Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei

Penicillium marneffei is an opportunistic pathogen of humans and displays a temperature dependent dimorphic transition. Like many fungi, exogenous DNA introduced by DNA mediated transformation is integrated randomly into the genome resulting in inefficient gene deletion and position-specific effects. To enhance successful gene targeting, the consequences of perturbing components of the non-homologous end joining recombination pathway have been examined. The deletion of the KU70 and LIG4 orthologs, pkuA and ligD, respectively, dramatically enhanced the observed homologous recombination frequency leading to efficient gene deletion. While ΔpkuA was associated with reduced genetic stability over-time, ΔligD represents a suitable recipient strain for downstream applications and combined with a modified Gateway? system for the rapid generation of gene deletion constructs, this represents an efficient pipeline for characterizing gene function in P. marneffei.     

The role of the soil seed bank in vegetation recovery on an oceanic island severely damaged by introduced goats

Question: Are the seed banks of an isolated subtropical oceanic island capable of naturally regenerating vegetation either with species of the historical forest community or with the existing grassland community after severe damage to the vegetation by goats? Location: Nakoudojima Island, Bonin Archipelago (Ogasawara Shoto), Japan. Methods: Soil samples were collected at 0–5 cm and 5–10 cm depths from seven plots in forests, grasslands, artificially matted areas and bare land. Soil seed banks were assessed using the seedling emergence method followed by the hand‐sorting of ungerminated seeds. We determined the size and composition of the seed banks in upper soil layers of plots and compared the seed banks to the standing vegetation. Results: A total of 12 220 seedlings belonging to 42 species from 20 families germinated. Total mean seed density (0–5 cm depth) was low in all plots within forest, grassland, and heavily degraded vegetation types (34.7 ± 8.6 to 693.5 ± 123.6, 58.6 ± 7.8 to 107.1 ± 10.0, and 1.1 ± 0.5 to 7.2 ± 2.3 seeds/m², respectively). Forbs and graminoids dominated the seed banks of grassland and forest plots including Cyperus brevifolius, Gnaphalium pensylvanicum, Oxalis corniculata and Solanum nigrum, and these alien species comprised 90% of the density of the seed bank. There was little correlation between seed banks and standing vegetation of the island (Sørensen similarity coefficient values 0.26 to 0.45). Conclusions: If natural regeneration occurs from the seed bank of the island, future vegetation will not move toward the original forest community, because the seed bank is dominated by non‐native herbaceous grassland species. Though isolated, a few forest remnants with low species richness could be an important source for the natural re‐establishment of forest on the island; however, seed availability may be limited by either poor dispersal or pollination so that woody species will probably recover very slowly on this goat‐impacted island.     

Mammography Screening in a Large Health System Following the U.S. Preventive Services Task Force Recommendations and the Affordable Care Act

Background

Practice recommendations for mammography screening were issued by the U.S. Preventive Services Task Force in 2009 and expansion of insurance coverage was provided under the Patient Protection and Affordable Care Act soon thereafter, yet the influence of these changes on screening practices in the United States is not known.

Methods

To determine changes in mammography screening and their associations with new practice recommendations and the Affordable Care Act, we examined patient-level data from 249,803 screening mammograms from January 1, 2008 through December 31, 2012 in a large community-based health system in the northwestern United States. Associations were determined by an intervention analysis of time-series data method.

Results

Among women screened, 64% were age 50-74 years; 84% self-identified as white race; 62% had commercial insurance; and 70% were seen in facilities located in metropolitan areas. Practice recommendations were associated with decreased screening volumes among women age <40 (-37.4 mammograms/month; -39.4% change; P<0.001), 40-49 (-106.0 mammograms/month; -11.2% change; P<0.001), and ≥75 (-54.7 mammograms/month; -10.0% change; P<0.001), but not women age 50-74. Implementation of the Affordable Care Act was associated with increased screening among women age 50-74 (+184.3 mammograms/month; +7.2% change; P=0.001), but not women <40 or ≥75; increases for age 40-49 were of borderline statistical significance (+56.9 mammograms/month; +6% change; P=0.06). Practice recommendations were also associated with decreased screening for women with commercial insurance, while the Affordable Care Act was associated with increased screening for women with Medicare, Medicaid, or other noncommercial sources of payment.

Conclusions

Mammography screening volumes in a large community health system decreased among women age <50 and ≥75 in association with new U.S. Preventive Services Task Force practice recommendations, while insurance coverage changes under the Affordable Care Act were associated with increased screening volumes among women age 50-74.     

Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation

Genetic factors impact liver injury susceptibility and disease progression. Prominent histological features of some chronic human liver diseases are hepatocyte ballooning and Mallory-Denk bodies. In mice, these features are induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in a strain-dependent manner, with the C57BL and C3H strains showing high and low susceptibility, respectively. To identify modifiers of DDC-induced liver injury, we compared C57BL and C3H mice using proteomic, biochemical, and cell biological tools. DDC elevated reactive oxygen species (ROS) and oxidative stress enzymes preferentially in C57BL livers and isolated hepatocytes. C57BL livers and hepatocytes also manifested significant down-regulation, aggregation, and nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH knockdown depleted bioenergetic and antioxidant enzymes and elevated hepatocyte ROS, whereas GAPDH overexpression decreased hepatocyte ROS. On the other hand, C3H livers had higher expression and activity of the energy-generating nucleoside-diphosphate kinase (NDPK), and knockdown of hepatocyte NDPK augmented DDC-induced ROS formation. Consistent with these findings, cirrhotic, but not normal, human livers contained GAPDH aggregates and NDPK complexes. We propose that GAPDH and NDPK are genetic modifiers of murine DDC-induced liver injury and potentially human liver disease.     

A Golgi-localized hexose transporter is involved in heterotrimeric G protein-mediated early development in Arabidopsis

Signal transduction involving heterotrimeric G proteins is universal among fungi, animals, and plants. In plants and fungi, the best understood function for the G protein complex is its modulation of cell proliferation and one of several important signals that are known to modulate the rate at which these cells proliferate is D-glucose. Arabidopsis thaliana seedlings lacking the beta subunit (AGB1) of the G protein complex have altered cell division in the hypocotyl and are D-glucose hypersensitive. With the aim to discover new elements in G protein signaling, we screened for gain-of-function suppressors of altered cell proliferation during early development in the agb1-2 mutant background. One agb1-2-dependent suppressor, designated sgb1-1(D) for suppressor of G protein beta1 (agb1-2), restored to wild type the altered cell division in the hypocotyl and sugar hypersensitivity of the agb1-2 mutant. Consistent with AGB1 localization, SGB1 is found at the highest steady-state level in tissues with active cell division, and this level increases in hypocotyls when grown on D-glucose and sucrose. SGB1 is shown here to be a Golgi-localized hexose transporter and acts genetically with AGB1 in early seedling development.     

A histone deacetylase-dependent screen in yeast

Histone deacetylase (HDAC) proteins are promising targets for cancer treatment, as shown by the recent FDA approval of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA, Vorinostat) for the treatment of cutaneous T-cell lymphoma. To identify additional potent inhibitors and characterize HDAC mutant proteins, there is interest to develop an inexpensive screening method dependent on native substrates. Here, we report the first yeast-based gene reporter screen dependent on the yeast Rpd3, which is a homolog of human class I HDAC proteins. The screen was sensitive to an inactive Rpd3 mutant and various inhibitors in qualitative, agar-based and quantitative, solution-phase formats. Interestingly, inclusion of the lytic enzyme zymolyase enhanced the inhibitor sensitivity of the screen. The gene reporter screen provides a tool to screen Rpd3 mutants and inhibitors of class I HDAC proteins.     

Global photosynthetic capacity is optimized to the environment

Earth system models (ESMs) use photosynthetic capacity, indexed by the maximum Rubisco carboxylation rate (V_cmax), to simulate carbon assimilation and typically rely on empirical estimates, including an assumed dependence on leaf nitrogen determined from soil fertility. In contrast, new theory, based on biochemical coordination and co‐optimization of carboxylation and water costs for photosynthesis, suggests that optimal V_cmax can be predicted from climate alone, irrespective of soil fertility. Here, we develop this theory and find it captures 64% of observed variability in a global, field‐measured V_cmax dataset for C₃ plants. Soil fertility indices explained substantially less variation (32%). These results indicate that environmentally regulated biophysical constraints and light availability are the first‐order drivers of global photosynthetic capacity. Through acclimation and adaptation, plants efficiently utilize resources at the leaf level, thus maximizing potential resource use for growth and reproduction. Our theory offers a robust strategy for dynamically predicting photosynthetic capacity in ESMs.     

O-linked-N-acetylglucosamine cycling and insulin signaling are required for the glucose stress response in Caenorhabditis elegans

In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. Here we demonstrate that O-GlcNAc cycling and insulin signaling are both essential components of the C. elegans response to glucose stress. A number of insulin-dependent processes were found to be sensitive to glucose stress, including fertility, reproductive timing, and dauer formation, yet each of these differed in their threshold of sensitivity to glucose excess. Our findings suggest that O-GlcNAc cycling and insulin signaling are both required for a robust and adaptable response to glucose stress, but these two pathways show complex and interdependent roles in the maintenance of glucose-insulin homeostasis.     

Thermal limits of leaf metabolism across biomes

High‐temperature tolerance in plants is important in a warming world, with extreme heat waves predicted to increase in frequency and duration, potentially leading to lethal heating of leaves. Global patterns of high‐temperature tolerance are documented in animals, but generally not in plants, limiting our ability to assess risks associated with climate warming. To assess whether there are global patterns in high‐temperature tolerance of leaf metabolism, we quantified T_crit (high temperature where minimal chlorophyll a fluorescence rises rapidly and thus photosystem II is disrupted) and T_max (temperature where leaf respiration in darkness is maximal, beyond which respiratory function rapidly declines) in upper canopy leaves of 218 plant species spanning seven biomes. Mean site‐based T_crit values ranged from 41.5 °C in the Alaskan arctic to 50.8 °C in lowland tropical rainforests of Peruvian Amazon. For T_max, the equivalent values were 51.0 and 60.6 °C in the Arctic and Amazon, respectively. T_crit and T_max followed similar biogeographic patterns, increasing linearly (?8 °C) from polar to equatorial regions. Such increases in high‐temperature tolerance are much less than expected based on the 20 °C span in high‐temperature extremes across the globe. Moreover, with only modest high‐temperature tolerance despite high summer temperature extremes, species in mid‐latitude (~20–50°) regions have the narrowest thermal safety margins in upper canopy leaves; these regions are at the greatest risk of damage due to extreme heat‐wave events, especially under conditions when leaf temperatures are further elevated by a lack of transpirational cooling. Using predicted heat‐wave events for 2050 and accounting for possible thermal acclimation of T_crit and T_max, we also found that these safety margins could shrink in a warmer world, as rising temperatures are likely to exceed thermal tolerance limits. Thus, increasing numbers of species in many biomes may be at risk as heat‐wave events become more severe with climate change.     

Synthesis and biological evaluation of histone deacetylase inhibitors that are based on FR235222: a cyclic tetrapeptide scaffold

We outline the synthesis of six novel derivatives that are based on a recently discovered HDAC inhibitor FR235222. Our work is the first report utilizing a novel binding element, guanidine, as metal coordinators in HDAC inhibitors. Further, we demonstrate that these compounds show cytotoxicity that parallels their ability to inhibit deacetylase activity, and that the most potent compounds maintain an L-Phe at position 1, and a D-Pro at position 4. Both inhibition of HDAC activity and cytotoxicity against the pancreatic cancer cell line BxPC3 are exhibited by these compounds, establishing that a guanidine unit can be utilized successfully to inhibit HDAC activity.