QuikChange shuffling: a convenient and robust method for site-directed mutagenesis and random recombination of homologous genes

Here we describe a robust method, termed QuikChange shuffling, for efficient site-directed mutagenesis and random recombination of homologous genes. The homologous genes are fragmented, and the random fragments are reassembled in a self-priming polymerase reaction to obtain chimeric genes. The product is then mixed with linearized vector and two pairs of complementary mutagenic primers, followed by assembly of the chimeric genes and linearized vector through QuikChange-like amplification to introduce recombinant plasmids with a site-directed mutation. The method, which can yield 100% chimeric genes after library construction, is more convenient and efficient than current DNA shuffling methods.     

A model of BmK CT in inhibiting glioma cell migration via matrix metalloproteinase-2 from experimental and molecular dynamics simulation study

The significance of BmK CT, a key chlorotoxin-like peptide isolated from the scorpion venom of Buthus martensii Karsch, is a novel blocker of the chloride ion channel and matrix metalloproteinase-2 (MMP-2). Site-directed mutagenesis of BmK CT, wound healing assay, gelatin zymography assay and computational simulation highlight the importance of electrostatic contribution to BmK CT–MMP-2 catalytic domain complex and a model of BmK CT–MMP-2 catalytic domain complex is therefore proposed. This is the first documentation of the structural mechanism of in the inhibition of glioma cell migration by BmK CT and may lead to the molecular design of specific inhibitors of MMP-2.     

Molecular cloning, tissue expression and SNP analysis in the goat nerve growth factor gene

In this study, we cloned the full coding region of NGF gene from the caprine ovary. Result showed the caprine NGF cDNA (GenBank Accession No. JQ308184) contained a 726 bp open reading frame encoding a protein with 241 amino acid residues. Bioinformatic analysis indicated that caprine NGF amino acid sequence was 83–99 % identical to that of mouse, pig, dog, human and bovine. It was predicted that caprine NGF contained nine serine phosphorylation loci, four threonine phosphorylation loci and nine specific PKC phosphorylation loci. The NGF mRNA expression pattern showed that NGF gene was expressed highly in ovary. This work provided an important experimental basis for further research on the function of NGF in goat. A single nucleotide polymorphism (A705G) in the coding region of NGF gene was detected by PCR–RFLP and DNA sequencing in 630 goats of three breeds. The frequencies of G allele were 0.52–0.61, and frequencies of A allele were 0.48–0.39 for SN, GZ and BG breeds, respectively. The does with GG genotype had higher litter size than those with GA and AA genotypes (P < 0.05). Hence, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the NGF gene could serve as a genetic marker for litter size in goat breeding.     

Recent advances in TMEM16A: Structure,function, and disease

TMEM16A (also known as anoctamin 1, ANO1) is the molecular basis of the calcium-activated chloride channels, with ten transmembrane segments. Recently, atomic structures of the transmembrane domains of mouse TMEM16A (mTMEM16A) were determined by single-particle electron cryomicroscopy. This gives us a solid ground to discuss the electrophysiological properties and functions of TMEM16A. TMEM16A is reported to be dually regulated by Ca²⁺ and voltage. In addition, the dysfunction of TMEM16A has been found to be involved in many diseases including cystic fibrosis, various cancers, hypertension, and gastrointestinal motility disorders. TMEM16A is overexpressed in many cancers, including gastrointestinal stromal tumors, gastric cancer, head and neck squamous cell carcinoma (HNSCC), colon cancer, pancreatic ductal adenocarcinoma, and esophageal cancer. Furthermore, overexpression of TMEM16A is related to the occurrence, proliferation, and migration of tumor cells. To date, several studies have shown that many natural compounds and synthetic compounds have regulatory effects on TMEM16A. These small molecule compounds might be novel drugs for the treatment of diseases caused by TMEM16A dysfunction in the future. In addition, recent studies have shown that TMEM16A plays different roles in different diseases through different signal transduction pathways. This review discusses the topology, electrophysiological properties, modulators and functions of TMEM16A in mediates nociception, gastrointestinal dysfunction, hypertension, and cancer and focuses on multiple regulatory mechanisms regarding TMEM16A.     

The Alzheimer Disease Protective Mutation A2T Modulates Kinetic and Thermodynamic Properties of Amyloid-β (Aβ) Aggregation

Missense mutations in alanine 673 of the amyloid precursor protein (APP), which corresponds to the second alanine of the amyloid β (Aβ) sequence, have dramatic impact on the risk for Alzheimer disease; A2V is causative, and A2T is protective. Assuming a crucial role of amyloid-Aβ in neurodegeneration, we hypothesized that both A2V and A2T mutations cause distinct changes in Aβ properties that may at least partially explain these completely different phenotypes. Using human APP-overexpressing primary neurons, we observed significantly decreased Aβ production in the A2T mutant along with an enhanced Aβ generation in the A2V mutant confirming earlier data from non-neuronal cell lines. More importantly, thioflavin T fluorescence assays revealed that the mutations, while having little effect on Aβ42 peptide aggregation, dramatically change the properties of the Aβ40 pool with A2V accelerating and A2T delaying aggregation of the Aβ peptides. In line with the kinetic data, Aβ A2T demonstrated an increase in the solubility at equilibrium, an effect that was also observed in all mixtures of the A2T mutant with the wild type Aβ40. We propose that in addition to the reduced β-secretase cleavage of APP, the impaired propensity to aggregate may be part of the protective effect conferred by A2T substitution. The interpretation of the protective effect of this mutation is thus much more complicated than proposed previously.     

Expression of bacterial beta-galactosidase in animal cells

A recombinant plasmid containing the gene for bacterial β-galactosidase, situated close to the simian virus 40 early promoter, has been constructed. Transfection of CHO, L, and COS-1 cells with this plasmid led to the expression and appearance of the enzyme. Using this system, we have developed a series of promoter cloning vehicles capable of accepting promoter signals for animal genes.     

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.