Aberrant Sites Research Articles

Abstract 1167: Osteolysis, splenic and hepatic extramedullary hematopoiesis, MDSCs, tumor growth, and metastases by orthotopic mammary tumors are increased by alcohol consumption and fatty diets

Abstract Fatty diets can induce low-grade inflammation that we report is increased by chronic alcohol consumption (CAC). CAC as 16.6% of total calories when administered in combination with the Lieber-DeCarli high-fat diet increases inflammation included hepatic and splenic extramedullary hematopoiesis (EMH) as assessed by flow cytometry, immunohistochemistry and a colony forming unit-granulocyte macrophage (CFU-GM) assay. Further, an increased number of hepatic myeloid derived suppressor cells (MDSCs) CD11b+Gr1+ cells that were predominantly Ly6cbr are observed. The increase in MDSCs is associated with an increased number of hepatic non-parenchymal cells including adipocytes (Oil Red O). Consistent with the increased number of hepatic MDSCs and EMH is a decrease in bone marrow cellularity and progenitor cells measured by flow cytometry (Lin-CD11b-Gr1-Sca-1+) and CFU-GM/femur. Unexpectedly, we observed demineralization and osteolytic lesions by micro computed tomography (micro CT) in all bones examined including femur, tibia, fibula and vertebral column that was associated with osteoclast activity (Trap+). Osteolysis was most notable in the fibula and vertebral spurs associated with osteoclast channels, and the demineralization appeared to be associated with areas of active myelopoiesis. The low grade, chronic inflammation associated with the Lieber-DeCarli fatty diet and CAC accelerated the induction of orthotopic 4T1 mammary tumors, resulting in extensive bone osteolysis, demineralization and increased metastases at aberrant sites including splenic, cardiac, hepatic, and extensive lymph node foci in addition to peritoneal and pleural effusions. The latter were haemorrhagic with a predominant nucleated cell infiltrate composed of bands, segs and myelocytes, supporting EMH. These results support the suggestion that a high-fat diet and CAC together increase tumor induction, metastasis and pathology in association with MDSC mobilization, expansion and EMH resulting in increased numbers of osteoclasts, associated bone demineralization and suppression of T-cell frequency and function. These results support the development of combination therapy strategies incorporating multiple molecular therapeutics that inhibit OCs, MDSCs and their associated mediators. Citation Format: Anand Dusad, Saraswoti Khadge, Geoffrey M. Thiele, Michael J. Duryee, Holly C. Britton, Lynell W. Klassen, Alicia J. Dafferner, Tracy Farrell, Timothy R. McGuire, Carlos D. Hunter, Karen C. Easterling, Karen J. O'Kane, John Graham Sharp, James E. Talmadge. Osteolysis, splenic and hepatic extramedullary hematopoiesis, MDSCs, tumor growth, and metastases by orthotopic mammary tumors are increased by alcohol consumption and fatty diets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1167. doi:10.1158/1538-7445.AM2014-1167

Frequent inactivation of the XAF1 tumor suppressor gene in human ovarian cancer by aberrant promoter CpG sites hypermethylation

Frequent inactivation of the XAF1 tumor suppressor gene in human ovarian cancer by aberrant promoter CpG sites hypermethylation

Abstract 2236: A selection strategy for the design of small proteins that can distinguish DNA repair intermediates.

Abstract Recognition of DNA damage by repair proteins is crucial for genomic stability. Several repair enzymes recognize aberrant DNA structural features, including nicks, gaps, and abasic sites, in a sequence-independent DNA manner. Reported here is a strategy to model these protein-DNA interactions using small proteins. A phage-displayed library of approximately one hundred million distinct zinc fingers was engineered at the DNA level based on the DNA binding domain of the repair protein poly(ADP-ribose) polymerase. Eight codons, representing amino acid residues in ‘loop’ regions of the zinc finger structure, were randomized using an NNK codon scheme. The DNA cassette was cloned into a commercially obtained T7 phagemid vector. The resultant phage library was exposed to nicked DNA molecules, which were attached to magnetic beads via a biotin-streptavidin linkage. In four rounds of selection that featured negative selection against non-nicked DNA and streptavidin coated beads, viral retention on the affinity resin increased from a negligible quantity to 62%. DNA sequencing following the final round revealed near convergence to a single, 39-mer peptide. This selected peptide will be generated by bacterial expression and assayed for binding activity using multiple strategies. While phage display of sequence specific DNA binding proteins is well-precedented, selection of peptides that bind specific DNA structures has not been reported. Molecules discovered by this strategy will help illuminate the structure-function relationships of proteins that recognize distinct DNA structures and may lead to new anticancer approaches. Citation Format: Rachel M. Guerra, Kristina M. Langenborg, Kayla M. Gross, Emily V. Sher, John W. Gilboy, Dylan Plaskon, Kevin P. Rice. A selection strategy for the design of small proteins that can distinguish DNA repair intermediates. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2236. doi:10.1158/1538-7445.AM2013-2236

Geographical distribution of lymph node metastases on MR lymphography in prostate cancer patients

PurposeTo investigate the pattern of lymph node spread on magnetic resonance lymphography (MRL) in prostate cancer patients and compare this pattern to the clinical target volume for elective pelvis irradiation as defined by the radiation therapy oncology group (RTOG-CTV). Methods and materialsThe charts of 60 intermediate and high risk prostate cancer patients with non-enlarged positive lymph nodes on MRL were reviewed. Positive lymph nodes were assigned to a lymph node region according to the guidelines for delineation of the RTOG-CTV. Five lymph node regions outside this RTOG-CTV were defined: the para-aortal, proximal common iliac, pararectal, paravesical and inguinal region. ResultsFifty-three percent of the patients had an MRL-positive lymph node in a lymph node region outside the RTOG-CTV. The most frequently involved aberrant sites were the proximal common iliac, the pararectal and para-aortal region, which were affected in 30%, 25% and 18% respectively. ConclusionMore than half of the patients had an MRL-positive lymph node outside the RTOG-CTV. To reduce geographical miss while minimizing the toxicity of radiotherapy, image based definition of an individual target volume seems to be necessary.

First record of Chrysomya albiceps Wiedemann, 1819 (Diptera: Calliphoridae) maggots from a sambar deer (Rusa unicolor) in Kerala, South India

Fully grown third stage larvae (LIII) of Chrysomya albiceps were recovered from aberrant sites viz. trachea and rumen during necropsy of a free-range sambar deer that had been observed to bear an inflamed tongue infested with maggots and subsequently died due to starvation. Five dead maggots of C. bezziana were also recovered from rumen. The aberrant locations of the recovery of the maggots indicated that they might have reached these sites accidentally. This is the first report of LIII of C. albiceps from a sambar deer from Kerala, South India.

Epididymo-orchitis: An unusual manifestation of salmonellosis

Salmonellosis continues to be a major public health problem, especially in developing countries. The formation of focal abscesses may occur following either hematogenous or lymphatic spread. There are large number of serious and life-threatening clinical manifestations of Salmonella spp., ranging from osteomyelitis to infective endocarditis and meningitis. However, even though Salmonella epidydimo-orchitis is a relatively rare clinical manifestion, it can present, most often in male babies and adolescent boys, following contact with nontyphoidal Salmonella. Here, we report a case of epididymo-orchitis due to Salmonella Paratyphi A that presented in an otherwise healthy 63-year-old man in order to highlight this organism's unusual clinical presentation. In countries such as India, where Salmonella infections are endemic, a high index of suspicion should be always be maintained and the possibility of a Salmonella infection at an aberrant site where it is hardly expected should not be ruled out.

Rcl1 Protein, a Novel Nuclease for 18 S Ribosomal RNA Production

In all forms of life, rRNAs for the small and large ribosomal subunit are co-transcribed as a single transcript. Although this ensures the equimolar production of rRNAs, it requires the endonucleolytic separation of pre-rRNAs to initiate rRNA production. In yeast, processing of the primary transcript encoding 18 S, 5.8 S, and 25 S rRNAs has been studied extensively. Nevertheless, most nucleases remain to be identified. Here, we show that Rcl1, conserved in all eukaryotes, cleaves pre-rRNA at so-called site A(2), a co-transcriptional cleavage step that separates rRNAs destined for the small and large subunit. Recombinant Rcl1 cleaves pre-rRNA mimics at site A(2) in a reaction that is sensitive to nearby RNA mutations that inhibit cleavage in vivo. Furthermore, mutations in Rcl1 disrupt rRNA processing at site A(2) in vivo and in vitro. Together, these results demonstrate that the role of Rcl1 in eukaryotic pre-rRNA processing is identical to that of RNase III in bacteria: to co-transcriptionally separate the pre-rRNAs destined for the small and large subunit. Furthermore, because Rcl1 has no homology to other known endonucleases, these data also establish a novel class of nucleases.

Salmonella-Salmonellosis-Rare presentations of a common pathogen

Salmonella are most commonly associated with gastroenteritis and enteric fever in humans. Occasionally, dissemination of bacilli throughout the body results in establishment of localized foci of persistent infection especially in patients with debilitating diseases and immunosuppressive states. Infection at various aberrant sites due to Salmonella has been reported relatively seldom. It has perfected the art of intracellular survival in niches from where they can cause myriad of effects. Six cases with Salmonella infection at unusual sites without any preexisting or underlying disease diagnosed over a period of two years are presented here. Salmonella etiology was not suspected in these patients and the diagnosis was made microbiologically only after culture isolation.

Value of sentinel node mapping in cancer of the cervix

Objectives To compare the relative value of two methods of detection for the sentinel lymphatic nodes (SLNs): colorimetric with Isosulfan blue (ISB) and radio-isotopic with Technetium-99 (Tc99), and to evaluate the concept of the SLN mapping applied to cervical cancer. Methods From October 2000 to December 2006, radical surgery was planned in 211 patients who presented early-stage cancer of the cervix. Both ISB and Tc99 were used to detect the SLNs. In all cases, we proceeded with laparoscopy for the identification and removal of the SLNs, followed by a complete pelvic lymphadenectomy with or without para-aortic node sampling. The SLNs were sent for frozen section (1 level) and were ultra-staged (6 levels) for final pathology. Detection rate, sensitivity and negative predictive value (NPV) were calculated. Results Among the 211 patients, ISB (n = 152) identified at least 1 SLN in 92.8% of the cases. With Tc99 (n = 166), the detection rate of SLN increased to 96.9%. When both techniques were used together (n = 107), Tc99 was significantly better than ISB by 7.8% (p = 0.0094) and at least 1 SLN (hot and/or blue) was found in 99.1% of the cases. In 16.7% of patients, a SLN was located in aberrant sites, including 3.8% in the para-aortic area. Thirty-three out of the 211 patients (15.6%) had lymph node metastases. When considering only the 181 patients with bilateral SLNs identified, the NPV of SLN is 100% after ultra staging on final pathology and 94.2% on frozen section (FS). Conclusion Sentinel node mapping is feasible using laparoscopy. The radio-isotopic technique adds significantly to the rate of detection. The main benefits of SLN mapping in cervical cancer are the detection of micro-metastases on ultra staging which might be missed on routine pathological evaluation, and identification of aberrant drainage sites. However, the current frozen section techniques lack sensitivity to identify very small metastases and need refinement. SLN mapping should become the standard of care in the modern management of cervical cancer and complete pelvic lymphadenectomy could be avoided when bilateral SLNs are detected in patients with lesions less than 2 cm.

In a Xenograft Mouse Model, Bethanechol, a Muscarinic Receptor Agonist, Attenuates the Growth of Human Colon Cancer Cells

hSRBC is a recently identified tumor suppressor located at the chromosomal region 11p15.4. hSRBC expression is frequently downregulated in many types of malignancies including colorectal cancer due to the aberrant promoter CpG sites hypermethylation. The hSRBC activation induces cell cycle arrest and apoptosis by enhancing the protein stability of p53. However, the molecular basis of hSRBC activation and its regulation has been poorly understood. To explore the implication of hSRBC abnormality in colorectal tumorigenesis and the molecular mechanism underlying its tumor suppressive function, the present study tested the possible regulation of hSRBC by TNFα.TNFα regulation of hSRBC expression was examined using semi-quantitative RT-PCR and immunoblot assays. It was found that mRNA expression of hSRBC is strongly activated by TNFa in a doseand time-associated manner in various human colonic epithelial cells, includingHCT15 andHT-29. An immunoblot assay also revealed that hSRBC protein expression is increased following TNFa treatment. TNFa induction of hSRBC was blocked by pretreatment of the cells with the NFkB inhibitor BAY11-7082 or transfection with a dominant negative mutant form of IkB, indicating that TNFα-induced hSRBC expression is mediated via the NFkB pathway. Flow cytometric analysis of sub-G1 phase using hSRBC-nonexpressing HCT15 cells revealed that ectopic expression of hSRBC greatly enhances apoptotic response to TNFα. Collectively, these data strongly suggest that hSRBC might be a novel transcription target of TNFα and play an important role in cellular response to TNFα. TNFα regulation of hSRBC expression was examined using semi-quantitative RT-PCR and immunoblot assays. It was found that mRNA expression of hSRBC is strongly activated by TNFα in a doseand time-associated manner in various human colonic epithelial cells, including HCT15 and HT-29. An immunoblot assay also revealed that hSRBC protein expression is increased following TNFa treatment. TNFa induction of hSRBC was blocked by pretreatment of the cells with the NFkB inhibitor BAY11-7082 or transfection with a dominant negative mutant form of IkB, indicating that TNFα-induced hSRBC expression is mediated via the NFkB pathway. Flow cytometric analysis of sub-G1 phase using hSRBC-nonexpressing HCT15 cells revealed that ectopic expression of hSRBC greatly enhances apoptotic response to TNFα. Collectively, these data strongly suggest that hSRBC might be a novel transcription target of TNFα and play an important role in cellular response to TNFα.

Not Quite Right Landscapes: Designing within Redundant Space in a Peri-urban Agriculture Region

In this case study design processes are used to explore potential new land-use patterns in redundant spaces in an intensive horticultural region, in peri-urban Melbourne, Victoria. Currently, a mismatch between title boundaries and commercial scale horticultural production methods has led to pockets of under-utilised land. The projects parameters were to increase efficiency, introduce cross programming and develop a strategy for recreation as a new activity in this region. The underlying purpose of this speculative design project was to develop design methods to work with aberrant and unusual sites without losing the very qualities that characterise these places. On this site the overriding imperative was to ensure that the outcome preserves the existing haunting beauty of this region. The case study offers a design framework that can be utilised in other circumstances where it is important to reorient land use without destroying the not-quite-right qualities of places that are so easily overlooked but so important to the genius loci.

Abstract 922: Association of the hSRBC gene polymorphism with endometrial cancer

Abstract Background: hSRBC is a putative tumor suppressor located at 11p15.4, at which frequent genomic loss has been observed in several human malignancies. We previously reported that hSRBC expression is frequently downregulated in many types of human cancers due to aberrant promoter CpG sites hypermethylation, and hSRBC activation induces cell cycle arrest and apoptosis by enhancing the protein stability of p53. Interestingly, our recent studies suggest that hSRBC might be a novel transcription target of TNFα, which plays a crucial role in inflammation and tumorigenesis. We explored the possible association of an intreaexonic SNP (T509C), which results in a Leu to Pro substitution, with risk for endometrial cancer development. Methods: TNFα regulation of hSRBC expression was examined using semi-quantitative RT-PCR and immunoblot assays. Endometrial tissues from 147 cancer patients and 191 healthy individuals were included for test for SNP T509C by restriction endonuclease PvuII-based genotyping. Allele frequencies in cancer specimens were compared with those in healthy controls. Results: Allelic frequencies of T and C at the T509C position was 30.27% and 69.73% in healthy control and 37.17% and 62.83% in cancer specimens, respectively. Allelic frequency at T509C in cancer specimens showed no significant difference compared to controls. However, genotype frequencies of TT, CT, and CC in healthy controls were 16.75%, 40.84%, and 42.412%, respectively, while cancer specimens displayed 14.97%, 30.61%, and 54.42%. The CC genotype frequency in cancer specimens was significantly higher (p < 0.05) compared with that in healthy controls. Conclusion: Although the number of specimens used in this study is not enough, this study raises the possibility that the T509C SNP of hSRBC, a putative novel transcription target of TNFα and proapoptotic tumor suppressor, might be associated with endometrial cancer development. Further genotype screening of several other SNPs within hSRBC in cancer patients would provide valuable diagnostic and prognostic information for human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 922.

Abstract 193: Dissecting satellite and imprinting genes methylation status in cloned bovine: Aberrant methylation in imprinting loci

Abstract In mammals, genome-wide epigenetic reprogramming systems exist in primordial germ cells and zygote. It plays a crucial role in regulating genome functions at critical stages of embryo development and confers stability of gene expression during mammalian development. Inappropriate reprogramming of donor nuclei in somatic cell resulted in neonatal death and preimplantation defects during animal cloning technique. Specific demethylation events in differentiated tissues could then lead to further changes in gene expression as needed. In this study, six cloned bovines created by ear fibroblast nuclear transfer with short life span and multiple organ defects were used as the experimental materials. We focus on three imprinting genes included two growth factor genes IGF-2, H19 and Xist (X chromosome inactive regulated gene) by combined bisulfite and restriction assay (COBRA). Bisulfite sequencing was also applied to analyze the aberrant CpG sites in different imprinted genes loci in these cloned bovine genomes. Our data show that loss of imprinting (LOI) phenomenon was frequently appeared in IGF-2, Xist and H19 imprinted loci in several tissues of these cloned bovines. Furthermore, we detected the cloned bovine NTG-2 expression of these imprinting genes with quantitative real time PCR (Q-PCR). The data suggested that IGF-2 and H19 are extremely overexpressed in liver, vein, ear, skin and uterus. The IGF-2 mRNA expression in uterus and liver of NTG-2 cow are almost 1,200 and 800 folds compared to WT cow. The H19 expression level in liver of NTG-2 also exhibited a 34,000-fold higher than that of WT cow. According to our research, the extensively overexpression of IGF-2 and H19, probably were the major reason to cause the cloned bovine NTG-2 not only organ defects, but also aberrant embryonic development. In conclusion, the death of clones may be due to aberrant DNA methylation at the loci of imprinting genes and disruption of incompletely reprogrammed after nuclear transfer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 193.

A Sliding Scale Governs Mismatch Recognition and Initiation of DNA Mismatch Repair

DNA mismatch repair (MMR) maintains genome integrity by correcting base pair errors and triggering cell cycle checkpoints following DNA damage. It follows that MMR proteins are critical for suppressing mutagenesis and defects in their function are linked to cancer. The challenging task of finding rare aberrant sites in DNA is performed by MutS proteins, which recognize mismatches/lesions in DNA and initiate a response. To understand the mechanism of action of MutS, we monitored S. cerevisiae MutSα (Msh2‐6) binding to DNA in real time and measured transient interactions that govern its search for target sites. We found that Msh2‐6 binds a multitude of sites at similar high rates (kON=107 M−1s−1), but releases them at rates that vary by 100‐fold; e.g., kOFF for G:T or O6‐MeG:T is 0.015 s−1 (t1/2=46 s), while kOFF for +3T insertion is 2 s−1 (t1/2=0.34 s). By tracking Msh2‐6 movement we found that the protein slides on DNA making rapid contact with bases, and the lifetimes of resulting Msh2‐6•DNA complexes determine their selection as target sites for MMR. Thus, the long‐lived G:T complex effectively stabilizes Msh2‐6 in an ATP‐bound form that signals MMR, whereas the short‐lived +3T complex has little effect on ATP hydrolysis. Our kinetic data reveal that Msh2‐6 literally uses a ‘sliding scale’ to distinguish target sites, and offer an underlying basis for the bias in MMR reported by previous in vivo studies. Funding: NSF.

Serum Response Factor Is An Essential Transcription Factor in Megakaryocytic Maturation.

Abstract 3652Poster Board III-588SRF is a MADS-box transcription factor first identified as an inducer of immediate-early gene expression, such as c-fos, Junb, Fosb and Egr1, in response to cytokines. It plays a critical role in cardiac and smooth muscle development and differentiation by regulating cell cycle, apoptosis, cell growth, and differentiation. SRF regulates expression of contractile and cytoskeletal genes. Its function in hematopoiesis has not yet been revealed. While no hematopoietic disease specific mutations of SRF have been identified, the region on chromosome 6 where the SRF gene is located, is a site of frequent chromosomal aberrations in MDS. MKL1, a target of the t(1;22) translocation in acute megakaryoblastic leukemia, is a cofactor for SRF mediated gene activation. In published work (Cheng EC et al., Blood 2009;113:2826), we showed that MKL1 expression increases with normal megakaryocytic (Mk) differentiation, that it promotes Mk differentiation by increasing the expression of Mk specific genes such as CD42b, and that mice lacking MKL1 have decreased platelet counts while showing increased numbers of low ploidy Mk in the BM. In addition, we showed that the effects of MKL1 on Mk differentiation require SRF. In order to test the role of SRF in Mk development, we crossed PF4-Cre mice (Tiedt R et al., Blood 2007;109:1503), which express Cre recombinase in cells committed to the Mk lineage, to SRFF/F mice (Miano JM et al., PNAS 2004;101:17132) in which functional SRF is no longer expressed after Cre-mediated excision. Our findings are quite surprising, as knockout of SRF in the Mk lineage leads to a far more profound phenotype than MKL1 KO. PF4-Cre x SRFF/F (PF4-Cre/SRF) mice are born with a normal mendelian frequency, but have significant macrothrombocytopenia. The platelet counts in WT and KO mice are 703 ± 33 × 103/μl vs 460 ± 23 × 103/μl, respectively. Despite the decreased platelet number, the BM has increased numbers and percentages of CD41+ Mk (WT: 0.41 ± 0.06% and KO: 1.92 ± 0.12%) with a predominance of Mk with hypolobated nuclei. Spleens of PF4-Cre/SRF mice show significantly increased numbers of Mk (mean of 4 and 15 Mk per high power field for WT and KO spleens, respectively). Ploidy as an assay of Mk maturation in the BM is significantly reduced in SRF −/− Mk. The percentage of CD41+ cells in SRF KO BM that is 2N is nearly 2-fold higher, and the percentage with greater than 8N ploidy is decreased by 70%. Hematopoietic stem and progenitor populations in PF4-Cre/SRF BM are unaffected, as expected due to stage and lineage specific knockout of SRF. In contrast, there is an increase in Mk progenitors in vivo in PF4-Cre/SRF mice, reflected both by FACS analysis and CFU-Mk in vitro analysis. The mechanism by which SRF disrupts Mk maturation was studied by assessment of Mk morphology, and gene expression analysis. Mk lacking SRF expression have abnormal stress fiber formation based on phalloidin staining, a phenotype parallel to that observed in mice lacking myosin heavy chain 9 (MYH9) expression, and MYH9 expression was decreased in SRF KO Mk. In summary, the data show that SRF is critical for normal Mk maturation including polyploidization and platelet production, and the mechanism by which loss of SRF disrupts megakaryocytopoiesis is, at least in part, by loss of normal expression of known targets of SRF, such as cytoskeletal genes including MYH9. Disclosures:No relevant conflicts of interest to declare.

Two-stage dynamic DNA quality check by xeroderma pigmentosum group C protein

Xeroderma pigmentosum group C (XPC) protein initiates the DNA excision repair of helix-distorting base lesions. To understand how this versatile subunit searches for aberrant sites within the vast background of normal genomic DNA, the real-time redistribution of fluorescent fusion constructs was monitored after high-resolution DNA damage induction. Bidirectional truncation analyses disclosed a surprisingly short recognition hotspot, comprising approximately 15% of human XPC, that includes two beta-hairpin domains with a preference for non-hydrogen-bonded bases in double-stranded DNA. However, to detect damaged sites in living cells, these DNA-attractive domains depend on the partially DNA-repulsive action of an adjacent beta-turn extension that promotes the mobility of XPC molecules searching for lesions. The key function of this dynamic interaction surface is shown by a site-directed charge inversion, which results in increased affinity for native DNA, retarded nuclear mobility and diminished repair efficiency. These studies reveal a two-stage discrimination process, whereby XPC protein first deploys a dynamic sensor interface to rapidly interrogate the double helix, thus forming a transient recognition intermediate before the final installation of a more static repair-initiating complex.

INFREQUENTLY METHYLATED EVENT AT SITES −181 TO −9 WITHIN THE 5′ CpG ISLAND OF E-CADHERIN IN NON-SMALL CELL LUNG CANCER

Epigenetic silencing of E-cadherin via aberrant methylation has been investigated in various human tumors, whereas evidence for elucidating mechanism underlying reduction of E-cadherin mRNA remains unclear in non-small cell lung cancer (NSCLC). The authors previously found that reduction of E-cadherin mRNA or protein expression has been frequently observed in NSCLC. In this study, the authors explore the contribution of E-cadherin methylation to the development and progression of NSCLC. The authors directly performed the bisulfite DNA sequencing to examine CpG methylation within the 5′ CpG island of E-cadherin in 35 tumor and paired normal tissue specimens from patients with primary NSCLC. Then, the authors measured the level of E-cadherin mRNA by real-time quantitative polymerase chain reaction (PCR) analysis. Despite of reduction in E-cadherin mRNA by 65.7% (23/35) and presence of methylation by 28.6% (10/35) in tumors, the authors found no association of reduction of E-cadherin mRNA level with methylation of 19 sites from −181 to −9 bp located upstream from the translation start of E-cadherin in NSCLC. In conclusion, the authors provide no evidence for the presence of aberrant methylation sites of E-cadherin in tumors from patients with NSCLC, which can explain decrease of E-cadherin mRNA. Decrease in E-cadherin mRNA may be regulated by methylation-independent pathways in NSCLC.

Phylogenetic reconstruction and the identification of ancient polymorphism in the Bovini tribe (Bovidae, Bovinae).

BackgroundThe Bovinae subfamily incorporates an array of antelope, buffalo and cattle species. All of the members of this subfamily have diverged recently. Not surprisingly, a number of phylogenetic studies from molecular and morphological data have resulted in ambiguous trees and relationships amongst species, especially for Yak and Bison species. A partial phylogenetic reconstruction of 13 extant members of the Bovini tribe (Bovidae, Bovinae) from 15 complete or partially sequenced autosomal genes is presented.ResultsWe identified 3 distinct lineages after the Bovini split from the Boselaphini and Tragelaphini tribes, which has lead to the (1) Buffalo clade (Bubalus and Syncerus species) and a more recent divergence leading to the (2) Banteng, Gaur and Mithan and (3) Domestic cattle clades. A fourth lineage may also exist that leads to Bison and Yak. However, there was some ambiguity as to whether this was a divergence from the Banteng/Gaur/Mithan or the Domestic cattle clade. From an analysis of approximately 30,000 sites that were amplified in all species 133 sites were identified with ambiguous inheritance, in that all trees implied more than one mutation at the same site. Closer examination of these sites has identified that they are the result of ancient polymorphisms that have subsequently undergone lineage sorting in the Bovini tribe, of which 53 have remained polymorphic since Bos and Bison species last shared a common ancestor with Bubalus between 5–8 million years ago (MYA).ConclusionUncertainty arises in our phylogenetic reconstructions because many species in the Bovini diverged over a short period of time. It appears that a number of sites with ambiguous inheritance have been maintained in subsequent populations by chance (lineage sorting) and that they have contributed to an association between Yak and Domestic cattle and an unreliable phylogenetic reconstruction for the Bison/Yak clade. Interestingly, a number of these aberrant sites are in coding sections of the genome and their identification may have important implications for studying the neutral rate of mutation at nonsynonymous sites. The presence of these sites could help account for the apparent contradiction between levels of polymorphism and effective population size in domesticated cattle.

Bilateral Tuboovarian Abscess Due to Salmonella paratyphi A

Local abscess formation may occur as a complication of any Salmonella infection by either hematogenous or lymphatic spread but a tuboovarian site of Salmonella infection is rare. Mostly, predisposing factors such as ovarian abnormalities like dermoid cyst, endometrioma, cystadenoma, or simple cyst are present where hematogenously disseminated Salmonella tend to localize. A case of bilateral tuboovarian abscess due to Salmonella paratyphi A is being reported in a 36-year-old woman having multicystic ovaries to highlight the unusual presentation of this organism. In countries like India, where Salmonella infections are endemic, differential diagnosis of Salmonella should always be kept in mind by the clinicians while dealing with the tuboovarian abscesses because there is a possibility of Salmonella infection at aberrant sites, especially in patients with history of persistent fever or gastroenteritis or any preexisting abnormality that makes the tissue or organ vulnerable.

Mrd1p binds to pre-rRNA early during transcription independent of U3 snoRNA and is required for compaction of the pre-rRNA into small subunit processomes

In Saccharomyces cerevisiae, synthesis of the small ribosomal subunit requires assembly of the 35S pre-rRNA into a 90S preribosomal complex. SnoRNAs, including U3 snoRNA, and many trans-acting proteins are required for the ordered assembly and function of the 90S preribosomal complex. Here, we show that the conserved protein Mrd1p binds to the pre-rRNA early during transcription and is required for compaction of the pre-18S rRNA into SSU processome particles. We have exploited the fact that an Mrd1p-GFP fusion protein is incorporated into the 90S preribosomal complex, where it acts as a partial loss-of-function mutation. When associated with the pre-rRNA, Mrd1p-GFP functionally interacts with the essential Pwp2, Mpp10 and U3 snoRNP subcomplexes that are functionally interconnected in the 90S preribosomal complex. The fusion protein can partially support 90S preribosome-mediated cleavages at the A0–A2 sites. At the same time, on a substantial fraction of transcripts, the composition and/or structure of the 90S preribosomal complex is perturbed by the fusion protein in such a way that cleavage of the 35S pre-rRNA is either blocked or shifted to aberrant sites. These results show that Mrd1p is required for establishing productive structures within the 90S preribosomal complex.