Subcellular localization of the APOBEC3 proteins during mitosis and implications for genomic DNA deamination

Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme’s capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.     

Rates and Factors Associated with Major Modifications to First-Line Combination Antiretroviral Therapy: Results from the Asia-Pacific Region

Background

In the Asia-Pacific region many countries have adopted the WHO’s public health approach to HIV care and treatment. We performed exploratory analyses of the factors associated with first major modification to first-line combination antiretroviral therapy (ART) in resource-rich and resource-limited countries in the region.

Methods

We selected treatment naive HIV-positive adults from the Australian HIV Observational Database (AHOD) and the TREAT Asia HIV Observational Database (TAHOD). We dichotomised each country’s per capita income into high/upper-middle (T-H) and lower-middle/low (T-L). Survival methods stratified by income were used to explore time to first major modification of first-line ART and associated factors. We defined a treatment modification as either initiation of a new class of antiretroviral (ARV) or a substitution of two or more ARV agents from within the same ARV class.

Results

A total of 4250 patients had 961 major modifications to first-line ART in the first five years of therapy. The cumulative incidence (95% CI) of treatment modification was 0.48 (0.44–0.52), 0.33 (0.30–0.36) and 0.21 (0.18–0.23) for AHOD, T-H and T-L respectively. We found no strong associations between typical patient characteristic factors and rates of treatment modification. In AHOD, relative to sites that monitor twice-yearly (both CD4 and HIV RNA-VL), quarterly monitoring corresponded with a doubling of the rate of treatment modifications. In T-H, relative to sites that monitor once-yearly (both CD4 and HIV RNA-VL), monitoring twice-yearly corresponded to a 1.8 factor increase in treatment modifications. In T-L, no sites on average monitored both CD4 & HIV RNA-VL concurrently once-yearly. We found no differences in rates of modifications for once- or twice-yearly CD4 count monitoring.

Conclusions

Low-income countries tended to have lower rates of major modifications made to first-line ART compared to higher-income countries. In higher-income countries, an increased rate of RNA-VL monitoring was associated with increased modifications to first-line ART.     

Enhancing the egg's natural defence against bacterial penetration by increasing cuticle deposition

The cuticle is a proteinaceous layer covering the avian egg and is believed to form a defence to microorganism ingress. In birds that lay eggs in challenging environments, the cuticle is thicker, suggesting evolutionary pressure; however, in poultry, selection pressure for this trait has been removed because of artificial incubation. This study aimed to quantify cuticle deposition and to estimate its genetic parameters and its role on trans‐shell penetration of bacteria. Additionally, cuticle proteins were characterised to establish whether alleles for these genes explained variation in deposition. A novel and reliable quantification was achieved using the difference in reflectance of the egg at 650 nm before and after staining with a specific dye. The heritability of this novel measurement was moderate (0.27), and bacteria penetration was dependent on the natural variation in cuticle deposition. Eggs with the best cuticle were never penetrated by bacteria (P < 0.001). The cuticle proteome consisted of six major proteins. A significant association was found between alleles of one of these protein genes, ovocleidin‐116 (MEPE), and cuticle deposition (P = 0.015) and also between alleles of estrogen receptor 1 (ESR1) gene and cuticle deposition (P = 0.008). With the heritability observed, genetic selection should be possible to increase cuticle deposition in commercial poultry, so reducing trans‐generational transmission of microorganisms and reversing the lack of selection pressure for this trait during recent domestication.     

Multispecies coexistence of trees in tropical forests: spatial signals of topographic niche differentiation increase with environmental heterogeneity

Neutral and niche theories give contrasting explanations for the maintenance of tropical tree species diversity. Both have some empirical support, but methods to disentangle their effects have not yet been developed. We applied a statistical measure of spatial structure to data from 14 large tropical forest plots to test a prediction of niche theory that is incompatible with neutral theory: that species in heterogeneous environments should separate out in space according to their niche preferences. We chose plots across a range of topographic heterogeneity, and tested whether pairwise spatial associations among species were more variable in more heterogeneous sites. We found strong support for this prediction, based on a strong positive relationship between variance in the spatial structure of species pairs and topographic heterogeneity across sites. We interpret this pattern as evidence of pervasive niche differentiation, which increases in importance with increasing environmental heterogeneity.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Lysosomal membrane permeabilization is involved in oxidative stress-induced apoptotic cell death in LAMP2-deficient iPSCs-derived cerebral cortical neurons

Patients with Danon disease may suffer from severe cardiomyopathy, skeletal muscle dysfunction as well as varying degrees of mental retardation, in which the primary deficiency of lysosomal membrane-associated protein-2 (LAMP2) is considerably associated. Owing to the scarcity of human neurons, the pathological role of LAMP2 deficiency in neural injury of humans remains largely elusive. However, the application of induced pluripotent stem cells (iPSCs) may shed light on overcoming such scarcity.In this study, we obtained iPSCs derived from a patient carrying a mutated LAMP2 gene that is associated with Danon disease. By differentiating such LAMP2-deficient iPSCs into cerebral cortical neurons and with the aid of various biochemical assays, we demonstrated that the LAMP2-deficient neurons are more susceptible to mild oxidative stress-induced injury.The data from MTT assay and apoptotic analysis demonstrated that there was no notable difference in cellular viability between the normal and LAMP2-deficient neurons under non-stressed condition. When exposed to mild oxidative stress (10 μM H₂O₂), the LAMP2-deficient neurons exhibited a significant increase in apoptosis. Surprisingly, we did not observe any aberrant accumulation of autophagic materials in the LAMP2-deficient neurons under such stress condition.Our results from cellular fractionation and inhibitor blockade experiments further revealed that oxidative stress-induced apoptosis in the LAMP2-deficient cortical neurons was caused by increased abundance of cytosolic cathepsin L. These results suggest the involvement of lysosomal membrane permeabilization in the LAMP2 deficiency associated neural injury.     

Fixing a snag in carbon emissions estimates from wildfires

Wildfire is an essential earth‐system process, impacting ecosystem processes and the carbon cycle. Forest fires are becoming more frequent and severe, yet gaps exist in the modeling of fire on vegetation and carbon dynamics. Strategies for reducing carbon dioxide (CO₂) emissions from wildfires include increasing tree harvest, largely based on the public assumption that fires burn live forests to the ground, despite observations indicating that less than 5% of mature tree biomass is actually consumed. This misconception is also reflected though excessive combustion of live trees in models. Here, we show that regional emissions estimates using widely implemented combustion coefficients are 59%–83% higher than emissions based on field observations. Using unique field datasets from before and after wildfires and an improved ecosystem model, we provide strong evidence that these large overestimates can be reduced by using realistic biomass combustion factors and by accurately quantifying biomass in standing dead trees that decompose over decades to centuries after fire (“snags”). Most model development focuses on area burned; our results reveal that accurately representing combustion is also essential for quantifying fire impacts on ecosystems. Using our improvements, we find that western US forest fires have emitted 851 ± 228 Tg CO₂ (~half of alternative estimates) over the last 17 years, which is minor compared to 16,200 Tg CO₂ from fossil fuels across the region.     

Molecular characterization of four Haemophilus influenzae serotype a strains isolated from patients in Quebec, Canada

Four epidemiologically unrelated Haemophilus influenzae serotype a (Hia) strains from patients in Quebec, Canada, were characterized and found to represent 3 distinct groups. One isolate, found to be biotype I and sequence type (ST)-62 by multilocus sequence typing, was shown to possess the copper- and zinc-containing superoxide dismutase gene, sodC, and was suspected to belong to clonal division II. The other 3 isolates were classified as clonal division I based on the absence of the sodC gene. Among the 3 sodC-negative Hia strains, 2 were biotype II and had related STs (ST-23 and ST-403) and highly similar DNA fingerprints, similar to a group of previously described Hia isolates causing invasive disease in Manitoba, Canada. The remaining sodC-negative strain belonged to biotype I and ST-4 and shared no common allele with ST-23, ST-403, or ST-62. This isolate also possessed the IS1016-bexA partial deletion, which is often associated with increased virulence. Despite the small number of isolates used in this study, our finding of 3 distinct groups shows the existence of a potential genetic diversity not previously described for Hia. Whether this genetic diversity is related to the severity and epidemiology of Hia disease requires further studies.