Nanopore analysis of cis-diols in fruits.

Natural fruits contain a large variety of cis-diols. However, due to the lack of a high-resolution sensor that can simultaneously identify all cis-diols without a need of complex sample pretreatment, direct and rapid analysis of fruits in a hand-held device has never been previously reported. Nanopore, a versatile single molecule sensor, can be specially engineered to perform this task. A hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore modified with a sole phenylboronic acid (PBA) adapter is prepared. This engineered MspA accurately recognizes 1,2-diphenols, alditols, α-hydroxy acids and saccharides in prune, grape, lemon, different varieties of kiwifruits and commercial juice products. Assisted with a custom machine learning program, an accuracy of 99.3% is reported and the sample pretreatment is significantly simplified. Enantiomers such as DL-malic acids can also be directly identified, enabling sensing of synthetic food additives. Though demonstrated with fruits, these results suggest wide applications of nanopore in food and drug administration uses.

Simultaneous Identification of Major Thyroid Hormones by a Nickel Immobilized Biological Nanopore.

Thyroid hormones (THs) are a variety of iodine-containing hormones that demonstrate critical physiological impacts on cellular activities. The assessment of thyroid function and the diagnosis of thyroid disorders require accurate measurement of TH levels. However, largely due to their structural similarities, the simultaneous discrimination of different THs is challenging. Nanopores, single-molecule sensors with a high resolution, are suitable for this task. In this paper, a hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore containing a single nickel ion immobilized to the pore constriction has enabled simultaneous identification of five representative THs including l-thyroxine (T4), 3,3',5-triiodo-l-thyronine (T3), 3,3',5'-triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2) and 3,3'-diiodo-l-thyronine (3,3'-T2). To automate event classification and avoid human bias, a machine learning algorithm was also developed, reporting an accuracy of 99.0%. This sensing strategy is also applied in the analysis of TH in a real human serum environment, suggesting its potential use in a clinical diagnosis.

Discrimination of Disaccharide Isomers of Different Glycosidic Linkages Using a Modified MspA Nanopore.

Disaccharides are composed of two monosaccharide subunits joined by a glycosidic linkage in an α or ß configuration. Different combinations of isomeric monosaccharide subunits and different glycosidic linkages result in different isomeric disaccharide products. Thus, direct discrimination of these disaccharide isomers from a mixture is extremely difficult. In this paper, a hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore conjugated with a phenylboronic acid (PBA) adapter was applied for disaccharide sensing, with which three most widely known disaccharides in nature, including sucrose, lactose and maltose, were clearly discriminated. Besides, all six isomeric α-D-glucopyranosyl-D-fructoses, differing only in their glycosidic linkages, were also well resolved. Assisted by a custom machine learning algorithm, a 0.99 discrimination accuracy is achieved. Nanopore discrimination of disaccharide isomers with different glycosidic linkages, which has never been previously demonstrated, is inspiring for nanopore saccharide sequencing. This sensing capacity was also applied in direct identification of isomaltulose additives in a commercial sucrose-free yogurt, from which isomaltulose, lactose and L-lactic acid were simultaneously detected.

Identification of nucleoside monophosphates and their epigenetic modifications using an engineered nanopore.

RNA modifications play critical roles in the regulation of various biological processes and are associated with many human diseases. Direct identification of RNA modifications by sequencing remains challenging, however. Nanopore sequencing is promising, but the current strategy is complicated by sequence decoding. Sequential nanopore identification of enzymatically cleaved nucleoside monophosphates may simultaneously provide accurate sequence and modification information. Here we show a phenylboronic acid-modified hetero-octameric Mycobacterium smegmatis porin A nanopore, with which direct distinguishing between monophosphates of canonical nucleosides, 5-methylcytidine, N6-methyladenosine, N7-methylguanosine, N1-methyladenosine, inosine, pseudouridine and dihydrouridine was achieved. A custom machine learning algorithm, which reports an accuracy of 0.996, was also applied to the quantitative analysis of modifications in microRNA and natural transfer RNA. It is generally suitable for sensing of a variety of other nucleoside or nucleotide derivatives and may bring new insights to epigenetic RNA sequencing.

Nanopore Identification of Alditol Epimers and Their Application in Rapid Analysis of Alditol-Containing Drinks and Healthcare Products.

Alditols, which have a sweet taste but produce much lower calories than natural sugars, are widely used as artificial sweeteners. Alditols are the reduced forms of monosaccharide aldoses, and different alditols are diastereomers or epimers of each other and direct and rapid identification by conventional methods is difficult. Nanopores, which are emerging single-molecule sensors with exceptional resolution when engineered appropriately, are useful for the recognition of diastereomers and epimers. In this work, direct distinguishing of alditols corresponding to all 15 monosaccharide aldoses was achieved by a boronic acid-appended hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore (MspA-PBA). Thirteen alditols including glycerol, erythritol, threitol, adonitol, arabitol, xylitol, mannitol, sorbitol, allitol, dulcitol, iditol, talitol, and gulitol (l-sorbitol) could be fully distinguished, and their sensing features constitute a complete nanopore alditol database. To automate event classification, a custom machine-learning algorithm was developed and delivered a 99.9% validation accuracy. This strategy was also used to identify alditol components in commercially available "zero-sugar" drinks and healthcare products, suggesting their use in rapid and sensitive quality control for the food and medical industry.

A Nanopore-Based Saccharide Sensor.

Saccharides play critical roles in many forms of cellular activities. Saccharide structures are however complicated and similar, setting a technical hurdle for direct identification. Nanopores, which are emerging single molecule tools sensitive to minor structural differences between analytes, can be engineered to identity saccharides. A hetero-octameric Mycobacterium smegmatis porin A nanopore containing a phenylboronic acid was prepared, and was able to clearly identify nine monosaccharide types, including D-fructose, D-galactose, D-mannose, D-glucose, L-sorbose, D-ribose, D-xylose, L-rhamnose and N-acetyl-D-galactosamine. Minor structural differences between saccharide epimers can also be distinguished. To assist automatic event classification, a machine learning algorithm was developed, with which a general accuracy score of 0.96 was achieved. This sensing strategy is generally suitable for other saccharide types and may bring new insights to nanopore saccharide sequencing.

Non-binary Encoded Nucleic Acid Barcodes Directly Readable by a Nanopore.

A large collection of unique molecular barcodes is useful in the simultaneous sensing or screening of molecular analytes. Though the sequence of DNA has been widely applied to encode for molecular barcodes, decoding of these barcodes is normally assisted by sequencing. We here demonstrate a barcode system based solely on self-assembly of synthetic nucleic acids and direct nanopore decoding. Each molecular barcode is composed of "n" distinct information nodes in a non-binary manner and can be sequentially scanned and decoded by a Mycobacterium smegmatis porin A (MspA) nanopore. Nanopore events containing step-shaped features were consistently reported. 14 unique information nodes were developed which in principle could encode for 14n unique molecular barcodes in a barcode containing "n" information nodes. These barcode probes were adapted to detect different antibody proteins or cancer-related microRNAs, suggesting their immediate application in a wide variety of sensing applications.

MicroRNA-129-5p promotes proliferation and metastasis of hepatocellular carcinoma by regulating the BMP2 gene.

Hepatocellular carcinoma (HCC) is a malignant tumor that poses a serious threat to human health. Due to its occult onset and rapid development, HCC is a challenge to diagnose early and effectively treat, and thus patients with HCC often have an unfavorable prognosis. MicroRNA (miR)-129 and its target gene play an important role in the regulation of various diseases. Therefore, the aim of the present study was to investigate the role and mechanism of action for miR-129-5p in the development of HCC. Quantitative results of clinical samples analyzed using reverse transcription-quantitative PCR suggested that miR-129-5p had a significantly lower expression level in tumoral tissues compared with corresponding peritumoral tissues. Overexpression of miR-129-5p in HCC cells was performed using a transfection technique, followed by MTT, Transwell, invasion and wound healing assays to detect the effect of miR-129-5p on the cell cytotoxicity and metastasis of liver cancer in vitro. The downstream target gene of miR-129-5p, bone morphogenetic protein 2 (BMP2), was determined using a luciferase reporter assay. Overexpression of miR-129-5p played a vital role in decreasing cytotoxicity and promoting metastasis of HCC, which may be attributed to its inhibitory effect on the expression of its target gene, BMP2. In clinical samples, miR-129-5p expression levels were found to be negatively correlated with BMP2 and closely associated with HCC metastasis and infiltration. Collectively, the results suggested that miR-129-5p may contribute to proliferation and metastasis of HCC through its target gene, BMP2, and thus may be a potential novel therapeutic target for the treatment of HCC.

Pseudomonas aeruginosa: mannose sensitive hemagglutinin inhibits the growth of human hepatocarcinoma cells via mannose-mediated apoptosis.

A vaccine derived from the outer membrane proteins of the Gram-negative bacterium Pseudomonas aeruginosa has been shown to have immune modulatory properties. An inactivated mutant strain of P. aeruginosa with mannose sensitive hemagglutinin fimbria (PA-MSHA) has been used for adjuvant therapy for malignant cancer. In this study, the growth of human hepatocellular carcinoma Hep G2 and BEL-7402 cells is inhibited by PA-MSHA, but not by mannose-cleaved PA-MSHA. PA-MSHA-treated cells arrested in the S phase of the cell cycle and underwent apoptosis. We hypothesize that apoptosis induced by treatment of Hep G2 and BEL-7402 cells with PA-MSHA is mediated by the mannose residues of PA-MSHA and is propagated through the extrinsic apoptosis pathway directly through caspase-8. These data provide mechanistic details for the potential application of PA-MSHA-based treatment of hepatocellular carcinoma.

Disparities in mortality patterns among Canadian immigrants and refugees, 1980-1998: results of a national cohort study.

This study examines mortality patterns among Canadian immigrants, including both refugees and non-refugees, 1980-1998. Records of a stratified random sample of Canadian immigrants landing between 1980-1990 (N = 369,936) were probabilistically linked to mortality data (1980-1998). Mortality rates among immigrants were compared to those of the general Canadian population, stratifying by age, sex, immigration category, region of birth and time in Canada. Multivariate analysis examined mortality risks for various immigrant subgroups. Although immigrants presented lower all-cause mortality than the general Canadian population (SMR between 0.34 and 0.58), some cause-specific mortality rates were elevated among immigrants, including mortality from stroke, diabetes, infectious diseases (AIDS and hepatitis among certain subgroups), and certain cancers (liver and nasopharynx). Mortality rates differed by region of birth, and were higher among refugees than other immigrants. These results support the need to consider the heterogeneity of immigrant populations and vulnerable subgroups when developing targeted interventions.

Personal Health Practices.

HEALTH ISSUE: There are differences in health practices and self-rated health among different socio-demographic groups of women. The relationship between socio-demographic status and a) a range of health behaviours and b) a combination of multiple risk and multiple health promoting practices were examined. The relationship between self-rated health and health practices was also assessed. KEY FINDINGS: There were geographic differences in health practices with women in British Columbia having the highest odds of engaging in multiple health promoting practices, while women in Quebec had the lowest. Reports of engaging in multiple risk behaviours were most common in Ontario. Women from Ontario had the highest odds of reporting very good/excellent health and women from British Columbia had among the lowest odds.The data supported a strong social gradient between an increase in income/education and healthy practices, especially those that are health promoting. However, women with higher education were more likely to be overweight and those with higher incomes were more likely to drink alcohol regularly.Immigrant women were less likely to engage in multiple health risk practices compared to Canadian-born women. However, they were less likely to report very good/ excellent health than non- immigrants. While marriage appeared to have a generally protective effect on women's health practices, single women were more likely to be physically active and have a normal weight. DATA GAPS AND RECOMMENDATIONS: More sensitive indicators need to be developed to better understand possible reasons for the socioeconomic gradient. Data collection should focus on both rural and Aboriginal populations.