Evaluating putative ecological drivers of microcystin spatiotemporal dynamics using metabarcoding and environmental data.

Microcystin is a cyanobacterial hepatotoxin of global concern. Understanding the environmental factors that cause high concentrations of microcystin is crucial to the development of lake management strategies that minimize harmful exposures. While the literature is replete with studies linking cyanobacterial production of microcystin to changes in various nutrients, abiotic stressors, grazers, and competitors, no single biotic or abiotic factor has been shown to be reliably predictive of microcystin concentrations in complex ecosystems. We performed random forest regression analyses with 16S and 18S rRNA gene sequencing data and environmental data to determine which putative ecological drivers best explained spatiotemporal variation in total microcystin and several individual congeners in a eutrophic freshwater reservoir. Model performance was best for predicting concentrations of the congener MC-LR, with ca. 88% of spatiotemporal variance explained. Most of the variance was associated with changes in the relative abundance of the cyanobacterial genus Microcystis. Follow-up RF regression analyses revealed that factors that were the most important in predicting MC-LR were also the most important in predicting Microcystis population dynamics. We discuss how these results relate to prevailing ecological hypotheses regarding the function of microcystin.

Accurate zygote-specific discrimination of single-nucleotide polymorphisms using microfluidic electrochemical DNA melting curves.

We report the first electrochemical system for the detection of single-nucleotide polymorphisms (SNPs) that can accurately discriminate homozygous and heterozygous genotypes using microfluidics technology. To achieve this, our system performs real-time melting-curve analysis of surface-immobilized hybridization probes. As an example, we used our sensor to analyze two SNPs in the apolipoprotein E (ApoE) gene, where homozygous and heterozygous mutations greatly affect the risk of late-onset Alzheimer's disease. Using probes specific for each SNP, we simultaneously acquired melting curves for probe-target duplexes at two different loci and thereby accurately distinguish all six possible ApoE allele combinations. Since the design of our device and probes can be readily adapted for targeting other loci, we believe that our method offers a modular platform for the diagnosis of SNP-based diseases and personalized medicine.

Acoustophoretic sorting of viable mammalian cells in a microfluidic device.

We report the first use of ultrasonic acoustophoresis for the label-free separation of viable and nonviable mammalian cells within a microfluidic device. Cells that have undergone apoptosis are physically smaller than viable cells, and our device exploits this fact to achieve efficient sorting based on the strong size dependence of acoustic radiation forces within a microchannel. As a model, we have selectively enriched viable MCF-7 breast tumor cells from heterogeneous mixtures of viable and nonviable cells. We found that this mode of separation is gentle and enables efficient, label-free isolation of viable cells from mixed samples containing 10(6) cells/mL at flow rates of up to 12 mL/h. We have extensively characterized the device, and we report the effects of piezoelectric voltage and sample flow rate on device performance and describe how these parameters can be tuned to optimize recovery, purity, or throughput.

Gel-based optical waveguides with live cell encapsulation and integrated microfluidics.

In this Letter, we demonstrate a biocompatible microscale optical device fabricated from agarose hydrogel that allows for encapsulation of cells inside an optical waveguide. This allows for better interaction between the light in the waveguide and biology, since it can interact with the direct optical mode rather than the evanescent field. We characterize the optical properties of the waveguide and further incorporate a microfluidic channel over the optical structure, thus developing an integrated optofluidic system fabricated entirely from agarose gel.

Optofluidic ring resonator switch for optical particle transport.

In this work, we demonstrate an optofluidic switch using a microring resonator architecture to direct particles trapped in the evanescent field of a solid-core waveguide. When excited at the resonant wavelength, light inserted into the bus waveguide becomes amplified within the ring structure. The resulting high optical intensities in the evanescent field of the ring generate a gradient force that diverts particles trapped on the bus to the ring portion of the device. We show that this increase in optical energy translates to an increase of 250% in the radiation pressure induced steady-state velocity of particles trapped on the ring. We also characterize the switching fraction of the device, showing that 80% of particles are diverted onto the ring when the device is at an on-resonance state. The optofluidic switch we present here demonstrates the versatility in exploiting planar optical devices for integrated particle manipulation applications.

Optical manipulation of nanoparticles and biomolecules in sub-wavelength slot waveguides.

The ability to manipulate nanoscopic matter precisely is critical for the development of active nanosystems. Optical tweezers are excellent tools for transporting particles ranging in size from several micrometres to a few hundred nanometres. Manipulation of dielectric objects with much smaller diameters, however, requires stronger optical confinement and higher intensities than can be provided by these diffraction-limited systems. Here we present an approach to optofluidic transport that overcomes these limitations, using sub-wavelength liquid-core slot waveguides. The technique simultaneously makes use of near-field optical forces to confine matter inside the waveguide and scattering/adsorption forces to transport it. The ability of the slot waveguide to condense the accessible electromagnetic energy to scales as small as 60 nm allows us also to overcome the fundamental diffraction problem. We apply the approach here to the trapping and transport of 75-nm dielectric nanoparticles and lambda-DNA molecules. Because trapping occurs along a line, rather than at a point as with traditional point traps, the method provides the ability to handle extended biomolecules directly. We also carry out a detailed numerical analysis that relates the near-field optical forces to release kinetics. We believe that the architecture demonstrated here will help to bridge the gap between optical manipulation and nanofluidics.

Forces and transport velocities for a particle in a slot waveguide.

Optofluidic transport seeks to exploit the high-intensity electromagnetic energy in waveguiding structures to manipulate nanoscopic matter using radiation pressure and optical trapping forces. In this paper, we present an analysis of optical trapping and transport of sub-100 nm polystyrene and gold nanoparticles in silicon slot waveguides. This study focuses on the effect of particle size, particle refractive index, and slot waveguide geometry on trapping stability and the resulting transport speed. Our results indicate that stable trapping and transport can be achieved for objects as small as 10 or 20 nm in diameter with as much as a 100 fold enhancement in trapping stiffness over the state of the art.

Nanobiosensors: optofluidic, electrical and mechanical approaches to biomolecular detection at the nanoscale.

Next generation biosensor platforms will require significant improvements in sensitivity, specificity and parallelity in order to meet the future needs of a variety of fields ranging from in vitro medical diagnostics, pharmaceutical discovery and pathogen detection. Nano-biosensors, which exploit some fundamental nanoscopic effect in order to detect a specific biomolecular interaction, have now been developed to a point where it is possible to determine in what cases their inherent advantages over traditional techniques (such as nucleic acid microarrays) more than offset the added complexity and cost involved constructing and assembling the devices. In this paper we will review the state of the art in nanoscale biosensor technologies, focusing primarily on optofluidic type devices but also covering those which exploit fundamental mechanical and electrical transduction mechanisms. A detailed overview of next generation requirements is presented yielding a series of metrics (namely limit of detection, multiplexibility, measurement limitations, and ease of fabrication/assembly) against which the various technologies are evaluated. Concluding remarks regarding the likely technological impact of some of the promising technologies are also provided.

Stability analysis of optofluidic transport on solid-core waveguiding structures.

Optofluidic transport involves the use of electromagnetic energy to transport nanoparticles through the exploitation of scattering, adsorption and gradient (polarization) based forces. This paper presents a new approach to stability analysis for a system of broad applicability to such transport, namely the optical trapping of dielectric particles in the evanescent field of low index (polymer) and high index (silicon) solid-core waveguide structures integrated with microfluidics. Three-dimensional finite element based simulations are used to determine the electromagnetic and hydrodynamic field variables for the system of interest. The net force acting on particles is determined through evaluation of the full Maxwell and flow shear stress tensors, and a trapping stability number is obtained by comparing the work required to remove a particle from the waveguide with available random thermal energy. These forces are correlated to controllable experimental parameters such as particle size, fluid velocity, and channel height, and a series of trapping stability diagrams is produced which detail the conditions under which optofluidic transport is possible.

Structure-based design of caspase-1 inhibitor containing a diphenyl ether sulfonamide.

A series of compounds was designed and prepared as inhibitors of interleukin-1beta converting enzyme (ICE), also known as caspase-1. These inhibitors, which employ a diphenyl ether sulfonamide, were designed to improve potency by forming favorable interactions between the diphenyl ether rings and the prime side hydrophobic region. An X-ray crystal structure of a representative member of the diphenyl ether sulfonamide series bound to the active site of caspase-1 was obtained.

Microsatellite instability in ovarian and other pelvic carcinomas.

Twenty-six cases of ovarian carcinoma and six cases of other pelvic neoplasms were analyzed for microsatellite instability (MSI) using frozen specimens, fluorescence technology, and four selected markers (D2S123 on chromosome 2, D18S58 on chromosome 18, BAT26 on chromosome 2, and BAT40 on chromosome 1). This procedure also allowed the detection of loss of heterogeneity (LOH) at the four selected loci. One of the cases of ovarian carcinoma exhibited MSI and this was evident at three loci. Of 44 informative loci, 7 exhibited LOH representing 3 cases of ovarian carcinoma, 3 of 4 cases of primary peritoneal carcinoma, and one case of unknown primary. These data support other findings that MSI is not a frequent occurrence in ovarian cancer; however, LOH is a more frequent event and may be a target for the development of diagnostic/prognostic procedures for ovarian and primary peritoneal carcinoma.

Mutation analysis of BRCA1, TP53, and KRAS2 in ovarian and related pelvic tumors.

Cancer may be viewed as a genetic disease resulting from critical mutations that disrupt normal cell growth. To characterize the involvement of the BRCA1 and TP53 tumor suppressor genes and of the KRAS2 protooncogene in gynecologic cancer, mutation analysis of these genes was conducted in pelvic tumors of 85 patients that included 49 epithelial ovarian carcinoma cases. The 85 pelvic tumors contained 5 tumors with BRCA1 mutations, 33 with TP53 mutations, and 1 with a KRAS2 mutation. Each of the BRCA1 and KRAS2 mutations, and 25 of the TP53 mutations, were in ovarian carcinomas. Four of the BRCA1 mutations were germline and 1 was somatic. The 4 patients with germline BRCA1 mutations had an early age of disease onset (33-48 years) relative to the mean age of onset (58 years) of all 49 ovarian carcinoma patients, and 3 of these 4 patients had a family history of ovarian or breast cancer. None of the 4 tumors with germline BRCA1 mutations had a KRAS2 mutation or a TP53 mutation, despite a 51% frequency of TP53 mutations in the 49 ovarian carcinomas. Three of the 4 tumors with germline BRCA1 mutations retained a wild-type BRCA1 allele. The tumor with the somatic BRCA1 mutation contained a TP53 mutation and had no evidence for wild-type BRCA1 and TP53 alleles. These data suggest that both BRCA1 and TP53 were inactivated in 1 of 49 ovarian carcinomas. Moreover, mutational inactivation of both BRCA1 and TP53 did not occur in 4 tumors with a germline BRCA1 mutation. It has been proposed that tumorigenesis in cells with a heterozygous BRCA1 mutation requires inactivation of the wild-type BRCA1 and TP53 alleles, which results in genomic instability and acquisition of mutations in protooncogenes. Clearly, mutational inactivation of TP53 and the wild-type BRCA1 allele in ovarian tumors with a heterozygous, germline BRCA1 mutation is not an absolute requirement for tumor formation. It is possible that these alleles may be inactivated by nonmutational mechanisms or that other tumor formation pathways exist.

Mutations in BRCA1 from fixed, paraffin-embedded tissue can be artifacts of preservation.

DNA isolated from paraffin-embedded tissues has been used for analysis of DNA alterations in disease states. Use of archival tissue can expedite the gathering of large numbers of specimens from rare disease subtypes that would take years to accumulate prospectively. Therefore, archival tissues from 70 ovarian cancer cases diagnosed before or at age 40 were retrieved for analysis of BRCA1 mutations. DNA was isolated from paraffin-embedded tissue of 70 ovarian cancer cases diagnosed before or at age 40. BRCA1 mutation analysis was conducted by single-strand conformation polymorphism analysis and DNA sequencing. Fifty-eight BRCA1 mutations were found in 34 of the 70 ovarian cancer cases. Twenty-two cases had one mutation each and 12 cases had multiple mutations. Multiple mutations found in histologically normal tissue of 2 cases were not present in matched tumor tissue. For another case, DNA from two separate blocks of normal tissue contained different mutations. These observations were anomalous and suggested that mutations detected in fixed tissues may be artifacts of tissue preservation and not present in the original unfixed tissues. To test this suggestion, blood was obtained from 2 patients for whom mutations were found in fixed, normal tissue. DNA from their unfixed lymphocytes did not contain the mutations found in fixed normal tissue. Thus, mutations found in fixed, paraffin-embedded tissues can be artifacts of tissue preservation. The reliability of DNA sequence data derived from such tissues must be questioned in the absence of corroborating data from unfixed tissues. This severely limits the use of fixed tissues as a source of DNA for retrospective research and for clinical genetic testing in families for which a disease-affected member is not alive.

Correlation of TP53 mutations and p53 expression in ovarian tumors.

To characterize the involvement of the TP53 tumor suppressor gene in ovarian cancer, mutation analysis of exons 2-11 of TP53 and immunodetection of its protein product, p53, were done in 48 ovarian tumors. Normally, p53 is not immunodetectable. Missense TP53 mutations have been reported to result in p53 accumulation and detection, but mutations generating premature stop codons have not. Mutations were identified in 19 of 41 malignant tumors but not in 5 benign tumors and 2 tumors of low malignant potential. Fifteen of the 19 tumors with mutations also stained positively by immunohistochemistry or Western blot or both. They included 11 missense mutations, 1 in-frame duplication (474ins6), and 3 frameshift mutations generating premature stop codons. The three tumors with frameshifts also had a wild-type TP53 allele and displayed normal size but not truncated p53 by Western blot. This indicates that these tumors express wild-type p53. The significance of TP53 mutations in the development of the three tumors is questionable unless there is a mechanism for inactivating wild-type p53. Nine of the 19 mutations found here, including the 3 frameshifts, were previously not reported in ovarian cancer. Thirteen of the 19 mutations were single nucleotide substitutions with 6 transitions and 7 transversions. The ratio of transversions to transitions (1.2) was different from literature reports (0.5) (P < 0.01). Thus, the spectrum of TP53 mutations in our study differed from other ovarian tumor reports. This difference may be due to population-based differences in the molecular epidemiology of TP53 mutations.

Binding of synthetic sulfated ligands by human splenic galectin 1, a beta-galactoside-binding lectin.

The carbohydrate-binding site of galectin 1, a vertebrate beta-galactoside-binding lectin, has a pronounced specificity for the betaGal(1-->3)- and betaGal(1-->4)GlcNAc sequences. The binding inhibition study reported herein was carried out to determine whether sulfation of saccharides would influence their binding by galectin 1. The presence of 6'-OSO3- on LacNAc greatly reduces the inhibitory potency relative to LacNAc. 3'-OSO3-LacNAc, 3'-OSO3-Galbeta(1-->3)GlcNAc(beta)1-OBzl and 3-OSO3-Galbeta1-OMe are more potent inhibitors than the non-sulfated parent compounds. Surprisingly, 2'-OSO3-LacNAc showed over 40 fold less inhibitory potency relative to LacNAc. Ovarian carcinoma A121 cells were shown to synthesize sulfated macromolecules that bind to galectin 1. Modulation in vivo of saccharide sulfation may lead to modulation of galectin 1 interaction with glycoconjugates; hence, sulfation could play a role in modulating lectin functions.

Genetic protocols review by Institutional Review Boards at National Cancer Institute-designated cancer centers.

Absent a clear and enforceable national policy on cancer genetic testing and genetic research, the responsibility may currently be on institutional review boards to regulate those activities at their institutions through protocol approval or disapproval. The survey reported here was carried out to gain information on National Cancer Institute-designated cancer center policies governing institutional review board review of genetic protocols and the preparedness of institutional review boards to review genetic protocols. Thirty-five responses (63% response rate) were received, of which 30 were evaluable. Twenty-four responders reported that they believed that there is a need for research that may lead to improved review of genetic research and genetic testing protocols. Only 14 responders felt adequately informed of current and developing issues and legislation relative to genetic testing and research. Seven responders reported that their cancer centers require an institutional review board-approved protocol for genetic testing activities. Five responders reported that their cancer centers have a formal written policy that guides institutional review board review of genetic testing protocols. About half of the responders reported that their cancer centers have no formal written policy that guides institutional review board review of genetic research protocols. Only three responders reported that institutional review board members receive formal training to prepare them to evaluate all of the issues associated with genetic protocols. We conclude that greater effort needs to be made to establish uniform policy governing cancer genetic testing and genetic research and greater effort should be made to prepare institutional review boards formally for the review of genetic-related protocols.

Assessment of family cancer history collection and utilization in patient care.

OBJECTIVES: This study was conducted to determine whether patients and accompanying persons visiting the Gynecologic Oncology Clinic were aware if a family cancer history was recorded and utilized in their medical care; whether they were aware of the importance of a family cancer history, and whether they would like to learn more about familial cancer. METHODS: Sixteen- and 17-item self-report questionnaires were administered to patients and their accompanying persons, respectively, who were visiting the Gynecologic Oncology Clinic. All responses were anonymous. RESULTS: Two hundred forty-four patient questionnaires and 114 accompanying person questionnaires were completed. Seventy-eight percent of the patients and 70% of the accompanying persons replied that a physician had inquired about their family history of cancer. Only 40% of those patients and 70% of those accompanying persons (31 and 49% of total patients and accompanying persons, respectively) replied that the inquiry was by their family physician. Sixty-seven percent of these patients and 63% of these accompanying persons reported that a written record was made of the family history. Thirty-one percent of the patients and 28% of accompanying persons knew that their family cancer history information had been used to aid in their medical care. Eighty-eight percent of the patients and 83% of the accompanying persons reported the occurrence of at least one relative with cancer; however, only 44% of the patients and 35% of the accompanying persons replied that a health care provider had ever provided teaching about the importance of a family cancer history. Seventy-five percent of the patients and 73% of the accompanying persons indicated that they would like to learn more about hereditary cancer and cancer genetics. CONCLUSIONS: This study demonstrates that patients desire information about cancer genetics and hereditary cancer. Therefore, health care providers should provide better education and information to their patients as well as improve their family history-taking skills.

Isolation of a melibiose-binding protein from human spleen.

A melibiose-binding protein was isolated from human spleen by serial affinity chromatography on lactose-, mannose-, and melibiose-Sepharose. The purified protein agglutinated rabbit erythrocytes and re-bound to melibiose, but did not bind to murine nor human laminin. The protein was composed of approximately 58 kDa and 26 kDa polypeptides. The polypeptides were detected in buffy coat cell extracts and they were synthesized in vitro by B lymphoblastoid cells. The polypeptides did not react with anti-galaptin, anti-C-reactive protein, anti-amyloid P, anti-keratin, and anti-rat lung lectin 29 sera. The 58 kDa polypeptide reacted very weakly with anti-core-specific lectin serum and reacted with anti-IgG serum. The data suggest that the major protein isolated is an anti-Ga1 alpha 1-->6 immunoglobulin.

Galaptin-mediated adhesion of human ovarian carcinoma A121 cells and detection of cellular galaptin-binding glycoproteins.

Previously, we have shown that galaptin, an endogenous beta-galactoside-binding lectin, is present in extracellular matrix where it may participate in the adhesion of A121 human ovarian carcinoma cells to extracellular matrix via interaction with specific cell surface carbohydrate receptors. We now report that A121 cells adhere to polystyrene plates coated with polymerized human splenic galaptin. The carbohydrate-mediated specificity of this adhesive interaction was demonstrated by inhibition with lactose. Additionally, treatment of A121 cells with neuraminidase increased cellular adherence by 30%, while beta-galactosidase treatment of cells decreased adherence by 65%. These findings prompted us to isolate and identify the cell surface galaptin receptor. In a Western blot of A121 cell extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 125I-labeled polymerized galaptin bound [corrected] to a unique cellular protein having a molecular mass of 110 kDa. This receptor was enriched by affinity chromatography using polymerized galaptin-Sepharose. Treatment of this material with N-glycanase ablated its galaptin-binding activity. In related studies, A121 cells metabolically labeled with [3H]glucosamine demonstrated a radiolabeled polymerized galaptin-binding protein with an identical molecular mass of 110 kDa. These studies confirmed the glycoprotein nature of this putative endogenous cellular galaptin receptor. Further studies with antibodies directed against two lysosomal associated membrane proteins, lamp-1 and lamp-2, demonstrated specific reactivity in Western blots with the 110-kDa glycoprotein. Additionally, 125I-polymerized galaptin recognized a 110-kDa protein in Western blots of material immunoprecipitated from A121 cell lysates by lamp-1 and lamp-2 antibodies. Finally, indirect immunofluorescence using antibodies directed against lamps detected cell surface antigenicity. Therefore, lamp-1 and/or lamp-2 appear to be the putative cell surface receptors involved in the adhesion of ovarian carcinoma cells to extracellular matrix mediated by galaptin.

Galaptin and galaptin-binding glycoconjugates in serum and effusions of carcinoma patients.

The objectives of this study were (1) to quantify galaptin, an endogenous lectin, and galaptin-binding glycoconjugates present in normal serum, (2) to determine if these components were altered in the serum and effusions of carcinoma patients with advanced disease, and (3) to determine if ovarian carcinoma cells synthesize and release soluble galaptin inhibitors. Serum from healthy females (n = 10) had a mean galaptin content of 96 +/- 40 ng/ml. Galaptin levels in carcinoma patient serum (n = 29) were depressed (mean 21 +/- 23 ng/ml; p < 0.0001). Galaptin was not detected in 6 of 8 ovarian carcinoma patient sera. Effusions (n = 17) had a mean galaptin content of 358 +/- 326 ng/ml. Assays involving inhibition of binding of galaptin-peroxidase conjugates to asialofetuin were carried out to evaluate the levels of galaptin-binding glycoconjugates in serum and effusions. The mean inhibition titer of normal serum (n = 12) was 75 +/- 38. Patient serum (n = 28) had an elevated inhibitor content (mean titer = 304 +/- 155; p < 0.0001). Effusions (n = 17) also had a higher inhibitor content relative to normal serum (mean titer = 247 +/- 202; p = 0.0037). Ovarian carcinoma cells isolated from effusions and cultured in vitro were shown to synthesize and release into the medium galaptin-binding glycoconjugates of molecular mass 100-200 kD. An ovarian carcinoma cell line, A121, released galaptin-binding glycoconjugates of molecular mass > or = 200 kD into the medium. The data presented show that the levels of soluble galaptin and galaptin-binding glycoconjugates in the serum of advanced cancer patients are perturbed relative to normal female serum.