Activation of the PI3K/AKT pathway correlates with prognosis in stage II colon cancer.

BACKGROUND: Patients with UICC/AJCC stage II colon cancer have a high 5-year overall survival rate after surgery. Nevertheless, a significant subgroup of patients develops tumour recurrence. Currently, there are no clinically established biomarkers available to identify this patient group. We applied reverse-phase protein arrays (RPPA) for phosphatidylinositide-3-kinase pathway activation mapping to stratify patients according to their risk of tumour recurrence after surgery. METHODS: Full-length proteins were extracted from formalin-fixed, paraffin-embedded tissue samples of 118 patients who underwent curative resection. RPPA technology was used to analyse expression and/or phosphorylation levels of six major factors of the phosphatidylinositide-3-kinase pathway. Oncogenic mutations of KRAS and BRAF, and DNA microsatellite status, currently discussed as prognostic markers, were analysed in parallel. RESULTS: Expression of phospho-AKT (HR=3.52; P=0.032), S6RP (HR=6.3; P=0.044), and phospho-4E-BP1 (HR=4.12; P=0.011) were prognostic factors for disease-free survival. None of the molecular genetic alterations were significantly associated with prognosis. CONCLUSIONS: Our data indicate that activation of the PI3K/AKT pathway evidenced on the protein level might be a valuable prognostic marker to stratify patients for their risk of tumour recurrence. Beside adjuvant chemotherapy targeting of upregulated PI3K/AKT signalling may be an attractive strategy for treatment of high-risk patients.

A rapid ex vivo tissue model for optimising drug detection and ionisation in MALDI imaging studies.

The aim of this study was to establish an ex vivo model for a faster optimisation of sample preparation procedures, for example matrix choice, in matrix-assisted laser desorption/ionisation (MALDI) drug imaging studies. The ionisation properties of four drugs, afatinib, erlotinib, irinotecan and pirfenidone, were determined in an ex vivo tissue experiment by spotting decreasing dilution series onto liver sections. Hereby, the drug signals were distinctly detectable using different matrix compounds, which allowed the selection of the optimal matrix for each drug. The analysis of afatinib and erlotinib yielded high drug signals with α-cyano-4-hydroxycinnamic acid matrix, whereas 2,3-dihydroxybenzoic acid was identified as optimal matrix for irinotecan and pirfenidone detection. Our method was validated by a MALDI drug imaging approach of in vivo treated mouse tissue resulting in corresponding findings, indicating the spotting method as an appropriate approach to determine the matrix of choice. The present study shows the accordance between the detection of ex vivo spotted drugs and in vivo administered drugs by MALDI-TOF and MALDI-FT-ICR imaging, which has not been demonstrated so far. Our data suggest the ex vivo tissue spotting method as an easy and reliable model to optimise MALDI imaging measurements and to predict drug detection in tissue sections derived from treated mice prior to the recruitment of laboratory animals, which helps to save animals, time and costs.

Could hyponatremia be a marker of anastomotic leakage after colorectal surgery? A single center analysis of 1,106 patients over 5 years.

PURPOSE: The aim of this study is to define the significance of hyponatremia as a marker of anastomotic leakage after colorectal surgery. METHODS: All anastomoses in colorectal surgery performed at a single institution between July 2007 and July 2012 (n = 1,106) were retrospectively identified. Serum sodium levels and leukocyte values measured when an anastomotic leak was diagnosed by CT scan and/or surgical reintervention (n = 81) were compared to the values preferably on postoperative day 5 in the absence of an anastomotic leak (n = 1,025). RESULTS: The leak rate in anastomoses of the rectum was 9.0 %, while the leak rate of the other anastomoses was 5.4 %. Mean serum sodium level was 138.8 mmol/l in the group with an anastomotic leak and 140.5 mmol/l in the group without. Hyponatremia (<136 mmol/l) was present in 23 % of patients in the group with an anastomotic leak and in 15 % in the group without (p < 0.001). In multivariate analysis, leukocytes and serum sodium level remained as significant markers of an anastomotic leak. As a marker of an anastomotic leak, hyponatremia had a specificity of 93 % and a sensitivity of 23 %, while the presence of either leukocytosis or hyponatremia had a sensitivity of 68 %, a specificity of 75 %, a positive predictive value of 18 %, and a negative predictive value of 97 %. CONCLUSIONS: Hyponatremia could be a specific and relevant marker of anastomotic leakage after colorectal surgery. If hyponatremia and leukocytosis are present after colorectal surgery, anastomotic leakage should be suspected and a CT scan with rectal contrast dye is recommended.

Specific activity of cyclin-dependent kinase I is a new potential predictor of tumour recurrence in stage II colon cancer.

BACKGROUND: There are no established biomarkers to identify tumour recurrence in stage II colon cancer. As shown previously, the enzymatic activity of the cyclin-dependent kinases 1 and 2 (CDK1 and CDK2) predicts outcome in breast cancer. Therefore, we investigated whether CDK activity identifies tumour recurrence in colon cancer. METHODS: In all, 254 patients with completely resected (R0) UICC stage II colon cancer were analysed retrospectively from two independent cohorts from Munich (Germany) and Leiden (Netherlands). None of the patients received adjuvant treatment. Development of distant metastasis was observed in 27 patients (median follow-up: 86 months). Protein expression and activity of CDKs were measured on fresh-frozen tumour samples. RESULTS: Specific activity (SA) of CDK1 (CDK1SA), but not CDK2, significantly predicted distant metastasis (concordance index=0.69, 95% confidence interval (CI): 0.55-0.79, P=0.036). Cutoff derivation by maximum log-rank statistics yielded a threshold of CDK1SA at 11 (SA units, P=0.029). Accordingly, 59% of patients were classified as high-risk (CDK1SA ≥11). Cox proportional hazard analysis revealed CDK1SA as independent prognostic variable (hazard ratio=6.2, 95% CI: 1.44-26.9, P=0.012). Moreover, CKD1SA was significantly elevated in microsatellite-stable tumours. CONCLUSION: Specific activity of CDK1 is a promising biomarker for metastasis risk in stage II colon cancer.

Enabling fiber optic serotyping of pathogenic bacteria through improved anti-fouling functional surfaces.

Significant research efforts are continually being directed towards the development of sensitive and accurate surface plasmon resonance biosensors for sequence specific DNA detection. These sensors hold great potential for applications in healthcare and diagnostics. However, the performance of these sensors in practical usage scenarios is often limited due to interference from the sample matrix. This work shows how the co-immobilization of glycol(PEG) diluents or 'back filling' of the DNA sensing layer can successfully address these problems. A novel SPR based melting assay is used for the analysis of a synthetic oligomer target as well as PCR amplified genomic DNA extracted from Legionella pneumophila. The benefits of sensing layer back filling on the assay performance are first demonstrated through melting analysis of the oligomer target and it is shown how back filling enables accurate discrimination of Legionella pneumophila serogroups directly from the PCR reaction product with complete suppression of sensor fouling.

Identification of a suppressor of the Dictyostelium profilin-minus phenotype as a CD36/LIMP-II homologue.

Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme-mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type-like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2 terminus and the other at the COOH terminus. It is homologous to mammalian CD36/LIMP-II and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ringlike structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is unclear how far upstream in a regulatory cascade DdLIMP might be positioned. However, the PIP2 binding of DdLIMP points towards a function via the phosphatidylinositol pathway, a major regulator of profilin.

An introduction to optical super-resolution microscopy for the adventurous biologist.

Ever since the inception of light microscopy, the laws of physics have seemingly thwarted every attempt to visualize the processes of life at its most fundamental, sub-cellular, level. The diffraction limit has restricted our view to length scales well above 250 nm and in doing so, severely compromised our ability to gain true insights into many biological systems. Fortunately, continuous advancements in optics, electronics and mathematics have since provided the means to once again make physics work to our advantage. Even though some of the fundamental concepts enabling super-resolution light microscopy have been known for quite some time, practically feasible implementations have long remained elusive. It should therefore not come as a surprise that the 2014 Nobel Prize in Chemistry was awarded to the scientists who, each in their own way, contributed to transforming super-resolution microscopy from a technological tour de force to a staple of the biologist's toolkit. By overcoming the diffraction barrier, light microscopy could once again be established as an indispensable tool in an age where the importance of understanding life at the molecular level cannot be overstated. This review strives to provide the aspiring life science researcher with an introduction to optical microscopy, starting from the fundamental concepts governing compound and fluorescent confocal microscopy to the current state-of-the-art of super-resolution microscopy techniques and their applications.

Inactivation of lmpA, encoding a LIMPII-related endosomal protein, suppresses the internalization and endosomal trafficking defects in profilin-null mutants.

Profilin is a key phosphoinositide and actin-binding protein connecting and coordinating changes in signal transduction pathways with alterations in the actin cytoskeleton. Using biochemical assays and microscopic approaches, we demonstrate that profilin-null cells are defective in macropinocytosis, fluid phase efflux, and secretion of lysosomal enzymes but are unexpectedly more efficient in phagocytosis than wild-type cells. Disruption of the lmpA gene encoding a protein (DdLIMP) belonging to the CD36/LIMPII family suppressed, to different degrees, most of the profilin-minus defects, including the increase in F-actin, but did not rescue the secretion defect. Immunofluorescence microscopy indicated that DdLIMP, which is also capable of binding phosphoinositides, was associated with macropinosomes but was not detected in the plasma membrane. Also, inactivation of the lmpA gene in wild-type strains resulted in defects in macropinocytosis and fluid phase efflux but not in phagocytosis. These results suggest an important role for profilin in regulating the internalization of fluid and particles and the movement of material along the endosomal pathway; they also demonstrate a functional interaction between profilin and DdLIMP that may connect phosphoinositide-based signaling through the actin cytoskeleton with endolysosomal membrane trafficking events.

Highly controllable direct femtosecond laser writing of gold nanostructures on titanium dioxide surfaces.

A highly reproducible and controllable deposition procedure for gold nanostructures on a titanium dioxide (TiO2) surface using femtosecond laser light has been demonstrated. This is realized by precisely focusing onto the TiO2 surface in the presence of a pure gold ion solution. The deposition is demonstrated both in dot arrays and line structures. Thanks to the multi-photon excitation, we observe that the deposition area of the nanostructures can be confined to a degree far greater than the diffraction limited focal spot. Finally, we demonstrate that catalytic activity with visible light irradiation is enhanced, proving the applicability of our new deposition technique to the catalytic field.

A porcine model of osteosarcoma.

We previously produced pigs with a latent oncogenic TP53 mutation. Humans with TP53 germline mutations are predisposed to a wide spectrum of early-onset cancers, predominantly breast, brain, adrenal gland cancer, soft tissue sarcomas and osteosarcomas. Loss of p53 function has been observed in >50% of human cancers. Here we demonstrate that porcine mesenchymal stem cells (MSCs) convert to a transformed phenotype after activation of latent oncogenic TP53(R167H) and KRAS(G12D), and overexpression of MYC promotes tumorigenesis. The process mimics key molecular aspects of human sarcomagenesis. Transformed porcine MSCs exhibit genomic instability, with complex karyotypes, and develop into sarcomas on transplantation into immune-deficient mice. In pigs, heterozygous knockout of TP53 was sufficient for spontaneous osteosarcoma development in older animals, whereas homozygous TP53 knockout resulted in multiple large osteosarcomas in 7-8-month-old animals. This is the first report that engineered mutation of an endogenous tumour-suppressor gene leads to invasive cancer in pigs. Unlike in Trp53 mutant mice, osteosarcoma developed in the long bones and skull, closely recapitulating the human disease. These animals thus promise a model for juvenile osteosarcoma, a relatively uncommon but devastating disease.

Dictyostelium discoideum: a genetic model system for the study of professional phagocytes. Profilin, phosphoinositides and the lmp gene family in Dictyostelium.

Profilin is a key regulator of actin polymerization, and plays a pivotal role at the interface of the phosphoinositide signal transduction pathway and the cytoskeleton. Recent evidence suggests the involvement of profilin in the regulation of phagocytosis and macropinocytosis, and the transport along the endosomal pathway. Disruption of profilin leads to a complex phenotype that includes abnormal cytokinesis, a block in development and defects in the endosomal pathway. Macropinocytosis, fluid phase efflux and secretion of lysosomal enzymes were reduced, whereas the rate of phagocytosis was increased as compared to wild-type cells. The lmpA gene, a homolog of the CD36/LIMPII family, was identified as a suppressor for most of the profilin-minus defects. This gene encodes an integral membrane protein, it localizes to lysosomes and macropinosomes, and binds to phosphoinositides. Even though phosphatidylinositol lipids constitute only a small fraction of total lipids in the membranes of eukaryotic cells, they play an important role in vesicle transport, signal transduction and cytoskeletal regulation. Disruption of lmpA in wild-type cells resulted in defects in fluid phase efflux and macropinocytosis, but not in phagocytosis. The discovery and initial characterization of two additional members of the CD36/LIMPII family in Dictyostelium, lmpB and lmpC, that exhibit intriguing differences in developmental regulation and their putative sorting signals, suggests that a set of lysosomal integral membrane proteins contribute to the crosstalk between vesicles and cytoskeletal proteins.

Cloning and characterization of beta-COP from Dictyostelium discoideum.

We have isolated a cDNA coding for beta-COP from Dictyostelium discoideum by polymerase chain reaction using degenerate primers derived from rat beta-COP. The complete cDNA clone has a size of 2.8 kb and codes for a protein with a calculated molecular mass of 102 kDa. Dictyostelium beta-COP exhibits highest homology to mammalian beta-COP, but it is considerably smaller due to a shortened variable region that is thought to form a linker between the highly conserved N- and C-terminal domains. Dictyostelium beta-COP is encoded by a single gene, which is transcribed at moderate levels into two RNAs that are present throughout development. To localize the protein, full-length beta-COP was fused to GFP and expressed in Dictyostelium cells. The fusion protein was detected on vesicles distributed all over the cells and was strongly enriched in the perinuclear region. Based on coimmunofluorescence studies with antibodies directed against the Golgi marker comitin, this compartment was identified as the Golgi apparatus. Beta-COP distribution in Dictyostelium was not brefeldin A sensitive being most likely due to the presence of a brefeldin A resistance gene. However, upon DMSO treatment we observed a reversible disassembly of the Golgi apparatus. In mammalian cells DMSO treatment had a similar effect on beta-COP distribution.

Truncation mutagenesis of the non-alpha-helical carboxyterminal tail domain of vimentin reveals contributions to cellular localization but not to filament assembly.

We have investigated the effect of stepwise truncating the carboxyterminal domain ("tail") of the intermediate filament (IF) protein vimentin of the clawed toad, Xenopus laevis, on filament assembly in vitro and, using cell transfection, in vivo and also on the cellular topology of the structures formed. All truncations examined, except the minimal one missing the last 11 amino acids which made the protein more sensitive to changes of ionic strength, did not significantly alter IF assembly in vitro, as judged by electron microscopy, viscometry and determination of viscoelastic properties with a laser-operated torsion pendulum. Stable transfections of vimentin-free mammalian cells with cDNAs encoding these mutations resulted at 28 degrees C, i.e. the permissive temperature for assembly of Xenopus vimentin, in the formation of extended IF bundle arrays. At 37 degrees C, however, the mutants lacking more than the last 35 amino acids could leave the cytoplasm and accumulated in the nucleus, indicating a certain topogenic element is located in the tail and directs cytoplasmic restriction in the wild-type protein although this does not form IFs under these conditions. Transfer to the nucleus is, however, abolished if the IF-consensus motif at the end of the rod domain is removed, suggesting that this part of the molecule also contributes to nuclear location. Similar results were obtained with human vimentin: While the rod entered the nucleus, headless vimentin, unable to form IFs, remained restricted to the cytoplasm owing to its tail domain. In contrast, tailless human vimentin and tailless mouse desmin, which are fully assembly-competent in vitro, both formed extensive IF arrays in the cytoplasm but did not accumulate in the nucleus. We conclude that in class III IF proteins stepwise deletions in the tail, while not considerably altering IF assembly in vitro, can change the topogenesis of IF proteins and structures in the living cell.

Expression of the crustacean hyperglycaemic hormones and the gonad-inhibiting hormone during the reproductive cycle of the female American lobster Homarus americanus.

Crustacean reproduction is regulated by a complex chain of hormonal interactions in which the crustacean hyperglycaemic hormones A and B (CHH-A and CHH-B) and the gonad-inhibiting hormone (GIH) play a primary role. These neurohormones are produced in the same neuroendocrine cells of the X-organ sinus gland complex, situated in the eyestalks of the American lobster, Homarus americanus. In order to obtain more information on the synthesis, storage, release and function of these three neuropeptides during the reproductive cycle, we studied the levels of their mRNAs in the X-organ, their peptide storage in the sinus gland and their concentration in the haemolymph at different stages of the female reproductive cycle. A high CHH-A mRNA level was found only in the previtellogenic stage, while elevated mRNA levels were determined for CHH-B in the mature as well as the previtellogenic stage. High CHH storage levels in the sinus gland were found during previtellogenesis. The total amount of CHH (CHH-A plus -B) in the haemolymph was significantly higher during maturation. A low level of GIH mRNA in the X-organ and a low amount of the GIH I isoform in the sinus gland were found only in the immature stage. In contrast, GIH haemolymph levels were high during the immature and previtellogenic stages. We conclude that CHH-A and -B are involved in triggering the onset of vitellogenesis and that CHH-B in particular is responsible for stimulating oocyte maturation before spawning, while GIH prevents the start of vitellogenesis in the ovary. Moreover, our results show that the balance between the haemolymph levels of the CHHs and GIH may tune the synchronization of reproduction and molting during the biannual reproductive cycle of the American lobster.

Comparative characterization of hyperglycemic neuropeptides from the lobster Homarus americanus.

With the use of a two-step HPLC purification procedure, two sets of two isoforms of the crustacean hyperglycemic hormone (CHH) were isolated from sinus glands of the lobster Homarus americanus. Structural differences between the two groups of isoforms were found in their amino acid sequences, amino acid compositions and precise molecular weights. Using peptide mapping, the difference between the isoforms in each group was located within the first eight amino acids at the N-termini. The nature of this difference remained unclear as all four peptides had the same N-terminal amino acid sequence unto residue 19.

Isolation and amino acid sequence of crustacean hyperglycemic hormone precursor-related peptides.

The crustacean hyperglycemic hormone (CHH) is synthesized as part of a larger preprohormone in which the sequence of CHH is N-terminally flanked by a peptide for which the name CPRP (CHH precursor-related peptide) is proposed. Both CHH and CPRP are present in the sinus gland, the neurohemal organ of neurosecretory cells located in the eyestalk of decapod crustaceans. This paper describes the isolation and sequence analysis of CPRPs isolated from sinus glands of the crab Carcinus maenas, the crayfish Orconectes limosus and the lobster Homarus americanus. The published sequence of "peptide H" isolated from the land crab, Cardisoma carnifex, has now been recognized as a CPRP in this species. Sequence comparison reveals a high level of identity for the N-terminal region (residues 1-13) between all four peptides, while identity in the C-terminal domain is high between lobster and crayfish CPRP on the one hand, and between both crab species on the other. Conserved N-terminal residues include a putative monobasic processing site at position 11, which suggests that CPRP may be a biosynthetic intermediate from which a potentially bioactive decapeptide can be derived.

Automated nanoflow liquid chromatography-tandem mass spectrometry for a differential display proteomic study on Xenopus laevis neuroendocrine cells.

Many proteomic projects based on a comparison of protein profiles displayed on two-dimensional polyacrylamide gel electrophoresis rely on the identification of these proteins using peptide mass fingerprinting on a matrix-assisted laser desorption/ionization mass spectrometer after tryptic digestion. However, this approach is limited to an organism of which genomic information is largely available, i.e. when the total genome sequence is known. For other organisms, mass spectrometric sequence analysis is necessary for protein identification. We established a nano-LC-MS-MS system based on a quadrupole time-of-flight mass spectrometer, which allows automated sequence analysis of tryptic digestion mixtures from single gel spots. This system is applied in a differential-display proteomic study to identify differentially expressed proteins in the neuroendocrine cells of the neurointermediate pituitary of black- and white-background adapted Xenopus laevis.

Thyroid function and deiodinase activities in rats with marginal iodine deficiency.

The hypothesis tested was whether marginal iodine deficiency for a period of 6 wk affects iodothyronine deiodinase activities in liver and brain of rats. Male rats were fed purified diets either deficient or sufficient in iodine; the diets were fed on a restricted basis (60% of ad libitum intake). Body weight gain of the two groups was comparable. Iodine deficiency was evidenced by increased thyroid weight (26%), reduced urinary iodine excretion (80%), and reduced plasma T4 concentrations (22%). Activities of liver type I and brain type III deiodinase were unchanged, but the activity of type II deiodinase in brain was increased (28%) in the iodine-deficient rats. Food restriction per se significantly lowered T3 (30%) and T4 (22%) concentrations in plasma and decreased type III deiodinase activity in brain (30%). These results indicate that in marginal iodine deficiency the activities of hepatic type I deiodinase and brain type III deiodinase are unchanged, whereas that of brain type II deiodinase is increased.

C/EBP homologous protein inhibits tissue repair in response to gut injury and is inversely regulated with chronic inflammation.

Loss of intestinal epithelial cell (IEC) homeostasis and apoptosis negatively affect intestinal barrier function. Uncontrolled activation of the unfolded protein response (UPR) in IEC contributes to an impaired barrier and is implicated in the pathogenesis of inflammatory bowel diseases. However, the contribution of the UPR target gene C/EBP homologous protein (CHOP), an apoptosis-associated transcription factor, to inflammation-related disease susceptibility remains unclear. Consistent with observations in patients with ulcerative colitis, we show that despite UPR activation in the epithelium, CHOP expression was reduced in mouse models of T-cell-mediated and bacteria-driven colitis. To elucidate the molecular mechanisms of IEC-specific CHOP expression, we generated a conditional transgenic mouse model (Chop(IEC Tg/Tg)). Chop overexpression increased the susceptibility toward dextran sodium sulfate (DSS)-induced intestinal inflammation and mucosal tissue injury. Furthermore, a delayed recovery from DSS-induced colitis and impaired closure of mechanically induced mucosal wounds was observed. Interestingly, these findings seemed to be independent of CHOP-mediated apoptosis. In vitro and in vivo cell cycle analyses rather indicated a role for CHOP in epithelial cell proliferation. In conclusion, these data show that IEC-specific overexpression impairs epithelial cell proliferation and mucosal tissue regeneration, suggesting an important role for CHOP beyond mediating apoptosis.

Fast and accurate peanut allergen detection with nanobead enhanced optical fiber SPR biosensor.

This paper is the first report of a fiber optic SPR biosensor with nanobead signal enhancement. We evaluated the system with a bioassay for the fast and accurate detection of peanut allergens in complex food matrices. Three approaches of an immunoassay to detect Ara h1 peanut allergens in chocolate candy bars were compared; a label-free assay, a secondary antibody sandwich assay and a nanobead enhanced assay. Although label-free detection is the most convenient, our results illustrate that functionalized nanobeads can offer a refined solution to improve the fiber SPR detection limit. By applying magnetite nanoparticles as a secondary label, the detection limit of the SPR bioassay for Ara h1 was improved by two orders of magnitude from 9 to 0.09 µg/mL. The super paramagnetic character of the nanoparticles ensured easy handling. The SPR fibers could be regenerated easily and one fiber could be reused for up to 35 times without loss of sensitivity. The results were benchmarked against a commercially available polyclonal ELISA kit. An excellent correlation was found between the Ara h1 concentrations obtained with the ELISA and the concentrations measured with the SPR fiber assay. In addition, with the SPR fiber we could measure the samples twice as fast as compared to the fastest ELISA protocol. Since the dipstick fiber has no need for microchannels that can become clogged, time consuming rinsing step could be avoided. The linear dynamic range of the presented sensor was between 0.1 and 2 µg/mL, which is considerably larger than the ELISA benchmark.