Image Quality of 3rd Generation Spiral Cranial Dual-Source CT in Combination with an Advanced Model Iterative Reconstruction Technique: A Prospective Intra-Individual Comparison Study to Standard Sequential Cranial CT Using Identical Radiation Dose

Objectives

To prospectively intra-individually compare image quality of a 3^rd generation Dual-Source-CT (DSCT) spiral cranial CT (cCT) to a sequential 4-slice Multi-Slice-CT (MSCT) while maintaining identical intra-individual radiation dose levels.

Methods

35 patients, who had a non-contrast enhanced sequential cCT examination on a 4-slice MDCT within the past 12 months, underwent a spiral cCT scan on a 3^rd generation DSCT. CTDI_vol identical to initial 4-slice MDCT was applied. Data was reconstructed using filtered backward projection (FBP) and 3^rd-generation iterative reconstruction (IR) algorithm at 5 different IR strength levels. Two neuroradiologists independently evaluated subjective image quality using a 4-point Likert-scale and objective image quality was assessed in white matter and nucleus caudatus with signal-to-noise ratios (SNR) being subsequently calculated.

Results

Subjective image quality of all spiral cCT datasets was rated significantly higher compared to the 4-slice MDCT sequential acquisitions (p<0.05). Mean SNR was significantly higher in all spiral compared to sequential cCT datasets with mean SNR improvement of 61.65% (p*_{Bonferroni0.05}<0.0024). Subjective image quality improved with increasing IR levels.

Conclusion

Combination of 3^rd-generation DSCT spiral cCT with an advanced model IR technique significantly improves subjective and objective image quality compared to a standard sequential cCT acquisition acquired at identical dose levels.     

The TatC component of the twin‐arginine protein translocase functions as an obligate oligomer

The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate‐bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild‐type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue‐native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild‐type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate‐induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.     

Regulation of p53 and Rb Links the Alternative NF-κB Pathway to EZH2 Expression and Cell Senescence

There are two major pathways leading to induction of NF-κB subunits. The classical (or canonical) pathway typically leads to the induction of RelA or c-Rel containing complexes, and involves the degradation of IκBα in a manner dependent on IκB kinase (IKK) β and the IKK regulatory subunit NEMO. The alternative (or non-canonical) pathway, involves the inducible processing of p100 to p52, leading to the induction of NF-κB2(p52)/RelB containing complexes, and is dependent on IKKα and NF-κB inducing kinase (NIK). Here we demonstrate that in primary human fibroblasts, the alternative NF-κB pathway subunits NF-κB2 and RelB have multiple, but distinct, effects on the expression of key regulators of the cell cycle, reactive oxygen species (ROS) generation and protein stability. Specifically, following siRNA knockdown, quantitative PCR, western blot analyses and chromatin immunoprecipitation (ChIP) show that NF-κB2 regulates the expression of CDK4 and CDK6, while RelB, through the regulation of genes such as PSMA5 and ANAPC1, regulates the stability of p21WAF1 and the tumour suppressor p53. These combine to regulate the activity of the retinoblastoma protein, Rb, leading to induction of polycomb protein EZH2 expression. Moreover, our ChIP analysis demonstrates that EZH2 is also a direct NF-κB target gene. Microarray analysis revealed that in fibroblasts, EZH2 antagonizes a subset of p53 target genes previously associated with the senescent cell phenotype, including DEK and RacGAP1. We show that this pathway provides the major route of crosstalk between the alternative NF-κB pathway and p53, a consequence of which is to suppress cell senescence. Importantly, we find that activation of NF-κB also induces EZH2 expression in CD40L stimulated cells from Chronic Lymphocytic Leukemia patients. We therefore propose that this pathway provides a mechanism through which microenvironment induced NF-κB can inhibit tumor suppressor function and promote tumorigenesis.     

Keratin 76 Is Required for Tight Junction Function and Maintenance of the Skin Barrier

Keratins are cytoskeletal intermediate filament proteins that are increasingly being recognised for their diverse cellular functions. Here we report the consequences of germ line inactivation of Keratin 76 (Krt76) in mice. Homozygous disruption of this epidermally expressed gene causes neonatal skin flaking, hyperpigmentation, inflammation, impaired wound healing, and death prior to 12 weeks of age. We show that this phenotype is associated with functionally defective tight junctions that are characterised by mislocalization of the integral protein CLDN1. We further demonstrate that KRT76 interacts with CLDN1 and propose that this interaction is necessary to correctly position CLDN1 in tight junctions. The mislocalization of CLDN1 has been associated in various dermopathies, including the inflammatory disease, psoriasis. These observations establish a previously unknown connection between the intermediate filament cytoskeleton network and tight junctions and showcase Krt76 null mice as a possible model to study aberrant tight junction driven skin diseases.     

In Vivo Transplantation of Neurosphere-Like Bodies Derived from the Human Postnatal and Adult Enteric Nervous System: A Pilot Study

Recent advances in the in vitro characterization of human adult enteric neural progenitor cells have opened new possibilities for cell-based therapies in gastrointestinal motility disorders. However, whether these cells are able to integrate within an in vivo gut environment is still unclear. In this study, we transplanted neural progenitor-containing neurosphere-like bodies (NLBs) in a mouse model of hypoganglionosis and analyzed cellular integration of NLB-derived cell types and functional improvement. NLBs were propagated from postnatal and adult human gut tissues. Cells were characterized by immunohistochemistry, quantitative PCR and subtelomere fluorescence in situ hybridization (FISH). For in vivo evaluation, the plexus of murine colon was damaged by the application of cationic surfactant benzalkonium chloride which was followed by the transplantation of NLBs in a fibrin matrix. After 4 weeks, grafted human cells were visualized by combined in situ hybridization (Alu) and immunohistochemistry (PGP9.5, GFAP, SMA). In addition, we determined nitric oxide synthase (NOS)-positive neurons and measured hypertrophic effects in the ENS and musculature. Contractility of treated guts was assessed in organ bath after electrical field stimulation. NLBs could be reproducibly generated without any signs of chromosomal alterations using subtelomere FISH. NLB-derived cells integrated within the host tissue and showed expected differentiated phenotypes i.e. enteric neurons, glia and smooth muscle-like cells following in vivo transplantation. Our data suggest biological effects of the transplanted NLB cells on tissue contractility, although robust statistical results could not be obtained due to the small sample size. Further, it is unclear, which of the NLB cell types including neural progenitors have direct restoring effects or, alternatively may act via ‘bystander’ mechanisms in vivo. Our findings provide further evidence that NLB transplantation can be considered as feasible tool to improve ENS function in a variety of gastrointestinal disorders.     

Spatio-Temporal Variability of Copepod Abundance along the 20°S Monitoring Transect in the Northern Benguela Upwelling System from 2005 to 2011

Long-term data sets are essential to understand climate-induced variability in marine ecosystems. This study provides the first comprehensive analysis of longer-term temporal and spatial variations in zooplankton abundance and copepod community structure in the northern Benguela upwelling system from 2005 to 2011. Samples were collected from the upper 200 m along a transect at 20°S perpendicular to the coast of Namibia to 70 nm offshore. Based on seasonal and interannual trends in surface temperature and salinity, three distinct time periods were discernible with stronger upwelling in spring and extensive warm-water intrusions in late summer, thus, high temperature amplitudes, in the years 2005/06 and 2010/11, and less intensive upwelling followed by weaker warm-water intrusions from 2008/09 to 2009/10. Zooplankton abundance reflected these changes with higher numbers in 2005/06 and 2010/11. In contrast, zooplankton density was lower in 2008/09 and 2009/10, when temperature gradients from spring to late summer were less pronounced. Spatially, copepod abundance tended to be highest between 30 and 60 nautical miles off the coast, coinciding with the shelf break and continental slope. The dominant larger calanoid copepods were Calanoides carinatus, Metridia lucens and Nannocalanus minor. On all three scales studied, i.e. spatially from the coast to offshore waters as well as temporally, both seasonally and interannually, maximum zooplankton abundance was not coupled to the coldest temperature regime, and hence strongest upwelling intensity. Pronounced temperature amplitudes, and therefore strong gradients within a year, were apparently important and resulted in higher zooplankton abundance.     

Dicer Regulates Differentiation and Viability during Mouse Pancreatic Cancer Initiation

miRNA levels are altered in pancreatic ductal adenocarcinoma (PDA), the most common and lethal pancreatic malignancy, and intact miRNA processing is essential for lineage specification during pancreatic development. However, the role of miRNA processing in PDA has not been explored. Here we study the role of miRNA biogenesis in PDA development by deleting the miRNA processing enzyme Dicer in a PDA mouse model driven by oncogenic Kras. We find that loss of Dicer accelerates Kras driven acinar dedifferentiation and acinar to ductal metaplasia (ADM), a process that has been shown to precede and promote the specification of PDA precursors. However, unconstrained ADM also displays high levels of apoptosis. Dicer loss does not accelerate development of Kras driven PDA precursors or PDA, but surprisingly, we observe that mouse PDA can develop without Dicer, although at the expense of proliferative capacity. Our data suggest that intact miRNA processing is involved in both constraining pro-tumorigenic changes in pancreatic differentiation as well as maintaining viability during PDA initiation.     

A Novel High-Resolution Single Locus Sequence Typing Scheme for Mixed Populations of Propionibacterium acnes In Vivo

The Gram-positive anaerobic bacterium Propionibacterium acnes is a prevalent member of the normal skin microbiota of human adults. In addition to its suspected role in acne vulgaris it is involved in a variety of opportunistic infections. Multi-locus sequence-typing (MLST) schemes identified distinct phylotypes associated with health and disease. Being based on 8 to 9 house-keeping genes these MLST schemes have a high discriminatory power, but their application is time- and cost-intensive. Here we describe a single-locus sequence typing (SLST) scheme for P. acnes. The target locus was identified with a genome mining approach that took advantage of the availability of representative genome sequences of all known phylotypes of P. acnes. We applied this SLST on a collection of 188 P. acnes strains and demonstrated a resolution comparable to that of existing MLST schemes. Phylogenetic analysis applied to the SLST locus resulted in clustering patterns identical to a reference tree based on core genome sequences. We further demonstrate that SLST can be applied to detect multiple phylotypes in complex microbial communities by a metagenomic pyrosequencing approach. The described SLST strategy may be applied to any bacterial species with a basically clonal population structure to achieve easy typing and mapping of multiple phylotypes in complex microbiotas. The P. acnes SLST database can be found at http://medbac.dk/slst/pacnes.