A Moonlighting Job for alpha-Globin in Blood Vessels.

Red blood cells (RBC) express high levels of hemoglobin A tetramer (HbA, 22) to facilitate oxygen (O2) transport. Hemoglobin and related proteins are also expressed at lower levels in other tissues across the animal kingdom. Physiological functions for most non-erythroid globins likely derive from their ability to catalyze reduction-oxidation (redox) reactions via electron transfer through heme-associated iron. An interesting example is illustrated by the recent discovery that -globin without -globin is expressed in some arteriolar endothelial cells (ECs). -Globin binds EC nitric oxide synthase (eNOS) and degrades its enzymatic product nitric oxide (NO), a potent vasodilator. Thus, depletion of -globin in ECs or inhibition of its association with eNOS causes arteriolar relaxation and lowering of blood pressure in mice. Some of these findings have been replicated in isolated human blood vessels and genetic studies are tractable in populations where -thalassemia alleles are prevalent. Two small studies identified associations between loss of -genes in humans and NO-regulated vascular responses elicited by local hypoxia-induced blood flow or thermal stimulation. In a few larger population-based studies, no associations were detected between loss of -globin genes and blood pressure, ischemic stroke or pulmonary hypertension. In contrast, a significant positive association between -globin gene copy number and kidney disease was detected in an African American cohort. Further studies are required to define comprehensively the expression of -globin and related globin proteins in different vascular beds and ascertain their overall impact on normal and pathological vascular physiology.

Inter-species lateral gene transfer focused on the Chlamydia plasticity zone identifies loci associated with immediate cytotoxicity and inclusion stability.

Chlamydia muridarum actively grows in murine mucosae and is a representative model of human chlamydial genital tract disease. In contrast, C. trachomatis infections in mice are limited and rarely cause disease. The factors that contribute to these differences in host adaptation and specificity remain elusive. Overall genomic similarity leads to challenges in the understanding of these significant differences in tropism. A region of major genetic divergence termed the plasticity zone (PZ) has been hypothesized to contribute to the host specificity. To evaluate this hypothesis, lateral gene transfer was used to generate multiple hetero-genomic strains that are predominately C. trachomatis but have replaced regions of the PZ with those from C. muridarum. In vitro analysis of these chimeras revealed C. trachomatis-like growth as well as poor mouse infection capabilities. Growth-independent cytotoxicity phenotypes have been ascribed to three large putative cytotoxins (LCT) encoded in the C. muridarum PZ. However, analysis of PZ chimeras supported that gene products other than the LCTs are responsible for cytopathic and cytotoxic phenotypes. Growth analysis of associated chimeras also led to the discovery of an inclusion protein, CTL0402 (CT147), and homolog TC0424, which was critical for the integrity of the inclusion and preventing apoptosis.

The miR-124 family of microRNAs is crucial for regeneration of the brain and visual system in the planarian Schmidtea mediterranea.

Brain regeneration in planarians is mediated by precise spatiotemporal control of gene expression and is crucial for multiple aspects of neurogenesis. However, the mechanisms underpinning the gene regulation essential for brain regeneration are largely unknown. Here, we investigated the role of the miR-124 family of microRNAs in planarian brain regeneration. The miR-124 family (miR-124) is highly conserved in animals and regulates neurogenesis by facilitating neural differentiation, yet its role in neural wiring and brain organization is not known. We developed a novel method for delivering anti-miRs using liposomes for the functional knockdown of microRNAs. Smed-miR-124 knockdown revealed a key role for these microRNAs in neuronal organization during planarian brain regeneration. Our results also demonstrated an essential role for miR-124 in the generation of eye progenitors. Additionally, miR-124 regulates Smed-slit-1, which encodes an axon guidance protein, either by targeting slit-1 mRNA or, potentially, by modulating the canonical Notch pathway. Together, our results reveal a role for miR-124 in regulating the regeneration of a functional brain and visual system.

Hybrid Inquiry-Based Laboratory Curriculum Highlights Scientific Method Using Bacterial Conjugation as a Model.

Undergraduate microbiology students are exposed to the theory of the scientific method throughout their undergraduate coursework, but laboratory course curricula often focus on technical skills rather than fully integrating scientific thinking as a component of competencies addressed. Here, we have designed a six-session inquiry-based laboratory (IBL) curriculum for an upper-level microbiology laboratory course that fully involves students in the scientific process using bacterial conjugation as the model system, including both online discussions and in-person laboratory sessions. The student learning objectives focus on the scientific method, experimental design, data analysis, bacterial conjugation mechanisms, and scientific communication. We hypothesized students would meet these learning objectives after completing this IBL and tracked student learning and surveyed students to provide an assessment of the structure of the IBL using pre- and post-IBL quizzes and the Laboratory Course Assessment Survey. Overall, our results show this IBL results in positive student learning gains.

A CURE for the COVID-19 Era: A Vaccine-Focused Online Immunology Laboratory.

During the COVID-19 pandemic, universities across the globe quickly shifted to online education. Laboratory courses faced unique challenges and were forced to reevaluate learning objectives and identify creative projects to engage students online. This study describes a newly developed online immunology laboratory curriculum focused on vaccine development. The course incorporated learning objectives to teach the scientific process, key experimental design components, and immunology techniques to evaluate vaccine efficacy. The curriculum, a course-based undergraduate research experience (CURE), asked students to engage in the research literature, propose a vaccine design and assessment, and interpret mock results. Instructor evaluation of student work as well as student self-evaluations demonstrated that students met the curriculum's learning objectives. Additionally, results from the laboratory course assessment survey (LCAS) indicate that this curriculum incorporated the CURE elements of collaboration, discovery and relevance, and iteration.

Transposon Mutagenesis in Chlamydia trachomatis Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer.

Transposon mutagenesis is a widely applied and powerful genetic tool for the discovery of genes associated with selected phenotypes. Chlamydia trachomatis is a clinically significant, obligate intracellular bacterium for which many conventional genetic tools and capabilities have been developed only recently. This report describes the successful development and application of a Himar transposon mutagenesis system for generating single-insertion mutant clones of C. trachomatis This system was used to generate a pool of 105 transposon mutant clones that included insertions in genes encoding flavin adenine dinucleotide (FAD)-dependent monooxygenase (C. trachomatis148 [ct148]), deubiquitinase (ct868), and competence-associated (ct339) proteins. A subset of Tn mutant clones was evaluated for growth differences under cell culture conditions, revealing that most phenocopied the parental strain; however, some strains displayed subtle and yet significant differences in infectious progeny production and inclusion sizes. Bacterial burden studies in mice also supported the idea that a FAD-dependent monooxygenase (ct148) and a deubiquitinase (ct868) were important for these infections. The ct339 gene encodes a hypothetical protein with limited sequence similarity to the DNA-uptake protein ComEC. A transposon insertion in ct339 rendered the mutant incapable of DNA acquisition during recombination experiments. This observation, along with in situ structural analysis, supports the idea that this protein is playing a role in the fundamental process of lateral gene transfer similar to that of ComEC. In all, the development of the Himar transposon system for Chlamydia provides an effective genetic tool for further discovery of genes that are important for basic biology and pathogenesis aspects.IMPORTANCEChlamydia trachomatis infections have an immense impact on public health; however, understanding the basic biology and pathogenesis of this organism has been stalled by the limited repertoire of genetic tools. This report describes the successful adaptation of an important tool that has been lacking in Chlamydia studies: transposon mutagenesis. This advance enabled the generation of 105 insertional mutants, demonstrating that numerous gene products are not essential for in vitro growth. Mammalian infections using these mutants revealed that several gene products are important for infections in vivo Moreover, this tool enabled the investigation and discovery of a gene critical for lateral gene transfer; a process fundamental to the evolution of bacteria and likely for Chlamydia as well. The development of transposon mutagenesis for Chlamydia has broad impact for the field and for the discovery of genes associated with selected phenotypes, providing an additional avenue for the discovery of molecular mechanisms used for pathogenesis and for a more thorough understanding of this important pathogen.