A comparison of three methods of decellularization of pig corneas to reduce immunogenicity.

AIM: To investigate whether decellularization using different techniques can reduce immunogenicity of the cornea, and to explore the decellularized cornea as a scaffold for cultured corneal endothelial cells (CECs). Transplantation of decellularized porcine corneas increases graft transparency and survival for longer periods compared with fresh grafts. METHODS: Six-month-old wild-type pig corneas were cut into 100-200 µm thickness, and then decellularized by three different methods: 1) 0.1% sodium dodecyl sulfate (SDS); 2) hypoxic nitrogen (N2); and 3) hypertonic NaCl. Thickness and transparency were assessed visually. Fresh and decellularized corneas were stained with hematoxylin/eosin (H&E), and for the presence of galactose-α1,3-galactose (Gal) and N-glycolylneuraminic acid (NeuGc, a nonGal antigen). Also, a human IgM/IgG binding assay was performed. Cultured porcine CECs were seeded on the surface of the decellularized cornea and examined after H&E staining. RESULTS: All three methods of decellularization reduced the number of keratocytes in the stromal tissue by >80% while the collagen structure remained preserved. No remaining nuclei stained positive for Gal or NeuGc, and expression of these oligosaccharides on collagen was also greatly decreased compared to expression on fresh corneas. Human IgM/IgG binding to decellularized corneal tissue was considerably reduced compared to fresh corneal tissue. The cultured CECs formed a confluent monolayer on the surface of decellularized tissue. CONCLUSION: Though incomplete, the significant reduction in the cellular component of the decellularized cornea should be associated with a significantly reduced in vivo immune response compared to fresh corneas

Process evaluation of a behaviour change approach to improving clinical practice for detecting hereditary cancer

© 2019 The Author(s). Background: This retrospective process evaluation reports on the application of a 1-year implementation program to increase identification and management of patients at high risk of a hereditary cancer syndrome. The project used the Theoretical Domains Framework Implementation (TDFI) approach, a promising implementation methodology, used successfully in the United Kingdom to address patient safety issues. This Australian project run at two large public hospitals aimed to increase referrals of patients flagged as being at risk of Lynch syndrome on the basis of a screening test to genetic services. At the end of the project, the pathologists' processes had changed, but the referral rate remained inconsistent and low. Methods: Semi-structured interviews explored participants' perceptions of the TDFI approach and Health services researchers wrote structured reflections. Interview transcripts and reflections were coded initially against implementation outcomes for the various TDFI approach activities: acceptability, appropriateness, feasibility, value for time cost, and adoption. On a second pass, themes were coded around challenges to the approach. Results: Interviews were held with nine key project participants including pathologists, oncologists, surgeons, genetic counsellors and an administrative officer. Two health services researchers wrote structured reflections. The first of two major themes was 'Theory-related challenges', with subthemes of accessibility of theory underpinning the TDFI, commitment to that theory-based approach, and the problem of complexity. The second theme was 'Practical challenges' with subthemes of stakeholder management, navigating the system, and perceptions of the problem. Health services researchers reflected on the benefits of bridging professional divides and facilitating collective learning and problem solving, but noted frustrations around clinicians' time constraints that led to sparse interactions with the team, and lack of authority to effect change themselves. Conclusions: Mixed success of adoption as an outcome was attributed to the complexity and highly nuanced nature of the setting. This made identifying the target behaviour, a key step in the TDFI approach, challenging. Introduced changes in the screening process led to new, unexpected issues yet to be addressed. Strategies to address challenges are presented, including using an internal facilitator with a focus on applying a theory-based implementation approach

Quick and sensitive determination of gene expression of fatty acid synthase in vitro by using real-time polymerase chain reaction amplification (PCR)

Obesity results from an imbalance between energy intake and energy expenditure, which leads to a pathological accumulation of adipose tissue, but the underlying mechanism at gene level, is far from being elucidated. The objective of this study was to investigate the correlation between mRNA express from fatty acid synthase (FAS) with a different glucose level in primary adipocytes by real-time polymerase chain reaction amplification (PCR), which can aid in the understanding of the mechanism of obesity in vitro. By using the following formula, this study was able to quantify the mRNA expression of FAS of unknown samples: Y = -3.156X + 41.21 (Y = threshold cycle, X = log starting quantity). The high concentrations of glucose group significantly improved the mRNA expression of FAS (P < 0.01) rather than 0.25 and 0% concentrations of glucose. These results provide significant data that confirm an association between different glucose level and FAS expression in preadipocytes. The glucose concentration of the high group substantially augmented the mRNA expression of FAS.Key words: Expression, fatty acid synthase, lipid deposition, real-time polymerase chain reaction amplification (PCR)

Using NASA Earth Observation Data in ArcGIS

The NASA Goddard Earth Sciences Data and Information Services Center archives tens of thousands of Earth Observation (EO) parameters for land, atmosphere, and ocean. To facilitate GIS users to easily find, visualize, obtain, and analyze these EO data through, we developed an ArcGIS infrastructure with the Server, image services, Portal, and AOL. We will show how this capability supports broad GIS applications. Use cases including water management and air quality analyses will be demonstrated

Cell voltage versus electrode potential range in aqueous supercapacitors

Supercapacitors with aqueous electrolytes and nanostructured composite electrodes are attractive because of their high charging-discharging speed, long cycle life, low environmental impact and wide commercial affordability. However, the energy capacity of aqueous supercapacitors is limited by the electrochemical window of water. In this paper, a recently reported engineering strategy is further developed and demonstrated to correlate the maximum charging voltage of a supercapacitor with the capacitive potential ranges and the capacitance ratio of the two electrodes. Beyond the maximum charging voltage, a supercapacitor may still operate, but at the expense of a reduced cycle life. In addition, it is shown that the supercapacitor performance is strongly affected by the initial and zero charge potentials of the electrodes. Further, the differences are highlighted and elaborated between freshly prepared, aged under open circuit conditions, and cycled electrodes of composites of conducting polymers and carbon nanotubes. The first voltammetric charging-discharging cycle has an electrode conditioning effect to change the electrodes from their initial potentials to the potential of zero voltage, and reduce the irreversibility

Internal genes of a highly pathogenic H5N1 influenza virus determine high viral replication in myeloid cells and severe outcome of infection in mice.

The highly pathogenic avian influenza (HPAI) H5N1 influenza virus has been a public health concern for more than a decade because of its frequent zoonoses and the high case fatality rate associated with human infections. Severe disease following H5N1 influenza infection is often associated with dysregulated host innate immune response also known as cytokine storm but the virological and cellular basis of these responses has not been clearly described. We rescued a series of 6:2 reassortant viruses that combined a PR8 HA/NA pairing with the internal gene segments from human adapted H1N1, H3N2, or avian H5N1 viruses and found that mice infected with the virus with H5N1 internal genes suffered severe weight loss associated with increased lung cytokines but not high viral load. This phenotype did not map to the NS gene segment, and NS1 protein of H5N1 virus functioned as a type I IFN antagonist as efficient as NS1 of H1N1 or H3N2 viruses. Instead we discovered that the internal genes of H5N1 virus supported a much higher level of replication of viral RNAs in myeloid cells in vitro but not in epithelial cells and that this was associated with high induction of type I IFN in myeloid cells. We also found that in vivo during H5N1 recombinant virus infection cells of haematopoetic origin were infected and produced type I IFN and proinflammatory cytokines. Taken together our data infer that human and avian influenza viruses are differently controlled by host factors in alternative cell types; internal gene segments of avian H5N1 virus uniquely drove high viral replication in myeloid cells, which triggered an excessive cytokine production, resulting in severe immunopathology

MicroRNAs targeting oncogenes are down-regulated in pancreatic malignant transformation from benign tumors

BACKGROUND MicroRNA (miRNA) expression profiles have been described in pancreatic ductal adenocarcinoma (PDAC), but these have not been compared with pre-malignant pancreatic tumors. We wished to compare the miRNA expression signatures in pancreatic benign cystic tumors (BCT) of low and high malignant potential with PDAC, in order to identify miRNAs deregulated during PDAC development. The mechanistic consequences of miRNA dysregulation were further evaluated. METHODS Tissue samples were obtained at a tertiary pancreatic unit from individuals with BCT and PDAC. MiRNA profiling was performed using a custom microarray and results were validated using RT-qPCR prior to evaluation of miRNA targets. RESULTS Widespread miRNA down-regulation was observed in PDAC compared to low malignant potential BCT. We show that amongst those miRNAs down-regulated, miR-16, miR-126 and let-7d regulate known PDAC oncogenes (targeting BCL2, CRK and KRAS respectively). Notably, miR-126 also directly targets the KRAS transcript at a "seedless" binding site within its 3'UTR. In clinical specimens, miR-126 was strongly down-regulated in PDAC tissues, with an associated elevation in KRAS and CRK proteins. Furthermore, miR-21, a known oncogenic miRNA in pancreatic and other cancers, was not elevated in PDAC compared to serous microcystic adenoma (SMCA), but in both groups it was up-regulated compared to normal pancreas, implicating early up-regulation during malignant change. CONCLUSIONS Expression profiling revealed 21 miRNAs down-regulated in PDAC compared to SMCA, the most benign lesion that rarely progresses to invasive carcinoma. It appears that miR-21 up-regulation is an early event in the transformation from normal pancreatic tissue. MiRNA expression has the potential to distinguish PDAC from normal pancreas and BCT. Mechanistically the down-regulation of miR-16, miR-126 and let-7d promotes PDAC transformation by post-transcriptional up-regulation of crucial PDAC oncogenes. We show that miR-126 is able to directly target KRAS; re-expression has the potential as a therapeutic strategy against PDAC and other KRAS-driven cancers

Lectin-like bacteriocins from pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins

CAR-T cell. the long and winding road to solid tumors

Adoptive cell therapy of solid tumors with reprogrammed T cells can be considered the "next generation" of cancer hallmarks. CAR-T cells fail to be as effective as in liquid tumors for the inability to reach and survive in the microenvironment surrounding the neoplastic foci. The intricate net of cross-interactions occurring between tumor components, stromal and immune cells leads to an ineffective anergic status favoring the evasion from the host's defenses. Our goal is hereby to trace the road imposed by solid tumors to CAR-T cells, highlighting pitfalls and strategies to be developed and refined to possibly overcome these hurdles