Engineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics.

Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation and cooling it for replication. The process uses a specialized hardware and exposes biomolecules to temperatures above 95 °C. Here, we engineer a PcrA M6 helicase with enhanced speed and processivity to replace the heating step by enzymatic DNA unwinding while retaining desired PCR characteristics. We name this isothermal amplification method SHARP (SSB-Helicase Assisted Rapid PCR) because it uses the engineered helicase and single-stranded DNA binding protein (SSB) in addition to standard PCR reagents. SHARP can generate amplicons with lengths of up to 6000 base pairs. SHARP can produce functional DNA, a plasmid that imparts cells with antibiotic resistance, and can amplify specific fragments from genomic DNA of human cells. We further use SHARP to assess the outcome of CRISPR-Cas9 editing at endogenous genomic sites.

Highly sensitive and selective detection of single-nucleotide polymorphisms using gold nanoparticle MutS enzymes and a micro cantilever resonator.

Mutant DNAs are important markers useful for the diagnosis of human disease. Single-nucleotide polymorphisms (SNPs) represent the most common types of DNA mutations. As there is only a one base pair change in a single nucleotide between the SNP and the wild-type DNA, it is difficult to distinguish the SNPs. In this report, a highly sensitive and selective detection and discrimination of SNPs is performed using MutS, gold nanoparticles (AuNP) and a resonator. A single mismatched base exists between the SNP mutation and the probe DNA on the resonator, and MutS binds to the DNA at the location of the mismatch. As MutS is attached to AuNP (MutS-AuNP), both MutS and AuNP are adsorbed onto the resonator. The detection is based on the resonance frequency shift of the resonator following the adsorption of MutS-AuNP on the resonator. Highly sensitive detection of DNA mutations was achieved using AuNPs that act as mass amplifiers, and the obtained limit of detected was 100 fM. Additionally, our proposed method detected mutations in the presence of as little as 0.1% wild-type, and discrimination of specific mutations was also achieved. The results obtained from our proposed method suggest its potential for diagnosing cancer patients.

Association between comorbidity and participation in breast and cervical cancer screening: A systematic review and meta-analysis.

BACKGROUND: Comorbidity is associated with poor outcomes for cancer patients but it is less clear how it influences cancer prevention and early detection. This review synthesizes evidence from studies that have quantified the association between comorbidity and participation in breast and cervical screening. METHODS: PubMed, CINAHL and EMBASE databases were systematically searched using key terms related to cancer screening and comorbidity for original research articles published between 1 January 1991 and 21 March 2016. Two reviewers independently screened 1283 studies that met eligibility criteria related to Population (adult, non-cancer populations), Exposure (comorbidity), Comparison (a 'no comorbidity' group), and Outcome (participation in breast cancer or cervical screening). Data was extracted and risk of bias assessed using a standardised tool from the 22 studies identified for inclusion (17 breast; 13 cervical). Meta-analyses were performed for participation in breast and cervical screening, stratified by important study characteristics. RESULTS: The majority of studies were conducted in the United States. Results of individual studies were variable. Most had medium to high risk of bias. Based on the three "low risk of bias" studies, mammography screening was less common among those with comorbidity (pooled Odds Ratio 0.66, 95%CI 0.44-0.88). The one "low risk of bias" study of cervical screening reported a negative association between comorbidity and participation. CONCLUSION: While a definitive conclusion could not be drawn, the results from high quality studies suggest that women with comorbidity are less likely to participate in breast, and possibly cervical, cancer screening.

A Study on the Dietary Intake and the Nutritional Status among the Pancreatic Cancer Surgical Patients.

The adequate dietary intake is important to maintain the nutritional status of the patients after pancreatic cancer surgery. This prospective study was designed to investigate the dietary intake and the nutritional status of the patients who had pancreatic cancer surgery. Thirty-one patients (15 men, 16 women) were enrolled and measured body weight, body mass index (BMI), nutritional risk index (NRI), and Malnutrition Universal Screening Tool (MUST). Actual oral intake with nutritional impact symptoms recorded on the clinical research foam at every meal and medical information were collected from electronic medical charts. The rates of malnutrition at admission were 45.1% (14/31) and 28.9% (9/31) by NRI and MUST method, respectively, but those were increased to 87% (27/31) and 86.6% (26/31) after operation on discharge. The median values of daily intake of energy, carbohydrates, fat, and protein were 588.1 kcal, 96.0 g, 11.8 g, and 27.0 g, respectively. Most patients (n = 20, 64.5%) experienced two or more symptoms such as anorexia, abdominal bloating and early satiety. There were negative correlations between C-reactive protein (CRP) levels and the intake of total energy, protein, fat, and zinc. The rates of malnutrition were increased sharply after surgery and the dietary intake also influenced the inflammatory indicators. The results suggested that need of considering special therapeutic diets for the patients who received pancreatic surgery.

Protein profiling in human sera for identification of potential lung cancer biomarkers using antibody microarray.

To identify potential biomarkers of lung cancer (LC), profiling of proteins in sera obtained from healthy and LC patients was determined using an antibody microarray. Based on our previous study on mRNA expression profiles between patients with LC and healthy persons, 19 proteins of interest were selected as targets for fabrication of an antibody microarray. Antibody to each protein and five nonspecific control antibodies were spotted onto a hydrogel-coated glass slide and used for profiling of proteins in sera of LC patients in a two-color fluorescence assay. Forty-eight human sera samples were analyzed, and expression profiling of proteins were represented by the internally normalized ratio method. Six proteins were distinctly down-regulated in sera of LC patients; this observation was validated by Wilcoxon test, false discovery rate, and Western blotting. Blind test of other 32 human sera using the antibody microarray followed by hierarchical clustering analysis revealed an approximate sensitivity of 88%, specificity of 80%, and an accuracy of 84%, respectively, in classifying the sera, which supports the potential of the six identified proteins as biomarkers for the prognosis of lung cancer.