Transcript Ratio Research Articles

The human ribosomal DNA array is composed of highly homogenized tandem clusters.

The structure of the human ribosomal DNA (rDNA) cluster has traditionally been hard to analyze owing to its highly repetitive nature. However, the recent development of long-read sequencing technology, such as Oxford Nanopore sequencing, has enabled us to study the large-scale structure of the genome. Using this technology, we found that human cells have a quite regular rDNA structure. Although each human rDNA copy has some variations in its noncoding region, contiguous copies of rDNA are similar, suggesting that homogenization through gene conversion frequently occurs between copies. Analysis of rDNA methylation by Nanopore sequencing further showed that all the noncoding regions are heavily methylated, whereas about half of the coding regions are clearly unmethylated. The ratio of unmethylated copies, which are speculated to be transcriptionally active, was lower in individuals with a higher rDNA copy number, suggesting that there is a mechanism that keeps the active copy number stable. In addition, the rDNA in progeroid syndrome patient cells with reduced DNA repair activity had more unstable copies compared with control normal cells, although the rate was much lower than previously reported using a fiber-FISH method. Collectively, our results clarify the view of rDNA stability and transcription regulation in human cells, indicating the presence of mechanisms for both homogenizations to ensure sequence quality and maintenance of active copies for cellular functions.

Hepatitis B Virus Induces Microtubule Stabilization to Promote Productive Infection through Upregulating Microtubule-associated Protein 1S.

Background and AimsContinuous release and transmission of hepatitis B virus (HBV) is one of the main factors leading to chronic hepatitis B (CHB) infection. However, the mechanism of HBV-host interaction for optimal viral transport is unclear. Hence, we aimed to explore how HBV manipulates microtubule-associated protein 1S (MAP1S) and microtubule (MT) to facilitate its transport and release.MethodsThe expression of MAP1S or acetylated MT was investigated by immunofluorescence, RT-PCR, immunoblotting, and plasmid transfection. MAP1S overexpression or knockdown was performed by lentiviral infection or sh-RNA transfection, respectively. HBV DNA was quantified using q-PCR.ResultsSignificantly higher level of MAP1S in HepG2215 cells compared with HepG2 cells was detected using RT-PCR (p<0.01) and immunoblotting (p<0.001). Notably, stronger MAP1S expression was observed in the liver tissues of patients with CHB than in healthy controls. MAP1S overexpression or knockdown demonstrated that MAP1S promoted MT acetylation and reduced the ratio of HBV DNA copies inside to outside cells. Further, transfection with the hepatitis B virus X protein (HBx)-expressing plasmids induced significantly higher level of MAP1S than that in controls (p<0.0001), whereas HBVX− mutant-encoding HBV proteins (surface antigen, core protein, and viral DNA polymerase) hardly affected its expression.ConclusionsThese results demonstrate that HBx induces the formation of stable MTs to promote the release of HBV particles through upregulating MAP1S. Thus, our studies delineate a unique molecular pathway through which HBV manipulates the cytoskeleton to facilitate its own transportation, and indicate the possibility of targeting MAP1S pathway for treatment of patients with CHB.

Detailed Reanalysis of 500 Breast Cancers With Equivocal HER2 Immunohistochemistry and Borderline ERBB2 Fluorescence In Situ Hybridization Results.

We investigated the impact of our laboratory's reflex testing process for resolving ERBB2 (HER2) status on breast cancer samples that require additional workup after fluorescence in situ hybridization (FISH), per guideline recommendations published in 2018 by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP). In total, 500 breast cancer specimens with ERBB2 FISH results in groups 2 through 4 (all reported as immunohistochemistry [IHC] equivocal [2+] at external laboratories) were resubmitted for IHC testing in our laboratory. Per the ASCO/CAP guideline, FISH was rescored when internal IHC was also equivocal (2+), targeted to tumor areas demonstrating more intense IHC staining, if observed. Reflex IHC/FISH testing changed the final reported ERBB2 status in 185 of 500 (37.0%) samples. Result changes included discordant IHC (n = 4 score 0, n = 132 score 1+, and n = 16 score 3+) and discordant FISH (n = 33). Numerical differences in FISH scores were comparable for targeted vs nontargeted FISH rescoring (P = .086 for ERBB2 copy number; P = .49 for ERBB2 ratio). Two cases showed larger differences in FISH scores, suggesting heterogeneity. Retesting of breast cancer samples with equivocal IHC frequently changes IHC results, but targeted reanalysis of borderline FISH results rarely identifies significant differences in ERBB2 copy number or ratio.

Longitudinal telomere length and body composition in healthy term-born infants during the first two years of life.

ObjectiveLeukocyte telomere length (LTL) is one of the markers of biological aging as shortening occurs over time. Shorter LTL has been associated with adiposity and a higher risk of cardiovascular diseases. The objective was to assess LTL and LTL shortening during the first 2 years of life in healthy, term-born infants and to associate LTL shortening with potential stressors and body composition.Study designIn 145 healthy, term-born infants (85 boys), we measured LTL in blood, expressed as telomere to single-gene copy ratio (T/S ratio), at 3 months and 2 years by quantitative PCR technique. Fat mass (FM) was assessed longitudinally by PEAPOD, DXA, and abdominal FM by ultrasound.ResultsLTL decreased by 8.5% from 3 months to 2 years (T/S ratio 4.10 vs 3.75, p<0.001). LTL shortening from 3 months to 2 years associated with FM%(R = 0.254), FM index(R = 0.243) and visceral FM(R = 0.287) at 2 years. LTL shortening tended to associate with gain in FM% from 3 to 6 months (R = 0.155, p = 0.11), in the critical window for adiposity programming. There was a trend to a shorter LTL in boys at 2 years(p = 0.056). LTL shortening from 3 months to 2 years was not different between sexes.ConclusionWe present longitudinal LTL values and show that LTL shortens considerably (8.5%) during the first 2 years of life. LTL shortening during first 2 years of life was associated with FM%, FMI and visceral FM at age 2 years, suggesting that adverse adiposity programming in early life could contribute to more LTL shortening.

Community Transmission of SARS-CoV-2 by Surfaces: Risks and Risk Reduction Strategies.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, is perceived to be primarily transmitted via person-to-person contact through droplets produced while talking, coughing, and sneezing. Transmission may also occur through other routes, including contaminated surfaces; nevertheless, the role that surfaces have on the spread of the disease remains contested. Here, we use the Quantitative Microbial Risk Assessment framework to examine the risks of community transmission of SARS-CoV-2 through surfaces and to evaluate the effectiveness of hand and surface disinfection as potential interventions. Using conservative assumptions on input parameters of the model (e.g., dose–response relationship, ratio of genome copies to infective virus), the average of the median risks for single hand-to-surface contact followed by hand-to-face contact range from 1.6 × 10–4 to 5.6 × 10–9 for modeled prevalence rates of 0.2%–5%. For observed prevalence rates (0.2%, 1%), this corresponds to a low risk of infection (<10–6). Hand disinfection substantially reduces risks of transmission independently of the disease’s prevalence and contact frequency. In contrast, the effectiveness of surface disinfection is highly dependent on the prevalence and the frequency of contacts. The work supports the current perception that contaminated surfaces are not a primary mode of transmission of SARS-CoV-2 and affirms the benefits of making hand disinfectants widely available.

Study of Occurrence Characteristics of Antibiotic Resistance Genes in Pig manure during Anaerobic Digestion and Digestate Fertilization

Antibiotic resistance genes (ARGs) existing in livestock and poultry manure have the risk to spread and proliferate. This might endanger people’s health. The common treatment of livestock and poultry manure is anaerobic digestion. But the change of ARGs during anaerobic digestion require further study, and the effect of digestate fertilization to the antibiotic resistance of cropland soil is still unclear. This study investigated the pig manure, biogas liquid, biogas residue, and cropland soils fertilized with and without digestate. The results showed that, the relative abundance of ARGs in biogas residue was much higher than other samples. The average relative abundance was 1.46×10-1 copy ratio (copy of ARG/copy of 16S rRNA gene), and the total relative abundance was 3.07 copy ratio. There were 21 ARGs detected in the 5 samples. 11 of them were shared by the 5 samples. The main ARGs were aminoglycoside, chloramphenicol, sulfonamide, tetracycline, and multidrug. Aminoglycoside had the highest relative abundance, and the total relative abundance in all samples was 1.18 copy ratio. Anaerobic digestion increased the total relative abundance of ARGs in pig manure from 1.14×10-1 to 1.70×10-1 copy ratio. Fertilization of digestate increased the total relative abundance of AGRs in soil from 3.27×10-1 to 7.29×10-1 copy ratio.

A new literature search model for multi data objective programming

Aiming at the problem that the current literature search algorithms only provide literature clues and original texts, which can not realize the analysis and evaluation, it is the formal comparison and cannot compare the actual contents of new papers and existing literatures; the objective programming target algorithm is introduced to calculate the “copy ratio” of the substantive content by minimizing the deviation between the objective function and the target value; small sample verification is carried out It is feasible and can open up a new idea for the upgrading of literature search tools.

Aberrant Telomere Length in Circulating Cell-Free DNA as Possible Blood Biomarker with High Diagnostic Performance in Endometrial Cancer.

To investigate the diagnostic performance of relative telomere length (RTL) in cell-free DNA (cfDNA) for endometrioid endometrial cancer (EC). We measured RTL in cfDNA of 40 EC patients (65 ± 12years) and 31 healthy controls (HC) (63 ± 13years), excluding in both groups other oncologic and severenon-oncologic diseases to limit confounders. Circulating cfDNA was extracted from serum using the QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). After the quantitative real-time polymerase chain reaction, telomere repeat copy number to single-gene copy number ratio was calculated. RTL in cfDNA was found to be significantly lower in EC patients than in HC (p < 0.0001). The diagnostic performance of cfDNA RTL was estimated with receiver operating characteristics (ROC) curve analysis, which showed a diagnostic accuracy for EC of 0.87 (95% CI: 0.79-0.95, p < 0.0001). The cutoff cfDNA RTL value of 2.505 (T/S copy ratio) reported a sensitivity of 80.0% (95% CI: 64.35-90.95) and a specificity of 80.65% (95% CI: 62.53-92.55). Significant differences of RTL among EC stages or grades (p = 0.85 and p = 0.89, respectively) were not observed. Our results suggest that cfDNA RTL analysis may be a diagnostic tool for EC detection since the early stage, whilst its diagnostic performance seems unsatisfactory for cancer progression, staging, and grading. However, further studies are needed to confirm these preliminary findings. In particular, future investigations should focus on high-risk patients (such as those with atypical endometrial hyperplasia) that may benefit from this tool, because TL shortening is not specific for EC and is influenced by other oncologic and non-oncologic diseases.

TMOD-13. MODELING THE GENETIC, TRANSCRIPTOMIC, AND CELLULAR HETEROGENEITY OF GLIOBLASTOMA USING TUMOR ORGANOIDS

Abstract Glioblastoma exhibits enormous genetic, transcriptional, and cellular heterogeneity at the macroscopic level across regions of the tumor as well as at the microscopic level between neighboring cells, all of which present significant challenges towards creating a definitive treatment for this devastating disease. We have developed a method of generating glioblastoma organoids (GBOs) from fresh tissue obtained directly from surgical resection and maintaining them in a defined medium without bFGF/EGF. Whole exome sequencing revealed that GBOs maintain the genomic landscape of their parent tumors. Somatic and copy number variants are present in the GBOs at similar allele frequencies or copy ratios as in the parent tumor, suggesting that the relative proportions of clonal populations are largely maintained in the organoids. Bulk transcriptomic analysis demonstrated strong gene expression correlations between the parent tumor and corresponding GBOs through 12 weeks of culture. Some tumors were sampled at multiple different anatomic regions, and the corresponding GBOs maintained region-specific gene expression signatures and genomic variants. EGFRvIII, a tumor-specific variant targeted in a number of emerging therapies, also remains present in the GBOs at similar transcript frequencies, reflecting the native heterogeneity of the parent tumor. Finally, we used single cell transcriptomics to examine cellular heterogeneity and find that GBOs contain many different cell types that exhibit similar gene expression profiles as the matching cell type in the corresponding parent tumor. Notably, these GBOs retain neoplastic as well as non-neoplastic cells, such as tumor associated macrophages / microglia, T-cells, endothelial cells, stromal cells, and oligodendrocytes. These GBOs preserve complex tumor heterogeneity an in vitro environment, creating opportunities for extended manipulation, characterization, and functional study for mechanistic investigation and therapeutic testing.

Telomere reprogramming during fetal life in low socioeconomic mothers

BackgroundRelative telomere length (RTL), the biological chronometer, varies considerably among individuals under the influence of multiple risk factors such as socioeconomic status (SES). It is anticipated that during fetal life, telomeres undergo reprogramming. The purpose of this study is to find the association between SES and telomere length of mother-newborn and genetic remodeling that occurs during fetal life.ResultsThe mean telomere/single gene copy (T/S) ratio and RTL (base pairs) among 250 mother-newborn dyads were higher in cord blood of newborns (1.18 ± 0.23) (6765 ± 1350 bp) (95% confidence level) compared to maternal blood (1.13 ± 0.18) (6432 ± 1350 bp) of all SES of the Pakistani population. A positive association (r = 0.396, p < 0.05) (F (2,238) = 9.229, p < 0.05) was found between maternal and newborn telomere length by using Spearman’s correlation and regression analyses. Calculated RTL by Kruskal Wallis was found significant in low SES maternal and cord blood (5916 ± 754-6214 ± 596) compared to high SES maternal and cord blood (6818 ± 1248-7471 ± 1851).ConclusionSignificantly longer RTL in cord blood than maternal blood was observed in the targeted Pakistani population, including the low socioeconomic group highlighting fetal telomere reprogramming. High education appears to have a strong determining factor for longer RTL.

HER2 testing: Insights from pathologists’ perspective on challenging HER2 cases.

e12512 Background: HER2 status dictates treatment decisions in breast cancer making the precision and accuracy of laboratory testing essential. While most HER2 testing is straightforward using ASCO-CAP interpretation guidelines, 5% of cases fall into the less common categories (groups 2-4) and can present unusual diagnostic dilemmas. 6 high volume HER2 FISH laboratories evaluated 10 challenging HER2 cases. The results highlight the challenges and potential for variability in interpreting rare cases with unusual results. Methods: Ten diagnostically challenging HER2 FISH cases were selected from our breast cancer archives (41 months/3863 cases). Unstained slides were evaluated by 6 high volume laboratories. Laboratories performed HER2 FISH testing (along with HER2 IHC as needed) and rendered an interpretation. Participants discussed results during a conference call. Cases included the following situations: low-level amplification (ratio ≥ 2.0, mean HER2 signals/cell between 4.0-5.9), HER2 mean copy number or ratio near a clinically actionable threshold, complex heterogeneity or clones, and a technical challenge (signal clustering). Results: All laboratories elected to perform HER2 IHC in conjunction with FISH in all cases. Only 3/10 cases resulted in 100% agreement between the 6 institutions, all of which were in the category addressing complex heterogeneity. During the study, we identified a problematic subset (low amplified FISH cases) which may have discordant (negative) IHC results. Another problematic scenario is the finding of a small, highly amplified HER2 clone. Lastly, we identified cases with high cell to cell variability, and we saw discordant values resulting in disparate overall HER2 interpretations. Conclusions: Oncologists and pathologists should be aware of these challenges and the potential for variability in interpretation of these rare case scenarios. Evaluation for concordance between IHC and FISH results, correlation with other clinico-pathologic features and additional steps to ensure the accuracy of test results can help resolve unusual results, but grey zones may still remain and should be discussed when making clinical decisions based on HER2 test results.

The role of cross-disciplinary academic language skills in disciplinary, source-based writing: investigating the role of core academic language skills in science summarization for middle grade writers

Learning to use language in order to complete source-based writing tasks is a challenge for middle grade writers worthy of additional study, especially given that these tasks are increasingly common in classrooms. Here, we examined the contribution of receptive and productive cross-disciplinary academic language skills to the writing quality of middle graders’ science summaries, after accounting for students’ sociodemographic characteristics, their comprehension of the science source text, and general summary features (i.e., length and copy ratio). Our results suggest that the ability to produce a high-quality science summary is predominately driven by the writers’ receptive academic language proficiency, comprehension of the source text and productive academic language used when writing, including text length. We contend that these results are illuminating because of the joint examination of students’ productive and receptive academic language skills. In addition, this study attempts to unpack the relation between summary length and quality, identifying levels of receptive academic language skills as an important moderator. These findings have the potential to advance the current understanding of malleable factors associated with science writing proficiency, as well as with those associated with source-based writing more generally.

Association between internet gaming addiction and leukocyte telomere length in Korean male adolescents

Internet gaming addiction (IGA) has been associated with many negative health outcomes, especially for youth. In particular, the potential association between IGA and leukocyte telomere length (LTL) has yet to be examined. In this study we compared LTL in Korean male adolescents with and without IGA and examined the association between LTL and autonomic functions. Specifically, plasma catecholamine, serum cortisol, and psychological stress levels were measured as autonomic functions. Data were collected using participant blood samples analyzed for LTL, catecholamine, and cortisol levels and a set of questionnaires to assess IGA and psychological stress levels of the participants. The LTL measurements were made using a qPCR-based technique, and the relative LTL was calculated as the telomere/single copy (T/S) ratio. T/S ratio was significantly shorter in the IGA group than in the non-IGA group (150.43 ± 6.20 and 187.23 ± 6.42, respectively; p < .001) after adjusting for age. In a univariate regression analysis, age, daily Internet gaming time, IGA score, and catecholamine level (epinephrine and norepinephrine) were significantly associated with T/S ratio. However, duration of Internet gaming exposure, dopamine, cortisol, and psychological stress levels were not found to be associated with T/S ratio. In the final multiple linear regression model, age, daily Internet gaming time, and epinephrine level showed statistically significant relationships with T/S ratio. Our results indicate that in addition to age, involvement in excessive Internet gaming may induce LTL shortening in male adolescents, which may be partially attributable to changes in autonomic function such as catecholamine level. These findings further understanding of the health effects of IGA and highlight the need for screening and intervention strategies for male adolescents with IGA.

A Mechanism Underlying Attenuation of Recombinant Influenza A Viruses Carrying Reporter Genes.

Influenza A viruses (IAV) carrying reporter genes provide a powerful tool to study viral infection and pathogenesis in vivo, however, incorporating a non-essential gene into the IAV genome often results in virus attenuation and genetic instability. Very few studies have systematically compared different reporter IAVs, and most optimization attempts seem to lack authentic directions. In this study, we evaluated the ratio of genome copies to the number of infectious unit of two reporter IAVs, PR8-NS1-Gluc and PR8-PB2-Gluc. As a result, PR8-NS1-Gluc and PR8-PB2-Gluc produced 41.4 and 3.8 genomes containing noninfectious particles respectively for every such particle produced by parental PR8 virus. RdRp assay demonstrated that modification of segment NS by inserting reporter genes can interfere with the replication competitive property of the corresponding vRNAs, and the balance of the 8 segments of the reporter IAVs were drastically impaired in infected cells. As a consequence, large amounts of NS-null noninfectious particles were produced during the PR8-NS1-Gluc packaging. In summary, we unravel a mechanism underlying attenuation of reporter IAVs, which suggests a new approach to restore infectivity and virulence by introducing extra mutations compensating for the impaired replication property of corresponding segments.

Uraemia-induced immune senescence and clinical outcomes in chronic kidney disease patients.

Patients with chronic kidney disease (CKD) are more prone to develop premature age-related diseases. Data on immune senescence are scarce in CKD populations, except in end-stage renal disease and dialysis. We designed a longitudinal prospective study to evaluate immune senescence at different CKD stages and its influence on CKD patient outcomes. Clinical and biological data collections were performed on 222 patients at different CKD stages [1-2 (n = 85), 4 (n = 53) and 5 (n = 84)]. Immune senescence biomarkers were measured by cytometry on T cells (CD28, CD57, CD45RA, CD31, γH2A.X) or by quantitative polymerase chain reaction [relative telomere length (RTL)] on peripheral blood mononuclear cells and analysed according to CKD stages and outcomes. CKD was associated with an increase in immune senescence and inflammation biomarkers, as follows: low thymic output (197 ± 25 versus 88 ± 13 versus 73 ± 21 CD4+CD45RA+CD31+ T cells/mm3), an increased proportion of terminally differentiated T cells (CD8+CD28-CD57+) (24 ± 18 versus 32 ± 17 versus 35 ± 19%) restricted to cytomegalovirus-positive patients, telomere shortening (1.11 ± 0.36 versus 0.78 ± 0.24 versus 0.97 ± 0.21 telomere:single copy ratio) and an increase in C-reactive protein levels [median 2.9 (range 1.8-4.9) versus 5.1 (27-9.6) versus 6.2 (3.4-10.5) mg/L]. In multivariate analysis, shorter RTL was associated with death {hazard ratio [HR] 4.12 [95% confidence interval (CI) 1.44-11.75]}. Low thymic output was associated with infections [HR 1.79 (95% CI (1.34-9.58)] and terminally differentiated CD8+ T-cell expansion with a risk of cardiovascular events [CEs; HR 4.86 (95% CI 1.72-13.72)]. CKD was associated with premature immune ageing. Each of these alterations increased the risk of specific age-related diseases, such as RTL and death, thymic function and infections and terminally differentiated CD8+ T-cell expansion and CEs.

Abstract 3660: Can the presence of fusion genes in circulating cell-free DNA serve as potential biomarkers for Ewing's sarcoma

Abstract Background: Circulating cell-free DNA (cfDNA) is fragmented DNA derived from tumors that circulates in the blood of patients. With liquid biopsy, cfDNA has the potential to be used to monitor tumor burden in real time. Most reports have described that tumor-specific alterations in cfDNA are mutations, although little has been reported for the detection of tumor specific gene fusions. The objective of this study was to identify whether cfDNA can be used to detect tumor specific gene fusions and whether this would reflect the tumor burden of patients with Ewing's sarcoma. Patients and Methods: Multiplex long-range genomic PCR was performed to detect patient-specific genomic breakpoints in eight patients. Probes for digital PCR were designed based on the genomic breakpoint of each patient. cfDNA was extracted from the serum of patients, which was drawn at multiple time-points (pretreatment, completion of the treatment, and/or relapse). cfDNA quantification was performed by digital PCR (BioRad Qx-200). Results: Gene fusions in the cfDNA were detected in six (75%) of eight patients with high specificity. The relative gene fusion copy ratio (gene fusion copy number/wild KRAS copy number) was higher in the patients with M1b (extrapulmonary metastases at diagnosis) compared to the patients with M1a (pulmonary metastases at diagnosis) or M0 (no metastasis), although no significant relationship was observed between the relative gene fusion copy ratio and chemosensitivity. In each patient, the relative gene fusion copy ratio was the highest in pretreatment samples and decreased as the treatment progressed. Patients with relapse displayed an elevated relative gene fusion copy ratio in advance of clinical manifestation or elevation of lactase dehydrogenase. Conclusion: The amount of cfDNA in the serum strongly correlated with the treatment stage and would be useful as an early biomarker for Ewing's sarcoma patients. Citation Format: Shintaro Iwata, Hajime Kageyama, Tsukasa Yonemoto, Makiko Itami. Can the presence of fusion genes in circulating cell-free DNA serve as potential biomarkers for Ewing's sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3660.

Temporal and spatial distribution of Microcystis biomass and genotype in bloom areas of Lake Taihu

Cyanobacterial blooms as a global environmental issue are of public health concern. In this study, we investigated the spatial (10 sites) and temporal (June, August and October) variations in: 1) their biomass based on chlorophyll-a (chl-a) concentration, 2) their toxic genotype based on gene copy ratio of mcyJ to cpcBA, and 3) their cpcBA genotype composition of Microcystis during cyanobacterial bloom in Lake Taihu. While spatial-temporal variations were found in chl-a and mcyJ/cpcBA ratio, only spatial variation was observed in cpcBA genotype composition. Samples from northwestern part had a higher chl-a, but mcyJ/cpcBA ratio didn't vary among the sites. High chl-a was observed in August, while mcyJ/cpcBA ratio and genotypic richness increased with time. The spatial variations in chl-a and mcyJ/cpcBA ratio and temporal variation in cpcBA genotype were correlated negatively with dissolved N and positively with dissolved P. Spatial distribution of Microcystis biomass was positively correlated with nitrite and P excluding October, but no correlation was found for spatial distribution of mcyJ/cpcBA ratio and cpcBA genotype. Spatial distribution of toxic and cpcBA genotypes may result from horizontal transport of Microcystis colonies, while spatial variation in Microcystis biomass was probably controlled by both nutrient-mediated growth and horizontal transport of Microcystis. The temporal variation in Microcystis biomass, toxic genotype and cpcBA genotype composition were related to nutrient levels, but cause-and-effect relationships require further study.

Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer

Excess telomere shortening has been observed in most cancer cells. The telomere quantitative polymerase chain reaction (qPCR) assay has become an important tool for epidemiological studies examining the effects of aging, stress, and other factors on the length of telomeres. Current telomere qPCR methods analyze the relative length of telomeres by amplifying telomere sequence products and normalizing with single-copy gene products. However, the current telomere qPCR does not always reflect absolute telomere length in cancer DNA. Because of genomic instability in cancer cells, we hypothesized that the use of single-copy genes (scg) is less accurate for normalizing data in cancer DNA and that new primer sets are required to better represent relative telomere length in cancer DNA. We first confirmed that cancer cells had a different copy ratio among different scg, implying that DNA is aneuploid. By using the new primer sets that amplify multiple-copy sequences (mcs) throughout the genome, the telomere qPCR results showed that the mcs primers were interchangeable with the scg primers as reference primers in normal DNA. By comparing results from the traditional southern blotting method (as kilobases) and results from monochrome multiplex qPCR using the mcs primers (as T/M ratios), we verified that the T/M ratio is highly correlated with absolute telomere length from the southern blot analysis. Together, the mcs primers were able to represent the telomere lengths accurately in cancer DNA samples. These results would allow for analyses of telomeres within cancerous DNA and the development of new, less invasive diagnostic tools for cancer.

Providing Reliable Communications over Static-node-assisted Vehicular Networks Using Distance-vector Routing

To support various practical applications that are expected to work over vehicular networks, it is important to develop a network infrastructure that provides quality communications on which applications can rely. However, since a vehicular network is essentially a delay tolerant network (DTN) in which connected paths to the destinations do not always exist, providing reliable communications on such a network is a challenging research goal. In this paper, we propose a new table-driven distance-vector routing protocol called RDV (Reliable Distance-Vector routing) for static-node-assisted vehicular networks. RDV provides the guarantee that the expected packet delivery ratio exceeds the preconfigured value by creating the necessary number of packet copies sent on the shortest path. Evaluation results show that RDV provides a stably high packet delivery ratio and outperforms other conventional routing schemes in both packet delivery ratio and overhead of packet copies.

Population and community structure shifts of ammonia oxidizers after four-year successive biochar application to agricultural acidic and alkaline soils

Long-term studies that advance our mechanistic understanding of biochar (BC)‑nitrogen (N) interactions in agricultural soils are lacking. In this study, soil potential nitrification rates (PNR), the abundance and composition of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) communities following 4-year of BC application were investigated using the shaken-slurry procedure and molecular sequencing techniques for an acidic Oxisol (QU) and an alkaline Cambisol (YU). Soils were obtained from an outdoor soil column experiment with straw-BC application rates of 0 (BC0), 2.25 (BC2.25) and 11.3 (BC11.3) Mgha−1 per cropping season for eight consecutive wheat/millet seasons. Quantitative polymerase chain reaction (qPCR) and 454 high-throughput pyrosequencing techniques were performed to quantify and sequence amoA gene copies and composition of AOA and AOB. Results showed that QU had lower PNR and a higher ratio of amoA gene copies of AOA to AOB than YU, PNR of QU with BC application was significantly associated with the amoA gene of AOB. Similar to previous short-term findings, BC application enhanced QU soil nitrification, which may be explained by the significant increase in AOB abundance and a shift in AOB community structure from Nitrosospira cluster 2 toward cluster 3, along with the disappearance of some obligate acidophile AOA groups, leading to the appearance of ammonia-oxidizers from neutral-alkaline soils in BC-amended acid soils. Canonical correspondence analysis (CCA) showed that soil pH was the most important factor driving shifts in ammonia-oxidizers composition. Although BC application did not have significant effects on PNR in YU, BC11.3 decreased AOA and AOB gene copies and influenced the relative abundance of community structure. Our findings represent the first investigation of long-term BC effects on AOA and AOB communities in agricultural soils using 454 high-throughput pyrosequencing, showing that BC application can alter soil characteristics and influence ammonia oxidizer community composition, abundance, especially in acid soils.