The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes

Glc7, the yeast protein phosphatase 1, is a component of the cleavage and polyadenylation factor (CPF). Here we show that downregulation of Glc7, or its dissociation from CPF in the absence of CPF subunits Ref2 or Swd2, results in similar snoRNA termination defects. Overexpressing a C-terminal fragment of Sen1, a superfamily I helicase required for snoRNA termination, suppresses the growth and termination defects associated with loss of Swd2 or Ref2, but not Glc7. Suppression by Sen1 requires nuclear localization and direct interaction with Glc7, which can dephosphorylate Sen1 in vitro. The suppressing fragment, and in a similar manner full-length Sen1, copurifies with the snoRNA termination factors Nrd1 and Nab3, suggesting loss of Glc7 from CPF can be compensated by recruiting Glc7 to Nrd1-Nab3 through Sen1. Swd2 is also a subunit of the Set1c histone H3K4 methyltransferase complex and is required for its stability and optimal methyltransferase activity.     

Herbivore-induced changes in plant carbon allocation: assessment of below-ground C fluxes using carbon-14

Effects of above-ground herbivory on short-term plant carbon allocation were studied using maize (Zea mays) and a generalist lubber grasshopper (Romalea guttata). We hypothesized that above-ground herbivory stimulates current net carbon assimilate allocation to below-ground components, such as roots, root exudation and root and soil respiration. Maize plants 24 days old were grazed (c. 25–50% leaf area removed) by caging grasshoppers around individual plants and 18 h later pulse-labelled with¹⁴CO₂. During the next 8 h,¹⁴C assimilates were traced to shoots, roots, root plus soil respiration, root exudates, rhizosphere soil, and bulk soil using carbon-14 techniques. Significant positive relationships were observed between herbivory and carbon allocated to roots, root exudates, and root and soil respiration, and a significant negative relationship between herbivory and carbon allocated to shoots. No relationship was observed between herbivory and¹⁴C recovered from soil. While herbivory increased root and soil respiration, the peak time for¹⁴CO₂ evolved as respiration was not altered, thereby suggesting that herbivory only increases the magnitude of respiration, not patterns of translocation through time. Although there was a trend for lower photosynthetic rates of grazed plants than photosynthetic rates of ungrazed plants, no significant differences were observed among grazed and ungrazed plants. We conclude that above-ground herbivory can increase plant carbon fluxes below ground (roots, root exudates, and rhizosphere respiration), thus increasing resources (e.g., root exudates) available to soil organisms, especially microbial populations.     

Efficient Organic Solar Cell with 16.88% Efficiency Enabled by Refined Acceptor Crystallization and Morphology with Improved Charge Transfer and Transport Properties

Single‐layered organic solar cells (OSCs) using nonfullerene acceptors have reached 16% efficiency. Such a breakthrough has inspired new sparks for the development of the next generation of OSC materials. In addition to the optimization of electronic structure, it is important to investigate the essential solid‐state structure that guides the high efficiency of bulk heterojunction blends, which provides insight in understanding how to pair an efficient donor–acceptor mixture and refine film morphology. In this study, a thorough analysis is executed to reveal morphology details, and the results demonstrate that Y6 can form a unique 2D packing with a polymer‐like conjugated backbone oriented normal to the substrate, controlled by the processing solvent and thermal annealing conditions. Such morphology provides improved carrier transport and ultrafast hole and electron transfer, leading to improved device performance, and the best optimized device shows a power conversion efficiency of 16.88% (16.4% certified). This work reveals the importance of film morphology and the mechanism by which it affects device performance. A full set of analytical methods and processing conditions are executed to achieve high efficiency solar cells from materials design to device optimization, which will be useful in future OSC technology development.     

A Site‐Specific Integration Reporter System That Enables Rapid Evaluation of CRISPR/Cas9‐Mediated Genome Editing Strategies in CHO Cells

Targeted gene knockout and site‐specific integration (SSI) are powerful genome editing techniques to improve the development of industrially relevant Chinese hamster ovary (CHO) cell lines. However, past efforts to perform SSI in CHO cells are characterized by low efficiencies. Moreover, numerous strategies proposed to boost SSI efficiency in mammalian cell types have yet to be evaluated head to head or in combination to appreciably boost efficiencies in CHO. To enable systematic and rapid optimization of genome editing methods, the SSIGNAL (s ite‐s pecific i ntegration and g en ome al teration) reporter system is developed. This tool can analyze CRISPR (clustered regularly interspaced palindromic repeats)/Cas9 (CRISPR‐associated protein 9)‐mediated disruption activity alone or in conjunction with SSI efficiency. The reporter system uses green and red dual‐fluorescence signals to indicate genotype states within four days following transfection, facilitating rapid data acquisition via standard flow cytometry instrumentation. In addition to describing the design and development of the system, two of its applications are demonstrated by first comparing transfection conditions to maximize CRISPR/Cas9 activity and subsequently assessing the efficiency of several promising SSI strategies. Due to its sensitivity and versatility, the SSIGNAL reporter system may serve as a tool to advance genome editing technology.     

The use and misuse of regression models in landscape genetic analyses

The field of landscape genetics has been rapidly evolving, adopting and adapting analytical frameworks to address research questions. Current studies are increasingly using regression‐based frameworks to infer the individual contributions of landscape and habitat variables on genetic differentiation. This paper outlines appropriate and inappropriate uses of multiple regression for these purposes, and demonstrates through simulation the limitations of different analytical frameworks for making correct inference. Of particular concern are recent studies seeking to explain genetic differences by fitting regression models with effective distance variables calculated independently on separate landscape resistance surfaces. When moving across the landscape, organisms cannot respond independently and uniquely to habitat and landscape features. Analyses seeking to understand how landscape features affect gene flow should model a single conductance or resistance surface as a parameterized function of relevant spatial covariates, and estimate the values of these parameters by linking a single set of resistance distances to observed genetic dissimilarity via a loss function. While this loss function may involve a regression‐like step, the associated nuisance parameters are not interpretable in terms of organismal movement and should not be conflated with what is actually of interest: the mapping between spatial covariates and conductance/resistance. The growth and evolution of landscape genetics as a field has been rapid and exciting. It is the goal of this paper to highlight past missteps and demonstrate limitations of current approaches to ensure that future use of regression models will appropriately consider the process being modeled, which will provide clarity to model interpretation.     

Natural Infection of Burkholderia pseudomallei in an Imported Pigtail Macaque (Macaca nemestrina) and Management of the Exposed Colony

Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.Abbreviations: CDC, Centers for Disease Control and Prevention; IHA, indirect hemagglutination assay; PEP, postexposure prophylacticBurkholderia pseudomallei, formerly known as Pseudomonas pseudomallei, is a gram-negative, aerobic, bipolar, motile, rod-shaped bacterium. B. pseudomallei infections (melioidosis) can be severe and even fatal in both humans and animals. This environmental saprophyte is endemic to Southeast Asia and northern Australia, but it has also been found in other tropical and subtropical areas of the world.^7,22,32,42 The bacterium is usually found in soil and water in endemic areas and is transmitted to humans and animals primarily through percutaneous inoculation, ingestion, or inhalation of a contaminated source.^{8, 22,28,32,42} Human-to-human, animal-to-animal, and animal-to-human spread are rare.^8,32 In December 2012, the National Select Agent Registry designated B. pseudomallei as a Tier 1 overlap select agent.³⁹ Organisms classified as Tier 1 agents present the highest risk of deliberate misuse, with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence. Select agents with this status have the potential to pose a severe threat to human and animal health or safety or the ability to be used as a biologic weapon.³⁹Melioidosis in humans can be challenging to diagnose and treat because the organism can remain latent for years and is resistant to many antibiotics.^12,37,41 B. pseudomallei can survive in phagocytic cells, a phenomenon that may be associated with latent infections.^19,38 The incubation period in naturally infected animals ranges from 1 d to many years, but symptoms typically appear 2 to 4 wk after exposure.^13,17,35,38 Disease generally presents in 1 of 2 forms: localized infection or septicemia.²² Multiple methods are used to diagnose melioidosis, including immunofluorescence, serology, and PCR analysis, but isolation of the bacteria from blood, urine, sputum, throat swabs, abscesses, skin, or tissue lesions remains the ‘gold standard.’^9,22,40,42 The prognosis varies based on presentation, time to diagnosis, initiation of appropriate antimicrobial treatment, and underlying comorbidities.^7,28,42 Currently, there is no licensed vaccine to prevent melioidosis.There are several published reports of naturally occurring melioidosis in a variety of nonhuman primates (NHP; 2,10,13,17,25,30,31,35 The first reported case of melioidosis in monkeys was recorded in 1932, and the first published case in a macaque species was in 1966.³⁰ In the United States, there have only been 7 documented cases of NHP with B. pseudomallei infection.^2,13,17 All of these cases occurred prior to the classification of B. pseudomallei as a select agent. Clinical signs in NHP range from subclinical or subacute illness to acute septicemia, localized infection, and chronic infection. NHP with melioidosis can be asymptomatic or exhibit clinical signs such as anorexia, wasting, purulent drainage, subcutaneous abscesses, and other soft tissue lesions. Lymphadenitis, lameness, osteomyelitis, paralysis and other CNS signs have also been reported.^{2,7,10,22,28,32} In comparison, human''s clinical signs range from abscesses, skin ulceration, fever, headache, joint pain, and muscle tenderness to abdominal pain, anorexia, respiratory distress, seizures, and septicemia.^7,9,21,22

Table 1.

Summary of reported cases of naturally occurring Burkholderia pseudomalleiinfections in nonhuman primates

Paired inspiratory-expiratory chest CT scans to assess for small airways disease in COPD

Background

Gas trapping quantified on chest CT scans has been proposed as a surrogate for small airway disease in COPD. We sought to determine if measurements using paired inspiratory and expiratory CT scans may be better able to separate gas trapping due to emphysema from gas trapping due to small airway disease.

Methods

Smokers with and without COPD from the COPDGene Study underwent inspiratory and expiratory chest CT scans. Emphysema was quantified by the percent of lung with attenuation < −950HU on inspiratory CT. Four gas trapping measures were defined: (1) Exp₋₈₅₆, the percent of lung < −856HU on expiratory imaging; (2) E/I MLA, the ratio of expiratory to inspiratory mean lung attenuation; (3) RVC_856-950, the difference between expiratory and inspiratory lung volumes with attenuation between −856 and −950 HU; and (4) Residuals from the regression of Exp₋₈₅₆ on percent emphysema.

Results

In 8517 subjects with complete data, Exp₋₈₅₆ was highly correlated with emphysema. The measures based on paired inspiratory and expiratory CT scans were less strongly correlated with emphysema. Exp₋₈₅₆, E/I MLA and RVC_856-950 were predictive of spirometry, exercise capacity and quality of life in all subjects and in subjects without emphysema. In subjects with severe emphysema, E/I MLA and RVC_856-950 showed the highest correlations with clinical variables.

Conclusions

Quantitative measures based on paired inspiratory and expiratory chest CT scans can be used as markers of small airway disease in smokers with and without COPD, but this will require that future studies acquire both inspiratory and expiratory CT scans.     

Quantifying Policy Options for Reducing Future Coronary Heart Disease Mortality in England: A Modelling Study

Aims

To estimate the number of coronary heart disease (CHD) deaths potentially preventable in England in 2020 comparing four risk factor change scenarios.

Methods and Results

Using 2007 as baseline, the IMPACT_SEC model was extended to estimate the potential number of CHD deaths preventable in England in 2020 by age, gender and Index of Multiple Deprivation 2007 quintiles given four risk factor change scenarios: (a) assuming recent trends will continue; (b) assuming optimal but feasible levels already achieved elsewhere; (c) an intermediate point, halfway between current and optimal levels; and (d) assuming plateauing or worsening levels, the worst case scenario. These four scenarios were compared to the baseline scenario with both risk factors and CHD mortality rates remaining at 2007 levels. This would result in approximately 97,000 CHD deaths in 2020. Assuming recent trends will continue would avert approximately 22,640 deaths (95% uncertainty interval: 20,390-24,980). There would be some 39,720 (37,120-41,900) fewer deaths in 2020 with optimal risk factor levels and 22,330 fewer (19,850-24,300) in the intermediate scenario. In the worst case scenario, 16,170 additional deaths (13,880-18,420) would occur. If optimal risk factor levels were achieved, the gap in CHD rates between the most and least deprived areas would halve with falls in systolic blood pressure, physical inactivity and total cholesterol providing the largest contributions to mortality gains.

Conclusions

CHD mortality reductions of up to 45%, accompanied by significant reductions in area deprivation mortality disparities, would be possible by implementing optimal preventive policies.     

Comparison between the effects of diallyl tetrasulfide on human retina pigment epithelial cells (ARPE-19) and HCT116 cells

Background

Diallyl mono- and polysulfanes from garlic are known to induce an adaptive cell response and the formation of antioxidants in cancer cells. In the case of a severe ER stress and a failure in the response, cancer cells eventually go into apoptosis. Only little is known about the response of normal cells upon treatment.

Methods

Normal ARPE-19 cells were treated with diallyl tetrasulfide to study their cellular response and the results were compared with those of HCT116 cancer cells. Cell viability was checked by an MTT assay and cytofluorimetry. The formation of superoxide radicals, H₂O₂ and thiols were determined and proteins involved in the ER stress response were also detected by Western blot analysis.

Results

We found that diallyl tetrasulfide induced reactive oxygen species (ROS) in normal cells similar to cancer cells in a time (0 to 60 min) and dose dependent manner (0 to 50 μM). The level of heme oxigenase-1 (HO-1) was up-regulated in both cell types. Initially, we found a decrease in the total thiol level in both cell types but in contrast to cancer cells, normal cells recovered from the decrease in the total thiol concentration within 60 min of treatment.

Conclusions

The recovery of the thiol concentration in normal cells treated with diallyl tetrasulfide seems to be responsible for the failure to induce the ER stress signalling pathway and finally apoptosis in normal cells.

General Significance

The difference in the recovery of the thiol status might be an explanation for the anti-carcinogenic effects of garlic compounds.