Improvement of the silver staining method for bacterial flagella.

Flagella staining is a common method used in microbial research to identify and mark morphological features of bacteria. We improved the Blendon staining method by adding two steps to the usual procedure, viz. "preparation of a pre-atomized microscope slide" and "stretching flagella in situ". The staining effects were then comparatively studied for this new, improved method on Bacillus subtilis, under four different culture conditions: 1) liquid culture medium, 2) aqueous solution at the bottom of slant medium, 3) solid culture medium adding water for stretching after culture, and 4) semi-solid culture medium adding water for stretching after culture. The results revealed that after the addition of these two steps to the usual procedure, the order of the staining effects for the four culture conditions from best to worst was as follows: semi-solid culture medium > solid culture medium > aqueous solution at the bottom of slant medium > liquid culture medium. Hence, the semi-solid culture medium brought about the best staining effect, with flagella stretching freely and not entangled with each other, while the liquid culture medium had the worst staining, owing to the serious background interference. This improved method is simple, low cost, and worthy of promotion.

Sediment records of polybrominated diphenyl ethers (PBDEs) in Huaihe River, China: Implications for historical production and household usage of PBDE-containing products.

In recent decades, rapid development of industrialization and urbanization caused adverse impact on the aqueous ecology and environment of the Huaihe River basin, China. In this work, three 210Pb-dated sediment cores extracted from the middle reach of Huaihe River in Anhui Province, China were analyzed to elucidate the temporal trends and sources of polybrominated diphenyl ethers (PBDEs). Source diagnostics indicated that commercial Deca-BDE, Penta-BDE and Octa-BDE products and debromination of higher brominated BDE compounds were likely the PBDE sources in the Huaihe River. The prevalence of BDE-47 in the sediment cores was attributed to the extensive use of commercial Bromkal 70-5DE and Bromkal DE-71 in the region. BDE-28 was another congener that was prevalent in all sediment samples, suggesting that reductive debromination occurred in the sediments. Dramatic increase of PBDE concentrations in both three cores since the post-1980s could be attributed to the rapid expansion of production of electronic and telecommunication equipment and household usage in China. PBDE temporal trends in core S1 located at rural area mainly reflected the regional and national inputs deriving from long distance atmospheric transport, and the positive correlations between PBDE concentration in core S1 and gross domestic product (GDP) and household appliances production volumes (HPVs) were observed. PBDE inputs at site S3 mainly include the transport of contaminated water and re-suspended fine sediment particles from the upstream site S2, which was located in the industrial area and adjacent to e-waste recycling area. The government efforts to protect the environment and improve the e-waste management resulted in the progressive decrease trends in PBDE concentrations in cores S2 and S3.

Walnut Shell Powder Can Limit Acid Mine Drainage Formation by Shaping the Bacterial Community Structure.

The formation of acid mine drainage (AMD), which results from the oxidation of sulfur minerals by air and water, can be accelerated by acidophilic and chemolithotrophic bacteria such as Acidithiobacillus ferrooxidans. Our previous study revealed that walnut shell powder and its phenolic component inhibit the growth of A. ferrooxidans. However, their inhibitory effect on AMD formation in the environment needs verification. We established a bioleaching system to test whether walnut shell powder and its phenolic component can limit AMD formation. Our results showed that lignin and cellulose isolated from walnut shell decreased metal ion concentrations through absorption, whereas the phenolic component increased pH by downregulating the expression of Fe2+-oxidizing genes and rus operon genes of A. ferrooxidans. Only walnut shell powder showed an excellent ability to curb AMD by binding metal ions and increasing the pH value. On probing deeper into the alteration of the bacterial community structure in the bioleaching system, we found that the bacterial community became more diverse-the amount of A. ferrooxidans decreased and that of some non-acidophilic bacteria increased. The bacterial community in samples treated with walnut shell powder or its phenolic component had low abundance in the pathways of metabolism and energy production, as determined by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt). In other words, preponderant microbes, mainly A. ferrooxidans, lacked energy to grow well in the treated samples. Our findings provide a practical applicability of walnut shell powder to reduce leaching from a complex environmental community.

Temporal trends of polybrominated diphenyl ethers in the sediment cores from different areas in China.

This paper presented the historical data on the temporal trends of polybrominated diphenyl ethers (PBDEs) in the sediment cores collected from the Huaihe River, Yellow River and Chaohu Lake, China. Among the 40 targeted PBDE congeners, only 10, 6, and 9 of them were detected respectively in the samples from the Huaihe River, Yellow River and Chaohu Lake. On average, the total PBDEs concentrations in sediments were highest in Chaohu Lake followed by the Huaihe River and the Yellow River. As compared to other PBDE congeners, BDE-209 had higher concentrations and detection rates. The similar down core variation between PBDEs and total organic carbon (TOC) suggests that TOC is an important factor influencing PBDEs distribution in the sediments. The total PBDEs concentrations showed an increasing trend from bottom to upper sediments before a decreasing trend in the topmost sediments. The rapid urbanization and industrialization of these regions in recent decades may cause the historically increasing concentrations of sedimentary PBDEs, especially BDE-209. The decreasing PBDEs concentrations in topmost sediments was probably related to the strict environmental policies at present.

Physical and Antibacterial Properties of Sodium Alginate-Sodium Carboxymethylcellulose Films Containing Lactococcus lactis.

Edible films have gradually become a research focus for food packaging materials due to a variety of benefits, including environmental friendliness, good barrier properties, and good carrying capacity. In this experimental study, we used sodium alginate as a film-forming substrate, sodium carboxymethylcellulose as a modifier, and glycerol as a plasticizer, then Lactococcus lactis was added to film solutions to form bacteriostatic films via the tape casting method. With the addition of Lactococcus lactis, the films did not significantly change thickness, while the transparency decreased and a significant increase in red and yellow hues was observed. Meanwhile, the dispersion of bacterial cells in film solutions destroyed intermolecular interactions in the solutions during film formation and increased the volume of voids in the Lactococcus lactis-containing films, thereby slightly decreasing the tensile strength of the films, but significantly increasing water vapor permeability. Moreover, the films with added Lactococcus lactis showed significant bacteriostatic activity against Staphylococcus aureus at 4 °C. In a seven-day bacteriostatic test, the films with Lactococcus lactis added at a level of 1.5 g/100 g resulted in a decrease in the viable cell count of Staphylococcus aureus by at least four logarithmic units. This study of Lactococcus lactis-containing films has provided a new method and strategy for antibacterial preservation of foods.

Preparation and properties of polylactic acid-tea polyphenol-chitosan composite membranes.

To study the properties of composite membranes consisting of polylactic acid (PLA), tea polyphenol (TP), and chitosan (CS), the stretch film method was employed to make PLA-TP- CS composite membranes of different concentrations. By testing the density, mechanical properties, heat-sealing performance, water vapor permeability, and solubility of the pure PLA membrane and the composite membranes, the comprehensive performance of the composite membranes were analyzed with regard to the actual use value. The results show that, compared with the pure PLA membrane, adding TP and CS significantly increases the heat-sealing strength, water vapor permeability, and solubility of the composite membrane. When the composite membrane is used for the preservation of cherries, it is found that the composite membrane with the mass ratio of TP to CS of 3:7 can decrease the rotting rate and mass loss rate significantly, postpone the consumption of soluble solids and vitamin C, maintain the quality of the cherries, and extend the shelf life, thus proving its potential for application in food packaging.

Altered leaf colour is associated with increased superoxide-scavenging activity in aureusidin-producing transgenic plants.

The health-promoting property of diets rich in fruits and vegetables is based, in part, on the additive and synergistic effects of multiple antioxidants. In an attempt to further enhance food quality, we introduced into crops the capability to synthesize a yellow antioxidant, aureusidin, that is normally produced only by some ornamental plants. For this purpose, the snapdragon (Antirrhinum majus) chalcone 4'-O-glucosyltransferase (Am4'CGT) and aureusidin synthase (AmAs1) genes, which catalyse the synthesis of aureusidin from chalcone, were expressed in tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa) plants that displayed a functionally active chalcone/flavanone biosynthetic pathway. Leaves of the resulting transgenic plants developed a yellow hue and displayed higher superoxide dismutase (SOD) inhibiting and oxygen radical absorbance capacity (ORAC) activities than control leaves. Our results suggest that the nutritional qualities of leafy vegetables can be enhanced through the introduction of aurone biosynthetic pathways.

Tuber-specific silencing of the acid invertase gene substantially lowers the acrylamide-forming potential of potato.

Some popular processed foods including French fries contain small amounts of toxic acrylamide. Efforts to lower the accumulation of this reactive compound by modifying the production process have a negative effect on sensory characteristics and are not broadly applicable. This study optimized a method developed more than a decade ago to lower the accumulation of the acrylamide precursors glucose and fructose in cold-stored tubers. In contrast to the original application, which lowered hexose content by one-third through constitutive expression of an antisense copy of the cold-inducible acid invertase (Inv) gene, the current approach was based on tuber-specific expression of an Inv-derived inverted repeat. Stored tubers of transgenic plants contained as little as 2% of the reducing sugars that accumulated in controls. This decline in glucose and fructose formation is counterbalanced by increased sucrose and starch levels. However, it did not trigger any phenotypic changes and also did not affect the formation of free asparagine, ascorbic acid, phenylalanine, and chlorogenic acid. Importantly, French fries from the low-invertase tubers contained up to 8-fold reduced amounts of acrylamide. Given the important role of processed potato products in the modern Western diet, a replacement of current varieties with the low-hexose potatoes would reduce the average daily intake of acrylamide by one-fourth.

Low-acrylamide French fries and potato chips.

SUMMARY: Acrylamide is produced in starchy foods that are baked, roasted or fried at high temperatures. Concerns about the potential health issues associated with the dietary intake of this reactive compound led us to reduce the accumulation of asparagine, one of its main precursors, in the tubers of potato (Solanum tuberosum). This metabolic change was accomplished by silencing two asparagine synthetase genes through 'all-native DNA' transformation. Glasshouse-grown tubers of the transformed intragenic plants contained up to 20-fold reduced levels of free asparagine. This metabolic change coincided with a small increase in the formation of glutamine and did not affect tuber shape or yield. Heat-processed products derived from the low-asparagine tubers were also indistinguishable from their untransformed counterparts in terms of sensory characteristics. However, both French fries and potato chips accumulated as little as 5% of the acrylamide present in wild-type controls. Given the important role of processed potato products in the modern Western diet, a replacement of current varieties with intragenic potatoes could reduce the average daily intake of acrylamide by almost one-third.

Engineered native pathways for high kaempferol and caffeoylquinate production in potato.

Flavonols and caffeoylquinates represent important groups of phenolic antioxidants with health-promoting activities. The genetic potential of potato (Solanum tuberosum) to produce high levels of these dietary compounds has not been realized in currently available commodity varieties. In this article, it is demonstrated that tuber-specific expression of the native and slightly modified MYB transcription factor gene StMtf1(M) activates the phenylpropanoid biosynthetic pathway. Compared with untransformed controls, transgenic tubers contained fourfold increased levels of caffeoylquinates, including chlorogenic acid (CGA) (1.80 mg/g dry weight), whilst also accumulating various flavonols and anthocyanins. Subsequent impairment of anthocyanin biosynthesis through silencing of the flavonoid-3',5'-hydroxylase (F3'5'h) gene resulted in the accumulation of kaempferol-rut (KAR) to levels that were approximately 100-fold higher than in controls (0.12 mg/g dry weight). The biochemical changes were associated with increased expression of both the CGA biosynthetic hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (Hqt) gene and the upstream chorismate mutase (Cm) and prephenate dehydratase (Pdh) genes. Field trials indicated that transgenic lines produced similar tuber yields to the original potato variety Bintje. Processed products of these lines retained most of their phenylpropanoids and were indistinguishable from untransformed controls in texture and taste.

Development of an in planta method for transformation of alfalfa (Medicago sativa).

Conventional methods in transforming alfalfa (Medicago sativa) require multiple tissue culture manipulations that are time-consuming and expensive, while applicable only to a few highly regenerable genotypes. Here, we describe a simple in planta method that makes it possible to transform a commercial variety without employing selectable marker genes. Basically, young seedlings are cut at the apical node, cold-treated, and vigorously vortexed in an Agrobacterium suspension also containing sand. About 7% of treated seedlings produced progenies segregating for the T-DNA. The vortex-mediated seedling transformation method was applied to transform alfalfa with an all-native transfer DNA comprising a silencing construct for the caffeic acid o-methyltransferase (Comt) gene. Resulting intragenic plants accumulated reduced levels of the indigestible fiber component lignin that lowers forage quality. The absence of both selectable marker genes and other foreign genetic elements may expedite the governmental approval process for quality-enhanced alfalfa.

Improving potato storage and processing characteristics through all-native DNA transformation.

The dominant potato (Solanum tuberosum) variety for French fry production in the United States is the 131-year-old Russet Burbank. Market penetration of the higher yielding and more uniform Ranger Russet variety is limited to about one-fifth of that of the Russet Burbank because of two storage deficits: black spot bruise sensitivity and high levels of cold-induced sweetening. Here, these trait weaknesses are turned into strengths by simultaneously lowering the expression of Ranger Russet's tuber-expressed polyphenol oxidase (Ppo), starch-associated R1, and phosphorylase-L (PhL) genes. This genetic modification was accomplished without inserting any foreign DNA into the plant genome. French fries from the intragenic potatoes also contained reduced amounts of the antinutritional compound acrylamide while, unexpectedly, displaying enhanced sensory characteristics.

New construct approaches for efficient gene silencing in plants.

An important component of conventional sense, antisense, and double-strand RNA-based gene silencing constructs is the transcriptional terminator. Here, we show that this regulatory element becomes obsolete when gene fragments are positioned between two oppositely oriented and functionally active promoters. The resulting convergent transcription triggers gene silencing that is at least as effective as unidirectional promoter-to-terminator transcription. In addition to short, variably sized, and nonpolyadenylated RNAs, terminator-free cassette produced rare, longer transcripts that reach into the flanking promoter. These read-through products did not influence the efficacy and expression levels of the neighboring hygromycin phosphotransferase gene. Replacement of gene fragments by promoter-derived sequences further increased the extent of gene silencing. This finding indicates that genomic DNA may be a more efficient target for gene silencing than gene transcripts.

Plant-derived transfer DNAs.

The transfer of DNA from Agrobacterium to plant cell nuclei is initiated by a cleavage reaction within the 25-bp right border of Ti plasmids. In an effort to develop all-native DNA transformation vectors, 50 putative right border alternatives were identified in both plant expressed sequence tags and genomic DNA. Efficacy tests in a tobacco (Nicotiana tabacum) model system demonstrated that 14 of these elements displayed at least 50% of the activity of conventional Agrobacterium transfer DNA borders. Four of the most effective plant-derived right border alternatives were found to be associated with intron-exon junctions. Additional elements were embedded within introns, exons, untranslated trailers, and intergenic DNA. Based on the identification of a single right border alternative in Arabidopsis and three in rice (Oryza sativa), the occurrence of this motif was estimated at a frequency of at least 0.8x10(-8). Modification of plasmid DNA sequences flanking the alternative borders demonstrated that both upstream and downstream sequences play an important role in initiating DNA transfer. Optimal DNA transfer required the elements to be preceded by pyrimidine residues interspaced by AC-rich trinucleotides. Alteration of this organization lowered transformation frequencies by 46% to 93%. Despite their weaker resemblance with left borders, right border alternatives also functioned effectively in terminating DNA transfer, if both associated with an upstream A[C/T]T[C/G]A[A/T]T[G/T][C/T][G/T][C/G]A[C/T][C/T][A/T] domain and tightly linked cytosine clusters at their junctions with downstream DNA. New insights in border region requirements were used to construct an all-native alfalfa (Medicago sativa) transfer DNA vector that can be used for the production of intragenic plants.

Crop improvement through modification of the plant's own genome.

Plant genetic engineering has, until now, relied on the incorporation of foreign DNA into plant genomes. Public concern about the extent to which transgenic crops differ from their traditionally bred counterparts has resulted in molecular strategies and gene choices that limit, but not eliminate, the introduction of foreign DNA. Here, we demonstrate that a plant-derived (P-) DNA fragment can be used to replace the universally employed Agrobacterium transfer (T-) DNA. Marker-free P-DNAs are transferred to plant cell nuclei together with conventional T-DNAs carrying a selectable marker gene. By subsequently linking a positive selection for temporary marker gene expression to a negative selection against marker gene integration, 29% of derived regeneration events contain P-DNA insertions but lack any copies of the T-DNA. Further refinements are accomplished by employing Omega-mutated virD2 and isopentenyl transferase cytokinin genes to impair T-DNA integration and select against backbone integration, respectively. The presented methods are used to produce hundreds of marker-free and backbone-free potato (Solanum tuberosum) plants displaying reduced expression of a tuber-specific polyphenol oxidase gene in potato. The modified plants represent the first example of genetically engineered plants that only contain native DNA.