966. CLAVIJA DOMINGENSIS

Clavija domingensis Urb. & Ekman was one of the many Haitian endemics that were described based on collections made by the great Swedish botanist Leonard Ekman between 1924 and 1928. The species is Critically Endangered sensu IUCN (criteria c2a(i); D) and it is currently the focus of conservation initiatives in Jardin Botanique des Cayes (Haiti), Jardín Botánico Nacional Dr. Rafael M. Moscoso (Dominican Republic), and Fairchild Tropical Botanic Garden (U.S.A.). Now known from only six localities from southern Haiti, each locality only represents a single individual. The species is illustrated based on plants grown in Fairchild Tropical Botanic Garden.     

Cryopreservation and storage of mussel (Mytilus spp.) haemocytes for latent analysis by the Comet assay

Estuarine and coastal habitats are known to be polluted by a range of chemical contaminants from both industrial and domestic sources. Blue mussels (Mytilus spp.), which inhabit these areas, are widely used as bio-indicators in eco-toxicological studies, because of their sedentary nature and their ability to bio-accumulate contaminants. The analysis of DNA damage in mussel haemocytes is a valuable tool for biomonitoring but sampling issues related to storage, handling and transportation have often limited its application in large-scale monitoring programmes. This study uses a trial and error method to evaluate and validate a suitable protocol for cryopreservation of mussel haemocytes, thereby allowing material collected in the field to be analysed later under controlled laboratory conditions. Three different cell-culture media, i.e. Leibovitz-15, Hank's balanced salt solution and mussel physiological saline, along with four different cryoprotectants, i.e. dimethyl sulphoxide (10% and 20%), 1,2-propanediol (10%), ethylene glycol (10%) and glycerol (10%) were tested to assess their suitability for cryopreservation of mussel haemocytes for analysis in the comet assay. Experimental studies where mussel haemocytes were also exposed to UV radiation or benzo(a)pyrene were conducted in order to mimic environmental stresses and to verify the effectiveness of newly defined cryopreservation protocols. The comet assay was used to demonstrate that mussel haemocytes could be preserved at cryogenic temperatures for a month without altering levels of DNA damage, which could possibly be used for lab or field studies where time constraints or facilities do not allow instant analysis.     

The value and meaning attached to genetic relatedness among Australian sperm donors

While in the context of western societies sperm is attributed with a wide range of meanings, in the context of assisted reproduction it has increasingly been treated as an alienable commodity. Yet despite attempts by medical professionals to encourage a disconnect between donors and their sperm, the latter continues in many instances to operate as a synecdoche for the former. This can be seen, for example, both in donor-conceived children's desire to know their donor and in donors' investments in the use of their sperm. This paper explores the latter example by providing a discourse analysis of the narratives of 30 Australian sperm donors, with a focus on how they accounted for the value and meaning of their sperm. Three broad themes are discussed: sperm as a marker of genetic legacy, responsibility for sperm as genetic material, and sperm as a “gift” to others. The implications of these understandings of sperm among donors are discussed in relation to outcomes for all parties involved in donor conception, and suggestions are made for recognizing the investments that donors may have in their sperm.     

Phylogeographic Reconstruction of African Yellow Fever Virus Isolates Indicates Recent Simultaneous Dispersal into East and West Africa

Yellow fever virus (YFV) is a mosquito-borne flavivirus that is a major public health problem in tropical areas of Africa and South America. There have been detailed studies on YFV ecology in West Africa and South America, but current understanding of YFV circulation on the African continent is incomplete. This inadequacy is especially notable for East and Central Africa, for which the unpredictability of human outbreaks is compounded by limitations in both historical and present surveillance efforts. Sparse availability of nucleotide sequence data makes it difficult to investigate the dispersal of YFV in these regions of the continent. To remedy this, we constructed Bayesian phylogenetic and geographic analyses utilizing 49 partial genomic sequences to infer the structure of YFV divergence across the known range of the virus on the African continent. Relaxed clock analysis demonstrated evidence for simultaneous divergence of YFV into east and west lineages, a finding that differs from previous hypotheses of YFV dispersal from reservoirs located on edges of the endemic range. Using discrete and continuous geographic diffusion models, we provide detailed structure of YFV lineage diversity. Significant transition links between extant East and West African lineages are presented, implying connection between areas of known sylvatic cycling. The results of demographic modeling reinforce the existence of a stably maintained population of YFV with spillover events into human populations occurring periodically. Geographically distinct foci of circulation are reconstructed, which have significant implications for studies of YFV ecology and emergence of human disease. We propose further incorporation of Bayesian phylogeography into formal GIS analyses to augment studies of arboviral disease.     

Structure of the Parainfluenza Virus 5 (PIV5) Hemagglutinin-Neuraminidase (HN) Ectodomain

Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.     

Outpatient Healthcare Settings and Transmission of Clostridium difficile

Background

Recent reports suggest that community-associated Clostridium difficile infection (CDI) (i.e., no healthcare facility admission within 90 days) may be increasing in frequency. We hypothesized that outpatient clinics could be an important source for acquisition of community-associated CDI.

Methods

We performed a 6-month prospective study of CDI patients to determine frequency of and risk factors for skin and environmental shedding during outpatient visits and to derive a prediction rule for positive cultures. We performed a point–prevalence culture survey to assess the frequency of C. difficile contamination in outpatient settings and evaluated the frequency of prior outpatient visits in patients with community-associated CDI.

Results

Of 67 CDI patients studied, 54 (81%) had 1 or more outpatient visits within 12 weeks after diagnosis. Of 44 patients cultured during outpatient visits, 14 (32%) had skin contamination and 12 (27%) contaminated environmental surfaces. Decreased mobility, fecal incontinence, and treatment with non-CDI antibiotics were associated with positive cultures, whereas vancomycin taper therapy was protective. In patients not on CDI therapy, a prediction rule including incontinence or decreased mobility was 90% sensitive and 79% specific for detection of spore shedding. Of 84 clinic and emergency department rooms cultured, 12 (14%) had 1 or more contaminated environmental sites. For 33 community-associated CDI cases, 31 (94%) had an outpatient visit during the 12 weeks prior to onset of diarrhea.

Conclusions

Patients with recent CDI present a significant risk for transmission of spores during outpatient visits. The outpatient setting may be an underappreciated source of community-associated CDI cases.