Duplex DNA from Sites of Helicase-Polymerase Uncoupling Links Non-B DNA Structure Formation to Replicative Stress

BACKGROUND: Replication impediments can produce helicase-polymerase uncoupling allowing lagging strand synthesis to continue for as much as 6 kb from the site of the impediment. MATERIALS AND METHODS: We developed a cloning procedure designed to recover fragments from lagging strand near the helicase halt site. RESULTS: A total of 62% of clones from a p53-deficient tumor cell line (PC3) and 33% of the clones from a primary cell line (HPS-19I) were within 5 kb of a G-quadruplex forming sequence. Analyses of a RACK7 gene sequence, that was cloned multiple times from the PC3 line, revealed multiple deletions in region about 1 kb from the cloned region that was present in a non-B conformation. Sequences from the region formed G-quadruplex and i-motif structures under physiological conditions. CONCLUSION: Defects in components of non-B structure suppression systems (e.g. p53 helicase targeting) promote replication-linked damage selectively targeted to sequences prone to G-quadruplex and i-motif formation

Assessing karyotype precision by microarray-based comparative genomic hybridization in the myelodysplastic/myeloproliferative syndromes

<p>Abstract</p> <p>Background</p> <p>Recent genome-wide microarray-based research investigations have revealed a high frequency of submicroscopic copy number alterations (CNAs) in the myelodysplastic syndromes (MDS), suggesting microarray-based comparative genomic hybridization (aCGH) has the potential to detect new clinically relevant genomic markers in a diagnostic laboratory.</p> <p>Results</p> <p>We performed an exploratory study on 30 cases of MDS, myeloproliferative neoplasia (MPN) or evolving acute myeloid leukemia (AML) (% bone marrow blasts ≤ 30%, range 0-30%, median, 8%) by aCGH, using a genome-wide bacterial artificial chromosome (BAC) microarray. The sample data were compared to corresponding cytogenetics, fluorescence <it>in situ </it>hybridization (FISH), and clinical-pathological findings. Previously unidentified imbalances, in particular those considered submicroscopic aberrations (< 10 Mb), were confirmed by FISH analysis. CNAs identified by aCGH were concordant with the cytogenetic/FISH results in 25/30 (83%) of the samples tested. aCGH revealed new CNAs in 14/30 (47%) patients, including 28 submicroscopic or hidden aberrations verified by FISH studies. Cryptic 344-kb <it>RUNX1 </it>deletions were found in three patients at time of AML transformation. Other hidden CNAs involved 3q26.2/EVI1, 5q22/APC, 5q32/TCERG1,12p13.1/EMP1, 12q21.3/KITLG, and 17q11.2/NF1. Gains of CCND2/12p13.32 were detected in two patients. aCGH failed to detect a balanced translocation (n = 1) and low-level clonality (n = 4) in five karyotypically aberrant samples, revealing clinically important assay limitations.</p> <p>Conclusions</p> <p>The detection of previously known and unknown genomic alterations suggests that aCGH has considerable promise for identification of both recurring microscopic and submicroscopic genomic imbalances that contribute to myeloid disease pathogenesis and progression. These findings suggest that development of higher-resolution microarray platforms could improve karyotyping in clinical practice.</p

Lineage-Specific Gene Families and Evolutionary Innovation

The mechanisms underlying organism evolution and innovation has long been studied. Currently there are three theories detailing how innovation is encoded in the genome. First, the gene duplication theory states that creation of new genomic material, ranging from a single exon to the entire genome, via duplication and subsequent mutational processes encodes for diversity. Second, the gene regulation theory states that mutation of a gene's regulatory sequences changes the temporal and spacial expression pattern of that gene and that, in turn, creates diversity. Finally, the de novo gene synthesis theory states that new genes are needed for creating new structures. Substantial progress has been made in understanding how conserved gene families, through both duplication and changes in regulatory sequences, encode for evolutionary innovation. However, very little is known about the function of de novo genes and their role in evolution. De novo genes fall within two categories. First, if de novo genes are found only within one particular species and share no homology with any other protein or domain, they are called orphan genes. Second, if orphan genes become fixed within the lineage they are called lineage-specific or taxonomically-restricted gene families. Here, we focused on lineage-specific gene families and investigated their significance in chordate and vertebrate evolution. We defined a set of vertebrate-specific gene families using two established protein homology databases, HomoloGene and TreeFam. We found that 20% of all known gene families are vertebrate-specific. Once we created a list of families, we identified biological processes with a significant overrepresentation of vertebrate-specific genes. We found that vertebrate-specific genes are enriched in the sensory, immune and neural systems, all of which are sites of vertebrate innovation. This suggests that vertebrate-specific genes are important in vertebrate diversity and innovation. Using the zebrafish (Danio rerio) as a model for vertebrate evolution, we identified a chordate-specific gene family, ponzr, and determined the role of ponzr1 in zebrafish kidney evolution. We found that ponzr1 is required for pronephric glomerular development, which is a vertebrate innovation. Furthermore, when ponzr1 is knocked down, the kidney retains some functionality. However, the filtering appears further posterior along the pronephric ducts. This suggests that, without ponzr1, a functional kidney is still patterned. However, the zebrafish patterns a simplified kidney demonstrating tubular secretion, similar to those seen in aglomerular fish. Finally, to make the zebrafish a more tractable model for vertebrate evolution and development and to create ponzr1 a knockout, we developed a technology for site-specific genome engineering in the zebrafish. Using a second generation transcription activator-like effector nuclease (TALEN) scaffold, GoldyTALEN, we saw increased efficiency in TALEN-created mutations. In 3 out of the 5 TALENs tested, we saw biallelic conversion in zebrafish larvae. The mutagenesis efficiency was sufficient to identify an F0 phenotype in larvae, similar to the phenotype in knockdown experiments. With this increased TALEN efficiency, we asked whether we could introduce exogenous sequences via homology directed repair. Using single stranded DNA as a template along with the ponzr1 GoldyTALEN, we were able to engineer an EcoRV and modified loxP site into the second exon of ponzr1. Furthermore, we were able to see germline transmission of the EcoRV sequence, creating the first successful HDR addition of an exogenous sequence in the zebrafish. In conclusion, this thesis demonstrates that lineage-specific gene families are important for evolutionary innovation. Using the zebrafish, we show that ponzr1 is necessary for pronephric glomerular innovation. Finally, we demonstrate that, using TALENs, we can engineer the zebrafish genome, making it a tractable model to study vertebrate innovation

Improving Functional Movement Patterns Reduces Pathomechanics in Competitive Distance Runners

Running related injuries are linked to faulty running biomechanics such as excessive hip adduction (HADD), hip internal rotation (HIR), knee valgus (KVAL), and contralateral pelvis drop (CPD). Functional movement pattern (FMP) training is a novel intervention used by clinicians to correct underlying FMPs (stepping, squatting) with the aim of affecting more sport specific mechanics. The purpose of this study was to evaluate the effectiveness of an 8-week FMP corrective program to reduce pathomechanics in six runners with dysfunctional FMPs. We used paired t-tests to evaluate changes in peak motion in degrees for HADD, HIR, KVAL, and CPD; and total Functional Movement Screen (FMS) scores out of 21. Five of seven FMS movements are scored bilaterally to evaluate asymmetries. Five male and one female adult competitive runners who ran at least 50 mpw (18.5+0.5 yrs, 1.7+0.1 m, 55.4+3.8 kg) and had dysfunctional FMPs as identified using the FMS participated in this study. They underwent 3D motion analysis of running biomechanics using a 10-camera motion capture system while running on a treadmill (3.10+0.3 m/s). An 8-week corrective exercise program based on their FMS results was incorporated into their team’s strength and conditioning sessions. Improvements were shown in FMS scores (pre 14.8+1.0 vs post 16.0+1.7, p\u3c.05), number of asymmetries (pre 4 vs post 2) and HIR (pre 16.2+3.60 vs post 10.4+3.60, p\u3c.05. There were no significant changes in HADD or CPD, p\u3e.05. Correcting underlying FMPs may reduce known running pathomechanics. This novel approach may be effective in developing injury prevention programs

Biomechanics Differ between Highest and Lowest Race Finishing Places of Collegiate Distance Runners: A Case Analysis

Faulty running biomechanics have been associated with injury, yet their role in performance is poorly understood. We sought to determine if gait and functional movement mechanics could differentiate between high and low performing distance runners. Three top and three bottom place finishers from a men’s university cross-country team were studied, [(top; 22.7+2.9 yrs, 63.0+4.0 kg, 1.73+0.04 m) (bottom; 19.7+4.6 yrs, 62.8+6.0 kg, 1.71+0.07 m)]. They were identified by reviewing six NCAA races for consistent performance in Fall 2016, and represent six of 11 team members. Participants underwent a 3D analysis using a camcorder and 10-camera motion capture system while running on an instrumented treadmill (2.86-3.28 m/s). Running technique was qualitatively and quantitatively examined for known faulty biomechanics such as excessive hip internal rotation (HIR), hip adduction (HADD), knee valgus (KVAL), rearfoot eversion (REV), and contralateral hip drop (CHD). Movement patterns were assessed using the Functional Movement Screen (FMS), a tool that categorizes seven movements as dysfunctional, compensatory, or adequate. Five of seven movements are scored bilaterally to evaluate asymmetries. Results showed both groups had one asymmetry and no dysfunctional scores on the FMS. However, 3/3 bottom placing runners displayed a CHD; 2/3 excessive HIR; 2/3 KVAL outside of normal limits; 1/3 excessive REV. Two of three top finishers showed a CHD and 1/3 excessive HADD. We conclude that the bottom finishers had a greater number of faulty biomechanics than the top finishers, while there were no differences in movement patterns. Coaches should promote proper mechanics to positively impact performance

Zebrafish: A Pharmacogenetic Model for Anesthesia

General anesthetics are small molecules that interact with and effect the function of many different proteins to promote loss of consciousness, amnesia, and sometimes, analgesia. Owing to the complexity of this state transition and the transient nature of these drug/protein interactions, anesthetics can be difficult to study. The zebrafish is an emerging model for the discovery of both new genes required for the response to and side effects of anesthesia. Here we discuss the tools available to manipulate the zebrafish genome, including both genetic screens and genome engineering approaches. Additionally, there are various robust behavior assays available to study anesthetic and other drug responses. These assays are available for single-gene study or high throughput for genetic or drug discovery. Finally, we present a case study of using propofol as an anesthetic in the zebrafish. These techniques and protocols make the zebrafish a powerful model to study anesthetic mechanisms and drug discovery

Molecular karyotypes of hodgkin and reed-sternberg cells at disease onset reveal distinct copy number alterations in chemosensitive versus refractory hodgkin lymphoma

Purpose: To determine the recurring DNA copy number alterations (CNA) in classical Hodgkin lymphoma (HL) by microarray-based comparative genomic hybridization (aCGH) using laser capture microdissected CD30+ Hodgkin and Reed-Sternberg (HRS) cells. Experimental Design: Archived tissues from 27 CD30+ HL plus control samples were analyzed by DNA microarrays. The HL molecular karyotypes were compared with the genomic profiles of germinal center B cells and treatment outcome (chemotherapy responsive vs. primary refractory disease). Results: Gains and losses observed in more than 35% of HL samples were localized to 22 and 12 chromosomal regions, respectively. Frequent gains (\u3e65%) were associated with growth and proliferation, NF-kB activation, cell-cycle control, apoptosis, and immune and lymphoid development. Frequent losses (\u3e40%) observed encompassed tumor suppressor genes (SPRY1, NELL1, and ID4, inhibitor of DNA binding 4), transcriptional repressors (TXNIP, thioredoxin interacting protein), SKP2 (S-phase kinase-associated protein 2; ubiquitin ligase component), and an antagonist of NF-kB activation (PPARGC1A). In comparison to the germinal center profiles, the most frequent imbalances in HL were losses in 5p13 (AMACR, GDNF, and SKP2), and gains in 7q36 (SHH, sonic hedgehog homolog) and 9q34 (ABL1, CDK9, LCN2, and PTGES). Gains (\u3e35%) in theHLchemoresponsive patients housed genes known to regulate T-cell trafficking or NF-κB activation (CCL22, CX3CL1, CCL17, DOK4, and IL10), whereas the refractory samples showed frequent loss of 4q27 (interleukin; IL21/IL2) and 17p12, and gain of 19q13.3 (BCL3/RELB). Conclusion: We identified nonrandom CNAs in the molecular karyotypes of classical HL. Several recurring genetic lesions correlated with disease outcome. These findings may be useful prognostic markers in the counseling and management of patients and for the development of novel therapeutic approaches in primary refractory HL. ©2011 AACR

Evaluation of chronic lymphocytic leukemia by BAC-based microarray analysis

<p>Abstract</p> <p>Background</p> <p>Chronic lymphocytic leukemia (CLL) is a highly variable disease with life expectancies ranging from months to decades. Cytogenetic findings play an integral role in defining the prognostic significance and treatment for individual patients.</p> <p>Results</p> <p>We have evaluated 25 clinical cases from a tertiary cancer center that have an established diagnosis of CLL and for which there was prior cytogenetic and/or fluorescence <it>in situ </it>hybridization (FISH) data. We performed microarray-based comparative genomic hybridization (aCGH) using a bacterial artificial chromosome (BAC)-based microarray designed for the detection of known constitutional genetic syndromes. In 15 of the 25 cases, aCGH detected all copy number imbalances identified by prior cytogenetic and/or FISH studies. For the majority of those not detected, the aberrations were present at low levels of mosaicism. Furthermore, for 15 of the 25 cases, additional abnormalities were detected. Four of those cases had deletions that mapped to intervals implicated in inherited predisposition to CLL. For most cases, aCGH was able to detect abnormalities present in as few as 10% of cells. Although changes in ploidy are not easily discernable by aCGH, results for two cases illustrate the detection of additional copy gains and losses present within a mosaic tetraploid cell population.</p> <p>Conclusions</p> <p>Our results illustrate the successful evaluation of CLL using a microarray optimized for the interrogation of inherited disorders and the identification of alterations with possible relevance to CLL susceptibility.</p