Artificial nanopores that mimic the transport selectivity of the nuclear pore complex.

Nuclear pore complexes (NPCs) act as effective and robust gateways between the nucleus and the cytoplasm, selecting for the passage of particular macromolecules across the nuclear envelope. NPCs comprise an elaborate scaffold that defines a approximately 30 nm diameter passageway connecting the nucleus and the cytoplasm. This scaffold anchors proteins termed 'phenylalanine-glycine' (FG)-nucleoporins, the natively disordered domains of which line the passageway and extend into its lumen. Passive diffusion through this lined passageway is hindered in a size-dependent manner. However, transport factors and their cargo-bound complexes overcome this restriction by transient binding to the FG-nucleoporins. To test whether a simple passageway and a lining of transport-factor-binding FG-nucleoporins are sufficient for selective transport, we designed a functionalized membrane that incorporates just these two elements. Here we demonstrate that this membrane functions as a nanoselective filter, efficiently passing transport factors and transport-factor-cargo complexes that specifically bind FG-nucleoporins, while significantly inhibiting the passage of proteins that do not. This inhibition is greatly enhanced when transport factor is present. Determinants of selectivity include the passageway diameter, the length of the nanopore region coated with FG-nucleoporins, the binding strength to FG-nucleoporins, and the antagonistic effect of transport factors on the passage of proteins that do not specifically bind FG-nucleoporins. We show that this artificial system faithfully reproduces key features of trafficking through the NPC, including transport-factor-mediated cargo import.

Nucleocytoplasmic transport: a role for nonspecific competition in karyopherin-nucleoporin interactions.

Nucleocytoplasmic transport occurs through the nuclear pore complex (NPC), which in yeast is a ~50 MDa complex consisting of ~30 different proteins. Small molecules can freely exchange through the NPC, but macromolecules larger than ~40 kDa must be aided across by transport factors, most of which belong to a related family of proteins termed karyopherins (Kaps). These transport factors bind to the disordered phenylalanine-glycine (FG) repeat domains in a family of NPC proteins termed FG nups, and this specific binding allows the transport factors to cross the NPC. However, we still know little in terms of the molecular and kinetic details regarding how this binding translates to selective passage of transport factors across the NPC. Here we show that the specific interactions between Kaps and FG nups are strongly modulated by the presence of a cellular milieu whose proteins appear to act as very weak competitors that nevertheless collectively can reduce Kap/FG nup affinities by several orders of magnitude. Without such modulation, the avidities between Kaps and FG nups measured in vitro are too tight to be compatible with the rapid transport kinetics observed in vivo. We modeled the multivalent interactions between the disordered repeat binding sites in the FG nups and multiple cognate binding sites on Kap, showing that they should indeed be sensitive to even weakly binding competitors; the introduction of such competition reduces the availability of these binding sites, dramatically lowering the avidity of their specific interactions and allowing rapid nuclear transport.

Connecting improvisational exercises and pharmacy communication skills: A how-to guide.

BACKGROUND AND PURPOSE: Medical improvisation (improv) is increasingly being used to teach physicians, nurses, and other caregivers how to better communicate with patients and other members of the healthcare team. This article describes how improvisational activities were incorporated within an existing pharmacy practice lab course and how to implement improv games targeting specific communication skills. EDUCATIONAL ACTIVITY AND SETTING: Three hours of improvisational activities were incorporated into a semester-long pharmacy practice lab course. Partner games such as "the mirror" and group games such as "out-of-order story" were played and connected to communication skills related to counseling or taking a patient's history. Additional activities were introduced targeting specific areas of weakness identified in a formative assessment. FINDINGS: A survey was used to assess student perceptions of the improv activities. Students were largely able to connect the skills learned in improv to pharmacy, and some provided examples of how they were already implementing these skills in practice. SUMMARY: This article provides a user manual so that faculty with little to no improv experience can include these activities in their communications courses.

Making a Case for Faculty Advisor-Advisee Concordant Pairs.

The Accreditation Council for Pharmacy Education Standards require schools and colleges of pharmacy to provide the needed resources for student success, including student advising. The faculty advisor's role in schools and colleges of pharmacy can be varied and may include modeling professional behavior, serving as students' advocate, and providing academic and professional guidance. This is especially important upon students entering pharmacy school when the risk of being overwhelmed has been documented. Various factors, including geographic distance from home, professional identity formation, and anxiety over career trajectory have been challenges to their perceived well-being. As in all human interactions, we believe that successful advisor/mentor-student relationships are more likely to develop if there is a deeper personal connection-some element(s) of shared demographic background-in addition to complementary professional, educational, or research backgrounds of the faculty advisor. Pharmacy students in Chinese and American universities and those in other health care disciplines have rated very highly their perceived value of the supporting role of the faculty advisor; hence, we postulated that using this criterion when matching faculty advisor-student pairs may produce better outcomes. In this commentary, we propose expanded criteria including demographics for producing faculty advisor-advisee concordant (or nearly so) pairs mindfully and strategically in a standardized stepwise process of advising aimed at facilitating success for students of all backgrounds, with the goal of promoting diversity, equity, and inclusion and the ultimate attendant benefits to patient care.

Mentorship Landscape and Common Practices in an Academic Pharmacy Association.

OBJECTIVES: To explore the landscape of mentorship within professional associations in pharmacy academia, including reviewing available literature and describing currently available programs within the American Association of Colleges of Pharmacy, and recommend key considerations for the development of mentorship programs within professional associations. FINDINGS: A literature review of mentorship programs within professional associations for pharmacy academics was conducted, with a total of 5 articles identified and summarized. Additionally, a survey was conducted to determine the landscape of available mentorship programs within American Association of Colleges of Pharmacy affinity groups to capture unpublished experiences. Information regarding common characteristics and assessment methods was collected for groups that have mentorship programs, while needs and barriers were collected for those who did not. SUMMARY: Literature, while limited, supports positive perceptions of mentorship programs within professional associations. Based on the responses and working group experience, several recommendations are proposed for mentorship program development, including the need for clearly defined goals, relevant program outcomes, association support to reduce redundancies and promote participation, and, in some cases, implementation of an association-wide program to ensure access to mentorship.

Simple kinetic relationships and nonspecific competition govern nuclear import rates in vivo.

Many cargoes destined for nuclear import carry nuclear localization signals that are recognized by karyopherins (Kaps). We present methods to quantitate import rates and measure Kap and cargo concentrations in single yeast cells in vivo, providing new insights into import kinetics. By systematically manipulating the amounts, types, and affinities of Kaps and cargos, we show that import rates in vivo are simply governed by the concentrations of Kaps and their cargo and the affinity between them. These rates fit to a straightforward pump-leak model for the import process. Unexpectedly, we deduced that the main limiting factor for import is the poor ability of Kaps and cargos to find each other in the cytoplasm in a background of overwhelming nonspecific competition, rather than other more obvious candidates such as the nuclear pore complex and Ran. It is likely that most of every import round is taken up by Kaps and nuclear localization signals sampling other cytoplasmic proteins as they locate each other in the cytoplasm.

A novel learning approach to pharmaceutical sciences research in a pharmacy research advanced pharmacy practice experience (APPE) elective course.

BACKGROUND AND PURPOSE: This project investigates the use of pharmacy student metacognitive learning in a laboratory-based science research advanced pharmacy practice experience (APPE). EDUCATIONAL ACTIVITY AND SETTING: We describe a five-week research APPE. This course is separated into two parts which run simultaneously. In part 1, students read and discuss papers from primary literature to learn the context of the project and the theory behind each laboratory procedure. In part 2, students perform experiments in the laboratory that contribute to the primary investigator's (PI's) ongoing research project and relate directly to the readings from part 1. Metacognitive processes allow students to better understand and evaluate the primary literature and to connect that information with the hands-on experiments being performed. FINDINGS: Currently, this APPE has run five times with a total of eight students. Student learning was assessed by several written and oral assignments graded with rubrics. Students' perceptions of their own learning and metacognitive development following the course was assessed using a survey. SUMMARY: This APPE seems to be a useful experience for both faculty and students. Students obtain laboratory and metacognitive skill development, while the collaborating laboratory is supplied with material required for further experiments. Importantly, the APPE preceptor is not the PI, so the preceptor is able to focus on the learning skills (both metacognition and hands-on) portion of the APPE.

Mechanism of Nonsense-Mediated mRNA Decay Stimulation by Splicing Factor SRSF1.

The splicing factor SRSF1 promotes nonsense-mediated mRNA decay (NMD), a quality control mechanism that degrades mRNAs with premature termination codons (PTCs). Here we show that transcript-bound SRSF1 increases the binding of NMD factor UPF1 to mRNAs while in, or associated with, the nucleus, bypassing UPF2 recruitment and promoting NMD. SRSF1 promotes NMD when positioned downstream of a PTC, which resembles the mode of action of exon junction complex (EJC) and NMD factors. Moreover, splicing and/or EJC deposition increase the effect of SRSF1 on NMD. Lastly, SRSF1 enhances NMD of PTC-containing endogenous transcripts that result from various events. Our findings reveal an alternative mechanism for UPF1 recruitment, uncovering an additional connection between splicing and NMD. SRSF1's role in the mRNA's journey from splicing to decay has broad implications for gene expression regulation and genetic diseases.

The molecular mechanism of nuclear transport revealed by atomic-scale measurements.

Nuclear pore complexes (NPCs) form a selective filter that allows the rapid passage of transport factors (TFs) and their cargoes across the nuclear envelope, while blocking the passage of other macromolecules. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG)-rich repeats line the pore and interact with TFs. However, the reason that transport can be both fast and specific remains undetermined, through lack of atomic-scale information on the behavior of FGs and their interaction with TFs. We used nuclear magnetic resonance spectroscopy to address these issues. We show that FG repeats are highly dynamic IDPs, stabilized by the cellular environment. Fast transport of TFs is supported because the rapid motion of FG motifs allows them to exchange on and off TFs extremely quickly through transient interactions. Because TFs uniquely carry multiple pockets for FG repeats, only they can form the many frequent interactions needed for specific passage between FG repeats to cross the NPC.

Mechanism of metal ion activation of the diphtheria toxin repressor DtxR.

The diphtheria toxin repressor (DtxR) is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear. Calorimetric techniques have demonstrated that although binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 x 10(-7), binding site 2 (primary) is a low-affinity binding site with a binding constant of 6.3 x 10(-4). These two binding sites act in an independent fashion, and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A,C102D), reported here, and the previously reported DtxR(H79A) have allowed us to propose a mechanism of metal activation for DtxR.

A method for the rapid and efficient elution of native affinity-purified protein A tagged complexes.

A problem faced in proteomics studies is the recovery of tagged protein complexes in their native and active form. Here we describe a peptide, Bio-Ox, that mimics the immunoglobulin G (IgG) binding interface of Staphylococcus aureus Protein A, and competitively displaces affinity-purified Protein A fusion proteins and protein complexes from IgG-Sepharose. We show that Bio-Ox elution is a robust method for the efficient and rapid recovery of native tagged proteins, and can be applied to a variety of structural genomics and proteomics studies.