Enhanced-acceptor fluorescence-based single cell ATP biosensor monitors ATP in heterogeneous cancer populations in real time

Current methods to monitor cellular ATP do not provide spatial or temporal localization of ATP in single cells in real time or they display imperfect specificity to ATP. Here, we have developed a single cell, Enhanced Acceptor Fluorescence (EAF)-based ATP biosensor to visualize ATP in real time. This biosensor utilizes a modified mimic of the ε-subunits of the Bacillus subtilis F₀F₁ synthase and is coupled to the EAF fluorophores pairs, GFP and YFP. The sensor was then used to monitor ATP in a heterogeneous glioblastoma multiform cancer cell population. We anticipate that this innovative technology and our better understanding of the ATP machinery will have substantial influence on future translational studies.     

A novel estrogen-regulated avian apolipoprotein

In search for yet uncharacterized proteins involved in lipid metabolism of the chicken, we have isolated a hitherto unknown protein from the serum lipoprotein fraction with a buoyant density of ≤1.063 g/ml. Data obtained by protein microsequencing and molecular cloning of cDNA defined a 537 bp cDNA encoding a precursor molecule of 178 residues. As determined by SDS-PAGE, the major circulating form of the protein, which we designate apolipoprotein-VLDL-IV (Apo-IV), has an apparent M_r of approximately 17 kDa. Northern Blot analysis of different tissues of laying hens revealed Apo-IV expression mainly in the liver and small intestine, compatible with an involvement of the protein in lipoprotein metabolism. To further investigate the biology of Apo-IV, we raised an antibody against a GST-Apo-IV fusion protein, which allowed the detection of the 17-kDa protein in rooster plasma, whereas in laying hens it was detectable only in the isolated ≤1.063 g/ml density lipoprotein fraction. Interestingly, estrogen treatment of roosters caused a reduction of Apo-IV in the liver and in the circulation to levels similar to those in mature hens. Furthermore, the antibody crossreacted with a 17-kDa protein in quail plasma, indicating conservation of Apo-IV in avian species. In search for mammalian counterparts of Apo-IV, alignment of the sequence of the novel chicken protein with those of different mammalian apolipoproteins revealed stretches with limited similarity to regions of ApoC-IV and possibly with ApoE from various mammalian species. These data suggest that Apo-IV is a newly identified avian apolipoprotein.     

Estradiol effects on behavior and serum oxytocin are modified by social status and polymorphisms in the serotonin transporter gene in female rhesus monkeys

Despite the well-documented relation between estradiol (E2) and behavior, exposure to stressors may modify sensitivity to E2. The effects of E2 on behavior are, in part, likely related to their modulation of the serotonin (5HT) and oxytocin systems. The short allele (s-variant) polymorphism found in the promoter region of the SLC6A4 gene that encodes the 5HT transporter (5HTT) modulates responsivity to stressors. The current study used ovariectomized adult female rhesus monkeys to evaluate how exposure to the psychosocial stressor of social subordination and polymorphisms in the gene encoding 5HTT influence the behavioral effects of E2 and immunoreactive serum oxytocin. Dominant females had higher levels of oxytocin than subordinate animals even though E2 increased immunoreactive serum oxytocin in all females. E2 increased affiliative behaviors in all animals, with even more of these prosocial behaviors directed at dominant females. S-variant females, regardless of social status, were more aggressive toward more subordinate cage mates and these behaviors too were increased by E2. Subordinate s-variant females are most often involved in agonistic behavior, less affiliative behavior, and were less responsive to the anxiolytic action of E2. The results show that the short allele of the 5HTT gene synergizes with psychosocial stress exposure to affect the behavioral efficacy of E2 while confirming the actions of E2 for producing generalized behavioral arousal in females. Whether differences in the central action of 5HT and/or oxytocin are responsible for this effect requires further study.     

Cancer cell survival following DNA damage-mediated premature senescence is regulated by mammalian target of rapamycin (mTOR)-dependent Inhibition of sirtuin 1

DNA-damaging agents can induce premature senescence in cancer cells, which contributes to the static effects of cancer. However, senescent cancer cells may re-enter the cell cycle and lead to tumor relapse. Understanding the mechanisms that control the viability of senescent cells may be helpful in eliminating these cells before they can regrow. Treating human squamous cell carcinoma (SCC) cells with the anti-cancer compounds, resveratrol and doxorubicin, triggered p53-independent premature senescence by invoking oxidative stress-mediated DNA damage. This process involved the mTOR-dependent phosphorylation of SIRT1 at serine 47, resulting in the inhibition of the deacetylase activity of SIRT1. SIRT1 phosphorylation caused concomitant increases in p65/RelA NF-κB acetylation and the expression of an anti-apoptotic Bfl-1/A1. SIRT1 physically interacts with the mTOR-Raptor complex, and a single amino acid substitution in the TOS (TOR signaling) motif in the SIRT1 prevented Ser-47 phosphorylation and Bfl-1/A1 induction. The pharmacologic and genetic inhibition of mTOR, unphosphorylatable S47A, or F474A TOS mutants restored SIRT1 deacetylase activity, blocked Bfl-1/A1 induction, and sensitized prematurely senescent SCC cells for apoptosis. We further show that the treatment of UVB-induced SCCs with doxorubicin transiently stabilized tumor growth but was followed by tumor regrowth upon drug removal in p53(+/-)/SKH-1 mice. The subsequent treatment of stabilized SCCs with rapamycin decreased tumor size and induced caspase-3 activation. These results demonstrate that the inhibition of SIRT1 by mTOR fosters survival of DNA damage-induced prematurely senescent SCC cells via Bfl-1/A1 in the absence of functional p53.     

Dissecting the effect of a heterogeneous environment on the interaction between host and parasite fitness traits

Environmental variation can alter the probability of parasitic infection or the fitness consequence of infection, and thus has the potential to dramatically alter the dynamics of host parasite coevolution. Here we investigated the effect of a changing temperature on host-parasite interactions using the crustacean Daphnia magna and its bacterial parasite Pasteuria ramosa. By reciprocally varying (1) the temperature at which exposure to parasites occurred and (2) the temperature at which within-host parasite growth occurred, and measuring several fitness-related traits, we show that while there are temperature combinations that favour either host or parasite, there are also conditions that favour neither, that is, negative fitness consequences for the host without fitness benefits for the parasite. This result highlights the importance of considering a heterogeneous rather than static environment in coevolutionary studies, while also showing support for an optimal virulence strategy in castrating parasites.     

Fermentation and microencapsulation of the nematophagous fungus <Emphasis Type="Italic">Hirsutella rhossiliensis</Emphasis> in a novel type of hollow beads

In this work, fermentation and formulation aspects of the nematophagous fungus Hirsutella rhossiliensis BBA were investigated. When incubated in 2% (w/w) glucose and 0.5% (w/w) yeast extract medium in a 1-L Erlenmeyer flask without baffles, heavy pellet formation was observed. Only 40% of the mycelium had a size less than 500 μm. When a flask with three baffles was used, the portion of mycelium <500 μm rose to 95%. In the next step, the influence of aeration rate and stirrer speed on production of finely dispersed mycelium in a stirred tank reactor was investigated. The best fermentation results were obtained at 0.4 vvm and 400 rpm stirrer speed with 90% mycelium <500 μm and 5 g/L biomass. Then, mycelium was microencapsulated in hollow beads based on sulfoethylcellulose (SEC). Experiments on the capsule nutrient reservoir showed that 15% (w/w) corn gluten and 0.5% (w/w) yeast extract could be replaced with 3% (w/w) autoclaved baker's yeast which was never used as capsule additive before. Radial growth of mycelium out of dried hollow beads containing 1% (w/w) biomass and 3% (w/w) baker's yeast was faster than for alginate beads containing equivalent amounts of biomass and yeast indicating a higher bio-control potential.     

Bacteria primed by antimicrobial peptides develop tolerance and persist

Antimicrobial peptides (AMPs) are key components of innate immune defenses. Because of the antibiotic crisis, AMPs have also come into focus as new drugs. Here, we explore whether prior exposure to sub-lethal doses of AMPs increases bacterial survival and abets the evolution of resistance. We show that Escherichia coli primed by sub-lethal doses of AMPs develop tolerance and increase persistence by producing curli or colanic acid, responses linked to biofilm formation. We develop a population dynamic model that predicts that priming delays the clearance of infections and fuels the evolution of resistance. The effects we describe should apply to many AMPs and other drugs that target the cell surface. The optimal strategy to tackle tolerant or persistent cells requires high concentrations of AMPs and fast and long-lasting expression. Our findings also offer a new understanding of non-inherited drug resistance as an adaptive response and could lead to measures that slow the evolution of resistance.     

Predicting the Mosquito Species and Vertebrate Species Involved in the Theoretical Transmission of Rift Valley Fever Virus in the United States

Rift Valley fever virus (RVFV) is a mosquito-borne virus in the family Bunyaviridiae that has spread throughout continental Africa to Madagascar and the Arabian Peninsula. The establishment of RVFV in North America would have serious consequences for human and animal health in addition to a significant economic impact on the livestock industry. Published and unpublished data on RVFV vector competence, vertebrate host competence, and mosquito feeding patterns from the United States were combined to quantitatively implicate mosquito vectors and vertebrate hosts that may be important to RVFV transmission in the United States. A viremia-vector competence relationship based on published mosquito transmission studies was used to calculate a vertebrate host competence index which was then combined with mosquito blood feeding patterns to approximate the vector and vertebrate amplification fraction, defined as the relative contribution of the mosquito or vertebrate host to pathogen transmission. Results implicate several Aedes spp. mosquitoes and vertebrates in the order Artiodactyla as important hosts for RVFV transmission in the U.S. Moreover, this study identifies critical gaps in knowledge which would be necessary to complete a comprehensive analysis identifying the different contributions of mosquitoes and vertebrates to potential RVFV transmission in the U.S. Future research should focus on (1) the dose-dependent relationship between viremic exposure and the subsequent infectiousness of key mosquito species, (2) evaluation of vertebrate host competence for RVFV among North American mammal species, with particular emphasis on the order Artiodactyla, and (3) identification of areas with a high risk for RVFV introduction so data on local vector and host populations can help generate geographically appropriate amplification fraction estimates.     

Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity

The midbody is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. Midbodies persist in cells long after division as midbody derivatives (MB(d)s), but their fate is unclear. Here we show that MB(d)s are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells, induced pluripotent stem cells and potential cancer 'stem cells' in vivo and in vitro. MB(d) loss accompanies stem-cell differentiation, and involves autophagic degradation mediated by binding of the autophagic receptor NBR1 to the midbody protein CEP55. Differentiating cells and normal dividing cells do not accumulate MB(d)s and possess high autophagic activity. Stem cells and cancer cells accumulate MB(d)s by evading autophagosome encapsulation and exhibit low autophagic activity. MB(d) enrichment enhances reprogramming to induced pluripotent stem cells and increases the in vitro tumorigenicity of cancer cells. These results indicate unexpected roles for MB(d)s in stem cells and cancer 'stem cells'.     

Excellent adherence to antiretrovirals in HIV+ Zambian children is compromised by disrupted routine, HIV nondisclosure, and paradoxical income effects

Introduction

A better understanding of pediatric antiretroviral therapy (ART) adherence in sub-Saharan Africa is necessary to develop interventions to sustain high levels of adherence.

Methodology/Principal Findings

Adherence among 96 HIV-infected Zambian children (median age 6, interquartile range [IQR] 2,9) initiating fixed-dose combination ART was measured prospectively (median 23 months; IQR 20,26) with caregiver report, clinic and unannounced home-based pill counts, and medication event monitoring systems (MEMS). HIV-1 RNA was determined at 48 weeks. Child and caregiver characteristics, socio-demographic status, and treatment-related factors were assessed as predictors of adherence. Median adherence was 97.4% (IQR 96.1,98.4%) by visual analog scale, 94.8% (IQR 86,100%) by caregiver-reported last missed dose, 96.9% (IQR 94.5,98.2%) by clinic pill count, 93.4% (IQR 90.2,96.7%) by unannounced home-based pill count, and 94.8% (IQR 87.8,97.7%) by MEMS. At 48 weeks, 72.6% of children had HIV-1 RNA <50 copies/ml. Agreement among adherence measures was poor; only MEMS was significantly associated with viral suppression (p = 0.013). Predictors of poor adherence included changing residence, school attendance, lack of HIV disclosure to children aged nine to 15 years, and increasing household income.

Conclusions/Significance

Adherence among children taking fixed-dose combination ART in sub-Saharan Africa is high and sustained over two years. However, certain groups are at risk for treatment failure, including children with disrupted routines, no knowledge of their HIV diagnosis among older children, and relatively high household income, possibly reflecting greater social support in the setting of greater poverty.