Health profession students' outlooks on the medical profession during the COVID-19 pandemic: a global perspective.

BACKGROUND: This article summarizes a global study of the effect of the COVID-19 pandemic on junior health professions students' outlook on medicine. The pandemic has significantly affected health professions education. There is limited understanding of how students' pandemic experiences will affect them, and what impact these events may have on their career paths or the future of the professions. This information is important as it impacts the future of medicine. METHODS: In the Fall 2020 semester, 219 health professions students at 14 medical universities worldwide responded to the question: 'Has this experience (with COVID-19) changed your outlook on medicine as a profession?'. Short essay responses were semantically coded and organized into themes and subthemes using an inductive approach to thematic analysis. RESULTS: 145 responses were submitted. Themes were identified: (1) students reflected on the interaction between politics and healthcare; (2) reported becoming more aware of the societal expectations placed on healthcare professionals, including undertaking high risks and the sacrifices that healthcare professionals must make; (3) found reassurance from the recognized importance of healthcare professionals and expressed pride to be entering the profession; and (4) reflected on the current state of healthcare, including its limitations and future. CONCLUSION: Most students, independent of the extent of the pandemic in their respective countries, noted a change in their outlook regarding medicine. An overall positive outlook was noted in most junior students. Educators need to work on nurturing these sentiments and attitudes to help young students maintain a healthy relationship towards their chosen profession.

A thematic analysis of students' discussions on death and body donation in international online focus groups.

Historically, Anatomy education is an in-person discipline involving exposure to human body donors that facilitates personal and professional growth through, in part, the initiation of reflection on the topic of death. However, during the COVID-19 pandemic the decreased exposure to cadaveric anatomy for many health professions students may have influenced the depth of their individual reflections on this topic. Accordingly, this study aimed to investigate the effect of an alternate approach-focus group discussions between peers with varying degrees of exposure to cadaveric material-that may offer one strategy to stimulate deep reflection on the topic of death. A programmatic intervention was introduced, wherein students (n = 221) from 13 international universities discussed differences in their anatomy courses during small focus group sessions as part of an online exchange program. An inductive semantic thematic analysis was conducted on responses to an open-ended text-response question on how the activity influenced students' reflections about death. Resulting themes were organized into categories that described the content and topics of the students' discussions as they grappled with this sensitive topic. The students reportedly engaged in deep reflection and expressed an increased sense of connectedness with their peers, despite their disparate exposure levels to cadaveric anatomy and being physically distanced. This demonstrates that focus groups with students experiencing different laboratory contexts can be used to help all students reflect on the topic of death and that interchanges between dissecting and non-dissecting students can initiate thoughts about death and body donation among non-dissecting students.

A tension-independent mechanism reduces Aurora B-mediated phosphorylation upon microtubule capture by CENP-E at the kinetochore.

During mitosis, Aurora B kinase is required for forming proper bi-oriented kinetochore-microtubule attachments. Current models suggest that tension exerted between a pair of sister-kinetochores (inter-kinetochore stretch) produces a spatial separation of Aurora B kinase from kinetochore-associated microtubule binding substrates, such as the Knl1-Mis12-Ndc80 (KMN) network, resulting in a decrease of phosphorylation and, thus, an increase of affinity for microtubules. Using Single-Molecule High-Resolution Colocalization (SHREC) microscopy analysis of the kinetochore-associated motor CENP-E, we now show that CENP-E undergoes structural rearrangements prior to and after tension generation at the kinetochore, and displays a bi-modal Gaussian distribution on a pair of bi-oriented sister kinetochores. The conformational change of CENP-E depends on its microtubule-stimulated motor motility and the highly flexible coiled-coil between its motor and kinetochore-binding tail domains. Chemical inhibition of the motor motility or perturbations of the coiled-coil domain of CENP-E increases Aurora B-mediated Ndc80 phosphorylation in a tension-independent manner. Metaphase chromosome misalignment caused by CENP-E inhibition can be rescued by chemical inhibition of Aurora B kinase. Furthermore, a pair of monotelic sister-kinetochores shows asymmetric levels of Aurora B-mediated phosphorylation in mono-polar spindles depending on CENP-E motor activity. These results collectively suggest a tension-independent mechanism to reduce Aurora B-mediated phosphorylation of outer kinetochore components in response to microtubule capture by CENP-E.

Nuclear Actin Polymerized by mDia2 Confines Centromere Movement during CENP-A Loading.

Centromeres are specialized chromosomal regions epigenetically defined by the histone H3 variant centromere protein A (CENP-A). CENP-A needs to be replenished in every cell cycle, but how new CENP-A is stably incorporated into centromeric chromatin remains unclear. We have discovered that a cytoskeletal protein, diaphanous formin mDia2, is essential for the stable incorporation of new CENP-A proteins into centromeric nucleosomes. Here we report that mDia2-mediated formation of dynamic and short nuclear actin filaments in G1 nucleus is required to maintain CENP-A levels at the centromere. Importantly, mDia2 and nuclear actin are required for constrained centromere movement during CENP-A loading, and depleting nuclear actin or MgcRacGAP, which lies upstream of mDia2, extends centromeric association of the CENP-A loading chaperone Holliday junction recognition protein (HJURP). Our findings thus suggest that nuclear actin polymerized by mDia2 contributes to the physical confinement of G1 centromeres so that HJURP-mediated CENP-A loading reactions can be productive, and centromere's epigenetic identity can be stably maintained.

Analysis of Mitotic Checkpoint Function in Xenopus Egg Extracts.

Accurate sister chromatid segregation is pivotal in the faithful transmission of genetic information during each cell division. To ensure accurate segregation, eukaryotic organisms have evolved a "mitotic (or spindle assembly) checkpoint" to prevent premature advance to anaphase before successful attachment of every chromosome to the microtubules of the mitotic spindle. An unattached kinetochore generates a diffusible signal that inhibits ubiquitination of substrates such as cyclin B and securins. This protocol presents an in vitro assay for studying the mitotic checkpoint using Xenopus laevis egg extracts. Meiotic spindles assembled around nuclei added to egg extracts are synchronized at metaphase by an endogenous activity known as cytostatic factor (CSF). Normally, the mitotic checkpoint results in continued metaphase arrest following inactivation of CSF activity (by addition of calcium) and disassembly of spindle microtubules (with a microtubule inhibitor such as nocodazole). Simple DAPI staining for chromatin structure or biochemical analysis of Cdc2/cyclin B (cyclin-dependent kinase) histone H1 kinase activity can be used to evaluate the stages of the cell cycle and the status of the mitotic checkpoint. This cell-free system derived from Xenopus eggs has been successfully used to unravel the mechanisms of mitosis, and it provides a distinct advantage over cell-based studies in which perturbing kinetochore functions often results in lethality.

Formin-mediated epigenetic maintenance of centromere identity.

Accurate chromosome segregation in mammalian cells is guided by the centromere, a specialized chromosome region defined by the histone H3 variant centromere protein A (CENP-A). It is not well understood how cells maintain CENP-A levels at centromeres while continuously going through genome replications and cell divisions. A MgcRacGAP-dependent small GTPase molecular switch has been shown as essential for centromeric CENP-A maintenance. By using quantitative imaging, pulse-chase and live cell analysis, a recent work has suggested that the diaphanous formin mDia2, a well-established small GTPase effector, functions downstream of this small GTPase pathway to maintain CENP-A levels at centromeres. A constitutively active mDia2 construct is able to rescue the CENP-A loading defect caused by MgcRacGAP depletion. This study has uncovered an unsuspected role of the cytoskeleton protein mDia2 as an effector of the MgcRacGAP-dependent small GTPase signaling inside the nucleus to participate in the epigenetic regulation of centromere maintenance during cell cycle.

Diaphanous formin mDia2 regulates CENP-A levels at centromeres.

Centromeres of higher eukaryotes are epigenetically defined by centromere protein A (CENP-A), a centromere-specific histone H3 variant. The incorporation of new CENP-A into centromeres to maintain the epigenetic marker after genome replication in S phase occurs in G1 phase; however, how new CENP-A is loaded and stabilized remains poorly understood. Here, we identify the formin mDia2 as essential for stable replenishment of new CENP-A at centromeres. Quantitative imaging, pulse-chase analysis, and high-resolution ratiometric live-cell studies demonstrate that mDia2 and its nuclear localization are required to maintain CENP-A levels at centromeres. Depletion of mDia2 results in a prolonged centromere association of holiday junction recognition protein (HJURP), the chaperone required for CENP-A loading. A constitutively active form of mDia2 rescues the defect in new CENP-A loading caused by depletion of male germ cell Rac GTPase-activating protein (MgcRacGAP), a component of the small GTPase pathway essential for CENP-A maintenance. Thus, the formin mDia2 functions downstream of the MgcRacGAP-dependent pathway in regulating assembly of new CENP-A containing nucleosomes at centromeres.

Meeting report - New York Symposium on Quantitative Biology of the Cell.

In the city that never sleeps, great science never takes a break. On 15 January 2016, the 'New York Symposium on Quantitative Biology of the Cell', a one-day local meeting of the American Society for Cell Biology (ASCB), took place at Columbia University Medical Center in upper Manhattan. Focusing on the quantitative understanding of cellular and multicellular systems, this meeting created an otherwise rare opportunity for interaction among scientists at various career levels with differing but complementary backgrounds. Highlighting cutting-edge experimental measurements and theoretical modeling, the symposium broke the barrier between disciplines and ignited a hopefully continuing regional dialogue on the emergent topic of quantitative biology of the cell.

A dynein independent role of Tctex-1 at the kinetochore.

Dynein light chains are accessory subunits of the cytoplasmic dynein complex, a minus-end directed microtubule motor. Here, we demonstrate that the dynein light chain Tctex-1 associates with unattached kinetochores and is essential for accurate chromosome segregation. Tctex-1 knockdown in cells does not affect the localization and function of dynein at the kinetochore, but produces a prolonged mitotic arrest with a few misaligned chromosomes, which are subsequently missegregated during anaphase. This function is independent of Tctex-1's association with dynein. The kinetochore localization of Tctex-1 is independent of the ZW10-dynein pathway, but requires the Ndc80 complex. Thus, our findings reveal a dynein independent role of Tctex-1 at the kinetochore to enhance the stability of kinetochore-microtubule attachment.

Aurora A Kinase Inhibition Selectively Synergizes with Histone Deacetylase Inhibitor through Cytokinesis Failure in T-cell Lymphoma.

PURPOSE: Aurora A kinase (AAK) is expressed exclusively during mitosis, and plays a critical role in centrosome duplication and spindle formation. Alisertib is a highly selective AAK inhibitor that has demonstrated marked clinical activity of alisertib across a spectrum of lymphomas, though particularly in patients with T-cell lymphoma (TCL). We sought to compare and contrast the activity of alisertib in preclinical models of B-cell lymphoma (BCL) and TCL, and identify combinations worthy of clinical study. High-throughput screening of pralatrexate, the proteasome inhibitor (ixazomib), and the histone deacetylase (HDAC) inhibitor (romidepsin) revealed that only romidepsin synergized with alisertib, and only in models of TCL. We discovered that the mechanism of synergy between AAK inhibitors and HDAC inhibitors appears to be mediated through cytokinesis failure. EXPERIMENTAL DESIGN: A high-throughput screening approach was used to identify drugs that were potentially synergistic in combination with alisertib. Live-cell imaging was used to explore the mechanistic basis for the drug: drug interaction between alisertib and romidepsin. An in vivo xenograft TCL model was used to confirm in vitro results. RESULTS: In vitro, alisertib exhibited concentration-dependent cytotoxicity in BCL and TCL cell lines. Alisertib was synergistic with romidepsin in a T-cell-specific fashion that was confirmed in vivo. Live-cell imaging demonstrated that the combination treatment resulted in profound cytokinesis failure. CONCLUSIONS: These data strongly suggest that the combination of alisertib and romidepsin is highly synergistic in TCL through modulation of cytokinesis and merits clinical development.

The novel kinesin spindle protein (KSP) inhibitor SB-743921 exhibits marked activity in in vivo and in vitro models of aggressive large B-cell lymphoma.

The kinesin spindle protein (KSP) is a mitotic protein essential for cell cycle control and motility. SB-743921 (hereafter SB-921) is an inhibitor that selectively targets the ATP-binding domain of the KSP. The preclinical activity of SB-921 was evaluated in models of diffuse large B-cell lymphoma (DLBCL). The cytotoxicity of SB-921 was evaluated in a series of germinal center (GC-DLBCL) and post-germinal center (ABC-DLBCL) DLBCL cell lines and a murine lymphoma xenograft model. GC-DLBCL lines generally demonstrated greater sensitivity to SB-921. IC50 values ranged between 1 nM and 900 nM for GC-DLBCL compared to 1 nM to 10 µM for ABC lines. SB-921 demonstrated marked activity in a xenograft model of Ly-1 (GC-DLBCL). While SB-921 was relatively more active in GC derived cell lines, ABC-derived lines still underwent apoptosis at higher concentrations. These results demonstrate that SB-921 inhibits proliferation and induces apoptosis in both GC-DLBCL and ABC-DLBCL.

New insights into the mechanism for chromosome alignment in metaphase.

During mitosis, duplicated sister chromatids are properly aligned at the metaphase plate of the mitotic spindle before being segregated into two daughter cells. This requires a complex process to ensure proper interactions between chromosomes and spindle microtubules. The kinetochore, the proteinaceous complex assembled at the centromere region on each chromosome, serves as the microtubule attachment site and powers chromosome movement in mitosis. Numerous proteins/protein complexes have been implicated in the connection between kinetochores and dynamic microtubules. Recent studies have advanced our understanding on the nature of the interface between kinetochores and microtubule plus ends in promoting and maintaining their stable attachment. These efforts have demonstrated the importance of this process to ensure accurate chromosome segregation, an issue which has great significance for understanding and controlling abnormal chromosome segregation (aneuploidy) in human genetic diseases and in cancer progression.

The MRN-CtIP pathway is required for metaphase chromosome alignment.

Faithful duplication of the genome in S phase followed by its accurate segregation in mitosis is essential to maintain genomic integrity. Recent studies have suggested that proteins involved in DNA transactions are also required for whole-chromosome stability. Here we demonstrate that the MRN (Mre11, Rad50, and Nbs1) complex and CtIP are required for accurate chromosome segregation. Depletion of Mre11 or CtIP, antibody-mediated inhibition of Mre11, or small-molecule inhibition of MRN using mirin results in metaphase chromosome alignment defects in Xenopus egg extracts. Similarly, loss of MRN function adversely affects spindle assembly around DNA-coated beads in egg extracts. Inhibition of MRN function in mammalian cells triggers a metaphase delay and disrupts the RCC1-dependent RanGTP gradient. Addition of the Mre11 inhibitor mirin to egg extracts and mammalian cells reduces RCC1 association with mitotic chromosomes. Thus, the MRN-CtIP pathway contributes to Ran-dependent mitotic spindle assembly by modulating RCC1 chromosome association.

CENP-E--dependent BubR1 autophosphorylation enhances chromosome alignment and the mitotic checkpoint.

How the state of spindle microtubule capture at the kinetochore is translated into mitotic checkpoint signaling remains largely unknown. In this paper, we demonstrate that the kinetochore-associated mitotic kinase BubR1 phosphorylates itself in human cells and that this autophosphorylation is dependent on its binding partner, the kinetochore motor CENP-E. This CENP-E-dependent BubR1 autophosphorylation at unattached kinetochores is important for a full-strength mitotic checkpoint to prevent single chromosome loss. Replacing endogenous BubR1 with a nonphosphorylatable BubR1 mutant, as well as depletion of CENP-E, the BubR1 kinase activator, results in metaphase chromosome misalignment and a decrease of Aurora B-mediated Ndc80 phosphorylation at kinetochores. Furthermore, expressing a phosphomimetic BubR1 mutant substantially reduces the incidence of polar chromosomes in CENP-E-depleted cells. Thus, the state of CENP-E-dependent BubR1 autophosphorylation in response to spindle microtubule capture by CENP-E is important for kinetochore function in achieving accurate chromosome segregation.

mDia3-EB1-APC: A connection between kinetochores and microtubule plus ends.

Kinetochores must continuously associate with dynamic microtubule plus ends, as they oscillate along the mitotic spindle. The molecular basis for the kinetochore to track microtubule plus ends remains unresolved. In a recent study, we have shown an essential role of the formin mDia3 in stable kinetochore microtubule attachment and metaphase chromosome alignment. This function is attributable to EB1-binding by mDia3, for replacing endogenous mDia3 with an EB1-binding deficient mutant results in chromosome misalignment. EB1 specifically targets to attached, antipoleward kinetochores with polymerizing microtubules during chromosome oscillation. Therefore, we speculate that the mDai3-EB1-APC complex formation may relay EB1 microtubule plus end-tracking activity to the kinetochore.

FORMIN a link between kinetochores and microtubule ends.

The mammalian diaphanous-related (mDia) formin proteins are well known for their actin-nucleation and filament-elongation activities in mediating actin dynamics. They also directly bind to microtubules and regulate microtubule stabilization at the leading edge of the cell during cell migration. Recently, the formin mDia3 was shown to associate with the kinetochore and to contribute to metaphase chromosome alignment, a process in which kinetochores form stable attachments with growing and shrinking microtubules. We suggest that the formin mDia3 could contribute to the regulation of kinetochore-bound microtubule dynamics, in coordination with attachment via its own microtubule-binding activity, as well as via its interaction with the tip-tracker EB1 (end-binding protein 1).

Formin mDia3: A novel target for Aurora B kinase.

Stable attachment of kinetochores to spindle microtubules is essential for accurate chromosome segregation. We have shown that a kinetochore-associated formin protein, mDia3, contributes to the generation of stable kinetochore-microtubule attachment. The published report reviewed here shows an essential role of mDia3 in achieving metaphase chromosome alignment, and this function is directly regulated by Aurora B phosphorylation. Aurora B is a central component during the capture of spindle microtubules by kinetochores, in which it selectively eliminates incorrect attachments by phosphorylating a group of microtubule binding proteins at kinetochores to reduce their microtubule binding affinity. Here, we discuss the roles of Aurora B kinase and its substrates in achieving proper kinetochore-microtubule attachment.

Aurora B regulates formin mDia3 in achieving metaphase chromosome alignment.

Proper bipolar attachment of sister kinetochores to the mitotic spindle is critical for accurate chromosome segregation in mitosis. Here we show an essential role of the formin mDia3 in achieving metaphase chromosome alignment. This function is independent of mDia3 actin nucleation activity, but is attributable to EB1-binding by mDia3. Furthermore, the microtubule binding FH2 domain of mDia3 is phosphorylated by Aurora B kinase in vitro, and cells expressing the nonphosphorylatable mDia3 mutant cannot position chromosomes at the metaphase plate. Purified recombinant mDia3 phosphorylated by Aurora B exhibits reduced ability to bind microtubules and stabilize microtubules against cold-induced disassembly in vitro. Cells expressing the phosphomimetic mDia3 mutant do not form stable kinetochore microtubule fibers; despite they are able to congress chromosomes to the metaphase plate. These findings reveal a key role for mDia3 and its regulation by Aurora B phosphorylation in achieving proper stable kinetochore microtubule attachment.

Emerging functions of force-producing kinetochore motors.

More than two decades of research has resulted in the identification of some 60 microtubule motor proteins, several of which have been implicated in mitosis. Although some kinesin super-family proteins function as microtubule depolymerases at kinetochores, such as Kinesin-8 and -13, it is now appreciated that there are only two force-producing kinetochore associated motors, the plus end-directed microtubule motor CENP-E and the minus end-directed microtubule motor cytoplasmic dynein. Defining their roles at kinetochores has been hampered by the complexity of mitosis itself, and a multiplicity of mitotic roles, at least for cytoplasmic dynein. Nonetheless, recent advances have served to define the primary roles of the two kinetochore motors in detail.

Oncogenic Adenomatous polyposis coli mutants impair the mitotic checkpoint through direct interaction with Mad2.

The majority of colorectal tumors are aneuploid because of the underlying chromosome instability (CIN) phenotype, in which a defective mitotic checkpoint is implicated. Adenomatous polyposis coli (APC), a tumor suppressor gene that is commonly mutated in colon cancers, has been suggested in causing CIN; however, the molecular mechanism remains unresolved. In this study, we report an interaction of tumor-associated N-terminal APC fragments (N-APC) with Mad2, an essential mitotic checkpoint protein, providing a direct molecular support for linking APC mutations to the generation of CIN. N-APC interacts with Mad2 in Xenopus egg extracts, colon cancer cells, and in vitro with purified components. The interaction between N-APC and Mad2 decreases the soluble pool of Mad2, which is essential for Mad2 cycling and releasing from unattached kinetochores to produce a diffusible |P;wait anaphase|P' signal. Addition of such an N-APC mutant of egg extracts inactivates the mitotic checkpoint. Expressing a tumor-associated N-APC mutant in mammalian cells with an intact mitotic checkpoint produces premature anaphase onset with missegregated chromosomes.