Thalassemia Intermedia: Chelator or Not?

Thalassemia is the most common genetic disorder worldwide. Thalassemia intermedia (TI) is non-transfusion-dependent thalassemia (NTDT), which includes ß-TI hemoglobin, E/ß-thalassemia and hemoglobin H (HbH) disease. Due to the availability of iron chelation therapy, the life expectancy of thalassemia major (TM) patients is now close to that of TI patients. Iron overload is noted in TI due to the increasing iron absorption from the intestine. Questions are raised regarding the relationship between iron chelation therapy and decreased patient morbidity/mortality, as well as the starting threshold for chelation therapy. Searching all the available articles up to 12 August 2022, iron-chelation-related TI was reviewed. In addition to splenectomized patients, osteoporosis was the most common morbidity among TI cases. Most study designs related to ferritin level and morbidities were cross-sectional and most were from the same Italian study groups. Intervention studies of iron chelation therapy included a subgroup of TI that required regular transfusion. Liver iron concentration (LIC) ≥ 5 mg/g/dw measured by MRI and ferritin level > 300 ng/mL were suggested as indicators to start iron chelation therapy, and iron chelation therapy was suggested to be stopped at a ferritin level ≤ 300 ng/mL. No studies showed improved overall survival rates by iron chelation therapy. TI morbidities and mortalities cannot be explained by iron overload alone. Hypoxemia and hemolysis may play a role. Head-to-head studies comparing different treatment methods, including hydroxyurea, fetal hemoglobin-inducing agents, hypertransfusion as well as iron chelation therapy are needed for TI, hopefully separating ß-TI and HbH disease. In addition, the target hemoglobin level should be determined for ß-TI and HbH disease.

Comparing different surface modifications of zinc oxide nanoparticles in the developmental toxicity of zebrafish embryos and larvae.

Nanotechnology allows for a greater quality of life, but may also cause environmental and organismic harm. Zinc oxide nanoparticles (ZnONPs) are one of the most commonly used metal oxide nanoparticles for commercial and industrial products. Due to its extensive use in various fields, there has already been much concern raised about the environmental health risks of ZnONPs. Many studies have investigated the toxicological profile of ZnONPs in zebrafish embryonic development; however, the specific characteristics of ZnONPs in zebrafish embryonic/larval developmental damage and their molecular toxic mechanisms of liver development are yet to be fully elucidated. This study aimed to reveal the hazard ranking of different surface modifications of ZnONPs on developing zebrafish and the toxicological mechanisms of these modified ZnONPs in liver tissue. The ~30 nm ZnONPs with amino- (NH2- ZnONPs) or carboxyl- (COOH-ZnONPs) modification were incorporated during the embryonic/larval stage of zebrafish. Severe toxicity was observed in both ZnONP groups, especially NH2-ZnONPs, which presented a higher toxicity in the low concentration groups. After prolonging the exposure time, the long-term toxicity assay showed a greater retardation in body length of zebrafish in the NH2-ZnONP group. Response data from multiple toxicity studies was integrated for the calculation of the EC50 values of bulk ZnO and ZnONPs, and the hazard levels were found to be decreasing in the order of NH2-, COOH-ZnONPs and bulk ZnO. Notably, NH2-ZnONPs induced ROS burden in the developing liver tissue, which activated autophagy-related gene and protein expression and finally induced liver cell apoptosis to reduce liver size. In conclusion, our findings are conducive to understanding the hazard risks of different surface modifications of ZnONPs in aquatic environments and will also be helpful for choosing the type of ZnONPs in future industrial applications.

Using Sankey diagrams to explore the trend of article citations in the field of bladder cancer: Research achievements in China higher than those in the United States.

BACKGROUND: Urology authors are required to evaluate research achievements (RAs) in the field of bladder cancer (BC). However, no such bibliometric indices were appropriately applied to quantify the contributions to BC in research. In this study, we examined 3 questions: whether RAs in China are higher than those in the United States, how the Sankey-based temporal bar graph (STBG) may be applied to the analysis of the trend of article citations in the BC field, and what subthemes were reflected in China's and the United States' proportional counts in BC articles. METHODS: Using the PubMed search engine to download data, we conducted citation analyses of BC articles authored by urology scholars since 2012. A total of 9885 articles were collected and analyzed using the relative citations ratios (RCRs) and the STBG. The 3 research goals were verified using the RCRs, the STBG, and medical subject headings (MesH terms). The choropleth map and the forest plot were used to 1 highlight the geographical distributions of publications and RCRs for countries/regions and 2 compare the differences in themes (denoted by major MeSH terms on proportional counts using social network analysis to cluster topics) between China and the United States. RESULTS: There was a significant rise over the years in RCRs within the 9885 BC articles. We found that the RCRs in China were substantially higher than those in the United States since 2017, the STBG successfully explored the RCR trend of BC articles and was easier and simpler than the traditional line charts, area plots, and TBGs, and the subtheme of genetics in China has a significantly higher proportion of articles than the United States. The most productive and influential countries/regions (denoted by RCRs) were {Japan, Germany, and Italy} and {Japan, Germany, New York}, respectively, when the US states and provinces/metropolitan cities/areas in China were separately compared to other countries/regions. CONCLUSIONS: With an overall increase in publications and RCRs on BC articles, research contributions assessed by the RCRs and visualized by the STBGs are suggested for use in future bibliographical studies.

Toxic Effects and Mechanisms of Silver and Zinc Oxide Nanoparticles on Zebrafish Embryos in Aquatic Ecosystems.

The global application of engineered nanomaterials and nanoparticles (ENPs) in commercial products, industry, and medical fields has raised some concerns about their safety. These nanoparticles may gain access into rivers and marine environments through industrial or household wastewater discharge and thereby affect the ecosystem. In this study, we investigated the effects of silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnONPs) on zebrafish embryos in aquatic environments. We aimed to characterize the AgNP and ZnONP aggregates in natural waters, such as lakes, reservoirs, and rivers, and to determine whether they are toxic to developing zebrafish embryos. Different toxic effects and mechanisms were investigated by measuring the survival rate, hatching rate, body length, reactive oxidative stress (ROS) level, apoptosis, and autophagy. Spiking AgNPs or ZnONPs into natural water samples led to significant acute toxicity to zebrafish embryos, whereas the level of acute toxicity was relatively low when compared to Milli-Q (MQ) water, indicating the interaction and transformation of AgNPs or ZnONPs with complex components in a water environment that led to reduced toxicity. ZnONPs, but not AgNPs, triggered a significant delay of embryo hatching. Zebrafish embryos exposed to filtered natural water spiked with AgNPs or ZnONPs exhibited increased ROS levels, apoptosis, and lysosomal activity, an indicator of autophagy. Since autophagy is considered as an early indicator of ENP interactions with cells and has been recognized as an important mechanism of ENP-induced toxicity, developing a transgenic zebrafish system to detect ENP-induced autophagy may be an ideal strategy for predicting possible ecotoxicity that can be applied in the future for the risk assessment of ENPs.

An app to classify a 5-year survival in patients with breast cancer using the convolutional neural networks (CNN) in Microsoft Excel: Development and usability study.

BACKGROUND: Breast cancer (BC) is the most common malignant cancer in women. A predictive model is required to predict the 5-year survival in patients with BC (5YSPBC) and improve the treatment quality by increasing their survival rate. However, no reports in literature about apps developed and designed in medical practice to classify the 5YSPBC. This study aimed to build a model to develop an app for an automatically accurate classification of the 5YSPBC. METHODS: A total of 1810 patients with BC were recruited in a hospital in Taiwan from the secondary data with codes on 53 characteristic variables that were endorsed by professional staff clerks as of December 31, 2019. Five models (i.e., revolution neural network [CNN], artificial neural network, Naïve Bayes, K-nearest Neighbors Algorithm, and Logistic regression) and 3 tasks (i.e., extraction of feature variables, model comparison in accuracy [ACC] and stability, and app development) were performed to achieve the goal of developing an app to predict the 5YSPBC. The sensitivity, specificity, and receiver operating characteristic curve (area under ROC curve) on models across 2 scenarios of training (70%) and testing (30%) sets were compared. An app predicting the 5YSPBC was developed involving the model estimated parameters for a website assessment. RESULTS: We observed that the 15-variable CNN model yields higher ACC rates (0.87 and 0.86) with area under ROC curves of 0.80 and 0.78 (95% confidence interval 0.78-82 and 0.74-81) based on 1357 training and 540 testing cases an available app for patients predicting the 5YSPBC was successfully developed and demonstrated in this study. CONCLUSION: The 15-variable CNN model with 38 parameters estimated using CNN for improving the ACC of the 5YSPBC has been particularly demonstrated in Microsoft Excel. An app developed for helping clinicians assess the 5YSPBC in clinical settings is required for application in the future.

BRCA Genes and Related Cancers: A Meta-Analysis from Epidemiological Cohort Studies.

Background and Objectives:BRCA1 and BRCA2 are genes located in different chromosomes that are disproportionately associated with hereditary breast and ovarian cancer syndrome. Their association with other cancers remains to be explored. Materials and Methods: We systematically reviewed cohort studies to explore the association of BRCA 1 and BRCA2 with various cancers except lung cancer. We searched PubMed, Medline (EBSCOhost) and relevant articles published up to 10 May 2021. The odds ratio, standardised morbidity rate and cancer-specific standardised incidence ratio were pooled together as relative risk (RR) estimates. Results: Twelve studies were included for analysis. BRCA mutation increased pancreatic and uterine cancers by around 3-5- and 1.5-fold, respectively. BRCA mutation did not increase brain cancer; colorectal cancer; prostate, bladder and kidney cancer; cervical cancer; or malignant melanoma. BRCA2 increased gastric cancer with RR = 2.15 (1.98-2.33). Conclusion: The meta-analysis results can provide clinicians and relevant families with information regarding increased specific cancer risk in BRCA1 and BRCA2 mutation carriers.

BRCA1 and BRCA2 Gene Mutations and Lung Cancer Sisk: A Meta-Analysis.

Background and objective: BRCA1 and BRCA2 are associated with many cancer types in addition to hereditary breast and ovarian cancers. However, their relation to lung cancer remains to be explored. Materials and Methods: Observation studies were systematically reviewed to explore the association of BRCA1 or BRCA2 with lung cancer. PubMed, MEDLINE [EBSCOhost], and relevant articles published up to 7 January 2020 were searched. Odd ratio (OR), standardized morbidity rate (SMR), and cancer-specific standardized incidence ratios (SIRs) were pooled together as relative risk (RR) estimates (95% confidence interval [CI], 0.66-1.40). Results: Thirteen studies were included for analysis. Results showed that the RR of BRCA2 is 0.76 (95% CI, 0.48-1.19), the overall RR is 0.96 (95% CI, 0.66-1.40), and that of BRCA1 is 0.66 (95% CI, 0.41-1.05), indicating that it was not associated with lung cancer. Conclusion: With the limitation of the retrospective study design and severe heterogeneity, these results inform clinicians and relevant families that BRCA1 and BRCA2 mutation carriers have no increased risk of lung cancer.

The Current Understanding of Autophagy in Nanomaterial Toxicity and Its Implementation in Safety Assessment-Related Alternative Testing Strategies.

Nanotechnology has rapidly promoted the development of a new generation of industrial and commercial products; however, it has also raised some concerns about human health and safety. To evaluate the toxicity of the great diversity of nanomaterials (NMs) in the traditional manner, a tremendous number of safety assessments and a very large number of animals would be required. For this reason, it is necessary to consider the use of alternative testing strategies or methods that reduce, refine, or replace (3Rs) the use of animals for assessing the toxicity of NMs. Autophagy is considered an early indicator of NM interactions with cells and has been recently recognized as an important form of cell death in nanoparticle-induced toxicity. Impairment of autophagy is related to the accelerated pathogenesis of diseases. By using mechanism-based high-throughput screening in vitro, we can predict the NMs that may lead to the generation of disease outcomes in vivo. Thus, a tiered testing strategy is suggested that includes a set of standardized assays in relevant human cell lines followed by critical validation studies carried out in animals or whole organism models such as C. elegans (Caenorhabditis elegans), zebrafish (Danio rerio), and Drosophila (Drosophila melanogaster)for improved screening of NM safety. A thorough understanding of the mechanisms by which NMs perturb biological systems, including autophagy induction, is critical for a more comprehensive elucidation of nanotoxicity. A more profound understanding of toxicity mechanisms will also facilitate the development of prevention and intervention policies against adverse outcomes induced by NMs. The development of a tiered testing strategy for NM hazard assessment not only promotes a more widespread adoption of non-rodent or 3R principles but also makes nanotoxicology testing more ethical, relevant, and cost- and time-efficient.

Which Should Be Used First for ALK-Positive Non-Small-Cell Lung Cancer: Chemotherapy or Targeted Therapy? A Meta-Analysis of Five Randomized Trials.

Background and objectives: Targeted therapy is widely used in the era of precision medicine. Whether the sequence in which targeted therapy and chemotherapy are performed matters, is however not known. We examined the impact of the sequential treatment of targeted therapy and chemotherapy among advanced anaplastic lymphoma kinase (ALK), non-small cell lung cancer (NSCLC) patients. Materials and Methods: Randomized controlled trials comparing the use of ALK inhibitors with chemotherapy were included in this meta-analysis. We estimated the hazard ratios (HRs) and 95% confidence intervals (CI), for progression-free survival (PFS) and overall survival (OS) from a random effects model. Two-sided statistical tests were used to determine the significance of these estimates. Results: In five eligible studies (1404 patients), ALK targeted therapy, in comparison with chemotherapy, had a significantly higher PFS (HR = 0.48; 95% CI, 0.42⁻0.55), but not significantly higher OS (HR = 0.88; 95% CI, 0.72⁻1.07). Crossover from chemotherapy to ALK inhibitors was allowed after progression in all trials. The sensitivity analysis of the use of ALK inhibitors as either the first- or second-line treatment, showed improvements in PFS but not in OS. Conclusions: Our results indicate that using targeted therapy first improved PFS, but that the sequence in which the treatments were performed did not cause a significant difference in overall survival.

Molecular Markers and Prognosis of Myelofibrosis in the Genomic Era: A Meta-analysis.

Molecular markers are important in guiding treatment and predicting outcome in the genomic era. Meta-analysis of molecular markers in myelofibrosis through a search of PubMed and Medline through October 31, 2017 was performed. Markers with more than 3 studies that compared overall survival (OS) and leukemia-free survival (LFS) were analyzed. A total of 16 studies were included. Hazard ratios (HRs) for OS were as follows: IDH 2.65 (95% confidence interval [CI], 1.66-4.21), SRSF2 2.12 (95% CI, 1.18-3.79), high-risk myeloma 2.11 (95% CI, 1.70-2.61), ASXL1 1.92 (95% CI, 1.60-2.32), EZH2 1.88 (95% CI, 1.32-2.67), JAK2 1.41 (95% CI, 1.04-1.93) in the univariate analysis and 1.49 (95% CI, 0.42-5.30) in the multivariate analysis. LFS of JAK2 and SRSF2 had HRs of 1.81 (95% CI, 0.42-5.30) and 0.36 (95% CI, 0.02-6.48), respectively. In conclusion, mutations in IDH, SRSF2, and ASXL1 had worse prognosis in OS with HRs around 2. JAK2 and SRSF2 mutation were not associated with increased leukemia transformation. The adverse effect of triple-negative, which was often compared with CALR mutation, needs to be explored.

An Outbreak of Streptococcus pyogenes in a Mental Health Facility: Advantage of Well-Timed Whole-Genome Sequencing Over emm Typing.

OBJECTIVEWe report the utility of whole-genome sequencing (WGS) conducted in a clinically relevant time frame (ie, sufficient for guiding management decision), in managing a Streptococcus pyogenes outbreak, and present a comparison of its performance with emm typing.SETTINGA 2,000-bed tertiary-care psychiatric hospital.METHODSActive surveillance was conducted to identify new cases of S. pyogenes. WGS guided targeted epidemiological investigations, and infection control measures were implemented. Single-nucleotide polymorphism (SNP)-based genome phylogeny, emm typing, and multilocus sequence typing (MLST) were performed. We compared the ability of WGS and emm typing to correctly identify person-to-person transmission and to guide the management of the outbreak.RESULTSThe study included 204 patients and 152 staff. We identified 35 patients and 2 staff members with S. pyogenes. WGS revealed polyclonal S. pyogenes infections with 3 genetically distinct phylogenetic clusters (C1-C3). Cluster C1 isolates were all emm type 4, sequence type 915 and had pairwise SNP differences of 0-5, which suggested recent person-to-person transmissions. Epidemiological investigation revealed that cluster C1 was mediated by dermal colonization and transmission of S. pyogenes in a male residential ward. Clusters C2 and C3 were genomically diverse, with pairwise SNP differences of 21-45 and 26-58, and emm 11 and mostly emm120, respectively. Clusters C2 and C3, which may have been considered person-to-person transmissions by emm typing, were shown by WGS to be unlikely by integrating pairwise SNP differences with epidemiology.CONCLUSIONSWGS had higher resolution than emm typing in identifying clusters with recent and ongoing person-to-person transmissions, which allowed implementation of targeted intervention to control the outbreak.Infect Control Hosp Epidemiol 2018;852-860.

Computerized Silent Reading Rate and Strategy Instruction for Fourth Graders at Risk in Silent Reading Rate.

Fourth graders whose silent word reading and/or sentence reading rate was, on average, two-thirds standard deviation below their oral reading of real and pseudowords and reading comprehension accuracy were randomly assigned to treatment (n=7) or wait-listed (n=7) control groups. Following nine sessions combining computerized rapid accelerated-reading program (RAP), which individually tailors rate of written text presentation to comprehension criterion (80%), and self-regulated strategies for attending and engaging, the treated group significantly outperformed the wait-listed group before treatment on (a) a grade-normed, silent sentence reading rate task requiring lexical- and syntactic level processing to decide which of three sentences makes sense; and (b) RAP presentation rates yoked to comprehension accuracy level. Each group improved significantly on these same outcomes from before to after instruction. Attention ratings and working memory for written words predicted post-treatment accuracy, which correlated significantly with the silent sentence reading rate score. Implications are discussed for (a) preventing silent reading disabilities during the transition to increasing emphasis on silent reading, (b) evidence-based approaches for making accommodation of extra time on timed tests requiring silent reading, and

A gene expression signature of epithelial tubulogenesis and a role for ASPM in pancreatic tumor progression.

BACKGROUND & AIMS: Many patients with pancreatic ductal adenocarcinoma (PDAC) develop recurrent or metastatic diseases after surgery, so it is important to identify those most likely to benefit from aggressive therapy. Disruption of tissue microarchitecture is an early step in pancreatic tumorigenesis and a parameter used in pathology grading of glandular tumors. We investigated whether changes in gene expression during pancreatic epithelial morphogenesis were associated with outcomes of patients with PDAC after surgery. METHODS: We generated architectures of human pancreatic duct epithelial cells in a 3-dimensional basement membrane matrix. We identified gene expression profiles of the cells during different stages of tubular morphogenesis (tubulogenesis) and of PANC-1 cells during spheroid formation. Differential expression of genes was confirmed by immunoblot analysis. We compared the gene expression profile associated with pancreatic epithelial tubulogenesis with that of PDAC samples from 27 patients, as well as with their outcomes after surgery. RESULTS: We identified a gene expression profile associated with tubulogenesis that resembled the profile of human pancreatic tissue with differentiated morphology and exocrine function. Patients with PDACs with this profile fared well after surgery. Based on this profile, we established a 6-28 gene tubulogenesis-specific signature that accurately determined the prognosis of independent cohorts of patients with PDAC (total n = 128; accuracy = 81.2%-95.0%). One gene, ASPM, was down-regulated during tubulogenesis but up-regulated in human PDAC cell lines and tumor samples; up-regulation correlated with patient outcomes (Cox regression P = .0028). Bioinformatic, genetic, biochemical, functional, and clinical correlative studies showed that ASPM promotes aggressiveness of PDAC by maintaining Wnt-ß-catenin signaling and stem cell features of PDAC cells. CONCLUSIONS: We identified a gene expression profile associated with pancreatic epithelial tubulogenesis and a tissue architecture-specific signature of PDAC cells that is associated with patient outcomes after surgery.

Essential role of ß-human 8-oxoguanine DNA glycosylase 1 in mitochondrial oxidative DNA repair.

8-Oxoguanine (8-OG) is the major mutagenic base lesion in DNA caused by reactive oxygen species (ROS) and accumulates in both nuclear and mitochondrial DNA (mtDNA). In humans, 8-OG is primarily removed by human 8-OG DNA glycosylase 1 (hOGG1) through the base excision repair (BER) pathway. There are two major hOGG1 isoforms, designated α- and ß-hOGG1, generated by alternative splicing, and they have distinct subcellular localization: cell nuclei and mitochondria, respectively. Using yeast two-hybrid screening assays, we found that ß- but not α-hOGG1 directly interacts with the mitochondrial protein NADH:ubiquinone oxidoreductase 1 beta subcomplex 10 (NDUFB10), an integral factor in Complex 1 on the mitochondrial inner membrane. Using coimmunoprecipitation and immunofluorescence studies, we found that this interaction was greatly increased by hydrogen peroxide-induced oxidative stress, suggesting that ß- but not α-hOGG1 is localized in the mitochondrial inner membrane. Analyses of nuclear and mtDNA damage showed that the ß- but not α- hogg1 knockdown (KD) cells were severely defective in mitochondrial BER, indicating an essential requirement of ß-hOGG1 for mtDNA repair. ß-hogg1 KD cells were also found to be mildly deficient in Complex I activity, suggesting that ß-hOGG1 is an accessory factor for the mitochondrial integral function for ATP synthesis. In summary, our findings define ß-hOGG1 as an important factor for mitochondrial BER and as an accessory factor in the mitochondrial Complex I function.

Teaching children with dyslexia to spell in a reading-writers' workshop.

To identify effective treatment for both the spelling and word decoding problems in dyslexia, 24 students with dyslexia in grades 4 to 9 were randomly assigned to treatments A (n=12) or B (n=12) in an after-school reading-writers' workshop at the university (thirty 1-h sessions twice a week over 5 months). First, both groups received step 1 treatment of grapheme-phoneme correspondences (gpc) for oral reading. At step 2, treatment A received gpc training for both oral reading and spelling, and treatment B received gpc training for oral reading and phonological awareness. At step 3, treatment A received orthographic spelling strategy and rapid accelerated reading program (RAP) training, and treatment B continued step 2 training. At step 4, treatment A received morphological strategies and RAP training, and treatment B received orthographic spelling strategy training. Each treatment also had the same integrated reading-writing activities, which many school assignments require. Both groups improved significantly in automatic letter writing, spelling real words, compositional fluency, and oral reading (decoding) rate. Treatment A significantly outperformed treatment B in decoding rate after step 3 orthographic training, which in turn uniquely predicted spelling real words. Letter processing rate increased during step 3 RAP training and correlated significantly with two silent reading fluency measures. Adding orthographic strategies with "working memory in mind" to phonics helps students with dyslexia spell and read English words.

Photodynamic inactivation of viruses using upconversion nanoparticles.

Photodynamic therapy (PDT) is a promising treatment modality that utilizes light of an appropriate wavelength to excite photosensitive materials called photosensitizers, which upon excitation, generate reactive oxygen species (ROS) that are cytocidal and virucidal. However, problems such as hydrophobicity of photosensitizers and limited tissue penetration ability of the current light sources impeded its promotion as a mainstay in medical technology. Here, by using near-infrared (NIR)-to-visible upconversion nanoparticles (UCNs), we demonstrate UCN-based photodynamic inactivation as a potential antiviral strategy. These UCNs are nanotransducers which not only act as carriers of photosensitizers but also active participants in PDT by transducing NIR radiation to visible emissions appropriate for excitation of the attached photosensitizers. The UCNs effectively reduced the infectious virus titers in vitro with no clear pathogenicity in murine model and increased target specificity to virus-infected cells. Hence, this is a promising antiviral approach with feasible applications in the treatments of virus-associated infections, lesions and cancers.

Characterization of a single-stranded DNA binding protein from Salmonella enterica serovar Typhimurium LT2.

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as DNA replication, repair, and recombination, and is essential for cell survival. We characterized the single-stranded DNA (ssDNA)-binding properties of Salmonella enterica serovar Typhimurium LT2 SSB (StSSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified StSSB by gel filtration chromatography showed a stable tetramer in solution. In fluorescence titrations, StSSB bound to 21-38 nucleotides (nt) per tetramer depending on the salt concentration. Using EMSA, we characterized the stoichiometry of StSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 22 ± 1 nt. Furthermore, EMSA results indicated that the dissociation constants of StSSB for the first tetramer were less than that for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding-mode of StSSB is expected to be noncooperative.

Characterization of a single-stranded DNA-binding protein from Pseudomonas aeruginosa PAO1.

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as in DNA replication, repair, and recombination, and is essential for cell survival. We characterized the single-stranded DNA (ssDNA)-binding properties of Pseudomonas aeruginosa PAO1 SSB (PaSSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified PaSSB by gel filtration chromatography revealed a stable tetramer in solution. In fluorescence titrations, PaSSB bound 22-32 nucleotides (nt) per tetramer depending on salt concentration. Using EMSA, we characterized the stoichiometry of PaSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 29 ± 1 nt. Furthermore, EMSA results indicated that the dissociation constants of PaSSB for the first tetramer were less than those for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding mode of PaSSB is expected to be noncooperative.

Evolution of the mammalian transcription factor binding repertoire via transposable elements.

Identification of lineage-specific innovations in genomic control elements is critical for understanding transcriptional regulatory networks and phenotypic heterogeneity. We analyzed, from an evolutionary perspective, the binding regions of seven mammalian transcription factors (ESR1, TP53, MYC, RELA, POU5F1, SOX2, and CTCF) identified on a genome-wide scale by different chromatin immunoprecipitation approaches and found that only a minority of sites appear to be conserved at the sequence level. Instead, we uncovered a pervasive association with genomic repeats by showing that a large fraction of the bona fide binding sites for five of the seven transcription factors (ESR1, TP53, POU5F1, SOX2, and CTCF) are embedded in distinctive families of transposable elements. Using the age of the repeats, we established that these repeat-associated binding sites (RABS) have been associated with significant regulatory expansions throughout the mammalian phylogeny. We validated the functional significance of these RABS by showing that they are over-represented in proximity of regulated genes and that the binding motifs within these repeats have undergone evolutionary selection. Our results demonstrate that transcriptional regulatory networks are highly dynamic in eukaryotic genomes and that transposable elements play an important role in expanding the repertoire of binding sites.

Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes.

The paired-end ditagging (PET) technique has been shown to be efficient and accurate for large-scale transcriptome and genome analysis. However, as with other DNA tag-based sequencing strategies, it is constrained by the current efficiency of Sanger technology. A recently developed multiplex sequencing method (454-sequencing) using picolitre-scale reactions has achieved a remarkable advance in efficiency, but suffers from short-read lengths, and a lack of paired-end information. To further enhance the efficiency of PET analysis and at the same time overcome the drawbacks of the new sequencing method, we coupled multiplex sequencing with paired-end ditagging (MS-PET) using modified PET procedures to simultaneously sequence 200,000 to 300,000 dimerized PET (diPET) templates, with an output of nearly half-a-million PET sequences in a single 4 h machine run. We demonstrate the utility and robustness of MS-PET by analyzing the transcriptome of human breast carcinoma cells, and by mapping p53 binding sites in the genome of human colorectal carcinoma cells. This combined sequencing strategy achieved an approximate 100-fold efficiency increase over the current standard for PET analysis, and furthermore enables the short-read-length multiplex sequencing procedure to acquire paired-end information from large DNA fragments.