Impact of metal pollution on fungal diversity and community structures

The impact of metal pollution on plant communities has been studied extensively in the past, but little is known about the effects of metal pollution on fungal communities that occur in metal‐polluted soils. Metal‐tolerant ecotypes of the ectomycorrhizal fungus Suillus luteus are frequently found in pioneer pine forests in the Campine region in Belgium on metal‐polluted soils. We hypothesized that metal pollution would play an important role in shaping below‐ground fungal communities that occur in these soils and that Suillus luteus would be a dominant player. To test these hypotheses, the fungal communities in a young pine plantation in soil polluted with zinc, and cadmium were studied using 454 amplicon pyrosequencing. Results show that zinc, cadmium and soil organic matter content were strongly correlated with the fungal community composition, but no effects on fungal diversity were observed. As hypothesized, S. luteus was found to be a dominant member of the studied fungal communities. However, other dominant fungal species, such as Sistotrema sp., Wilcoxina mikolae and Cadophora finlandica were found as well. Their presence in metal‐polluted sites is discussed.     

The Value of In Vitro Diagnostic Testing in Medical Practice: A Status Report

BackgroundIn vitro diagnostic (IVD) investigations are indispensable for routine patient management. Appropriate testing allows early-stage interventions, reducing late-stage healthcare expenditure (HCE).AimTo investigate HCE on IVDs in two developed markets and to assess the perceived value of IVDs on clinical decision-making. Physician-perceived HCE on IVD was evaluated, as well as desired features of new diagnostic markers.MethodsPast and current HCE on IVD was calculated for the US and Germany. A total of 79 US/German oncologists and cardiologists were interviewed to assess the number of cases where: physicians ask for IVDs; IVDs are used for initial diagnosis, treatment monitoring, or post-treatment; and decision-making is based on an IVD test result. A sample of 201 US and German oncologists and cardiologists was questioned regarding the proportion of HCE they believed to be attributable to IVD testing. After disclosing the actual IVD HCE, the physician’s perception of the appropriateness of the amount was captured. Finally, the association between physician-rated impact of IVD on decision-making and perceived contribution of IVD expenditure on overall HCE was assessed.ResultsIVD costs account for 2.3% and 1.4% of total HCE in the US and Germany. Most physicians (81%) believed that the actual HCE on IVDs was >5%; 19% rated the spending correctly (0–4%, p<0.001). When informed of the actual amount, 64% of physicians rated this as appropriate (p<0.0001); 66% of decision-making was based on IVD. Significantly, more physicians asked for either additional clinical or combined clinical/health economic data than for the product (test/platform) alone (p<0.0001).ConclusionsOur results indicate a poor awareness of actual HCE on IVD, but a high attributable value of diagnostic procedures for patient management. New markers should deliver actionable and medically relevant information, to guide decision-making and foster improved patient outcomes.     

Tuberculosis Disease during Pregnancy and Treatment Outcomes in HIV-Infected and Uninfected Women at a Referral Hospital in Cape Town

BackgroundTuberculosis during pregnancy and treatment outcomes are poorly defined in high prevalence tuberculosis and HIV settings.MethodsA prospective cohort study of pregnant and postpartum women identified to be routinely on antituberculosis treatment was conducted at Tygerberg Hospital, Cape Town, South Africa, from January 2011 through December 2011. Maternal tuberculosis disease spectrum and tuberculosis-exposed newborns were characterized by maternal HIV status. Maternal tuberculosis treatment outcomes were documented and a multivariable regression model identified predictors of unfavourable tuberculosis treatment outcomes. Infant outcomes were also described.ResultsSeventy-four women with tuberculosis, 53 (72%) HIV-infected, were consecutively enrolled; 35 (47%) were diagnosed at delivery or postpartum and 22 (30%) of women reported previous antituberculosis treatment. HIV-infected women were 5.67 times more likely to have extrapulmonary tuberculosis (95% CI 1.18–27.25, p = 0.03). All 5 maternal deaths were amongst HIV-infected women. Birth outcomes were available for 75 newborns (2 sets of twins, missing data for 1 stillbirth). Of the 75 newborns, 49 (65%) were premature and 44 (59%) were low birth weight (LBW; <2500 grams). All 6 infants who died and the 4 stillbirths were born to HIV-infected women. Unfavourable tuberculosis treatment outcomes were documented in 33/74 (45%) women. Unfavourable maternal tuberculosis outcome was associated with delivery of LBW infants (OR 3.83; 95% CI 1.40–10.53, p = 0.009).ConclusionsA large number of pregnant women with tuberculosis presented at a provincial referral hospital. All maternal and infant deaths occurred in HIV-infected women and their newborns. Maternal tuberculosis treatment outcomes were poor.     

NMR structure of the Vibrio vulnificus ribosomal protein S1 domains D3 and D4 provides insights into molecular recognition of single-stranded RNAs

The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1–D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical β2-β3-β5 interfaces. Evidently, the 3′-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.     

The Origin and Early Radiation of Archosauriforms: Integrating the Skeletal and Footprint Record

We present a holistic approach to the study of early archosauriform evolution by integrating body and track records. The ichnological record supports a Late Permian–Early Triassic radiation of archosauriforms not well documented by skeletal material, and new footprints from the Upper Permian of the southern Alps (Italy) provide evidence for a diversity not yet sampled by body fossils. The integrative study of body fossil and footprint data supports the hypothesis that archosauriforms had already undergone substantial taxonomic diversification by the Late Permian and that by the Early Triassic archosauromorphs attained a broad geographical distribution over most parts of Pangea. Analysis of body size, as deduced from track size, suggests that archosauriform average body size did not change significantly from the Late Permian to the Early Triassic. A survey of facies yielding both skeletal and track record indicate an ecological preference for inland fluvial (lacustrine) environments for early archosauromorphs. Finally, although more data is needed, Late Permian chirotheriid imprints suggest a shift from sprawling to erect posture in archosauriforms before the end-Permian mass extinction event. We highlight the importance of approaching palaeobiological questions by using all available sources of data, specifically through integrating the body and track fossil record.     

Specific immuno capturing of the Staphylococcal superantigen toxic‐shock syndrome toxin‐1 in plasma

Toxic‐shock syndrome is primarily caused by the Toxic‐shock syndrome toxin 1 (TSST‐1), which is secreted by the Gram‐positive bacterium Staphylococcus aureus. The toxin belongs to a family of superantigens (SAgs) which exhibit several shared biological properties, including the induction of massive cytokine release and V_β‐specific T‐cell proliferation. In this study we explored the possibility to use monoclonal Variable domains of Llama Heavy‐chain antibodies (VHH) in the immuno capturing of TSST‐1 from plasma. Data is presented that the selected VHHs are highly specific for TSST‐1 and can be efficiently produced in large amounts in yeast. In view of affinity chromatography, the VHHs are easily coupled to beads, and are able to deplete TSST‐1 from plasma at very low, for example, pathologically relevant, concentrations. When spiked with 4 ng/mL TSST‐1 more than 96% of TSST‐1 was depleted from pig plasma. These data pave the way to further explore application of high‐affinity columns in the specific immuno depletion of SAgs in experimental sepsis models and in sepsis in humans. Biotechnol. Bioeng. 2009; 104: 143–151 © 2009 Wiley Periodicals, Inc.     

Analysis of the influence of disc degeneration on the mechanical behaviour of a lumbar motion segment using the finite element method

Compared to a healthy intervertebral disc, the geometry and the material properties of the involved tissues are altered in a degenerated disc. It is not completely understood how this affects the mechanical behaviour of a motion segment. In order to study the influence of disc degeneration on motion segment mechanics a three-dimensional, nonlinear finite element model of the L3/L4 functional unit was used. Different grades of disc degeneration were simulated by varying disc height and bulk modulus of the nucleus pulposus. The model was loaded with pure moments of 10Nm in the three main anatomic planes. The finite element model predicted the same trends for intersegmental rotation and intradiscal pressure as described in the literature for in vitro studies. A comparison between calculated intersegmental rotation and experimental data showed a mean difference of 1.9 degrees while the mean standard deviation was 2.5 degrees . A mildly degenerated disc increases intersegmental rotation for all loading cases. With further increasing disc degeneration intersegmental rotation is decreased. For axial rotation the decrease takes place in the final stage. Intradiscal pressure is lower while facet joint force and maximum von Mises stress in the annulus are higher in a degenerated compared to a healthy disc.     

Early Selection in Gag by Protective HLA Alleles Contributes to Reduced HIV-1 Replication Capacity That May Be Largely Compensated for in Chronic Infection

Mutations that allow escape from CD8 T-cell responses are common in HIV-1 and may attenuate pathogenesis by reducing viral fitness. While this has been demonstrated for individual cases, a systematic investigation of the consequence of HLA class I-mediated selection on HIV-1 in vitro replication capacity (RC) has not been undertaken. We examined this question by generating recombinant viruses expressing plasma HIV-1 RNA-derived Gag-Protease sequences from 66 acute/early and 803 chronic untreated subtype B-infected individuals in an NL4-3 background and measuring their RCs using a green fluorescent protein (GFP) reporter CD4 T-cell assay. In acute/early infection, viruses derived from individuals expressing the protective alleles HLA-B*57, -B*5801, and/or -B*13 displayed significantly lower RCs than did viruses from individuals lacking these alleles (P < 0.05). Furthermore, acute/early RC inversely correlated with the presence of HLA-B-associated Gag polymorphisms (R = −0.27; P = 0.03), suggesting a cumulative effect of primary escape mutations on fitness during the first months of infection. At the chronic stage of infection, no strong correlations were observed between RC and protective HLA-B alleles or with the presence of HLA-B-associated polymorphisms restricted by protective alleles despite increased statistical power to detect these associations. However, RC correlated positively with the presence of known compensatory mutations in chronic viruses from B*57-expressing individuals harboring the Gag T242N mutation (n = 50; R = 0.36; P = 0.01), suggesting that the rescue of fitness defects occurred through mutations at secondary sites. Additional mutations in Gag that may modulate the impact of the T242N mutation on RC were identified. A modest inverse correlation was observed between RC and CD4 cell count in chronic infection (R = −0.17; P < 0.0001), suggesting that Gag-Protease RC could increase over the disease course. Notably, this association was stronger for individuals who expressed B*57, B*58, or B*13 (R = −0.27; P = 0.004). Taken together, these data indicate that certain protective HLA alleles contribute to early defects in HIV-1 fitness through the selection of detrimental mutations in Gag; however, these effects wane as compensatory mutations accumulate in chronic infection. The long-term control of HIV-1 in some persons who express protective alleles suggests that early fitness hits may provide lasting benefits.The host immune response elicited by CD8⁺ cytotoxic T lymphocytes (CTLs) is a major contributor to viral control following human immunodeficiency virus type 1 (HIV-1) infection (6, 39), but antiviral pressure exerted by CTLs is diminished by the selection of escape mutations in targeted regions throughout the viral proteome (7, 18, 29, 35, 41, 45, 57). A comprehensive identification of HLA-associated viral polymorphisms has recently been achieved through population-based analyses of HIV-1 sequences and HLA class I types from different cohorts worldwide (3, 8, 13-15, 34, 43, 50, 56, 63). However, despite improved characterization of the sites and pathways of immune escape, effective ways to incorporate these findings into immunogen design remain an area of debate. A better understanding of the impact of escape mutations on viral fitness may provide novel directions for HIV-1 vaccines that are designed to attenuate pathogenesis.The development of innovative vaccine strategies that can overcome the extreme diversity of HIV is a key priority (4). One proposed approach is to target the most conserved T-cell epitopes, which presumably cannot escape from CTL pressure easily due to structural or functional constraints on the viral protein (55). Complementary approaches include the design of polyvalent and/or mosaic immunogens that incorporate commonly observed viral diversity (4, 38) or the specific targeting of vulnerable regions of the viral proteome that do escape but only at a substantial cost to viral replication capacity (RC) (1, 40). A chief target of such vaccine approaches is the major HIV-1 structural protein Gag, which is known to be highly immunogenic and to elicit CTL responses that correlate with the natural control of infection (22, 36, 66). Indeed, several lines of evidence support a relationship between the selection of CTL escape mutations and reduced HIV-1 fitness. These include the reversion of escape mutations following transmission to an HLA-mismatched recipient who cannot target the epitope (19, 24, 41) as well as reduced plasma viral load (pVL) set point following the transmission of certain escape variants from donors who expressed protective HLA alleles (17, 27). Notably, these in vivo observations have been made most often for variations within Gag that are attributed to CTL responses restricted by the protective alleles HLA-B*57 and -B*5801 (17, 19, 27, 41). Most recently, reduced in vitro RCs of clinical isolates and/or engineered strains encoding single or multiple escape mutations in Gag selected in the context of certain protective HLA alleles, including B*57, B*5801, B*27, and B*13, have been demonstrated (9, 10, 42, 53, 59, 62). Despite these efforts, the goal of a T-cell vaccine that targets highly conserved and attenuation-inducing sites is hampered by a lack of knowledge concerning the contribution of most escape mutations to HIV-1 fitness as well as a poor understanding of the relative influence of HLA on the viral RC at different stages of infection.The mutability of HIV-1 permits the generation of progeny viruses encoding compensatory mutations that restore normal protein function and/or viral fitness. Detailed studies have demonstrated that the in vitro RC of escape variants in human and primate immunodeficiency viruses can be enhanced by the addition of secondary mutations outside the targeted epitope (10, 20, 52, 59, 65). Thus, vaccine strategies aimed at attenuating HIV-1 must also consider, among other factors, the frequency, time course, and extent to which compensation might overcome attenuation mediated by CTL-induced escape. Despite its anticipated utility for HIV-1 vaccine design, systematic studies to examine the consequences of naturally occurring CTL escape and compensatory mutations on viral RC have not been undertaken.We have described previously an in vitro recombinant viral assay to examine the impact of Gag-Protease mutations on HIV-1 RC (47, 49). Gag and protease have been included in each virus to minimize the impact of sequence polymorphisms at Gag cleavage sites, which coevolve with changes in protease (5, 37). Using this approach, we have demonstrated that viruses derived from HIV-1 controllers replicated significantly less well than those derived from noncontrollers and that these differences were detectable at both the acute/early (49) and chronic (47) stages. Escape mutations in Gag associated with the protective HLA-B*57 allele, as well as putative compensatory mutations outside known CTL epitopes, contributed to this difference in RC (47). However, substantial variability was observed for viruses from controllers and noncontrollers, indicating that additional factors were likely to be involved. Benefits of this assay include its relatively high-throughput capacity as well as the fact that clinically derived HIV-1 sequences are used in their entirety. Thus, it is possible to examine a large number of “real-world” Gag-Protease sequences, to define an RC value for each one, and to identify sequences within the population of recombinant strains that are responsible for RC differences.Here, we use this recombinant virus approach to examine the contribution of HLA-associated immune pressure on Gag-Protease RC during acute/early (n = 66) and chronic (n = 803) infections in the context of naturally occurring HIV-1 subtype B isolates from untreated individuals. In a recent report (64), we employed this system to examine the Gag-Protease RC in a similar cohort of chronic HIV-1 subtype C-infected individuals. The results of these studies provide important insights into the roles of immune pressure and fitness constraints on HIV-1 evolution that may contribute to the rational design of an effective vaccine.     

Cysteine engineering of polyproteins for single-molecule force spectroscopy

Single-molecule methods such as force spectroscopy give experimental access to the mechanical properties of protein molecules. So far, owing to the limitations of recombinant construction of polyproteins, experimental access has been limited to mostly the N-to-C terminal direction of force application. This protocol gives a fast and simple alternative to current recombinant strategies for preparing polyproteins. We describe in detail the method to construct polyproteins with precisely controlled linkage topologies, based on the pairwise introduction of cysteines into protein structure and subsequent polymerization in solution. Stretching such constructed polyproteins in an atomic force microscope allows mechanical force application to a single protein structure via two precisely controlled amino acid positions in the functional three-dimensional protein structure. The capability for site-directed force application can provide valuable information about both protein structure and directional protein mechanics. This protocol should be applicable to almost any protein that can be point mutated. Given correct setup of all necessary reagents, this protocol can be accomplished in fewer than 10 d.