Bushmeat genetics: setting up a reference framework for the DNA typing of African forest bushmeat

The bushmeat trade in tropical Africa represents illegal, unsustainable off‐takes of millions of tons of wild game – mostly mammals – per year. We sequenced four mitochondrial gene fragments (cyt b, COI, 12S, 16S) in >300 bushmeat items representing nine mammalian orders and 59 morphological species from five western and central African countries (Guinea, Ghana, Nigeria, Cameroon and Equatorial Guinea). Our objectives were to assess the efficiency of cross‐species PCR amplification and to evaluate the usefulness of our multilocus approach for reliable bushmeat species identification. We provide a straightforward amplification protocol using a single ‘universal’ primer pair per gene that generally yielded >90% PCR success rates across orders and was robust to different types of meat preprocessing and DNA extraction protocols. For taxonomic identification, we set up a decision pipeline combining similarity‐ and tree‐based approaches with an assessment of taxonomic expertise and coverage of the GENBANK database. Our multilocus approach permitted us to: (i) adjust for existing taxonomic gaps in GENBANK databases, (ii) assign to the species level 67% of the morphological species hypotheses and (iii) successfully identify samples with uncertain taxonomic attribution (preprocessed carcasses and cryptic lineages). High levels of genetic polymorphism across genes and taxa, together with the excellent resolution observed among species‐level clusters (neighbour‐joining trees and Klee diagrams) advocate the usefulness of our markers for bushmeat DNA typing. We formalize our DNA typing decision pipeline through an expert‐curated query database – DNAbushmeat – that shall permit the automated identification of African forest bushmeat items.     

Evaluating the Impact of Zimbabwe’s Prevention of Mother-to-Child HIV Transmission Program: Population-Level Estimates of HIV-Free Infant Survival Pre-Option A

Objective

We estimated HIV-free infant survival and mother-to-child HIV transmission (MTCT) rates in Zimbabwe, some of the first community-based estimates from a UNAIDS priority country.

Methods

In 2012 we surveyed mother-infant pairs residing in the catchment areas of 157 health facilities randomly selected from 5 of 10 provinces in Zimbabwe. Enrolled infants were born 9–18 months before the survey. We collected questionnaires, blood samples for HIV testing, and verbal autopsies for deceased mothers/infants. Estimates were assessed among i) all HIV-exposed infants, as part of an impact evaluation of Option A of the 2010 WHO guidelines (rolled out in Zimbabwe in 2011), and ii) the subgroup of infants unexposed to Option A. We compared province-level MTCT rates measured among women in the community with MTCT rates measured using program monitoring data from facilities serving those communities.

Findings

Among 8568 women with known HIV serostatus, 1107 (12.9%) were HIV-infected. Among all HIV-exposed infants, HIV-free infant survival was 90.9% (95% confidence interval (CI): 88.7–92.7) and MTCT was 8.8% (95% CI: 6.9–11.1). Sixty-six percent of HIV-exposed infants were still breastfeeding. Among the 762 infants born before Option A was implemented, 90.5% (95% CI: 88.1–92.5) were alive and HIV-uninfected at 9–18 months of age, and 9.1% (95%CI: 7.1–11.7) were HIV-infected. In four provinces, the community-based MTCT rate was higher than the facility-based MTCT rate. In Harare, the community and facility-based rates were 6.0% and 9.1%, respectively.

Conclusion

By 2012 Zimbabwe had made substantial progress towards the elimination of MTCT. Our HIV-free infant survival and MTCT estimates capture HIV transmissions during pregnancy, delivery and breastfeeding regardless of whether or not mothers accessed health services. These estimates also provide a baseline against which to measure the impact of Option A guidelines (and subsequently Option B+).     

Lipid Classes and Fatty Acids in Ophryotrocha cyclops,a Dorvilleid from Newfoundland Aquaculture Sites

A new opportunistic annelid (Ophryotrocha cyclops) discovered on benthic substrates underneath finfish aquaculture sites in Newfoundland (NL) may be involved in the remediation of organic wastes. At those aquaculture sites, bacterial mats and O. cyclops often coexist and are used as indicators of organic enrichment. Little is known on the trophic strategies used by these annelids, including whether they might consume bacteria or other aquaculture-derived wastes. We studied the lipid and fatty acid composition of the annelids and their potential food sources (degraded flocculent organic matter, fresh fish pellets and bacterial mats) to investigate feeding relationships in these habitats and compared the lipid and fatty acid composition of annelids before and after starvation. Fish pellets were rich in lipids, mainly terrestrially derived C₁₈ fatty acids (18:1ω9, 18:2ω6, 18:3ω3), while bacterial samples were mainly composed of ω7 fatty acids, and flocculent matter appeared to be a mixture of fresh and degrading fish pellets, feces and bacteria. Ophryotrocha cyclops did not appear to store excessive amounts of lipids (13%) but showed a high concentration of ω3 and ω6 fatty acids, as well as a high proportion of the main fatty acids contained in fresh fish pellets and bacterial mats. The dorvilleids and all potential food sources differed significantly in their lipid and fatty acid composition. Interestingly, while all food sources contained low proportions of 20:5ω3 and 20:2ω6, the annelids showed high concentrations of these two fatty acids, along with 20:4ω6. A starvation period of 13 days did not result in a major decrease in total lipid content; however, microscopic observations revealed that very few visible lipid droplets remained in the gut epithelium after three months of starvation. Ophryotrocha cyclops appears well adapted to extreme environments and may rely on lipid-rich organic matter for survival and dispersal in cold environments.     

Escape of HIV-1-Infected Dendritic Cells from TRAIL-Mediated NK Cell Cytotoxicity during NK-DC Cross-Talk—A Pivotal Role of HMGB1

Early stages of Human Immunodeficiency Virus-1 (HIV-1) infection are associated with local recruitment and activation of important effectors of innate immunity, i.e. natural killer (NK) cells and dendritic cells (DCs). Immature DCs (iDCs) capture HIV-1 through specific receptors and can disseminate the infection to lymphoid tissues following their migration, which is associated to a maturation process. This process is dependent on NK cells, whose role is to keep in check the quality and the quantity of DCs undergoing maturation. If DC maturation is inappropriate, NK cells will kill them (“editing process”) at sites of tissue inflammation, thus optimizing the adaptive immunity. In the context of a viral infection, NK-dependent killing of infected-DCs is a crucial event required for early elimination of infected target cells. Here, we report that NK-mediated editing of iDCs is impaired if DCs are infected with HIV-1. We first addressed the question of the mechanisms involved in iDC editing, and we show that cognate NK-iDC interaction triggers apoptosis via the TNF-related apoptosis-inducing ligand (TRAIL)-Death Receptor 4 (DR4) pathway and not via the perforin pathway. Nevertheless, once infected with HIV-1, DC_HIV become resistant to NK-induced TRAIL-mediated apoptosis. This resistance occurs despite normal amounts of TRAIL released by NK cells and comparable DR4 expression on DC_HIV. The escape of DC_HIV from NK killing is due to the upregulation of two anti-apoptotic molecules, the cellular-Flice like inhibitory protein (c-FLIP) and the cellular inhibitor of apoptosis 2 (c-IAP2), induced by NK-DC_HIV cognate interaction. High-mobility group box 1 (HMGB1), an alarmin and a key mediator of NK-DC cross-talk, was found to play a pivotal role in NK-dependent upregulation of c-FLIP and c-IAP2 in DC_HIV. Finally, we demonstrate that restoration of DC_HIV susceptibility to NK-induced TRAIL killing can be obtained either by silencing c-FLIP and c-IAP2 by specific siRNA, or by inhibiting HMGB1 with blocking antibodies or glycyrrhizin, arguing for a key role of HMGB1 in TRAIL resistance and DC_HIV survival. These findings provide evidence for a new strategy developed by HIV to escape immune attack, they challenge the question of the involvement of HMGB1 in the establishment of viral reservoirs in DCs, and they identify potential therapeutic targets to eliminate infected DCs.     

Living together: behavior and welfare in single and mixed species groups of capuchin (Cebus apella) and squirrel monkeys (Saimiri sciureus)

There are potential advantages of housing primates in mixed species exhibits for both the visiting public and the primates themselves. If the primates naturally associate in the wild, it may be more educational and enjoyable for the public to view. Increases in social complexity and stimulation may be enriching for the primates. However, mixed species exhibits might also create welfare problems such as stress from interspecific aggression. We present data on the behavior of single and mixed species groups of capuchin monkeys (Cebus apella) and squirrel monkeys (Saimiri sciureus) housed at the Living Links to Human Evolution Research Centre in the Royal Zoological Society of Scotland's Edinburgh Zoo. These species associate in the wild, gaining foraging benefits and decreased predation. But Cebus are also predators themselves with potential risks for the smaller Saimiri. To study their living together we took scan samples at ≥15 min intervals on single (n=109) and mixed species groups (n=152), and all occurrences of intraspecific aggression and interspecific interactions were recorded. We found no evidence of chronic stress and Saimiri actively chose to associate with Cebus. On 79% of scans, the two species simultaneously occupied the same part of their enclosure. No vertical displacement was observed. Interspecific interactions were common (>2.5/hr), and equally divided among mildly aggressive, neutral, and affiliative interactions such as play. Only one aggressive interaction involved physical contact and was non‐injurious. Aggressive interactions were mostly (65%) displacements and vocal exchanges, initiated almost equally by Cebus and Saimiri. Modifications to the enclosure were successful in reducing these mildly aggressive interactions with affiliative interactions increasing in frequency and diversity. Our data suggest that in carefully designed, large enclosures, naturally associating monkeys are able to live harmoniously and are enriched by each other. Am. J. Primatol. 72:33–47, 2010. © 2009 Wiley‐Liss, Inc.     

Changes in cholesterol levels in the plasma membrane modulate cell signaling and regulate cell adhesion and migration on fibronectin

The number and distribution of lipid molecules, including cholesterol in particular, in the plasma membrane, may play a key role in regulating several physiological processes in cells. We investigated the role of membrane cholesterol in regulating cell shape, adhesion and motility. The acute depletion of cholesterol from the plasma membrane of cells that were well spread and motile on fibronectin caused the rounding of these cells and decreased their adhesion to and motility on fibronectin. These modifications were less pronounced in cells plated on laminin, vitronectin or plastic, indicating that cholesterol-mediated changes in adhesion and motility are more specific for adhesion mediated by fibronectin-specific integrins, such as alpha5beta1. These changes were accompanied by remodeling of the actin cytoskeleton, the spatial reorganization of paxillin in the membrane, and changes to the dynamics of alpha5 integrin and paxillin-rich focal adhesions. Levels of tyrosine phosphorylation at position 576/577 of FAK and Erk1/Erk2 MAP-kinase activity levels were both lower in cholesterol-depleted than in control cells. These levels normalized only on fibronectin when cholesterol was reincorporated into the cell membrane. Thus, membrane cholesterol content has a specific effect on certain signaling pathways specifically involved in regulating cell motility on fibronectin and organization of the actin cytoskeleton.     

The anti-biofilm activity secreted by a marine Pseudoalteromonas strain

Bacterial biofilms occur on all submerged structures in marine environments. The authors previously reported that the marine bacterium Pseudoalteromonas sp. 3J6 secretes antibiofilm activity. Here, it was discovered that another Pseudoalteromonas sp. strain, D41, inhibited the development of strain 3J6 in mixed biofilms. Confocal laser scanning microscope observations revealed that the culture supernatant of strain D41 impaired biofilm formation of strain 3J6 and another marine bacterium. A microtiter plate assay of the antibiofilm activity was set up and validated with culture supernatants of Pseudoalteromonas sp. 3J6. This assay was used to determine the spectra of action of strains D41 and 3J6. Each culture supernatant impaired the biofilm development of 13 marine bacteria out of 18. However, differences in the spectra of action and the physical behaviours of the antibiofilm molecules suggest that the latter are not identical. They nevertheless share the originality of being devoid of antibacterial activity against planktonic bacteria.     

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

We previously reported that HSV-2 R1, the R1 subunit (ICP10; UL39) of herpes simplex virus type-2 ribonucleotide reductase, protects cells against apoptosis induced by the death receptor (DR) ligands tumor necrosis factor-alpha- (TNFα) and Fas ligand (FasL) by interrupting DR-mediated signaling at, or upstream of, caspase-8 activation. Further investigation of the molecular mechanism underlying HSV-2 R1 protection showed that extracellular-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3-K)/Akt, NF-κB and JNK survival pathways do not play a major role in this antiapoptotic function. Interaction studies revealed that HSV-2 R1 interacted constitutively with caspase-8. The HSV-2 R1 deletion mutant R1(1-834)-GFP and Epstein–Barr virus (EBV) R1, which did not protect against apoptosis induced by DR ligands, did not interact with caspase-8, indicating that interaction is required for protection. HSV-2 R1 impaired caspase-8 activation induced by caspase-8 over-expression, suggesting that interaction between the two proteins prevents caspase-8 dimerization/activation. HSV-2 R1 bound to caspase-8 directly through its prodomain but did not interact with either its caspase domain or Fas-associated death domain protein (FADD). Interaction between HSV-2 R1 and caspase-8 disrupted FADD-caspase-8 binding. We further demonstrated that individually expressed HSV-1 R1 (ICP6) shares, with HSV-2 R1, the ability to bind caspase-8 and to protect cells against DR-induced apoptosis. Finally, as the long-lived Fas protein remained stable during the early period of infection, experiments with the HSV-1 UL39 deletion mutant ICP6∆ showed that HSV-1 R1 could be essential for the protection of HSV-1-infected cells against FasL.     

Regulation of apoptotic mediators reveals dynamic responses to thermal stress in the reef building coral Acropora millepora

Background

Mass coral bleaching is increasing in scale and frequency across the world''s coral reefs and is being driven primarily by increased levels of thermal stress arising from global warming. In order to understand the impacts of projected climate change upon corals reefs, it is important to elucidate the underlying cellular mechanisms that operate during coral bleaching and subsequent mortality. In this respect, increased apoptotic cell death activity is an important cellular process that is associated with the breakdown of the mutualistic symbiosis between the cnidarian host and their dinoflagellate symbionts.

Methodology/Principal Findings

The present study reports the impacts of different stressors (colchicine and heat stress) on three phases of apoptosis: (i) the potential initiation by differential expression of Bcl-2 members, (ii) the execution of apoptotic events by activation of caspase 3-like proteases and (iii) and finally, the cell disposal indicated by DNA fragmentation in the reef building coral Acropora millepora. In corals incubated with colchicine, an increase in caspase 3-like activity and DNA fragmentation was associated with a relative down-regulation of Bcl-2, suggesting that the initiation of apoptosis may be mediated by the suppression of an anti-apoptotic mechanism. In contrast, in the early steps of heat stress, the induction of caspase-dependent apoptosis was related to a relative up-regulation of Bcl-2 consecutively followed by a delayed decrease in apoptosis activity.

Conclusions/Significance

In the light of these results, we propose a model of heat stress in coral hosts whereby increasing temperatures engage activation of caspase 3-dependent apoptosis in cells designated for termination, but also the onset of a delayed protective response involving overexpression of Bcl-2 in surviving cells. This mitigating response to thermal stress could conceivably be an important regulatory mechanism for cell survival in corals exposed to sudden environmental changes.