Biosensors based on peptide exposure show single molecule conformations in live cells.

We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. "Binder/tag" is based on incorporation of a 7-mer peptide (the tag) into a protein where its solvent exposure is controlled by protein conformation. Only upon exposure can the peptide specifically interact with a reporter protein (the binder). Thus, simple fluorescence localization reflects protein conformation. Through direct excitation of bright dyes, the trajectory and conformation of individual proteins can be followed. Simple protein engineering provides highly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn, tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.

A post-invasion role for Chlamydia type III effector TarP in modulating the dynamics and organization of host cell focal adhesions.

The human pathogen Chlamydia trachomatis targets epithelial cells lining the genital mucosa. We observed that infection of various cell types, including fibroblasts and epithelial cells resulted in the formation of unusually stable and mature focal adhesions that resisted disassembly induced by the myosin II inhibitor, blebbistatin. Superresolution microscopy revealed in infected cells the vertical displacement of paxillin and focal adhesion kinase from the signaling layer of focal adhesions, whereas vinculin remained in its normal position within the force transduction layer. The candidate type III effector TarP, which localized to focal adhesions during infection and when expressed ectopically, was sufficient to mimic both the reorganization and blebbistatin-resistant phenotypes. These effects of TarP, including its localization to focal adhesions, required a post-invasion interaction with the host protein vinculin through a specific domain at the C terminus of TarP. This interaction is repurposed from an actin-recruiting and -remodeling complex to one that mediates nanoarchitectural and dynamic changes of focal adhesions. The consequence of Chlamydia-stabilized focal adhesions was restricted cell motility and enhanced attachment to the extracellular matrix. Thus, via a novel mechanism, Chlamydia inserts TarP within focal adhesions to alter their organization and stability.

Manipulation of the Host Cell Cytoskeleton by Chlamydia.

Chlamydiae are obligate intracellular pathogens. They undergo a biphasic developmental cycle differentiating between the infectious but metabolically quiescent elementary body and the vegetative, but non-infectious reticulate body. Chlamydia spends a significant portion of its development in the non-infectious stage, demanding an effective strategy of manipulating the host cells to ensure its intracellular survival and replication. A common target of all Chlamydia species studied so far is the host cell cytoskeleton, with past and recent findings revealing crucial roles in invasion, inclusion maintenance, nutrient acquisition, and egress. The molecular details of how Chlamydia co-opts the cytoskeleton is becoming clearer, with bacterial factors and their corresponding host cell targets identified.

The Chlamydia effector TarP mimics the mammalian leucine-aspartic acid motif of paxillin to subvert the focal adhesion kinase during invasion.

Host cell signal transduction pathways are often targets of bacterial pathogens, especially during the process of invasion when robust actin remodeling is required. We demonstrate that the host cell focal adhesion kinase (FAK) was necessary for the invasion by the obligate intracellular pathogen Chlamydia caviae. Bacterial adhesion triggered the transient recruitment of FAK to the plasma membrane to mediate a Cdc42- and Arp2/3-dependent actin assembly. FAK recruitment was via binding to a domain within the virulence factor TarP that mimicked the LD2 motif of the FAK binding partner paxillin. Importantly, bacterial two-hybrid and quantitative imaging assays revealed a similar level of interaction between paxillin-LD2 and TarP-LD. The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34(Arc), and actin to the plasma membrane. In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction. Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector.

Citizenship status and career self-efficacy: An intersectional study of biomedical trainees in the United States.

This study examines the intersectional role of citizenship and gender with career self-efficacy amongst 10,803 doctoral and postdoctoral trainees in US universities. These biomedical trainees completed surveys administered by 17 US institutions that participated in the National Institutes of Health Broadening Experiences in Scientific Training (NIH BEST) Programs. Findings indicate that career self-efficacy of non-citizen trainees is significantly lower than that of US citizen trainees. While lower career efficacy was observed in women compared with men, it was even lower for non-citizen female trainees. Results suggest that specific career interests may be related to career self-efficacy. Relative to US citizen trainees, both male and female non-citizen trainees showed higher interest in pursuing a career as an academic research investigator. In comparison with non-citizen female trainees and citizen trainees of all genders, non-citizen male trainees expressed the highest interest in research-intensive (and especially principal investigator) careers. The authors discuss potential causes for these results and offer recommendations for increasing trainee career self-efficacy which can be incorporated into graduate and postdoctoral training.

Fluorogenic RNA-based biomaterials for imaging and tracking the cargo of extracellular vesicles.

Extracellular vesicles (EVs), or exosomes, play important roles in physiological and pathological cellular communication and have gained substantial traction as biological drug carriers. EVs contain both short and long non-coding RNAs that regulate gene expression and epigenetic processes. To fully capitalize on the potential of EVs as drug carriers, it is important to study and understand the intricacies of EV function and EV RNA-based communication. Here we developed a genetically encodable RNA-based biomaterial, termed EXO-Probe, for tracking EV RNAs. The EXO-Probe comprises an EV-loading RNA sequence (EXO-Code), fused to a fluorogenic RNA Mango aptamer for RNA imaging. This fusion construct allowed the visualization and tracking of EV RNA and colocalization with markers of multivesicular bodies; imaging RNA within EVs, and non-destructive quantification of EVs. Overall, the new RNA-based biomaterial provides a useful and versatile means to interrogate the role of EVs in cellular communication via RNA trafficking to EVs and to study cellular sorting decisions. The system will also help lay the foundation to further improve the therapeutic efficacy of EVs as drug carriers.

Development and Assessment of a Sustainable PhD Internship Program Supporting Diverse Biomedical Career Outcomes.

A doctoral-level internship program was developed at the University of North Carolina at Chapel Hill with the intent to create customizable experiential learning opportunities for biomedical trainees to support career exploration, preparation, and transition into their post-graduate professional roles. We report the outcomes of this program over a five-year period. During that 5-year period, 123 internships took place at over 70 partner sites, representing at least 20 academic, for-profit, and non-profit career paths in the life sciences. A major goal of the program was to enhance trainees' skill development and expertise in careers of interest. The benefits of the internship program for interns, host/employer, and supervisor/principal investigator were assessed using a mixed-methods approach, including surveys with closed- and open-ended responses as well as focus group interviews. Balancing stakeholder interests is key to creating a sustainable program with widespread support; hence, the level of support from internship hosts and faculty members were key metrics analyzed throughout. We hypothesized that once a successful internship program was implemented, faculty culture might shift to be more accepting of internships; indeed, the data quantifying faculty attitudes support this. Furthermore, host motivation and performance expectations of interns were compared with results achieved, and this data revealed both expected and surprising benefits to hosts. Data suggests a myriad of benefits for each stakeholder group, and themes are cataloged and discussed. Program outcomes, evaluation data, policies, resources, and best practices developed through the implementation of this program are shared to provide resources that facilitate the creation of similar internship programs at other institutions. Program development was initially spurred by National Institutes of Health pilot funding, thereafter, successfully transitioning from a grant-supported model, to an institutionally supported funding model to achieve long-term programmatic sustainability.

Career self-efficacy disparities in underrepresented biomedical scientist trainees.

The present study examines racial, ethnic, and gender disparities in career self-efficacy amongst 6077 US citizens and US naturalized graduate and postdoctoral trainees. Respondents from biomedical fields completed surveys administered by the National Institutes of Health Broadening Experiences in Scientific Training (NIH BEST) programs across 17 US institutional sites. Graduate and postdoctoral demographic and survey response data were examined to evaluate the impact of intersectional identities on trainee career self-efficacy. The study hypothesized that race, ethnicity and gender, and the relations between these identities, would impact trainee career self-efficacy. The analysis demonstrated that racial and ethnic group, gender, specific career interests (academic principal investigator vs. other careers), and seniority (junior vs. senior trainee level) were, to various degrees, all associated with trainee career self-efficacy and the effects were consistent across graduate and postdoctoral respondents. Implications for differing levels of self-efficacy are discussed, including factors and events during training that may contribute to (or undermine) career self-efficacy. The importance of mentorship for building research and career self-efficacy of trainees is discussed, especially with respect to those identifying as women and belonging to racial/ethnic populations underrepresented in biomedical sciences. The results underscore the need for change in the biomedical academic research community in order to retain a diverse biomedical workforce.

C-N linked donor type porphyrin derivatives: unrevealed hole-transporting materials for efficient hybrid perovskite solar cells.

A new series of Zn(II) and Cu(II)-based porphyrin complexes 5a and 5b doubly functionalised with carbazole units were developed to be used as hole-transporting materials (HTMs) in perovskite solar cells (PSCs). These complexes were obtained via a nucleophilic substitution reaction mediated by PhI(OAc)2/NaAuCl4·2H2O, or using C-N transition metal-assisted coupling. The hole extraction capability of 5a and 5b was assessed using cyclic voltammetry; this study confirmed the better alignment of the Zn(II) complex 5a with the perovskite valence band level, compared to the Cu(II) complex 5b. The optimised geometry and molecular orbitals of both complexes also corroborate the higher potential of 5a as a HTM. Photoluminescence characterisation showed that the presence of 5a and 5b as HTMs on the perovskite surface resulted in the quenching of the emission, matching the hole transfer phenomenon. The photovoltaic performance was evaluated and compared with those of reference cells made with the standard HTM spiro-OMeTAD. The optimised 5-based devices showed improvements in all photovoltaic characteristics; their open circuit voltage (Voc) reached close to 1 V and short-circuit current density (Jsc) values were 13.79 and 9.14 mA cm-2 for 5a and 5b, respectively, disclosing the effect of the metallic centre. A maximum power conversion efficiency (PCE) of 10.01% was attained for 5a, which is 65% of the PCE generated by using the spiro-OMeTAD reference. This study demonstrates that C-N linked donor-type porphyrin derivatives are promising novel HTMs for developing efficient and reproducible PSCs.

Actin nano-architecture of phagocytic podosomes.

Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterize the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy, a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy and localization-based super-resolution microscopy. Phagocytic podosomes are observed during frustrated phagocytosis, a model in which cells attempt to engulf micropatterned IgG antibodies. For circular patterns, this results in regular arrays of podosomes with well-defined geometry. Using persistent homology, we develop a pipeline for semi-automatic identification and measurement of podosome features. These studies reveal an hourglass shape of the podosome actin core, a protruding knob at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, α-actinin, cortactin, and microtubules relative to actin are characterized.

A multi-functional microfluidic device compatible with widefield and light sheet microscopy.

We present a microfluidic device compatible with high resolution light sheet and super-resolution microscopy. The device is a 150 µm thick chamber with a transparent fluorinated ethylene propylene (FEP) cover that has a similar refractive index (1.34) to water (1.33), making it compatible with top-down imaging used in light sheet microscopy. We provide a detailed fabrication protocol and characterize the optical performance of the device. We demonstrate that the device supports long-term imaging of cell growth and differentiation as well as the rapid addition and removal of reagents while simultaneously maintaining sterile culture conditions by physically isolating the sample from the dipping lenses used for imaging. Finally, we demonstrate that the device can be used for super-resolution imaging using lattice light sheet structured illumination microscopy (LLS-SIM) and DNA PAINT. We anticipate that FEP-based microfluidics, as shown here, will be broadly useful to researchers using light sheet microscopy due to the ability to switch reagents, image weakly adherent cells, maintain sterility, and physically isolate the specimen from the optics of the instruments.

Characterization of the Growth of Chlamydia trachomatis in In Vitro-Generated Stratified Epithelium.

Chlamydia infection targets the mucosal epithelium, where squamous and columnar epithelia can be found. Research on Chlamydia-epithelia interaction has predominantly focused on columnar epithelia, with very little known on how Chlamydia interacts with the squamous epithelium. The stratification and differentiation processes found in the squamous epithelium might influence chlamydial growth and infection dissemination. For this reason, three-dimensional (3D) organotypic stratified squamous epithelial cultures were adapted to mimic the stratified squamous epithelium and chlamydial infection was characterized. Chlamydia trachomatis infection in monolayers and 3D cultures were monitored by immunofluorescence and transmission electron microscopy to evaluate inclusion growth and chlamydial interconversion between elementary and reticulate body. We observed that the stratified epithelium varied in susceptibility to C. trachomatis serovars L2 and D infection. The undifferentiated basal cells were susceptible to infection by both serovars, while the terminally differentiated upper layers were resistant. The differentiating suprabasal cells exhibited different susceptibilities to serovars L2 and D, with the latter unable to establish a successful infection in this layer. Mature elementary body-containing inclusions were much more prevalent in these permissive basal layers, while the uppermost differentiated layers consistently harbored very few reticulate bodies with no elementary bodies, indicative of severely limited bacterial replication and development. For serovar D, the differentiation state of the host cell was a determining factor, as calcium-induced differentiation of cells in a monolayer negatively affected growth of this serovar, in contrast to serovar L2. The apparent completion of the developmental cycle in the basal layers of the 3D cultures correlated with the greater degree of dissemination within and the level of disruption of the stratified epithelium. Our studies indicate that the squamous epithelium is a suboptimal environment for growth, and thus potentially contributing to the protection of the lower genital tract from infection. The relatively more fastidious serovar D exhibited more limited growth than the faster-growing and more invasive L2 strain. However, if given access to the more hospitable basal cell layer, both strains were able to produce mature inclusions, replicate, and complete their developmental cycle.