Multiplex SNP discrimination

Multiplex hybridization reactions of perfectly matched duplexes and duplexes containing a single basepair mismatch (SNPs) were investigated on DNA microarrays. Effects of duplex length, G-C percentage, and relative position of the SNP on duplex hybridization and SNP resolution were determined. Our theoretical model of multiplex hybridization accurately predicts observed results and implicates target concentration as a critical variable in multiplex SNP detection.     

Dynamic L-type CaV1.2 channel trafficking facilitates CaV1.2 clustering and cooperative gating

L-type Ca_V1.2 channels are key regulators of gene expression, cell excitability and muscle contraction. Ca_V1.2 channels organize in clusters throughout the plasma membrane. This channel organization has been suggested to contribute to the concerted activation of adjacent Ca_V1.2 channels (e.g. cooperative gating). Here, we tested the hypothesis that dynamic intracellular and perimembrane trafficking of Ca_V1.2 channels is critical for formation and dissolution of functional channel clusters mediating cooperative gating. We found that Ca_V1.2 moves in vesicular structures of circular and tubular shape with diverse intracellular and submembrane trafficking patterns. Both microtubules and actin filaments are required for dynamic movement of Ca_V1.2 vesicles. These vesicles undergo constitutive homotypic fusion and fission events that sustain Ca_V1.2 clustering, channel activity and cooperative gating. Our study suggests that Ca_V1.2 clusters and activity can be modulated by diverse and unique intracellular and perimembrane vesicular dynamics to fine-tune Ca²⁺ signals.     

Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.     

Outcomes associated with planned place of birth among women with low-risk pregnancies

Background:Previous studies have shown that planned home birth is associated with a decreased likelihood of intrapartum intervention with no difference in neonatal outcomes compared with planned hospital birth. The purpose of our study was to evaluate different birth settings by comparing neonatal mortality, morbidity and rates of birth interventions between planned home and planned hospital births in Ontario, Canada.Methods:We used a provincial database of all midwifery-booked pregnancies between 2006 and 2009 to compare women who planned home birth at the onset of labour to a matched cohort of women with low-risk pregnancies who had planned hospital births attended by midwives. We conducted subgroup analyses by parity. Our primary outcome was stillbirth, neonatal death (< 28 d) or serious morbidity (Apgar score < 4 at 5 min or resuscitation with positive pressure ventilation and cardiac compressions).Results:We compared 11 493 planned home births and 11 493 planned hospital births. The risk of our primary outcome did not differ significantly by planned place of birth (relative risk [RR] 1.03, 95% confidence interval [CI] 0.68–1.55). These findings held true for both nulliparous (RR 1.04, 95% CI 0.62–1.73) and multiparous women (RR 1.00, 95% CI 0.49–2.05). All intrapartum interventions were lower among planned home births.Interpretation:Compared with planned hospital birth, planned home birth attended by midwives in a jurisdiction where home birth is well-integrated into the health care system was not associated with a difference in serious adverse neonatal outcomes but was associated with fewer intrapartum interventions.In Ontario, Canada, the College of Midwives of Ontario has regulated midwifery since 1994, and increasing numbers of women with low obstetrical risk and their newborns receive care in a publicly funded, midwifery-led continuity of care model.¹ Midwives have admission and discharge privileges at their local hospitals and are able to consult or transfer care to other health care providers if required. In Ontario, midwives attend a small proportion of all births in the province (10%), and about 20% of the births they attend take place at home.² A comprehensive record is maintained for every woman and infant in a midwife’s care. Until 2009, this record was submitted to the provincial Ministry of Health and Long-term Care (MOHLTC) through the Ontario Midwifery Program to access reimbursement for care provided.In the last century, Western culture has come to view hospital birth as safer than home birth.³ Recently, however, the value of hospital birth for all women with low-risk pregnancies has come into question; it has been suggested that in the absence of benefit, a planned hospital birth for this population may increase the use of intrapartum interventions, including cesarean delivery.^4–7 Even though recent studies comparing planned home and hospital births have had moderate sample sizes, they are individually limited in their ability to report definitively on rare outcomes such as death. Owing to a lack of evidence from randomized controlled trials (RCTs) to show that restricting a woman’s freedom to choose a place of birth prevents harm, the authors of a 2012 Cochrane review of planned hospital versus planned home births concluded that home birth services with collaborative medical backup should be established and offered to women with low-risk pregnancies in all jurisdictions.⁸ This conclusion, along with findings from the large English Birthplace Cohort Study,⁴ may be what prompted the National Institute for Health and Care Excellence (NICE) in England to update its intrapartum care guidelines to recommend that, for women at low risk of birth-associated complications, home birth should be considered a generally safe option.⁹ With the paucity of information derived from RCTs,⁸ observational studies are essential to continue to inform and monitor maternal and infant outcomes for women at low obstetrical risk who plan home or hospital birth, and to continue to provide pregnant women with quality information about choice of birthplace.The primary purpose of this retrospective cohort study was to determine the risk of stillbirth or neonatal death or serious neonatal morbidity among women at low obstetrical risk whose deliveries were attended by midwives and who had planned a home birth at the onset of labour, compared with women at low obstetrical risk who planned a hospital birth at the onset of labour. In addition, we also compared the incidence of maternal death and morbidity, birth interventions and breastfeeding between planned home births and planned hospital births.     

Effects of competition and season on survival and maturation of young bank vole females

In territorial microtines intra-specific density dependent processes can limit the maturation of individuals during the summer of their birth. This may have demographic consequences by affecting the number and the age distribution of breeding individuals in the population. Little is known about this process on a community level, though populations of many northern microtine species fluctuate in synchrony and are known to interfere socially with each other. We experimentally studied the influence of the field vole Microtus agrestis on maturation, breeding, space use and survival of weanling bank voles, Clethrionomys glareolus. Two additive competition experiments on bank vole populations were conducted in large outdoor enclosures, half of them additionally housing a field vole population. In a mid-summer experiment low population density and absence of older breeding females minimised intra-specific competition. Survival was not affected by the presence of field voles. Season had a significant effect on both the probability of maturation and breeding of the weanlings. Competition with field voles significantly delayed breeding, and coupled with seasonal effects decreased the probability of breeding. In a late-summer experiment breeding and survival of bank vole weanlings were studied for three weeks as part of a high density breeding bank vole population. Weanlings did not mature at all nor were their space use and survival affected by the presence of field voles. Our results show that competition with other species can also have an impact on breeding of immatures. In an extreme seasonal environment, even a short delay of breeding may decrease survival chances of offspring. Seasonal and competition effects together may thus limit the contribution of year born females to reproductive output of the population. Other studies have shown that adult breeding bank voles suffer lower survival in the presence of field voles, but this study showed no survival effects on the weanlings. Thus it might be beneficial for weanlings to stay immature especially in the end of the breeding season and postpone reproduction to the next breeding season if densities of competing species are high.     

Molecular cloning of a cDNA for human pregnancy-specific beta 1-glycoprotein:homology with human carcinoembryonic antigen and related proteins

Human pregnancy-specific beta 1-glycoprotein (SP1) plays an essential role in normal pregnancy. It is also a well-characterized oncodevelopmental antigen, expressed aberrantly by all trophoblastic tumors and some other malignant cell types. Here we report the identification of a human placental cDNA encoding the SP1 polypeptide sequence. The coding sequence shows 95% identity at the nucleotide level with a distinct, recently published SP1 cDNA sequence (PSG16). Unexpectedly, the sequence is also highly homologous to the published sequence of human carcinoembryonic antigen (CEA). SP1, CEA and CEA-related nonspecific cross-reacting species thus belong to a group of closely related though antigenically diverse tumor-associated glycoproteins. Comparison of the deduced amino acid sequence of the SP1 cDNA with that of CEA provides insight into the modular nature of these related proteins. This may have implications for the genomic organization and evolution of the CEA gene family.     

Limited contemporary gene flow and high self‐replenishment drives peripheral isolation in an endemic coral reef fish

Extensive ongoing degradation of coral reef habitats worldwide has lead to declines in abundance of coral reef fishes and local extinction of some species. Those most vulnerable are ecological specialists and endemic species. Determining connectivity between locations is vital to understanding recovery and long‐term persistence of these species following local extinction. This study explored population connectivity in the ecologically‐specialized endemic three‐striped butterflyfish (Chaetodon tricinctus) using mt and msatDNA (nuclear microsatellites) to distinguish evolutionary versus contemporary gene flow, estimate self‐replenishment and measure genetic diversity among locations at the remote Australian offshore coral reefs of Middleton Reef (MR), Elizabeth Reef (ER), Lord Howe Island (LHI), and Norfolk Island (NI). Mt and msatDNA suggested genetic differentiation of the most peripheral location (NI) from the remaining three locations (MR, ER, LHI). Despite high levels of mtDNA gene flow, there is limited msatDNA gene flow with evidence of high levels of self‐replenishment (≥76%) at all four locations. Taken together, this suggests prolonged population recovery times following population declines. The peripheral population (NI) is most vulnerable to local extinction due to its relative isolation, extreme levels of self‐replenishment (95%), and low contemporary abundance.     

Role of Streptococcus thermophilus MR-1C Capsular Exopolysaccharide in Cheese Moisture Retention

Recent work by our group has shown that an exopolysaccharide (EPS)-producing starter pair, Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii subsp. bulgaricus MR-1R, can significantly increase moisture retention in low-fat mozzarella (D. B. Perry, D. J. McMahon, and C. J. Oberg, J. Dairy Sci. 80:799–805, 1997). The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. Analysis of low-fat mozzarella made with different combinations of MR-1C, MR-1R, and the non-EPS-producing starter culture strains S. thermophilus TA061 and Lactobacillus helveticus LH100 showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level. To investigate the role of the S. thermophilus MR-1C EPS in cheese moisture retention, the epsE gene in this bacterium was inactivated by gene replacement. Low-fat mozzarella made with L. helveticus LH100 plus the non-EPS-producing mutant S. thermophilus DM10 had a significantly lower moisture content than did cheese made with strains LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-1C EPS indicated that the polymer has a novel basic repeating unit composed of d-galactose, l-rhamnose, and l-fucose in a ratio of 5:2:1.Lactic acid bacteria (LAB) are a diverse group of industrially important, gram-positive, non-spore-forming microbes that produce lactic acid as a major product of carbohydrate fermentation. Many strains of LAB produce extracellular polysaccharides which may be tightly associated with the bacterial cell wall as capsules or liberated into the growth medium as a loose slime (5). The term exopolysaccharide (EPS) has been used to refer to either type of external polysaccharide. EPSs may be homopolysaccharides, composed of a single type of sugar monomer, or heteropolysaccharides, containing several types of sugar monomers (25). Extracellular homopolysaccharides are made by such LAB as Leuconostoc mesenteroides and Streptococcus mutans, while extracellular heteropolysaccharides are produced by several other species of LAB, including Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (6).The ability to produce EPS is unstable in LAB and may be lost following numerous transfers, prolonged periods of storage, or incubation at temperatures above that optimal for growth (6, 24). This instability of EPS production in mesophilic LAB has been attributed to the fact that the genes involved in polymer production are plasmid encoded. In contrast, genes for EPS production in thermophilic LAB, such as S. thermophilus and L. delbrueckii subsp. bulgaricus, are believed to be chromosomally encoded (6). Consequently, the unstable nature of the EPS phenotype in thermophilic strains is not understood, but it may be related to mobile genetic elements or genomic instability (24).Because of the ability of EPSs to act as viscosifying, stabilizing, or water-binding agents in various foods, these polymers can act as effective natural alternatives to commercial stabilizers (6). For example, EPS-producing (EPS⁺) LAB are commonly used as starter cultures for yogurt manufacture because EPS improves the viscosity and texture of yogurt and decreases its susceptibility to syneresis (loss of whey from the curd) (14, 28).Analysis of cheese microstructure has shown that in full-fat or part-skim mozzarella, the fat and a large portion of the water are located within channels that are formed by fat globules when the cheese curd is heated and stretched (18, 20). In low-fat mozzarella, however, there are very few fat globules to break up the protein matrix, resulting in less space for water retention (20). As a consequence, the cheese has a tough and rubbery texture and requires more heat for melting (19). Merrill et al. showed that procedures which increased moisture levels in reduced- and low-fat mozzarella improved the body, texture, and functional properties of the cheese (19). In addition to enhanced functionality, the ability to increase cheese moisture level (even by as little as 1%) gives processors an important economic advantage in the highly competitive mozzarella industry (27).Since EPS has the capacity to bind significant amounts of water, it was the hypothesis of our group that EPS⁺ LAB may be useful for the production of reduced- and low-fat mozzarella. Work by Perry et al. (21) recently showed that an EPS⁺ starter pair, S. thermophilus MR-1C and L. delbrueckii subsp. bulgaricus MR-1R, could be used to significantly increase moisture levels in low-fat mozzarella. The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. The results showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level and demonstrated that this effect required the bacterium’s capsular EPS.(Part of this research was presented at the 92nd Annual Meeting of the American Dairy Science Association, Guelph, Ontario, Canada, 22 to 25 June 1997.)     

Exploring the effect of chronotype on hippocampal volume and shape: A combined approach

Current evidence suggests that acute depression is associated with reduced total hippocampal volume and regional atrophy. Here, using structural magnetic resonance imaging, we assayed linear effects of chronotype on total hippocampal volume and morphology. Later chronotype was associated with localised atrophy in the subiculum region of the right hippocampus in the absence of changes in total volume. The hippocampus forms a key node in a network of brain regions implicated in emotional regulation and alterations in the structure of this region may underpin, in part, the increased vulnerability for depression in late chronotype individuals.