Identification of PSME3 as a novel serum tumor marker for colorectal cancer by combining two-dimensional polyacrylamide gel electrophoresis with a strictly mass spectrometry-based approach for data analysis

The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC). Proteins from matched CRC and adjacent normal tissue samples were resolved by two-dimensional gel electrophoresis. From each gel all spots were excised, and enveloped proteins were identified by MS. By comparison of the resulting protein profiles, dysregulated proteins can be identified. A list of all identified proteins and validation of five exemplarily selected proteins, elevated in CRC was reported previously (Roessler, M., Rollinger, W., Palme, S., Hagmann, M. L., Berndt, P., Engel, A. M., Schneidinger, B., Pfeffer, M., Andres, H., Karl, J., Bodenmuller, H., Ruschoff, J., Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W., and Tacke, M. (2005) Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin. Cancer Res. 11, 6550-6557). Here we describe identification and initial validation of another potential marker protein for CRC. Comparison of tissue protein profiles revealed strong elevation of proteasome activator complex subunit 3 (PSME3) expression in CRC tissue. This dysregulation was not detectable based on the spot pattern. The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.     

Endothelial toll‐like receptor 4 maintains lung integrity via epigenetic suppression of p16INK4a

We previously reported that the canonical innate immune receptor toll‐like receptor 4 (TLR4) is critical in maintaining lung integrity. However, the molecular mechanisms via which TLR4 mediates its effect remained unclear. In the present study, we identified distinct contributions of lung endothelial cells (Ec) and epithelial cells TLR4 to pulmonary homeostasis using genetic‐specific, lung‐ and cell‐targeted in vivo methods. Emphysema was significantly prevented via the reconstituting of human TLR4 expression in the lung Ec of TLR4?/? mice. Lung Ec‐silencing of TLR4 in wild‐type mice induced emphysema, highlighting the specific and distinct role of Ec‐expressed TLR4 in maintaining lung integrity. We also identified a previously unrecognized role of TLR4 in preventing expression of p16^INK4a, a senescence‐associated gene. Lung Ec‐p16^INK4a‐silencing prevented TLR4?/? induced emphysema, revealing a new functional role for p16^INK4ain lungs. TLR4 suppressed endogenous p16^INK4a expression via HDAC2‐mediated deacetylation of histone H4. These findings suggest a novel role for TLR4 in maintaining of lung homeostasis via epigenetic regulation of senescence‐related gene expression.     

Highly conserved piggyBac elements in noctuid species of Lepidoptera

The piggyBac transposable element was originally discovered in a Trichoplusia ni cell line and nearly identical elements were subsequently discovered in the tephritid fly, Bactrocera dorsalis. This suggested the existence of piggyBac in additional insects and this study shows highly conserved, though not identical, piggyBac sequences in the noctuid species Heliocoverpa armigera, H. zea, and Spodoptera frugiperda, as well as new piggyBac sequences from the T. ni organismal genome. Genomic piggyBac elements could not be unambiguously identified in several other moth species indicating a discontinuous presence of piggyBac in the Lepidoptera. Most sequences have greater than 95% nucleotide identity to the original IFP2 piggyBac, except for a more diverged sequence in S. frugiperda, having approximately 78% identity. Variants of 1.3 and 0.8kb sequences found in both H. armigera and H. zea most likely became established by interbreeding, supporting the notion that the species are conspecific. None of the independent piggyBac sequences isolated from T. ni larval genomes are identical to IFP2, though all have an uninterrupted reading frame with the potential for encoding a functional transposase. The piggyBac sequences from T. ni and the Helicoverpa species, as well as those previously reported from B. dorsalis, all share three common nucleotide substitutions resulting in a single amino acid substitution in the transposase. This suggests that the original IFP2 piggyBac is a related variant of a predecessor element that became widespread. The existence of conserved piggyBac elements, some of which may have been transmitted horizontally between lepidopteran species, raises important considerations for the stability and practical use of piggyBac transformation vectors.     

Potentially Modifiable Factors Associated with Death of Infants and Children with Severe Pneumonia Routinely Managed in District Hospitals in Malawi

Objective

To investigate recognised co-morbidities and clinical management associated with inpatient pneumonia mortality in Malawian district hospitals.

Methods

Prospective cohort study, of patient records, carried out in Malawi between 1^st October 2000 and 30^th June 2003. The study included all children aged 0-59 months admitted to the paediatric wards in sixteen district hospitals throughout Malawi with severe and very severe pneumonia. We compared individual factors between those that survived (n = 14 076) and those that died (n = 1 633).

Results

From logistic regression analysis, predictors of death in hospital, adjusted for age, sex and severity grade included comorbid conditions of meningitis (OR =2.49, 95% CI 1.50-4.15), malnutrition (OR =2.37, 95% CI 1.94-2.88) and severe anaemia (OR =1.41, 95% CI 1.03-1.92). Requiring supplementary oxygen (OR =2.16, 95% CI 1.85-2.51) and intravenous fluids (OR =3.02, 95% CI 2.13-4.28) were associated with death while blood transfusion was no longer significant (OR =1.10, 95% CI 0.77-1.57) when the model included severe anaemia.

Conclusions

This study identified a number of challenges to improve outcome for Malawian infants and children hospitalised with pneumonia. These included improved assessment of co-morbidities and more rigorous application of standard case management.     

A comprehensive proteome map of the lipid-requiring nosocomial pathogen Corynebacterium jeikeium K411

Corynebacterium jeikeium is a lipid-requiring pathogen that is considered as part of the normal microflora of the human skin and associated with severe nosocomial infections. Systematic reference maps of the cytoplasmic, cell surface-associated, and extracellular proteome fractions of the clinical isolate C. jeikeium K411 were examined by 2-DE coupled with MALDI-TOF MS. A sum total of 555 protein spots were identified by PMF, corresponding to 358 different proteins that were classified into functional categories and integrated into metabolic pathways. The majority of the proteins were linked to housekeeping functions in energy production and translation and to physiological processes in amino acid, carbohydrate, nucleotide, and lipid metabolism. A complete enzymatic machinery necessary to utilize exogenous fatty acids by beta-oxidation was detected in the cytoplasmic proteome fraction. In addition, several predicted virulence factors of C. jeikeium K411 were identified in the cell surface-associated and extracellular subproteome, including the cell surface proteins SurA and SurB, the surface-anchored pilus subunits SapA and SapB, the surface-anchored collagen adhesin CbpA, the cholesterol esterase Che, and the acid phosphatase AcpA.     

The oncogenic role of Epstein–Barr virus‐encoded microRNAs in Epstein–Barr virus‐associated gastric carcinoma

Epstein–Barr virus (EBV) infection is detected in various epithelial malignancies, such as nasopharyngeal carcinoma (NPC) and gastric cancer (GC). EBV comprises some unique molecular features and encodes viral genes and microRNAs (miRNAs) by its own DNA sequence. EBV genes are required to maintain latency and contribute to oncogenic property. miRNAs encoded by EBV have been shown to contribute to initiation and progression of EBV‐related malignancies. By a number of genomic profiling studies, some EBV miRNAs were confirmed to be highly expressed in EBV‐associated gastric cancer (EBVaGC) samples and cell lines. The majority host targets of the EBV miRNAs are important for promoting cell growth and inhibiting apoptosis, facilitating cell survival and immune evasion. However, the integrated molecular mechanisms related to EBV miRNAs remain to be investigated. In this review, we summarized the crucial role of EBV miRNAs in epithelial malignancies, especially in EBVaGC. Collectively, EBV miRNAs play a significant role in the viral and host gene regulation network. Understanding the comprehensive potential targets and relevant functions of EBV miRNAs in gastric carcinogenesis might provide better clinical translation.     

Foraging in surfperches: resource partitioning or individualistic responses?

Synopsis On Naples Reef off southern California, five sympatric species of surfperch (Embiotocidae) eat small animal prey associated with algal turf and other benthos. Although they tend to select different foods in different microhabitats, their different foraging traits are probably individualistic responses (non-interactive or autecological species responses to resources) because the species' pairwise resource overlaps are neither uniformly small nor generally complementary. Mean overlap in both food and foraging microhabitat was significantly greater than random as most species converged in resource use. Only two exploiters of extremes in the available spectra of prey types and microhabitats had significantly narrow overlap. Thus, resource overlaps may simply reflect similarities and differences in the species' independently evolved foraging traits, which were rendered more or less inflexible as different morphological specializations for picking, crunching, or winnowing prey. Therefore, any one species may occur about the reef to its best advantage independently of the others. Its distribution and abundance may simply reflect its tolerance of the local environment. The evidence indicates that the surfperches are not concertedly partitioning resources on Naples Reef, i.e., that the five species had not all coevolved to avoid interspecific competition in the past nor have they all mutually shifted their niches to minimize it in the present.     

An Implantable Vascularized Protein Gel Construct That Supports Human Fetal Hepatoblast Survival and Infection by Hepatitis C Virus in Mice

Background

Widely accessible small animal models suitable for the study of hepatitis C virus (HCV) in vivo are lacking, primarily because rodent hepatocytes cannot be productively infected and because human hepatocytes are not easily engrafted in immunodeficient mice.

Methodology/Principal Findings

We report here on a novel approach for human hepatocyte engraftment that involves subcutaneous implantation of primary human fetal hepatoblasts (HFH) within a vascularized rat collagen type I/human fibronectin (rCI/hFN) gel containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) in severe combined immunodeficient X beige (SCID/bg) mice. Maturing hepatic epithelial cells in HFH/Bcl-2-HUVEC co-implants displayed endocytotic activity at the basolateral surface, canalicular microvilli and apical tight junctions between adjacent cells assessed by transmission electron microscopy. Some primary HFH, but not Huh-7.5 hepatoma cells, appeared to differentiate towards a cholangiocyte lineage within the gels, based on histological appearance and cytokeratin 7 (CK7) mRNA and protein expression. Levels of human albumin and hepatic nuclear factor 4α (HNF4α) mRNA expression in gel implants and plasma human albumin levels in mice engrafted with HFH and Bcl-2-HUVEC were somewhat enhanced by including murine liver-like basement membrane (mLBM) components and/or hepatocyte growth factor (HGF)-HUVEC within the gel matrix. Following ex vivo viral adsorption, both HFH/Bcl-2-HUVEC and Huh-7.5/Bcl-2-HUVEC co-implants sustained HCV Jc1 infection for at least 2 weeks in vivo, based on qRT-PCR and immunoelectron microscopic (IEM) analyses of gel tissue.

Conclusion/Significance

The system described here thus provides the basis for a simple and robust small animal model of HFH engraftment that is applicable to the study of HCV infections in vivo.     

Molecular and microscopic analysis of the gut contents of abundant rove beetle species (Coleoptera,?Staphylinidae) in the boreal balsam fir forest of Quebec,?Canada

Experimental research on beetle responses to removal of logging residues following clearcut harvesting in the boreal balsam fir forest of Quebec revealed several abundant rove beetle (Staphylinidae) species potentially important for long-term monitoring. To understand the trophic affiliations of these species in forest ecosystems, it was necessary to analyze their gut contents. We used microscopic and molecular (DNA) methods to identify the gut contents of the following rove beetles: Atheta capsularis Klimaszewski, Atheta klagesi Bernhauer, Oxypoda grandipennis (Casey), Bryophacis smetanai Campbell, Ischnosoma longicorne (Mäklin), Mycetoporus montanus Luze, Tachinus frigidus Erichson, Tachinus fumipennis (Say), Tachinus quebecensis Robert, and Pseudopsis subulata Herman. We found no apparent arthropod fragments within the guts; however, a number of fungi were identified by DNA sequences, including filamentous fungi and budding yeasts [Ascomycota: Candida derodonti Suh & Blackwell (accession number FJ623605), Candida mesenterica (Geiger) Diddens & Lodder (accession number FM178362), Candida railenensis Ramirez and Gonzáles (accession number JX455763), Candida sophie-reginae Ramirez & González (accession number HQ652073), Candida sp. (accession number AY498864), Pichia delftensis Beech (accession number AY923246), Pichia membranifaciens Hansen (accession number JQ26345), Pichia misumaiensis Y. Sasaki and Tak. Yoshida ex Kurtzman 2000 (accession number U73581), Pichia sp. (accession number AM261630), Cladosporium sp. (accession number KF367501), Acremoniumpsammosporum W. Gams (accession number GU566287), Alternaria sp. (accession number GU584946), Aspergillus versicolor Bubak (accession number AJ937750), and Aspergillusamstelodami (L. Mangin) Thom and Church (accession number HQ728257)]. In addition, two species of bacteria [Bradyrhizobium japonicum (Kirchner) Jordan (accession number BA000040) and Serratia marcescens Bizio accession number CP003942] were found in the guts. These results not only provide evidence of the consumer-resource relations of these beetles but also clarify the relationship between rove beetles, woody debris and fungi. Predominance of yeast-feeding by abundant rove beetles suggests that it may play an important role in their dietary requirements.