The one-component system ArnR: a membrane-bound activator of the crenarchaeal archaellum.

Linking the motility apparatus to signal transduction systems enables microbes to precisely control their swimming behaviour according to environmental conditions. Bacteria have therefore evolved a complex chemotaxis machinery, which has presumably spread through lateral gene transfer into the euryarchaeal subkingdom. By contrast Crenarchaeota encode no chemotaxis-like proteins but are nevertheless able to connect external stimuli to archaellar derived motility. This raises fundamental questions about the underlying regulatory mechanisms. Recently, we reported that the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius becomes motile upon nutrient starvation by promoting transcription of flaB encoding the filament forming subunits. Here we describe two transcriptional activators as paralogous one-component-systems Saci_1180 and Saci_1171 (ArnR and ArnR1). Deletions of arnR and arnR1 resulted in diminished flaB expression and accordingly the deletion mutants revealed impaired swimming motility. We further identified two inverted repeat sequences located upstream of the flaB core promoter of S. acidocaldarius. These cis-regulatory elements were shown to be critical for ArnR and ArnR1 mediated flaB gene expression in vivo. Finally, bioinformatic analysis revealed ArnR to be conserved not only in Sulfolobales but also in the crenarchaeal order of Desulfurococcales and thus might represent a more general control mechanism of archaeal motility.

Functional interaction of osteogenic transcription factors Runx2 and Vdr in transcriptional regulation of Opn during soft tissue calcification.

Loss of Abcc6 gene expression was identified to be responsible for dystrophic calcification of the heart (DCC) or vessels after acute injury in several strains of laboratory mice. This calcification shares features with osteogenesis and may involve osteogenic factors. Tissue expression of osteopontin (Opn) and 11 osteogenic transcription factors were studied in vivo in mouse models for DCC and in vitro using luciferase reporter gene assays. Compared with DCC-resistant C57BL/6 mice, a significant increase in Opn transcription was demonstrated in necrotic lesions of both DCC-susceptible C3H/He and B6.C3H(Dyscalc1) congenic mice at day 3 after injury. Significant increases in gene expression were also demonstrated for the transcription factors runt domain-containing transcription factor 2 (Runx2), vitamin D receptor (Vdr), SRY (sex-determining region Y)-box 9 protein, and Nfkb1 in C3H/He mice versus C57BL/6 controls. However, only Runx2 remained significantly increased in the B6.C3H(Dyscalc1) congenic mice, which carry only the Dyscalc1 locus with functional Abcc6 deletion on a C57BL/6 genetic background. Luciferase assay use increased Opn promoter activity, which was demonstrated after overexpression of Runx2. A poly-T stretch insertion was identified to stabilize the binding of Runx2, thus significantly enhancing Opn promoter activity. This Runx2-mediated activation was further enhanced by cotransfection with Vdr. Our data suggest a key role of Runx2 in the regulation of Opn in a model of cardiovascular calcification and demonstrate a synergistic cooperation of Runx2 and Vdr.

Ultrafine mapping of Dyscalc1 to an 80-kb chromosomal segment on chromosome 7 in mice susceptible for dystrophic calcification.

In mice, dystrophic cardiovascular calcification (DCC) is controlled by a major locus on proximal mouse chromosome 7 named Dyscalc1. Here we present a strategy that combines in silico analysis, expression analysis, and extensive sequencing for ultrafine mapping of the Dyscalc1 locus. We subjected 15 laboratory mouse strains to freeze-thaw injury of the heart, and association with respective genotypes allowed condensation of the Dyscalc1 locus to 1 Mb. Within this region, 51 known and predicted genes were studied in DCC-susceptible C3H/He and DCC-resistant C57BL/6 mice with respect to mRNA expression in response to injury. Five genes displayed differential expression. Genotyping of seven novel single nucleotide polymorphisms (SNPs) within these genes revealed an 80-Kb region in NZB mice that were found positive for calcification though carrying otherwise alleles from DCC-resistant mice. This microheterogeneity in NZB mice was evolutionary conserved in all DCC-susceptible mouse strains and contains the genes EMP-3, BC013491, and Abcc6 (partially). The flanking SNPs are rs3703247 and NT_039420.5_2757991. mRNA levels of EMP-3 were found to be upregulated in response to injury in both C57BL/6 and C3H/He mice. Sequencing of EMP-3 revealed an SNP leading to an amino acid substitution (p.T153I) that was found in all mouse strains susceptible for DCC but not in resistant strains such as C57BL/6 mice. Thus, the p.T153I changes might affect the biological function of EMP-3 gene product after injury. Using this combined approach, we ultrafine-mapped the Dyscalc1 locus to an 80-Kb region and identified EMP-3 as a new candidate gene for DCC.

Chitosan-film enhanced chitosan nerve guides for long-distance regeneration of peripheral nerves.

Biosynthetic nerve grafts are developed in order to complement or replace autologous nerve grafts for peripheral nerve reconstruction. Artificial nerve guides currently approved for clinical use are not widely applied in reconstructive surgery as they still have limitations especially when it comes to critical distance repair. Here we report a comprehensive analysis of fine-tuned chitosan nerve guides (CNGs) enhanced by introduction of a longitudinal chitosan film to reconstruct critical length 15 mm sciatic nerve defects in adult healthy Wistar or diabetic Goto-Kakizaki rats. Short and long term investigations demonstrated that the CNGs enhanced by the guiding structure of the introduced chitosan film significantly improved functional and morphological results of nerve regeneration in comparison to simple hollow CNGs. Importantly, this was detectable both in healthy and in diabetic rats (short term) and the regeneration outcome almost reached the outcome after autologous nerve grafting (long term). Hollow CNGs provide properties likely leading to a wider clinical acceptance than other artificial nerve guides and their performance can be increased by simple introduction of a chitosan film with the same advantageous properties. Therefore, the chitosan film enhanced CNGs represent a new generation medical device for peripheral nerve reconstruction.

Peripheral Nerve Regeneration Through Hydrogel-Enriched Chitosan Conduits Containing Engineered Schwann Cells for Drug Delivery.

Critical length nerve defects in the rat sciatic nerve model were reconstructed with chitosan nerve guides filled with Schwann cells (SCs) containing hydrogel. The transplanted SCs were naive or had been genetically modified to overexpress neurotrophic factors, thus providing a cellular neurotrophic factor delivery system. Prior to the assessment in vivo, in vitro studies evaluating the properties of engineered SCs overexpressing glial cell line-derived neurotrophic factor (GDNF) or fibroblast growth factor 2 (FGF-2(18kDa)) demonstrated their neurite outgrowth inductive bioactivity for sympathetic PC-12 cells as well as for dissociated dorsal root ganglion cell drop cultures. SCs within NVR-hydrogel, which is mainly composed of hyaluronic acid and laminin, were delivered into the lumen of chitosan hollow conduits with a 5% degree of acetylation. The viability and neurotrophic factor production by engineered SCs within NVR-Gel inside the chitosan nerve guides was further demonstrated in vitro. In vivo we studied the outcome of peripheral nerve regeneration after reconstruction of 15-mm nerve gaps with either chitosan/NVR-Gel/SCs composite nerve guides or autologous nerve grafts (ANGs). While ANGs did guarantee for functional sensory and motor regeneration in 100% of the animals, delivery of NVR-Gel into the chitosan nerve guides obviously impaired sufficient axonal outgrowth. This obstacle was overcome to a remarkable extent when the NVR-Gel was enriched with FGF-2(18kDa) overexpressing SCs.

In vitro evaluation of cell-seeded chitosan films for peripheral nerve tissue engineering.

Natural biomaterials have attracted an increasing interest in the field of tissue-engineered nerve grafts, representing a possible alternative to autologous nerve transplantation. With the prospect of developing a novel entubulation strategy for transected nerves with cell-seeded chitosan films, we examined the biocompatibility of such films in vitro. Different types of rat Schwann cells (SCs)--immortalized, neonatal, and adult-of the chitosan substrate. Both cell types were viable on the biomaterial and showed different metabolic activities and proliferation behavior, indicating cell-type-specific cell-biomaterial interaction. Moreover, the cell types also displayed their typical morphology. In cocultures adult SCs used the BMSCs as a feeder layer and no negative interactions between both cell types were detected. Further, the chitosan films allow neurite outgrowth from dissociated sensory neurons, which is additionally supported on film preseeded with SC-BMSC cocultures. The presented chitosan films therefore demonstrate high potential for their use in tissue-engineered nerve grafts.

Nanotechnology versus stem cell engineering: in vitro comparison of neurite inductive potentials.

PURPOSE: Innovative nerve conduits for peripheral nerve reconstruction are needed in order to specifically support peripheral nerve regeneration (PNR) whenever nerve autotransplantation is not an option. Specific support of PNR could be achieved by neurotrophic factor delivery within the nerve conduits via nanotechnology or stem cell engineering and transplantation. METHODS: Here, we comparatively investigated the bioactivity of selected neurotrophic factors conjugated to iron oxide nanoparticles (np-NTFs) and of bone marrow-derived stem cells genetically engineered to overexpress those neurotrophic factors (NTF-BMSCs). The neurite outgrowth inductive activity was monitored in culture systems of adult and neonatal rat sensory dorsal root ganglion neurons as well as in the cell line from rat pheochromocytoma (PC-12) cell sympathetic culture model system. RESULTS: We demonstrate that np-NTFs reliably support numeric neurite outgrowth in all utilized culture models. In some aspects, especially with regard to their long-term bioactivity, np-NTFs are even superior to free NTFs. Engineered NTF-BMSCs proved to be less effective in induction of sensory neurite outgrowth but demonstrated an increased bioactivity in the PC-12 cell culture system. In contrast, primary nontransfected BMSCs were as effective as np-NTFs in sensory neurite induction and demonstrated an impairment of neuronal differentiation in the PC-12 cell system. CONCLUSION: Our results evidence that nanotechnology as used in our setup is superior over stem cell engineering when it comes to in vitro models for PNR. Furthermore, np-NTFs can easily be suspended in regenerative hydrogel matrix and could be delivered that way to nerve conduits for future in vivo studies and medical application.

Projections of costs, financing, and additional resource requirements for low- and lower middle-income country immunization programs over the decade, 2011-2020.

The Decade of Vaccines Global Vaccine Action Plan has outlined a set of ambitious goals to broaden the impact and reach of immunization across the globe. A projections exercise has been undertaken to assess the costs, financing availability, and additional resource requirements to achieve these goals through the delivery of vaccines against 19 diseases across 94 low- and middle-income countries for the period 2011-2020. The exercise draws upon data from existing published and unpublished global forecasts, country immunization plans, and costing studies. A combination of an ingredients-based approach and use of approximations based on past spending has been used to generate vaccine and non-vaccine delivery costs for routine programs, as well as supplementary immunization activities (SIAs). Financing projections focused primarily on support from governments and the GAVI Alliance. Cost and financing projections are presented in constant 2010 US dollars (US$). Cumulative total costs for the decade are projected to be US$57.5 billion, with 85% for routine programs and the remaining 15% for SIAs. Delivery costs account for 54% of total cumulative costs, and vaccine costs make up the remainder. A conservative estimate of total financing for immunization programs is projected to be $34.3 billion over the decade, with country governments financing 65%. These projections imply a cumulative funding gap of $23.2 billion. About 57% of the total resources required to close the funding gap are needed just to maintain existing programs and scale up other currently available vaccines (i.e., before adding in the additional costs of vaccines still in development). Efforts to mobilize additional resources, manage program costs, and establish mutual accountability between countries and development partners will all be necessary to ensure the goals of the Decade of Vaccines are achieved. Establishing or building on existing mechanisms to more comprehensively track resources and commitments for immunization will help facilitate these efforts.

Chitosan tubes of varying degrees of acetylation for bridging peripheral nerve defects.

Biosynthetic nerve grafts are desired as alternative to autologous nerve grafts in peripheral nerve reconstruction. Artificial nerve conduits still have their limitations and are not widely accepted in the clinical setting. Here we report an analysis of fine-tuned chitosan tubes used to reconstruct 10 mm nerve defects in the adult rat. The chitosan tubes displayed low, medium and high degrees of acetylation (DAI: ≈ 2%, DA: ≈ 5%, DAIII: ≈ 20%) and therefore different degradability and microenvironments for the regenerating nerve tissue. Short and long term investigations were performed demonstrating that the chitosan tubes allowed functional and morphological nerve regeneration similar to autologous nerve grafts. Irrespective of the DA growth factor regulation demonstrated to be the same as in controls. Analyses of stereological parameters as well as the immunological tissue response at the implantation site and in the regenerated nerves, revealed that DAI and DAIII chitosan tubes displayed some limitations in the support of axonal regeneration and a high speed of degradation accompanied with low mechanical stability, respectively. The chitosan tubes combine several pre-requisites for a clinical acceptance and DAII chitosan tubes have to be judged as the most supportive for peripheral nerve regeneration.

The potential global market size and public health value of an HIV-1 vaccine in a complex global market.

An effective HIV vaccine will be essential for the control of the HIV pandemic. This study evaluated the potential global market size and value of a hypothetical HIV vaccine and considered clade diversity, disease burden, partial prevention of acquisition, impact of a reduction in viral load resulting in a decrease in transmission and delay to treatment, health care system differences regarding access, and HIV screening and vaccination, across all public and private markets. Vaccine product profiles varied from a vaccine that would have no effect on preventing infection to a vaccine that would effectively prevent infection and reduce viral load. High disease burden countries (HDBC; HIV prevalence > or = 1%) were assumed to routinely vaccinate pre-sexually active adolescents (10 years old), whereas low disease burden countries (LDBC; HIV prevalence rate <1%) were assumed to routinely vaccinate higher risk populations only. At steady state, routine vaccination demand for vaccines that would prevent infection only was 22-61 million annual doses with a potential market value of $210 million to $2.7 billion, depending on the vaccine product profile. If one-time catch-up campaigns were included (11-14 years old for HDBC and higher risk groups for LDBC), the additional cumulative approximately 70-237 million doses were needed over a 10-year period with a potential market value of approximately $695 million to $13.4 billion, depending on the vaccine product profile. Market size and value varied across market segments with the majority of the value in high income countries and the majority of the demand in low income countries. However, the value of the potential market in low income countries is still significant with up to $550 million annually for routine vaccination only and up to $1.7 billion for a one-time only catch-up campaign in 11-14 years old. In the most detail to date, this study evaluated market size and value of a potential multi-clade HIV vaccine, accounting for differences in disease burden, product profile and health care complexities. These findings provide donors and suppliers highly credible new data to consider in their continued efforts to develop an HIV-1 vaccine to address the worldwide disease burden.

Novel missense mutations (p.T596M and p.P1797H) in NOTCH1 in patients with bicuspid aortic valve.

The bicuspid aortic valve (BAV) is the most common congenital cardiac malformation, occurring in 1-2% of the population. In a recent report, mutations in NOTCH1 a signaling and transcriptional regulator have been shown to cause BAV in two families. This study provides data on systematic sequencing in search for novel mutations in NOTCH1 gene in a large sample BAV. For the first time, we report results of a systematic mutation-analysis based on DNA-sequencing of all coding exons and adjacent splice consensus sequences of NOTCH1 gene. Our analyses revealed 57 NOTCH1 sequence variants. Twenty-one variants are located within exons and 36 within intronic or 5'-UTR sequences. Thirty-five variants were described previously as polymorphisms. The remaining 22, however, were neither listed in public SNP databases nor in the literature and were therefore considered novel. Seventeen variants were found only once (MAF = 1%), of these 15 were novel. Two sequence variants led to amino acid substitutions (p.T596M and p.P1797H) and are located in highly conserved regions of the NOTCH1 protein. In addition, these two mutations could not be detected in at least 327 healthy controls by using RFLP-analysis. The functional relevance of the other 13 novel and rare variants could not be proven without further functional examination. In this study, we provide a new evidence that the mutations in the NOTCH1 gene may trigger the underlying mechanism causing the valve calcification, especially in BAV. In conclusion, NOTCH1 gene mutations do not only play a role in familiar BAV, but can also be observed in approximately 4% of sporadic cases.