SEA-PHAGES and SEA-GENES: Advancing Virology and Science Education.

Research opportunities for undergraduate students are strongly advantageous, but implementation at a large scale presents numerous challenges. The enormous diversity of the bacteriophage population and a supportive programmatic structure provide opportunities to engage early-career undergraduates in phage discovery, genomics, and genetics. The Science Education Alliance (SEA) is an inclusive Research-Education Community (iREC) providing centralized programmatic support for students and faculty without prior experience in virology at institutions from community colleges to research-active universities to participate in two course-based projects, SEA-PHAGES (SEA Phage Hunters Advancing Genomic and Evolutionary Science) and SEA-GENES (SEA Gene-function Exploration by a Network of Emerging Scientists). Since 2008, the SEA has supported more than 50,000 undergraduate researchers who have isolated more than 23,000 bacteriophages of which more than 4,500 are fully sequenced and annotated. Students have functionally characterized hundreds of phage genes, and the phage collection has fueled the therapeutic use of phages for treatment of Mycobacterium infections. Participation in the SEA promotes student persistence in science education, and its inclusivity promotes a more equitable scientific community.

Immunofluorescence Microscopy.

Visualizing fluorescence-tagged molecules is a powerful strategy that can reveal the complex dynamics of the cell. One robust and broadly applicable method is immunofluorescence microscopy, in which a fluorescence-labeled antibody binds the molecule of interest and then the location of the antibody is determined by fluorescence microscopy. The effective application of this technique includes several considerations, such as the nature of the antigen, specificity of the antibody, permeabilization and fixation of the specimen, and fluorescence imaging of the cell. Although each protocol will require fine-tuning depending on the cell type, antibody, and antigen, there are steps common to nearly all applications. This article provides protocols for staining the cytoskeleton and organelles in two very different kinds of cells: flat, adherent fibroblasts and thick, free-swimming Tetrahymena cells. Additional protocols enable visualization with widefield, laser scanning confocal, and eSRRF super-resolution fluorescence microscopy. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence staining of adherent cells such as fibroblasts Basic Protocol 2: Immunofluorescence of suspension cells such as Tetrahymena Basic Protocol 3: Visualizing samples with a widefield fluorescence microscope Alternate Protocol 1: Staining suspension cells adhered to poly-l-lysine-coated coverslips Alternate Protocol 2: Visualizing samples with a laser scanning confocal microscope Alternate Protocol 3: Generating super-resolution images with SRRF microscopy.

Evaluation of a Culturally Responsive Mentorship Education Program for the Advisers of Howard Hughes Medical Institute Gilliam Program Graduate Students.

Effective mentorship is critical to the success of trainees in research career pathways, significantly impacting their research productivity, academic and research self-efficacy, and career satisfaction. Research faculty may be unaware of or unprepared to address mentor-mentee dynamics in mentoring relationships, especially those that go beyond traditional scientific skill development. Addressing mentorship dynamics can be even more challenging for mentors from well-represented backgrounds working with mentees from historically excluded racial/ethnic groups. The Howard Hughes Medical Institute supports programmatic interventions, like the Mentorship Skills Development (MSD) course, an innovative program that aims to advance the mentorship competencies and cultural diversity awareness of mentors. Between 2015 and 2020, more than 200 faculty mentors participated in the MSD. Quantitative and qualitative data reveal significant gains in mentorship skills and cultural awareness, with mentors reporting increases in their confidence to have conversations around race and culture with their mentees. More than 85% reported actual or intended changes to their cultural responsiveness or mentorship behaviors. Importantly, behavioral changes were also observed by their mentees. These data indicate that culturally responsive mentorship education can increase knowledge and efficacy in effective mentorship practices and improve mentorship experiences of both mentors and mentees.

Broadening Access to STEM through the Community College: Investigating the Role of Course-Based Research Experiences (CREs).

Broadening access to science, technology, engineering, and mathematics (STEM) professions through the provision of early-career research experiences for a wide range of demographic groups is important for the diversification of the STEM workforce. The size and diversity of the community college system make it a prime educational site for achieving this aim. However, some evidence shows that women and Black, Latinx, and Native American student groups have been hindered in STEM at the community college level. One option for enhancing persistence in STEM is to incorporate the course-based research experiences (CREs) into the curriculum as a replacement for the prevalent traditional laboratory. This can be achieved through the integration of community colleges within extant, multi-institutional CREs such as the SEA-PHAGES program. Using a propensity score-matching technique, students in a CRE and traditional laboratory were compared on a range of psychosocial variables (project ownership, self-efficacy, science identity, scientific community values, and networking). Results revealed higher ratings for women and persons excluded because of their ethnicity or race (PEERs) in the SEA-PHAGES program on important predictors of persistence such as project ownership and science identity. This suggests that the usage of CREs at community colleges could have positive effects in addressing the gender gap for women and enhance inclusiveness for PEER students in STEM.

Race Matters.

Despite their initial high interest in science, students who belong to excluded racial and ethnic groups leave science at unacceptably high rates. "Fixing the student" approaches are not sufficient at stemming the loss. It is time to change the culture of science by putting inclusive diversity at the center.

An inclusive Research Education Community (iREC): Impact of the SEA-PHAGES program on research outcomes and student learning.

Engaging undergraduate students in scientific research promises substantial benefits, but it is not accessible to all students and is rarely implemented early in college education, when it will have the greatest impact. An inclusive Research Education Community (iREC) provides a centralized scientific and administrative infrastructure enabling engagement of large numbers of students at different types of institutions. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) is an iREC that promotes engagement and continued involvement in science among beginning undergraduate students. The SEA-PHAGES students show strong gains correlated with persistence relative to those in traditional laboratory courses regardless of academic, ethnic, gender, and socioeconomic profiles. This persistent involvement in science is reflected in key measures, including project ownership, scientific community values, science identity, and scientific networking.

From HHMI: Doubling Down on Diversity.

In spite of modest gains in the past four decades, the United States has not been able to substantially improve on the pervasive underrepresentation of minorities in postsecondary science, technology, engineering, and mathematics (STEM) pathways. We suggest a way to guide a national effort to double the persistence of underrepresented minorities in STEM in the next decade.

Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity.

The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery.

Partnered research experiences for junior faculty at minority-serving institutions enhance professional success.

Scientific workforce diversity is critical to ensuring the realization of our national research goals and minority-serving institutions play a vital role in preparing undergraduate students for science careers. This paper summarizes the outcomes of supporting career training and research practices by faculty from teaching-intensive, minority-serving institutions. Support of these faculty members is predicted to lead to: 1) increases in the numbers of refereed publications, 2) increases in federal grant funding, and 3) a positive impact on professional activities and curricular practices at their home institutions that support student training. The results presented show increased productivity is evident as early as 1 yr following completion of the program, with participants being more independently productive than their matched peers in key areas that serve as measures of academic success. These outcomes are consistent with the goals of the Visiting Professorship Program to enhance scientific practices impacting undergraduate student training. Furthermore, the outcomes demonstrate the benefits of training support for research activities at minority-serving institutions that can lead to increased engagement of students from diverse backgrounds. The practices and results presented demonstrate a successful generalizable approach for stimulating junior faculty development and can serve as a basis for long-term faculty career development strategies that support scientific workforce diversity.

Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control.

BACKGROUND: Ichthyophthirius multifiliis, commonly known as Ich, is a highly pathogenic ciliate responsible for 'white spot', a disease causing significant economic losses to the global aquaculture industry. Options for disease control are extremely limited, and Ich's obligate parasitic lifestyle makes experimental studies challenging. Unlike most well-studied protozoan parasites, Ich belongs to a phylum composed primarily of free-living members. Indeed, it is closely related to the model organism Tetrahymena thermophila. Genomic studies represent a promising strategy to reduce the impact of this disease and to understand the evolutionary transition to parasitism. RESULTS: We report the sequencing, assembly and annotation of the Ich macronuclear genome. Compared with its free-living relative T. thermophila, the Ich genome is reduced approximately two-fold in length and gene density and three-fold in gene content. We analyzed in detail several gene classes with diverse functions in behavior, cellular function and host immunogenicity, including protein kinases, membrane transporters, proteases, surface antigens and cytoskeletal components and regulators. We also mapped by orthology Ich's metabolic pathways in comparison with other ciliates and a potential host organism, the zebrafish Danio rerio. CONCLUSIONS: Knowledge of the complete protein-coding and metabolic potential of Ich opens avenues for rational testing of therapeutic drugs that target functions essential to this parasite but not to its fish hosts. Also, a catalog of surface protein-encoding genes will facilitate development of more effective vaccines. The potential to use T. thermophila as a surrogate model offers promise toward controlling 'white spot' disease and understanding the adaptation to a parasitic lifestyle.

Analysis of properties of cilia using Tetrahymena thermophila.

Cilia and eukaryotic flagella are important structures required for the motility of cells, the movement of medium across the surfaces of cells, and the connections between the receptor and synthetic portions of sensory cells. The axoneme forms the cytoskeleton of the cilium comprising several hundreds of proteins that assemble into the 9 + 2 arrangement of outer doublet and central pair microtubules, the inner and outer rows of dynein arms, and many other structures. Tetrahymena thermophila is an excellent model organism for the study of cilia and ciliogenesis. The cell is covered by about 1,000 cilia which are essential for survival. Additionally, the Tetrahymena genome is available and targeted genetic manipulations are straightforward. In this chapter, we describe five protocols that examine properties of cilia: (a) measuring mRNA levels to see the effect of deciliation on gene expression; (b) swimming velocity and linearity; (c) ciliary length and density; (d) phagocytosis that occurs through the ciliated oral apparatus; and (e) depolarization-induced ciliary reversal.

Dynein-2 and ciliogenesis in Tetrahymena.

Dynein-2 is the motor responsible for retrograde intraflagellar transport. In situ, dynein-2 comprises four subunits: the dynein-2 heavy chain (DYH2); the dynein-2 intermediate chain; the dynein-2 light-intermediate chain (D2LIC); and dynein light chain 8 (Rompolas et al. 2007. Chlamydomonas FAP133 is a dynein intermediate chain associated with the retrograde intraflagellar transport motor. J Cell Sci 120:3653-3665). In contrast to what has been reported in other model organisms, when the DYH2 gene or the D2LIC gene was disrupted in Tetrahymena, the cells continued to produce motile cilia that were not swollen or filled with material [Rajagopalan et al.2009. Dynein-2 affects the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena thermophila. Mol Biol Cell 20:708-720]. When compared to wildtype cells, the dynein-2 mutants were found to have cilia that were at a lower density, shorter, and much more variable in length. One possible explanation for these effects is that the dynein-2 knockout cells grow cilia too slowly to enable them to achieve normal length and density before the cell divides. In the present study, dynein-2 knockout cells were deciliated and then allowed to regrow their cilia for 22 hr under conditions in which the cells did not divide. When dynein-2 was disabled, three effects were observed: (1) a decreased rate of cilia growth; (2) a lower cilia density that did not change over time; and (3) a wide distribution of cilia lengths that increased over time. These results confirm the importance of dynein-2 in regulating ciliary length in Tetrahymena. Cell Motil. Cytoskeleton, 2009. (c) 2009 Wiley-Liss, Inc.

Dynein-2 affects the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena thermophila.

Eukaryotic cilia and flagella are assembled and maintained by the bidirectional intraflagellar transport (IFT). Studies in alga, nematode, and mouse have shown that the heavy chain (Dyh2) and the light intermediate chain (D2LIC) of the cytoplasmic dynein-2 complex are essential for retrograde intraflagellar transport. In these organisms, disruption of either dynein-2 component results in short cilia/flagella with bulbous tips in which excess IFT particles have accumulated. In Tetrahymena, the expression of the DYH2 and D2LIC genes increases during reciliation, consistent with their roles in IFT. However, the targeted elimination of either DYH2 or D2LIC gene resulted in only a mild phenotype. Both knockout cell lines assembled motile cilia, but the cilia were of more variable lengths and less numerous than wild-type controls. Electron microscopy revealed normally shaped cilia with no swelling and no obvious accumulations of material in the distal ciliary tip. These results demonstrate that dynein-2 contributes to the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena.

Identification and characterization of dynein genes in Tetrahymena.

We describe the protocol through which we identify and characterize dynein subunit genes in the ciliated protozoan Tetrahymena thermophila. The gene(s) of interest is found by searching the Tetrahymena genome, and it is characterized in silico including the prediction of the open reading frame and identification of likely introns. The gene is then characterized experimentally, including the confirmation of the exon-intron organization of the gene and the measurement of the expression of the gene in nondeciliated and reciliating cells. In order to understand the function of the gene product, the gene is modified-for example, deleted, overexpressed, or epitope-tagged-using the straightforward gene replacement strategies available with Tetrahymena. The effect(s) of the dynein gene modification is evaluated by examining transformants for ciliary traits including cell motility, ciliogenesis, cell division, and the engulfment of particles through the oral apparatus. The multistepped protocol enables undergraduate students to engage in short- and long-term experiments. In our laboratory during the last 6 years, more than two dozen undergraduate students have used these methods to investigate dynein subunit genes.

Twenty-five dyneins in Tetrahymena: A re-examination of the multidynein hypothesis.

Dyneins are responsible for essential movements in eukaryotic cells. The motor activity of each dynein complex resides in its complement of heavy chains. In the present study, we examined 136 heavy chain sequences from the completed genomes of 11 diverse model organisms, including examples from Viridiplantae, Excavata, Chromalveolata, and Metazoa. In many cases, we discovered dynein heavy chains previously not identified. For example, Tetrahymena expresses a total of 25 DYH genes rather than the previously identified 14. The Tetrahymena DYH genes are nonaxonemal DYH1 and DYH2; axonemal outer arm alpha, beta, and gamma; axonemal two-headed inner arm 1alpha and 1beta; and 18 single-headed inner arm heavy chains. The heavy chains divide into nine classes; six of these are highly conserved in sequence and number of isoforms in a given organism. The other three are single-headed inner arm dyneins, whose numbers vary significantly in different organisms. These findings lead to two conclusions. One, the last common ancestor of all eukaryotes expressed nine different dynein heavy chains. Two, subsequent to the divergences leading to different organisms, additional dynein heavy chains emerged. These newer dyneins are not well conserved across species and the variation may reflect different motility requirements in different organisms. Together, these results suggest that each of the nine classes of dyneins is functionally distinct, but members within some of the classes are not specialized. An understanding of the relationships among the various dynein heavy chains is important when deducing functions across species.

Dynein light chain family in Tetrahymena thermophila.

Dyneins are large protein complexes that produce directed movement on microtubules. In situ, dyneins comprise combinations of heavy, intermediate, light-intermediate, and light chains. The light chains regulate the locations and activities of dyneins but their functions are not completely understood. We have searched the recently sequenced Tetrahymena thermophila macronuclear genome to describe the entire family of dynein light chains expressed in this organism. We identified fourteen genes encoding putative dynein light chains and seven genes encoding light chain-like proteins. RNA-directed PCR revealed that all 21 genes were expressed. Quantitative real time reverse transcription PCR showed that many of these genes were upregulated after deciliation, indicating that these proteins are present in cilia. Using the nomenclature developed in Chlamydomonas, Tetrahymena expresses two isoforms each of LC2, LC4, LC7, and Tctex1, three isoforms of p28, and six LC8/LC8-like isoforms. Tetrahymena also expresses two LC3-like genes. No Tetrahymena orthologue was found for Chlamydomonas LC5 or LC6. This study provides a complete description of the different genes and isoforms of the dynein light chains that are expressed in Tetrahymena, a model organism in which the targeted manipulation of genes is straightforward.

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote.

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.

The two cytoplasmic dynein-2 isoforms in Leishmania mexicana perform separate functions.

Eukaryotic organisms with cilia or flagella typically express two non-axonemal or "cytoplasmic" dyneins, dynein-1 and dynein-2. Interestingly, we find that Leishmania mexicana is unusual and contains two distinct cytoplasmic dynein-2 heavy chain genes (designated LmxDHC2.1 and LmxDHC2.2) along with a single dynein-1 heavy chain (LmxDHC1). Disruption of LmxDHC2.2 resulted in immotile parasites that had a rounded cell body. Although they assume amastigote morphology, immunoblot analysis of these cells demonstrates protein expression consistent with the promastigote stage. Ultrastructural analysis revealed non-emergent flagella that lacked the paraflagellar rod and an axoneme with deficiencies in several components. We confirmed the absence of paraflagellar rod proteins PFR1 and PFR2. These results show that LmxDHC2.2 is required for flagellar assembly and also participates in the maintenance of promastigote cell shape. In contrast to the results with LmxDHC2.2, we were unable to generate homologous disruptions of LmxDHC2.1. This result suggests that, unlike LmxDHC2.2, LmxDHC2.1 is an essential gene in Leishmania. Together, these findings demonstrate that the two dynein-2 heavy chain isoforms in Leishmania perform distinct functions. The observation that the genomes of Leishmania major, Leishmania infantum and Trypanosoma brucei also contain two dynein-2 isoforms suggests that this unusual aspect of cytoplasmic dynein is a conserved feature of the kinetoplastids.