Strong Positive Signal Research Articles

Comparative evaluation of specific methods for labeling of Encephalitozoon cuniculi in paraffin wax–embedded tissue samples

Detection of the microsporidian Encephalitozoon cuniculi in tissue samples is considered difficult. The aim of the current study was to determine whether immunohistochemistry (IHC) and in situ hybridization (ISH) represent reliable methods for the detection of E. cuniculi in postmortem tissue samples of rabbits. Paraffin-embedded tissue sections of brain and kidneys of 48 naturally infected pet rabbits, 10 negative controls, and the eyes of 3 further rabbits were used for all investigations. By IHC in 19 animals (37.3%), spores could be clearly detected and were all equally stained. By ISH using a digoxigenin-labeled oligonucleotide probe, only 6 animals (11.8%) proved undoubtedly positive. In these cases, many parasite-like objects revealed strong typical purple-black positive signals. However, several of the examined samples showed only partial staining of the pathogen or unclear results. Thus, in order to find an explanation for these inconsistent ISH results and to take a more detailed look at the different developmental stages of the organism, electron microscopy was applied. Empty spores, which had already discharged their polar filaments, prevailed in total number. Taken together, both techniques are rather insensitive, but under the condition that sufficient numbers of microsporidia are present, IHC can be recommended for specific identification of E. cuniculi in tissue samples. In contrast, ISH failed to detect some developmental stages of the organism, and, as such, ISH is therefore considered an inappropriate diagnostic method.

득량만의 조개류 생산량과 환경요인 관계 분석

This study was carried out to determine marine environments and phytoplankton community in Deukryang Bay during the period of summer in 1987-2010. Water temperature, salinity, pH and dissolved oxygen were shown in much yearly fluctuations. In August, water temperatures in surface and on bottom were the highest, compared with average surface (24.54<TEX>$^{\circ}C$</TEX>) and bottom (22.90<TEX>$^{\circ}C$</TEX>) water temperature for 18 years in Deukryang Bay. The main reason is assumed to longer duration of sunshine during the period of August. Although the amount of the rainfall in August was the highest, significant impact of marine environment did not show. Most of dissolved inorganic nitrogen and phosphate in Deukryang were lower concentration during summer and N:P ratio also showed below 18 in Redfield. In particular, extreme increasing of N:P ratio in August was occurred by intensive precipitation. Distribution of phytoplankton community was a consistent occurrence for 18 years. The genus of Chaetoceros, Cosinodisucs and Skeletonema were regarded as the represent diatom, whereas the highest occurrence of genus among dinofagellates was Ceratium. It is thought that the relationship between phytoplankton and nutrient has a strong positive signal, although nutrients persist a little concentration and much fluctuations in marine environments were observed. High availability in phytoplankton is contributed to consistently provide the food organism of shellfish. Consequently, recent decreasing production of shellfish and seed are probably associated with higher temperature during the period of summer. However, higher temperature is also occurred ago and after 2000. On the basis of geography, Deukryang Bay had a small mouth and long channel, which is attributed to decreasing genetic diversity. It is assumed that higher temperature and lower genetic diversity have a extreme impact of larvae and shellfish for reproduction in Deukryang. It is necessary to persistently monitor based on water quality and phytoplankton community.

Activity and immunodetection of lysozyme in earthworm Dendrobaena veneta (Annelida)

In the present study, lysozyme-like activity against Micrococcus luteus was detected in the coelomic fluid, the extract from coelomocytes, intestine and in the homogenates from cocoons of Dendrobaena veneta. Four hours after immunization with Escherichia coli, the lysozyme activity in the coelomic fluid increased about three times and in the extract of coelomocytes – four times, in comparison to the control. In three cases: of the coelomic fluid, the homogenates from cocoons and the extract from coelomocytes, the antibody against HEWL (hen egg white lysozyme) recognized only one protein with a molecular mass of about 14.4kDa. In the coelomic fluid, apart from the protein with molecular mass of 14.4kDa the antibody directed against human lysozyme recognized an additional protein of 22kDa. Using the bioautography technique after electrophoretic resolution of native proteins in acidic polyacrylamide gels, two lytic zones of M. luteus were observed in the case of the coelomic fluid and three after the analysis of the extract of coelomocytes and the egg homogenates. The results indicated the existence of several forms of lysozyme with a different electric charge in the analyzed D. veneta samples. The highest lysozyme activity in the intestine of D. veneta was observed in the midgut. The antibody directed against human lysozyme indicated a strong positive signal in epidermal and midgut cells of earthworm.

Betaine homocysteine methyl transferase 1, a novel auto-antigen associated with anti-Golgi immune reactivity

Anti-Golgi antibodies are rare autoantibodies that have been described in systemic autoimmune diseases. Not all Golgi auto-antigens are known. The objective of this study was to identify a novel auto-antigen associated with anti-Golgi immune reactivity.Sera from a patient with Golgi immune reactivity and from a control individual were used for Western blotting after 2-dimensional gel separation of a rat Golgi-enriched extract.Betaine homocysteine S-methyltransferase 1 (BHMT1) was identified as an auto-antigen by MALDI-TOF/TOF mass spectrometry. Using human recombinant BHMT1, a strong positive blotting signal was obtained with serum from the patient but not from a control. Pre-absorption of the serum sample with reactivity to BHMT1 with recombinant human BHMT1 resulted in decreased reactivity on Western blotting and in disappearance of the Golgi-like pattern on indirect immunofluorescence. Using immunocytochemistry, we confirmed the subcellular localization of BHMT1 to the Golgi apparatus. Antibodies to BHMT1 were found in four of 80 samples with a Golgi-pattern on indirect immunofluorescence. The antibodies were not associated with a specific clinical condition.We identified BHMT1 as a novel auto-antigen associated with anti-Golgi immune reactivity.

Expression of secreted mucins (MUC2, MUC5AC, MUC5B, and MUC6) and membrane-bound mucin (MUC4) in the lungs of pigs experimentally infected with Actinobacillus pleuropneumoniae

The expression patterns of different secreted (MUC2, MUC5AC, MUC5B, and MUC6) and membrane-bound (MUC4) mucins were determined immunohistochemically in the lungs of pigs experimentally infected with Actinobacillus pleuropneumoniae. Forty-seven-week-old colostrum-deprived pigs were randomly allocated to infected (n=20) or control groups (n=20). Five infected and uninfected pigs were euthanized at 0, 6, 12, and 48h post-inoculation (hpi). In the infected pigs, the expression of both types of mucins, which were invariably observed, was associated with bronchiolar and respiratory bronchiolar lesions. Strong positive mucin signals were seen on the surface of bronchiolar and respiratory bronchiolar epithelium with neutrophil infiltration. The mean mucin-positive area peaked at 6hpi and decreased significantly to control levels by 48hpi on the surface of the bronchiolar and respiratory bronchiolar epithelium. Further studies are needed to establish the functional relationship between mucin expression and the host defense mechanism against A. pleuropneumoniae in the lungs of infected pigs.

Vascular Gene Transfer of SDF-1 Promotes Endothelial Progenitor Cell Engraftment and Enhances Angiogenesis in Ischemic Muscle

Gene therapy approaches to enhance endothelial progenitor cell (EPC) homing may augment cell engraftment to ischemic tissue and lead to a greater therapeutic response. Therefore, we assessed the effects of ultrasound-mediated (UM) transfection of the chemokine stromal cell-derived factor-1 (SDF-1) on homing and engraftment of intravenously administered EPCs and the subsequent angiogenic response in chronically ischemic skeletal muscle. Bone marrow-derived EPCs were isolated from donor Fisher 344 rats, cultured and labeled in preparation for injection into recipient animals via a jugular vein. Using a model of chronic hindlimb ischemia in rats, we demonstrated that UM destruction of intravenous carrier microbubbles loaded with SDF-1 plasmid DNA resulted in targeted transfection of the vascular endothelium within ischemic muscle and greater local engraftment of EPCs. The combination of SDF-1gene therapy and EPCs lead to the greatest increase in tissue perfusion and microvascular density within ischemic muscle, compared to no treatment or either monotherapy alone. Our results demonstrate that UM transfection of SDF-1 improves EPC targeting to chronically ischemic tissue, enhancing vascular engraftment and leading to a more robust neovascularization response.

The defensin gene family expansion in the tick Ixodes scapularis

Ixodid ticks transmit a variety of pathogens by blood feeding. Here, we report computational identification of two multigene families of defensin-like peptides (DLPs) in the Ixodes scapularis genome, one corresponding to scapularisin and the other named scasin. Members in the scapularisin family share high sequence similarity to some antibacterial ancient invertebrate-type defensins (AITDs) isolated from primitive insects, arachnids, bivalvia, and fungi whereas scasins represent a novel family of DLPs identified by their overall acidic molecular surface and low sequence similarity to any known defensins. Codon-substitution models support neutral evolution in scapularisins but strong positive selection signal was found throughout the molecules of scasins. The synthetic γ-core region of scapularisin-20 exhibits a wide-spectrum of antimicrobial activity at micromolar concentrations. The finding of extensive gene expansion of DLPs in a vector arachnida may be valuable in the understanding of its role in pathogen transmission.

Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration

Several adult reptiles, such as Gekko japonicus, have the ability to precisely re-create a missing tail after amputation. To ascertain the associated acquisition of positional information from blastemal cells and the underlying molecular mechanism of tail regeneration, a candidate molecule CD59 was isolated from gecko. CD59 transcripts displayed a graded expression in the adult gecko spinal cord with the highest level in the anterior segment, with a stable expression along the normal tail. After tail amputation, CD59 transcripts in the spinal cord proximal to the injury sites increased markedly at 1 day and 2 weeks; whereas in the regenerating blastema, strong CD59 positive signals were detected in the blastemal cells anterior to the blastema, with a gradual decrease along the proximodistal (PD) axis. When treated with RA following amputation, CD59 transcripts in the blastema were up-regulated. PD confrontation assays revealed that the proximal blastema engulfed the distal one after in vitro culture, and rabbit-anti human CD59 antibody was able to block this PD engulfment. Overexpression of the CD59 during tail regeneration causes distal blastemal cells to translocate to a more proximal location. Our results suggest that position identity is not restricted to amphibian limb regeneration, but has already been established in tail blastema of reptiles. The CD59, a cell surface molecule, acted as a determinant of proximal–distal cell identity.

Identification of Fusarium Species in Formalin-Fixed and Paraffin-Embedded Sections by In Situ Hybridization Using Peptide Nucleic Acid Probes

Fusarium has recently emerged as an opportunistic pathogen of humans, but the histological differentiation of Fusarium from Aspergillus and Scedosporium is particularly difficult because these fungi may induce similar clinical features and exhibit filamentous development in host tissues. Thus, there is a need to establish rapid and reliable methods that are applicable to pathological diagnoses. The aim of this study was to evaluate and establish in situ hybridization (ISH) using peptide nucleic acid (PNA) probes targeting the 28S rRNA to identify Fusarium species in tissue sections. This technique was validated using both formalin-fixed and paraffin-embedded pulmonary tissues from mice infected with seven different species of fungi and cell blocks from fungal cultures of 30 strains. As a result, strong positive signals were observed within fungal organisms present in tissues of the lung from mice infected with Fusarium solani. Furthermore, this probe reacted strongly with both F. solani and Fusarium oxysporum in sections from cell blocks. Although some cross-reactivity occurred with the Pseudallescheria boydii in sections from cell blocks, the signal intensity was low and most hyphae were not reactive. In conclusion, it was confirmed that ISH with PNA probes is accurate and is a valuable tool for identifying Fusarium spp. among organisms that have identical morphological features in formalin-fixed and paraffin-embedded sections.

Tyrosine Kinase Receptor Flt/VEGFR Family: Its Characterization Related to Angiogenesis and Cancer

Ligands and their tyrosine kinase (TK) receptors regulate a variety of biological systems in animals. Vascular endothelial growth factor (VEGF) and its receptor (Flt/VEGFR family) system play a crucial role not only in physiological but also in most parts of pathological angiogenesis including cancer. Flt-1/VEGFR-1 and KDR/VEGFR-2 bind VEGF-A but have different functions on angiogenesis at early embryogenesis: Flt-1 has a negative role by trapping ligands, whereas KDR (Flk1 in mice) exerts a strong positive signal, resulting in a balance in blood vessel formation. At adult stages, however, both VEGFRs contribute to pathological angiogenesis either directly or through stimulation of migration/activation of macrophage lineage cells and stimulate tumor growth, metastasis, and inflammation. VEGFRs activate downstream signaling of the phospholipase Cγ-protein kinase C-MAP kinase pathway but not Ras pathway for cell proliferation. The VEGF-C/D and Flt-4/VEGFR-3 system regulates lymphangiogenesis. Thus, VEGFs as well as these receptor TKs are attractive targets for suppressing pathological angiogenesis.

Detection of five common cell culture contaminant Molllcutes using rolling circle amplification

Objective To develop a novel padlock probe and rolling circle amplification(RCA) to detect 5 common cell culture contaminant Mollicute( Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycomplasma orale, and Acholeplasma laidlawii ). Methods Universal primers ( SPS1, SPA2 and SPS2, SPA1) were used to amplify the Mollicute 16S-23S rRNA intergenic spacer region.Amplicon was ligate Mollicute specific padlock probe. Probe amplified and monitored using a Corbett RotorGeneTM 6000 machine. Results Five reference Mollicute strains were correctly detected by RCA method.There was no cross-reaction. RCA method can sensitive detect 10 copies templates and show strong positive signal. Sixty-two cell culture specimens were detected. Thirty-seven samples were contained single specie Mollicute and 14 samples contained two Mollicutes. Eleven samples did not contained Mollicute. RCA detection results were concordant with previously species-specific PCR. Conclusion RCA can rapid, sensitive and specific detect contamination Mollicute. Key words: Rolling circle amplification; Padlock probe

A possible rare cause of renal failure in streptococcal infection

To the best of our knowledge, this is the first biopsy-proven case of streptococcal infection-associated acute interstitial nephritis (AIN) with existence of streptococcal pyrogenic exotoxin B (SPE B) by a controlled immunohistochemical method. Both the intact tubular epithelial cells and oedematous interstitium had strong positive signals, whereas only interstitial inflammation was dominant without tubular necrosis. Reflective of the nature of AIN is that the injury from the hypersensitivity reaction was specific for renal interstitium instead of tubules. SPE B is potentially allergenic and may confuse the clinicians due to its clinical mimicry of drug-induced AIN. Although very rare, AIN might be included into the differential diagnosis of patients with streptococcal sepsis and acute renal failure.

Developmental expression of sorting nexin 3 in the mouse central nervous system

We previously reported that sorting nexin 3 (SNX3), a protein belonging to the sorting nexin family, regulates neurite outgrowth in mouse N1E-115 neuroblastoma cells. The snx3 gene is disrupted in patients with microcephaly, microphthalmia, ectrodactyly, and prognathism (MMEP) and mental retardation, demonstrating that SNX3 plays an important role in the genesis of these organs during development. The present study was designed to determine the expression pattern of snx3 mRNA, particularly in the mouse central nervous system (CNS), from the embryonic stage to adulthood. Whole mount in situ hybridization of embryonic day (E) 9.5 and 10.5 mouse embryos revealed strong positive signals for snx3 mRNA in the forebrain, pharyngeal arches, eyes, and limb buds. In situ hybridization analyses of embryonic and neonatal brain sections revealed that snx3 mRNA is mainly expressed in the cerebral cortex, hippocampus, piriform cortex, cerebellum, and spinal cord. In adulthood, the expression of snx3 mRNA is observed in the cerebral cortex, hippocampus, piriform cortex, and cerebellar neurons. Thus, snx3 mRNA is expressed during neural development and in adult neural tissues, suggesting that SNX3 may play an important role in the development and function of the CNS.

Imaging crustal structure in south central Costa Rica with receiver functions

An array of broadband seismometers transecting the Talamanca Range in southern Costa Rica was operated from 2005 until 2007. In combination with data from a short‐period network near Quepos in central Costa Rica, this data is analyzed by the receiver function method to image the crustal structure in south‐central Costa Rica. Two strong positive signals are seen in the migrated images, interpreted as the Moho (at around 35 km depth) and an intra‐crustal discontinuity (15 km depth). A relatively flat crustal and Moho interface underneath the north‐east flank of the Talamanca Range can be followed for a lateral distance of about 50 km parallel to the trench, with only slight changes in the overall geometry. Closer to the coast, the topography of the discontinuities shows several features, most notably a deeper Moho underneath the Talamanca Mountain Range and volcanic arc. Under the highest part of the mountain ranges, the Moho reaches a depth of about 50 km, which indicates that the mountain ranges are approximately isostatically compensated. Local deviations from the crustal thickness expected for isostatic equilibrium occur under the active volcanic arc and in south Costa Rica. In the transition region between the active volcanic arc and the Talamanca Range, both the Moho and intracrustal discontinuity appear distorted, possibly related to the southern edge of the active volcanic zone and deformation within the southern part of the Central Costa Rica Deformed Belt. Near the volcanoes Irazu and Turrialba, a shallow converter occurs, correlating with a low‐velocity, low‐density body seen in tomography and gravimetry. Applying a grid search for the crustal interface depth and vp/vs ratio cannot constrain vp/vs values well, but points to generally low values (&lt;1.7) in the upper crust. This is consistent with quartz‐rich rocks forming the mountain range.

Production, characterization and application of monoclonal antibody to spherulocytes: A subpopulation of coelomocytes of Apostichopus japonicus

One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA), Western blotting and fluorescenceactivated cell sorter (FACS), mAb 3F6 showed specific for spherulocytes of A. japonicus. The mAb 3F6 recognized an antigen of molecular weight 136 kDa in Western blotting. Isotype analysis revealed mAb 3F6 as IgG type. The flow cytometry assay confirmed the microscopy observations and showed coelomocytes positive to mAb 3F6. The antigencity of haemocytes or coelomocytes of Hemicentrotus pulcherrimus, Scapharca subcrenata, Asterina pectinifera, Asterias rollestoni, Ruditapes philipinarum, Patinopecten yessoensis and Mytilus edulis was compared and the result showed that none of them was positive with mAb 3F6. Most of cells free in polian vesicle were positive with mAb 3F6. The positive cells are in spherical shape, 5–7 μm in diameter, smaller than coelomic spherulocytes of A. japonicus. The result of immunofluorescent staining with cells in hemal vessel showed that there were strong positive signals on cytoplasm of some spherical cells with diameter of 7–8 μm. Some other cells with higher nucelo-plasmic ratio, about 5–6 μm in diameter showed weak positive signals on membrane. Immunohistochemistry assay revealed that positive signals were mostly observed in the lumen structure of rete mirabile and haemocoel of the respiratory tree. In addition, the outer epidermis of body wall and tentacle also showed positive.

Comparison of the Binding Modes of [Ru(2,2'-bipyridine)3]2+

The <TEX>$[Ru(tpy)_2]Cl_2$</TEX> (tpy:2,2':6',2"-terpyridine) complex was synthesized and its structure was confirmed by <TEX>$^1H$</TEX>-NMR and elemental analysis. Its binding mode toward DNA was compared with the well-known <TEX>$[Ru(bpy)_3]Cl_2$</TEX> (bpy:2,2-bipyridyl), using isotropic absorption, linear dichroism(LD) spectroscopy, and an energy minimization study. Compared to <TEX>$[Ru(bpy)_3]^{2+}$</TEX>, the <TEX>$[Ru(tpy)_2]^{2+}$</TEX> complex exhibited very little change in its absorption pattern, especially in the MLCT band, upon binding to DNA. Furthermore, upon DNA binding, both Ru(II) complexes induced a decrease in the LD magnitude in the DNA absorption region. The <TEX>$[Ru(tpy)_2]^{2+}$</TEX> complex produced a strong positive LD signal in the ligand absorption region, which is in contrast with the <TEX>$[Ru(bpy)_3]^{2+}$</TEX> complex. Observed spectral properties led to the conclusion that the interaction between the ligands and DNA bases is negligible for the <TEX>$[Ru(tpy)_2]^{2+}$</TEX> complex, although it formed an adduct with DNA. This conclusion implies that both complexes bind to the surface of DNA, most likely to negatively charged phosphate groups via a simple electrostatic interaction, thereby orienting to exhibit the LD signal. The energy minimization calculation also supported this conclusion.

Abstract CN07-03: CD47 is an adverse prognostic factor and therapeutic antibody target on human AML stem cells

Abstract Human acute myeloid leukemia (AML) is organized as a cellular hierarchy initiated and maintained by rare self-renewing leukemia stem cells (LSC), which must be eliminated in order to cure the patient. We identified increased expression of CD47 on human AML LSC compared to their normal counterparts. CD47 is a cell surface molecule that serves as the ligand for SIRP-alpha on the surface of phagocytes, which in turn transmits a dominant inhibitory signal for phagocytosis. In this way, CD47 essentially functions as a “don't eat me” signal. We hypothesized that increased CD47 expression contributes to pathogenesis by inhibiting phagocytosis of AML LSC. Consistent with this hypothesis, we found that increased CD47 expression predicted worse overall survival in 3 independent cohorts of adult AML patients. Furthermore, we predicted that disruption of the interaction of CD47 with SIRP-alpha would result in phagocytosis and elimination of AML LSC. We found that blocking monoclonal antibodies directed against CD47 enabled phagocytosis of AML LSC, but not normal CD34+ human bone marrow progenitor cells, by human macrophages in vitro. Additionally, coating of human AML LSC with anti-CD47 monoclonal antibodies inhibited their engraftment in vivo in a xenotransplantation assay. Finally, analogous to a clinical therapy, treatment of human AML-engrafted mice with anti-CD47 antibody eliminated AML cells in the peripheral blood and bone marrow and targeted LSC. In summary, increased CD47 expression is an independent poor prognostic factor that can be targeted on human AML stem cells with monoclonal antibodies capable of stimulating phagocytosis and elimination of LSC. Targeting of CD47 with blocking monoclonal antibodies to induce phagocytosis is a novel mechanism for antibody cancer therapy. While anti-CD47 antibodies can be effective monotherapy for human AML, such antibodies may be equally, if not more, effective as part of a combination strategy. The combination of an anti-CD47 antibody, able to block a strong inhibitory signal for phagocytosis, with a second antibody able to bind an LSC-specific molecule and engage Fc receptors on phagocytes, thereby delivering a strong positive signal for phagocytosis, may result in a synergistic stimulus for phagocytosis and specific elimination of AML LSC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN07-03.

In-Depth Exploration of Cow’s Whey Proteome via Combinatorial Peptide Ligand Libraries

The use of combinatorial peptide ligand libraries, containing hexapeptides terminating with a primary amine, or modified with a terminal carboxyl group, allowed discovering and identifying a large number of previously unreported proteins in cow's whey. Whereas comprehensive whey protein lists progressively increased in the last 6 years from 17 unique gene products to more than 100, our findings have considerably expanded this list to a total of 149 unique protein species, of which 100 were not described in previous proteomics studies. As an additional interesting result, a polymorphic alkaline protein was observed with a strong positive signal when blotted from an isoelectric focusing separation in gel and tested with sera of allergic patients. This polymorphic protein, found only after treatment with the peptide library, was identified as an immunoglobulin (Ig), a minor allergen that had been largely amplified. The list of cow's whey components here reported is by far the most comprehensive at present and could serve as a starting point for the functional characterization of low-abundance proteins possibly having novel pharmaceutical, diagnostic, and biomedical applications.

RNA Colony Blot Hybridization Method for Enumeration of Culturable Vibrio cholerae and Vibrio mimicus Bacteria

A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria in environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of V. cholerae and V. mimicus was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse V. cholerae-V. mimicus strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by Grimontia (Vibrio) hollisae, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent V. cholerae strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when G. hollisae was not present. When the protocol was applied to laboratory microcosms containing V. cholerae attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of V. cholerae per copepod. The protocol was also used to analyze pond water samples collected in an area of cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable V. cholerae bacteria 1 to 2 months prior to observed increases in pathogenic V. cholerae and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of V. cholerae in the environment. The RNA blot hybridization protocol can also be applied to detection of other gram-negative bacteria for taxon-specific enumeration.

Clinicopathological significance of DEK overexpression in serous ovarian tumors

To investigate the significance of DEK protein expression in ovarian lesions, a total of 113 ovarian serous tumors, including 62 serous cystadenocarcinomas and 19 serous borderline tumors, were studied on immunohistochemistry. For comparison, 32 benign serous tumors, including 12 serous papillary cystadenomas, 10 serous cystadenomas, and 10 serous surface papillomas, were also included. DEK was positive in 93.5% of serous cystadenocarcinomas (58/62), 63.2% of serous borderline tumors (12/19), and weakly positive in 15.6% of benign serous tumors (5/32). The strong positive signal was detected only in serous adenocarcinomas (80.6%, 50/62) and borderline tumors (21.1%, 4/19), but no serous benign tumors were strongly positive (0%, 0/32). Meanwhile, the strong positivity rate of DEK protein was significantly higher in grade 2 and grade 3 than in grade 1 ovarian cancers (P < 0.05), but there was no significant association between DEK protein expression level and International Federation of Gynecology and Obstetrics (FIGO) stage of serous ovarian adenocarcinoma (P > 0.05). In summary, DEK plays an important role in the progression of ovarian serous cancers. The detection of DEK protein expression should be useful for the diagnosis and prognosis of ovarian serous cancers, and DEK might be a useful molecular target for ovarian cancer therapy.