Genetic engineering of crops as potential source of genetic hazard in the human diet.

The benefits of genetic engineering of crop plants to improve the reliability and quality of the world food supply have been contrasted with public concerns raised about the food safety of the resulting products. Debates have concentrated on the possible unforeseen risks associated with the accumulation of new metabolites in crop plants that may contribute to toxins, allergens and genetic hazards in the human diet. This review examines the various molecular and biochemical mechanisms by which new hazards may appear in foods as a direct consequence of genetic engineering in crop plants. Such hazards may arise from the expression products of the inserted genes, secondary or pleiotropic effects of transgene expression, and random insertional mutagenic effects resulting from transgene integration into plant genomes. However, when traditional plant breeding is evaluated in the same context, these mechanisms are no different from those that have been widely accepted from the past use of new cultivars in agriculture. The risks associated with the introduction of new genes via genetic engineering must be considered alongside the common breeding practice of introgressing large fragments of chromatin from related wild species into crop cultivars. The large proportion of such introgressed DNA involves genes of unknown function linked to the trait of interest such as pest or disease resistance. In this context, the potential risks of introducing new food hazards from the applications of genetic engineering are no different from the risks that might be anticipated from genetic manipulation of crops via traditional breeding. In many respects, the precise manner in which genetic engineering can control the nature and expression of the transferred DNA offers greater confidence for producing the desired outcome compared with traditional breeding.     

Genotype accounts for intraspecific variation in the timing and duration of multiple,sequential life-cycle events in a willow species

Premise

Phenological variation among individuals within populations is common and has a variety of ecological and evolutionary consequences, including forming the basis for population-level responses to environmental change. Although the timing of life-cycle events has genetic underpinnings, whether intraspecific variation in the duration of life-cycle events reflects genetic differences among individuals is poorly understood.

Methods

We used a common garden experiment with 10 genotypes of Salix hookeriana (coastal willow) from northern California, United States to investigate the extent to which genetic variation explains intraspecific variation in the timing and duration of multiple, sequential life-cycle events: flowering, leaf budbreak, leaf expansion, fruiting, and fall leaf coloration. We used seven clones of each genotype, for a total of 70 individual trees.

Results

Genotype affected each sequential life-cycle event independently and explained on average 62% of the variation in the timing and duration of vegetative and reproductive life-cycle events. All events were significantly heritable. A single genotype tended to be “early” or “late” across life-cycle events, but for event durations, there was no consistent response within genotypes.

Conclusions

This research demonstrates that genetic variation can be a major component underlying intraspecific variation in the timing and duration of life-cycle events. It is often assumed that the environment affects durations, but we show that genetic factors also play a role. Because the timing and duration of events are independent of one another, our results suggest that the effects of environmental change on one event will not necessarily cascade to subsequent events.     

Methacarn (methanol-Carnoy) fixation

Summary According to chemical data, methanol raises the shrinkage temperature of collagen significantly more than ethanol (86° C versus 70° C). Since increase of shrinkage temperature appears desirable in tissues to be embedded in paraffin, methanol was substituted for ethanol in Carnoy's fluid. This methanol-Carnoy mixture is referred to as methacarn solution. The fixation-embedding procedure was similar to that described in the study of Carnoy fixation. Methacarn-fixed sections showed little or no shrinkage and compared well with material fixed in Carnoy's or Zenker's fluid. Myofibrils, especially in endothelial and epithelial cells, were more prominent in methacarn- than in Carnoy-fixed tissues.A review of the chemical literature showed that methanol, ethanol and chloroform stabilize or even enhance helical conformations of proteins, presumably by strengthening of hydrogen bonds. Interference with hydrophobic bonds causes unfolding and/or structural rearrangements in globular proteins. The twin-helical structure of DNA collapses in alcoholic solutions. Hence, methacarn fixation can be expected to preserve the helical proteins in myofibrils and collagen, but the conformations of globular proteins and DNA will be significantly altered. Literature on conformational effects produced by fixatives used in electron microscopy was also reviewed. Glutaraldehyde and OsO₄ cause considerable loss of helix (22–29% and 39–66% respectively). KMnO₄ and glutaraldehyde followed by OsO₄ produce extensive transitions from helical to random-coil conformations similar to those seen in powerful denaturants such as 8 M urea. Evidently these fixatives are unsuitable for studies of helical proteins. In contrast ethylene glycol preserves helical conformations.     

IL-17A Induces Pendrin Expression and Chloride-Bicarbonate Exchange in Human Bronchial Epithelial Cells

The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.     

Ecological genetics of an induced plant defense against herbivores: additive genetic variance and costs of phenotypic plasticity

Abstract.— Adaptive phenotypic plasticity in chemical defense is thought to play a major role in plant-herbivore interactions. We investigated genetic variation for inducibility of defensive traits in wild radish plants and asked if the evolution of induction is constrained by costs of phenotypic plasticity. In a greenhouse experiment using paternal half-sibling families, we show additive genetic variation for plasticity in glucosinolate concentration. Genetic variation for glucosinolates was not detected in undamaged plants, but was significant following herbivory by a specialist herbivore, Pieris rapae . On average, damaged plants had 55% higher concentrations of glucosinolates compared to controls. In addition, we found significant narrow-sense heritabilities for leaf size, trichome number, flowering phenology, and lifetime fruit production. In a second experiment, we found evidence of genetic variation in induced plant resistance to P. rapae . Although overall there was little evidence for genetic correlations between the defensive and life-history traits we measured, we show that more plastic families had lower fitness than less plastic families in the absence of herbivory (i.e., evidence for genetic costs of plasticity). Thus, there is genetic variation for induction of defense in wild radish, and the evolution of inducibility may be constrained by costs of plasticity.     

Decreased Soluble Adenylyl Cyclase Activity in Cystic Fibrosis Is Related to Defective Apical Bicarbonate Exchange and Affects Ciliary Beat Frequency Regulation

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO₃⁻/CO₂ stimulation to increase ciliary beat frequency (CBF). Because apical HCO₃⁻ exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO₃⁻/CO₂ exposure in part because of greater intracellular acidification from unbalanced CO₂ influx (estimated by 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence). Importantly, ciliated cell-specific cAMP production (estimated by FRET fluorescence ratio changes of tagged cAMP-dependent protein kinase (PKA) subunits expressed under a ciliated cell-specific promoter) in response to increased apical HCO₃⁻/CO₂ perfusion was higher in normal compared with CF cells. Inhibition of bicarbonate influx via CFTR (CFTR_inh172) and inhibition of sAC (KH7) and PKA activation (H89) led to larger CBF declines in normal cells, now comparable with changes seen in CF cells. These inhibitors also reduced FRET changes in normal cells to the level of CF cells with the expected exception of H89, which does not prevent dissociation of the fluorescently tagged PKA subunits. Basolateral permeabilization and subsequent perfusion with HCO₃⁻/CO₂ rescued CBF and FRET changes in CF cells to the level of normal cells. These results suggest that CBF regulation by sAC-produced cAMP could be impaired in CF, thereby possibly contributing to mucociliary dysfunction in this disease, at least during disease exacerbations when airway acidification is common.     

Biochemical regulation of mammalian AMP-activated protein kinase activity by NAD and NADH

AMP-activated protein kinase (AMPK) serves as an energy-sensing protein kinase that is activated by a variety of metabolic stresses that lower cellular energy levels. When activated, AMPK modulates a network of metabolic pathways that result in net increased substrate oxidation, generation of reduced nucleotide cofactors, and production of ATP. AMPK is activated by a high AMP:ATP ratio and phosphorylation on threonine 172 by an upstream kinase. Recent studies suggest that mechanisms that do not involve changes in adenine nucleotide levels can activate AMPK. Another sensor of the metabolic state of the cell is the NAD/NADH redox potential. To test whether the redox state might have an effect on AMPK activity, we examined the effect of beta-NAD and NADH on this enzyme. The recombinant T172D-AMPK, which was mutated to mimic the phosphorylated state, was activated by beta-NAD in a dose-dependent manner, whereas NADH inhibited its activity. We explored the effect of NADH on AMPK by systematically varying the concentrations of ATP, NADH, peptide substrate, and AMP. Based on our findings and established activation of AMPK by AMP, we proposed a model for the regulation by NADH. Key features of this model are as follows. (a) NADH has an apparent competitive behavior with respect to ATP and uncompetitive behavior with respect to AMP resulting in improved binding constant in the presence of AMP, and (b) the binding of the peptide is not significantly altered by NADH. In the absence of AMP, the binding constant of NADH becomes higher than physiologically relevant. We conclude that AMPK senses both components of cellular energy status, redox potential, and phosphorylation potential.     

Endovascular Treatment with Stent-Retriever Devices for Acute Ischemic Stroke: A Meta-Analysis of Randomized Controlled Trials

Importance

Acute ischemic stroke is a leading cause of death and disability worldwide. Several recent clinical trials have shown that endovascular treatment improves clinical outcomes among patients with acute ischemic stroke.

Objective

To provide an overall and precise estimate of the efficacy of endovascular treatment predominantly using second-generation mechanical thrombectomy devices (stent-retriever devices) compared to medical management on clinical and functional outcomes among patients with acute ischemic stroke.

Data Sources

MEDLINE, EMBASE, Cochrane Collaboration Central Register of Controlled Clinical Trials, Web of Science, and NIH ClinicalTrials.gov were searched through November 2015.

Study Selection

Searches returned 3,045 articles. After removal of duplicates, two authors independently screened titles and abstracts to assess eligibility of 2,495 potentially relevant publications. From these, 38 full-text publications were more closely assessed. Finally, 5 randomized controlled trials of endovascular treatment with predominant use of retrievable stents were selected.

Data Extraction and Synthesis

Three authors independently extracted information on participant and trial characteristics and clinical events using a standardized protocol. Random effects models were used to pool endovascular treatment effects across outcomes.

Main Outcomes and Measures

The primary outcome was better functional outcome as measured on the modified Rankin Scale at 90 days of follow-up. Secondary outcomes included all-cause mortality and symptomatic intra-cerebral hemorrhage.

Results

Five trials representing 1,287 patients were included. Overall, patients randomized to endovascular therapy experienced 2.22 times greater odds of better functional outcome compared to those randomized to medical management (95% CI, 1.66 to 2.98; P < 0.0001). Endovascular therapy was not associated with mortality [OR (95% CI), 0.78 (0.54, 1.12); P = 0.1056] or symptomatic intracerebral hemorrhage [OR (95% CI), 1.19 (0.69, 2.05); P = 0.5348]. Meta-regression analysis suggested that shorter times from stroke onset to groin puncture and from stroke onset to reperfusion result in better functional outcomes in ischemic stroke patients (P = 0.0077 and P = 0.0089). There were no significant differences in the beneficial effects of endovascular treatment on functional outcomes across categories of gender, age, stroke severity, ischemic changes on computed tomography, or intravenous tissue plasminogen activator administration.

Conclusions and Relevance

This meta-analysis demonstrated superior functional outcomes in subjects receiving endovascular treatment compared to medical management. Further, this analysis showed that acute ischemic stroke patients may receive enhanced functional benefit from earlier endovascular treatment.     

Decreased Expression Of apM1 in Omental and Subcutaneous Adipose Tissue of Humans With Type 2 Diabetes

We have screened a subtracted cDNA library in order to identify differentially expressed genes in omental adipose tissue of human patients with Type 2 diabetes. One clone (#1738) showed a marked reduction in omental adipose tissue from patients with Type 2 diabetes. Sequencing and BLAST analysis revealed clone #1738 was the adipocyte-specific secreted protein gene apM1 (synonyms ACRP30, AdipoQ, GBP28). Consistent with the murine orthologue, apM1 mRNA was expressed in cultured human adipocytes and not in preadipocytes. Using RT-PCR we confirmed that apM1 mRNA levels were significantly reduced in omental adipose tissue of obese patients with Type 2 diabetes compared with lean and obese normoglycemic subjects. Although less pronounced, apM1 mRNA levels were reduced in subcutaneous adipose tissue of Type 2 diabetic patients. Whereas the biological function of apM1 is presently unknown, the tissue specific expression, structural similarities to TNFα and the dysregulated expression observed in obese Type 2 diabetic patients suggest that this factor may play a role in the pathogenesis of insulin resistance and Type 2 diabetes.