Development and Application of a Real-Time PCR Approach for Quantification of Uncultured Bacteria in the Central Baltic Sea

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the “Epsilonproteobacteria” related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 × 10³ to 4.4 × 10⁹ copies ml⁻¹ or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 × 10¹ to 2.2 ×10⁶ copies ml⁻¹ or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml⁻¹. The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.     

Diversity patterns and activity of uncultured marine heterotrophic flagellates unveiled with pyrosequencing

Flagellated heterotrophic microeukaryotes have key roles for the functioning of marine ecosystems as they channel large amounts of organic carbon to the upper trophic levels and control the population sizes of bacteria and archaea. Still, we know very little on the diversity patterns of most groups constituting this evolutionary heterogeneous assemblage. Here, we investigate 11 groups of uncultured flagellates known as MArine STramenopiles (MASTs). MASTs are ecologically very important and branch at the base of stramenopiles. We explored the diversity patterns of MASTs using pyrosequencing (18S rDNA) in coastal European waters. We found that MAST groups range from highly to lowly diversified. Pyrosequencing (hereafter ‘454'') allowed us to approach to the limits of taxonomic diversity for all MAST groups, which varied in one order of magnitude (tens to hundreds) in terms of operational taxonomic units (98% similarity). We did not evidence large differences in activity, as indicated by ratios of DNA:RNA-reads. Most groups were strictly planktonic, although we found some groups that were active in sediments and even in anoxic waters. The proportion of reads per size fraction indicated that most groups were composed of very small cells (∼2–5 μm). In addition, phylogenetically different assemblages appeared to be present in different size fractions, depths and geographic zones. Thus, MAST diversity seems to be highly partitioned in spatial scales. Altogether, our results shed light on these ecologically very important but poorly known groups of uncultured marine flagellates.     

RNAi-mediated Hip1R silencing results in stable association between the endocytic machinery and the actin assembly machinery

Actin filaments transiently associate with the endocytic machinery during clathrin-coated vesicle formation. Although several proteins that might mediate or regulate this association have been identified, in vivo demonstration of such an activity has not been achieved. Huntingtin interacting protein 1R (Hip1R) is a candidate cytoskeletal-endocytic linker or regulator because it binds to clathrin and actin. Here, Hip1R levels were lowered by RNA interference (RNAi). Surprisingly, rather than disrupting the transient association between endocytic and cytoskeletal proteins, clathrin-coated structures (CCSs) and their endocytic cargo became stably associated with dynamin, actin, the Arp2/3 complex, and its activator, cortactin. RNAi double-depletion experiments demonstrated that accumulation of the cortical actin-endocytic complexes depended on cortactin. Fluorescence recovery after photobleaching showed that dynamic actin filament assembly can occur at CCSs. Our results provide evidence that Hip1R helps to make the interaction between actin and the endocytic machinery functional and transient.     

Incidence of AIDS-Defining Opportunistic Infections and Mortality during Antiretroviral Therapy in a Cohort of Adult HIV-Infected Individuals in Hanoi, 2007-2014

Background

Although the prognosis for HIV-infected individuals has improved after antiretroviral therapy (ART) scale-up, limited data exist on the incidence of AIDS-defining opportunistic infections (ADIs) and mortality during ART in resource-limited settings.

Methods

HIV-infected adults in two large hospitals in urban Hanoi were enrolled to the prospective cohort, from October 2007 through December 2013. Those who started ART less than one year before enrollment were assigned to the survival analysis. Data on ART history and ADIs were collected retrospectively at enrollment and followed-up prospectively until April 2014.

Results

Of 2,070 cohort participants, 1,197 were eligible for analysis and provided 3,446 person-years (PYs) of being on ART. Overall, 161 ADIs episodes were noted at a median of 3.20 months after ART initiation (range 0.03–75.8) with an incidence 46.7/1,000 PYs (95% confidence interval [CI] 39.8–54.5). The most common ADI was tuberculosis with an incidence of 29.9/1,000 PYs. Mortality after ART initiation was 8.68/1,000 PYs and 45% (19/45) died of AIDS-related illnesses. Age over 50 years at ART initiation was significantly associated with shorter survival after controlling for baseline CD4 count, but neither having injection drug use (IDU) history nor previous ADIs were associated with poor survival. Semi-competing risks analysis in 951 patients without ADIs history prior to ART showed those who developed ADIs after starting ART were at higher risk of death in the first six months than after six months.

Conclusion

ADIs were not rare in spite of being on effective ART. Age over 50 years, but not IDU history, was associated with shorter survival in the cohort. This study provides in-depth data on the prognosis of patients on ART in Vietnam during the first decade of ART scale-up.     

Identification of Cardiac Myosin-binding Protein C as a Candidate Biomarker of Myocardial Infarction by Proteomics Analysis

Acute myocardial infarction (AMI) is a common cause of death for which effective treatments are available provided that diagnosis is rapid. The current diagnostic gold standards are circulating cardiac troponins I and T. However, their slow release delays diagnosis, and their persistence limits their utility in the identification of reinfarction. The aim was to identify candidate biomarkers of AMI. Isolated mouse hearts were perfused with oxygenated protein-free buffer, and coronary effluent was collected after ischemia or during matched normoxic perfusion. Effluents were analyzed using proteomics approaches based on one- or two-dimensional initial separation. Of the 459 proteins identified after ischemia with one-dimensional separation, 320 were not detected in the control coronary effluent. Among these were all classic existing biomarkers of AMI. We also identified the cardiac isoform of myosin-binding protein C in its full-length form and as a 40-kDa degradation product. This protein was not detected in the other murine organs examined, increased markedly with even trivial myocardial infarction, and could be detected in the plasma after myocardial infarction in vivo, a profile compatible with a biomarker of AMI. Two-dimensional fluorescence DIGE of ischemic and control coronary effluents identified more than 200 asymmetric spots verified by swapping dyes. Once again existing biomarkers of injury were confirmed as well as posttranslational modifications of antioxidant proteins such as peroxiredoxins. Perfusing hearts with protein-free buffers provides a platform of graded ischemic injury that allows detailed analysis of protein release and identification of candidate cardiac biomarkers like myosin-binding protein C.Acute myocardial infarction (AMI)1 is a common cause of death for which effective treatments are available provided that the condition is rapidly diagnosed. The modern diagnosis of AMI relies on the rise and fall of a specific serum biomarker accompanied by an appropriate circumstance such as chest pain or revascularization. In this accepted paradigm, the diagnosis cannot be ruled in or ruled out without the definite presence or definite absence of a serum biomarker. The ideal biomarker of cardiac injury should be cardiac specific and released rapidly after myocardial injury in direct proportion to the extent of damage. Furthermore, the biomarker should have a high sensitivity and specificity (1). Several biomarkers of AMI have been described in the literature, but only a few, none of which are ideal, have found their way into routine clinical practice. For example, CK-MB starts to increase 4–8 h after coronary artery occlusion and returns to base line within 2–3 days (2). However, its use is limited by its presence in skeletal muscle and normal serum and by sensitivity of the assay to interference, causing some to question its utility (3). Myoglobin is another cytoplasmic protein found in cardiac and skeletal, but not smooth, muscle. It is released even earlier within 1–2 h of AMI and peaks within 5–6 h (2). Unfortunately, any injury to skeletal muscle also causes elevated levels of myoglobin, reducing specificity. Fatty acid-binding proteins (FABPs) are small (15-kDa) cytoplasmic proteins expressed in all tissues with active fatty acid metabolism. Among the nine proteins, heart-specific FABP (H-FABP) is found in heart but also kidney, brain, skeletal muscle, and placenta (4). Following acute myocardial infarction, H-FABP can be detected within 20 min and peaks at 4 h, considerably faster even than CK/CK-MB in the same patient cohort. Although H-FABP concentrations in normal plasma are low, they are known to rise nonspecifically during physical exertion (without a troponin rise), kidney injury, and stroke (5).The most specific and sensitive cardiac proteins released after acute myocardial infarction are cardiac troponins I and T. Both troponins I and T are released slowly, peaking ∼18 h after myocardial infarction, and remain elevated for 7–10 days (2). This slow release is likely the result of their relatively inaccessible cellular location compared with CK-MB, myoglobin, and H-FABP. Troponins regulate the physical interaction of actin and myosin and thus are found almost entirely associated within the crystalline structure of the sarcomere of striated muscle cells (6). The troponin complex is composed of three forms: I, T, and C. Troponins I and T exist as cardiac specific isoforms with epitopes that differ from the corresponding skeletal isoforms. In addition, the absent or extremely low normal circulating levels of troponin provide the greatest dynamic range of any of the currently available biomarkers (7). Although there is no doubt troponins have revolutionized the detection and management of patients with AMI (8), they do have disadvantages. The slow release of troponin delays diagnosis and the initiation of specific treatments that could salvage heart tissue in those in whom it is raised. Similarly, patients in whom it is absent and who are ultimately reassured and discharged are admitted to the hospital unnecessarily. Furthermore, the persistence of troponins limits their utility in the diagnosis of reinfarction.It is therefore widely accepted that there is a need for new biomarkers that can diagnose AMI earlier during its natural history and/or that have a short plasma half-life, allowing use in diagnosis and quantification of reinfarction. The purpose of this study was to use the platform of the crystalloid perfused mouse hearts to perform a systematic proteomics analysis of the coronary effluent after minimal AMI to identify new potential biomarkers (9).     

Poly(methyl metacrylate) conductive fiber optic transducers as dual biosensor platforms

Herein the development of an alternative optic-conductive fiber configuration applied for the construction of biosensing platforms. This new approach is based on applying the chemical polymerization of pyrrole onto the surface of polymethyl metacrylate (PMMA) fibers to create a polymer—a conductive surface, onto which an additional photoactive polypyrrole-benzophenone (PpyBz) film is electrochemically generated upon the fiber surface. Irradiation of the benzophenone groups embedded in the Ppy films with UV radiation (350 nm) formed active radicals that allowed the covalent attachment of the desired bioreceptors. Characterization of the amperometric biosensing matrix was accomplished by using a model Urease (Urs) through electrochemical impedance spectroscopy (EIS) and amperometry. Both techniques have shown a low charge transfer resistance (340 kΩ) and a high sensitivity (12.3 μA mM⁻¹ cm⁻²). Thereafter, the construction of an optical biosensing matrix based on horseradish peroxidase (HRP) production of photons was carried out. The high signal to noise (S/N) ratio (1600) indicated clearly that this approach can serve as a new platform to replace glass optical fibers based on biosensors.     

Structure-based library design and the discovery of a potent and selective mast cell β-tryptase inhibitor as an oral therapeutic agent

A solid phase combinatorial library was designed based on X-ray structures and in-silico models to explore an inducible S4+ pocket, which is formed by a simple side-chain rotation of Tyr95. This inducible S4+ pocket is unique to β-tryptase and does not exist for other trypsin-like serine proteases of interest. Therefore, inhibitors utilizing this pocket have inherent advantages for being selective against other proteases in the same family. A member of this library was found to be a potent and selective β-tryptase inhibitor with a suitable pharmacokinetic profile for further clinical evaluation.     

Genomic Patterns of Positive Selection at the Origin of Rust Fungi

Understanding the origin and evolution of pathogenicity and biotrophic life-style of rust fungi has remained a conundrum for decades. Research on the molecular mechanisms responsible for rust fungi evolution has been hampered by their biotrophic life-style until the sequencing of some rust fungi genomes. With the availability of multiple whole genomes and EST data for this group, it is now possible to employ genome-wide surveys and investigate how natural selection shaped their evolution. In this work, we employed a phylogenomics approach to search for positive selection and genes undergoing accelerated evolution at the origin of rust fungi on an assembly of single copy genes conserved across a broad range of basidiomycetes. Up to 985 genes were screened for positive selection on the phylogenetic branch leading to rusts, revealing a pervasive signal of positive selection throughout the data set with the proportion of positively selected genes ranging between 19.6–33.3%. Additionally, 30 genes were found to be under accelerated evolution at the origin of rust fungi, probably due to a mixture of positive selection and relaxation of purifying selection. Functional annotation of the positively selected genes revealed an enrichment in genes involved in the biosynthesis of secondary metabolites and several metabolism and transporter classes.