Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA

Background

Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns.

Methodology/Principal Findings

In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level.

Conclusions

Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.     

Effect of meal size on excess post-exercise oxygen consumption in fishes with different locomotive and digestive performance

Effects of feeding on pre-exercise VO₂ and excess post-exercise oxygen consumption (EPOC) after exhaustive exercise were investigated in sedentary southern catfish, active herbivorous grass carp, omnivorous crucian carp, and sluggish omnivorous darkbarbel catfish to test whether feeding had different effects on EPOC and to compare EPOC in fishes with different ecological habits. For fasting fish, the pre-exercise and peak post-exercise VO₂ were higher and recovery rates were faster in crucian carp and grass carp compared to those of darkbarbel catfish and southern catfish. EPOC magnitudes of grass carp and southern catfish were significantly larger than those of crucian carp and darkbarbel catfish. Feeding had no significant effect on peak post-exercise VO₂, recovery rate, and EPOC magnitude in grass carp. Both the pre-exercise and peak post-exercise VO₂ increased with meal size, while the EPOC magnitude and duration decreased significantly in the larger meal size groups of crucian carp and southern catfish. In darkbarbel catfish, both the pre-exercise and peak post-exercise VO₂ increased with meal size, but the VO₂ increment elicited by exercise was larger in feeding groups compared with the fasting group. These results suggest that (1) the characteristics of the post-exercise VO₂ profile, such as peak post-exercise VO₂ and recovery rate, were closely related to the activity of fishes, whereas the EPOC magnitude was not and (2) the effects of feeding on EPOC were more closely related to the postprandial increase in VO₂.     

Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers

Despite a long-suspected role in the development of human colorectal cancer (CRC), the composition of gut microbiota in CRC patients has not been adequately described. In this study, fecal bacterial diversity in CRC patients (n=46) and healthy volunteers (n=56) were profiled by 454 pyrosequencing of the V3 region of the 16S ribosomal RNA gene. Both principal component analysis and UniFrac analysis showed structural segregation between the two populations. Forty-eight operational taxonomic units (OTUs) were identified by redundancy analysis as key variables significantly associated with the structural difference. One OTU closely related to Bacteroides fragilis was enriched in the gut microbiota of CRC patients, whereas three OTUs related to Bacteroides vulgatus and Bacteroides uniformis were enriched in that of healthy volunteers. A total of 11 OTUs belonging to the genera Enterococcus, Escherichia/Shigella, Klebsiella, Streptococcus and Peptostreptococcus were significantly more abundant in the gut microbiota of CRC patients, and 5 OTUs belonging to the genus Roseburia and other butyrate-producing bacteria of the family Lachnospiraceae were less abundant. Real-time quantitative PCR further validated the significant reduction of butyrate-producing bacteria in the gut microbiota of CRC patients by measuring the copy numbers of butyryl-coenzyme A CoA transferase genes (Mann–Whitney test, P<0.01). Reduction of butyrate producers and increase of opportunistic pathogens may constitute a major structural imbalance of gut microbiota in CRC patients.     

Inhibition of integrin β3, a binding partner of kallistatin,leads to reduced viability,invasion and proliferation in NCI-H446 cells

Background

Kallistatin is a serine proteinase inhibitor and heparin-binding protein. It is considered an endogenous angiogenic inhibitor. In addition, multiple studies demonstrated that kallistatin directly inhibits cancer cell growth. However, the molecular mechanisms underlying these effects remain unclear.

Methods

Pull-down, immunoprecipitation, and immunoblotting were used for binding experiments. To elucidate the mechanisms, integrin β3 knockdown (siRNA) or blockage (antibody treatment) on the cell surface of small the cell lung cancer NCI-H446 cell line was used.

Results

Interestingly, kallistatin was capable of binding integrin β3 on the cell surface of NCI-H446 cells. Meanwhile, integrin β3 knockdown or blockage resulted in loss of antitumor activities induced by kallistatin. Furthermore, kallistatin suppressed tyrosine phosphorylation of integrin β3 and its downstream signaling pathways, including FAK/-Src, AKT and Erk/MAPK. Viability, proliferation and migration of NCI-H446 cells were inhibited by kallistatin, with Bcl-2 and Grb2 downregulation, and Bax, cleaved caspase-9 and caspase 3 upregulation.

Conclusions

These findings reveal a novel role for kallistatin in preventing small cell lung cancer growth and mobility, by direct interaction with integrin β3, leading to blockade of the related signaling pathway.

     

A simple and efficient method for generating Nurr1-positive neuronal stem cells from human wisdom teeth (tNSC) and the potential of tNSC for stroke therapy

Background aimsWe have isolated human neuronal stem cells from exfoliated third molars (wisdom teeth) using a simple and efficient method. The cultured neuronal stem cells (designated tNSC) expressed embryonic and adult stem cell markers, markers for chemotatic factor and its corresponding ligand, as well as neuron proteins. The tNSC expressed genes of Nurr1, NF-M and nestin. They were used to treat middle cerebral artery occlusion (MCAO) surgery-inflicted Sprague–Dawley (SD) rats to assess their therapeutic potential for stroke therapy.MethodsFor each tNSC cell line, a normal human impacted wisdom tooth was collected from a donor with consent. The tooth was cleaned thoroughly with normal saline. The molar was vigorously shaken or vortexed for 30 min in a 50-mL conical tube with 15–20 mL normal saline. The mixture of dental pulp was collected by centrifugation and cultured in a 25-cm² tissue culture flask with 4–5 mL Medium 199 supplemented with 5–10% fetal calf serum. The tNSC harvested from tissue culture, at a concentration of 1–2 × 10⁵, were suspended in 3 µL saline solution and injected into the right dorsolateral striatum of experimental animals inflicted with MCAO.ResultsBehavioral measurements of the tNSC-treated SD rats showed a significant recovery from neurologic dysfunction after MCAO treatment. In contrast, a sham group of SD rats failed to recover from the surgery. Immunohistochemistry analysis of brain sections of the tNSC-treated SD rats showed survival of the transplanted cells.ConclusionsThese results suggest that adult neuronal stem cells may be procured from third molars, and tNSC thus cultivated have potential for treatment of stroke-inflicted rats.     

Investigating factors associated with success of breastfeeding in first-time mothers undergoing epidural analgesia: a prospective cohort study

Background

We investigated the possible risk factors that could influence the likelihood of breastfeeding at 5 to 9 weeks postpartum with our primary aim being to analyse the associations between psychological vulnerabilities, such as peripartum depression and anxiety, and continued breastfeeding. Our secondary aim was to investigate other non-psychological factors’ influence on continued breastfeeding.

Methods

A prospective cohort study was conducted in KK Women’s and Children’s Hospital in Singapore. Healthy nulliparous parturients at ≥36 weeks gestation with a singleton fetus who received epidural analgesia were recruited. Demographic and anaesthetic data were obtained. Self-reported psychological and pain determinants such as anxiety (State-Trait Anxiety Inventory), depression (Edinburgh Postnatal Depression Scale), stress (Perceived Stress Scale), pain susceptibility (Pain Catastrophizing Scale) and pain perception (McGill Pain Questionnaire) were also recorded at baseline. A phone interview was then performed at 5 to 9 weeks postpartum to obtain information on breastfeeding status.

Results

329 participants were included into this study, of which 263 (79.9%) of them were still breastfeeding at 5 weeks postpartum. Multivariate logistic regression analysis showed that a higher State-Trait Anxiety Inventory score (Adjusted Odds Ratio [AOR] 0.97; 95% Confidence Interval [CI] 0.94, 1.00) at baseline, higher intrapartum blood loss (AOR 0.76; 95% CI 0.61, 0.93), and occurrence of fetal anomalies (AOR 0.15; 95% CI 0.03, 0.72) were associated with reduced likelihood of breastfeeding at 5 to 9 weeks postpartum. Indians (AOR 0.56; 95% CI 0.20, 1.53), Malays (AOR 0.30; 95% CI 0.14, 0.62) and other ethnicities (AOR 0.36; 95% CI 0.16, 0.83) were less likely to continue breastfeeding compared to Chinese participants. On the other hand, receiving any support services on breastfeeding during the participants’ hospital stay was 3.3 times more likely (AOR 3.30; 95% CI 1.21, 9.02) to increase the likelihood of breastfeeding at 5 to 9 weeks postpartum.

Conclusion

We identified 5 independent association factors that could have significant influences on breastfeeding at 5 to 9 weeks postpartum. Healthcare providers could utilize this risk stratification to identify parturients likely to have poorer breastfeeding outcomes and undertake interventions that may help safeguard optimization of breastfeeding outcomes and parturient care.

Trial registration

Clinicaltrials.gov NCT02278601. Registered 26 October 2014.