Gene Manipulation of a Heavy Metal Hyperaccumulator Species Thlaspi caerulescens L. via Agrobacterium-mediated Transformation

Thlaspi caerulescens L. is well known as a Zn/Cd hyperaccumulator. The genetic manipulation of T. caerulescens through transgenic technology can modify plant features for use in phytoremediation. Here, we describe the efficient transformation of T. caerulescens using Agrobacterium tumefaciens strain EHA105 harboring a binary vector pBI121 with the nptII gene as a selectable marker, the gus gene as a reporter and a foreign catalase gene. Based on the optimal concentration of growth regulators, the shoot cluster regeneration system via callus phase provided the basis of the genetic transformation in T. caerulescens. The key variables in transformation were examined, such as co-cultivation period and bacterial suspension density. Optimizing factors for T-DNA delivery resulted in kanamycin-resistant transgenic shoots with transformation efficiency more than 20%, proven by histochemical GUS assay and PCR analysis. Southern analysis of nptII and RT-PCR of catalase gene demonstrated that the foreign genes were integrated in the genome of transformed plantlets. Moreover, the activity of catalase enzyme in transgenic plants was obviously higher than in wild-type plants. This method offers new prospects for the genetic engineering of this important hyperaccumulator species.     

Photothermal treatment of human carcinoma cells using liposome-encapsulated gold nanoshells

We report the application of liposome-encapsulated gold nanoshells for in vitro photo-induced hyperthermia in human mammary carcinoma cells. In addition to evaluating their effects in vitro, we compared the application liposome-encapsulated gold nanoshells and free-standing gold nanoshells for NanoPhotoTherapy (NPT). NPT-induced hyperthermia was performed using a 785-nm near-infrared light from a diode laser and the in vitro effects were evaluated using nucleic acid molecular probes by fluorescence microscopy. Additionally, we monitored the effectiveness of NPT by detecting apoptosis via capase-9 activity.     

PCR-Activated Cell Sorting for Cultivation-Free Enrichment and Sequencing of Rare Microbes

Microbial systems often exhibit staggering diversity, making the study of rare, interesting species challenging. For example, metagenomic analyses of mixed-cell populations are often dominated by the sequences of the most abundant organisms, while those of rare microbes are detected only at low levels, if at all. To overcome this, selective cultivation or fluorescence-activated cell sorting (FACS) can be used to enrich for the target species prior to sequence analysis; however, since most microbes cannot be grown in the lab, cultivation strategies often fail, while cell sorting requires techniques to uniquely label the cell type of interest, which is often not possible with uncultivable microbes. Here, we introduce a culture-independent strategy for sorting microbial cells based on genomic content, which we term PCR-activated cell sorting (PACS). This technology, which utilizes the power of droplet-based microfluidics, is similar to FACS in that it uses a fluorescent signal to uniquely identify and sort target species. However, PACS differs importantly from FACS in that the signal is generated by performing PCR assays on the cells in microfluidic droplets, allowing target cells to be identified with high specificity with suitable design of PCR primers and TaqMan probes. The PACS assay is general, requires minimal optimization and, unlike antibody methods, can be developed without access to microbial antigens. Compared to non-specific methods in which cells are sorted based on size, granularity, or the ability to take up dye, PACS enables genetic sequence-specific sorting and recovery of the cell genomes. In addition to sorting microbes, PACS can be applied to eukaryotic cells, viruses, and naked nucleic acids.     

Prediction of Percentage Body Fat in Rural Thai Population Using Simple Anthropometric Measurements

Objective: To develop and validate sex‐specific equations for predicting percentage body fat (%BF) in rural Thai population, based on BMI and anthropometric measurements. Research Methods and Procedures: %BF (DXA; GE Lunar Corp., Madison, WI) was measured in 181 men and 255 women who were healthy and between 20 and 84 years old. Anthropometric measures such as weight (kilograms), height (centimeters), BMI (kilograms per meter squared), waist circumference (centimeters), hip circumference (centimeters), thickness at triceps skinfold (millimeters), biceps skinfold (millimeters), subscapular skinfold (millimeters), and suprailiac skinfold (millimeters) were also measured. The sample was randomly divided into a development group (98 men and 125 women) and a validation group (83 men and 130 women). Regression equations of %BF derived from the development group were then evaluated for accuracy in the validation group. Results: The equation for estimating %BF in men was: %BF(men) = 0.42 × subscapular skinfold + 0.62 × BMI ? 0.28 × biceps skinfold + 0.17 × waist circumference ? 18.47, and in women: %BF(women) = 0.42 × hip circumference + 0.17 × suprailiac skinfold + 0.46 × BMI ? 23.75. The coefficient of determination (R²) for both equations was 0.68. Without anthropometric variables, the predictive equation using BMI, age, and sex was: %BF = 1.65 × BMI + 0.06 × age ? 15.3 × sex ? 10.67 (where sex = 1 for men and sex = 0 for women), with R² = 0.83. When these equations were applied to the validation sample, the difference between measured and predicted %BF ranged between ±9%, and the positive predictive values were above 0.9. Discussion: These results suggest that simple, noninvasive, and inexpensive anthropometric variables may provide an accurate estimate of %BF and could potentially aid the diagnosis of obesity in rural Thais.     

Characterization of bacteriophage phi C69 of Saccharopolyspora erythraea and demonstration of heterologous actinophage propagation by transfection of Streptomyces and Saccharopolyspora

A bacteriophage, designated phi C69, isolated from a culture of Saccharopolyspora erythraea was characterized. The phage propagates on Sac. erythraea NRRL 2338 but does not infect 10 Streptomyces or 3 Micromonospora species tested. It infects Sac. erythraea NRRL 2359 but does not produce infectious phage particles in this host. phi C69 is approximately 40 kb in length and contains cohesive ends. A cos fragment containing ligated phage DNA ends was cloned in Escherichia coli. Restriction maps of the phage DNA and the cos fragment for several enzymes are shown. Transfection of both Sac. erythraea and Streptomyces lividans with phi C69 resulted in approximately equal titres of infectious phage particles produced from approximately the same number of regenerating cells. Transfection of Sac. erythraea with DNA from Streptomyces phages SH10 and KC404 also resulted in the production of infectious phage particles. The basis for differences among hosts in susceptibility to infection by various actinophages is discussed.     

Characterization of <Emphasis Type="Italic">Flavobacterium aquimarinum</Emphasis> sp. nov., a halotolerant bacterium isolated from seawater

A novel, aerobic, Gram-stain-negative, non-motile, non-spore forming, rod-shaped bacterium, designated strain Dol 15-39^T, was isolated from a seawater sample near Geoje Island in the South Sea, Republic of Korea. The strain was found to be oxidase-negative and catalase-positive. The isolate was observed to grow at temperatures from 4 to 37°C, at salinities of up to 7%, and at pH levels from 6 to 9; moreover, it was not able to degrade starch, DNA, esculin, or tyrosine. Phylogenetic analysis based on 16S rRNA gene sequences showed that Dol 15-39^T was most closely related to Flavobacterium jumunjinense HME7102^T with a sequence similarity of 97.3%. However, the levels of DNA-DNA relatedness between Dol 15-39^T and the most closely related species were much lower than 70%, confirming that they represented distinct genomic species. The genomic DNA G + C content of Dol 15-39^T was calculated to be 32.6 mol%. MK-6 was the predominant respiratory quinine, while iso-C_15:0 (25.0%), iso-C_15:1 G (17.0%), and iso-C_17:0 3-OH (10.4%) were the major cellular fatty acids. Phosphatidylethanolamine was identified as a major polar lipid, while various unidentified aminolipids and polar lipids were also detected. Based on polyphasic taxonomic data, Dol 15-39^T represents a novel species of the genus Flavobacterium, for which the name F. aquimarinum sp. nov. is proposed. The type strain is accessible under the culture collection numbers (KEMB 9005-617^T = JCM 31930^T).     

Proposal of three novel species of soil bacteria, <Emphasis Type="Italic">Variovorax ureilyticus,Variovorax rhizosphaerae</Emphasis>, and <Emphasis Type="Italic">Variovorax robiniae</Emphasis>, in the family <Emphasis Type="Italic">Comamonadaceae</Emphasis>

Three novel bacterial strains (UCM-2^T, UCM-G28^T, and UCM-G35^T) were obtained while isolating soil bacteria for the development of antibiotics. Cells of these strains were Gram-negative, non-spore forming, motile by means of a single flagellum, and rod shaped. In all strains, the predominant isoprenoid quinone was ubiquinone-8 (Q-8). Cells contained C_16:0, summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), summed feature 8 (C_18:1ω7c and/or C_18:1ω6c), and C_17:0 cyclo as the major fatty acids, and C_10:0 3-OH as the major hydroxy fatty acid. The polar lipid profiles of the three novel strains were dominated by diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The genomic DNA G + C contents of strains UCM-2^T, UCM-G28^T, and UCMG35^T were 67.5, 65.9, and 66.4 mol%, respectively. Phylogenetic analyses based on 16S rRNA sequences showed that strain UCM-2^T was most closely related to Variovorax soli NBRC 106424^T, whereas strains UCM-G28^T and UCM-G35^T were most similar to Variovorax ginsengisoli Gsoil 3165^T. Values indicating DNA-DNA hybridization between the novel isolates and closely related species in the genus Variovorax were lower than the 70% cut-off point. These phenotypic, chemotaxonomic, and phylogenetic data indicate that the three isolates should be classified as new members of the genus Variovorax, for which the names Variovorax ureilyticus sp. nov., Variovorax rhizosphaerae sp. nov., and Variovorax robiniae sp. nov. are proposed. The type strains are UCM-2^T (= KACC 18899^T = NBRC 112306^T), UCMG28^T (= KACC 18900^T = NBRC 112307^T), and UCM-G35^T (= KACC 18901^T = NBRC 112308^T), respectively.     

Detoxification of Fusarium-contaminated maize with sodium sulphite – in vivo efficacy with special emphasis on mycotoxin residues and piglet health

A feeding experiment with piglets was performed to examine the efficacy of a wet preservation of Fusarium (FUS)-contaminated maize with sodium sulphite (SoS) based on deoxynivalenol (DON) and zearalenone (ZEN) residue levels in urine, bile and liquor and health traits of piglets. For this purpose, 80 castrated male piglets (7.57 ± 0.92 kg BW) were assigned to four treatment groups: CON? (control diet, with 0.09 mg DON and <0.01 mg ZEN/kg diet), CON+ (diet CON?, wet-preserved with 5 g SoS/kg maize; containing 0.05 mg DON and <0.01 mg ZEN/kg diet), FUS? (diet with mycotoxin-contaminated maize; containing 5.36 mg DON and 0.29 mg ZEN/kg diet), and FUS+ (diet FUS?, wet-preserved with 5 g SoS/kg maize; resulting in 0.83 mg DON and 0.27 mg ZEN/kg diet). After 42 d, 40 piglets (n = 10 per group) were sampled. A clear reduction of DON levels by approximately 75% was detected in all specimens of pigs fed diet FUS+. ZEN was detected in all urine, bile and liquor samples, while their metabolites were only detectable in urine and bile. Additionally, their concentrations were not influenced by SoS treatment. Among the health-related traits, feeding of FUS diets increased the total counts of leukocytes and segmented neutrophil granulocytes irrespective of SoS treatment. SoS treatment increased the total blood protein content slightly with a similar numerical trend in albumin concentration. These effects occurred at an obviously lower level in FUS-fed groups. Moreover, SoS treatment recovered the reduction of NO production induced by feeding diet FUS? indicating an effect on the redox level. As this effect only occurred in group FUS+, it is obviously related to the adverse effects of the Fusarium toxins. In conclusion, treatment of FUS-contaminated maize with SoS decreased the inner exposure with DON as indicated by the lower DON levels in various piglet specimens. However, health-related traits did not consistently reflect this decreased exposure.