proteins (DEPs), including 106 down-regulated and 42 up-regulated proteins in PT, were identified. Of these proteins, 32 were identified through 2-DE and 116 were generated by iTRAQ. It is noteworthy that only three proteins could be detected via both iTRAQ and 2-DE, and most of the identified DEPs were not newly produced proteins but proteins with altered abundance. These results indicated that many DEPs could be detected in the proteome of PT maize seeds and the corresponding wild type after overexpression of the target gene, but the changes in these proteins were not substantial. Functional classification revealed many DEPs involved in posttranscriptional modifications and some ribosomal proteins and heat-shock proteins that may generate adaptive effects in response to the insertion of exogenous genes. Protein-protein interaction analysis demonstrated that the detected interacting proteins were mainly ribosomal proteins and heat-shock proteins. Our data provided new information on such unintended effects through a proteomic analysis of maize seeds.

Comparative Proteomics of Leaves from Phytase-Transgenic Maize and Its Non-transgenic Isogenic Variety.

To investigate unintended effects in genetically modified crops (GMCs), a comparative proteomic analysis between the leaves of the phytase-transgenic maize and the non-transgenic plants was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 57 differentially expressed proteins (DEPs) were successfully identified, which represents 44 unique proteins. Functional classification of the identified proteins showed that these DEPs were predominantly involved in carbohydrate transport and metabolism category, followed by post-translational modification. KEGG pathway analysis revealed that most of the DEPs participated in carbon fixation in photosynthesis. Among them, 15 proteins were found to show protein-protein interactions with each other, and these proteins were mainly participated in glycolysis and carbon fixation. Comparison of the changes in the protein and tanscript levels of the identified proteins showed that most proteins had a similar pattern of changes between proteins and transcripts. Our results suggested that although some significant differences were observed, the proteomic patterns were not substantially different between the leaves of the phytase-transgenic maize and the non-transgenic isogenic type. Moreover, none of the DEPs was identified as a new toxic protein or an allergenic protein. The differences between the leaf proteome might be attributed to both genetic modification and hybrid influence.

Comparative proteomics of Bt-transgenic and non-transgenic cotton leaves.

BACKGROUND: As the rapid growth of the commercialized acreage in genetically modified (GM) crops, the unintended effects of GM crops' biosafety assessment have been given much attention. To investigate whether transgenic events cause unintended effects, comparative proteomics of cotton leaves between the commercial transgenic Bt + CpTI cotton SGK321 (BT) clone and its non-transgenic parental counterpart SY321 wild type (WT) was performed. RESULTS: Using enzyme linked immunosorbent assay (ELISA), Cry1Ac toxin protein was detected in the BT leaves, while its content was only 0.31 pg/g. By 2-DE, 58 differentially expressed proteins (DEPs) were detected. Among them 35 were identified by MS. These identified DEPs were mainly involved in carbohydrate transport and metabolism, chaperones related to post-translational modification and energy production. Pathway analysis revealed that most of the DEPs were implicated in carbon fixation and photosynthesis, glyoxylate and dicarboxylate metabolism, and oxidative pentose phosphate pathway. Thirteen identified proteins were involved in protein-protein interaction. The protein interactions were mainly involved in photosynthesis and energy metabolite pathway. CONCLUSIONS: Our study demonstrated that exogenous DNA in a host cotton genome can affect the plant growth and photosynthesis. Although some unintended variations of proteins were found between BT and WT cotton, no toxic proteins or allergens were detected. This study verified genetically modified operation did not sharply alter cotton leaf proteome, and the target proteins were hardly checked by traditional proteomic analysis.

Pre-operative education and counselling are associated with reduced anxiety symptoms following carotid endarterectomy: a randomized and open-label study.

BACKGROUND: Post-operative anxiety is common and may have significant impact on the post-operative recovery of the patients. Theatre nurse visits before surgery has been shown to reduce patient's anxiety levels following general surgery. AIM: To investigate the effect of pre-operative visits and counselling by intensive care unit (ICU) nurses on patient's anxiety levels following carotid endarterectomy. METHODS: This is an open-label and randomized clinical trial. Patients undergoing carotid endarterectomy were divided into study (n=60) and control group (n=60). For the study group, in addition to routine pre-operational counselling by the surgeons, ICU nurses visited the patients and provided a structured counselling the day before surgery. For the control group, only routine pre-operative counselling was provided. Anxiety levels were assessed by Zung self-rating anxiety scale (SAS) the day before surgery and on the day after being discharged from ICU to the ward. RESULTS: The two groups were comparable in age, sex, surgical methods, and duration of ICU stays. Following the surgery, the mean SAS score in the control group increased from 50.5±5.4 to 58.5±7.3 (p=0.03), whereas the mean SAS score in the study group reduced from 51.5±4.3 to 45.1±6.5 (p=0.02). The proportion of patients with anxiety symptoms in the control group was higher than in the study group following the surgery (58.3% vs. 33.3%, p=0.001). CONCLUSIONS: Pre-operative visits and counselling by ICU nurses could reduce patient's anxiety levels following carotid endarterectomy.

Recent advances on the GAP promoter derived expression system of Pichia pastoris.

Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins and the most important feature of P. pastoris is the existence of a strong and tightly regulated promoter from the alcohol oxidase I (AOX1) gene. The AOX1 promoter (pAOX1) has been used to express foreign genes and to produce a variety of recombinant proteins in P. pastoris. However, some efforts have been made to develop new alternative promoters to pAOX1 to avoid the use of methanol. The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated. Some important developments and features of this expression system will be summarized in this review.