DICER1 Hotspot Mutations in Pleuropulmonary Blastoma: A Case Series From a Tertiary Center.

Pleuropulmonary blastoma (PPB) is a potentially aggressive, rare childhood neoplasia. We investigated histopathological features, survival, and DICER1 hotspot mutations among PPB patients. Archive records at our institution were reviewed, covering a 20-year period. Thirteen children (6 males and 7 females) with a mean age of 30.5 (range 6-83) months were included. The tumor subtypes were type I in 6 (46%), type II in 4 (31%), and type III in 3 (23%). Only tumors with type II and type III histology showed anaplasia (4/7, 57%). Median follow-up was 28 (range 9-216) months. Three-year overall survival rate was 83.3% and 3-year progression-free survival rate was 25%. Progression was seen in 60% (3/5) of type I and 66.7% (4/6) of type II and type III cases. Two patients died of disseminated disease at 9 and 44 months. Hotspot missense mutations on DICER1 gene were detected in all 11 patients with available tumor tissue. We found an additional novel germline loss-of-function mutation (c.5436dupT; p.E1813*) in 1 case. To the best of our knowledge, this is the first study to investigate hotspot missense mutations on DICER1 gene among the largest series of Turkish children with PPB.

Sex-specific genotype-by-environment interactions for cuticular hydrocarbon expression in decorated crickets, Gryllodes sigillatus: implications for the evolution of signal reliability.

Phenotypic traits that convey information about individual identity or quality are important in animal social interactions, and the degree to which such traits are influenced by environmental variation can have profound effects on the reliability of these cues. Using inbred genetic lines of the decorated cricket, Gryllodes sigillatus, we manipulated diet quality to test how the cuticular hydrocarbon (CHC) profiles of males and females respond across two different nutritional rearing environments. There were significant differences between lines in the CHC profiles of females, but the effect of diet was not quite statistically significant. There was no significant genotype-by-environment interaction (GEI), suggesting that environmental effects on phenotypic variation in female CHCs are independent of genotype. There was, however, a significant effect of GEI for males, with changes in both signal quantity and content, suggesting that environmental effects on phenotypic expression of male CHCs are dependent on genotype. The differential response of male and female CHC expression to variation in the nutritional environment suggests that these chemical cues may be under sex-specific selection for signal reliability. Female CHCs show the characteristics of reliable cues of identity: high genetic variability, low condition dependence and a high degree of genetic determination. This supports earlier work showing that female CHCs are used in self-recognition to identify previous mates and facilitate polyandry. In contrast, male CHCs show the characteristics of reliable cues of quality: condition dependence and a relatively higher degree of environmental determination. This suggests that male CHCs are likely to function as cues of underlying quality during mate choice and/or male dominance interactions.

Induction of a melanoma-specific antibody response by a monovalent, but not a divalent, synthetic GM2 neoglycopeptide.

The GM2 ganglioside represents an important target for specific anticancer immunotherapy. We designed and synthesized a neoglycopeptide immunogen displaying one or two copies of the GM2 tetrasaccharidic moiety. These glycopeptides were prepared using the Huisgen cycloaddition, which enables the efficient ligation of the alkyne-functionalized biosynthesized GM2 with an azido CD4(+) T cell epitope peptide. It is worth noting that the GM2 can be produced on a gram scale in bacteria, which can be advantageous for a scale-up of the process. We show here for the first time that a fully synthetic glycopeptide, which is based on a ganglioside carbohydrate moiety, can induce human tumor cell-specific antibodies after immunization in mice. Interestingly, the monovalent, but not the divalent, form of GM2 peptide construct induced antimelanoma antibodies. Unlike traditional vaccines, this vaccine is a pure chemically-defined entity, a key quality for consistent studies and safe clinical evaluation. Therefore, such carbohydrate-peptide conjugate represents a promising cancer vaccine strategy for active immunotherapy targeting gangliosides.

Toxicity of urban highway runoff with respect to storm duration.

The toxicity of stormwater runoff during various time-based stages was measured in both grab and composite samples collected from three highly urbanized highway sites in Los Angeles, California between 2002 and 2005. Stormwater runoff samples were tested for toxicity using three freshwater species (the water flea Ceriodaphnia dubia, the fathead minnow Pimephales promelas, and the green algae Pseudokirchneriella subcapitatum) and two marine species (the purple sea urchin Strongylocentrotus purpuratus, and the luminescent bacteria Photobacterium phosphoreum using Microtox. Toxicity results varied substantially throughout the storm events for both freshwater and marine species toxicity tests. In general, however, the first few samples were found to be more toxic compared with those collected during later stages of each storm event. In most cases, more than 40% of the toxicity was associated with the first 20% of discharged runoff volume. Furthermore, on average, 90% of the toxicity was observed during the first 30% of storm duration. Toxicity identification evaluation results found copper and zinc to be the primary cause of toxicity in about 90% of the samples evaluated with these procedures. Surfactants were also found to be the cause of toxicity in less than 10% of the samples.

Effects of dietary selenomethionine on larval rainbow trout (Oncorhynchus mykiss).

Increased selenium (Se) concentrations in water (>10 microg/L) have been measured in the San Diego Creek, which is a tributary of the Upper Newport Bay in Orange County, CA. The objective of this study was to develop tissue- and dietary-based thresholds for Se in resident fish species in San Diego Creek. A 90-day dietary experiment was conducted to determine the effects of seleno-L-methionine (SeMe) on the growth, survival, and whole-body Se accumulation in larval (24-day-old) rainbow trout. Decreased and oxidized glutathione (GSH-to-GSSG ratio) and thiobarbituric acid-reactive substances (TBARS) were also measured in livers of exposed animals to assess oxidative damage caused by Se. Fish food was spiked with SeMe to contain 4.6, 12, and 18 microg/g (dry weight) of Se. Fish exposed to SeMe for 90 days exhibited a significant decrease in body weight and fork length in the 4.6 and 12 microg/g Se treatments compared with controls. Whole-body total Se concentrations increased significantly in fish fed 12 and 18 microg/g SeMe after 90 days compared with controls. Lipid peroxidation (TBARS) and GSH-to-GSSG ratios were unchanged by SeMe treatment. Based on decreased growth after 90 days, a dietary Se lowest observed-effect concentration (LOEC) value of 4.6 microg/g and a Se body burden LOEC of 1.20 microg/g (wet weight) were estimated.

Toxicity of parking lot runoff after application of simulated rainfall.

Stormwater runoff is an important source of toxic substances to the marine environment, but the effects of antecedent dry period, rainfall intensity, and duration on the toxicity of runoff are not well understood. In this study, simulated rainfall was applied to parking lots to examine the toxicity of runoff while controlling for antecedent period, intensity, and duration of rainfall. Parking areas were divided into high and low use and maintained and unmaintained treatments. The parking stalls were cleaned by pressure washing at time zero. Simulated rainfall was then applied to subplots of the parking lots so that antecedent periods of 1, 2, and 3 months were achieved, and all of the runoff was collected for analysis. On a separate parking lot, rainfall was applied at a variety of intensities and durations after a 3-month antecedent period. Runoff samples were tested for toxicity using the purple sea urchin fertilization test. Every runoff sample tested was found to be toxic. Mean toxicity for the sea urchin fertilization test ranged from 2.0 to 12.1 acute toxic units. The toxicity increased rapidly during the first month but then decreased approximately to precleaning levels and remained there. No difference in toxicity was found between the different levels of use or maintenance treatments. The intensity and duration of rainfall were inversely related to degree of toxicity. For all intensities tested, toxicity was always greatest in the first sampling time interval. Dissolved zinc was most likely the primary cause of toxicity based on toxicant characterization of selected runoff samples.

Temporal aggregation bias and inference of causal regulatory networks.

Time course experiments with microarrays have begun to provide a glimpse into the dynamic behavior of gene expression. In a typical experiment, scientists use microarrays to measure the abundance of mRNA at discrete time points after the onset of a stimulus. Recently, there has been much work on using these data to infer causal regulatory networks that model how genes influence each other. However, microarray studies typically have slow sampling rates that can lead to temporal aggregation of the signal. That is, each successive sampling point represents the sum of all signal changes since the previous sample. In this paper, we show that temporal aggregation can bias algorithms for causal inference and lead them to discover spurious relations that would not be found if the signal were sampled at a much faster rate. We discuss the implications of temporal aggregation on inference, the problems it creates, and potential directions for solutions.

Induction of carbohydrate-specific antibodies in HLA-DR transgenic mice by a synthetic glycopeptide: a potential anti cancer vaccine for human use.

Over the last few years, anticancer immunotherapy has emerged as a new exciting area for controlling tumors. In particular, vaccination using synthetic tumor-associated antigens (TAA), such as carbohydrate antigens hold promise for generating a specific antitumor response by targeting the immune system to cancer cells. However, development of synthetic vaccines for human use is hampered by the extreme polymorphism of human leukocyte-associated antigens (HLA). In order to stimulate a T-cell dependent anticarbohydrate response, and to bypass the HLA polymorphism of the human population, we designed and synthesized a glycopeptide vaccine containing a cluster of a carbohydrate TAA B-cell epitope (Tn antigen: alpha-GalNAc-Ser) covalently linked to peptides corresponding to the Pan DR 'universal' T-helper epitope (PADRE) and to a cytotoxic T lymphocyte (CTL) epitope from the carcinoembryonic antigen (CEA). The immunogenicity of the construct was evaluated in outbred mice as well as in HLA transgenic mice (HLA-DR1, and HLA-DR4). A strong T-cell dependent antibody response specific for the Tn antigen was elicited in both outbred and HLA transgenic mice. The antibodies induced by the glycopeptide construct efficiently recognized a human tumor cell line underlying the biological relevance of the response. The rational design and synthesis of the glycopeptide construct presented herein, together with its efficacy to induce antibodies specific for native tumor carbohydrate antigens, demonstrate the potential of a such synthetic molecule as an anticancer vaccine candidate for human use.

Revising regulatory networks: from expression data to linear causal models.

Discovering the complex regulatory networks that govern mRNA expression is an important but difficult problem. Many current approaches use only expression data from microarrays to infer the likely network structure. However, this ignores much existing knowledge because for a given organism and system under study, a biologist may already have a partial model of gene regulation. We propose a method for revising and improving these initial models, which may be incomplete or partially incorrect, with expression data. We demonstrate our approach by revising a model of photosynthesis regulation proposed by a biologist for Cyanobacteria. Applied to wild type expression data, our system suggested several modifications consistent with biological knowledge. Applied to a mutant strain, our system correctly modified the disabled gene. Power experiments with synthetic data that indicate that reliable revision is feasible even with a small number of samples.

Concentrations of methyl- tert-butyl ether (MTBE) in inputs and receiving waters of Southern California.

The occurrence and concentration of the fuel additive methyl-tert-butyl ether (MTBE) were measured in dry weather runoff, municipal wastewater and industrial effluents, and coastal receiving waters in southern California. Combined, refineries and sewage treatment plants release approximately 214 kg day(-1) of MTBE into the marine environment, with Santa Monica Bay receiving most (98%) of this discharge. Dry weather urban runoff was analysed for samples collected from 25 streams and rivers, and accounted for less than 0.5% of the mass of MTBE discharged to coastal waters. Receiving water samples were collected from 23 stations in Santa Monica Bay, Los Angeles Harbour and Mission Bay or San Diego Bay. MTBE was detected at low concentrations near effluent discharges, however there was no evidence of baywide MTBE contamination related to these outfalls. Marinas and areas used intensively for recreational boating had the highest average MTBE concentration (8.8 microg l(-1)). Surface water contamination was most widespread in San Diego Bay and Mission Bay, areas with no refinery or sewage treatment plant inputs.

A monoclonal antibody, MIN/3/60, that recognizes the sulpho-Lewis(x) and sulpho-Lewis(a) sequences detects a sub-population of epithelial glycans in the crypts of human colonic epithelium.

Monoclonal antibodies (MAbs) directed to Lewis(x) (Le(x)) and related carbohydrate sequences have been invaluable in anticipating biological roles for these oligosaccharides by detecting the remarkable changes that occur in their expression from the earliest stages of embryogenesis, through development and sequential stages of cell differentiation and maturation. A notable impact has been in the molecular dissection of ligand-receptor interactions in key cell adhesion events at the initial stages of leukocyte recruitment in inflammation, and almost certainly in the metastasis of epithelial tumours. Antibodies that recognise Le(x) and the 3'-sialyl forms were observed to identify leukocyte subsets; these were subsequently found to match those recognized by the leukocyte-endothelium adhesion molecules, the E- and P-selectins. We now describe a MAb (rat hybridoma MIN/3/60) raised to 3'-sulpho-Le(x), a carbohydrate sequence which, in vitro, is bound not only by the E-, L-, and P-selectins, but also by the cysteine-rich domain of the macrophage endocytosis receptor. We observe that MIN/3/60 is bispecific, however; it binds 3'-sulpho-Le(a) as well as 3'-sulpho-Le(x). Nevertheless, our exploratory studies reveal that it may be a useful histochemical reagent when used in conjunction with a monospecific antibody to 3'-sulpho-Le(a). The MIN/3/60 antibody reveals a sub-population of epithelial glycans in the crypts of Lieberkühn in normal human colon.

Anti-tumor immunity provided by a synthetic multiple antigenic glycopeptide displaying a tri-Tn glycotope.

In many cancer cells the alteration of glycosylation processes leads to the expression of cryptic carbohydrate moieties, which make them good targets for immune intervention. Identification of cancer-associated glycotopes as well as progress in chemical synthesis have opened up the way for the development of fully synthetic immunogens that can induce anti-saccharide immune responses. Here, we synthesized a dendrimeric multiple antigenic glycopeptide (MAG) containing the Tn Ag O:-linked to a CD4(+) T cell epitope. This MAG is based on three consecutive Tn moieties (tri-Tn) corresponding to the glycotope recognized by an mAb (MLS 128) produced against the LS180 colon carcinoma cell line. The Abs induced by this MAG recognized murine and human tumor cell lines expressing the Tn Ag. Prophylactic vaccination using MAG provided protection of mice against tumor challenge. When used in active specific immunotherapy, the MAG carrying the tri-Tn glycotope was much more efficient than the mono-Tn analogue in promoting the survival of tumor-bearing mice. Furthermore, in active specific immunotherapy, a linear glycopeptide carrying two copies of the tri-Tn glycotope was shown to be poorly efficient compared with the dendrimeric MAG. Therefore, both the clustering of carbohydrate Ags and the way they are displayed seem to be important parameters for stimulating efficient anti-saccharide immune responses.

Production and functional characterization of two mouse/human chimeric antibodies with specificity for the tumor-associated Tn-antigen.

In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.

Construction and evaluation of a capillary array DNA sequencer based on a micromachined sheath-flow cuvette.

A capillary array electrophoresis DNA sequencer is reported based on a micromachined sheath-flow cuvette as the detection chamber. This cuvette is equipped with a set of micromachined features that hold the capillaries in precise registration to ensure uniform spacing between the capillaries, in order to generate uniform hydrodynamic flow in the cuvette. A laser beam excites all of the samples simultaneously, and a microscope objective images fluorescence onto a set of avalanche photodiodes, which operate in the analog mode. A high-gain transimpedance amplifier is used for each photodiode, providing high duty-cycle detection of fluorescence.

Short synthetic glycopeptides successfully induce antibody responses to carcinoma-associated Tn antigen.

Glycopeptides containing a tumor-associated carbohydrate antigen (mono-, tri- or hexa-Tn antigen) as a B-cell epitope and a CD4+ T-cell epitope (PV: poliovirus or TT: tetanus toxin) were prepared for immunological studies. Several Tn antigen residues [FmocSer/Thr (alpha-GalNAc)-OH] were successively incorporated into the peptide sequence with unprotected carbohydrate groups. The tri- and hexa-Tn glycopeptides were recognized by MLS128, a Tn-specific monoclonal antibody. The position of the tri-Tn motif in the peptide sequence and the peptide backbone itself do not alter its antigenicity. As demonstrated by both ELISA and FACS analysis, the glycopeptides induced high titers of anti-Tn antibodies in mice, in the absence of a carrier molecule. In addition, the generated antibodies recognized the native Tn antigen on cancer cells. The antibody response obtained with a D-(Tn3)-PV glycopeptide containing three alpha-GalNAc-D-serine residues is similar that obtained with the Tn6-PV glycopeptide. These results demonstrate that short synthetic glycopeptides are able to induce anticancer antibody responses.

Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy.

Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti-Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.

Biochemical and functional characterization of the Tn-specific lectin from Salvia sclarea seeds.

SSL, the lectin isolated from Salvia sclarea seeds, recognizes the Tn antigen (GalNAcalpha-O-Ser/Thr), a specific marker of many human carcinomas. Two-dimensional electrophoresis, amino-acid and amino-sugar analysis, and MALDI-TOF MS showed that SSL is an acidic (pI 5.5), 60-61-kDa dimeric glycoprotein composed of apparently identical subunits linked by a single disulfide bond. The apparent molecular mass of SSL in solution determined by equilibrium sedimentation analytical ultracentrifugation was 59 +/- 9 kDa. This value did not change in the pH range 2.5-8.5, indicating that SSL does not associate into higher order structures. Tandem mass spectrometry and methylation analysis of N-glycans released from SSL by hydrazinolysis indicated that SSL possesses 2-3 glycosylation sites occupied with the typical plant glycans Manalpha1-6[(Manalpha1-3)(Xylbeta1-2)]Manbeta1-4 -GlcNAcbeta1-4(Fucalp ha1-3)GlcNAc and [(Manalpha1-3/6)(Xylbeta1-2)]Manbeta1-4-GlcNAcbeta1 -4(Fucalpha1-3)Glc NAc. The influence of adjacent Tn structures on the binding of two Tn-specific lectins (SSL and the isolectin B4 from Vicia villosa) and an anti-Tn monoclonal antibody (mAb 83D4) was evaluated using synthetic Tn glycopeptides. The binding of both lectins to the synthetic Tn glycopeptides was independent of the density of Tn structures. On the other hand, mAb 83D4 only reacted with glycopeptides displaying two or three consecutive Tn structures.

DNA sequencing in a monolithic microchannel device.

We present 50 cm long microchannels in a monolithic device for high resolution, long read-length DNA sequencing. These devices were fabricated and bonded in borofloat glass using unconventional photolithography techniques with 48-188 independent, straight microchannels. The microchannel DNA separation was tested with POP-6 polymer and a DNA sequencing ladder separated at room temperature and 200 V/cm. Single-base resolution greater than 600 bases was achieved and the sequence base called to 640 bases with 98% accuracy. Under the same experimental conditions, the performance of the microchip was identical to a fused-silica capillary with similar cross-sectional area.

Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus.

The phylogenetic relationships among 36 validly described species or subspecies within the genus Staphylococcus were investigated by cloning and sequencing their 60 kDa heat-shock protein (HSP60) genes using a set of universal degenerate HSP60 PCR primers. The cloned partial HSP60 DNA sequences from nine Staphylococcus aureus strains were highly conserved (97-100% DNA sequence similarity; mean 98%), indicating that the HSP60 gene of multiple isolates within the same species have little microheterogeneity. At the subspecies level, DNA sequence similarity among members of S. aureus, Staphylococcus schleiferi, Staphylococcus cohnii and Staphylococcus capitis ranged from 91 to 98%. At the interspecies level, sequence similarity among 23 distinct species of staphylococci ranged from 74 to 93% (mean 82%). By comparison, the highest sequence similarity of Bacillus subtilis and Escherichia coli with members within the genus Staphylococcus was only 70 and 59%, respectively. Importantly, phylogenetic analysis based on the neighbour-joining distance method revealed remarkable concordance between the tree derived from partial HSP60 gene sequences and that based on genomic DNA-DNA hybridization, while 16S rRNA gene sequences correlated less well. The results demonstrate that DNA sequences from the highly conserved and ubiquitous HSP60 gene offer a convenient and accurate tool for species-specific identification and phylogenetic analysis of staphylococci.

A fully synthetic immunogen carrying a carcinoma-associated carbohydrate for active specific immunotherapy.

Aberrant glycosylation of mucins leads to the exposure of cryptic carbohydrate antigens at the surface of carcinoma cells, which, therefore, represent potent targets for anticancer therapeutic vaccines. To date, the development of immunogens to stimulate immune response to such saccharidic antigens is based on carbohydrate conjugation to carrier proteins. However, these traditional protein conjugates are poorly defined in chemical composition and structure. As an alternative, we synthesized a multiple antigenic O-linked glycopeptide (MAG) carrying the carbohydrate Tn antigen associated with a CD4+ T-cell epitope (MAG:Tn-PV). This fully synthetic immunogen is highly defined in composition and carries a high saccharidic epitope ratio over the entire molecule. The MAG:Tn-PV was able to induce anti-Tn IgG antibodies that recognize human tumor cell lines. A therapeutic immunization protocol performed with this fully synthetic immunogen increased the survival of tumor-bearing mice. Thus, the accurately defined and versatile MAG system represents an efficient strategy to induce carbohydrate-specific antitumor immune responses but may also be applicable to the prevention of infectious diseases, if it is based on bacterial oligosaccharides.