Evaluation of Marker-Assisted Introgression of Yield QTL Alleles into Adapted Soybean

Genetic diversity is limited in southern elite soybean [Glycine max (L.) Merrill]. Introgression of diverse alleles for yield may increase the rate of yield improvement. Beneficial yield alleles at three quantitative trait loci (QTL) from the northern cultivar Archer have been tagged with molecular markers. The objective of this research was to assess the value of the three Archer alleles for increased yield in southern environments and genetic backgrounds. Four sets of near isogenic lines (NIL) for each quantitative trait locus (QTL) were derived from heterozygous F6 plants identified from the crosses of Archer X Asgrow A5403 and Archer X Pioneer 9641. The NIL sets were tested at four environments across 2 yr. Data was collected on yield, height, and maturity. None of the marker effects were significant for any of the three QTL for any trait, when averaged over all sets or for individual sets. The results suggest that the Archer alleles are not superior to the southern alleles when tested in southern environments. Archer has low relative yield in the South, while in the original mapping study ARcher was the high-yield adapted parent. The superior genetic value assigned to the Archer yield QTL to may not be readily transported to populations or environments where Archer is inferior. Recombination and epistasis may also have affected the ability of the Archer markers and QTL to improve yield. Our results indicate that it may be difficult to capture the value assigned to QTL alleles derived from diverse parents with variable relative genetic value when the alleles are introgressed into populations with different genetic backgrounds, or when tested in different environments

Rapid verification of terminators using the pGR-blue plasmid and golden gate assembly

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed specifically for this protocol and allows for the quantification of terminator efficiency. As a proof of concept, six terminators were bioinformatically identified in the mycobacteriophage Bernal13. Once identified, terminators were then made as oligonucleotides with the appropriate sticky ends and annealed together. Using Golden Gate Assembly (GGA), terminators were then cloned into pGR-Blue. Under visible light, false positive colonies appear blue and positively transformed colonies are white/yellow. After induction of an arabinose inducible promoter (pBad) with arabinose, colony strength can be determined by measuring the ratio of green fluorescent protein (GFP) produced to red fluorescent protein (RFP) produced. With pGR-Blue, the protocol can be completed in as little as three days and is ideal in an educational setting. Additionally, results show that this protocol is useful as a means for understanding in silico predictions of terminator efficiency related to the regulation of transcription

Twitter: More than Tweets for Undergraduate Student Researchers

During the COVID-19 pandemic, biology educators were forced to think of ways to communicate with their students, engaging them in science and with the scientific community. For educators using course-based undergraduate research experiences (CUREs), the challenge to have students perform real science, analyze their work, and present their results to a larger scientific audience was difficult as the world moved online. Many instructors were able to adapt CUREs utilizing online data analysis and virtual meeting software for class discussions and synchronous learning. However, interaction with the larger scientific community, an integral component of making science relevant for students and allowing them to network with other young scientists and experts in their fields, was still missing. Even before COVID-19, a subset of students would travel to regional or national meetings to present their work, but most did not have these opportunities. With over 300 million active users, Twitter provided a unique platform for students to present their work to a large and varied audience. The Cell Biology Education Consortium hosted an innovative scientific poster session entirely on Twitter to engage undergraduate researchers with one another and with the much broader community. The format for posting on this popular social media platform challenged students to simplify their science and make their points using only a few words and slides. Nineteen institutions and over one hundred students participated in this event. Even though these practices emerged as a necessity during the COVID-19 pandemic, the Twitter presentation strategy shared in this paper can be used widely

From Genetics to Biotechnology: Synthetic Biology as a Flexible Course-embedded Research Experience

The need for changing how science is taught and the expansion of undergraduate research experiences is essential to foster critical thinking in the Natural Sciences. Most faculty research programs only involve a small number of upper-level undergraduate students each semester. The course-based undergraduate research experience (CURE) model enables more students to take ownership over an independent project and experience authentic research. Further, by creating projects that fit into a curriculum\u27s learning goals and student-oriented outcomes, departments help strengthen critical thinking skills in the classroom. Here, we report on the incorporation of a synthetic biology CURE into a mid-level cellular biology course and two advanced level genetics/molecular biology courses. Synthetic biology involves systematic engineering of novel organisms, such as bacteria and plants, to work as functional devices to solve problems in medicine, agriculture, and manufacturing. The value of synthetic biology and its ultimate utility as a teaching tool relies on reusable, standard genetic parts that can be interchanged using common genetic engineering principles. This Synthetic biology CURE effectively achieves five essential goals: (1) a sense of project ownership; (2) self-efficacy: mastery of a manageable number of techniques; (3) increased tolerance for obstacles through challenging research; (4) increased communication skills; and (5) a sense of belonging in a larger scientific community. Based upon our student assessment data, we demonstrate that this course-based synthetic biology laboratory engages students directly in an authentic research experience and models important elements of collaboration, discovery, iteration, and critical thinking

Circulating Exosomal microRNAs as Predictive Biomarkers of Neoadjuvant Chemotherapy Response in Breast Cancer

Background: Neoadjuvant chemotherapy (NACT) is an increasingly used approach for treatment of breast cancer. The pathological complete response (pCR) is considered a good predictor of disease-specific survival. This study investigated whether circulating exosomal microRNAs could predict pCR in breast cancer patients treated with NACT. Method: Plasma samples of 20 breast cancer patients treated with NACT were collected prior to and after the first cycle. RNA sequencing was used to determine microRNA profiling. The Cancer Genome Atlas (TCGA) was used to explore the expression patterns and survivability of the candidate miRNAs, and their potential targets based on the expression levels and copy number variation (CNV) data. Results: Three miRNAs before that NACT (miR-30b, miR-328 and miR-423) predicted pCR in all of the analyzed samples. Upregulation of miR-127 correlated with pCR in triple-negative breast cancer (TNBC). After the first NACT dose, pCR was predicted by exo-miR-141, while miR-34a, exo-miR182, and exo-miR-183 predicted non-pCR. A significant correlation between the candidate miRNAs and the overall survival, subtype, and metastasis in breast cancer, suggesting their potential role as predictive biomarkers of pCR. Conclusions: If the miRNAs identified in this study are validated in a large cohort of patients, they might serve as predictive non-invasive liquid biopsy biomarkers for monitoring pCR to NACT in breast cancer

Gene expression and data analysis pipeline using cancer BioPortal in the classroom

At institutions with an emphasis on authentic research experiences as an integral part of the biology curriculum, COVID created a huge challenge for course instructors whose learning objectives were designed for such experiences. Moving such laboratory experiences online when remote learning became necessary has resulted in a new model for CUREs that utilizes free online databases to provide not only a novel research experience for students, but also the opportunity to engage in big data analysis. Cancer BioPortal (cBioPortal) is an open-access collective cancer research resource for storing and exploring clinical, genomic, proteomic, and transcriptomic data. cBioPortal eliminates the computational barrier of interpreting complex genomic data by providing easily understandable visualization that can be interpreted and translated into relevant biological insights. Because no prior computational knowledge is required, cBioPortal is an ideal educational tool for either in-person or distance learning environments. We developed a pedagogical approach, video tutorials, and data analysis workflows centered on using cBioPortal. Pedagogically, students develop an initial research outline that is continually updated and graded throughout the project. Progress during the project or course is assessed by a series of student presentations that are 5 to 15 minutes in length and are aimed at explaining the approach used in data acquisition, interpretation of the data, and relevance to the initial hypothesis. While cancer-specific, this analysis platform appeals to a wide range of classes and student interests. Further, the project has been successfully done both as an independent research experience and as part of a virtual class-based research project

Transitioning cell culture CURE labs from campus to online: Novel strategies for a novel time

Course-based undergraduate research experiences (CUREs) provide a way for students to gain research experience in a classroom setting. Few examples of cell culture CUREs or online CUREs exist in the literature. The Cell Biology Education Consortium (CBEC) provides a network and resources for instructors working to incorporate cell-culture based research into the classroom. In this article, we provide examples from six instructors from the CBEC network on how they structure their cell-culture CUREs and how they transitioned the labs to online in the spring semester of 2020. We intend for these examples to provide instructors with ideas for strategies to set up cell culture CUREs, how to change that design mid-term, and for creating online CUREs in the future

Glioma-derived exosomes drive the differentiation of neural stem cells to astrocytes

Exosomes appear to be effective inter-cellular communicators delivering several types of molecules, such as proteins and RNAs, suggesting that they could influence neural stem cell (NSC) differentiation. Our RNA sequencing studies demonstrated that the RNAs related to cell proliferation and astrocyte differentiation were upregulated in human mesenchymal stem cells (hMSC) when co-cultured with exosomes obtained from the culture medium of human glioma cells (U87). Metallothionein 3 and elastin genes, which are related to cell proliferation, increased 10 and 7.2 fold, respectively. Expression of genes for astrocyte differentiation, such as tumor growth factor alpha, induced protein 3 of the NOTCH1 family, colony stimulating factor and interleukin 6 of the STAT3 family and Hes family bHLH transcription factor 1 also increased by 2.3, 10, 4.7 and 2.9 fold, respectively. We further examined the effects of these exosomes on rat fetal neural stem cell (rNSC) differentiation using the secreted exosomes from U87 glioma cells or exosomes from U87 cells that were stimulated with interleukin 1β (IL-1β). The rNSCs, extracted from rat brains at embryonic day 14 (E14), underwent a culture protocol that normally leads to predominant (∼90%) differentiation to ODCs. However, in the presence of the exosomes from untreated or IL-1β-treated U87 cells, significantly more cells differentiated into astrocytes, especially in the presence of exosomes obtained from the IL-1β-challenged glioma cells. Moreover, glioma-derived exosomes appeared to inhibit rNSC differentiation into ODCs or astrocytes as indicated by a significantly increased population of unlabeled cells. A portion of the resulting astrocytes coexpressed both CD133 and glial fibrillary acidic protein (GFAP) suggesting that exosomes from U87 cells could promote astrocytic differentiation of NSCs with features expected from a transformed cell. Our data clearly demonstrated that exosomes secreted by human glioma cells provide a strong driving force for rat neural stem cells to differentiate into astrocytes, uncovering potential pathways and therapeutic targets that might control this aggressive tumor type

Genome sequences of four cluster P mycobacteriophages

Four bacteriophages infecting Mycobacterium smegmatis mc2155 (three belonging to subcluster P1 and one belonging to subcluster P2) were isolated from soil and sequenced. All four phages are similar in the left arm of their genomes, but the P2 phage differs in the right arm. All four genomes contain features of temperate phages

Genomic diversity of bacteriophages infecting Microbacterium spp

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics