The molecular biology of HIV cloning and characterization of shiv-1157ipel

Diese Arbeit beschreibt die Konstuktion eines neuartigen Simian-Human Immunodeficiency Virus (SHIV1157ipEL). Die Evaluation der Funktionstüchtigkeit (Replikations-, Infektionsfähigkeit und Neutralisationseigenschaft) in verschiedenen Zellkulturen konnte zeigen dass diese Konstruktion voll funktionsfähig ist und ein neues Tool für HIV-Vaccine Studien darbietet

R5 Clade C SHIV Strains with Tier 1 or 2 Neutralization Sensitivity: Tools to Dissect Env Evolution and to Develop AIDS Vaccines in Primate Models

Background: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. Methodology/Principal Findings: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an “early,” recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a “late” form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to “late” SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4$^+$ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. Conclusions/Significance: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates

A Novel sample preparation technology for sepsis diagnosis

Eine der größten Herausforderungen, die sich unsere Gesellschaft heute und in Zukunft stellen muss, ist die Eindämmung des Aufkommens multiresistenter Keime und die damit einhergehende erhöhte Mortalitätsund Morbiditätsrate durch Sepsis. Auch politisch, von Seiten der Obama Administration, der Europäischen Kommission und der G7, wurde diese Herausforderung erkannt und Aktionspläne sowie Strategiepapiere entwickelt. Eine Voraussetzung im Kampf gegen multiresistente Keime ist eine schnelle und sensitive Detektion und Charakterisierung von Pathogenen, um eine frühe, zielgerichtete Therapie zu initiieren. Allerdings scheitern moderne Entwicklungen aus Forschung und Industrie immer noch bei der Ablöse der langwierigen Blutkultur als Gold-Standard. Die größte Hürde hierbei, die hohe Komplexität der Blutprobe und die marginale Anzahl an Analyten, wurde zwar bereits von Unternehmen und Forschungsgruppen identifiziert, aber noch nicht befriedigend bewältigt. Als Antwort auf diese Notwendigkeit konzentriert sich die in dieser Dissertationsschrift vorgestellte Arbeit auf die Entwicklung einer innovativen Technologie, die ein großes Potenzial für eine neue Art der Probenvorbereitung bietet und hier an Hand eines Labor-Demonstrators gezeigt wird. Durch die Verwendung von elektrischen Feldern wird der Unterschied in der Anfälligkeit von menschlichen Zellen und prokaryotischen Zellen verwendet, um menschliche Blutzellen in einer schnellen, spezifischen, kostengünstigen und vollautomatischen Weise in einem mikrofluidischen Durchfluss-Chip effizient zu lysieren, um eventuelle, im Blut befindliche Pathogene aufreinigen und aufkonzentrieren zu können. Um die theoretische Zelltyp-Spezifität von elektrischen Feldern in einer mikrofluidischen Konstruktion zu realisieren, werden die elektrochemischen Effekte an der Elektroden/Elektrolyt-Grenzfläche durch die Entwicklung einer neuartigen und vielversprechenden Passivierungsstrategie der Elektroden deutlich reduziert. Für das Erreichen dieses Ziels wurden die elektrischen Eigenschaften verschiedener Passivierungsstrategien in analytischer Weise untersucht, optimiert und führten zur Anwendung von dielektrischen Dünnfilmen mit hoher Permittivität. In Bezug auf die klassischen und weit verbreiteten Ansätze, die noch von nicht passivierten Elektroden Gebrauch machen, ist die Einführung von Passivierungsschichten mit hoher Permittivität für zukünftige Elektrochemie-freie elektrische Feldanwendungen in der Biotechnologie von potenziell großer Bedeutung.The fight against antimicrobial resistance together with sepsis associated mortality, morbidity and health care costs is one of the biggest global challenges society is facing today and in future. Efforts to develop action plans, concepts and strategies to combat this challenge have therefore been major focus points for the Obama administration, the European Commissions Directorate for Health Research and the G7 summit in 2015. A fundamental prerequisite for the urgently needed targeted therapy is fast and sensitive pathogen detection and characterization. Modern technologies, however, struggle to sufficiently meet the requirements to replace blood culture, todays tedious gold standard. The biggest hurdle, which is the complexity of the sample blood and the marginal amount of analyte, has already been identified by companies and academia, but is not yet addressed in a satisfying manner. In answer to this need, the work presented in this thesis focusses on the development of an innovative technology bearing a huge potential as a new sample preparation strategy and is demonstrated in a lab-scale demonstrator. By using electric fields, the difference in the susceptibility of human cells and prokaryotic cells is used to efficiently lyse human blood cells in a fast, specific, cost efficient and fully automated manner in a microfluidic flow-through chip. Thus, blood-borne pathogens can be purified and concentrated for highly sensitive and specific detection. To gain cell-type specificity of electric fields in a microfluidic design, superimposing electrochemical effects at the electrode/electrolyte interface are significantly reduced by the development of a novel and promising electrode passivation design. Thus the electrical characteristics of different passivation strategies were investigated and resulted in the application of high-k dielectric thin films. In respect to the classical approaches which still make use of bare metal electrodes, the introduction of high-k dielectric passivation strategies is of utmost importance for future electrochemistry-liberated electric field applications in biotechnology.12

Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells

10.1111/acel.12484AGING CELL154744-75

Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells

Damage to cells and tissues is one of the driving forces of aging and age-related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self-renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor-age-dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR-31 as a crucial component. We demonstrated that miR-31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR-31 is secreted within senescent cell-derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled-3. Therefore, we suggest that microvesicular miR-31 in the plasma of elderly might play a role in the pathogenesis of age-related impaired bone formation and that miR-31 might be a valuable plasma-based biomarker for aging and for a systemic environment that does not favor cell-based therapies whenever osteogenesis is a limiting factor

Figure 5

<p>(A) Sequence alignment of V1/V2 sites of and SHIV-1157ipEL-p, SHIV-1157ipEL-pΔ3N and SHIV-1157ipd3N4 (numbering according to HXB2). (B) Molecular modeling of SHIV-1157ipEL-p and SHIV-1157ipd3N4 sequences was performed using the X-ray structure of the CD4-bound YU2 gp120 core <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011689#pone.0011689-Kwong1" target="_blank">[20]</a>; PDB code 1RZK). The V1, V2, and V3 loops were modeled onto the core. Mutations inducing structural heterogeneity in the V1 and V2 loops were identified. (C and D) Illustration of three-dimensional (3D) gp120 of SHIV-1157ipEL-p (white), SHIV-1157ipd3N4 (green), and (D) SHIV-1157ipEL-pΔ3N (yellow) showing that access to the CD4 binding site (red) is more restricted for SHIV-1157ipEL-pΔ3N and SHIV-1157ipd3N4 than for SHIV-1157ipEL-p.</p