Impaired monocyte maturation in response to CpG oligodeoxynucleotide is related to viral RNA levels in human immunodeficiency virus disease and is at least partially mediated by deficiencies in alpha/beta interferon responsiveness and production

The biological activity of CpG oligodeoxynucleotide 2216 (ODN2216), a Toll-like receptor 9 agonist, was investigated with monocytes from human immunodeficiency virus (HIV)-negative and HIV-positive (HIV+) donors. Exposure of peripheral blood mononuclear cells to CpG ODN2216 led to decreased expression of the monocyte marker CD14 and increased expression of the dendritic cell marker CD83, as well as increased expression of HLA-DR, CD40, CD80, and CD86 among the monocytes. Several features of the CpG ODN-induced maturation were diminished in monocytes from HIV+ donors, and these deficiencies were related to increased viremia but not to CD4 cell counts. Alpha interferon (IFN-alpha) was implicated as at least a partial mediator of the CpG ODN-induced monocyte maturation. Reduced production of IFN-alpha in response to CpG ODN and reduced frequencies of plasmacytoid dendritic cells, the principal IFN-alpha-producing cell type in peripheral blood, were observed in peripheral blood mononuclear cells from HIV+ donors. These deficiencies also were related to levels of plasma HIV RNA. Responses of monocytes from HIV+ donors to direct stimulation with IFN-alpha also were partially impaired. Thus, reduced production of IFN-alpha and reduced IFN-alpha responsiveness may contribute to diminished functional responses to CpG ODN in HIV disease. Application of CpG ODNs in HIV disease for adjuvant or immunoregulatory purposes may be particularly useful for HIV+ donors without high-level viremia.     

The choroid plexus removes beta-amyloid from brain cerebrospinal fluid

beta-Amyloid (Abeta) concentration in the cerebrospinal fluid (CSF) of the brain may be regulated by the choroid plexus, which forms a barrier between blood and brain CSF. Abeta uptake from CSF was determined as its volume of distribution (V(D)) into isolated rat choroid plexus tissue. The V(D) of [125I]Abeta1-40 was corrected by subtraction of the V(D) of [14C]sucrose, a marker for extracellular space and diffusion. Abeta uptake into choroid plexus was time and temperature dependent. Uptake of [125I]Abeta was saturable. Abeta uptake was not affected by addition of transthyretin or apolipoprotein E3. In studies with primary culture monolayers of choroidal epithelial cells in Transwells, Abeta permeability across cells, corrected by [(14)C]sucrose, was greater from the CSF-facing membrane than from the blood-facing membrane. Similarly, cellular accumulation of [125I]Abeta was concentrative from both directions and was greater from the CSF-facing membrane, suggesting a bias for efflux. Overall, these results suggest the choroid plexus selectively cleanses Abeta from the CSF by an undetermined mechanism(s), potentially reducing Abeta from normal brains and the brains of Alzheimer's disease patients.     

Vital capacity, respiratory muscle strength, and pulmonary gas exchange during long-duration exposure to microgravity.

Extended exposure to microgravity (microG) is known to reduce strength in weight-bearing muscles and was also reported to reduce respiratory muscle strength. Short- duration exposure to microG reduces vital capacity (VC), a surrogate measure for respiratory muscle strength, for the first few days, with little change in O2 uptake, ventilation, or end-tidal partial pressures. Accordingly we measured VC, maximum inspiratory and expiratory pressures, and indexes of pulmonary gas exchange in 10 normal subjects (9 men, 1 woman, 39-52 yr) who lived on the International Space Station for 130-196 days in a normoxic, normobaric atmosphere. Subjects were studied four times in the standing and supine postures preflight at sea level at 1 G, approximately monthly in microG, and multiple times postflight. VC in microG was essentially unchanged compared with preflight standing [5.28 +/- 0.08 liters (mean +/- SE), n = 187; 5.24 +/- 0.09, n = 117, respectively; P = 0.03] and considerably greater than that measured supine in 1G (4.96 +/- 0.10, n = 114, P < 0.001). There was a trend for VC to decrease after the first 2 mo of microG, but there were no changes postflight. Maximum respiratory pressures in microG were generally intermediate to those standing and supine in 1G, and importantly they showed no decrease with time spent in microG. O2 uptake and CO2 production were reduced (approximately 12%) in extended microG, but inhomogeneity in the lung was not different compared with short-duration exposure to microG. The results show that VC is essentially unchanged and respiratory muscle strength is maintained during extended exposure to microG, and metabolic rate is reduced.     

Asymmetric oceanographic processes mediate connectivity and population genetic structure,as revealed by RADseq,in a highly dispersive marine invertebrate (Parastichopus californicus)

Marine populations are typically characterized by weak genetic differentiation due to the potential for long‐distance dispersal favouring high levels of gene flow. However, strong directional advection of water masses or retentive hydrodynamic forces can influence the degree of genetic exchange among marine populations. To determine the oceanographic drivers of genetic structure in a highly dispersive marine invertebrate, the giant California sea cucumber (Parastichopus californicus), we first tested for the presence of genetic discontinuities along the coast of North America in the northeastern Pacific Ocean. Then, we tested two hypotheses regarding spatial processes influencing population structure: (i) isolation by distance (IBD: genetic structure is explained by geographic distance) and (ii) isolation by resistance (IBR: genetic structure is driven by ocean circulation). Using RADseq, we genotyped 717 individuals from 24 sampling locations across 2,719 neutral SNPs to assess the degree of population differentiation and integrated estimates of genetic variation with inferred connectivity probabilities from a biophysical model of larval dispersal mediated by ocean currents. We identified two clusters separating north and south regions, as well as significant, albeit weak, substructure within regions (F_ST = 0.002, p = .001). After modelling the asymmetric nature of ocean currents, we demonstrated that local oceanography (IBR) was a better predictor of genetic variation (R² = .49) than geographic distance (IBD) (R² = .18), and directional processes played an important role in shaping fine‐scale structure. Our study contributes to the growing body of literature identifying significant population structure in marine systems and has important implications for the spatial management of P. californicus and other exploited marine species.     

Characteristics and preferences of volunteers in a large national bird conservation program in Australia

Volunteers and citizen scientists can make an important contribution to bird monitoring and threatened species conservation projects. Members of BirdLife Australia’s Threatened Bird Network (TBN), a programme which encouraged community participation in conservation projects for threatened birds, were surveyed in 2013 to gain an insight into their demographics and volunteering motivation and preferences. In the 2013 survey, a large proportion of survey respondents were aged between 55 and 74 years old and over half were retired, representing a higher proportion of retired participants than found in a similar study of TBN members in 2000. A large proportion (69%) of respondents had volunteered with TBN projects (occasionally or at least once per year), despite being involved with other environmental groups. Respondents that volunteered mostly preferred short‐term (one day) field‐based volunteer activities, with nearly half also preferring to travel 50 km or less to participate in a project. Less than one third of respondents had never volunteered with TBN projects; this was attributed to not enough projects close to home, time restrictions and lack of transport. Preferences of volunteers in the 2013 survey were similar to those undertaken in 2000 for the majority of responses, including the preference for the activity occurring outdoors, the importance of regular feedback from the organiser, the moderate importance placed on seeing target species and the lack of importance for the provision of transport. A higher importance was placed on the following factors in the 2013 survey responses compared to 2000 (potentially influenced by the higher number of retirees): Having activities closer to home, the activity not being too physically demanding, the opportunity for free time during the activity, good weather was predicted on the day of the activity, existing skills were adequate for the activity, and accommodation was provided if required.     

High-performance liquid chromatography–electrospray ionization mass spectrometry method for the measurement of moclobemide and two metabolites in plasma

A rapid and sensitive liquid chromatography–electrospray ionisation mass spectrometry (HPLC–ESI-MS) assay has been developed for the measurement of moclobemide and metabolites, Ro12-5637 and Ro12-8095, in human plasma. Sample preparation (0.5 ml plasma) involves solid-phase extraction using C₁₈ cartridges. A Nova-Pak phenyl column (Waters, 4 μm, 150×2 mm I.D.) was employed for analyte separation with a mixture of 0.2 M ammonium formate buffer, pH 3.57 and acetonitrile as the mobile phase. The within- and between-day precisions of the assay were <18% and the limit of quantification for all analytes was 0.01 μg/ml. The total run-time was 6 min. The method described was used to measure moclobemide, Ro12-5637 and Ro12-8095 in human plasma following an oral 300 mg dose.     

Energetic Consequences of Human Sociality: Walking Speed Choices among Friendly Dyads

Research has shown that individuals have an optimal walking speed–a speed which minimizes energy expenditure for a given distance. Because the optimal walking speed varies with mass and lower limb length, it also varies with sex, with males in any given population tending to have faster optimal walking speeds. This potentially creates an energetic dilemma for mixed-sex walking groups. Here we examine speed choices made by individuals of varying stature, mass, and sex walking together. Individuals (N = 22) walked around a track alone, with a significant other (with and without holding hands), and with friends of the same and opposite sex while their speeds were recorded every 100 m. Our findings show that males walk at a significantly slower pace to match the females’ paces (p = 0.009), when the female is their romantic partner. The paces of friends of either same or mixed sex walking together did not significantly change (p>0.05). Thus significant pace adjustment appears to be limited to romantic partners. These findings have implications for both mobility and reproductive strategies of groups. Because the male carries the energetic burden by adjusting his pace (slowing down 7%), the female is spared the potentially increased caloric cost required to walk together. In energetically demanding environments, we will expect to find gender segregation in group composition, particularly when travelling longer distances.     

The M33 Chemokine Receptor Homolog of Murine Cytomegalovirus Exhibits a Differential Tissue-Specific Role during In Vivo Replication and Latency

M33, encoded by murine cytomegalovirus (MCMV), is a member of the UL33 homolog G-protein-coupled receptor (GPCR) family and is conserved across all the betaherpesviruses. Infection of mice with recombinant viruses lacking M33 or containing specific signaling domain mutations in M33 results in significantly diminished MCMV infection of the salivary glands. To determine the role of M33 in viral dissemination and/or infection in other tissues, viral infection with wild-type K181 virus and an M33 mutant virus, ΔM33B_T2, was characterized using two different routes of inoculation. Following both intraperitoneal (i.p.) and intranasal (i.n.) inoculation, M33 was attenuated for infection of the spleen and pancreas as early as 7 days after infection. Following i.p. inoculation, ΔM33B_T2 exhibited a severe defect in latency as measured by a diminished capacity to reactivate from spleens and lungs in reactivation assays (P < 0.001). Subsequent PCR analysis revealed markedly reduced ΔM33B_T2 viral DNA levels in the latently infected spleens, lungs, and bone marrow. Following i.n. inoculation, latent ΔM33B_T2 viral DNA was significantly reduced in the spleen and, in agreement with results from i.p. inoculation, did not reactivate from the spleen (P < 0.001). Furthermore, in vivo complementation of ΔM33B_T2 virus replication and/or dissemination to the salivary glands and pancreas was achieved by coinfection with wild-type virus. Overall, our data suggest a critical tissue-specific role for M33 during infection in the salivary glands, spleen, and pancreas but not the lungs. Our data suggest that M33 contributes to the efficient establishment or maintenance of long-term latent MCMV infection.Since the discovery of the G protein-coupled receptors (GPCRs) encoded by the betaherpesviruses, there has been intense speculation on the biological role these viral proteins play during infection (15, 16, 22). Human cytomegalovirus (HCMV), a betaherpesvirus, is a ubiquitous pathogen that asymptomatically infects humans and establishes a long-term persistent infection. HCMV is life-threatening, however, to immunocompromised individuals, such as neonates, AIDS patients, and transplant recipients. HCMV, similar to a number of herpesviruses, encodes viral genes that are predicted to impact virus-host interactions that may promote efficient long-term infection of the host. The CMVs encode genes for proteins that potentially enhance viral dissemination and replication and promote immune evasion by mimicry of host functions that influence the conditions of primary infection, the virus-specific immune response, and even long-term host control of persistent or latent infection (reviewed in references 1, 44, and 68).HCMV encodes four GPCRs (UL33, UL78, US28, and US27) which share homology to host chemokine receptors (16). This suggests that these virally encoded chemokine receptors may function similarly to their cellular receptor counterparts. Chemokines are chemoattractant cytokines that bind and activate chemokine receptors that are on the surfaces of cells. Host chemokine receptors then mediate the activation of cellular signaling pathways and cell migration to sites of inflammation by transmitting signals through G proteins (56, 70). In humans, approximately 50 chemokines and 20 chemokine receptors have been identified, many of which have close homologs in mice and other species (39). Chemokines are divided into two classes, lymphoid chemokines, which are constitutively expressed and involved in lymphoid tissue organization, and inflammatory chemokines, which are induced following infection and part of the inflammatory response (21, 39, 51). Growing evidence indicates that chemokines play a critical role in the host response to infection and inflammation during both the innate and adaptive immune responses (26), thus suggesting that the betaherpesviruses have “hijacked” the chemokine receptors from the host genome to subvert or alter these responses during infection. Besides chemokine receptors, HCMV also encodes a CXC chemokine (UL146) that induces the migration of neutrophils (48); a second CXC chemokine homolog (UL147) whose function is not yet known; a viral CC chemokine (UL131) that is critical for infection of macrophages, endothelial cells, and epithelial cells (25, 57, 73); and a RANTES decoy protein (72). A CC chemokine (vMCK or m131/129) is also encoded by murine CMV (MCMV), and a homolog in rat CMV ([RCMV] r131) that promotes monocyte-associated viremia (20, 37, 59, 60). The MCMV m131/129 chemokine was shown to recruit myelomonocytic progenitors from the bone marrow, perhaps to facilitate cell-type-specific viremia (46). Clearly, the CMVs have invested a great deal of effort into manipulating or subverting the host chemokine system, thus making it reasonable to speculate that these viral members of the chemokine system play an important role during CMV pathogenesis.Of the HCMV-encoded GPCRs, US28 has been well characterized in vitro and functions as a bona fide chemokine receptor, whereas much less is known about the receptor activity of US27, UL33, and UL78. US28 binds and sequesters CC chemokines, induces smooth-muscle cell migration, and constitutively activates signaling pathways (5, 7-9, 42, 52, 64, 67, 71). US28 and US27 are found only in primate CMVs, whereas both UL33 and UL78 are highly conserved across all betaherpesvirus genomes, suggesting an important evolutionary function for UL33 and UL78 during CMV infection. Two other betaherpesviruses, human herpesviruses 6 and 7 (HHV6 and HHV7), encode homologs to the UL33 and UL78 receptors, U12 and U51, respectively. The U12 receptors of HHV6 and HHV7 (34, 45, 66) and the HHV6-encoded U51 receptor (22) exhibit chemokine binding activity. UL33, along with its rodent CMV homologs, M33 (MCMV) and R33 (RCMV), constitutively activates signaling pathways (13, 23, 71). M33 induces smooth-muscle cell migration (39), similar to US28-mediated smooth-muscle cell migration (64). Thus, members of the UL33 family potentially function during viral infection by modulating or influencing the composition of leukocytes at sites of infection, the migration of infected cells or infiltrating leukocytes, or modulation of intracellular signaling pathways.Due to the species specificity of CMV, the in vivo role of the HCMV-encoded GPCRs cannot be addressed. However, the importance of UL33 and UL78 for viral dissemination and virulence in vivo has been indicated by disruption of the viral homologs in MCMV and RCMV (6, 19, 36, 47). Disruption of the UL33, M33, and R33 genes demonstrated that they are dispensable for replication in vitro, indicating that the UL33 family members are not required for replication or cell entry in at least some cell types (6, 19, 40). Infection of mice with M33-deficient MCMV or infection of rats with R33-deficient RCMV results in highly attenuated viruses and diminished infection of the salivary glands. The RCMV R33 protein also appears to play a role in virulence since rats infected with an R33 deletion virus had a lower mortality rate (6). More recently, constitutive M33-mediated activation of signaling pathways was shown to be essential for MCMV infection of salivary glands (14). Significantly, the UL33 protein partially rescued the defect in salivary gland infection attributed to disruption of M33, indicating the evolutionary conservation of function between the HCMV (UL33) and MCMV (M33) chemokine receptor homologs.In this paper, the role of M33 is further investigated using two routes of infection to assess viral dissemination and viral replication kinetics at different tissue sites, the numbers of infected cells following infection, and the possibility that M33 plays a role during latent infection. In addition to the critical role that M33 plays in salivary gland infection, this study reveals that M33 is important for MCMV infection of the spleen and the pancreas but not the lungs. Significantly, our studies provide preliminary evidence that disruption of M33 leads to reduced latent viral load in the spleen, lungs, and bone marrow, perhaps due to defects in the establishment and/or maintenance of latent infection. Lastly, we demonstrate that the tissue defects observed during acute infection with an M33 mutant virus (ΔM33B_T2) can be complemented in vivo when mice are coinfected with ΔM33B_T2 virus and wild-type MCMV. Taken together, our findings indicate that M33 plays a critical tissue-specific role during acute MCMV infection and, importantly, contributes to the efficient establishment or maintenance of latent MCMV infection.     

A molecular and physiological survey of a diverse collection of hydrothermal vent Thermococcus and Pyrococcus isolates

Strains of hyperthermophilic anaerobic hydrothermal vent archaea maintained in the culture collection assembled by Holger Jannasch at the Woods Hole Oceanographic Institution between 1984 and 1998 were identified and partially characterized by Denaturing Gradient Gel Electrophoresis, 16S rRNA gene sequencing, and by growth tests at different temperatures and on different organic carbon and nitrogen sources. All strains were members of the genera Thermococcus and Pyrococcus. The greatest phylogenetic diversity was found in strains from a single Guaymas Basin core isolated by serial dilution from four different depth horizons of heated sediment incubated at the corresponding in situ temperatures. In contrast, geographically distinct vent locations and sample materials yielded a lower diversity of isolates when enriched under uniform temperature regimes and without prior dilution of the source material.