Enhanced mismatch selectivity of T4 DNA ligase far above the probe: Target duplex dissociation temperature

T4 DNA ligase is a widely used ligase in many applications; yet in single nucleotide polymorphism analysis, it has been found generally lacking owing to its tendency to ligate mismatches quite efficiently. To address this lack of selectivity, we explored the effect of temperature on the selectivity of the ligase in discriminating single base pair mismatches at the 3′‐terminus of the ligating strand using short ligation probes (9‐mers). Remarkably, we observe outstanding selectivities when the assay temperature is increased to 7 °C to 13 °C above the dissociation temperature of the matched probe:target duplexes using commercially available enzyme at low concentration. Higher enzyme concentration shifts the temperature range to 13 °C to 19 °C above the probe:target dissociation temperatures. Finally, substituting the 5′‐phosphate terminus with an abasic nucleotide decreases the optimal temperature range to 7 °C to 10 °C above the matched probe:target duplex. We compare the temperature dependence of the T4 DNA ligase catalyzed ligation and a nonenzymatic ligation system to contrast the origin of their modes of selectivity. For the latter, temperatures above the probe:target duplex dissociation lead to lower ligation conversions even for the perfect matched system. This difference between the two ligation systems reveals the uniqueness of the T4 DNA ligase's ability to maintain excellent ligation yields for the matched system at elevated temperatures. Although our observations are consistent with previous mechanistic work on T4 DNA ligase, by mapping out the temperature dependence for different ligase concentrations and probe modifications, we identify simple strategies for introducing greater selectivity into SNP discrimination based on ligation yields.     

Effects of Ethylene Glycol Monomethyl Ether and Its Metabolite, 2‐Methoxyacetic Acid,on Organogenesis Stage Mouse Limbs In Vitro

Exposure to ethylene glycol monomethyl ether (EGME), a glycol ether compound found in numerous industrial products, or to its active metabolite, 2‐methoxyacetic acid (2‐MAA), increases the incidence of developmental defects. Using an in vitro limb bud culture system, we tested the hypothesis that the effects of EGME on limb development are mediated by 2‐MAA‐induced alterations in acetylation programming. Murine gestation day 12 embryonic forelimbs were exposed to 3, 10, or 30 mM EGME or 2‐MAA in culture for 6 days to examine effects on limb morphology; limbs were cultured for 1 to 24 hr to monitor effects on the acetylation of histones (H3K9 and H4K12), a nonhistone protein, p53 (p53K379), and markers for cell cycle arrest (p21) and apoptosis (cleaved caspase‐3). EGME had little effect on limb morphology and no significant effects on the acetylation of histones or p53 or on biomarkers for cell cycle arrest or apoptosis. In contrast, 2‐MAA exposure resulted in a significant concentration‐dependent increase in limb abnormalities. 2‐MAA induced the hyperacetylation of histones H3K9Ac and H4K12Ac at all concentrations tested (3, 10, and 30 mM). Exposure to 10 or 30 mM 2‐MAA significantly increased acetylation of p53 at K379, p21 expression, and caspase‐3 cleavage. Thus, 2‐MAA, the proximate metabolite of EGME, disrupts limb development in vitro, modifies acetylation programming, and induces biomarkers of cell cycle arrest and apoptosis     

Factors Affecting Family Satisfaction with Inpatient End-of-Life Care

Background

Little data exists addressing satisfaction with end-of-life care among hospitalized patients, as they and their family members are systematically excluded from routine satisfaction surveys. It is imperative that we closely examine patient and institution factors associated with quality end-of-life care and determine high-priority target areas for quality improvement.

Methods

Between September 1, 2010 and January 1, 2012 the Canadian Health care Evaluation Project (CANHELP) Bereavement Questionnaire was mailed to the next-of-kin of recently deceased inpatients to seek factors associated with satisfaction with end-of-life care. The primary outcome was the global rating of satisfaction. Secondary outcomes included rates of actual versus preferred location of death, associations between demographic factors and global satisfaction, and identification of targets for quality improvement.

Results

Response rate was 33% among 275 valid addresses. Overall, 67.4% of respondents were very or completely satisfied with the overall quality of care their relative received. However, 71.4% of respondents who thought their relative did not die in their preferred location favoured an out-of-hospital location of death. A common location of death was the intensive care unit (45.7%); however, this was not the preferred location of death for 47.6% of such patients. Multivariate Poisson regression analysis showed respondents who believed their relative died in their preferred location were 1.7 times more likely to be satisfied with the end-of-life care that was provided (p = 0.001). Items identified as high-priority targets for improvement included: relationships with, and characteristics of health care professionals; illness management; communication; and end-of-life decision-making.

Interpretation

Nearly three-quarters of recently deceased inpatients would have preferred an out-of-hospital death. Intensive care units were a common, but not preferred, location of in-hospital deaths. Family satisfaction with end-of-life care was strongly associated with their relative dying in their preferred location. Improved communication regarding end-of-life care preferences should be a high-priority quality improvement target.     

JNK activation is responsible for mucus overproduction in smoke inhalation injury

Background

Increased mucus secretion is one of the important characteristics of the response to smoke inhalation injuries. We hypothesized that gel-forming mucins may contribute to the increased mucus production in a smoke inhalation injury. We investigated the role of c-Jun N-terminal kinase (JNK) in modulating smoke-induced mucus secretion.

Methods

We intubated mice and exposed them to smoke from burning cotton for 15 min. Their lungs were then isolated 4 and 24 h after inhalation injury. Three groups of mice were subjected to the smoke inhalation injury: (1) wild-type (WT) mice, (2) mice lacking JNK1 (JNK1-/- mice), and (3) WT mice administered a JNK inhibitor. The JNK inhibitor (SP-600125) was injected into the mice 1 h after injury.

Results

Smoke exposure caused an increase in the production of mucus in the airway epithelium of the mice along with an increase in MUC5AC gene and protein expression, while the expression of MUC5B was not increased compared with control. We found increased MUC5AC protein expression in the airway epithelium of the WT mice groups both 4 and 24 h after smoke inhalation injury. However, overproduction of mucus and increased MUC5AC protein expression induced by smoke inhalation was suppressed in the JNK inhibitor-treated mice and the JNK1 knockout mice. Smoke exposure did not alter the expression of MUC1 and MUC4 proteins in all 3 groups compared with control.

Conclusion

An increase in epithelial MUC5AC protein expression is associated with the overproduction of mucus in smoke inhalation injury, and that its expression is related on JNK1 signaling.     

Inhibition of a calcium-activated, non-selective cation channel, in a rat insulinoma cell line, by adenine derivatives

The effects of adenosine and adenine nucleotides on a calcium-activated non-selective cation channel, present in the plasma membrane of an insulin-secreting cell line CRI-Gl were investigated. Single-channel currents were recorded from inside-out membrane patches and the adenine derivatives applied to the solution bathing the cytoplasmic aspect of the membrane surface. The activity of this channel is shown to be inhibited by all the derivatives tested. The potency sequence for inhibition was found to be AMP greater than ADP greater than ATP greater than adenosine.     

Induction of sexual forms in the rose aphid Macrosiphum rosae

The rose aphid, Macrosiphum rosae (L.), was tested for its ability to produce sexual forms on rose leaves, after induction by environmental conditions in the field and in the laboratory. Clones were collected in Tübingen, Federal Republic of Germany, and in south-eastern New South Wales, Australia. The German clones all produced sexual forms, while the response of the Australian clones was weak. There appeared to be a bias in the sex ratio towards females: This is discussed in developmental terms. The environmental and genetic factors influencing the response are considered.     

Immunochemical detection and characterization of pregnancy-associated endometrial alpha 1- and alpha 2-globulins secreted by human endometrium and decidua

Antisera raised against the soluble antigens of the endometrium of early pregnancy detected two antigenic proteins of alpha 1 and alpha 2 mobility in extracts of this tissue and were termed antigens A and B. Neither antigen was detected in pregnancy sera or extracts of proliferative endometrium, but antigen B was detected in extracts of secretory endometrium and both were present in amniotic fluid and medium from in-vitro incubations of pregnancy endometrium. Fractionation of radiolabelled medium on ion-exchange chromatography demonstrated that antigens A and B co-eluted with the proteins from which EP14 and EP15 were derived and which were the major secretory polypeptides of pregnancy endometrium in vitro. Further biochemical purification revealed that EP14 (Mr 32 000) was derived from a protein of native molecular weight 36 000 which existed in two forms, whereas EP15 (Mr 28 000) was derived from a dimeric glycoprotein of native molecular weight 56 000. Immunochemical studies demonstrated that antigens A and B are identical to these two secretory proteins and have been termed pregnancy-associated endometrial alpha 1- and alpha 2-globulins (alpha 1- and alpha 2-PEG).