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1.  Increased Bisecting N-Acetylglucosamine and Decreased Branched Chain Glycans of N-linked Glycoproteins in Expressed Prostatic Secretions Associated with Prostate Cancer Progression 
Proteomics. Clinical applications  2013;10.1002/prca.201200134.
Using prostatic fluids rich in glycoproteins like prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) , the goal of this study was to identify the structural types and relative abundance of glycans associated with prostate cancer status for subsequent use in emerging mass spectrometry-based glycopeptide analysis platforms.
Experimental Design
A series of pooled samples of expressed prostatic secretions (EPS) and exosomes reflecting different stages of prostate cancer disease were used for N-linked glycan profiling by three complementary methods, MALDI-TOF profiling, normal-phase HPLC separation, and triple quadropole MS analysis of PAP glycopeptides.
Glycan profiling of N-linked glycans from different EPS fluids indicated a global decrease in larger branched tri- and tetra-antennary glycans. Differential exoglycosidase treatments indicated a substantial increase in bisecting N-acetylglucosamines correlated with disease severity. A triple quadrupole MS analysis of the N-linked glycopeptides sites from PAP in aggressive prostate cancer pools was done to cross-reference with the glycan profiling data.
Conclusion and clinical relevance
Changes in glycosylation as detected in EPS fluids reflect the clinical status of prostate cancer. Defining these molecular signatures at the glycopeptide level in individual samples could improve current approaches of diagnosis and prognosis.
PMCID: PMC3969874  PMID: 23775902
Glycomics; N-linked glycosylation; Prostatic acid phosphatase; Urine Exosome; Prostate Cancer; Expressed Prostatic Secretion
2.  A MALDI Imaging Mass Spectrometry Workflow for Spatial Profiling Analysis of N-linked Glycan Expression in Tissues 
Analytical chemistry  2013;85(20):9799-9806.
A new Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in tissues is described. Application of an endoglycosidase, peptide N-glycosidase F (PNGaseF), directly on tissues followed by incubation releases N-linked glycan species amenable to detection by MALDI-IMS. The method has been designed to simultaneously profile the multiple glycan species released from intracellular organelle and cell surface glycoproteins, while maintaining histopathology compatible preparation workflows. A recombinant PNGaseF enzyme was sprayed uniformly across mouse brain tissue slides, incubated for two hours, then sprayed with 2,5-dihydroxybenzoic acid matrix for MALDI-IMS analysis. Using this basic approach, global snapshots of major cellular N-linked glycoforms were detected, including their tissue localization and distribution, structure and relative abundance. Off-tissue extraction and modification of glycans from similarly processed tissues and further mass spectrometry or HPLC analysis was done to assign structural designations. MALDI-IMS has primarily been utilized to spatially profile proteins, lipids, drug and small molecule metabolites in tissues, but it has not been previously applied to N-linked glycan analysis. The translatable MALDI-IMS glycan profiling workflow described herein can readily be applied to any tissue type of interest. From a clinical diagnostics perspective, the ability to differentially profile N glycans and correlate their molecular expression to histopathological changes can offer new approaches to identifying novel disease related targets for biomarker and therapeutic applications.
PMCID: PMC3969840  PMID: 24050758
3.  MALDI Imaging Mass Spectrometry Profiling of N-Glycans in Formalin-Fixed Paraffin Embedded Clinical Tissue Blocks and Tissue Microarrays 
PLoS ONE  2014;9(9):e106255.
A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.
PMCID: PMC4153616  PMID: 25184632
4.  Diversity of Bacteria Carried by Pinewood Nematode in USA and Phylogenetic Comparison with Isolates from Other Countries 
PLoS ONE  2014;9(8):e105190.
Pine wilt disease (PWD) is native to North America and has spread to Asia and Europe. Lately, mutualistic relationship has been suggested between the pinewood nematode (PWN), Bursaphelenchus xylophilus the causal nematode agent of PWD, and bacteria. In countries where PWN occurs, nematodes from diseased trees were reported to carry bacteria from several genera. However no data exists for the United States. The objective of this study was to evaluate the diversity of the bacterial community carried by B. xylophilus, isolated from different Pinus spp. with PWD in Nebraska, United States. The bacteria carried by PWN belonged to Gammaproteobacteria (79.9%), Betaproteobacteria (11.7%), Bacilli (5.0%), Alphaproteobacteria (1.7%) and Flavobacteriia (1.7%). Strains from the genera Chryseobacterium and Pigmentiphaga were found associated with the nematode for the first time. These results were compared to results from similar studies conducted from other countries of three continents in order to assess the diversity of bacteria with associated with PWN. The isolates from the United States, Portugal and China belonged to 25 different genera and only strains from the genus Pseudomonas were found in nematodes from all countries. The strains from China were closely related to P. fluorescens and the strains isolated from Portugal and USA were phylogenetically related to P. mohnii and P. lutea. Nematodes from the different countries are associated with bacteria of different species, not supporting a relationship between PWN with a particular bacterial species. Moreover, the diversity of the bacteria carried by the pinewood nematode seems to be related to the geographic area and the Pinus species. The roles these bacteria play within the pine trees or when associated with the nematodes, might be independent of the presence of the nematode in the tree and only related on the bacteria's relationship with the tree.
PMCID: PMC4134288  PMID: 25127255
5.  Nematode Spatial and Ecological Patterns from Tropical and Temperate Rainforests 
PLoS ONE  2012;7(9):e44641.
Large scale diversity patterns are well established for terrestrial macrobiota (e.g. plants and vertebrates), but not for microscopic organisms (e.g. nematodes). Due to small size, high abundance, and extensive dispersal, microbiota are assumed to exhibit cosmopolitan distributions with no biogeographical patterns. This assumption has been extrapolated from local spatial scale studies of a few taxonomic groups utilizing morphological approaches. Recent molecularly-based studies, however, suggest something quite opposite. Nematodes are the most abundant metazoans on earth, but their diversity patterns are largely unknown. We conducted a survey of nematode diversity within three vertical strata (soil, litter, and canopy) of rainforests at two contrasting latitudes in the North American meridian (temperate: the Olympic National Forest, WA, U.S.A and tropical: La Selva Biological Station, Costa Rica) using standardized sampling designs and sample processing protocols. To describe nematode diversity, we applied an ecometagenetic approach using 454 pyrosequencing. We observed that: 1) nematode communities were unique without even a single common species between the two rainforests, 2) nematode communities were unique among habitats in both rainforests, 3) total species richness was 300% more in the tropical than in the temperate rainforest, 4) 80% of the species in the temperate rainforest resided in the soil, whereas only 20% in the tropics, 5) more than 90% of identified species were novel. Overall, our data provided no support for cosmopolitanism at both local (habitats) and large (rainforests) spatial scales. In addition, our data indicated that biogeographical patterns typical of macrobiota also exist for microbiota.
PMCID: PMC3439412  PMID: 22984536
6.  Reflections on Plant and Soil Nematode Ecology: Past, Present and Future 
Journal of Nematology  2012;44(2):115-126.
The purpose of this review is to highlight key developments in nematode ecology from its beginnings to where it stands today as a discipline within nematology. Emerging areas of research appear to be driven by crop production constraints, environmental health concerns, and advances in technology. In contrast to past ecological studies which mainly focused on management of plant-parasitic nematodes, current studies reflect differential sensitivity of nematode faunae. These differences, identified in both aquatic and terrestrial environments include response to stressors, environmental conditions, and management practices. Methodological advances will continue to influence the role nematodes have in addressing the nature of interactions between organisms, and of organisms with their environments. In particular, the C. elegans genetic model, nematode faunal analysis and nematode metagenetic analysis can be used by ecologists generally and not restricted to nematologists.
PMCID: PMC3578467  PMID: 23482864
ecology; future; history; interaction; management; molecular biology; nematology; plant; soil
7.  MOTUs, Morphology, and Biodiversity Estimation: A Case Study Using Nematodes of the Suborder Criconematina and a Conserved 18S DNA Barcode 
Journal of Nematology  2011;43(1):35-48.
DNA barcodes are increasingly used to provide an estimate of biodiversity for small, cryptic organisms like nematodes. Nucleotide sequences generated by the barcoding process are often grouped, based on similarity, into molecular operational taxonomic units (MOTUs). In order to get a better understanding of the taxonomic resolution of a 3' 592-bp 18S rDNA barcode, we have analyzed 100 MOTUs generated from 214 specimens in the nematode suborder Criconematina. Previous research has demonstrated that the primer set for this barcode reliably amplifies all nematodes in the Phylum Nematoda. Included among the Criconematina specimens were 25 morphologically described species representing 12 genera. Using the most stringent definition of MOTU membership, where a single nucleotide difference is sufficient for the creation of a new MOTU, it was found that an MOTU can represent a subgroup of a species (e.g. Discocriconemella limitanea), a single species (Bakernema inaequale), or a species complex (MOTU 76). A maximum likelihood phylogenetic analysis of the MOTU dataset generated four major clades that were further analyzed by character-based barcode analysis. Fourteen of the 25 morphologically identified species had at least one putative diagnostic nucleotide identified by this character-based approach. These diagnostic nucleotides could be useful in biodiversity assessments when ambiguous results are encountered in database searches that use a distance-based metric for nucleotide sequence comparisons. Information and images regarding specimens examined during this study are available online.
PMCID: PMC3380479  PMID: 22791913
Criconematidae; DNA taxonomy; phylogeny; barcode analysis; plant parasitic nematodes; nematode diversity
8.  Vittatidera zeaphila (Nematoda: Heteroderidae), a new genus and species of cyst nematode parasitic on corn (Zea mays) 
Journal of Nematology  2010;42(2):139-150.
A new genus and species of cyst nematode, Vittatidera zeaphila, is described from Tennessee. The new genus is superficially similar to Cactodera but is distinguished from other cyst-forming taxa in having a persistent lateral field in females and cysts, persistent vulval lips covering a circumfenestrate vulva, and subventral gland nuclei of the female contained in a separate small lobe. Infective juveniles (J2) are distinguished from all previously described Cactodera spp. by the short stylet in the second-stage juvenile (14-17 μm); J2 of Cactodera spp. have stylets at least 18 μm long. The new species also is unusual in that the females produce large egg masses. Known hosts are corn and goosegrass. DNA analysis suggests that Vittatidera forms a separate group apart from other cyst-forming genera within Heteroderinae.
PMCID: PMC3380471  PMID: 22736850
cyst nematode; Eleusine indica; goosegrass; maize; molecular analysis; new genus; taxonomy; Vittatidera zeaphila; Zea mays
9.  Morphological and Molecular Characterization of Discocriconemella inarata, an Endemic Nematode from North American Native Tallgrass Prairies 
Journal of Nematology  2010;42(1):35-45.
Discocriconemella inarata, a plant parasitic nematode species originally discovered in a virgin tallgrass prairie in northwest Iowa, was re-examined by molecular and morphological analyses of topotype material. This species has never been recorded in cultivated fields and could potentially serve as an indicator for high quality prairie habitats. DNA sequence from a conserved 3’ portion of the 18S ribosomal gene exhibited an identical match between D. inarata topotype specimens and topotype specimens of Mesocriconema xenoplax from Fresno, California. Higher resolution sequence analyses using the internal transcribed spacer 1 (ITS1) and a portion of the mitochondrial gene cytochrome b (cytb) allowed discrimination of D. inarata apart from M. xenoplax. This pair of species formed a well-supported clade with other Mesocriconema species exclusive of tropical Discocriconemella species. Scanning electron microscopy confirmed the absence of submedian lobes on D. inarata, suggesting a secondary loss of this defining morphological characteristic for Mesocriconema. Observations and measurements of D. inarata juveniles were added for the first time. Surveys of other prairies within the Great Plains expanded the known distribution of this species.
PMCID: PMC3380506  PMID: 22736835
Criconematidae Discocriconemella inarata; DNA taxonomy; endemic nematode; environmental indicator; native habitats; plant parasitic nematodes; ring nematodes; tallgrass prairie
10.  Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1 
Journal of Nematology  1997;29(3):255-267.
The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).
PMCID: PMC2619784  PMID: 19274157
clover cyst nematode; corn cyst nematode; diagnosis; Heterodera glycines; Heterodera goettingiana; Heterodera schachtii; Heterodera trifolii; Heterodera zeae; molecular nematology; nematode PCR-RFLP; pea cyst nematode; rDNA variation; regulatory nematology; soybean cyst nematode; sugar beet cyst nematode
11.  Characterization of Heterorhabditis Isolates by PCR Amplification of Segments of mtDNA and rDNA Genes 
Journal of Nematology  1994;26(3):260-270.
Restriction digests of amplified DNA from the mitochondrial genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H. bacteriophora complex. The H. bacteriophora complex could be further resolved into three genotypes represented by H. zealandica, the H. bacteriophora, Brecon (Australian) type isolate for H. bacteriophora, and a grouping composed of isolates NC1, V16, HI82, and HP88. All cultures obtained of the H. megidis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhabditis as well as allowing a rapid initial assessment of taxonomic grouping.
PMCID: PMC2619499  PMID: 19279891
entomopathogenic nematode; Heterorhabditis; molecular diagnostics; mtDNA; nematode; ribosomal DNA
12.  Estimation of Genetic Divergence in Meloidogyne Mitochondrial DNA 
Journal of Nematology  1988;20(4):505-511.
Restriction fragments from purified mitochondrial DNA can be readily detected following rapid end-labeling with [α-³²]nucleoside triphosphates and separation by gel electrophoresis. Mitochondrial DNA from 12 populations of Meloidogyne species was digested with 12 restriction enzymes producing more than 60 restriction fragments for each species. The mitochondrial genome of M. arenaria is the most genetically distinct of the four species compared. M. arenaria shows approximately 2.1-3.1% nucleotide sequence divergence from the mitochondrial genomes of M. javanica, M. incognita, and M. hapla. Among the latter three species, interspecific estimates of sequence divergence range from 0.7 to 2.3%. Relatively high intraspecific variation in mitochondrial restriction fragment patterns was observed in M. hapla. Intraspecific variation in M. incognita resulted in sequence divergence estimates of 0.5-1.0%. Such polymorphisms can serve as genetic markers for discerning mitochondrial DNA genotypes in nematode populations in the same way that allozymes have been used to discern nuclear DNA genotypes.
PMCID: PMC2618847  PMID: 19290247
Meloidogyne spp.; mitochondrial DNA; molecular evolution; nucleotide sequence divergence
13.  Species-Specific Restriction Site Polymorphism in Root-knot Nematode Mitochondrial DNA 
Journal of Nematology  1986;18(3):288-293.
Research was initiated to physically characterize the mitochondrial genomes of several Meloidogyne spp. and host-races, to address questions regarding their systematics and dispersal, and to assess the possibility of developing molecular diagnostics for these nematodes. Techniques were developed for purification and rapid detection of mitochondrial DNA from root-knot nematodes. Mitochondrial DNAs among Meloidogyne spp. were demonstrated to exhibit extensive divergence. The potential for using the rapidly diverging mitochondrial genomes as a diagnostic assay for M. incognita, M. hapla, M. arenaria, and M. javanica is discussed.
PMCID: PMC2618552  PMID: 19294180
Meloidogyne spp.; molecular diagnostics; hybridization probe
14.  Taxonomic Limits of the Genus Nagelus (Thorne and Malek, 1968 Siddiqi, 1979 with a Description of Nagelus borealis n.sp. from Alaska 
Journal of Nematology  1983;15(4):582-593.
The genus Nagelus (Thorne and Malek, 1968) Siddiqi, 1979 is modified and a new species from Alaska is described. The combination of scanning electron microscopy and light microscopy permits the characterization of Nagelus spp. as having a broadly oval face pattern, no longitudinal striations on the lip region, deirids surrounded by six incisures, and an irregularly tapering tail with a large hyaline region. Nagelus leptus (Allen, 1955) Siddiqi, 1979, N. alpensis Doucet and Luc, 1981, N. camelliae (Kheiri, 1972) Siddiqi, 1979, N. jamelensis (Nesterov, 1973) Siddiqi, 1979, and N. obscurus (Allen, 1955) n. comb. are retained on this basis. Nagelus borealis n. sp. is characterized by a styler length of 30 μm or more, an irregularly scalloped perioral disc, and a proportionately larger basal bulb. Nagelus abalosi (Doucet, 1978) Doucet. 1980 and N. virginalis (Doucet, 1978) Doucet, 1980 are synonymized with N. leptus. Nine other species from Nagelus are transferred to Merlinius Siddiqi, 1970.
PMCID: PMC2618320  PMID: 19295852
Merliniinae; taxonomy; Tylenchorhynchidae; scanning electron microscopy

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