Molecular authentication of the medicinal herb Ruta graveolens (Rutaceae) and an adulterant using nuclear and chloroplast DNA markers

Dried parts of different plant species often look alike, especially in powdered form, making them very difficult to identify. Ruta graveolens, sold as a dried medicinal herb, can be adulterated with Euphorbia dracunculoides. The genomic DNA was isolated from the leaf powder (100 mg each) using the modified CTAB method. Internal transcribed spacer sequences of nuclear ribosomal DNA (nrDNA-ITS), and chloroplast spacer sequences (rpoB and rpoC1) are regarded as potential genes for plant DNA barcoding. We amplified and sequenced these spacer sequences and confirmed the sequences with a BLAST search. Sequence alignment was performed using ClustalX to look for differences in the sequences. A DNA marker was developed based on rpoB and rpoC1 of the nrDNA-ITS for the identification of the adulterant E. dracunculoides in samples of R. graveolens that are sold in local herbal markets. Sequence-characterized amplified region markers of 289 and 264 bp for R. graveolens and 424 bp for E. dracunculoides were developed from dissimilar sequences of this nrDNA-ITS to speed up the authentication process. This marker successfully distinguished these species in extracted samples with as little as 5 ng DNA/μL extract.     

Molecular diversity analysis of eggplant (Solanum melongena) genetic resources

Eggplant (Solanum melongena), a vegetable that is cultivated worldwide, is of considerable importance to agriculture in China. We analyzed the diversity of this plant using inter-simple sequence repeat (ISSR) and RAPD procedures to subdivide 143 Chinese-cultivated eggplants based on coefficient of parentage, genetic diversity index (GDI) and canonical discriminant analysis. ISSR markers were more effective than RAPD markers for detecting genetic diversity, which ranged from 0.10-0.51, slightly lower than what is known from other crops. Our ISSR/RAPD data provide molecular evidence that coincides with morphological-based classification into three varieties and further subdivision into eight groups, except for two groups. Intensive use of elite parents and extensive crossing within groups have resulted in increased coefficient of parentage and proportional contribution but decreased GDI during the past decades. The mean coefficient of parentage and proportional contribution increased from 0.05 to 0.10% and from 3.22 to 6.46% during 1980-1991 and 1992-2003, respectively. The GDI of landraces was 0.21, higher than the 0.09 and 0.08 calculated for the hybrid cultivars released during the two periods. The recent introduction of alien genotypes into eggplant breeding programs may broaden the genetic base.     

Childhood obesity and parental smoking as risk factors for childhood ADHD in Liverpool children

ADHD prevalence has risen in parallel with rising prevalence of pregnancy smoking and childhood obesity. The objective was to determine the epidemiological association of pregnancy smoking and childhood obesity with ADHD. A cross-sectional community study was conducted in 2006 using a parental questionnaire. A total of 1,074 schoolchildren aged 5-11 years were enrolled from 15 primary schools in a lower socio-economic area of Merseyside. ADHD was defined by the question "does your child have Attention Deficit Hyperactivity Disorder, (ADHD), which has been diagnosed by a doctor?" The prevalence estimates for childhood obesity, maternal smoking during pregnancy and childhood ADHD were 14.9% (116/777), 28.0% (269/955), and 3.4% (32/945), respectively. ADHD prevalence increased fivefold in children with obesity (RR, 4.80, 95% CI 2.2-10.4, P < 0.001) and more than twofold in children of mothers who smoked during pregnancy (RR, 2.44, 95% CI 1.2-4.9, P = 0.02). Regression analysis adjusting for obesity, overweight, maternal smoking during pregnancy, heavy maternal smoking, household member smoking during pregnancy, doctor-diagnosed asthma, preterm birth, and low birthweight showed significant independent associations of ADHD prevalence with obesity (AOR, 4.66, 95% CI 1.57-13.89, P = 0.006) and pregnancy smoking (AOR, 3.19, 95% CI 1.08-9.49, P = 0.04). There was a positive dose-response association of ADHD with the number of maternal cigarettes smoked during pregnancy. Measures to reduce both smoking among pregnant women and childhood obesity might reduce prevalence of childhood ADHD.     

In vivo SPECT reporter gene imaging of regulatory T cells

Regulatory T cells (Tregs) were identified several years ago and are key in controlling autoimmune diseases and limiting immune responses to foreign antigens, including alloantigens. In vivo imaging techniques including intravital microscopy as well as whole body imaging using bioluminescence probes have contributed to the understanding of in vivo Treg function, their mechanisms of action and target cells. Imaging of the human sodium/iodide symporter via Single Photon Emission Computed Tomography (SPECT) has been used to image various cell types in vivo. It has several advantages over the aforementioned imaging techniques including high sensitivity, it allows non-invasive whole body studies of viable cell migration and localisation of cells over time and lastly it may offer the possibility to be translated to the clinic. This study addresses whether SPECT/CT imaging can be used to visualise the migratory pattern of Tregs in vivo. Treg lines derived from CD4(+)CD25(+)FoxP3(+) cells were retrovirally transduced with a construct encoding for the human Sodium Iodide Symporter (NIS) and the fluorescent protein mCherry and stimulated with autologous DCs. NIS expressing self-specific Tregs were specifically radiolabelled in vitro with Technetium-99m pertechnetate ((99m)TcO(4)(-)) and exposure of these cells to radioactivity did not affect cell viability, phenotype or function. In addition adoptively transferred Treg-NIS cells were imaged in vivo in C57BL/6 (BL/6) mice by SPECT/CT using (99m)TcO(4)(-). After 24 hours NIS expressing Tregs were observed in the spleen and their localisation was further confirmed by organ biodistribution studies and flow cytometry analysis. The data presented here suggests that SPECT/CT imaging can be utilised in preclinical imaging studies of adoptively transferred Tregs without affecting Treg function and viability thereby allowing longitudinal studies within disease models.     

Investigation of cellular and molecular responses to pulsed focused ultrasound in a mouse model

Continuous focused ultrasound (cFUS) has been widely used for thermal ablation of tissues, relying on continuous exposures to generate temperatures necessary to induce coagulative necrosis. Pulsed FUS (pFUS) employs non-continuous exposures that lower the rate of energy deposition and allow cooling to occur between pulses, thereby minimizing thermal effects and emphasizing effects created by non-thermal mechanisms of FUS (i.e., acoustic radiation forces and acoustic cavitation). pFUS has shown promise for a variety of applications including drug and nanoparticle delivery; however, little is understood about the effects these exposures have on tissue, especially with regard to cellular pro-homing factors (growth factors, cytokines, and cell adhesion molecules). We examined changes in murine hamstring muscle following pFUS or cFUS and demonstrate that pFUS, unlike cFUS, has little effect on the histological integrity of muscle and does not induce cell death. Infiltration of macrophages was observed 3 and 8 days following pFUS or cFUS exposures. pFUS increased expression of several cytokines (e.g., IL-1α, IL-1β, TNFα, INFγ, MIP-1α, MCP-1, and GMCSF) creating a local cytokine gradient on days 0 and 1 post-pFUS that returns to baseline levels by day 3 post-pFUS. pFUS exposures induced upregulation of other signaling molecules (e.g., VEGF, FGF, PlGF, HGF, and SDF-1α) and cell adhesion molecules (e.g., ICAM-1 and VCAM-1) on muscle vasculature. The observed molecular changes in muscle following pFUS may be utilized to target cellular therapies by increasing homing to areas of pathology.     

Long-term effects of foetal undernutrition on intermediary metabolism in growing lambs

The objective of this study was to investigate the effects of foetal undernutrition on the metabolism in growing lambs. Seven-month-old lambs whose mothers had been fed either restrictively (RN; n = 14) or adequately (AN; n = 6) in late gestation were fasted for three days. One hour before fasting and after 48 h and 72 h fasting, changes in plasma concentrations of metabolites, i.e. glucose, nonesterified fatty acids (NEFA), 3-beta-hydroxybutyrate (BOHB) and urea as well as hormones, i.e. insulin, the insulin-like growth factor (IGF-I) and leptin, were determined. Blood glucose, NEFA, urea, insulin, IGF-I and leptin were not different between the two groups of lambs. Unexpectedly, at the end of the 3 d fasting, in spite of lower NEFA concentration (1.6 +/- 0.03 vs. 1.9 +/- 0.05 mM in Groups RN and AN, respectively), the BOHB concentration in RN lambs (0.94 +/- 0.02 mM) was significantly higher than that in AN lambs (0.78 +/- 0.04 mM). This higher rate of BOHB production might be interpreted as perturbations in ketone body metabolism potentially induced by undernutrition during foetal life. However, more investigations are necessary to clarify this interrelationship.     

Selective inhibition of GluN2D-containing N-methyl-D-aspartate receptors prevents tissue plasminogen activator-promoted neurotoxicity both in vitro and in vivo

Background

Tau protein is the principal component of the neurofibrillary tangles found in Alzheimer's disease, where it is hyperphosphorylated on serine and threonine residues, and recently phosphotyrosine has been demonstrated. The Src-family kinase Fyn has been linked circumstantially to the pathology of Alzheimer's disease, and shown to phosphorylate Tyr18. Recently another Src-family kinase, Lck, has been identified as a genetic risk factor for this disease.

Results

In this study we show that Lck is a tau kinase. In vitro, comparison of Lck and Fyn showed that while both kinases phosphorylated Tyr18 preferentially, Lck phosphorylated other tyrosines somewhat better than Fyn. In co-transfected COS-7 cells, mutating any one of the five tyrosines in tau to phenylalanine reduced the apparent level of tau tyrosine phosphorylation to 25-40% of that given by wild-type tau. Consistent with this, tau mutants with only one remaining tyrosine gave poor phosphorylation; however, Tyr18 was phosphorylated better than the others.

Conclusions

Fyn and Lck have subtle differences in their properties as tau kinases, and the phosphorylation of tau is one mechanism by which the genetic risk associated with Lck might be expressed pathogenically.     

Signaling-mediated cross-talk modulates swarming and biofilm formation in a coral pathogen Serratia marcescens

Interactions within microbial communities associated with marine holobionts contribute importantly to the health of these symbiotic organisms formed by invertebrates, dinoflagellates and bacteria. However, mechanisms that control invertebrate-associated microbiota are not yet fully understood. Hydrophobic compounds that were isolated from surfaces of asymptomatic corals inhibited biofilm formation by the white pox pathogen Serratia marcescens PDL100, indicating that signals capable of affecting the associated microbiota are produced in situ. However, neither the origin nor structures of these signals are currently known. A functional survey of bacteria recovered from coral mucus and from cultures of the dinoflagellate Symbiodinium spp. revealed that they could alter swarming and biofilm formation in S. marcescens. As swarming and biofilm formation are inversely regulated, the ability of some native α-proteobacteria to affect both behaviors suggests that the α-proteobacterial signal(s) target a global regulatory switch controlling the behaviors in the pathogen. Isolates of Marinobacter sp. inhibited both biofilm formation and swarming in S. marcescens PDL100, without affecting growth of the coral pathogen, indicative of the production of multiple inhibitors, likely targeting lower level regulatory genes or functions. A multi-species cocktail containing these strains inhibited progression of a disease caused by S. marcescens in a model polyp Aiptasia pallida. An α-proteobacterial isolate 44B9 had a similar effect. Even though ∼4% of native holobiont-associated bacteria produced compounds capable of triggering responses in well-characterized N-acyl homoserine lactone (AHL) biosensors, there was no strong correlation between the production of AHL-like signals and disruption of biofilms or swarming in S. marcescens.