Atg11 tethers Atg9 vesicles to initiate selective autophagy.

Autophagy recycles cytoplasmic components by sequestering them in double membrane-surrounded autophagosomes. The two proteins Atg11 and Atg17 are scaffolding components of the Atg1 kinase complex. Atg17 recruits and tethers Atg9-donor vesicles, and the corresponding Atg1 kinase complex induces the formation of nonselective autophagosomes. Atg11 initiates selective autophagy and coordinates the switch to nonselective autophagy by recruiting Atg17. The molecular function of Atg11 remained, however, less well understood. Here, we demonstrate that Atg11 is activated by cargo through a direct interaction with autophagy receptors. Activated Atg11 dimerizes and tethers Atg9 vesicles, which leads to the nucleation of phagophores in direct vicinity of cargo. Starvation reciprocally regulates the activity of both tethering factors by initiating the degradation of Atg11 while Atg17 is activated. This allows Atg17 to sequester and tether Atg9 vesicles independent of cargo to nucleate nonselective phagophores. Our data reveal insights into the molecular mechanisms governing cargo selection and specificity in autophagy.

The relevance of neutral arm positioning for true ap-view X-ray to provide true projection of the humeral head shaft angle.

INTRODUCTION: Textbooks commonly recommend using the true anterior-posterior (ap)-view with the patient's arm in a sling and therefore in internal rotation (IR) for radiologic diagnostic assessment of the proximal humerus after trauma. However, IR or external rotation (ER) may affect the projection of the head shaft angle (HSA) and therefore bias the diagnostic conclusion significantly. We hypothesized that neutral rotation (NR) of the arm is mandatory for true ap-view to provide true projection of the HSA. MATERIALS AND METHODS: A simplified geometrical model of the proximal humerus was used to examine the influence of different arm positions and angulations of the central ray in relation to the projection of the HSA. RESULTS: Both ER and IR misleadingly suggested an increased valgus angle. Simulating the true ap-view with the central ray in cranio-caudal direction, IR changed the projection of the HSA substantially. CONCLUSION: In conclusion, standard fixation of the patient's arm in a shoulder sling in IR for true ap-view may result in an oblique projection, potentially leading to incorrect surgical implications. To prevent misdiagnosed valgus or varus angulation, NR of the arm should be obeyed when performing true ap-view X-ray. We, therefore, highly recommend to overcome the traditionally arm position, ensuring the true amount of dislocation to assure correct surgical implications and comparable follow-up examinations.

Five-year results of the uncemented RM pressfit cup clinical evaluation and migration measurements by EBRA.

This was a prospective consecutive, study of 50 patients (mean age 72 years, 56% males) treated unilaterally for primary osteoarthrosis using the uncemented, isoelastic, monoblock RM Pressfit Cup. Migration and wear were assessed using the EBRA (Ein-Bild-Röntgen-Analyse) software (201 radiographs from 36 patients). Mean migration was 0.82 mm after 2 years and 1.25 mm after 5 years. A decreasing migration rate over the period was observed. The mean annual wear rate was 0.09 mm/y. Five years after surgery, 84% of patients reached good or excellent Harris hip score values. No revisions related to the acetabular component were necessary. All cups appeared to have high primary stability.

Use of minicircle plasmids for gene therapy.

A large number of cancer gene therapy clinical trials are currently being performed that are attempting to evaluate novel approaches to eliminate tumor cells by the introduction of genetic material into patients. One of the most important objectives in gene therapy is the development of highly safe and efficient vector systems for gene transfer in eukaryotic cells. Currently, viral and nonviral vector systems are used, both having their advantages and limitations. Minicircles are novel supercoiled minimal expression cassettes, derived from conventional plasmid DNA by site-specific recombination in vivo in Escherichia coli for the use in nonviral gene therapy and vaccination. Minicircle DNA lacks the bacterial backbone sequence consisting of an antibiotic resistance gene, an origin of replication, and inflammatory sequences intrinsic to bacterial DNA. In addition to their improved safety profile, minicircles have been shown to greatly increase the efficiency oftransgene expression in various in vitro and in vivo studies. In this chapter, we describe the production, purification, and application of minicircle DNA and discuss the rationale of the improved gene transfer efficiencies compared to conventional plasmid DNA.

Reconstituting Autophagy Initiation from Purified Components.

The hallmark of macroautophagy is the de novo generation of a membrane structure that collects cytoplasmic material and delivers it to lysosomes for degradation. The nucleation of this precursor membrane, termed phagophore, involves the coordinated assembly of the Atg1-kinase complex and the recruitment of Atg9 vesicles. The latter represents one important membrane source in order to produce phagophores in vivo. We explain how the process of phagophore nucleation can be reconstituted from purified components in vitro. We describe the assembly of the ~500 kDa pentameric Atg1-kinase complex from its purified subunits. We also explain how Atg9-donor vesicles are generated in vitro to study the interaction of Atg9 and Atg1-kinase complexes by floatation experiments.

Minicircle-DNA production by site specific recombination and protein-DNA interaction chromatography.

BACKGROUND: Conventional plasmid-DNA (pDNA) used in gene therapy and vaccination can be subdivided into a bacterial backbone and a transcription unit. Bacterial backbone sequences are needed for pDNA production in bacteria. However, for gene transfer application, these sequences are dispensable, reduce the overall efficiency of the DNA agent and, most importantly, represent a biological safety risk. For example, the dissemination of antibiotic resistance genes, as well as the uncontrolled expression of backbone sequences, may have profound detrimental effects and unmethylated CpG motifs have been shown to contribute to silencing of episomal transgene expression. Therefore, an important goal in nonviral vector development is to produce supercoiled pDNA lacking bacterial backbone sequences. METHODS: A method is described to provide circular, supercoiled minimal expression cassettes (minicircle-DNA) based on two processes: (i) an inducible, sequence specific, in vivo recombination process that is almost 100% efficient and (2) a novel affinity-based chromatographic purification approach for the isolation of the minicircle-DNA. RESULTS: Quantitative real-time polymerase chain reaction analysis, capillary gel electrophoresis and restriction analysis of the recombination products, and the minicircle-DNA revealed a recombination efficiency greater than 99.5% and a purity of the isolated minicircle-DNA of more than 98.5%. CONCLUSIONS: The results obtained in the present study demonstrate that the described technology facilitates the production of highly pure minicircle-DNA for direct application in gene therapy and vaccination. The process described is efficient, stable and suitable for further scale-up in industrial large-scale manufacturing.

Passing membranes to autophagy: Unconventional membrane tethering by Atg17.

Macroautophagy delivers cytoplasmic material to lysosomal/vacuolar compartments for degradation. Conserved multisubunit complexes, composed of autophagy-related (Atg) proteins, initiate the formation of membrane precursors, termed phagophores. Under physiological conditions these cup-shaped structures can capture cytoplasmic material highly selectively. Starvation or cytotoxic stresses, however, initiate the formation of much larger phagophores to enclose cytoplasm nonselectively. The biogenesis of nonselective autophagosomes is initiated by the hierarchical assembly of the Atg1 kinase complex and the recruitment of Atg9 vesicles at the phagophore assembly site (PAS). In this punctum we summarize our recent findings regarding tethering of Atg9 vesicles by the Atg1 kinase complex. We discuss membrane tethering by and activation of its central subunit Atg17 in the context of other canonical membrane tethering factors. Our results show that Atg17 suffices to bind and tether Atg9 vesicles. The Atg31-Atg29 subcomplex inhibits Atg17 activity, and activation of Atg17 depends on the formation of the Atg1 kinase complex that involves recruiting Atg1-Atg13. Our studies lead to a model of unconventional membrane tethering in autophagy.

Tubulins interact with porcine and human S proteins of the genus Alphacoronavirus and support successful assembly and release of infectious viral particles.

Coronavirus spike proteins mediate host-cell-attachment and virus entry. Virus replication takes place within the host cell cytosol, whereas assembly and budding occur at the endoplasmic reticulum-Golgi intermediate compartment. In this study we demonstrated that the last 39 amino acid stretches of Alphacoronavirus spike cytoplasmic domains of the human coronavirus 229E, NL63, and the porcine transmissible gastroenteritis virus TGEV interact with tubulin alpha and beta chains. In addition, a partial co-localization of TGEV spike proteins with authentic host cell ß-tubulin was observed. Furthermore, drug-induced microtubule depolymerization led to changes in spike protein distribution, a reduction in the release of infectious virus particles and less amount of spike protein incorporated into virions. These data demonstrate that interaction of Alphacoronavirus spike proteins with tubulin supports S protein transport and incorporation into virus particles.

Immobilization of plasmid DNA in bacterial ghosts.

The development of novel delivery vehicles is crucial for the improvement of DNA vaccine efficiency. In this report, we describe a new platform technology, which is based on the immobilization of plasmid DNA in the cytoplasmic membrane of a bacterial carrier. This technology retains plasmid DNA (Self-Immobilizing Plasmid, pSIP) in the host envelope complex due to a specific protein/DNA interaction during and after protein E-mediated lysis. The resulting bacterial ghosts (empty bacterial envelopes) loaded with pDNA were analyzed in detail by real time PCR assays. We could verify that pSIP plasmids were retained in the pellets of lysed Escherichia coli cultures indicating that they are efficiently anchored in the inner membrane of bacterial ghosts. In contrast, a high percentage of control plasmids that lack essential features of the self-immobilization system were expelled in the culture broth during the lysis process. We believe that the combination of this plasmid immobilization procedure and the protein E-mediated lysis technology represents an efficient in vivo technique for the production of non-living DNA carrier vehicles. In conclusion, we present a "self-loading", non-living bacterial DNA delivery vector for vaccination endowed with intrinsic adjuvant properties of the Gram-negative bacterial cell envelope.

Minicircle DNA immobilized in bacterial ghosts: in vivo production of safe non-viral DNA delivery vehicles.

DNA as an active agent is among the most promising technologies for vaccination and therapy. However, plasmid backbone sequences needed for the production of pDNA in bacteria are dispensable, reduce the efficiency of the DNA agent and, most importantly, represent a biological safety risk. In this report we describe a novel technique where a site-specific recombination system based on the ParA resolvase was applied to a self-immobilizing plasmid system (SIP). In addition, this system was combined with the protein E-specific lysis technology to produce non-living bacterial carrier vehicles loaded with minicircle DNA. The in vivo recombination process completely divided an origin plasmid into a minicircle and a miniplasmid. The replicative miniplasmid containing the origin of replication and the antibiotic resistance gene was lost during the subsequently induced PhiX174 gene E-mediated lysis process, which results in bacterial ghosts. The minicircle DNA was retained in these empty bacterial cell envelopes during the lysis process via the specific interaction of a membrane anchored protein with the minicircle DNA. Using this novel platform technology, a DNA delivery vehicle--consisting of a safe bacterial carrier with known adjuvant properties and minicircle DNA with an optimized safety profile--can be produced in vivo in a continuous process. Furthermore, this study provides the basis for the development of an efficient in vitro minicircle purification process.

Antigen discovery and delivery of subunit vaccines by nonliving bacterial ghost vectors.

The bacterial ghost (BG) platform system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. BGs are nonliving Gram-negative bacterial cell envelopes which are devoid of their cytoplasmic contents, yet maintain their cellular morphology and antigenic structures, including bioadhesive properties. The main advantages of BGs as carriers of subunit vaccines include their ability to stimulate a high immune response and to target the carrier itself to primary antigen-presenting cells. The intrinsic adjuvant properties of BGs enhance the immune response to target antigens, including T-cell activation and mucosal immunity. Since native and foreign antigens can be carried in the envelope complex of BGs, combination vaccines with multiple antigens of diverse origin can be presented to the immune system simultaneously. Beside the capacity of BGs to function as carriers of protein antigens, they also have a high loading capacity for DNA. Thus, loading BGs with recombinant DNA takes advantage of the excellent bioavailability for DNA-based vaccines and the high expression rates of the DNA-encoded antigens in target cell types such as macrophages and dendritic cells. There are many spaces within BGs including the inner and outer membranes, the periplasmic space and the internal lumen which can carry antigens, DNA or mediators of the immune response. All can be used for subunit antigen to design new vaccine candidates with particle presentation technology. In addition, the fact that BGs can also carry piggyback large-size foreign antigen particles, increases the technologic usefulness of BGs as combination vaccines against viral and bacterial pathogens. Furthermore, the BG antigen carriers can be stored as freeze-dried preparations at room temperature for extended periods without loss of efficacy. The potency, safety and relatively low production cost of BGs offer a significant technical advantage over currently utilized vaccine technologies.