Deepening biomedical research training: Community-Building Wellness Workshops for Post-Baccalaureate Research Education Program (PREP) Trainees.

Problem: All trainees, especially those from historically minoritized backgrounds, experience stresses that may reduce their continuation in science, technology, engineering, math, and medicine (STEMM) careers. The Johns Hopkins University School of Medicine is one of ~45 institutions with a National Institutes of Health funded Postbaccalaureate Research Education Program (PREP) that provides mentoring and a year of fulltime research to prepare students from historically excluded groups for graduate school. Having experienced the conflation of stresses during the COVID-19 pandemic and related shutdown, we realized our program lacked a component that explicitly helped PREP Scholars recognize and cope with non-academic stresses (financial, familial, social, mental) that might threaten their confidence and success as scientists and future in STEMM. Intervention: We developed an early-intervention program to help Scholars develop life-long skills to become successful and resilient scientists. We developed a year-long series comprised of 9 workshops focused on community, introspection, financial fitness, emotional intelligence, mental health, and soft-skills. We recruited and compensated a cohort of PhD students and postdoctoral fellows to serve as Peer Mentors, to provide a community and the safest 'space' for Scholars to discuss personal concerns. Peer Mentors were responsible for developing and facilitating these Community-Building Wellness Workshops (CBWW). Context: CBWW were created and exectued as part of the larger PREP program. Workshops included a PowerPoint presentation by Peer Mentors that featured several case studies that prompted discussion and provided time for small-group discussions between Scholars and Peer Mentors. We also included pre- and post-work for each workshop. These touch-points helped Scholars cultivate the habit of introspection. Impact: The CBWW exceeded our goals. Both Peer Mentors and Scholars experienced strong mutual support, and Scholars developed life-long skills. Notably, several Scholars who had been experiencing financial, mental or mentor-related stress immediately brought this to the attention of program leadership, allowing early and successful intervention. At the completion of CBWW, PREP Scholars reported implementing many workshop skills into practice, were reshaping their criteria for choosing future mentors, and evaluating career decisions. Strikingly, Peer Mentors found they also benefitted from the program as well, suggesting a potential larger scope for the role of CBWW in academia. Lessons Learned: Peer Mentors were essential in creating a safe supportive environment that facilitated discussions, self-reflection, and self-care. Providing fair compensation to Peer Mentors for their professional mentoring and teaching contributions was essential and contributed meaningfully to the positive energy and impact of this program.

A Multi-Institutional Description of Processes and Outcomes of Postbaccalaureate Research Education Programs in the Mid-Atlantic Region.

ABSTRACT: Outcome data from 6 National Institutes of Health-funded Postbaccalaureate Research Education Programs (PREPs) in the Mid-Atlantic region were combined to give a multi-institutional perspective on their scholars' characteristics and progress through biomedical research training. The institutions hosting these programs were Johns Hopkins University School of Medicine, the Medical University of South Carolina, the University of Maryland School of Medicine, the University of North Carolina at Chapel Hill, Virginia Commonwealth University, and Virginia Polytechnic Institute and State University. The authors summarize the institutional pathways, demographics, undergraduate institutions, and graduate institutions for a total of 384 PREP scholars who completed the programs by June 2021. A total of 228 (59.4%) of these PREP scholars identified as Black or African American, 116 (30.2%) as Hispanic or Latinx, and 269 (70.0%) as female. The authors found that 376 of 384 scholars (97.9%) who started PREP finished their program, 319 of 376 (84.8%) who finished PREP matriculated into PhD or MD/PhD programs, and 284 of 319 (89.0%) who matriculated have obtained their PhD or are successfully making progress toward their PhD.

Lamins: The backbone of the nucleocytoskeleton interface.

The nuclear lamina (NL) is a crucial component of the inner nuclear membrane (INM) and consists of lamin filaments and associated proteins. Lamins are type V intermediate filament proteins essential for maintaining the integrity and mechanical properties of the nucleus. In human cells, 'B-type' lamins (lamin B1 and lamin B2) are ubiquitously expressed, while 'A-type' lamins (lamin A, lamin C, and minor isoforms) are expressed in a tissue- and development-specific manner. Lamins homopolymerize to form filaments that localize primarily near the INM, but A-type lamins also localize to and function in the nucleoplasm. Lamins play central roles in the assembly, structure, positioning, and mechanics of the nucleus, modulating cell signaling and influencing development, differentiation, and other activities. This review highlights recent findings on the structure and regulation of lamin filaments, providing insights into their multifaceted functions, including their role as "mechanosensors", delving into the emerging significance of lamin filaments as vital links between cytoskeletal and nuclear structures, chromatin organization, and the genome.

Native lamin A/C proteomes and novel partners from heart and skeletal muscle in a mouse chronic inflammation model of human frailty.

Clinical frailty affects ∼10% of people over age 65 and is studied in a chronically inflamed (Interleukin-10 knockout; "IL10-KO") mouse model. Frailty phenotypes overlap the spectrum of diseases ("laminopathies") caused by mutations in LMNA. LMNA encodes nuclear intermediate filament proteins lamin A and lamin C ("lamin A/C"), important for tissue-specific signaling, metabolism and chromatin regulation. We hypothesized that wildtype lamin A/C associations with tissue-specific partners are perturbed by chronic inflammation, potentially contributing to dysfunction in frailty. To test this idea we immunoprecipitated native lamin A/C and associated proteins from skeletal muscle, hearts and brains of old (21-22 months) IL10-KO versus control C57Bl/6 female mice, and labeled with Tandem Mass Tags for identification and quantitation by mass spectrometry. We identified 502 candidate lamin-binding proteins from skeletal muscle, and 340 from heart, including 62 proteins identified in both tissues. Candidates included frailty phenotype-relevant proteins Perm1 and Fam210a, and nuclear membrane protein Tmem38a, required for muscle-specific genome organization. These and most other candidates were unaffected by IL10-KO, but still important as potential lamin A/C-binding proteins in native heart or muscle. A subset of candidates (21 in skeletal muscle, 30 in heart) showed significantly different lamin A/C-association in an IL10-KO tissue (p < 0.05), including AldoA and Gins3 affected in heart, and Lmcd1 and Fabp4 affected in skeletal muscle. To screen for binding, eleven candidates plus prelamin A and emerin controls were arrayed as synthetic 20-mer peptides (7-residue stagger) and incubated with recombinant purified lamin A "tail" residues 385-646 under relatively stringent conditions. We detected strong lamin A binding to peptides solvent exposed in Lmcd1, AldoA, Perm1, and Tmem38a, and plausible binding to Csrp3 (muscle LIM protein). These results validated both proteomes as sources for native lamin A/C-binding proteins in heart and muscle, identified four candidate genes for Emery-Dreifuss muscular dystrophy (CSRP3, LMCD1, ALDOA, and PERM1), support a lamin A-interactive molecular role for Tmem38A, and supported the hypothesis that lamin A/C interactions with at least two partners (AldoA in heart, transcription factor Lmcd1 in muscle) are altered in the IL10-KO model of frailty.

Choreography of lamina-associated domains: structure meets dynamics.

Lamina-associated domains are large regions of heterochromatin positioned at the nuclear periphery. These domains have been implicated in gene repression, especially in the context of development. In mammals, LAD organization is dependent on nuclear lamins, inner nuclear membrane proteins, and chromatin state. In addition, chromatin readers and modifier proteins have been implicated in this organization, potentially serving as molecular tethers that interact with both nuclear envelope proteins and chromatin. More recent studies have focused on teasing apart the rules that govern dynamic LAD organization and how LAD organization, in turn, relates to gene regulation and overall 3D genome organization. This review highlights recent studies in mammalian cells uncovering factors that instruct the choreography of LAD organization, re-organization, and dynamics at the nuclear lamina, including LAD dynamics in interphase and through mitotic exit, when LAD organization is re-established, as well as intra-LAD subdomain variations.

Acute downregulation of emerin alters actomyosin cytoskeleton connectivity and function.

Fetal lung fibroblasts contribute dynamic infrastructure for the developing lung. These cells undergo dynamic mechanical transitions, including cyclic stretch and spreading, which are integral to lung growth in utero. We investigated the role of the nuclear envelope protein emerin in cellular responses to these dynamic mechanical transitions. In contrast to control cells, which briskly realigned their nuclei, actin cytoskeleton, and extracellular matrices in response to cyclic stretch, fibroblasts that were acutely downregulated for emerin showed incomplete reorientation of both nuclei and actin cytoskeleton. Emerin-downregulated fibroblasts were also aberrantly circular in contrast to the spindle-shaped controls and exhibited an altered pattern of filamentous actin organization that was disconnected from the nucleus. Emerin knockdown was also associated with reduced myosin light chain phosphorylation during cell spreading. Interestingly, emerin-downregulated fibroblasts also demonstrated reduced fibronectin fibrillogenesis and production. These findings indicate that nuclear-cytoskeletal coupling serves a role in the dynamic regulation of cytoskeletal structure and function and may also impact the transmission of traction force to the extracellular matrix microenvironment.

Genomic Action of Sigma-1 Receptor Chaperone Relates to Neuropathic Pain.

Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) chaperones implicated in neuropathic pain. Here we examine if the Sig-1R may relate to neuropathic pain at the level of dorsal root ganglia (DRG). We focus on the neuronal excitability of DRG in a "spare nerve injury" (SNI) model of neuropathic pain in rats and find that Sig-1Rs likely contribute to the genesis of DRG neuronal excitability by decreasing the protein level of voltage-gated Cav2.2 as a translational inhibitor of mRNA. Specifically, during SNI, Sig-1Rs translocate from ER to the nuclear envelope via a trafficking protein Sec61ß. At the nucleus, the Sig-1R interacts with cFos and binds to the promoter of 4E-BP1, leading to an upregulation of 4E-BP1 that binds and prevents eIF4E from initiating the mRNA translation for Cav2.2. Interestingly, in Sig-1R knockout HEK cells, Cav2.2 is upregulated. In accordance with those findings, we find that intra-DRG injection of Sig-1R agonist (+)pentazocine increases frequency of action potentials via regulation of voltage-gated Ca2+ channels. Conversely, intra-DRG injection of Sig-1R antagonist BD1047 attenuates neuropathic pain. Hence, we discover that the Sig-1R chaperone causes neuropathic pain indirectly as a translational inhibitor.

Helping Scholars Overcome Socioeconomic Barriers to Medical and Biomedical Careers: Creating a Pipeline Initiative.

Problem: To achieve their potential in medical and biomedical careers, students (scholars) from under-resourced backgrounds must build sophisticated skills and develop confidence and professionalism. To flourish in an advanced educational system that may be unfamiliar, these scholars also need networks of mentors and role models. These challenges can affect scholars at multiple stages of their education. Intervention: To meet these challenges, we created a broad and innovative biomedical research-focused pipeline program: the Johns Hopkins Initiative for Careers in Science in Medicine (CSM Initiative). This initiative targets three levels: high school, undergraduate, and post-baccalaureate/pre-doctoral (graduate and medical). We provide training in essential academic, research, professional, and social skills to meet the unique challenges of our scholars from under-resourced backgrounds. Scholars also build relationships with mentors who provide career guidance and support. We present an overview of the training and assessment at each level of this initiative. Context: The initiative took place at an institution located in the greater Baltimore area and that is endowed with exceptional doctoral and postdoctoral trainees, staff, and faculty including clinicians, physician-scientists, and scientists who served as key role models and mentors. Our pipeline program draws from local high school students and a local and national pool of undergraduates and post-baccalaureates preparing for medical or graduate school. Impact: Our goals for the high school scholars are significant improvement in academic skills, increased confidence, and matriculation into higher education systems. Currently, at least 83% of high school scholars have matriculated into four-year college programs and 73% have chosen science, technology, engineering, math, and medicine (STEMM)-related majors. Among undergraduate participants, 42% have matriculated thus far into medical or biomedical graduate programs and this number is expected to rise as more scholars graduate from college and either enter graduate training or pursue STEMM careers. Another 25% have returned to our post-baccalaureate program. Among post-baccalaureate scholars, 71% have now matriculated into doctoral-level graduate biomedical programs (medical or graduate school) and the remaining 29% are pursuing careers in STEMM-related fields such as biomedical research with some still aiming at graduate-level education. Our long-term goal is to see a large majority of our scholars become successful professionals in medicine, biomedical research, allied healthcare, or other STEMM fields. Analysis of the early phases of the CSM initiative demonstrates such outcomes are attainable. Lessons Learned: This program provides experiences in which scholars develop and practice core competencies essential for developing their self-identity as scientists and professionals. The most important lesson learned is that mentorship teams must be highly dynamic, flexible, thoughtful, and personal in responding to the wide range of challenges and obstacles that scholars from under-resourced backgrounds must overcome to achieve career success.

Rare BANF1 Alleles and Relatively Frequent EMD Alleles Including 'Healthy Lipid' Emerin p.D149H in the ExAC Cohort.

Emerin (EMD) and barrier to autointegration factor 1 (BANF1) each bind A-type lamins (LMNA) as fundamental components of nuclear lamina structure. Mutations in LMNA, EMD and BANF1 are genetically linked to many tissue-specific disorders including Emery-Dreifuss muscular dystrophy and cardiomyopathy (LMNA, EMD), lipodystrophy, insulin resistance and type 2 diabetes (LMNA) and progeria (LMNA, BANF1). To explore human genetic variation in these genes, we analyzed EMD and BANF1 alleles in the Exome Aggregation Consortium (ExAC) cohort of 60,706 unrelated individuals. We identified 13 rare heterozygous BANF1 missense variants (p.T2S, p.H7Y, p.D9N, p.S22R, p.G25E, p.D55N, p.D57Y, p.L63P, p.N70T, p.K72R, p.R75W, p.R75Q, p.G79R), and one homozygous variant (p.D9H). Several variants are known (p.G25E) or predicted (e.g., p.D9H, p.D9N, p.L63P) to perturb BANF1 and warrant further study. Analysis of EMD revealed two previously identified variants associated with adult-onset cardiomyopathy (p.K37del, p.E35K) and one deemed 'benign' in an Emery-Dreifuss patient (p.D149H). Interestingly p.D149H was the most frequent emerin variant in ExAC, identified in 58 individuals (overall allele frequency 0.06645%), of whom 55 were East Asian (allele frequency 0.8297%). Furthermore, p.D149H associated with four 'healthy' traits: reduced triglycerides (-0.336; p = 0.0368), reduced waist circumference (-0.321; p = 0.0486), reduced cholesterol (-0.572; p = 0.000346) and reduced LDL cholesterol (-0.599; p = 0.000272). These traits are distinct from LMNA-associated metabolic disorders and provide the first insight that emerin influences metabolism. We also identified one novel in-frame deletion (p.F39del) and 62 novel emerin missense variants, many of which were relatively frequent and potentially disruptive including p.N91S and p.S143F (∼0.041% and ∼0.034% of non-Finnish Europeans, respectively), p.G156S (∼0.39% of Africans), p.R204G (∼0.18% of Latinx), p.R207P (∼0.08% of South Asians) and p.R221L (∼0.15% of Latinx). Many novel BANF1 variants are predicted to disrupt dimerization or binding to DNA, histones, emerin or A-type lamins. Many novel emerin variants are predicted to disrupt emerin filament dynamics or binding to BANF1, HDAC3, A-type lamins or other partners. These new human variants provide a foundational resource for future studies to test the molecular mechanisms of BANF1 and emerin function, and to understand the link between emerin variant p.D149H and a 'healthy' lipid profile.

Nuclear import pathway key to rescuing dominant progerin phenotypes.

In this issue of Science Signaling, Larrieu et al show that an acetyltransferase inhibitor that rescues many dominant nuclear phenotypes caused by progerin, a truncated form of lamin A, does so by releasing the specialized nuclear import receptor TNPO1 from sequestration by microtubules. This release enables TNPO1-dependent import of specific cargoes, including the nuclear pore protein Nup153 and the heterogeneous nuclear ribonucleoprotein hnRNPA1, thus restoring the functionality of nuclear pore complexes, rebalancing the nucleocytoplasmic Ran gradient, and normalizing gene expression.

OGT (O-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A.

The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in LMNA cause over 12 diseases ('laminopathies'). Lamins A and C are identical for their first 566 residues. However, they form separate filaments in vivo, with apparently distinct roles. We report that lamin A is ß-O-linked N-acetylglucosamine-(O-GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified O-GlcNAc transferase (OGT) enzyme showed robust O-GlcNAcylation of recombinant mature lamin A tails (residues 385⁻646), with no detectable modification of lamin B1, lamin C, or 'progerin' (Δ50) tails. Using mass spectrometry, we identified 11 O-GlcNAc sites in a 'sweet spot' unique to lamin A, with up to seven sugars per peptide. Most sites were unpredicted by current algorithms. Double-mutant (S612A/T643A) lamin A tails were still robustly O-GlcNAc-modified at seven sites. By contrast, O-GlcNAcylation was undetectable on tails bearing deletion Δ50, which causes Hutchinson⁻Gilford progeria syndrome, and greatly reduced by deletion Δ35. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion Δ35, which does not remove the majority of identified O-GlcNAc sites, does remove potential OGT-association motifs (lamin A residues 622⁻625 and 639⁻645) homologous to that in mouse Tet1. These biochemical results are significant because they identify a novel molecular pathway that may profoundly influence lamin A function. The hypothesis that lamin A is selectively regulated by OGT warrants future testing in vivo, along with two predictions: genetic variants may contribute to disease by perturbing OGT-dependent regulation, and nutrient or other stresses might cause OGT to misregulate wildtype lamin A.

LMNA Sequences of 60,706 Unrelated Individuals Reveal 132 Novel Missense Variants in A-Type Lamins and Suggest a Link between Variant p.G602S and Type 2 Diabetes.

Mutations in LMNA, encoding nuclear intermediate filament proteins lamins A and C, cause multiple diseases ('laminopathies') including muscular dystrophy, dilated cardiomyopathy, familial partial lipodystrophy (FPLD2), insulin resistance syndrome and progeria. To assess the prevalence of LMNA missense mutations ('variants') in a broad, ethnically diverse population, we compared missense alleles found among 60,706 unrelated individuals in the ExAC cohort to those identified in 1,404 individuals in the laminopathy database (UMD-LMNA). We identified 169 variants in the ExAC cohort, of which 37 (∼22%) are disease-associated including p.I299V (allele frequency 0.0402%), p.G602S (allele frequency 0.0262%) and p.R644C (allele frequency 0.124%), suggesting certain LMNA mutations are more common than previously recognized. Independent analysis of LMNA variants via the type 2 diabetes (T2D) Knowledge Portal showed that variant p.G602S associated significantly with type 2 diabetes (p = 0.02; odds ratio = 4.58), and was more frequent in African Americans (allele frequency 0.297%). The FPLD2-associated variant I299V was most prevalent in Latinos (allele frequency 0.347%). The ExAC cohort also revealed 132 novel LMNA missense variants including p.K108E (limited to individuals with psychiatric disease; predicted to perturb coil-1B), p.R397C and p.R427C (predicted to perturb filament biogenesis), p.G638R and p.N660D (predicted to perturb prelamin A processing), and numerous Ig-fold variants predicted to perturb phenotypically characteristic protein-protein interactions. Overall, this two-pronged strategy- mining a large database for missense variants in a single gene (LMNA), coupled to knowledge about the structure, biogenesis and functions of A-type lamins- revealed an unexpected number of LMNA variants, including novel variants predicted to perturb lamin assembly or function. Interestingly, this study also correlated novel variant p.K108E with psychiatric disease, identified known variant p.I299V as a potential risk factor for metabolic disease in Latinos, linked variant p.G602 with type 2 diabetes, and identified p.G602S as a predictor of diabetes risk in African Americans.

Simple Separation of Functionally Distinct Populations of Lamin-Binding Proteins.

The inner membrane of the nuclear envelope (NE) is home to hundreds of integral membrane proteins (NE transmembrane proteins, "NETs") with conserved or tissue-specific roles in genome organization and nuclear function. Nearly all characterized NETs bind A- or B-type lamins directly. However, hundreds of NETs remain uncharacterized, collectively posing an enormous gap that must be bridged to understand nuclear function and genome biology. We provide technically simple protocols for the separation and recovery of functionally distinct populations of NETs and A-type lamins. This protocol was developed for emerin, an inner nuclear membrane protein that binds lamins and barrier-to-autointegration factor (BANF1) as a component of nuclear lamina structure, and has diverse roles in nuclear assembly, signaling, and gene regulation. This protocol separates easily solubilized ("easy") populations of nuclear lamina proteins (emerin, lamin A, BAF) from "sonication-dependent" populations. Depending on cell type, the "easy" and "sonication-dependent" fractions each contain up to about half the available emerin, A-type lamins, and BAF, whereas B-type lamins and histone H3 are predominantly sonication dependent. The two populations of emerin have distinct posttranslational modifications, and only one population associates with BAF. This method may be useful for functional screening or analysis of other lamin-associated proteins, including novel NETs emerging from proteomic studies.

SINC, a type III secreted protein of Chlamydia psittaci, targets the inner nuclear membrane of infected cells and uninfected neighbors.

SINC, a new type III secreted protein of the avian and human pathogen Chlamydia psittaci, uniquely targets the nuclear envelope of C. psittaci-infected cells and uninfected neighboring cells. Digitonin-permeabilization studies of SINC-GFP-transfected HeLa cells indicate that SINC targets the inner nuclear membrane. SINC localization at the nuclear envelope was blocked by importazole, confirming SINC import into the nucleus. Candidate partners were identified by proximity to biotin ligase-fused SINC in HEK293 cells and mass spectrometry (BioID). This strategy identified 22 candidates with high confidence, including the nucleoporin ELYS, lamin B1, and four proteins (emerin, MAN1, LAP1, and LBR) of the inner nuclear membrane, suggesting that SINC interacts with host proteins that control nuclear structure, signaling, chromatin organization, and gene silencing. GFP-SINC association with the native LEM-domain protein emerin, a conserved component of nuclear "lamina" structure, or with a complex containing emerin was confirmed by GFP pull down. Our findings identify SINC as a novel bacterial protein that targets the nuclear envelope with the capability of globally altering nuclear envelope functions in the infected host cell and neighboring uninfected cells. These properties may contribute to the aggressive virulence of C. psittaci.

The molecular basis of emerin-emerin and emerin-BAF interactions.

Emerin is a conserved membrane component of nuclear lamina structure. Here, we report an advance in understanding the molecular basis of emerin function: intermolecular emerin-emerin association. There were two modes: one mediated by association of residues 170-220 in one emerin molecule to residues 170-220 in another, and the second involving residues 170-220 and 1-132. Deletion analysis showed residues 187-220 contain a positive element essential for intermolecular association in cells. By contrast, deletion of residues 168-186 inactivated a proposed negative element, required to limit or control association. Association of GFP-emerin with nuclear BAF in cells required the LEM domain (residues 1-47) and the positive element. Emerin peptide arrays revealed direct binding of residues 170-220 to residues 206-225 (the proposed positive element), residues 147-174 (particularly P(153)MYGRDSAYQSITHYRP(169)) and the LEM domain. Emerin residues 1-132 and 159-220 were each sufficient to bind lamin A or B1 tails in vitro, identifying two independent regions of molecular contact with lamins. These results, and predicted emerin intrinsic disorder, support the hypothesis that there are multiple 'backbone' and LEM-domain configurations in a proposed intermolecular emerin network at the nuclear envelope.