An european pupil project linked to the scientific aims of the experiment AQUARIUS-XENOPUS on the taxi Soyuz flight Andromede to ISS

The German-French biological experiment AQUARIUS-XENOPUS which flew on the Soyuz flight Andromede to the International Space Station ISS (launched October 21, 2001 in Baikonour/Kazakhstan) was extended by an outreach project. Pupils of class 10 to 12 from Ulm/D and Nancy-Tomblaine/F studied swimming behavior of Xenopus tadpoles on ground. They were instructed to perform all experimental steps following the protocol of similar video recordings on ISS. After the flight, they evaluated the kinetics of swimming of both ground controls and space animals. The pupil project included theoretical components to introduce them to the field of gravitational biology. One feature of the project was the exchange of ideas between pupils by meetings which took place in Ulm (June 2001), Nancy (February 2002) and Paris (May 2002). We consider our approach as a successful way to include young people in space experiments on a cheap cost level and to bring ideas of gravitational biology into the curricula of European schools.     

Biological controls upon the physical taphonomy of exceptionally preserved salamanders from the Miocene of Rubielos de Mora,northeast Spain

McNamara, M.E., Orr, P.J., Manzocchi, T., Alcalá, L., Anadón, P. & Peñalver, E. 2011: Biological controls upon the physical taphonomy of exceptionally preserved salamanders from the Miocene of Rubielos de Mora, northeast Spain. Lethaia, Vol. 45, pp. 210–226. The middle Miocene Rubielos de Mora Konservat‐Lagerstätte of northeast Spain is hosted within profundal, finely laminated, lacustrine mudstones. The diverse biota includes abundant salamanders. Most individuals died during separate episodes and sank rapidly postmortem. Specimens are typically preserved in dorso‐ventral aspect, the most hydrodynamically stable orientation. The near‐cylindrical morphology of the body, however, allowed some carcasses to settle in or subsequently re‐orientate into, lateral orientations. Loss of skeletal elements (i.e. reduced completeness) reflects their location within the body and followed a distal to proximal trend. Two stages are identified: initial loss of a small number of phalanges, followed by loss of more proximal limb bones plus additional phalanges. Disarticulation is more complex: it occurred via several mechanisms (notably, abdominal rupture and re‐orientation of part of the body and limbs during decay) and shows no consistent pattern among specimens. The physical taphonomy of the salamanders is controlled predominantly by intrinsic biological factors, i.e. the geometry of the body and of individual skeletal elements, the orientation, inherent strength and location of specific joints and the extent to which soft tissues, particularly the skin, persist during decay. These biological factors probably control patterns of physical taphonomy of other fossil tetrapods with a similar skeletal configuration. □Articulation, completeness, Konservat‐Lagerstätten, orientation, quantitative taphonomy, salamanders.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles

Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteins trans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, these trans-complemented HCV particles (HCV_TCP) penetrate target cells in a CD81 receptor-dependent fashion. Since HCV_TCP production was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCV_TCP stocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 10⁶ tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excluding cis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCV_TCP may prove valuable for gene delivery or vaccination approaches.Hepatitis C virus (HCV) is an enveloped plus-strand RNA virus of the genus Hepacivirus within the family Flaviviridae (34). The HCV genome is approximately 9.6 kb in length and consists of a single open reading frame encoding a polyprotein of ca. 3,000 amino acids and nontranslated regions (NTRs) located at the 5′ and 3′ termini. These NTRs are highly structured RNA segments encompassing critical cis-active RNA elements essential for genome replication and RNA translation (31). Viral proteins are expressed in a cap-independent manner by means of an internal ribosome entry site (IRES) located in the 5′ NTR. Co- and posttranslational cleavages liberate 10 viral proteins: core; envelope protein 1 (E1) and E2, representing the structural proteins that constitute the virion; a small membrane-associated ion channel protein designated p7 that is essential for virus assembly (16, 22, 43, 57); and six nonstructural (NS) proteins (NSs 2, 3, 4A, 4B, 5A, and 5B). HCV proteins NS3 to NS5B are both necessary and sufficient to establish membrane-bound replication complexes catalyzing RNA replication (13, 36). More recent data indicate that the NS2 protease that catalyzes cleavage at the NS2-NS3 site in addition participates in assembly and release of infectious viruses (22). Finally, ribosomal frame-shifting and internal translation initiation yield a group of additional proteins designated ARFP (alternative reading frame protein) or core+1 proteins. However, their function for the HCV replication cycle is currently not known.One hallmark of HCV is its high propensity to establish a persistent infection, which frequently causes progressive morbidity ranging from hepatic fibrosis to cirrhosis and hepatocellular carcinoma (20). Despite considerable progress in the treatment of HCV infection, the currently available therapy (a combination of pegylated interferon alpha with ribavirin) is not well tolerated and is efficacious in only ca. 50% of patients infected with the most prevalent genotype 1 (38). Therapeutic or prophylactic vaccines are not available. For these reasons and with currently ca. 170 million persistently infected individuals, HCV infection represents a considerable global health problem necessitating pertinent basic and applied research efforts.In recent years three major advances enabled analysis of the HCV replication cycle in tissue culture. First, Lohmann and colleagues developed subgenomic HCV replicons (36). These autonomously replicating RNA molecules carry all the genetic elements necessary for self-replication (the NTRs and NS3 to NS5B), including a selectable marker or a reporter gene in place of the viral structural proteins, and an internal IRES for expression of the HCV replicase genes (reviewed in reference 45). Second, HCV pseudotype particles, i.e., retroviral particles surrounded by an envelope carrying HCV E1-E2 complexes in place of their cognate envelope proteins, were established (3, 21). As these particles carry functional HCV glycoprotein complexes on their surface, HCV pseudotype particles have been instrumental for the analysis of E1-E2 receptor interactions and the early events of HCV infection (reviewed in reference 2). Finally, in 2005 fully permissive cell culture systems which are based on the JFH1 clone were described (33, 66, 72). This isolate replicates with unprecedented efficiency in transfected Huh7 human hepatoma cells and produces particles infectious both in vitro and in vivo, thus providing a model system reproducing the complete HCV replication cycle.Use of these novel models has considerably expanded our knowledge of viral and host cell factors involved in HCV replication (for a recent review, see reference 59). It is now known that similar to virtually all other plus-strand RNA viruses, HCV induces intracellular membrane alterations and replicates its genome in conjunction with vesicular membrane structures, the so-called “membranous web” (10, 13). Presumably as a consequence of this specific, rather secluded architecture of the membrane-associated replication machinery, all viral proteins involved in RNA replication, with the exception of NS5A function in cis, cannot be complemented in trans (1). Restricted trans-complementation of viral replicase proteins has been observed for other plus-strand RNA viruses as well, thus indicating that a rather “closed” replication machinery is a shared feature of these viruses (15, 27, 60). In contrast, for a number of plus-strand RNA viruses from diverse virus families like Picornaviridae (poliovirus), Alphaviridae (Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus), Coronaviridae (human coronavirus E229), and Flaviviridae (tick-borne encephalitis virus, Kunjin virus, West Nile virus, and yellow fever virus), assembly of progeny viruses can be achieved when structural proteins are expressed in trans and independent from the RNA molecule that encodes the replicase proteins. Similarly, Miyanari recently reported that HCV genomes with lethal mutations in core protein can be rescued by ectopic expression of functional core protein (39). This flexibility has been extensively used to create viral vectors for gene delivery as well as viral vector-based immunization approaches (32, 48, 49, 61, 68) (for a recent review on alphaviral vectors, the most frequently used among plus strand RNA vectors, see reference 37). In these systems the viral genome region encoding the structural proteins is replaced by a transgene. The resulting defective vector genomes are capable of RNA replication but due to the lack of structural proteins are unable to produce progeny virus particles. This defect is rescued by expression of the structural proteins in trans via helper viruses (28, 55) or, in some cases, by DNA constructs stably expressed in packaging cell lines (17). The resulting virus-like particles are infectious but support only single-round infection and are unable to spread, thus improving the safety of the viral transduction system.Given the success of plus-strand RNA vector technology for basic and applied clinical research, in this study we developed a trans-complementation system for HCV that provided new insights into the basic principles of HCV particle assembly.     

Infrared Thermography and Ultrasonography to Indirectly Monitor the Influence of Liner Type and Overmilking on Teat Tissue Recovery

Eight Danish Holstein cows were milked with a 1-mm thick specially designed soft liner on their right rear teat and a standard liner mounted under extra high tension on their left rear teat. Four of the animals were overmilked for 5 min. Rear teats were subjected to ultrasound examination on the first day and to infrared thermography on the second day. Teats were submersed in ethanol 20 min post-milking on the second day. Ultrasonography measurements showed that teat canal length increased by 30–41% during milking. Twenty minutes after milking, teats milked with modified standard liners still had elongated teat canals while teats milked with the soft liner were normalized. Overmilking tended to increase teat wall thickness. Approximately 80% of variability in teat canal length, from before teat preparation to after milking, could be explained by changes during teat preparation. Thermography indicated a general drop in teat temperature during teat preparation. Teat temperature increased during milking and continued to increase until the ethanol challenge induced a significant drop. Temperatures approached pre-challenge rather than pre-milking temperatures within 10 minutes after challenge. Teat temperatures were dependent on type of liner. Mid-teat temperatures post-challenge relative to pre-teat preparation were dependent on overmilking. Thermography and ultrasound were considered useful methods to indirectly and non invasively evaluate teat tissue integrity.     

重组腺相关病毒2型转导人外周血单核细胞来源树突状细胞的研究

To find out the infection efficiency of recombinant adeno-as sociated virus 2-mediated exogenou s genes in human peripheral blood monocyte-derived dendritic cells(D Cs),the process of transfection wa s investigated by FITC-labeled rAA V2,and observed under confocal mic roscope.Newly separated dendritic cells were tranfected by rAAV2-luc and rAAV2-GFP at different MOI,and transfection efficiency were detec ted by luminometer for rAAV2-luc a nd flow cytometry for rAAV2-GFP.Th e results were elucidated in four different assay systems:(1)60min w as needed for rAAV2 to bind on den dritic cells,and got into cells in the following 10min;(2)the express ion of luc could be detected at th e MOI as low as 1×10~5v.g/cell,and the expression plateau was reached by the MOI of 10~6～10~7v.g/cell,furt her increase of MOI had no functio n on expression level;(3)transgene expression was detected after 48h, and maintained a higher expression level from 96h to 240h after infec tion;(4)7days postinfection of rAA V2-GFP,5%～18% dendritic cells were GFP positive. These data suggest th at rAAV2 vector can efficiently in fect monocyte-derived dendritic ce lls and mediate exogenous gene exp ression,and that the application o f rAAV2 as vector may be useful fo r gene transfer to dendritic cells in ex vivo immunotherapy.     

Evolution of body condition‐dependent dispersal in metapopulations

Body condition‐dependent dispersal strategies are common in nature. Although it is obvious that environmental constraints may induce a positive relationship between body condition and dispersal, it is not clear whether positive body conditional dispersal strategies may evolve as a strategy in metapopulations. We have developed an individual‐based simulation model to investigate how body condition–dispersal reaction norms evolve in metapopulations that are characterized by different levels of environmental stochasticity and dispersal mortality. In the model, body condition is related to fecundity and determined either by environmental conditions during juvenile development (adult dispersal) or by those experienced by the mother (natal dispersal). Evolutionarily stable reaction norms strongly depend on metapopulation conditions: positive body condition dependency of dispersal evolved in metapopulation conditions with low levels of dispersal mortality and high levels of environmental stochasticity. Negative body condition‐dependent dispersal evolved in metapopulations with high dispersal mortality and low environmental stochasticity. The latter strategy is responsible for higher dispersal rates under kin competition when dispersal decisions are based on body condition reached at the adult life stage. The evolution of both positive and negative body condition‐dependent dispersal strategies is consequently likely in metapopulations and depends on the prevalent environmental conditions.     

Characterization of Determinants Important for Hepatitis C Virus p7 Function in Morphogenesis by Using trans-Complementation

Hepatitis C virus (HCV) p7 is an integral membrane protein that forms ion channels in vitro and that is crucial for the efficient assembly and release of infectious virions. Due to these properties, p7 was included in the family of viroporins that comprises proteins like influenza A virus M2 and human immunodeficiency virus type 1 (HIV-1) vpu, which alter membrane permeability and facilitate the release of infectious viruses. p7 from different HCV isolates sustains virus production with variable efficiency. Moreover, p7 determinants modulate processing at the E2/p7 and the p7/NS2 signal peptidase cleavage sites, and E2/p7 cleavage is incomplete. Consequently, it was unclear if a differential ability to sustain virus production was due to variable ion channel activity or due to alternate processing at these sites. Therefore, we developed a trans-complementation assay permitting the analysis of p7 outside of the HCV polyprotein and thus independently of processing. The rescue of p7-defective HCV genomes was accomplished by providing E2, p7, and NS2, or, in some cases, by p7 alone both in a transient complementation assay as well as in stable cell lines. In contrast, neither influenza A virus M2 nor HIV-1 vpu compensated for defective p7 in HCV morphogenesis. Thus, p7 is absolutely essential for the production of infectious HCV particles. Moreover, our data indicate that p7 can operate independently of an upstream signal sequence, and that a tyrosine residue close to the conserved dibasic motif of p7 is important for optimal virus production in the context of genotype 2a viruses. The experimental system described here should be helpful to investigate further key determinants of p7 that are essential for its structure and function in the absence of secondary effects caused by altered polyprotein processing.Hepatitis C virus (HCV) is a highly variable enveloped virus. It is the sole member of the genus Hepacivirus within the family Flaviviridae (36). Based on sequence homology, patient isolates are classified into seven genotypes and more than 100 subtypes (17, 52).The genome of HCV is a single-stranded RNA molecule of positive polarity with a size of ∼9.6 kb. It encodes a polyprotein of ca. 3,000 amino acids and contains nontranslated regions (NTRs) at both the 5′ and 3′ termini that are required for translation and RNA replication (33). Cellular and two viral proteases, NS2-3 and NS3-4A, liberate the individual viral proteins. The N-terminal portion of the polyprotein contains the structural proteins core and envelope glycoproteins 1 and 2 (E1, E2), which constitute the virus particle. These proteins are cleaved from the polyprotein by the host cell signal peptidase (18, 24). In the case of the core protein, an additional cleavage step mediated by the signal peptide peptidase liberates its mature C terminus (41). Further downstream of the structural proteins the polyprotein harbors p7, a short membrane-associated polypeptide required for virus assembly and release (27, 55), and the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Proteins NS3 to NS5B are the minimal components of the membrane-bound replication complexes that catalyze RNA replication (16, 38).Using the novel JFH1-based HCV infection model (35, 61, 65), it has been demonstrated recently that besides the canonical structural proteins core, E1, and E2, NS5A, p7, NS3, and NS2 also are crucial for the production of infectious HCV particles (1, 26, 27, 39, 40, 55, 57). These data highlight that HCV assembly and release is a coordinated process involving both structural and nonstructural proteins. However, how the aforementioned proteins contribute to the production of infectious virus particles remains poorly understood.HCV p7 comprises two helical domains connected by a polar loop. Studies with epitope-tagged p7 variants indicate that both termini of the protein are resident in the lumen of the endoplasmic reticulum (ER) (4) or that, in addition, a second alternative topology with the C terminus exposed to the cytoplasm can be adopted (25). Using such constructs for fluorescent microscopy, a complex localization of p7 was revealed. While most prominent staining generally was observed at the ER (4, 19, 23), pools of p7 also were detected at mitochondria (19) and at the plasma membrane (4). These data suggest that p7 influences virus replication at various sites within infected cells, and that the function and/or localization of p7 is regulated by different trafficking signals that could be exposed in a topology-dependent manner. However, caution is warranted since, due to the lack of antibodies, epitope-tagged p7 variants had to be employed for most analyses, and since localization studies of virus-producing cells with functional p7 still are lacking.One hallmark of p7 is its ability to form cation-selective channels in artificial membranes (20, 46, 49), a property that likely depends on the oligomerization of the protein (7, 21). There are intriguing correlations that link p7''s function as an ion channel protein in vitro to its role in the assembly and release of infectious HCV particles in tissue culture. First, the mutation of the conserved dibasic motif in the polar loop of p7 abrogates ion channel activity and interferes with virus production in tissue culture (20, 27, 55). Second, iminosugars coupled to long alkyl chains like N-nonyl deoxygalactonojirimycin (NN-DGJ) not only interfere with ion channel activity but also repress the release of infectious particles from transfected Huh-7 cells (46, 56). Taken together, these data suggest that the ion channel activity of p7 is crucial for its role in the late steps of the HCV replication cycle, and that this function is amenable to the development of selective inhibitors for antiviral therapy. However, presently it is unknown how mechanistically p7, as an ion channel protein, facilitates HCV assembly and release or if p7 also is a component of virus particles and participates in entry.Besides its function as an ion channel, p7 harbors a signal-like sequence in its C-terminal domain that directs the insertion of the N terminus of NS2 into the lumen of the ER (4). Strikingly, due to structural determinants within the C terminus of E2, p7, and the N terminus of NS2, signalase cleavages at the E2/p7 and the p7/NS2 sites are incomplete, thus yielding E2-p7-NS2 and E2-p7 precursor proteins (3, 18, 34, 42). Although these precursors are not absolutely essential for the production of infectious HCV particles (26, 27), a defined ratio between mature and precursor proteins might play a role to orchestrate optimal virus assembly. Given these circumstances, genetic studies of p7 function are complicated, since mutations may, on the one hand, affect ion channel activity, and on the other hand influence processing at the E2-p7 and p7-NS2 junctions.To circumvent this problem, in this study we developed a complementation system that permits the rescue of genomes with defects in p7 by the ectopic expression of p7 in trans. This enabled us to directly assess the function of p7 in the absence of secondary effects caused by aberrant polyprotein cleavage. Using this approach, we analyzed the role of the native signal sequence of p7 and p7-containing precursor proteins. In addition, we investigated key determinants that are essential for the optimal function of p7 in the course of HCV infectious particle production.