Foot mucus is a good source for non-destructive genetic sampling in Polyplacophora

A non-destructive method for collecting samples for DNA analysis from the mucus of molluscs was successfully adapted for use with the genus Ischnochiton. DNA was extracted using a Chelex-based method and the COI subunit of the mtDNA was amplified and sequenced. Sequences from the mucus were crosschecked against sequences from the foot tissue of the same animal and were found to be identical. This method provides a non-destructive way of carrying out larger studies of the genetics of rare organisms and may be of general use for genetic-based field studies of molluscs.     

Crops and agriculture during the Iron Age and late antiquity in Cerdanyola del Vallès (Catalonia,Spain)

Various sites in the valley of the Sant Cugat stream in Cerdanyola del Vallès (Catalonia) were subject to systematic archaeobotanical sampling to obtain an overview of the crops and agriculture of the area during the Iron Age and late antiquity. In all cases, the most numerous taxa were crop plants. Among these, cereals were clearly predominant at all sites investigated, especially Hordeum vulgare var. vulgare (hulled barley) and Triticum aestivum/durum (bread or macaroni wheat), both in numbers and frequency. Other cereals, such as Triticum dicoccum (emmer) or Setaria italica (foxtail bristle-grass), were regularly present in considerably lower numbers but in fairly high frequencies. Pulses were much less numerous, although their presence increases in terms of frequency. Among them, clearly the best represented was Lens culinaris (lentil). The results show that the agriculture in the period considered was principally based on winter cereals, with a gradual substitution of hulled barley by bread/hard wheat, accompanied by other cereals of minor importance, led by Triticum dicoccum (emmer), and pulses. The appearance of Vitis (grapevine) in the Iberian period is one of the important characteristics of agriculture in the Iberian world. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biofilm formation by exopolysaccharide mutants of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> strain NRRL B-1355

Leuconostoc mesenteroides strain NRRL B-1355 produces the soluble exopolysaccharides alternan and dextran in planktonic cultures. Mutants of this strain are available that are deficient in the production of alternan, dextran, or both. Another mutant of NRRL B-1355, strain R1510, produces an insoluble glucan in place of alternan and dextran. To test the effect of exopolysaccharide production on biofilm formation, these strains were cultured in a biofilm reactor. All strains grew well as biofilms, with comparable cell densities, including strain NRRL B-21414, which produces neither alternan nor dextran in planktonic cultures. However, the exopolysaccharide phenotype clearly affected the appearance of the biofilms and the sloughed-off biofilm material produced by these biofilms. For all strains, soluble glucansucrases and soluble polysaccharides produced by biofilm cultures appeared to be similar to those produced by planktonic cultures. Biofilms from all strains also contained insoluble polysaccharides. Strain R1510 biofilms contained an insoluble polysaccharide similar to that produced by planktonic cultures. For most other strains, the insoluble biofilm polysaccharides resembled a mixture of alternan and dextran. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Allogeneic tumor lysate can serve as both antigen source and protein supplementation for dendritic cell culture

BACKGROUND: Recent preclinical and clinical evidence suggests the use of allogeneic tumor as a source of antigen for DC-based immunotherapy against cancer. We hypothesized that addition of allogeneic tumor lysate to monocyte-derived DC culture could serve a dual purpose: (1) antigen source and (2) protein supplementation of DC culture media. Protein supplementation whether of known origin (human serum/plasma, fetal bovine serum, human serum albumin) or undeclared origin ("serum-free" media) is a source of variability and bias. We addressed the question whether protein supplementation can be omitted in the presence of allogeneic tumor lysate. MATERIALS AND METHODS: Human DC cultured in the presence of lysate from medullary thyroid carcinoma (MTC) cell line SHER-I (TuLy-DC) and DC pulsed with the same lysate but cultured in the presence of FBS (FBS-DC) were assessed for morphology, phenotype, maturation and functional properties. RESULTS: In comparison of FBS-DC/TuLy-DC no significant differences in morphology, phenotype and maturation could be detected. Both culture conditions produced CD1a(high), CD14(low) DC with high expression of costimulatory molecules and CD83 upon stimulation. TuLy-DC gave significantly better yields and produced more IL12p70. DC showed high (allo)stimulatory capacity toward T-cells. TuLy-DC induced more intracellular IFNgamma in CD8+T-cells of vaccinated MTC patients. Both types of DC induced killing of SHER-I after short in vitro restimulation. Tumor lysate from SHER-I can substitute for further protein supplementation in DC culture. Allogeneic tumor lysates should be taken into consideration as both source of antigen and protein supplementation in monocyte-derived DC culture.     

Acetylcholine inhibits long-term hypoxia-induced apoptosis by suppressing the oxidative stress-mediated MAPKs activation as well as regulation of Bcl-2, c-IAPs,and caspase-3 in mouse embryonic stem cells

This study examined the effect of acetylcholine (ACh) on the hypoxia-induced apoptosis of mouse embryonic stem (ES) cells. Hypoxia (60 h) decreased both the cell viability and level of [³H] thymidine incorporation, which were prevented by a pretreatment with ACh. However, the atropine (ACh receptor [AChR] inhibitor) treatment blocked the protective effect of ACh. Hypoxia (90 min) increased the intracellular level of reactive oxygen species (ROS). On the other hand, ACh inhibited the hypoxia-induced increase in ROS, which was blocked by an atropine treatment. Subsequently, the hypoxia-induced ROS increased the level of p38 mitogen activated protein kinase (MAPK) and Jun-N-terminal kinase (JNK) phosphorylation, which were inhibited by the ACh pretreatment. Moreover, hypoxic exposure (90 min) increased the level of nuclear factor-κB (NF-κB) phosphorylation, which was blocked by a pretreatment with SB 203580 (p38 MAPK inhibitor) or SP 600125 (JNK inhibitor). However, hypoxia (60 h) decreased the protein levels of Bcl-2 and c-IAPs (cellular inhibitor of apoptosis proteins) but increased the level of caspase-3 activation. All these effects were inhibited by a pretreatment with ACh. In conclusion, ACh prevented the hypoxia-induced apoptosis of mouse ES cells by inhibiting the ROS-mediated p38 MAPK and JNK activation as well as the regulation of Bcl-2, c-IAPs, and caspase-3. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Using genetic and morphological analysis to distinguish endangered taxa from their hybrids with the cultivated exotic pest plant <Emphasis Type="Italic">Lantana strigocamara</Emphasis> (syn: <Emphasis Type="Italic">Lantana camara</Emphasis>)

Because highly invasive species can rapidly assimilate rare taxa, we questioned whether two Florida endangered Lantana depressa varieties existed 21 years after Sanders documented their widespread hybridization with exotic Lantana strigocamara, and whether morphological traits could accurately discriminate genetic individuals. Stepwise discriminant analysis of morphological characters discriminated the three taxa, correctly classifying 98, 91, 89% of L. strigocamara, L. depressa var. depressa, and var. floridana. Hybrids blurred taxonomic distinctions of varieties and reduced classification accuracy by 7–17%. Species-specific Random Fragment Length Polymorphism (RFLP-PCR) confirmed hybridization has occurred. Intersimple Sequence Repeat (ISSR) fingerprints analyzed with STRUCTURE identified three groups indicating introgression. Morphological traits significantly, but weakly correlated with q ratios (P = 0.0001; r ² = 0.45). Although L. strigocamara introgression is widespread and ongoing, wild populations contain individuals that are predominantly L. depressa genome, supporting actions to remove adventive L. strigocamara, prevent its sale, and promote sales of genetically confirmed natives.     

Spectrum of <Emphasis Type="Italic">CREBBP</Emphasis> mutations in Indian patients with Rubinstein-Taybi syndrome

Rubinstein-Taybi syndrome (RSTS), a developmental disorder comprising abnormalities that include mental retardation, an unusual facial appearance, broad thumbs and big toes is frequently associated with molecular lesions in the CREB-binding protein gene, CREBBP. The objective of the present study was to identify and analyse CREBBP mutations in Indian RSTS patients on which there are no data. Direct sequencing of CREBBP performed in 13 RSTS patients identified the three zinc fingers (CH1, CH2, CH3) and HAT domain as mutational hotspots in which ten novel pathogenic mutations were localized. Functional analysis revealed that three of these mutations affecting amino acids Glu1459, Leu1668 and Glu1724 were critical for histone acetyltransferase activity. Twenty-eight novel CREBBP single-nucleotide polymorphisms (SNPs) were also identified in the Indian population. Linkage disequilibrium studies revealed associations between (i) SNP (rs129974/c.3836-206G>C) and mutation (p.Asp1340Ala); (ii) (rs130002) with mutation (p.Asn435Lys) and (iii) SNPs rs129974, rs130002 and SNP (c.3836-206G>C) signifying a disease affection status. In conclusion, the present study reports the highest detection rate of CREBBP mutations (76.9%) in RSTS patients to date, of which ten are predicted to be pathogenic and three critical for histone acetyltransferase activity. Moreover, identification of the association of CREBBP polymorphisms with disease susceptibility could be an important risk factor for the pathogenesis of RSTS.