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Background The unique physicochemical properties of multi-walled carbon nanotubes (MWCNT) have led to many industrial applications. Due to their low density and small size, MWCNT are easily aerosolized in the workplace making respiratory exposures likely in workers. The International Agency for Research on Cancer designated the pristine Mitsui-7 MWCNT (MWCNT-7) as a Group 2B carcinogen, but there was insufficient data to classify all other MWCNT. Previously, MWCNT exposed to high temperature (MWCNT-HT) or synthesized with nitrogen (MWCNT-ND) have been found to elicit attenuated toxicity; however, their genotoxic and carcinogenic potential are not known. Our aim was to measure the genotoxicity of MWCNT-7 compared to these two physicochemically-altered MWCNTs in human lung epithelial cells (BEAS-2B & SAEC). Results Dose-dependent partitioning of individual nanotubes in the cell nuclei was observed for each MWCNT material and was greatest for MWCNT-7. Exposure to each MWCNT led to significantly increased mitotic aberrations with multi- and monopolar spindle morphologies and fragmented centrosomes. Quantitative analysis of the spindle pole demonstrated significantly increased centrosome fragmentation from 0.024–2.4 μg/mL of each MWCNT. Significant aneuploidy was measured in a dose-response from each MWCNT-7, HT, and ND; the highest dose of 24 μg/mL produced 67, 61, and 55%, respectively. Chromosome analysis demonstrated significantly increased centromere fragmentation and translocations from each MWCNT at each dose. Following 24 h of exposure to MWCNT-7, ND and/or HT in BEAS-2B a significant arrest in the G1/S phase in the cell cycle occurred, whereas the MWCNT-ND also induced a G2 arrest. Primary SAEC exposed for 24 h to each MWCNT elicited a significantly greater arrest in the G1 and G2 phases. However, SAEC arrested in the G1/S phase after 72 h of exposure. Lastly, a significant increase in clonal growth was observed one month after exposure to 0.024 μg/mL MWCNT-HT & ND. Conclusions Although MWCNT-HT & ND cause a lower incidence of genotoxicity, all three MWCNTs cause the same type of mitotic and chromosomal disruptions. Chromosomal fragmentation and translocations have not been observed with other nanomaterials. Because in vitro genotoxicity is correlated with in vivo genotoxic response, these studies in primary human lung cells may predict the genotoxic potency in exposed human populations.

The hyline tribe Lophyohylini includes 87 species of treefrogs, of which cytogenetics aspects have been studied in less than 20% of them. In order to evaluate the evolution of some of its chromosome characters (NOR position, C-bands, and DAPI/CMA3 bands), we studied the karyotypes of 21 lophyohylines, 16 of them for the first time, and analyzed them in a phylogenetic context. Most species showed similar karyotypes regarding chromosome number (2n = 24) and morphology (FN = 48), excepting Phyllodytes edelmoi and Osteocephalus buckleyi with 2n = 22 (FN = 44) and 2n = 28 (FN = 50), respectively. The NOR location was variable among species and provided valuable phylogenetic information. This marker was located in pair 11 in all species of Trachycephalus, Itapotihyla langsdorffii, and Nyctimantis arapapa, representing the plesiomorphic condition of Lophyohylini. Besides, other apomorphic states were recovered for the clades comprising N. rugiceps and N. siemersi (NOR in pair 5), and Dryaderces pearsoni, Osteocephalus, and Osteopilus (NOR in pair 9). Phyllodytes presented variation for NORs position; they were in pair 2 in P. edelmoi, pair 7 in P. melanomystax, and pair 8 in P. gyrinaethes and P. praeceptor. Polymorphisms in size, number, and activity of this marker were observed for N. siemersi, Osteocephalus fuscifacies, and some species of Trachycephalus. Remarkably, in N. siemersi NORs were detected on a single chromosome in the two specimens studied by this technique, raising the question of how this complex polymorphism is maintained. Interstitial telomeric sequences were found in P. edelmoi,P. melanomystax, and Osteocephalus buckleyi, and their presence seems to be not related to the chromosome reorganization events. Finally, some species showed spontaneous rearrangements, possibly as a consequence of an uncommon phenomenon in anuran cytogenetics: the presence of fragile sites or secondary constrictions not associated with NORs. We propose that this rare feature would have played an important role in the evolution of this group of frogs. From the evidence obtained in this and previous studies, we conclude that Lophyohylini presents a complex chromosome evolution. Fil: Suarez, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Ferro, Juan Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Nagamachi, Cleusa Y.. Universidade Federal do Pará; Brasil Fil: Cardozo, Dario Elbio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Blasco zuñiga, Ailin. Pontificia Universidad Católica del Ecuador; Ecuador Fil: Silva, Jéssica B.. Universidade Federal do Pará; Brasil Fil: Marciano Jr, Euvaldo. Universidade Estadual de Santa Cruz; Brasil Fil: Costa, Marco A.. Universidade Estadual de Santa Cruz; Brasil Fil: Orrico, Victor G.D.. Universidade Estadual de Santa Cruz; Brasil Fil: Solé, Mirco. Universidade Estadual de Santa Cruz; Brasil Fil: Roberto, Igor J.. Universidad Federal del Amazonas.; Brasil Fil: Rivera, Miryan. Pontificia Universidad Católica del Ecuador; Ecuador Fil: Wiley, John E.. University School of Medicine; Estados Unidos Fil: Faivovich, Julián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; Argentina Fil: Baldo, Juan Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Pieczarka, Julio. Universidade Federal do Pará; Brasil

Fil: Ferro, Juan Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico (Nordeste). Instituto de Biología Subtropical; Argentina. Fil: Ferro, Juan Martín. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Química y Naturales. Instituto de Biología Subtropical; Argentina. Fil: Ferro, Juan Martín. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Química y Naturales. Laboratorio de Genética Evolutiva; Argentina. Fil: Cardozo, Dario Elbio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico (Nordeste). Instituto de Biología Subtropical; Argentina. Fil: Cardozo, Dario Elbio. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Química y Naturales. Instituto de Biología Subtropical; Argentina. Fil: Cardozo, Dario Elbio. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Química y Naturales. Laboratorio de Genética Evolutiva; Argentina. Fil: Suarez, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico (Nordeste). Instituto de Biología Subtropical; Argentina. Fil: Suarez, Pablo. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Química y Naturales. Instituto de Biología Subtropical; Argentina. Fil: Boeris, Juan Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico (Nordeste). Instituto de Biología Subtropical; Argentina. Fil: Boeris, Juan Martín. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Química y Naturales. Instituto de Biología Subtropical; Argentina. Fil: Boeris, Juan Martín. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Química y Naturales. Laboratorio de Genética Evolutiva; Argentina. Fil: Blasco-Zúñiga, Ailin. Pontificia Universidad Católica del Ecuador. Centro de Investigación para la Salud en América Latina. Laboratorio de Investigación en Citogenética y Biomoléculas de Anfibios; Ecuador. Fil: Barbero, Gastón Alexis. Universidad Maimónides. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y Diagnóstico; Argentina. Fil: Barbero, Gastón Alexis Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y Diagnóstico; Argentina. Fil: Gomes, Anderson. Instituto Federal de Educación, Ciencia y Tecnología de Pará; Brasil. Fil: Gazoni, Thiago. Universidad Estatal Paulista Julio de Mesquita Filho. Instituto de Biociencias. Departamento de Biología; Brasil. Fil: Costa, William. Universidad Estatal de Campinas. Instituto de Biología. Departamento de Biología Estructural y Funcional; Brasil. Fil: Nagamachi, Cleusa Yoshiko. Universidad Federal do Pará. Instituto de Ciencias Biologicas. Centro de Estudios Avanzados de Biodiversidad. Laboratorio de Citogenética; Brasil. Fil: Rivera, Miryan. Pontificia Universidad Católica del Ecuador. Centro de Investigación para la Salud en América Latina. Laboratorio de Investigación en Citogenética y Biomoléculas de Anfibios; Ecuador. The hylid tribe Cophomantini is a diverse clade of Neotropical treefrogs composed of the genera Aplastodiscus, Boana, Bokermannohyla, Hyloscirtus, and Myersiohyla. The phylogenetic relationships of Cophomantini have been comprehensively reviewed in the literature, providing a suitable framework for the study of chromosome evolution. Employing different banding techniques, we studied the chromosomes of 25 species of Boana and 3 of Hyloscirtus; thus providing, for the first time, data for Hyloscirtus and for 15 species of Boana. Most species showed karyotypes with 2n = 2x = 24 chromosomes; some species of the B. albopunctata group have 2n = 2x = 22, and H. alytolylax has 2n = 2x = 20. Karyotypes are all bi-armed in most species presented, with the exception of H. larinopygion (FN = 46) and H. alytolylax (FN = 38), with karyotypes that have a single pair of small telocentric chromosomes. In most species of Boana, NORs are observed in a single pair of chromosomes, mostly in the small chromosomes, although in some species of the B. albopunctata, B. pulchella, and B. semilineata groups, this marker occurs on the larger pairs 8, 1, and 7, respectively. In Hyloscirtus, NOR position differs in the three studied species: H. alytolylax (4p), H. palmeri (4q), and H. larinopygion (1p). Heterochromatin is a variable marker that could provide valuable evidence, but it would be necesserary to understand the molecular composition of the C-bands that are observed in different species in order to test its putative homology. In H. alytolylax, a centromeric DAPI+ band was observed on one homologue of chromosome pair 2. The band was present in males but absent in females, providing evidence for an XX/XY sex determining system in this species. We review and discuss the importance of the different chromosome markers (NOR position, C-bands, and DAPI/CMA3 patterns) for their impact on the taxonomy and karyotype evolution in Cophomantini.

Background Pierpont syndrome was first described in 1998 with key characteristics including developmental delay, dysmorphic facial features, fat pads on hands and feet, and feeding difficulties. To date the mechanism of inheritance is unknown. Nine out of ten previously described patients with Pierpont syndrome were boys. This is the first report of a case of a non-white patient with Pierpont syndrome and she is the second female patient to be described as having Pierpont syndrome. Case presentation Our patient is a 16-month-old Hispanic girl with extreme developmental delay, microcephaly, large ears, short and thick upper lip, broad philtrum, widely spaced teeth, constipation, dysphagia, fat pads on feet and hands, autistic behavior and seizure-like episodes. She had a normal karyotype (46,XX), and array testing showed greater than 8 % homozygosity with otherwise normal results. Genes within these areas of homozygosity may provide clues to an etiology and suggest autosomal recessive inheritance. This case report highlights the possibility of ethnic variations in this syndrome’s presentation, which may have ramifications in uncovering the pathogenesis as well as expanding the phenotype. Conclusion Pierpont syndrome should be considered in the evaluation of children with the described features, regardless of their gender and ethnicity.

Mitochondrial translation initiation factor 2 (MTIF2) is nuclear-encoded and functions in mitochondria to initiate the translation of proteins encoded by the mitochondrial genome. To gain insight into mechanisms that regulate MTIF2 gene expression, the genomic copy and the 5' and 3' flanking regions of MTIF2 were isolated using a combination of genomic library screening and polymerase chain reaction (PCR). MTIF2 is approximately 33.5-kb long and contains 16 exons, confirming data from the Human Genome Project. With RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we mapped the transcription start point in human heart tissue to a cytosine residue 296 bp upstream from the translation initiation site. The region surrounding the transcription start point contains consensus binding sites for transcription factors Sp1, nuclear respiratory factor 2 (NRF-2) and estrogen receptor, while enhancer binding sites were identified upstream. Promoter constructs were prepared in a luciferase reporter vector and transiently transfected into 293T cells. The minimal promoter gave an expression level 3.5x higher than the SV40 control (P=0.001), while the construct containing the minimal promoter plus the enhancer region gave a 3.8x higher level of expression compared to the control (P

The pine woods treefrog, Hyla femoralis, is unique among North American hylid frogs in having a metacentric chromosome 6 and heteromorphic sex chromosomes of the XY/XX type. The X chromosome is distinguished by having a nucleolar organizing region (NOR) in the short arm. The Y chromosome does not possess an NOR. Until the present study, it was not known if the NOR was not present on the Y chromosome or inactive and therefore not detectable by conventional cytogenetic methods like silver staining. Exclusive of its unique features the karyotype of H. femoralis closely resembles those of North American frogs with karyotypes like H. chrysoscelis. We used replication banding and fluorescence in situ hybridization (FISH) with a DNA probe to the 18S + 28S ribosomal genes, which are located at the NOR, to characterize the H. femoralis karyotype. Our analysis revealed that the 18S + 28S ribosomal genes are not present on the Y chromosome, and that the karyotype of H. femoralis was derived from an H. chrysoscelis-like karyotype by relocation of the NOR to the X chromosome from chromosome 6 and either a concurrent or subsequent pericentric inversion of chromosome 6.

Populations of the diploid-tetraploid treefrogs Hyla chrysoscelis and H. versicolor can be defined by the polymorphic positions of the nucleolar organizing regions (NORs) on their chromosomes. Evidence from NOR positions and interstitial telomere sequence data shows that gene flow between H. chrysoscelis populations appears to be restricted, with contact occurring only in narrow “hybrid” zones. Hyla versicolor appears to have had multiple origins from H. chrysoscelis populations, and this, too, is reflected in the NOR positions. We used replication banding to determine if genetic isolation of H. chrysoscelis populations was accompanied by karyotype evolution in the populations or in contact zones. We also sought to detect karyotype alteration or replication differences associated with polyploidy in H. versicolor. Homologous chromosome pairs of all H. chrysoscelis studied displayed no differences in replication banding patterns, nor did they differ from those of H. versicolor. Although NOR positions differed between the populations studied, no disturbance of the replication banding patterns was found, indicating that structural rearrangements were not involved in creating the multiple NOR positions seen in populations of H. versicolor and H. chrysoscelis.

To present proof-of-principle application for employing micro(mi)RNAs as diagnostic markers for colon cancer, we carried out global microarray expression studies on stool samples obtained from fifteen individuals (three controls, and three each with TNM stage 0-1, stage 2, stage 3, and stage 4 colon cancer), using Affymetrix GeneChip miRNA 3.0 Array, to select for a panel of miRNA genes for subsequent focused semi-quantitative polymerase chain reaction (PCR) analysis studies. Microarray results showed 202 preferentially expressed miRNA genes that were either increased (141 miRNAs), or reduced (61 miRNAs) in expression. We then conducted a stem-loop reverse transcriptase (RT)-TaqMan® minor groove binding (MGB) probes, followed by a modified qPCR expression study on 20 selected miRNAs. Twelve of the miRNAs exhibited increased and 8 decreased expression in stool from 60 individuals (20 controls, 20 with tumor-lymph node-metastatic (TNM) stage 0-1, 10 with stage 2, five with stage 3, and 5 with stage 4 colon cancer) to quantitatively monitor miRNA changes at various TNM stages of colon cancer progression. We also used laser-capture microdissection (LCM) of colon mucosal epithelial tissue samples (three control samples, and three samples from each of the four stages of colon cancer, for a total of 15 samples) to find concordance or lack thereof with stool findings. The reference housekeeping pseudogene-free ribosomal gene (18S rRNA), which shows little variation in expression, was employed as a normalization standard for relative PCR quantification. Results of the PCR analyses confirmed that twelve miRNAs (miR-7, miR-17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p and miR214) had an increased expression in the stool of patients with colon cancer, and that later TNM carcinoma stages exhibited a more pronounced expression than did adenomas. On the other hand, eight miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, miR-222 and miR-938) had decreased expression in the stool of patients with colon cancer, which was also more pronounced from early to later TNM stages. Results from colon mucosal tissues were similar to those from stool samples, although with more apparent changes in expression. Cytological studies on purified stool colonocytes that employed Giemsa staining showed 80% sensitivity for detecting tumor cells in stool smears. The performance characteristics of the test confirmed that stool is a medium well-suited for colon cancer screening, and that the quantitative changes in the expression of few mature miRNA molecules in stool associated with colon cancer progression provided for more sensitive and specific non-invasive diagnostic markers than tests currently available on the market. Thus, a larger prospective and properly randomized validation study of control individuals and patients exhibiting various stages of colon cancer progression (TNM stages 0-IV) is now needed in order to standardize test conditions, and provide a means for determining the true sensitivity and specificity of a miRNA screening approach in stool for the non-invasive detection of colon cancer, particularly at an early stage (0-I). Eventually, we will develop a chip to enhance molecular screening for colon cancer, as has been accomplished for the detection of genetically-modified organisms (GMOs) in foods.

We carried out this study to present proof-of-principal application, showing that by using a global microarray expression analysis, followed by quantitative stem-loop reverse transcriptase in conjunction with TaqMan® polymerase chain reaction (PCR) analysis of micro(mi)RNA genes, on limited number of plasma and tissue samples obtained from 20 individuals (five healthy, five TNM stage 0-1 colon cancer, five stage 2 and five stage 3), we were able to quantitatively monitor miRNA changes at the various TNM stages of colon cancer progression, particularly at the early, pre-malignant adenoma stage (e.g. polyps ≥ 1 cm with high grade dysplasia). The expression of some of the tested miRNAs showed less variability in tissue than in plasma. Nevertheless, our limited preliminary data on the plasma by itself show that plasma is well-suited for screening, and that the quantitative changes in the expression of a few cell-free circulatory mature miRNA molecules in plasma, that are associated with colon cancer progression, would provide for more sensitive and specific markers than those tests currently available on the market. In addition, analysis of miRNA molecules offers a quantitative and cost-effective non-invasive diagnostic approach for screening, than currently employed methods in a prevalent cancer that can be cured if it is detected at the early TNM stages, and that becomes deadly if not diagnosed before metastasis. Thus, a larger prospective and properly randomized clinical study using plasma derived from many control individuals and at various stages of colon cancer (TNM stages 0-IV) from patients, in order to corroborate the initial results, is now urgently needed in order to allow for a statistically valid analysis, standardizing test conditions which will provide a means for determining the true sensitivity and specificity of a miRNA-screening approach. This approach, when combined with bioinformatics analysis to correlate miRNA seed data with mRNA target data, would allow for a mechanistic understanding of how miRNAs regulate mRNA gene expression, and would offer a better comprehensive diagnostic screening test for early-detection of colon cancer non-invasively.

Chimerism in humans appears to be a rare phenomenon that is usually discovered by accident. Here we describe a stillborn male fetus with multiple congenital anomalies which was found to have two cytogenetically abnormal cell lines, 47,XY, +21 and 47, XX, +12. The difference in sex chromosome constitution between the cell lines indicated that the fetus had dispermic chimerism. To our knowledge, this is the first report of chimerism with two abnormal cell lines.

A small clinical survey was undertaken at East Carolina University School of Medicine to examine the factors which influenced the decisions of five families to continue pregnancies after a chromosomal abnormality was detected. Little has been published concerning the psychosocial effects after continuing pregnancies in which the fetus was diagnosed with a chromosome abnormality by amniocentesis. In order to identify the factors that influenced their decisions, an interview with each couple was undertaken using a 25-part questionnaire. This paper addresses the method of interviewing, case material, and background concerning each couple and the summary of the results.

This study presents proof-of-principle application showing that label-free affinity enrichment surface plasmon resonance (SPR) biosensor binding is able to semiquantitatively detect molecular DNA-protein interactions in crude cellular extracts in a real-time ligand fishing analysis study. Crude cell extracts obtained from a confluent HT-28 human adenocarcinoma cell line, synchronized to the G(0)/G(1) phase of the cell cycle, were extracted in a chaotropic medium and cryopreserved in liquid nitrogen. Various immunoprecipitation antibodies were used against defective human excision and mismatch repair genes, hDDB2 and hMSH2, respectively, which theoretically allow for protein binding to DNA ligands in their native conformation. A set of biotinylated DNA target sequence heteroduplexes were also utilized for binding hDDB2 and hMSH2, prepared by heating a biotinylated oligonucleotide strand with an equimolar amount of the complementary strand to form a DNA duplex for hMSH2; a UV-irradiated duplex was employed for hDDB2 instead of an irradiated single-strand DNA to enhance binding. SDS was used to regenerate heteroduplex-modified chips that were used in a BIAcore 2000 SPR instrument at 25°C. Results showed that hMSH2 does not bind preferentially to the heteroduplex-complementary pair. In contrast, hDDB2 was found to bind preferentially to the UV-irradiated version of the heteroduplex-complementary pair. It is concluded that the choice of antibodies with appropriate epitopes is crucial to the success of these SPR binding studies because of enhanced specificity.

Complex DNA damage may manifest in double-strand breaks (DSBs) and non-DSB, bistranded, oxidatively induced clustered DNA lesions (OCDLs). Although the carcinogen benzo[alpha]pyrene (B[alpha]P) has been shown to induce chromosomal aberrations and transformation of mammary cells, it is not known whether this compound engenders clustered DNA damage. Normal primary breast tissue-derived cells were treated with B[alpha]P, and the levels of DNA lesions, chromosomal aberrations, total antioxidant capacity (TAC), and reactive oxygen species (ROS) were determined. DNA from cells treated with 2 and 8 microM B[alpha]P exhibited increases of 3- and 4-fold in APE1 (p0.001), 11- and 19-fold in Endo III (p0.001), and 8- and 15-fold in hOGG1 (p0.001) OCDLs, respectively, compared to the 0 microM B[alpha]P-treated (control) group. Mammary cells treated with 8 microM B[alpha]P produced 0.12 aberrations per cell (p0.05) and there was a strong positive correlation (r=0.91) between the levels of OCDLs and those of chromosomal aberrations. Finally, TAC was decreased by 25% (p0.02), whereas ROS production increased by 2-fold (p0.02) in cells treated with 8 microM B[alpha]P compared to the control group. In conclusion, oxidatively induced clustered DNA damage mediated through differential expression of APE1, reduced TAC, and increased ROS may play a significant role in the chemically induced transformation of normal primary mammary cells.

Expression of the immunostimulatory xenoantigen alphaGal on malignant cells is being investigated as a means to formulate anticancer vaccines. Expression methods have been limited to gene transfer using viral vectors and enzymatic manipulation. We report here a novel method using polyethylene glycol (PEG) to induce plasma membrane fusion between malignant human hematological cells and alphaGal(+) porcine blood cells (PBC) in order to display alphaGal antigens on human cells.Freshly isolated white blood cells (WBC) were obtained from patients with malignant hematological disease and combined with diluted PBC. Cell mixtures were labeled with human CD mAbs, followed by IB4 lectin or M86 mAb to detect alphaGal antigens and then co-incubated with PEG. Back-gated, dual-color flow cytometry was used to detect alphaGal on human cells.alphaGal antigens were detected on sizeable numbers of human WBC (approximately 45%) after incubation with PEG. Antigen expression was profuse as assessed by the strong fluorescent intensity demonstrated by IB4-FITC and M86 labeling. Human cells combined with PBC without PEG were not reactive with IB4-FITC or M86.Our method provides an effective, highly reproducible means to efficiently express alphaGal antigens on cells obtained from patients with a spectrum of hematological malignancies. This method can provide a simple, safe alternative to viral-mediated gene transfer or enzymatic alteration to express alphaGal antigens on human tumor cells. By virtue of its simplicity, our technique presents a novel approach to the preparation of polyvalent autologous or syngeneic anticancer vaccines.

We carried out this in vitro molecular study to investigate the effect of two clinical X-irradiation modalities (a two-dimensional external beam radiotherapy referred to in this article as conventional RT, and a three dimensional conformal intensity-modulated radiation therapy (IMRT) on a colon adenocarcinoma HT-29 cell line. Cells were synchronized by serum deprivation 48 h before irradiation so that90% of them were in the G(0)/G(1) phase of the cell cycle. Cells were allowed to recover 3 h after irradiation before total RNA extraction. Two types of arrays, namely Affymetrix Human HG U133A 2.0 oligonucleotide microarrays and Ambion mirVana bioarrays, were employed to study mRNA and microRNA expressions, respectively. Three flasks were used per irradiation dose, and an additional three unirradiated flasks served as control. Microarray data were validated by reverse transcriptase quantitative polymerase chain reaction, and proteins of some expressed genes were determined by Western blots. Results showed the existence of differences in expression profiles between the two irradiation modalities. IMRT appeared to influence expression of some DNA repair genes, whereas in conventional RT, some DNA repair and cell cycle-related genes that initially seemed to be preferentially expressed dwindled to normal levels. Earlier in vitro experiments using cell survival to study sublethal damage repair support our conclusions. Bioinformatic investigation revealed a correlation of gene expression with derepression effects of microRNA molecules. We have presented opinions as to how microRNAs might influence gene expression during radiation-induced stress and have suggested future avenues for research.

Individual specimens of Bufo terrestris were discovered that possessed ribosomal gene locations in addition to those normally found. Every specimen from an island population that was examined had extra sites, whereas fewer individuals from coastal mainland populations and none from inland populations had them. Although the extra ribosomal gene locations probably did not arise through gross structural chromosome rearrangements, their origin remains unclear.

Normal human foreskin fibroblasts (HSF4) were transfected using the pSV3-neo plasmid. A pool of 10 G418-resistant colonies, HSF4-T12, showed a progressive increase in the expression of a number of in vitro transformation markers with passage in culture and became immortalized. Although no tumors were formed when cells were injected subcutaneously into nude mice, this cell line produced progressive tumors when cells were injected into preimplanted Gelfoam sponges in the mice. When these tumors were cultured in vitro and subsequently injected subcutaneously, progressive tumors were produced with median latency periods as short as 4 weeks. Three phases of cytogenetic change could be distinguished. At early passages after transfection. HSF4-T12 exhibited many random chromosomal changes. At a time just after immortalization, both flow karyotype and G-banded analyses showed the appearance of balanced clonal rearrangements. These included t(2;4), t(2;14), t(3;?), 6p-, i(6p), 8p-, t(14;15), i(15), and t(18;?). These clonal rearrangements were stable with passage in culture, and less variability from cell to cell was noted. The only consistent chromosomal loss observed was -Y. Analysis of three independent tumors showed characteristic loss of chromosomal material rather than balanced chromosomal rearrangements. Frequent loss of 6q and chromosomes #13, 15, 20, and Y was noted.

The intrachromosomal distribution of non-telomeric sites of the (TTAGGG)n telomeric repeat was determined for 100 vertebrate species. The most common non-telomeric location of this sequence was in the pericentric regions of chromosomes. A variety of species showed relatively large amounts of this sequence present within regions of constitutive heterochromatin. We discuss possible relationships between the non-telomeric distribution of the (TTAGGG)n sequence and the process of karyotype evolution, during which these sites may provide potential new telomeres.

We demonstrated that different numbered human chromosomes could be discriminated and sorted with confocal Raman-tweezers spectroscopy, and the result was confirmed with G-band technique.

A pancreatoblastoma from a 4-year old male was examined by flow cytometric ploidy analysis and cytogenetics. To detect differences within the tumor, the specimen was divided into four portions and sampled separately. Flow analysis revealed that each sample contained a diploid and a tetraploid population of tumor cells. These findings correlated well with the cytogenetic analysis, which also revealed differences in structural rearrangements between samples. A t(13;22)(q10;q10) was the only rearrangement found in near-diploid cells as well as one near-tetraploid line. Other common structural changes in near-tetraploid cells included t(13;13)(q10;q10), i(6p)(p10), and del(1). Chromosomes 1, 6, 13, and 22 were consistently missing from all near-tetraploid cells lines. To our knowledge, this is the first flow cytometric and cytogenetic study of a pancreatoblastoma.

We report on a patient with mosaic tetrasomy 8p [46,XX/47,XX+i(8p)]. The patient has 2 fused vertebrae, abnormal ribs, congenital heart defects, agenesis of corpus callosum, hypotonia, and delayed development. The patient's developmental delays are most marked in receptive and expressive language skills, with more moderate delays on cognitive, sensorimotor, and motor skill testing. These findings are similar to those of the 3 previously reported patients with mosaic i(8p). © 1993 Wiley-Liss, Inc.

We report on an infant girl born with findings of CHARGE association who proved to be a genetic male (46, XY) on cytogenetic study. Further investigation of the genitalia demonstrated partially female internal organs but absence of gonads by ultrasonography, hormone studies, and absence of ZFY by DNA probe of Yp. Pelvic exploration confirmed lack of gonadal tissue and uterus. Facial phenotype was compatible with CHARGE appearance.

We evaluated the diagnostic contribution of adjunct studies performed on aspirated material in the work-up of pediatric fine-needle aspiration (FNA) biopsies. Ancillary studies were performed on 54 of 136 (39.7%) pediatric FNA biopsies during a 5-year period. In 23 (16.9%) cases, immunocytochemical (ICC) studies, consisting of immunoperoxidase staining of direct smears and/or cell blocks or flow cytometric immunophenotyping, were performed. The studies were adequate in 14 cases (60.9%), suboptimal in five cases (21.7%), and inadequate in four cases (17.4%). Of the adequate and suboptimal cases, the ICC data helped to narrow the differential diagnosis or classify the disease process in eight cases (42.1%), confirmed cytologic impression in nine cases (47.4%), and gave contradictory results in two cases (10.5%). Adequate material for electron microscopy (EM) was obtained in 14/19 cases (73.7%). Ultrastructural studies were diagnostic, or helped classify the disease process in five cases (35.7%), confirmed the cytologic impression in four cases (28.6%), helped exclude diagnostic considerations in three cases (21.4%), and were judged to be non-contributory in two cases (14.3%). Cytogenetic studies revealed six of seven cases (all neoplasms) to have abnormal karyotypes. Special stains for organisms performed on smears from 25 cases including Ziehl-Neelsen, Gomori methenamine silver (GMS), Gram, and Warthin-Starry (WS) were negative except for 1/16 GMS and 4/9 Gram stains. In summary, we found that with appropriate case selection, ancillary studies performed on aspirated material can provide useful information in pediatric FNA cytology.

Objectives The reasons for the increasing incidence of esophageal adenocarcinoma are not clear. A causal relation between gastroesophageal reflux disease and esophageal adenocarcinoma has been suggested. Support for this comes from the development of esophageal adenocarcinoma in the rat reflux model. However, to date, no systematic characterization of the tumors derived from this model has been reported. Methods We induced biliary reflux by creating esophagojejunal anastomoses in 12 Sprague–Dawley rats. The experiment was terminated at 9 months, and rat esophagi were harvested for histopathologic documentation of reflux-associated changes and evidence of tumor formation. Three cell lines were established from 2 of the reflux-associated tumors. We tested the ability of these cells to grow in vitro in tissue culture and in vivo as xenografts in an orthotopic location at the gastroesophageal junction. Furthermore, we performed a cytogenetic analysis and determined the array-based gene expression profiles of these 3 rodent carcinoma lines compared with normal esophageal mucosa. Results At 9 months, 12 of 12 rodents had histologic features of metaplastic columnar epithelium in the esophagus, with 7 having invasive carcinomas with glandular differentiation (either adenocarcinomas or adenosquamous carcinomas). The 3 cell lines established from 2 reflux-associated tumors were capable of sustained in vitro propagation and grew successfully as xenografts in both subcutaneous and orthotopic locations, confirming the tumorigenic nature of these lines. Despite their establishment from primary tumors with glandular features, the histology of the xenografts was that of well-differentiated squamous carcinomas. Karyotype analyses demonstrated cytogenetic heterogeneity and aneuploidy; furthermore, translocation (7:11) was present in all 3 lines. Array-based gene expression profiling confirmed upregulation of several cancer-related genes important in human esophageal cancer. Quantitative reverse transcription–polymerase chain reaction was used to confirm the differential expression of selected transcripts (vascular endothelial growth factor [ VEGF ], polo-like kinases [ PLK ], cyclin dependent kinase 4 [ CDK4 ], hypoxia-inducible factor 1α [ HIF1α ], and insulin-like growth factor 1 [ IGF-1 ]) in comparison with nonneoplastic esophageal mucosal scrapings. Conclusions The rodent reflux model is capable of inducing metaplastic epithelial changes simulating Barrett esophagus, as well as subsequent neoplastic transformation, at a high frequency. Cell lines have been established from these tumors that are capable of in vitro and in vivo passaging. The rodent reflux model should be a valuable model for studying therapy and chemoprevention efforts for Barrett esophagus, whereas the established cell lines provide a useful resource for drug discovery and other high-throughput studies.

The authors of the work: 'Chromosomal analysis and identification based on optical tweezers and raman spectroscopy' [Opt. Express 14, 5385 (2006], claim that they have been able to identify and differentiate between three human chromosomes with an optical-tweezer - Raman Spectroscopic experimental (LTRS) set-up. The results and conclusions as they are presented in the paper are questionable, however, when the spectral data and data analysis are studied in greater detail.

Constitutional nonreciprocal translocations are extremely rare, and even their existence is controversial. We report on a newborn infant with a de novo nonreciprocal translocation between chromosomes 1 and 8 resulting in 1q42.3 deletion syndrome. Fluorescent in situ hybridization with whole chromosome paints confirmed the conventional cytogenetic diagnosis.

Thank you for the opportunity to comment on the letter from Dr. Qumsiyeh and Dr. Peppers. The 1;8 unbalanced translocation described in our report was unequivocally diagnosed by conventional cytogenetic studies prior to our analysis with whole chromosome paints (WCP). Examination of at least 50 metaphases with band levels of 550 or more failed to detect any chromosome 1 material on the deleted chromosome 8. Although the partial karyotype in our report is a little dark, it is obvious that the translocation is not balanced. The clinical findings in our patient alone support a diagnosis of 1q42.3 deletion syndrome. 6 refs.

The chromosomes of the diploid Hyla chrysoscelis and its tetraploid sibling species H. versicolor were studied with AgNO3 staining and in situ hybridization to determine the chromosome location of the 18S and 28S ribosomal RNA (rRNA) genes. A total of 236 Hyla chrysoscelis from 34 localities in 15 U.S. states and 100 H. versicolor from 15 localities in 12 states were examined. The rRNA gene sites were extremely variable in H. chrysoscelis, and also variable, but to a lesser extent, in H. versicolor. The most common rRNA gene site in both H. chrysoscelis and H. versicolor was on the short arm of chromosome 6. All of the rRNA gene locations seen in H. versicolor were also seen in H. chrysoscelis, supporting the hypothesis that the tetraploid H. versicolor arose from H. chrysoscelis. Although polymorphic rRNA gene sites in H. versicolor may reflect the positions of the rRNA genes in H. chrysoscelis ancestors, the origin of the extreme variability of such sites in H. chrysoscelis seems more obscure. Possible explanations include inversions, translocations, mobile genetic elements or a combination of some or all of these.

Two white females, age 2 1/2 and 33 years, respectively, were investigated because of severe mental retardation associated with neurologic abnormalities, coarse face, and soft tissue syndactyly involving upper and lower limbs. Each had cytogenetic findings of a mosaic variant of Ullrich-Turner syndrome with X ring chromosome in peripheral lymphocyte and skin fibroblasts. Early X replication occurred in one-third of the X ring chromosomes; there was no evidence for X-autosome translocation involving either X and an autosomal duplication; results of studies for fragility of the X chromosomes were unremarkable. In situ hybridization with an X centromere probe was positive for the ring. To our knowledge, the unusual constellation of cytogenetic, physical, and mental findings seen in these 2 individuals has not been reported previously.

The phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was used as a comitogen with the plant lectins phytohemagglutinin (PHA) and concanavalin A (ConA) in short-term cultures of whole blood from nonmammalian vertebrates. Stimulation with TPA in addition to standard mitogens resulted in a synergistic effect, consistently yielding more metaphases than cultures stimulated with either PHA, ConA, or TPA alone and is successful with blood samples as small as 0.1 ml. The increased mitotic index makes it possible to use different banding procedures for systematic studies. Also, because the amount of blood needed is so small, this procedure, unlike other published techniques, does not require the destruction of smaller animals to do chromosome studies.

Six different techniques were evaluated to define better those technical factors that are most critical for obtaining prometaphase cells for banding analysis. Our results demonstrate: colcemid exposures of 30 min or less have no effect on increasing the yield of prometaphase cells, colcemid exposures of greater than 0.1 microgram/ml can be toxic, methotrexate depresses the mitotic index significantly and seems to increase the incidence of prometaphase cells only because it suppresses later forms; and (d) the optimum number of cytogenetically satisfactory prometaphase cells can be obtained with a 4-h exposure to a combination of low concentration actinomycin D (0.5 microgram/ml) and colcemid (0.1 microgram/ml). This technique inhibits chromosome condensation while permitting prometaphase cells to accumulate for 4 h.