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At least 379 records · Page 21
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Outgrowth of erlotinib-resistant subpopulations recapitulated in patient-derived lung tumor spheroids and organoids

A model that recapitulates development of acquired therapeutic resistance is needed to improve oncology drug development and patient outcomes. To achieve this end, we established methods for the preparation and growth of spheroids from primary human lung adenocarcinomas, including methods to culture, passage, monitor growth, and evaluate changes in mutational profile over time. Primary lung tumor spheroids were cultured in Matrigel® with varying concentrations of erlotinib, a small molecule kinase inhibitor of epidermal growth factor receptor (EGFR) that is ineffective against KRAS mutant cells. Subtle changes in spheroid size and number were observed within the first two weeks of culture. Spheroids were cultured for up to 24 weeks, during which time interactions between different cell types, movement, and assembly into heterogeneous organoid structures were documented. Allele-specific competitive blocker PCR (ACB-PCR) was used to quantify low frequency BRAF V600E, KRAS G12D, KRAS G12V, and PIK3CA H1047R mutant subpopulations in tumor tissue residue (TR) samples and cultured spheroids. Mutant subpopulations, including multiple mutant subpopulations, were quite prevalent. Twelve examples of mutant enrichment were found in eight of the 14 tumors analyzed, based on the criteria that a statistically-significant increase in mutant fraction was observed relative to both the TR and the no-erlotinib control. Of the mutants quantified in erlotinib-treated cultures, PIK3CA H1047 mutant subpopulations increased most often (5/14 tumors), which is consistent with clinical observations. Thus, this ex vivo lung tumor spheroid model replicates the cellular and mutational tumor heterogeneity of human lung adenocarcinomas and can be used to assess the outgrowth of mutant subpopulations. Spheroid cultures with characterized mutant subpopulations could be used to investigate the efficacy of lung cancer combination therapies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Evaluation of a parasite-density based pooled targeted amplicon deep sequencing (TADS) method for molecular surveillance of Plasmodium falciparum drug resistance genes in Haiti

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes ( crt , dhps , dhfr , and mdr1 ), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

Louha, Swarnali (ORCID:0000000207778507)↗
DOE OSTI2015

Recovery Efficiency, False Negative Rate, and Limit of Detection Performance of a Validated Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates

The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in. × 2 in.) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest for vinyl tile (50.8% with BAS, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm 2 , where CFU denotes ‘colony forming units’). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results will be discussed in a subsequent report.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Recovery Efficiency, False Negative Rate, and Limit of Detection Performance of a Validated Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates

The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in × 2 in) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest for vinyl tile (50.8% with BAS, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2, where CFU denotes ‘colony forming units’). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results are discussed in a separate report.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Evaluation of Three RNA Extraction Kits with a 2019-nCoV Assay

The following characteristics of three RNA extraction kits (Norgen Biotek Total RNA Purification Kit, #17200; Bioneer AccuPrep Viral RNA Extraction Kit, #K-3033; and Promega Maxwell HT Viral TNA Kit, #AX2340) were evaluated: Limits of detection for quantitative synthetic RNA using the Centers for Disease Control and Prevention’s (CDC) 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel and the Applied Biosystems 7500 Fast Dx system; Limits of detection for positive clinical specimen using the Centers for Disease Control and Prevention’s (CDC) 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel and the Applied Biosystems 7500 Fast Dx system; Ability of the lysis buffers in each kit to inactivate the virus as measured by a cell-based infectivity assay.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Data-Driven Buy Clean: Decarbonization and Beyond

This report was compiled to provide recommendations on the availability of public background data from the U.S. Federal life cycle assessment (LCA) Data Commons to be conformant with the Association for Life Cycle Assessment (ACLCA) 2022 Product Category Rule (PCR) Open Standard to build technical tools that can assist industry in creating more comparable Type II Environmental Product Declarations (EPDs) for Federal Buy Clean and sustainability initiatives. The Federal LCA Commons is not only a public data source but also a consistently structured, self-referencing mega-repository for data developed by federal agency experts (in agency repositories) and by academia, nonprofit organizations, and industry (via the US Life Cycle Inventory Database). The Federal LCA Commons Technical Working Group is continuously improving the standardization of data documentation, formatting, and nomenclature to ensure lossless data loading and accurate data representation. This report and appendixes include the following: 1) An introduction to data-driven Buy Clean and decarbonization initiatives at the federal level; 2) The current status and associated challenges with LCA data and EPD standards and comparability; 3) Opportunities for the Federal LCA Commons to support conformance with the ACLCA 2022 PCR Open Standard and provide resources to implement the Federal Sustainability Plan, Buy Clean Program, and Inflation Reduction Act (IRA) sustainability goals and objectives. To date, the Federal LCA Commons is the result of coordinated work by National Renewable Energy Laboratory (NREL), the U.S. Department of Agriculture (USDA), the Environmental Protection Agency (EPA), the National Energy Technology Laboratory (NETL), the Argonne National Laboratory (ANL), the U.S. Army Corps of Engineers (USACE), the Federal Highway Administration (FHWA), the U.S. Forest Service (USFS), the Federal Aviation Administration (FAA), the Department of Defense (DoD) and the National Institute of Standards and Technologies (NIST). The Federal LCA Commons will continue to combine databases from the collaborating agencies while remaining a public resource. There are several initiatives among the collaborating agencies to expand the Federal LCA Commons and dedicated federal funding and resources could accelerate and strengthen these initiatives.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗
DOE OSTI2025

Nucleic Acid-Based Detection Protease Activity

Proteases include clinically relevant markers for clotting disorders, certain cancers as well as toxins. Assays for protease activity often use designed peptides mimicking natural substrates and detection with colorometric and fluorescence-based detection that is difficult to multiplex without expensive and resource demanding instruments. This work demonstrates detection of proteolytic activity using PCR and sequencing-readable reporter molecules. The assay development focused on binding the constructed peptide-oligonucleotide chimera to immobilized streptavidin. Thrombin, an essential component of the clotting cascade, was used as a model system for testing peptide substrate recognition and release of a designed oligonucleotide for detection. Detection of protease activity was demonstrated in a concentration-dependent manner using MALDI-MS, RT-PCR and DNA sequencing.

Wunschel, David S [Pacific Northwest National Labo↗
DOE OSTI2015

Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.

Alphaproteobacteria↗
DOE OSTI2016

Characterization of Shiga toxin subtypes and virulence genes in porcine Shiga toxin-producing Escherichia coli

Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx 2e (81%) was the most common Stx variant, followed by stx 1a (14%), stx 2d (3%), and stx 1c (1%). The STEC serogroups that carried stx 2d were O15:H27, O159:H16 and O159:H-. Similar to stx 2a and stx 2c , the stx 2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfA O113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfA O26 , lpfA O157 , fedA, orfA, and orfB. Furthermore, the present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2

Zika virus (ZIKV) has had detrimental effects on global public health in recent years. This is because the management of the disease has been limited, in part because its pathogenic mechanisms are not yet completely understood. Infectious clones are an important tool that utilize reverse genetics; these can be used to modify the ZIKV genomic RNA at the DNA level. A homologous recombination clone was used to construct pWSK29, a low copy plasmid that contained sequences for a T7 promoter, the whole genome of ZIKV ZKC2 strain, and a hepatitis delta virus ribozyme. High fidelity PCR was then used to amplify the T7 transcription template. The transcript was then transfected into susceptible cells via lipofection to recover the ZIKV ZKC2 strain. Finally, the virulence of rZKC2 was evaluated both in vitro and in vivo . The rZKC2 was successfully obtained and it showed the same virulence as its parent, the ZIKV ZKC2 strain (pZKC2), both in vitro and in vivo . The 3730 (NS2A-D62G) mutation site was identified as being important, since it had significant impacts on rZKC2 recovery. The 4015 (NS2A, A157V) mutation may reduce virus production by increasing the interferon type I response. In this study, one of the earliest strains of ZIKV that was imported into China was used for infectious clone construction and one possible site for antiviral medication development was discovered. The use of homologous recombination clones, of PCR products as templates for T7 transcription, and of lipofection for large RNA transfection could increase the efficiency of infectious clone construction. Our infectious clone provides an effective tool which can be used to explore the life cycle and medical treatment of ZIKV.

Qin, Zhiran↗
DOE OSTI2022

A triton X-100 assisted PMAxx-qPCR assay for rapid assessment of infectious African swine fever virus

Introduction African Swine Fever (ASF) is a highly infectious disease of pigs, caused by African swine fever virus (ASFV). The lack of vaccines and drugs makes strict disinfection practices to be one of the main measurements to curb the transmission of ASF. Therefore, it is important to assess if all viruses are inactivated after disinfection or after long time exposure in their natural conditions. Currently, the infectivity of ASFV is determined by virus isolation and culture in a biosafety level 3 (BSL-3) laboratory. However, BSL-3 laboratories are not readily available, need skilled expertise and may be time consuming. Methods In this study, a Triton X-100 assisted PMAxx-qPCR method was developed for rapid assessment of infectious ASFV in samples. PMAxx, an improved version of propidium monoazide (PMA), can covalently cross-link with naked ASFV-DNA or DNA inside inactivated ASFV virions under assistance of 0.1% (v/v) TritonX-100, but not with ASFV-DNA inside live virions. Formation of PMAxx-DNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, the limit of detection of the PMAxx-qPCR assay was 2.32log 10 HAD 50 /mL of infectious ASFV. Testing different samples showed that the PMAxx-qPCR assay was effective to evaluate intact ASFV virions after treatment by heat or chemical disinfectants and in simulated samples such as swine tissue homogenate, swine saliva swabs, and environmental swabs. However, whole-blood and saliva need to be diluted before testing because they may inhibit the PCR reaction or the cross-linking of PMAxx with DNA. Conclusion The Triton X-100 assisted PMAxx-qPCR assay took less than 3 h from sample to result, offering an easier and faster way for assessing infectious ASFV in samples from places like pig farms and pork markets.

Liu, Huan↗
DOE OSTI2022

One-Step Regeneration of Hairy Roots to Induce High Tanshinone Plants in Salvia miltiorrhiza

Salvia miltiorrhiza is a traditional Chinese medicinal plant of Labiatae, which has been widely utilized to treat a variety of cardiovascular and cerebrovascular diseases. However, due to the long growth cycle, low content of active ingredients, and serious quality deterioration of S. miltiorrhiza , the use of biotechnology to improve S. miltiorrhiza to meet the growing demand for clinical applications has become a research hotspot. In this study, a novel one-step hairy root regeneration method was developed, which could rapidly obtain hairy roots and regenerated plants with high tanshinone content. By optimizing the parameters of Agrobacterium rhizogenes transformation in S. miltiorrhiza , it was finally established that the explants were infected in Ar.qual (OD 600 = 0.6) for 10 min, co-cultured for 3 days, and then screened on the screening medium containing 7.5 mg/l hygromycin, the maximum transformation frequency can reach 73.85%. GFP and PCR detection yielded a total of 9 positive transgenic hairy root lines and 11 positive transgenic regenerated plants. SmGGPPS1 was successfully overexpressed in positive transgenic regenerated plants, according to the results of qRT-PCR. The content of tanshinone IIA and cryptotanshinone were dramatically enhanced in transgenic regenerated plants and hairy roots by Ultra Performance Liquid Chromatography analysis. Based on the Agrobacterium-mediated transformation of S. miltiorrhiza , this study developed a new method for regenerating plants with transgenic hairy roots. This method provides a foundation for the breeding of S. miltiorrhiza and the sustainable development of medicinal plant resources, as well as provides a useful reference for the application of other species.

Su, Yuekai↗
DOE OSTI2024

Inactivation of highly transmissible livestock and avian viruses including influenza A and Newcastle disease virus for molecular diagnostics

There is a critical need for an inactivation method that completely inactivates pathogens at the time of sample collection while maintaining the nucleic acid quality required for diagnostic PCR testing. This inactivation method is required to alleviate concerns about transmission potential, minimize shipping complications and cost, and enable testing in lower containment laboratories, thereby enhancing disease diagnostics through improved turn-around time. This study evaluated a panel of 10 surrogate viruses that represent highly pathogenic animal diseases. These results showed that a commercial PrimeStore® molecular transport media (PSMTM) completely inactivated all viruses tested by >99.99%, as determined by infectivity and serial passage assays. However, the detection of viral nucleic acid by qRT-PCR was comparable in PSMTM and control-treated conditions. These results were consistent when viruses were evaluated in the presence of biological material such as sera and cloacal swabs to mimic diagnostic sample conditions for non-avian and avian viruses, respectively. The results of this study may be utilized by diagnostic testing laboratories for highly pathogenic agents affecting animal and human populations. These results may be used to revise guidance for select agent diagnostic testing and the shipment of infectious substances.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Groundwater Depth Overrides Tree-Species Effects on the Structure of Soil Microbial Communities Involved in Nitrogen Cycling in Plantation Forests

Microbial communities found in soil ecosystems play important roles in decomposing organic materials and recycling nutrients. A clear understanding on how biotic and abiotic factors influence the microbial community and its functional role in ecosystems is fundamental to terrestrial biogeochemistry and plant production. The purpose of this study was to investigate microbial communities and functional genes involved in nitrogen cycling as a function of groundwater depth (deep and shallow), tree species (pine and eucalypt), and season (spring and fall). Soil fungal, bacterial, and archaeal communities were determined by length heterogeneity polymerase chain reaction (LH-PCR). Soil ammonia oxidation archaeal (AOA) amoA gene, ammonia oxidation bacterial (AOB) amoA gene, nitrite oxidoreductase nrxA gene, and denitrifying bacterial narG, nirK, nirS, and nosZ genes were further studied using PCR and denaturing gradient gel electrophoresis (DGGE). Soil fungal and bacterial communities remained similar between tree species and groundwater depths, respectively, regardless of season. Soil archaeal communities remained similar between tree species but differed between groundwater depths in the spring only. Archaeal amoA for nitrification and bacterial nirK and nosZ genes for denitrification were detected in DGGE, whereas bacterial amoA and nrxA for nitrification and bacterial narG and nirS genes for denitrification were undetectable. The detected nitrification and denitrification communities varied significantly with groundwater depth. There was no significant difference of nitrifying archaeal amoA or denitrifying nirK communities between different tree species regardless of season. The seasonal difference in microbial communities and functional genes involved in nitrogen cycling suggests microorganisms exhibit seasonal dynamics that likely impact relative rates of nitrification and denitrification.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2018

Biomarkers’ Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM

Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR) rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC) containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1TM, including several reductive dehalogenases (RDases) and a Ni-Fe hydrogenase, Hup. Different KB-1™ DMC strains could be resolved at the RNA and protein level through differences in the sequence of a common RDase (DET1545-like homologs) and differences in expression of their vinyl chloride-respiring RDases. The dominant strain expresses VcrA, whereas the minor strain utilizes BvcA. We then used quantitative reverse-transcriptase PCR (qRT-PCR) as a targeted approach for quantifying transcript copies in the KB-1TM consortium operated under a range of TCE respiration rates in continuously-fed, pseudo-steady-state reactors. These candidate biomarkers from KB-1TM demonstrated a variety of trends in terms of transcript abundance as a function of respiration rate over the range: 7.7 × 10-12 to 5.9 × 10-10 microelectron equivalents per cell per hour (μeeq/cell∙h). Power law trends were observed between the respiration rate and transcript abundance for the main DMC RDase (VcrA) and the hydrogenase HupL (R2 = 0.83 and 0.88, respectively), but not transcripts for 16S rRNA or three other RDases examined: TceA, BvcA or the RDase DET1545 homologs in KB1TM. Overall, HupL transcripts appear to be the most robust activity biomarker across multiple DMC strains and in mixed communities including DMC co-cultures such as KB1TM. The addition of oxygen induced cell stress that caused respiration rates to decline immediately (>95% decline within one hour). Although transcript levels did decline, they did so more slowly than the respiration rate observed (transcript decay rates between 0.02 and 0.03 per hour). Data from strain-specific probes on the pangenome array strains suggest that a minor DMC strain in KB-1™ that harbors a bvcA homolog preferentially recovered following oxygen stress relative to the dominant, vcrA-containing strain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Investigation and Expression Analysis of R2R3-MYBs and Anthocyanin Biosynthesis-Related Genes during Seed Color Development of Common Bean ( Phaseolus vulgaris )

Anthocyanins are responsible for the coloration of common bean seeds, and their accumulation is positively correlated with the expression level of anthocyanin biosynthetic genes. The MBW (MYB-bHLH-WD40) complex is thought to regulate the expression of these genes, and MYB proteins, which are a key factor in activating anthocyanin pathway genes, have been identified in several plants. This study demonstrated gene structures, chromosomal placements, gene duplications of R2R3-MYBs, miRNAs associated with R2R3-MYBs, and the interaction of these genes with other flavonoid regulatory genes. qRT-PCR was used to investigate the role of specific R2R3-MYBs and flavonoid genes in common bean seed color development. As a result of a comprehensive analysis with the help of in silico tools, we identified 160 R2R3-MYB genes in the common bean genome. We divided these genes into 16 classes on the basis of their intron-exon and motif structures. Except for three, the rest of the common bean R2R3-MYB members were distributed to all chromosomes with different densities, primarily located on chromosomes 3 and 8. We identified a total of 44 duplicated gene pairs dispersed across 11 chromosomes and evolved under purifying selection (Ka/Ks < 1), 19 of which were derived from a whole-genome duplication. Our research uncovered 25 putative repressor PvMYB proteins that contain the EAR motif. Additionally, fifty different cis-regulatory elements regulated by light, stress, and hormone were identified. Within the genome of the common bean, we discovered a total of 36 microRNAs that target a total of 72 R2R3-MYB transcripts. The effect of 16 R2R3-MYB genes and 16 phenylpropanoid pathway genes, selected on the basis of their interaction in the protein-protein interaction map, playing role in the regulation of seed coat color development was evaluated using qRT-PCR in 5 different tissues at different developmental stages. The results revealed that these specific genes have different expression levels during different developmental periods, with higher levels in the pod filling and early pod stages than in the rest of the developmental periods. Furthermore, it was shown that PvTT8 (bHLH), PvTT2 (PvMYB42), PvMYB113, PvTTG1, and PvWD68 genes have effects on the regulation of seed coat color. The findings of this study, which is the first to use whole-genome analysis to identify and characterize the R2R3-MYB genes in common bean, may serve as a reference for future functional research in the legume.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Sequence Length of HIV-1 Subtype B Increases over Time: Analysis of a Cohort of Patients with Hemophilia over 30 Years

We aimed to investigate whether the sequence length of HIV-1 increases over time. We performed a longitudinal analysis of full-length coding region sequences (FLs) during an HIV-1 outbreak among patients with hemophilia and local controls infected with the Korean subclade B of HIV-1 (KSB). Genes were amplified by overlapping RT-PCR or nested PCR and subjected to direct sequencing. Overall, 141 FLs were sequentially determined over 30 years in 62 KSB-infected patients. Phylogenetic analysis indicated that within KSB, two FLs from plasma donors O and P comprised two clusters, together with 8 and 12 patients with hemophilia, respectively. Signature pattern analysis of the KSB of HIV-1 revealed 91 signature nucleotide residues (1.1%). In total, 48 and 43 signature nucleotides originated from clusters O and P, respectively. Six positions contained 100% specific nucleotide(s) in clusters O and P. In-depth FL analysis for over 30 years indicated that the KSB FL significantly increased over time before combination antiretroviral therapy (cART) and decreased with cART. This increase occurred due to the significant increase in env and nef genes, originating in the variable regions of both genes. The increase in sequence length of HIV-1 over time suggests an evolutionary direction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

59 BASIC BIOLOGICAL SCIENCES↗