Artificial and natural duplicates in pyrosequencing reads of metagenomic data

Background  

Artificial duplicates from pyrosequencing reads may lead to incorrect interpretation of the abundance of species and genes in metagenomic studies. Duplicated reads were filtered out in many metagenomic projects. However, since the duplicated reads observed in a pyrosequencing run also include natural (non-artificial) duplicates, simply removing all duplicates may also cause underestimation of abundance associated with natural duplicates.     

Low light and low ammonium are key factors for guayule leaf tissue shoot organogenesis and transformation

A new method has been developed for guayule tissue culture and transformation. Guayule leaf explants have a poor survival rate when placed on normal MS medium and under normal culture room light conditions. Low light and low ammonium treatment greatly improved shoot organogenesis and transformation from leaf tissues. Using this method, a 35S promoter driven BAR gene and an ubiquitin-3 promoter driven GUS gene (with intron) have been successfully introduced into guayule. These transgenic guayule plants were resistant to the herbicide ammonium-glufosinate and were positive to GUS staining. Molecular analysis showed the expected band and signal in all GUS positive transformants. The transformation efficiency with glufosinate selection ranged from 3 to 6%. Transformation with a pBIN19-based plasmid containing a NPTII gene and then selection with kanamycin also works well using this method. The ratio of kanamycin-resistant calli to total starting explants reached 50% in some experiments.     

Neuron-derived IgG Protects Neurons from Complement-dependent Cytotoxicity

Passive immunity of the nervous system has traditionally been thought to be predominantly due to the blood-brain barrier. This concept must now be revisited based on the existence of neuron-derived IgG. The conventional concept is that IgG is produced solely by mature B lymphocytes, but it has now been found to be synthesized by murine and human neurons. However, the function of this endogenous IgG is poorly understood. In this study, we confirm IgG production by rat cortical neurons at the protein and mRNA levels, with 69.0 ± 5.8% of cortical neurons IgG-positive. Injury to primary-culture neurons was induced by complement leading to increases in IgG production. Blockage of neuron-derived IgG resulted in more neuronal death and early apoptosis in the presence of complement. In addition, FcγRI was found in microglia and astrocytes. Expression of FcγR I in microglia was increased by exposure to neuron-derived IgG. Release of NO from microglia triggered by complement was attenuated by neuron-derived IgG, and this attenuation could be reversed by IgG neutralization. These data demonstrate that neuron-derived IgG is protective of neurons against injury induced by complement and microglial activation. IgG appears to play an important role in maintaining the stability of the nervous system.     

Identification and Characterization of <Emphasis Type="Italic">sanH</Emphasis> and <Emphasis Type="Italic">sanI</Emphasis> Involved in the Hydroxylation of Pyridyl Residue During Nikkomycin Biosynthesis in <Emphasis Type="Italic">Streptomyces ansochromogenes</Emphasis>

Nikkomycins are highly potent inhibitors of chitin synthase. The nikkomycin biosynthetic gene cluster has been cloned from Streptomyces asochromogenes. Two cytochrome P450 monooxygenase genes (sanQ, sanH) and one ferredoxin gene (sanI) were found in the cluster. It was reported that SanQ is involved in the hydroxylation of l-His, a key step in 4-formyl-4-imidazolin-2-one base biosynthesis. Here, we have studied the function of sanH and sanI. Disruption of sanH abolished the production of nikkomycin X and Z, but it accumulated one dominant component nikkomycin Lx, which is the nikkomycin X analog lacking the hydroxy group at the pyridyl residue. The sanI disruption mutant accumulated predominantly nikkomycin Lx in addition to nikkomycin X and Z. The nikkomycin production profile of the sanH and sanI double disruption mutant was the same as that of the sanH disruption mutant. These results confirmed that SanH is essential for the hydroxylation of pyridyl residue in nikkomycin biosynthesis of S. ansochromogenes and first demonstrated that SanI is an effective electron donor for SanH, but not for SanQ in vivo.     

Ellagic acid from acorn fringe by enzymatic hydrolysis and combined effects of operational variables and enzymes on yield of the production

The individual effects of three different enzyme types -- one single enzyme (ellagitannin acyl hydrolase) and two combinations of enzymes (ellagitannin acyl hydrolase-beta-glucosidase-polyphenol oxidase and ellagitannin acyl hydrolase-cellulase-xylanase) -- on ellagic acid yield, combined with other process parameters -- enzyme concentration, hydrolysis time, particle size and solid-to-liquid ratio -- were evaluated by response surface methodology. The selection of the enzymes for the study was based on preliminary experiments that showed higher increments in ellagic acid yield. The quantitative parameters studied were enzyme concentration (0.1, 0.45, 2 w/w or %), solid-to-liquid ratio (0.05, 0.15, 0.2), particle size (220, 445, 900 microm) and hydrolysis time (60, 89, 132 min). Experimental data for ellagic acid yield obtained with a single enzyme and two combination enzymes correlated very well with process parameters (P<0.0001), resulting in models with high coefficient of determination for ellagic acid yield (r(2)=0.9636). The combinations of enzymes appeared more effective for ellagic acid production than the single enzyme did. The yield of ellagic acid from non-heat-treated acorn fringe by the use of enzymes in general increased, compared with that from heat-treated material. The research opens a technological-efficient way and develop easily-available renewable raw material for ellagic acid production.     

Molecular imaging of cell-based cancer immunotherapy

Cell-based cancer immunotherapy represents a new and powerful weapon in the arsenal of anticancer treatments. Non-invasive monitoring of the disposition, migration and destination of therapeutic cells will facilitate the development of cell based therapy. The therapeutic cells can be modified intrinsically by a reporter gene or labeled extrinsically by introducing imaging probes into the cells or on the cell surface before transplant. Various advanced non-invasive molecular imaging techniques are playing important roles in optimizing cellular therapy by tracking cells and monitoring the therapeutic effects of transplanted cells in vivo. This review will summarize the application of multiple molecular imaging modalities in cell-based cancer immunotherapy.     

Targeted introgression of a wheat stem rust resistance gene by DNA marker-assisted chromosome engineering

Chromosome engineering is a useful strategy for transfer of alien genes from wild relatives into modern crops. However, this strategy has not been extensively used for alien gene introgression in most crops due to low efficiency of conventional cytogenetic techniques. Here, we report an improved scheme of chromosome engineering for efficient elimination of a large amount of goatgrass (Aegilops speltoides) chromatin surrounding Sr39, a gene that provides resistance to multiple stem rust races, including Ug99 (TTKSK) in wheat. The wheat ph1b mutation, which promotes meiotic pairing between homoeologous chromosomes, was employed to induce recombination between wheat chromosome 2B and goatgrass 2S chromatin using a backcross scheme favorable for inducing and detecting the homoeologous recombinants with small goatgrass chromosome segments. Forty recombinants with Sr39 with reduced surrounding goatgrass chromatin were quickly identified from 1048 backcross progenies through disease screening and molecular marker analysis. Four of the recombinants carrying Sr39 with a minimal amount of goatgrass chromatin (2.87-9.15% of the translocated chromosomes) were verified using genomic in situ hybridization. Approximately 97% of the goatgrass chromatin was eliminated in one of the recombinants, in which a tiny goatgrass chromosome segment containing Sr39 was retained in the wheat genome. Localization of the goatgrass chromatin in the recombinants led to rapid development of three molecular markers tightly linked to Sr39. The new wheat lines and markers provide useful resources for the ongoing global effort to combat Ug99. This study has demonstrated great potential of chromosome engineering in genome manipulation for plant improvement.     

Processing of DNA structures via DNA unwinding and branch migration by the S. cerevisiae Mph1 protein

The budding yeast Mph1 protein, the putative ortholog of human FANCM, possesses a 3' to 5' DNA helicase activity and is capable of disrupting the D-loop structure to suppress chromosome arm crossovers in mitotic homologous recombination. Similar to FANCM, genetic studies have implicated Mph1 in DNA replication fork repair. Consistent with this genetic finding, we show here that Mph1 is able to mediate replication fork reversal, and to process the Holliday junction via DNA branch migration. Moreover, Mph1 unwinds 3' and 5' DNA Flap structures that bear key features of the D-loop. These biochemical results not only provide validation for a role of Mph1 in the repair of damaged replication forks, but they also offer mechanistic insights as to its ability to efficiently disrupt the D-loop intermediate.     

COI and ITS2 sequences delimit species, reveal cryptic taxa and host specificity of fig-associated Sycophila (Hymenoptera, Eurytomidae)

Although the genus Sycophila has broad host preferences, some species are specifically associated with figs as nonpollinator wasps. Because of their sexual dimorphism, morphological plasticity, cryptic mating behaviour and poorly known biology, species identifications are often uncertain. It is particularly difficult to match conspecific females and males. In this study, we employed two molecular markers, mitochondrial COI and nuclear ITS2, to identify Sycophila from six Chinese fig species. Morphological studies revealed 25 female and male morphs, while sequence results for both genes were consistent in supporting the presence of 15 species, of which 13 were host specialists and two used dual hosts. A single species of Sycophila was respectively found on four fig species, but six species were isolated from Ficus benjamina and a same number was reared from Ficus microcarpa. Sequence results revealed three male morphs in one species and detected two species that were overlooked by morphological analysis.