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Cultivated potato is a clonally propagated autotetraploid species with a highly heterogeneous genome. Phased assemblies of six cultivars including two chromosome-scale phased genome assemblies revealed extensive allelic diversity, including altered coding and transcript sequences, preferential allele expression, and structural variation that collectively result in a highly complex transcriptome and predicted proteome, which are distributed across the homologous chromosomes. Wild species contribute to the extensive allelic diversity in tetraploid cultivars, demonstrating ancestral introgressions predating modern breeding efforts. As a clonally propagated autotetraploid that undergoes limited meiosis, dysfunctional and deleterious alleles are not purged in tetraploid potato. Nearly a quarter of the loci bore mutations are predicted to have a high negative impact on protein function, complicating breeder's efforts to reduce genetic load. The StCDF1 locus controls maturity, and analysis of six tetraploid genomes revealed that 12 allelic variants of StCDF1 are correlated with maturity in a dosage-dependent manner. Knowledge of the complexity of the tetraploid potato genome with its rampant structural variation and embedded deleterious and dysfunctional alleles will be key not only to implementing precision breeding of tetraploid cultivars but also to the construction of homozygous, diploid potato germplasm containing favorable alleles to capitalize on heterosis in F1 hybrids., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

Introduction: Nosocomial infections (NIs) are a major source of iatrogenic harm in neonatal intensive care units (NICUs). The influence of the infrastructure of NICUs on NIs is not well documented. This study aims to examine the effect of single-room units (SRU) versus open-bay units (OBU) on the incidence of NIs, including central-line-associated bloodstream infections (CLABSI), in preterm neonates., Methods: All preterm neonates (< 32 weeks gestational age) admitted to our NICU were included. Two study periods were compared: one prior to (May 2015-May 2017) and one following (May 2017-May 2019) transition from OBU to SRU. Incidence density (number of infections per 1000 patient-days) and cumulative incidence (number of infections per 100 neonates) for NIs were calculated. CLABSIs were calculated per 1000 central-line days. U chart analysis was performed to determine special-cause variation in quarterly CLABSI and NI rates. Multivariate competing risk regression was performed to identify independent NI risk factors., Results: Of the 712 included infants, 164 (23%) infants acquired ≥ 1 NIs. No differences were found in incidence density (13.68 vs. 12.62, p = 0.62) or cumulative incidence of NI (23.97 vs. 22.02, p = 0.59) between OBU and SRU. CLABSIs showed a similar non-significant reduction after the move (14.00 vs. 10.59, p = 0.51). U chart analysis did not identify unit transition as a potential source of special-cause variation for CLABSI and NI. Competing risks regression analysis revealed longer duration of invasive mechanical ventilation as a significant risk factor for NI (subhazards ratio: 1.03 per day on ventilation, p = 0.01)., Conclusion: Single-rooms are not associated with a significant reduction in NIs in the NICU. This study therefore does not add evidence that could support the transition to SRUs if based only on a large multimodal infection control strategy. Recommendations to build SRUs would require a wider justification, also taking into account other SRU benefits.

Introduction: Nosocomial infections (NIs) are a major source of iatrogenic harm in neonatal intensive care units (NICUs). The influence of the infrastructure of NICUs on NIs is not well documented. This study aims to examine the effect of single-room units (SRU) versus open-bay units (OBU) on the incidence of NIs, including central-line-associated bloodstream infections (CLABSI), in preterm neonates., Methods: All preterm neonates (< 32 weeks gestational age) admitted to our NICU were included. Two study periods were compared: one prior to (May 2015-May 2017) and one following (May 2017-May 2019) transition from OBU to SRU. Incidence density (number of infections per 1000 patient-days) and cumulative incidence (number of infections per 100 neonates) for NIs were calculated. CLABSIs were calculated per 1000 central-line days. U chart analysis was performed to determine special-cause variation in quarterly CLABSI and NI rates. Multivariate competing risk regression was performed to identify independent NI risk factors., Results: Of the 712 included infants, 164 (23%) infants acquired ≥ 1 NIs. No differences were found in incidence density (13.68 vs. 12.62, p  = 0.62) or cumulative incidence of NI (23.97 vs. 22.02, p  = 0.59) between OBU and SRU. CLABSIs showed a similar non-significant reduction after the move (14.00 vs. 10.59, p  = 0.51). U chart analysis did not identify unit transition as a potential source of special-cause variation for CLABSI and NI. Competing risks regression analysis revealed longer duration of invasive mechanical ventilation as a significant risk factor for NI (subhazards ratio: 1.03 per day on ventilation, p  = 0.01)., Conclusion: Single-rooms are not associated with a significant reduction in NIs in the NICU. This study therefore does not add evidence that could support the transition to SRUs if based only on a large multimodal infection control strategy. Recommendations to build SRUs would require a wider justification, also taking into account other SRU benefits., Supplementary Information: The online version contains supplementary material available at 10.1007/s40121-020-00380-9., (© The Author(s) 2020.)

Objective: Impaired ventricular performance in patients who underwent surgery for congenital heart defect (CHD) may ultimately result in clinical heart failure. It is therefore important to regularly evaluate the left ventricular (LV) and right ventricular (RV) performance. We evaluated tissue Doppler imaging (TDI) and speckle tracking echocardiography (STE) before and after surgical correction of a CHD., Study Design: Patients with CHD undergoing biventricular corrective surgery were included in this retrospective study. In addition, a group of healthy controls was included. The performance of LV, RV and interventricular septum (IVS) was assessed using mitral inflow velocities, TDI peak systolic (S'), peak early (E') and peak late (A') diastolic velocity. LV and RV global longitudinal strain (GLS) were assessed using STE. Measurements were performed preoperatively and at discharge., Results: A total of 204 patients and 78 controls were included. All TDI measurements were significantly decreased after surgery. Nonetheless, LV S' and A' were still within normal limits. LV and RV GLS were significantly reduced after surgery and significantly lower compared to controls. A longer aortic cross-clamp time and cardiopulmonary bypass (CPB) time were associated with significantly lower TDI and strain parameters in the RV and IVS, but not in the LV., Conclusion: TDI and STE results generally imply a significantly lower ventricular performance in patients with CHD postoperatively. Furthermore, a negative correlation was observed between aortic cross-clamping and CPB time and several RV performance parameters. As this association was not observed in the LV, this could implicate less vulnerability of the LV to cardiopulmonary bypass., (© 2017, Wiley Periodicals, Inc.)

Background: In flowering plants it has been shown that de novo genome assemblies of different species and genera show a significant drop in the proportion of alignable sequence. Within a plant species, however, it is assumed that different haplotypes of the same chromosome align well. In this paper we have compared three de novo assemblies of potato chromosome 5 and report on the sequence variation and the proportion of sequence that can be aligned., Results: For the diploid potato clone RH89-039-16 (RH) we produced two linkage phase controlled and haplotype-specific assemblies of chromosome 5 based on BAC-by-BAC sequencing, which were aligned to each other and compared to the 52 Mb chromosome 5 reference sequence of the doubled monoploid clone DM 1-3 516 R44 (DM). We identified 17.0 Mb of non-redundant sequence scaffolds derived from euchromatic regions of RH and 38.4 Mb from the pericentromeric heterochromatin. For 32.7 Mb of the RH sequences the correct position and order on chromosome 5 was determined, using genetic markers, fluorescence in situ hybridisation and alignment to the DM reference genome. This ordered fraction of the RH sequences is situated in the euchromatic arms and in the heterochromatin borders. In the euchromatic regions, the sequence collinearity between the three chromosomal homologs is good, but interruption of collinearity occurs at nine gene clusters. Towards and into the heterochromatin borders, absence of collinearity due to structural variation was more extensive and was caused by hemizygous and poorly aligning regions of up to 450 kb in length. In the most central heterochromatin, a total of 22.7 Mb sequence from both RH haplotypes remained unordered. These RH sequences have very few syntenic regions and represent a non-alignable region between the RH and DM heterochromatin haplotypes of chromosome 5., Conclusions: Our results show that among homologous potato chromosomes large regions are present with dramatic loss of sequence collinearity. This stresses the need for more de novo reference assemblies in order to capture genome diversity in this crop. The discovery of three highly diverged pericentric heterochromatin haplotypes within one species is a novelty in plant genome analysis. The possible origin and cytogenetic implication of this heterochromatin haplotype diversity are discussed.

One of the most powerful technologies in unraveling the organization of a eukaryotic plant genome is high-resolution Fluorescent in situ hybridization of repeats and single copy DNA sequences on pachytene chromosomes. This technology allows the integration of physical mapping information with chromosomal positions, including centromeres, telomeres, nucleolar-organizing region, and euchromatin and heterochromatin. In this report, we established chromosomal positions of different repeat fractions of the potato genomic DNA (Cot100, Cot500 and Cot1000) on the chromosomes. We also analysed various repeat elements that are unique to potato including the moderately repetitive P5 and REP2 elements, where the REP2 is part of a larger Gypsy-type LTR retrotransposon and cover most chromosome regions, with some brighter fluorescing spots in the heterochromatin. The most abundant tandem repeat is the potato genomic repeat 1 that covers subtelomeric regions of most chromosome arms. Extensive multiple alignments of these repetitive sequences in the assembled RH89-039-16 potato BACs and the draft assembly of the DM1-3 516 R44 genome shed light on the conservation of these repeats within the potato genome. The consensus sequences thus obtained revealed the native complete transposable elements from which they were derived.

Potato (Solanum tuberosum L.) originates from the Andes and evolved short-day-dependent tuber formation as a vegetative propagation strategy. Here we describe the identification of a central regulator underlying a major-effect quantitative trait locus for plant maturity and initiation of tuber development. We show that this gene belongs to the family of DOF (DNA-binding with one finger) transcription factors and regulates tuberization and plant life cycle length, by acting as a mediator between the circadian clock and the StSP6A mobile tuberization signal. We also show that natural allelic variants evade post-translational light regulation, allowing cultivation outside the geographical centre of origin of potato. Potato is a member of the Solanaceae family and is one of the world's most important food crops. This annual plant originates from the Andean regions of South America. Potato develops tubers from underground stems called stolons. Its equatorial origin makes potato essentially short-day dependent for tuberization and potato will not make tubers in the long-day conditions of spring and summer in the northern latitudes. When introduced in temperate zones, wild material will form tubers in the course of the autumnal shortening of day-length. Thus, one of the first selected traits in potato leading to a European potato type is likely to have been long-day acclimation for tuberization. Potato breeders can exploit the naturally occurring variation in tuberization onset and life cycle length, allowing varietal breeding for different latitudes, harvest times and markets.

Background: The presence of an inflammatory response resulting from bowel perforation or anastomotic leakage has been suggested to enhance recurrence rates in colorectal cancer patients. Currently, it is unknown if bowel perforation or anastomotic leakage has prognostic significance in early stage colon cancer patients. In this study, the impact of peri-operative bowel perforation including anastomotic leakage on disease-free survival of stage I/II colon cancer patients was investigated., Methods: Prospective follow up data of 448 patients with stages I/II colon cancer that underwent resection were included. Patients who died within 3 months after initial surgery were excluded., Results: Median follow up was 56.0 months. Patients with peri-operative bowel perforation (n = 25) had a higher recurrence rate compared to patients without perforation (n = 423), 36.0 % vs. 16.1 % (p = 0.01). Disease-free survival was significantly worse for the perforation group compared to patients without perforation (p = 0.004). Multivariate analysis including T-stage, histological grade, and adjuvant chemotherapy showed peri-operative bowel perforation to be an independent factor significantly associated with disease recurrence (odds ratio, 2.7; 95 % CI, 1.1-6.7)., Conclusion: Peri-operative bowel perforation is associated with increased recurrence rates and impaired disease-free survival in early-stage colon cancer patients.

Background: Potato is the world's third most important food crop, yet cultivar improvement and genomic research in general remain difficult because of the heterozygous and tetraploid nature of its genome. The development of physical map resources that can facilitate genomic analyses in potato has so far been very limited. Here we present the methods of construction and the general statistics of the first two genome-wide BAC physical maps of potato, which were made from the heterozygous diploid clone RH89-039-16 (RH)., Results: First, a gel electrophoresis-based physical map was made by AFLP fingerprinting of 64478 BAC clones, which were aligned into 4150 contigs with an estimated total length of 1361 Mb. Screening of BAC pools, followed by the KeyMaps in silico anchoring procedure, identified 1725 AFLP markers in the physical map, and 1252 BAC contigs were anchored the ultradense potato genetic map. A second, sequence-tag-based physical map was constructed from 65919 whole genome profiling (WGP) BAC fingerprints and these were aligned into 3601 BAC contigs spanning 1396 Mb. The 39733 BAC clones that overlap between both physical maps provided anchors to 1127 contigs in the WGP physical map, and reduced the number of contigs to around 2800 in each map separately. Both physical maps were 1.64 times longer than the 850 Mb potato genome. Genome heterozygosity and incomplete merging of BAC contigs are two factors that can explain this map inflation. The contig information of both physical maps was united in a single table that describes hybrid potato physical map., Conclusions: The AFLP physical map has already been used by the Potato Genome Sequencing Consortium for sequencing 10% of the heterozygous genome of clone RH on a BAC-by-BAC basis. By layering a new WGP physical map on top of the AFLP physical map, a genetically anchored genome-wide framework of 322434 sequence tags has been created. This reference framework can be used for anchoring and ordering of genomic sequences of clone RH (and other potato genotypes), and opens the possibility to finish sequencing of the RH genome in a more efficient way via high throughput next generation approaches.

A cytogenetic map has been developed for diploid potato (Solanum tuberosum), in which the arms of the 12 potato bivalents can be identified in pachytene complements using multicolor fluorescence in situ hybridization (FISH) with a set of 60 genetically anchored bacterial artificial chromosome (BAC) clones from the RHPOTKEY BAC library. This diagnostic set of selected BACs (five per chromosome) hybridizes to euchromatic regions and corresponds to well-defined loci in the ultradense genetic map, and with these probes a new detailed and reliable pachytene karyotype could be established. Chromosome size has been estimated both from microscopic length measurements and from 4',6-diamidino-2-phenylindole fluorescence-based DNA content measurements. In both approaches, chromosome 1 is the largest (100-115 Mb) and chromosome 11 the smallest (49-53 Mb). Detailed measurements of mega-base-pair to micrometer ratios have been obtained for chromosome 5, with average values of 1.07 Mb/mum for euchromatin and 3.67 Mb/mum for heterochromatin. In addition, our FISH results helped to solve two discrepancies in the potato genetic map related to chromosomes 8 and 12. Finally, we discuss the significance of the potato cytogenetic map for extended FISH studies in potato and related Solanaceae, which will be especially beneficial for the potato genome-sequencing project.

Summary A microarray was printed containing cDNAs from a library made from cytoplasm microaspirated from the oesophageal gland cell region of parasitic stages of the soybean cyst nematode, Heterodera glycines. The array contained both previously described clones (Wang et al. Mol. Plant-Microbe Interact. 2001, 14, 536-544) and uncharacterized cDNAs. Fluorescent probes for array hybridization were prepared using RNA polymerase amplification of nematode mRNA. Developmental expression profiles of the arrayed cDNAs were determined by hybridizing the microarray with probes from parasitic and non-parasitic H. glycines life stages. Distinct patterns of developmental expression were ascertained for the previously described gland expressed genes. In addition, four H. glycines cDNAs (SCN1018, SCN1020, SCN1028 and SCN1167) were identified that showed up-regulation in one or more parasitic life stages. Clone SCN1018 encodes a C-type lectin domain and is expressed in the hypodermis of females. Clone SCN1020 encodes a probable S-adenosylmethionine synthetase. Clone SCN1028 encodes a piwi protein with high similarity to the germ-line-specific protein R06C7.1 of Caenorhabditis elegans. The sequence of SCN1167 had no similarity to known genes. This paper describes the first use of cDNA microarrays to analyse genes of a plant-parasitic nematode and establishes a functional method to mine nematode cDNA libraries.

We report the cloning of a Heterodera glycines cDNA that has 72% identity at the amino acid level to a pectate lyase from Globodera rostochiensis. In situ hybridizations showed that the corresponding gene (Hg-pel-1) is expressed in the subventral esophageal gland cells of second-stage juveniles. The deduced amino acid sequence of the H. glycines cDNA shows homology to class III pectate lyases of bacterial and fungal origin.

Secretions from the esophageal gland cells of plant-parasitic nematodes play critical roles in the nematode-parasitic cycle. A novel method to isolate cDNA encoding putative nematode secretory proteins was developed that utilizes mRNA for reverse transcription-polymerase chain reaction derived from microaspiration of the esophageal gland cell contents of parasitic stages of the soybean cyst nematode Heterodera glycines. The resulting H. glycines gland cell cDNA was cloned into the pRK18 vector, and plasmid DNA was transformed into a mutated yeast host for specific selection of cDNA inserts that encode proteins with functional signal peptides. Of the 223 cDNA clones recovered from selection in yeast, 97% of the clones encoded a predicted signal peptide. Fourteen unique cDNA clones hybridized to genomic DNA of H. glycines on Southern blots and, among them, nine cDNA clones encoded putative extracellular proteins, as predicted by PSORT II computer analysis. Four cDNA clones hybridized to transcripts within the dorsal esophageal gland cell of parasitic stages of H. glycines, and in situ hybridization within H. glycines was not detected for eight cDNA clones. The protocol provides a direct means to isolate potential plant-parasitic nematode esophageal gland secretory protein genes.

Two beta-1,4-endoglucanase (EGase) cDNAs were isolated from Globodera tabacum, the tobacco cyst nematode, and have been designated as GT-eng-1 and GT-eng-2. GT-eng-1 and GT-eng-2 encode precursor proteins with a predicted secretion signal sequence, cellulolytic catalytic domain, and a linker domain. The protein product GT-ENG-1 contains an additional 95 amino acid carboxy terminal sequence with strong similarity to type II cellulose binding domains. Riboprobes and polyclonal antibodies raised to recombinant cyst nematode EGases were used to follow expression patterns of EGase transcripts and proteins throughout the nematode life cycle. EGase transcripts and proteins were specifically detected within the subventral esophageal gland cells of G. tabacum second-stage juveniles (J2) within eggs prior to hatching, in preparasitic J2, and in parasitic J2 that had invaded tobacco roots. EGase transcripts and proteins were not detected in G. tabacum after the molt to the sedentary J3, J4, and adult female life stages. Interestingly, EGase transcription and translation resumed in the subventral esophageal glands of late J4 males. It is hypothesized that secreted EGases play a major role to facilitate intracellular migration of G. tabacum within tobacco roots.

Two beta-1,4-endoglucanases (EGases), Hg-eng-1 and Hg-eng-2, were recently cloned from the soybean cyst nematode, Heterodera glycines, and their expression was shown in the subventral esophageal glands of hatched second-stage juveniles (J2). We examined the expression of these EGases in the subventral glands of all post-embryonic life stages of H. glycines by in situ hybridization and immunolocalization. The first detectable accumulation of EGase mRNAs occurred in the subventral glands of unhatched J2. EGase transcripts remained detectable in J2 after hatching and during subsequent root invasion. However, in late parasitic J2 and third-stage juveniles (J3), the percentage of individuals that showed EGase transcripts decreased. In female fourth-stage juveniles and adult females, EGase transcripts were no longer detected in the subventral glands. EGase hybridization signal reappeared in unhatched males coiled within the J3 cuticle, and transcripts were also present in the subventral glands of migratory adult males. Immunofluorescence labeling showed that EGase translation products are most abundantly present in the subventral glands of preparasitic J2, migratory parasitic J2, and adult males. The presence of EGases predominantly in the migratory stages suggests that the enzymes are used by the nematodes to soften the walls of root cells during penetration and intracellular migration.

A beta-1,4-endoglucanase encoding cDNA (EGases, E.C. 3.2.1.4), named Mi-eng-1, was cloned from Meloidogyne incognita second-stage juveniles (J2). The deduced amino acid sequence contains a catalytic domain and a cellulose-binding domain separated by a linker. In M. incognita, the gene is transcribed in the migratory J2, in males, and in the sedentary adult females. In pre-parasitic J2, endoglucanase transcripts are located in the cytoplasm of the subventral esophageal glands. The presence of beta-1,4-endoglucanase transcripts in adult females could be related to the expression of the gene in esophageal glands at this stage. However, cellulase activity within the egg matrix of adult females suggests that the endoglucanase may also be synthesized in the rectal glands and involved in the extrusion of the eggs onto the root surface. The maximum identity of the predicted MI-ENG-1 catalytic domain with the recently cloned cyst nematode beta-1,4-endoglucanases is 52.5%. In contrast to cyst nematodes, M. incognita pre-parasitic J2 were not found to express a beta-1,4-endoglucanase devoid of a cellulose-binding domain.

A method is presented for in-situ hybridization to mRNA in second-stage juveniles (J2) of the soybean cyst nematode Heterodera glycines. The protocol was developed using a digoxigenin-labeled RNA probe transcribed from cDNA of a cellulase gene that was known to be expressed in the subventral esophageal glands of H. glycines. Formaldehyde-fixed J2 were cut into sections with a vibrating razor blade to make the inside of the nematodes accessible for probing. These nematode fragments then were hybridized in suspension with riboprobe, and labeled with an alkaline phosphatase-conjugated antibody to digoxigenin. Staining with nitroblue tetrazolium and bromo-chloro-indolyl phosphate revealed a highly specific hybridization signal to mRNA within the cytoplasm of the subventral gland cells, using this specific antisense probe. This in-situ hybridization protocol will be useful for the characterization and identification of esophageal gland secretion genes in plant-parasitic nematodes, among other applications.

beta-1,4-Endoglucanases (EGases, EC 3.2.1.4) degrade polysaccharides possessing beta-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37-44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi, Clostridium acetobutylicum, and Bacillus subtilis. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3' end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.

Expression of single-chain antibody fragments (scFvs) in the plant cytosol is often cumbersome. It was unexpectedly shown that addition at the C-terminus of the ER retention signal KDEL resulted in significantly improved expression levels. In this report the cytosolic location of the scFv-CK was confirmed, excluding possible mistranslocation to other subcellular compartments. It was shown that expression of several other scFvs was also improved in tobacco protoplasts. In addition expression was improved in transgenic potato. Changing from KDEL to KDEI did not affect the enhanced protein expression level. Addition of the KDEL motif is a simple and straightforward tool to stabilize in planta cytosolic expression of many scFvs.

ABSTRACT In preparasitic second-stage juveniles (J(2)) of potato cyst nematode Globodera rostochiensis, six proteins with molecular masses of 30, 31a/b, 32, 39, and 49 kDa were recognized on Western blots by a monoclonal antibody (MGR48) specific for the subventral esophageal glands. All of these subventral gland proteins (svp's) focused in the basic range (pI 6.8 to 8.6) of an immobilized pH gradient. Western blotting showed that the svp's were present in preparasitic and parasitic J(2) and not in later juvenile stages and adult females. Minor svp quantities also were observed in adult males. Immunogold labeling of preparasitic J(2) showed that the svp's were localized in the rough endoplasmic reticulum and secretory granules of the subventral esophageal glands. Potato root diffusate triggered the secretion of svp's through the stylet, and 5-methoxy-N,N-dimethyltryptamine-hydrogen-oxalate had only a quantitative, additional effect. The forward flow of svp's through the metacorporal pump chamber was confirmed by the presence of svp's in the circular lumen of the esophagus (procorpus), as established by immunoelectron microscopy. Our data provide conclusive evidence that secretory proteins of the subventral glands of G. rostochiensis can be secreted through the stylet and support the hypothesis that the subventral esophageal glands play an important role in the early events of this nematode-plant interaction.

Sodium dodecyl sulfate-extracted proteins from second-stage juveniles (J2) of the potato cyst nematode Globodera rostochiensis were fractionated by preparative continuous flow electrophoresis, and monoclonal antibodies (MAbs) were raised against the 38- to 40.5-kDa protein fraction. Screening of the hybridoma culture fluids by immunofluorescence microscopy of J2 resulted in the identification of 12 MAbs that bound specifically to the subventral esophageal glands. On Western blots of J2 these MAbs identified four protein bands with apparent molecular masses of 30, 31, 39, and 49 kDa. Immunoelectron microscopy with one of these MAbs showed an intense labeling of the electron dense core of the secretory granules in the subventral gland cells of J2. It is concluded that one or more of these proteins are localized within these secretory granules. Immunofluorescence microscopy of J2 from other plant parasitic nematode species showed that most of these MAbs also bind to the subventral glands of G. pallida and G. tabacum but not of Heterodera schachtii, H. glycines, Meloidogyne incognita, or M. hapla.

From patients with bacterial vaginosis motile, anaerobic, comma-shaped bacteria can be isolated, which have recently been placed into the new genus Mobiluncus. In this study, electron microscopy was used to examine the in situ adherence of these motile curved rods to detached epithelial cells (comma cells) in vaginal fluid from two patients with bacterial vaginosis. Thin sections showed that the curved rods attached both directly to the epithelial cell surface and at various distances from it. It is concluded that after initial attachment these motile bacteria can grow at the epithelial cell surface in sessile microcolonies. Ruthenium red staining demonstrated a coating of precipitated glycocalyx material both on the surface of the curved rods and on their flagella. This may indicate that in situ the adherent curved rods were enclosed in a very hydrated matrix of exopolysaccharides. Conspicuous was the ability of the curved rods to attach to the epithelial cell surface via their cell tips. However, in situ no specialized bacteria cell surface structures were seen that might explain this polar attachment. Electron microscopy of pure cultures demonstrated that both Mobiluncus curtisii subsp. curtisii and Mobiluncus mulieris can produce a glycocalyx in vitro.