A Heparin-Coated Circuit Reduces Complement Activation and the Release of Leukocyte Inflammatory Mediators During Extracorporeal Circulation in a Rabbit

Heparin coating modifies complement activation during extracorporeal circulation much more effcclively than systemically administered heparin. This rabbit study was undertaken to address possible mechanisms responsible for this difference. We evaluated the effect of heparin coating on complement activation and subsequently the release of leukocyte inflammatory mediators during extracorporeal circulation through a simplified circuit. We found in the heparin-coated group a significantly reduced complement hemolytic activity (CH 50 ), remaining higher leukocyte numbers, significantly decreased release of -glucuronidase, and most strikingly a complete prevention of tumor necrosis factor (TNF) formation. The significantly reduced CH 50 activity in the heparin-coated groups indicates the reduction of one or more native classical complement products. This could be explained by the absorption of complement components by the circuit, which results in reduced activity of the complement cascade. We conclude therefore that heparin coating reduces complement activation and consequently reduces the release of leukocyte inflammatory mediators.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73454/1/j.1525-1594.1992.tb00533.x.pd

Evaluation of the right atrial venous oxygen saturation as a physiologic monitor in a neonatal model

Pulmonary artery (PA) mixed venous saturation (SO2) has become a crucial monitor in the adult intensive care unit, but is not used in neonates because of the difficulty in PA catheterization. We evaluated the possibility of utilizing the right atrial venous oxygen saturation (RAO2), which is easily accessed in the neonate, as a monitor of the effects of mechanical ventilation and intravascular volume in an animal model selected to be the size of the human neonate. A continuous RAO2 monitoring catheter was placed into the right atrium of 16 normal rabbits (2.2 to 4.1 kg). Oxygen delivery was manipulated by alterations in peak inspiratory pressure (PIP) (n = 6), positive end-expiratory pressure (PEEP) (n = 6), or by progressive hypovolemia (n = 4). RAO2 decreased with onset of mechanical ventilation alone from 69% +/- 6% to 61% +/- 5% (P 2O, the RAO2 progressively decreased from 59% +/- 4% to 49% +/- 6% (P 2O, the RAO2 progressively decreased from 64% +/- 5% to 33% +/- 16% (P 2 approached baseline after return to continuous positive airway pressure (CPAP) of 3 cm H2O. Progressive phlebotomy to a total of 10 mL/kg resulted in a decrease in RAO2 from 70% +/- 6% to 27% +/- 5% (P 2 to near baseline. Peripheral arterial oxygen saturation remained at a constant 100% throughout each protocol. With oxygen consumption stable, the RAO2 is an excellent monitor of the effect of airway pressure or of hypovolemia on oxygen delivery. Use of the peripheral arterial oxygen saturation alone as a parameter for monitoring ventilator or intravascular volume effect is not adequate. RAO2 monitoring may be a powerful tool in the management of the critically ill neonates.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30724/1/0000373.pd

The effect of two different health messages on physical activity levels and health in sedentary overweight, middle-aged women

Background: Most public health guidelines recommend that adults need to participate in 30 minutes of moderate intensity physical activity on most days of the week to maintain good health. Achieving the recommended 30 minutes of exercise a day can be difficult in middle aged, overweight women. This 12 week study evaluated whether a 10,000 steps per day message was more effective than a 30 minutes a day message in increasing physical activity in low active, overweight women. Methods: Thirty participants were randomized into 2 groups: Group 1 was asked to undertake 30 minutes of walking/day, whereas Group 2 was asked to accumulate 10,000 steps/day using their pedometers. Results: Results showed that there were no changes in anthropometric and blood pressure measures between or within groups. However, the 10,000 step and the 30 minutes groups’ daily average number of steps/day were significantly higher than baseline at week 6 (p = 0.038 and p = 0.039 respectively) and at week 12 (p = 0.028 and p = 0.038 respectively). At week 12, the 10,000 steps group were taking an average of 4616 steps per day more (43% increase) than at baseline and the 30 minutes group were taking an average of 2761 steps per day more (35% increase) than at baseline. There was a significant difference in the number of steps with the 10,000 steps group versus 30 minutes group at 12 weeks (p = 0.045).Conclusions: This study found that low active, overweight women undertook significantly more physical activity when they had a daily 10,000 step goal using a pedometer, than when they were asked to achieve 30 minutes of walking/day. Therefore we suggest that a public health recommendation of “10,000 steps/day”, rather than the “30 min/day” could be applied to promote increased physical activity in sedentary middle aged women

A putative antiviral role of plant cytidine deaminases

[EN] Background: A mechanism of innate antiviral immunity operating against viruses infecting mammalian cells has been described during the last decade. Host cytidine deaminases (e.g., APOBEC3 proteins) edit viral genomes, giving rise to hypermutated nonfunctional viruses; consequently, viral fitness is reduced through lethal mutagenesis. By contrast, sub-lethal hypermutagenesis may contribute to virus evolvability by increasing population diversity. To prevent genome editing, some viruses have evolved proteins that mediate APOBEC3 degradation. The model plant Arabidopsis thaliana genome encodes nine cytidine deaminases (AtCDAs), raising the question of whether deamination is an antiviral mechanism in plants as well. Methods: Here we tested the effects of expression of AtCDAs on the pararetrovirus Cauliflower mosaic virus (CaMV). Two different experiments were carried out. First, we transiently overexpressed each one of the nine A. thaliana AtCDA genes in Nicotiana bigelovii plants infected with CaMV, and characterized the resulting mutational spectra, comparing them with those generated under normal conditions. Secondly, we created A. thaliana transgenic plants expressing an artificial microRNA designed to knock-out the expression of up to six AtCDA genes. This and control plants were then infected with CaMV. Virus accumulation and mutational spectra where characterized in both types of plants. Results: We have shown that the A. thaliana AtCDA1 gene product exerts a mutagenic activity, significantly increasing the number of G to A mutations in vivo, with a concomitant reduction in the amount of CaMV genomes accumulated. Furthermore, the magnitude of this mutagenic effect on CaMV accumulation is positively correlated with the level of AtCDA1 mRNA expression in the plant. Conclusions: Our results suggest that deamination of viral genomes may also work as an antiviral mechanism in plants.This work was supported by the former Spanish Ministerio de Ciencia e Innovación-FEDER grant BFU2009-06993 to SFE. JMC was supported by the CSIC JAE-doc program/Fondo Social Europeo. AG-P was supported by a grant for Scientific and Technical Activities and by grant P10-CVI-65651, both from Junta de Andalucía.Martín, S.; Cuevas, J.; Grande-Perez, A.; Elena Fito, SF. (2017). A putative antiviral role of plant cytidine deaminases. F1000Research. 1-14. https://doi.org/10.12688/f1000research.11111.2S11

Characterization of the Interaction of Full-Length HIV-1 Vif Protein with its Key Regulator CBFβ and CRL5 E3 Ubiquitin Ligase Components

Human immunodeficiency virus-1 (HIV-1) viral infectivity factor (Vif) is essential for viral replication because of its ability to eliminate the host's antiviral response to HIV-1 that is mediated by the APOBEC3 family of cellular cytidine deaminases. Vif targets these proteins, including APOBEC3G, for polyubiquitination and subsequent proteasome-mediated degradation via the formation of a Cullin5-ElonginB/C-based E3 ubiquitin ligase. Determining how the cellular components of this E3 ligase complex interact with Vif is critical to the intelligent design of new antiviral drugs. However, structural studies of Vif, both alone and in complex with cellular partners, have been hampered by an inability to express soluble full-length Vif protein. Here we demonstrate that a newly identified host regulator of Vif, core-binding factor-beta (CBFβ), interacts directly with Vif, including various isoforms and a truncated form of this regulator. In addition, carboxyl-terminal truncations of Vif lacking the BC-box and cullin box motifs were sufficient for CBFβ interaction. Furthermore, association of Vif with CBFβ, alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif. Finally, a stable complex containing Vif-CBFβ-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5). This efficient strategy for purifying Vif-Cul5-CBFβ-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates

Multi-Scale Modeling of HIV Infection in vitro and APOBEC3G-Based Anti-Retroviral Therapy

The human APOBEC3G is an innate restriction factor that, in the absence of Vif, restricts HIV-1 replication by inducing excessive deamination of cytidine residues in nascent reverse transcripts and inhibiting reverse transcription and integration. To shed light on impact of A3G-Vif interactions on HIV replication, we developed a multi-scale computational system consisting of intracellular (single-cell), cellular and extracellular (multicellular) events by using ordinary differential equations. The single-cell model describes molecular-level events within individual cells (such as production and degradation of host and viral proteins, and assembly and release of new virions), whereas the multicellular model describes the viral dynamics and multiple cycles of infection within a population of cells. We estimated the model parameters either directly from previously published experimental data or by running simulations to find the optimum values. We validated our integrated model by reproducing the results of in vitro T cell culture experiments. Crucially, both downstream effects of A3G (hypermutation and reduction of viral burst size) were necessary to replicate the experimental results in silico. We also used the model to study anti-HIV capability of several possible therapeutic strategies including: an antibody to Vif; upregulation of A3G; and mutated forms of A3G. According to our simulations, A3G with a mutated Vif binding site is predicted to be significantly more effective than other molecules at the same dose. Ultimately, we performed sensitivity analysis to identify important model parameters. The results showed that the timing of particle formation and virus release had the highest impacts on HIV replication. The model also predicted that the degradation of A3G by Vif is not a crucial step in HIV pathogenesis

Evolution of the Primate APOBEC3A Cytidine Deaminase Gene and Identification of Related Coding Regions

The APOBEC3 gene cluster encodes six cytidine deaminases (A3A-C, A3DE, A3F-H) with single stranded DNA (ssDNA) substrate specificity. For the moment A3A is the only enzyme that can initiate catabolism of both mitochondrial and nuclear DNA. Human A3A expression is initiated from two different methionine codons M1 or M13, both of which are in adequate but sub-optimal Kozak environments. In the present study, we have analyzed the genetic diversity among A3A genes across a wide range of 12 primates including New World monkeys, Old World monkeys and Hominids. Sequence variation was observed in exons 1–4 in all primates with up to 31% overall amino acid variation. Importantly for 3 hominids codon M1 was mutated to a threonine codon or valine codon, while for 5/12 primates strong Kozak M1 or M13 codons were found. Positive selection was apparent along a few branches which differed compared to positive selection in the carboxy-terminal of A3G that clusters with A3A among human cytidine deaminases. In the course of analyses, two novel non-functional A3A-related fragments were identified on chromosome 4 and 8 kb upstream of the A3 locus. This qualitative and quantitative variation among primate A3A genes suggest that subtle differences in function might ensue as more light is shed on this increasingly important enzyme